Anti-coronavirus antibodies and their uses

Abstract

The present disclosure relates to anti-coronavirus (e.g., SARS-CoV-2) antibodies or antigen-binding fragments and uses thereof.

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 012,751, filed on April 20, 2020, and U.S. Provisional Application No. 63 / 069,610, filed on August 24, 2020. The entire contents of the foregoing applications are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to anti-coronavirus antibodies or antigen-binding fragments and uses thereof. Background Art

[0004] Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses with mammalian and avian hosts. Previous coronaviruses known to infect humans include 229E, NL63, OC43, HKU1, SARS-CoV, and MERS CoV, which can cause a range of mild seasonal illnesses to severe outbreaks. It is worth noting that past outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) (2012) were caused by coronaviruses SARS-CoV and MERS-CoV, respectively (see Human Coronavirus Types: Centers for Disease Control and Prevention). SARS-CoV-2 is the seventh known coronavirus to infect humans, and is the third coronavirus to cross the species barrier and cause severe respiratory infections in humans in less than two decades after SARS and MERS. SARS-CoV-2 causes coronavirus disease 2019 (COVID-19) (see Okba NMA, Muller MA, Li W, et al. Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease 2019 Patients. Emerg Infect Dis. 2020 Apr 8;26(7)), which is more contagious than SARS-CoV and MERS-CoV.

[0005] The high infection rate and global impact caused by the disease have led the World Health Organization to declare COVID-19 an epidemic. As of April 13, 2020, the virus has been confirmed to infect more than 1.8 million people worldwide, with nearly 120,000 confirmed deaths and an estimated mortality rate of 6.34% (see Coronavirus disease (COVID-19) Pandemic World Health Organization: World Health Organization). The identification of viral etiology, the publication of research, and international collaborative efforts have led to the rapid development of real-time PCR diagnostic assays that support case determination and tracking of COVID-19 outbreaks. However, there is still a lack of validated serological diagnostic assays and therapeutic agents for SARS-CoV-2, and the serological diagnostic assays and therapeutic agents have become an urgent need to combat COVID-19. Summary of the invention

[0006] The present disclosure relates to anti-coronavirus S protein antibodies, antigen-binding fragments thereof, and uses thereof.

[0007] In one aspect, an antibody or antigen-binding fragment thereof is provided herein, which binds to a coronavirus S protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH), wherein the VHH comprises complementarity determining regions (CDRs) 1, 2, and 3. In some embodiments, the VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence. In some embodiments, the selected VHH CDR1, 2, 3 amino acid sequence is one of the following:

[0008] (1) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1, 2, and 3, respectively;

[0009] (2) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4, 5 and 6, respectively;

[0010] (3) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 7, 8 and 9, respectively;

[0011] (4) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 10, 11 and 12, respectively;

[0012] (5) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13, 14 and 15, respectively;

[0013] (6) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 16, 17 and 18, respectively;

[0014] (7) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 19, 20 and 21, respectively;

[0015] (8) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 22, 23 and 24, respectively;

[0016] (9) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 25, 26 and 27, respectively;

[0017] (10) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 28, 29 and 30, respectively;

[0018] (11) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 31, 32 and 33, respectively;

[0019] (12) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 34, 35 and 36, respectively;

[0020] (13) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 37, 38 and 39, respectively;

[0021] (14) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 40, 41 and 42, respectively;

[0022] (15) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 43, 44 and 45, respectively;

[0023] (16) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 46, 47 and 48, respectively;

[0024] (17) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 49, 50 and 51, respectively;

[0025] (18) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 52, 53 and 54, respectively;

[0026] (19) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 55, 56 and 57, respectively;

[0027] (20) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 58, 59, and 60, respectively;

[0028] (21) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 61, 62 and 63, respectively;

[0029] (22) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 64, 65 and 66, respectively;

[0030] (23) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 67, 68, and 69, respectively;

[0031] (24) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 70, 71 and 72, respectively;

[0032] (25) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 73, 74 and 75, respectively;

[0033] (26) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 76, 77, and 78, respectively;

[0034] (27) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 79, 80, and 81, respectively;

[0035] (28) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 82, 83 and 84, respectively;

[0036] (29) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 85, 86, and 87, respectively;

[0037] (30) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 88, 89 and 90, respectively;

[0038] (31) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 91, 92 and 93, respectively;

[0039] (32) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 94, 95, and 96, respectively;

[0040] (33) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 97, 98, and 99, respectively;

[0041] (34) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 100, 101 and 102, respectively;

[0042] (35) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 103, 104 and 105, respectively;

[0043] (36) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 106, 107, and 108, respectively;

[0044] (37) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 109, 110, and 111, respectively;

[0045] (38) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 112, 113, and 114, respectively;

[0046] (39) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 115, 116, and 117, respectively;

[0047] (40) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 118, 119, and 120, respectively;

[0048] (41) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 121, 122, and 123, respectively;

[0049] (42) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 124, 125, and 126, respectively;

[0050] (43) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 127, 128, and 129, respectively;

[0051] (44) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 130, 131, and 132, respectively;

[0052] (45) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 133, 134, and 135, respectively;

[0053] (46) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 136, 137, and 138, respectively;

[0054] (47) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 139, 140, and 141, respectively;

[0055] (48) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 142, 143, and 144, respectively;

[0056] (49) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 145, 146, and 147, respectively;

[0057] (50) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 148, 149, and 150, respectively;

[0058] (51) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 151, 152 and 153, respectively;

[0059] (52) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 154, 155, and 156, respectively;

[0060] (53) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 157, 158, and 159, respectively;

[0061] (54) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 160, 161, and 162, respectively;

[0062] (55) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 163, 164, and 165, respectively;

[0063] (56) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 166, 167, and 168, respectively;

[0064] (57) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 169, 170, and 171, respectively;

[0065] (58) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 172, 173, and 174, respectively;

[0066] (59) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 175, 176, and 177, respectively;

[0067] (60) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 178, 179, and 180, respectively;

[0068] (61) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 181, 182 and 183, respectively;

[0069] (62) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 184, 185, and 186, respectively;

[0070] (63) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 187, 188, and 189, respectively;

[0071] (64) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 190, 191, and 192, respectively;

[0072] (65) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 193, 194, and 195, respectively;

[0073] (66) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 196, 197, and 198, respectively;

[0074] (67) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 199, 200, and 201, respectively;

[0075] (68) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 202, 203, and 204, respectively;

[0076] (69) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 205, 206, and 207, respectively;

[0077] (70) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 208, 209, and 210, respectively;

[0078] (71) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 211, 212 and 213, respectively;

[0079] (72) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 214, 215, and 216, respectively;

[0080] (73) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 217, 218, and 219, respectively;

[0081] (74) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 220, 221, and 222, respectively;

[0082] (75) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 223, 224 and 225, respectively;

[0083] (76) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 226, 227, and 228, respectively;

[0084] (77) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 229, 230, and 231, respectively;

[0085] (78) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 232, 233, and 234, respectively;

[0086] (79) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 235, 236, and 237, respectively;

[0087] (80) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 238, 239, and 240, respectively;

[0088] (81) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 241, 242, and 243, respectively;

[0089] (82) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 244, 245, and 246, respectively;

[0090] (83) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 247, 248, and 249, respectively;

[0091] (84) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 250, 251, and 252, respectively;

[0092] (85) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 253, 254, and 255, respectively;

[0093] (86) The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 256, 257 and 258, respectively; and

[0094] (87) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 259, 260, and 261, respectively.

[0095] In some embodiments, the VHH comprises CDR1, 2, 3 whose amino acid sequences are shown in SEQ ID NO: 1, 2, and 3, respectively.

[0096] In one aspect, an antibody or antigen-binding fragment thereof is provided herein, wherein the antibody or antigen-binding fragment thereof binds to a coronavirus S protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain single variable region (VHH), wherein the VHH comprises an amino acid sequence at least 80% identical to a selected VHH sequence. In some embodiments, the selected VHH sequence is selected from the group consisting of SEQ ID NO: 262-348. In some embodiments, the VHH comprises the sequence of SEQ ID NO: 262.

[0097] In some embodiments, the antibody or antigen-binding fragment specifically binds to the coronavirus S protein.

[0098] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0099] In one aspect, provided herein is an antibody or an antigen-binding fragment thereof, comprising the VHH CDR1, 2, 3 of the antibody or antigen-binding fragment thereof as described herein.

[0100] In some embodiments, the antibody or antigen-binding fragment comprises human IgG Fc.

[0101] In some embodiments, the antibody or antigen-binding fragment comprises two or more heavy chain single variable domains.

[0102] In one aspect, the present invention provides a nucleic acid comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof as described herein. In some embodiments, the nucleic acid is a cDNA.

[0103] In one aspect, provided herein is a vector comprising one or more of the nucleic acids described herein.

[0104] In one aspect, a cell is provided herein, comprising a vector as described herein. In some embodiments, the cell is a CHO cell. In one aspect, a cell is provided herein, comprising one or more nucleic acids in a nucleic acid as described herein.

[0105] In one aspect, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, the method comprising: (a) culturing the cell as described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; and (b) collecting the antibody or the antigen-binding fragment produced by the cell.

[0106] In one aspect, provided herein is a method for treating a subject suffering from a coronavirus-related disease, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein.

[0107] In one aspect, provided herein is a method for neutralizing a coronavirus, the method comprising contacting the coronavirus with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein.

[0108] In one aspect, the present invention provides a method for blocking the internalization of a coronavirus by a cell, the method comprising contacting the coronavirus with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein.

[0109] On the one hand, the present invention provides a method for identifying a subject suffering from coronavirus disease, the method comprising detecting a sample collected from the subject suffering from the coronavirus by an antibody or antigen-binding fragment thereof as described herein, thereby identifying that the subject suffers from coronavirus infection.

[0110] In some embodiments, the sample is a blood sample, a saliva sample, a stool sample, or a liquid sample from the respiratory tract of the subject. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the coronavirus is MERS-CoV. In some embodiments, the coronavirus is SARS-CoV.

[0111] In one aspect, provided herein is a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier.

[0112] In one aspect, provided herein is an antibody or antigen-binding fragment thereof that cross-competes with an antibody or antigen-binding fragment thereof as described herein.

[0113] As used herein, the term "antibody" refers to any antigen binding molecule that contains at least one (e.g., one, two, three, four, five or six) complementary determining regions (CDRs) (e.g., any one of the three CDRs from an immunoglobulin light chain or any one of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, single variable domain (VHH) antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, bifunctional antibodies, linear antibodies, and multispecific antibodies formed by antibody fragments.

[0114] As used herein, the term "antigen binding fragment" refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, an antigen binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain or VHH). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0115] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification, and describe the animal, human or non-human being provided for treatment according to the method of the present invention. Veterinary and non-veterinary applications are contemplated in the present disclosure. Human patients can be adults or adolescents (e.g., people under 18 years of age). In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs and primates. For example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.) are included, rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, porcine animals (e.g., pigs, miniature pigs), horses, dogs, cats, cattle and other livestock animals, farm animals and zoo animals are included.

[0116] As used herein, when referring to antibodies, the phrase "specifically binding / specifically binds" refers to the interaction of an antibody with its target molecule, preferably with other molecules, because the interaction depends on the presence of a specific structure (i.e., antigenic determinant or epitope) on the target molecule; in other words, the agent generally recognizes molecules containing a specific structure rather than all molecules and binds to the molecule. An antibody that specifically binds to a target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to an S protein may be referred to as an S protein-specific antibody or an anti-S protein antibody.

[0117] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length of at least two amino acids.

[0118] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to a polymer of nucleotides of any length of at least two nucleotides, and include but are not limited to DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0119] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0120] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1 Schematic diagram showing the workflow of anti-SARS-CoV-2 antibody development. A natural library was constructed using peripheral blood mononuclear cells (PBMCs) from 65 llamas, and a humanized library was constructed from the natural VHH library, in which the VHH framework was partially humanized and the CDR1, 2, and 3 of the VHH were reorganized.

[0122] Figure 2A shows a phylogenetic tree of 69 unique VHH binders.

[0123] FIG. 2B is a schematic diagram showing an ELISA assay to assess binding of VHHs to recombinant SARS-CoV-2 S protein.

[0124] FIG. 3A is a schematic diagram showing an ACE2 competition assay.

[0125] FIG. 3B is a table showing a list of 9 unique S / ACE2 blockers.

[0126] Figure 4 is a table showing the results of ACE2 competition assays using pairwise combinations of 9 S / ACE2 blockers. (-): >= 100%, (+): 80%-100%, and (++): remaining signal < 80% compared to single VHH addition.

[0127] Figure 5A is a schematic diagram showing the receptor binding domain (RBD) of the spike protein binding to the ACE2 receptor.

[0128] Figure 5B is a schematic diagram showing the structural organization of bispecific and trispecific llama VHH Nanobody-Fc molecules.The design process utilized CAAD to optimize the characteristics of Nanobody-Fc.

[0129] Figure 6 The proposed therapeutic mechanism of Nanobody-Fc is shown.

[0130] Figure 7 Exemplary diagnostic utilization of humanized llama VHHs as single or combined probes is shown.

[0131] Figure 8A shows the results of the epitope binning assay. Epitope binning of VHH-Fc was assessed on a Gator (Probe Life) using an RBD sensor loaded with 2A-Fc, which quantified the wavelength shift (indicative of binding signal) over time.

[0132] Figure 8B is a table showing the results of epitope binning based on ELISA. SARS-CoV-2S1 protein was incubated with 1B-2A-Fc and 3F-Fc, and competition for binding with VHH was performed, followed by detection of biotinylation. The difference percentage of the competing pairs relative to the VHH-Fc signal alone is indicated. VHH association percentages of the 1B-2A-Fc group exceeding 90% are likely to be high VHH competitors, and some competitors exceeding 60%. VHH association percentages of the 3F-Fc group exceeding 60% are competitors.

[0133] FIG8C shows two groups of VHHs classified based on binding to an epitope on the S1 RBD.

[0134] Figure 9A shows the ACE2 binding residues on the SARS-CoV-2 S1 RBD. ACE2 binding residues on SARS-CoV-2S1 RBD were determined based on protein-protein interactions from Protein Data Bank (PDB) 6M0J. The residues under the arrow symbol are predicted ACE2 interactors.

[0135] Figure 9B shows the SARS-CoV-2S1 RBD protein sequence and deletion map schematic diagram. The boxed area in the upper figure represents the deletion, and the lower figure is a schematic diagram of the deletion map of each deletion mutant.

[0136] Figure 9C shows the percentage of FITC-positive Expi293 cells expressing SARS-CoV-2S1 wild-type (WT) or mutant proteins (del1-del5). The binding of VHH-Fc or ACE2 to Expi293 cells was assessed by flow cytometry, followed by FITC-conjugated secondary antibody treatment. Isotype control antibodies and FACS buffer were used as negative controls.

[0137] Figure 9D is a table showing the percent binding of each VHH-Fc relative to S1 WT. The percent binding was calculated in the context of each deletion mutant.

[0138] Fig.9E Shown by Schrodinger Software-generated docking model between SARS-CoV-2S1 RBD and lead VHHs (1B, 3F, and 2A).

[0139] Fig. 10ABinding of multispecific, bispecific and monoclonal VHH-Fc to SARS-CoV-2 S1 protein at different concentrations in duplicate using ELISA method is shown. Binding signal is based on fluorescence and is expressed in relative fluorescence units (RFU). Error bars represent standard deviation.

[0140] Fig. 10B Shown is a graph of the binding kinetics of the trispecific VHH-Fc 1B-3F-2A-Fc.

[0141] Fig. 10C Shown is a graph of the binding kinetics of the trispecific VHH-Fc 3F-1B-2A-Fc.

[0142] Fig. 10D Blocking of SARS-CoV-2S / ACE2 interaction by multispecific, bispecific and monoclonal VHH-Fc at different concentrations in duplicate using ELISA method is shown. The percentage of inhibition is calculated based on the blocking signal expressed in RFU for each VHH-Fc treatment. Error bars represent standard deviation.

[0143] Fig. 10E is a table showing the developability characteristics of the biophysical and chemical properties of VHH-Fc examined using DLS (dynamic light scattering), DSF (differential scanning fluorimetry) and SLS (static light scattering).

[0144] Figure 11A shows blocking of SARS-CoV-2 pseudovirus infection by combined treatment of VHH-Fc 3F-1B-2A-Fc, 1B-3F-2A-Fc and VHH-Fc 1B, 3F and 2A. HEK293ACE2 / TMPRSS2 cells were incubated with SARS-CoV-2 pseudovirus and 1:5 serial dilutions of VHH-Fc starting from 1000nM in triplicate. The percentage of inhibition was calculated based on the luminescent signal represented by RFU for each VHH-Fc treatment. Error bars represent the standard error of the mean.

[0145] Figure 11B shows the calculated cell death percentage based on the percentage of cells treated with VHH-Fc relative to the percentage of cells of isotype control. The ADCC function of the trispecific VHH-Fc (3F-1B-2A, 3A-3F-2A and isotype control antibody) was evaluated in duplicate. Error bars represent standard deviations.

[0146] Figures 12A-12C is a structural docking model showing how 3F-1B-2A-Fc interacts with SARS-CoV-2S1 RBD. A 3D docking model of SARS-CoV-2S1 RBD with trispecific VHH-Fc 3F-1B-2A was generated. In the software, the SARS-CoV-2RBD spike protein trimer (PDB 6X2A) was divided into three monomer forms (chains A, B, and C). Then, the 1B / 3F / RBD model structure was aligned with chain A of PDB 6X2A to create group 1, and the 2A / RBD model structure was aligned with chain B of PDB 6X2A to create group 2. Group 1, group 2, and chain C were then merged to produce the final structure. The S1 RBD / VHH docking structure is represented by a surface structure (Figure 12A) and a ribbon structure (Figure 12B). The enlarged S1 RBD / VHH docking structure is shown on the right (Figure 12C).

[0147] Figure 13A shows the binding of trispecific VHH-Fc3A-3F-2A and 3F-1B-2A to SARS-CoV-2S1 protein at different concentrations in duplicate using ELISA method. The binding signal is based on fluorescence and is expressed in relative fluorescence units (RFU). Error bars represent standard deviations.

[0148] Figure 13B shows the blocking of SARS-CoV-2S / ACE2 interaction by VHH-Fc 3A-3F-2A and 3F-1B-2A at different concentrations in duplicate using ELISA method. The inhibition percentage is calculated based on the blocking signal represented by RFU for each VHH-Fc treatment. The error bars represent standard deviation.

[0149] Fig.14 Schematic administration strategy of 3F-1B-2A-Fc anti-SARS-CoV-2 trispecific antibody (TriAb) in transgenic mice expressing human ACE2 is shown. In treatment groups G2-G4, antibodies were administered by intranasal (IN) or intraperitoneal (IP) routes. No antibodies were administered in control group G1. The term "hpi" means hours after infection.

[0150] Fig.15 Shown are SARS-CoV-2 virus titers in the lungs collected from control mice (G1) and treated mice (G2-G4) 3 days after SARS-CoV-2 infection.

[0151] Fig.16 Relative mean body weights of mice in the control group (G1) and treatment groups (G2-G4) relative to the mean initial body weight are shown.

[0152] Fig.17A Shown is a graph of the binding kinetics of the 3F-1B-2A-Fc anti-SARS-CoV-2 trispecific antibody (TriAb) binding to the S protein with wild-type RBD.

[0153] Fig. 17B Shown is a graph of the binding kinetics of the 3F-1B-2A-Fc anti-SARS-CoV-2 trispecific antibody (TriAb) binding to the S protein with three mutations in the RBD (K417N / E484K / N501Y) or RBD tri-mut.

[0154] Fig.18 Shown is the blocking of RBD / ACE2 interaction or RBD tri-mut (K417N / E484K / N501Y) / ACE2 interaction by trispecific 3F-1B-2A-Fc and monoclonal VHH-Fc (3F+1B+2A) at different concentrations in duplicate using ELISA method. The inhibition percentage is calculated based on the blocking signal expressed in RFU for each VHH-Fc treatment.

[0155] Fig.19 Shown are graphs of binding kinetics determined by biolayer interferometry (BLI) using the trispecific antibody 3F-1B-2A-Fc heated at 45°C for 2 weeks.

[0156] Fig. 20 TABLE 1 is a table showing the developability characteristics of the biophysical and chemical properties of the trispecific antibody 3F-1B-2A-Fc using DLS (dynamic light scattering), DSF (differential scanning fluorescence) and SLS (static light scattering). 3F-1B-2A-Fc was kept at 45°C for 2 or 3 weeks prior to measurement. Tm stands for melting temperature. Tagg stands for aggregation temperature.

[0157] Fig.21 The CDR sequences of anti-SARS-CoV-2S protein antibodies are listed.

[0158] Fig. 22 The amino acid sequences of VHH of anti-SARS-CoV-2 S protein antibodies are listed. The underlined sequences are CDR sequences.

[0159] Fig.23 Specific combinations of Group 1 - Group 2 - Group 1 VHH are listed.

[0160] Fig.24 Specific combinations of Group 2-Group 1-Group 1 VHHs are listed.

[0161] Fig.25 Specific combinations of Group 2-Group 2-Group 1 VHH are listed. DETAILED DESCRIPTION

[0162] SARS-CoV-2 is a newly emerged coronavirus that causes COVID-19, which has had adverse effects on human health and caused a pandemic. Due to the severity of SARS-CoV-2 and the lack of treatment options, there is an unmet need to develop therapeutics against SARS-CoV-2. A promising approach to combat COVID-19 is to neutralize SARS-CoV-2 through therapeutic antibodies.

[0163] SARS-CoV2 is a coronavirus that causes the human disease COVID-19, which is contagious and can spread rapidly to cause mild to severe infection, including death (CDC). The spread of this emerging virus has reached epidemic levels and has had a significant public impact on the world, resulting in more than 12 million infections and more than 500,000 deaths worldwide (World Health Organization (WHO)). In addition to threatening human health, COVID-19 has also caused significant socioeconomic impacts around the world (United Nations).

[0164] Although there are relatively successful diagnostic methods to detect SARS-CoV-2 infection in humans, there are currently no successful therapeutics that can prevent viral infection. However, recent research results show that the small molecule antiviral drug Remdesivir (Gilead), which inhibits the RNA-dependent RNA polymerase of SARS-CoV-2, shortens the recovery time of patients with COVID-19, but it is likely that it will not completely prevent or prevent SARS-CoV-2 infection in humans. In addition, there are no approved vaccines to prevent SARS-CoV-2 infection in humans, even though some groups are currently developing such vaccines (WHO). Therefore, the rapid development of therapeutics and prevention strategies has become a fundamental and urgent need to combat the COVID-19 epidemic.

[0165] The trimeric spike (S) protein that protrudes through the envelope of the SARS-CoV-2 virion mediates viral entry into host cells by interacting with the ACE2 human receptor. Therefore, the main goal of the anti-SARS-CoV-2 neutralizing antibodies under development is to block the interaction of the SARS-CoV-2S1 protein with ACE2. Specifically, two popular strategies have been used to discover and develop monoclonal IgG antibodies that can mainly recognize the SARS-CoV-2S1 protein by binding to its receptor binding domain (RBD). The first commonly used method is to clone the antibody V gene from the B cells of surviving COVID-19 patients who have developed a natural immune response to SARS-CoV-2. This strategy has produced many neutralizing monoclonal antibodies; however, it should be noted that the patient's antibody reserve status and the time of blood sample collection play a key role in its success. Another recognized and classic method for antibody production is to immunize humanized mice. In addition, the most noteworthy method for generating new SARS-CoV-2 antibodies is developed by screening cross-neutralizing antibodies of SARS-CoV-2S1 protein binders from the antibodies initially tested or developed to treat SARS by blocking SARS-CoV S / ACE2 or MERS, by blocking CoV S / CD26 interactions. One of the cross-binding substances is a single domain antibody / nanobody (VHH) produced by SARS-CoV S-immunized llamas. In addition, VHHs against SARS-CoV-2 have also been generated from llama VHH libraries. The method of using camel antibody VHHs is advantageous because the VHH region is easy to produce, stable and small in size, which increases the possibility of targeting unique epitopes that conventional VH / VL antibodies cannot reach.

[0166] The SARS-CoV-2 virus particle consists of a helical capsid formed by a nucleocapsid (N) protein bound to an RNA genome, surrounded by a membrane (M) protein, an envelope (E) protein, and a trimeric spike (S) protein that gives it a "crown-like" appearance (see Zhou P, Yang XL, Wang XG et al. Apneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020 Mar;579(7798):270-273). The S protein receptor binding domain (RBD) in the S1 subunit binds to angiotensin-converting enzyme (angiotensin I converting enzyme 2; ACE2) on the cell membrane of type 2 pneumocytes and intestinal epithelial cells. After binding, the S protein is cleaved by the host cell transmembrane serine protease 2 (TMPRSS2), which facilitates the subsequent entry of the virus into the host cell (see Hoffmann M, Kleine Weber H, Schroeder S et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell, March 4, 2020).

[0167] It is known that therapeutic antibodies neutralize viral infection through two mechanisms of action, namely, an Fc-independent function that blocks capsid / host receptor interactions and induces viral aggregation, and an Fc-dependent function that causes Fc-FcR interactions to activate immune cells to kill the virus (see Klasse PJ. Neutralization of Virus Infectivity by Antibodies: Old Problems in New Perspectives. Adv Biol. 2014; 2014). In general, polyclonal antibodies show better virus neutralization ability than monoclonal antibodies. However, the isolation of immunoglobulins from COVID-19 survivors is limited by the lack of availability of plasma from donors. As an alternative treatment method, combination therapy with several monoclonal antibodies is also limited due to high production costs and potential toxicity. Therefore, an approach to block the interaction of SARS-CoV-2 S protein with ACE2 using humanized llama antibodies was adopted with the goal of rapidly developing high affinity and avidity bi- or tri-specific therapeutic antibodies that neutralize SARS-CoV-2 before it infects cells.

[0168] Previous reports have shown that if the virus binds to low-titer therapeutic antibodies with low affinity and avidity, then Fc-FcR interactions may trigger antibody-dependent enhancement (ADE) of viral entry into host cells (see Zellweger RM, Prestwood TR, Shresta S. Enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue disease. Cell Host Microbe 2010 Feb 18;7(2):128-39). Therefore, ADE should be circumvented by developing high-titer neutralizing antibodies.

[0169] The present disclosure provides a strategy for rapidly identifying and generating llama nanobodies (VHH) from natural and synthetic humanized VHH phage libraries, which specifically bind to the S1 SARS-CoV-2 spike protein and block the interaction with the ACE2 human receptor. Based on the epitope prediction of the lead VHH, computer-aided design is used to construct a multi-specific VHH antibody fused to the human IgG1 Fc domain. The resulting trispecific VHH-Fc antibody shows more potent S1 binding, S1 / ACE2 blocking, and SARS-CoV-2 pseudovirus neutralization than the combination of bispecific VHH-Fc or monoclonal VHH-Fc alone. In addition, the protein stability analysis of VHH-Fc shows good developability characteristics, which enables it to be quickly and successfully developed into a treatment for COVID-19.

[0170] The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to the S protein of a coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV). These antibodies or their antigen-binding fragments are high-titer neutralizing antibodies or their antigen-binding fragments. In some embodiments, the antibody or its antigen-binding fragment comprises or consists of one, two, three, four, five or more humanized llama heavy chain single variable domains (VHH).

[0171] Specifically, the present disclosure also provides antibodies or antigen-binding fragments having two or more VHHs, which can further provide at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 5-fold or 10-fold improvement in binding affinity and / or avidity to coronavirus S protein compared to a similar antibody or antigen-binding fragment thereof having a single VHH.

[0172] The present disclosure further provides methods for treating COVID-19 using the antibodies or antigen-binding fragments thereof as described herein, and methods for diagnosing COVID-19 using the antibodies or antigen-binding fragments thereof as described herein.

[0173] Heavy chain single variable domain (VHH) antibodies

[0174] Monoclonal antibodies and recombinant antibodies are important tools in medicine and biotechnology. Like all mammals, Camelidae (e.g., llamas) can produce conventional antibodies consisting of two heavy chains and two light chains that are bound together in a Y shape (e.g., IgG1) with a disulfide bond. However, Camelidae also produces two unique IgG subclasses: IgG2 and IgG3, also referred to as heavy chain IgG. These antibodies are composed of only two heavy chains that lack the CH1 region but still carry the antigen-binding domain known as VHH (or nanobody) at its N-terminus. Conventional Ig requires the association of the variable regions from both heavy and light chains to allow for the high diversity of antigen-antibody interactions. Although the separated heavy and light chains still show this ability, when compared with paired heavy and light chains, they show very low affinity. The unique feature of heavy chain IgG is that its monomeric antigen-binding region is capable of binding antigens with specificity, affinity, and diversity in particular, which is comparable to conventional antibodies that do not need to be paired with another region. This feature is mainly due to some major variations in the amino acid sequence of the variable regions of the two heavy chains, which induce profound conformational changes compared to conventional Ig. The major substitutions in the variable regions prevent the light chain from binding to the heavy chain, but also prevent the unbound heavy chain from being retrieved by the immunoglobulin binding protein.

[0175] The single variable domain (named as VHH, sdAb or nanobody) of these antibodies is the minimum antigen binding domain produced by the adaptive immune system. It has been found that the third complementarity determining region (CDR3) of the variable region of these antibodies is twice as long as conventional antibodies. This increases the interaction surface with the antigen and the diversity of antigen-antibody interactions, which compensates for the lack of light chain. In the case of long complementarity determining region 3 (CDR3), VHH can extend into the protein cleft that conventional antibodies cannot reach, including the site of interest in function, such as the active site of an enzyme or the receptor binding gorge on the viral surface. In addition, other cysteine ​​residues make the structure more stable, thus increasing the intensity of interaction.

[0176] Compared with conventional antibodies carrying the variable domains (VH and VL) of conventional antibodies, VHH provides many other advantages, including higher stability, solubility, expression yield and refolding ability and better in vivo tissue penetration. In addition, compared with the VH domain of conventional antibodies, VHH does not show an inherent tendency to bind to the light chain. This is conducive to inducing heavy chain antibodies in the presence of functional light chain loci. Further, since VHH does not bind to the VL domain, it is much easier to reformat VHH into a bispecific antibody construct than to reformat it into a construct containing a conventional VH-VL pair or a single domain based on the VH domain.

[0177] The present disclosure provides, for example, anti-coronavirus (e.g., SARS-CoV-2, SARS-CoV or MERS-CoV) S protein antibodies, modified antibodies thereof, chimeric antibodies thereof, and humanized antibodies thereof.

[0178] The CDR sequences of Covid19-E2A3 and Covid19-E2A3-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 1, 2 and 3, respectively.

[0179] The CDR sequences of Covid19-E2A6 and Covid19-E2A6-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 4, 5 and 6, respectively.

[0180] The CDR sequences of Covid19-E2A8 and Covid19-E2A8-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 7, 8 and 9, respectively.

[0181] The CDR sequences of Covid19-E2B3 and Covid19-E2B3-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 10, 11 and 12, respectively.

[0182] The CDR sequences of Covid19-E2B7 and Covid19-E2B7-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 13, 14 and 15, respectively.

[0183] The CDR sequences of Covid19-E2B10 and Covid19-E2B10-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 16, 17 and 18, respectively.

[0184] The CDR sequences of Covid19-E2C6 and Covid19-E2C6-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 19, 20 and 21, respectively.

[0185] The CDR sequences of Covid19-E2C7 and Covid19-E2C7-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 22, 23 and 24, respectively.

[0186] The CDR sequences of Covid19-E2C9 and Covid19-E2C9-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domains as shown in SEQ ID NOs: 25, 26 and 27, respectively.

[0187] The CDR sequences of Covid19-E2D4 and Covid19-E2D4-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 28, 29 and 30, respectively.

[0188] The CDR sequences of Covid19-E2E2 and Covid19-E2E2-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 31, 32 and 33, respectively.

[0189] The CDR sequences of Covid19-E2E3 and Covid19-E2E3-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 34, 35 and 36, respectively.

[0190] The CDR sequences of Covid19-E2F3 and Covid19-E2F3-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 37, 38 and 39, respectively.

[0191] The CDR sequences of Covid19-E2F6 and Covid19-E2F6-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 40, 41 and 42, respectively.

[0192] The CDR sequences of Covid19-E2G8 and Covid19-E2G8-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 43, 44 and 45, respectively.

[0193] The CDR sequences of Covid19-E2H2 and Covid19-E2H2-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domains as shown in SEQ ID NOs: 46, 47 and 48, respectively.

[0194] The CDR sequences of Covid19-E2P2B12 and Covid19-E2P2B12-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 49, 50 and 51, respectively.

[0195] The CDR sequences of Covid19-E2P2F11 and Covid19-E2P2F11-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 52, 53 and 54, respectively.

[0196] The CDR sequences of Covid19-E2P2G1 and Covid19-E2P2G1-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 55, 56 and 57, respectively.

[0197] The CDR sequences of Covid19-E2P2G4 and Covid19-E2P2G4-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 58, 59 and 60, respectively.

[0198] The CDR sequences of Covid19-E2P2H4 and Covid19-E2P2H4-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 61, 62 and 63, respectively.

[0199] The CDR sequences of Covid19-S1A4 and Covid19-S1A4-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 64, 65 and 66, respectively.

[0200] The CDR sequences of Covid19-S1A9 and Covid19-S1A9-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 67, 68 and 69, respectively.

[0201] The CDR sequences of Covid19-S1A10 and Covid19-S1A10-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 70, 71 and 72, respectively.

[0202] The CDR sequences of Covid19-S1B4 and Covid19-S1B4-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 73, 74 and 75, respectively.

[0203] The CDR sequences of Covid19-S1B5 and Covid19-S1B5-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 76, 77 and 78, respectively.

[0204] The CDR sequences of Covid19-S1B10 and Covid19-S1B10-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 79, 80 and 81, respectively.

[0205] The CDR sequences of Covid19-S1B12 and Covid19-S1B12-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 82, 83 and 84, respectively.

[0206] The CDR sequences of Covid19-S1C3 and Covid19-S1C3-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 85, 86 and 87, respectively.

[0207] The CDR sequences of Covid19-S1C8 and Covid19-S1C8-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 88, 89 and 90, respectively.

[0208] The CDR sequences of Covid19-S1C10 and Covid19-S1C10-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 91, 92 and 93, respectively.

[0209] The CDR sequences of Covid19-S1D2 and Covid19-S1D2-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domains as shown in SEQ ID NOs: 94, 95 and 96, respectively.

[0210] The CDR sequences of Covid19-S1D6 and Covid19-S1D6-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domains as shown in SEQ ID NOs: 97, 98 and 99, respectively.

[0211] The CDR sequences of Covid19-S1D8 and Covid19-S1D8-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 100, 101 and 102, respectively.

[0212] The CDR sequences of Covid19-S1D12 and Covid19-S1D12-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 103, 104 and 105, respectively.

[0213] The CDR sequences of Covid19-S1E1 and Covid19-S1E1 derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 106, 107 and 108, respectively.

[0214] The CDR sequences of Covid19-S1E6 and Covid19-S1E6-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 109, 110 and 111, respectively.

[0215] The CDR sequences of Covid19-S1E8 and Covid19-S1E8-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 112, 113 and 114, respectively.

[0216] The CDR sequences of Covid19-S1F5 and Covid19-S1F5-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 115, 116 and 117, respectively.

[0217] The CDR sequences of Covid19-S1F9 and Covid19-S1F9-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 118, 119 and 120, respectively.

[0218] The CDR sequences of Covid19-S1F11 and Covid19-S1F11-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 121, 122 and 123, respectively.

[0219] The CDR sequences of Covid19-S1F12 and Covid19-S1F12-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 124, 125 and 126, respectively.

[0220] The CDR sequences of Covid19-S1G4 and Covid19-S1G4-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 127, 128 and 129, respectively.

[0221] The CDR sequences of Covid19-S1G5 and Covid19-S1G5-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 130, 131 and 132, respectively.

[0222] The CDR sequences of Covid19-S1G6 and Covid19-S1G6-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 133, 134 and 135, respectively.

[0223] The CDR sequences of Covid19-S1G7 and Covid19-S1G7-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 136, 137 and 138, respectively.

[0224] The CDR sequences of Covid19-S1G10 and Covid19-S1G10-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 139, 140 and 141, respectively.

[0225] The CDR sequences of Covid19-S1H1 and Covid19-S1H1-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 142, 143 and 144, respectively.

[0226] The CDR sequences of Covid19-S1H3 and Covid19-S1H3-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 145, 146 and 147, respectively.

[0227] The CDR sequences of Covid19-S1H6 and Covid19-S1H6-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 148, 149 and 150, respectively.

[0228] The CDR sequences of Covid19-S1H7 and Covid19-S1H7-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 151, 152 and 153, respectively.

[0229] The CDR sequences of Covid19-S1H8 and Covid19-S1H8-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 154, 155 and 156, respectively.

[0230] The CDR sequences of Covid19-S1P2A10 and Covid19-S1P2A10-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 157, 158 and 159, respectively.

[0231] The CDR sequences of Covid19-S1P2A12 and Covid19-S1P2A12-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 160, 161 and 162, respectively.

[0232] The CDR sequences of Covid19-S1P2C8 and Covid19-S1P2C8-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 163, 164 and 165, respectively.

[0233] The CDR sequences of Covid19-S1P2F5 and Covid19-S1P2F5-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 166, 167 and 168, respectively.

[0234] The CDR sequences of Covid19-S1P2F12 and Covid19-S1P2F12-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 169, 170 and 171, respectively.

[0235] The CDR sequences of Covid19-S1P2H4 and Covid19-S1P2H4-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 172, 173 and 174, respectively.

[0236] The CDR sequences of Covid19-S1P2H5 and Covid19-S1P2H5-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 175, 176 and 177, respectively.

[0237] The CDR sequences of Covid19-S1P2H6 and Covid19-S1P2H6-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 178, 179 and 180, respectively.

[0238] The CDR sequences of Covid19-S1P2H9 and Covid19-S1P2H9-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 181, 182 and 183, respectively.

[0239] The CDR sequences of Covid19-S1P2H10 and Covid19-S1P2H10-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 184, 185 and 186, respectively.

[0240] The CDR sequences of Covid19-S2A3 and Covid19-S2A3-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 187, 188 and 189, respectively.

[0241] The CDR sequences of Covid19-1B6 and Covid19-1B6-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 190, 191 and 192, respectively.

[0242] The CDR sequences of Covid19-2A4 and Covid19-2A4-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 193, 194 and 195, respectively.

[0243] The CDR sequences of Covid19-3A11 and Covid19-3A11-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 196, 197 and 198, respectively.

[0244] The CDR sequences of Covid19-3F2 and Covid19-3F2-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 199, 200 and 201, respectively.

[0245] The CDR sequences of Covid19-3F10 and Covid19-3F10-derived antibodies (e.g., humanized antibodies) contain the CDRs of the VHH domain as shown in SEQ ID NOs: 202, 203 and 204, respectively.

[0246] The CDR sequences of 1D7 and 1D7-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 205, 206, and 207, respectively.

[0247] The CDR sequences of 1C11 and 1C11-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 208, 209, and 210, respectively.

[0248] The CDR sequences of 1F12 and 1F12-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 211, 212, and 213, respectively.

[0249] The CDR sequences of 2B4 and 2B4-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 214, 215, and 216, respectively.

[0250] The CDR sequences of 2E8 and 2E8-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 217, 218, and 219, respectively.

[0251] The CDR sequences of 3A4 and 3A4-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 220, 221, and 222, respectively.

[0252] The CDR sequences of 3G7 and 3G7-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 223, 224, and 225, respectively.

[0253] The CDR sequences of 3B11 and 3B11-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 226, 227, and 228, respectively.

[0254] The CDR sequences of 3B12 and 3B12-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 229, 230, and 231, respectively.

[0255] The CDR sequences of 4A9 and 4A9-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 232, 233, and 234, respectively.

[0256] The CDR sequences of 4F7 and 4F7-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 235, 236, and 237, respectively.

[0257] The CDR sequences of 4C12 and 4C12-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 238, 239, and 240, respectively.

[0258] The CDR sequences of 4F12 and 4F12-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as set forth in SEQ ID NOs: 241, 242, and 243, respectively.

[0259] The CDR sequences of 1F11_B and 1F11_B-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as shown in SEQ ID NOs: 244, 245, and 246, respectively.

[0260] The CDR sequences of 1F12_B and 1F12_B-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as shown in SEQ ID NOs: 247, 248, and 249, respectively.

[0261] The CDR sequences of 1G12_B and 1G12_B-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as shown in SEQ ID NOs: 250, 251 and 252, respectively.

[0262] The CDR sequences of 2A4_B and 2A4_B-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as shown in SEQ ID NOs: 253, 254, and 255, respectively.

[0263] The CDR sequences of 2A5_B and 2A5_B-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as shown in SEQ ID NOs: 256, 257, and 258, respectively.

[0264] The CDR sequences of 2D12_B and 2D12_B-derived antibodies (eg, humanized antibodies) comprise the CDRs of the VHH domain as shown in SEQ ID NOs: 259, 260, and 261, respectively.

[0265] The amino acid sequence of the VHH domain of the Covid19-E2A3 antibody is shown in SEQ ID NO:262.

[0266] The amino acid sequence of the VHH domain of the Covid19-E2A6 antibody is shown in SEQ ID NO:263.

[0267] The amino acid sequence of the VHH domain of the Covid19-E2A8 antibody is shown in SEQ ID NO:264.

[0268] The amino acid sequence of the VHH domain of the Covid19-E2B3 antibody is shown in SEQ ID NO:265.

[0269] The amino acid sequence of the VHH domain of the Covid19-E2B7 antibody is shown in SEQ ID NO:266.

[0270] The amino acid sequence of the VHH domain of the Covid19-E2B10 antibody is shown in SEQ ID NO:267.

[0271] The amino acid sequence of the VHH domain of the Covid19-E2C6 antibody is shown in SEQ ID NO:268.

[0272] The amino acid sequence of the VHH domain of the Covid19-E2C7 antibody is shown in SEQ ID NO:269.

[0273] The amino acid sequence of the VHH domain of the Covid19-E2C9 antibody is shown in SEQ ID NO:270.

[0274] The amino acid sequence of the VHH domain of the Covid19-E2D4 antibody is shown in SEQ ID NO:271.

[0275] The amino acid sequence of the VHH domain of the Covid19-E2E2 antibody is shown in SEQ ID NO:272.

[0276] The amino acid sequence of the VHH domain of the Covid19-E2E3 antibody is shown in SEQ ID NO:273.

[0277] The amino acid sequence of the VHH domain of the Covid19-E2F3 antibody is shown in SEQ ID NO:274.

[0278] The amino acid sequence of the VHH domain of the Covid19-E2F6 antibody is shown in SEQ ID NO:275.

[0279] The amino acid sequence of the VHH domain of the Covid19-E2G8 antibody is shown in SEQ ID NO:276.

[0280] The amino acid sequence of the VHH domain of the Covid19-E2H2 antibody is shown in SEQ ID NO: 277. The amino acid sequence of the VHH domain of the Covid19-E2P2B12 antibody is shown in SEQ ID NO: 278. The amino acid sequence of the VHH domain of the Covid19-E2P2F11 antibody is shown in SEQ ID NO: 279. The amino acid sequence of the VHH domain of the Covid19-E2P2G1 antibody is shown in SEQ ID NO: 280. The amino acid sequence of the VHH domain of the Covid19-E2P2G4 antibody is shown in SEQ ID NO: 281. The amino acid sequence of the VHH domain of the Covid19-E2P2H4 antibody is shown in SEQ ID NO: 282. The amino acid sequence of the VHH domain of the Covid19-S1A4 antibody is shown in SEQ ID NO: 283. The amino acid sequence of the VHH domain of the Covid19-S1A9 antibody is shown in SEQ ID NO: 284. The amino acid sequence of the VHH domain of the Covid19-S1A10 antibody is shown in SEQ ID NO: 285. The amino acid sequence of the VHH domain of the Covid19-S1B4 antibody is shown in SEQ ID NO: 286. The amino acid sequence of the VHH domain of the Covid19-S1B5 antibody is shown in SEQ ID NO: 287. The amino acid sequence of the VHH domain of the Covid19-S1B10 antibody is shown in SEQ ID NO: 288. The amino acid sequence of the VHH domain of the Covid19-S1B12 antibody is shown in SEQ ID NO: 289. The amino acid sequence of the VHH domain of the Covid19-S1C3 antibody is shown in SEQ ID NO: 290. The amino acid sequence of the VHH domain of the Covid19-S1C8 antibody is shown in SEQ ID NO: 291. The amino acid sequence of the VHH domain of the Covid19-S1C10 antibody is shown in SEQ ID NO: 292. The amino acid sequence of the VHH domain of the Covid19-S1D2 antibody is shown in SEQ ID NO: 293. The amino acid sequence of the VHH domain of the Covid19-S1D6 antibody is shown in SEQ ID NO: 294. The amino acid sequence of the VHH domain of the Covid19-S1D8 antibody is shown in SEQ ID NO: 295. The amino acid sequence of the VHH domain of the Covid19-S1D12 antibody is shown in SEQ ID NO: 296. The amino acid sequence of the VHH domain of the Covid19-S1E1 antibody is shown in SEQ ID NO: 297.The amino acid sequence of the VHH domain of the Covid19-S1E6 antibody is shown in SEQ ID NO: 298. The amino acid sequence of the VHH domain of the Covid19-S1E8 antibody is shown in SEQ ID NO: 299. The amino acid sequence of the VHH domain of the Covid19-S1F5 antibody is shown in SEQ ID NO: 300. The amino acid sequence of the VHH domain of the Covid19-S1F9 antibody is shown in SEQ ID NO: 301. The amino acid sequence of the VHH domain of the Covid19-S1F11 antibody is shown in SEQ ID NO: 302. The amino acid sequence of the VHH domain of the Covid19-S1F12 antibody is shown in SEQ ID NO: 303. The amino acid sequence of the VHH domain of the Covid19-S1G4 antibody is shown in SEQ ID NO: 304. The amino acid sequence of the VHH domain of the Covid19-S1G5 antibody is shown in SEQ ID NO: 305. The amino acid sequence of the VHH domain of the Covid19-S1G6 antibody is shown in SEQ ID NO: 306. The amino acid sequence of the VHH domain of the Covid19-S1G7 antibody is shown in SEQ ID NO: 307. The amino acid sequence of the VHH domain of the Covid19-S1G10 antibody is shown in SEQ ID NO: 308. The amino acid sequence of the VHH domain of the Covid19-S1H1 antibody is shown in SEQ ID NO: 309. The amino acid sequence of the VHH domain of the Covid19-S1H3 antibody is shown in SEQ ID NO: 310. The amino acid sequence of the VHH domain of the Covid19-S1H6 antibody is shown in SEQ ID NO: 311. The amino acid sequence of the VHH domain of the Covid19-S1H7 antibody is shown in SEQ ID NO: 312. The amino acid sequence of the VHH domain of the Covid19-S1H8 antibody is shown in SEQ ID NO: 313. The amino acid sequence of the VHH domain of the Covid19-S1P2A10 antibody is shown in SEQ ID NO: 314. The amino acid sequence of the VHH domain of the Covid19-S1P2A12 antibody is shown in SEQ ID NO: 315. The amino acid sequence of the VHH domain of the Covid19-S1P2C8 antibody is shown in SEQ ID NO: 316. The amino acid sequence of the VHH domain of the Covid19-S1P2F5 antibody is shown in SEQ ID NO: 317. The amino acid sequence of the VHH domain of the Covid19-S1P2F12 antibody is shown in SEQ ID NO: 318.The amino acid sequence of the VHH domain of the Covid19-S1P2H4 antibody is shown in SEQ ID NO: 319. The amino acid sequence of the VHH domain of the Covid19-S1P2H5 antibody is shown in SEQ ID NO: 320. The amino acid sequence of the VHH domain of the Covid19-S1P2H6 antibody is shown in SEQ ID NO: 321. The amino acid sequence of the VHH domain of the Covid19-S1P2H9 antibody is shown in SEQ ID NO: 322. The amino acid sequence of the VHH domain of the Covid19-S1P2H10 antibody is shown in SEQ ID NO: 323. The amino acid sequence of the VHH domain of the Covid19-S2A3 antibody is shown in SEQ ID NO: 324. The amino acid sequence of the VHH domain of the Covid19-1B6 antibody is shown in SEQ ID NO: 325. The amino acid sequence of the VHH domain of the Covid19-2A4 antibody is shown in SEQ ID NO: 326. The amino acid sequence of the VHH domain of the Covid19-3A11 antibody is shown in SEQ ID NO: 327. The amino acid sequence of the VHH domain of the Covid19-3F2 antibody is shown in SEQ ID NO: 328. The amino acid sequence of the VHH domain of the Covid19-3F10 antibody is shown in SEQ ID NO: 329.

[0281] The amino acid sequence of the VHH domain of the 1D7 antibody is shown in SEQ ID NO:330.

[0282] The amino acid sequence of the VHH domain of the 1C11 antibody is shown in SEQ ID NO:331.

[0283] The amino acid sequence of the VHH domain of the 1F12 antibody is shown in SEQ ID NO:332.

[0284] The amino acid sequence of the VHH domain of the 2B4 antibody is shown in SEQ ID NO:333.

[0285] The amino acid sequence of the VHH domain of the 2E8 antibody is shown in SEQ ID NO:334.

[0286] The amino acid sequence of the VHH domain of the 3A4 antibody is shown in SEQ ID NO:335.

[0287] The amino acid sequence of the VHH domain of the 3G7 antibody is shown in SEQ ID NO:336.

[0288] The amino acid sequence of the VHH domain of the 3B11 antibody is shown in SEQ ID NO:337.

[0289] The amino acid sequence of the VHH domain of the 3B12 antibody is shown in SEQ ID NO:338.

[0290] The amino acid sequence of the VHH domain of the 4A9 antibody is shown in SEQ ID NO:339.

[0291] The amino acid sequence of the VHH domain of the 4F7 antibody is shown in SEQ ID NO:340.

[0292] The amino acid sequence of the VHH domain of the 4C12 antibody is shown in SEQ ID NO:341.

[0293] The amino acid sequence of the VHH domain of the 4F12 antibody is shown in SEQ ID NO:342.

[0294] The amino acid sequence of the VHH domain of the 1F11_B antibody is shown in SEQ ID NO:343.

[0295] The amino acid sequence of the VHH domain of the 1F12_B antibody is shown in SEQ ID NO:344.

[0296] The amino acid sequence of the VHH domain of the 1G12_B antibody is shown in SEQ ID NO:345.

[0297] The amino acid sequence of the VHH domain of the 2A4_B antibody is shown in SEQ ID NO:346.

[0298] The amino acid sequence of the VHH domain of the 2A5_B antibody is shown in SEQ ID NO:347.

[0299] The amino acid sequence of the VHH domain of the 2D12_B antibody is shown in SEQ ID NO:348.

[0300] Also provided are various modified or humanized VHH amino acid sequences. Because there are different ways to modify or humanize llama antibodies (e.g., different amino acids can be substituted for the modified sequence), the heavy and light chains of the antibodies can have more than one version of the humanized sequence. In some embodiments, the humanized VHH domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any sequence of SEQ ID NO: 262-348.

[0301] In addition, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may further contain one, two or three VHH domain CDRs selected from the group consisting of SEQ ID NO: 1-3, SEQ ID NO: 4-6, SEQ ID NO: 7-9, SEQ ID NO: 10-12, SEQ ID NO: 13-15, SEQ ID NO: 16-18, SEQ ID NO: 19-21, SEQ ID NO: 22-24, SEQ ID NO: 25-27, SEQ ID NO: 28-30, SEQ ID NO: 31-33, SEQ ID NO: 34-36, SEQ ID NO: 37-39, SEQ ID NO: 40-42, SEQ ID NO: 43-45, SEQ ID NO: 46-48, SEQ ID NO: 49-51, SEQ ID NO: 52-54, SEQ ID NO: 55-57, SEQ ID NO: 58-60, SEQ ID NO: 61-63, SEQ ID NO: ID NO:64-66, SEQ ID NO:67-69, SEQ ID NO:70-72, SEQ ID NO:73-75, SEQ ID NO:76-78, SEQ ID NO:79-81, SEQ ID NO:82-84, SEQ ID NO:85-87, SEQ ID NO:88-90, SEQ ID NO:91-93, SEQ ID NO:94-96, SEQ ID NO:97-99, SEQ ID NO:100-102, SEQ ID NO:103-105, SEQ ID NO:106-108, SEQ ID NO:109-111, SEQ ID NO:112-114, SEQ ID NO:115-117, SEQ ID NO:118-120, SEQ ID NO:121-123, SEQ ID NO:124-126, SEQ ID NO:127-129, SEQ ID NO:130-132, SEQ ID NO:133-135, SEQ ID NO:136-138, SEQ ID NO:139-141, SEQ ID NO:142-144, SEQ ID NO:145-147, SEQ ID NO:148-150, SEQ ID NO:151-153, SEQ ID NO:154-156, SEQ ID NO:157-159, SEQ ID NO:160-162, SEQ ID NO:163-165, SEQ IDNO:166-168, SEQ ID NO:169-171, SEQ ID NO:172-174, SEQ ID NO:175-177, SEQ ID NO:178-180, SEQ ID NO:181-183, SEQ ID NO:184-186, SEQ ID NO:187-189, SEQ ID NO:190-192, SEQ ID NO:193-195, SEQ ID NO:196-198, SEQ ID NO:199-201, SEQ ID NO:202-204, SEQ ID NO:205-207, SEQ ID NO:208-210, SEQ ID NO:211-213, SEQ ID NO:214-216, SEQ ID NO:217-219, SEQ ID NO:220-222、SEQ ID NO:223-225、SEQ ID NO:226-228, SEQ ID NO:229-231, SEQ ID NO:232-234, SEQ ID NO:235-237, SEQ ID NO:238-240, SEQ ID NO:241-243, SEQ ID NO:244-246, SEQ ID NO:247-249, SEQ ID NO:250-252, SEQ ID NO:253-255, SEQ ID NO:256-258 and SEQ ID NO:259-261.

[0302] In some embodiments, the antibody may have a heavy chain single variable domain (VHH), said VHH comprising complementarity determining regions (CDR) 1, 2 and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VHH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VHH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VHH CDR3 amino acid sequence. Fig.21 Selected VHH CDR1, 2, 3 amino acid sequences are shown in .

[0303] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two or three of the following: a VHH CDR1 having zero, one or two amino acid insertions, deletions or substitutions; a VHH CDR2 having zero, one or two amino acid insertions, deletions or substitutions; a VHH CDR3 having zero, one or two amino acid insertions, deletions or substitutions, wherein VHH CDR1, VHH CDR2 and VHH CDR3 are selected from Fig.21 CDRs in .

[0304] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two or three of the following: CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions or substitutions; CDRs of SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions or substitutions; CDRs of SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions or substitutions.

[0305] Insertions, deletions and substitutions may be within a CDR sequence, or at one or both ends of a CDR sequence. In some embodiments, CDRs are determined based on the Kabat numbering scheme. In some embodiments, CDRs are determined based on a combined numbering scheme.

[0306] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to coronavirus (e.g., SARS-CoV-2, SARS-CoV or MERS-CoV) S proteins. The antibody or antigen-binding fragment thereof contains a heavy chain single variable region (VHH), and the VHH includes an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VHH sequence or consists of the amino acid sequence. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 262. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 263. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 264. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 265. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 266. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 267. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 268. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 269. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 270. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 271. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 272. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 273. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 274. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 275. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 276. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 277. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 278. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 279. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 280. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 281. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 282. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 283. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 284. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 285.In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 286. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 287. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 288. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 289. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 290. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 291. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 292. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 293. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 294. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 295. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 296. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 297. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 298. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 299. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 300. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 301. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 302. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 303. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 304. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 305. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 306. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 307. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 308. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 309. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 310. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 311. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 312. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 313.In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 314. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 315. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 316. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 317. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 318. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 319. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 320. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 321. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 322. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 323. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 324. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 325. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 326. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 327. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 328. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 329. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 330. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 331. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 332. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 333. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 334. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 335. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 336. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 337. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 338. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 339. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 340. In some embodiments, the selected VHH sequence is set forth in SEQ ID NO: 341.In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 342. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 343. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 344. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 345. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 346. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 347. In some embodiments, the selected VHH sequence is shown in SEQ ID NO: 348.

[0307] In order to determine the identity percentage of two amino acid sequences or two nucleotide sequences, the sequences are compared for the purpose of best comparison (for example, a gap can be introduced in one or both of the first amino acid and the second amino acid or nucleotide sequence for best comparison, and for the purpose of comparison, non-homologous sequences can be ignored). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the amino acid residue or nucleotide identical to the corresponding position in the second sequence, these molecules are identical at the position. The identity percentage between the two sequences is a function of the number of identical positions shared by the sequences taking into account the number of gaps and the length of each gap, and the function needs to be introduced to perform the best comparison of the two sequences. For the purpose of illustration, the comparison of the sequence and the determination of the identity percentage between the two sequences can be completed, for example, using the Blossum 62 scoring matrix, wherein the gap penalty is 12, the gap extension penalty is 4 and the frameshift gap penalty is 5.

[0308] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising immunoglobulin heavy chain single variable domains (VHH). VHH comprises Fig.21 The CDRs shown, or having Fig. 22 sequence shown.

[0309] The antibody and antigen-binding fragment can also be an antibody or antibody fragment and an antibody variant (comprising derivatives and conjugates) of a multispecific (e.g., bispecific) antibody or antibody fragment. Other antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multispecific antibodies (multimeric antibodies, e.g., bispecific antibodies), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, antibodies made in cells (i.e., intracellular antibodies) and antigen-binding fragments thereof.

[0310] In some embodiments, the antibody or antigen-binding fragment thereof includes an Fc domain, which may be derived from various types (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclasses. In some embodiments, the Fc domain is derived from an IgG antibody or antigen-binding fragment thereof. In some embodiments, the Fc domain includes one, two, three, four, or more heavy chain constant regions.

[0311] The disclosure also provides antibodies or antigen binding fragments thereof that cross-compete with any antibody or antigen binding fragment as described herein. Cross-competition assays are known in the art and are described in, for example, Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein" Journal of Virology 70.3 (1996): 1863-1872, which is incorporated herein by reference in its entirety. On the one hand, the disclosure also provides antibodies or antigen binding fragments thereof that bind to the same epitope or region as any antibody or antigen binding fragment as described herein. Epitope binning assays are known in the art and are described, for example, in Estep et al., "High throughput solution-based measurement of antibody-antigen affinity and epitope binning," MAb Vol. 5, No. 2, Taylor & Francis, 2013, which is incorporated herein by reference in its entirety.

[0312] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR1 selected from SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 2, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, or 259.

[0313] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR2 selected from SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 3, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257 or 260.

[0314] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR3 selected from SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 4, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, or 261.

[0315] Antibody characteristics

[0316] The antibodies or antigen-binding fragments thereof described herein can block the binding between the coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) S protein and ACE2. In some embodiments, the antibodies can neutralize the coronavirus by binding to the coronavirus S protein. In some embodiments, the antibodies can promote viral aggregation. In some embodiments, the antibodies can induce Fc-dependent antiviral functions. In some embodiments, the antibodies can inhibit the cleavage of the S protein by the host cell TMPRSS2. In some embodiments, the antibodies can block the virus from entering the host cell.

[0317] The present disclosure provides antibodies or antigen-binding fragments thereof that block the interaction of coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) S protein with ACE2 (S / ACE) such that the remaining S / ACE binding is less than or about 95%, less than or about 90%, less than or about 85%, less than or about 80%, less than or about 75%, less than or about 70%, less than or about 65%, less than or about 60%, less than or about 55%, less than or about 50%, less than or about 45%, less than or about 40%, less than or about 35%, less than or about 30%, less than or about 25%, less than or about 20%, less than or about 15%, less than or about 10%, less than or about 10%, or less than or about 5% compared to the S / ACE binding in the absence of the antibody or antigen-binding fragment thereof as described herein.

[0318] The present disclosure provides antibodies or antigen-binding fragments thereof that neutralize coronaviruses (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) such that the neutralized coronaviruses account for at least or about 5%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, or at least or about 95% of the total coronaviruses.

[0319] The present disclosure provides antibodies or antigen-binding fragments thereof that promote aggregation of coronaviruses (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) by at least or about 1 fold, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8 fold, at least or about 9 fold, at least or about 10 fold, at least or about 20 fold, at least or about 30 fold, at least or about 40 fold, at least or about 50 fold, or at least or about 100 fold compared to in the absence of the antibody or antigen-binding fragment thereof as described herein.

[0320] The present disclosure provides antibodies or antigen-binding fragments thereof, which include a human Fc domain, which induce Fc-dependent antiviral function at least or about 1 fold, at least or about 2 fold, at least or about 3 fold, at least or about 4 fold, at least or about 5 fold, at least or about 6 fold, at least or about 7 fold, at least or about 8 fold, at least or about 9 fold, at least or about 10 fold, at least or about 20 fold, at least or about 30 fold, at least or about 40 fold, at least or about 50 fold, or at least or about 100 fold compared to in the absence of the antibody or antigen-binding fragment thereof as described herein.

[0321] The present disclosure provides antibodies or antigen-binding fragments thereof, which include a human Fc domain, which induce a host immune response that is at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, or at least or about 100-fold greater than in the absence of the antibody or antigen-binding fragment thereof as described herein.

[0322] The present disclosure provides antibodies or antigen-binding fragments thereof that block viral entry (or internalization) into host cells such that the internalization rate is less than or about 50%, less than or about 45%, less than or about 40%, less than or about 35%, less than or about 30%, less than or about 25%, less than or about 20%, less than or about 15%, less than or about 10%, less than or about 10%, or less than or about 5%, compared to the internalization rate in the absence of the antibody or antigen-binding fragment thereof as described herein.

[0323] In some embodiments, antibodies or antigen-binding fragments thereof comprising a single heavy chain are provided herein. In some embodiments, antibodies or antigen-binding fragments thereof comprising a pair of heavy chains are provided herein. In some embodiments, the heavy chain pair is connected by a disulfide bond. In some embodiments, the heavy chain pair comprises a knob-in-hole modification. In some embodiments, the heavy chain comprises a human IgG Fc domain. In some embodiments, the antibody or antigen-binding fragment thereof comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 VHH domains in each heavy chain. In some embodiments, the VHH domains in each heavy chain specifically bind to the same epitope. In some embodiments, the VHH domains in each heavy chain specifically bind to different epitopes. In some embodiments, the VHH domains in each heavy chain specifically bind to at least 1, 2, 3, 4 or 5 different epitopes. In some embodiments, the epitope is located in the receptor binding domain of the S protein of a coronavirus (e.g., SARS-CoV-2, SARS-CoV or MERS-CoV).

[0324] In some embodiments, the antibody or antigen-binding fragment thereof is a trispecific antibody. In some embodiments, the trispecific antibody is a trispecific VHH-Fc. In some embodiments, the trispecific antibody comprises the same VHH. In some embodiments, the trispecific antibody comprises different VHH. In some embodiments, the VHH binds to the same epitope (e.g., the same region of the SARS-CoV-2S protein RBD). In some embodiments, the VHH binds to different epitopes (e.g., different regions of the SARS-CoV-2S protein RBD). In some embodiments, the trispecific antibody comprises three VHHs, and the three VHHs are selected from the VHHs in group 1 (or G1; e.g., 1B, 2A, 1C, 1F, 4F, and G4) and group 2 (or G2; e.g., 3F and 3A) as shown in Figure 8C. In some embodiments, the three VHHs are connected in the following order (e.g., from N-terminus to C-terminus): G1-G1-G1, G1-G1-G2, G1-G2-G1, G1-G2-G2, G2-G1-G1, G2-G1-G2, G2-G2-G1 or G2-G2-G2. In some embodiments, the specific combination of the VHHs (e.g., from N-terminus to C-terminus) is as follows: Figure 23-25 One of the combinations shown.

[0325] In some embodiments, the antibody or antigen-binding fragment thereof has 4 VHHs. In some embodiments, in order to increase the developability of anti-SARS-CoV-2 multispecific antibodies, 4 VHHs are combined to construct a tetraspecific VHH without adding an IgG Fc domain. These molecules will have the additional advantage of increased affinity and avidity for the SARS-CoV-2 S1 protein compared to bispecific and trispecific VHH-Fc, even in the absence of Fc effector functions. These tetraspecific antibodies will be ideally suited as antibody prophylactics to prevent SARS-CoV-2 infection in humans, as the tetraspecific antibodies will have increased thermal stability, easier binding capacity, and the possibility of easy large-scale manufacturing using cost-effective expression systems such as yeast due to the structure of only llama VHHs. In some embodiments, the four VHHs are linked in the following order (e.g., from N-terminus to C-terminus): G1-G1-G1-G1, G1-G1-G1-G2, G1-G1-G2-G1, G1-G1-G2-G2, G1-G2-G1-G1, G1-G2-G1-G2, G1-G2-G1-G1, G1-G2-G1-G2, G1-G2-G2-G1, G1-G2-G2-G2, G2-G1-G1-G1, G2-G1-G1-G2, G2-G1-G2-G1, G2-G1-G2-G2, G2-G2-G1-G1, G2-G2-G1-G2, G2-G2-G2-G1, or G2-G2-G2-G2.

[0326] In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody or a trispecific antibody. In some embodiments, the antibody or antigen-binding fragment thereof can specifically bind to at least 4, 5 or 6 antigens.

[0327] In some embodiments, the antibody (or antigen-binding fragment thereof) is activated in less than 0.1 s -1 Less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1 The dissociation rate (koff) of the S protein (e.g., SARS-CoV-2 S protein, SARS-CoV S protein or MERS-CoV S protein) is specifically bound to the S protein. In some embodiments, the dissociation rate (koff) is greater than 0.01s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0328] In some embodiments, the kinetic association rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0329] Affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10 -10In some embodiments, the KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, or 0.05 nM. In some embodiments, the KD is greater than 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10 -11 M or greater than 1×10 -12 M.

[0330] In some embodiments, the antibody (or antigen-binding fragment thereof) has an IC of less than 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. 50 The IC value specifically binds to the S protein (e.g., SARS-CoV-2 S protein, SARS-CoV S protein, or MERS-CoV S protein). In some embodiments, the IC 50 The value is less than 1μg / ml, 0.9μg / ml, 0.8μg / ml, 0.7μg / ml, 0.6μg / ml, 0.5μg / ml, 0.4μg / ml, 0.3μg / ml, 0.2μg / ml or 0.1μg / ml.

[0331] In some embodiments, the trispecific VHH antibodies described herein have a lower aggregation potential (e.g., based on DLS) than bispecific VHH antibodies comprising at least one of the same VHH of the trispecific VHH antibodies. In some embodiments, the trispecific VHH antibodies described herein have a lower aggregation potential (e.g., based on DLS) than VHH antibodies comprising the same VHH of the trispecific VHH antibodies.

[0332] In some embodiments, the trispecific VHH antibodies described herein are more thermostable than bispecific VHH antibodies comprising at least one of the same VHH of the trispecific VHH antibody. In some embodiments, the trispecific VHH antibodies described herein are more thermostable than VHH antibodies comprising the same VHH of the trispecific VHH antibody.

[0333] In some embodiments, the trispecific VHH antibody (e.g., trispecific VHH-Fc) binds to a coronavirus S protein (e.g., SARS-CoV-2 S1 protein RBD) with a binding affinity that is at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, or more times greater than the binding affinity of a bispecific or monospecific VHH antibody that includes at least one of the same VHH of the trispecific VHH antibody. In some embodiments, the trispecific VHH antibody (e.g., trispecific VHH-Fc) blocks the coronavirus S protein / ACE2 interaction with a blocking efficacy that is at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, or more greater than the blocking efficacy of a bispecific or monospecific VHH antibody that includes at least one of the same VHH of the trispecific VHH antibody.

[0334] In some embodiments, the coronavirus S protein (e.g., SARS-CoV-2 S1 protein RBD) binding properties and / or coronavirus S protein / ACE2 blocking ability of the multispecific VHH antibody (e.g., trispecific VHH antibody) are determined by the physical arrangement and / or binding orientation of the multispecific VHH antibody.

[0335] In some embodiments, the antibodies or antigen-binding fragments thereof described herein address more than one mechanism to neutralize coronavirus infection.

[0336] Avidity specifically refers to strengthening binding through more than one interaction point. This effect can be quantified as the ratio of the dissociation constant of intrinsic affinity to the dissociation constant of functional affinity. Sometimes, the term avidity is used as affinity in a looser sense, and in practice is especially a synonym of functional affinity, as are the terms avidity determination and avidity index. In some embodiments, the antibody or antigen-binding fragment thereof comprises more than one antigen-binding unit (e.g., VHH domain), which confers better affinity and avidity than an antibody consisting of a single antigen-binding unit.

[0337] Common techniques for measuring the affinity and / or avidity of antibodies to antigens include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to SARS-CoV-2 S protein, SARS-CoV S protein, MERS-CoV S protein, or other coronavirus S protein. In some embodiments, the antibody does not bind to other coronavirus S proteins.

[0338] In some embodiments, thermal stability is determined. The Tm (melting temperature) of an antibody or antigen-binding fragment as described herein may be greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C or 95°C. In some embodiments, the Tm is less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94 or 95°C. The Tagg (aggregation temperature, e.g., Tagg at 266 nm (Tagg266) or Tagg at 473 nm (Tagg473)) of an antibody or antigen-binding fragment as described herein may be greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94 or 95°C. In some embodiments, Tagg is less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C or 95°C.

[0339] In some embodiments, the antibody or antigen-binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is phagocytosis.

[0340] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

[0341] In some embodiments, the antibody or antigen-binding fragment does not have an Fc region. For example, the antibody (or antigen-binding fragment thereof) is a polypeptide comprising one or more VHH domains, and the VHH domains are interconnected by a linker peptide. In some embodiments, the antibody comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 domains. In some embodiments, the VHH domains specifically bind to the same epitope. In some embodiments, the VHH domains specifically bind to different epitopes. In some embodiments, the VHH domains specifically bind to at least 1, 2, 3, 4 or 5 different epitopes.

[0342] In some embodiments, the antibody or antigen-binding fragment does not have a functional Fc region. In some embodiments, the Fc region has a LALA mutation (L234A and L235A mutations in EU numbering) or a LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering).

[0343] Method for preparing anti-coronavirus S protein antibodies

[0344] Variants of antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into DNA encoding human antibodies, humanized antibodies or chimeric antibodies or their antigen-binding fragments described herein or by peptide synthesis. Such variants include, for example, deletions, insertions or substitutions of residues in the amino acid sequence of the antigen-binding site or antigen-binding domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for target proteins (e.g., SARS-CoV-2S proteins). Any combination of deletions, insertions and / or combinations can be performed to obtain antibodies or their antigen-binding fragments with increased binding affinity to the target. Amino acid changes introduced into antibodies or antigen-binding fragments can also change antibodies or antigen-binding fragments or introduce new post-translational modifications into antibodies or antigen-binding fragments, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation sites (e.g., changing the amino acid sequence so that different sugars are connected by enzymes present in the cell) or introducing new glycosylation sites.

[0345] The VHH domains described herein may be derived from Camelidae (eg, llamas and camels) or cartilaginous fish (eg, sharks, rays / skates, and sawfish).

[0346] Humanized antibodies include antibodies with variable regions and constant regions of human germline immunoglobulin sequences derived from human immunoglobulin scaffold sequences (or with amino acid sequences identical to amino acid sequences of human germline immunoglobulin sequences derived from human immunoglobulin scaffold sequences). Humanized antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). Therefore, "humanized" antibodies are chimeric antibodies in which sequences from non-human species are replaced by corresponding human sequences.

[0347] Typically, amino acid sequence variants of human antibodies, humanized antibodies, or chimeric anti-coronavirus (e.g., SARS-CoV-2) S protein antibodies will contain an amino acid sequence having a percent identity of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with the sequence present in the VHH domain of the original antibody.

[0348] Identity or homology with respect to the original sequence is typically the percentage of amino acid residues present in the candidate sequence that are identical to the sequence present in a human antibody, humanized antibody, or chimeric anti-coronavirus (e.g., SARS-CoV-2) S protein antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity and not taking into account conservative substitutions as part of the sequence identity.

[0349] Anti-coronavirus S protein antibodies or antigen binding fragments can be additionally modified. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The resulting homodimeric antibodies can have any increased half-life in vitro and / or in vivo. Homodimeric antibodies with increased half-life in vitro and / or in vivo can also be prepared using heterologous bifunctional cross-linkers such as described in, for example, Wolff et al. Wolff et al. ("Monoclonal antibody homodimers: enhanced antitumor activity in nude mice" Cancer research 53.11 (1993): 2560-2565). Alternatively, antibodies with dual Fc regions can be engineered.

[0350] In some embodiments, anti-coronavirus (e.g., SARS-CoV-2) S protein antibodies or antigen-binding fragments thereof can be covalently modified. These covalent modifications can be performed by chemical synthesis or enzymatic synthesis or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting the targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that is capable of reacting with a selected side chain or N-terminal residue or C-terminal residue.

[0351] In some embodiments, antibody variants are provided as having a carbohydrate structure lacking fucose connected to the Fc region (directly or indirectly). For example, the amount of fucose in such antibody compositions can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) connected to Asn 297 as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (Eu numbering of Fc region residues; or position 314 in Kabat numbering) in the Fc region; however, due to minor sequence changes in antibodies, Asn297 can also be located at approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function.In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody may be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0352] The present disclosure also provides a recombinant vector (e.g., an expression vector) comprising an isolated polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), a host cell into which the recombinant vector is introduced (i.e., such that the host cell contains the polynucleotide and / or a vector comprising the polynucleotide), and the production of a recombinant antibody polypeptide or fragment thereof by recombinant technology.

[0353] As used herein, "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. "Expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector has been introduced. Therefore, in an expression vector, the polynucleotide of interest is positioned to be expressed in the vector by: being operably connected to regulatory elements such as promoters, enhancers and / or poly-A tails in the vector or in the genome of the host cell located at or near the integration site of the polynucleotide of interest or flanking the integration site, so that the polynucleotide of interest is translated in the host cell introduced with the expression vector.

[0354] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., using a recombinant virus). Therefore, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with a cationic condensing agent.

[0355] In some embodiments, polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses), which may involve the use of non-pathogenic (defective), replication-competent viruses, or replication-defective viruses may be used. In the latter case, viral propagation will generally occur only in complementing viral packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proceedings of the National Academy of Sciences of the United States of America (Natl. Acad. Sci. USA) 86:317-321; Flexner et al., 1989, Ann. NY Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner, Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci., 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci., 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA can also be "naked", as described, for example, in Ulmer et al., 1993, Science, 259: 1745-1749 and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads, which are efficiently transported into cells.

[0356] For expression, a DNA insert comprising a polynucleotide encoding an antibody or a polynucleotide encoding a polypeptide disclosed herein can be operably linked to an appropriate promoter (e.g., a heterologous promoter), such as the bacteriophage λ PL promoter, E. coli lac, trp and tac promoters, SV40 early and late promoters, and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled person. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression construct may further contain sites for transcription initiation and termination, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct may include a translation starting at the beginning and a stop codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0357] As indicated, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase resistance or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include but are not limited to bacterial cells, such as Escherichia coli, Streptomyces (Streptomyces) and Salmonella typhimurium (Salmonella typhimurium) cells; fungal cells, such as yeast cells; insect cells, such as fruit fly S2 and noctuid Sf9 cells; animal cells, such as CHO, COS, Bowes melanoma and HK 293 cells; and plant cells. Suitable culture media and conditions for host cells described herein are known in the art.

[0358] Non-limiting vectors for use in bacteria include: pQE70, pQE60, and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, available from Pharmacia. Non-limiting eukaryotic vectors include: pWLNEO, pSV2CAT, pOG44, pXT1, and pSG, available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL, available from Pharmacia. Other suitable vectors will be apparent to the skilled artisan.

[0359] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters of retroviral LTRs, such as the promoter of Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.

[0360] In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH are available.

[0361] The introduction of constructs into host cells can be accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0362] Transcription of the DNA encoding the antibodies of the present disclosure by higher eukaryotic organisms can be increased by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, typically about 10 to 300 bp, that acts to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located on the rear side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the rear side of the replication origin, and adenovirus enhancers.

[0363] In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, a suitable secretion signal may be incorporated into the expressed polypeptide. The signal may be endogenous to the polypeptide, or it may also be a heterologous signal.

[0364] Polypeptides (e.g., antibodies) can be expressed in a modified form, such as a fusion protein (e.g., GST-fusion) or with a histidine tag, and can contain not only a secretion signal, but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or during subsequent processing and storage. Similarly, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final preparation of the polypeptide. Adding peptide moieties to polypeptides to cause secretion or excretion, improve stability and facilitate purification (among other things) is well known and conventional in the art.

[0365] The present disclosure also provides nucleic acids that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 200%, Sequences and amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence as described herein. In some embodiments, the disclosure relates to nucleotide sequences encoding any peptide described herein or any amino acid sequence encoded by any nucleotide sequence as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

[0366] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any of the sequences described herein.

[0367] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any of the sequences described herein.

[0368] In some embodiments, the antibody or antigen-binding fragment thereof is expressed in yeast, insect cells, or mammalian cells (eg, CHO cells).

[0369] In some embodiments, the generation, optimization, and testing of the multispecific VHH antibodies (e.g., trispecific VHH-Fc) described herein are completed in less than 6 months, 5 months, 4 months, 3 months, 2 months, or less.

[0370] In some embodiments, the binding epitope of VHH-Fc (e.g., 1B, 2A, 1C, 1F, 4F, G4, B38, CB6, or P2B-2F6) is within the del3, del4, and / or del5 region of the S1 RBD as shown in Figure 9A. In some embodiments, the binding epitope of VHH-Fc (e.g., 1B, 2A, 1C, 1F, 4F, G4, B38, CB6, or P2B-2F6) is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of the del3, del4, and / or del5 region of the S1 RBD as shown in Figure 9A.

[0371] In some embodiments, the binding epitope of VHH-Fc (e.g., 3F, 3A, or CB6) is within the del2 region of S1 RBD as shown in Figure 9A. In some embodiments, the binding epitope of VHH-Fc (e.g., 3F, 3A, or CB6) is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of the del2 region of S1 RBD as shown in Figure 9A.

[0372] In some embodiments, the binding epitope of VHH-Fc (e.g., S309 or BD-23) is within the del1 region of S1 RBD as shown in Figure 9A. In some embodiments, the binding epitope of VHH-Fc (e.g., S309 or BD-23) is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the del1 region of S1 RBD as shown in Figure 9A.

[0373] Treatment and diagnostic methods

[0374] The antibodies or antigen-binding fragments thereof disclosed herein can be used for various therapeutic purposes.

[0375] On the one hand, the present disclosure provides methods for treating coronavirus-related diseases in subjects, methods for neutralizing coronaviruses, methods for blocking coronavirus / ACE2 interactions, methods for promoting coronavirus aggregation, methods for inducing Fc-dependent antiviral functions, methods for blocking coronaviruses from being internalized by cells, and methods for identifying subjects with coronavirus-related diseases. In some embodiments, treatment can stop, slow, delay, or inhibit the progression of coronavirus-related diseases. In some embodiments, treatment can reduce the number, severity, and / or duration of one or more symptoms of coronavirus-related diseases in subjects.

[0376] In one aspect, the disclosure features methods comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having, identified or diagnosed as having, a coronavirus-related disease).

[0377] In some embodiments, the coronavirus-related disease is COVID-19 (coronavirus disease 2019), severe acute respiratory syndrome (SARS), or Middle East respiratory syndrome (MERS).

[0378] In some embodiments, the coronavirus causing the coronavirus-related disease is SARS-CoV, SARS-CoV-2, MERS-CoV, or other types of coronaviruses having one or more S proteins. In some embodiments, the amino acid sequence of the S protein of the coronavirus described herein includes a sequence that is at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, or at least or about 98% identical to the receptor binding domain sequence of the SARS-CoV-2 S protein.

[0379] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for coronavirus-related diseases. Patients with coronavirus-related diseases can be identified using various methods known in the art.

[0380] As used herein, "effective amount" means an amount or dosage sufficient to produce a beneficial or desired result, including stopping, slowing, delaying or inhibiting the progression of a disease (e.g., a coronavirus-related disease). The effective amount will vary according to, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vectors including polynucleotides and / or compositions thereof are to be administered, the severity of the symptoms, and the route of administration, and therefore administration can be determined according to individual circumstances.

[0381] The effective amount can be administered in one or more administrations. For example, the effective amount of an antibody or antigen binding fragment is an amount sufficient to improve, stop, stabilize, reverse, inhibit, slow down and / or delay the progression of a patient's coronavirus-related disease. As is known in the art, the effective amount of an antibody or antigen binding fragment can vary, particularly depending on the patient's medical history and other factors, such as the type (and / or dosage) of the antibody used.

[0382] The effective amount and regimen of administering the antibodies, polynucleotides encoding antibodies and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of the art. It will be appreciated by those skilled in the art that the dosage that must be administered will vary, for example, depending on the mammal to which the antibodies, polynucleotides encoding antibodies and / or compositions disclosed herein will be administered, the route of administration, the specific type of antibodies, polynucleotides encoding antibodies, antigen-binding fragments and / or compositions disclosed herein used, and other drugs administered to the mammal.

[0383] The typical daily dosage of an effective amount of antibody is 0.01 mg / kg to 100 mg / kg (milligrams per kilogram of patient body weight). In some embodiments, the dosage can be less than 100 mg / kg, 50 mg / kg, 25 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg or 0.1 mg / kg. In some embodiments, the dosage can be greater than 25 mg / kg, 20 mg / kg, 15 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg or 0.01 mg / kg. In some embodiments, the dosage is about 25 mg / kg, 20 mg / kg, 15 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg or 0.1 mg / kg. In some embodiments, the multispecific VHH antibody (e.g., trispecific VHH-Fc) is administered at the dosages described herein without affecting the weight and / or survival rate of the subject.

[0384] In any of the methods described herein, at least one antibody, antigen binding fragment thereof, or pharmaceutical composition (e.g., any of the antibodies, antigen binding fragments, or pharmaceutical compositions described herein) and optionally at least one additional therapeutic agent may be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen binding fragments are administered with the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen binding fragment and at least one additional therapeutic agent are administered with the same composition (e.g., a liquid composition containing at least one antibody or antigen binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, the at least one additional therapeutic agent is administered in the form of a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered with a sustained release oral formulation.

[0385] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that the biological activity periods of the one or more additional therapeutic agents and at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject overlap.

[0386] In some embodiments, at least one antibody, antigen-binding antibody fragment or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments or pharmaceutical compositions described herein) may be administered to a subject over an extended period of time (e.g., at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years or 5 years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or tracking treatment effectiveness (e.g., observing at least one symptom of a coronavirus-related disease). As described herein, a skilled medical professional may also change (e.g., increase or decrease) the properties and quantity of the antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject, and may also adjust (e.g., increase or decrease) the dosage or frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject based on an evaluation of treatment effectiveness (e.g., using any of the methods described herein and known in the art).

[0387] In some embodiments, the antibodies or antigen-binding fragments thereof can be used to detect coronaviruses (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) in a subject (e.g., a human) or to diagnose a coronavirus-related disease. Methods known in the art (e.g., ELISA) can be designed to produce diagnostic test kits. In some embodiments, one or more antibodies or antigen-binding fragments comprising any one of the heavy chain single variable domains as described herein can be used.

[0388] In some embodiments, a multispecific VHH antibody (e.g., a trispecific VHH-Fc) is administered to a subject (e.g., a human) at a lower concentration than a bispecific or monospecific VHH antibody comprising at least one VHH domain identical to the multispecific VHH antibody.

[0389] In some embodiments, a multispecific VHH antibody (e.g., a trispecific VHH-Fc) binds to at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) coronavirus S protein (e.g., SARS-CoV-2 S protein) from the same virus. In some embodiments, a multispecific VHH antibody (e.g., a trispecific VHH-Fc) binds to at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) coronavirus S protein (e.g., SARS-CoV-2 S protein) from different viruses. In some embodiments, a single multispecific VHH antibody (e.g., a trispecific VHH-Fc) binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 coronaviruses. In some embodiments, the multispecific VHH antibody (e.g., trispecific VHH-Fc) binds to at least one mutated coronavirus S protein (e.g., mutated SARS-CoV-2 S protein). In some embodiments, the multispecific VHH antibody (e.g., trispecific VHH-Fc) binds to at least one mutated coronavirus S protein and at least one wild-type coronavirus S protein (e.g., SARS-CoV-2 S protein).

[0390] In some embodiments, the multispecific VHH antibody (e.g., trispecific VHH-Fc) can be delivered to a subject by intranasal administration or intraperitoneal administration. In some embodiments, administration of the multispecific VHH antibody reduces the viral titer in the subject (e.g., in the lung) to less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1%, compared to the viral titer in a subject not administered. In some embodiments, the viral titer is about 10 11 , about 10 10 , about 10 9 , about 10 8 , about 10 7 , about 10 6 , about 10 5 , about 10 4 , about 10 3 , about 10 2Or about 10 copies / gram of collected sample (eg, lung). In some embodiments, viral titer is determined 1 day, 2 days, 3 days, 4 days, or 5 days after viral infection.

[0391] In some embodiments, the S protein has one or more mutations, for example in the RBD. In some embodiments, the one or more mutations are located at K417, E484, N501, D614, and / or N501 of the wild-type S protein. In some embodiments, the one or more mutations are K417N, E484K, N501Y, D614G, and / or N501Y. In some embodiments, the multispecific VHH antibodies (e.g., trispecific VHH-Fc) described herein have similar binding function and ACE2 blocking function to S proteins having one or more mutations (e.g., K417N, E484K, N501Y, D614G, and / or N501Y). In some embodiments, the KD value of a multispecific VHH antibody (e.g., a trispecific VHH-Fc) described herein that binds to an S protein having one or more mutations (e.g., K417N, E484K, N501Y, D614G and / or N501Y) is 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% greater than the KD value for binding to a wild-type S protein.

[0392] Administration via the respiratory tract

[0393] Compositions as described herein can be administered via the respiratory tract by various means, such as intranasal administration, intranasal instillation, insufflation (e.g., nasal spray), inhalation (through the nose or mouth), intrapulmonary administration, intratracheal administration, or any combination thereof. As used herein, the term "intranasal instillation" refers to a procedure for delivering a therapeutic agent directly into the nose and onto the nasal membranes, wherein a portion of the therapeutic agent may pass through the trachea and be delivered to the lungs.

[0394] Because the function of the lungs to be treated is occasionally limited, therapeutic agents are sometimes unable to be effectively delivered to target sites (e.g., bronchioles or alveoli) in the lungs by respiratory administration. In these cases, agents that can clear the airways can be first administered to the subject. In some embodiments, these agents can induce bronchial passage dilation and / or muscle vasodilation. Such agents include, but are not limited to, beta 2 adrenergic receptor agonists, anticholinergics, corticosteroids. In some embodiments, agents for the treatment of asthma can be used.

[0395] Pharmaceutical compositions suitable for administration via the respiratory tract may comprise, for example, liquid solutions, aqueous solutions (in which water is soluble), dispersions, etc. In some embodiments, these compositions may include one or more surfactants.

[0396] As used herein, the term "respiratory tract" refers to the air passages from the nose to the alveoli, including the nose, throat, pharynx, larynx, trachea, bronchi, and any part of the lungs. In some embodiments, the composition is administered to the lungs or any part of the respiratory system.

[0397] In some embodiments, the composition can be applied to the subject by a delivery system (e.g., atomizer, vaporizer, nasal sprayer, inhaler, soft mist inhaler, jet nebulizer, ultrasonic nebulizer, pressurized metered dose inhaler, breath-activated pressurized metered dose inhaler or vibrating mesh device) that can convert the composition into an aerosol form. As used herein, the term "inhaler" refers to a device for applying a composition in the form of a spray or dry powder, which is inhaled (naturally or mechanically forcibly inhaled into the lungs) by the nose or mouth. In certain embodiments, the inhaler includes, for example, a passive or active ventilator (mechanical with or without an endotracheal tube), a nebulizer, a dry powder inhaler, a metered dose inhaler, and a pressurized metered dose inhaler. Once the antibody (or its Fab) is deposited or positioned near the cell, a subset of the antibody can neutralize the coronavirus as described herein.

[0398] In some embodiments, the device can use air (e.g., oxygen, compressed air) or ultrasonic power to break up solutions and suspensions into small aerosol particles (e.g., droplets) that can be inhaled directly from the mouthpiece of the device. In some embodiments, the device uses a mesh / membrane with laser-drilled holes (e.g., 1000 to 7000 holes) that vibrates on top of a reservoir and thereby presses a mist of very fine droplets through the holes.

[0399] The delivery system can also have a unit dose delivery system. The volume of the solution or suspension delivered per dose can be from about 5 to about 2000 microliters, from about 10 to about 1000 microliters, or from about 50 to about 500 microliters. The delivery systems of these different dosage forms can be dropper bottles, plastic extrusion units, vaporizers, nebulizers, or pharmaceutical aerosols in unit dose or multidose packaging.

[0400] In some embodiments, the device is a small, hard bottle to which a metered dose sprayer is attached. The metered dose can be delivered by inhaling the composition into a volume-limited chamber having an orifice sized to atomize the aerosol formulation by forming a spray when the liquid in the chamber is compressed. The chamber is compressed to administer the composition. In some devices, the chamber is a piston arrangement. Such devices are commercially available.

[0401] Alternatively, a squeeze bottle having an orifice or opening sized to atomize the aerosol formulation by forming a spray when squeezed can be used. The opening is typically located at the top of the bottle, and the top is typically tapered to partially fit into the nasal passages to effectively administer the aerosol formulation. Preferably, the nasal inhaler can provide a metered amount of the aerosol formulation to administer a measured dose of the therapeutic agent.

[0402] In some embodiments, aerosolization of a liquid formulation for inhalation into the lungs involves a propellant. The propellant may be any propellant commonly used in the art. Specific non-limiting examples of such useful propellants are chlorofluorocarbons, hydrofluorocarbons, hydrochlorofluorocarbons or hydrocarbons, including trifluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane or a combination thereof.

[0403] Pharmaceutically acceptable diluents in such aerosol formulations include, but are not limited to, sterile water, saline, buffered saline, glucose solution, etc. In certain embodiments, the diluent that can be used in the present invention or pharmaceutical formulation is phosphate buffered saline or a buffered saline solution or water typically in the pH range of 7.0-8.0 (e.g., pH 7.4).

[0404] Aerosol formulations may also optionally contain pharmaceutically acceptable carriers, diluents, solubilizers or emulsifiers, surfactants and excipients.

[0405] The present disclosure further contemplates an aerosol formulation comprising a composition as described herein and another therapeutically effective agent.

[0406] The total amount of the composition delivered to the subject will depend on several factors, including the total amount aerosolized, the type of nebulizer, the particle size, the subject's breathing pattern, the severity of the lung disease and the concentration in the aerosolized solution and the duration of the inhalation therapy. The amount of the composition measured in the alveoli can be substantially less than the amount expected for the amount of the composition present in the aerosol, because a large portion of the composition can be exhaled by the subject or trapped on the internal surfaces of the nebulizer device.

[0407] A skilled practitioner will be able to easily design an effective regimen, particularly where the particle size of the aerosol is optimized. In some instances, it is useful to administer a higher dose when treating more severe conditions. If necessary, the treatment can be repeated on a specific basis, depending on the results achieved. If the treatment is repeated, the mammalian host can be monitored to ensure that there is no adverse immune response to the treatment. The frequency of treatment depends on many factors, such as the amount of the composition administered per dose and the health and medical history of the subject.

[0408] Pharmaceutical composition

[0409] Also provided herein are pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein may be present in the pharmaceutical composition in any combination. The pharmaceutical composition may be formulated in any manner known in the art.

[0410] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal). The composition may include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvent, an antibacterial or antifungal agent, such as benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate or phosphate; and an isotonic agent, such as a sugar (e.g., glucose), a polyol (e.g., mannitol or sorbitol) or a salt (e.g., sodium chloride) or any combination thereof. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. The formulation of the composition may be formulated and packaged in an ampoule, a disposable syringe or a multiple dose vial. Where necessary (e.g., in injectable formulations), appropriate fluidity can be maintained, for example, by using a coating such as lecithin or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be extended by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems that can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0411] Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound to facilitate administration and uniformity of dosage).

[0412] The pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic and manufactured under good manufacturing practice (GMP) conditions. The pharmaceutical composition can be provided in a unit dosage form (i.e., a single dose). The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the selected route of administration. For injection, the antibody can be formulated in an aqueous solution, preferably in a physiologically compatible buffer to reduce discomfort at the injection site. The solution may contain a formulation agent, such as a suspending agent, a stabilizer, and / or a dispersant. Alternatively, the antibody can be in a lyophilized form to be constructed with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[0413] Data obtained from cell culture assays and animal studies can be used to formulate the appropriate dose of any given agent for a subject (e.g., human). The therapeutically effective amount of one or more (e.g., one, two, three or four) antibodies or their antigen-binding fragments (e.g., any antibody or antibody fragment described herein) will be in a subject (e.g., identified as a human subject with COVID-19) or identified as a subject at risk of developing a disease (e.g., previously infected with coronavirus but now cured) to treat the subject's disease (e.g., suppress coronavirus), reduce the severity, frequency and / or duration of one or more symptoms of the subject's (e.g., human) disease. The effectiveness and dosage of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art and by observing one or more symptoms of the subject's (e.g., human) disease. Certain factors may affect the dosage and time course required for the effective treatment of a subject (e.g., the severity of the disease or condition, previous treatment, the overall health and / or age of the subject, and the presence of other diseases).

[0414] Exemplary dosages include any of the antibodies or antigen-binding fragments described herein in milligram or microgram amounts per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; about 1 μg / kg to about 50 μg / kg; about 1 mg / kg to about 10 mg / kg; or about 1 mg / kg to about 5 mg / kg). Although these dosages cover a wide range, it will be appreciated by one of ordinary skill in the art that the efficacy of therapeutic agents (including antibodies and antigen-binding fragments thereof) varies, and effective amounts can be determined by methods known in the art. Typically, a relatively low dose is initially administered, and the attending health care professional or veterinary professional (in the case of therapeutic applications) or researcher (when still working in the research and development phase) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it should be understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound employed, the age, weight, general health, sex and diet of the subject, time of administration, route of administration, rate of excretion and half-life of the antibody or antibody fragment in vivo.

[0415] The pharmaceutical composition may be contained in a container, pack, or dispenser together with instructions for administration. The present disclosure also provides methods of making antibodies or antigen-binding fragments thereof for the various uses as described herein.

[0416] Examples

[0417] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0418] Materials and methods

[0419] Cell lines and transfection

[0420] The cell lines used in this study were cultured in the following media. Expi293 (Thermo Fisher Scientific)-Expi293 expression medium (Thermo Fisher Scientific), NK-92-CD16 (natural killer cell line expressing CD16) cells (ATCC)-RPMI 1640, 10% fetal calf serum (FCS), 40ng / ml IL-2. The cells were maintained in a humidified chamber at 37°C under 8% CO2. Expi293 cells were transiently transfected with a plasmid expressing the SARS-CoV-2 S1 protein using ExpiFectamine 293 transfection reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, cells were transfected at 1.7×10 6 The density of individual cells / ml is plated in the 30ml fresh medium in the 125ml shake bottle and spends the night. On the second day, 30 μ g DNA and 80 μ l ExpiFectamine293 are mixed among the 1.5ml Opti-MEM respectively, and at room temperature incubation is 3 minutes. Then, ExpiFectamine293 and DNA mixture are mixed, and at room temperature incubation is other 20 minutes. Finally, it is added in the flask that contains the cell, and at 37 ℃ at 8% CO2 incubation in the humidification chamber. Cell is incubated overnight or in liquid N2 and is used for experiment after freezing.

[0421] VHH-Fc expression and purification

[0422] As described above, Expi293 cells were transiently transfected with a plasmid expressing VHH-Fc according to the manufacturer's instructions. Enhancers were added to the cells 17 hours after transfection, and centrifuged at 3000g for 10 minutes 72 hours after transfection. The supernatant was then filtered with a 0.45 μm filter, and the antibody concentration was determined using a protein A probe on Gator (probe detection company). VHH-Fc was then purified using a protein A column on an AKTA Explorer 100 purification system (buffer A: PBS, pH = 7.4; buffer B: 0.1 M glycine, pH = 2.5) and dialyzed twice in PBS. The antibody was then filtered again with a 0.22 μM filter and used in the experiment.

[0423] Epitope binning (competition) assay

[0424] The initial assay was performed using the Gator system (Probe Detection, Inc.). After pre-wetting the SARS-CoV-2S1 RBD sensor in Q buffer (Probe Detection, Inc.), the sensor captured 30 μg / ml of the first monoclonal VHH-Fc 2A for approximately 300 seconds, and then the loaded sensor captured 10 μg / ml of the second monoclonal VHH-Fc (1B, 3F, or 2A), which was quantified by Gator over time.

[0425] VHH-Fc 1B-2A and 3F were subsequently determined by ELISA. 96-well plates were coated with SARS-CoV-2S1 protein at a final concentration of 1 μg / ml and placed overnight at 4°C. Then, 60 μg / ml 1B-2A-Fc or 3F-Fc was premixed with each competitive c-Myc-tagged VHH from the periplasmic supernatant at a ratio of 1: 1. After another hour of incubation, biotinylated anti-c-Myc antibody (9E10) was added, and the samples were incubated for another hour. Then, streptavidin-HRP was added, followed by treatment with Amplex Red (Thermo Fisher Scientific) and a developing solution containing 30% H2O2. The emitted signal of each sample was detected by using a fluorescent plate reader (SpectraMax Gemini XPS). The percentage difference of the signal of the competing pair relative to that of VHH-Fc alone was calculated using the following formula: VHH-Fc signal difference %=(1-((signal of the competing pair-signal of no antibody) / (signal of VHH-Fc alone-signal of no antibody))*100.

[0426] In vitro S1 protein binding assay

[0427] 96-well ELISA plates (Greiner Bio-One) were directly coated with SARS-CoV-2 S1 protein (Acro Biosystems) diluted at 1 μg / ml in PBS and incubated overnight at 4°C. The plates were then washed with PBS containing 0.5% Tween 20 (PBST) and blocked with PBS containing 1% bovine serum albumin (BSA) for one hour at room temperature. The plates were washed again with PBST and incubated with the test antibodies for one hour at room temperature. Antibodies were used at a 1:5 serial dilution. The plates were washed with PBST, followed by the addition of anti-human Fc antibodies conjugated to horseradish peroxidase (HRP) (Jackson ImmunoResearch) diluted 1:5000 in PBST, and the plates were incubated at room temperature for 1 hour. After washing again with PBST, the plates were treated with an ELISA developing buffer solution containing Amplex Red and 30% H2O2. The emitted binding signal of each sample was detected by using a fluorescence plate reader. Blocking was measured in relative fluorescence units (RFU), and % inhibition was calculated as follows: % inhibition = (1-(mean of experimental values / mean of no antibody controls))*100.

[0428] S / ACE2 blocking assay

[0429] 96-well ELISA plates (Grena First Biochemical International) were coated with SARS-CoV-2S1 protein (Bipsys Biotech Co., Ltd.) and incubated overnight as described above. The plates were then washed with PBST and blocked with PBS containing 2% BSA for one hour at room temperature. The plates were washed again with PBST and incubated with the test antibodies for 45 minutes at room temperature. Antibodies were used at a 1:5 serial dilution. Recombinant biotinylated ACE2 (Bipsys Biotech Co., Ltd.) was then added directly to the plates at 4.65 μg / ul and incubated for another 45 minutes at room temperature. The plates were washed with PBST, followed by the addition of HRP-conjugated streptavidin diluted 1:1000 in PBST. The plates were incubated for another 45 minutes at room temperature. The plates were then washed with PBST and treated with ELISA developing buffer. The emitted binding signal for each sample was detected by using a fluorescent plate reader.

[0430] Analysis of physical properties of VHH-Fc

[0431] The thermal stability of purified VHH-Fc was analyzed by the UNcle system (Unchained Labs) using differential scanning fluorimetry (DSF) and static light scattering (SLS), and its aggregation potential was analyzed using dynamic light scattering (DLS) assay. DLS was measured at 25°C, and the data was analyzed using UNcle Analysis Software. For DSF / DLS assays, a temperature ramp of 1°C / min was performed with monitoring from 25°C to 95°C. SLS was measured by UNcle at 266nm and 473nm. Tm and Tagg were analyzed and calculated by UNcle analysis software.

[0432] Pseudovirus neutralization assay

[0433] Pseudovirus neutralization assays were performed in collaboration with GenScript Biotech (Piscataway, NJ). Briefly, pseudoviruses expressing luciferase and containing SARS-CoV-2S1 as an envelope glycoprotein in a lentiviral vector were produced in HEK293T cells, and virus titration was determined by ELISA. HEK293 cells expressing ACE2 receptor and transmembrane serine protease 2 (TMPRSS2) were used as target cells and seeded in 96-well plates. Pseudoviruses with serial dilutions of antibodies were then mixed with target cells. The cells were incubated at 37°C for 48 hours, and 30 μl of the cell suspension was transferred to the assay plate. The assay plate was incubated with 1% paraformaldehyde from Bio-Glo TM The luciferase detection reagent of the luciferase assay system (Promega) was mixed and incubated at room temperature for 5-10 minutes. Then, the luminescence was measured by a plate reader. The background RLU was subtracted from the RLU of the experimental sample. The value of inhibition % was derived from the RLU as follows: inhibition % = (1-(average of experimental values-average of cells treated with buffer only) / (average of cells treated with SARS-CoV-2 only))*100.

[0434] Antibody-dependent cellular cytotoxicity (ADCC) assay

[0435] Target Expi293 cells expressing S1 protein (293SProt) were washed with RPMI medium containing 10% horse serum and 40 ng / ml IL-2 and incubated at 1×10 4 The cells were plated in 96-well plates at a density of 10 cells / well. The cells were then mixed with antibodies at a final concentration of 40 μg / ml. NK-92-CD16 cells expressing GFP were then plated at a density of 3×10 4The density of cells / well (effector: target -3: 1) was added to the wells, and the plate was incubated overnight at 37°C under 8% CO2. Then, the cells were washed twice and resuspended in DPBS containing 2% FBS. The cells were evaluated by flow cytometry using a FACSCalibur cytometer (BD Biosciences). 293SProt and GFP-NK-92-CD16 cells were each used as a reference to establish overall target cell gating and establish a GFP-positive NK-92-CD16 population, thereby allowing differentiation between NK-92-CD16 effector cells and 293SProt target cells. The GFP-negative 293SProt cell percentage of all samples was assessed. Then, the percentage of cell death was calculated as follows: cell death %=(1-(average value of antibody-treated cell percentage / isotype control percentage))*100.

[0436] Statistical analysis

[0437] Four-parameter nonlinear regression analysis from Prism software version 8.4.3 was used for all binding and blocking curves, which also contained the IC of the blocking assay. 50 Unless otherwise stated, all error bars represented in the data are based on standard deviation.

[0438] Example 1. Discovery of Anti-SARS-CoV-2 Antibodies

[0439] A natural and a designed synthetic llama VHH library were used in this approach, as Figure 1 As shown. PBMCs were obtained from 65 llamas, and RNA was isolated to produce cDNA by reverse transcription. Then, VHH genes were amplified by two rounds of PCR, and the genes were cloned into phage display vectors to construct natural VHH libraries. Synthetic (e.g., humanized) VHH libraries were prepared by incorporating the shuffled VHH CDR1, 2, and 3 generated by overlapping PCR into modified human VH scaffolds to produce enhanced diversity and maintain low immunogenicity.

[0440] Two llama VHH libraries were panned for recombinant SARS-CoV-2S protein. Specifically, the VHH phage library was used to pan SARS-CoV-2S1 fused to mouse Fc protein as the target antigen. Anti-mouse Fc coated wells were used to fix the antigen, and 3 rounds of phage panning were performed with reduced antigen concentrations in each round. After panning, 91 high-affinity VHH hits for SARS-CoV-2S protein binding were obtained, of which 69 were unique sequences (Fig. 2A). The ability of VHH to bind to recombinant SARS-CoV-2S protein was assessed by ELISA. As shown in Fig. 2B, the plate was coated with SARS-CoV-2S1, the bound VHH was detected by biotinylated anti-c-Myc antibody, and streptavidin-HRP was subsequently added. By adding substrate, a chemiluminescent signal was induced by HRP.

[0441] The ability of VHH to block the interaction of SARS-CoV-2 S protein with ACE2 (S / ACE2) in vitro was also evaluated by ACE2 competition assay. As shown in FIG. 3A , the ACE2 competition assay was performed by ELISA by coating the plate with SARS-CoV-2 S1 as described in FIG. 2B and adding VHH in the presence of biotinylated ACE2. The S1 / ACE2 blocking function was determined by the reduction of HRP-induced chemiluminescent signal. The results showed that 9 of the 69 unique S protein binders exhibited S / ACE2 blocking function, as listed in FIG. 3B .

[0442] Using the same assay, subsequent studies demonstrated that pairwise combinations of some of the nine VHH blockers (eg, clones 2+5; or clones 3+5) resulted in synergistic blocking efficacy of the S / ACE2 interaction. Figure 4 The results are shown in .

[0443] Example 2. Design of multispecific nanobody-Fc for SARS-CoV-2 treatment

[0444] By selecting two or three different humanized VHH sequences that target different but adjacent S protein RBD epitopes (Figure 5A) and fusing them into a single multispecific antibody with affinity and avidity, the overall S protein binding affinity and avidity can be improved. Such therapeutic antibodies can also exhibit increased S / ACE2 blocking function. As shown in Figure 5B, this design can be assisted by computer simulation using modeling structure analysis of different VHH combinations. CAAD can also be used to further improve the VHH sequence so that the VHH has low immunogenicity in humans and high developability and manufacturability. The designed candidates can be analyzed in vitro by binding and blocking assays to determine their S protein binding and S / ACE2 blocking abilities, respectively. Fc-fused versions of such multispecific VHH antibodies can be produced and their SARS-CoV-2 neutralization abilities can be explored. Figure 6 As shown, these molecules can neutralize SARS-CoV-2 through multiple mechanisms of action, such as blocking S / ACE2 interaction and subsequent viral internalization, promoting viral aggregation and / or inducing Fc-dependent antiviral functions.

[0445] Example 3. Diagnostic application of anti-SARS-CoV-2 antibodies

[0446] All 69 unique S protein binders can be evaluated to explore whether they can be used in diagnostic applications to detect serum S protein and / or SARS-CoV-2 virions as individual VHHs or in combination ( Figure 7 ).

[0447] Example 4. Identification of VHHs that bind to different epitopes of the SARS-CoV-2 S1 protein RBD

[0448] Assumptions Figure 4 The synergistic effect shown is caused by the binding of the VHH to different epitopes within the S1 RBD. To test this, a selected number of candidates were subjected to epitope binning assays by biolayer interferometry (Figure 8A) or ELISA (Figure 8B).

[0449] First, in an epitope binning assay (Figure 8A), 2A-Fc was captured using an S1 RBD sensor and subsequently incubated with lead candidates 1B-Fc, 3F-Fc, or 2A-Fc, which were fused to a human IgG1 Fc domain to present Fc effector functions against SARS-CoV-2. This analysis showed that 3F-Fc significantly increased the signal compared to the 2A-Fc control, while 1B-Fc significantly reduced the signal compared to the control. This indicates that 3F-Fc does not compete with 2A-Fc and that it is likely to bind to a different S1 RBD epitope. In contrast, 1B-Fc competes with 2A-Fc, indicating that it competes for binding to the same S1 RBD epitope (Figure 8A). These results confirm the hypothesis and indicate that the VHH blocking S / ACE2 binds to at least two separate unique epitopes within the S1 RBD.

[0450] Next, an ELISA-based epitope binning assay was performed to evaluate five additional VHHs (1C, 1F, 3A, 4F, and G4) that were not fused to Fc, but were previously evaluated to block SARS-CoV-2S / ACE2 interactions. The evaluation of more VHHs classified several other VHHs into binding groups, which helps in multispecific antibody design and construction. In this ELISA, wells were coated with SARS-CoV-2S1 and incubated with bispecific VHH-Fc 1B-2A (based on previous data, 1B and 2A may bind to the same epitope) or monoclonal VHH-Fc 3F-Fc (based on previous data, this binds to an epitope different from 1B or 2A), pre-mixed with VHH candidates. The resulting relative fluorescence signal obtained for each sample (relative to the VHH-Fc signal alone) was calculated to reflect the percentage difference from the 1C, 1F, 3A, 4F or G4 signal when VHH was combined with 1B-2A-Fc or 3F-Fc (Figure 8B). The results show that VHH-Fc 1C, 1F and 4F are almost 100% different from 1B-2A-Fc, indicating that they compete for the same epitope. However, G4 may compete with the 1B-2A-Fc portion, while 3A is unlikely to compete for the same epitope. In addition, these results indicate that 3A can compete with the 3F portion (Figure 8B). In summary, the 8 VHH blockers of S / ACE2 interaction can be divided into 2 major categories based on their epitope binding; Group 1 consists of 6 VHHs, while Group 2 consists of 2 VHHs (Figure 8C).

[0451] In the example, 3F, 1B, 2A, G4, 4F, 1F, 1C and 3A correspond to Fig. 22 Covid19-3F2, Covid19-1B6, Covid19-E2A6, Covid19-S1G4, 4F12, 1F12, 1C11 and 3A4.

[0452] Example 5. Elucidation of the epitope on S1 RBD that binds to VHH-Fc

[0453] To elucidate the structural basis of the newly discovered epitope binding group, Schrodinger The software computationally generates structural models of 1B, 3F and 2A VHHs and docks them with the SARS-CoV-2S1RBD structure derived from PDB 6MOJ. Figure 9A shows the ACE2 binding residues of the SARS-CoV-2S1 protein (under the arrow symbol). This method produces a series of poses for each S1 RBD / VHH complex structure, which allows further analysis of the interface of these poses with good PIPER cluster size. Five regions in the RBD were identified that may interact with VHH 1B, 2A and 3F, respectively ( Figures 9A-9B ). Next, 5 different S1 RBD deletion mutants were generated to validate the computationally mapped epitopes in vitro and thus select the best docking model for molecular analysis. In fact, these S1RBD deletion regions have been shown to block the S1 RBD / ACE2 interaction (Table 1).

[0454] Table 1S1 List of published antibodies that have RBD deletions and utilize the deleted region to block S / ACE2 interaction

[0455] SARS-CoV-2 S1 RBD deletion Using known antibodies to the deleted region S1 RBD del 1 S309, BD-23 S1 RBD del 2 CB6 S1 RBD del 3 B38, CB6, P2B-2F6 S1 RBD del 4 B38、CB6 P2B-2F6 S1 RBD del 5 B38、CB6

[0456] The wild type and all S1 deletion mutants were tested to evaluate their binding profiles with selected VHH-Fc and ACE2 from groups 1 and 2. The binding of VHH-Fc candidates from both groups 1 and 2 and ACE2 to S1 RBD was affected after the deletion of region 1 (del1). This result may be caused by conformational changes or reductions in S1 protein expression after the deletion of region 1. Because the missing domain is not part of the S1 RBD / ACE2 interface based on the crystal structure of the RBD / ACE2 complex (PDB 6MOJ). The deletion of region 2 (del2) does not prevent the binding of group 1 VHH-Fc to S1 RBD, and in fact shows a slight increase in binding. In addition, the deletion of region 2 does not prevent the binding of ACE2 to S1 RBD. However, the deletion affects the binding of group 2 VHH-FC to S1RBD, and it is also adjacent to the epitope domain predicted computationally in region 1. The deletion of regions 3, 4, and 5 all reduced the binding of both group 1 and group 2 VHH-Fc to S1 RBD. However, these regions are more critical for group 1 than for group 2 in terms of their binding. In addition, these regions are critical for ACE2 binding to S1 RBD. In summary, the binding epitopes of group 1 are more associated with del3, 4, and 5 regions, while the epitopes of group 2 are located closer to del2. In addition, binding changes were observed in group 1. For example, 2A has a greater reduction in binding to del1, del3, del4, and del5 than 1B. This suggests that the epitopes of 2A and 1B are not the same, even though they compete with each other and were initially characterized as being within the same binding group 1 ( Figures 9C-9D Based on the binding and epitope binning data, a 3D docking model was constructed to predict the interactions between SARS-CoV-2S1RBD, ACE2 and lead VHH-Fc ( Fig.9E ). In summary, this analysis confirmed the existence of two major binding groups (Group 1 and Group 2) and showed the possible binding regions of each VHH on the SARS-CoV-2S1 protein.

[0457] Example 6. Trispecific VHH-Fc shows potent S1 RBD binding activity and S / ACE2 blocking activity

[0458] Next, it was tested whether the combination of a single VHH binding to different S1 RBD epitopes and a bispecific antibody molecule would produce a synergistic effect in SARS-CoV-2 binding and S / ACE2 blocking. As expected, the resulting bispecific VHH-Fc 1B-3F showed strong binding to S1 RBD and S / ACE2 blocking compared to the single component VHH-Fc. Since the SARS-CoV-2S protein forms a trimer, it was tested whether a trispecific antibody with two binding units from group 1 and another binding unit from group 2 (or vice versa) has better binding and blocking functions than a bispecific antibody. Here, only trispecific VHH-Fc was tested, because any larger multispecific molecule may affect the developability of the Fc fusion protein. A trispecific VHH-Fc was constructed. Computer-assisted antibody design was used, which enabled it to be effectively constructed and optimized. Then, trispecific, bispecific and monospecific VHH-Fc were tested to determine their ability to bind to SARS-CoV-2S1 protein and block S / ACE2 in vitro ( Fig. 10A and Fig. 10D As expected, the multispecific antibodies showed higher binding affinity to the SARS-CoV-2 S1 protein RBD in vitro, with the trispecific VHH-Fc 3F-1B-2A (KD of about 0.047 nM) and 1B-3F-2A (KD of about 0.095 nM) showing more potent binding than the bispecific VHH-Fc 1B-3F ( Figures 10A-10C , 10E). The binding affinity of the trispecific VHH-Fc was higher than the binding affinity of the individual components VHH-Fc 1B, 3F and 2A used in combination, and the binding affinity of 1B-3F-Fc was higher than the binding affinity of the individual components VHH-Fc 1B and 3F used in combination ( Fig. 10A In addition, 3F-1B-2A and 1B-3F-2A showed potent blockade of SARS-CoV-2S / ACE2 interaction, with IC 50 The values ​​were 0.71 nM and 0.74 nM, respectively, and the complete inhibition of both was about 10 nM, which was superior to using the individual components VHH-Fc as a combination (IC 50 In addition, 3F-1B-2A and 1B-3F-2A were more effective than the bispecific VHH-Fc 1B-3F in blocking the SARS-CoV-2S / ACE2 interaction ( Fig. 10D ). Specifically, the trispecific VHH-Fc 2A-1B-3F exhibited lower S / ACE2 blocking ability, indicating that the physical arrangement and / or binding orientation of the VHH in the multispecific antibody is important for its binding and blocking ( Fig. 10D). In summary, this result shows that trispecific VHH-Fc has higher synergistic efficacy in binding and blocking S1 or S1 / ACE2 interaction than bispecific or monospecific antibodies.

[0459] Example 7. Trispecific VHH-Fc has good developability characteristics

[0460] During the computer-aided design process, several developmental enhancements were incorporated into the structure of the VHH-Fc. Therefore, the physicochemical properties of the lead bispecific and trispecific antibodies were analyzed using DLS and DSF / SLS methods to determine whether they had favorable properties for large-scale manufacturing, which is critical for the commercial development of antibodies ( Fig.10E The data showed that the lead trispecific VHH-Fc 3F-1B-2A had a low aggregation potential based on the DLS method and was thermally stable based on the DSF / SLS method ( Fig.10E ).

[0461] Example 8. Trispecific VHH-Fc 3F-1B-2A neutralizes SARS-CoV-2 infection in cells

[0462] Multispecific VHH-Fc was tested to determine its ability to target SARS-CoV-2 in cell biological functional assays. First, the virus neutralization ability of the antibodies was analyzed using pseudoviruses expressing the SARS-CoV-2S1 protein. Trispecific VHH-Fc3F-1B-2A, 1B-3F-2A, and the monospecific combination of VHH (1B-Fc+3F-Fc+2A-Fc) prevented pseudovirus infection of human cells (Figure 11A). Based on the SARS-CoV-2S / ACE2 blocking data, the trispecific VHH-Fc was more effective in neutralizing pseudovirus infection compared to the combination treatment of VHH-Fc 1B, 3F, and 2A, with an IC of 3F-1B-2A. 50 The IC value of 1B-3F-2A is 3.00nM. 50 The value was 6.44 nM, and the IC 50 The value was 29.19 nM (Figure 11A). This pseudovirus data confirmed the synergistic effect of the trispecific antibodies, and the results showed that the trispecific antibodies were effective in preventing SARS-CoV-2 infection.

[0463] Since VHH-Fc contains the Fc domain of human IgG1, it may be able to trigger Fc-dependent functions to clear the virus from the body. To test this, a cell line was used to transiently express the SARS-CoV-2 S1 protein. Then, the ability of the multispecific VHH-Fc to promote antibody-dependent cellular cytotoxicity (ADCC) was evaluated, suggesting that the antibody has Fc-dependent functions. In addition to the lead trispecific VHH-Fc antibody 3F-1B-2A-Fc, another trispecific antibody 3A-3F-2A-Fc ( Figures 13A-13B ). As expected, VHH-Fc was able to induce ADCC in cells (Figure 11B). This suggests that these VHH-Fc can bind to immune cells through their Fc domains and trigger Fc-dependent functions, thereby allowing multiple mechanisms of action against SARS-CoV-2, including binding to SARS-CoV-S1 and blocking S1 / ACE2 interactions.

[0464] In this study, llama-derived multispecific nanobodies were developed and characterized with resulting data strongly indicating their effectiveness against SARS-CoV-2, the virus that causes COVID-19. The COVID-19 pandemic has caused widespread health and social problems worldwide, and therapeutic approaches that can effectively block and prevent SARS-CoV-2 infection are needed. Several monoclonal antibodies against SARS-CoV-2 have been proposed and are being tested as antiviral therapies, either as single agents or in combination; however, this is the first study to introduce and demonstrate the efficacy of a multispecific antibody against SARS-CoV-2.

[0465] In order to successfully design and construct multispecific VHH binders, epitope information for each individual VHH clone is necessary. Here, instead of obtaining the crystal structure of each antigen / antibody complex, a new method was used for epitope identification. Gator was used for epitope binning, and the VHHs that blocked S / ACE2 were classified into 2 groups. The VHHs in each group competed, but not with the VHHs of the other group, strongly indicating that group 1 and group 2 VHHs are two separate binding groups. Then, a computational VHH model was constructed and docked to the S1 RBD structure obtained from the publicly available crystal structure of SARS-COV-2S1 RBD / ACE2, respectively, and the docking structure (with a higher pose cluster size) was used to predict the possible epitopes of individual VHHs. In order to verify the involvement of the predicted epitopes in VHH / S1 RBD binding, the binding ability of each VHH to the wild-type S1 RBD or five deletion mutants with each predicted missing epitope was compared. As Figures 9C-9DAs shown, group 1 VHH may bind to regions del3, del4, and del5, which overlap with the ACE2 binding interface of S1 RBD. However, group 2 VHH may bind tightly to region del2, which does not overlap with the ACE2 binding interface of S1 RBD. Once the region close to del2 is bound by group 2 VHH, the conformation of the entire RBD will be changed, ultimately blocking its ability to bind to ACE2. Currently, many structures of S1 RBD / antibody complexes have been published. Analysis of these structures suggests that there may be 2 major "hot" antibody binding regions in S1 RBD: one may be in the N-terminal region (del1), and the other may be in the ACE2 binding interface (del3, del4, del5). The selected VHH binders in the trispecific antibody may cover both regions ( Figures 12A-12C ). Based on this information, a lead trispecific VHH-Fc format can be defined, including the linker length and the order of the VHH binders.

[0466] Trispecific antibodies are advantageous as therapeutic agents because they simultaneously bind to multiple epitopes within the S1 protein RBD, which increases their antigen-binding affinity and avidity ( Figures 12A-12C ). The VHH 1B and 3F of the bispecific antibody bind to two different epitopes in the RBD of the S1 protein. In the trispecific antibody design, VHH 2A, which has almost the same epitope as 1B, was incorporated. These VHHs can bind to the same or similar epitopes in different orientations, or to the corresponding epitopes in another S1 protein in the trimer, thereby increasing the binding and blocking efficacy of the trispecific VHH-Fc. In fact, this phenomenon has been shown previously in other multispecific antibodies. For example, in clinical development, CD20-TCB (Roche), a T cell engager antibody targeting CD20 with two CD20 binding domains (2:1 molecular format), has enhanced efficacy compared to other bispecific antibodies that bind to CD20 (see Bacac, M. et al. CD20-TCB with Obinutuzumab Pretreatment as Next-Generation Treatment of Hematologic Malignancies. Clin Cancer Res 24, 4785-4797, doi: 10.1158 / 1078-0432. CCR-18-0455 (2018)). Consistent with this hypothesis, the resulting trispecific VHH-Fc showed very potent properties in terms of S binding and S / ACE2 blocking efficacy, which is the best among the currently published anti-SARS-CoV-2 therapeutic antibodies (Table 1).

[0467] Due to these characteristics, trispecific VHH-Fc can be used for therapeutic applications at low concentrations, which will potentially reduce its toxicity to humans. In addition, the strong binding of antibodies to viral particles will minimize the risk of antibody-dependent enhancement (ADE) caused by suboptimal antigen-antibody interactions and promoting enhanced viral infection. The multispecific targeting method also minimizes the loss of antibody binding to viral antigens due to viral mutations. It is known that RNA viruses mutate, and in this sense, coronaviruses may lose binding to antibodies relatively easily due to structural changes in viral components. However, VHH multispecific antibodies will still bind to mutated viruses because other VHHs in the trispecific antibodies will bind to unmutated epitopes of the virus. Another advantage of the VHH multispecific platform is the ability to target multiple viruses. For example, it is possible to adjoin VHHs that bind to other coronaviruses (such as SARS-CoV and MERS-CoV) and construct a pan-coronavirus trispecific VHH-Fc that can effectively prevent and treat a wide spectrum of coronaviruses.

[0468] Multispecific antibody designs link human IgG1 Fc domains to bispecific or trispecific VHHs. Having an Fc domain in the antibody structure confers Fc-dependent cytotoxic functions such as ADCC, complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). In addition to blocking viral entry and possible viral aggregation, these additional Fc-dependent functions will also provide VHH-Fc with multiple mechanisms of action, making it more effective in neutralizing coronaviruses. In fact, the lead trispecific VHH-Fc 3F-1B-2A showed potent neutralization of SARS-CoV-2 pseudovirus infection in human cells.

[0469] One question in the field of antibody therapeutics is whether the use of multispecific single molecules is superior to using combinations of monoclonal antibodies that collectively target the same epitope. Here, it was shown that multispecific antibodies are more effective than combinations of monoclonal antibodies in blocking host-virus interactions. The trispecific VHH-Fc 3F-1B-A2 was more effective in blocking the SARS-CoV-2S / ACE2 interaction than VHH-Fc 3F, 1B, and A2 alone as a combination. Physically combining VHHs may increase the overall association constant (K on value) and reduce the overall dissociation constant (K off value), thereby producing a lower binding constant, thereby increasing the affinity of the antibody for the antigen. This also increases the avidity of the antibody, making it more effective in neutralizing the virus.

[0470] One of the characteristics of successful therapeutic antibodies is their developability characteristics. Especially during epidemics such as COVID-19, when rapid production of large quantities of antibodies is crucial, the developability and manufacturability of antibodies even play a crucial role. The design described in this article has the advantage of using llama VHH nanobodies with high stability. In fact, the biochemical and biophysical properties of multispecific VHH-Fc show that it can be purified in large quantities, has better resistance to aggregation and good thermal stability. In addition, antibodies have high developability because the multispecific design combines individual VHHs into a single molecule rather than a combination, making it easier to manufacture. An alternative strategy to increase the developability of anti-SARS-CoV-2 multispecific antibodies is to combine 4 VHHs to construct a four-specific VHH without adding an IgG Fc domain. Compared with bispecific and trispecific VHH-Fc, these molecules will have the additional advantage of increased affinity and avidity for the SARS-CoV-2S1 protein, even if the Fc effector function is lacking. These tetraspecific antibodies would be ideally suited as antibody prophylactics to prevent SARS-CoV-2 infection in humans, as the tetraspecific antibodies would have increased thermal stability due to the structure of only llama VHHs, easier binding capacity, and the possibility of easy large-scale manufacturing using cost-effective expression systems such as yeast.

[0471] One of the key features of the therapeutic antibodies described herein is the use of computer-aided design, which greatly reduces its development time and enhances its optimization efficiency. For example, starting from this project, it was possible to produce, optimize and test the lead tri-specific VHH-Fc in less than three months. This shows that this strategy is powerful for generating new therapeutic antibodies for time-sensitive unmet needs and can be used for future epidemics that require rapid development of antibody therapeutics.

[0472] Example 9. Trispecific antibody 3F-1B-2A-Fc prevents and treats COVID-19 infection in transgenic mice expressing human ACE2

[0473] like Fig.14As shown, NM-KI-200272CAG-human ACE2-IRES-luciferase-WPRE-polyA mice (Shanghai Southern Model Biological Technology Development Co., Ltd. (Shanghai Model Organisms)) were inoculated with 1000 PFU SARS-CoV-2. The mice were then randomly divided into a control group (G1) and three treatment groups (G2-G4). All mice were inoculated with SARS-CoV-2 on day 0 (inoculation day), and the experiment was terminated on day 4 (4 days after inoculation, or the end of the study). Specifically, in the control group G1, 5 mice were inoculated with SARS-CoV-2 on day 0 without antibody administration; in the treatment group G2, 4 mice were treated by intranasal (IN) administration of 25 mg / kg trispecific antibody 3F-1B-2A-Fc 10 hours before inoculation (-10 hpi) and then inoculated with SARS-CoV-2 on day 0; in the treatment group G3, 6 mice were inoculated with SARS-CoV-2 on day 0 and then treated by intranasal (IN) administration of 25 mg / kg 3F-1B-2A-Fc 2 hours after infection (+2 hpi); in the treatment group G4, 6 mice were inoculated with SARS-CoV-2 on day 0 and then treated by intraperitoneal (IP) administration of 10 mg / kg 3F-1B-2A-Fc 2 hours after infection (+2 hpi).

[0474] On day 3 (3 days after inoculation), SARS-CoV-2 virus titers in lungs collected from mice were determined by RT-PCR. Fig.15 As shown, compared with the control group (G1), the two treatment strategies including the administration route (e.g., IN and IP) used in the G2-G4 groups all showed the efficacy of reducing the viral titer. Specifically, the G2 group mice showed a significant reduction in viral titer, wherein the viral titer level was undetectable in 3 of the 4 mice.

[0475] From day 0 to day 3, the body weight of mice in each group was measured daily, and Fig.16 The results are shown in . During the measurement period, the body weight and survival rate of the treated group mice (G2-G4) were comparable to those of the control group mice (G1), indicating that the trispecific antibody 3F-1B-2A-Fc has good tolerability and safety for the treatment of COVID-19.

[0476] Example 10. Trispecific antibody 3F-1B-2A-Fc maintains potent binding and ACE2 blocking function against SARS-CoV-2 S protein mutants

[0477] To investigate whether the trispecific antibody 3F-1B-2A-Fc can maintain binding and ACE2 blocking functions against SARS-CoV-2 S protein mutants, S protein mutants with three mutations (i.e., K417N, E484K, and N501Y) in the receptor binding domain (RBD) were purified. S protein mutants are referred to as "tri-mut" or "RBD tri-mut". Figures 17A-17B As shown, the binding affinity of the trispecific antibody 3F-1B-2A-Fc to RBD tri-mut (KD of about 0.249 nM) was reduced compared to the S protein with wild-type RBD (KD of about 0.0446 nM). Fig.18 The results shown show that 3F-1B-2A-Fc exhibited a blocking effect on the RBD tri-mut / ACE2 interaction comparable to that of the S protein (with wild-type RBD) / ACE2 interaction. In addition, the results also showed that the blocking effect of the trispecific antibody 3F-1B-2A-Fc on the RBD tri-mut / ACE2 interaction was more potent than the blocking effect of the combination of VHH-Fc 3F, 1B and 2A alone.

[0478] Other S protein mutants, such as D614G and N501Y, were also tested, and 3F-1B-2A-Fc showed similar binding affinity to the mutant S proteins compared to the wild-type S protein.

[0479] Example 11. Thermal stability test of trispecific antibody 3F-1B-2A-Fc

[0480] The thermal stability of 3F-1B-2A-Fc was tested. Specifically, the antibody was kept at 45°C for 2 weeks, and then its binding to the SARS-CoV-2 S protein was determined by biolayer interferometry (BLI). Fig.19 BLI data are shown in Figure 2. Binding kinetic parameter K on , K off and KD were calculated to be 1.82×10 5 / Ms, 9.90×10 -5 s -1 and 5.43×10 -10 M.

[0481] In addition, the physicochemical properties of 3F-1B-2A-Fc were analyzed using DLS and DSF / SLS methods after the antibody was kept at 45°C for 2 or 3 weeks. Fig. 20 The results are shown in . The results show that the heated 3F-1B-2A-Fc antibody has acceptable Tm and Tagg values ​​compared to those determined using the unheated antibody (see Fig.10E ).

[0482] The above results show that 3F-1B-2A-Fc maintains strong binding to SARS-CoV-2S protein after heating at 45°C for 2-3 weeks. The heated protein has low aggregation potential based on the DLS method and is thermally stable based on the DSF / SLS method. The great thermal stability allows room temperature shipping and distribution.

[0483] Other embodiments

[0484] It should be understood that although the invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. An antibody or an antigen-binding fragment thereof that binds to the S protein of coronavirus SARS-CoV-2, the antibody or the antigen-binding fragment thereof comprising: a heavy chain single variable domain (VHH), said VHH comprising complementarity determining regions (CDRs) 1, 2 and 3; The VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 190, 191 and 192, respectively.

2. An antibody or an antigen-binding fragment thereof that binds to the S protein of the coronavirus SARS-CoV-2, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH), wherein the VHH sequence is shown by SEQ ID NO:

325.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the antibody or antigen-binding fragment comprises human IgG Fc.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the antibody or antigen-binding fragment comprises three heavy chain single variable domains VHH1, VHH2 and VHH3; The three heavy chain single variable domains VHH1, VHH2 and VHH3 complementarity determining regions (CDRs) 1, 2 and 3 are shown below: The VHH1 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 199, 200 and 201, respectively; The VHH2 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 190, 191 and 192, respectively; The VHH3 CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4, 5 and 6, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the VHH1 sequence is represented by SEQ ID NO: 328; The VHH2 sequence is shown by SEQ ID NO: 325; The VHH3 sequence is shown by SEQ ID NO:

263.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody structure is VHH1-VHH2-VHH3-IgG Fc.

8. A nucleic acid comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. The nucleic acid of claim 8, wherein the nucleic acid is cDNA.

10. A vector comprising the nucleic acid according to claim 8. A cell comprising the vector according to claim 10.

12. The cell according to claim 11, wherein the cell is a CHO cell.

13. A cell comprising the nucleic acid according to claim 8.

14. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising: (a) culturing the cell according to claim 11 under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; as well as (b) collecting the antibody or the antigen-binding fragment produced by the cell.

15. Use of a composition for the preparation of a medicament for treating a subject suffering from a coronavirus-related disease, wherein the composition comprises an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the coronavirus is SARS-CoV-2.

16. Use of a composition in the preparation of a medicament for preventing or treating coronavirus infection, wherein the composition comprises an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the coronavirus is SARS-CoV-2.

17. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in the preparation of a kit for identifying a subject suffering from a coronavirus disease, wherein the coronavirus is SARS-CoV-2.

18. The use according to claim 17, wherein the sample required for identification is a blood sample, a saliva sample, or a stool sample from the respiratory tract of the subject.

19. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

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