WO2022161472A1

WO2022161472A1 - Sars-cov-2 binding molecules and uses thereof

Info

Publication number: WO2022161472A1
Application number: PCT/CN2022/074735
Authority: WO
Inventors: Xiaohu FAN; Qiuchuan ZHUANG; Xu Fang; Jianrui ZHU; Hefei HOU
Original assignee: Nanjing Legend Biotech Co., Ltd.
Priority date: 2021-01-29
Filing date: 2022-01-28
Publication date: 2022-08-04

Abstract

The present disclosure provides single domain antibodies that bind to SARS-CoV-2 S1, and fusion proteins comprising same. Pharmaceutical compositions, kits and methods of preventing or treating a disease or disorder such as a respiratory disease are also provided.

Description

SARS-COV-2 BINDING MOLECULES AND USES THEREOF

1. CROSS REFERENCE

This application claims benefit of priority of International Patent Application No. PCT/CN2021/074429 filed on January 29, 2021, the content of which is incorporated herein by reference in its entirety.

2. SEQUENCE LISTING

This application incorporates by reference a Sequence Listing submitted with this application as a text format, entitled “14651-037-228_SEQ_LISTING. txt, ” created on January 17, 2022 having a size of 174, 943 bytes.

3. FIELD

The present disclosure relates to single domain antibodies targeting SARS-CoV-2 S1 and methods of use thereof.

4. BACKGROUND

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly-emergent coronavirus which causes a coronavirus disease 2019 (COVID-19) . Clinical features of COVID-19 include fever, dry cough, and fatigue, and the disease can cause respiratory failure resulting in death. Moreover, SARS-CoV-2 spreads rapidly in the

human population, the current COVID-19 pandemic has presented an unprecedented challenge to modern human society. In view of the continuing threat to human health, there is an urgent need for preventive and therapeutic antiviral therapies for SARS-CoV-2 control.

5. SUMMARY

In one aspect, provided herein is a single domain antibody (sdAb) such as a VHH domain that binds to the S protein of SARS-CoV-2. In some embodiments, the sdAb provided herein binds to the S1 subunit of the S protein (i.e., SARS-CoV-2 S1) . In some embodiments, the VHH domain provided herein binds to the receptor binding domain (RBD) of SARS-CoV-2. In some embodiments, the anti-SARS-CoV-2 S1 sdAb provided herein comprises (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or (iv) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments, provided herein is an anti-SARS-CoV-2 S1 single domain antibody (sdAb) comprising (i) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 13; (ii) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 14; (iii) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 15; (iv) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 16; (v) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 17; (vi) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 18; (vii) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 19; (viii) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 20; (ix) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 21; (x) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 22; (xi) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 23; or (xii) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 24. In some embodiments, the CDR1, CDR2 or CDR3 are determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof.

In some embodiments, the anti-SARS-CoV-2 S1 sdAb provided herein further comprises one or more FR regions as set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and/or SEQ ID NO: 24.

In some embodiments, the anti-SARS-CoV-2 S1 sdAb provided herein comprises the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

In some embodiments, the anti-SARS-CoV-2 S1 sdAb provided herein is a camelid sdAb. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is a humanized sdAb. In some embodiments, the sdAb is a VHH domain.

In another aspect, provided herein is a fusion protein comprising an sdAb provided herein.

In some embodiments, the anti-SARS-CoV-2 S1 sdAb is genetically fused or chemically conjugated to an agent. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is fused to an Fc region. In some embodiments, the Fc region is a human IgG1 Fc region or a variant thereof comprising the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63.

In some embodiments, the fusion protein provided herein comprises the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36.

In some embodiments, provided herein is a fusion protein comprising a first anti-SARS-CoV-2 S1 sdAb and a second anti-SARS-CoV-2 S1 sdAb, wherein each of the first and the second anti-SARS-CoV-2 S1 sdAb is a VHH domain.

In some embodiments, the first anti-SARS-CoV-2 S1 sdAb and the second anti-SARS-CoV-2 S1 sdAb are each independently an sdAb provided herein.

In some embodiments, the first anti-SARS-CoV-2 S1 sdAb and the second anti-SARS-CoV-2 S1 sdAb are the same. In other embodiments, the first anti-SARS-CoV-2 S1 sdAb and the second anti-SARS-CoV-2 S1 sdAb are different. In some embodiments, the first anti-SARS-CoV-2 S1 sdAb is at the N terminus of the second anti-SARS-CoV-2 S1 sdAb. In other embodiments, the first anti-SARS-CoV-2 S1 sdAb is at the C terminus of the second anti-SARS-CoV-2 S1 sdAb.

In some embodiments, the fusion protein further comprises one or more additional agent (s) . In some embodiments, the fusion protein further comprises a domain comprising a human IgG1 Fc region or a variant thereof. In some embodiments, the domain comprises the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63. In some embodiments, the fusion protein comprises from N terminus to C terminus: the first anti-SARS-CoV-2 S1 sdAb-IgG1 Fc-the second anti-SARS-CoV-2 S1 sdAb. In other embodiments, the fusion protein comprises from N terminus to C terminus: the first anti-SARS-CoV-2 S1 sdAb-the second anti-SARS-CoV-2 S1 sdAb-IgG1 Fc. In some embodiments, two or more regions in the fusion protein are linked directly or via a peptide linker.

In some embodiments, the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10;and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12; and wherein the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12; and wherein the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12; and wherein the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the fusion protein provided herein comprises the amino acid sequence of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60, or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.

In another aspect, provided herein is an isolated nucleic acid comprising a nucleic acid sequence encoding the anti-SARS-CoV-2 S1 sdAb provided herein or the fusion protein provided herein. In yet another aspect, provided herein is a vector comprising the isolated nucleic acid provided herein.

In yet another aspect, provided herein is a pharmaceutical composition, comprising the anti-SARS-CoV-2 S1 sdAb, the fusion protein, or the vector provided herein, and a pharmaceutically acceptable excipient.

In yet another aspect, provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the anti-SARS-CoV-2 S1 sdAb, the fusion protein, the vector, or the pharmaceutical composition provided herein. In some embodiments, the disease or disorder is a SARS-CoV-2 associated disease or disorder. In some embodiments, the disease or disorder is a respiratory disease. In some embodiments, the respiratory disease is severe acute respiratory syndrome (SARS) or coronavirus disease (COVID-19) . In some embodiments, the disease or disorder is a cardiovascular disease.

6. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the potency of chimeric single domain antibodies blocking the binding of S1-His or RBD-His to HeLa-ACE2 cells. RBD-His indicates SARS-CoV-2 S protein RBD expressed with a His tag. S1-His indicates SARS-CoV-2 S1 protein with a His tag. Conc. is short for concentration.

FIG. 2 shows the potency of chimeric single domain antibodies blocking the binding of SARS-CoV-2 S protein variants to HeLa-ACE2 cells.

FIGs. 3A-3E show the resistance of chimeric single domain antibodies to alkali, acid, oxidative stress, as well as temperature. FZ-3rd/5th cycle indicates antibodies underwent 3 or 5 freeze-and-thaw cycles; 500 mM NH4HCO3-14h/22h/38h indicates 500 mM NH4HCO3 was used as alkali disrupting agent and antibodies were treated at 37℃ for 14, 22 or 38 hours; Low pH-2h/4h indicates pH 3.5 citric acid was used as acid disrupting agent and antibodies were treated at 37℃ for 2 or 4 hours; H2O2-4h/8h indicates 1%hydrogen peroxide was used as the oxidant, 4 or 8 hours treatment at room temperature; 40C-D7/D14/D28 indicates antibodies were stored at 40℃ for 1 week, 2 weeks or 4 weeks.

FIG. 4 shows the potency of chimeric Bi-VHH antibodies for blocking the binding of SARS-CoV-2 S1 protein to HeLa-ACE2 cells. S1-His indicates SARS-CoV-2 S1 protein with a His tag. Conc. is short for concentration.

FIG. 5 shows the potency of humanized chimeric antibodies for blocking the binding of SARS-CoV-2 S1 protein to HeLa-ACE2 cells. S1-His indicates SARS-CoV-2 S1 protein with a His tag. Conc. is short for concentration.

7. DETAILED DESCRIPTION

The present disclosure is based in part on the novel single domain antibodies (e.g., VHH domains) that bind to SARS-CoV-2 (e.g., subunit S1 of the S protein or more specifically RBD) , binding molecules comprising same, and improved properties thereof.

Like other coronaviruses, SARS-CoV-2 has four structural proteins, known as the S (spike) , E (envelope) , M (membrane) , and N (nucleocapsid) proteins; the N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. The S protein, which has been imaged at the atomic level using cryogenic electron microscopy, is the protein responsible for allowing the virus to attach to and fuse with the membrane of a host cell; specifically, its S1 subunit catalyzes attachment, and its S2 subunit promotes fusion (Wu C, etal. Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods. Acta Pharm Sin B. 2020 May; 10 (5) : 766-788. ) . During infection, the S protein is cleaved into the N-terminal S1 subunit and C-terminal S2 subunit by host proteases such as transmembrane protease, serine 2 (TMPRSS2) , and changes conformation from the prefusion to the postfusion state. S1 and S2 comprise the extracellular domain (ECD; 1 to 1208 amino acids) and a single transmembrane helix and mediate receptor binding and membrane fusion, respectively. S1, which consists of the N-terminal domain (NTD) and the receptor binding domain (RBD) , is critical in determining tissue tropism and host ranges (Chi X, etal.. A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science. 2020 Aug 7; 369 (6504) : 650-655. ) . The RBD of SARS-CoV-2 binds tightly to the extracellular domain of angiotensin-converting enzyme 2 (ACE2) on human cells as a mechanism of cell entry (Huo J, etal., Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2. Nat Struct Mol Biol. 2020 Sep; 27 (9) : 846-854. ) . The SARS-CoV-2 S protein-targeting monoclonal antibodies (mAbs) with potent neutralizing activity are a focus in the development of therapeutic interventions for COVID-19. However, the RBD-targeting mAbs, applied individually, might induce resistance mutations in the virus (Chi X, etal.. A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science. 2020 Aug 7; 369 (6504) : 650-655. ) . Thus, there is a need in the art for improved binding molecules for SARS-CoV-2, particularly targeting S1 or more specifically RBD. The single domain antibodies (e.g., V _HH) with neutralization activity against S1 protein or RBD of SARS-CoV-2 provided herein can be used both as research tools and potential therapeutics in the prevention and treatment of COVID-19.

5.1. Definitions

Techniques and procedures described or referenced herein include those that are generally well understood and/or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001) ; Current Protocols in Molecular Biology (Ausubel et al. eds., 2003) ; Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009) ; Monoclonal Antibodies : Methods and Protocols (Albitar ed. 2010) ; and

Antibody

Engineering Vols 1 and 2 (Kontermann and Dübel eds., 2d ed. 2010) . Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

The term “antibody, ” “immunoglobulin, ” or “Ig” is used interchangeably herein, and is used in the broadest sense and specifically covers, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies) , antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) , formed from at least two intact antibodies, single chain antibodies, and fragments thereof (e.g., domain antibodies) , as described below. An antibody can be human, humanized, chimeric and/or affinity matured, as well as an antibody from other species, for example, mouse, rabbit, llama, etc. The term “antibody” is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) , each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed. 1995) ; and Kuby, Immunology (3d ed. 1997) . Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies including from Camelidae species (e.g., llama or alpaca) or their humanized variants, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc. ) , Fab fragments, F (ab’) fragments, F (ab) ₂ fragments, F (ab’) ₂ fragments, disulfide-linked Fvs (dsFv) , Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody) . Such antibody fragments can be found in, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989) ; Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995) ; Huston et al., 1993, Cell Biophysics 22: 189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178: 497-515; and Day, Advanced Immunochemistry (2d ed. 1990) . The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. Antibodies may be agonistic antibodies or antagonistic antibodies _. Antibodies may be neither agonistic nor antagonistic.

An “antigen” is a structure to which an antibody can selectively bind. A target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, an antigen is associated with a cell, for example, is present on or in a cell.

An “intact” antibody is one comprising an antigen-binding site as well as a CL and at least heavy chain constant regions, CH1, CH2 and CH3. The constant regions may include human constant regions or amino acid sequence variants thereof. In certain embodiments, an intact antibody has one or more effector functions.

The term “heavy chain-only antibody” or “HCAb” refers to a functional antibody, which comprises heavy chains, but lacks the light chains usually found in 4-chain antibodies. Camelidanimals (such as camels, llamas, or alpacas) are known to produce HCAbs.

“Single domain antibody” or “sdAb” as used herein refers to a single monomeric variable antibody domain and which is capable of antigen binding (e.g., single domain antibodies that bind to SARS-CoV-2 S1 (e.g., RBD) ) . Single domain antibodies include VHH domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies naturally devoid of light chains such as those from Camelidae species (e.g., llama) , single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, and bovine. For example, a single domain antibody can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco, as described herein. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; VHHs derived from such other species are within the scope of the disclosure. In some embodiments, the single domain antibody (e.g., VHH domain) provided herein has a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies may be genetically fused or chemically conjugated to another molecule (e.g., an agent) as described herein. Single domain antibodies may be part of a bigger binding molecule (e.g., an Fc fusion protein or multispecific binding molecules comprising two or more binding domains) .

The terms “binds” or “binding” refer to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The ratio of dissociation rate (k _off) to association rate (k _on) of a binding molecule (e.g., an antibody) to a monovalent antigen (k _off/k _on) is the dissociation constant K _D, which is inversely related to affinity. The lower the K _D value, the higher the affinity of the antibody. The value of K _D varies for different complexes of antibody and antigen and depends on both k _on and k _off. The dissociation constant K _D for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art.

In certain embodiments, the binding molecules or antigen binding domains can comprise “chimeric” sequences in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-55) . Chimeric sequences may include humanized sequences.

In certain embodiments, the binding molecules or antigen binding domains can comprise portions of “humanized” forms of nonhuman (e.g., camelid, murine, non-human primate) antibodies that include sequences from human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as camelid, mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin sequences are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. A humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin. For further details, see, Jones et al., Nature 321: 522-25 (1986) ; Riechmann et al., Nature 332: 323-29 (1988) ; Presta, Curr. Op. Struct. Biol. 2: 593-96 (1992) ; Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285-89 (1992) ; U.S. Pat. Nos: 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

In certain embodiments, the binding molecules or antigen binding domains can comprise portions of a “fully human antibody” or “human antibody, ” wherein the terms are used interchangeably herein and refer to an antibody that comprises a human variable region and, for example, a human constant region. The binding molecules may comprise a single domain antibody sequence. In specific embodiments, the terms refer to an antibody that comprises a variable region and constant region of human origin. “Fully human” antibodies, in certain embodiments, can also encompass antibodies which bind polypeptides and are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence. The term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) . A “human antibody” is one that possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991) ; Marks et al., J. Mol. Biol. 222: 581 (1991) ) and yeast display libraries (Chao et al., Nature Protocols 1: 755-68 (2006) ) . Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985) ; Boerner et al., J. Immunol. 147 (1) : 86-95 (1991) ; and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) . Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., mice (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6 (5) : 561-66 (1995) ; Brüggemann and Taussing, Curr. Opin. Biotechnol. 8 (4) : 455-58 (1997) ; and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE ^TM technology) . See also, for example, Li et al., Proc. Natl. Acad. Sci. USA 103: 3557-62 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

In certain embodiments, the binding molecules or antigen binding domains can comprise portions of a “recombinant human antibody, ” wherein the phrase includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, L.D. et al., Nucl. Acids Res. 20: 6287-6295 (1992) ) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (See Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) . In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

In certain embodiments, the binding molecules or antigen binding domains can comprise a portion of a “monoclonal antibody, ” wherein the term as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts or well-known post-translational modifications such as amino acid iomerizatio or deamidation, methionine oxidation or asparagine or glutamine deamidation, each monoclonal antibody will typically recognize a single epitope on the antigen. In specific embodiments, a “monoclonal antibody, ” as used herein, is an antibody produced by a single hybridoma or other cell. The term “monoclonal” is not limited to any particular method for making the antibody. For example, the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., Nature 256: 495 (1975) , or may be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567) . The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352: 624-28 (1991) and Marks et al., J. Mol. Biol. 222: 581-97 (1991) , for example. Other methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are well known in the art. See, e.g., Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002) .

A typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain of the heavy chain (CH1) . Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994) ; and Immunobiology (Janeway et al. eds., 5 ^th ed. 2001) .

The term “variable region, ” “variable domain, ” “V region, ” or “V domain” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as “VH. ” The variable region of the light chain may be referred to as “VL. ” The term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long. The variable regions of heavy and light chains each comprise four FRs, largely adopting a β sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the β sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991) ) . The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) . The variable regions differ extensively in sequence between different antibodies. In specific embodiments, the variable region is a human variable region.

The term “variable region residue numbering according to Kabat” or “amino acid position numbering as in Kabat” , and variations thereof, refer to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, an FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 and three inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra) . The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra) . The “EU index as in Kabat” refers to the residue numbering of the human IgG 1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy-terminal portion includes a constant region. The constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha (α) , delta (δ) , epsilon (ε) , gamma (γ) , and mu (μ) , based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4.

The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy-terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (κ) or lambda (λ) based on the amino acid sequence of the constant domains.

As used herein, the terms “hypervariable region, ” “HVR, ” “Complementarity Determining Region, ” and “CDR” are used interchangeably. A “CDR” refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL β-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences.

CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems. For example, the Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra; Deschacht et al., J Immunol 2010; 184: 5696-5704) . Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196: 901-17 (1987) ) . The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34) . The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dübel eds., 2d ed. 2010) ) . The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. Another universal numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information

(Lafranc et al., Dev. Comp. Immunol. 27 (1) : 55-77 (2003) ) . IMGT is an integrated information system specializing in immunoglobulins (IG) , T-cell receptors (TCR) , and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger and Plückthun, J. Mol. Biol. 309: 657-70 (2001) .

Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra) . The residues from each of these hypervariable regions or CDRs are exemplified in Table 1 below.

Table 1. Exemplary CDRs According to Various Numbering Systems

The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms “CDR” and “complementary determining region” of a given antibody or region thereof, such as a variable region, as well as individual CDRs (e.g., CDR-H1, CDR-H2) of the antibody or region thereof, should be understood to encompass the complementary determining region as defined by any of the known schemes described herein above. In some instances, the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR is given. It should be noted CDR regions may also be defined by a combination of various numbering systems, e.g., a combination of Kabat and Chothia numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, the term such as “a CDR as set forth in a specific VH or VHH” includes any CDR1 as defined by the exemplary CDR numbering systems described above, but is not limited thereby. Once a variable region (e.g., a VHH, VH or VL) is given, those skilled in the art would understand that CDRs within the region can be defined by different numbering systems or combinations thereof.

Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (L1) , 46-56 or 50-56 (L2) , and 89-97 or 89-96 (L3) in the VL, and 26-35 or 26-35A (H1) , 50-65 or 49-65 (H2) , and 93-102, 94-102, or 95-102 (H3) in the VH.

In some embodiments, the amino acid residues of a single-domain antibody (such as V _HH) are numbered according to the general numbering for V _H domains given by Kabat et al. ( “Sequence of proteins of immunological interest” , US Public Health Services, NIH Bethesda, Md., Publication No. 91) , as applied to V _HH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2) : 185-195 and Deschacht et al., J Immunol 2010; 184: 5696-5704. According to this numbering, FR1 of a V _HH comprises the amino acid residues at positions 1-30, CDR1 of a V _HH comprises the amino acid residues at positions 31-35, FR2 of a V _HH comprises the amino acids at positions 36-49, CDR2 of a V _HH comprises the amino acid residues at positions 50-65, FR3 of a V _HH comprises the amino acid residues at positions 66-94, CDR3 of a V _HH comprises the amino acid residues at positions 95-102, and FR4 of a V _HH comprises the amino acid residues at positions 103-113. In this respect, it should be noted that-as is well known in the art for V _H domains and for V _HH domains-the total number of amino acid residues in each of the CDR's may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering) . See, e.g., Deschacht et al., 2010. J Immunol 184: 5696-704 for an exemplary numbering for VHH domains according to Kabat.

The term “constant region” or “constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. The term refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site. The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

The term “framework” or “FR” refers to those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies (e.g., single domain antibodies) , diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.

The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor) , etc. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays known to those skilled in the art. A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion) . In certain embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide. The variant Fc region herein can possess at least about 80%homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90%homology therewith, for example, at least about 95%homology therewith.

As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody comprising a single domain antibody sequence) can specifically bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a “conformational, ” “non-linear” or “discontinuous” epitope) . It will be appreciated by one of skill in the art that, in general, a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids regardless of whether they are folded in a natural three dimensional protein structure. In other embodiments, a binding molecule requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.

“Percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN ^TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

An “isolated nucleic acid” is a nucleic acid, for example, a RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding a single domain antibody or an antibody as described herein are isolated or purified. The term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure molecule may include isolated forms of the molecule. Specifically, an “isolated” nucleic acid molecule encoding a fusion protein or an sdAb described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced.

The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, in order to introduce a nucleic acid sequence into a host cell.

The term “host” as used herein refers to an animal, such as a mammal (e.g., a human) .

The term “host cell” as used herein refers to a particular subject cell that may be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

The term “effective amount” or “therapeutically effective amount” as used herein refers to the amount of a single domain antibody or a therapeutic molecule comprising an agent and the single domain antibody or pharmaceutical composition provided herein which is sufficient to result in the desired outcome.

The terms “subject” and “patient” may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate or a primate (e.g., human) . In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In another embodiment, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder.

“Administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art.

As used herein, the terms “treat, ” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or condition resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and/or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder. The term “treating” includes both managing and ameliorating the disease. The terms “manage, ” “managing, ” and “management” refer to the beneficial effects that a subject derives from a therapy which does not necessarily result in a cure of the disease.

The terms “prevent, ” “preventing, ” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom (s) .

The term “SARS-CoV-2 associated disease or disorder” as used herein refers to a disease or disorder caused directly or indirectly and at least in part by the infection of SARS-CoV-2. Such diseases or disorders include but not limited to respiratory diseases and cardiovascular diseases.

The terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

As used in the present disclosure and claims, the singular forms “a” , “an” and “the” include plural forms unless the context clearly dictates otherwise.

It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of” otherwise analogous embodiments described in terms of “consisting of” are also provided.

The term “between” as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.

The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone) ; and B (alone) . Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone) ; B (alone) ; and C (alone) .

5.2. Single Domain Antibodies

5.2.1. Single Domain Antibodies that Bind to SARS-CoV-2 S1

In one aspect, provided herein are single domain antibodies (e.g., VHH domains) capable of binding to SARS-CoV-2. More specifically, provided herein are sdAbs such as VHH domains capable of binding to the S protein of SARS-CoV-2, e.g., the subunit S1 (i.e., SARS-CoV-2 S1) or a variant thereof. In more specific embodiments, the sdAbs provided herein (e.g., VHH domains) bind to RBD of the subunit S1 of the S protein of SARS-CoV-2 or a variant thereof. In some embodiments, the sdAb that binds SARS-CoV-2 S1 also binds one or more of SARS-CoV-2 S protein RBD (N354D, D364Y) , SARS-CoV-2 S protein RBD (V367F) , SARS-CoV-2 S protein RBD (N354D) , SARS-CoV-2 S protein RBD (W436R) , SARS-CoV-2 S protein RBD (R408I) , SARS-CoV-2 S protein RBD (G476S) , SARS-CoV-2 S protein RBD (V483A) , and SARS-CoV-2 S1 protein (D614G) . In some embodiments, provided herein is a sdAb (e.g., a VHH domain) that binds all of SARS-CoV-2 S protein RBD (N354D, D364Y) , SARS-CoV-2 S protein RBD (V367F) , SARS-CoV-2 S protein RBD (N354D) , SARS-CoV-2 S protein RBD (W436R) , SARS-CoV-2 S protein RBD (R408I) , SARS-CoV-2 S protein RBD (G476S) , SARS-CoV-2 S protein RBD (V483A) , and SARS-CoV-2 S1 protein (D614G) .

In some embodiments, the anti-SARS-CoV-2 S1 single domain antibody provided herein binds to SARS-CoV-2 S1 (e.g., RBD) with a dissociation constant (K _D) of ≤ 1μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g. 10 ^-8 M or less, e.g. from 10 ^-8 M to 10 ^-13 M, e.g., from 10 ^-9 M to 10 ^-13 M) . A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, including by RIA, for example, performed with the Fab version of an antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293: 865-81) ; by biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by

using, for example, an

system, or by

using, for example, a

or a

An “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the

the

or the

system.

In some embodiments, the anti-SARS-CoV-2 S1 single domain antibodies provided herein are VHH domains. Exemplary VHH domains provided herein are generated as described below in Section 6, including those VHH domains referred to as 77NCOVP01, 77NCOVP02, 77NCOVP03, 77NCOVP04, 77NCOVP01H1, 77NCOVP01H2, 77NCOVP02H1, 77NCOVP02H2, 77NCOVP03H1, 77NCOVP03H2, 77NCOVP04H1, and 77NCOVP04H2 (as also shown in Table 4 and Table 16) .

Thus, in some embodiments, the single domain antibody provided herein comprises one or more CDR sequences of any one of 77NCOVP01, 77NCOVP02, 77NCOVP03, 77NCOVP04, 77NCOVP01H1, 77NCOVP01H2, 77NCOVP02H1, 77NCOVP02H2, 77NCOVP03H1, 77NCOVP03H2, 77NCOVP04H1, and 77NCOVP04H2. In some embodiments, provided herein is a single domain antibody that binds to SARS-CoV-2 S1 (e.g., RBD) comprising the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the CDR sequences are selected from those in 77NCOVP01, 77NCOVP02, 77NCOVP03, 77NCOVP04, 77NCOVP01H1, 77NCOVP01H2, 77NCOVP02H1, 77NCOVP02H2, 77NCOVP03H1, 77NCOVP03H2, 77NCOVP04H1, and 77NCOVP04H2.

In some embodiments, there is provided an anti-SARS-CoV-2 S1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 13. In some embodiments, there is provided an anti-SARS-CoV-2 S1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided an anti-SARS-CoV-2 S1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 15. In some embodiments, there is provided an anti-SARS-CoV-2 S1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided an anti-SARS-CoV-2 S1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 17. In some embodiments, there is provided an anti-SARS-CoV-2 S1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 18. In some embodiments, there is provided an anti-SARS-CoV-2 S1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 19. In some embodiments, there is provided an anti-SARS-CoV-2 S1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 20. In some embodiments, there is provided an anti-SARS-CoV-2 S1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 21. In some embodiments, there is provided an anti-SARS-CoV-2 S1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 22. In some embodiments, there is provided an anti-SARS-CoV-2 S1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 23. In some embodiments, there is provided an anti-SARS-CoV-2 S1 sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb has a CDR1 having the amino acid sequence of the CDR1 as set forth in SEQ ID NO: 13. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 13. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 13. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 13. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 13. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 13. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 13. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb has a CDR1 having the amino acid sequence of the CDR1 as set forth in SEQ ID NO: 14. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 14. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 14. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 14. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 14. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 14. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 14. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb has a CDR1 having the amino acid sequence of the CDR1 as set forth in SEQ ID NO: 15. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 15. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 15. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 15. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 15. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 15. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 15. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb has a CDR1 having the amino acid sequence of the CDR1 as set forth in SEQ ID NO: 16. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 16. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 16. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 16. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 16. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 16. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 16. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb has a CDR1 having the amino acid sequence of the CDR1 as set forth in SEQ ID NO: 17. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 17. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 17. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 17. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 17. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 17. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 17. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb has a CDR1 having the amino acid sequence of the CDR1 as set forth in SEQ ID NO: 18. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 18. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 18. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 18. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 18. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 18. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 18. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb has a CDR1 having the amino acid sequence of the CDR1 as set forth in SEQ ID NO: 19. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 19. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 19. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 19. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 19. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 19. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 19. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb has a CDR1 having the amino acid sequence of the CDR1 as set forth in SEQ ID NO: 20. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 20. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 20. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 20. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 20. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 20. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 20. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb has a CDR1 having the amino acid sequence of the CDR1 as set forth in SEQ ID NO: 21. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 21. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 21. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 21. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 21. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 21. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 21. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb has a CDR1 having the amino acid sequence of the CDR1 as set forth in SEQ ID NO: 22. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 22. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 22. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 22. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 22. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 22. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 22. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti- SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb has a CDR1 having the amino acid sequence of the CDR1 as set forth in SEQ ID NO: 23. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 23. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 23. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 23. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 23. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 23. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 23. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb has a CDR1 having the amino acid sequence of the CDR1 as set forth in SEQ ID NO: 24. In some embodiments, the sdAb has a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 24. In other embodiments, the sdAb has a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 24. In some embodiments, the sdAb has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 24. In some embodiments, the sdAb has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 24. In some embodiments, the sdAb has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 24. In some embodiments, the sdAb has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 24. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, provided herein is a sdAb that binds to SARS-CoV-2 S1 (e.g., RBD) comprising the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein (i) the CDR1 comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; (ii) the CDR2 comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; and/or (iii) the CDR3 comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In other embodiments, provided herein is an sdAb that binds to SARS-CoV-2 S1 (e.g., RBD) comprising the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein (i) the CDR1 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; (ii) the CDR2 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; and/or (iii) the CDR3 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, provided herein is an anti-SARS-CoV-2 S1 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, provided herein is an anti-SARS-CoV-2 S1 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, provided herein is an anti-SARS-CoV-2 S1 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, provided herein is an anti-SARS-CoV-2 S1 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is camelid. In some embodiments, the anti-SARS-CoV-2 S1 sdAb is humanized. In some embodiments, the anti-SARS-CoV-2 S1 sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, the sdAb further comprises one or more framework region (s) of 77NCOVP01, 77NCOVP02, 77NCOVP03, 77NCOVP04, 77NCOVP01H1, 77NCOVP01H2, 77NCOVP02H1, 77NCOVP02H2, 77NCOVP03H1, 77NCOVP03H2, 77NCOVP04H1, and/or 77NCOVP04H2. In some embodiments, the sdAb comprises one or more framework (s) derived from a VHH domain comprising the sequence of SEQ ID NO: 13. In some embodiments, the sdAb comprises one or more framework (s) derived from a VHH domain comprising the sequence of SEQ ID NO: 14. In some embodiments, the sdAb comprises one or more framework (s) derived from a VHH domain comprising the sequence of SEQ ID NO: 15. In some embodiments, the sdAb comprises one or more framework (s) derived from a VHH domain comprising the sequence of SEQ ID NO: 16. In some embodiments, the sdAb comprises one or more framework (s) derived from a VHH domain comprising the sequence of SEQ ID NO: 17. In some embodiments, the sdAb comprises one or more framework (s) derived from a VHH domain comprising the sequence of SEQ ID NO: 18. In some embodiments, the sdAb comprises one or more framework (s) derived from a VHH domain comprising the sequence of SEQ ID NO: 19. In some embodiments, the sdAb comprises one or more framework (s) derived from a VHH domain comprising the sequence of SEQ ID NO: 20. In some embodiments, the sdAb comprises one or more framework (s) derived from a VHH domain comprising the sequence of SEQ ID NO: 21. In some embodiments, the sdAb comprises one or more framework (s) derived from a VHH domain comprising the sequence of SEQ ID NO: 22. In some embodiments, the sdAb comprises one or more framework (s) derived from a VHH domain comprising the sequence of SEQ ID NO: 23. In some embodiments, the sdAb comprises one or more framework (s) derived from a VHH domain comprising the sequence of SEQ ID NO: 24.

In some embodiments, the sdAb provided herein is a humanized sdAb. In some embodiments, humanized single domain antibodies can be generated using the method exemplified in the Section 6 below or the methods described in the section below.

Framework regions described herein are determined based upon the boundaries of the CDR numbering system. In other words, if the CDRs are determined by, e.g., Kabat, IMGT, or Chothia, then the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format, from the N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N-terminal to the CDR1 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residues between CDR1 and CDR2 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residues between CDR2 and CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, and FR4 is defined as the amino acid residues C-terminal to the CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

In some embodiments, there is provided an isolated anti-SARS-CoV-2 S1 sdAb comprising a VHH domain having the amino acid sequence of SEQ ID NO: 13. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, there is provided an isolated anti-SARS-CoV-2 S1 sdAb comprising a VHH domain having the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, there is provided an isolated anti-SARS-CoV-2 S1 sdAb comprising a VHH domain having the amino acid sequence of SEQ ID NO: 15. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, there is provided an isolated anti-SARS-CoV-2 S1 sdAb comprising a VHH domain having the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, there is provided an isolated anti-SARS-CoV-2 S1 sdAb comprising a VHH domain having the amino acid sequence of SEQ ID NO: 17. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, there is provided an isolated anti-SARS-CoV-2 S1 sdAb comprising a VHH domain having the amino acid sequence of SEQ ID NO: 18. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, there is provided an isolated anti-SARS-CoV-2 S1 sdAb comprising a VHH domain having the amino acid sequence of SEQ ID NO: 19. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, there is provided an isolated anti-SARS-CoV-2 S1 sdAb comprising a VHH domain having the amino acid sequence of SEQ ID NO: 20. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, there is provided an isolated anti-SARS-CoV-2 S1 sdAb comprising a VHH domain having the amino acid sequence of SEQ ID NO: 21. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, there is provided an isolated anti-SARS-CoV-2 S1 sdAb comprising a VHH domain having the amino acid sequence of SEQ ID NO: 22. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, there is provided an isolated anti-SARS-CoV-2 S1 sdAb comprising a VHH domain having the amino acid sequence of SEQ ID NO: 23. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, there is provided an isolated anti-SARS-CoV-2 S1 sdAb comprising a VHH domain having the amino acid sequence of SEQ ID NO: 24. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO: 24.

In certain embodiments, an antibody described herein or an antigen-binding fragment thereof comprises amino acid sequences with certain percent identity relative to any one of antibodies 77NCOVP01, 77NCOVP02, 77NCOVP03, 77NCOVP04, 77NCOVP01H1, 77NCOVP01H2, 77NCOVP02H1, 77NCOVP02H2, 77NCOVP03H1, 77NCOVP03H2, 77NCOVP04H1, and 77NCOVP04H2.

The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 87: 2264 2268 (1990) , modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 90: 5873 5877 (1993) . Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215: 403 (1990) . BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, word length=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, word length=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25: 3389 3402 (1997) . Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id. ) . When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi. nlm. nih. gov) . Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 4: 11-17 (1998) . Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

In some embodiments, there is provided an anti-SARS-CoV-2 S1 single domain antibody comprising a VHH domain having at least about any one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of a sdAb generated in Section 6 below including those in Table 4 and Table 16. In some embodiments, a VHH sequence having at least about any one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the reference sequence, but the anti-SARS-CoV-2 S1 single domain antibody comprising that sequence retains the ability to bind to SARS-CoV-2 S1 (e.g., RBD) . In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in an amino acid sequence of an sdAb generated in Section 6 below including those in Table 4 and Table 16. In some embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs) . Optionally, the anti-SARS-CoV-2 S1 single domain antibody comprises an amino acid sequence of an sdAb generated in Section 6 below including those in Table 4 and Table 16, including post-translational modifications of that sequence.

In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 13, wherein the single domain antibody binds to SARS-CoV-2 S1 (e.g., RBD) . In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 14, wherein the single domain antibody binds to SARS-CoV-2 S1 (e.g., RBD) . In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 15, wherein the single domain antibody binds to SARS-CoV-2 S1 (e.g., RBD) . In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 16, wherein the single domain antibody binds to SARS-CoV-2 S1 (e.g., RBD) . In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 17, wherein the single domain antibody binds to SARS-CoV-2 S1 (e.g., RBD) . In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 18, wherein the single domain antibody binds to SARS-CoV-2 S1 (e.g., RBD) . In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 19, wherein the single domain antibody binds to SARS-CoV-2 S1 (e.g., RBD) . In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 20, wherein the single domain antibody binds to SARS-CoV-2 S1 (e.g., RBD) . In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 21, wherein the single domain antibody binds to SARS-CoV-2 S1 (e.g., RBD) . In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 22, wherein the single domain antibody binds to SARS-CoV-2 S1 (e.g., RBD) . In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 23, wherein the single domain antibody binds to SARS-CoV-2 S1 (e.g., RBD) . In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 24, wherein the single domain antibody binds to SARS-CoV-2 S1 (e.g., RBD) .

In some embodiments, functional epitopes can be mapped, e.g., by combinatorial alanine scanning, to identify amino acids in the SARS-CoV-2 S1 (e.g., RBD) protein that are necessary for interaction with anti-SARS-CoV-2 S1 single domain antibodies provided herein. In some embodiments, conformational and crystal structure of anti-SARS-CoV-2 S1 single domain antibody bound to SARS-CoV-2 S1 (e.g., RBD) may be employed to identify the epitopes. In some embodiments, the present disclosure provides an antibody that specifically binds to the same epitope as any of the anti-SARS-CoV-2 S1 single domain antibodies provided herein. For example, in some embodiments, an antibody is provided that binds to the same epitope as an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, an antibody is provided that binds to the same epitope as an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, an antibody is provided that binds to the same epitope as an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, an antibody is provided that binds to the same epitope as an anti- SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, an antibody is provided that binds to the same epitope as an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, an antibody is provided that binds to the same epitope as an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, an antibody is provided that binds to the same epitope as an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, an antibody is provided that binds to the same epitope as an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, an antibody is provided that binds to the same epitope as an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, an antibody is provided that binds to the same epitope as an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, an antibody is provided that binds to the same epitope as an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, an antibody is provided that binds to the same epitope as an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 24.

In some embodiments, provided herein is an anti-SARS-CoV-2 S1 antibody, or antigen binding fragment thereof, that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with any one of the anti-SARS-CoV-2 S1 single domain antibodies described herein. In some embodiments, competitive binding may be determined using an ELISA assay. For example, in some embodiments, an antibody is provided that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, an antibody is provided that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, an antibody is provided that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, an antibody is provided that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, an antibody is provided that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, an antibody is provided that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, an antibody is provided that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, an antibody is provided that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, an antibody is provided that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, an antibody is provided that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, an antibody is provided that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, an antibody is provided that specifically binds to SARS-CoV-2 S1 (e.g., RBD) competitively with an anti-SARS-CoV-2 S1 single domain antibody comprising the amino acid sequence of SEQ ID NO: 24.

In some embodiments, provided herein is a SARS-CoV-2 S1 (e.g., RBD) binding protein comprising any one of the anti-SARS-CoV-2 S1 single domain antibodies described above. In some embodiments, the SARS-CoV-2 S1 (e.g., RBD) binding protein is a monoclonal antibody, including a camelid, chimeric, humanized or human antibody. In some embodiments, the anti-SARS-CoV-2 S1 antibody is an antibody fragment, e.g., a VHH fragment. In some embodiments, the anti-SARS-CoV-2 S1 antibody is a full-length heavy-chain only antibody comprising an Fc region of any antibody class or isotype, such as IgG1 or IgG4. In some embodiments, the Fc region has reduced or minimized effector function. In some embodiments, the SARS-CoV-2 S1 (e.g., RBD) binding protein is a fusion protein comprising the anti-SARS-CoV-2 S1 single domain antibody provided herein. In other embodiments, the SARS-CoV-2 S1 (e.g., RBD) binding protein is a multispecific antibody comprising one or more anti-SARS-CoV-2 S1 single domain antibody provided herein. Other exemplary SARS-CoV-2 S1 (e.g., RBD) binding molecules are described in more detail in the following sections.

In some embodiments, the anti-SARS-CoV-2 S1 antibody or antigen binding protein according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 5.2.2 to 5.2.6 below.

5.2.2. Humanized Single Domain Antibodies

The single domain antibodies described herein include humanized single domain antibodies. General strategies to humanize single domain antibodies from Camelidae species have been described (see, e.g., Vincke et al., J. Biol. Chem., 284 (5) : 3273-3284 (2009) ) and may be useful for producing humanized VHH domains as disclosed herein. The design of humanized single domain antibodies from Camelidae species may include the hallmark residues in the VHH, such as residues 11, 37, 44, 45 and 47 (residue numbering according to Kabat) (Muyldermans, Reviews Mol Biotech 74: 277-302 (2001) .

Humanized antibodies, such as the humanized single domain antibodies disclosed herein can also be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239, 400; International publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089) , veneering or resurfacing (European Patent Nos. EP 592, 106 and EP 519, 596; Padlan, Molecular Immunology 28 (4/5) : 489-498 (1991) ; Studnicka et al., Protein Engineering 7 (6) : 805-814 (1994) ; and Roguska et al., PNAS 91: 969-973 (1994) ) , chain shuffling (U.S. Patent No. 5,565,332) , and techniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO 9317105, Tan et al., J. Immunol. 169: 1119 25 (2002) , Caldas et al., Protein Eng. 13 (5) : 353-60 (2000) , Morea et al., Methods 20 (3) : 267 79 (2000) , Baca et al., J. Biol. Chem. 272 (16) : 10678-84 (1997) , Roguska et al., Protein Eng. 9 (10) : 895 904 (1996) , Couto et al., Cancer Res. 55 (23 Supp) : 5973s-5977s (1995) , Couto et al., Cancer Res. 55 (8) : 1717-22 (1995) , Sandhu JS, Gene 150 (2) : 409-10 (1994) , and Pedersen et al., J. Mol. Biol. 235 (3) : 959-73 (1994) . See also U.S. Patent Pub. No. US 2005/0042664 A1 (Feb. 24, 2005) , each of which is incorporated by reference herein in its entirety.

In some embodiments, single domain antibodies provided herein can be humanized single domain antibodies that bind to SARS-CoV-2 S1 (e.g., RBD) . For example, humanized single domain antibodies of the present disclosure may comprise one or more CDRs set forth in SEQ ID NOs: 13-24. Exemplary humanized sdAbs include those in Table 16 below. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization may be performed, for example, following the method of Jones et al., Nature 321: 522-25 (1986) ; Riechmann et al., Nature 332: 323-27 (1988) ; and Verhoeyen et al., Science 239: 1534-36 (1988) ) , by substituting hypervariable region sequences for the corresponding sequences of a human antibody. In a specific embodiment, humanization of the single domain antibody provided herein is performed as described in Section 6 below.

In some cases, the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the CDRs of the parent non-human antibody are grafted onto a human antibody framework, as described in Padlan et al., FASEB J. 9: 133-39 (1995) , and Kashmiri et al., Methods 36: 25-34 (2005) . The choice of human variable domains to be used in making the humanized antibodies can be important to reduce antigenicity, as described in Sims et al., J. Immunol. 151: 2296-308 (1993) ; and Chothia et al., J. Mol. Biol. 196: 901-17 (1987) , Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285-89 (1992) ; and Presta et al., J. Immunol. 151: 2623-32 (1993) . In an alternative paradigm based on comparison of CDRs, called superhumanization, FR homology is irrelevant (see, e.g., Tan et al., J. Immunol. 169: 1119-25 (2002) ) . It is further generally desirable that antibodies be humanized with retention of their affinity for the antigen and other favorable biological properties using various technologies known in the art (see, e.g., Whitelegg and Rees, Protein Eng. 13: 819-24 (2002) , Sali and Blundell, J. Mol. Biol. 234: 779-815 (1993) , and Guex and Peitsch, Electrophoresis 18: 2714-23 (1997) ) . Another method for antibody humanization is based on a metric of antibody humanness termed Human String Content (HSC) (see Lazar et al., Mol. Immunol. 44: 1986-98 (2007) ) . In addition to the methods described above, empirical methods may be used to generate and select humanized antibodies (see, e.g., Hoogenboom, Nat. Biotechnol. 23: 1105-16 (2005) ; Dufner et al., Trends Biotechnol. 24: 523-29 (2006) ; Feldhaus et al., Nat. Biotechnol. 21: 163-70 (2003) ; and Schlapschy et al., Protein Eng. Des. Sel. 17: 847-60 (2004) ) . In the FR library approach, a collection of residue variants are introduced at specific positions in the FR followed by screening of the library to select the FR that best supports the grafted CDR (see, e.g., Foote and Winter, J. Mol. Biol. 224: 487-99 (1992) ) , or from the more limited set of target residues identified by Baca et al. J. Biol. Chem. 272: 10678-84 (1997) . In FR shuffling, whole FRs are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g., Dall’Acqua et al., Methods 36: 43-60 (2005) ) . A one-step FR shuffling process may be used (see, e.g., Damschroder et al., Mol. Immunol. 44: 3049-60 (2007) ) . The “humaneering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non-human fragments into libraries of human FRs and assessment of binding. The “human engineering” method involves altering a non-human antibody or antibody fragment by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies. Techniques for making human engineered proteins are described in greater detail in Studnicka et al., Protein Engineering 7: 805-14 (1994) ; U.S. Pat. Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619; and PCT Publication WO 93/11794. A composite human antibody can be generated using, for example, Composite Human Antibody ^TM technology (Antitope Ltd., Cambridge, United Kingdom) . A deimmunized antibody is an antibody in which T-cell epitopes have been removed. Methods for making deimmunized antibodies have been described. See, e.g., Jones et al., Methods Mol Biol. 525: 405-23 (2009) , xiv, and De Groot et al., Cell. Immunol. 244: 148-153 (2006) ) .

5.2.3. Single Domain Antibody Variants

In some embodiments, amino acid sequence modification (s) of the single domain antibodies that bind to SARS-CoV-2 S1 (e.g., RBD) described herein are contemplated. For example, it may be desirable to optimize the binding affinity and/or other biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector functions, glycosylation, reduced immunogenicity, or solubility. Thus, in addition to the single domain antibodies that bind to SARS-CoV-2 S1 (e.g., RBD) described herein, it is contemplated that variants of the single domain antibodies that bind to SARS-CoV-2 S1 (e.g., RBD) described herein can be prepared. For example, single domain antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Those skilled in the art who appreciate that amino acid changes may alter post-translational processes of the single domain antibody.

Chemical Modifications

In some embodiments, the single domain antibodies provided herein are chemically modified, for example, by the covalent attachment of any type of molecule to the single domain antibody. The antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or conjugation to one or more immunoglobulin domains (e.g., Fc or a portion of an Fc) . Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody may contain one or more non-classical amino acids.

In some embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. In some embodiments, the single domain antibody provided herein is fused to an Fc region, which may be modified, details of which is described in Section 5.3 below.

In some embodiments, it may be desirable to create cysteine engineered antibodies, in which one or more residues of an antibody are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein.

Substitutions, Deletions, or Insertions

Variations may be a substitution, deletion, or insertion of one or more codons encoding the single domain antibody or polypeptide that results in a change in the amino acid sequence as compared with the original antibody or polypeptide. Sites of interest for substitutional mutagenesis include the CDRs and FRs.

Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In certain embodiments, the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In a specific embodiment, the substitution is a conservative amino acid substitution made at one or more predicted non-essential amino acid residues. The variation allowed may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the parental antibodies.

Amino acid sequence insertions include amino-and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue.

Single domain antibodies generated by conservative amino acid substitutions are included in the present disclosure. In a conservative amino acid substitution, an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. As described above, families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined. Conservative (e.g., within an amino acid group with similar properties and/or side chains) substitutions may be made, so as to maintain or not significantly change the properties. Exemplary substitutions are shown in Table 2 below.

Table 2. Amino Acid Substitutions

Amino acids may be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975) ) : (1) non-polar: Ala (A) , Val (V) , Leu (L) , Ile (I) , Pro (P) , Phe (F) , Trp (W) , Met (M) ; (2) uncharged polar: Gly (G) , Ser (S) , Thr (T) , Cys (C) , Tyr (Y) , Asn (N) , Gln (Q) ; (3) acidic: Asp (D) , Glu (E) ; and (4) basic: Lys (K) , Arg (R) , His (H) . Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. For example, any cysteine residue not involved in maintaining the proper conformation of the single domain antibody also may be substituted, for example, with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and to prevent aberrant crosslinking. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody) . Generally, the resulting variant (s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity) .

Alterations (e.g., substitutions) may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR “hotspots, ” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008) ) , and/or SDRs (a-CDRs) , with the resulting variant antibody or fragment thereof being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al.in Methods in Molecular Biology 178: 1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001) . ) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis) . A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. More detailed description regarding affinity maturation is provided in the section below.

In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. In some embodiments of the variant VHH sequences provided herein, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells, Science, 244: 1081-1085 (1989) . In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino-and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N-or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, Biochem J. 237: 1-7 (1986) ; and Zoller et al., Nucl. Acids Res. 10: 6487-500 (1982) ) , cassette mutagenesis (see, e.g., Wells et al., Gene 34: 315-23 (1985) ) , or other known techniques can be performed on the cloned DNA to produce the single domain antibody variant DNA.

5.2.4. In vitro Affinity Maturation

In some embodiments, antibody variants having an improved property such as affinity, stability, or expression level as compared to a parent antibody may be prepared by in vitro affinity maturation. Like the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. Libraries of antibodies are displayed on the surface of an organism (e.g., phage, bacteria, yeast, or mammalian cell) or in association (e.g., covalently or non-covalently) with their encoding mRNA or DNA. Affinity selection of the displayed antibodies allows isolation of organisms or complexes carrying the genetic information encoding the antibodies. Two or three rounds of mutation and selection using display methods such as phage display usually results in antibody fragments with affinities in the low nanomolar range. Affinity matured antibodies can have nanomolar or even picomolar affinities for the target antigen.

Phage display is a widespread method for display and selection of antibodies. The antibodies are displayed on the surface of Fd or M13 bacteriophages as fusions to the bacteriophage coat protein. Selection involves exposure to antigen to allow phage-displayed antibodies to bind their targets, a process referred to as “panning. ” Phage bound to antigen are recovered and used to infect bacteria to produce phage for further rounds of selection. For review, see, for example, Hoogenboom, Methods. Mol. Biol. 178: 1-37 (2002) ; and Bradbury and Marks, J. Immunol. Methods 290: 29-49 (2004) .

In a yeast display system (see, e.g., Boder et al., Nat. Biotech. 15: 553–57 (1997) ; and Chao et al., Nat. Protocols 1: 755-68 (2006) ) , the antibody may be fused to the adhesion subunit of the yeast agglutinin protein Aga2p, which attaches to the yeast cell wall through disulfide bonds to Aga1p. Display of a protein via Aga2p projects the protein away from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen the library to select for antibodies with improved affinity or stability. Binding to a soluble antigen of interest is determined by labeling of yeast with biotinylated antigen and a secondary reagent such as streptavidin conjugated to a fluorophore. Variations in surface expression of the antibody can be measured through immunofluorescence labeling of either the hemagglutinin or c-Myc epitope tag flanking the single chain antibody (e.g., scFv) . Expression has been shown to correlate with the stability of the displayed protein, and thus antibodies can be selected for improved stability as well as affinity (see, e.g., Shusta et al., J. Mol. Biol. 292: 949-56 (1999) ) . An additional advantage of yeast display is that displayed proteins are folded in the endoplasmic reticulum of the eukaryotic yeast cells, taking advantage of endoplasmic reticulum chaperones and quality-control machinery. Once maturation is complete, antibody affinity can be conveniently “titrated” while displayed on the surface of the yeast, eliminating the need for expression and purification of each clone. A theoretical limitation of yeast surface display is the potentially smaller functional library size than that of other display methods; however, a recent approach uses the yeast cells’ mating system to create combinatorial diversity estimated to be 10 ¹⁴ in size (see, e.g., U.S. Pat. Publication 2003/0186374; and Blaise et al., Gene 342: 211–18 (2004) ) .

In ribosome display, antibody-ribosome-mRNA (ARM) complexes are generated for selection in a cell-free system. The DNA library coding for a particular library of antibodies is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The resulting complex of mRNA, ribosome, and protein can bind to surface-bound ligand, allowing simultaneous isolation of the antibody and its encoding mRNA through affinity capture with the ligand. The ribosome-bound mRNA is then reverse transcribed back into cDNA, which can then undergo mutagenesis and be used in the next round of selection (see, e.g., Fukuda et al., Nucleic Acids Res. 34: e127 (2006) ) . In mRNA display, a covalent bond between antibody and mRNA is established using puromycin as an adaptor molecule (Wilson et al., Proc. Natl. Acad. Sci. USA 98: 3750-55 (2001) ) .

As these methods are performed entirely in vitro, they provide two main advantages over other selection technologies. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be introduced easily after each selection round, for example, by non-proofreading polymerases, as no library must be transformed after any diversification step.

In some embodiments, mammalian display systems may be used.

Diversity may also be introduced into the CDRs of the antibody libraries in a targeted manner or via random introduction. The former approach includes sequentially targeting all the CDRs of an antibody via a high or low level of mutagenesis or targeting isolated hot spots of somatic hypermutations (see, e.g., Ho et al., J. Biol. Chem. 280: 607-17 (2005) ) or residues suspected of affecting affinity on experimental basis or structural reasons. Diversity may also be introduced by replacement of regions that are naturally diverse via DNA shuffling or similar techniques (see, e.g., Lu et al., J. Biol. Chem. 278: 43496-507 (2003) ; U.S. Pat. Nos. 5,565,332 and 6,989,250) . Alternative techniques target hypervariable loops extending into framework-region residues (see, e.g., Bond et al., J. Mol. Biol. 348: 699-709 (2005) ) employ loop deletions and insertions in CDRs or use hybridization-based diversification (see, e.g., U.S. Pat. Publication No. 2004/0005709) . Additional methods of generating diversity in CDRs are disclosed, for example, in U.S. Pat. No. 7,985,840. Further methods that can be used to generate antibody libraries and/or antibody affinity maturation are disclosed, e.g., in U.S. Patent Nos. 8,685,897 and 8,603,930, and U.S. Publ. Nos. 2014/0170705, 2014/0094392, 2012/0028301, 2011/0183855, and 2009/0075378, each of which are incorporated herein by reference.

Screening of the libraries can be accomplished by various techniques known in the art. For example, single domain antibodies can be immobilized onto solid supports, columns, pins, or cellulose/poly (vinylidene fluoride) membranes/other filters, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads or used in any other method for panning display libraries.

For review of in vitro affinity maturation methods, see, e.g., Hoogenboom, Nature Biotechnology 23: 1105-16 (2005) ; Quiroz and Sinclair, Revista Ingeneria Biomedia 4: 39-51 (2010) ; and references therein.

5.2.5. Modifications of Single Domain Antibodies

Covalent modifications of single domain antibodies are included within the scope of the present disclosure. Covalent modifications include reacting targeted amino acid residues of a single domain antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of the single domain antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983) ) , acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Other types of covalent modification of the single domain antibody included within the scope of this present disclosure include altering the native glycosylation pattern of the antibody or polypeptide as described above (see, e.g., Beck et al., Curr. Pharm. Biotechnol. 9: 482-501 (2008) ; and Walsh, Drug Discov. Today 15: 773-80 (2010) ) , and linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG) , polypropylene glycol, or polyoxyalkylenes, in the manner set forth, for example, in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. The single domain antibody that binds to SARS-CoV-2 S1 (e.g., RBD) of the disclosure may also be genetically fused or conjugated to one or more immunoglobulin constant regions or portions thereof (e.g., Fc) to extend half-life and/or to impart known Fc-mediated effector functions (which is described in more detail below) .

The single domain antibody that binds to SARS-CoV-2 S1 (e.g., RBD) of the present disclosure may also be modified to form chimeric molecules comprising the single domain antibody that binds to SARS-CoV-2 S1 (e.g., RBD) fused to another, heterologous polypeptide or amino acid sequence, for example, an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60: 523-33 (2003) ) or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999) ) . The single domain antibody that binds to SARS-CoV-2 S1 (e.g., RBD) may be used to generate SARS-CoV-2 S1 (e.g., RBD) binding fusion proteins, as described in more detail below.

Also provided herein are panels of antibodies that bind to a SARS-CoV-2 S1 (e.g., RBD) antigen. In specific embodiments, the panels of antibodies have different association rates, different dissociation rates, different affinities for a SARS-CoV-2 S1 (e.g., RBD) antigen, and/or different specificities for a SARS-CoV-2 S1 (e.g., RBD) antigen. In some embodiments, the panels comprise or consist of about 10 to about 1000 antibodies or more. Panels of antibodies can be used, for example, in 96-well or 384-well plates, for assays such as ELISAs.

5.2.6. Preparation of Single Domain Antibodies

Methods of preparing single domain antibodies have been described. See, e.g., Els Pardon et al, Nature Protocol, 9 (3) : 674 (2014) . Single domain antibodies (such as VHHs) may be obtained using methods known in the art such as by immunizing a Camelid species (such as camel or llama) and obtaining hybridomas therefrom, or by cloning a library of single domain antibodies using molecular biology techniques known in the art and subsequent selection by ELISA with individual clones of unselected libraries or by using phage display.

Single domain antibodies provided herein may be produced by culturing cells transformed or transfected with a vector containing a single domain antibody-encoding nucleic acids. Polynucleotide sequences encoding polypeptide components of the antibody of the present disclosure can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridomas cells or B cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in host cells. Many vectors that are available and known in the art can be used for the purpose of the present disclosure. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Host cells suitable for expressing antibodies of the present disclosure include prokaryotes such as Archaebacteria and Eubacteria, including Gram-negative or Gram-positive organisms, eukaryotic microbes such as filamentous fungi or yeast, invertebrate cells such as insect or plant cells, and vertebrate cells such as mammalian host cell lines. Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Antibodies produced by the host cells are purified using standard protein purification methods as known in the art.

Methods for antibody production including vector construction, expression, and purification are further described in Plückthun et al., Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation 203-52 (McCafferty et al. eds., 1996) ; Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, in Current Protocols in Protein Science (2009) ; Tachibana and Takekoshi, Production of Antibody Fab Fragments in Escherichia coli, in Antibody Expression and Production (Al-Rubeai ed., 2011) ; and Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed., 2009) .

It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare anti-SARS-CoV-2 S1 single domain antibodies. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis (1969) ; and Merrifield, J. Am. Chem. Soc. 85: 2149-54 (1963) ) . In vitro protein synthesis may be performed using manual techniques or by automation. Various portions of the anti-SARS-CoV-2 S1 antibody may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-SARS-CoV-2 S1 antibody. Alternatively, antibodies may be purified from cells or bodily fluids, such as milk, of a transgenic animal engineered to express the antibody, as disclosed, for example, in U.S. Pat. Nos. 5,545,807 and 5,827,690.

Specifically, the single domain antibodies, or other SARS-CoV-2 S1 (e.g., RBD) binders provided herein, can be generated by immunizing llamas, performing single B-cell sorting, undertaking V-gene extraction, cloning the SARS-CoV-2 S1 (e.g., RBD) binders, such as VHH-Fc fusions, and then performing small scale expression and purification. Additional screening of the single domain antibodies and other molecules that bind to SARS-CoV-2 S1 (e.g., RBD) can be performed, including one or more of selecting for ELISA-positive, BLI-positive, and K _D less than 100 nM. These selection criteria can be combined as described in Section 6 below. Additionally, individual VHH binders (and other molecules that bind to SARS-CoV-2 S1 (e.g., RBD) ) can be assayed for their ability to bind to cells expressing SARS-CoV-2 S1 (e.g., RBD) . Such assay can be performed using FACS analysis with cells expressing SARS-CoV-2 S1 (e.g., RBD) , and measuring the mean fluorescence intensity (MFI) of fluorescently-labeled VHH molecules. Polyclonal Antibodies and monoclonal antibodies may be prepared according known methods in the art.

Polynucleic acid sequences encoding the antibodies of the present disclosure can be obtained in prokaryotic cells using standard recombinant techniques. Desired polynucleic acid sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present disclosure. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. The vector components generally include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS) , a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence.

In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. The expression vector of the present application may comprise two or more promoter-cistron pairs, encoding each of the polypeptide components. Prokaryotic host cells suitable for expressing the antibodies of the present disclosure include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Prokaryotic cells used to produce the antibodies of the present application are grown in media known in the art and suitable for culture of the selected host cells. Any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source. If an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for the activation of the promoter. The expressed antibodies of the present disclosure are secreted into and recovered from the periplasm of the host cells. Alternatively, protein production is conducted in large quantity by a fermentation process. To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive) , certain host strains deficient for proteolytic enzymes can be used for the present invention. The antibodies produced herein can be further purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art can be employed.

For eukaryotic expression, the vector components generally include, but are not limited to, one or more of the following, a signal sequence, an origin of replication, one or more marker genes, and enhancer element, a promoter, and a transcription termination sequence. Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the desired polypeptide sequences. Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryote cells known in the art, including vertebrate host cells. Host cells can be transformed with the above-described expression or cloning vectors for antibodies production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The protein composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography.

5.3. Binding Molecules Comprising sdAbs

In another aspect, provided herein is a binding molecule comprising one or more single domain antibodies provided herein (e.g., those described above in Section 5.2) .

In various embodiments, the single domain antibody provided herein can be genetically fused or chemically conjugated to another agent, for example, protein-based entities. The single domain antibody may be chemically-conjugated to the agent, or otherwise non-covalently conjugated to the agent. The agent can be a peptide or antibody (or a fragment thereof) .

Thus, in some embodiments, provided herein are single domain antibodies (e.g., VHH domains) that are recombinantly fused or chemically conjugated (covalent or non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, for example, to a polypeptide of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450 or about 500 amino acids, or over 500 amino acids) to generate fusion proteins, as well as uses thereof. In particular, provided herein are fusion proteins comprising an antigen-binding fragment of the single domain antibody provided herein (e.g., CDR1, CDR2, and/or CDR3) and a heterologous protein, polypeptide, or peptide.

5.3.1. SdAb Based Multivalent Molecules that Bind to SARS-CoV-2 S1

In some embodiments, provided herein is a fusion protein that comprises two or more sdAbs (e.g., VHH domains) provided herein. In a specific embodiment, the fusion protein comprises two sdAbs (bivalent sdAb or Bi-sdAb) provided herein. In a specific embodiment, the fusion protein comprises two VHH domains (bivalent VHH or Bi-VHH domains) provided herein.

In some embodiments, each of the VHH domains in the fusion protein is an sdAb described in Section 5.2 above. The multiple VHH domains in the fusion protein provided herein can be the same or different, and can be arranged in different orders. For example, in case there are two VHH domains in the fusion protein, the first anti-SARS-CoV-2 S1 sdAb can be at the N terminus of the second anti-SARS-CoV-2 S1 sdAb; or the first anti-SARS-CoV-2 S1 sdAb can be at the C terminus of the second anti-SARS-CoV-2 S1 sdAb.

More specifically, in some embodiments, each of the VHH domains in the fusion protein is a sdAb comprising (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or (iv) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the fusion protein comprises two VHH domains both having the same set of CDRs, i.e., CDR set (i) , (ii) , (iii) or (iv) described above. In other embodiments, the two VHH domains have different set of CDRs, for example, one having CDR set (i) and the other having CDR set (ii) ; one having CDR set (i) and the other having CDR set (iii) ; one having CDR set (i) and the other having CDR set (iv) ; one having CDR set (ii) and the other having CDR set (iii) ; one having CDR set (ii) and the other having CDR set (iv) ; or one having CDR set (iii) and the other having CDR set (iv) , without limitation to the order of the two VHH domains in the fusion protein (the position of N terminus or C terminus in the fusion protein) .

In some embodiments, each of the VHH domains in the fusion protein is an sdAb comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In some embodiments, the fusion protein comprises two VHH domains both having a SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In other embodiments, the two VHH domains comprise different sequences, for example, a pair of SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 13 and SEQ ID NO: 15; SEQ ID NO: 13 and SEQ ID NO: 16; SEQ ID NO: 13 and SEQ ID NO: 17; SEQ ID NO: 13 and SEQ ID NO: 18; SEQ ID NO: 13 and SEQ ID NO: 19; SEQ ID NO: 13 and SEQ ID NO: 20; SEQ ID NO: 13 and SEQ ID NO: 21; SEQ ID NO: 13 and SEQ ID NO: 22; SEQ ID NO: 13 and SEQ ID NO: 23; SEQ ID NO: 13 and SEQ ID NO: 24; SEQ ID NO: 14 and SEQ ID NO: 15; SEQ ID NO: 14 and SEQ ID NO: 16; SEQ ID NO: 14 and SEQ ID NO: 17; SEQ ID NO: 14 and SEQ ID NO: 18; SEQ ID NO: 14 and SEQ ID NO: 19; SEQ ID NO: 14 and SEQ ID NO: 20; SEQ ID NO: 14 and SEQ ID NO: 21; SEQ ID NO: 14 and SEQ ID NO: 22; SEQ ID NO: 14 and SEQ ID NO: 23; SEQ ID NO: 14 and SEQ ID NO: 24; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 15 and SEQ ID NO: 17; SEQ ID NO: 15 and SEQ ID NO: 18; SEQ ID NO: 15 and SEQ ID NO: 19; SEQ ID NO: 15 and SEQ ID NO: 20; SEQ ID NO: 15 and SEQ ID NO: 21; SEQ ID NO: 15 and SEQ ID NO: 22; SEQ ID NO: 15 and SEQ ID NO: 23; SEQ ID NO: 15 and SEQ ID NO: 24; SEQ ID NO: 16 and SEQ ID NO: 17; SEQ ID NO: 16 and SEQ ID NO: 18; SEQ ID NO: 16 and SEQ ID NO: 19; SEQ ID NO: 16 and SEQ ID NO: 20; SEQ ID NO: 16 and SEQ ID NO: 21; SEQ ID NO: 16 and SEQ ID NO: 22; SEQ ID NO: 16 and SEQ ID NO: 23; SEQ ID NO: 16 and SEQ ID NO: 24; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 17 and SEQ ID NO: 19; SEQ ID NO: 17 and SEQ ID NO: 20; SEQ ID NO: 17 and SEQ ID NO: 21; SEQ ID NO: 17 and SEQ ID NO: 22; SEQ ID NO: 17 and SEQ ID NO: 23; SEQ ID NO: 17 and SEQ ID NO: 24; SEQ ID NO: 18 and SEQ ID NO: 19; SEQ ID NO: 18 and SEQ ID NO: 20; SEQ ID NO: 18 and SEQ ID NO: 21; SEQ ID NO: 18 and SEQ ID NO: 22; SEQ ID NO: 18 and SEQ ID NO: 23; SEQ ID NO: 18 and SEQ ID NO: 24; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 19 and SEQ ID NO: 21; SEQ ID NO: 19 and SEQ ID NO: 22; SEQ ID NO: 19 and SEQ ID NO: 23; SEQ ID NO: 19 and SEQ ID NO: 24; SEQ ID NO: 20 and SEQ ID NO: 21; SEQ ID NO: 20 and SEQ ID NO: 22; SEQ ID NO: 20 and SEQ ID NO: 23; SEQ ID NO: 20 and SEQ ID NO: 24; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO: 21 and SEQ ID NO: 23; SEQ ID NO: 21 and SEQ ID NO: 24; SEQ ID NO: 22 and SEQ ID NO: 23; SEQ ID NO: 22 and SEQ ID NO: 24; SEQ ID NO: 23 and SEQ ID NO: 24, without limitation to the order of the two VHH domains in the fusion protein (the position of N terminus or C terminus in the fusion protein) .

In some embodiments, the fusion protein further comprises one or more additional agent (s) . In some embodiments, the fusion protein further comprises a domain comprising a human IgG1 Fc region or a variant thereof. In some embodiments, the domain comprises the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63. In some embodiments, the fusion protein comprises from N terminus to C terminus: the first anti-SARS-CoV-2 S1 sdAb-IgG1 Fc-the second anti-SARS-CoV-2 S1 sdAb. In other embodiments, the fusion protein comprises from N terminus to C terminus: the first anti-SARS-CoV-2 S1 sdAb-the second anti-SARS-CoV-2 S1 sdAb-IgG1 Fc. Additional fusion proteins (including Fc fusion proteins) are described in sections below.

In some embodiments, two or more regions in the fusion protein are linked directly. In other embodiments, the two or more regions in the fusion protein are linked via a peptide linker. The peptide linkers connecting different various regions may be the same or different.

Each peptide linker may have the same or different length and/or sequence depending on the structural and/or functional features of the single domain antibodies and/or the various domains. Each peptide linker may be selected and optimized independently. The length, the degree of flexibility and/or other properties of the peptide linker (s) used in the fusion proteins may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes. For example, longer peptide linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another. In some embodiment, a peptide linker comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids long. In some embodiments, the peptide linker is no more than about any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids long. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acids to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include but not limited to glycine polymers (G) _n, glycine-serine polymers (including, for example, (GS) _n, (GSGGS) _n, (GGGS) _n, and (GGGGS) _n, where n is an integer of at least one) , glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Exemplary peptide linkers are listed in the table below. In a specific embodiment, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 64) . In another specific embodiment, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 65) .

Table 3. Exemplary Peptide Linkers

Sequences	SEQ ID NO
(GS) _n, n is an integer including, e.g., 1, 2, 3, 4, 5, and 6.	SEQ ID NO: 66
(GSGGS) _n, n is an integer including, e.g., 1, 2, 3, 4, 5, and 6.	SEQ ID NO: 67
(GGGS) _n, n is an integer including, e.g., 1, 2, 3, 4, 5, and 6.	SEQ ID NO: 68
GGGGSGGGGSGGGGGGSGSGGGGSGGGGSGGGGS	SEQ ID NO: 69
(GGGGS) _n, n is an integer including, e.g., 1, 2, 3, 4, 5, and 6.	SEQ ID NO: 70
DGGGS	SEQ ID NO: 71
TGEKP	SEQ ID NO: 72
GGRR	SEQ ID NO: 73
GGGGSGGGGSGGGGGGSGSGGGGS	SEQ ID NO: 74
EGKSSGSGSESKVD	SEQ ID NO: 75
KESGSVSSEQLAQFRS	SEQ ID NO: 76
GGRRGGGS	SEQ ID NO: 77
LRQRDGERP	SEQ ID NO: 78
LRQKDGGGSERP	SEQ ID NO: 79

Sequences	SEQ ID NO
LRQKDGGGSGGGSERP	SEQ ID NO: 80
GSTSGSGKPGSGEGST	SEQ ID NO: 81
GSTSGSGKSSEGKG	SEQ ID NO: 82
KESGSVSSEQLAQFRSLD	SEQ ID NO: 83

Other linkers known in the art, for example, as described in WO2016014789, WO2015158671, WO2016102965, US20150299317, WO2018067992, US7741465, Colcher et al., J. Nat. Cancer Inst. 82: 1191-1197 (1990) , and Bird et al., Science 242: 423-426 (1988) may also be included in the fusion proteins provided herein, the disclosure of each of which is incorporated herein by reference.

In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 37. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 38. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 39. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 40. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 41. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 42. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 43. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 44. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 45. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 46. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 47. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 48. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 49. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 50. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 51. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 52. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 53. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 54. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 55. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 56. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 57. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 58. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 59. In some specific embodiments, the fusion protein provided herein comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 60.

5.3.2. Fc Fusion Proteins

In some embodiments, the binding molecule provided herein is a fusion protein comprising at least one (such as 1, 2, 3 or more) sdAb (s) provided herein and an Fc region. Any sdAbs described in Section 5.2 and any bi-VHH domains described in Section 5.3.1 may be included in the present Fc fusion proteins, including, e.g., a sdAb comprising (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9; (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10; (iii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or (iv) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12; or specifically an sdAb comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.

In some specific embodiments, the Fc region is a human IgG1 Fc region or a variant thereof. In some embodiment, the Fc region provided herein comprises the amino acid sequence of SEQ ID NO: 62. In other embodiments, the Fc region provided herein comprises the amino acid sequence of SEQ ID NO: 63.

In more specific embodiments, the fusion protein provided herein comprises the amino acid sequence of having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 25. In more specific embodiments, the fusion protein provided herein comprises the amino acid sequence of having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 26. In more specific embodiments, the fusion protein provided herein comprises the amino acid sequence of having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 27. In more specific embodiments, the fusion protein provided herein comprises the amino acid sequence of having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 28. In more specific embodiments, the fusion protein provided herein comprises the amino acid sequence of having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 29. In more specific embodiments, the fusion protein provided herein comprises the amino acid sequence of having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 30. In more specific embodiments, the fusion protein provided herein comprises the amino acid sequence of having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 31. In more specific embodiments, the fusion protein provided herein comprises the amino acid sequence of having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 32. In more specific embodiments, the fusion protein provided herein comprises the amino acid sequence of having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 33. In more specific embodiments, the fusion protein provided herein comprises the amino acid sequence of having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 34. In more specific embodiments, the fusion protein provided herein comprises the amino acid sequence of having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 35. In more specific embodiments, the fusion protein provided herein comprises the amino acid sequence of having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity or 100%identical to SEQ ID NO: 36.

When the single domain antibody provided herein is fused to an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15: 26-32 (1997) . The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc) , galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in the binding molecules provided herein may be made in order to create variants with certain improved properties.

In other embodiments, when the single domain antibody provided herein is fused to an Fc region, antibody variants provided herein may have a carbohydrate structure that lacks fucose attached (directly or indirectly) to said Fc region. For example, the amount of fucose in such antibody may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues) ; however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 and US 2004/0093621. Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004) ; Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) . Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986) ; US Patent Application No. US 2003/0157108; and WO 2004/056312, especially at Example 11) , and knockout cell lines, such as alpha-1, 6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) ; Kanda, Y. et al., Biotechnol. Bioeng., 94 (4) : 680-688 (2006) ; and WO2003/085107) .

The binding molecules comprising a single domain antibody provided herein are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and/or improved ADCC function. Examples of such variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al. ) ; US Patent No. 6,602,684 (Umana et al. ) ; and US 2005/0123546 (Umana et al.) . Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such variants may have improved CDC function. Such variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

In molecules that comprise the present single domain antibody and an Fc region, one or more amino acid modifications may be introduced into the Fc region, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

In some embodiments, the present application contemplates variants that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the binding molecule in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the binding molecule lacks FcγR binding (hence likely lacking ADCC activity) , but retains FcRn binding ability. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83: 7059-7063 (1986) ) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82: 1499-1502 (1985) ; 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987) ) . Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI ^TM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox

non-radioactive cytotoxicity assay (Promega, Madison, WI) . Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95: 652-656 (1998) . C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) ; Cragg, M.S. et al., Blood 101: 1045-1052 (2003) ; and Cragg, M.S. and M.J. Glennie, Blood 103: 2738-2743 (2004) ) . FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18 (12) : 1759-1769 (2006) ) .

Binding molecules with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) . Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581) .

Certain variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9 (2) : 6591-6604 (2001) . )

In some embodiments, a variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues) . In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC) , e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000) .

Binding molecules with increased half lives and improved binding to the neonatal Fc receptor (FcRn) , which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994) ) , are described in US2005/0014934A1 (Hinton et al. ) . Those molecules comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 234, 235, 238, 239, 241, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826) . See also Duncan &Winter, Nature 322: 738-40 (1988) ; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.

In some specific embodiments, certain Fc region mutations are introduced to reduce antibody-dependent enhancement (ADE) . ADE effect may cause acute lung injury. See Shi R &Shan C, Nature 584: 120-124 (2020) . For example, in some more specific embodiments, mutations L14A (L234A) and L15A (L235A) are introduced into human IgG1 Fc region of SEQ ID NO: 62 to reduce ADE. In a specific embodiment, the Fc region provided herein is a human IgG1 Fc region variant comprising SEQ ID NO: 63.

5.3.3. Other Fusion Proteins

In addition to the above described fusion proteins, the sdAbs provided herein may be fused to other agents for various purposes. For example, antibodies provided herein can be fused to marker or “tag” sequences, such as a peptide, to facilitate purification. In specific embodiments, the marker or tag amino acid sequence is a hexa-histidine peptide, hemagglutinin ( “HA” ) tag, and “FLAG” tag.

Methods for fusing or conjugating moieties (including polypeptides) to antibodies are known (see, e.g., Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, in Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al. eds., 1985) ; Hellstrom et al., Antibodies for Drug Delivery, in Controlled Drug Delivery 623-53 (Robinson et al. eds., 2d ed. 1987) ; Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, in Monoclonal Antibodies: Biological and Clinical Applications 475-506 (Pinchera et al. eds., 1985) ; Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, in Monoclonal Antibodies for Cancer Detection and Therapy 303-16 (Baldwin et al. eds., 1985) ; Thorpe et al., Immunol. Rev. 62: 119-58 (1982) ; U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,723,125; 5,783,181; 5,908,626; 5,844,095; and 5,112,946; EP 307, 434; EP 367, 166; EP 394, 827; PCT publications WO 91/06570, WO 96/04388, WO 96/22024, WO 97/34631, and WO 99/04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-39 (1991) ; Traunecker et al., Nature, 331: 84-86 (1988) ; Zheng et al., J. Immunol. 154: 5590-600 (1995) ; and Vil et al., Proc. Natl. Acad. Sci. USA 89: 11337-41 (1992) ) .

Fusion proteins may be generated, for example, through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling” ) . DNA shuffling may be employed to alter the activities of the single domain antibodies as provided herein, including, for example, antibodies with higher affinities and lower dissociation rates (see, e.g., U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458; Patten et al., Curr. Opinion Biotechnol. 8: 724-33 (1997) ; Harayama, Trends Biotechnol. 16 (2) : 76-82 (1998) ; Hansson et al., J. Mol. Biol. 287: 265-76 (1999) ; and Lorenzo and Blasco, Biotechniques 24 (2) : 308-13 (1998) ) . Antibodies, or the encoded antibodies, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. A polynucleotide encoding an antibody provided herein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

In some embodiments, a single domain antibody provided herein (e.g., VHH domain) is conjugated to a second antibody to form an antibody heteroconjugate.

In various embodiments, the single domain antibody is genetically fused to the agent. Genetic fusion may be accomplished by placing a linker (e.g., a polypeptide) between the single domain antibody and the agent. The linker may be a flexible linker.

In various embodiments, the single domain antibody is genetically conjugated to a therapeutic molecule, with a hinge region linking the single domain antibody to the therapeutic molecule.

Also provided herein are methods for making the various fusion proteins provided herein. The various methods described in Section 5.2.6 above may also be utilized to make the fusion proteins provided herein.

In a specific embodiment, the fusion protein provided herein is recombinantly expressed. Recombinant expression of a fusion protein provided herein may require construction of an expression vector containing a polynucleotide that encodes the protein or a fragment thereof. Once a polynucleotide encoding a protein provided herein or a fragment thereof has been obtained, the vector for the production of the molecule may be produced by recombinant DNA technology using techniques well-known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriptional and translational control signals. Also provided are replicable vectors comprising a nucleotide sequence encoding a fusion protein provided herein, or a fragment thereof, or a CDR, operably linked to a promoter. The expression vector can be transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce a fusion protein provided herein. Thus, also provided herein are host cells containing a polynucleotide encoding a fusion protein provided herein or fragments thereof operably linked to a heterologous promoter.

A variety of host-expression vector systems may be utilized to express the fusion protein provided herein. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express a fusion protein provided herein in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) . Bacterial cells such as Escherichia coli, or, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, can be used for the expression of a recombinant fusion protein. For example, mammalian cells such as Chinese hamster ovary cells (CHO) , in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies or variants thereof. In a specific embodiment, the expression of nucleotide sequences encoding the fusion proteins provided herein is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. For long-term, high-yield production of recombinant proteins, stable expression can be utilized. Known selection systems may be used. The expression level of a fusion protein can be increased by vector amplification.

Once a fusion protein provided herein has been produced by recombinant expression, it may be purified by any method known in the art for purification of a polypeptide (e.g., an immunoglobulin molecule) , for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, sizing column chromatography, and Kappa select affinity chromatography) , centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the fusion protein molecules provided herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

5.3.4. Immunoconjugates

In some embodiments, the present disclosure also provides immunoconjugates comprising one or more of any of the antibodies (such as anti-SARS-CoV-2 S1 single domain antibodies) described herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof) , or radioactive isotopes.

In some embodiments, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1) ; an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298) ; a dolastatin; a calicheamicin or derivative thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53: 3336-3342 (1993) ; and Lode et al., Cancer Res. 58: 2925-2928 (1998) ) ; an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13: 477-523 (2006) ; Jeffrey et al., Bioorganic &Med. Chem. Letters 16: 358-362 (2006) ; Torgov et al., Bioconj. Chem. 16: 717-721 (2005) ; Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000) ; Dubowchik et al., Bioorg. &Med. Chem. Letters 12: 1529-1532 (2002) ; King et al., J. Med. Chem. 45: 4336-4343 (2002) ; and U.S. Patent No. 6,630,579) ; methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.

In some embodiments, an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa) , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S) , momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In some embodiments, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include At ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹², P ³², Pb ²¹² and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) , such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) , succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) , iminothiolane (IT) , bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl) , active esters (such as disuccinimidyl suberate) , aldehydes (such as glutaraldehyde) , bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine) , bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine) , diisocyanates (such as toluene 2, 6-diisocyanate) , and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) . For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987) . Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.

The linker may be a “cleavable linker” facilitating release of the conjugated agent in the cell, but non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates of the present disclosure include, without limitation, acid labile linkers (e.g., hydrazone linkers) , disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers comprising amino acids, for example, valine and/or citrulline such as citrulline-valine or phenylalanine-lysine) , photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to evade multidrug transporter-mediated resistance.

The immunuoconjugates or ADCs herein contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A) .

In other embodiments, antibodies provided herein are conjugated or recombinantly fused, e.g., to a diagnostic molecule. Such diagnosis and detection can be accomplished, for example, by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/biotin or avidin/biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to, luciferase, luciferin, or aequorin; chemiluminescent material, such as, 225Acγ-emitting, Auger-emitting, β-emitting, an alpha-emitting or positron-emitting radioactive isotope.

5.4. Polynucleotides and Vectors

In certain embodiments, the disclosure provides polynucleotides that encode the single domain antibodies that bind to SARS-CoV-2 S1 (e.g., RBD) and fusion proteins comprising the single domain antibodies that bind to SARS-CoV-2 S1 (e.g., RBD) described herein. The polynucleotides of the disclosure can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

In exemplary embodiments, the nucleic acid molecule provided herein comprises a sequence that encodes the single domain antibody having the sequence of any one of SEQ ID NOs: 13-24. In other exemplary embodiments, the nucleic acid molecule provided herein comprises a sequence that encodes a fusion protein having the sequence of any one of SEQ ID NOs: 25-60.

The present disclosure further relates to variants of the polynucleotides described herein, wherein the variant encodes, for example, fragments, analogs, and/or derivatives of the single domain antibody or fusion protein that binds SARS-CoV-2 S1 (e.g., RBD) of the disclosure. In certain embodiments, the present disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence at least about 75%identical, at least about 80%identical, at least about 85%identical, at least about 90%identical, at least about 95%identical, and in some embodiments, at least about 96%, 97%, 98%or 99%identical to a polynucleotide encoding the single domain antibody or fusion protein that binds SARS-CoV-2 S1 (e.g., RBD) of the disclosure. As used herein, the phrase “a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence” is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5%of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code) . Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (i.e., change codons in the human mRNA to those preferred by a bacterial host such as E. coli) . In some embodiments, a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.

In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.

Also provided are vectors comprising the nucleic acid molecules described herein. In an embodiment, the nucleic acid molecules can be incorporated into a recombinant expression vector. The present disclosure provides recombinant expression vectors comprising any of the nucleic acids of the disclosure. As used herein, the term “recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors described herein are not naturally-occurring as a whole; however, parts of the vectors can be naturally-occurring. The described recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring or non-naturally-occurring internucleotide linkages, or both types of linkages. The non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.

In an embodiment, the recombinant expression vector of the disclosure can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, Md. ) , the pBluescript series (Stratagene, LaJolla, Calif. ) , the pET series (Novagen, Madison, Wis. ) , the pGEX series (Pharmacia Biotech, Uppsala, Sweden) , and the pEX series (Clontech, Palo Alto, Calif. ) . Bacteriophage vectors, such as λGT10, λGT11, λEMBL4, and λNM1149, λZapII (Stratagene) can be used. Examples of plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech) . Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech) . The recombinant expression vector may be a viral vector, e.g., a retroviral vector, e.g., a gamma retroviral vector.

In an embodiment, the recombinant expression vectors are prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColE1, SV40, 2μ plasmid, λ, bovine papilloma virus, and the like.

The recombinant expression vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, plant, fungus, or animal) into which the vector is to be introduced, as appropriate, and taking into consideration whether the vector is DNA-or RNA-based.

The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the described expression vectors include, for instance, neomycin/G418 resistance genes, histidinol x resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or normative promoter operably linked to the nucleotide sequence of the disclosure. The selection of promoters, e.g., strong, weak, tissue-specific, inducible and developmental-specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an RSV promoter, an SV40 promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus.

The recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression.

Further, the recombinant expression vectors can be made to include a suicide gene. As used herein, the term “suicide gene” refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art and include, for example, the Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

In certain embodiments, a polynucleotide is isolated. In certain embodiments, a polynucleotide is substantially pure.

Also provided are host cells comprising the nucleic acid molecules described herein. The host cell may be any cell that contains a heterologous nucleic acid. The heterologous nucleic acid can be a vector (e.g., an expression vector) . For example, a host cell can be a cell from any organism that is selected, modified, transformed, grown, used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. An appropriate host may be determined. For example, the host cell may be selected based on the vector backbone and the desired result. By way of example, a plasmid or cosmid can be introduced into a prokaryote host cell for replication of several types of vectors. Bacterial cells such as, but not limited to DH5α, JM109, and KCB,

Competent Cells, and SOLOPACK Gold Cells, can be used as host cells for vector replication and/or expression. Additionally, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to yeast (e.g., YPH499, YPH500 and YPH501) , insects and mammals. Examples of mammalian eukaryotic host cells for replication and/or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, Saos, PC12, SP2/0 (American Type Culture Collection (ATCC) , Manassas, VA, CRL-1581) , NS0 (European Collection of Cell Cultures (ECACC) , Salisbury, Wiltshire, UK, ECACC No. 85110503) , FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196) . Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologics, Walkersville, MD) , CHO-K1 (ATCC CRL-61) or DG44.

5.5. Pharmaceutical Compositions

In one aspect, the present disclosure further provides pharmaceutical compositions comprising a single domain antibody, a binding molecule or therapeutic molecule comprising one or more single domain antibodies of the present disclosure. In other embodiments, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid provided herein, e.g., in a vector, and a pharmaceutically acceptable excipient, e.g., suitable for gene therapy.

In a specific embodiment, the term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) , carrier or vehicle. Pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions will contain a prophylactically or therapeutically effective amount of the active ingredient provided herein, such as in purified form, together with a suitable amount of excipient so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In some embodiments, the choice of excipient is determined in part by the particular binding molecule, and/or antibody, and/or by the method of administration. Accordingly, there are a variety of suitable formulations.

Typically, acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g. Zn-protein complexes) ; chelating agents such as EDTA and/or non-ionic surfactants.

Buffers may be used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Preservatives may be added to retard microbial growth. Tonicity agents, sometimes known as “stabilizers” can be present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter and intra-molecular interactions. Additional exemplary excipients include: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) agents preventing denaturation or adherence to the container wall. Non-ionic surfactants or detergents (also known as “wetting agents” ) may be present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. In order for the pharmaceutical compositions to be used for in vivo administration, they are preferably sterile.

The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means. In another embodiment, a pharmaceutical composition can be provided as a controlled release or sustained release system.

The pharmaceutical compositions described herein may also contain more than one active compound or agent as necessary for the particular indication being treated. Alternatively, or in addition, the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

Various compositions and delivery systems are known and can be used with the therapeutic agents provided herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the single domain antibody or therapeutic molecule provided herein, construction of a nucleic acid as part of a retroviral or other vector, etc.

In some embodiments, the pharmaceutical composition provided herein contains the binding molecules in amounts effective to treat or prevent the disease or disorder, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined.

5.6. Methods and Uses

In another aspect, provided herein are methods for using and uses of the SARS-CoV-2 S1 (e.g., RBD) binding molecules provided herein, including the anti-SARS-CoV-2 S1 sdAbs and fusion proteins comprising same provided herein.

5.6.1. Therapeutic Methods and Uses

Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules or compositions containing the same, to a subject having a disease, condition, or disorder associated with SARS-CoV-2. In some embodiments, the molecule and/or composition is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the antibodies and fusion proteins in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the antibodies or compositions comprising the same, to the subject having or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or disorder in the subject.

In some embodiments, the treatment provided herein cause complete or partial amelioration or reduction of a disease or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms include, but do not imply, complete curing of a disease or complete elimination of any symptom or effect (s) on all symptoms or outcomes.

As used herein, in some embodiments, the treatment provided herein delay development of a disease or disorder, e.g., defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease or disorder. In other embodiments, the method or the use provided herein prevents a disease or disorder.

In some embodiments, the disease or disorder is a SARS-CoV-2 associated disease or disorder. In some embodiments, the disease or disorder is a respiratory disease. In some embodiments, the disease or disorder is a severe acute respiratory syndrome (SARS) , or coronavirus disease (COVID-19) .

In some embodiments, the subject, to whom the compositions are administered is a primate, such as a human. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some examples, the subject is a validated animal model for disease and/or for assessing toxic outcomes.

The SARS-CoV-2 S1 (e.g., RBD) -binding molecules, such as VHH domains and fusion proteins comprising the VHH domains, can be administered by any suitable means, for example, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

The amount of a prophylactic or therapeutic agent provided herein that will be effective in the prevention and/or treatment of a disease or condition can be determined by standard clinical techniques. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For the prevention or treatment of disease, the appropriate dosage of the binding molecule or cell may depend on the type of disease or disorder to be treated, the type of binding molecule, the severity and course of the disease or disorder, whether the therapeutic agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician. The compositions, molecules and cells are in some embodiments suitably administered to the patient at one time or over a series of treatments.

For example, depending on the type and severity of the disease, dosages of antibodies for fusion proteins may include about 10 ug/kg to 100 mg/kg or more. Multiple doses may be administered intermittently. An initial higher loading dose, followed by one or more lower doses may be administered. In some embodiments, wherein the pharmaceutical composition comprises any one of the single domain antibodies described herein or fusion proteins comprising same, the pharmaceutical composition is administered at a dosage of about 10 ng/kg up to about 100 mg/kg of body weight of the individual or more per day, for example, at about 1 mg/kg/day to 10 mg/kg/day, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature (see, e.g., U.S. Pat. Nos. 4,657,760; 5,206,344; and 5,225,212) .

In some embodiments, the pharmaceutical composition is administered for a single time. In some embodiments, the pharmaceutical composition is administered for multiple times (such as any of 2, 3, 4, 5, 6, or more times) . In some embodiments, the pharmaceutical composition is administered once or multiple times during a dosing cycle. A dosing cycle can be, e.g., 1, 2, 3, 4, 5 or more week (s) , or 1, 2, 3, 4, 5, or more month (s) . The optimal dosage and treatment regime for a particular patient can be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

In some embodiments, the compositions are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as another antibody or receptor or agent, such as a cytotoxic or therapeutic agent.

In some embodiments, the compositions are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the compositions are co-administered with another therapy sufficiently close in time such that the compositions enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the compositions are administered prior to the one or more additional therapeutic agents. In some embodiments, the compositions are administered after to the one or more additional therapeutic agents.

In some specific embodiments, provided herein is a method for treating a disease or disorder in a subject comprising administering to the subject a binding molecule comprising a single domain antibody that binds to SARS-CoV-2 S1 (e.g., RBD) as described in Section 5.2 above, including, e.g., those with CDRs in Table 4 and Table 16, those comprising the amino acid sequence of any one of SEQ ID NOs: 13-24, and those comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identify to any one of SEQ ID NOs: 13-24. In some embodiments, the disease or disorder is a SARS-CoV-2 associated disease or disorder. In some embodiments, the disease or disorder is a respiratory disease.

In other specific embodiments, provided herein is a method for treating a disease or disorder in a subject comprising administering to the subject a fusion protein comprising a single domain antibody that binds to SARS-CoV-2 S1 (e.g., RBD) , including, e.g., fusion proteins described in Section 5.3, and those comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identify to any one of SEQ ID NOs: 25-60. In some embodiments, the disease or disorder is a SARS-CoV-2 associated disease or disorder. In some embodiments, the disease or disorder is a respiratory disease.

5.6.2. Diagnostic and Detection Methods and Uses

In another aspect, provided herein are methods involving use of the binding molecules provided herein, e.g., VHH domains that bind SARS-CoV-2 S1 (e.g., RBD) and molecules (such as fusion proteins/conjugates and complexes) containing such VHH domains, for detection, prognosis, diagnosis, staging, determining binding of a particular treatment to one or more tissues or cell types, and/or informing treatment decisions in a subject, such as by the detection of SARS-CoV-2 S1 (e.g., RBD) and/or the presence of an epitope thereof recognized by the antibody.

In some embodiments, an anti-SARS-CoV-2 S1 antibody (such as any one of the anti-SARS-CoV-2 S1 single domain antibodies described herein) for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of SARS-CoV-2 in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of SARS-CoV-2 S1 (e.g., RBD) protein in a biological sample.

The methods in some embodiments include incubating and/or probing a biological sample with the antibody and/or administering the antibody to a subject. In certain embodiments, a biological sample includes a cell or tissue or portion thereof. In certain embodiments, the contacting is under conditions permissive for binding of the anti-SARS-CoV-2 S1 antibody to SARS-CoV present in the sample. In some embodiments, the methods further include detecting whether a complex is formed between the anti-SARS-CoV-2 S1 antibody and a SARS-CoV-2 protein in the sample, such as detecting the presence or absence or level of such binding. Such a method may be an in vitro or in vivo method. In one embodiment, an anti-SARS-CoV-2 S1 antibody is used to select subjects eligible for therapy with an anti-SARS-CoV-2 S1 antibody or fusion protein comprising same, e.g., where SARS-CoV-2 S1 (e.g., RBD) is a biomarker for selection of patients.

In some embodiments, a sample, such as a cell, tissue sample, lysate, composition, or other sample derived therefrom is contacted with the anti-SARS-CoV-2 S1 antibody and binding or formation of a complex between the antibody and the sample (e.g., SARS-CoV-2 S1 (e.g., RBD) in the sample) is determined or detected. When binding in the test sample is demonstrated or detected as compared to a reference cell of the same tissue type, it may indicate the presence of an associated disease or disorder, and/or that a therapeutic containing the antibody will specifically bind to a tissue or cell that is the same as or is of the same type as the tissue or cell or other biological material from which the sample is derived. In some embodiments, the sample is from human tissues and may be from diseased and/or normal tissue, e.g., from a subject having the disease or disorder to be treated and/or from a subject of the same species as such subject but that does not have the disease or disorder to be treated.

Various methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays include fluorescence polarization immunoassay (FPIA) , fluorescence immunoassay (FIA) , enzyme immunoassay (EIA) , nephelometric inhibition immunoassay (NIA) , enzyme linked immunosorbent assay (ELISA) , and radioimmunoassay (RIA) . An indicator moiety, or label group, can be used so as to meet the needs of various uses of the method which are often dictated by the availability of assay equipment and compatible immunoassay procedures. Exemplary labels include radionuclides (e.g. ¹²⁵I, ¹³¹I, ³⁵S, ³H, or ³²P and/or chromium ( ⁵¹Cr) , cobalt ( ⁵⁷Co) , fluorine ( ¹⁸F) , gadolinium ( ¹⁵³Gd, ¹⁵⁹Gd) , germanium ( ⁶⁸Ge) , holmium ( ¹⁶⁶Ho) , indium ( ¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In) , iodine ( ¹²⁵I, ¹²³I, ¹²¹I) , lanthanium ( ¹⁴⁰La) , lutetium ( ¹⁷⁷Lu) , manganese ( ⁵⁴Mn) , molybdenum ( ⁹⁹Mo) , palladium ( ¹⁰³Pd) , phosphorous ( ³²P) , praseodymium ( ¹⁴²Pr) , promethium ( ¹⁴⁹Pm) , rhenium (186Re, 188Re) , rhodium (105Rh) , rutheroium (97Ru) , samarium ( ¹⁵³Sm) , scandium ( ⁴⁷Sc) , selenium ( ⁷⁵Se) , ( ⁸⁵Sr) , sulphur ( ³⁵S) , technetium ( ⁹⁹Tc) , thallium ( ²⁰¹Ti) tin ( ¹¹³Sn, ¹¹⁷Sn) , tritium (3H) , xenon ( ¹³³Xe) , ytterbium ( ¹⁶⁹Yb, ¹⁷⁵Yb) , yttrium ( ⁹⁰Y) , ) , enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or β-glactosidase) , fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP) , or luminescent moieties (e.g., Qdot ^TM nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif. ) . Various general techniques to be used in performing the various immunoassays noted above are known.

In certain embodiments, labeled antibodies (such as anti-SARS-CoV-2 S1 single domain antibodies) are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels) , as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. In other embodiments, antibodies are not labeled, and the presence thereof can be detected using a labeled antibody which binds to any of the antibodies.

5.7. Kits and Articles of Manufacture

Further provided are kits, unit dosages, and articles of manufacture comprising any of the single domain antibodies, or the fusion proteins described herein. In some embodiments, a kit is provided which contains any one of the pharmaceutical compositions described herein and preferably provides instructions for its use.

The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) , and the like. Kits may optionally provide additional components such as buffers and interpretative information. The present application thus also provides articles of manufacture, which include vials (such as sealed vials) , bottles, jars, flexible packaging, and the like.

The article of manufacture can comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. Generally, the container holds a composition which is effective for treating a disease or disorder described herein, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) . The label or package insert indicates that the composition is used for treating the particular condition in an individual. The label or package insert will further comprise instructions for administering the composition to the individual. The label may indicate directions for reconstitution and/or use. The container holding the pharmaceutical composition may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation. Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The kits or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.

The disclosure is generally disclosed herein using affirmative language to describe the numerous embodiments. The disclosure also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the disclosure is generally not expressed herein in terms of what the disclosure does not include, aspects that are not expressly included in the disclosure are nevertheless disclosed herein.

A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, the following examples are intended to illustrate but not limit the scope of disclosure described in the claims.

8. EXAMPLES

The following is a description of various methods and materials used in the studies, and are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like associated with the teachings of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc. ) , but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees

Centigrade, and pressure is at or near atmospheric.

6.1. Example 1-Generation of V _HH against SARS-CoV-2 S protein RBD

The RBD of SARS-CoV-2 is responsible for binding to ACE2 on human cells, which results in cell entry. Antibodies targeting RBD of SARS-CoV-2 could block the RBD-ACE2 interaction. To develop single domain antibodies with high binding affinity to the RBD of SARS-CoV-2, camels were immunized, a phage-display library was constructed and two rounds of phage display were preformed followed by an ELISA binding screen.

6.1.1. Cell lines construction

A HeLa cell line (ATCC, Cat. No. CCL-2) stably expressing human ACE2 (Uniprot No. Q9BYF1. SEQ ID NO: 61) was established by Genscript and was named HeLa-ACE2. Extracellular expression of ACE2 was monitored via flow cytometry. Biotinylated SARS-CoV-2 S protein RBD (ACRO, Cat. No. SPD-C82E9) was used as the detection binding domain and PE/Cy5 Streptavidin (Biolegend, Cat. No. 405205) was selected as second antibody. Following this method, the mean fluorescence intensity (MFI) of labeled HeLa-ACE2 cells was 498.22 folds higher than that of HeLa cells (MFI values 1120000 and 2248, respectively) .

6.1.2. Animal immunization and immune response testing

One adult male camel (Camelus bactrian) was immunized subcutaneously with SARS-CoV-2 S protein RBD-His (Genscript, Cat. No. Z03479) (a protein composed of the RBD of SARS-CoV-2 expressed with a His tag) for five times with two-week intervals. Blood was collected on pre-immune day (Pre) and last immunization day (TB) . Immune response of the camel was evaluated by ELISA, in which the serum samples were tested for binding to immobilized immunogens. A series of diluted sera were added to the plate. HRP-conjugated anti-llama lgG secondary antibody (BETHYL, Cat. No. A160100P) was added as the detection second antibody. From the ELISA results, a robust immune response was induced upon antigen injection to the animal, with the serum titer >1: 243 k. It indicted the animal immunization was successful.

To test competitive inhibition of serum, SARS-CoV-2 S protein RBD-mFc (Genscript, Cat. No. Z03491) was coated on plates. Pre-serum or TB-serum was co-incubated with human ACE2-His tag protein (Kactus, Cat. No. ACE-HM401) and then added to plates. At the same time, a fully human anti-SARS-CoV-2 S protein RBD antibody (Sanyoubio, Cat. No. AHA001) was co-incubated with human ACE2 as benchmark group. HRP-conjugated anti-His tag secondary antibody (Genscript, Cat. No. A00612) was added as the detection second antibody. From the ELISA results, TB-serum showed obvious competitive inhibition of human ACE2 to SARS-CoV-2 S protein RBD-mFc.

Three to five days after the last immunization, 100 mL of blood was collected from the jugular vein as production bleed. Peripheral blood lymphocytes (PBLs) were isolated from the blood according to the procedure of Lymphoprep.

6.1.3. V _HH phage display library construction

Total RNA was extracted from the isolated lymphocytes (from Section 6.1.2) according to the manual of

Reagent (Thermofisher, Cat. No. 15596026) . After quality testing, total RNA was reverse transcribed into cDNA using oligo (dT) ₂₀ primer according to the manual of PrimeScript ^TM 1st Strand cDNA Synthesis Kit (Takara, Cat. No. 6110A) . Nested PCR primers (Refer to patent CN105555310B) were designed for the amplification of V _HH fragments, with two SfiI restriction sites introduced. The V _HH fragment was amplified using a two-step polymerase chain reaction (PCR) , and the PCR products were digested with SfiI and gel purified, and then inserted into phagemid vector pFL249 (see CN105555310B) , which were electro-transferred into E. coli cells to generate the phage display V _HH immune library.

A small portion of the transformed cells was diluted and streaked on 2×YT plates supplemented with 100 μg/mL ampicillin. The colonies were counted to calculate the library size. Positive clones were randomly picked and sequenced to assess the quality of the library. The rest of the transformed cells were streaked onto 245-mm square 2 ×YT-agar dishes supplemented with 100 μg/mL ampicillin and 2%glucose. Lawns of colonies were scraped off the dishes. A small aliquot of the cells were used for library plasmids isolation. The rest was supplemented with glycerol and stored at ‐80℃ as stock.

Finally, a V _HH library was constructed. The positive clones were sequencing and V _HH domains from the library had a good CDR3 diversity.

6.1.4. Phage-display panning

After infection with helper phage, recombinant phage particles which display V _HH as gene III fusion proteins on the surface were produced. Phage particles were prepared according to standard methods and stored after filter sterilization at 4℃ for further research. Before each round of panning, V _HH-displaying phages particles were rescued and amplified by adding helper phages.

Phage libraries were used for different panning strategies. For solid panning, in a first and second round, SARS-CoV-2 S protein RBD-His (Genscript, Cat. No. Z03483) and SARS-CoV-2 S protein RBD-mFc (Sino, Cat. No. 40592-V05H) was incubated with the phage libraries. Following extensive washing, bound phages were eluted with triethylamine. After two round panning, phage enrichment was observed.

For liquid panning, biotinylated SARS-CoV-2 S protein RBD-His (ACRO, Cat. No. SPD-C82E9) and biotinylated SARS-CoV-2 S protein RBD-His (KACTUS, Cat. No. COV-VM4BD) was incubated with the phage libraries and subsequently captured on Streptavidin Dynabeads (Invitogen) . Following extensive washing, bound phages were eluted with triethylamine. After two round panning, phage enrichment was observed.

6.1.5. ELISA screening

Individual library clones were inoculated and induced for expression in 96‐deep‐well plates. ELISA screening was performed to isolate V _HH clones which recognize SARS-CoV-2 S protein RBD-His (ACRO, Cat. No. SPD-C82E9) specifically.

After screening, clones with high binding capacities were selected and sequenced. The CDR and V _HH sequences were summarized in Table 4.

Table 4. Sequence ID number of CDRs and V _HH domains (Kabat Numbering)

Clone ID	CDR1	CDR2	CDR3	V _HH domain
77NCOVP01	SEQ ID NO: 1	SEQ ID NO: 5	SEQ ID NO: 9	SEQ ID NO: 13
77NCOVP02	SEQ ID NO: 2	SEQ ID NO: 6	SEQ ID NO: 10	SEQ ID NO: 14
77NCOVP03	SEQ ID NO: 3	SEQ ID NO: 7	SEQ ID NO: 11	SEQ ID NO: 15
77NCOVP04	SEQ ID NO: 4	SEQ ID NO: 8	SEQ ID NO: 12	SEQ ID NO: 16

6.2. Example 2-Preparation of chimeric single domain antibodies against SARS-CoV-2 S protein RBD

6.2.1. Construction of V _HH-expression plasmid

The V _HH coding sequences for the selected antibodies were optimized for human codon biased expression with GenScript OptimumGene ^TM-Codon Optimization, synthesized and fused to human IgG1Fc (SEQ ID NO: 62) coding sequence for transient expression in chimeric formats. The chimeric antibody coding sequence were cloned into pcDNA3.4-based mammalian expression system plasmids and the plasmids were maxi-prepared for protein production by GenScript with general molecular biology techniques known in the art. At the same time, heavy and light chain sequences of CB6 (GenBank No. MT470196 and MT470197) against SARS-CoV-2 were selected and expressed as whole IgG. It was named as JSCB6 as a benchmark antibody. The chimeric antibodies were constructed and are shown in Table 5.

Table 5. Amino Acid Sequence ID Numbers of chimeric antibodies and corresponding V _HH domains

6.2.2. Expression and purification of chimeric antibodies

HEK293F cells (ThermoFisher, Cat. No. A14528) were grown in serum-free FreeStyle ^TM 293 Expression Medium. The cells were maintained in Erlenmeyer Flasks at 37℃with 5%CO ₂ on an orbital shaker. One day before transfection, cells were seeded at an appropriate density in Corning Erlenmeyer Flasks. For transfection, DNA and polyetherimide (PEI) were mixed at an optimal ratio and added into the flask with cells. The supernatant was collected on day 6 and used for purification. Cell culture broth was centrifuged followed by filtration. Filtered supernatant was loaded onto a 1 mL HiTrap ^TM Protein A column (GE Healthcare, Cat. No. 17040201) at 0.5 mL/min. After washing and elution with appropriate buffer, the antibody fractions were collected and neutralized. The purity of proteins was evaluated by SDS-PAGE (GenScript Cat. No. M42012) . The concentration was determined by Bradford method. Representative data of protein expression was summarized in Table 6.

Table 6. Expression of chimeric antibodies

6.3. Example 3-Characterization of chimeric single domain antibodies

6.3.1. Affinity by SPR

The binding kinetics of the antibodies to recombinant SARS-CoV-2 S protein RBD-His (Genscript, Cat. No. Z03483) were measured by the surface plasmon resonance (SPR) (e.g., Biacore) . Binding rate (ka) and dissociation rates (kd) were calculated using a simple one-to-one Langmuir binding model (Biacore Evaluation Software) . The equilibrium dissociation constant (KD) is calculated as the ratio kd/ka. The measured binding affinities of chimeric antibodies are shown in Table 7.

Table 7. Affinity measurement of chimeric antibodies to SARS-CoV-2 S protein RBD

Ligand	Analyte	Chi ² (RU ²)	ka (1/Ms)	kd (1/s)	KD (M)	Rmax (RU)
77NCOVP05	RBD	2.42E-01	1.24E+06	6.88E-04	5.55E-10	29.4
77NCOVP06	RBD	2.26E-01	8.36E+06	5.17E-04	6.18E-11	31.8
77NCOVP07	RBD	2.80E-01	7.68E+06	9.98E-04	1.30E-10	21.7
77NCOVP08	RBD	1.96E-01	2.69E+07	7.53E-04	2.79E-11	24.2
JSCB6	RBD	2.12E-01	4.36E+05	7.42E-04	1.70E-09	34.9

6.3.2. Blocking inhibition of SARS-CoV-2 S1 protein by FACS

5×10 ⁵ HeLa-ACE2 cells were prepared for each tube. High, middle and low concentration of SARS-CoV-2 S1-His protein (ACRO, Cat. No. S1N-C52H4, a protein composed of the S1 subunit of SARS-CoV-2 expressed with a His tag) or SARS-CoV-2 S protein RBD-His protein (Genscript, Cat. No. Z03483) was added to corresponding tube, respectively. After that, gradient dilution of antibodies were added to each experimental group and PBS was added to S1 protein max binding group or RBD max binding group. After incubating on ice for 30 min and washing for three times, PE anti-His Tag Antibody (Biolegend, Cat. No. 362603) was added as the detection antibody, and the fluorescence intensity was detected by flow cytometry after incubation on ice for 30 minutes. The formula for calculating Blocking inhibition: Blocking ratio%=100 × (1-MFI _{experimental group}/MFI _{max binding group}) .

GraphPad Prism software was used for data processing. By four-parameter fitting, the blocking cure and IC ₅₀ value of antibodies on the effect between ACE2 expressed on HeLa-ACE2 cells and S1-His (SARS-CoV-2 S1-His protein) or between ACE2 expressed on HeLa cells and RBD-His (SARS-CoV-2 S protein RBD-His) were obtained. The results are shown in FIG. 1 and Table 8. Compared with JSCB6, all chimeric single domain antibodies could effectively block the interaction between ACE2 expressed on HeLa cells and S1-His or between ACE2 expressed on HeLa cells and RBD-His with lower IC ₅₀ value.

Table 8. IC ₅₀ of antibodies blocking S1-His or RBD-His binding to HeLa-ACE2 cells

6.3.3. Cross-neutralization of antibodies to different SARS-CoV-2 S protein variants by FACS

5×10 ⁵ HeLa-ACE2 cells were prepared for each tube. Referring to epidemiological analysis, eight kinds of SARS-CoV-2 S protein variants were selected and added to corresponding tube with concentrations of EC ₈₀ (effect concentration of S protein and HeLa-ACE2 cells) , respectively. Information of SARS-CoV-2 S protein variants reference to Lou et al., Cross-neutralization antibodies against SARS-CoV-2 and RBD mutations from convalescent patient antibody libraries, bioRxiv (June 6, 2020) is listed in Table 9. After that, gradient dilution of antibodies were added to each experimental group and PBS was added to S protein max binding group. After incubating on ice for 30 min and washing for three times, PE anti-His Tag Antibody (Biolegend, Cat. No. 362603) was added as the detection antibody, and the fluorescence intensity was detected by flow cytometry after incubation on ice for 30 minutes. The formula for calculating Blocking inhibition: Blocking ratio %=100 × (1-MFI _{experimental group}/MFI _max binding _group) .

GraphPad Prism software was used for data processing. By four-parameter fitting, the blocking cure and IC ₅₀ value of antibodies on the effect between ACE2 expressed on HeLa-ACE2 cells and SARS-CoV-2 S protein variants were obtained. The results are shown in FIG. 2 and Table 10. Compared with JSCB6, all anti-SARS-CoV-2 S single domain antibodies had obvious cross-neutralization with lower IC ₅₀ value.

Table 9. Information of SARS-CoV-2 S protein variants

Table 10. IC ₅₀ of antibodies blocking SARS-CoV-2 S protein variants binding to HeLa-ACE2 cells

6.3.4. Epitope binning

Epitope binning of anti-SARS-CoV-2 S antibodies were analyzed by SPR (Biacore 8K, GE Healthcare) . SARS-CoV-2 S protein RBD-His protein (Genscript, Cat. No. Z03483) was covalently immobilized to a CM5 sensor chip via amine groups in 10 mM sodium acetate buffer (pH 4.5) for a final RU around 50. Two antibodies were sequentially injected and monitored for binding activity to determine whether the two mAbs recognized separate or closely-situated epitopes. Blocking efficacy was determined by comparison of response units with and without same antibody incubation. Based on the competitive binding result, the antibodies are assigned to two epitope groups (Table 11) . 77NCOVP05 binds the different epitope of SARS-CoV-2 S protein RBD-His with 77NCOVP06, 77NCOVP07, 77NCOVP08, and JSCB6.

Table 11. Epitope Binning of anti-SARS-CoV-2 S Antibodies

Group 1	Group 2
77NCOVP06	77NCOVP05
77NCOVP07
77NCOVP08
JSCB6

6.4. Example 4-Stability test of chimeric anti-SARS-CoV-2 S antibodies

6.4.1. Resistance of antibodies to alkali, acid and oxidative stress

500 mM ammonium bicarbonate was used as alkali disrupting agent and anti-SARS-CoV-2 S antibodies were treated at 37℃ for 14, 22 or 38 hours. pH 3.5 citric acid was used as acid disrupting agent and anti-SARS-CoV-2 S antibodies were treated at 37℃ for 2 or 4 hours. 1%hydrogen peroxide was used as the oxidant, 4 or 8 hours treatment at room temperature.

The changes in the biological activity of the anti-SARS-CoV-2 S antibodies obtained by the above Examples before and after treatment were measured using FACS blocking assay as method in Section 6.3.2 and untreated antibody (UT) served as a control. The SARS-CoV-2 S1-His protein (ACRO, Cat. No. S1N-C52H4) at a concentration of 3.33 μg/mL and serial diluted antibodies were added respectively to 1×10 ⁵ HeLa-ACE2 cells. As can be seen in FIGs. 3A-3E and Table 12, alkali and acid treatments did not affect the activity of the anti-SARS-CoV-2 S antibodies. After treated by 1%hydrogen peroxide, unlike significantly reduced blocking capability of JSCB6 (IC ₅₀ from 9.47 nM to 211.30 nM and 261.40 nM) , all chimeric anti-SARS-CoV-2 S single domain antibodies still maintain remarkable blocking capability to SARS-COV-2 S1 protein.

6.4.2. Resistance of antibodies to temperature

To determine the thermal stability of anti-SARS-CoV-2 S antibodies. All antibodies were stored at 40℃ for 1 week, 2 weeks or 4 weeks. The samples underwent 3 or 5 freeze-and-thaw cycles were also measured.

The changes in the biological activity of the anti-SARS-CoV-2 S antibodies obtained by the above Examples before and after treatment were measured using FACS blocking assay as method in Section 6.3.2 and untreated antibody (UT) served as a control. The SARS-CoV-2 S1-His protein (ACRO, Cat. No. S1N-C52H4) at a concentration of 3.33 μg/mL and serial diluted anti-SARS-CoV-2 S antibodies were added respectively to 1×10 ⁵ HeLa-ACE2 cells. As can be seen in FIGs. 3A-3E and Table 12, all antibodies had obvious thermal stability.

Table 12. IC ₅₀ (nM) of antibodies after different processes blocking SARS-CoV-2 S1-His protein binding to HeLa-ACE2 cells

Process	77NCOVP05	77NCOVP06	77NCOVP07	77NCOVP08	JSCB6
UT	5.70	4.94	3.00	4.20	9.47
FZ-3rd cycle	5.92	5.18	2.11	2.91	10.81
FZ-5th cycle	6.38	5.10	4.97	4.92	12.3
NH ₄HCO ₃-14h	4.91	4.51	6.94	4.84	6.39
NH ₄HCO ₃-22h	6.81	5.24	7.66	4.86	7.09
NH ₄HCO ₃-38h	6.38	7.39	10.59	8.52	14.36
Low pH-2h	8.27	5.13	3.86	3.58	5.78
Low pH-4h	7.56	5.52	4.05	3.97	6.27
H ₂O ₂-4h	7.52	6.40	3.99	3.33	211.30
H ₂O ₂-8h	7.97	7.21	5.42	8.21	261.40
40C-D7	3.70	7.93	11.78	6.81	10.47
40C-D14	3.38	7.27	9.30	4.39	8.83
40C-D28	8.84	6.90	7.82	5.86	11.23

6.5. Example 5-Preparation and characterization of chimeric anti-SARS-CoV-2 S Bi-V _HH antibodies

6.5.1. Antibodies expression and purification

Two antibody formats were selected. In the first format, the first VHH (V _HH1) was cloned into N domain of human IgG1 Fc variant sequence (SEQ ID NO: 63) and the second VHH (V _HH2) was cloned into C domain of human IgG1 Fc variant sequence (e.g., via a linker having a SEQ ID NO: 64) . In the second format, V _HH1 was linked with V _HH2 firstly using a linker (SEQ ID NO: 65) and then cloned into N domain of human IgG1 Fc variant sequences. 77NCOVP01, 77NCOVP02, 77NCOVP03, 77NCOVP04 was randomly paired to format bivalent or bi-epitope structure. Coding sequences for the selected antibodies were optimized for human codon biased expression with GenScript OptimumGene ^TM-Codon Optimization and coding sequence were cloned into pcDNA3.4-based mammalian expression system plasmids and the plasmids were maxi-prepared for protein production by GenScript with general molecular biology techniques known in the art. Exemplary chimeric Bi-V _HH antibodies of the disclosure are shown in Table 13, as well as their corresponding V _HH domains.

Table 13. The structure of chimeric Bi-V _HH antibodies and their corresponding V _HH domains

The chimeric Bi-V _HH antibodies were expressed and the purity was evaluated by SDS- PAGE (GenScript Cat. No. M42012) . The concentration was determined by Bradford method. Representative data of protein expression was summarized in Table 14.

Table 14. Expression of chimeric Bi-V _HH antibodies

6.5.2. Blocking inhibition of SARS-CoV-2 S1 protein by FACS

The biological activity of the anti-SARS-CoV-2 S chimeric Bi-V _HH antibodies obtained by the above Examples were measured using FACS blocking assay as method in Section 6.3.2. The SARS-CoV-2 S1-His protein (ACRO, Cat. No. S1N-C52H4) at a concentration of 3.33 μg/mL and serial diluted chimeric Bi-V _HH antibodies were added respectively to 1×10 ⁵ HeLa-ACE2 cells. As can be seen in FIG. 4 and Table 15, compared with single chimeric V _HH antibodies (77NCOVP05 IC ₅₀: 6.71nM, 77NCOVP06 IC ₅₀: 6.52nM, 77NCOVP07 IC ₅₀: 4.58nM and 77NCOVP08 IC ₅₀: 4.3nM) , chimeric Bi-V _HH antibodies had more robust blocking ability, especially, 77NCNV109 (IC ₅₀: 0.48nM) and 77NCNV112 (IC ₅₀: 0.57nM) .

Table 15. IC ₅₀ of chimeric Bi-V _HH antibodies blocking SARS-CoV-2 S1 protein binding to HeLa-ACE2 cells

Sample ID

IC ₅₀ (nM)

Sample ID

IC ₅₀ (nM)

77NCNV101	4.82	77NCNV102	4.99
77NCNV104	5.37	77NCNV107	3.74
77NCNV201	6.81	77NCNV202	5.76
77NCNV204	7.06	77NCNV207	3.39
77NCNV103	2.81	77NCNV105	2.57
77NCNV110	4.84	77NCNV108	1.71
77NCNV203	4.91	77NCNV205	1.63
77NCNV210	5.66	77NCNV208	2.83
77NCNV106	2.08	77NCNV109	0.48
77NCNV111	1.45	77NCNV112	0.57
77NCNV206	1.89	77NCNV209	1.81
77NCNV211	2.35	77NCNV212	2.67

6.6. Example 6-Preparation and characterization of anti-SARS-CoV-2 S humanized antibodies

6.6.1. Humanization design

To humanize antibodies containing binding moieties with high binding affinity, V _HH amino acid residues from Example 1 were humanized according to the description by Ce′cile Vincke et al (J. Biol. Chem. 2009, 284: 3273-3284) and resurfacing framework of V _HH antibodies.

The homologous modeling of camelid V _HH was performed using the modeling software MODELLER. The reference homologous sequence of 77NCOVP01 was a camelid VHH HL6 antibody fragment (Protein Data Bank, PDB code: 1OP9) . The reference homologous sequence of 77NCOVP02 and 77NCOVP04 was a Synthetic nanobody (Protein Data Bank, PDB code: 5M13) . The reference homologous sequence of 77NCOVP03 was a Fab fragment of a Charcot-Leyden (Protein Data Bank, PDB code: 6GKU) .

According to alignment with human germline gene, IGHV3-66*01 was chosen as human acceptor for 77NCOVP01 and 77NCOVP02. IGHV3-23*04 was chosen as human acceptor for 77NCOVP03. IGHV3-30*02 was chosen as human acceptor for 77NCOVP04. Relative solvent accessibility of the amino acids is calculated according to the three-dimensional structure of the protein. If one of the amino acids of V _HH is exposed to a solvent, it was replaced with the original amino acid. Exemplary humanized V _HH domains of the disclosure are shown in Table 16.

Table 16. Sequence ID number of humanized V _HH domains (Kabat Numbering)

Clone ID	CDR1	CDR2	CDR3	V _HH domain
77NCOVP01H1	SEQ ID NO: 1	SEQ ID NO: 5	SEQ ID NO: 9	SEQ ID NO: 17
77NCOVP01H2	SEQ ID NO: 1	SEQ ID NO: 5	SEQ ID NO: 9	SEQ ID NO: 18
77NCOVP02H1	SEQ ID NO: 2	SEQ ID NO: 6	SEQ ID NO: 10	SEQ ID NO: 19

77NCOVP02H2	SEQ ID NO: 2	SEQ ID NO: 6	SEQ ID NO: 10	SEQ ID NO: 20
77NCOVP03H1	SEQ ID NO: 3	SEQ ID NO: 7	SEQ ID NO: 11	SEQ ID NO: 21
77NCOVP03H2	SEQ ID NO: 3	SEQ ID NO: 7	SEQ ID NO: 11	SEQ ID NO: 22
77NCOVP04H1	SEQ ID NO: 4	SEQ ID NO: 8	SEQ ID NO: 12	SEQ ID NO: 23
77NCOVP04H2	SEQ ID NO: 4	SEQ ID NO: 8	SEQ ID NO: 12	SEQ ID NO: 24

6.6.2. Antibodies expression and purification

The humanized V _HH coding sequences for the selected antibodies were optimized for human codon biased expression with GenScript OptimumGene ^TM-Codon Optimization, synthesized and fused to human IgG1Fc (SEQ ID NO: 62) coding sequence for transient expression in chimeric formats. The chimeric antibody coding sequence were cloned into pcDNA3.4-based mammalian expression system plasmids and the plasmids were maxi-prepared for protein production by GenScript with general molecular biology techniques known in the art. Exemplary humanized chimeric antibodies of the disclosure are shown in Table 17, as well as their corresponding humanized V _HH domains.

Table 17. Amino Acid Sequence ID Number of humanized chimeric antibodies and corresponding humanized V _HH domains

The humanized chimeric antibodies were expressed and the purity of proteins was evaluated by SDS-PAGE (GenScript Cat. No. M42012) . The concentration was determined by Bradford method. Representative data of protein expression was summarized in Table 18.

Table 18. Expression of humanized chimeric antibodies

6.6.3. Blocking inhibition of SARS-CoV-2 S1 protein by FACS

The biological activity of the humanized chimeric antibodies obtained by the above Examples were measured using FACS blocking assay as method in Section 6.3.2. The SARS-CoV-2 S1-His protein (ACRO, Cat. No. S1N-C52H4) at a concentration of 3.33 μg/mL and serial diluted humanized anti-SARS-CoV-2 S chimeric antibodies were added respectively to 1×10 ⁵ HeLa-ACE2 cells. As can be seen in FIG. 5 and Table 19, compared with parental antibodies, humanized chimeric antibodies had closely blocking ability. It indicated that humanization was successful.

Table 19. IC ₅₀ of humanized chimeric antibodies vs. their parental antibodies blocking SARS-CoV-2 S1 protein binding to HeLa-ACE2 cells

Sample ID	IC ₅₀ (nM)	Sample ID	IC ₅₀ (nM)
77NCOVP05	7.16	77NCOVP06	8.08
77NCOVP09	10.69	77NCOVP11	8.90
77NCOVP10	8.44	77NCOVP12	9.89
77NCOVP07	3.89	77NCOVP08	7.15
77NCOVP13	4.88	77NCOVP15	9.01
77NCOVP14	5.69	77NCOVP16	8.20

The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

From the foregoing, it will be appreciated that, although specific embodiments have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of what is provided herein. All of the references referred to above are incorporated herein by reference in their entireties.

SEQUENCE LISTING

Claims

An anti-SARS-CoV-2 S1 single domain antibody (sdAb) , wherein optionally the sdAb comprises:

(i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9;

(ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10;

(iii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or

(iv) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12.
An anti-SARS-CoV-2 S1 single domain antibody (sdAb) comprising:

(i) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 13;

(ii) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 14;

(iii) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 15;

(iv) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 16;

(v) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 17;

(vi) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 18;

(vii) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 19;

(viii) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 20;

(ix) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 21;

(x) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 22;

(xi) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 23; or

(xii) a CDR1, a CDR2, and a CDR3 having the amino acid sequences of the CDR1, CDR2, and CDR3, respectively, as set forth in SEQ ID NO: 24.
The anti-SARS-CoV-2 S1 sdAb of claim 2, wherein the CDR1, CDR2 or CDR3 are determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or a combination thereof.
The anti-SARS-CoV-2 S1 sdAb of any one of claims 1 to 3, further comprising one or more FR regions as set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and/or SEQ ID NO: 24.
The anti-SARS-CoV-2 S1 sdAb of any one of claims 1 to 4, comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
The anti-SARS-CoV-2 S1 sdAb of any one of claims 1 to 4, wherein the anti-SARS-CoV-2 S1 sdAb comprises or consists of the amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with the sequence of SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
The anti-SARS-CoV-2 S1 sdAb of claim 1 or claim 2, wherein the anti-SARS-CoV-2 S1 sdAb is a camelid sdAb or wherein the anti-SARS-CoV-2 S1 sdAb is a humanized sdAb.
The anti-SARS-CoV-2 S1 sdAb of claim 1 or claim 2, wherein the anti-SARS-CoV-2 S1 sdAb is a VHH domain, wherein the VHH domain optionally binds to the receptor binding domain (RBD) of SARS-CoV-2 or a variant thereof.
The anti-SARS-CoV-2 S1 sdAb of any one of claims 1 to 8, wherein the anti-SARS-CoV-2 S1 sdAb is genetically fused or chemically conjugated to an agent.
The anti-SARS-CoV-2 S1 sdAb of any one of claims 1 to 8, wherein the anti-SARS- CoV-2 S1 sdAb is fused to an Fc region.
The anti-SARS-CoV-2 S1 sdAb of claim 10, wherein the Fc region is a human IgG1 Fc region or a variant thereof comprising the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63.
A fusion protein comprising the anti-SARS-CoV-2 S1 sdAb of any one of claims 1 to 8 and an Fc region, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, or the amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36.
A fusion protein comprising a first anti-SARS-CoV-2 S1 sdAb and a second anti-SARS-CoV-2 S1 sdAb, wherein each of the first and the second anti-SARS-CoV-2 S1 sdAb is a VHH domain.
The fusion protein of claim 13, wherein the first anti-SARS-CoV-2 S1 sdAb and the second anti-SARS-CoV-2 S1 sdAb are each independently an sdAb of any one of claims 1 to 8.
The fusion protein of claim 13 or claim 14, wherein the first anti-SARS-CoV-2 S1 sdAb and the second anti-SARS-CoV-2 S1 sdAb are the same.
The fusion protein of claim 13 or claim 14, wherein the first anti-SARS-CoV-2 S1 sdAb and the second anti-SARS-CoV-2 S1 sdAb are different.
The fusion protein of any one of claims 13 to 16, wherein the first anti-SARS-CoV-2 S1 sdAb is at the N terminus of the second anti-SARS-CoV-2 S1 sdAb.
The fusion protein of any one of claims 13 to 16, wherein the first anti-SARS-CoV-2 S1 sdAb is at the C terminus of the second anti-SARS-CoV-2 S1 sdAb.
The fusion protein of any one of claims 13 to 18, wherein the fusion protein further comprises one or more additional agent (s) .
The fusion protein of any one of claims 13 to 18, wherein the fusion protein further comprises a domain comprising a human IgG1 Fc region or a variant thereof.
The fusion protein of claim 20, wherein the domain comprises the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63.
The fusion protein of claim 20 or claim 21, wherein the fusion protein comprises from N terminus to C terminus: the first anti-SARS-CoV-2 S1 sdAb-IgG1 Fc-the second anti-SARS-CoV-2 S1 sdAb.
The fusion protein of claim 20 or claim 21, wherein the fusion protein comprises from N terminus to C terminus: the first anti-SARS-CoV-2 S1 sdAb-the second anti-SARS-CoV-2 S1 sdAb-IgG1 Fc.
The fusion protein of claim 22 or claim 23, wherein two or more regions in the fusion protein are linked directly or via a peptide linker.
The fusion protein of any one of claims 13 to 20, wherein

(i) the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10;

(ii) the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11;

(iii) the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12;

(iv) the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9;

(v) the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11;

(vi) the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12;

(vii) the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9;

(viii) the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10;

(ix) the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12;

(x) the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12; and wherein the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9;

(xi) the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12; and wherein the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a CDR2 comprising the amino acid sequence of SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10; or

(xii) the first anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12; and wherein the second anti-SARS-CoV-2 S1 sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11.
The fusion protein of claim 20 or claim 21, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60, or the amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.
An isolated nucleic acid comprising a nucleic acid sequence encoding the anti-SARS-CoV-2 S1 sdAb of any one of claims 1 to 11 or the fusion protein of any one of claims 12 to 26.
A vector comprising the isolated nucleic acid of claim 27.
A pharmaceutical composition, comprising the anti-SARS-CoV-2 S1 sdAb of any one of claims 1 to 11, the fusion protein of any one of claims 12 to 26, or the vector of claim 28, and a pharmaceutically acceptable excipient.
A method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the anti-SARS-CoV-2 S1 sdAb of any one of claims 1 to 11, the fusion protein of any one of claims 12 to 26, the vector of claim 28, or the pharmaceutical composition of claim 29.
The method of claim 30, wherein the disease or disorder is a SARS-CoV-2 associated disease or disorder, optionally being a respiratory disease.
The method of claim 31, wherein the respiratory disease is severe acute respiratory syndrome (SARS) or coronavirus disease (COVID-19) .
The method of claim 30, wherein the disease or disorder is a cardiovascular disease.