Antigen-binding molecules targeting SARS-COV-2

Abstract

The present disclosure provides, in various embodiments, polypeptides (e.g., antibodies and antigen-binding fragments thereof) that specifically bind to the S2 domain of a betacoronavirus spike glycoprotein, such as the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disclosure also provides, in various embodiments, fusion proteins comprising one or more of the polypeptides, polynucleotides encoding the polypeptides, vectors and host cells suitable for expressing the polypeptides, and methods for treating viral infections (e.g., COVID-19).

Description

[Technical Field]

[0001] Related Applications This application is a continuation of U.S. Provisional Application No. 63 / 364,331 filed May 6, 2022, U.S. Provisional Application No. 63 / 364,328 filed May 6, 2022, U.S. Provisional Application No. 63 / 381,131 filed October 26, 2022, U.S. Provisional Application No. 63 / 381,132 filed October 26, 2022, U.S. Provisional Application No. 63 / 424,945 filed November 13, 2022, and U.S. Provisional Application No. 63 / 424,945 filed November 13, 2022. This application claims the benefit of U.S. Provisional Application No. 63 / 383,695, filed November 14, 2022, U.S. Provisional Application No. 63 / 385,957, filed December 2, 2022, U.S. Provisional Application No. 63 / 478,650, filed January 5, 2023, U.S. Provisional Application No. 63 / 480,903, filed January 20, 2023, and U.S. Provisional Application No. 63 / 492,206, filed March 24, 2023. The entire teachings of the above applications are incorporated herein by reference.

[0002] Including material by reference in XML This application incorporates by reference the sequence listing contained in the following extensible markup language (XML) file filed concurrently herewith: a) File name: 57081065006.xml, created on May 4, 2023, size 306,430 bytes. [Background technology]

[0003] The novel coronavirus SARS-coronavirus 2 (SARS-CoV-2) first caused a cluster of pneumonia cases (COVID-19) in Wuhan, China. As of March 1, 2020, 79,968 patients in China had tested positive for COVID-19, and 2,873 had died, corresponding to a mortality rate of 3.6% (95% CI 3.5-3.7) (Baud et al., "Real estimates of mortality following COVID-19 infection," Lancet Infect Dis. 20(7):773 (2020)). However, this figure may underestimate the potential threat of COVID-19 among symptomatic patients (ibid.).

[0004] COVID-19 has rapidly spread worldwide, resulting in a pandemic. The World Health Organization's (WHO) Situation Report on Coronavirus Disease 2019 (COVID-19) released on April 21, 2020, reported 2,397,216 confirmed cases and 162,956 deaths. Of these, 83,006 new cases and 5,109 deaths were added within the past 24 hours. Preventing the spread of the disease and providing supportive care for those who become ill relies on quarantine, isolation, and infection control measures (Baden & Rubin, Covid-19—The Search for Effective Therapy, N Engl J Med. 382(19):1851-52 (2020)). Despite the development and use of vaccines and therapeutics, SARS-CoV2 outbreaks continue, and SARS-CoV2 variants continue to evolve and evade these prevention and treatment strategies. Thus, there is a need for additional therapeutics that can be rapidly deployed, preferably those that combat escape variants, e.g., through broad neutralizing activity, and retain therapeutic efficacy. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Baud et al.Real estimates of mortality following COVID-19 infection,Lancet Infect Dis.20(7):773(2020) [Non-patent document 2] Baden & Rubin, Covid-19-The Search for Effective Therapy,N Engl J Med.382(19):1851-52(2020) Summary of the Invention [Means for solving the problem]

[0006] There is a great need to develop specific antiviral therapeutics to prevent the transmission of COVID-19 and treat COVID-19 patients, preferably those that retain activity against new and emerging variants with broad neutralizing activity. The present disclosure provides such therapeutics.

[0007] The disclosure provided herein is based in part on the discovery that the polypeptides disclosed herein specifically bind to the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-spike). The disclosure provided herein is based in part on the discovery that the polypeptides disclosed herein exhibit robust neutralizing activity against SARS-CoV-2 variants in vitro and in vivo. Accordingly, the present disclosure generally relates to compositions (e.g., polypeptides, pharmaceutical compositions) and methods useful for reducing viral entry into cells mediated by the spike (e.g., SARS-CoV-2-spike).

[0008] In particular, provided herein are polypeptides (e.g., antibodies and antigen-binding fragments thereof) that specifically bind to an epitope in the S2 domain of a betacoronavirus spike glycoprotein (e.g., an epitope in the S2 domain of the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)). In some embodiments, the polypeptides have one or more properties selected from broad neutralizing activity against multiple known and predicted betacoronaviruses (e.g., past, present, emerging, and future betacoronaviruses) and binding affinity for an epitope in the S2 domain that is highly conserved across multiple betacoronaviruses. In some embodiments, the polypeptides have broad neutralizing activity against multiple known and predicted betacoronaviruses and binding affinity for an epitope in the S2 domain that is highly conserved across multiple betacoronaviruses.

[0009] The present disclosure provides, inter alia, a polypeptide that specifically binds to SARS-CoV-2-spike, the polypeptide comprising a V selected from: H / V L containing a paratope substantially similar to the paratope of an antibody containing pair: SEQ ID NO: 4 and SEQ ID NO: 51 (AB-1), SEQ ID NO: 5 and SEQ ID NO: 52 (AB-2), SEQ ID NO: 6 and SEQ ID NO: 53 (AB-3), SEQ ID NO: 7 and SEQ ID NO: 54 (AB-4), SEQ ID NO: 8 and SEQ ID NO: 51 (AB-5), SEQ ID NO: 9 and SEQ ID NO: 55 (AB-6), SEQ ID NO: 10 and SEQ ID NO: 56 (AB-7), SEQ ID NO: 11 and SEQ ID NO: 57 (AB-8), SEQ ID NO: 12 and SEQ ID NO: 58 (AB-9), SEQ ID NO: 13 and SEQ ID NO: 59 (AB-10), SEQ ID NO: 14 and SEQ ID NO: 60 (AB-11), SEQ ID NO: 15 and SEQ ID NO: 56 (AB-12), SEQ ID NO: 16 and SEQ ID NO: 51 (AB-13), SEQ ID NO: 10 and SEQ ID NO: 50 (AB-14), SEQ ID NO: 17 and SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 and SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 and SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 and SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 and SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 and SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 and SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 and SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 and SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 and SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 and SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 and SEQ ID NO: 51 (AB-26), SEQ ID NO: 26 and SEQ ID NO: 56 (AB-27), SEQ ID NO: 27 and SEQ ID NO: 61 (AB-28), SEQ ID NO: 28 and SEQ ID NO: 56 (AB-29), SEQ ID NO: 28 and SEQ ID NO: 69 (AB-30), SEQ ID NO: 29 and SEQ ID NO: 70 (AB-31), SEQ ID NO: 30 and SEQ ID NO: 71 (AB-32), SEQ ID NO: 31 and SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 and SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 and SEQ ID NO: 56 (AB-35), SEQ ID NO: 34 and SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 and SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 and SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 and SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 and SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 and SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 and SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 and SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 and SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 and SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 and SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 and SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 and SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 and SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 and SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 and SEQ ID NO: 56 (AB-51), or Any combination of the above.

[0010] The present disclosure also provides, inter alia, a polypeptide that specifically binds to SARS-CoV-2-spike, the polypeptide comprising: An immunoglobulin heavy chain variable domain (V) comprising heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and / or heavy chain complementarity determining region 3 (HCDR3), each substantially similar to HCDR1, HCDR2, and HCDR3 of any one of the amino acid sequences of SEQ ID NOs: 4 to 48. H ) an amino acid sequence; An immunoglobulin light chain variable domain (V) comprising LCDR1, LCDR2, and LCDR3, each of which is substantially similar to the light chain complementarity determining region 1 (LCDR1), the light chain complementarity determining region 2 (LCDR2), and / or the light chain complementarity determining region 3 (LCDR3) of any one of the amino acid sequences of SEQ ID NOs: 51 to 76. L ) amino acid sequence.

[0011] In some embodiments, the polypeptides disclosed herein comprise the HCDR1, HCDR2, and / or HCDR3, and / or LCDR1, LCDR2, and / or LCDR3 of an antibody comprising an amino acid sequence selected from the following: SEQ ID NO: 4 and SEQ ID NO: 51 (AB-1), SEQ ID NO: 5 and SEQ ID NO: 52 (AB-2), SEQ ID NO: 6 and SEQ ID NO: 53 (AB-3), SEQ ID NO: 7 and SEQ ID NO: 54 (AB-4), SEQ ID NO: 8 and SEQ ID NO: 51 (AB-5), SEQ ID NO: 9 and SEQ ID NO: 55 (AB-6), SEQ ID NO: 10 and SEQ ID NO: 56 (AB-7), SEQ ID NO: 11 and SEQ ID NO: 57 (AB-8), SEQ ID NO: 12 and SEQ ID NO: 58 (AB-9), SEQ ID NO: 13 and SEQ ID NO: 59 (AB-10), SEQ ID NO: 14 and SEQ ID NO: 60 (AB-11), SEQ ID NO: 15 and SEQ ID NO: 56 (AB-12), SEQ ID NO: 16 and SEQ ID NO: 51 (AB-13), SEQ ID NO: 10 and SEQ ID NO: 50 (AB-14), SEQ ID NO: 17 and SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 and SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 and SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 and SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 and SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 and SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 and SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 and SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 and SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 and SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 and SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 and SEQ ID NO: 51 (AB-26), SEQ ID NO: 26 and SEQ ID NO: 56 (AB-27), SEQ ID NO: 27 and SEQ ID NO: 61 (AB-28), SEQ ID NO: 28 and SEQ ID NO: 56 (AB-29), SEQ ID NO: 28 and SEQ ID NO: 69 (AB-30), SEQ ID NO: 29 and SEQ ID NO: 70 (AB-31), SEQ ID NO: 30 and SEQ ID NO: 71 (AB-32), SEQ ID NO: 31 and SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 and SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 and SEQ ID NO: 56 (AB-35), SEQ ID NO: 34 and SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 and SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 and SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 and SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 and SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 and SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 and SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 and SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 and SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 and SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 and SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 and SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 and SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 and SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 and SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 and SEQ ID NO: 56 (AB-51).

[0012] In some embodiments, the polypeptides disclosed herein comprise a paratope that is identical to the paratope of an antibody comprising an amino acid sequence selected from the following: SEQ ID NO: 4 and SEQ ID NO: 51 (AB-1), SEQ ID NO: 5 and SEQ ID NO: 52 (AB-2), SEQ ID NO: 6 and SEQ ID NO: 53 (AB-3), SEQ ID NO: 7 and SEQ ID NO: 54 (AB-4), SEQ ID NO: 8 and SEQ ID NO: 51 (AB-5), SEQ ID NO: 9 and SEQ ID NO: 55 (AB-6), SEQ ID NO: 10 and SEQ ID NO: 56 (AB-7), SEQ ID NO: 11 and SEQ ID NO: 57 (AB-8), SEQ ID NO: 12 and SEQ ID NO: 58 (AB-9), SEQ ID NO: 13 and SEQ ID NO: 59 (AB-10), SEQ ID NO: 14 and SEQ ID NO: 60 (AB-11), SEQ ID NO: 15 and SEQ ID NO: 56 (AB-12), SEQ ID NO: 16 and SEQ ID NO: 51 (AB-13), SEQ ID NO: 10 and SEQ ID NO: 50 (AB-14), SEQ ID NO: 17 and SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 and SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 and SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 and SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 and SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 and SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 and SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 and SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 and SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 and SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 and SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 and SEQ ID NO: 51 (AB-26), SEQ ID NO: 26 and SEQ ID NO: 56 (AB-27), SEQ ID NO: 27 and SEQ ID NO: 61 (AB-28), SEQ ID NO: 28 and SEQ ID NO: 56 (AB-29), SEQ ID NO: 28 and SEQ ID NO: 69 (AB-30), SEQ ID NO: 29 and SEQ ID NO: 70 (AB-31), SEQ ID NO: 30 and SEQ ID NO: 71 (AB-32), SEQ ID NO: 31 and SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 and SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 and SEQ ID NO: 56 (AB-35), SEQ ID NO: 34 and SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 and SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 and SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 and SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 and SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 and SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 and SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 and SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 and SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 and SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 and SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 and SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 and SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 and SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 and SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 and SEQ ID NO: 56 (AB-51).

[0013] The present disclosure relates to, inter alia, V comprising SEQ ID NO:2 H Further provided is a polypeptide comprising: X1 is not S, X2 is not D, X3 is not T, X4 is not L, The X5 is not an S. The X6 is not an N. The X7 is not a G. X8 is not V, or X9 is not Q, Or any combination of the above.

[0014] In some embodiments, the polypeptides disclosed herein comprise a V comprising SEQ ID NO:49. L Including, X 10 is not Q, X 11 is not G, X 12 is not S, X 13 is not S, X 14 is not N, X 15 is not S, X 16 is not F, or X 17 is not Y, Or any combination of the above.

[0015] In some embodiments, the present disclosure provides a polypeptide that specifically binds to SARS-CoV-2-spike, comprising: V having at least 70% sequence identity to SEQ ID NO:3 H array, V having at least 70% sequence identity to SEQ ID NO: 50 L an array, or and combinations thereof, V H The sequence does not include SEQ ID NO: 3 or V L The sequence does not include SEQ ID NO: 50, or both.

[0016] In some embodiments, the polypeptides disclosed herein are fusion proteins.

[0017] In some embodiments, the present disclosure provides polynucleotides encoding the polypeptides disclosed herein, vectors comprising such polynucleotides, and host cells comprising such polynucleotides and / or vectors.

[0018] In some embodiments, the present disclosure provides methods of treating a patient and / or subject in need thereof (e.g., a subject having a SARS-CoV infection, such as COVID-19) comprising administering to the subject an effective amount (e.g., a therapeutically effective amount) of one or more polypeptides disclosed herein and / or a composition (e.g., a pharmaceutical composition) comprising one or more polypeptides disclosed herein.

[0019] In some embodiments, the present disclosure provides a method of neutralizing a SARS-CoV-2 variant in a cell (e.g., a cell of a subject), comprising contacting the cell with an effective amount of a composition comprising a polypeptide disclosed herein and / or a composition (e.g., a pharmaceutical composition) comprising a polypeptide disclosed herein.

[0020] In some embodiments, the present disclosure provides a method of neutralizing a SARS-CoV-2 variant in a subject, the method comprising providing to the subject an effective amount of a composition comprising a polypeptide disclosed herein and / or a composition (e.g., a pharmaceutical composition) comprising a polypeptide disclosed herein.

[0021] The foregoing will become apparent from the following more particular description of exemplary embodiments, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the embodiments.

[0022] In the figures, "ref" refers to the reference antibody. [Brief explanation of the drawings]

[0023] [Figure 1] 1 shows the amino acid sequence of the epitope (bold) within the S2 domain (underlined) of SARS-CoV-2 spike (SEQ ID NO: 1). Epitope residues bound by the reference antibodies disclosed herein are indicated by asterisks. [Figure 2]Alignment of the heavy chain variable domain (VH) amino acid sequences of the reference antibody and AB-1 is shown. The heavy chain complementarity determining region (HCDR) amino acid sequences, as determined by ImMunoGeneTics (IMGT) numbering, are indicated using underlining. Bold indicates variable residues (designated "Xn" throughout this disclosure) in the reference antibody and AB-1 through AB-51. "*" indicates paratope residues. See also the VH consensus sequence in Table 1. The paratope location was defined as the antibody residues in the reference that are within 5 angstroms of the antigen when bound to the S2 domain of the SARS-CoV-2 spike. [Figure 3] Figure 1 shows an alignment of the light chain variable domain (VL) amino acid sequences of the reference antibody and AB-1. The light chain complementarity determining region (LCDR) amino acid sequence, as determined by IMGT numbering, is shown using underlining. Bold indicates variable residues (designated "Xn" throughout this disclosure) in the reference antibody and AB-1 through AB-51. "*" indicates paratope residues. See also the VL consensus sequence in Table 2. The paratope location was defined as the antibody residues in the reference antibody that are within 5 angstroms of the antigen when bound to the S2 domain of the SARS-CoV-2 spike. [Figure 4A] Binding data expressed as a composite score across the indicated viruses is shown. "Design" refers to the test antibody, and "Seed" refers to the test antibody selected for Project Learning. ADG20, REGN10933, and REGN10987 are control antibodies. [Figure 4B] Binding data expressed as a composite score across the indicated viruses is shown. "Design" refers to the test antibody, and "Seed" refers to the test antibody selected for Project Learning. ADG20, REGN10933, and REGN10987 are control antibodies. [Figure 5A] Binding and neutralization data expressed as a composite score across the indicated viruses are shown. [Figure 5B] Binding and neutralization data expressed as a composite score across the indicated viruses are shown. [Figure 6A] Tables summarizing data for screening hits, clinical-stage molecules, and reference antibodies are shown. Composite Neutralization Score: A score that takes into account all neutralization data for each antibody and represents them as a single value. The higher the value, the better the overall neutralization profile for that specific antibody. Composite Binding Score: A score that takes into account all binding data for each antibody and represents them as a single value. The higher the value, the better the overall binding profile for that specific antibody. Neutralized Delta Lentiluminescence 3: Percentage neutralization of Delta pseudovirus by each antibody at 0.16 μg / ml. Neutralized Omicron BA.2 Lentiluminescence 3: Percentage neutralization of Omicron BA.2 pseudovirus by each antibody at 0.16 μg / ml. Pseudovirus neutralization data was obtained as described in Materials and Methods. [Figure 6B] Tables summarizing data for screening hits, clinical-stage molecules, and reference antibodies are shown. Composite Neutralization Score: A score that takes into account all neutralization data for each antibody and represents them as a single value. The higher the value, the better the overall neutralization profile for that specific antibody. Composite Binding Score: A score that takes into account all binding data for each antibody and represents them as a single value. The higher the value, the better the overall binding profile for that specific antibody. Neutralized Delta Lentiluminescence 3: Percentage neutralization of Delta pseudovirus by each antibody at 0.16 μg / ml. Neutralized Omicron BA.2 Lentiluminescence 3: Percentage neutralization of Omicron BA.2 pseudovirus by each antibody at 0.16 μg / ml. Pseudovirus neutralization data was obtained as described in Materials and Methods. [Figure 7A] Neutralization curves of AB-1 and the clinical-stage control antibody sotrovimab against the indicated pseudoviruses are shown. Y-axis, % neutralization. X-axis, antibody concentration in μg / ml. [Figure 7B] Neutralization curves of AB-1 and the clinical-stage control antibody sotrovimab against the indicated pseudoviruses are shown. Y-axis, % neutralization. X-axis, antibody concentration in μg / ml. [Figure 8A]Neutralization curves of AB-41 and the clinical-stage control antibody sotrovimab against the indicated pseudoviruses are shown. Y-axis, % neutralization. X-axis, antibody concentration in μg / ml. [Figure 8B] Neutralization curves of AB-41 and the clinical-stage control antibody sotrovimab against the indicated pseudoviruses are shown. Y-axis, % neutralization. X-axis, antibody concentration in μg / ml. [Figure 9A] Neutralization curves of AB-17, AB-15, AB-1, and reference antibodies from high-throughput production, and the clinical-stage control antibody sotrovimab, against the indicated pseudoviruses are shown. Y-axis, % neutralization. X-axis, antibody concentration in μg / ml. [Figure 9B] Neutralization curves of AB-17, AB-15, AB-1, and reference antibodies from high-throughput production, and the clinical-stage control antibody sotrovimab, against the indicated pseudoviruses are shown. Y-axis, % neutralization. X-axis, antibody concentration in μg / ml. [Figure 10A] The three lead molecules demonstrate robust and comparable neutralization of multiple pseudoviruses. Neutralization experiments were performed with the indicated pseudoviruses and the following antibodies: an isotype control (Synagis), a reference antibody, a clinical-stage control (a human IgG1 molecule expressing the same variable region of sotrovimab, two antibodies comprising Evasheld [AZD1061 and AZD8895]), and lead molecules from large-scale production (AB-1, AB-17, and AB-15). Results are representative of at least eight technical replicates across at least two biological replicates and are shown as the mean ± SEM. [Figure 10B] The three lead molecules demonstrate robust and comparable neutralization of multiple pseudoviruses. Neutralization experiments were performed with the indicated pseudoviruses and the following antibodies: an isotype control (Synagis), a reference antibody, a clinical-stage control (a human IgG1 molecule expressing the same variable region of sotrovimab, two antibodies comprising Evasheld [AZD1061 and AZD8895]), and lead molecules from large-scale production (AB-1, AB-17, and AB-15). Results are representative of at least eight technical replicates across at least two biological replicates and are shown as the mean ± SEM. [Figure 11] 1 is a graph showing that AB-1, AB-17, and AB-15 neutralize live viruses. Neutralization experiments were performed with the indicated live viruses and the following antibodies: isotype control (Synagis), reference antibody, clinical-stage control (a human IgG1 molecule expressing the same variable region of sotrovimab), AB-1, AB-17, and AB-15. Results are representative of three replicates and are shown as the mean ± SEM. [Figure 12] 1 is a chart presenting feasibility study data obtained for AB-1, AB-15, and AB-17. [Figure 13A] 1 is a graph showing improved neutralization profile by combining AB-1 with a clinical-stage Class 3 anti-RBD antibody or Class 4 anti-RBD monoclonal antibody. The combination of AB-1 with a clinical-stage Class 3 anti-RBD antibody (sotrovimanb or bebuterovimab) or a Class 4 anti-RBD monoclonal antibody (anti-RBD4 mAb) shows an enhanced neutralization profile (as measured by area under the curve (AUC) and efficacy (maximal neutralization)) compared to AB-1 alone. [Figure 13B] 1 is a graph showing improved neutralization profile by combining AB-1 with a clinical-stage Class 3 anti-RBD antibody or Class 4 anti-RBD monoclonal antibody. The combination of AB-1 with a clinical-stage Class 3 anti-RBD antibody (sotrovimanb or bebuterovimab) or a Class 4 anti-RBD monoclonal antibody (anti-RBD4 mAb) shows an enhanced neutralization profile (as measured by area under the curve (AUC) and efficacy (maximal neutralization)) compared to AB-1 alone. [Figure 14A] 10 is a graph showing that the combination of a Class 4 anti-RBD monoclonal antibody with AB-1 improves the neutralization profile against Omicron variants. [Figure 14B] 10 is a graph showing that the combination of a Class 4 anti-RBD monoclonal antibody with AB-1 improves the neutralization profile against Omicron variants. [Figure 14C]1 is a chart showing that the combination of a Class 4 anti-RBD monoclonal antibody with AB-1 improves the neutralization profile against Omicron variants. [Figure 15-1] These graphs show that AB-1 protects against SARS-CoV-2 delta infection in hamsters. Two hamster studies involved prophylactic administration (day -1) of AB-1 expressed as human IgG1 (Figures 15A-15D) or hamster IgG2a (Figures 15E-15H) and challenge with SARS-CoV-2 delta (day 0). Both AB-1 formats demonstrate dose-dependent protection against delta-induced weight loss (Figures 15A-15B and 15E-15F), delta-induced lung weight gain (a surrogate of lung inflammation, Figures 15C and 15G), and an effect on viral titers (Figures 15D and 15H). The effect appears more pronounced at day 7 compared to earlier time points and to a slightly lesser extent compared to sotrovimab. 15A and 15E) Hamster weights were recorded up to 7 days post-challenge and expressed as percentage change relative to day 0 (pre-challenge) weight. Isotype control was delivered at 25 mg / kg, and sotrovimab at 5 mg / kg. Results are shown as mean ± SEM. 15B and 15F) Presentation of percentage weight change on day 7. Each point represents an individual hamster, and the horizontal line represents the median. 15C and 15G) Lung weights on day 7. 15D and 15H) Lung virus titers on day 4. Each point represents an individual hamster, and the horizontal line represents the median. N = 12 (days 0-4) or 6 (days 5-7) hamsters per group. Data were analyzed by one-way ANOVA (Dunnett's test) corrected for multiple comparisons. Black asterisks indicate comparisons with hamsters treated with isotype control. **p<0.01. [Figure 15-2] Same as above. [Figure 16]These graphs show that AB-1 protects against SARS-CoV-2 omicron BA.2 infection in hamsters. Hamsters were challenged with SARS-CoV-2 omicron BA.2 (day 0) following prophylactic administration (day -1) of AB-1, expressed as hamster IgG2a. AB-1 demonstrated dose-dependent protection against omicron BA.2-induced weight loss (Figures 16A-16B), dose-dependent protection against omicron BA.2-induced lung weight gain (a surrogate for lung inflammation, Figure 16C), and effects on viral titers (Figure 16D). 16A) Hamster weights were recorded up to 7 days post-challenge and expressed as a percentage change from day 0 (pre-challenge) weight. Isotype control was delivered at 25 mg / kg, and sotrovimab at 5 mg / kg. Results are shown as mean ± SEM. 16B) Presentation of the percentage weight change on day 7. Each point represents an individual hamster, and the horizontal line represents the median. 16C) Lung weight on day 7. 16D) Lung virus titer on day 4. Each point represents an individual hamster, and the horizontal line represents the median. N = 12 (days 0-4) or 6 (days 5-7) hamsters per group. Data were analyzed by one-way ANOVA (Dunnett's test) corrected for multiple comparisons. Black asterisks indicate comparison with hamsters treated with isotype control. **p<0.01. [Figure 17A] 17A-17C are graphs showing that AB-1 combined with Class 4 anti-RBD antibody 3a ("RD Class 4 mAb-3a" or "R-AB-3a") demonstrated improved neutralization potency and efficacy against Omicron BQ.1.1 pseudovirus. Figures 17A-17C show the neutralization profiles. Synagis was used as an isotype control. [Figure 17B] 17A-17C are graphs showing that AB-1 combined with Class 4 anti-RBD antibody 3a ("RD Class 4 mAb-3a" or "R-AB-3a") demonstrated improved neutralization potency and efficacy against Omicron BQ.1.1 pseudovirus. Figures 17A-17C show the neutralization profiles. Synagis was used as an isotype control. [Figure 17C]17A-17C are graphs showing that AB-1 combined with Class 4 anti-RBD antibody 3a ("RD Class 4 mAb-3a" or "R-AB-3a") demonstrated improved neutralization potency and efficacy against Omicron BQ.1.1 pseudovirus. Figures 17A-17C show the neutralization profiles. Synagis was used as an isotype control. [Figure 17D] Figure 17D shows the results of the neutralization profile shown in Figure 17B and reported as efficacy (% neutralization at 18 μg / ml). [Figure 17E] Figure 17E is a chart showing that AB-1 combined with class 4 anti-RBD antibody 3a ("RD class 4 mAb-3a" or "R-AB-3a") demonstrated improved neutralization potency and efficacy against Omicron-BQ.1.1 pseudovirus. Figure 17E reports the results shown in Figure 17B as EC50 (95% CI) and median % neutralization at 18 μg / ml (95% CI) values. Standard deviation (SD) is shown as the error bars in Figure 17B, and standard error of the mean (SEM) is shown as the error bars in Figure 17C. [Figure 18-1] 18A and 18B show the neutralization profiles against live SARS-CoV-2 Delta and BA.5 viruses, respectively. Synagis was used as an isotype control. Standard deviations (SD) are shown as error bars. [Figure 18-2]18A-18B are charts showing that the combination of AB-1 with class 4 anti-RBD antibody 4a ("RD class 4 mAb-4a" or "R-AB-4a") or sotrovimab improves the neutralization profile against Delta and Omicron BA.5 live viruses. Synagis was used as an isotype control. The results shown in Figures 18A-18B are reported as EC50 (95% CI) and median % neutralization at 18 μg / ml (95% CI) values. [Figure 19] 1 is a chart showing the in vitro neutralization potency of AB-1 against multiple variants of SARS-CoV-2. Results were obtained from pseudovirus neutralization assays. [Figure 20] Neutralization IC50 data expressed as log fold change relative to control antibody are shown for BQ.1.1 (x-axis) and XBB.1.5 (y-axis) VSV-dG pseudoviruses. "Design" refers to the test antibody, "Seed" refers to the test antibody selected for Project Learning, and "Benchmark" refers to the control antibody. [Figure 21] Binding of AB-1 and reference antibodies to the SARS-CoV-2 spike S2 ​​peptide by DELFIA. Binding of AB-1, reference antibodies, and isotype controls to the SARS-CoV-2 spike S2 ​​peptide (indicated by "S:" followed by the amino acid position) and an HIV-1 envelope negative control peptide was assessed by DELFIA. Results are expressed as AUC values ​​and shown as bars representing the mean ± standard deviation (representative of two independent experiments, each with three technical replicates). Statistical comparisons between AUC values ​​for AB-1 and the reference antibody were performed using two-way ANOVA corrected for multiple comparisons (** indicates P ≤ 0.01). [Figure 22]Binding of AB-1 and reference antibodies to the SARS-CoV-2 spike S2 ​​peptide by SPR. Binding of AB-1 and reference antibodies to the SARS-CoV-2 spike S2 ​​peptide (indicated by "S:" followed by the amino acid position) and an HIV-1 envelope negative control peptide was assessed by SPR at 25°C. Antibody binding responses were normalized to the capture level of the biotinylated peptide and are reported as normalized binding responses. Results are representative of one experiment with no technical replicates. [Figure 23A] Sensorgram of AB-1 Fab binding to the SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide by SPR. Sensorgram of AB-1 Fab binding to the SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide is shown. The real-time binding sensorgram is shown as the black curve, and the fit generated by globally fitting the data to a 1:1 binding model with mass transport limitations is shown as the dashed curve. Results are representative of one experiment, with no technical replicates. [Figure 23B] Sensorgram of the Fab of a reference antibody binding to the SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide by SPR. Sensorgram of the Fab of a reference antibody binding to the SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide is shown. The real-time binding sensorgram is shown as the black curve, and the fit generated by globally fitting the data to a 1:1 binding model with mass transport limitations is shown as the dashed curve. Results are representative of one experiment, with no technical replicates. [Figure 23C] Sensorgram of AB-1 Fab binding to the SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide by SPR. Sensorgram of AB-1 Fab binding to an HIV-1 envelope negative control peptide by SPR is shown. The real-time binding sensorgram is shown as the black curve, and the fit generated by globally fitting the data to a 1:1 binding model with mass transport limitations is shown as the dashed curve. Results are representative of one experiment with no technical replicates. [Figure 23D] Sensorgram of the Fab of the reference antibody binding to the SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide by SPR. Sensorgram of the Fab of the reference antibody binding to the HIV-1 envelope negative control peptide by SPR is shown. The real-time binding sensorgram is shown as the black curve, and the fit generated by globally fitting the data to a 1:1 binding model with mass transport limitations is shown as the dashed curve. Results are representative of one experiment with no technical replicates. [Figure 24A] The Fab of AB-1 binds to the SARS-CoV-2 spike with an apparent stoichiometry of Fab per spike. Low-pass filtered cryo-EM map of the SARS-CoV-2 BA.1 spike trimer, with the Fab of R-AB-3a binding to the RBD and AB-1 binding to the S2 stem helix. The map shows a side profile and a view from below. Each of the putative Fabs is labeled. [Figure 24B] AB-1 Fabs bind to SARS-CoV-2 spikes with an apparent stoichiometry of Fabs per spike. Density-docked atomic model of the SARS-CoV-2 spike assembly with bound Fabs. Spike trimer: High-resolution structure of the SARS-CoV-2 spike with resolved S2 stem helices. R-AB-3a: Designed model for R-AB-3a Fabs. Bottom: Three AB-1 Fabs docked into a propeller-shaped density around the S2 stem helix. Maps were low-pass filtered as in Figure 24A. [Figure 24C]The Fab of AB-1 binds to SARS-CoV-2 spikes with an apparent stoichiometry of Fab per spike. Published cryo-electron tomography map of SARS-CoV-2 spike trimers in virus-like particles bound to the Fab of a reference antibody. The map was low-pass filtered as in Figure 24A. Two copies of the Fab of the reference antibody are evident in the map. The map was low-pass filtered as in Figure 24A. Two copies of the Fab of the reference antibody are evident in the map. [Figure 25] Observed mutation frequencies in the region of interest. Distribution of observed relative frequencies of mutations falling into "HR1," "HR2," "epitope," and "epitope-adjacent residues" over three different time intervals ending on March 1, 2023: earliest [January 6, 2020 to March 1, 2023], three months [December 1, 2022 to March 1, 2023], and one month [February 1, 2023 to March 1, 2023]. Each data point corresponds to one mutation. Dashed lines indicate two thresholds that may allow for inclusion of a mutation in the reported set: 0.001 for the overall relative frequency and 0.01 for the recent (past 3 months) relative frequency. [Figure 26] Mutations in an epitope. Each panel shows the change in the relative frequency of one mutation over time. All mutations occurring within the epitope that exceed a relative frequency threshold are shown. The x-axis represents the time dimension. Each value along the axis is the end date of a lookback period. For example, "2023-01" represents the period ending on January 1, 2023. The y-axis represents the relative frequency of the mutation. Each colored line graph corresponds to a rolling lookback period of a specific length (earliest [starting January 6, 2020], 3 months [starting 3 months before the end date], 1 month [starting 1 month before the end date]). Each point along the line graph represents the relative frequency of the mutation over a specific lookback period ending on the end date. The dashed lines indicate two thresholds that may allow for the inclusion of a mutation in the reported set: 0.001 for the overall relative frequency and 0.01 for the recent (past 3 months) relative frequency. [Figure 27A]Mutations in HR1. All mutations occurring within HR1 that exceed a relative frequency threshold are shown. See brief description of Figure 26 for figure format. [Figure 27B] Mutations in HR1. All mutations occurring within HR1 that exceed a relative frequency threshold are shown. See brief description of Figure 26 for figure format. [Figure 28] Mutations in HR2. All mutations occurring within HR2 that exceed the relative frequency threshold are shown. See brief description of Figure 26 for figure format. [Figure 29] Relative frequency of the most prevalent lineages. Relative frequency of the overall most prevalent lineages occurring in three different time intervals ending on March 1, 2023: earliest [January 6, 2020 to March 1, 2023], three months [December 1, 2022 to March 1, 2023], and one month [February 1, 2023 to March 1, 2023]. The five most prevalent lineages are shown, with all other lineages grouped together as "other." [Figure 30] Relative frequency of targeted mutations in the most prevalent lineages, regardless of epitope on AB-1. Relative frequency of targeted mutations (reported in Table 2) in designated sequences of the top five most prevalent lineages (reported in Figure 5) over different time periods (earliest [January 6, 2020 to March 1, 2023], 3 months [December 1, 2022 to March 1, 2023], 1 month [February 1, 2023 to March 1, 2023]). [Figure 31A] Binding of AB-1 to SARS-CoV-2 and non-SARS-CoV-2 spike trimers by DELFIA. Binding of AB-1 and reference antibodies to spike trimers (indicated by "S:" followed by the respective virus name) representative of the following: Figure 31A, major SARS-CoV-2 variant (SARS-CoV-2). Results are expressed as EC50 values ​​(μg / mL) and shown as means and 95% confidence intervals, representative of two independent experiments, each with four technical replicates. The dashed line represents the upper limit of the 95% confidence interval for AB-1 and reference antibodies binding to the reference trimer (SARS-CoV-2 D614G for Figures 31A and 31B). [Figure 31B] Binding of AB-1 to SARS-CoV-2 and non-SARS-CoV-2 spike trimers by DELFIA. Binding of AB-1 and reference antibodies to spike trimers (indicated by "S:" followed by the respective virus name) representative of the following: Figure 31B, non-SARS-CoV-2 sarbecovirus (Sarbecov.). Results are expressed as EC50 values ​​(μg / mL) and shown as means and 95% confidence intervals, representative of two independent experiments, each with four technical replicates. The dashed line represents the upper limit of the 95% confidence interval for AB-1 and the reference antibody binding to the reference trimer (SARS-CoV-2 D614G for Figures 31A and 31B). [Figure 31C] Binding of AB-1 to SARS-CoV-2 and non-SARS-CoV-2 spike trimers by DELFIA. Binding of AB-1 and reference antibodies to spike trimers (indicated by "S:" followed by the respective virus name) representing: (Figure 31C) SARS-CoV-2 variants with polymorphisms in the AB-1 epitope (SARS-CoV-2 BA.2+P1162L and BA.2+P1162S) and their parental strain (SARS-CoV-2 BA.2(parent)). Results are expressed as EC50 values ​​(μg / mL) and shown as means and 95% confidence intervals, representative of two independent experiments, each with four technical replicates. The dashed line represents the upper limit of the 95% confidence interval for AB-1 and reference antibodies binding to the reference trimer (SARS-CoV-2 BA.2(parent) for Figure 31C). [Figure 32A] Sensorgrams of AB-1 Fab binding to a panel of SARS-CoV-2 spike trimers by SPR at 25°C. Sensorgrams of AB-1 Fab binding to SARS-CoV-2 spike trimers D614G, Delta, BA.4, BA.5, BQ.1.1, XBB.1.5, and MERS spike trimer (negative control) at 25°C from one independent experiment are shown. Real-time binding sensorgrams at different concentrations and fits generated by globally fitting the data to a 1:1 binding model with mass transport limitations are shown. [Figure 32B]Sensorgrams of reference antibody Fabs binding to a panel of SARS-CoV-2 spike trimers by SPR at 25°C. Sensorgrams of reference antibody Fabs binding to SARS-CoV-2 spike trimers D614G, Delta, BA.4, BA.5, BQ.1.1, XBB.1.5, and MERS spike trimer (negative control) at 25°C from one independent experiment are shown. Real-time binding sensorgrams at different concentrations and fits generated by globally fitting the data to a 1:1 binding model with mass transport limitations are shown. [Figure 32C] Sensorgrams of AB-1 Fab binding to a panel of SARS-CoV-2 spike trimers by SPR at 25°C. Sensorgrams of AB-1 Fab binding to SARS-CoV-2 spike trimers BA.2 (parent), BA.2+P1162L, and BA.2+P1162S at 25°C from one independent experiment are shown. Real-time binding sensorgrams at different concentrations and fits generated by globally fitting the data to a 1:1 binding model with mass transport limitations are shown. [Figure 32D] Sensorgrams of reference antibody Fabs binding to a panel of SARS-CoV-2 spike trimers by SPR at 25°C. Sensorgrams of reference antibody Fabs binding to SARS-CoV-2 spike trimers BA.2 (parent), BA.2+P1162L, and BA.2+P1162S at 25°C from one independent experiment are shown. Real-time binding sensorgrams at different concentrations and fits generated by globally fitting the data to a 1:1 binding model with mass transport limitations are shown. [Figure 33A]Sensorgrams of AB-1 Fab binding to a panel of SARS-CoV-2 spike trimers by SPR at 37°C. Sensorgrams of AB-1 Fab binding to SARS-CoV-2 spike trimers D614G, Delta, BA.4, BA.5, BQ.1.1, XBB.1.5, and MERS spike trimer (negative control) at 37°C from one independent experiment are shown. Real-time binding sensorgrams at different concentrations and fits generated by globally fitting the data to a 1:1 binding model with mass transport limitations are shown. [Figure 33B] Sensorgrams of reference antibody Fabs binding to a panel of SARS-CoV-2 spike trimers by SPR at 37°C. Sensorgrams of reference antibody Fabs binding to SARS-CoV-2 spike trimers D614G, Delta, BA.4, BA.5, BQ.1.1, XBB.1.5, and MERS spike trimer (negative control) at 37°C from one independent experiment are shown. Real-time binding sensorgrams at different concentrations are shown, as well as fits generated by globally fitting the data to a 1:1 binding model with mass transport limitations. [Figure 33C] Sensorgrams of AB-1 Fab binding to a panel of SARS-CoV-2 spike trimers by SPR at 37°C. Sensorgrams of AB-1 Fab binding to SARS-CoV-2 spike trimers BA.2 (parent), BA.2+P1162L, and BA.2+P1162S at 37°C from one independent experiment are shown. Real-time binding sensorgrams at different concentrations and fits generated by globally fitting the data to a 1:1 binding model with mass transport limitations are shown. [Figure 33D]Sensorgrams of reference antibody Fab binding to a panel of SARS-CoV-2 spike trimers by SPR at 37°C. Sensorgrams of reference antibody Fab binding to SARS-CoV-2 spike trimers BA.2 (parent), BA.2+P1162L, and BA.2+P1162S at 37°C from one independent experiment are shown. Real-time binding sensorgrams at different concentrations and fits generated by globally fitting the data to a 1:1 binding model with mass transport limitations are shown. [Figure 34] AB-1 neutralization of SARS-CoV-2 variants and non-SARS-CoV-2 sarbecoviruses. Neutralization profiles of AB-1, bebterovimab, and isotype control antibodies against pseudoviruses representing SARS-CoV-2 variants and non-SARS-CoV-2 sarbecoviruses. Results are reported as % neutralization and shown as mean ± standard deviation (representative of 3–6 independent experiments, each with 4 technical replicates). [Figure 35A] Neutralization of SARS-CoV-2 variants by AB-1 in combination with R-AB-2b. Neutralization profiles of AB-1 + R-AB-2b (tested at two different ratios), AB-1, R-AB-2b, and bebuterovimab as single agents, and an isotype control antibody against pseudoviruses representing the indicated SARS-CoV-2 variants using TMPRSS2-Vero E6 cells. Anti-spike antibodies tested as single agents were combined with the same concentration of isotype to control for the total mass of anti-spike antibodies tested in combination. The x-axis indicates the concentration of each individual antibody in the combination, or, if concentrations differ, the concentration of the antibody with the highest concentration. Results are reported as % neutralization and shown as the mean ± standard deviation (representative of three or four independent experiments, each with four technical replicates). [Figure 35B]Neutralization of SARS-CoV-2 variants by AB-1 in combination with R-AB-2b. Neutralization profiles of AB-1 + R-AB-2b (tested at two different ratios), AB-1, R-AB-2b, and bebuterovimab as single agents, and an isotype control antibody were measured against pseudoviruses representing the indicated SARS-CoV-2 variants using Vero E6 cells. Anti-spike antibodies tested as single agents were combined with the same concentration of isotype to control for the total mass of anti-spike antibodies tested in combination. The x-axis indicates the concentration of each individual antibody in the combination, or, if concentrations differ, the concentration of the antibody with the highest concentration. Results are reported as % neutralization and shown as the mean ± standard deviation (representative of three or four independent experiments, each with four technical replicates). [Figure 36] Neutralization of SARS-CoV-2 variants by AB-1 in combination with sotrovimab VH / VL-huIgG1-LS. Neutralization profiles of AB-1 + sotrovimab VH / VL-huIgG1-LS (tested at two different ratios), AB-1, sotrovimab VH / VL-huIgG1-LS, and bebuterovimab as single agents, and an isotype control antibody were measured using Vero E6 cells against pseudoviruses representing the indicated SARS-CoV-2 variants. Anti-spike antibodies tested as single agents were combined with the same concentration of isotype to control for the total mass of anti-spike antibodies tested in combination. The x-axis indicates the concentration of each individual antibody in the combination, or, if concentrations differ, the concentration of the antibody with the highest concentration. Results are expressed as % neutralization and shown as the mean ± standard deviation (representative of three or four independent experiments, each with four technical replicates). [Figure 37] Evaluation of the neutralization profiles of parental and surrogate antibodies. Neutralization profiles of AB-1, sotrovimab VH / VL-huIgG1-LS, and isotype control antibodies, and their respective surrogate human IgG1 and hamster IgG2a molecules, against pseudoviruses representing the ancestral and BA.2 variants of SARS-CoV-2 harboring the D614G mutation. Results are expressed as % neutralization and shown as the mean ± standard deviation (representative of three independent experiments, each with four technical replicates). [Figure 38] Infectivity of SARS-CoV-2 pseudoviruses in Fc gamma receptor-bearing cells in the presence of AB-1. Monocyte cell lines (U-937, THP-1) and human primary PBMCs were infected with SARS-CoV-2 D614G pseudoviruses in the presence of AB-1, AB-1 VH / VL-huIgG1, bebuterovimab, and an isotype control. TMPRSS2-Vero E6 cells were used as a positive control for infection. Results are expressed in relative luminescence units and shown as the mean ± standard deviation (representative of three independent experiments, each with three technical replicates). [Figure 39] Neutralization of SARS-CoV-2 live virus variants by AB-1 in combination with R-AB-2b. Neutralization profiles of AB-1 + R-AB-2b, AB-1, R-AB-2b, and bebuterovimab as single agents, and an isotype control antibody against SARS-CoV-2 ancestral strain (Eng20 (WT)), Delta, BQ.1.1, and XBB.1.1 live viruses. Anti-spike antibodies tested as single agents were combined with the same concentration of isotype to control for the total mass of anti-spike antibodies tested in combination. The x-axis indicates the concentration of each individual antibody in the combination, or, if concentrations differ, the concentration of the antibody with the highest concentration. Results are reported as % neutralization and shown as the mean ± standard deviation (one experiment, representative of 2–3 technical replicates). DETAILED DESCRIPTION OF THE INVENTION

[0024] A description of exemplary embodiments follows.

[0025] Some aspects of the present disclosure are described below with reference to examples for illustrative purposes only. It should be understood that several specific details, relationships, and methods are set forth to provide a thorough understanding of the present disclosure. However, one of ordinary skill in the art will readily recognize that the present disclosure can be practiced without one or more of the specific details, or can be practiced using other methods, protocols, reagents, cell lines, and animals. The present disclosure is not limited to the order of acts or events illustrated, as some acts may occur in a different order and / or concurrently with other acts or events. Furthermore, not all illustrated acts, steps, or events are required to implement a methodology in accordance with the present disclosure.

[0026] Polypeptides that specifically bind to the spike protein of betacoronavirus In particular, provided herein are polypeptides that specifically bind to the S2 domain of the spike glycoprotein of a betacoronavirus. In some embodiments, the polypeptides have one or more properties selected from broad neutralizing activity against multiple known and predicted betacoronaviruses (e.g., past, current, emerging, and future betacoronaviruses), binding affinity for an epitope in the S2 domain that is highly conserved across multiple betacoronaviruses, and inhibitory activity against potentially emerging betacoronavirus escape variants. In some embodiments, the polypeptides specifically bind to the S2 domain of the spike protein of a sarbecovirus (e.g., SARS-CoV-1 virus, SARS-CoV-2 virus). In some embodiments, the polypeptides specifically bind to the S2 domain of the spike protein of a SARS-CoV-1 virus (e.g., multiple SARS-CoV-1 variants). In some embodiments, the polypeptides specifically bind to the S2 domain of the spike protein of a SARS-CoV-2 virus (e.g., multiple SARS-CoV-2 variants). In some embodiments, the polypeptide specifically binds to the S2 domain of the spike protein of the SARS-CoV-1 virus (e.g., multiple SARS-CoV-1 variants) and the S2 domain of the spike protein of the SARS-CoV-2 virus (e.g., multiple SARS-CoV-2 variants).

[0027] In some embodiments, the polypeptides disclosed herein have broad neutralizing activity against multiple betacoronaviruses (e.g., as measured using neutralization assays described herein or otherwise known to one of skill in the art). In some embodiments, the polypeptides have neutralizing activity against multiple sarbecoviruses (e.g., SARS-CoV-1 virus, SARS-CoV-2 virus). In some embodiments, the polypeptides have neutralizing activity against multiple SARS-CoV-1 viruses (e.g., multiple SARS-CoV-1 variants). In some embodiments, the polypeptides have neutralizing activity against multiple SARS-CoV-2 viruses (e.g., multiple SARS-CoV-2 variants). In some embodiments, the polypeptides have neutralizing activity against multiple SARS-CoV-1 viruses (e.g., multiple SARS-CoV-1 variants) and multiple SARS-CoV-2 viruses (e.g., multiple SARS-CoV-2 variants).

[0028] In some embodiments, the polypeptides disclosed herein have binding affinity for an epitope in the S2 domain that is conserved (e.g., highly conserved) across multiple betacoronaviruses. In some embodiments, the epitope in the S2 domain is highly conserved across multiple sarbecoviruses (e.g., SARS-CoV-1 virus, SARS-CoV-2 virus). In some embodiments, the epitope in the S2 domain is highly conserved across multiple SARS-CoV-1 viruses (e.g., multiple SARS-CoV-1 variants). In some embodiments, the epitope in the S2 domain is highly conserved across multiple SARS-CoV-2 viruses (e.g., multiple SARS-CoV-2 variants). In some embodiments, the epitope in the S2 domain is highly conserved across multiple SARS-CoV-1 viruses (e.g., multiple SARS-CoV-1 variants) and multiple SARS-CoV-2 viruses (e.g., multiple SARS-CoV-2 variants).

[0029] SARS-CoV-2 is the causative agent of COVID-19. The SARS-CoV-2 genome encodes 16 nonstructural proteins, as well as a nucleoprotein (N), membrane glycoprotein (M), small envelope glycoprotein (E), and spike protein (S) (Song et al., Cytokine storm induced by SARS-CoV-2, Clin Chim Acta. 509:280-7 (2020)). The SARS-CoV-2 spike, or S of SARS-CoV-2, facilitates the entry of the SARS-CoV-2 virus into host cells, including human host cells. The S is a trimer with a protomer composed of S1 and S2 subunits. S1 contains the receptor-binding domain (RBD) that binds to the ACE2 receptor, and S2 is required for viral fusion with the host membrane.

[0030] A non-limiting example of a wild-type SARS-CoV-2-spike (S) sequence is NCBI RefSeq YP_009724390 (SEQ ID NO: 1).

[0031]

[0032] As used herein, SARS-CoV-2 spike includes the wild-type SARS-CoV-2 spike protein (e.g., SEQ ID NO: 1 (RefSeq YP_009724390) or homologs thereof) and truncated forms thereof, mutants and engineered versions of the full-length and truncated SARS-CoV-2 spike protein, and modified forms (e.g., post-translationally modified forms) of the full-length and truncated SARS-CoV-2 spike protein.

[0033] In some embodiments, the polypeptides disclosed herein bind to the spike protein of SARS-CoV-2 comprising SEQ ID NO:1.

[0034] In some embodiments, the polypeptide binds to a mutant, engineered, and / or modified form of the SARS-CoV-2 spike. In some embodiments, the mutant, engineered, and / or modified form of the SARS-CoV-2 spike comprises an amino acid sequence having at least about 90% sequence identity to the wild-type SARS-CoV-2 spike sequence (e.g., SEQ ID NO: 1), e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the wild-type SARS-CoV-2 spike sequence. In some embodiments, the sequence identity is about 90-99.9%, 90-99.8%, 92-99.8%, 92-99.6%, 94-99.6%, 94-99.5%, 95-99.5%, 95-99.4%, 96-99.4%, 96-99.2%, 97-99.2%, or 97-99%.

[0035] In some embodiments, the mutant, engineered, and / or modified form of the SARS-CoV-2-spike comprises one or more mutations selected from the following relative to SEQ ID NO: 1: L5F, S13I, T19R, A67V, del69, del70, del69-70, D80G, T95I, G142D, del142-144, del144, Y145D, W152C, E 154K, F157S, del211, L212I, ins214EPE, A222V, D253G, G261D, G339D, V367F, S371L, S371L, S373P, S3 75F, K417N, N439K, N440K, G446S, L452R, Y453F, S477N, T478K, E484A, E484K, E484Q, F486L, S494P, Q49 3R, G496S, Q498R, N501T, N501Y, Y505H, T547K, F565L, A570D, H655Y, D614G, Q677H, N679K, P681H, P68 1R, A701V, T716I, N764K, D796Y, T859N, N856K, F888L, D950N, Q954H, Q957R, N969K, L981F, S982A, Q107 1H, V1176F, D1118H, K1191N, or combinations thereof, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or more.

[0036] In some embodiments, mutant, engineered, and / or modified forms of the SARS-CoV-2 spike comprise one or more mutations selected from the following, or a combination thereof, relative to SEQ ID NO: 1. In some embodiments, mutant, engineered, and / or modified forms of the SARS-CoV-2 spike comprise 69del, 70del, 144del, E484K, S494P, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, or K1191N. In some embodiments, the mutant, engineered, and / or modified form of the SARS-CoV-2-spike further comprises E484K, S494P, or K1191N, or a combination thereof.

[0037] In some embodiments, mutant, engineered, and / or modified forms of the SARS-CoV-2 spike comprise one or more mutations selected from the following, or a combination thereof, relative to SEQ ID NO: 1. In some embodiments, mutant, engineered, and / or modified forms of the SARS-CoV-2 spike comprise D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G, and A701V.

[0038] In some embodiments, mutant, engineered, and / or modified forms of the SARS-CoV-2 spike comprise one or more mutations selected from the following, or a combination thereof, relative to SEQ ID NO: 1: T19R, G142D, 156del, 157del, R158G, L452R, T478K, D614G, P681R, or D950N. In some embodiments, mutant, engineered, and / or modified forms of the SARS-CoV-2 spike comprise T19R, 156del, 157del, R158G, L452R, T478K, D614G, P681R, and D950N. In some embodiments, mutant, engineered, and / or modified forms of the SARS-CoV-2 spike further comprise G142D.

[0039] In some embodiments, the modified SARS-CoV-2 spike protein comprises one or more mutations selected from the following with respect to SEQ ID NO: 1: A67V, del69-70, T95I, del142-144, Y145D, del211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, or L981F, or a combination thereof.

[0040] In some embodiments, the modified SARS-CoV-2 spike protein comprises one or more mutations relative to SEQ ID NO: 1 selected from the following: T19I, del24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, or N969K, or a combination thereof.

[0041] In some embodiments, the modified SARS-CoV-2 spike protein comprises one or more mutations relative to SEQ ID NO: 1 selected from the following: T19I, del24-26, A27S, del69-70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, or N969K, or a combination thereof.

[0042] In some embodiments, the mutant, engineered, and / or modified form of the SARS-CoV-2-spike comprises one or more mutations selected from the following relative to SEQ ID NO: 1: 69del, 70del, 144del, A222V, G261D, V367F, K417N, N439K, Y453F, S477N, E484K, F486L, N501T, N501Y, A570D, or D614G, or a combination thereof.

[0043] In some embodiments, the mutant, engineered, and / or modified form of the SARS-CoV-2-spike comprises one or more mutations selected from the following, relative to SEQ ID NO: 1: E484K, N501Y, or D614G, or a combination thereof.

[0044] In some embodiments, the mutant, engineered, and / or modified form of the spike protein of SARS-CoV-2 comprises one or more mutations selected from the following, relative to SEQ ID NO: 1: F817P, A892P, A899P, A942P, K986P, or V987P, or a combination thereof.

[0045] In some embodiments, the mutant, engineered, and / or modified form of the spike protein of SARS-CoV-2 comprises one or more mutations selected from the following, relative to SEQ ID NO: 1: L452R, F486V, or R493Q, or a combination thereof.

[0046] In some embodiments, the mutant, engineered, and / or modified form of the spike protein of SARS-CoV-2 comprises one or more mutations selected from the following relative to SEQ ID NO: 1: A67V, del69-70, T95I, del142-144, Y145D, del211, L212I, ins214EPE, G339D, S371L, S 373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, or L981F, or a combination thereof.

[0047] In some embodiments, the modified SARS-CoV-2 spike protein comprises one or more mutations relative to SEQ ID NO: 1 selected from the following: T19I, del24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, or N969K, or a combination thereof.

[0048] In some embodiments, the modified SARS-CoV-2 spike protein comprises one or more mutations relative to SEQ ID NO: 1 selected from the following: T19I, del24-26, A27S, del69-70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, or N969K, or a combination thereof.

[0049] In some embodiments, the modified SARS-CoV-2 spike protein comprises one or more mutations relative to SEQ ID NO: 1 selected from the following: T19I, del24-26, A27S, del69-70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, or N969K, or a combination thereof.

[0050] In some embodiments, the modified SARS-CoV-2 spike protein comprises one or more mutations relative to SEQ ID NO: 1 selected from the following: T19I, del24-26, A27S, del69-70, G142D, V213G, G339D, R346T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T, L452R, N460K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, or N969K, or a combination thereof.

[0051] Additional modified SARS-CoV-2 spike proteins can be found at https: / / covariants.org / shared-mutations, the contents of which are incorporated herein by reference. Non-limiting examples include alpha, beta, gamma, delta, kappa, epsilon, eta, iota, lambda, mu, and / or omicron, e.g., AY.3, AY.4, AY.41, AY.44, AY.64, AY.103, B.1, B.1.1, B.1.1.1, B.1.1.529, B.1.1.7, B.1.177, B.1.2, B.1.351, B.1.427 / 429, B.1.525, B.1.526, B.1.533, B.1.617.1, B1.617.2, B1.621, BA.1, BA.1.1, BA.1.15, BA.1.17.2, BA.2, BA.2+P1162L, and BA.2+P1162S, BA.2.3.20, BA.2.10, BA.2.12.1, BA.2.75, BA.2.75.2, BA.3, BA.4, BA.4 / 5, BA.4 / 5+K444T, BA.4.6, BA.5, BA.5.2.6, BA.5.8, BF.7, BF.11, BN.1, BQ.1, BQ.1.1, C.37, CH.1.1, CH.1.1.1, D.2, GA.5, GR / 484A, P.1, P.1.17, P.1.10, P.2, P.3, Q.3, Q.4, Q.7, XBB, XBB.1.1, XBB.1.16, XBB.1.5, and / or XBB.1.9.1.

[0052] In some embodiments, the polypeptides disclosed herein bind to the S2 domain of the spike (S) protein of SARS-CoV-2. As used herein, the S2 domain includes the full-length S2 domain (e.g., having the amino acid sequence of SEQ ID NO: 193 or a homolog thereof) and truncated forms thereof, mutants and engineered versions of the full-length and truncated S2 domain (e.g., epitopes within the S2 domain (e.g., S2 (Figure 1)), and modified forms (e.g., post-translationally modified forms) of the full-length and truncated S2 domain.

[0053] PLQPELDSFKEELDKYFKNHTSPDVDL (SEQ ID NO: 193).

[0054] In some embodiments, the polypeptides disclosed herein bind to mutant, engineered, and / or modified forms of the S2 domain. In some embodiments, the mutant, engineered, and / or modified forms of the S2 domain comprise an amino acid sequence having at least about 90% sequence identity to a wild-type full-length S2 domain (e.g., SEQ ID NO: 193), e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity. In some embodiments, the sequence identity is about 90-99.9%, 90-99.8%, 92-99.8%, 92-99.6%, 94-99.6%, 94-99.5%, 95-99.5%, 95-99.4%, 96-99.4%, 96-99.2%, 97-99.2%, or 97-99%.

[0055] In some embodiments, a polypeptide disclosed herein binds to the spike protein of SARS-CoV-2 (e.g., SEQ ID NO:1 or SEQ ID NO:193) and comprises an immunoglobulin light chain variable domain, an immunoglobulin heavy chain variable domain, or an immunoglobulin light chain variable domain and an immunoglobulin heavy chain variable domain, wherein the polypeptide does not comprise SEQ ID NO:3 or SEQ ID NO:50, or both SEQ ID NO:3 and SEQ ID NO:50.

[0056] In some embodiments, the polypeptides disclosed herein comprise the V of SEQ ID NO:3. H Amino acid sequence and V of SEQ ID NO: 50 LThe polypeptides disclosed herein do not contain all six CDRs of the antibody comprising the amino acid sequence. In some embodiments, the polypeptides disclosed herein contain one, two, three, four, or five CDRs selected from SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:90, SEQ ID NO:133, SEQ ID NO:141, and SEQ ID NO:143. In some embodiments, the polypeptides disclosed herein contain one, two, or three CDRs selected from SEQ ID NO:79, SEQ ID NO:90, SEQ ID NO:133, and SEQ ID NO:143.

[0057] In some embodiments, the antibodies disclosed herein comprise the V H Amino acid sequence and V of SEQ ID NO: 50 L The antibodies disclosed herein do not contain all six CDRs of the antibody comprising the amino acid sequence. In some embodiments, the antibodies disclosed herein contain one, two, three, four, or five CDRs selected from SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:90, SEQ ID NO:133, SEQ ID NO:141, and SEQ ID NO:143. In some embodiments, the antibodies disclosed herein contain one, two, or three CDRs selected from SEQ ID NO:79, SEQ ID NO:90, SEQ ID NO:133, and SEQ ID NO:143.

[0058] In some embodiments, the present disclosure provides a polypeptide that specifically binds to the spike protein of SARS-CoV-2, the polypeptide comprising: a) an immunoglobulin heavy chain variable domain (V) comprising an amino acid sequence having at least 55% (e.g., at least 60, 65, 70, 75, 80, 85, 90, 95, 98, or 99%) sequence identity to SEQ ID NO:3; H ), b) an immunoglobulin light chain variable domain (V) comprising an amino acid sequence having at least 55% (e.g., at least 60, 65, 70, 75, 80, 85, 90, 95, 98, or 99%) sequence identity to SEQ ID NO: 50; L ), or It includes both a) and b), The polypeptide is V of SEQ ID NO:3 H Amino acid sequence and V of SEQ ID NO: 50 LIt does not include all six CDRs of an antibody containing amino acid sequence.

[0059] In some embodiments, the polypeptides disclosed herein do not comprise all four of the sequences of SEQ ID NO: 79, SEQ ID NO: 90, SEQ ID NO: 133, and SEQ ID NO: 143. In some embodiments, the polypeptides disclosed herein comprise one, two, or three CDRs selected from SEQ ID NO: 79, SEQ ID NO: 90, SEQ ID NO: 133, and SEQ ID NO: 143.

[0060] In some embodiments, the polypeptides disclosed herein bind to the spike protein of wild-type SARS-CoV-2 (e.g., SEQ ID NO: 1). In some embodiments, the polypeptides disclosed herein bind to one or more epitope residues of the spike protein of wild-type SARS-CoV-2 (e.g., one or more epitope residues within the S2 subunit of SARS-CoV-2).

[0061] As used herein, the term "comparator" or "comparator polypeptide" refers to a polypeptide (e.g., an immunoglobulin molecule) that specifically binds to SARS-CoV-2 and is not a polypeptide disclosed herein. The sequences of a comparator polypeptide and a polypeptide disclosed herein can be compared to illustrate structural differences therebetween (e.g., differences at one or more amino acid positions, such as amino acid substitutions). A polypeptide disclosed herein has more than a non-substantial difference (e.g., one or more substantial differences) compared to a comparator polypeptide, such that the polypeptide disclosed herein exhibits one or more (i.e., one, two, or all three) of a different function, a different manner, or achieves a different result compared to a comparator polypeptide under controlled conditions. A comparator polypeptide differs from a polypeptide disclosed herein by one or more amino acids, e.g., in some embodiments, by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, a comparator polypeptide differs from a polypeptide provided by the present disclosure by at least about 0.4, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55% or more amino acid identity.

[0062] In some embodiments, the comparator polypeptide comprises a V polypeptide comprising the amino acid sequence of SEQ ID NO:3. H Domain V comprising the amino acid sequence of SEQ ID NO: 50 LThe antibody referred to herein as the "reference antibody" comprises a domain, a heavy chain comprising the amino acid sequence of SEQ ID NO: 191, and a light chain comprising the amino acid sequence of SEQ ID NO: 192. The reference antibody is an antibody that binds to S2 of SARS-CoV-2 and neutralizes SARS-CoV-2 variants. For additional information regarding reference antibodies, see, e.g., PDB:7NAB_A, PDB:7NAB_B, 7NAB_C, and Jennewein et al., "Isolation and characterization of cross-neutralizing coronavirus antibodies from COVID-19." + See, Cell Rep. 36(2):109353 (2021). The reference antibody has the following heavy and light chain amino acid sequences:

[0063] EVQLVESGAEVKKPGESLKISCKGSGYTFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGHVTISADKSISTAYLQWNSLKASDTAMYYCARLPQYCSNGVCQRWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS (SEQ ID NO: 191)

[0064] EIVLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNSFPYTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE (SEQ ID NO: 192)

[0065] As used herein, the term "sequence identity" refers to the degree, expressed as a percentage, that two nucleotide sequences or two amino acid sequences have the same residues at the same positions when the sequences are aligned to achieve the maximum level of identity. For sequence alignment and comparison, typically, one sequence is designated as a reference sequence to which a test sequence is compared. Sequence identity is expressed as the percentage of positions over the entire length of the reference sequence that share the same nucleotide or amino acid when the reference and test sequences are aligned to achieve the maximum level of identity. As an example, if the test sequence has the same nucleotide or amino acid residue at 70% of the same positions over the entire length of the reference sequence when aligned to achieve the maximum level of identity, the two sequences are considered to have 70% sequence identity.

[0066] Alignment of sequences for comparison to achieve the maximum level of identity can be readily performed by one of skill in the art using an appropriate alignment method or algorithm, in some instances, the alignment may include gaps introduced to achieve the maximum level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see, e.g., generally, Ausubel et al., Current Protocols in Molecular Biology).

[0067] When using sequence comparison algorithm, test and reference sequences are input into computer, and if necessary, coordinates are subsequently designated, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence (s) to reference sequence based on designated program parameters.The tool commonly used to determine percent sequence identity is the Protein Basic Local Alignment Search Tool (BLASTP), available from the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health (USA) (Altschul et al., 1990).

[0068] The terms "polypeptide," "peptide," or "protein" refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Proteins, peptides, or polypeptides can contain any suitable L- and / or D-amino acids, including common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statins), and unusual amino acids (e.g., citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). Amino, carboxyl, and / or other functional groups on the peptide can be free (e.g., unmodified) or protected with suitable protecting groups. Suitable protecting groups for amino and carboxyl groups, as well as methods for adding or removing protecting groups, are known in the art and are disclosed, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis," John Wiley and Sons, 1991. Functional groups of proteins, peptides, or polypeptides can be derivatized (e.g., alkylated) or labeled (e.g., with a detectable label such as a fluorogen or hapten) using methods known in the art. Proteins, peptides, or polypeptides can contain one or more modifications, if desired (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifications (e.g., cyclization modifications), N-methyl-α-amino group substitutions). In addition, proteins, peptides, or polypeptides can be analogs of known and / or naturally occurring peptides, e.g., peptide analogs having conservative amino acid residue substitution(s).

[0069] In some embodiments, the present disclosure provides a polypeptide that specifically binds to SARS-CoV-2-spike, wherein the polypeptide comprises a V selected from: H / V L containing a paratope substantially similar to the paratope of an antibody containing pair: SEQ ID NO: 4 and SEQ ID NO: 51 (AB-1), SEQ ID NO: 5 and SEQ ID NO: 52 (AB-2), SEQ ID NO: 6 and SEQ ID NO: 53 (AB-3), SEQ ID NO: 7 and SEQ ID NO: 54 (AB-4), SEQ ID NO: 8 and SEQ ID NO: 51 (AB-5), SEQ ID NO: 9 and SEQ ID NO: 55 (AB-6), SEQ ID NO: 10 and SEQ ID NO: 56 (AB-7), SEQ ID NO: 11 and SEQ ID NO: 57 (AB-8), SEQ ID NO: 12 and SEQ ID NO: 58 (AB-9), SEQ ID NO: 13 and SEQ ID NO: 59 (AB-10), SEQ ID NO: 14 and SEQ ID NO: 60 (AB-11), SEQ ID NO: 15 and SEQ ID NO: 56 (AB-12), SEQ ID NO: 16 and SEQ ID NO: 51 (AB-13), SEQ ID NO: 10 and SEQ ID NO: 50 (AB-14), SEQ ID NO: 17 and SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 and SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 and SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 and SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 and SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 and SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 and SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 and SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 and SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 and SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 and SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 and SEQ ID NO: 51 (AB-26), SEQ ID NO: 26 and SEQ ID NO: 56 (AB-27), SEQ ID NO: 27 and SEQ ID NO: 61 (AB-28), SEQ ID NO: 28 and SEQ ID NO: 56 (AB-29), SEQ ID NO: 28 and SEQ ID NO: 69 (AB-30), SEQ ID NO: 29 and SEQ ID NO: 70 (AB-31), SEQ ID NO: 30 and SEQ ID NO: 71 (AB-32), SEQ ID NO: 31 and SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 and SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 and SEQ ID NO: 56 (AB-35), SEQ ID NO: 34 and SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 and SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 and SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 and SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 and SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 and SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 and SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 and SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 and SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 and SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 and SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 and SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 and SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 and SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 and SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 and SEQ ID NO: 56 (AB-51), or Any combination of them.

[0070] In some embodiments, the present disclosure provides a polypeptide that specifically binds to SARS-CoV-2-spike, wherein the polypeptide comprises a V selected from: H / V L Contains a paratope that is identical to the paratope of the antibody containing the pair: SEQ ID NO: 4 and SEQ ID NO: 51 (AB-1), SEQ ID NO: 5 and SEQ ID NO: 52 (AB-2), SEQ ID NO: 6 and SEQ ID NO: 53 (AB-3), SEQ ID NO: 7 and SEQ ID NO: 54 (AB-4), SEQ ID NO: 8 and SEQ ID NO: 51 (AB-5), SEQ ID NO: 9 and SEQ ID NO: 55 (AB-6), SEQ ID NO: 10 and SEQ ID NO: 56 (AB-7), SEQ ID NO: 11 and SEQ ID NO: 57 (AB-8), SEQ ID NO: 12 and SEQ ID NO: 58 (AB-9), SEQ ID NO: 13 and SEQ ID NO: 59 (AB-10), SEQ ID NO: 14 and SEQ ID NO: 60 (AB-11), SEQ ID NO: 15 and SEQ ID NO: 56 (AB-12), SEQ ID NO: 16 and SEQ ID NO: 51 (AB-13), SEQ ID NO: 10 and SEQ ID NO: 50 (AB-14), SEQ ID NO: 17 and SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 and SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 and SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 and SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 and SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 and SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 and SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 and SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 and SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 and SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 and SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 and SEQ ID NO: 51 (AB-26), SEQ ID NO: 26 and SEQ ID NO: 56 (AB-27), SEQ ID NO: 27 and SEQ ID NO: 61 (AB-28), SEQ ID NO: 28 and SEQ ID NO: 56 (AB-29), SEQ ID NO: 28 and SEQ ID NO: 69 (AB-30), SEQ ID NO: 29 and SEQ ID NO: 70 (AB-31), SEQ ID NO: 30 and SEQ ID NO: 71 (AB-32), SEQ ID NO: 31 and SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 and SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 and SEQ ID NO: 56 (AB-35), SEQ ID NO: 34 and SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 and SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 and SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 and SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 and SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 and SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 and SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 and SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 and SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 and SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 and SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 and SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 and SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 and SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 and SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 and SEQ ID NO: 56 (AB-51).

[0071] See Table 1 for SEQ ID NOs: 4 to 48, Table 2 for SEQ ID NOs: 51 to 76, Figure 2 for the paratope residues of antibodies comprising SEQ ID NOs: 4 to 48, and Figure 3 for the paratope residues of antibodies comprising SEQ ID NOs: 51 to 76.

[0072] The amino acid residues of the paratope contribute to the interaction of the antibody with its target protein epitope. The interaction can be hydrogen bonding, salt bridges, van der Waals interactions, electrostatic interactions, hydrophobic interactions, pi-interaction effects, ionic bonds, and / or any combination thereof. The interaction can be direct or indirect, for example, via a cooperating intermediate molecule such as an ion or water. The residues of the paratope, in some embodiments, include only residues that are part of a defined CDR. In some embodiments, the residues of the paratope further include one or more residues that are not part of a defined CDR (e.g., residues in a defined framework region).

[0073] In some embodiments, the paratope is less than about 5.0 angstroms from the epitope on the target antigen when the polypeptide binds to the target antigen, e.g., less than about 4.5, 4.0, 3.5, 3.0, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, or 0.9 angstroms from the epitope, or less than about 0. 9 to 5.0, 0.9 to 4.8, 1.0 to 5, 1.0 to 4.5, 1.0 to 4.0, 1.0 to 3.5, 1.1 to 3.5, 1.1 to 3.0, 1.2 to 3.0, 1.2 to 2.5, 1.3 to 2.5, 1.3 to 2.4, 1.4 to 2.4, 1.4 to 2.3, 1.5 to 2.3, 1.5 to 2.2, 1.6 to 2.2, 1.6 to 2.1, 1.7 to 2.1, 1.7 to 2.0, or 1.8 to 2.0 angstroms. In some embodiments, fewer than all of the amino acid residues within the paratope (e.g., about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the amino acid residues) that make up the paratope are oriented less than about 5.0 angstroms from the epitope on the target antigen when the polypeptide binds to the target antigen.

[0074] As used herein, the term "substantially similar to" refers to a polypeptide disclosed herein that is substantially similar in amino acid sequence (e.g., has at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid residue amino acid sequence identity) and that substantially preserves one or more functional properties of a particular polypeptide disclosed herein. In some embodiments, the one or more functional properties are selected from, but are not limited to, substantially similar binding affinity, substantially similar binding specificity, substantially similar inhibitory activity, substantially similar neutralizing activity, and substantially similar self-association properties.

[0075] In some embodiments, a polypeptide disclosed herein comprises a paratope substantially similar to a paratope of a polypeptide selected from any one of AB-1 to AB-51. In some embodiments, a polypeptide comprises a paratope that comprises only conservative substitutions (e.g., only highly conservative substitutions) relative to a paratope of a polypeptide selected from any one of AB-1 to AB-51. In some embodiments, a polypeptide comprises a paratope that comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative substitutions (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 highly conservative substitutions) relative to a paratope of a polypeptide selected from any one of AB-1 to AB-51. In some embodiments, a polypeptide comprises a paratope that has 100% sequence identity to a paratope of a polypeptide selected from any one of AB-1 to AB-51.

[0076] In some embodiments, the paratope comprises amino acid residues corresponding to each of T28, T30, R31, Y32, W33, Y52, G54, D55, X1 (position 57), K74, R98, X4 (position 99), P100, Q101, Y102, C103, X7 (position 106), C108, R110, and W111 of SEQ ID NO:2, and L46 of SEQ ID NO:49, or a subset thereof.

[0077] In some embodiments, the polypeptide comprising a paratope disclosed herein is an immunoglobulin heavy chain variable domain (V H ) and immunoglobulin light chain variable domain (V L In some embodiments, the paratope residues comprise the V H and V L Included within.

[0078] In some embodiments, the polypeptide comprises an immunoglobulin light chain variable region, an immunoglobulin heavy chain variable region, or an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region. In some embodiments, the polypeptide comprises six CDRs of a polypeptide disclosed herein. In some embodiments, the polypeptide has fewer than six (e.g., 1, 2, 3, 4, or 5) of the CDRs of a polypeptide disclosed herein.

[0079] In some embodiments, the present disclosure provides a polypeptide that specifically binds to SARS-CoV-2-spike, the polypeptide comprising: V shown in any of SEQ ID NOs: 4 to 48 H V containing HCDR1, HCDR2, and HCDR3 that are substantially similar in amino acid sequence to heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3), respectively. H an amino acid sequence; V shown in any of SEQ ID NOs: 51 to 76 LV comprising LCDR1, LCDR2, and LCDR3 that are substantially similar in amino acid sequence to light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3), respectively. L and an amino acid sequence.

[0080] In some embodiments, the polypeptide comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that substantially preserve one or more functional characteristics of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of a polypeptide selected from any one of AB-1 to AB-51.

[0081] In some embodiments, the polypeptide comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that comprise only one or more conservative substitutions (e.g., only one or more highly conservative substitutions) relative to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of a polypeptide selected from any one of AB-1 to AB-51.

[0082] In some embodiments, the polypeptide comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 that comprise up to 1, 2, or 3 conservative substitutions (e.g., up to 1, 2, or 3 highly conservative substitutions) relative to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of a polypeptide selected from any one of AB-1 to AB-51.

[0083] In some embodiments, the polypeptides disclosed herein comprise HCDR1, HCDR2, HCDR3, HCDR3, LCDR1, LCDR2, and LCDR3 that have 100% sequence identity to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of a polypeptide selected from any one of AB-1 to AB-51.

[0084] In some embodiments, the polypeptide comprises HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of an antibody comprising an amino acid sequence selected from the following: SEQ ID NO: 4 and SEQ ID NO: 51 (AB-1), SEQ ID NO: 5 and SEQ ID NO: 52 (AB-2), SEQ ID NO: 6 and SEQ ID NO: 53 (AB-3), SEQ ID NO: 7 and SEQ ID NO: 54 (AB-4), SEQ ID NO: 8 and SEQ ID NO: 51 (AB-5), SEQ ID NO: 9 and SEQ ID NO: 55 (AB-6), SEQ ID NO: 10 and SEQ ID NO: 56 (AB-7), SEQ ID NO: 11 and SEQ ID NO: 57 (AB-8), SEQ ID NO: 12 and SEQ ID NO: 58 (AB-9), SEQ ID NO: 13 and SEQ ID NO: 59 (AB-10), SEQ ID NO: 14 and SEQ ID NO: 60 (AB-11), SEQ ID NO: 15 and SEQ ID NO: 56 (AB-12), SEQ ID NO: 16 and SEQ ID NO: 51 (AB-13), SEQ ID NO: 10 and SEQ ID NO: 50 (AB-14), SEQ ID NO: 17 and SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 and SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 and SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 and SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 and SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 and SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 and SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 and SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 and SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 and SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 and SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 and SEQ ID NO: 51 (AB-26), SEQ ID NO: 26 and SEQ ID NO: 56 (AB-27), SEQ ID NO: 27 and SEQ ID NO: 61 (AB-28), SEQ ID NO: 28 and SEQ ID NO: 56 (AB-29), SEQ ID NO: 28 and SEQ ID NO: 69 (AB-30), SEQ ID NO: 29 and SEQ ID NO: 70 (AB-31), SEQ ID NO: 30 and SEQ ID NO: 71 (AB-32), SEQ ID NO: 31 and SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 and SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 and SEQ ID NO: 56 (AB-35), SEQ ID NO: 34 and SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 and SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 and SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 and SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 and SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 and SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 and SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 and SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 and SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 and SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 and SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 and SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 and SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 and SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 and SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 and SEQ ID NO: 56 (AB-51).

[0085] The CDRs (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3) can be CDRs defined by any art-recognized method for identifying CDR residues of an antibody, as further described herein (e.g., CDRs defined by Kabat, CDRs defined by Chothia, or CDRs defined by ImMunoGeneTics (IMGT) numbering (www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html, also accessible at www.imgt.org / )). In certain embodiments, the CDRs are defined by IMGT numbering. Examples of CDRs defined by IMGT numbering include the CDRs disclosed herein for the polypeptides of the present disclosure.

[0086] In some embodiments, the polypeptide comprises a V selected from: H / V L containing a paratope substantially similar to the paratope of an antibody containing pair: SEQ ID NO: 4 and SEQ ID NO: 51 (AB-1), SEQ ID NO: 5 and SEQ ID NO: 52 (AB-2), SEQ ID NO: 6 and SEQ ID NO: 53 (AB-3), SEQ ID NO: 7 and SEQ ID NO: 54 (AB-4), SEQ ID NO: 8 and SEQ ID NO: 51 (AB-5), SEQ ID NO: 9 and SEQ ID NO: 55 (AB-6), SEQ ID NO: 10 and SEQ ID NO: 56 (AB-7), SEQ ID NO: 11 and SEQ ID NO: 57 (AB-8), SEQ ID NO: 12 and SEQ ID NO: 58 (AB-9), SEQ ID NO: 13 and SEQ ID NO: 59 (AB-10), SEQ ID NO: 14 and SEQ ID NO: 60 (AB-11), SEQ ID NO: 15 and SEQ ID NO: 56 (AB-12), SEQ ID NO: 16 and SEQ ID NO: 51 (AB-13), SEQ ID NO: 10 and SEQ ID NO: 50 (AB-14), SEQ ID NO: 17 and SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 and SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 and SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 and SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 and SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 and SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 and SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 and SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 and SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 and SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 and SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 and SEQ ID NO: 51 (AB-26), SEQ ID NO: 26 and SEQ ID NO: 56 (AB-27), SEQ ID NO: 27 and SEQ ID NO: 61 (AB-28), SEQ ID NO: 28 and SEQ ID NO: 56 (AB-29), SEQ ID NO: 28 and SEQ ID NO: 69 (AB-30), SEQ ID NO: 29 and SEQ ID NO: 70 (AB-31), SEQ ID NO: 30 and SEQ ID NO: 71 (AB-32), SEQ ID NO: 31 and SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 and SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 and SEQ ID NO: 56 (AB-35), SEQ ID NO: 34 and SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 and SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 and SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 and SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 and SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 and SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 and SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 and SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 and SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 and SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 and SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 and SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 and SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 and SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 and SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 and SEQ ID NO: 56 (AB-51), or Any combination of them.

[0087] In some embodiments, the polypeptide comprises a V selected from: H / V L Contains a paratope that is identical to the paratope of the antibody containing the pair: SEQ ID NO: 4 and SEQ ID NO: 51 (AB-1), SEQ ID NO: 5 and SEQ ID NO: 52 (AB-2), SEQ ID NO: 6 and SEQ ID NO: 53 (AB-3), SEQ ID NO: 7 and SEQ ID NO: 54 (AB-4), SEQ ID NO: 8 and SEQ ID NO: 51 (AB-5), SEQ ID NO: 9 and SEQ ID NO: 55 (AB-6), SEQ ID NO: 10 and SEQ ID NO: 56 (AB-7), SEQ ID NO: 11 and SEQ ID NO: 57 (AB-8), SEQ ID NO: 12 and SEQ ID NO: 58 (AB-9), SEQ ID NO: 13 and SEQ ID NO: 59 (AB-10), SEQ ID NO: 14 and SEQ ID NO: 60 (AB-11), SEQ ID NO: 15 and SEQ ID NO: 56 (AB-12), SEQ ID NO: 16 and SEQ ID NO: 51 (AB-13), SEQ ID NO: 10 and SEQ ID NO: 50 (AB-14), SEQ ID NO: 17 and SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 and SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 and SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 and SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 and SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 and SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 and SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 and SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 and SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 and SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 and SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 and SEQ ID NO: 51 (AB-26), SEQ ID NO: 26 and SEQ ID NO: 56 (AB-27), SEQ ID NO: 27 and SEQ ID NO: 61 (AB-28), SEQ ID NO: 28 and SEQ ID NO: 56 (AB-29), SEQ ID NO: 28 and SEQ ID NO: 69 (AB-30), SEQ ID NO: 29 and SEQ ID NO: 70 (AB-31), SEQ ID NO: 30 and SEQ ID NO: 71 (AB-32), SEQ ID NO: 31 and SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 and SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 and SEQ ID NO: 56 (AB-35), SEQ ID NO: 34 and SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 and SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 and SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 and SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 and SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 and SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 and SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 and SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 and SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 and SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 and SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 and SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 and SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 and SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 and SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 and SEQ ID NO: 56 (AB-51).

[0088] In some embodiments, the present disclosure provides a V H The present invention provides a polypeptide that specifically binds to SARS-CoV-2 spike, comprising: X1 is not S, X2 is not D, X3 is not T, X4 is not L, The X5 is not an S. The X6 is not an N. The X7 is not a G. X8 is not V, or X9 is not Q, Or any combination of the above.

[0089] Consensus V for SEQ ID NOs: 3 to 48 herein H The sequence, identified as SEQ ID NO:2, is shown in Table 1.

[0090] In some embodiments, the polypeptides disclosed herein are L In some embodiments, the polypeptide further comprises a V comprising the amino acid sequence of SEQ ID NO:49. L Including, X 10 is not Q, X 11 is not G, X 12 is not S, X 13 is not S, X 14 is not N, X 15 is not S, X 16 is not F, or X 17 is not Y, Or any combination of the above.

[0091] Consensus V for SEQ ID NOs: 50 to 76 herein L The sequence, identified as SEQ ID NO: 49, is shown in Table 2.

[0092] In some embodiments, X1 is S, N, A, R, L, or F; X2 is D or E; X3 is T or V; X4 is L or V; X5 is S, Q, R, K, Y, D, or E; X6 is N, K, A, S, R, or E; X7 is G, N, or L; X8 is V, I, S, or K; or X9 is Q, Y, K, F, or H; Or any combination of the above.

[0093] In some embodiments, X1 is N, A, R, L, or F; X2 is E, X3 is V, X4 is V, X5 is Q, R, K, Y, D, or E; X6 is K, A, S, R, or E; X7 is N or L; X8 is I, S, or K; or X9 is Y, K, F or H; Or any combination of the above.

[0094] In some embodiments, X1 is not S. In some embodiments, X1 is S, N, A, R, L, or F. In some embodiments, X1 is N, A, R, L, or F. In some embodiments, X1 is S. In some embodiments, X1 is N. In some embodiments, X1 is A. In some embodiments, X1 is R. In some embodiments, X1 is L. In some embodiments, X1 is F.

[0095] In some embodiments, X2 is not D. In some embodiments, X2 is D or E. In some embodiments, X2 is D. In some embodiments, X2 is E.

[0096] In some embodiments, X3 is not T. In some embodiments, X3 is T or V. In some embodiments, X3 is T. In some embodiments, X3 is V.

[0097] In some embodiments, X4 is not L. In some embodiments, X4 is L or V. In some embodiments, X4 is L. In some embodiments, X4 is V.

[0098] In some embodiments, X5 is not S. In some embodiments, X5 is S, Q, R, K, Y, D, or E. In some embodiments, X5 is Q, R, K, Y, D, or E. In some embodiments, X5 is S. In some embodiments, X5 is Q. In some embodiments, X5 is R. In some embodiments, X5 is K. In some embodiments, X5 is Y. In some embodiments, X5 is D. In some embodiments, X5 is E.

[0099] In some embodiments, X6 is not N. In some embodiments, X6 is N, K, A, S, R, or E. In some embodiments, X6 is K, A, S, R, or E. In some embodiments, X6 is N. In some embodiments, X6 is K. In some embodiments, X6 is A. In some embodiments, X6 is S. In some embodiments, X6 is R. In some embodiments, X6 is E.

[0100] In some embodiments, X7 is not G. In some embodiments, X7 is G, N, or L. In some embodiments, X7 is N or L. In some embodiments, X7 is G. In some embodiments, X7 is N. In some embodiments, X7 is L.

[0101] In some embodiments, X8 is not V. In some embodiments, X8 is V, I, S, or K. In some embodiments, X8 is I, S, or K. In some embodiments, X8 is V. In some embodiments, X8 is I. In some embodiments, X8 is S. In some embodiments, X8 is K.

[0102] In some embodiments, X9 is not Q. In some embodiments, X9 is Q, Y, K, F, or H. In some embodiments, X9 is Y, K, F, or H. In some embodiments, X9 is Q. In some embodiments, X9 is Y. In some embodiments, X9 is K. In some embodiments, X9 is F. In some embodiments, X9 is H.

[0103] In some embodiments, X 10 is Q, K, or I, X 11 is G or S, X 12 is S, R, or V; X13 is S or N, X 14 is N, H, D, Y, or S; X 15 is S or Q, X 16 is F, Y, L, V, T, or D, or X 17 is Y or L; Or any combination of the above.

[0104] In some embodiments, X 10 is K or I, X 11 is S, X 12 is R or V, X 13 is N, X 14 is H, D, Y, or S; X 15 is Q, X 16 is Y, L, V, T, or D, or X 17 is L, Or any combination of the above.

[0105] In some embodiments, X 10 is not Q. In some embodiments, X 10 is Q, K, or I. In some embodiments, X 10 is K or I. In some embodiments, X 10 is Q. In some embodiments, X 10 is K. In some embodiments, X 10 is I.

[0106] In some embodiments, X 11 is not G. In some embodiments, X 11 is G or S. In some embodiments, X 11is G. In some embodiments, X 11 is S.

[0107] In some embodiments, X 12 is not S. In some embodiments, X 12 is S, R, or V. In some embodiments, X 12 is S. In some embodiments, X 12 is R. In some embodiments, X 12 is V.

[0108] In some embodiments, X 13 is not S. In some embodiments, X 13 is S or N. In some embodiments, X 13 is S. In some embodiments, X 13 is N.

[0109] In some embodiments, X 14 is not N. In some embodiments, X 14 is N, H, D, Y, or S. In some embodiments, X 14 is H, D, Y, or S. In some embodiments, X 14 is N. In some embodiments, X 14 is H. In some embodiments, X 14 is D. In some embodiments, X 14 is Y. In some embodiments, X 14 is S.

[0110] In some embodiments, X 15 is not S. In some embodiments, X 15 is S or Q. In some embodiments, X 15 is S. In some embodiments, X 15 is Q.

[0111] In some embodiments, X 16is not F. In some embodiments, X 16 is F, Y, L, V, T, or D. In some embodiments, X 16 is Y, L, V, T, or D. In some embodiments, X 16 is F. In some embodiments, X 16 is Y. In some embodiments, X 16 is L. In some embodiments, X 16 is V. In some embodiments, X 16 is T. In some embodiments, X 16 is D.

[0112] In some embodiments, X 17 is not Y. In some embodiments, X 17 is Y or L. In some embodiments, X 17 is Y. In some embodiments, X 17 is L.

[0113] In some embodiments, X1 is S, X2 is D, X3 is V, X4 is L, X5 is S, X6 is K, X7 is G, X8 is V, X9 is Y, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is H, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-1), X1 is S, X2 is D, X3 is T, X4 is L, X5 is Q, X6 is N, X7 is G, X8 is I, X9 is K, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14is D, X 15 is Q, X 16 is Y or X 17 is L, or a combination thereof (AB-2), X1 is N, X2 is D, X3 is V, X4 is L, X5 is R, X6 is K, X7 is G, X8 is I, X9 is K, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is D, X 15 is Q, X 16 is L, or X 17 is Y, or a combination thereof (AB-3), X1 is S, X2 is D, X3 is T, X4 is L, X5 is R, X6 is N, X7 is G, X8 is V, X9 is Y, X 10 is K, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is V or X 17 is Y, or a combination thereof (AB-4), X1 is S, X2 is D, X3 is T, X4 is L, X5 is R, X6 is A, X7 is N, X8 is V, X9 is F, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is H, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-5), X1 is S, X2 is D, X3 is V, X4 is L, X5 is K, X6 is K, X7 is L, X8 is I, X9 is K, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14 is D, X 15 is Q, X 16 is L, or X 17 is Y, or a combination thereof (AB-6), X1 is S, X2 is D, X3 is T, X4 is L, X5 is R, X6 is N, X7 is G, X8 is V, X9 is Q, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-7), X1 is N, X2 is D, X3 is V, X4 is L, X5 is S, X6 is A, X7 is G, X8 is S, X9 is F, X 10 is I,X 11 is G, X 12 is R, X 13 is N, X 14 is Y, X 15 is S, X 16 is Y or X 19 is Y, or a combination thereof (AB-8), X1 is N, X2 is D, X3 is V, X4 is L, X5 is R, X6 is S, X7 is G, X8 is I, X9 is K, X 10 is Q, X 11 is G, X 12 is S, X13 is S, X 14 is D, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-9), X1 is S, X2 is D, X3 is V, X4 is L, X5 is S, X6 is A, X7 is G, X8 is V, X9 is F, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is S, X 16 is Y or X 17 is Y, or a combination thereof (AB-10), X1 is S, X2 is D, X3 is T, X4 is L, X5 is R, X6 is N, X7 is G, X8 is V, X9 is H, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14 is N, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-11), X1 is S, X2 is D, X3 is T, X4 is L, X5 is S, X6 is R, X7 is G, X8 is V, X9 is K, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-12), X1 is S, X2 is D, X3 is V, X4 is L, X5 is R, X6 is K, X7 is G, X8 is V, X9 is H, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is H, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-13), X1 is S, X2 is D, X3 is T, X4 is L, X5 is R, X6 is N, X7 is G, X8 is V, X9 is Q, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-14), X1 is S, X2 is D, X3 is T, X4 is L, X5 is Y, X6 is A, X7 is G, X8 is V, X9 is H, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14 is H, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-15), X1 is S, X2 is D, X3 is V, X4 is L, X5 is R, X6 is S, X7 is G, X8 is S, X9 is H, X 10 is Q, X 11 is G, X 12 is R, X13 is S, X 14 is Y, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-16), X1 is N, X2 is D, X3 is V, X4 is L, X5 is R, X6 is K, X7 is G, X8 is I, X9 is K, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is H, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-17), X1 is S, X2 is D, X3 is T, X4 is L, X5 is R, X6 is N, X7 is G, X8 is S, X9 is K, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14 is D, X 15 is Q, X 16 is F or X 17 is L, or a combination thereof (AB-18), X1 is S, X2 is D, X3 is V, X4 is L, X5 is S, X6 is K, X7 is G, X8 is V, X9 is Y, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14 is H, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-19), or X1 is S, X2 is D, X3 is T, X4 is L, X5 is R, X6 is A, X7 is G, X8 is V, X9 is H, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14 is H, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-20), X1 is A, X2 is D, X3 is V, X4 is L, X5 is R, X6 is K, X7 is G, X8 is V, X9 is Y, X 10 is Q, X 11 is G, X 12 is V, X 13 is S, X 14 is H, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-21), X1 is S, X2 is D, X3 is T, X4 is L, X5 is R, X6 is S, X7 is G, X8 is V, X9 is Q, X 10 is Q, X 11 is S, X 12 is S, X 13 is S, X 14 is S, X 15 is Q, X 16 is T or X 17 is Y, or a combination thereof (AB-22), X1 is S, X2 is D, X3 is V, X4 is L, X5 is S, X6 is K, X7 is G, X8 is V, X9 is Y, X 10 is Q, X 11 is G, X 12 is R, X13 is S, X 14 is H, X 15 is S, X 16 is T or X 17 is Y, or a combination thereof (AB-23), X1 is S, X2 is D, X3 is T, X4 is L, X5 is R, X6 is S, X7 is G, X8 is S, X9 is Y, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-24), X1 is S, X2 is D, X3 is T, X4 is L, X5 is Y, X6 is A, X7 is G, X8 is V, X9 is K, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14 is Y, X 15 is S, X 16 is V or X 17 is Y, or a combination thereof (AB-25), X1 is S, X2 is D, X3 is T, X4 is V, X5 is R, X6 is S, X7 is G, X8 is V, X9 is F, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is H, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-26), X1 is S, X2 is E, X3 is T, X4 is V, X5 is S, X6 is R, X7 is G, X8 is V, X9 is Y, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-27), X1 is S, X2 is D, X3 is T, X4 is L, X5 is Y, X6 is R, X7 is G, X8 is V, X9 is K, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14 is H, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-28), X1 is R, X2 is D, X3 is T, X4 is L, X5 is S, X6 is R, X7 is G, X8 is V, X9 is F, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-29), X1 is R, X2 is D, X3 is T, X4 is L, X5 is S, X6 is R, X7 is G, X8 is V, X9 is F, X 10 is Q, X 11 is G, X 12 is S, X13 is S, X 14 is N, X 15 is Q, X 16 is V or X 17 is Y, or a combination thereof (AB-30), X1 is L, X2 is D, X3 is T, X4 is L, X5 is Y, X6 is K, X7 is G, X8 is V, X9 is K, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14 is H, X 15 is S, X 16 is V or X 17 is Y, or a combination thereof (AB-31), X1 is S, X2 is E, X3 is T, X4 is L, X5 is R, X6 is R, X7 is G, X8 is V, X9 is K, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is L, or X 17 is Y, or a combination thereof (AB-32), X1 is S, X2 is D, X3 is T, X4 is L, X5 is R, X6 is A, X7 is G, X8 is K, X9 is K, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is Y or X 17 is Y, or a combination thereof (AB-33), X1 is A, X2 is D, X3 is V, X4 is L, X5 is R, X6 is K, X7 is G, X8 is V, X9 is K, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14 is H, X 15 is S, X 16 is T or X 17 is Y, or a combination thereof (AB-34), X1 is A, X2 is D, X3 is T, X4 is L, X5 is S, X6 is R, X7 is G, X8 is V, X9 is Q, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-35), X1 is A, X2 is D, X3 is T, X4 is L, X5 is S, X6 is K, X7 is G, X8 is V, X9 is Y, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is H, X 15 is S, X 16 is T or X 17 is Y, or a combination thereof (AB-36), X1 is F, X2 is D, X3 is T, X4 is L, X5 is R, X6 is R, X7 is G, X8 is I, X9 is F, X 10 is Q, X 11 is G, X 12 is S, X13 is S, X 14 is H, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-37), X1 is S, X2 is D, X3 is T, X4 is L, X5 is Q, X6 is R, X7 is G, X8 is V, X9 is Q, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-38), X1 is A, X2 is D, X3 is V, X4 is L, X5 is R, X6 is K, X7 is G, X8 is K, X9 is K, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is H, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-39), X1 is S, X2 is E, X3 is T, X4 is L, X5 is R, X6 is S, X7 is G, X8 is V, X9 is Q, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is V or X 17 is Y, or a combination thereof (AB-40), X1 is N, X2 is D, X3 is V, X4 is L, X5 is S, X6 is A, X7 is G, X8 is V, X9 is F, X 10 is Q, X 11 is G, X 12 is S, X 13 is N, X 14 is Y, X 15 is S, X 16 is Y or X 17 is Y, or a combination thereof (AB-41), X1 is S, X2 is D, X3 is T, X4 is L, X5 is Q, X6 is S, X7 is G, X8 is V, X9 is K, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14 is D, X 15 is Q, X 16 is Y or X 17 is L, or a combination thereof (AB-42), X1 is S, X2 is D, X3 is T, X4 is L, X5 is S, X6 is A, X7 is G, X8 is V, X9 is F, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is H, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-43), X1 is L, X2 is D, X3 is V, X4 is L, X5 is R, X6 is E, X7 is G, X8 is K, X9 is H, X 10 is Q, X 11 is G, X 12 is R, X13 is N, X 14 is D, X 15 is Q, X 16 is D or X 17 is L, or a combination thereof (AB-44), X1 is S, X2 is D, X3 is T, X4 is L, X5 is S, X6 is S, X7 is G, X8 is V, X9 is Q, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-45), X1 is S, X2 is D, X3 is T, X4 is L, X5 is S, X6 is S, X7 is G, X8 is V, X9 is H, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is H, X 15 is S, X 16 is F or X 17 is Y, or a combination thereof (AB-46), X1 is S, X2 is D, X3 is T, X4 is L, X5 is Q, X6 is N, X7 is G, X8 is V, X9 is H, X 10 is Q, X 11 is G, X 12 is R, X 13 is N, X 14 is D, X 15 is Q, X 16 is D or X 17 is L, or a combination thereof (AB-47), X1 is N, X2 is D, X3 is V, X4 is L, X5 is D, X6 is K, X7 is G, X8 is I, X9 is K, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is D, X 15 is Q, X 16 is L, or X 17 is Y, or a combination thereof (AB-48), X1 is S, X2 is D, X3 is T, X4 is L, X5 is E, X6 is N, X7 is G, X8 is I, X9 is K, X 10 is Q, X 11 is G, X 12 is R, X 13 is S, X 14 is D, X 15 is Q, X 16 is Y or X 17 is L, or a combination thereof (AB-49), X1 is A, X2 is D, X3 is V, X4 is L, X5 is S, X6 is S, X7 is G, X8 is S, X9 is F, X 10 is Q, X 11 is G, X 12 is S, X 13 is S, X 14 is Y, X 15 is S, X 16 is T or X 17 is Y, or a combination thereof (AB-50), or X1 is S, X2 is D, X3 is T, X4 is L, X5 is S, X6 is N, X7 is G, X8 is V, X9 is Q, X 10 is Q, X 11 is G, X 12is S, X 13 is S, X 14 is N, X 15 is Q, X 16 is F or X 17 is Y, or a combination thereof (AB-51).

[0114] In some embodiments, the polypeptides disclosed herein comprise a V H V comprising HCDR1, HCDR2, and HCDR3 that are substantially similar in amino acid sequence to HCDR1, HCDR2, and HCDR3, respectively. H (See Table 1 for SEQ ID NOs: 4-48, and see Table 3 and Figure 2 for non-limiting examples of corresponding HCDR1, HCDR2, and HCDR3 sequences).

[0115] In some embodiments, the polypeptides disclosed herein comprise a V L V comprising LCDR1, LCDR2, and LCDR3 that are substantially similar in amino acid sequence to LCDR1, LCDR2, and LCDR3, respectively. L (See Table 2 for SEQ ID NOs: 51-76, and see Table 3 and Figure 3 for non-limiting examples of corresponding LCDR1, LCDR2, and LCDR3 sequences).

[0116] In some embodiments, the polypeptides disclosed herein comprise a V H V containing HCDR1, HCDR2, and HCDR3, which have the same amino acid sequences as HCDR1, HCDR2, and HCDR3, respectively. H Includes.

[0117] In some embodiments, the polypeptides disclosed herein comprise a V LV comprising LCDR1, LCDR2, and LCDR3, which have amino acid sequences identical to LCDR1, LCDR2, and LCDR3, respectively. L Includes:

[0118] In some embodiments, the polypeptides disclosed herein comprise a V selected from the following: H / V Land paratopes substantially similar to the paratopes of the following combinations: SEQ ID NO:4 / SEQ ID NO:51 (AB-1), SEQ ID NO:5 / SEQ ID NO:52 (AB-2), SEQ ID NO:6 / SEQ ID NO:53 (AB-3), SEQ ID NO:7 / SEQ ID NO:54 (AB-4), SEQ ID NO:8 / SEQ ID NO:51 (AB-5), SEQ ID NO:9 / SEQ ID NO:55 (AB-6), SEQ ID NO:10 / SEQ ID NO:56 (AB-7), SEQ ID NO:11 / SEQ ID NO:57 (AB-8), SEQ ID NO:12 / SEQ ID NO:58 (AB-9), SEQ ID NO:13 / SEQ ID NO:59 (AB-10), SEQ ID NO:14 / SEQ ID NO:60 (AB-11), SEQ ID NO: No. 15 / SEQ ID NO:56 (AB-12), SEQ ID NO:16 / SEQ ID NO:51 (AB-13), SEQ ID NO:10 / SEQ ID NO:50 (AB-14), SEQ ID NO:17 / SEQ ID NO:61 (AB-15), SEQ ID NO:18 / SEQ ID NO:62 (AB-16), SEQ ID NO:6 / SEQ ID NO:63 (AB-17), SEQ ID NO:19 / SEQ ID NO:64 (AB-18), SEQ ID NO:4 / SEQ ID NO:61 (AB-19), SEQ ID NO:20 / SEQ ID NO:61 (AB-20), SEQ ID NO:21 / SEQ ID NO:65 (AB-21), SEQ ID NO:22 / SEQ ID NO:66 (AB-22), SEQ ID NO:4 / SEQ ID NO:67 (AB-23), SEQ ID NO: 23 / SEQ ID NO:56 (AB-24), SEQ ID NO:24 / SEQ ID NO:68 (AB-25), SEQ ID NO:25 / SEQ ID NO:51 (AB-26), SEQ ID NO:26 / SEQ ID NO:56 (AB-27), SEQ ID NO:27 / SEQ ID NO:61 (AB-28), SEQ ID NO:28 / SEQ ID NO:56 (AB-29), SEQ ID NO:28 / SEQ ID NO:69 (AB-30), SEQ ID NO:29 / SEQ ID NO:70 (AB-31), SEQ ID NO:30 / SEQ ID NO:71 (AB-32), SEQ ID NO:31 / SEQ ID NO:72 (AB-33), SEQ ID NO:32 / SEQ ID NO:67 (AB-34), SEQ ID NO:33 / SEQ ID NO:56 (AB-35), SEQ ID NO: No. 34 / SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 / SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 / SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 / SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 / SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 / SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 / SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 / SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 / SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 / SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 / SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 / SEQ ID NO: 75 (AB-47),SEQ ID NO: 46 / SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 / SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 / SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 / SEQ ID NO: 56 (AB-51).

[0119] In some embodiments, the polypeptides disclosed herein comprise a V selected from the following: H / V LThe combinations include paratopes identical to the paratopes of: SEQ ID NO:4 / SEQ ID NO:51 (AB-1), SEQ ID NO:5 / SEQ ID NO:52 (AB-2), SEQ ID NO:6 / SEQ ID NO:53 (AB-3), SEQ ID NO:7 / SEQ ID NO:54 (AB-4), SEQ ID NO:8 / SEQ ID NO:51 (AB-5), SEQ ID NO:9 / SEQ ID NO:55 (AB-6), SEQ ID NO:10 / SEQ ID NO:56 (AB-7), SEQ ID NO:11 / SEQ ID NO:57 (AB-8), SEQ ID NO:12 / SEQ ID NO:58 (AB-9), SEQ ID NO:13 / SEQ ID NO:59 (AB-10), SEQ ID NO:14 / SEQ ID NO:60 (AB-11), SEQ ID NO:15 / SEQ ID NO: 56 (AB-12), SEQ ID NO: 16 / SEQ ID NO: 51 (AB-13), SEQ ID NO: 10 / SEQ ID NO: 50 (AB-14), SEQ ID NO: 17 / SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 / SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 / SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 / SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 / SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 / SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 / SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 / SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 / SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 / SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 / SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 / SEQ ID NO: 51 (AB-26), SEQ ID NO: 26 / SEQ ID NO: 56 (AB-27), SEQ ID NO: 27 / SEQ ID NO: 61 (AB-28), SEQ ID NO: 28 / SEQ ID NO: 56 (AB-29), SEQ ID NO: 28 / SEQ ID NO: 69 (AB-30), SEQ ID NO: 29 / SEQ ID NO: 70 (AB-31), SEQ ID NO: 30 / SEQ ID NO: 71 (AB-32), SEQ ID NO: 31 / SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 / SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 / SEQ ID NO: 56 (AB-35), SEQ ID NO: No. 34 / SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 / SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 / SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 / SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 / SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 / SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 / SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 / SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 / SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 / SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 / SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 / SEQ ID NO: 75 (AB-47),SEQ ID NO: 46 / SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 / SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 / SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 / SEQ ID NO: 56 (AB-51).

[0120] In some embodiments, the polypeptides disclosed herein comprise a V selected from the following: H / V LParatopes of the combinations include paratopes that differ by one to three (e.g., one, two, or three) residue substitutions (e.g., conservative, such as highly conservative substitutions): SEQ ID NO:4 / SEQ ID NO:51 (AB-1), SEQ ID NO:5 / SEQ ID NO:52 (AB-2), SEQ ID NO:6 / SEQ ID NO:53 (AB-3), SEQ ID NO:7 / SEQ ID NO:54 (AB-4), SEQ ID NO:8 / SEQ ID NO:51 (AB-5), SEQ ID NO:9 / SEQ ID NO:55 (AB-6), SEQ ID NO:10 / SEQ ID NO:56 (AB-7), SEQ ID NO:11 / SEQ ID NO:57 (AB-8), SEQ ID NO:12 / SEQ ID NO:58 (AB-9). -9), SEQ ID NO:13 / SEQ ID NO:59 (AB-10), SEQ ID NO:14 / SEQ ID NO:60 (AB-11), SEQ ID NO:15 / SEQ ID NO:56 (AB-12), SEQ ID NO:16 / SEQ ID NO:51 (AB-13), SEQ ID NO:10 / SEQ ID NO:50 (AB-14), SEQ ID NO:17 / SEQ ID NO:61 (AB-15), SEQ ID NO:18 / SEQ ID NO:62 (AB-16), SEQ ID NO:6 / SEQ ID NO:63 (AB-17), SEQ ID NO:19 / SEQ ID NO:64 (AB-18), SEQ ID NO:4 / SEQ ID NO:61 (AB-19), SEQ ID NO:20 / SEQ ID NO:61 (AB-20), SEQ ID NO:21 / SEQ ID NO:6 5 (AB-21), SEQ ID NO:22 / SEQ ID NO:66 (AB-22), SEQ ID NO:4 / SEQ ID NO:67 (AB-23), SEQ ID NO:23 / SEQ ID NO:56 (AB-24), SEQ ID NO:24 / SEQ ID NO:68 (AB-25), SEQ ID NO:25 / SEQ ID NO:51 (AB-26), SEQ ID NO:26 / SEQ ID NO:56 (AB-27), SEQ ID NO:27 / SEQ ID NO:61 (AB-28), SEQ ID NO:28 / SEQ ID NO:56 (AB-29), SEQ ID NO:28 / SEQ ID NO:69 (AB-30), SEQ ID NO:29 / SEQ ID NO:70 (AB-31), SEQ ID NO:30 / SEQ ID NO:71 (AB-32), SEQ ID NO:31 / SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 / SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 / SEQ ID NO: 56 (AB-35), SEQ ID NO: 34 / SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 / SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 / SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 / SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 / SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 / SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 / SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 / SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 / SEQ ID NO: 75 (AB-44),SEQ ID NO: 43 / SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 / SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 / SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 / SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 / SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 / SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 / SEQ ID NO: 56 (AB-51).

[0121] In some embodiments, the polypeptides disclosed herein comprise a V selected from the following: H / V L The paratope is identical to that of the combination: SEQ ID NO:3 / SEQ ID NO:50.

[0122] In some embodiments, the polypeptides disclosed herein comprise a V selected from the following: H / V L It contains a paratope that is identical to that of the combination: SEQ ID NO: 4 / SEQ ID NO: 51 (AB-1).

[0123] In some embodiments, the polypeptides disclosed herein comprise a V having at least about 70% sequence identity to the amino acid sequence of SEQ ID NO:3. H For example, V H may have at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, V H has at least about 85% or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 3. H The sequence corresponding to the domain, identified as SEQ ID NO: 3, is shown in Table 1.

[0124] In some embodiments, the polypeptides disclosed herein comprise a V having at least about 70% sequence identity to the amino acid sequence of SEQ ID NO:50. L For example, VL may have at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, V L has at least about 85% or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 50. L The sequence corresponding to the domain, identified as SEQ ID NO: 50, is shown in Table 2.

[0125] In some embodiments, the polypeptides disclosed herein comprise a V having at least about 70% sequence identity to any one or more amino acid sequences of SEQ ID NOs: 4-48. H For example, V H may have at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one or more amino acid sequences of SEQ ID NOs: 4-48. H has at least about 85% or at least about 90% sequence identity to any one or more amino acid sequences of SEQ ID NOs: 4 to 48.

[0126] In some embodiments, the polypeptides disclosed herein comprise a V having at least about 70% sequence identity to any one or more amino acid sequences of SEQ ID NOs: 51-76. L For example, V Lmay have at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one or more amino acid sequences of SEQ ID NOs: 51-76. L has at least about 85% or at least about 90% sequence identity to any one or more amino acid sequences of SEQ ID NOs: 51 to 76.

[0127] In some embodiments, the polypeptides disclosed herein comprise a V having at least about 70% sequence identity to the amino acid sequence of SEQ ID NO:4. H For example, V H may have at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, V H has at least about 85% or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 4. The sequence identified as SEQ ID NO: 4 is shown in Table 1.

[0128] In some embodiments, the polypeptides disclosed herein comprise a V having at least about 70% sequence identity to the amino acid sequence of SEQ ID NO:51. L For example, V L may have at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 51. In some embodiments, V Lhas at least about 85% or at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 51. The sequence identified as SEQ ID NO: 51 is shown in Table 2.

[0129] In some embodiments, the polypeptides disclosed herein comprise a V that contains at least one amino acid substitution (e.g., at least one conservative substitution, such as a highly conservative amino acid substitution) relative to the amino acid sequence of SEQ ID NO:3. H For example, the number of amino acid substitutions is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or about 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11, or 9-11. H contains about 1-10 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, at least one amino acid substitution replaces only a residue in HCDR1, HCDR2, and / or HCDR3 of SEQ ID NO: 3. In some embodiments, at least one amino acid substitution replaces only a residue in a non-CDR of SEQ ID NO: 3 (e.g., within a framework region).

[0130] In some embodiments, the polypeptides disclosed herein comprise a V that contains at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO: 50. L For example, the number of amino acid substitutions is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or about 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11, or 9-11. Lcontains about 1-10 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 50. In some embodiments, at least one amino acid substitution replaces only a residue in LCDR1, LCDR2, and / or LCDR3 of SEQ ID NO: 50. In some embodiments, at least one amino acid substitution replaces only a residue in a non-CDR of SEQ ID NO: 50 (e.g., within a framework region).

[0131] In some embodiments, the amino acid substitution is a conservative substitution. The term "conservative amino acid substitution(s)" or "conservative substitution(s)" refers to an amino acid substitution having a value of 0 or greater in BLOSUM62.

[0132] In some embodiments, the amino acid substitution is a highly conservative substitution. The term "highly conservative amino acid substitution(s)" or "highly conservative substitution(s)" refers to an amino acid substitution having a value of at least 1 (e.g., at least 2) in BLOSUM62.

[0133] In some embodiments, the polypeptides disclosed herein comprise a V that contains at least one amino acid substitution relative to the amino acid sequence of any one or more of SEQ ID NOs: 4-48. H For example, the number of amino acid substitutions is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or about 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11, or 9-11. H contains about 1 to 10 amino acid substitutions with respect to any one or more amino acid sequences of SEQ ID NOs: 4 to 48.

[0134] In some embodiments, at least one amino acid substitution replaces only a residue in HCDR1, HCDR2, and / or HCDR3 of any one or more of SEQ ID NOs: 4-48. In some embodiments, at least one amino acid substitution replaces only a residue in a non-CDR (e.g., within a framework region) of any one or more of SEQ ID NOs: 4-48.

[0135] In some embodiments, the polypeptides disclosed herein comprise a V that contains at least one amino acid substitution relative to the amino acid sequence of any one or more of SEQ ID NOs: 51-76. L For example, the number of amino acid substitutions is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or about 1-20, 1-19, 2-19, 2-18, 2-17, 3-17, 3-16, 4-16, 4-15, 5-15, 5-14, 6-14, 6-13, 7-13, 7-12, 8-12, 8-11, or 9-11. L contains about 1 to 10 amino acid substitutions with respect to any one or more amino acid sequences of SEQ ID NOs: 51 to 76.

[0136] In some embodiments, at least one amino acid substitution replaces only an LCDR1, LCDR2, and / or LCDR3 residue in any one or more of SEQ ID NOs: 51-76. In some embodiments, at least one amino acid substitution replaces only a non-CDR residue (e.g., within a framework region) in any one or more of SEQ ID NOs: 51-76.

[0137] In some embodiments, the polypeptide comprises: a) an HCDR1 comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO: 77; b) HCDR2 containing at least one amino acid substitution with respect to at least one amino acid sequence shown in SEQ ID NOs: 79 to 88 (for example, at least one amino acid sequence shown in SEQ ID NOs: 80 to 88); c) HCDR3 containing at least one amino acid substitution with respect to at least one amino acid sequence shown in SEQ ID NOs: 90 to 131 (for example, at least one amino acid sequence shown in SEQ ID NOs: 91 to 131); d) LCDR1 containing at least one amino acid substitution with respect to the amino acid sequence shown in SEQ ID NOs: 133 to 140 (for example, at least one amino acid sequence shown in SEQ ID NOs: 134 to 140); e) LCDR2 comprising at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO: 141; f) LCDR3 containing at least one amino acid substitution with respect to at least one amino acid sequence shown in SEQ ID NOs: 143 to 162 (for example, at least one amino acid sequence shown in SEQ ID NOs: 144 to 162); Or any combination of the foregoing.

[0138] In some embodiments, the polypeptide comprises: a) HCDR2 containing at least one amino acid substitution with respect to at least one amino acid sequence shown in SEQ ID NOs: 79 to 88 (for example, at least one amino acid sequence shown in SEQ ID NOs: 80 to 88); b) HCDR3 containing at least one amino acid substitution with respect to at least one amino acid sequence shown in SEQ ID NOs: 90 to 131 (for example, at least one amino acid sequence shown in SEQ ID NOs: 91 to 131); c) LCDR1 containing at least one amino acid substitution with respect to the amino acid sequence shown in SEQ ID NOs: 133 to 140 (for example, at least one amino acid sequence shown in SEQ ID NOs: 134 to 140), or d) LCDR3 containing at least one amino acid substitution with respect to at least one amino acid sequence shown in SEQ ID NOs: 143 to 162 (for example, at least one amino acid sequence shown in SEQ ID NOs: 144 to 162); Or any combination of the foregoing.

[0139] In some embodiments, the polypeptides disclosed herein comprise a V comprising the amino acid sequence of SEQ ID NO:3.H In some embodiments, the polypeptides disclosed herein comprise a V comprising the amino acid sequence of any one of SEQ ID NOs: 4-48. H In some embodiments, the polypeptides disclosed herein comprise a V comprising the amino acid sequence of SEQ ID NO:4. H Includes:

[0140] In some embodiments, the polypeptide disclosed herein comprises a V comprising the amino acid sequence of SEQ ID NO: 50. L In some embodiments, the polypeptides disclosed herein comprise a V comprising the amino acid sequence of any one of SEQ ID NOs: 51-76. L In some embodiments, the polypeptide disclosed herein comprises a V comprising the amino acid sequence of SEQ ID NO:51. L Includes:

[0141] In some embodiments, the polypeptides disclosed herein comprise: V comprising any one of the amino acid sequences of SEQ ID NOs: 4 to 48 H , and V comprising the amino acid sequence of SEQ ID NO: 50 L .

[0142] In some embodiments, the polypeptides disclosed herein comprise: V comprising the amino acid sequence of SEQ ID NO: 3 H , and V comprising any one of the amino acid sequences of SEQ ID NOs: 51 to 76 L .

[0143] In some embodiments, the polypeptides disclosed herein comprise: V comprising any one of the amino acid sequences of SEQ ID NOs: 4 to 48 H , and V comprising any one of the amino acid sequences of SEQ ID NOs: 51 to 76 L .

[0144] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 4, and b)V L comprises the amino acid sequence of SEQ ID NO: 51 (AB-1).

[0145] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 5, and b)V L comprises the amino acid sequence of SEQ ID NO: 52 (AB-2).

[0146] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 6, and b)V L comprises the amino acid sequence of SEQ ID NO: 53 (AB-3).

[0147] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 7, and b)V L comprises the amino acid sequence of SEQ ID NO: 54 (AB-4).

[0148] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 8, and b)V L comprises the amino acid sequence of SEQ ID NO: 51 (AB-5).

[0149] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 9, and b)VL comprises the amino acid sequence of SEQ ID NO: 55 (AB-6).

[0150] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 10, and b)V L comprises the amino acid sequence of SEQ ID NO: 56 (AB-7).

[0151] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 11, and b)V L comprises the amino acid sequence of SEQ ID NO: 57 (AB-8).

[0152] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 13, and b)V L comprises the amino acid sequence of SEQ ID NO: 59 (AB-10).

[0153] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 14, and b)V L comprises the amino acid sequence of SEQ ID NO: 60 (AB-11).

[0154] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 15, and b)V L comprises the amino acid sequence of SEQ ID NO: 56 (AB-12).

[0155] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 16, and b)V L comprises the amino acid sequence of SEQ ID NO: 51 (AB-13).

[0156] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 10, and b)V L comprises the amino acid sequence of SEQ ID NO: 50 (AB-14).

[0157] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 17, and b)V L comprises the amino acid sequence of SEQ ID NO: 61 (AB-15).

[0158] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 18, and b)V L comprises the amino acid sequence of SEQ ID NO: 62 (AB-16).

[0159] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 6, and b)V L comprises the amino acid sequence of SEQ ID NO: 63 (AB-17).

[0160] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 19, and b)VL comprises the amino acid sequence of SEQ ID NO: 64 (AB-18).

[0161] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 4, and b)V L comprises the amino acid sequence of SEQ ID NO: 61 (AB-19).

[0162] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 20, and b)V L comprises the amino acid sequence of SEQ ID NO: 61 (AB-20).

[0163] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 21, and b)V L comprises the amino acid sequence of SEQ ID NO: 65 (AB-21).

[0164] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 22, and b)V L comprises the amino acid sequence of SEQ ID NO: 66 (AB-22).

[0165] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 4, and b)V L comprises the amino acid sequence of SEQ ID NO: 67 (AB-23).

[0166] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 23, and b)V L comprises the amino acid sequence of SEQ ID NO: 56 (AB-24).

[0167] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 24, and b)V L comprises the amino acid sequence of SEQ ID NO: 68 (AB-25).

[0168] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 25, and b)V L comprises the amino acid sequence of SEQ ID NO: 51 (AB-26).

[0169] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 26, and b)V L comprises the amino acid sequence of SEQ ID NO: 56 (AB-27).

[0170] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 27, and b)V L comprises the amino acid sequence of SEQ ID NO: 61 (AB-28).

[0171] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 28, and b)V L comprises the amino acid sequence of SEQ ID NO: 56 (AB-29).

[0172] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 28, and b)V L comprises the amino acid sequence of SEQ ID NO: 69 (AB-30).

[0173] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 29, and b)V L comprises the amino acid sequence of SEQ ID NO: 70 (AB-31).

[0174] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 30, and b)V L comprises the amino acid sequence of SEQ ID NO: 71 (AB-32).

[0175] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 31, and b)V L comprises the amino acid sequence of SEQ ID NO: 72 (AB-33).

[0176] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 32, and b)V L comprises the amino acid sequence of SEQ ID NO: 67 (AB-34).

[0177] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 33, and b)V L comprises the amino acid sequence of SEQ ID NO: 56 (AB-35).

[0178] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 34, and b)V L comprises the amino acid sequence of SEQ ID NO: 73 (AB-36).

[0179] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 35, and b)V L comprises the amino acid sequence of SEQ ID NO: 51 (AB-37).

[0180] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 36, and b)V L comprises the amino acid sequence of SEQ ID NO: 56 (AB-38).

[0181] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 37, and b)V L comprises the amino acid sequence of SEQ ID NO: 63 (AB-39).

[0182] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 38, and b)V L comprises the amino acid sequence of SEQ ID NO: 69 (AB-40).

[0183] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 39, and b)V L comprises the amino acid sequence of SEQ ID NO: 74 (AB-41).

[0184] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 40, and b)V L comprises the amino acid sequence of SEQ ID NO: 52 (AB-42).

[0185] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 41, and b)V L comprises the amino acid sequence of SEQ ID NO: 51 (AB-43).

[0186] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 42, and b)V L comprises the amino acid sequence of SEQ ID NO: 75 (AB-44).

[0187] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 43, and b)V L comprises the amino acid sequence of SEQ ID NO: 56 (AB-45).

[0188] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 44, and b)V L comprises the amino acid sequence of SEQ ID NO: 51 (AB-46).

[0189] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 45, and b)V L comprises the amino acid sequence of SEQ ID NO: 75 (AB-47).

[0190] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 46, and b)V L comprises the amino acid sequence of SEQ ID NO: 53 (AB-48).

[0191] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 47, and b)V L comprises the amino acid sequence of SEQ ID NO: 52 (AB-49).

[0192] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 48, and b)V L comprises the amino acid sequence of SEQ ID NO: 76 (AB-50).

[0193] In some embodiments, the polypeptides disclosed herein comprise: a)V H comprises the amino acid sequence of SEQ ID NO: 3, and b)VL comprises the amino acid sequence of SEQ ID NO: 56 (AB-51).

[0194] In some embodiments, the polypeptides disclosed herein include V sequences that are humanized, include human framework regions, or a combination thereof. H and V L Includes:

[0195] In some embodiments, a polypeptide disclosed herein is an immunoglobulin molecule, e.g., an antibody (e.g., a whole antibody, an intact antibody) or an antigen-binding fragment of an antibody (e.g., a Fab, F(ab')2, Fab', scFv, or Fv). As used herein, the term "antibody" refers to an immunoglobulin molecule or portion thereof that can specifically bind to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen recognition site located in the variable domain of the immunoglobulin molecule. In some embodiments, the antibody is a whole antibody or an intact antibody.

[0196] In some embodiments, the polypeptides disclosed herein are single domain antibodies or antigen-binding fragments thereof. As used herein, the term "single domain antibody (sdAb)" or "nanobody" refers to an immunoglobulin molecule that consists of a single monomeric variable antibody domain and is capable of specifically binding to a target. Single domain antibodies can be of any species, such as murine, human, or humanized antibodies.

[0197] In some embodiments, the polypeptides disclosed herein are heavy chain antibodies, or antigen-binding fragments thereof, that comprise two or more heavy chains but lack light chains. Non-limiting examples of heavy chain antibodies include Camelidae Vhh (VHH or VHH). H Camelidae antibodies are antibodies from the camelid family of mammals, which includes llamas, camels, and alpacas.

[0198] In some embodiments, the polypeptides disclosed herein are antibodies or multimers thereof (e.g., IgM) comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable domain (V H ) and a heavy chain constant domain (comprising domains CH1, hinge CH2, and CH3). Each light chain contains a light chain variable domain (V L ) and a light chain constant domain (CL). H and V L The region can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), which are interspersed within the framework regions (FRs). H and V L Each of the CDRs contains three CDRs and four FR segments, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antibody can be of any species, such as a murine antibody, a human antibody, or a humanized antibody.

[0199] The extent of the framework regions and CDRs of an antibody can be identified using one of several suitable methodologies well known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition. Public and / or commercially available tools for identifying framework and / or CDR regions include IgBlast (accessible at www.ncbi.nlm.nih.gov / igblast / ), Scaligner (available from drugdesigntech at www.scaligner.com / ), IMGT rules and / or tools (see, e.g., www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html (also accessible at www.imgt.org / )), Chothia Canonical Assignment (accessible at www.bioinf.org.uk / abs / chothia.html), Antigen Receptor Numbering and Receptor Classification (ABR) (see, e.g., www.bioinf.org.uk / abs / chothia.html), and the Antigen Receptor Numbering and Receptor Classification (ABR). Nucleic Acids Research (ANARCI, available at opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / anarci / ), or the Paratome web server (available at www.ofranlab.org / paratome / ; see Vered Kunik, et al, Nucleic Acids Research, Volume 40, Issue W1, 1 July 2012, Pages W521-W524).

[0200] As used herein, "CDR" encompasses any CDR defined by art-recognized methods for identifying CDR residues on an antibody. See, e.g., Kabat, E.A., et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242; Chothia et al., (1989) Nature 342:877; Chothia, C. et al., (1987) J. Mol. Biol. 196:901-917; Al-lazikani et al., (1997) J. Mol. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. Two antibodies are determined to have the same CDRs as each other with respect to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 if the identity of the CDRs is determined for both antibodies using the same method.

[0201] In some embodiments, the polypeptides disclosed herein are antigen-binding fragments of antibodies. The term "antigen-binding fragment" refers to a portion of an immunoglobulin molecule (e.g., an antibody) that retains the antigen-binding properties of a full-length reference antibody. Non-limiting examples of antigen-binding fragments include V H area, V L region, Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment, and one V H Domain or one V L Examples include domain antibodies (dAbs) that consist of domains. H and V L The domains are linked together via synthetic linkers to form V H / V L Domains are intramolecular or V H and V LWhen the domains are expressed by separate chains, they can pair intermolecularly to form monovalent antigen-binding sites, e.g., single-chain Fvs (scFvs) or diabodies, forming various types of single-chain antibody designs. In some embodiments, the polypeptides disclosed herein are antigen-binding fragments selected from Fab, F(ab')2, Fab', scFv, or Fv. In some embodiments, the polypeptide is an scFv.

[0202] In some embodiments, a polypeptide (e.g., an antibody or antigen-binding fragment) disclosed herein is incorporated into a cell-based therapy. In some embodiments, the polypeptide is an engineered T cell receptor. In some embodiments, the polypeptide is a chimeric antigen receptor (CAR) (e.g., expressed on a T (CAR-T) cell, a natural killer (CAR-NK) cell, or a macrophage (CAR-M) cell). In some embodiments, the CAR comprises a transmembrane domain and an antigen recognition moiety, wherein the antigen recognition moiety binds to SARS-CoV-2 (e.g., an epitope within S2).

[0203] In some embodiments, the polypeptide is an antibody mimetic. The term "antibody mimetic" refers to a polypeptide that can mimic the ability of an antibody to bind to an antigen but that is structurally distinct from the natural antibody structure. Non-limiting examples of antibody mimetics include adnectins, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, nanobodies, nanoCLAMPs, and versabodies.

[0204] In some embodiments, the polypeptides disclosed herein compete with a comparator antibody (e.g., a reference antibody, sotrovimab) for binding to wild-type SARS-CoV-2 spike, a SARS-CoV-2 spike variant, or a combination thereof, where the comparator antibody specifically binds to wild-type SARS-CoV-2 spike (e.g., S2). The terms "specifically binding" or "specifically binds" refer to a preferential interaction, i.e., a significantly higher binding affinity, between an antibody or antigen-binding fragment thereof and its epitope compared to other antigens or amino acid sequences.

[0205] In some embodiments, the comparator antibody is V of SEQ ID NO: 3 H Sequence and V of SEQ ID NO: 50 L In some embodiments, the comparator antibody comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 90, respectively. In some embodiments, the reference antibody comprises the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 133, SEQ ID NO: 141, and SEQ ID NO: 143, respectively. In some embodiments, the comparator antibody comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 90, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 133, SEQ ID NO: 141, and SEQ ID NO: 143, respectively.

[0206] In some embodiments, the polypeptides disclosed herein comprise: a) antibody heavy chain constant domain sequences; b) an antibody light chain constant domain sequence, or c) Both an antibody heavy chain constant domain sequence and an antibody light chain constant domain sequence.

[0207] In some embodiments, the polypeptides disclosed herein comprise an antibody heavy chain constant domain sequence. In some embodiments, the antibody heavy chain constant domain is selected from the group consisting of an IgA constant domain, an IgD constant domain, an IgE constant domain, an IgG constant domain, and an IgM constant domain. In some embodiments, the IgG constant domain is an IgG1 constant domain, an IgG2 constant domain, an IgG3 constant domain, or an IgG4 constant domain. In some embodiments, the IgG2 constant domain is an IgG2a, IgG2b constant domain, or IgG2c constant domain. In some embodiments, the IgA constant domain is an IgA1 constant domain or an IgA2 constant domain. In some embodiments, the antibody heavy chain constant domain is an IgG1 constant domain (e.g., IGHV1-5 or IGHV5-51).

[0208] In some embodiments, the polypeptides disclosed herein comprise an immunoglobulin light chain variable domain (V L ) included. V H and V L The domains are linked together via a linker (e.g., a synthetic linker) to form a V H / V L Domains are intramolecular or V H and V L When the domains are expressed by separate chains, they can pair intermolecularly to form a monovalent antigen-binding site, creating a variety of single-chain antibody designs.

[0209] In some embodiments, the polypeptides disclosed herein comprise an antibody light chain constant domain sequence. In some embodiments, the antibody light chain constant domain is selected from the group consisting of a kappa constant domain and a lambda constant domain. In some embodiments, the antibody heavy chain constant domain is an IgG1 constant domain and the antibody light chain constant domain is a kappa constant domain.

[0210] In some embodiments, the antibody heavy chain constant domain sequence has at least about 60% sequence identity to the amino acid sequence of SEQ ID NO: 194. For example, the antibody heavy chain constant domain sequence may have at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 194. In some embodiments, the antibody heavy chain constant domain sequence has at least about 70% or at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 194. The sequence identified as SEQ ID NO: 194 is shown below:

[0211] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 194).

[0212] In some embodiments, the antibody light chain constant domain sequence has at least about 60% sequence identity to the amino acid sequence of SEQ ID NO: 195 or SEQ ID NO: 196. For example, the antibody light chain constant domain sequence may have at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 195 or SEQ ID NO: 196. In some embodiments, the antibody light chain constant domain sequence has at least about 70% or at least about 80% sequence identity to SEQ ID NO: 195 or SEQ ID NO: 196. The sequences identified as SEQ ID NO: 195 and SEQ ID NO: 196 are shown below:

[0213] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 195).

[0214] GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 196).

[0215] In some embodiments, the antibody heavy chain constant domain sequence comprises at least one amino acid substitution with respect to the amino acid sequence of SEQ ID NO: 194. For example, the number of amino acid substitutions can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or about 1 to 20, 1 to 19, 2 to 19, 2 to 18, 2 to 17, 3 to 17, 3 to 16, 4 to 16, 4 to 15, 5 to 15, 5 to 14, 6 to 14, 6 to 13, 7 to 13, 7 to 12, 8 to 12, 8 to 11, or 9 to 11. In some embodiments, the antibody heavy chain constant domain sequence comprises about 1 to 10 amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 194.

[0216] In some embodiments, the antibody light chain constant domain sequence comprises at least one amino acid substitution with respect to the amino acid sequence of SEQ ID NO: 195 or SEQ ID NO: 196. For example, the number of amino acid substitutions can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or about 1 to 20, 1 to 19, 2 to 19, 2 to 18, 2 to 17, 3 to 17, 3 to 16, 4 to 16, 4 to 15, 5 to 15, 5 to 14, 6 to 14, 6 to 13, 7 to 13, 7 to 12, 8 to 12, 8 to 11, or 9 to 11. In some embodiments, the antibody light chain constant domain sequence comprises about 1 to 10 amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 195 or SEQ ID NO: 196.

[0217] In some embodiments, the amino acid substitutions are conservative substitutions. In some embodiments, the amino acid substitutions are highly conservative substitutions.

[0218] In some embodiments, the polypeptides disclosed herein are isolated polypeptides. In some embodiments, the isolated polypeptides are recombinantly produced. In some embodiments, the isolated polypeptides are synthetically produced.

[0219] In some embodiments, the polypeptides disclosed herein are linked to a second polypeptide. The term "linked" means attached via a covalent bond or a non-covalent interaction. Conjugation can be achieved using a suitable linking agent. Non-limiting examples include peptide linkers, compound linkers, and chemical cross-linkers. In some embodiments, the linker is a disulfide bond.

[0220] In some embodiments, the polypeptides disclosed herein are conjugated to a heterologous moiety. The term "conjugated" refers to attachment via covalent or non-covalent interactions. Conjugation can use any suitable linking agent. Non-limiting examples include peptide linkers, compound linkers, and chemical cross-linkers.

[0221] In some embodiments, the heterologous moiety is a therapeutic agent, a diagnostic agent, or a combination thereof, hi some embodiments, the heterologous moiety is polyethylene glycol (PEG), hexadecanoic acid, a hydrogel, a nanoparticle, a multimerization domain, and a carrier peptide.

[0222] In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are polymeric nanoparticles. In some embodiments, the polymer is an amphiphilic polymer. In some embodiments, the polymer is a hydrophobic or hydrophilic polymer. Non-limiting examples of polymers include poly(lactic acid)-poly(ethylene glycol), poly(lactic acid-co-glycolic acid)-poly(ethylene glycol), poly(lactic-co-glycolic) acid (PLGA), poly(lactic acid-co-glycolic)-d-α-tocopheryl polyethylene glycol succinate, poly(lactic acid-co-glycolic)-ethylene oxide fumarate, poly(glycolic acid)-poly(ethylene glycol), polycaprolactone-poly(ethylene glycol), or any salt thereof. In some embodiments, the polymeric nanoparticles comprise poly(lactic-co-glycolic) acid (PLGA).

[0223] In some embodiments, the carrier polypeptide is albumin or an Fc polypeptide.

[0224] In some embodiments, the polypeptide is a) capable of binding to an epitope within the S2 domain of the SARS-CoV-2 spike, b) a K of 10 μM or less for SARS-CoV-1 and / or SARS-CoV-2 D Combine with c) neutralizing SARS-CoV-1 and / or SARS-CoV-2 infection of human host cells; d) has weaker non-specific binding than the reference antibody; e) has weaker self-association than the reference antibody; Or any combination of the foregoing.

[0225] In some embodiments, the polypeptide can bind to one or more epitope residues within the S2 domain of the SARS-CoV-2 spike, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 residues of the S2 domain. In some embodiments, the polypeptide can bind to one or more epitope residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 residues) selected from F1148, K1149, E1150, L1152, D1153, K1154, F1156, K1157, N1158, H1159, T1160, S1161, P1162, D1163, V1164, and D1165 of SEQ ID NO:1.

[0226] In some embodiments, the polypeptide has a binding constant (K D ) and binds to the SARS-CoV-2 spike. As used herein, the "K" also referred to as the "binding constant," "equilibrium dissociation constant," or "affinity constant" DThe term "reversible binding affinity" is a measure of the degree of reversible association between two molecular species (e.g., an antibody and a target protein) and includes both actual and apparent binding affinity. Binding affinity can be determined using methods known in the art, including, for example, measuring surface plasmon resonance using biolayer interferometry (Octet, ForteBio) or surface plasmon resonance (Biacore) systems and assays. A reference comparing various surface technologies for measuring binding affinity and kinetics is Yang, D., Singh, A., Wu, H., & Kroe-Barrett, R., Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics, Analytical Biochemistry 508:78-96 (2016), the contents of which are incorporated herein by reference in their entirety.

[0227] In some embodiments, the polypeptides include SARS-CoV-1-spike (e.g., of CoV-1 and / or WIV1) and / or SARS-CoV-2-spike (e.g., alpha, beta, gamma, delta, kappa, epsilon, eta, iota, lambda, mu, and / or omicron, e.g., AY.3, AY.4, AY.41, AY.44, AY.64, AY.103, B.1, B.1.1, B.2, B.3, B.4, B.5, B.6, B.7, B.8, B.9, B.10, B.11, B.12, B.13, B.14, B.15, B.16, B.17, B.18, B.19, B.20, B.21, B.22, B.23, B.24, B.25, B.26, B.27, B.28, B.29, B.29, B.30, B.31, B.32, B.33, B.34, B.35, B.36, B.37, B.38, B.39, B.40, B.41, B.42, B.43, B.44, B.45, B.46, B.47, B.48, B.49, B.50, B.51, B.52, B.53, B.54, B.55, B.56, B.57, B.58, B.59, B.60, B.61, B.62, B.63, B.64, B.65, B.66, B.67, B.68, B.69, B.70, B.71, B.72, B.73, B.74, B.7 .1.1.1, B.1.1.529, B.1.1.7, B.1.177, B.1.2, B.1.351, B.1.427 / 429, B.1.525, B.1.526, B.1.533, B.1.617.1 , B.1.617.2, B.1.621, BA.1, BA.1.1, BA.1.15, BA.1.17.2, BA.2, BA.2+P1162L, and BA.2+P1162S, BA.2.3.20, BA. 2.10, BA.2.12.1, BA.2.75, BA.2.75.2, BA.3, BA.4, BA.4 / 5, BA.4 / 5+K444T, BA.4.6, BA.5, BA.5.2.6, BA.5.8, B F.7, BF.11, BN.1, BQ.1, BQ.1.1, C.37, CH.1.1, CH.1.1.1, D.2, GA.5, GR / 484A, P.1, P.1.17, P.1.10, P.2, P.3, Q. 1.3, Q.4, Q.7, XBB, XBB.1.1, XBB.1.16, XBB.1.5, and / or XBB.1.9.1), or fragments thereof (e.g., the S2 domain and / or epitopes of the SARS-CoV-2 spike in Figure 1) with a K of about 5 μM, 2 μM, 1 μM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, or 0.1 nM or less. DIn some embodiments, the polypeptide binds to the SARS-CoV-1 spike (e.g., of CoV-1 and / or WIV1) and / or the SARS-CoV-2 spike (e.g., alpha, beta, gamma, delta, kappa, epsilon, eta, iota, lambda, mu, and / or omicron, e.g., AY.3, AY.4, AY.41, AY.44, AY.64, AY.103, B.1, B.1.1, B.1.1.1, B.1.1.529, B.1.1.7, B.1.177, B.1.2, B.1.351, B.1.427 / 429, B.1.52 5, B.1.526, B.1.533, B.1.617.1, B.1.617.2, B.1.621, BA.1, BA.1.1, BA.1.15, BA.1.17.2, BA.2, BA. 2+P1162L, and BA.2+P1162S, BA.2.3.20, BA.2.10, BA.2.12.1, BA.2.75, BA.2.75.2, BA.3, BA.4, BA.4 / 5, BA.4 / 5+K444T, BA.4.6, BA.5, BA.5.2.6, BA.5.8, BF.7, BF.11, BN.1, BQ.1, BQ.1.1, C.37, CH.1.1, or fragments thereof (e.g., the S2 domain and / or epitopes of the SARS-CoV-2 spike in Figure 1 ) with a K of 100 nM or less. D Combine with.

[0228] In some embodiments, the polypeptides are SARS-CoV-1-spike (e.g., of CoV-1 and / or WIV1) and / or SARS-CoV-2-spike (e.g., alpha, beta, gamma, delta, kappa, epsilon, eta, iota, lambda, mu, and / or omicron, e.g., B.1, B.1.1, B.1.1.1, B.1.1.529, B.1.1.7, B.1.177, B.1.2, B.1.351, B.1.427 / 429, B.1.525, B.1.526, B.1.617.1, 1, B.1.617.2, B.1.621, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.2.75.2, BA.3, BA.4, BA.4 / 5, BA.4 / 5+K444T, BA.4.6, BA5, BA5.2.6, BF.7, BF.11, BN.1, BQ.1, BQ.1.1, C.37, D.2, GA.5, GR / 484A, P.1, P.3, and / or XBB), or a fragment thereof (e.g., the S2 domain and / or epitope of the SARS-CoV-2 spike in Figure 1), -10 ~10 -5 M, 10 -10 ~5x10 -6 Medium, 2x10 -10 ~5x10 -6 Medium, 2x10 -10 ~2x10 -6 Medium, 5x10 -10 ~2x10 -6 Medium, 5x10 -10 ~10 -7 M, 10 -9 ~10 -7 M, 10 -9 ~5x10 -8 Medium, 2x10 -9 ~5x10 -8 Medium, 2x10 -9 ~2x10 -8 Medium, 5x10 -9 ~2x10 -8 M, or 5x10 -9 ~10 -8 K of M D Combine with.

[0229] In some embodiments, the polypeptide (e.g., a full-length IgG1 antibody) is a SARS-CoV-1-spike (e.g., of CoV-1 and / or WIV1) and / or SARS-CoV-2-spike (e.g., alpha, beta, gamma, delta, kappa, epsilon, eta, iota, lambda, mu, and / or omicron, e.g., AY.3, AY.4, AY.41, AY.44, AY.64, AY.103, B.1, B.1.1, B.1.1.1, B.1.1.529, B.1.1.7, B.1.177, B.1.2, B.1.351, B.1.427 / 429, B.1.525, B.1.526, B.1.533, B.1.617.1, B.1.617.2, B.1.621, BA.1, BA.1.1, BA.1.15, BA.1.17. 2, BA.2, BA.2+P1162L, and BA.2+P1162S, BA.2.3.20, BA.2.10, BA.2.12.1, BA.2.75, BA.2.75.2, BA.3, BA.4, BA.4 / 5, BA.4 / 5+K444T, BA.4.6, BA.5, BA.5.2.6, BA.5.8, BF.7, BF.11, BN.1, BQ.1, BQ.1.1, C.37 , CH.1.1, CH.1.1.1, D.2, GA.5, GR / 484A, P.1, P.1.17, P.1.10, P2, P3, Q.3, Q.4, Q.7, XBB, XBB.1.1, XBB.1.16, XBB.1.5, and / or XBB.1.9.1), or fragments thereof (e.g., the S2 domain and / or epitopes of the SARS-CoV-2 spike in Figure 1), -6 K below M D , for example, about 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, or 0.1 nM or less, or about 10 -10 ~10 -6 M, 10 -10 ~5x10 -7 Medium, 2x10 -10 ~5x10 -7 Medium, 2x10 -10 ~2x10 -7 Medium, 5x10 -10 ~2x10 -7 Medium, 5x10 -10 ~10 -7M, 10 -9 ~10 -7 M, 10 -9 ~5x10 -8 Medium, 2x10 -9 ~5x10 -8 Medium, 2x10 -9 ~2x10 -8 Medium, 5x10 -9 ~2x10 -8 M or 5x10 -9 ~10 -8 Join with M.

[0230] In some embodiments, the polypeptide competes with a reference antibody for binding to the SARS-CoV-2 spike (e.g., the S2 domain). Techniques and assays for assessing competition between antibodies are known in the art.

[0231] In some embodiments, the polypeptides are selected from the group consisting of SARS-CoV-1 (e.g., CoV-1 and / or WIV1) and / or SARS-CoV-2 (e.g., alpha, beta, gamma, delta, kappa, epsilon, eta, iota, lambda, mu, and / or omicron, e.g., AY.3, AY.4, AY.41, AY.44, AY.64, AY.103, B.1, B.1.1, B.1.1.1, B.1.1.529, B.1.1.7, B.1.177, B.1.2, B.1.351, B.1.427 / 429, B.1.525, B.1.526, B.1.533, B.1.617.1, B.1.617.2, B.1.621, BA.1, BA.1.1, BA.1.15, BA.1.17.2, BA.2, BA.2+P1162L, and BA.2+P1162S, BA.2.3.20, BA.2.10, BA.2.12.1, BA.2.75 , BA.2.75.2, BA.3, BA.4, BA.4 / 5, BA.4 / 5+K444T, BA.4.6, BA.5, BA.5.2.6, BA.5.8, BF.7, BF .11, BN.1, BQ.1, BQ.1.1, C.37, CH.1.1, CH.1.1.1, D.2, GA.5, GR / 484A, P.1, P.1.17, P1.10, P2, P3, Q.3, Q.4, Q.7, XBB, XBB.1.1, XBB.1.16, XBB.1.5, and / or XBB.1.9.1) with an IC of 10 μM or less. 50 Neutralize with.

[0232] In some embodiments, the polypeptide (e.g., a full-length IgG1 antibody) inhibits SARS-CoV-2 (e.g., alpha, beta, gamma, delta, kappa, epsilon, eta, iota, lambda, mu, and / or omicron, e.g., AY.3, AY.4, AY.41, AY.44, AY.64, AY.103, B.1, B.1.1, B1.1.1, B1.1.529, B1.1.7, B1.177, B1.2, B.1.351, B.1.427 / 429, B.1.525, B1.526, B1.533, B1.617.1, B1.617.2, B1.621, BA.1, BA.1, BA.1.1, BA.1.15, BA.1.17.2, BA. 2, BA.2+P1162L, and BA.2+P1162S, BA.2.3.20, BA.2.10, BA.2.12.1, BA.2.75, BA.2.75.2, B A.3, BA.4, BA.4 / 5, BA.4 / 5+K444T, BA.4.6, BA.5, BA.5.2.6, BA.5.8, BF.7, BF.11, BN.1, BQ. 1, BQ.1.1, C.37, CH.1.1, CH.1.1.1, D.2, GA.5, GR / 484A, P.1, P1.17, P1.10, P2, P3, Q.3, Q.4, Q.7, XBB, XBB.1.1, XBB.1.16, XBB.1.5, and / or XBB.1.9.1) infections at an IC of about 25,000 ng / mL or less. 50 , for example, about 20,000ng / mL, 15,000ng / mL, 10,000ng / mL, 5,000ng / mL, 2,500ng / mL, 1,000ng / mL, 750ng / mL, 500ng / mL, 250ng / mL, 100ng / mL, 75ng / mL, 50ng / mL, 25ng / mL, or 10ng / mL or less, for example, about 10 to 25,000ng / mL, 10 to 20,000ng / mL, 2 Neutralize with 5 to 20,000ng / mL, 25 to 15,000ng / mL, 50 to 15,000ng / mL, 50 to 10,000ng / mL, 75 to 10,000ng / mL, 75 to 5,000ng / mL, 100 to 5,000ng / mL, 100 to 2,500ng / mL, 250 to 2,500ng / mL, 250 to 1,000ng / mL, 500 to 1,000ng / mL, or 500 to 750ng / mL.

[0233] In some embodiments, the polypeptide (e.g., a full-length IgG1 antibody) is capable of inhibiting SARS-CoV-2 (e.g., alpha, beta, gamma, delta, kappa, epsilon, eta, iota, lambda, mu, and / or omicron, e.g., AY.3, AY.4, AY.41, AY.44, AY.64, AY.103, B.1, B.1.1, B1.1.1, B.1.1.529, B.1.1.7, B.1.177, B.1.2, B.1.351, B.1.427 / 429, B.1.525, B.1.526, B.1.533, B.1.617.1, B.1.617.2, B.1.621, BA.1, BA.11, BA.1.15, BA.1.17.2, BA.1.16, BA.1.17.3, BA.1.16 ... .2, BA.2+P1162L, and BA.2+P1162S, BA.2.3.20, BA.2.10, BA.2.12.1, BA.2.75, BA.2.75.2, BA.3, BA.4, BA.4 / 5, BA.4 / 5+K444T, BA.4.6, BA.5, BA.5.2.6, BA.5.8, BF.7, BF.11, BN.1, BQ .1, BQ.1.1, C.37, CH.1.1, CH1.1.1, D.2, GA.5, GR / 484A, P.1, P1.17, P1.10, P2, P3, Q.3, Q.4, Q.7, XBB, XBB.1.1, XBB.1.16, XBB.1.5, and / or XBB.1.9.1) infections at an IC of about 50,000 ng / mL or less. 80, for example, about 25,000ng / mL, 15,000ng / mL, 10,000ng / mL, 5,000ng / mL, 2,500ng / mL, 1,000ng / mL, 750ng / mL, 500ng / mL, 250ng / mL, 100ng / mL, 75ng / mL, 50ng / mL, 25ng / mL, or 10ng / mL or less, for example, about 10 to 50,000ng / mL, 10 to 25,000ng / mL, 2 Neutralize with 5 to 25,000ng / mL, 25 to 15,000ng / mL, 50 to 15,000ng / mL, 50 to 10,000ng / mL, 75 to 10,000ng / mL, 75 to 5,000ng / mL, 100 to 5,000ng / mL, 100 to 2,500ng / mL, 250 to 2,500ng / mL, 250 to 1,000ng / mL, 500 to 1,000ng / mL, or 500 to 750ng / mL.

[0234] In some embodiments, the polypeptide (e.g., a full-length IgG1 antibody) inhibits SARS-CoV-1 (e.g., CoV-1 and / or WIV1) infection of human host cells with an IC of about 25,000 ng / mL or less. 50 , for example, about 20,000ng / mL, 15,000ng / mL, 10,000ng / mL, 5,000ng / mL, 2,500ng / mL, 1,000ng / mL, 750ng / mL, 500ng / mL, 250ng / mL, 100ng / mL, 75ng / mL, 50ng / mL, 25ng / mL, or 10ng / mL or less, for example, about 10 to 25,000ng / mL, 10 to 20,000ng / mL, 2 Neutralize with 5 to 20,000ng / mL, 25 to 15,000ng / mL, 50 to 15,000ng / mL, 50 to 10,000ng / mL, 75 to 10,000ng / mL, 75 to 5,000ng / mL, 100 to 5,000ng / mL, 100 to 2,500ng / mL, 250 to 2,500ng / mL, 250 to 1,000ng / mL, 500 to 1,000ng / mL, or 500 to 750ng / mL.

[0235] In some embodiments, the polypeptide (e.g., a full-length IgG1 antibody) inhibits SARS-CoV-1 (e.g., CoV-1 and / or WIV1) infection of human host cells with an IC of about 50,000 ng / mL or less. 80 , for example, about 25,000ng / mL, 15,000ng / mL, 10,000ng / mL, 5,000ng / mL, 2,500ng / mL, 1,000ng / mL, 750ng / mL, 500ng / mL, 250ng / mL, 100ng / mL, 75ng / mL, 50ng / mL, 25ng / mL, or 10ng / mL or less, for example, about 10 to 50,000ng / mL, 10 to 25,000ng / mL, 2 Neutralize with 5 to 25,000ng / mL, 25 to 15,000ng / mL, 50 to 15,000ng / mL, 50 to 10,000ng / mL, 75 to 10,000ng / mL, 75 to 5,000ng / mL, 100 to 5,000ng / mL, 100 to 2,500ng / mL, 250 to 2,500ng / mL, 250 to 1,000ng / mL, 500 to 1,000ng / mL, or 500 to 750ng / mL.

[0236] In some embodiments, the polypeptide reduces betacoronavirus (e.g., SARS-CoV-2) infectivity of a host cell (e.g., a human host cell). In some embodiments, the polypeptide reduces betacoronavirus (e.g., SARS-CoV-2) infectivity of a host cell (e.g., a human host cell) by at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the polypeptide reduces betacoronavirus (e.g., SARS-CoV-2) infectivity of a human cell by at least about 30%.

[0237] In some embodiments, the polypeptide is capable of inhibiting a betacoronavirus (e.g., SARS-CoV-2 (e.g., alpha, beta, gamma, delta, kappa, epsilon, eta, iota, lambda, mu, and / or omicron, e.g., AY.3, AY.4, AY.41, AY.44, AY.64, AY.103, B.1, B.1.1, B1.1.1, B1.1.529, B1.1.7, B1.177, B1.2, B1.351, B.1.427 / 429, B.1.525, B1.526, B1.533, B1.617.1, B1.617.2, B1.621, BA.1, BA11, BA1.15, BA1.17) in a host cell. .2, BA.2, BA.2+P1162L, and BA.2+P1162S, BA.2.3.20, BA.2.10, BA.2.12.1, BA.2.75, BA.2 .75.2, BA.3, BA.4, BA.4 / 5, BA.4 / 5+K444T, BA.4.6, BA.5, BA.5.2.6, BA.5.8, BF.7, BF.11, Reduce reinfection with BN.1, BQ.1, BQ.1.1, C.37, CH.1.1, CH.1.1.1, D.2, GA.5, GR / 484A, P.1, P.1.17, P.1.10, P.2, P3, Q.3, Q.4, Q.7, XBB, XBB.1.1, XBB.1.16, XBB.1.5, and / or XBB.1.9.1.In some embodiments, the polypeptide is capable of inhibiting the proliferation of a betacoronavirus (e.g., SARS-CoV-2 (e.g., alpha, beta, gamma, delta, kappa, epsilon, eta, iota, lambda, mu, and / or omicron, e.g., AY.3, AY.4, AY.41, AY.44, AY.64, AY.103, B.1, B.1.1, B.1.1.1, B.1. 1.529, B.1.1.7, B.1.177, B.1.2, B.1.351, B.1.427 / 429, B.1.525, B.1.526, B.1.533, B.1.617.1, B.1.617.2 , B.1.621, BA.1, BA.1.1, BA.1.15, BA.1.17.2, BA.2, BA.2+P1162L, and BA.2+P1162S, BA.2.3.20, BA.2.10, BA. 2.12.1, BA.2.75, BA.2.75.2, BA.3, BA.4, BA.4 / 5, BA.4 / 5+K444T, BA.4.6, BA.5, BA.5.2.6, BA.5.8, BF.7, BF. 11, BN.1, BQ.1, BQ.1.1, C.37, CH.1.1, CH.1.1.1, D.2, GA.5, GR / 484A, P.1, P.1.17, P.1.10, P.2, P.3, Q.3, Q.4 , Q.7, XBB, XBB.1.1, XBB.1.16, XBB.1.5, and / or XBB.1.9.1)) reduce reinfection by at least about 10%, for example, by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.In some embodiments, the polypeptide is capable of inhibiting a betacoronavirus (e.g., SARS-CoV-2 (e.g., alpha, beta, gamma, delta, kappa, epsilon, eta, iota, lambda, mu, and / or omicron, e.g., AY.3, AY.4, AY.41, AY.44, AY.64, AY.103, B.1, B.1.1, B1.1.1, B1.1.529, B1.1.7, B.1.177, B.1.2, B.1.351, B.1.427 / 429, B.1.525, B1.526, B1.533, B1.617.1, B1.617.2, B1.621, BA.1, BA.11, BA.1.15, BA.1.17.2, BA.1.17.3, BA.1.17.4, BA.1.17.5, BA.1.17.6, BA.1.17.7, BA.1.17.8, BA.1.17.9, BA.1.17.10, BA.1.17.1 ...12, BA.1.17.13, BA.1.17.14, BA.1.17.15, BA.1.17.16, BA .2, BA.2+P1162L, and BA.2+P1162S, BA.2.3.20, BA.2.10, BA.2.12.1, BA.2.75, BA.2.75.2, BA.3, BA.4, BA.4 / 5, BA.4 / 5+K444T, BA.4.6, BA.5, BA.5.2.6, BA.5.8, BF.7, BF.11, BN.1, BQ .1, BQ.1.1, C.37, CH.1.1, CH.1.1.1, D.2, GA.5, GR / 484A, P.1, P.1.17, P.1.10, P.2, P.3, Q.3, Q.4, Q.7, XBB, XBB.1.1, XBB.1.16, XBB.1.5, and / or XBB.1.9.1)) reduce reinfection by at least about 30%.

[0238] Infectivity or reinfection can be measured using techniques such as pseudovirus neutralization assays or live virus neutralization assays (see, e.g., Pinto et al., Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody, Nature 583:290-95 (2020), the contents of which are incorporated herein by reference). Kits, such as the GenScript cPass™ SARS-CoV-2 Neutralizing Antibody Detection Kit, can be used according to the manufacturer's protocol.

[0239] In some embodiments, the polypeptide reduces SARS-CoV-1 (e.g., CoV-1 and / or WIV1) infectivity of a host cell (e.g., a human host cell). In some embodiments, the polypeptide reduces SARS-CoV-1 (e.g., CoV-1 and / or WIV1) infectivity of a host cell (e.g., a human host cell) by at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the polypeptide reduces SARS-CoV-1 (e.g., CoV-1 and / or WIV1) infectivity of a human cell by at least about 30%.

[0240] In some embodiments, the polypeptide reduces SARS-CoV-1 (e.g., CoV-1 and / or WIV1) reinfection of host cells (e.g., human host cells). In some embodiments, the polypeptide reduces SARS-CoV-1 (e.g., CoV-1 and / or WIV1) reinfection of host cells (e.g., human host cells) by at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the polypeptide reduces SARS-CoV-1 (e.g., CoV-1 and / or WIV1) reinfection of human cells by at least about 30%.

[0241] In some embodiments, the host cells are selected from the group consisting of pulmonary type II pneumocytes, ileal absorptive enterocytes, nasal goblet secretory cells, and combinations thereof.

[0242] In some embodiments, the polypeptide has weaker self-association than a reference antibody, as determined, for example, by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS) values. The AC-SINS value is the shift in the wavelength of maximum absorbance in the absorption spectrum of the coated nanoparticle compared to the spectrum of the nanoparticle alone. Thus, the greater the shift in wavelength of maximum absorbance, the greater the self-interaction of the antibody coated on the nanoparticle. Self-association is an undesirable property that correlates with poor viscosity and poor PK properties. Techniques and assays for assessing protein self-association are known in the art. See, for example, Patro & Przybycien, Biotechnol Bioeng. 52(2)193-203 (1996), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the polypeptide has weaker self-association than a reference antibody.

[0243] In some embodiments, the polypeptide has an AC-SINS value of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 20, 21, 22, 23, 24, or 25 or less. In some embodiments, the polypeptide has an AC-SINS value of about 14 or less. In some embodiments, the polypeptide has an AC-SINS value of about 8 or less. In some embodiments, the polypeptide has an AC-SINS value of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 20, 21, 22, 23, 24, or 25 or less. In some embodiments, the polypeptide has an AC-SINS value of about 0 to 25, e.g., 0 to 20, 0 to 15, 0 to 10, 0 to 8, 0 to 5, 2 to 20, 2 to 15, 2 to 10, 2 to 8, 2 to 5, 5 to 20, 5 to 15, 5 to 10, 5 to 8, 7 to 8, or 13 to 15. In some embodiments, the polypeptide has an AC-SINS value of about 13 to 14, 13 to 15, 7 to 9, or 7 to 8. In some embodiments, the polypeptide has an AC-SINS value of about 8 or 14.

[0244] In some embodiments, the polypeptide has improved developability (e.g., reduced AC-SINS) compared to a reference antibody. In some embodiments, the self-association of the polypeptide is at least about 10% lower than that of the reference antibody, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower than that of the reference antibody. In some embodiments, the self-association of the polypeptide is at least about 30% lower than that of the reference antibody.

[0245] In some embodiments, the self-association of the polypeptide is less than about 90% of that of the reference antibody, e.g., less than about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that of the reference antibody.

[0246] In some embodiments, the self-association of the polypeptide is about 1-90% relative to that of a reference antibody, e.g., about 2-90%, 2-85%, 3-85%, 3-80%, 4-80%, 4-75%, 5-75%, 5-70%, 6-70%, 6-65%, 7-65%, 7-60%, 8-60%, 8-55%, 9-55%, 9-50%, 10-50%, 10-45%, 15-45%, 15-40%, 20-40%, 20-35%, 25-35%, or 25-30% relative to that of the reference antibody.

[0247] In some embodiments, the reduction in self-association compared to a reference antibody is at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0248] Fusion proteins In some embodiments, the present disclosure provides fusion proteins comprising one or more of the polypeptides described herein.

[0249] The term "fusion protein" refers to a synthetic, semi-synthetic, or recombinant single protein molecule. A fusion protein can contain all or part of two or more different proteins and / or polypeptides attached by a covalent bond (e.g., a peptide bond). For example, a fusion protein can contain a full-length polypeptide disclosed herein (e.g., a whole antibody) or a fragment thereof (e.g., an antigen-binding fragment of an antibody). The heterologous partner can be a full-length protein or a fragment thereof (e.g., a truncated protein).

[0250] Fusion proteins can be produced recombinantly or synthetically using routine methods and reagents well known in the art. For example, the fusion proteins disclosed herein can be produced recombinantly in suitable host cells (e.g., bacteria) according to methods known in the art. See, for example, Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, and Molecular Cloning: a Laboratory Manual, 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. For example, a nucleic acid molecule containing a nucleotide sequence encoding a fusion protein described herein can be introduced into a suitable host cell (e.g., E. coli) and expressed, and the expressed fusion protein can be isolated / purified from the host cell (e.g., within inclusion bodies) using routine methods and readily available reagents. For example, DNA fragments encoding different protein sequences (e.g., photoresponsive domains, heterologous peptide components) can be ligated together in frame according to conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. In some embodiments, PCR amplification of nucleic acid fragments can be performed using anchor primers that generate complementary overhangs between two consecutive nucleic acid fragments that can then be annealed and reamplified to generate a chimeric nucleic acid sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992).

[0251] Nucleic acids, vectors, and host cells In some embodiments, the present disclosure provides one or more polynucleotides (e.g., DNA, RNA, or an analog of either, e.g., optionally comprising one or more modified nucleotides; the polynucleotide can be linear or circular, e.g., linear or circular RNA) encoding any one of the polypeptides or fusion proteins described herein. In some embodiments, a polypeptide or fusion protein disclosed herein is encoded by a single polynucleotide. In some embodiments, a polypeptide or fusion protein disclosed herein is encoded by multiple polynucleotides.

[0252] In some embodiments, the polynucleotide comprises a nucleotide sequence that is codon-optimized for a selected host cell.

[0253] In some embodiments, the present disclosure provides vectors (eg, expression vectors, including viral delivery vectors) comprising any one or more of the polynucleotides described herein.

[0254] The term "expression vector" refers to a replicable nucleic acid from which one or more proteins can be expressed when the expression vector is transformed into a suitable expression host cell.

[0255] In some embodiments, a vector (e.g., an expression vector) comprises an expression control polynucleotide sequence, a polynucleotide sequence encoding a selectable marker, or both, operably linked to a polynucleotide. In some embodiments, the expression control polynucleotide sequence comprises a promoter sequence, an enhancer sequence, or both. In some embodiments, the expression control polynucleotide sequence comprises an inducible promoter sequence. The term "promoter" refers to a region of DNA at which RNA polymerase binds to a gene and initiates transcription of the gene. The term "operably linked" means that a nucleic acid is positioned within a recombinant polynucleotide, e.g., a vector, in a manner that allows expression of the nucleic acid under the control of the element (e.g., promoter) to which it is linked. The term "selectable marker element" is an element that confers a trait suitable for artificial selection. The selectable marker element can be a negative or positive selectable marker.

[0256] In some embodiments, the present disclosure provides an expression host cell comprising any one or more of the polynucleotides or expression vectors described herein.

[0257] The term "expression host cell" refers to a cell useful for receiving, maintaining, replicating, and / or propagating a vector.

[0258] Non-limiting examples of expression host cells include mammalian cells such as hybridoma cells, Chinese hamster ovary (CHO) cells, COS cells, human embryonic kidney (HEK), yeast cells such as Pichia pastoris cells, or bacterial cells such as E. coli, including DH5α.

[0259] composition In some embodiments, the present disclosure provides a composition comprising any one of the polypeptides or fusion proteins described herein. In some embodiments, the composition is a pharmaceutical composition.

[0260] In some embodiments, the composition (e.g., pharmaceutical composition) comprises a pharmaceutically acceptable carrier, excipient, stabilizer, diluent, or enhancer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Suitable pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents, or enhancers include buffers (e.g., phosphate, citrate, histidine), antioxidants (e.g., ascorbic acid or methionine), preservatives, proteins (e.g., serum albumin, gelatin, or immunoglobulins), hydrophilic polymers, amino acids, carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose, or dextrins), chelating agents (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (e.g., sodium), metal complexes (e.g., Zn-protein complexes), non-ionic surfactants (e.g., Tween®), PLURONICS™, and polyethylene glycol (PEG).

[0261] In some embodiments, the compositions (e.g., pharmaceutical compositions) disclosed herein are formulated for a suitable administration schedule and route. Non-limiting examples of administration routes include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous, and topical. In some embodiments, the compositions (e.g., pharmaceutical compositions) disclosed herein are stored in the form of an aqueous solution or a dried formulation (e.g., lyophilized).

[0262] In some embodiments, the compositions are formulated to be administered by injection (eg, intravenous injection).

[0263] In some embodiments, the composition is formulated to be administered with a second therapeutic agent as a combination therapy. In some embodiments, the second therapeutic agent is any one of the polypeptides described herein. In some embodiments, the second therapeutic agent comprises bamlanivimab, etesevimab, casirivimab, imdevimab, silgavimab, tixagevimab, AZD7442 (tixagevimab-silgavimab), regdanvimab, or sotrovimab. In some embodiments, the second therapeutic agent comprises sotrovimab.

[0264] How to use In some embodiments, the present disclosure provides a method of neutralizing a SARS-CoV-2 infection in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as an active ingredient, any one of the polypeptides or fusion proteins described herein.

[0265] In some embodiments, the likelihood of SARS-CoV-2 infection in a subject is reduced by at least about 10%, e.g., by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0266] In some embodiments, the likelihood of SARS-CoV-2 infection in a subject in the presence of the polypeptide is about 1-90%, e.g., about 2-90%, 2-85%, 3-85%, 3-80%, 4-80%, 4-75%, 5-75%, 5-70%, 6-70%, 6-65%, 7-65%, 7-60%, 8-60%, 8-55%, 9-55%, 9-50%, 10-50%, 10-45%, 15-45%, 15-40%, 20-40%, 20-35%, 25-35%, or 25-30% of the likelihood in the absence of the polypeptide.

[0267] The terms "subject" and "patient" are used interchangeably herein to refer to an animal (e.g., a mammal such as a human) treated according to the methods disclosed herein. A subject treated according to the methods described herein may be a subject diagnosed with a particular condition (e.g., COVID-19) or a subject at risk for developing such a condition. Diagnosis may be performed by any method or technique known in the art. One of skill in the art will understand that a subject treated according to the present disclosure may be subjected to standard testing, or may be identified without testing, as being at risk due to the presence of one or more risk factors associated with a disease or condition.

[0268] In some embodiments, the subject has or is suspected of having COVID-19 (e.g., confirmed by testing, e.g., PCR or rapid testing). In some embodiments, the subject has COVID-19. In some embodiments, the subject has been diagnosed with COVID-19. In some embodiments, the subject is at risk of developing COVID-19.

[0269] In some embodiments, the subject is a mammal. In some embodiments, the subject is a mammal selected from the group consisting of dog, cat, mouse, rat, hamster, guinea pig, horse, pig, sheep, cow, chimpanzee, macaque, cynomolgus monkey, and human. In some embodiments, the subject is a primate. In some embodiments, the subject is human.

[0270] In some embodiments, the subject has a cardiac disease. In some embodiments, the subject has a cardiac disease selected from the group consisting of congenital heart disease, coronary artery disease, hypertensive heart disease, inflammatory heart disease, pulmonary heart disease, rheumatic heart disease, valvular heart disease, cardiomyopathy, heart failure, and combinations thereof. In some embodiments, the subject has congestive heart failure. In some embodiments, the subject has an inflammatory heart disease selected from the group consisting of endocarditis, cardiomegaly, myocarditis, and combinations thereof.

[0271] In some embodiments, the subject has diabetes.

[0272] In some embodiments, the subject has a pulmonary disease, non-limiting examples of which include acute respiratory distress syndrome, asthma, bronchitis, COPD, emphysema, lung tumors, pleural cavity diseases (e.g., pleural mesothelioma or tension pneumothorax), pulmonary vascular diseases (e.g., embolism, edema, arterial hypertension, or hemorrhage), and respiratory tract infections (e.g., pneumonia or other upper or lower respiratory tract infections).

[0273] In some embodiments, the subject is a tobacco smoker.

[0274] In some embodiments, the subject is immunocompromised (eg, has an underlying disorder or is undergoing immunosuppressive therapy).

[0275] In some embodiments, the subject is 40 years of age or older, e.g., at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 years of age.

[0276] A "therapeutically effective amount," "effective amount," or "effective dosage" is an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result (e.g., treatment, cure, inhibition or amelioration of a physiological response or symptom, reduction in infectivity, reduction in infection upon exposure, prophylaxis, reduction in viral load, etc.). The therapeutic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. A therapeutically effective amount can vary depending on factors such as the disease state, age, sex, and weight of the mammal, mode of administration, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual.

[0277] The effective amount of drug to be administered can be determined by a clinician of ordinary skill using the guidance provided herein and other methods known in the art. Relevant factors include the given drug, pharmaceutical formulation, route of administration, type of disease or disorder, and the identity (e.g., age, sex, and body weight) of the subject or host being treated. For example, a suitable dosage may be about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.01 mg / kg to about 1 mg / kg body weight per treatment. Determining the dosage for a particular drug, subject, and disease is well within the capabilities of one of ordinary skill in the art. Preferably, the dosage will cause no or minimal adverse side effects.

[0278] The desired response or desired outcome can include an effect at the cellular level, tissue level, or clinical outcome. Thus, a "therapeutically effective amount" or its equivalents depends on the context in which it is applied. For example, in some embodiments, it is an amount of a composition sufficient to achieve a therapeutic and / or prophylactic response compared to a response obtained without administration of the composition. In some embodiments, it is an amount that produces a beneficial or desired result in a subject compared to a control. As defined herein, a therapeutically effective amount of a composition disclosed herein can be readily determined by one of skill in the art by routine methods known in the art. Dosage regimens and routes of administration may be adjusted to provide the optimal therapeutic response.

[0279] In some embodiments, the methods disclosed herein are used for prophylactic therapy. In some embodiments, the effective dose is sufficient to prevent a subject from becoming infected with SARS-CoV-2.

[0280] In some embodiments, the methods disclosed herein are used to treat a SARS-CoV-2 infection. The term "treating" or "treatment" refers to the medical management of a subject intended to ameliorate, improve, stabilize (i.e., not worsen), prevent, or cure a disease, pathological condition, infection, or disorder, such as the specific indications exemplified herein. This term includes active treatment (treatment to ameliorate the disease, pathological condition, infection, or disorder), causal treatment (treatment directed at the cause of the associated disease, pathological condition, infection, or disorder), palliative treatment (treatment designed to relieve symptoms), preventative (e.g., prophylactic) treatment (treatment to minimize or partially or completely inhibit the onset of the associated disease, pathological condition, infection, or disorder), and supportive treatment (treatment used to complement another therapy). Treatment also includes the reduction in the extent of a disease or symptom, whether detectable or undetectable, the prevention of the spread of a disease or symptom, the delay or slowing of the progression of a disease or symptom, the improvement or palliation of a disease or symptom, and remission (partial or complete). "Ameliorating" or "alleviating" a disease or condition means reducing the severity and / or undesirable clinical symptoms of the disease, disorder, or condition and / or slowing or prolonging the time course of progression compared to the severity or time course in the absence of treatment. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition, infection, or disorder, as well as those prone to have the condition or disorder, or those in whom the condition, infection, or disorder is to be prevented.

[0281] In some embodiments, an effective dose is sufficient to reduce viral load in a subject, ie, by at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the reduction in viral load is about 10 to 99%, e.g., about 10 to 98%, 15 to 98%, 15 to 97%, 20 to 97%, 20 to 96%, 25 to 96%, 25 to 95%, 30 to 95%, 30 to 94%, 35 to 94%, 35 to 93%, 40 to 93%, 40 to 92%, 45 to 92%, 45 to 91%, 50 to 91%, 50 to 90%, 55 to 90%, 55 to 85%, 60 to 85%, 60 to 80%, 65 to 80%, 65 to 75%, or 70 to 75%.

[0282] In some embodiments, an effective dose is sufficient to inhibit binding of the virus to its target protein, target cell, or both, hi some embodiments, the reduction in binding is at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the reduction in binding is about 10-99%, e.g., about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0283] In some embodiments, an effective dose is sufficient to inhibit virus-mediated fusion with target cells, ie, the reduction in fusion is at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the reduction in fusion is about 10-99%, e.g., about 10-98%, 15-98%, 15-97%, 20-97%, 20-96%, 25-96%, 25-95%, 30-95%, 30-94%, 35-94%, 35-93%, 40-93%, 40-92%, 45-92%, 45-91%, 50-91%, 50-90%, 55-90%, 55-85%, 60-85%, 60-80%, 65-80%, 65-75%, or 70-75%.

[0284] In some embodiments, an effective dosage is sufficient to interfere with a conformational change in a viral envelope protein necessary for cell infectivity.

[0285] The therapeutic agents described herein can be administered via a variety of routes of administration, depending on the compound and the particular disease being treated, including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intraarterial, intravenous, intramuscular, subcutaneous injection, intradermal injection), intravenous infusion, and inhalation (e.g., intrabronchial, intranasal, or oral inhalation, intranasal drops) routes of administration. Administration can be local or systemic, as indicated. The preferred mode of administration can vary depending on the particular compound selected.

[0286] In some embodiments, a polypeptide, composition, or pharmaceutical composition disclosed herein is administered to a subject as a monotherapy.

[0287] In some embodiments, a polypeptide, composition, or pharmaceutical composition disclosed herein is administered to a subject in combination with one or more additional therapeutic agents (e.g., simultaneously or sequentially with one or more additional therapeutic agents) or prophylactic agents (e.g., simultaneously or sequentially with one or more prophylactic agents). In some embodiments, the subject has previously been treated with one or more therapeutic agents prior to being administered a polypeptide, composition, or pharmaceutical composition disclosed herein. In some embodiments, the methods disclosed herein comprise administering a therapeutically effective amount of one or more additional therapeutic agents to the subject simultaneously with or following administration of a polypeptide, composition, or pharmaceutical composition disclosed herein. In some embodiments, the methods disclosed herein comprise administering a therapeutically effective amount of one or more prophylactic agents to the subject prior to, simultaneously with, or following administration of a polypeptide, composition, or pharmaceutical composition disclosed herein. In some embodiments, the subject has previously received a therapeutic or prophylactic agent. In some embodiments, the subject has previously been infected with a betacoronavirus, such as SARS-CoV-2.

[0288] Non-limiting examples of additional therapeutic agents include antibiotics (e.g., azithromycin), antibodies or antigen-binding fragments thereof (e.g., other SARS-CoV-2 binding antibodies or antigen-binding fragments), antimalarials (e.g., chloroquine or hydroxychloroquine), antivirals (e.g., molnupiravir (LAGEVRIO, Merck), PF-07817883 (Pfizer), STI-1558 (Sorrento Therapeutics), PBI-0451 (Pardes Biosciences), EDP-235 (Enanta Pharmaceuticals), favipiravir, lopinavir, and / or ritonavir), cytokines (e.g., type 1 interferons such as interferon beta-1a), nucleotide analogs (e.g., remdesivir), protease inhibitors (e.g., danoprevir), renin-angiotensin-aldosterone system inhibitors (e.g., ACE2 inhibitors or angiotensin receptor blockers (ARBs)).

[0289] In some embodiments, the antiviral agent is selected from the group consisting of molnupiravir (LAGEVRIO, Merck), PF-07817883 (Pfizer), STI-1558 (Sorrento Therapeutics), PBI-0451 (Pardes Biosciences), EDP-235 (Enanta Pharmaceuticals), amantadine, favipiravir, lopinavir, oseltamivir (Tamiflu), pleconaril, rimantadine, ritonavir, antisense RNA against SARS-CoV-2, siRNA against SARS-CoV-2, additional anti-SARS-CoV-2 monoclonal antibodies, and combinations thereof.

[0290] In some embodiments, the antiviral agent is selected from the group consisting of molnupiravir (LAGEVRIO, Merck), PF-07817883 (Pfizer), STI-1558 (Sorrento Therapeutics), PBI-0451 (Pardes Biosciences), EDP-235 (Enanta Pharmaceuticals), and combinations thereof.

[0291] In some embodiments, the additional anti-SARS-CoV-2 antibody targets the S1 domain of the spike protein of SARS-CoV-2. In some embodiments, the additional anti-SARS-CoV-2 antibody targets the class 4 region of the S1 domain. In some embodiments, the additional anti-SARS-CoV-2 monoclonal antibody targets the RBD of the S1 domain of SARS-CoV-2 (e.g., an RBD class 1, 2, 3, or 4 epitope). In some embodiments, the additional anti-SARS-CoV-2 antibody targets the class 3 region of the RBD domain. In some embodiments, the additional anti-SARS-CoV-2 antibody targets the N-terminal domain (NTD) non-supersite region of the S1 domain. In some embodiments, the additional anti-SARS-CoV-2 antibody targets the SD1 region of the S1 domain.

[0292] In some embodiments, the additional anti-SARS-CoV-2 monoclonal antibody targets (e.g., binds to) the S2 domain of the spike protein of SARS-CoV-2. In some embodiments, the additional anti-SARS-CoV-2 monoclonal antibody is a neutralizing monoclonal antibody (e.g., as determined using a neutralization assay described herein or otherwise known in the art). Non-limiting examples of anti-SARS-CoV-2 monoclonal antibodies include bamlanivimab (LY-CoV555 or LY3819253), etesevimab (LY-CoV016 or LY3832479), bebuterovimab (LY-CoV1404, LY3853113), casirivimab (REGN10933), imdevimab (REGN10987), silgavimab, tixagevimab, AZD7442 / Evasheld (tixagevimab-silgavimab), regdanvimab, sotrovimab (Vir Biotechnology, Inc.), ADG20 (Adagio Therapeutics, Inc.), ensovivep (MP0420) (DARPin, Novartis), P2G3 (Aerium Tx), and S2X259 (Tortorici MA, et al. Broad sarbecovirus neutralization by a human monoclonal antibody. Nature. 2021 Sep;597(7874):103-108. doi:10.1038 / s41586-021-03817-4. Epub 2021 Jul 19 PMID:34280951). Additional examples of anti-SARS-CoV-2 antibodies include those described in U.S. Pat. No. 11,168,128, U.S. Pat. No. 11,192,940, WO2022 / 010912 A1, WO2022 / 010921 A1, WO2022 / 047033 A1, WO2021 / 173753 A1, WO2021 / 158521 A1, WO2021 / 203053 A1, WO2021 / 211775 A1, WO2021 / 226560 A1, the contents of which are incorporated herein by reference.Further examples of anti-SARS-CoV-2 antibodies are provided at www.covid19treatmentguidelines.nih.gov / therapies / anti-sars-cov-2-antibody-products / anti-sars-cov-2-monoclonal-antibodies.

[0293] In some embodiments, the subject is further treated (prior, concurrently, or sequentially) with one or more RBD class 4 antibodies (or antigen-binding fragments thereof), such as (e.g., an effective amount of) S2X259 or a variant thereof. Additional examples of SARS-CoV-2 RBD class 4 antibodies include, for example, those described herein as RBD class 4 mAb-1a, RBD class 4 mAb-1b, RBD class 4 mAb-1c, RBD class 4 mAb-1d, RBD class 4 mAb-2a, RBD class 4 mAb-2b, RBD class 4 mAb-2c, RBD class 4 mAb-2d, RBD class 4 mAb-3a, RBD class 4 mAb-3b, RBD class 4 mAb-3c, and RBD class 4 mAb-3d. (having the VH and VL sequences in Tables 10 and 11 herein, respectively), as well as those described in U.S. Patent Application Nos. 63 / 424,947 (filed November 13, 2022), 63 / 383,699 (filed November 14, 2022), 63 / 480,919 (filed January 20, 2023), and 63 / 492,211 (filed March 24, 2023), the entire contents of which are incorporated herein by reference. In some embodiments, the subject is treated with (e.g., an effective amount of) S2X259.

[0294] In some embodiments, the subject is further treated (prior, concurrently, or sequentially) with one or more SARS-CoV-2 RBD class 3 antibodies (or antigen-binding fragments thereof), such as, for example, one or more of sotrovimab, bebuterovimab, AZD1061, P2G3, and eversheld. In some embodiments, the subject is treated with (e.g., an effective amount of) bebuterovimab. In some embodiments, the subject is treated with (e.g., an effective amount of) eversheld. In some embodiments, the subject is treated with (e.g., an effective amount of) tixagevimab. In some embodiments, the subject is treated with (e.g., an effective amount of) silgavimab.

[0295] In some embodiments, the subject is further treated (prior, concurrently, or sequentially) with one or more SARS-CoV-2 N-terminal domain (NTD) non-supersite antibodies (or antigen-binding fragments thereof), such as C1520 and C1717.

[0296] In some embodiments, the subject is further treated (prior, concurrently, or sequentially) with one or more SARS-CoV-2 SD1 antibodies (or antigen-binding fragments thereof), such as S3H3 and P008_60.

[0297] In some embodiments, the ACE2 inhibitor is selected from the group consisting of RNAi against ACE2, siRNA against ACE2, CRISPR-based inhibitors of ACE2, soluble ACE2, soluble ACE2 variants, anti-ACE2 antibodies, vaccines, and combinations thereof. In some embodiments, the antibiotic is azithromycin. In some embodiments, the antimalarial agent comprises chloroquine or hydroxychloroquine. In some embodiments, the vaccine is a nucleic acid vaccine or an inactivated virus vaccine. In some embodiments, the vaccine is mrna-1273, BNT162, INO-4800, AZD1222, Ad5-nCoV, PiCoVacc, NVX-CoV2373, JNJ-78436735, or a combination thereof.

[0298] Administration of two or more therapeutic agents includes co-administration of the therapeutic agents in a substantially simultaneous manner, such as a pharmaceutical combination. In some embodiments, such administration includes co-administration in multiple containers, or in separate containers for each therapeutic agent (e.g., capsules, powders, and liquids). Such administration also includes use of various therapeutic agents in a sequential manner, either at about the same time or at different times. The compositions described herein and the second therapeutic agent may be administered via the same route of administration or via different routes of administration.

[0299] In some embodiments, the present disclosure provides methods for preventing SARS-CoV-2 infection in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as an active ingredient, any one of the polypeptides or fusion proteins described herein.

[0300] In some embodiments, the present disclosure provides methods of treating a SARS-CoV-2 infection in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as an active ingredient, any of the polypeptides or fusion proteins described herein.

[0301] In some embodiments, the present disclosure provides methods for reducing SARS-CoV-2 viral load in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as an active ingredient, any of the polypeptides or fusion proteins described herein.

[0302] In some embodiments, the present disclosure provides a method of inhibiting binding of SARS-CoV-2 to a target cell, comprising contacting the target cell with an effective amount of any of the polypeptides or fusion proteins described herein.

[0303] In some embodiments, the present disclosure provides a method of inhibiting binding of SARS-CoV-2 to a target protein on a target cell, comprising contacting the target cell with an effective amount of any of the polypeptides or fusion proteins described herein.

[0304] In some embodiments, the present disclosure provides a method of inhibiting virus-mediated fusion with a target cell, comprising contacting the target cell with an effective amount of any of the polypeptides or fusion proteins described herein.

[0305] Unless otherwise defined, all technical terms, notations, and other scientific terms or terms used herein are intended to have the meaning commonly understood by those skilled in the art to which this disclosure pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be construed to have a meaning consistent with their meaning in the context of the relevant art and / or as otherwise defined herein.

[0306] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0307] As used herein, the indefinite articles "a," "an," and "the" are to be understood to include plural references unless the context clearly indicates otherwise.

[0308] Unless the context requires otherwise, throughout this specification and in the claims that follow, the word "comprise," as well as variations such as "comprises" or "comprising," will be understood to imply, for example, the inclusion of a stated element or step or group of elements or steps, but not the exclusion of any other element or step or group of elements or steps. As used herein, the term "comprising" can be substituted with the words "containing" or "including."

[0309] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of aspects or embodiments disclosed herein, any of the terms "comprising," "containing," "including," and "having" may, in some embodiments, be substituted with the terms "consisting of" or "essentially consisting of" to vary the scope disclosed herein.

[0310] As used herein, the conjunction term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or," the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to be within the meaning and thus meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the options is also understood to be within the meaning and thus meets the requirements of the term "and / or."

[0311] Where lists are presented, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment unless expressly stated otherwise. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C." For all numerical boundaries describing any parameter in this application (e.g., "about," "at least," "less than," "fewer than," and "more than"), the description necessarily encompasses any range bounded by the recited values. Thus, for example, a description of "at least 1, 2, 3, 4, or 5" also describes ranges such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, and 4 to 5, among others.

[0312] The headings used in this application are for convenience only and have no effect on the interpretation of this application.

[0313] Preferred features of each of the aspects or embodiments provided by the present invention are applicable mutatis mutandis to all other aspects or embodiments of the present invention, as exemplified, without limitation, by the dependent claims, and encompass combinations and permutations of individual features (e.g., elements (including numerical ranges and exemplary embodiments)) of specific embodiments and aspects (including examples) of the present invention. For example, specific experimental parameters illustrated in the examples can be adapted, piecemeal, for use in the claimed invention without departing from the invention. For example, for the disclosed materials, specific reference to each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, but each is specifically contemplated and described herein. Thus, if a class of elements A, B, and C, and a class of elements D, E, and F are disclosed, and an example combination of element A-D is disclosed, each is individually and collectively contemplated, even though each is not individually exemplified. Thus, in this example, each of the combinations AE, AF, BD, BE, BF, CD, CE, and CF is specifically contemplated and should be considered disclosed based on the disclosure of A, B, and C; D, E, and F; and the exemplary combination AD. Likewise, any subset or combination of these is specifically contemplated and disclosed. Thus, for example, subgroups of AE, BF, and CE are specifically contemplated and should be considered disclosed based on the disclosure of A, B, and C; D, E, and F; and the exemplary combination AD. This concept applies to all aspects of the application, including the elements of the subject compositions and steps in methods of making or using the compositions.

[0314] The foregoing aspects of the invention may be claimed in any combination or permutation so long as it is novel and non-obvious over the prior art as would be recognized by one of ordinary skill in the art following the teachings herein, and thus, to the extent that an element is described in one or more references known to those of ordinary skill in the art, it may be excluded from the claimed invention by, among other things, a negative disclaimer or disclaimer of a feature or combination of features. Embodiment 1. A polypeptide that specifically binds to the S2 domain of a spike glycoprotein of a betacoronavirus, the polypeptide having one or more properties selected from the following: Broad neutralizing activity against multiple known and predicted betacoronaviruses, Binding affinity to an epitope in the S2 domain that is highly conserved across multiple betacoronaviruses, and Inhibitory activity against potentially emerging betacoronavirus escape variants. 2. A polypeptide that specifically binds to the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-spike), wherein the polypeptide comprises a paratope substantially similar to the paratope of an antibody comprising an amino acid sequence selected from the following: SEQ ID NO: 4 and SEQ ID NO: 51 (AB-1), SEQ ID NO: 5 and SEQ ID NO: 52 (AB-2), SEQ ID NO: 6 and SEQ ID NO: 53 (AB-3), SEQ ID NO: 7 and SEQ ID NO: 54 (AB-4), SEQ ID NO: 8 and SEQ ID NO: 51 (AB-5), SEQ ID NO: 9 and SEQ ID NO: 55 (AB-6), SEQ ID NO: 10 and SEQ ID NO: 56 (AB-7), SEQ ID NO: 11 and SEQ ID NO: 57 (AB-8), SEQ ID NO: 12 and SEQ ID NO: 58 (AB-9), SEQ ID NO: 13 and SEQ ID NO: 59 (AB-10), SEQ ID NO: 14 and SEQ ID NO: 60 (AB-11), SEQ ID NO: 15 and SEQ ID NO: 56 (AB-12), SEQ ID NO: 16 and SEQ ID NO: 51 (AB-13), SEQ ID NO: 10 and SEQ ID NO: 50 (AB-14), SEQ ID NO: 17 and SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 and SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 and SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 and SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 and SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 and SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 and SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 and SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 and SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 and SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 and SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 and SEQ ID NO: 51 (AB-26), SEQ ID NO: 26 and SEQ ID NO: 56 (AB-27), SEQ ID NO: 27 and SEQ ID NO: 61 (AB-28), SEQ ID NO: 28 and SEQ ID NO: 56 (AB-29), SEQ ID NO: 28 and SEQ ID NO: 69 (AB-30), SEQ ID NO: 29 and SEQ ID NO: 70 (AB-31), SEQ ID NO: 30 and SEQ ID NO: 71 (AB-32), SEQ ID NO: 31 and SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 and SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 and SEQ ID NO: 56 (AB-35), SEQ ID NO: 34 and SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 and SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 and SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 and SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 and SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 and SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 and SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 and SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 and SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 and SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 and SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 and SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 and SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 and SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 and SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 and SEQ ID NO: 56 (AB-51), or Any combination of them. 3. Immunoglobulin heavy chain variable domain (V H ) and immunoglobulin light chain variable domain (V L 3. The polypeptide of embodiment 1 or 2, comprising: 4. A polypeptide that specifically binds to the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-spike), a) an immunoglobulin heavy chain variable domain (V) comprising heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3), each of which is substantially similar to HCDR1, HCDR2, and HCDR3 of any one of the amino acid sequences of SEQ ID NOs: 4 to 48, respectively; H ) an amino acid sequence; b) an immunoglobulin light chain variable domain (V) comprising LCDR1, LCDR2, and LCDR3 that are substantially similar to the light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3), respectively, of any one of the amino acid sequences of SEQ ID NOs: 51 to 76; L ) an amino acid sequence; and 5. The polypeptide of any one of embodiments 1 to 4, comprising HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of an antibody comprising an amino acid sequence selected from the following: SEQ ID NO: 4 and SEQ ID NO: 51 (AB-1), SEQ ID NO: 5 and SEQ ID NO: 52 (AB-2), SEQ ID NO: 6 and SEQ ID NO: 53 (AB-3), SEQ ID NO: 7 and SEQ ID NO: 54 (AB-4), SEQ ID NO: 8 and SEQ ID NO: 51 (AB-5), SEQ ID NO: 9 and SEQ ID NO: 55 (AB-6), SEQ ID NO: 10 and SEQ ID NO: 56 (AB-7), SEQ ID NO: 11 and SEQ ID NO: 57 (AB-8), SEQ ID NO: 12 and SEQ ID NO: 58 (AB-9), SEQ ID NO: 13 and SEQ ID NO: 59 (AB-10), SEQ ID NO: 14 and SEQ ID NO: 60 (AB-11), SEQ ID NO: 15 and SEQ ID NO: 56 (AB-12), SEQ ID NO: 16 and SEQ ID NO: 51 (AB-13), SEQ ID NO: 10 and SEQ ID NO: 50 (AB-14), SEQ ID NO: 17 and SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 and SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 and SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 and SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 and SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 and SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 and SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 and SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 and SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 and SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 and SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 and SEQ ID NO: 51 (AB-26), SEQ ID NO: 26 and SEQ ID NO: 56 (AB-27), SEQ ID NO: 27 and SEQ ID NO: 61 (AB-28), SEQ ID NO: 28 and SEQ ID NO: 56 (AB-29), SEQ ID NO: 28 and SEQ ID NO: 69 (AB-30), SEQ ID NO: 29 and SEQ ID NO: 70 (AB-31), SEQ ID NO: 30 and SEQ ID NO: 71 (AB-32), SEQ ID NO: 31 and SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 and SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 and SEQ ID NO: 56 (AB-35), SEQ ID NO: 34 and SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 and SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 and SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 and SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 and SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 and SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 and SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 and SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 and SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 and SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 and SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 and SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 and SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 and SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 and SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 and SEQ ID NO: 56 (AB-51). 6. The polypeptide of embodiment 4 or 5, comprising a paratope identical to a paratope of an antibody comprising an amino acid sequence selected from the following: SEQ ID NO: 4 and SEQ ID NO: 51 (AB-1), SEQ ID NO: 5 and SEQ ID NO: 52 (AB-2), SEQ ID NO: 6 and SEQ ID NO: 53 (AB-3), SEQ ID NO: 7 and SEQ ID NO: 54 (AB-4), SEQ ID NO: 8 and SEQ ID NO: 51 (AB-5), SEQ ID NO: 9 and SEQ ID NO: 55 (AB-6), SEQ ID NO: 10 and SEQ ID NO: 56 (AB-7), SEQ ID NO: 11 and SEQ ID NO: 57 (AB-8), SEQ ID NO: 12 and SEQ ID NO: 58 (AB-9), SEQ ID NO: 13 and SEQ ID NO: 59 (AB-10), SEQ ID NO: 14 and SEQ ID NO: 60 (AB-11), SEQ ID NO: 15 and SEQ ID NO: 56 (AB-12), SEQ ID NO: 16 and SEQ ID NO: 51 (AB-13), SEQ ID NO: 10 and SEQ ID NO: 50 (AB-14), SEQ ID NO: 17 and SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 and SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 and SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 and SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 and SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 and SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 and SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 and SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 and SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 and SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 and SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 and SEQ ID NO: 51 (AB-26), SEQ ID NO: 26 and SEQ ID NO: 56 (AB-27), SEQ ID NO: 27 and SEQ ID NO: 61 (AB-28), SEQ ID NO: 28 and SEQ ID NO: 56 (AB-29), SEQ ID NO: 28 and SEQ ID NO: 69 (AB-30), SEQ ID NO: 29 and SEQ ID NO: 70 (AB-31), SEQ ID NO: 30 and SEQ ID NO: 71 (AB-32), SEQ ID NO: 31 and SEQ ID NO: 72 (AB-33), SEQ ID NO: 32 and SEQ ID NO: 67 (AB-34), SEQ ID NO: 33 and SEQ ID NO: 56 (AB-35), SEQ ID NO: 34 and SEQ ID NO: 73 (AB-36), SEQ ID NO: 35 and SEQ ID NO: 51 (AB-37), SEQ ID NO: 36 and SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 and SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 and SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 and SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 and SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 and SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 and SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 and SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 and SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 and SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 and SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 and SEQ ID NO: 52 (AB-49), SEQ ID NO: 48 and SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 and SEQ ID NO: 56 (AB-51). 7. An immunoglobulin heavy chain variable domain (V) comprising the amino acid sequence of SEQ ID NO: 2 H 1. A polypeptide that specifically binds to the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-spike), comprising: X1 is not S, X2 is not D, X3 is not T, X4 is not L, The X5 is not an S. The X6 is not an N. The X7 is not a G. X8 is not V, or X9 is not Q, or any combination of the foregoing. 8. An immunoglobulin light chain variable domain (V) comprising the amino acid sequence of SEQ ID NO: 49 L ), X 10 is not Q, X 11is not G, X 12 is not S, X 13 is not S, X 14 is not N, X 15 is not S, X 16 is not F, or X 17 is not Y, 8. The polypeptide of embodiment 7, wherein the polypeptide is any combination of the foregoing. 9. An immunoglobulin heavy chain variable domain (V) comprising the amino acid sequence of SEQ ID NO: 2 H 1. A polypeptide that specifically binds to the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-spike), comprising: X1 is S, N, A, R, L, or F; X2 is D or E; X3 is T or V; X4 is L or V; X5 is S, Q, R, K, Y, D, or E; X6 is N, K, A, S, R, or E; X7 is G, N, or L; X8 is V, I, S, or K; or X9 is Q, Y, K, F, or H; or any combination of the foregoing. 10. X1 is N, A, R, L, or F; X2 is E, X3 is V, X4 is V, X5 is Q, R, K, Y, D, or E; X6 is K, A, S, R, or E; X7 is N or L; X8 is I, S, or K; or X9 is Y, K, F or H; 10. The polypeptide of embodiment 9, wherein the polypeptide is any combination of the foregoing. 11. An immunoglobulin light chain variable domain (V) comprising the amino acid sequence of SEQ ID NO: 49 L ), X 10 is Q, K, or I, X 11 is G or S, X 12 is S, R, or V; X 13 is S or N, X 14 is N, H, D, Y, or S; X 15 is S or Q, X 16 is F, Y, L, V, T, or D, or X 17 is Y or L; 11. The polypeptide of embodiment 9 or 10, or any combination of the foregoing. 12. X 10 is K or I, X 11 is S, X 12 is R or V, X 13 is N, X 14 is H, D, Y, or S; X 15 is Q, X 16 is Y, L, V, T, or D, or X 17 is L, 12. The polypeptide of embodiment 11, or any combination of the foregoing. 13.V HThe polypeptide according to any one of embodiments 1 to 4 and 6 to 12, comprising heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3), whose amino acid sequences are identical to those of heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3), respectively, of any one of SEQ ID NOs: 4 to 48. 14.V L The polypeptide according to any one of embodiments 1 to 4 and 6 to 13, comprising LCDR1, LCDR2, and LCDR3 whose amino acid sequences are identical to light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3), respectively, of any one of SEQ ID NOs: 51 to 76. 15. The polypeptide is selected from SEQ ID NO: 4 / SEQ ID NO: 51 (AB-1), SEQ ID NO: 5 / SEQ ID NO:52 (AB-2), SEQ ID NO:6 / SEQ ID NO:53 (AB-3), SEQ ID NO:7 / SEQ ID NO:54 (AB-4), SEQ ID NO:8 / SEQ ID NO:51 (AB-5), SEQ ID NO:9 / SEQ ID NO:55 (AB-6), SEQ ID NO:10 / SEQ ID NO:56 (AB-7), SEQ ID NO:11 / SEQ ID NO:57 (AB-8), SEQ ID NO:12 / SEQ ID NO:58 (AB-9), SEQ ID NO:13 / SEQ ID NO:59 (AB-10), SEQ ID NO:14 / SEQ ID NO:60 (AB-11), SEQ ID NO:15 / SEQ ID NO:56 (AB-12), SEQ ID NO:16 / SEQ ID NO:51 (AB-13), SEQ ID NO:10 / SEQ ID NO:50 (AB-14), SEQ ID NO: 17 / SEQ ID NO: 61 (AB-15), SEQ ID NO: 18 / SEQ ID NO: 62 (AB-16), SEQ ID NO: 6 / SEQ ID NO: 63 (AB-17), SEQ ID NO: 19 / SEQ ID NO: 64 (AB-18), SEQ ID NO: 4 / SEQ ID NO: 61 (AB-19), SEQ ID NO: 20 / SEQ ID NO: 61 (AB-20), SEQ ID NO: 21 / SEQ ID NO: 65 (AB-21), SEQ ID NO: 22 / SEQ ID NO: 66 (AB-22), SEQ ID NO: 4 / SEQ ID NO: 67 (AB-23), SEQ ID NO: 23 / SEQ ID NO: 56 (AB-24), SEQ ID NO: 24 / SEQ ID NO: 68 (AB-25), SEQ ID NO: 25 / SEQ ID NO: 51 (AB-26), SEQ ID NO:26 / SEQ ID NO:56 (AB-27), SEQ ID NO:27 / SEQ ID NO:61 (AB-28), SEQ ID NO:28 / SEQ ID NO:56 (AB-29), SEQ ID NO:28 / SEQ ID NO:69 (AB-30), SEQ ID NO:29 / SEQ ID NO:70 (AB-31), SEQ ID NO:30 / SEQ ID NO:71 (AB-32), SEQ ID NO:31 / SEQ ID NO:72 (AB-33), SEQ ID NO:32 / SEQ ID NO:67 (AB-34), SEQ ID NO:33 / SEQ ID NO:56 (AB-35), SEQ ID NO:34 / SEQ ID NO:73 (AB-36), SEQ ID NO:35 / SEQ ID NO:51 (AB-37), SEQ ID NO:3 6 / SEQ ID NO: 56 (AB-38), SEQ ID NO: 37 / SEQ ID NO: 63 (AB-39), SEQ ID NO: 38 / SEQ ID NO: 69 (AB-40), SEQ ID NO: 39 / SEQ ID NO: 74 (AB-41), SEQ ID NO: 40 / SEQ ID NO: 52 (AB-42), SEQ ID NO: 41 / SEQ ID NO: 51 (AB-43), SEQ ID NO: 42 / SEQ ID NO: 75 (AB-44), SEQ ID NO: 43 / SEQ ID NO: 56 (AB-45), SEQ ID NO: 44 / SEQ ID NO: 51 (AB-46), SEQ ID NO: 45 / SEQ ID NO: 75 (AB-47), SEQ ID NO: 46 / SEQ ID NO: 53 (AB-48), SEQ ID NO: 47 / SEQ ID NO: 52 (AB-49),V selected from the amino acid sequences of SEQ ID NO: 48 / SEQ ID NO: 76 (AB-50), or SEQ ID NO: 3 / SEQ ID NO: 56 (AB-51), H / V L 15. The polypeptide of any one of embodiments 1 to 5 and 7 to 14, comprising a paratope that is identical to the paratope of the combination of 16.V H 16. The polypeptide of any one of embodiments 1 to 15, wherein the polypeptide has at least 85% sequence identity to any one or more amino acid sequences of SEQ ID NOs: 4 to 48. 17.V H 17. The polypeptide of any one of embodiments 1 to 16, comprising about 1 to 10 amino acid substitutions relative to any one or more amino acid sequences of SEQ ID NOs: 4 to 48. 18.V L 18. The polypeptide of any one of embodiments 1 to 17, wherein the polypeptide has at least 85% sequence identity to any one or more amino acid sequences of SEQ ID NOs: 51 to 76. 19.V L 19. The polypeptide of any one of embodiments 1 to 18, comprising about 1 to 10 amino acid substitutions relative to the amino acid sequence of any one or more of SEQ ID NOs: 51 to 76. 20. The polypeptide of embodiment 17 or 19, wherein the amino acid substitutions are conservative substitutions. 21. The polypeptide of embodiment 20, wherein the amino acid substitutions are highly conservative substitutions. twenty two. a)V H comprises the amino acid sequence of SEQ ID NO: 4, b)V L 21. The polypeptide according to any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 51 (AB-1). twenty three. a)V H comprises the amino acid sequence of SEQ ID NO:5, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 52 (AB-2). twenty four. a)V H comprises the amino acid sequence of SEQ ID NO: 6, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 53 (AB-3). twenty five. a)V H comprises the amino acid sequence of SEQ ID NO: 7, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 54 (AB-4). 26. a)V H comprises the amino acid sequence of SEQ ID NO: 8, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 51 (AB-5). 27. a)V H comprises the amino acid sequence of SEQ ID NO: 9, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 55 (AB-6). 28. a)V H comprises the amino acid sequence of SEQ ID NO: 10; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 56 (AB-7). 29. a)V H comprises the amino acid sequence of SEQ ID NO: 11; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 57 (AB-8). 30. a)V H comprises the amino acid sequence of SEQ ID NO: 12; b)V L21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 30 (AB-9). 31. a)V H comprises the amino acid sequence of SEQ ID NO: 13, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 59 (AB-10). 32. a)V H comprises the amino acid sequence of SEQ ID NO: 14, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 60 (AB-11). 33. a)V H comprises the amino acid sequence of SEQ ID NO: 15; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 56 (AB-12). 34. a)V H comprises the amino acid sequence of SEQ ID NO: 16; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 51 (AB-13). 35. a)V H comprises the amino acid sequence of SEQ ID NO: 10; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 50 (AB-14). 36. a)V H comprises the amino acid sequence of SEQ ID NO: 17, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 61 (AB-15). 37. a)V Hcomprises the amino acid sequence of SEQ ID NO: 18; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 62 (AB-16). 38. a)V H comprises the amino acid sequence of SEQ ID NO: 6, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 63 (AB-17). 39. a)V H comprises the amino acid sequence of SEQ ID NO: 19, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 64 (AB-18). 40. a)V H comprises the amino acid sequence of SEQ ID NO: 4, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 61 (AB-19). 41. a)V H comprises the amino acid sequence of SEQ ID NO: 20; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 61 (AB-20). 42. a)V H comprises the amino acid sequence of SEQ ID NO: 21; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 65 (AB-21). 43. a)V H comprises the amino acid sequence of SEQ ID NO: 22; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 66 (AB-22). 44. a)V H comprises the amino acid sequence of SEQ ID NO: 4, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 67 (AB-23). 45. a)V H comprises the amino acid sequence of SEQ ID NO: 23, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 56 (AB-24). 46. a)V H comprises the amino acid sequence of SEQ ID NO: 24, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 68 (AB-25). 47. a)V H comprises the amino acid sequence of SEQ ID NO: 25; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 51 (AB-26). 48. a)V H comprises the amino acid sequence of SEQ ID NO: 26; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 56 (AB-27). 49. a)V H comprises the amino acid sequence of SEQ ID NO: 27; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 61 (AB-28). 50. a)V H comprises the amino acid sequence of SEQ ID NO: 28; b)V L21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 56 (AB-29). 51. a)V H comprises the amino acid sequence of SEQ ID NO: 28; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 69 (AB-30). 52. a)V H comprises the amino acid sequence of SEQ ID NO: 29; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 70 (AB-31). 53. a)V H comprises the amino acid sequence of SEQ ID NO: 30; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 71 (AB-32). 54. a)V H comprises the amino acid sequence of SEQ ID NO: 31; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 72 (AB-33). 55. a)V H comprises the amino acid sequence of SEQ ID NO: 32; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 67 (AB-34). 56. a)V H comprises the amino acid sequence of SEQ ID NO: 33; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 56 (AB-35). 57. a)V Hcomprises the amino acid sequence of SEQ ID NO: 34; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 73 (AB-36). 58. a)V H comprises the amino acid sequence of SEQ ID NO: 35; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 51 (AB-37). 59. a)V H comprises the amino acid sequence of SEQ ID NO: 36; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 56 (AB-38). 60. a)V H comprises the amino acid sequence of SEQ ID NO: 37; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 63 (AB-39). 61. a)V H comprises the amino acid sequence of SEQ ID NO: 38; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 69 (AB-40). 62. a)V H comprises the amino acid sequence of SEQ ID NO: 39; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 74 (AB-41). 63. a)V H comprises the amino acid sequence of SEQ ID NO: 40; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 52 (AB-42). 64. a)V H comprises the amino acid sequence of SEQ ID NO: 41; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 51 (AB-43). 65. a)V H comprises the amino acid sequence of SEQ ID NO: 42; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 75 (AB-44). 66. a)V H comprises the amino acid sequence of SEQ ID NO: 43; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 56 (AB-45). 67. a)V H comprises the amino acid sequence of SEQ ID NO: 44; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 51 (AB-46). 68. a)V H comprises the amino acid sequence of SEQ ID NO: 45; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 75 (AB-47). 69. a)V H comprises the amino acid sequence of SEQ ID NO: 46; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 53 (AB-48). 70. a)V H comprises the amino acid sequence of SEQ ID NO: 47; b)V L21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 52 (AB-49). 71. a)V H comprises the amino acid sequence of SEQ ID NO: 48; b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 76 (AB-50). 72. a)V H comprises the amino acid sequence of SEQ ID NO: 3, b)V L 21. The polypeptide of any one of embodiments 1 to 20, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 56 (AB-51). 73.V H and V L 73. The polypeptide of any one of embodiments 3 to 72, wherein said polypeptide is humanized, comprises human framework regions, or a combination thereof. 74. The polypeptide of any one of embodiments 1-73, wherein the polypeptide is an antibody or an antigen-binding fragment thereof. 75. The polypeptide of embodiment 74, wherein the antigen-binding fragment is selected from Fab, F(ab')2, Fab', scFv, or Fv. 76. The polypeptide of embodiment 74, comprising an antibody heavy chain constant domain sequence, an antibody light chain constant domain sequence, or both an antibody heavy chain constant domain sequence and an antibody light chain constant domain sequence. 77. The polypeptide of embodiment 76, wherein the antibody heavy chain constant domain is selected from the group consisting of an IgA constant domain, an IgD constant domain, an IgE constant domain, an IgG constant domain, and an IgM constant domain. 78. The polypeptide of embodiment 77, wherein the antibody heavy chain constant domain is an IgG1 heavy chain constant domain. 79. The polypeptide of any one of embodiments 76-78, comprising an antibody light chain constant domain selected from the group consisting of a kappa constant domain or a lambda constant domain. 80. The polypeptide of embodiment 79, wherein the antibody light chain constant domain is a kappa light chain constant domain. 81. The polypeptide of any one of embodiments 1-80, wherein the polypeptide is conjugated to a heterologous moiety. 82. The polypeptide of embodiment 81, wherein the heterologous moiety is a therapeutic agent, a diagnostic agent, or a combination thereof. 83. The polypeptide of embodiment 81, wherein the heterologous moiety is selected from the group consisting of polyethylene glycol (PEG), hexadecanoic acid, hydrogels, lipid nanoparticles, polymer nanoparticles, and heterologous polypeptide sequences, or combinations thereof. 84. The polypeptide of embodiment 83, wherein the polymer nanoparticles comprise poly(lactic-co-glycolic) acid (PLGA). 85. The polypeptide of embodiment 81, wherein the heterologous polypeptide sequence comprises a carrier polypeptide. 86. The polypeptide of embodiment 85, wherein the carrier polypeptide is albumin or an Fc polypeptide. 87. A polypeptide is a) SARS-CoV-2 K of 1 μM or less D Combine with b) SARS-CoV-2 infection of human host cells with an IC of approximately 25,000 ng / mL or less 50 Neutralize with c) reducing the infectivity of SARS-CoV-2 in human cells; 87. The polypeptide of any one of embodiments 1 to 86, wherein the polypeptide is any one of the foregoing combinations. 88. The polypeptide of embodiment 87, wherein the SARS-CoV-2 is a variant comprising T19R, 156del, 157del, R158G, L452R, T478K, D614G, P681R, and D950N, and optionally G142D. 89. The polypeptide binds to SARS-CoV-2 with a K of 100 nM or less. D 89. The polypeptide of embodiment 87 or 88, wherein the polypeptide binds at 90. The polypeptide inhibits SARS-CoV-2 infection of human host cells with an IC of about 25,000 ng / mL or less. 50 88. The polypeptide of embodiment 87, wherein said polypeptide is neutralized by 91. The polypeptide of embodiment 87, wherein the polypeptide reduces the infectivity of SARS-CoV-2 in human cells by at least about 30%. 92. A fusion protein comprising a polypeptide according to any one of embodiments 1 to 91. 93. A polynucleotide (e.g., DNA or RNA, linear or circular, optionally containing one or more modified nucleotides) comprising a sequence encoding a polypeptide according to any one of embodiments 1 to 80 or a fusion protein according to embodiment 92. 94. A vector (e.g., an expression vector, including a viral delivery vector) comprising the polynucleotide of embodiment 93. 95. A host cell comprising a polynucleotide according to embodiment 93 or a vector according to embodiment 94. 96. A composition comprising a polypeptide according to any one of embodiments 1 to 91, or a fusion protein according to embodiment 92, or a polynucleotide according to embodiment 93. 97. The composition of embodiment 96, comprising one or more pharmaceutical excipients, diluents, or carriers. 98. A method of treating a subject in need thereof, comprising administering to the subject an effective amount of the composition of embodiment 96 or 97. 99. A method for reducing the infectivity of a betacoronavirius, such as SARS-CoV-2, in a cell in a subject, comprising contacting the cell with an effective amount of the composition of embodiment 96 or 97. 100. The method of embodiment 98 or 99, wherein the subject has (e.g., confirmed by testing, e.g., PCR or rapid testing) or is suspected of having COVID-19. 101. The method of embodiment 98 or 99, wherein the subject is at risk of developing COVID-19. 102. The method of any one of embodiments 98-101, wherein the subject is a human. 103. The method of any one of embodiments 98-102, wherein the subject has a heart disease. 104. The method of embodiment 103, wherein the cardiac disease is selected from the group consisting of congestive heart disease, coronary artery disease, hypertensive heart disease, inflammatory heart disease, pulmonary heart disease, rheumatic heart disease, valvular heart disease, cardiomyopathy, heart failure, and combinations thereof. 105. The method of embodiment 104, wherein the heart failure is congestive heart failure. 106. The method of embodiment 104, wherein the inflammatory heart disease is selected from the group consisting of endocarditis, cardiac hypertrophy, myocarditis, and combinations thereof. 107. The method of any one of embodiments 98-106, wherein the subject has diabetes. 108. The method of any one of embodiments 98-107, wherein the subject has a pulmonary disease. 109. The method of embodiment 108, wherein the pulmonary disease is selected from the group consisting of acute respiratory distress syndrome, asthma, bronchitis, COPD, emphysema, lung tumors, pleural cavity diseases, pulmonary vascular diseases, respiratory tract infections, and combinations thereof. 110. a) the respiratory tract infection is an upper respiratory tract infection, a lower respiratory tract infection, or pneumonia; b) The pleural cavity disease is pleural mesothelioma or tension pneumothorax. c) the pulmonary vascular disease is embolic, edema, arterial hypertension, or hemorrhage; or d) The method of embodiment 109, which is a combination thereof. 111. The method of any one of embodiments 98-110, wherein the subject is a tobacco smoker. 112. The method of any one of embodiments 98-111, wherein the subject has a weakened immune system. 113. The method of embodiment 112, wherein the subject is undergoing immunosuppressive therapy. 114. The method of any one of embodiments 98-113, wherein the subject is 40 years of age or older. 115. The method of any one of embodiments 98-114, comprising administering to the subject a therapeutically effective amount of an additional therapeutic or prophylactic agent. 116. The method of embodiment 115, wherein the additional therapeutic agent is selected from the group consisting of an antiviral agent, an ACE2 inhibitor, an additional SARS-CoV-2-spike binding antibody, an antibiotic, an antimalarial agent, a vaccine, and combinations thereof. 117. a) the additional SARS-CoV-2-spike binding antibody is selected from the group consisting of bamlanivimab, etesevimab, bebuterovimab, casirivimab, imdevimab, silgavimab, tixagevimab, AZD7442 (tixagevimab-silgavimab), regdanvimab, sotrovimab, and combinations thereof; b) the antiviral agent is selected from the group consisting of molnupiravir (LAGEVRIO, Merck), PF-07817883 (Pfizer), STI-1558 (Sorrento Therapeutics), PBI-0451 (Pardes Biosciences), EDP-235 (Enanta Pharmaceuticals), oseltamivir (Tamiflu), favipiravir, amantadine, remdesivir, rimantadine, pleconaril, antisense RNA against SARS-CoV-2, siRNA against SARS-CoV-2, and combinations thereof; c) the ACE2 inhibitor is selected from the group consisting of RNAi against ACE2, siRNA against ACE2, CRISPR-based inhibitor of ACE2, soluble ACE2, soluble ACE2 variant, anti-ACE2 antibody, and combinations thereof; d) the antibiotic comprises azithromycin; e) the antimalarial agent comprises chloroquine; f) the vaccine is a nucleic acid vaccine or an inactivated virus vaccine; or g) The method of embodiment 116, which is a combination thereof. 118. The method of embodiment 117, wherein the vaccine is mrna-1273, BNT162, INO-4800, AZD1222, Ad5-nCoV, PiCoVacc, NVX-CoV2373, JNJ-78436735, or a combination thereof. 119. The method of any one of embodiments 98 to 118, wherein the subject is further treated (prior, concurrently, or sequentially) with one or more RBD class 4 antibodies (or antigen-binding fragments thereof). 120. The one or more RBD class 4 antibodies include S2X259, RBD class 4 mAb-1a having a VH comprising SEQ ID NO: 174 and a VL comprising SEQ ID NO: 178, RBD class 4 mAb-1b having a VH comprising SEQ ID NO: 174 and a VL comprising SEQ ID NO: 179, RBD class 4 mAb-1c having a VH comprising SEQ ID NO: 174 and a VL comprising SEQ ID NO: 180, RBD class 4 mAb-1d having a VH comprising SEQ ID NO: 174 and a VL comprising SEQ ID NO: 181, RBD class 4 mAb-2a having a VH comprising SEQ ID NO: 175 and a VL comprising SEQ ID NO: 182, RBD class 4 mAb-2b having a VH comprising SEQ ID NO: 175 and a VL comprising SEQ ID NO: 183, RBD class 4 mAb-2c having a VH comprising SEQ ID NO: 175 and a VL comprising SEQ ID NO: 184, b-2c, RBD class 4 mAb-2d having a VH comprising SEQ ID NO: 175 and a VL comprising SEQ ID NO: 185, RBD class 4 mAb-3a having a VH comprising SEQ ID NO: 176 and a VL comprising SEQ ID NO: 186, RBD class 4 mAb-3b having a VH comprising SEQ ID NO: 176 and a VL comprising SEQ ID NO: 187, RBD class 4 mAb-3c having a VH comprising SEQ ID NO: 176 and a VL comprising SEQ ID NO: 188, RBD class 4 mAb-3d having a VH comprising SEQ ID NO: 176 and a VL comprising SEQ ID NO: 189, and RBD class 4 mAb-4a having a VH comprising SEQ ID NO: 177 and a VL comprising SEQ ID NO: 190, a variant of any of the foregoing, or a combination of any of the foregoing. 121. The method of any one of embodiments 98-120, wherein the subject has previously received a therapeutic or prophylactic agent. 122. The method of any one of embodiments 98 to 121, wherein the subject has previously been infected with a betacoronavirus, such as SARS-CoV-2. 123. The method of any one of embodiments 98-122, comprising administering to the subject an effective amount of a combination of AB-1 and sotrovimab. 124. The method of any one of embodiments 98-122, comprising administering to the subject an effective amount of a combination of AB-1 and bebuterovimab. 125. The method of any one of embodiments 98-122, comprising administering to the subject an effective amount of a combination of AB-1 and Evasheld. 126. The method of any one of embodiments 98-122, comprising administering to the subject an effective amount of a combination of AB-1 and AZD-1061. 127. The method of any one of embodiments 98-122, comprising administering to the subject an effective amount of a combination of AB-1 and P2G3. 128. The method of any one of embodiments 98-122, comprising administering to the subject an effective amount of a combination of AB-1 and RBD class 4 mAb-1a.

[0315] Example Example 1. Materials and Methods Binding assay (DELFIA) Polystyrene MaxiSorp plates (ThermoFisher, Waltham, MA, catalog number 460372) were coated with 5 μg / ml of spike protein diluted in phosphate-buffered saline (PBS) and incubated overnight at 4°C. The plates were washed with Tris-buffered saline Tween® 20 buffer (TBS-T) (ThermoFisher, Waltham, MA, catalog number 28360) and blocked with assay diluent (BioLegend, San Diego, CA, catalog number 421205) for 1 hour at room temperature. Serial dilutions (1:4) of antibodies were made in PBS / bovine serum albumin (BSA) starting at 4.5 μg / ml. After one wash with TBS-T, the serially diluted antibodies were transferred to the pre-coated plates and incubated for 1 hour at room temperature. Plates were then washed three times with TBS-T, and europium-labeled secondary antibodies (PerkinElmer, Waltham, MA, catalog number 1244-330) were added for 30 minutes at room temperature. After incubation, plates were washed three times with TBS-T, and enhancement solution (PerkinElmer, Waltham, MA, catalog number 4001-0010) was applied. Time-resolved fluorescence was read at 615 nm using an EnVision plate reader (PerkinElmer, Waltham, MA).

[0316] Pseudovirus neutralization assay For pseudovirus neutralization, Vero-TMPRSS2 cells were plated at 3.5 × 10 in tissue culture-treated 384-well plates (ThermoFisher, Waltham, MA, catalog no. 164610). 3Cells were plated at a density of 1000 cells / well in a volume of 20 μl. Plates were briefly spun down at <50 g and incubated for 2-4 hours at 37°C, 5% CO2. To generate 5-, 10-, or 12-point titration curves, antibodies were serially diluted 3-, 4-, or 6-fold in PBS / 0.2% BSA / 1x penicillin-streptomycin (Pen-Strep) buffer starting at 72 μg / ml or 18 μg / ml (4x final concentration) in round-bottom 96-well plates (ThermoFisher, Waltham, MA, catalog no. 268200). The antibodies were mixed with an equal volume of diluted SARS-CoV-2 pseudovirus (lentivirus pseudotyped with SARS-CoV-2 delta, BA.1, SARS-CoV-1, or WIV1 spikes, and VSV-dG pseudotyped with SARS-CoV-2 D614G, delta, BA.2, BA.2.12.1, BA.4 / 5, BA.4 / 5+K444T, BQ.1, BQ.1.1, XBB.1.5, SARS-CoV-1, or WIV1 spikes). The antibody-virus mixture was incubated at 37°C and 5% CO for 30–60 min. Then, 20 μl of the antibody-virus mixture was transferred to a 384-well plate pre-seeded with Vero-TMPRSS2 cells. The 384-well plates were briefly spun down at <50g and incubated at 37°C, 5% CO2 for 24 hours (VSV-dG) or 72 hours (lentivirus). At the end of the incubation, 40 μl of luciferase detection buffer (BPS Bioscience ONE-Step™ Luciferase Assay System, BPS Bioscience, San Diego, CA, catalog number 60690-3) was added to each well of the cell culture plate. The plates were centrifuged at 50g for <5 seconds and incubated for 15 minutes with gentle rocking. Luminescence signals were recorded using an Envision plate reader. Results were expressed as percentage neutralization and analyzed using Prism 9. Curves were generated by fitting the data using the following equation: log (inhibitor) vs. normalized response—variable slope (4 parameters).

[0317] SARS-CoV-2 infection in vivo In vivo experiments were performed at Bioqual. Male hamsters were injected intraperitoneally with the indicated antibodies (variable regions [VH / VL] expressed as human IgG1 [huIgG1] or hamster IgG2a [hamIgG2a]) one day before intranasal inoculation with SARS-CoV-2 delta. Body weights were recorded daily until day 7 and reported as percentage change relative to day 0 body weight. On day 7, hamsters were sacrificed, lungs were harvested, and their weights were recorded. Viral titers in the nasal cavity and lungs (day 4 post-infection) were recorded, and lung histopathology was performed (day 7 post-infection).

[0318] Live virus neutralization assay Live virus neutralization assays were performed at the Virology Research Service. SARS-CoV-2 Delta, BA.1, or BA.5 live virus was incubated with 3- or 4-fold serial dilutions of each antibody for 1 hour, after which the mixture was added to Vero cells. Antiviral activity was determined after 6 hours using an immunofluorescence-based assay. Results were expressed as percentage neutralization and analyzed using Prism 9. Curves were generated by fitting the data using the following equation: log(inhibitor) vs. normalized response—variable slope (4 parameters).

[0319] Example 2. Generation and characterization of S2-binding polypeptides The S2 domain of the SARS-CoV-2 spike protein contains the fusion machinery and a sequence that is similarly conserved across SARS-CoV-2 variants and the Sarbecovirus subgenus. One of these sequences is a stem-helix peptide that is targeted by antibodies with neutralizing activity, yet exhibits low neutralizing potency in vitro and efficacy at relatively high doses in in vivo models of SARS-CoV-2 infection. Notably, these antibodies bind to the stem-helix peptide from different angles, providing an opportunity to examine the effects of both sequence variation and binding orientation on antibody function.

[0320] The generation campaign selected a human IgG1 antibody isolated from a convalescent donor and targeting the stem-helix peptide targeted by a reference antibody, whose target epitope is highly conserved across SARS-CoV-2 genomes sequenced to date, with mutations occurring at very low frequency (the most frequent mutations over the past three months were P1162L and P1162S, which were detected in only 0.4% and 0.2% of SARS-CoV-2 genome sequences, respectively). A set of 182 antibody variants was generated, and the following datasets were obtained: (1) developability: PSR, AC-SINS, SEC; (2) spike protein binding: SARS-CoV-2-related viruses (Delta, BA.1, BA.2), SARS-CoV-1-related viruses (SARS-CoV-1, WIV1); and (3) pseudotyped virus neutralization: SARS-CoV-2-related viruses (Delta, BA.2), SARS-CoV-1-related viruses (SARS-CoV-1, WIV1).

[0321] Many of the generated antibodies bound with high affinity to the spike proteins of SARS-CoV-1- and SARS-CoV-2-related viruses (Figures 4A-4B), and some of them also neutralized SARS-CoV-2 delta, BA.2 (Figures 5A-5B), XBB.1.5, and BQ.1.1 (Figure 21). Compared to the clinical-stage molecule, sotrovimab used in these experiments does not have the LS mutation and has three additional amino acid mutations due to differential IgG allele usage. ADG20 used in these experiments has the LS mutation in the Fc region, while the clinical-stage molecule has the LA mutation.

[0322] The binding and neutralization data were combined into a composite fitness score to rank the top 20 screening hits. Twelve molecules with acceptable developability parameters (AC-SINS<20, SEC>90%, PSR<10) were then further refined as seeds for the next round of Project Learning. Notably, the reference antibody expressed six sequence liabilities (four if two cysteines were not considered for bond formation), which posed a low-to-moderate risk for process development, and several seeds exhibited already reduced numbers of liabilities. As part of our Project Learning campaign, the objective was to improve antibody function and further reduce the number of sequence liabilities.

[0323] A handful of seeds also showed promising characteristics as lead molecules. AB-1, which potently neutralized past (SARS-CoV-1), current (SARS-CoV-2 delta, BA.2), and potentially emerging (WIV1) sarbecoviruses (Figures 6A-6B), differed from the reference antibody by four mutations and had only one sequence liability outside of the two cysteines. AB-2, while not as potent as AB-1 (Figures 7A-7B), differed from the reference antibody by seven mutations, had optimal AC-SINS (<10), and no liability outside of the two cysteines.

[0324] Using seed molecules identified by screening this variant set, such as those shown in Figures 6 and 7, we generated a new variant set of 364 antibodies. The following datasets were obtained as part of the screening campaign: (1) developability: PSR, AC-SINS, SEC; (2) spike protein binding: SARS-CoV-2-related viruses (Delta, BA.1, BA.2), SARS-CoV-1-related viruses (SARS-CoV-1, WIV1); and (3) pseudotyped virus neutralization: SARS-CoV-2-related viruses (Delta, BA.2). Antibodies were ranked based on their combined neutralization scores, and the top 20 were selected (Figures 8A-8B). Of these, three antibodies (AB-17, AB-15, and AB-1) were further refined based on their optimal binding, neutralization, and developability profiles (Figures 9A-9B and Table 4). These antibodies were then tested in additional neutralization assays (SARS-CoV-2 BA.2, BA.2.12.1, and BA.4 / 5 VSV-dG, SARS-CoV-2 Delta, SARS-CoV-1, and WIV1 pseudotyped lentiviruses) and benchmarked against reference antibodies (ref Abs) and sotrovimab (Synagis was used as an isotype control) (Figures 9A-9B and Table 4). AB-17, AB-15, and AB-1 demonstrated increased neutralization potency and efficacy compared to the reference Abs across all neutralization assays. They also demonstrated neutralization activity that was either greater than (SARS-CoV-2 BA.2, BA.2.12.1, and BA.4 / 5 VSV-dG) or comparable to sotrovimab (SARS-CoV-2 Delta, SARS-CoV-1, and WIV1). The isotype control was inactive across all assays.

[0325] Example 3. Selection and characterization of development candidates Monoclonal antibodies (mAbs) targeting the spike protein of SARS-CoV-2 have proven effective in preventing and treating COVID-19. Several mAbs targeting different regions of the spike protein (receptor-binding domain [RBD], N-terminal domain [NTD], and S2) have been characterized to date, and several anti-RBD mAbs have received emergency use authorization over the past two years based on their ability to neutralize circulating SARS-CoV-2 variants in vitro and in vivo. However, SARS-CoV-2 variants harbor several mutations in the RBD that confer escape from many mAbs. Therefore, there is a need to develop mAbs targeting conserved regions of the spike protein with robust neutralizing activity against multiple SARS-CoV-2 variants.

[0326] The S2 domain contains highly conserved epitopes, namely the fusion peptide and stem-helix peptide. Antibodies targeting these epitopes have been isolated from convalescent / vaccinated donors and immunized animals and exhibit neutralizing activity, albeit with low potency. However, if an anti-S2 neutralizing antibody with sufficient potency were discovered, it would potentially be highly valuable due to the broad conservation of the S2 epitope across coronaviruses. The reference antibody was a human IgG1 anti-S2 antibody isolated from a convalescent donor, which showed promising neutralizing potency against coronaviruses of the Sarbecovirus subgenus (Li et al., Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern, Cell Reports 38(2):110-210 (2021)). The binding mode is known from the co-crystal structure with the stem-helix peptide. Additionally, the target epitope of the reference antibody is highly conserved across all SARS-CoV-2 genomes sequenced to date, with only two mutations at position P1162 occurring at very low frequency (P1162L and P1162S, respectively, in 0.4% and 0.2% of SARS-CoV-2 genome sequences from the past three months). We hypothesized that by exploring the antibody sequence space compatible with this binding mode, it may be possible to identify sequences with improved neutralization potency compared to the reference antibody.

[0327] Novel anti-S2 stem-helix binders were generated using an in-house computational protein design protocol. Starting from the co-crystal structure of a reference antibody bound to an S2 stem-helix peptide, an in-house machine learning (ML) model was used to predict sequence landscapes compatible with the bound conformation (Ingraham et al., "Generative models for graph-based protein design," 33rd Conference on Neural Information Processing Systems (NeurIPS 2019), Vancouver, Canada; Zhou et al., "A general-purpose protein design framework based on mining sequence-structure relationships in known protein structures," Proc Natl Acad Sci USA. 117(2):1059-68 (2020)). 182 diverse sequences were sampled from them for experimental testing.

[0328] To test these designs, antibody variants were generated in a human IgG1 format and screened for function (neutralization of pseudoviruses), affinity (sarbecovirus spike protein binding estimated using DELFIA), and developability properties (self-association propensity [AC-SINS], monomericity [aSEC], and polyspecific reactivity [PSR]). Neutralization of SARS-CoV-2 variants Delta, BA.1, and BA.2, as well as SARS-CoV-1 and WIV-1 pseudoviruses, was measured across five different titration concentrations. Affinity for the spike proteins of SARS-CoV-1, WIV-1, and SARS-CoV-2 variants Delta, BA.1, and BA.2 was measured across eight different titration concentrations. Based on these experiments, a subset of antibodies with promising neutralization and binding profiles was identified.

[0329] To further improve the neutralization potency of the candidate sequences, a second round of computational design was performed, in which experimental measurements were used to train a model that predicted potency and affinity from the sequence. The resulting model was then used to co-optimize predicted potency and affinity in the context of the sequence landscape described above to generate a set of 364 second-round sequences.

[0330] After experimental characterization of all antibody variants, lead antibody sequences were selected based on functional, binding, and developability properties measured across 533 unique sequences generated from two rounds of sequence design.

[0331] Based on the screening data, we selected three lead molecules with optimal functional profiles: AB-1, AB-17, and AB-15, which exhibited 4, 8, and 5 mutations, respectively, compared to the reference antibody. These molecules were tested against a panel of pseudoviruses representing SARS-CoV-2 variants (Delta, Omicron BA.1, BA.2, BA.2.12.1, and BA.4 / 5) and other sarbecoviruses (SARS-CoV-1, WIV1) along with an isotype control (Synagis), a reference antibody, and clinical-stage benchmarks (an IgG1 molecule expressing the variable region of sotrovimab and two molecules comprising Evasheld [AZD8895, AZD1061]) (Figures 10A-10B). The three lead molecules exhibited comparable neutralization profiles across all pseudoviruses and overall improved neutralization potency (IC) compared to the reference antibody. 50 The three lead molecules demonstrated improved neutralization potency and efficacy against the BA.2 sublineage (BA.2, BA.2.12.1, and BA.4 / 5) compared with sotrovimab, and a BA.4 / 5 neutralization profile comparable to that of AZD1061 (Figures 10A-10B, Tables 5 and 6). Preliminary experiments using live virus also confirmed the neutralization of SARS-CoV-2 delta and omicron BA.1 by the three lead molecules (Figure 11, Table 7).

[0332] The FDA fact sheet for clinical-stage antibodies reports that if the ratio between the neutralization potencies against two viruses is <5, their neutralization profiles are considered equivalent. To compare neutralization potency across experiments, the neutralization potency of the three lead molecules against each virus was expressed as a ratio relative to the neutralization potency of sotrovimab against SARS-CoV-2 delta. A ratio <5 was interpreted as equivalent neutralization to sotrovimab neutralization of SARS-CoV-2 delta. This may be a relevant metric because sotrovimab has demonstrated clinical efficacy against SARS-CoV-2 delta, and a ratio <5 may indicate human efficacy for our lead molecule at the clinical dose of sotrovimab (500 mg IV). Notably, the ratios calculated for the three lead molecules across all viruses were <5, confirming equivalent neutralization potency to sotrovimab neutralization of delta (Table 8).

[0333] Overall, the three lead candidates demonstrated robust and comparable neutralization profiles against various coronaviruses. To select a development candidate from the three lead candidates, we evaluated developability parameters. AB-1 was selected as a development candidate because it exhibited optimal PSR and acceptable AC-SISN (Figure 12).

[0334] The neutralizing activity of AB-1 was further validated in a hamster model of SARS-CoV-2 delta challenge. For these in vivo experiments, the variable region (V) of AB-1, an isotype (Synagis), and a clinical-stage (sotrovimab) control were used. H / V L) were expressed as human IgG1 (huIgG1, Figures 15A-15D) and hamster IgG2a (hamIgG2a, Figures 15E-15H) to account for interaction with hamster Fcγ receptors. Hamsters were injected with the indicated antibodies one day before infection with SARS-CoV-2 delta. Body weights were recorded daily and expressed as percentage change relative to day 0 (pre-infection) body weight. On day 7, hamsters were sacrificed, lungs were harvested, and their weights were recorded (as a proxy for lung inflammation). AB-1 expressed as human IgG1 or hamster IgG2a protected against weight loss in a dose-dependent manner (Figures 15A-B and E-F), and these results correlated with reduced lung weight (Figures 15C and G).

[0335] The neutralizing activity of AB-1 was further validated in a hamster model of SARS-CoV-2 Omicron BA.2 challenge. For these in vivo experiments, the variable region (V) of AB-1, an isotype (Synagis), and a clinical-stage (sotrovimab) control were used. H / V L ) was expressed as hamster IgG2a (hamIgG2a, Figures 16A-16D) to account for interaction with hamster Fcγ receptors. Hamsters were injected with the indicated antibodies one day before infection with SARS-CoV-2 Omicron BA.2. Body weights were recorded daily and expressed as percentage change relative to day 0 (preinfection) body weight. On day 7, hamsters were sacrificed, lungs were harvested, and their weights were recorded (as a surrogate for lung inflammation). AB-1 expressed as hamster IgG2a protected against weight loss in a dose-dependent manner (Figures 16A-16B), and these results correlated with reduced lung weight (Figure 16C). Thus, further in vivo characterization of AB-1 confirmed protection from weight loss in the Omicron BA.2 model of infection, and protection measured by weight loss did not correlate with reduced viral load in the lungs (Figures 15D&H and 16D).

[0336] Figures 17A-17D, 18A-18C, and 19 show that AB-1, either alone or in combination with another antibody (e.g., a Class 4 anti-RBD antibody), demonstrated significant neutralizing potency and efficacy against multiple variants, including Omicron BQ.1.1, Omicron BA.5, and Delta.

[0337] Overall, these results demonstrate that AB-1 has robust neutralizing activity against SARS-CoV-2 variants in vitro and in vivo.

[0338] Recently, many SARS-CoV-2 variants have emerged, with remarkable escape from clinical-stage antibodies. Some of these variants show signs of convergent evolution, such as mutations at position K444. Therefore, we evaluated the neutralization of BA.4 / 5 pseudoviruses with and without the K444T mutation (BA.4 / 5+K444T) by AB-1 and the clinical-stage antibodies sotrovimab and bebuterovimab. IC 50 As measured by β-actin, sotrovimab poorly neutralizes both pseudoviruses, while bebuterovimab shows a significant impairment in neutralizing the BA.4 / 5+K444T pseudovirus. AB-1 neutralizes BA.4 / 5 and BA.4 / 5+K444T pseudoviruses with comparable neutralizing efficacy (Table 9).

[0339] AB-1 targets an S2 stem helix epitope that does not overlap with the epitope targeted by anti-RBD antibodies. Combinations of AB-1 with clinical-stage Class 3 anti-RBD antibodies (sotrovimab, bebuterovimab) or Class 4 anti-RBD antibodies (anti-RBD4) were tested in neutralization assays using the BA.4 / 5+K444T pseudovirus, which abolishes the neutralizing activity of sotrovimab and bebuterovimab. All combinations demonstrate enhanced neutralization profiles compared to AB-1 alone, as assessed by AUC (area under the curve) and efficacy (% neutralization at maximum concentration) (Figures 13A-13B).

[0340] Example 4. Pseudovirus Neutralization Assay Vero-TMPRSS2 cells were plated at 3.5 × 10 in tissue culture-treated 384-well plates (ThermoFisher, Waltham, MA, catalog no. 164610). 3 Cells were plated at a density of 10 ... Then, 20 μl of the antibody-virus mixture was transferred to a 384-well plate pre-seeded with Vero-TMPRSS2 cells. The 384-well plate was briefly spun down at 50 g for <5 seconds and incubated at 37°C, 5% CO2 for 24 hours. At the end of the incubation, 40 μl of luciferase detection buffer (BPS Bioscience ONE-Step™ Luciferase Assay System, BPS Bioscience, San Diego, CA, Catalog No. 60690-3) was added to each well of the cell culture plate. The plate was centrifuged at 50 g for <5 seconds and incubated for 15 minutes with gentle rocking. Luminescence signals were recorded using an Envision plate reader. Results were expressed as percentage neutralization and analyzed using Prism 9. Curves were generated by fitting the data using the following equation: log(inhibitor) vs. normalized response - variable slope (4 parameters).

[0341] As shown in Figure 19, AB-1 exhibits therapeutically meaningful in vitro neutralization potency across multiple variants, suggesting that it can be used to address a range of variants by binding to low-immunogenic, highly conserved regions of SARS-CoV-2.

[0342] Pseudoviruses (VSV-dG pseudotyped with the spike protein) representing SARS-CoV-2 variants tracked by the CDC or experiencing increasing incidence worldwide, such as delta, BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4 / 5, BA.2.75, BA.2.75.2, BA.4.6, BA.5.2.6, BF.7, BF.11, BN.1, BQ.1, BQ.1.1, and XBB, as well as the non-SARS-CoV-2 sarbecoviruses SARS-CoV-1 and WIV1, are generated. The pseudotyping system is used to evaluate the neutralizing activity of polypeptides disclosed herein (e.g., AB-1) against current and potential novel SARS-CoV-2 variants. These results are confirmed using live virus. Pseudoviruses carrying mutations in the spike epitope that may result in reduced neutralizing activity of the polypeptide (e.g., AB-1) are developed and evaluated. These mutations are selected based on structural and / or biochemical evaluation of the binding epitope, analysis of mutations in selected sequences reported in public databases, and escape experiments using replication-competent VSV, or a combination of the above. Finally, the neutralizing activity of the polypeptide (e.g., AB-1) is tested against pseudoviruses carrying mutations that result in reduced neutralizing activity against clinical-stage monoclonal antibodies. For mutations already represented in the Omicron sublineage, neutralizing activity is tested against the Omicron sublineage instead of pseudoviruses generated with the selected escape mutations.

[0343] Example 5. Biochemical characterization of the AB-1 epitope The monoclonal antibody (mAb) AB-1 was developed using an in-house machine learning model and leveraging the co-crystal structure of a human-derived reference antibody (Hurlburt 2022, Jennewein 2021, Ullah 2021, Li 2022). AB-1 is a human IgG1 mAb that targets the SARS-CoV-2 spike S2 ​​stem-helix peptide with an LS mutation in the Fc region to extend half-life and promote translocation to mucosal tissues. AB-1 has demonstrated robust neutralization against all major SARS-CoV-2 variants, as well as other sarbecoviruses associated with previous epidemics (SARS-CoV-1) or pandemic potential (WIV1, bat SARS-like coronavirus). AB-1 is hypothesized to be effective as a single agent and potentially in combination with one or more other anti-spike mAbs for the prevention of COVID-19 in high-risk populations across all current omicron sublineages and future variants of concern.

[0344] The goal of Example 5 was to evaluate the binding properties of AB-1 to the SARS-CoV-2 spike S2 ​​stem helix peptide (the target epitope of the reference antibody). Using dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) and surface plasmon resonance (SPR), we evaluated the binding of AB-1 and the reference antibody to biotinylated peptides reported in Hurlburt 2022: a partially overlapping peptide spanning the target epitope of the reference antibody (aa 1133-1162), a peptide representing the C-terminus of the stem helix (aa 1149-1167), and a control 15-mer peptide derived from the HIV-1 envelope protein. Additional biotinylated peptides were designed to encompass the SARS-CoV-2 spike S2 ​​stem helix (aa 1143-1162).

[0345] Biochemical characterization of the AB-1 epitope by A. DELFIA Biotinylated peptides: SARS-CoV-2 spike S2 ​​peptides 1133–1147 (Biosynth, Gardner, MA, Lot No. LP10933), 1137–1151 (Biosynth, Lot No. LP10934), 1141–1155 (Biosynth, Lot No. LP10935), 1145–1159 (Biosynth, Lot No. LP10936), 1149–1163 (Biosynth) Biosynth, Lot No. LP10945), 1153-1167 (Biosynth, Lot No. LP10938), 1157-1171 (Biosynth, Lot No. LP10939), 1149-1167 (Biosynth, Lot No. LP10937), 1143-1162 (Biosynth, Lot No. BU17943), HIV-1 envelope peptide (Biosynth, Lot No. LP10940). See, e.g., Table 36. Antibodies: AB-1 (Lonza, Lot No. 1100-130922-01), Reference Antibody (GenScript USA, Inc., Piscataway, NJ, Lot No. U737NHI220, variable region of the reference antibody expressed as human IgG1 with LS mutations in the Fc region), Isotype (GenScript USA, Inc., Lot No. U799WHJ270-3, variable region of palivizumab targeting the RSV F protein and expressed as human IgG1 with LS mutations in the Fc region). • Neutravidin: Neutravidin protein (Thermo Fisher Scientific, Waltham, MA, catalog number 31000). ● Dilution plates: 96-well plates, round bottom, non-treated, polypropylene (Corning, Corning, NY, Cat. No. 3365; Greiner, Kremsmuenster, Austria, Cat. No. 650201). ●DELFIA Plates: 384-well plates white, MaxiSorp, polystyrene (Thermo Fisher Scientific, catalog number 460372). TBS-T: 20x TBS Tween®-20 (Thermo Fisher Scientific, Catalog No. 28360), diluted 1:20 in ddH2O Blocking solution: ELISA Assay Diluent B (5X) (BioLegend, San Diego, CA, Catalog No. 421205), diluted 1:5 in PBS. ● Secondary antibody: DELFIA Eu-N1 anti-human IgG (PerkinElmer, Waltham, MA, catalog number 1244-330). • Enhancement solution: DELFIA Enhancement Solution (PerkinElmer, Cat. No. 4001-0010).

[0346] DELFIA (time-resolved fluorescence (TRF) intensity technique) was used to determine the binding of AB-1 to different SARS-CoV-2 spike S2 ​​stem-helix peptides. DELFIA assays are designed to detect the presence of an analyte of interest using a lanthanide (e.g., europium [Eu]) chelate-labeled reagent. Upon completion of the immune reaction, Eu ions are dissociated from the labeled immune component bound to the solid phase by adding an enhancement solution. Eu fluorescence is then measured by TRF.

[0347] A 384-well plate was coated with neutravidin (2 μg / mL) overnight at room temperature. The plate was washed and coated with biotinylated peptide (50 nM) for 1 hour at room temperature. The plate was then washed, incubated with blocking solution for 2 hours at room temperature, washed again, and incubated with antibodies (AB-1, reference antibody, and isotype control) for 1 hour at room temperature. Antibody binding was assessed in technical triplicates using a 12-point titration curve (1:4 serial dilutions were prepared in blocking solution, starting at 18 μg / mL). Following this, the plate was washed and incubated with Eu-labeled anti-human IgG secondary antibody (0.1 μg / mL) for 30 minutes at room temperature. Finally, the plate was washed and incubated with DELFIA enhancement solution for 15 minutes. TRF was recorded at 615 nm using a PerkinElmer EnVision plate reader.

[0348] Statistical analysis was performed using Prism 9.5.0 software using data from two independent replicates. Area under the curve (AUC) values ​​and 95% confidence intervals were derived for log-transformed antibody titration curves using the trapezoidal rule. Statistical comparisons between AUC values ​​for AB-1 and the reference antibody were performed using two-way ANOVA corrected for multiple comparisons, as reported in the software documentation (https: / / www.graphpad.com / support / faqid / 2031 / ).

[0349] B. Biochemical characterization of the AB-1 epitope by SPR • Biacore 8K+ (Cytiva, Marlborough, MA, serial number 2873569). ● Biacore 8K control software (Cytiva, version 4.0.8.19879). • Biacore Insight evaluation software (Cytiva, version 4.0.8.19879). ●GraphPad Prism (version 9.5.0). Biotinylated peptides: SARS-CoV-2 spike S2 ​​peptides 1133–1147 (Biosynth, Lot No. LP10933), 1137–1151 (Biosynth, Lot No. LP10934), 1141–1155 (Biosynth, Lot No. LP10935), 1145–1159 (Biosynth, Lot No. LP10936), 1149–1163 (Biosynth, Lot No. LP10937). (Biosynth, Lot No. LP10945), 1153-1167 (Biosynth, Lot No. LP10938), 1157-1171 (Biosynth, Lot No. LP10939), 1149-1167 (Biosynth, Lot No. LP10937), 1143-1162 (Biosynth, Lot No. BU17943), HIV-1 envelope peptide (Biosynth, Lot No. LP10940). See, e.g., Table 36. ●Fab: AB-1 (prepared in-house), reference antibody (prepared in-house). Antibody: AB-1 (produced in-house), variable domain of reference antibody (GenScript USA, Inc., Lot No. U3767HH180-11, variable domain of reference antibody expressed as human IgG1 with LS mutations in the Fc region). • Series S Biotin CAPture Kit (Cytiva, catalog number 28920234), containing Biotin CAPture Reagent, 50 μg / mL in HBS-EP, Regeneration Stock 1 (8 M guanidine hydrochloride), and Regeneration Stock 2 (1 M sodium hydroxide). • 20× HBS-EP+, pH 7.6 (Teknova, Hollister, CA, catalog no. H8022) is composed of 0.2 M HEPES (pH 7.4), 3 M NaCl, 60 mM EDTA, and 1% surfactant P20, prepared at 1×. • Microplate, 96W, deep well, U-bottom, PP, 2mL (Porvair, Ashland, VA, catalog number 219020). ●Microplate Foil 96W (Cytiva, catalog number 28975816).

[0350] Biacore is a label-free platform that uses SPR to measure binding interactions in real time. Kinetic and affinity parameters are extracted from experimental data by an iterative process of finding the best fit to a set of equations that describe the interaction. The association rate constant, k a (M -1 seconds -1 ) governs the rate at which the complex forms. The dissociation rate constant, k d (seconds -1 ) governs the rate at which the complex dissociates. The equilibrium dissociation constant, K D (M) describes the strength of the interaction.

[0351] To evaluate the response units (RU) of AB-1 and reference antibodies binding to peptides spanning the SARS-CoV-2 spike S2 ​​stem-helix region, a multi-cycle kinetic approach was implemented using Biacore 8K+. Biotinylated peptides were captured using the Biotin CAPture kit. Biotin CAPture reagent was added in 2 μL min. -1 The biotinylated peptide (referred to as the ligand) was injected at a flow rate of 10 μL for 180 seconds. -1 The AB-1 and reference antibodies (referred to as analytes) were injected at 100 nM in 30 μL min for 10 seconds at a flow rate of 100 nM each, achieving a capture level of approximately 10-20 RU. -1 The surface was then injected with 10 μL of HCl for 180 seconds at a flow rate of 10 μL / min and the complexes were allowed to dissociate for 900 seconds using 1×HBS-EP+ as the running buffer. -1 The assay was regenerated by injecting a solution of 3 parts regeneration stock 1 with 1 part regeneration stock 2 for 120 seconds at a flow rate of 0.05%. The assay was performed at 25°C. Reporting points for capture levels were taken 25 seconds after the end of the ligand injection, and reporting points for binding responses were taken 5 seconds before the end of the analyte injection. Binding responses were normalized to ligand capture levels using GraphPad Prism and are reported as normalized binding responses.

[0352] To assess the kinetics of AB-1 and reference antibody Fab binding to peptides spanning the SARS-CoV-2 spike S2 ​​stem-helix region, a single-cycle kinetic approach was implemented using Biacore 8K+. Biotinylated peptides were captured using the Biotin CAPture kit. Biotin CAPture reagent was added in 2 μL min. -1 The biotinylated peptide (referred to as the ligand) was injected at a flow rate of 10 μL for 180 seconds. -1 The AB-1 and reference antibody Fab (referred to as analyte) were injected in 30 μL min for 180 s at a flow rate of 10 μL / min for 10 s to achieve a capture level of approximately 10-20 RU. -1 Analyte concentrations were sequentially injected over the surface starting with low to high at a flow rate of 100 nM, and the complexes were allowed to dissociate for 900 seconds using 1× HBS-EP+ as the running buffer. The concentration series consisted of 3-fold serial dilutions starting from 300 nM for a total of six concentrations. The surface was then filled with 10 μL of -1 The antibody was regenerated by injecting a solution of 3 parts regeneration stock 1 with 1 part regeneration stock 2 for 120 seconds at a flow rate of 0.05%. The assay was performed at 25 °C. The kinetic parameters of the concentration series were obtained by double-referencing the data and globally fitting them to a 1:1 binding model with mass transport limitations using Biacore Insight evaluation software. The kinetics of AB-1 and the reference antibody Fab binding to the SARS-CoV-2 spike S2 ​​peptide (representing the C-terminus of the stem helix (aa 1149-1167)) are reported and the resulting sensorgrams are shown.

[0353] C. Results DELFIA and SPR data show the binding profile of AB-1 to the SARS-CoV-2 spike S2 ​​peptide (Figure 21, Figure 22, Table 13). The specificity of these results is confirmed by the lack of binding of the isotype control in the DELFIA experiments, as well as the lack of binding of AB-1 and the reference antibody to the HIV-1 envelope peptide used as a negative control in both the DELFIA and SPR experiments. Notably, the Fab of AB-1 binds with higher affinity to the SARS-CoV-2 spike S2 ​​peptide represented by the C-terminus of the stem helix (aa 1149-1167) compared to the reference antibody (K). D Values: AB-1, 0.9 nM; reference antibody, 5.4 nM (Table 14, Figures 23A-23D).

[0354] In conclusion, Example 5 shows that AB-1 binds to the SARS-CoV-2 spike S2 ​​stem helix with higher affinity than the reference antibody.

[0355] Example 6. Structural characterization of the AB-1 epitope The goal of Example 6 was to collect structural information on AB-1 in complex with both the SARS-CoV-2 BA.1 spike trimer and spike S2 ​​(aa 1149-1167) stem helix peptide and define its binding site.

[0356] Using X-ray crystallography, we determined the high-resolution structure of the AB-1 Fab in complex with the spike S2 ​​(aa 1149–1167) stem-helix peptide. Cryo-electron microscopy (cryo-EM) was used to determine the structure of the AB-1 Fab in complex with the SARS-CoV-2 BA.1 spike trimer. As observed in previous structure determination attempts, the coil-coil S2 stem-helix peptide of the SARS-CoV-2 BA.1 spike trimer is highly flexible and can be very difficult to resolve by cryo-EM. To stabilize motion in this region, we used the Fab region of an internally discovered monoclonal antibody (R-AB-3a) targeting a class 4 epitope in the spike receptor-binding domain (RBD) in complex with the SARS-CoV-2 BA.1 spike trimer bound to the AB-1 Fab.

[0357] A. Structural characterization of the AB-1 epitope by cryo-EM. Antigen: SARS-CoV-2 BA.1 spike trimer (produced in-house) ●Fab: AB-1 (in-house production), R-AB-3a (in-house production) Expi293F™ cells (Thermo Fisher Scientific, catalog number A14527) Gibco Expi293™ Expression Medium (Thermo Fisher Scientific, Catalog No. A14351-01) ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific, Catalog No. A14525) Nickel Sepharose Excel (Cytiva, Catalog No. 17371201, Lot No. 10313877) LambdaFabSelect (Cytiva, catalog number 17548201, lot number 10302825) Capto™ L (Cytiva, catalog number 17547802, lot number 10305645) Quantifoil Gold R1.2 / 3 Grids (Electron Microscopy Sciences, Catalog No. 261655, Lot No. Q82801)

[0358] Equipment: Kuhner shaker (Kuhner, Model: ISF1-ZC Peltier) ●Akta Pure (Cytiva) ●SRT-C SEC-500 (Sepax, PN:235500-4630) Superose 6 Increase 10 / 300GL (Cytiva, Catalog No. 29091596, Lot No. 10325571) NanoDrop One (Thermo Fisher Scientific, model NanoDrop One) HPLC 1260 Infinity II (Agilent, TT number: 1581T8) ●Pelco easiGLOW (Pelco, model number: 91000) Vitrobot Mark IV (Thermo Fisher Scientific, serial number 220301059) Glacios (Thermo Fisher Scientific, serial number 9956936)

[0359] software: ●EPU (Thermo Fisher Scientific, v3.2) ●cryoSPARC (Structura Biotechnology, v4.1.1) ChimeraX (UCSF RVBI, v1.5)

[0360] a. Expression and purification of SARS-CoV-2 BA.1 spike trimer The SARS-CoV-2 BA.1 spike trimer was expressed in Expi293™ cells according to the Gibco™ Expi293™ Expression System protocol. Briefly, 3 million cells were transfected with approximately 1 mg of plasmid DNA. Cells were incubated at 37°C on an orbital shaker at 150 RPM with 80% relative humidity and 8% CO2. Four days after transfection, cells were harvested and pelleted at 3,900 x g for 30 minutes at 4°C. The supernatant was decanted onto a 0.22 μm filter unit and stored at 4°C until purification. The SARS-CoV-2 BA.1 spike trimer was purified using nickel-Sepharose Excel resin. The supernatant was incubated with 2 mL of nickel-Sepharose Excel resin overnight at 4°C and purified by gravity flow.

[0361] b. Expression and purification of AB-1 and R-AB-3a Fabs AB-1 and R-AB-3a Fabs were expressed in Expi293™ cells according to the same protocol used for the SARS-CoV-2 BA.1 spike trimer. Supernatants from both Fabs were incubated overnight at 4°C with either Capto™ L resin (AB-1 Fab) or LambdaFabSelect resin (R-AB-3a Fab). The mixture of supernatant and resin was loaded onto a 10 mL disposable column equilibrated with 1x PBS (pH 7.4). The column was then washed with 10 CV of 1x PBS (pH 7.4). AB-1 and R-AB-3a were eluted with 50 mM glycine (pH 2.5). The proteins were immediately neutralized with 1 M Tris-HCl (pH 8.0). Each protein was buffer-exchanged using a PD-10 desalting column and eluted with 1x PBS (pH 7.4). The purified protein was concentrated and kept at 4°C until complexed with the SARS-CoV-2 BA.1 spike trimer.

[0362] c. Complexation of SARS-CoV-2 BA.1 spike trimer with AB-1 and R-AB-3a Fabs The SARS-CoV-2 BA.1 spike trimer was incubated with AB-1 and R-AB-3a Fabs at a 1:2:2 molar ratio overnight at 4°C with gentle mixing. The complex was purified by size-exclusion chromatography on a Superose 6 Increase 10 / 300GL column equilibrated with 50 mM HEPES (pH 8.0), 150 mM NaCl. Prior to injection, the incubated sample was spun at 1699 x g. One mL of the complex was injected onto the column via a 1 mL loop. The flow rate throughout the run was 0.5 mL / min. Each elution peak was analyzed by SDS-PAGE to determine which fractions contained the complex between the SARS-CoV-2 BA.1 spike trimer and the AB-1 and R-AB-3a Fabs. These fractions were further analyzed by aSEC on an SRT-C SEC-500 column equilibrated with 50 mM HEPES (pH 8.0), 150 mM NaCl.

[0363] d. Cryo-EM sample preparation Four microliters of sample was applied to a Quantifoil gold grid that was glow discharged for 30 seconds with a plasma current of 0.15 mA with negative polarity.

[0364] e. Cryo-EM data collection Cryo-EM images were acquired on a Glacios cryo-TEM using EPU software (v3.2). The Glacios was operated at 200 kV with a Falcon4i direct electron detector and a Selectris energy filter with a 10 eV zero-loss slit width. 5160 movies were collected at 130,000x magnification and a pixel size of 0.876 Å. The total dose per movie was 51.3 electrons per square angstrom. The targeted defocus range was 0.5–2.4 μm.

[0365] f. Cryo-EM data processing All computational steps were performed using the cryoSPARC (v4.1.1) software suite and ChimeraX (v1.5) molecular visualization software. EER-format movies were imported, divided into 40 frames, and sampled at the physical pixel size. Beam-induced motion correction, per-frame dose weighting, and CTF estimation were performed using the Patch Motion Correction and Patch CTF jobs in cryoSPARC (v4.1.1). Exposures were selected semi-automatically using the interactive exposure curation tool. After setting stringent cutoffs for CTF-fit decomposition, defocus, and relative ice thickness, 2,921 images were selected for subsequent processing.

[0366] Template-based particle picking was performed using projections from a 3D map of the SARS-CoV-2 BA.1 spike trimer low-pass filtered to 20 Å. 466,074 particles were extracted from 2,921 micrographs. 2D classification was performed with 200 classes to identify incorrectly picked or damaged particles, and 87,564 particles were retained for further processing.

[0367] Ab initio reconstruction using the three classes was performed to obtain an initial 3D map of the expected size and shape of the SARS-CoV-2 BA.1 spike trimer from a subset of 51,996 particles. Subsequent heterogeneous 3D refinement of these particles in cryoSPARC yielded a "consensus map" at 3.6 Å resolution by the gold standard FSC criteria.

[0368] g. Focused classification and model fitting An atomic model of the SARS-CoV-2 BA.1 spike trimer without bound Fab was computationally docked to the consensus map using the "fit in map" tool in ChimeraX (v1.5). The map closely fits the density, except for several unknown densities in the RBD and S2 stem helix regions of the map. Gaussian low-pass filtering of the map revealed that these densities are dumbbell-shaped with a central hole, as expected for Fab molecules. Using the "Segment Map" tool in ChimeraX and the volume tool in cryoSPARC, we created a focused mask around the S2 stem helix surrounding the propeller-shaped putative Fab density. A focused 3D classification without alignment in cryoSPARC was performed with 10 classes to identify a subset of 9,822 particles with stronger density of putative Fab bound to the S2 stem helix. This subset was refined using heterogeneous refinement to 5.7 Å resolution by the gold standard FSC criteria, without applying symmetry.

[0369] Local resolution estimation in cryoSPARC indicated that the Fab bound to the S2 stem helix had lower local resolution than the core of the SARS-CoV-2 BA.1 spike trimer. The CDR-containing domain of the Fab had a local resolution of approximately 8 Å, while the flexibly associated framework domain of the Fab had a local resolution of approximately 14–20 Å. For this reason, a 3 Å wide Gaussian low-pass filter was applied in ChimeraX (v1.5). One Fab was clearly weaker than the other two. Nevertheless, these densities were sufficient to unambiguously dock the three copies of the AB-1 Fab-S2 stem helix complex crystal structure (described below) to each of the three S2 stem helix Fab densities using the ChimeraX "fit-in map" tool. The structure of the R-AB-3a Fab bound to the RBD was also docked to the dumbbell-shaped density at the spike RBD.

[0370] BStructural characterization of the AB-1 epitope by X-ray crystallography ●Fab: AB-1 (prepared in-house). • Fabalactica Midispin Fab Digestion Kit (Genovis, catalog number A2-AFK-100). ●Peptide: SARS-CoV-2 spike S2 ​​peptides 1149–1167 (Biosynth, lot number LP10941). • MCSG-3 crystallization screening (Anatrace, part number MCSG-3). ●16 / 900 Superdex 200pg column (custom-made).

[0371] a. Production of AB-1 Fab and SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide AB-1 was produced by Lonza, and monomer content was assessed by SE-HPLC. Fab was digested and purified from IgG using the Genovis Fabalactica Midispin Fab Digestion Kit according to the manufacturer's instructions. The SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide (H2N-KEELDKYFKNHTSPDVDLG-OH) (SEQ ID NO: 197) was synthesized by Biosynth, and quality was verified using HPLC, mass spectrometry, and amino acid analysis. Binding of AB-1 to the SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide was assessed using AB-1-001-PD.

[0372] b. AB-1 Fab: SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide co-crystallization trial: AB-1 Fab and SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide were added at a 1:1 molar ratio, incubated on ice for 20 minutes, concentrated, and injected onto a 16 / 900 Superdex 200pg column (buffer: 25 mM HEPES, pH 7.5, 150 mM NaCl). Peak fractions were analyzed by SDS-PAGE, combined, and concentrated to 10 mg / mL. The ARI Crystallization Griffon droplet setting robot used a 96-3 Intelliplate to set crystallization droplets of the complex at 10 mg / mL with three ratios of complex to crystallization conditions (1:1, 2:1, and 3:1). A 96-well MCSG-3 commercial screening setup was set up and incubated at 4 and 20 °C.

[0373] c.AB-1 Fab: SARS-CoV-2 spike S2 ​​(aa1149-1167) peptide data collection and structure determination Crystals from MCSG-2 A2 (1:1 ratio) were harvested at 4°C and cryoprotected in 20% glycerol. Crystals were sent to the NSLS2 synchrotron, and X-ray datasets were collected at the AMX beamline equipped with an Eiger X 9M detector. Data were processed to 1.93 Å using DIALS and XDS. Once processed, the dataset was phased by molecular replacement using Phaser_MR with an AlphaFold model of the AB-1 Fab and the peptide reference antibody. Several rounds of refinement were performed using Phenix and Coot.

[0374] C. Results Structural characterization of the AB-1 Fab:SARS-CoV-2 BA.1 spike trimer complex by cryo-EM and the AB-1 Fab:SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide complex by X-ray crystallography reveals that AB-1 binds to the S2 peptide (Figures 24A-24C). Additionally, the results show that three copies of AB-1 Fab can bind to the SARS-CoV-2 BA.1 spike trimer.

[0375] In conclusion, structural characterization of the Fab of AB-1 in complex with the SARS-CoV-2 BA.1 spike trimer and the SARS-CoV-2 spike S2 ​​(aa 1149–1167) peptide revealed that AB-1 binds to the SARS-CoV-2 spike S2 ​​stem-helix peptide.

[0376] Example 7. Mutational analysis of the AB-1 epitope and flanking sequences The goal of Example 7 was to assess sequence conservation of regions of the SARS-CoV-2 spike protein that may potentially affect the neutralization efficacy of AB-1 by defining the relative frequency of mutations in the AB-1 epitope, epitope-flanking regions (within 5 Å of the epitope in the protein structure), heptad repeat 1 (HR1), and heptad repeat 2 (HR2) among publicly reported SARS-CoV-2 spike protein sequences over the course of the pandemic (January 6, 2020 - March 1, 2023).

[0377] A. Defining the Region of Interest In this study, the four regions of interest in the SARS-CoV-2 spike protein are the AB-1 epitope (referred to as the "epitope"), residues adjacent to the epitope (referred to as "epitope-flanking"), HR1, and HR2. Using the epitope residues and the publicly available structure of the SARS-CoV-2 spike protein (PDB ID: 6VSB), we defined "epitope-flanking" residues as residues containing any atom within 5 Å of any epitope residue atom. The residues corresponding to HR1 and HR2 are defined in the NCBI entry for SARS-CoV-2 surface glycoprotein YP_009724390.1. The locations of each of these four regions in the SARS-CoV-2 spike sequence are summarized in Table 15. If an epitope intersects with HR2, the intersecting region was defined as part of the "epitope" category and excluded from the HR2 category throughout the analysis to avoid double counting residues present in both regions. Similarly, epitope-adjacent positions also intersect with HR2. This intersection is defined as part of HR2 and excluded from the epitope-adjacent category for analysis.

[0378] B. Identification of Associated Mutations in the AB-1 Epitope, Flanking Regions, and Heptad Repeats The covSPECTRUM API (application programming interface) was used to query the GenBank database for mutations occurring in each of the four regions of interest (Chen 2021). For each mutation, the relative frequency of the mutation was calculated for periods ending in each month from January 6, 2020, to March 1, 2023, using three different lookback periods: monthly (1 month, 3 months), and overall (from January 6, 2020 onward). Relative frequency was determined by dividing the number of sequences with the mutation of interest observed during a given time interval by the total number of sequences observed during the same time interval.

[0379] Mutations were identified that met the following criteria: - Located within either the "epitope," "epitope adjacent," "HR1," or "HR2." a), b), or both are met: a) Overall relative frequency: Has a relative frequency of at least 0.001 among all sequences deposited from the start of data availability (January 6, 2020) through March 1, 2023. b) Recent relative frequency: It has a relative frequency of at least 0.01 among all sequences deposited between January 1, 2023 and March 1, 2023, and has been observed at least 100 times during that period.

[0380] The relative frequency data was plotted over the course of the pandemic to see whether mutations currently appear to be increasing or decreasing in incidence.

[0381] C. Identifying the most prevalent lineages among sequences carrying mutations To identify the most prevalent strains among sequences carrying each mutation of interest, we queried covSPECTRUM for the set of sequences carrying each mutation observed from the start of data availability on January 6, 2020, through March 1, 2023. For each mutation, we tallied the lineage assignments (nextcladePangoLineage assignments reported by covSPECTRUM) for all sequences carrying that mutation and ranked the lineages according to absolute frequency. For each mutation and lineage, to convert counts to the relative frequency of the strain among all sequences carrying that mutation, we divided the number of lineage sequences carrying that mutation by the total number of sequences carrying the mutation observed from January 6, 2020, through March 1, 2023. This analysis reports the four most prevalent lineages associated with each mutation.

[0382] D. Relative frequency of the mutation of interest in the most prevalent lineages The five most prevalent lineages were determined for each of three time periods ending March 1, 2023 (1 month,...

Claims

1. A polypeptide that specifically binds to the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-spike), A heavy chain variable domain (VH) comprising a heavy chain complementarity determining region (HCDR) 1 containing the amino acid sequence of SEQ ID NO: 77, HCDR 2 containing the amino acid sequence of SEQ ID NO: 80, and HCDR 3 containing the amino acid sequence of SEQ ID NO: 91, A light chain variable domain (V L) comprising a light chain complementarity determining region (LCDR) 1 containing the amino acid sequence of SEQ ID NO: 133, an LCDR 2 containing the amino acid sequence of SEQ ID NO: 141, and an LCDR 3 containing the amino acid sequence of SEQ ID NO: 144, The polypeptide is an antibody or an antigen-binding fragment thereof, and if necessary, The HCDR1 consists of the amino acid sequence of SEQ ID NO: 77, the HCDR2 consists of the amino acid sequence of SEQ ID NO: 80, the HCDR3 consists of the amino acid sequence of SEQ ID NO: 91, and The LCDR1 comprises the amino acid sequence of SEQ ID NO: 133, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 141, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 144, forming a polypeptide.

2. a) The VH comprises the amino acid sequence of Sequence ID No. 4, b) The polypeptide according to claim 1, wherein V L comprises the amino acid sequence of SEQ ID NO: 51 (AB-1).

3. The polypeptide according to claim 1, wherein the polypeptide is an antigen-binding fragment of an antibody, and optionally the antigen-binding fragment comprises a single-strand variable fragment (scFv), an antigen-binding fragment (Fab), Fab', or F(ab')2.

4. The polypeptide according to claim 1, comprising an antibody heavy chain constant domain, an antibody light chain constant domain, or both an antibody heavy chain constant domain and an antibody light chain constant domain.

5. The antibody heavy chain constant domain is an IgG1, IgG2, IgG3, or IgG4 constant domain, and if necessary, the antibody heavy chain constant domain is a) It is an IgG1 constant domain, and / or b) The polypeptide according to claim 4, comprising one or more mutations that increase the serum half-life of the antibody or its antigen-binding fragment in humans.

6. a) an antibody heavy chain (HC) constant domain comprising the amino acid sequence of Sequence ID No. 194, and b) Antibody light chain (LC) constant domain containing the amino acid sequence of SEQ ID NO: 195 The polypeptide according to claim 1, comprising:

7. a) an antibody heavy chain (HC) constant domain comprising the amino acid sequence of Sequence ID No. 194, and b) Antibody light chain (LC) constant domain containing the amino acid sequence of SEQ ID NO: 196 The polypeptide according to claim 1, comprising:

8. A polynucleotide comprising a sequence encoding the polypeptide described in any one of claims 1 to 7.

9. A host cell comprising the polynucleotide described in Claim 8.

10. A composition comprising a polypeptide according to any one of claims 1 to 7, and one or more pharmaceutical excipients, diluents, or carriers.

11. The composition according to claim 10 for use in a method of treating COVID-19 in a subject requiring treatment for COVID-19.

12. The composition according to claim 10 for use in a method for reducing the infectivity of betacoronavirus in a subject where reduction of the infectivity of betacoronavirus is required.

13. A composition according to claim 10 for use in a method for treating COVID-19 or reducing the infectivity of a betacoronavirus in a subject requiring treatment of COVID-19 or reduction of the infectivity of a betacoronavirus, further comprising the use of an additional therapeutic or prophylactic agent.

14. The additional therapeutic agent is selected from the group consisting of antiviral agents, ACE2 inhibitors, additional SARS-CoV-2 spike-binding antibodies, antibiotics, antimalarial agents, vaccines, and combinations thereof, and if necessary, a) The additional SARS-CoV-2 spike-binding antibody is selected from the group consisting of bamranivimab, etesevimab, bebuterobimab, casilibimab, imdevimab, silgabimab, tixagevimab, AZD7442, regdanvimab, sotrovimab, and combinations thereof. b) The antiviral agent is selected from the group consisting of mornupiravir, PF-07817883, STI-1558, PBI-0451, EDP-235, oseltamivir, favipiravir, amantadine, remdesivir, rimantadine, preconalil, antisense RNA against SARS-CoV-2, siRNA against SARS-CoV-2, and combinations thereof. c) The ACE2 inhibitor is selected from the group consisting of RNAi against ACE2, siRNA against ACE2, CRISPR-based inhibitors of ACE2, soluble ACE2, soluble ACE2 variants, anti-ACE2 antibodies, and combinations thereof. d) The antibiotic includes azithromycin, e) The antimalarial agent contains chloroquine, f) The vaccine is a nucleic acid vaccine or an inactivated virus vaccine, g) A combination thereof, the composition for use according to claim 13.

15. The composition according to claim 10 for use in a method for treating COVID-19 or reducing the infectivity of betacoronavirus in a subject requiring treatment of COVID-19 or reduction of the infectivity of betacoronavirus, a) The subject has COVID-19, is suspected of having COVID-19, or is at risk of developing COVID-19, and / or b) A composition in which the subject has heart disease, diabetes, lung disease, weakened immune function, is receiving immunosuppressive therapy, or a combination thereof.

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