Antigen binding molecules targeting SARSCOV-2

Abstract

In various embodiments, the present disclosure provides polypeptides (e.g., antibodies and antigen-binding fragments thereof) that specifically bind to the S2 domain of beta coronavirus spike glycoproteins, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoproteins. In various embodiments, the disclosure also provides fusion proteins comprising one or more of a polypeptide, a polynucleotide encoding the polypeptide, a vector and a host cell adapted to express the polypeptide, and methods for treating a viral infection (e.g., COVID-19).

Description

[0001] Related Applications

[0002] This application claims the benefit 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, 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.

[0003] Incorporation by Reference of Material in XML Format

[0004] This application incorporates by reference the Sequence Listing contained in the following Extensible Markup Language (XML) file, which was submitted concurrently herewith:

[0005] a) File name: 57081065006.xml; created on May 4, 2023, and having a size of 306,430 bytes. BACKGROUND

[0006] SARS-coronavirus-2 (SARS-CoV-2) is a novel coronavirus that causes pneumonia (COVID-19).

[0007] COVID-19 has spread rapidly around the world, causing a pandemic. The World Health Organization's (WHO) Coronavirus Disease (COVID-2019) Situation Report, dated 21 April 2020, reported 2,397,216 confirmed infections and 162,956 deaths. Of these, 83,006 new cases and 5,109 deaths were reported in the past 24 hours. Quarantine, isolation, and infection control measures have been used to prevent the spread of the disease and to provide supportive care for those who are ill (Baden and Rubin, Covid-19: The Search for Effective Therapy, New England Journal of Medicine 382(19):1851-52(2020)).

[0008] Despite the development and use of vaccines and therapeutics, the SARS-CoV-2 outbreak continues, and SARS-CoV-2 mutants continue to evolve and evade these preventative and therapeutic measures. Therefore, there is a need for additional therapeutics that can be rapidly deployed, preferably those that combat evading mutants while maintaining therapeutic efficacy (e.g., through broad-spectrum neutralizing activity). Summary of the Invention

[0009] There is an urgent need to develop specific antiviral therapeutics for the prevention of the spread of COVID-19 and for the treatment of COVID-19 patients, preferably wherein such therapeutics retain activity against new and emerging variants and have broad neutralizing activity. This disclosure provides such therapeutics.

[0010] The disclosure herein is partly based on the finding that the disclosed peptides specifically bind to the spike glycoprotein of SARS-CoV-2 (SARS-CoV-2-spike). The disclosure herein is also partly based on the finding that the disclosed peptides exhibit robust neutralizing activity against SARS-CoV-2 variants in vitro and in vivo. Therefore, this disclosure generally relates to compositions (e.g., peptides, pharmaceutical compositions) and methods for reducing spike-mediated viral entry into cells (e.g., SARS-CoV-2-spike).

[0011] Among other things, this document provides peptides (e.g., antibodies and their antigen-binding fragments) that specifically bind to S2 domain epitopes of β-coronavirus spike glycoproteins (e.g., S2 domain epitopes of the SARS-CoV-2 spike glycoprotein). In some embodiments, the peptides have one or more properties selected from: broad neutralizing activity against a variety of known and predicted β-coronaviruses (e.g., past, present, emerging, and future β-coronaviruses), and binding affinity to highly conserved S2 domain epitopes across a variety of β-coronaviruses. In some embodiments, the peptides have broad neutralizing activity against a variety of known and predicted β-coronaviruses, and binding affinity to highly conserved S2 domain epitopes across a variety of β-coronaviruses.

[0012] Among other things, this disclosure provides a polypeptide that specifically binds to the SARS-CoV-2 spike protein, wherein the polypeptide contains a complementary site substantially similar to the complementary site of an antibody, and the antibody contains V... H / V L right:

[0013] SEQ ID NO:4 and SEQ ID NO:51(AB-1);

[0014] SEQ ID NO:5 and SEQ ID NO:52(AB-2);

[0015] SEQ ID NO:6 and SEQ ID NO:53(AB-3);

[0016] SEQ ID NO:7 and SEQ ID NO:54(AB-4);

[0017] SEQ ID NO:8 and SEQ ID NO:51(AB-5);

[0018] SEQ ID NO:9 and SEQ ID NO:55(AB-6);

[0019] SEQ ID NO:10 and SEQ ID NO:56(AB-7);

[0020] SEQ ID NO:11 and SEQ ID NO:57(AB-8);

[0021] SEQ ID NO:12 and SEQ ID NO:58(AB-9);

[0022] SEQ ID NO:13 and SEQ ID NO:59(AB-10);

[0023] SEQ ID NO:14 and SEQ ID NO:60(AB-11);

[0024] SEQ ID NO:15 and SEQ ID NO:56(AB-12);

[0025] SEQ ID NO:16 and SEQ ID NO:51(AB-13);

[0026] SEQ ID NO:10 and SEQ ID NO:50 (AB-14);

[0027] SEQ ID NO:17 and SEQ ID NO:61(AB-15);

[0028] SEQ ID NO:18 and SEQ ID NO:62(AB-16);

[0029] SEQ ID NO:6 and SEQ ID NO:63(AB-17);

[0030] SEQ ID NO:19 and SEQ ID NO:64(AB-18);

[0031] SEQ ID NO:4 and SEQ ID NO:61(AB-19);

[0032] SEQ ID NO:20 and SEQ ID NO:61(AB-20);

[0033] SEQ ID NO:21 and SEQ ID NO:65(AB-21);

[0034] SEQ ID NO:22 and SEQ ID NO:66(AB-22);

[0035] SEQ ID NO:4 and SEQ ID NO:67(AB-23);

[0036] SEQ ID NO:23 and SEQ ID NO:56(AB-24);

[0037] SEQ ID NO:24 and SEQ ID NO:68(AB-25);

[0038] SEQ ID NO:25 and SEQ ID NO:51(AB-26);

[0039] SEQ ID NO:26 and SEQ ID NO:56(AB-27);

[0040] SEQ ID NO:27 and SEQ ID NO:61(AB-28);

[0041] SEQ ID NO:28 and SEQ ID NO:56(AB-29);

[0042] SEQ ID NO:28 and SEQ ID NO:69(AB-30);

[0043] SEQ ID NO:29 and SEQ ID NO:70(AB-31);

[0044] SEQ ID NO:30 and SEQ ID NO:71(AB-32);

[0045] SEQ ID NO:31 and SEQ ID NO:72(AB-33);

[0046] SEQ ID NO:32 and SEQ ID NO:67(AB-34);

[0047] SEQ ID NO:33 and SEQ ID NO:56(AB-35);

[0048] SEQ ID NO:34 and SEQ ID NO:73(AB-36);

[0049] SEQ ID NO:35 and SEQ ID NO:51(AB-37);

[0050] SEQ ID NO:36 and SEQ ID NO:56(AB-38);

[0051] SEQ ID NO:37 and SEQ ID NO:63(AB-39);

[0052] SEQ ID NO:38 and SEQ ID NO:69 (AB-40);

[0053] SEQ ID NO:39 and SEQ ID NO:74(AB-41);

[0054] SEQ ID NO:40 and SEQ ID NO:52 (AB-42);

[0055] SEQ ID NO:41 and SEQ ID NO:51(AB-43);

[0056] SEQ ID NO:42 and SEQ ID NO:75(AB-44);

[0057] SEQ ID NO:43 and SEQ ID NO:56(AB-45);

[0058] SEQ ID NO:44 and SEQ ID NO:51(AB-46);

[0059] SEQ ID NO:45 and SEQ ID NO:75(AB-47);

[0060] SEQ ID NO:46 and SEQ ID NO:53 (AB-48);

[0061] SEQ ID NO:47 and SEQ ID NO:52 (AB-49);

[0062] SEQ ID NO:48 and SEQ ID NO:76 (AB-50); or

[0063] SEQ ID NO:3 and SEQ ID NO:56(AB-51), or

[0064] Any of the aforementioned combinations.

[0065] Among other things, this disclosure also provides a polypeptide that specifically binds to the SARS-CoV-2 spike protein, wherein the polypeptide comprises:

[0066] Variable domains of immunoglobulin heavy chain (V H ) amino acid sequence, the V H The amino acid sequence comprises HCDR1, HCDR2, and HCDR3, which are substantially similar to the heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and / or 3 (HCDR3) of any of the amino acid sequences in SEQ ID NO:4-48, respectively; and

[0067] Variable domains of immunoglobulin light chains (V L ) amino acid sequence, the V L The amino acid sequences include LCDR1, LCDR2 and LCDR3, which are substantially similar to the light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2) and / or 3 (LCDR3) of the amino acid sequences of any of SEQ ID NO:51-76, respectively.

[0068] In some embodiments, the polypeptides disclosed herein comprise HCDR1, HCDR2 and / or HCDR3 and / or LCDR1, LCDR2 and / or LCDR3 of an antibody, said antibody comprising an amino acid sequence selected from:

[0069] SEQ ID NO:4 and SEQ ID NO:51(AB-1);

[0070] SEQ ID NO:5 and SEQ ID NO:52(AB-2);

[0071] SEQ ID NO:6 and SEQ ID NO:53(AB-3);

[0072] SEQ ID NO:7 and SEQ ID NO:54(AB-4);

[0073] SEQ ID NO:8 and SEQ ID NO:51(AB-5);

[0074] SEQ ID NO:9 and SEQ ID NO:55(AB-6);

[0075] SEQ ID NO:10 and SEQ ID NO:56(AB-7);

[0076] SEQ ID NO:11 and SEQ ID NO:57(AB-8);

[0077] SEQ ID NO:12 and SEQ ID NO:58(AB-9);

[0078] SEQ ID NO:13 and SEQ ID NO:59(AB-10);

[0079] SEQ ID NO:14 and SEQ ID NO:60(AB-11);

[0080] SEQ ID NO:15 and SEQ ID NO:56(AB-12);

[0081] SEQ ID NO:16 and SEQ ID NO:51(AB-13);

[0082] SEQ ID NO:10 and SEQ ID NO:50 (AB-14);

[0083] SEQ ID NO:17 and SEQ ID NO:61(AB-15);

[0084] SEQ ID NO:18 and SEQ ID NO:62(AB-16);

[0085] SEQ ID NO:6 and SEQ ID NO:63(AB-17);

[0086] SEQ ID NO:19 and SEQ ID NO:64(AB-18);

[0087] SEQ ID NO:4 and SEQ ID NO:61(AB-19);

[0088] SEQ ID NO:20 and SEQ ID NO:61(AB-20);

[0089] SEQ ID NO:21 and SEQ ID NO:65(AB-21);

[0090] SEQ ID NO:22 and SEQ ID NO:66(AB-22);

[0091] SEQ ID NO:4 and SEQ ID NO:67(AB-23);

[0092] SEQ ID NO:23 and SEQ ID NO:56(AB-24);

[0093] SEQ ID NO:24 and SEQ ID NO:68(AB-25);

[0094] SEQ ID NO:25 and SEQ ID NO:51(AB-26);

[0095] SEQ ID NO:26 and SEQ ID NO:56(AB-27);

[0096] SEQ ID NO:27 and SEQ ID NO:61(AB-28);

[0097] SEQ ID NO:28 and SEQ ID NO:56(AB-29);

[0098] SEQ ID NO:28 and SEQ ID NO:69(AB-30);

[0099] SEQ ID NO:29 and SEQ ID NO:70(AB-31);

[0100] SEQ ID NO:30 and SEQ ID NO:71(AB-32);

[0101] SEQ ID NO:31 and SEQ ID NO:72(AB-33);

[0102] SEQ ID NO:32 and SEQ ID NO:67(AB-34);

[0103] SEQ ID NO:33 and SEQ ID NO:56(AB-35);

[0104] SEQ ID NO:34 and SEQ ID NO:73(AB-36);

[0105] SEQ ID NO:35 and SEQ ID NO:51(AB-37);

[0106] SEQ ID NO:36 and SEQ ID NO:56(AB-38);

[0107] SEQ ID NO:37 and SEQ ID NO:63(AB-39);

[0108] SEQ ID NO:38 and SEQ ID NO:69 (AB-40);

[0109] SEQ ID NO:39 and SEQ ID NO:74(AB-41);

[0110] SEQ ID NO:40 and SEQ ID NO:52 (AB-42);

[0111] SEQ ID NO:41 and SEQ ID NO:51(AB-43);

[0112] SEQ ID NO:42 and SEQ ID NO:75(AB-44);

[0113] SEQ ID NO:43 and SEQ ID NO:56(AB-45);

[0114] SEQ ID NO:44 and SEQ ID NO:51(AB-46);

[0115] SEQ ID NO:45 and SEQ ID NO:75(AB-47);

[0116] SEQ ID NO:46 and SEQ ID NO:53 (AB-48);

[0117] SEQ ID NO:47 and SEQ ID NO:52 (AB-49);

[0118] SEQ ID NO:48 and SEQ ID NO:76 (AB-50); or

[0119] SEQ ID NO:3 and SEQ ID NO:56(AB-51).

[0120] In some embodiments, the polypeptide disclosed herein contains the same complementary site as the antibody, which contains an amino acid sequence selected from:

[0121] SEQ ID NO:4 and SEQ ID NO:51(AB-1);

[0122] SEQ ID NO:5 and SEQ ID NO:52(AB-2);

[0123] SEQ ID NO:6 and SEQ ID NO:53(AB-3);

[0124] SEQ ID NO:7 and SEQ ID NO:54(AB-4);

[0125] SEQ ID NO:8 and SEQ ID NO:51(AB-5);

[0126] SEQ ID NO:9 and SEQ ID NO:55(AB-6);

[0127] SEQ ID NO:10 and SEQ ID NO:56(AB-7);

[0128] SEQ ID NO:11 and SEQ ID NO:57(AB-8);

[0129] SEQ ID NO:12 and SEQ ID NO:58(AB-9);

[0130] SEQ ID NO:13 and SEQ ID NO:59(AB-10);

[0131] SEQ ID NO:14 and SEQ ID NO:60(AB-11);

[0132] SEQ ID NO:15 and SEQ ID NO:56(AB-12);

[0133] SEQ ID NO:16 and SEQ ID NO:51(AB-13);

[0134] SEQ ID NO:10 and SEQ ID NO:50 (AB-14);

[0135] SEQ ID NO:17 and SEQ ID NO:61(AB-15);

[0136] SEQ ID NO:18 and SEQ ID NO:62(AB-16);

[0137] SEQ ID NO:6 and SEQ ID NO:63(AB-17);

[0138] SEQ ID NO:19 and SEQ ID NO:64(AB-18);

[0139] SEQ ID NO:4 and SEQ ID NO:61(AB-19);

[0140] SEQ ID NO:20 and SEQ ID NO:61(AB-20);

[0141] SEQ ID NO:21 and SEQ ID NO:65(AB-21);

[0142] SEQ ID NO:22 and SEQ ID NO:66(AB-22);

[0143] SEQ ID NO:4 and SEQ ID NO:67(AB-23);

[0144] SEQ ID NO:23 and SEQ ID NO:56(AB-24);

[0145] SEQ ID NO:24 and SEQ ID NO:68(AB-25);

[0146] SEQ ID NO:25 and SEQ ID NO:51(AB-26);

[0147] SEQ ID NO:26 and SEQ ID NO:56(AB-27);

[0148] SEQ ID NO:27 and SEQ ID NO:61(AB-28);

[0149] SEQ ID NO:28 and SEQ ID NO:56(AB-29);

[0150] SEQ ID NO:28 and SEQ ID NO:69(AB-30);

[0151] SEQ ID NO:29 and SEQ ID NO:70(AB-31);

[0152] SEQ ID NO:30 and SEQ ID NO:71(AB-32);

[0153] SEQ ID NO:31 and SEQ ID NO:72(AB-33);

[0154] SEQ ID NO:32 and SEQ ID NO:67(AB-34);

[0155] SEQ ID NO:33 and SEQ ID NO:56(AB-35);

[0156] SEQ ID NO:34 and SEQ ID NO:73(AB-36);

[0157] SEQ ID NO:35 and SEQ ID NO:51(AB-37);

[0158] SEQ ID NO:36 and SEQ ID NO:56(AB-38);

[0159] SEQ ID NO:37 and SEQ ID NO:63(AB-39);

[0160] SEQ ID NO:38 and SEQ ID NO:69 (AB-40);

[0161] SEQ ID NO:39 and SEQ ID NO:74(AB-41);

[0162] SEQ ID NO:40 and SEQ ID NO:52 (AB-42);

[0163] SEQ ID NO:41 and SEQ ID NO:51(AB-43);

[0164] SEQ ID NO:42 and SEQ ID NO:75(AB-44);

[0165] SEQ ID NO:43 and SEQ ID NO:56(AB-45);

[0166] SEQ ID NO:44 and SEQ ID NO:51(AB-46);

[0167] SEQ ID NO:45 and SEQ ID NO:75(AB-47);

[0168] SEQ ID NO:46 and SEQ ID NO:53 (AB-48);

[0169] SEQ ID NO:47 and SEQ ID NO:52 (AB-49);

[0170] SEQ ID NO:48 and SEQ ID NO:76 (AB-50); or

[0171] SEQ ID NO:3 and SEQ ID NO:56(AB-51).

[0172] Among other things, this disclosure further provides a polypeptide comprising V H The V H Includes SEQ ID NO:2, where:

[0173] X1 is not S;

[0174] X2 is not D;

[0175] X3 is not T;

[0176] X4 is not L;

[0177] X5 is not S;

[0178] X6 is not N;

[0179] X7 is not G;

[0180] X8 is not V; or

[0181] X9 is not Q.

[0182] Or any combination thereof.

[0183] In some embodiments, the polypeptide disclosed herein comprises V L The V L Includes SEQ ID NO:49, where:

[0184] X 10 Not Q;

[0185] X 11 Not G;

[0186] X 12 Not S;

[0187] X 13 Not S;

[0188] X 14 Not N;

[0189] X 15 Not S;

[0190] X 16 Not F; or

[0191] X 17 Not Y,

[0192] Or any combination thereof.

[0193] In some embodiments, this disclosure provides a polypeptide that specifically binds to the SARS-CoV-2 spike protein, comprising:

[0194] V, which has at least 70% sequence identity with SEQ ID NO:3 H sequence;

[0195] V, which has at least 70% sequence identity with SEQ ID NO:50 L Sequence; or

[0196] Its combination,

[0197] Wherein V H The sequence does not contain SEQ ID NO:3, the V L The sequence does not contain SEQ ID NO:50 or both.

[0198] In some embodiments, the polypeptides disclosed herein are fusion proteins.

[0199] In some embodiments, this disclosure provides a polynucleotide encoding a polypeptide disclosed herein, a vector containing such a polynucleotide, and a host cell containing such a polynucleotide and / or the vector.

[0200] In some embodiments, this disclosure provides a method of treating a patient and / or subject in need (e.g., a subject with 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 comprising one or more polypeptides disclosed herein (e.g., a pharmaceutical composition).

[0201] In some embodiments, this disclosure provides a method for neutralizing SARS-CoV-2 variants in cells (e.g., cells in a subject's body), the method comprising contacting the cells with an effective amount of a composition comprising a polypeptide disclosed herein and / or a composition comprising a polypeptide disclosed herein (e.g., a pharmaceutical composition).

[0202] In some embodiments, this disclosure provides a method for neutralizing SARS-CoV-2 variants in a subject, the method comprising providing the subject with an effective amount of a composition comprising a peptide disclosed herein and / or a composition comprising a peptide disclosed herein (e.g., a pharmaceutical composition). Attached Figure Description

[0203] The foregoing will become clear from the following more specific description of exemplary embodiments, as illustrated in the accompanying drawings, in which similar reference numerals refer to the same parts in different views. The drawings are not necessarily drawn to scale, but are intended to illustrate the embodiments.

[0204] In the attached figure, "reference" refers to the reference antibody.

[0205] Figure 1 The amino acid sequences of epitopes (bold letters) within the S2 domain (underlined) of the SARS-CoV-2 spike (SEQ ID NO:1) are depicted. Epitope residues bound by the reference antibody disclosed herein are indicated by asterisks.

[0206] Figure 2 The heavy chain variable domain (V) of the reference antibody and AB-1 was described. H Amino acid sequence alignment. Heavy chain complementarity-determining region (HCDR) amino acid sequences, as determined by ImMunoGeneTics (IMGT) numbering, are indicated by underlining. Bold letters indicate reference antibodies and variable residues in AB-1 through AB-51 (marked as “Xn” in this disclosure). * indicates complementary residues. See also Table 1, V. H Common sequence. The complementary position is defined as the antibody residue in the reference that is within 5 angstroms of the antigen when it binds to the S2 domain of the SARS-CoV-2 spike.

[0207] Figure 3 The light chain variable domain (V) of the reference antibody and AB-1 was described. L Amino acid sequence alignment. The amino acid sequence of the light chain complementarity-determining region (LCDR) as determined by IMGT number is indicated by underlining. Bold letters indicate reference antibodies and variable residues in AB-1 to AB-51 (marked by “Xn” in this disclosure). “*” indicates complementary residues. See also Table 2, V. L Common sequence. The complementary position is defined as the antibody residue in the reference antibody that is within 5 angstroms of the antigen when it binds to the S2 domain of the SARS-CoV-2 spike.

[0208] Figures 4A-4B The combined data, expressed as a composite score for the indicated virus, are 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.

[0209] Figures 5A-5B The combined and neutral data, expressed as a comprehensive score for the indicated virus, are shown.

[0210] Figures 6A-6BA table summarizing data from screening hits, clinical-stage molecules, and reference antibodies is shown. Overall Neutralization Score: A score representing all neutralization data for each antibody, expressed as a single value. A higher value indicates a better overall neutralization curve for that specific antibody. Overall Binding Score: A score representing all binding data for each antibody, expressed as a single value. A higher value indicates a better overall binding curve for that specific antibody. Neutralization δ Slow Emission 3: The percentage of δ pseudovirus neutralized by each antibody at 0.16 μg / ml. Neutralization oBA.2 Slow Emission 3: The percentage of oBA.2 pseudovirus neutralized by each antibody at 0.16 μg / ml. Pseudovirus neutralization data were obtained as described in Materials and Methods.

[0211] Figures 7A-7B Neutralization curves for AB-1 and the clinical-stage control antibody sotrovimab against the indicated pseudovirus are shown. Y-axis: neutralization %; X-axis: antibody concentration expressed in μg / ml.

[0212] Figures 8A-8B Neutralization curves for AB-41 and the clinical-stage control antibody sotopemumab against the indicated pseudovirus are shown. Y-axis: neutralization %; X-axis: antibody concentration expressed in μg / ml.

[0213] Figures 9A-9B Neutralization curves are shown for high-throughput produced AB-17, AB-15, AB-1, and reference antibodies, as well as the clinical-stage control antibody sotopemumab against the indicated pseudovirus. Y-axis: neutralization %; X-axis: antibody concentration expressed in μg / ml.

[0214] Figures 10A-10B Three lead molecules showed robustness and considerable neutralization against a variety of pseudoviruses. Neutralization assays were performed using the indicated pseudoviruses and the following antibodies: isotype control (Synagis), reference antibody, clinical-phase control (human IgG1 molecules expressing the same variable region as sotopimazole, two antibodies constituting Evusheld [AZD1061 and AZD8895]), and lead molecules from mass production (AB-1, AB-17, AB-15). Results represent at least eight technical replicas within at least two biological replicas and are shown as mean ± SEM.

[0215] Figure 11 This is a graph showing the neutralization of live virus by AB-1, AB-17, and AB-15. Neutralization experiments were performed using the indicated live virus and the following antibodies: isotype control (Sinakis), reference antibody, clinical phase control (human IgG1 molecule expressing the same variable region of sotopemumab), AB-1, AB-17, and AB-15. Results represent three replicates and are shown as mean ± SEM.

[0216] Figure 12 It presents charts showing the exploitation study data for AB-1, AB-15, and AB-17.

[0217] Figure 13A and 13B This graph shows the improved neutralization curves achieved by combining AB-1 with either a clinical-stage class 3 anti-RBD antibody or a class 4 anti-RBD monoclonal antibody. Compared to AB-1 alone, the combination of AB-1 with either a clinical-stage class 3 anti-RBD antibody (sotovizumab or betelovirimab) or a class 4 anti-RBD monoclonal antibody (anti-RBD4mAb) showed enhanced neutralization curves (as measured by area under the curve (AUC) and efficacy (maximum neutralization)).

[0218] Figures 14A-14C Figures (14A, 14B) and graph (14C) show how the combination of four types of anti-RBD monoclonal antibodies and AB-1 improved the neutralization curves against the o variant.

[0219] Figures 15A-15H This figure illustrates how AB-1 protects hamsters from SARS-CoV-2δ infection. Two hamster studies showed that prophylactic administration (day -1) resulted in the expression of human IgG1 (…). Figures 15A-15D ) or hamster IgG2a ( Figures 15E-15H The AB-1 formulation was used, and SARS-CoV-2 was challenged with δ (day 0). Both AB-1 forms showed dose-dependent protection against δ-induced weight loss. Figures 15A-15B (and 15E-15F), dose-dependent protection against delta-induced lung weight increase (a representative of lung inflammation, Figure 15C and 15G And its impact on viral titer ( Figure 15D and 15HThe effect was more pronounced on day 7 compared to earlier time points, and its magnitude appeared slightly lower compared to sotopimumab. Hamster body weight was recorded up to day 7 after challenge and expressed as a percentage change from day 0 (pre-challenge). Allotype controls were delivered at 25 mg / kg, and sotopimumab at 5 mg / kg. Results are shown as mean ± SEM. Percentage change in body weight on day 7 (15B and 15F). Each point represents an individual hamster, and the horizontal line represents the median. Lung weight on day 7 (15C and 15G). Lung viral titer on day 4 (15D and 15H). Each point represents an individual hamster, and the horizontal line represents the median. N = 12 hamsters (days 0–4) or 6 hamsters (days 5–7) per group. Data were analyzed by one-way ANOVA corrected for multiple comparisons (Dunnett's test). Black asterisks indicate comparisons with hamsters treated with the same type of control. **p<0.01.

[0220] Figures 16A-16D This figure illustrates how AB-1 protects hamsters from SARS-CoV-2oBA.2 infection. A hamster study was conducted in which AB-1, expressing hamster IgG2a, was administered prophylactically (day -1) and challenged with SARS-CoV-2oBA.2 (day 0). AB-1 showed dose-dependent protection against oBA.2-induced weight loss. Figures 16A-16B ), dose-dependent protection against oBA.2-induced lung weight increase (a representative of lung inflammation, Figure 16C And its impact on viral titer ( Figure 16D 16A) Hamster body weight was recorded up to day 7 after challenge and expressed as a percentage change from day 0 (before challenge). Allotype controls were delivered at 25 mg / kg, and sotopemumab at 5 mg / kg. Results are shown as mean ± SEM. 16B) Percentage change in body weight on day 7. Each point represents an individual hamster, and the horizontal line represents the median. 16C) Lung weight on day 7. 16D) Lung viral titer on day 4. Each point represents an individual hamster, and the horizontal line represents the median. N = 12 hamsters (days 0–4) or 6 hamsters (days 5–7) per group. Data were analyzed by one-way ANOVA adjusted for multiple comparisons (Dunnett's test). Black asterisks indicate comparisons with allotype control hamsters. **p < 0.01.

[0221] Figures 17A-17D It is a picture, and Figure 17E The chart shows that the combination of AB-1 with class 4 anti-RBD antibody 3a (“RD class 4 mAb-3a” or “R-AB-3a”) demonstrates enhanced neutralizing potency and efficacy against oBQ.1.1 pseudoviruses.Figures 17A-17C Neutralization curves are shown. Sinakis was used as an isotype control. Figure 17D It shows Figure 17B The results of the neutralization curves shown are reported as efficacy (neutralization % at 18 μg / ml). Figure 17E Reported Figure 17B The results shown are for EC 50 (95% CI) values ​​and median neutralization % values ​​at 18 μg / ml (95% CI). Standard deviation (SD) is applied as an error bar. Figure 17B In the mean, the standard error (SEM) of the mean is used as an error bar. Figure 17C middle.

[0222] Figure 18A and 18B It is a picture, and Figure 18C The graph shows the neutralization curves against δ and oBA.5 live viruses when the combination of AB-1 and class 4 anti-RBD antibody 4a (“RD class 4 mAb-4a” or “R-AB-4a”) or sotopemumab is improved. Figure 18A and 18B Neutralization curves against SARS-CoV-2δ and BA.5 live virus are shown separately. Sinakis was used as an isotype control. Figure 18C Reported Figures 18A-18B The results shown are for EC 50 (95% CI) values ​​and median neutral % values ​​at 18 μg / ml (95% CI). Standard deviation (SD) is used as an error bar in 18A-18B.

[0223] Figure 19 This is a graph showing the in vitro neutralizing efficacy of AB-1 against multiple variants of SARS-CoV-2. Results were obtained from a pseudovirus neutralization assay.

[0224] Figure 20 The neutralizing IC is shown 50 Data are expressed as the logarithmic fold change of the reference antibody against the 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.

[0225] Figure 21The binding of AB-1 and reference antibody to the SARS-CoV-2 spike S2 ​​peptide was assessed by DELFIA. The binding of AB-1, reference antibody, and allotype control to the SARS-CoV-2 spike S2 ​​peptide (indicated as “S:” followed by the amino acid position) and the HIV-1Env negative control peptide was evaluated by DELFIA. Results are expressed as AUC values ​​and are shown in bars representing mean ± standard deviation (representing two independent experiments, three technical replicas per experiment). Statistical comparisons between AB-1 and reference antibody AUC values ​​were performed using a two-dimensional ANOVA corrected for multiple comparisons (**indicating P ≤ 0.01).

[0226] Figure 22 The binding of AB-1 and reference antibodies to the SARS-CoV-2 spike S2 ​​peptide was assessed by SPR. The binding of AB-1 and reference antibodies to the SARS-CoV-2 spike S2 ​​peptide (indicated as "S:", followed by the amino acid position) and the HIV-1Env negative control peptide was evaluated by SPR at 25 °C. Antibody binding responses were normalized relative to biotinylated peptide capture levels and reported as normalized binding responses. Results represent an experiment without technical replicas.

[0227] Figures 23A-23D Sensing map of Fab binding to the SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide by SPR AB-1 and reference antibody. AB-1 (aa 1149-1167) peptide bound to the SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide obtained by SPR. Figure 23A ) and reference antibody ( Figure 23B The sensor map of Fab, and AB-1 (which binds to the HIV-1Env negative control peptide). Figure 23C ) and reference antibody ( Figure 23D The sensor plots of the Fab are shown. Real-time combined sensor plots are displayed as black curves, while the fit produced by globally fitting the data to a 1:1 combined model with quality transmission limitations is displayed as dashed curves. The results represent an experiment without technical replication.

[0228] Figures 24A-24C AB-1Fab binds to the SARS-CoV-2 spike, with each spike having an apparent stoichiometry of three Fabs. Figure 24A Low-pass filtered Cryo-EM plot of the SARS-CoV-2BA.1 spike trimer with R-AB-3a Fab binding RBD and AB-1 binding S2 stem helix. The plot is shown from the side profile and from below. Each hypothetical Fab is labeled. Figure 24BDensity-docking atomic model of the SARS-CoV-2 spike assembly with bound Fabs. Spike trimer: High-resolution structure of the SARS-CoV-2 spike, with the S2 stem-helix resolved. R-AB-3a: Model of the design of the R-AB-3a Fab. Bottom: Three AB-1 Fabs docked in a propeller-shaped density around the S2 stem-helix. Figure 24C : A publicly available cryo-electron tomography image of SARS-CoV-2 spike trimers in virus-like particles bound to a reference antibody Fab. (Compared to...) Figure 24A The same applies to the graph, which has been low-pass filtered. The two copies of the reference antibody's Fab are clearly visible in the graph.

[0229] Figure 25 Frequency of mutations observed in the region of interest. Distribution of the relative frequencies of mutations within “HR1”, “HR2”, “epitope”, and “adjacent epitope residues” observed over three different time intervals up to March 1, 2023 (earliest [January 6, 2020 to March 1, 2023], 3 months [December 1, 2022 to March 1, 2023], and 1 month [February 1, 2023 to March 1, 2023]). Each data point corresponds to one mutation. Dashed lines indicate two thresholds that allow mutations to be included in the reported set: 0.001 represents the relative frequency over all time periods, and 0.01 represents the recent (last 3 months) relative frequency.

[0230] Figure 26 Mutations in a tabletop. Each small plot shows how the relative frequency of a mutation changes over time. All mutations that occurred in the tabletop and exceeded the relative frequency threshold are shown. The X-axis represents the time dimension: each value along the axis is the end date of the backtracking period; for example, "2023-01" represents the period ending on January 1, 2023. The Y-axis represents the relative frequency of mutations. Each colored line graph is paired with a rolling backtracking period of a specific length (earliest [starting from January 6, 2020], 3 months [starting from 3 months before the end date], 1 month [starting from 1 month before the end date]). Each point along the line graph represents the relative frequency of the mutation within a specific backtracking period ending on the end date. Dashed lines indicate two thresholds that allow mutations to be included in the reported set: 0.001 represents the relative frequency over all time, and 0.01 represents the most recent (last 3 months) relative frequency.

[0231] Figures 27A-27B Mutations in HR1. This shows all mutations that occurred in HR1 and exceeded the relative frequency threshold. See the diagram for the format. Figure 26 A brief explanation.

[0232] Figure 28Mutations in HR2. This shows all mutations that occurred in HR2 and exceeded the relative frequency threshold. See the diagram for the format. Figure 26 A brief explanation.

[0233] Figure 29 Relative frequencies of the most prevalent lineages. The relative frequencies of the overall most prevalent lineages appearing over three different time intervals up to March 1, 2023 (earliest [January 6, 2020 to March 1, 2023], 3 months [December 1, 2022 to March 1, 2023], and 1 month [February 1, 2023 to March 1, 2023]). Five of the most common lineages are shown; all other lineages are grouped together as "Other".

[0234] Figure 30 The relative frequencies of mutations of interest in the most prevalent lineages, regardless of the AB-1 epitope. The relative frequencies of mutations of interest in sequences assigned to 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], and 1 month [February 1, 2023 to March 1, 2023]) (reported in Table 2).

[0235] Figures 31A-31C The binding of AB-1 to the DELFIA of the spike trimer via AB-1 and a reference antibody, and the binding of AB-1 to both SARS-CoV-2 and non-SARS-CoV-2 spike trimers (indicated by "S:", followed by the corresponding virus name) are shown as follows: Figure 31A The main SARS-CoV-2 variant (SARS-CoV-2); Figure 31B It is not SARS-CoV-2 Sarbecovirus (Sarbecov.); Figure 31C The study included SARS-CoV-2 variants (SARS-CoV-2BA.2+P1162L and BA.2+P1162S) exhibiting polymorphism in the AB-1 epitope, as well as their parent strain (SARS-CoV-2BA.2 (parent)). The results showed EC... 50 Values ​​(μg / mL) are shown as mean and 95% confidence intervals and represent two independent experiments, each with four technical replicas. The dashed line represents the upper limit of the 95% confidence interval for AB-1 binding to the reference trimer and the reference antibody. Figure 31A and Figure 31B It is SARS-CoV-2D614G. Figure 31C (This is SARS-CoV-2BA.2 (parental)).

[0236] Figures 32A-32DSensing plots of AB-1 and reference antibody binding to a group of SARS-CoV-2 spike trimers via SPR at 25°C. A diagram from an independent experiment at 25°C is shown. Figure 32A ) and reference antibody ( Figure 32B Sensing plots of Fab binding to SARS-CoV-2 spike trimer D614G, δ, BA.4, BA.5, BQ.1.1, XBB.1.5 and MERS spike trimer (negative control). A fumed Fab assay is shown from an independent experiment at 25°C. Figure 32C ) and reference antibody ( Figure 32D Sensing plots of Fab binding to the SARS-CoV-2 spike trimer BA.2 (parental), BA.2+P1162L, and BA.2+P1162S are shown. Real-time binding sensing plots at different concentrations are presented, along with the fits obtained by globally fitting the data to a 1:1 binding model with mass transfer constraints.

[0237] Figures 33A-33D Sensing plots of AB-1 and reference antibody binding to a group of SARS-CoV-2 spike trimers via SPR at 37°C. A diagram from an independent experiment at 37°C is shown. Figure 33A ) and reference antibody ( Figure 33B Sensing plots of Fab binding to SARS-CoV-2 spike trimer D614G, δ, BA.4, BA.5, BQ.1.1, XBB.1.5 and MERS spike trimer (negative control). A sensory map of AB-1 binding to SARS-CoV-2 spike trimer D614G, δ, BA.4, BA.5, BQ.1.1, XBB.1.5 and MERS spike trimer (negative control) from an independent experiment is shown. Figure 33C ) and reference antibody ( Figure 33D Sensing plots of Fab binding to the SARS-CoV-2 spike trimer BA.2 (parental), BA.2+P1162L, and BA.2+P1162S are shown. Real-time binding sensing plots at different concentrations are presented, along with the fits obtained by globally fitting the data to a 1:1 binding model with mass transfer constraints.

[0238] Figure 34 AB-1 neutralization of SARS-CoV-2 variants and non-SARS-CoV-2 sabeclovirus. Neutralization curves for AB-1, betronidazole, and isotype control antibodies against pseudoviruses representing SARS-CoV-2 variants and non-SARS-CoV-2 sabeclovirus. Results are reported as % neutralization and are shown as mean ± standard deviation (representing three to six independent experiments, four technical replicas per experiment).

[0239] Figures 35A-35BNeutralization of SARS-CoV-2 variants via a combination of AB-1 and R-AB-2b. Using TMPRSS2-Vero E6 ( Figure 35A ) or Vero E6 ( Figure 35B Neutralization curves for AB-1+R-AB-2b (tested at two different ratios), AB-1, R-AB-2b, and betelvirumab as single agents, and an isotype control antibody against a pseudovirus representing the indicated SARS-CoV-2 variant. Anti-spike antibodies tested as single agents were combined with isotypes at the same concentration to control the total mass of anti-spike antibodies tested as combinations. 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 mean ± standard deviation (representing three or four independent experiments, each with four technical replicas).

[0240] Figure 36 Neutralization of SARS-CoV-2 variants by combination of AB-1 and sotopimumab VH / VL-huIgG1-LS. Neutralization curves were constructed using Vero E6 cells with AB-1 + sotopimumab VH / VL-huIgG1-LS (tested at two different ratios), AB-1, sotopimumab VH / VL-huIgG1-LS, and betelvirumab as single agents, and an isotype control antibody against a pseudovirus representing the indicated SARS-CoV-2 variant. Anti-spike antibodies tested as single agents were combined with isotypes at the same concentration to control the total mass of anti-spike antibodies tested as combinations. 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 are shown as mean ± standard deviation (representing three or four independent experiments, four technical replicas per experiment).

[0241] Figure 37 Evaluation of neutralization curves for parental and alternative antibodies. Neutralization curves for AB-1, sotopimumab VH / VL-huIgG1-LS, and allotype control antibodies, along with their corresponding alternative human IgG1 and hamster IgG2a molecules, against a pseudovirus representing a SARS-CoV-2 ancestor with the D614G mutation and BA.2 variant. Results are expressed as % neutralization and are shown as mean ± standard deviation (representing three independent experiments, four technical replicas per experiment).

[0242] Figure 38SARS-CoV-2 pseudoviral infectivity in cells carrying the Fcγ receptor in the presence of AB-1. Mononuclear cell lines (U-937, THP-1) and human primary PBMCs were infected with the SARS-CoV-2 D614G pseudovirus in the presence of AB-1, AB-1VH / VL-huIgG1, betelvirumab, and an isotype control. TMPRSS2-Vero E6 cells were used as a positive control for infection. Results are expressed in relative luminescent units and are shown as mean ± standard deviation (representing three independent experiments, three technical replicas per experiment).

[0243] Figure 39 Neutralization of live SARS-CoV-2 virus variants via the combination of AB-1 and R-AB-2b. Neutralization curves of AB-1+R-AB-2b, AB-1, R-AB-2b, and betronidazole as single agents, and isotype control antibodies against the ancestral SARS-CoV-2 strain (Eng20(WT)), δ, BQ.1.1, and XBB.1.1 live virus. Anti-spike antibodies tested as single agents were combined with isotypes at the same concentration to control the total mass of anti-spike antibodies tested as combinations. The x-axis indicates the concentration of each individual antibody in the combination, or, if the concentrations are different, the concentration of the antibody with the highest concentration. Results are reported as % neutralization and shown as mean ± standard deviation (representing one experiment, two to three technical replicas). Detailed Implementation

[0244] The following is a description of an example embodiment.

[0245] The following description of several aspects of this disclosure is for illustrative purposes only and with reference to examples. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of this disclosure. However, those skilled in the art will readily recognize that this disclosure can be practiced without one or more of the specific details described, or with other methods, protocols, reagents, cell lines, and animals. This disclosure is not limited to the order of the activities or events shown, as some activities may occur in a different order and / or simultaneously with other activities or events. Furthermore, implementing the methods according to this disclosure does not require all the activities, steps, or events shown.

[0246] Peptides that specifically bind to the β-coronavirus spike protein

[0247] Among other things, this document provides peptides that specifically bind to the S2 domain of the β-coronavirus spike glycoprotein. In some embodiments, the peptides have one or more properties selected from the following: broad neutralizing activity against a variety of known and predicted β-coronaviruses (e.g., past, present, emerging, and future β-coronaviruses), binding affinity to a highly conserved S2 domain epitope across a variety of β-coronaviruses, and inhibitory activity against potential emerging β-coronavirus escape variants. In some embodiments, the peptides specifically bind to the S2 domain of the spike protein of sabecloviruses (e.g., SARS-CoV-1 virus, SARS-CoV-2 virus). In some embodiments, the peptides specifically bind to the S2 domain of the spike protein of SARS-CoV-1 virus (e.g., various SARS-CoV-1 variants). In some embodiments, the peptides specifically bind to the S2 domain of the spike protein of SARS-CoV-2 virus (e.g., various SARS-CoV-2 variants). In some embodiments, the peptide specifically binds to the S2 domain of the spike protein of SARS-CoV-1 virus (e.g., various SARS-CoV-1 variants) and the S2 domain of the spike protein of SARS-CoV-2 virus (e.g., various SARS-CoV-2 variants).

[0248] In some embodiments, the peptides disclosed herein exhibit broad neutralizing activity against multiple beta coronaviruses (e.g., as measured using the neutralization assay described herein or other methods known to those skilled in the art). In some embodiments, the peptides exhibit neutralizing activity against multiple sabecloviruses (e.g., SARS-CoV-1 virus, SARS-CoV-2 virus). In some embodiments, the peptides exhibit neutralizing activity against multiple SARS-CoV-1 viruses (e.g., multiple SARS-CoV-1 variants). In some embodiments, the peptides exhibit neutralizing activity against multiple SARS-CoV-2 viruses (e.g., multiple SARS-CoV-2 variants). In some embodiments, the peptides exhibit neutralizing activity against both 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).

[0249] In some embodiments, the peptides disclosed herein have binding affinity for S2 domain epitopes conserved (e.g., highly conserved) across multiple β-coronaviruses. In some embodiments, the S2 domain epitopes are highly conserved across multiple β-coronaviruses (e.g., SARS-CoV-1 and SARS-CoV-2 viruses). In some embodiments, the S2 domain epitopes are highly conserved across multiple SARS-CoV-1 viruses (e.g., multiple SARS-CoV-1 variants). In some embodiments, the S2 domain epitopes are highly conserved across multiple SARS-CoV-2 viruses (e.g., multiple SARS-CoV-2 variants). In some embodiments, the S2 domain epitopes are 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).

[0250] SARS-CoV-2 is the pathogen of COVID-19. In addition to encoding 16 non-structural proteins, the SARS-CoV-2 genome also encodes a nucleoprotein (N), a membrane glycoprotein (M), a small envelope glycoprotein (E), and a 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 SARS-CoV-2 S, facilitates the entry of the SARS-CoV-2 virus into host cells, such as human host cells. S is a trimer composed of S1 and S2 subunits. S1 contains a receptor-binding domain (RBD) that binds to the ACE2 receptor, and S2 is essential for viral-host membrane fusion.

[0251] A non-restrictive example of a wild-type SARS-CoV-2 spike (S) sequence is NCBI RefSeq YP_009724390 (SEQ ID NO:1).

[0252] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS

[0253] NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLI

[0254] VNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFL

[0255] MDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINIT

[0256] RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD

[0257] PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWN

[0258] RKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPG

[0259] QTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT(SEQ ID NO:1)。

[0260] As used herein, the SARS-CoV-2 spike protein includes the SARS-CoV-2 spike protein (e.g., SEQ ID NO:1 (RefSeq YP_009724390) or its homologs) and its truncated forms, mutants and engineered versions of the full-length and truncated SARS-CoV-2 spike protein, and modified forms (e.g., post-translational modified forms) of the full-length and truncated SARS-CoV-2 spike protein.

[0261] In some embodiments, the peptide disclosed herein binds to the SARS-CoV-2 spike protein comprising SEQ ID NO:1.

[0262] In some embodiments, the peptide binds to a mutant, engineered, and / or modified form of the SARS-CoV-2 spike protein. In some embodiments, the mutant, engineered, and / or modified form of the SARS-CoV-2 spike protein comprises an amino acid sequence having at least about 90% sequence identity with the wild-type SARS-CoV-2 spike protein sequence (e.g., SEQ ID NO: 1), for example, having 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 with the wild-type SARS-CoV-2 spike protein sequence. In some embodiments, sequence identity is approximately: 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%.

[0263] In some embodiments, relative to SEQ ID NO:1, the mutant, engineered and / or modified form of the SARS-CoV-2 spike protein comprises one or more mutations selected from the following: L5F, S13I, T19R, A67V, del69, del70, del69-70, D80G, T95I, G142D, del142-144, del144, Y145D, W152C, E154K, F157S, del211, L212I, ins214E PE, A222V, D253G, G261D, G339D, V367F, S371L, S371L, S373P, S375F, K417N, N439K, N440K, G446S, L452 R, Y453F, S477N, T478K, E484A, E484K, E484Q, F486L, S494P, Q493R, G496S, Q498R, N501T, N501Y, Y505H, T547K, F565L, A570D, H655Y, D614G, Q677H, N679K, P681H, P681R, A701V, T716I, N764K, D796Y, T859N, N856K, F888L, D950N, Q954H, Q957R, N969K, L981F, S982A, Q1071H, V1176F, D1118H, K1191N or combinations thereof, for example, 1, 2 Types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, 9 types, 10 types, 11 types, 12 types, 13 types, 14 types, 15 types, 16 types, 17 types, 18 types, 19 types, 20 types, 21 types, 22 types, 23 types, 24 types, 25 types, 26 types, 27 types, 28 types, 29 types, 30 types, 31 types, 32 types, 33 types, 34 types, 35 types, 36 types, 37 types, 38 types, 39 types, 40 types, 41 types, 42 types, 43 types, 44 types, 45 types, or more types.

[0264] In some embodiments, relative to SEQ ID NO:1, the mutant, engineered, and / or modified form of the SARS-CoV-2 spike protein comprises one or more mutations selected from the group consisting of: 69del, 70del, 144del, E484K, S494P, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, or K1191N, or combinations thereof. In some embodiments, the mutant, engineered, and / or modified form of the SARS-CoV-2 spike protein comprises 69del, 70del, 144del, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H. In some embodiments, the mutant, engineered, and / or modified form of the SARS-CoV-2 spike protein further comprises E484K, S494P, or K1191N, or combinations thereof.

[0265] In some embodiments, relative to SEQ ID NO:1, the mutant, engineered, and / or modified form of the SARS-CoV-2 spike protein comprises one or more mutations selected from the group consisting of D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G, or A701V, or combinations thereof. In some embodiments, the mutant, engineered, and / or modified form of the SARS-CoV-2 spike protein comprises D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G, and A701V.

[0266] In some embodiments, relative to SEQ ID NO:1, the mutant, engineered, and / or modified form of the SARS-CoV-2 spike protein comprises one or more mutations selected from the group consisting of T19R, G142D, 156del, 157del, R158G, L452R, T478K, D614G, P681R, or D950N, or combinations thereof. In some embodiments, the mutant, engineered, and / or modified form of the SARS-CoV-2 spike protein comprises T19R, 156del, 157del, R158G, L452R, T478K, D614G, P681R, and D950N. In some embodiments, the mutant, engineered, and / or modified form of the SARS-CoV-2 spike protein further comprises G142D.

[0267] In some embodiments, relative to SEQ ID NO:1, the modified SARS-CoV-2 spike protein comprises one or more mutations selected from the following: 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 combinations thereof.

[0268] In some embodiments, relative to SEQ ID NO:1, the modified SARS-CoV-2 spike protein comprises one or more mutations 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 combinations thereof.

[0269] In some embodiments, relative to SEQ ID NO:1, the modified SARS-CoV-2 spike protein comprises one or more mutations 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 combinations thereof.

[0270] In some embodiments, relative to SEQ ID NO:1, the mutant, engineered and / or modified form of the SARS-CoV-2 spike protein comprises one or more mutations selected from the following: 69del, 70del, 144del, A222V, G261D, V367F, K417N, N439K, Y453F, S477N, E484K, F486L, N501T, N501Y, A570D or D614G or combinations thereof.

[0271] In some embodiments, the mutant, engineered and / or modified form of the SARS-CoV-2 spike protein, relative to SEQ ID NO:1, comprises one or more mutations selected from E484K, N501Y or D614G or combinations thereof.

[0272] In some embodiments, the mutant, engineered and / or modified form of the SARS-CoV-2 spike protein, relative to SEQ ID NO:1, comprises one or more mutations selected from F817P, A892P, A899P, A942P, K986P or V987P or combinations thereof.

[0273] In some embodiments, the mutant, engineered and / or modified form of the SARS-CoV-2 spike protein, relative to SEQ ID NO:1, comprises one or more mutations selected from L452R, F486V or R493Q or combinations thereof.

[0274] In some embodiments, relative to SEQ ID NO:1, the mutant, engineered and / or modified form of the SARS-CoV-2 spike protein comprises one or more mutations selected from the following: 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 combinations thereof.

[0275] In some embodiments, relative to SEQ ID NO:1, the modified SARS-CoV-2 spike protein comprises one or more mutations 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 combinations thereof.

[0276] In some embodiments, relative to SEQ ID NO:1, the modified SARS-CoV-2 spike protein comprises one or more mutations 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 combinations thereof.

[0277] In some embodiments, relative to SEQ ID NO:1, the modified SARS-CoV-2 spike protein comprises one or more mutations 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 combinations thereof.

[0278] In some embodiments, relative to SEQ ID NO:1, the modified SARS-CoV-2 spike protein comprises one or more mutations 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 combinations thereof.

[0279] Other 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 α, β, γ, δ, κ, ε, η, ι, λ, μ and / or ο, for example, 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.1.1 0, 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.

[0280] In some embodiments, the peptides disclosed herein bind to the S2 domain of the SARS-CoV-2 spike (S) protein. As used herein, the S2 domain includes the full-length S2 domain (e.g., an amino acid sequence having SEQ ID NO: 193 or a homologue thereof) and its truncated form, mutants of the full-length and truncated S2 domains, and engineered versions (e.g., epitopes within the S2 domain, e.g., S2( Figure 1 The modified forms of the full-length and truncated S2 domains (e.g., the post-translation modified forms).

[0281] PLQPELDSFKEELDKYFKNHTSPDVDL (SEQ ID NO: 193).

[0282] In some embodiments, the polypeptide disclosed herein binds 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 with the wild-type full-length S2 domain (e.g., SEQ ID NO: 193), for example, having 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, sequence identity is approximately: 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%.

[0283] In some embodiments, the polypeptide disclosed herein binds to the SARS-CoV-2 spike protein (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 both an immunoglobulin light chain variable domain and an immunoglobulin heavy chain variable domain, wherein the polypeptide does not contain SEQ ID NO:3 or SEQ ID NO:50 or both SEQ ID NO:3 and SEQ ID NO:50.

[0284] In some embodiments, the peptides disclosed herein do not contain all six CDRs of an antibody, said antibody containing V of SEQ ID NO:3. H Amino acid sequence and V of SEQ ID NO:50 LAmino acid sequence. In some embodiments, the polypeptide disclosed herein comprises 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 polypeptide disclosed herein comprises one, two, or three CDRs selected from SEQ ID NO:79, SEQ ID NO:90, SEQ ID NO:133, and SEQ ID NO:143.

[0285] In some embodiments, the antibodies disclosed herein do not contain all six CDRs of the antibody, said antibody containing V of SEQ ID NO:3. H Amino acid sequence and V of SEQ ID NO:50 L Amino acid sequence. In some embodiments, the antibodies disclosed herein comprise 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 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.

[0286] In some embodiments, this disclosure provides a polypeptide that specifically binds to the SARS-CoV-2 spike protein, wherein the polypeptide comprises:

[0287] a) Variable domains of immunoglobulin heavy chain (V H ), the V H Contains 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 with SEQ ID NO:3;

[0288] b) Variable domains of immunoglobulin light chains (V L ), the V L Contains 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 with SEQ ID NO:50; or

[0289] Both a) and b)

[0290] The polypeptide does not contain all 6 CDRs of the antibody, and the antibody contains V of SEQ ID NO:3. H Amino acid sequence and V of SEQ ID NO:50 L Amino acid sequence.

[0291] In some embodiments, the polypeptides disclosed herein do not contain all four 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 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.

[0292] In some embodiments, the peptides disclosed herein bind to the wild-type SARS-CoV-2 spike protein (e.g., SEQ ID NO: 1). In some embodiments, the peptides disclosed herein bind to one or more epitope residues of the wild-type SARS-CoV-2 spike protein (e.g., one or more epitope residues in the SARS-CoV-2 S2 subunit).

[0293] As used herein, the term "comparative peptide" or "comparative peptide polypeptide" refers to a polypeptide (e.g., an immunoglobulin molecule) that specifically binds to SARS-CoV-2 and is not the polypeptide disclosed herein. The sequences of the comparative peptide and the polypeptide disclosed herein can be compared to illustrate structural differences between them (e.g., differences in the positions of one or more amino acids, such as amino acid substitutions). The polypeptide disclosed herein has more than non-substantial differences (e.g., one or more substantial differences) compared to the comparative peptide, such that the polypeptide disclosed herein, under controlled conditions, will exhibit one or more different functions (i.e., one, two, or all three) in different ways compared to the comparative peptide to achieve different results. The comparative peptide will differ from the polypeptide disclosed herein by one or more amino acids, for example, 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, the difference between the comparative peptide and the peptide provided in this disclosure is 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.

[0294] In some embodiments, the comparative peptide is an antibody, referred to herein as a "reference antibody," which comprises a V amino acid sequence including SEQ ID NO:3. H The V domain, including the amino acid sequence of SEQ ID NO:50 L The reference antibody comprises a domain, a heavy chain including the amino acid sequence of SEQ ID NO:191, and a light chain including the amino acid sequence of SEQ ID NO:192. The reference antibody is an antibody that binds to SARS-CoV-2S2 and neutralizes SARS-CoV-2 variants. Further information about the reference antibody can be found, for example, in PDB:7NAB_A, PDB:7NAB_B, 7NAB_C, and Jennewein et al., from COVID-19 + Isolation and characterization of cross-neutralizing coronavirus antibodies from COVID-19 in subjects + (subjects), Cell Reports, 36(2):109353(2021). The reference antibody has the following heavy and light chain amino acid sequences:

[0295] EVQLVESGAEVKKPGESLKISCKGSGYTFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGHVTISADKSISTAYLQWNSLKASDTAMYYCARLPQYCSNGVCQRWFDPW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS(SEQ ID NO:191)

[0296] EIVLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNSFPYTFGQGTNLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE(SEQ IDNO:192)

[0297] As used herein, the term "sequence identity" refers, expressed as a percentage, to the extent that two nucleotide sequences or two amino acid sequences have identical residues at the same positions when sequences are aligned to achieve the highest level of identity. For sequence alignment and comparison, a sequence is typically designated as a reference sequence to be compared with the test sequence. Sequence identity is expressed as a percentage of positions over the entire length of the reference sequence where the reference and test sequences share the same nucleotides or amino acids when aligned to achieve the highest level of identity. For example, if the test sequence has the same nucleotide or amino acid residues at 70% of the same positions over the entire length of the reference sequence when aligned to achieve the highest level of identity, the two sequences are considered to have 70% sequence identity.

[0298] Those skilled in the art can readily perform sequence alignments for comparison to achieve the highest level of identity using appropriate alignment methods or algorithms. In some cases, the alignment may include introduced gaps to provide the highest level of identity. Examples include Smith and Waterman's local homology algorithm in Adv. Appl. Math. 2:482 (1981), Needleman and Wunsch's homology alignment algorithm in J. Mol. Biol. 48:443 (1970), and Pearson and Lipman's similarity search method in Proceedings of the National Academy of Sciences of the United States of America 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, Wisconsin), and visual inspection (generally see Ausubel et al., Current Protocols in Molecular Biology Experiments). Molecular Biology)

[0299] When using sequence comparison algorithms, the test and reference sequences are input into the computer, and subsequent coordinates are specified if necessary, along with the sequence algorithm program parameters. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the specified program parameters. A commonly used tool for determining the percentage of sequence identity is the Basic Local Alignment Search Tool for Proteins (BLASTP), which is available from the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health (Altschul et al., 1990).

[0300] The terms “polypeptide,” “peptide,” or “protein” refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). Proteins, peptides, or polypeptides may contain any suitable L- and / or D-amino acids, such as common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., β-alanine, 4-aminobutyric acid, 6-aminohexanoic acid, sarcosine, pepsinic acid), and unusual amino acids (e.g., citrulline, homocitrulline, homoserine, leucine, valine, ornithine). The amino, carboxyl, and / or other functional groups on the peptide may be free (e.g., unmodified) or protected with suitable protecting groups. Suitable protecting groups for the amino and carboxyl groups, and methods for adding or removing protecting groups, are known in the art and disclosed, for example, in Green and Wuts, “Protecting Groups in Organic Synthesis,” John Wiley and Sons, 1991. The functional groups of proteins, peptides, or polypeptides may also be derived (e.g., alkylation) or labeled (e.g., with detectable labels such as fluorophores or haptens) using methods known in the art. Proteins, peptides, or polypeptides may contain one or more modifications if desired (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal regulators (e.g., cyclization), N-methyl-□-amino substitution). Additionally, proteins, peptides, or polypeptides may be analogs of known and / or naturally occurring peptides, such as peptide analogs with conserved amino acid residue substitutions.

[0301] In some embodiments, this disclosure provides a polypeptide that specifically binds to the SARS-CoV-2 spike protein, wherein the polypeptide contains a complementary site substantially similar to the complementary site of an antibody, and the antibody contains V... H / V L right:

[0302] SEQ ID NO:4 and SEQ ID NO:51(AB-1);

[0303] SEQ ID NO:5 and SEQ ID NO:52(AB-2);

[0304] SEQ ID NO:6 and SEQ ID NO:53(AB-3);

[0305] SEQ ID NO:7 and SEQ ID NO:54(AB-4);

[0306] SEQ ID NO:8 and SEQ ID NO:51(AB-5);

[0307] SEQ ID NO:9 and SEQ ID NO:55(AB-6);

[0308] SEQ ID NO:10 and SEQ ID NO:56(AB-7);

[0309] SEQ ID NO:11 and SEQ ID NO:57(AB-8);

[0310] SEQ ID NO:12 and SEQ ID NO:58(AB-9);

[0311] SEQ ID NO:13 and SEQ ID NO:59(AB-10);

[0312] SEQ ID NO:14 and SEQ ID NO:60(AB-11);

[0313] SEQ ID NO:15 and SEQ ID NO:56(AB-12);

[0314] SEQ ID NO:16 and SEQ ID NO:51(AB-13);

[0315] SEQ ID NO:10 and SEQ ID NO:50 (AB-14);

[0316] SEQ ID NO:17 and SEQ ID NO:61(AB-15);

[0317] SEQ ID NO:18 and SEQ ID NO:62(AB-16);

[0318] SEQ ID NO:6 and SEQ ID NO:63(AB-17);

[0319] SEQ ID NO:19 and SEQ ID NO:64(AB-18);

[0320] SEQ ID NO:4 and SEQ ID NO:61(AB-19);

[0321] SEQ ID NO:20 and SEQ ID NO:61(AB-20);

[0322] SEQ ID NO:21 and SEQ ID NO:65(AB-21);

[0323] SEQ ID NO:22 and SEQ ID NO:66(AB-22);

[0324] SEQ ID NO:4 and SEQ ID NO:67(AB-23);

[0325] SEQ ID NO:23 and SEQ ID NO:56(AB-24);

[0326] SEQ ID NO:24 and SEQ ID NO:68(AB-25);

[0327] SEQ ID NO:25 and SEQ ID NO:51(AB-26);

[0328] SEQ ID NO:26 and SEQ ID NO:56(AB-27);

[0329] SEQ ID NO:27 and SEQ ID NO:61(AB-28);

[0330] SEQ ID NO:28 and SEQ ID NO:56(AB-29);

[0331] SEQ ID NO:28 and SEQ ID NO:69(AB-30);

[0332] SEQ ID NO:29 and SEQ ID NO:70(AB-31);

[0333] SEQ ID NO:30 and SEQ ID NO:71(AB-32);

[0334] SEQ ID NO:31 and SEQ ID NO:72(AB-33);

[0335] SEQ ID NO:32 and SEQ ID NO:67(AB-34);

[0336] SEQ ID NO:33 and SEQ ID NO:56(AB-35);

[0337] SEQ ID NO:34 and SEQ ID NO:73(AB-36);

[0338] SEQ ID NO:35 and SEQ ID NO:51(AB-37);

[0339] SEQ ID NO:36 and SEQ ID NO:56(AB-38);

[0340] SEQ ID NO:37 and SEQ ID NO:63(AB-39);

[0341] SEQ ID NO:38 and SEQ ID NO:69 (AB-40);

[0342] SEQ ID NO:39 and SEQ ID NO:74(AB-41);

[0343] SEQ ID NO:40 and SEQ ID NO:52 (AB-42);

[0344] SEQ ID NO:41 and SEQ ID NO:51(AB-43);

[0345] SEQ ID NO:42 and SEQ ID NO:75(AB-44);

[0346] SEQ ID NO:43 and SEQ ID NO:56(AB-45);

[0347] SEQ ID NO:44 and SEQ ID NO:51(AB-46);

[0348] SEQ ID NO:45 and SEQ ID NO:75(AB-47);

[0349] SEQ ID NO:46 and SEQ ID NO:53 (AB-48);

[0350] SEQ ID NO:47 and SEQ ID NO:52 (AB-49);

[0351] SEQ ID NO:48 and SEQ ID NO:76 (AB-50); or

[0352] SEQ ID NO:3 and SEQ ID NO:56(AB-51), or

[0353] Any combination thereof.

[0354] In some embodiments, this disclosure provides a polypeptide that specifically binds to the SARS-CoV-2 spike protein, wherein the polypeptide contains a complementary site identical to the complementary site of an antibody, and the antibody contains a subset of V... H / V L right:

[0355] SEQ ID NO:4 and SEQ ID NO:51(AB-1);

[0356] SEQ ID NO:5 and SEQ ID NO:52(AB-2);

[0357] SEQ ID NO:6 and SEQ ID NO:53(AB-3);

[0358] SEQ ID NO:7 and SEQ ID NO:54(AB-4);

[0359] SEQ ID NO:8 and SEQ ID NO:51(AB-5);

[0360] SEQ ID NO:9 and SEQ ID NO:55(AB-6);

[0361] SEQ ID NO:10 and SEQ ID NO:56(AB-7);

[0362] SEQ ID NO:11 and SEQ ID NO:57(AB-8);

[0363] SEQ ID NO:12 and SEQ ID NO:58(AB-9);

[0364] SEQ ID NO:13 and SEQ ID NO:59(AB-10);

[0365] SEQ ID NO:14 and SEQ ID NO:60(AB-11);

[0366] SEQ ID NO:15 and SEQ ID NO:56(AB-12);

[0367] SEQ ID NO:16 and SEQ ID NO:51(AB-13);

[0368] SEQ ID NO:10 and SEQ ID NO:50 (AB-14);

[0369] SEQ ID NO:17 and SEQ ID NO:61(AB-15);

[0370] SEQ ID NO:18 and SEQ ID NO:62(AB-16);

[0371] SEQ ID NO:6 and SEQ ID NO:63(AB-17);

[0372] SEQ ID NO:19 and SEQ ID NO:64(AB-18);

[0373] SEQ ID NO:4 and SEQ ID NO:61(AB-19);

[0374] SEQ ID NO:20 and SEQ ID NO:61(AB-20);

[0375] SEQ ID NO:21 and SEQ ID NO:65(AB-21);

[0376] SEQ ID NO:22 and SEQ ID NO:66(AB-22);

[0377] SEQ ID NO:4 and SEQ ID NO:67(AB-23);

[0378] SEQ ID NO:23 and SEQ ID NO:56(AB-24);

[0379] SEQ ID NO:24 and SEQ ID NO:68(AB-25);

[0380] SEQ ID NO:25 and SEQ ID NO:51(AB-26);

[0381] SEQ ID NO:26 and SEQ ID NO:56(AB-27);

[0382] SEQ ID NO:27 and SEQ ID NO:61(AB-28);

[0383] SEQ ID NO:28 and SEQ ID NO:56(AB-29);

[0384] SEQ ID NO:28 and SEQ ID NO:69(AB-30);

[0385] SEQ ID NO:29 and SEQ ID NO:70(AB-31);

[0386] SEQ ID NO:30 and SEQ ID NO:71(AB-32);

[0387] SEQ ID NO:31 and SEQ ID NO:72(AB-33);

[0388] SEQ ID NO:32 and SEQ ID NO:67(AB-34);

[0389] SEQ ID NO:33 and SEQ ID NO:56(AB-35);

[0390] SEQ ID NO:34 and SEQ ID NO:73(AB-36);

[0391] SEQ ID NO:35 and SEQ ID NO:51(AB-37);

[0392] SEQ ID NO:36 and SEQ ID NO:56(AB-38);

[0393] SEQ ID NO:37 and SEQ ID NO:63(AB-39);

[0394] SEQ ID NO:38 and SEQ ID NO:69 (AB-40);

[0395] SEQ ID NO:39 and SEQ ID NO:74(AB-41);

[0396] SEQ ID NO:40 and SEQ ID NO:52 (AB-42);

[0397] SEQ ID NO:41 and SEQ ID NO:51(AB-43);

[0398] SEQ ID NO:42 and SEQ ID NO:75(AB-44);

[0399] SEQ ID NO:43 and SEQ ID NO:56(AB-45);

[0400] SEQ ID NO:44 and SEQ ID NO:51(AB-46);

[0401] SEQ ID NO:45 and SEQ ID NO:75(AB-47);

[0402] SEQ ID NO:46 and SEQ ID NO:53 (AB-48);

[0403] SEQ ID NO:47 and SEQ ID NO:52 (AB-49);

[0404] SEQ ID NO:48 and SEQ ID NO:76 (AB-50); or

[0405] SEQ ID NO:3 and SEQ ID NO:56(AB-51).

[0406] See Table 1 for SEQ ID NO:4-48, and Table 2 for SEQ ID NO:51-76. The complementary residues of antibodies containing SEQ ID NO:4-48 are also listed in Table 2. Figure 2 And see the complementary residues of the antibody containing SEQ ID NO:51-76. Figure 3 .

[0407] The amino acid residues at the complementary site facilitate the epitope interaction between the antibody and its target protein. The interaction can be a hydrogen bond, salt bridge, van der Waals interaction, electrostatic interaction, hydrophobic interaction, π-interaction effect, ionic bond, and / or any combination thereof. The interaction can be direct or indirect, such as through a coordinating intermediate molecule, like an ion or water. In some embodiments, the residues at the complementary site contain only residues that are part of a defined CDR. In some embodiments, the residues at the complementary site further contain one or more residues that are not part of a defined CDR (e.g., residues within a defined frame region).

[0408] In some embodiments, when the polypeptide binds to the target antigen, the orientation of the complementary site is less than about 5.0 Å from the epitope on the target antigen, for example, 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 Å, or about 0.9-5.0, 0.9-4 Å from the epitope. 0.8, 1.0-5, 1.0-4.5, 1.0-4.0, 1.0-3.5, 1.1-3.5, 1.1-3.0, 1.2-3.0, 1.2-2.5, 1.3-2.5, 1.3-2.4, 1.4-2.4, 1.4-2.3, 1.5-2.3, 1.5-2.2, 1.6-2.2, 1.6-2.1, 1.7-2.1, 1.7-2.0 or 1.8-2.0 angstroms. In some embodiments, when the polypeptide binds to the target antigen, the orientation of less than all of the amino acid residues constituting the complementary site (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) at the complementary site is less than about 5.0 Å from the epitope on the target antigen.

[0409] As used herein, the term "substantially similar" means that the polypeptides disclosed herein have substantially similar amino acid sequences (e.g., having 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 sequence identity) and substantially retain one or more functional properties of the specific polypeptide disclosed herein. In some embodiments, said 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.

[0410] In some embodiments, the polypeptide disclosed herein includes a complementary site substantially similar to the complementary site of a polypeptide selected from any of AB-1 to AB-51. In some embodiments, the polypeptide includes a complementary site containing only conserved substitutions (e.g., only highly conserved substitutions) relative to the complementary site of a polypeptide selected from any of AB-1 to AB-51. In some embodiments, the polypeptide includes a complementary site containing up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conserved substitutions (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 highly conserved substitutions) relative to the complementary site of a polypeptide selected from any of AB-1 to AB-51. In some embodiments, the polypeptide includes a complementary site having 100% sequence identity with the complementary site of a polypeptide selected from any of AB-1 to AB-51.

[0411] In some embodiments, the complementary site comprises amino acid residues corresponding to each of the following: 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 or a subset thereof of SEQ ID NO:49.

[0412] In some embodiments, the polypeptide comprising the complementary site disclosed herein includes an immunoglobulin heavy chain variable domain (V... H ) and immunoglobulin light chain variable domain (V L In some embodiments, complementary residues are contained in the V of the polypeptide. H and V L Inside.

[0413] In some embodiments, the polypeptide comprises an immunoglobulin light chain variable region, an immunoglobulin heavy chain variable region, or both an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region. In some embodiments, the polypeptide comprises six CDRs of the polypeptide disclosed herein. In some embodiments, the polypeptide has fewer than six (e.g., 1, 2, 3, 4, or 5) CDRs of the polypeptide disclosed herein.

[0414] In some embodiments, this disclosure provides a polypeptide that specifically binds to the SARS-CoV-2 spike protein, wherein the polypeptide comprises:

[0415] V H amino acid sequence, the V HThe amino acid sequence contains the V that corresponds to any one of the amino acid sequences in SEQ ID NO:4-48. H The heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of the amino acid sequences are substantially similar to HCDR1, HCDR2, and HCDR3; and

[0416] V L amino acid sequence, the V L The amino acid sequence includes the V that corresponds to any one of the amino acid sequences in SEQ ID NO:51-76. L The light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) of the amino acid sequence are substantially similar to those of LCDR1, LCDR2, and LCDR3.

[0417] In some embodiments, the polypeptide comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which substantially retain one or more functional properties of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the polypeptide selected from any of AB-1 to AB-51.

[0418] In some embodiments, relative to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of any of the peptides selected from AB-1 to AB-51, the peptide comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing only one or more conserved substitutions (e.g., only one or more highly conserved substitutions).

[0419] In some embodiments, relative to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the polypeptide selected from any of AB-1 to AB-51, the polypeptide comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 including up to one, two, or three conserved substitutions (e.g., up to one, two, or three highly conserved substitutions).

[0420] In some embodiments, the polypeptides disclosed herein comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having 100% sequence identity with HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the polypeptide selected from any of AB-1 to AB-51.

[0421] In some embodiments, the polypeptide comprises HCDR1, HCDR2, and HCDR3 of an antibody, as well as LCDR1, LCDR2, and LCDR3, wherein the antibody comprises an amino acid sequence selected from the following:

[0422] SEQ ID NO:4 and SEQ ID NO:51(AB-1);

[0423] SEQ ID NO:5 and SEQ ID NO:52(AB-2);

[0424] SEQ ID NO:6 and SEQ ID NO:53(AB-3);

[0425] SEQ ID NO:7 and SEQ ID NO:54(AB-4);

[0426] SEQ ID NO:8 and SEQ ID NO:51(AB-5);

[0427] SEQ ID NO:9 and SEQ ID NO:55(AB-6);

[0428] SEQ ID NO:10 and SEQ ID NO:56(AB-7);

[0429] SEQ ID NO:11 and SEQ ID NO:57(AB-8);

[0430] SEQ ID NO:12 and SEQ ID NO:58(AB-9);

[0431] SEQ ID NO:13 and SEQ ID NO:59(AB-10);

[0432] SEQ ID NO:14 and SEQ ID NO:60(AB-11);

[0433] SEQ ID NO:15 and SEQ ID NO:56(AB-12);

[0434] SEQ ID NO:16 and SEQ ID NO:51(AB-13);

[0435] SEQ ID NO:10 and SEQ ID NO:50 (AB-14);

[0436] SEQ ID NO:17 and SEQ ID NO:61(AB-15);

[0437] SEQ ID NO:18 and SEQ ID NO:62(AB-16);

[0438] SEQ ID NO:6 and SEQ ID NO:63(AB-17);

[0439] SEQ ID NO:19 and SEQ ID NO:64(AB-18);

[0440] SEQ ID NO:4 and SEQ ID NO:61(AB-19);

[0441] SEQ ID NO:20 and SEQ ID NO:61(AB-20);

[0442] SEQ ID NO:21 and SEQ ID NO:65(AB-21);

[0443] SEQ ID NO:22 and SEQ ID NO:66(AB-22);

[0444] SEQ ID NO:4 and SEQ ID NO:67(AB-23);

[0445] SEQ ID NO:23 and SEQ ID NO:56(AB-24);

[0446] SEQ ID NO:24 and SEQ ID NO:68(AB-25);

[0447] SEQ ID NO:25 and SEQ ID NO:51(AB-26);

[0448] SEQ ID NO:26 and SEQ ID NO:56(AB-27);

[0449] SEQ ID NO:27 and SEQ ID NO:61(AB-28);

[0450] SEQ ID NO:28 and SEQ ID NO:56(AB-29);

[0451] SEQ ID NO:28 and SEQ ID NO:69(AB-30);

[0452] SEQ ID NO:29 and SEQ ID NO:70(AB-31);

[0453] SEQ ID NO:30 and SEQ ID NO:71(AB-32);

[0454] SEQ ID NO:31 and SEQ ID NO:72(AB-33);

[0455] SEQ ID NO:32 and SEQ ID NO:67(AB-34);

[0456] SEQ ID NO:33 and SEQ ID NO:56(AB-35);

[0457] SEQ ID NO:34 and SEQ ID NO:73(AB-36);

[0458] SEQ ID NO:35 and SEQ ID NO:51(AB-37);

[0459] SEQ ID NO:36 and SEQ ID NO:56(AB-38);

[0460] SEQ ID NO:37 and SEQ ID NO:63(AB-39);

[0461] SEQ ID NO:38 and SEQ ID NO:69 (AB-40);

[0462] SEQ ID NO:39 and SEQ ID NO:74(AB-41);

[0463] SEQ ID NO:40 and SEQ ID NO:52 (AB-42);

[0464] SEQ ID NO:41 and SEQ ID NO:51(AB-43);

[0465] SEQ ID NO:42 and SEQ ID NO:75(AB-44);

[0466] SEQ ID NO:43 and SEQ ID NO:56(AB-45);

[0467] SEQ ID NO:44 and SEQ ID NO:51(AB-46);

[0468] SEQ ID NO:45 and SEQ ID NO:75(AB-47);

[0469] SEQ ID NO:46 and SEQ ID NO:53 (AB-48);

[0470] SEQ ID NO:47 and SEQ ID NO:52 (AB-49);

[0471] SEQ ID NO:48 and SEQ ID NO:76 (AB-50); or

[0472] SEQ ID NO:3 and SEQ ID NO:56(AB-51).

[0473] CDRs (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3) can be defined by any method recognized in the art for identifying CDR residues of antibodies, as further described herein (e.g., CDRs defined by Kabat, CDRs defined by Chothia, or CDRs defined by ImMunoGeneTics (IMGT) numbers).

[0474] (www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html, also accessible at www.imgt.org / ). In certain embodiments, the CDR is defined by an IMGT number. Examples of CDRs defined by IMGT numbers include the CDRs of the peptides disclosed herein for use in this disclosure.

[0475] In some embodiments, the polypeptide includes a complementary site substantially similar to the complementary site of an antibody, the antibody comprising a V group selected from the group consisting of... H / V L right:

[0476] SEQ ID NO:4 and SEQ ID NO:51(AB-1);

[0477] SEQ ID NO:5 and SEQ ID NO:52(AB-2);

[0478] SEQ ID NO:6 and SEQ ID NO:53(AB-3);

[0479] SEQ ID NO:7 and SEQ ID NO:54(AB-4);

[0480] SEQ ID NO:8 and SEQ ID NO:51(AB-5);

[0481] SEQ ID NO:9 and SEQ ID NO:55(AB-6);

[0482] SEQ ID NO:10 and SEQ ID NO:56(AB-7);

[0483] SEQ ID NO:11 and SEQ ID NO:57(AB-8);

[0484] SEQ ID NO:12 and SEQ ID NO:58(AB-9);

[0485] SEQ ID NO:13 and SEQ ID NO:59(AB-10);

[0486] SEQ ID NO:14 and SEQ ID NO:60(AB-11);

[0487] SEQ ID NO:15 and SEQ ID NO:56(AB-12);

[0488] SEQ ID NO:16 and SEQ ID NO:51(AB-13);

[0489] SEQ ID NO:10 and SEQ ID NO:50 (AB-14);

[0490] SEQ ID NO:17 and SEQ ID NO:61(AB-15);

[0491] SEQ ID NO:18 and SEQ ID NO:62(AB-16);

[0492] SEQ ID NO:6 and SEQ ID NO:63(AB-17);

[0493] SEQ ID NO:19 and SEQ ID NO:64(AB-18);

[0494] SEQ ID NO:4 and SEQ ID NO:61(AB-19);

[0495] SEQ ID NO:20 and SEQ ID NO:61(AB-20);

[0496] SEQ ID NO:21 and SEQ ID NO:65(AB-21);

[0497] SEQ ID NO:22 and SEQ ID NO:66(AB-22);

[0498] SEQ ID NO:4 and SEQ ID NO:67(AB-23);

[0499] SEQ ID NO:23 and SEQ ID NO:56(AB-24);

[0500] SEQ ID NO:24 and SEQ ID NO:68(AB-25);

[0501] SEQ ID NO:25 and SEQ ID NO:51(AB-26);

[0502] SEQ ID NO:26 and SEQ ID NO:56(AB-27);

[0503] SEQ ID NO:27 and SEQ ID NO:61(AB-28);

[0504] SEQ ID NO:28 and SEQ ID NO:56(AB-29);

[0505] SEQ ID NO:28 and SEQ ID NO:69(AB-30);

[0506] SEQ ID NO:29 and SEQ ID NO:70(AB-31);

[0507] SEQ ID NO:30 and SEQ ID NO:71(AB-32);

[0508] SEQ ID NO:31 and SEQ ID NO:72(AB-33);

[0509] SEQ ID NO:32 and SEQ ID NO:67(AB-34);

[0510] SEQ ID NO:33 and SEQ ID NO:56(AB-35);

[0511] SEQ ID NO:34 and SEQ ID NO:73(AB-36);

[0512] SEQ ID NO:35 and SEQ ID NO:51(AB-37);

[0513] SEQ ID NO:36 and SEQ ID NO:56(AB-38);

[0514] SEQ ID NO:37 and SEQ ID NO:63(AB-39);

[0515] SEQ ID NO:38 and SEQ ID NO:69 (AB-40);

[0516] SEQ ID NO:39 and SEQ ID NO:74(AB-41);

[0517] SEQ ID NO:40 and SEQ ID NO:52 (AB-42);

[0518] SEQ ID NO:41 and SEQ ID NO:51(AB-43);

[0519] SEQ ID NO:42 and SEQ ID NO:75(AB-44);

[0520] SEQ ID NO:43 and SEQ ID NO:56(AB-45);

[0521] SEQ ID NO:44 and SEQ ID NO:51(AB-46);

[0522] SEQ ID NO:45 and SEQ ID NO:75(AB-47);

[0523] SEQ ID NO:46 and SEQ ID NO:53 (AB-48);

[0524] SEQ ID NO:47 and SEQ ID NO:52 (AB-49);

[0525] SEQ ID NO:48 and SEQ ID NO:76 (AB-50); or

[0526] SEQ ID NO:3 and SEQ ID NO:56(AB-51), or

[0527] Any combination thereof.

[0528] In some embodiments, the polypeptide contains the same complementary site as the antibody, the antibody containing V selected from the group consisting of... H / V L right:

[0529] SEQ ID NO:4 and SEQ ID NO:51(AB-1);

[0530] SEQ ID NO:5 and SEQ ID NO:52(AB-2);

[0531] SEQ ID NO:6 and SEQ ID NO:53(AB-3);

[0532] SEQ ID NO:7 and SEQ ID NO:54(AB-4);

[0533] SEQ ID NO:8 and SEQ ID NO:51(AB-5);

[0534] SEQ ID NO:9 and SEQ ID NO:55(AB-6);

[0535] SEQ ID NO:10 and SEQ ID NO:56(AB-7);

[0536] SEQ ID NO:11 and SEQ ID NO:57(AB-8);

[0537] SEQ ID NO:12 and SEQ ID NO:58(AB-9);

[0538] SEQ ID NO:13 and SEQ ID NO:59(AB-10);

[0539] SEQ ID NO:14 and SEQ ID NO:60(AB-11);

[0540] SEQ ID NO:15 and SEQ ID NO:56(AB-12);

[0541] SEQ ID NO:16 and SEQ ID NO:51(AB-13);

[0542] SEQ ID NO:10 and SEQ ID NO:50 (AB-14);

[0543] SEQ ID NO:17 and SEQ ID NO:61(AB-15);

[0544] SEQ ID NO:18 and SEQ ID NO:62(AB-16);

[0545] SEQ ID NO:6 and SEQ ID NO:63(AB-17);

[0546] SEQ ID NO:19 and SEQ ID NO:64(AB-18);

[0547] SEQ ID NO:4 and SEQ ID NO:61(AB-19);

[0548] SEQ ID NO:20 and SEQ ID NO:61(AB-20);

[0549] SEQ ID NO:21 and SEQ ID NO:65(AB-21);

[0550] SEQ ID NO:22 and SEQ ID NO:66(AB-22);

[0551] SEQ ID NO:4 and SEQ ID NO:67(AB-23);

[0552] SEQ ID NO:23 and SEQ ID NO:56(AB-24);

[0553] SEQ ID NO:24 and SEQ ID NO:68(AB-25);

[0554] SEQ ID NO:25 and SEQ ID NO:51(AB-26);

[0555] SEQ ID NO:26 and SEQ ID NO:56(AB-27);

[0556] SEQ ID NO:27 and SEQ ID NO:61(AB-28);

[0557] SEQ ID NO:28 and SEQ ID NO:56(AB-29);

[0558] SEQ ID NO:28 and SEQ ID NO:69(AB-30);

[0559] SEQ ID NO:29 and SEQ ID NO:70(AB-31);

[0560] SEQ ID NO:30 and SEQ ID NO:71(AB-32);

[0561] SEQ ID NO:31 and SEQ ID NO:72(AB-33);

[0562] SEQ ID NO:32 and SEQ ID NO:67(AB-34);

[0563] SEQ ID NO:33 and SEQ ID NO:56(AB-35);

[0564] SEQ ID NO:34 and SEQ ID NO:73(AB-36);

[0565] SEQ ID NO:35 and SEQ ID NO:51(AB-37);

[0566] SEQ ID NO:36 and SEQ ID NO:56(AB-38);

[0567] SEQ ID NO:37 and SEQ ID NO:63(AB-39);

[0568] SEQ ID NO:38 and SEQ ID NO:69 (AB-40);

[0569] SEQ ID NO:39 and SEQ ID NO:74(AB-41);

[0570] SEQ ID NO:40 and SEQ ID NO:52 (AB-42);

[0571] SEQ ID NO:41 and SEQ ID NO:51(AB-43);

[0572] SEQ ID NO:42 and SEQ ID NO:75(AB-44);

[0573] SEQ ID NO:43 and SEQ ID NO:56(AB-45);

[0574] SEQ ID NO:44 and SEQ ID NO:51(AB-46);

[0575] SEQ ID NO:45 and SEQ ID NO:75(AB-47);

[0576] SEQ ID NO:46 and SEQ ID NO:53 (AB-48);

[0577] SEQ ID NO:47 and SEQ ID NO:52 (AB-49);

[0578] SEQ ID NO:48 and SEQ ID NO:76 (AB-50); or

[0579] SEQ ID NO:3 and SEQ ID NO:56(AB-51).

[0580] In some embodiments, this disclosure provides a polypeptide that specifically binds to the SARS-CoV-2 spike protein, comprising V, an amino acid sequence including SEQ ID NO:2. H ,in:

[0581] X1 is not S;

[0582] X2 is not D;

[0583] X3 is not T;

[0584] X4 is not L;

[0585] X5 is not S;

[0586] X6 is not N;

[0587] X7 is not G;

[0588] X8 is not V; or

[0589] X9 is not Q.

[0590] Or any combination thereof.

[0591] Table 1 shows the sequence identified as SEQ ID NO:2, which is a common sequence of SEQ ID NO:3-48 in this paper. H sequence.

[0592] In some embodiments, the polypeptide disclosed herein further comprises V L In some embodiments, the polypeptide comprises V, which includes the amino acid sequence of SEQ ID NO:49. L ,in:

[0593] X 10 Not Q;

[0594] X 11 Not G;

[0595] X 12 Not S;

[0596] X 13 Not S;

[0597] X 14 Not N;

[0598] X 15 Not S;

[0599] X 16 Not F; or

[0600] X 17 Not Y,

[0601] Or any combination thereof.

[0602] Table 2 shows the sequence identified as SEQ ID NO:49, which is a common sequence of SEQ ID NO:50-76 in this paper. L sequence.

[0603] In some embodiments:

[0604] X1 is S, N, A, R, L, or F;

[0605] X2 is either D or E;

[0606] X3 is either T or V;

[0607] X4 is either L or V;

[0608] X5 is S, Q, R, K, Y, D, or E;

[0609] X6 is N, K, A, S, R, or E;

[0610] X7 is G, N, or L;

[0611] X8 is V, I, S, or K; or

[0612] X9 is Q, Y, K, F, or H.

[0613] Or any combination thereof.

[0614] In some embodiments:

[0615] X1 is N, A, R, L, or F;

[0616] X2 is E;

[0617] X3 is V;

[0618] X4 is V;

[0619] X5 is Q, R, K, Y, D, or E;

[0620] X6 is K, A, S, R, or E;

[0621] X7 is either N or L;

[0622] X8 is I, S, or K; or

[0623] X9 is Y, K, F, or H.

[0624] Or any combination thereof.

[0625] 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.

[0626] 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.

[0627] 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.

[0628] 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.

[0629] 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.

[0630] 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.

[0631] 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.

[0632] 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.

[0633] 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.

[0634] In some embodiments:

[0635] X 10 It is Q, K, or I;

[0636] X 11 Is it G or S;

[0637] X 12 Is it S, R, or V;

[0638] X 13 It is S or N;

[0639] X 14 It is N, H, D, Y, or S;

[0640] X15 Is it S or Q?

[0641] X 16 It is F, Y, L, V, T, or D; or

[0642] X 17 Is it Y or L?

[0643] Or any combination thereof.

[0644] In some embodiments:

[0645] X 10 It is K or I;

[0646] X 11 It is S;

[0647] X 12 It is R or V;

[0648] X 13 It is N;

[0649] X 14 It is H, D, Y, or S;

[0650] X 15 It's Q;

[0651] X 16 It is Y, L, V, T, or D; or

[0652] X 17 It is L.

[0653] Or any combination thereof.

[0654] In some embodiments, X 10 Not Q. In some embodiments, X 10 It is Q, K, or I. In some embodiments, X 10 It is K or I. In some embodiments, X 10 It is Q. In some embodiments, X 10 It is K. In some embodiments, X 10 It's I.

[0655] In some embodiments, X 11 Not G. In some embodiments, X 11 It is G or S. In some embodiments, X 11 It is G. In some embodiments, X 11 It is S.

[0656] In some embodiments, X 12 Not S. In some embodiments, X 12 It is S, R, or V. In some embodiments, X 12It is S. In some embodiments, X 12 It is R. In some embodiments, X 12 It is V.

[0657] In some embodiments, X 13 Not S. In some embodiments, X 13 It is S or N. In some embodiments, X 13 It is S. In some embodiments, X 13 It is N.

[0658] In some embodiments, X 14 Not N. In some embodiments, X 14 It is N, H, D, Y, or S. In some embodiments, X 14 It is H, D, Y, or S. In some embodiments, X 14 It is N. In some embodiments, X 14 It is H. In some embodiments, X 14 It is D. In some embodiments, X 14 It is Y. In some embodiments, X 14 It is S.

[0659] In some embodiments, X 15 Not S. In some embodiments, X 15 It is S or Q. In some embodiments, X 15 It is S. In some embodiments, X 15 It's Q.

[0660] In some embodiments, X 16 Not F. In some embodiments, X 16 It is F, Y, L, V, T, or D. In some embodiments, X 16 It is Y, L, V, T, or D. In some embodiments, X 16 It is F. In some embodiments, X 16 It is Y. In some embodiments, X 16 It is L. In some embodiments, X 16 It is V. In some embodiments, X 16 It is T. In some embodiments, X 16 It is D.

[0661] In some embodiments, X 17 Not Y. In some embodiments, X 17 It is Y or L. In some embodiments, X 17 It is Y. In some embodiments, X 17 It is L.

[0662] In some embodiments,

[0663] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-1);

[0664] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is D, X 15 Is it Q, X 16 Is it Y or X? 17 It is L or a combination thereof (AB-2);

[0665] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is D, X 15 Is it Q, X 16 Is it L or X? 17 It is Y or a combination thereof (AB-3);

[0666] 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 It is K, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it V or X? 17 It is Y or a combination thereof (AB-4);

[0667] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X14 It is H, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-5);

[0668] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is D, X 15 Is it Q, X 16 Is it L or X? 17 It is Y or a combination thereof (AB-6);

[0669] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-7);

[0670] 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 It is I, X 11 It is G, X 12 It is R, X 13 It is N, X 14 It is Y, X 15 It is S, X 16 Is it Y or X? 19 It is Y or a combination thereof (AB-8);

[0671] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is D, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-9);

[0672] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 It is S, X 16 Is it Y or X? 17 It is Y or a combination thereof (AB-10);

[0673] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-11);

[0674] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-12);

[0675] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-13);

[0676] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14It is N, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-14);

[0677] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-15);

[0678] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is Y, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-16);

[0679] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is H, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-17);

[0680] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is D, X 15 Is it Q, X 16 Is it F or X? 17 It is L or a combination thereof (AB-18);

[0681] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-19); or

[0682] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-20);

[0683] 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 it Q, X 11 It is G, X 12 It is V, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-21);

[0684] 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 it Q, X 11 It is S, X 12 It is S, X 13 It is S, X 14 It is S, X 15 Is it Q, X 16 Is it T or X? 17 It is Y or a combination thereof (AB-22);

[0685] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14It is H, X 15 It is S, X 16 Is it T or X? 17 It is Y or a combination thereof (AB-23);

[0686] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-24);

[0687] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is Y, X 15 It is S, X 16 Is it V or X? 17 It is Y or a combination thereof (AB-25);

[0688] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-26);

[0689] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-27);

[0690] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-28);

[0691] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-29);

[0692] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it V or X? 17 It is Y or a combination thereof (AB-30);

[0693] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it V or X? 17 It is Y or a combination thereof (AB-31);

[0694] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14It is N, X 15 Is it Q, X 16 Is it L or X? 17 It is Y or a combination thereof (AB-32);

[0695] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it Y or X? 17 It is Y or a combination thereof (AB-33);

[0696] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it T or X? 17 It is Y or a combination thereof (AB-34);

[0697] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-35);

[0698] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it T or X? 17 It is Y or a combination thereof (AB-36);

[0699] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-37);

[0700] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-38);

[0701] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is H, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-39);

[0702] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it V or X? 17 It is Y or a combination thereof (AB-40);

[0703] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is N, X 14It is Y, X 15 It is S, X 16 Is it Y or X? 17 It is Y or a combination thereof (AB-41);

[0704] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is D, X 15 Is it Q, X 16 Is it Y or X? 17 It is L or a combination thereof (AB-42);

[0705] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-43);

[0706] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is N, X 14 It is D, X 15 Is it Q, X 16 Is it D or X? 17 It is L or a combination thereof (AB-44);

[0707] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-45);

[0708] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is H, X 15 It is S, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-46);

[0709] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is N, X 14 It is D, X 15 Is it Q, X 16 Is it D or X? 17 It is L or a combination thereof (AB-47);

[0710] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is D, X 15 Is it Q, X 16 Is it L or X? 17 It is Y or a combination thereof (AB-48);

[0711] 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 it Q, X 11 It is G, X 12 It is R, X 13 It is S, X 14 It is D, X 15 Is it Q, X 16 Is it Y or X? 17 It is L or a combination thereof (AB-49);

[0712] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14It is Y, X 15 It is S, X 16 Is it T or X? 17 It is Y or a combination thereof (AB-50); or

[0713] 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 it Q, X 11 It is G, X 12 It is S, X 13 It is S, X 14 It is N, X 15 Is it Q, X 16 Is it F or X? 17 It is Y or a combination thereof (AB-51).

[0714] In some embodiments, the polypeptide disclosed herein comprises V H The V H Non-limiting examples of the amino acid sequences contained in SEQ ID NO:4-48 (see Table 1 for SEQ ID NO:4-48, and see Table 3 for corresponding HCDR1, HCDR2, and HCDR3 sequences) are respectively shown in the amino acid sequences. Figure 2 V shown by any of the following: H The amino acid sequences of HCDR1, HCDR2, and HCDR3 are basically similar.

[0715] In some embodiments, the polypeptide disclosed herein comprises V L The V L Non-limiting examples of the amino acid sequences contained in SEQ ID NO:51-76 (see Table 2 for SEQ ID NO:51-76, and see Table 3 for corresponding LCDR1, LCDR2, and LCDR3 sequences) are respectively shown in the amino acid sequences. Figure 3 V shown by any of the following: L The amino acid sequences of LCDR1, LCDR2, and LCDR3 are basically similar.

[0716] In some embodiments, the polypeptide disclosed herein comprises V H The V H The V contained in the amino acid sequence is respectively associated with any of the amino acid sequences in SEQ ID NO:4-48 H The amino acid sequences of HCDR1, HCDR2, and HCDR3 are identical to those of HCDR3.

[0717] In some embodiments, the polypeptide disclosed herein comprises V L The VL The V contained in the amino acid sequence is respectively shown in any of SEQ ID NO:51-76 L The amino acid sequences of LCDR1, LCDR2, and LCDR3 are identical to those of LCDR1, LCDR2, and LCDR3.

[0718] In some embodiments, the polypeptides disclosed herein comprise a subset of V selected from the following H / V LComplementary positions of the combination: Complementary positions that are substantially similar: 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:5, 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 IDNO: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 / SEQID 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).

[0719] In some embodiments, the polypeptides disclosed herein comprise a subset of V selected from the following H / V LComplementary positions with the same complementary positions in combination: 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 IDNO: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).

[0720] In some embodiments, the polypeptides disclosed herein comprise a subset of V selected from the following H / V LComplementary positions with different combinations of complementary positions: 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 IDNO: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 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), by substitution of 1 to 3 (e.g., 1, 2, or 3) residues (e.g., conservative, such as highly conservative substitution).

[0721] In some embodiments, the polypeptides disclosed herein comprise a subset of V selected from the following H / V L The complementary bits of the combination are the same: SEQ ID NO:3 / SEQ ID NO:50.

[0722] In some embodiments, the polypeptides disclosed herein comprise a subset of V selected from the following H / V L The complementary bits of the combination are the same: SEQ ID NO:4 / SEQ ID NO:51(AB-1).

[0723] In some embodiments, the polypeptide disclosed herein comprises V having at least about 70% sequence identity with the amino acid sequence of SEQ ID NO:3. H For example, V H It 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 with the amino acid sequence of SEQ ID NO:3. In some embodiments, V H It has at least about 85% or at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:3. Table 1 shows the sequence identified as SEQ ID NO:3, which is similar to human V... H Corresponding to the structural domain.

[0724] In some embodiments, the polypeptide disclosed herein comprises V having at least about 70% sequence identity with the amino acid sequence of SEQ ID NO:50. L For example, V L It 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 with the amino acid sequence of SEQ ID NO:50. In some embodiments, V L It has at least about 85% or at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:50. Table 2 shows the sequence identified as SEQ ID NO:50, which is similar to human V L Corresponding to the structural domain.

[0725] In some embodiments, the polypeptides disclosed herein comprise V having at least about 70% sequence identity with any one or more of the amino acid sequences in SEQ ID NO:4-48. H For example, V H The amino acid sequence 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 with any one or more of the amino acid sequences in SEQ ID NO:4-48. In some embodiments, VH It has at least about 85% or at least about 90% sequence identity with any or more of the amino acid sequences in SEQ ID NO:4-48.

[0726] In some embodiments, the polypeptides disclosed herein comprise V having at least about 70% sequence identity with any one or more of the amino acid sequences in SEQ ID NO:51-76. L For example, V L The amino acid sequence 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 with any one or more of SEQ ID NO:51-76. In some embodiments, V L It has at least about 85% or at least about 90% sequence identity with any or more of the amino acid sequences in SEQ ID NO:51-76.

[0727] In some embodiments, the polypeptide disclosed herein comprises V having at least about 70% sequence identity with the amino acid sequence of SEQ ID NO:4. H For example, V H It 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 with the amino acid sequence of SEQ ID NO:4. In some embodiments, V H It has at least about 85% or at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:4. The sequence identified as SEQ ID NO:4 is shown in Table 1.

[0728] In some embodiments, the polypeptide disclosed herein comprises V having at least about 70% sequence identity with the amino acid sequence of SEQ ID NO:51. L For example, V LIt 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 with the amino acid sequence of SEQ ID NO:51. In some embodiments, V L It has at least about 85% or at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:51. The sequence identified as SEQ ID NO:51 is shown in Table 2.

[0729] In some embodiments, the polypeptide disclosed herein comprises a V that includes at least one amino acid substitution (e.g., at least one conserved substitution, such as a highly conserved amino acid substitution) relative to the amino acid sequence of SEQ ID NO:3. H 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: 1-20, 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. In some embodiments, V, relative to the amino acid sequence of SEQ ID NO:3, H It contains approximately 1-10 amino acid substitutions. In some embodiments, at least one amino acid substitution replaces only the HCDR1, HCDR2, and / or HCDR3 residues of SEQ ID NO:3. In some embodiments, at least one amino acid substitution replaces only the non-CDR residues of SEQ ID NO:3 (e.g., within the frame region).

[0730] In some embodiments, the polypeptide disclosed herein comprises a V with at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:50. LFor 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: 1-20, 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. In some embodiments, V, relative to the amino acid sequence of SEQ ID NO:50, L It contains approximately 1-10 amino acid substitutions. In some embodiments, at least one amino acid substitution replaces only the LCDR1, LCDR2, and / or LCDR3 residues of SEQ ID NO:50. In some embodiments, at least one amino acid substitution replaces only the non-CDR residues of SEQ ID NO:50 (e.g., within the frame region).

[0731] In some embodiments, the amino acid substitution is a conservative substitution. The term "conservative amino acid substitution" or "conservative substitution" refers to an amino acid substitution with a value of 0 or more in BLOSUM62.

[0732] In some embodiments, the amino acid substitution is a highly conserved substitution. The term "highly conserved amino acid substitution" or "highly conserved substitution" refers to an amino acid substitution with a value of at least one (e.g., at least two) in BLOSUM62.

[0733] In some embodiments, the polypeptides disclosed herein comprise a V amino acid sequence comprising at least one amino acid substitution relative to any one or more of SEQ ID NO:4-48. H 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: 1-20, 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. In some embodiments, V represents the amino acid sequence relative to any one or more of the amino acid sequences in SEQ ID NO:4-48. H It contains approximately 1-10 amino acid substitutions.

[0734] In some embodiments, at least one amino acid substitution replaces only the HCDR1, HCDR2, and / or HCDR3 residues of any one or more of SEQ ID NO:4-48. In some embodiments, at least one amino acid substitution replaces only the non-CDR residues of any one or more of SEQ ID NO:4-48 (e.g., within the frame region).

[0735] In some embodiments, the polypeptides disclosed herein comprise a V amino acid sequence comprising at least one amino acid substitution relative to any one or more of SEQ ID NO:51-76. L 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: 1-20, 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. In some embodiments, V represents the amino acid sequence relative to any of SEQ ID NO: 51-76. L It contains approximately 1-10 amino acid substitutions.

[0736] In some embodiments, at least one amino acid substitution replaces only the LCDR1, LCDR2, and / or LCDR3 residues of any one or more of SEQ ID NO:51-76. In some embodiments, at least one amino acid substitution replaces only the non-CDR residues of any one or more of SEQ ID NO:51-76 (e.g., within the frame region).

[0737] In some embodiments, the polypeptide comprises:

[0738] a) HCDR1 containing at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:77;

[0739] b) HCDR2 containing at least one amino acid substitution relative to at least one amino acid sequence shown in SEQ ID NO:79-88 (e.g., at least one amino acid sequence shown in SEQ ID NO:80-88);

[0740] c) HCDR3 containing at least one amino acid substitution relative to at least one amino acid sequence shown in SEQ ID NO:90-131 (e.g., at least one amino acid sequence shown in SEQ ID NO:91-131);

[0741] d) LCDR1 containing at least one amino acid substitution relative to the amino acid sequences shown in SEQ ID NO:133-140 (e.g., at least one amino acid sequence shown in SEQ ID NO:134-140);

[0742] e) LCDR2 containing at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:141;

[0743] f) LCDR3 containing at least one amino acid substitution relative to at least one amino acid sequence shown in SEQ ID NO:143-162 (e.g., at least one amino acid sequence shown in SEQ ID NO:144-162);

[0744] Or any combination thereof.

[0745] In some embodiments, the polypeptide comprises:

[0746] a) HCDR2 containing at least one amino acid substitution relative to at least one amino acid sequence shown in SEQ ID NO:79-88 (e.g., at least one amino acid sequence shown in SEQ ID NO:80-88);

[0747] b) HCDR3 containing at least one amino acid substitution relative to at least one amino acid sequence shown in SEQ ID NO:90-131 (e.g., at least one amino acid sequence shown in SEQ ID NO:91-131);

[0748] c) LCDR1 containing at least one amino acid substitution relative to the amino acid sequences shown in SEQ ID NO:133-140 (e.g., at least one amino acid sequence shown in SEQ ID NO:134-140); or

[0749] d) LCDR3 containing at least one amino acid substitution relative to at least one amino acid sequence shown in SEQ ID NO:143-162 (e.g., at least one amino acid sequence shown in SEQ ID NO:144-162);

[0750] Or any combination thereof.

[0751] In some embodiments, the polypeptide disclosed herein comprises V, which includes the amino acid sequence of SEQ ID NO:3. H In some embodiments, the polypeptides disclosed herein comprise a V amino acid sequence including any of SEQ ID NO:4-48. H In some embodiments, the polypeptide disclosed herein comprises V, which includes the amino acid sequence of SEQ ID NO:4.H .

[0752] In some embodiments, the polypeptide disclosed herein comprises V, including the amino acid sequence of SEQ ID NO:50. L In some embodiments, the polypeptides disclosed herein comprise a V amino acid sequence including any of SEQ ID NO:51-76. L In some embodiments, the polypeptide disclosed herein comprises V, which includes the amino acid sequence of SEQ ID NO:51. L .

[0753] In some embodiments, the polypeptides disclosed herein comprise:

[0754] V containing the amino acid sequence of any one of SEQ ID NO:4-48 H ;as well as

[0755] V containing the amino acid sequence of SEQ ID NO:50 L .

[0756] In some embodiments, the polypeptides disclosed herein comprise:

[0757] V containing the amino acid sequence of SEQ ID NO:3 H ;as well as

[0758] V containing the amino acid sequence of any one of SEQ ID NO:51-76 L .

[0759] In some embodiments, the polypeptides disclosed herein comprise:

[0760] V containing the amino acid sequence of any one of SEQ ID NO:4-48 H ;as well as

[0761] V containing the amino acid sequence of any one of SEQ ID NO:51-76 L .

[0762] In some embodiments, the polypeptides disclosed herein comprise:

[0763] a) The V H Contains the amino acid sequence of SEQ ID NO:4; and

[0764] b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-1).

[0765] In some embodiments, the polypeptides disclosed herein comprise:

[0766] a) The V H Contains the amino acid sequence of SEQ ID NO:5; and

[0767] b) The V L The amino acid sequence containing SEQ ID NO:52 (AB-2).

[0768] In some embodiments, the polypeptides disclosed herein comprise:

[0769] a) The V H Contains the amino acid sequence of SEQ ID NO:6; and

[0770] b) The V L The amino acid sequence containing SEQ ID NO:53 (AB-3).

[0771] In some embodiments, the polypeptides disclosed herein comprise:

[0772] a) The V H Contains the amino acid sequence of SEQ ID NO:7; and

[0773] b) The V L The amino acid sequence containing SEQ ID NO:54 (AB-4).

[0774] In some embodiments, the polypeptides disclosed herein comprise:

[0775] a) The V H Contains the amino acid sequence of SEQ ID NO:8; and

[0776] b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-5).

[0777] In some embodiments, the polypeptides disclosed herein comprise:

[0778] a) The V H Contains the amino acid sequence of SEQ ID NO:9; and

[0779] b) The V L The amino acid sequence containing SEQ ID NO:55 (AB-6).

[0780] In some embodiments, the polypeptides disclosed herein comprise:

[0781] a) The V H Contains the amino acid sequence of SEQ ID NO:10; and

[0782] b) The V LThe amino acid sequence comprising SEQ ID NO:56 (AB-7). In some embodiments, the polypeptide disclosed herein comprises:

[0783] a) The V H Contains the amino acid sequence of SEQ ID NO:11; and

[0784] b) The V L An amino acid sequence comprising SEQ ID NO:57 (AB-8). In some embodiments, the polypeptide disclosed herein comprises:

[0785] a) The V H Contains the amino acid sequence of SEQ ID NO:13; and

[0786] b) The V L The amino acid sequence comprising SEQ ID NO:59 (AB-10). In some embodiments, the polypeptide disclosed herein comprises:

[0787] a) The V H Contains the amino acid sequence of SEQ ID NO:14; and

[0788] b) The V L The amino acid sequence comprising SEQ ID NO:60 (AB-11). In some embodiments, the polypeptide disclosed herein comprises:

[0789] a) The V H Contains the amino acid sequence of SEQ ID NO:15; and

[0790] b) The V L The amino acid sequence comprising SEQ ID NO:56 (AB-12). In some embodiments, the polypeptide disclosed herein comprises:

[0791] a) The V H Contains the amino acid sequence of SEQ ID NO:16; and

[0792] b) The V L The amino acid sequence comprising SEQ ID NO:51 (AB-13). In some embodiments, the polypeptide disclosed herein comprises:

[0793] a) The V H Contains the amino acid sequence of SEQ ID NO:10; and

[0794] b) The V L The amino acid sequence comprising SEQ ID NO:50 (AB-14). In some embodiments, the polypeptide disclosed herein comprises:

[0795] a) The V H Contains the amino acid sequence of SEQ ID NO:17; and

[0796] b) The V L The amino acid sequence comprising SEQ ID NO:61 (AB-15). In some embodiments, the polypeptide disclosed herein comprises:

[0797] a) The V H Contains the amino acid sequence of SEQ ID NO:18; and

[0798] b) The V L The amino acid sequence comprising SEQ ID NO:62 (AB-16). In some embodiments, the polypeptide disclosed herein comprises:

[0799] a) The V H Contains the amino acid sequence of SEQ ID NO:6; and

[0800] b) The V L The amino acid sequence comprising SEQ ID NO:63 (AB-17). In some embodiments, the polypeptide disclosed herein comprises:

[0801] a) The V H Contains the amino acid sequence of SEQ ID NO:19; and

[0802] b) The V L The amino acid sequence comprising SEQ ID NO:64 (AB-18). In some embodiments, the polypeptide disclosed herein comprises:

[0803] a) The V H Contains the amino acid sequence of SEQ ID NO:4; and

[0804] b) The V L The amino acid sequence comprising SEQ ID NO:61 (AB-19). In some embodiments, the polypeptide disclosed herein comprises:

[0805] a) The V H Contains the amino acid sequence of SEQ ID NO:20; and

[0806] b) The V L The amino acid sequence comprising SEQ ID NO:61 (AB-20). In some embodiments, the polypeptide disclosed herein comprises:

[0807] a) The V HContains the amino acid sequence of SEQ ID NO:21; and

[0808] b) The V L The amino acid sequence comprising SEQ ID NO:65 (AB-21). In some embodiments, the polypeptide disclosed herein comprises:

[0809] a) The V H Contains the amino acid sequence of SEQ ID NO:22; and

[0810] b) The V L The amino acid sequence comprising SEQ ID NO:66 (AB-22). In some embodiments, the polypeptide disclosed herein comprises:

[0811] a) The V H Contains the amino acid sequence of SEQ ID NO:4; and

[0812] b) The V L The amino acid sequence comprising SEQ ID NO:67 (AB-23). ​​In some embodiments, the polypeptide disclosed herein comprises:

[0813] a) The V H Contains the amino acid sequence of SEQ ID NO:23; and

[0814] b) The V L The amino acid sequence comprising SEQ ID NO:56 (AB-24). In some embodiments, the polypeptide disclosed herein comprises:

[0815] a) The V H Contains the amino acid sequence of SEQ ID NO:24; and

[0816] b) The V L An amino acid sequence comprising SEQ ID NO:68 (AB-25). In some embodiments, the polypeptide disclosed herein comprises:

[0817] a) The V H Contains the amino acid sequence of SEQ ID NO:25; and

[0818] b) The V L The amino acid sequence comprising SEQ ID NO:51 (AB-26). In some embodiments, the polypeptide disclosed herein comprises:

[0819] a) The V H Contains the amino acid sequence of SEQ ID NO:26; and

[0820] b) The VL The amino acid sequence comprising SEQ ID NO:56 (AB-27). In some embodiments, the polypeptide disclosed herein comprises:

[0821] a) The V H Contains the amino acid sequence of SEQ ID NO:27; and

[0822] b) The V L The amino acid sequence comprising SEQ ID NO:61 (AB-28). In some embodiments, the polypeptide disclosed herein comprises:

[0823] a) The V H Contains the amino acid sequence of SEQ ID NO:28; and

[0824] b) The V L The amino acid sequence comprising SEQ ID NO:56 (AB-29). In some embodiments, the polypeptide disclosed herein comprises:

[0825] a) The V H Contains the amino acid sequence of SEQ ID NO:28; and

[0826] b) The V L The amino acid sequence comprising SEQ ID NO:69 (AB-30). In some embodiments, the polypeptide disclosed herein comprises:

[0827] a) The V H Contains the amino acid sequence of SEQ ID NO:29; and

[0828] b) The V L The amino acid sequence comprising SEQ ID NO:70 (AB-31). In some embodiments, the polypeptide disclosed herein comprises:

[0829] a) The V H Contains the amino acid sequence of SEQ ID NO:30; and

[0830] b) The V L The amino acid sequence comprising SEQ ID NO:71 (AB-32). In some embodiments, the polypeptide disclosed herein comprises:

[0831] a) The V H Contains the amino acid sequence of SEQ ID NO:31; and

[0832] b) The V LThe amino acid sequence comprising SEQ ID NO:72 (AB-33). In some embodiments, the polypeptide disclosed herein comprises:

[0833] a) The V H Contains the amino acid sequence of SEQ ID NO:32; and

[0834] b) The V L The amino acid sequence comprising SEQ ID NO:67 (AB-34). In some embodiments, the polypeptides disclosed herein comprise:

[0835] a) The V H The amino acid sequence containing SEQ ID NO:33; and

[0836] b) The V L The amino acid sequence comprising SEQ ID NO:56 (AB-35). In some embodiments, the polypeptide disclosed herein comprises:

[0837] a) The V H The amino acid sequence containing SEQ ID NO:34; and

[0838] b) The V L The amino acid sequence comprising SEQ ID NO:73 (AB-36). In some embodiments, the polypeptide disclosed herein comprises:

[0839] a) The V H Contains the amino acid sequence of SEQ ID NO:35; and

[0840] b) The V L The amino acid sequence comprising SEQ ID NO:51 (AB-37). In some embodiments, the polypeptide disclosed herein comprises:

[0841] a) The V H Contains the amino acid sequence of SEQ ID NO:36; and

[0842] b) The V L The amino acid sequence comprising SEQ ID NO:56 (AB-38). In some embodiments, the polypeptide disclosed herein comprises:

[0843] a) The V H The amino acid sequence containing SEQ ID NO:37; and

[0844] b) The V L The amino acid sequence comprising SEQ ID NO:63 (AB-39). In some embodiments, the polypeptide disclosed herein comprises:

[0845] a) The V H The amino acid sequence containing SEQ ID NO:38; and

[0846] b) The V L The amino acid sequence comprising SEQ ID NO:69 (AB-40). In some embodiments, the polypeptide disclosed herein comprises:

[0847] a) The V H Contains the amino acid sequence of SEQ ID NO:39; and

[0848] b) The V L The amino acid sequence comprising SEQ ID NO:74 (AB-41). In some embodiments, the polypeptide disclosed herein comprises:

[0849] a) The V H Contains the amino acid sequence of SEQ ID NO:40; and

[0850] b) The V L An amino acid sequence comprising SEQ ID NO:52 (AB-42). In some embodiments, the polypeptide disclosed herein comprises:

[0851] a) The V H Contains the amino acid sequence of SEQ ID NO:41; and

[0852] b) The V L The amino acid sequence comprising SEQ ID NO:51 (AB-43). In some embodiments, the polypeptide disclosed herein comprises:

[0853] a) The V H Contains the amino acid sequence of SEQ ID NO:42; and

[0854] b) The V L The amino acid sequence comprising SEQ ID NO:75 (AB-44). In some embodiments, the polypeptide disclosed herein comprises:

[0855] a) The V H Contains the amino acid sequence of SEQ ID NO:43; and

[0856] b) The V L The amino acid sequence comprising SEQ ID NO:56 (AB-45). In some embodiments, the polypeptide disclosed herein comprises:

[0857] a) The V HContains the amino acid sequence of SEQ ID NO:44; and

[0858] b) The V L The amino acid sequence comprising SEQ ID NO:51 (AB-46). In some embodiments, the polypeptide disclosed herein comprises:

[0859] a) The V H Contains the amino acid sequence of SEQ ID NO:45; and

[0860] b) The V L The amino acid sequence comprising SEQ ID NO:75 (AB-47). In some embodiments, the polypeptide disclosed herein comprises:

[0861] a) The V H Contains the amino acid sequence of SEQ ID NO:46; and

[0862] b) The V L The amino acid sequence comprising SEQ ID NO:53 (AB-48). In some embodiments, the polypeptide disclosed herein comprises:

[0863] a) The V H The amino acid sequence containing SEQ ID NO:47; and

[0864] b) The V L The amino acid sequence containing SEQ ID NO:52 (AB-49).

[0865] In some embodiments, the polypeptides disclosed herein comprise:

[0866] a) The V H The amino acid sequence containing SEQ ID NO:48; and

[0867] b) The V L The amino acid sequence containing SEQ ID NO:76 (AB-50).

[0868] In some embodiments, the polypeptides disclosed herein comprise:

[0869] a) The V H Contains the amino acid sequence of SEQ ID NO:3; and

[0870] b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-51).

[0871] In some embodiments, the peptides disclosed herein comprise humanized V H and VL It contains a human frame region or a combination thereof.

[0872] In some embodiments, the polypeptides disclosed herein are immunoglobulin molecules, such as antibodies (e.g., whole antibodies, intact antibodies) or antigen-binding fragments of antibodies (e.g., Fab, F(ab')2, Fab', scFv, or Fv). As used herein, the term "antibody" refers to an immunoglobulin molecule or a portion thereof capable of specifically binding to a target (e.g., carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site located in a variable domain of an immunoglobulin molecule. In some embodiments, the antibody is a whole or intact antibody.

[0873] In some embodiments, the polypeptides disclosed herein are single-domain antibodies or antigen-binding fragments thereof. As used herein, the terms "single-domain antibody (sdAb)" or "nanobody" refer to an immunoglobulin molecule composed of a single monomeric variable antibody domain and capable of specifically binding to a target. Single-domain antibodies can be antibodies of any species, such as murine antibodies, human antibodies, or humanized antibodies.

[0874] In some embodiments, the polypeptides disclosed herein are heavy chain antibodies comprising two or more heavy chains but lacking light chains or their antigen-binding fragments. Non-limiting examples of heavy chain antibodies include Camelidae Vhh (also known as VHH or V). H H) antibodies. Cameloid antibodies are antibodies derived from mammals of the Camelidae family (including llamas, camels, and alpacas).

[0875] In some embodiments, the polypeptide disclosed herein is an antibody comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds or their polymers (e.g., IgM). Each heavy chain comprises a heavy chain variable domain (V... H The heavy chain contains constant structural domains (including domain CH1, hinges CH2 and CH3). Each light chain contains a light chain variable structural domain (V). L ) and light chain constant structural domains (CL). V H District and V L The region can be further subdivided into hypervariable regions called complementary determinant regions (CDRs), which are distributed within the frame region (FR). V H and V L Each antibody contains three CDRs and four FR segments arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Antibodies can be from any species, such as murine antibodies, human antibodies, or humanized antibodies.

[0876] The extent of the frame region and CDR of an antibody can be identified using one of several suitable methods well known in the art, such as the Kabat definition, Chothia definition, AbM definition, and / or contact definition. Publicly available and / or commercially available tools for identifying the frame region and / or CDR include IgBlast (available in […]).

[0877] Accessible via www.ncbi.nlm.nih.gov / igblast / , Scaligner (available from drugdesigntech's website www.scaligner.com / ), IMGT rules and / or tools (see, for example, www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html, also accessible at www.imgt.org / ), Chothia canonical alignment (available at www.bioinf.org.uk / abs / chothia.html), antigen receptor numbering and receptor classification (ANARCI, available at www.bioinf.org.uk / abs / chothia.html), and other related technologies.

[0878] Accessible at opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / anarci / or the Paratome web server (accessible at www.ofranlab.org / paratome / , see Vered Kunik et al., Nucleic Acids Research, Vol. 40, No. W1, July 1, 2012, pp. W521–W524).

[0879] As used herein, “CDR” encompasses any CDR defined by methods recognized in the art for identifying CDR residues on antibodies. See, for example, Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, et al., (1989) Nature 342:877; Chothia, C. et al., (1987) Journal of Molecular Biology 196:901-917; Allazikani et al., (1997) Journal of Molecular Biology 273:927-948; and Almagro, Journal of Molecular Recognition 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. When the same method is used to determine the identity of the CDRs of two antibodies, the two antibodies are determined to have the same CDRs relative to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3.

[0880] 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 District, V L Region, Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment and a V H A structural domain or a V L Domain-specific antibodies (dAbs) composed of structural domains, etc. V H and V L The domains can be linked together via synthetic linkers to form various types of single-chain antibody designs, in which V H / V L Domains pair intramolecularly or intermolecularly to enable V H and V L When expressed as a single chain, the domain forms a monovalent antigen-binding site, such as a single-chain Fv (scFv) or a bifunctional antibody. In some embodiments, the polypeptide disclosed herein is an antigen-binding fragment selected from Fab, F(ab')2, Fab', scFv, or Fv. In some embodiments, the polypeptide is scFv.

[0881] In some embodiments, the peptides disclosed herein (e.g., antibodies or antigen-binding fragments) are incorporated into cell-based therapies. In some embodiments, the peptide is an engineered T-cell receptor. In some embodiments, the peptide is a chimeric antigen receptor (CAR) (e.g., expressed on T (CAR-T) cells, natural killer (CAR-NK) cells, or macrophage (CAR-M) cells). In some embodiments, the CAR includes a transmembrane domain and an antigen recognition portion, wherein the antigen recognition portion binds to SARS-CoV-2 (e.g., an epitope within S2).

[0882] In some embodiments, the polypeptide is an antibody mimic. The term "antibody mimic" refers to a polypeptide that mimics the ability of an antibody to bind to an antigen but is structurally different from that of a natural antibody. Non-limiting examples of antibody mimics include adnectin, affibody, affilin, affimer, affitin, alpha body, anticarrier protein, high-affinity multimer (Avimer), DARPin, fynomer, Kunitz domain peptide, monomer, nanobody, nanoCLAMP, and versabody.

[0883] In some embodiments, the peptides disclosed herein compete with comparative antibodies (e.g., reference antibody, sotopimab) for binding to wild-type SARS-CoV-2 spike, SARS-CoV-2 spike mutant, or combinations thereof, wherein the comparative 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 between an antibody or its antigen-binding fragment and its epitope relative to other antigens or amino acid sequences, i.e., significantly higher binding affinity.

[0884] In some embodiments, the comparative antibody comprises V of SEQ ID NO:3 H Sequence and V of SEQ ID NO:50 LSequences. In some embodiments, the comparative 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 comparative antibody comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO:77, SEQ ID NO:79, and SEQ ID NO:90, respectively; and comprises the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO:133, SEQ ID NO:141, and SEQ ID NO:143, respectively.

[0885] In some embodiments, the polypeptides disclosed herein comprise:

[0886] a) Antibody heavy chain constant domain sequence;

[0887] b) Antibody light chain constant domain sequence; or

[0888] c) Antibody heavy chain constant domain sequence and antibody light chain constant domain sequence.

[0889] 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, an IgG2b, or an 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).

[0890] In some embodiments, the polypeptides disclosed herein comprise immunoglobulin light chain variable domains (V... L V H and V L Domains can be linked together via adapters (e.g., synthetic adapters) to form various types of single-chain antibody designs, in which V H / V L Domains pair intramolecularly or intermolecularly to enable V Hand V L When the domain is expressed by a single strand, it forms a monovalent antigen binding site.

[0891] 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 κ constant domain and a λ 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 κ constant domain.

[0892] In some embodiments, the antibody heavy chain constant domain sequence has at least about 60% sequence identity with 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 with 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 with the amino acid sequence of SEQ ID NO:194. The sequence identified as SEQ ID NO:194 is shown below:

[0893] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:194).

[0894] In some embodiments, the antibody light chain constant domain sequence has at least about 60% sequence identity with 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 with 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 with 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:

[0895] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 195).

[0896] GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 196).

[0897] In some embodiments, the antibody heavy chain constant domain sequence contains at least one amino acid substitution relative 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: 1-20, 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. In some embodiments, the antibody heavy chain constant domain sequence contains about 1-10 amino acid substitutions relative to the amino acid sequence of SEQ ID NO:194.

[0898] In some embodiments, the antibody light chain constant domain sequence contains at least one amino acid substitution relative 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: 1-20, 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. In some embodiments, the antibody light chain constant domain sequence contains about 1-10 amino acid substitutions relative to the amino acid sequence of SEQ ID NO:195 or SEQ ID NO:196.

[0899] In some embodiments, the amino acid substitution is a conserved substitution. In some embodiments, the amino acid substitution is a highly conserved substitution.

[0900] In some embodiments, the polypeptides disclosed herein are isolated polypeptides. In some embodiments, the isolated polypeptides are recombinantly generated. In some embodiments, the isolated polypeptides are synthetically generated.

[0901] In some embodiments, the polypeptide disclosed herein is linked to a second polypeptide. The term "link" refers to attachment via covalent or non-covalent interactions. The linker can be a suitable linker. Non-limiting examples include peptide linkers, compound linkers, and chemical crosslinking agents. In some embodiments, the linker is a disulfide bond.

[0902] In some embodiments, the polypeptides disclosed herein are conjugated to heterologous moieties. The term "conjugation" refers to attachment via covalent or non-covalent interactions. Conjugation can be performed using any suitable linker. Non-limiting examples include peptide linkers, compound linkers, and chemical crosslinking agents.

[0903] In some embodiments, the heterologous portion is a therapeutic agent, a diagnostic agent, or a combination thereof. In some embodiments, the heterologous portion is polyethylene glycol (PEG), hexadecanoic acid, hydrogel, nanoparticles, polymerized domains, and carrier peptides.

[0904] In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the nanoparticles are polymer 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 acid-co-glycolic acid) acid (PLGA), poly(lactic acid-co-glycolic acid)-d-α-tocopherol polyethylene glycol succinate, poly(lactic acid-co-glycolic acid)-ethylene oxide fumarate, poly(glycolic acid)-poly(ethylene glycol), polycaprolactone-poly(ethylene glycol), or any salt thereof. In some embodiments, the polymer nanoparticles comprise poly(lactic acid-co-glycolic acid) acid (PLGA).

[0905] In some embodiments, the carrier peptide is albumin or Fc peptide.

[0906] In some embodiments, polypeptide:

[0907] a) It can bind to epitopes in the S2 domain of the SARS-CoV-2 spike protein;

[0908] b) Using 10 μM or less K D Binds to SARS-CoV-1 and / or SARS-CoV-2;

[0909] c) Neutralizing SARS-CoV-1 and / or SARS-CoV-2 infections in human host cells;

[0910] d) It has a weaker nonspecific binding than the reference antibody;

[0911] e) It exhibits weaker self-association than the reference antibody;

[0912] Or a combination of any of the aforementioned.

[0913] In some embodiments, the peptide can bind to one or more epitope residues in 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 in 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.

[0914] In some embodiments, the peptide binds at a binding constant (K) of about 10 μM or less. D It binds to the SARS-CoV-2 spike protein. As used herein, the term "K" refers to the SARS-CoV-2 spike protein. D The "binding affinity," also known as the "binding constant," "equilibrium dissociation constant," or "affinity constant," is a measure of the degree of reversible association between two molecular species (e.g., antibody and target protein) and includes both actual binding affinity and apparent binding affinity. Binding affinity can be determined using methods known in the art, including, for example, by measuring surface plasmon resonances, such as using biolayer interferometry (Octet, ForteBio) or surface plasmon resonance (Biacore) systems and assays. A reference comparing various surface techniques used to measure binding affinity and kinetics is Yang, D., Singh, A., Wu, H., and Kroe-Barrett, R., "Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen bindingkinetics," Analytical Biochemistry 508:78-96 (2016), the contents of which are incorporated herein by reference in their entirety.

[0915] In some embodiments, the polypeptide is in the form of about the following K DWith SARS-CoV-1 spike (e.g., CoV-1 and / or WIV1) and / or SARS-CoV-2 spike (e.g., α, β, γ, δ, κ, ε, η, ι, λ, μ and / or ο, 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.3 7. 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) or fragments thereof (e.g., the S2 domain of the SARS-CoV-2 spike and / or Figure 1 Epitope binding: 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. In some embodiments, the peptide is in 100 nM or less K DWith SARS-CoV-1 spike (e.g., CoV-1 and / or WIV1) and / or SARS-CoV-2 spike (e.g., α, β, γ, δ, κ, ε, η, ι, λ, μ and / or ο, 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.3 7. 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) or fragments thereof (e.g., the S2 domain of the SARS-CoV-2 spike and / or Figure 1 (epitopes in the text) are combined.

[0916] In some embodiments, the polypeptide is in the form of about the following K DWith SARS-CoV-1 spike (e.g., CoV-1 and / or WIV1) and / or SARS-CoV-2 spike (e.g., α, β, γ, δ, κ, ε, η, ι, λ, μ and / or ο, 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, 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, BA.5, BA.5.2.6, 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 fragments thereof (e.g., the S2 domain of the SARS-CoV-2 spike and / or Figure 1 Epitopes in the text) combination: 10 -10 -10 -5 M, 10 -10 -5×10 -6 M, 2×10 -10 -5×10 -6 M, 2×10 -10 -2×10 -6 M, 5×10 -10 -2×10 -6 M, 5×10 -10 -10 -7 M, 10 -9 -10 -7 M, 10 -9 -5×10 -8 M, 2×10 -9 -5×10 - 8 M, 2×10 -9 -2×10 -8 M, 5×10 -9 -2×10 -8 M or 5×10 -9 -10 -8 M.

[0917] In some embodiments, the peptide (e.g., a full-length IgG1 antibody) is in a concentration of about 10... -6 M or smaller K DWith SARS-CoV-1 spike (e.g., CoV-1 and / or WIV1) and / or SARS-CoV-2 spike (e.g., α, β, γ, δ, κ, ε, η, ι, λ, μ and / or ο, 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.3 7. 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) or fragments thereof (e.g., the S2 domain of the SARS-CoV-2 spike and / or Figure 1 (epitopes in the text) binding, 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 -5×10 -7 M, 2×10 -10 -5×10 -7 M, 2×10 -10 -2×10 -7 M, 5×10 -10 -2×10 -7 M, 5×10 -10 -10 -7 M, 10 -9 -10 -7 M, 10 -9 -5×10 -8 M, 2×10 -9 -5×10 -8 M, 2×10-9 -2×10 -8 M, 5×10 -9 -2×10 -8 M or 5×10 -9 -10 -8 M.

[0918] In some embodiments, the peptide competes with a reference antibody for binding to the SARS-CoV-2 spike protein (e.g., the S2 domain). Techniques and assays for assessing competition between antibodies are known in the art.

[0919] In some embodiments, the peptide is in an IC50 concentration of 10 μM or less. 50 Neutralizes SARS-CoV-1 (e.g., CoV-1 and / or WIV1) and / or SARS-CoV-2 (e.g., α, β, γ, δ, κ, ε, η, ι, λ, μ and / or ο, 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, 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, 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).

[0920] In some embodiments, the peptide (e.g., a full-length IgG1 antibody) is administered at an IC50 concentration of about 25,000 ng / mL or less. 50Neutralizes SARS-CoV-2 (e.g., α, β, γ, δ, κ, ε, η, ι, λ, μ, and / or o, 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.1.1 5. 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. Infection of human host cells by, for example, 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, for example, approximately: 20,000 ng / mL, 15,000 ng / mL, 10,000 ng / mL, 5,000 ng / mL, 2,500 ng / mL, 1,000 ng / mL, 750 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 75 ng / mL, 50 ng / mL, 25 ng / mL or 10 ng / mL. / mL or less, for example, about: 10-25,000 ng / mL, 10-20,000 ng / mL, 25-20,000 ng / mL, 25-15,000 ng / mL, 50-15,000 ng / mL, 50-10,000 ng / mL, 75-10,000 ng / mL, 75-5,000 ng / mL, 100-5,000 ng / mL, 100-2,500 ng / mL, 250-2,500 ng / mL, 250-1,000 ng / mL, 500-1,000 ng / mL or 500-750 ng / mL.

[0921] In some embodiments, the peptide (e.g., a full-length IgG1 antibody) is administered at an IC50 concentration of about 50,000 ng / mL or less. 80Neutralizes SARS-CoV-2 (e.g., α, β, γ, δ, κ, ε, η, ι, λ, μ, and / or o, 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.1.1 5. 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. Infection of human host cells by, for example, 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, for example, approximately: 25,000 ng / mL, 15,000 ng / mL, 10,000 ng / mL, 5,000 ng / mL, 2,500 ng / mL, 1,000 ng / mL, 750 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 75 ng / mL, 50 ng / mL, 25 ng / mL or 10 ng / mL. / mL or less, for example, approximately: 10-50,000 ng / mL, 10-25,000 ng / mL, 25-25,000 ng / mL, 25-15,000 ng / mL, 50-15,000 ng / mL, 50-10,000 ng / mL, 75-10,000 ng / mL, 75-5,000 ng / mL, 100-5,000 ng / mL, 100-2,500 ng / mL, 250-2,500 ng / mL, 250-1,000 ng / mL, 500-1,000 ng / mL, or 500-750 ng / mL.

[0922] In some embodiments, the peptide (e.g., a full-length IgG1 antibody) is administered at an IC50 concentration of about 25,000 ng / mL or less. 50Neutralizes SARS-CoV-1 (e.g., CoV-1 and / or WIV1) infection of human host cells, for example, approximately: 20,000 ng / mL, 15,000 ng / mL, 10,000 ng / mL, 5,000 ng / mL, 2,500 ng / mL, 1,000 ng / mL, 750 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 75 ng / mL, 50 ng / mL, 25 ng / mL, or 10 ng / mL or less, for example, approximately: 10-25,000 ng / mL, 10-20,000ng / mL, 25-20,000ng / mL, 25-15,000ng / mL, 50-15,000ng / mL, 50-10,000ng / mL, 75-10,000ng / mL, 75- 5,000ng / mL, 100-5,000ng / mL, 100-2,500ng / mL, 250-2,500ng / mL, 250-1,000ng / mL, 500-1,000ng / mL or 500-750ng / mL.

[0923] In some embodiments, the peptide (e.g., a full-length IgG1 antibody) is administered at an IC50 concentration of about 50,000 ng / mL or less. 80 Neutralizes SARS-CoV-1 (e.g., CoV-1 and / or WIV1) infection of human host cells, for example, approximately: 25,000 ng / mL, 15,000 ng / mL, 10,000 ng / mL, 5,000 ng / mL, 2,500 ng / mL, 1,000 ng / mL, 750 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 75 ng / mL, 50 ng / mL, 25 ng / mL, or 10 ng / mL or less, for example, approximately: 10-50,000 ng / mL, 10-25,000ng / mL, 25-25,000ng / mL, 25-15,000ng / mL, 50-15,000ng / mL, 50-10,000ng / mL, 75-10,000ng / mL, 75- 5,000ng / mL, 100-5,000ng / mL, 100-2,500ng / mL, 250-2,500ng / mL, 250-1,000ng / mL, 500-1,000ng / mL or 500-750ng / mL.

[0924] In some embodiments, the peptide reduces the infectivity of a beta-coronavirus (e.g., SARS-CoV-2) in a host cell (e.g., a human host cell). In some embodiments, the peptide reduces the infectivity of a beta-coronavirus (e.g., SARS-CoV-2) in a host cell (e.g., a human host cell) by at least about 10%, for example, 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 peptide reduces the infectivity of a beta-coronavirus (e.g., SARS-CoV-2) in human cells by at least about 30%.

[0925] In some embodiments, the peptide reduces the levels of β-coronaviruses (such as SARS-CoV-2) (e.g., α, β, γ, δ, κ, ε, η, ι, λ, μ and / or ο) in host cells (e.g., human host cells), such as 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)) reinfection.In some embodiments, the polypeptide delivers a beta coronavirus (such as SARS-CoV-2) of the host cell (e.g., human host cells) (e.g., α, β, γ, δ, κ, ε, η, ι, λ, μ and / or ο, 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.1.3) to the host cell (e.g., human host cells). 51. 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, B Q.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 approximately 10%, for example, at least approximately: 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 peptide contains human cellular beta coronaviruses (such as SARS-CoV-2) (e.g., α, β, γ, δ, κ, ε, η, ι, λ, μ and / or ο, 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). The risk of reinfection is reduced by at least approximately 30% for the following: 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.

[0926] Techniques such as pseudovirus neutralization assays or live virus neutralization assays can be used to measure infectivity or reinfection (see, for example, 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 GenScriptcPass, can also be used. TM SARS-CoV-2 neutralizing antibody test kits can be used according to the manufacturer's instructions.

[0927] In some embodiments, the peptide reduces the infectivity of host cells (e.g., human host cells) for SARS-CoV-1 (e.g., CoV-1 and / or WIV1). In some embodiments, the peptide reduces the infectivity of host cells (e.g., human host cells) for SARS-CoV-1 (e.g., CoV-1 and / or WIV1) by at least about 10%, for example, 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 peptide reduces the infectivity of human cells for SARS-CoV-1 (e.g., CoV-1 and / or WIV1) by at least about 30%.

[0928] In some embodiments, the peptide reduces SARS-CoV-1 (e.g., CoV-1 and / or WIV1) reinfection in host cells (e.g., human host cells). In some embodiments, the peptide reduces SARS-CoV-1 (e.g., CoV-1 and / or WIV1) reinfection in host cells (e.g., human host cells) by at least about 10%, for example, 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 peptide reduces SARS-CoV-1 (e.g., CoV-1 and / or WIV1) reinfection in human cells by at least about 30%.

[0929] In some embodiments, the host cells are selected from the group consisting of: type II lung cells, ileal absorptive intestinal cells, nasal goblet cells, and combinations thereof.

[0930] In some embodiments, the peptide exhibits weaker self-association than a reference antibody, for example, as determined by affinity-captured self-interacting nanoparticle spectrometry (AC-SINS) values. The AC-SINS value is the change in the wavelength of maximum absorbance in the absorption spectrum of the coated nanoparticle compared to the spectrum of the nanoparticle alone. Therefore, the greater the change in the wavelength of maximum absorbance, the greater the self-interaction of the antibody coated on the nanoparticle. Self-association is an undesirable property, associated with poor viscosity and poor PK properties. Techniques and assays for assessing protein self-association are known in the art. See, for example, Patro and Przybycien, Biotechnol Bioeng. 52(2):193-203 (1996), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the peptide exhibits weaker self-association than a reference antibody.

[0931] In some embodiments, the AC-SINS value of the peptide does not exceed 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. In some embodiments, the AC-SINS value of the peptide does not exceed about 14. In some embodiments, the AC-SINS value of the peptide does not exceed about 8. In some embodiments, the AC-SINS value of the peptide is 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. In some embodiments, the AC-SINS value of the peptide is about 0-25, for example, 0-20, 0-15, 0-10, 0-8, 0-5, 2-20, 2-15, 2-10, 2-8, 2-5, 5-20, 5-15, 5-10, 5-8, 7-8, or 13-15. In some embodiments, the AC-SINS value of the peptide is about 13-14, 13-15, 7-9, or 7-8. In some embodiments, the AC-SINS value of the peptide is about 8 or 14.

[0932] In some embodiments, the peptide exhibits improved developability (e.g., reduced AC-SINS) relative to a reference antibody. In some embodiments, the peptide exhibits at least about 10% lower self-association than the reference antibody, for example, 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 peptide exhibits at least about 30% lower self-association than the reference antibody.

[0933] In some embodiments, the self-association of the polypeptide is less than about 90% of the self-association of the reference antibody, for example, 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%.

[0934] In some embodiments, the self-association of the peptide relative to the self-association of the reference antibody is about 1-90%, for example, 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%.

[0935] In some embodiments, the self-association is reduced by at least about 10% relative to the reference antibody, 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%.

[0936] Fusion protein

[0937] In some embodiments, this disclosure provides a fusion protein comprising one or more polypeptides described herein.

[0938] The term "fusion protein" refers to a single protein molecule that is synthetic, semi-synthetic, or recombinant. A fusion protein may comprise all or a portion of two or more different proteins and / or polypeptides attached by covalent bonds (e.g., peptide bonds). For example, a fusion protein may comprise a full-length polypeptide (e.g., a full antibody) or a fragment thereof (e.g., an antigen-binding fragment of an antibody) disclosed herein. A heterocouple may be a full-length protein or a fragment thereof (e.g., a truncated protein).

[0939] Fusion proteins can be recombined or synthesized using conventional methods and reagents well known in the art. For example, the fusion proteins disclosed herein can be recombined in suitable host cells (e.g., bacteria) according to methods known in the art. See, for example, *Current Protocols in Molecular Biology*, 2nd edition, edited by Ausubel et al., John Wiley & Sons, 1992; and *Molecular Cloning: a Laboratory Manual*, 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. For example, nucleic acid molecules containing nucleotide sequences encoding the fusion proteins described herein can be introduced and expressed in suitable host cells (e.g., *E. coli*), and the expressed fusion proteins can be isolated / purified from the host cells (e.g., in inclusion bodies) using conventional methods and readily available reagents. For example, DNA fragments encoding different protein sequences (e.g., light-responsive domains, heterologous peptide components) can be linked together within a frame according to conventional techniques. In another embodiment, the fusion gene can be synthesized using conventional techniques, including automated DNA synthesizers. In some embodiments, PCR amplification of the nucleic acid fragments can be performed using anchor primers that generate complementary overhangs between two consecutive nucleic acid fragments, which can then be annealed and re-amplified to produce a chimeric nucleic acid sequence (see Ausubel et al., *Molecular Biology: A Laboratory Manual*, 1992).

[0940] Nucleic acid, vector, host cell

[0941] In some embodiments, this disclosure provides one or more polynucleotides (e.g., DNA, RNA, or analogues thereof, optionally including one or more modified nucleotides; the polynucleotides may be linear or circular, e.g., linear or circular RNA). In some embodiments, the polypeptides or fusion proteins disclosed herein are encoded by a single polynucleotide. In some embodiments, the polypeptides or fusion proteins disclosed herein are encoded by multiple polynucleotides.

[0942] In some embodiments, the polynucleotide comprises a nucleotide sequence codon-optimized for a selected host cell.

[0943] In some embodiments, this disclosure provides a vector (e.g., an expression vector, including a viral delivery vector) that contains one or more polynucleotides of the polynucleotides described herein.

[0944] The term "expression vector" refers to a reproducible nucleic acid that can express one or more proteins when it is transformed into a suitable expression host cell.

[0945] In some embodiments, the vector (e.g., an expression vector) comprises an expression control polynucleotide sequence, a polynucleotide sequence encoding an optional marker, or both, operatively 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 to which an RNA polymerase binds and initiates transcription of a gene. The term "operatively linked" means that the nucleic acid is located in a recombinant polynucleotide (e.g., a vector) in a manner that allows the nucleic acid to be expressed under the control of an element (e.g., a promoter) to which it is linked. The term "optional marker element" is an element that confers traits suitable for artificial selection. An optional marker element can be a negative or positive selection marker.

[0946] In some embodiments, this disclosure provides an expression host cell that comprises any or more of the polynucleotides or expression vectors described herein.

[0947] The term "expression host cell" refers to a cell that can be used to receive, maintain, replicate, and / or amplify a vector.

[0948] Non-restricted examples of expression of 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 Escherichia coli), including DH5α, etc.

[0949] Composition

[0950] In some embodiments, this disclosure provides a composition comprising any of the polypeptides or fusion proteins described herein. In some embodiments, the composition is a pharmaceutical composition.

[0951] In some embodiments, the composition (e.g., a pharmaceutical composition) comprises a pharmaceutically acceptable carrier, excipient, stabilizer, diluent, or supplement (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). A suitable pharmaceutically acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dose and concentration employed. Non-limiting examples of pharmaceutically acceptable carriers, excipients, stabilizers, diluents, or supplements include buffers (e.g., phosphates, citrates, histidines), 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 dextrin); chelating agents (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (e.g., sodium), metal complexes (e.g., zinc protein complexes); nonionic surfactants (e.g., Tween), and PLURONICS. TM And polyethylene glycol (PEG).

[0952] In some embodiments, the compositions disclosed herein (e.g., pharmaceutical compositions) are formulated for a suitable schedule and route of administration. Non-limiting examples of routes of administration include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous, and topical administration. In some embodiments, the compositions disclosed herein (e.g., pharmaceutical compositions) are stored in the form of an aqueous solution or a dried formulation (e.g., lyophilized).

[0953] In some embodiments, the composition is formulated for administration by infusion (e.g., intravenous infusion).

[0954] In some embodiments, the composition is formulated for administration as a combination therapy together with a second therapeutic agent. In some embodiments, the second therapeutic agent is any of the polypeptides described herein. In some embodiments, the second therapeutic agent comprises bamlanivimab, etesevimab, casirivimab, imdevimab, cilgavimab, tixagevimab, AZD7442 (tesaxagvirmab-cigarib), regdanvimab, or sotopimumab. In some embodiments, the second therapeutic agent comprises sotopimumab.

[0955] How to use

[0956] In some embodiments, this disclosure provides a method for neutralizing SARS-CoV-2 infection in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and wherein the active ingredient is either a polypeptide or a fusion protein described herein.

[0957] In some embodiments, the likelihood of a subject being infected with SARS-CoV-2 is reduced by at least about 10%, for example, 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%.

[0958] In some embodiments, the probability of a subject being infected with SARS-CoV-2 in the presence of a peptide is approximately 1-90%, for example, approximately: 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 the probability in the absence of the peptide.

[0959] The terms “subject” and “patient” are used interchangeably herein to refer to an animal (e.g., a mammal, such as a human) receiving treatment according to the methods disclosed herein. A subject treated according to the methods described herein may be a subject diagnosed with a specific condition (e.g., COVID-19) or a subject at risk of developing such a condition. Diagnosis can be made by any method or technique known in the art. Those skilled in the art will understand that a subject to be treated according to this disclosure may have already undergone standard testing or may be identified unexamined as being at risk due to the presence of one or more risk factors associated with a disease or condition.

[0960] In some embodiments, the subject has (e.g., confirmed by testing, such as by PCR or rapid testing) or is suspected of having COVID-19. In some embodiments, the subject has COVID-19. In some embodiments, the subject is diagnosed with COVID-19. In some embodiments, the subject is at risk of developing COVID-19.

[0961] In some embodiments, the subject is a mammal. In some embodiments, the subject is a mammal selected from the group consisting of: dogs, cats, mice, rats, hamsters, guinea pigs, horses, pigs, sheep, cattle, chimpanzees, macaques, cynomolgus monkeys, and humans. In some embodiments, the subject is a primate. In some embodiments, the subject is a human.

[0962] In some embodiments, the subject has heart disease. In some embodiments, the subject has heart 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 inflammatory heart disease selected from the group consisting of: endocarditis, cardiac hypertrophy, myocarditis, and combinations thereof.

[0963] In some embodiments, the subjects had diabetes.

[0964] In some embodiments, the subject has a lung disease. Non-limiting examples of lung diseases 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).

[0965] In some embodiments, the subjects are smokers.

[0966] In some embodiments, the subject has an impaired immune system (e.g., has an underlying medical condition or is receiving immunosuppressive therapy).

[0967] In some embodiments, the subjects are 40 years of age or older, for example, at least: 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years of age.

[0968] "Therapeutic effective dose," "effective dose," or "effective amount" refers to the amount that effectively achieves the desired therapeutic outcome (e.g., treatment, cure, suppression or improvement of physiological response or symptoms, reduction of infectivity, reduction of post-exposure infection, prevention, reduction of viral load, etc.) within the necessary dose and time period. Therapeutic effects do not necessarily occur with the administration of a single dose and can occur after only a series of doses. Therefore, therapeutic effective doses can be administered in a single or multiple-dose manner. Therapeutic effective doses can vary in an individual based on factors such as the mammalian's disease state, age, sex, and weight, the mode of administration, and the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response.

[0969] Those skilled in the art can determine the effective amount of a drug to be administered using the guidance provided herein and other methods known in the art. Relevant factors include the given drug, the drug formulation, the route of administration, the type of disease or condition, the identity of the subject (e.g., age, sex, weight), or the host receiving treatment. For example, appropriate doses for each treatment may be from about 0.001 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, or from about 0.01 mg / kg to about 1 mg / kg body weight. Determining the dosage for a specific drug, subject, and disease is entirely within the capabilities of those skilled in the art. Preferably, the dosage will not cause or will produce minimal adverse side effects.

[0970] The desired response or expected outcome includes effects at the cellular, tissue, or clinical levels. Therefore, "therapeuticly effective amount" or its synonyms depend on the context of its application. For example, in some embodiments, the amount of composition is sufficient to achieve a therapeutic and / or preventative response compared to a response obtained without composition administration. In some embodiments, it is the amount that produces a beneficial or desired outcome in a subject compared to a control. As defined herein, a therapeutically effective amount of the compositions disclosed herein can be readily determined by those skilled in the art using conventional methods known in the art. Dosing regimens and routes of administration can be adjusted to provide an optimal therapeutic response.

[0971] In some embodiments, the methods disclosed herein are used as a preventative therapy. In some embodiments, the effective dose is sufficient to prevent the subject from contracting SARS-CoV-2.

[0972] In some embodiments, the methods disclosed herein are used to treat SARS-CoV-2.

[0973] The term "treating" or "treatment" refers to the medical administration of a subject with the aim of improving, alleviating, stabilizing (i.e., preventing worsening), preventing, or curing a disease, pathological symptom, infection, or condition, as illustrated in the specific indications herein. This term includes active treatment (treatment aimed at improving a disease, pathological symptom, infection, or condition), etiological treatment (treatment targeting the cause of the associated disease, pathological symptom, infection, or condition), palliative treatment (treatment aimed at relieving symptoms), and preventive (e.g., prophylactic) treatment (treatment aimed at minimizing or partially or completely suppressing the development of an associated disease, pathological symptom, infection, or condition); as well as supportive treatment (treatment used to supplement another therapy). Treatment also includes reducing the severity of a disease or symptom; preventing the spread of a disease or symptom; delaying or slowing the progression of a disease or symptom; improving or alleviating a disease or symptom; and detectable or undetectable remission (whether partial or complete). "Improvement" or "remission" of a disease or condition means a reduction in the severity and / or undesirable clinical presentation and / or a slowing or prolonging of its progression compared to a condition without treatment. "Treatment" can also mean an extension of life expectancy compared to the expected life expectancy without treatment. People in need of treatment include those who already have a condition, infection, or illness, as well as those who are susceptible to a condition or illness, or those who need preventative measures against a condition, infection, or illness.

[0974] In some embodiments, the effective dose is sufficient to reduce the viral load in the subject. In some embodiments, the viral load is reduced by at least about 10%, for example, 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 viral load is reduced by approximately 10-99%, for example, approximately: 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%.

[0975] In some embodiments, the effective dose is sufficient to inhibit the binding of the virus to its target proteins, target cells, or both. In some embodiments, the binding is reduced by at least about 10%, for example, 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 is approximately 10-99%, for example, approximately: 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%.

[0976] In some embodiments, the effective dose is sufficient to inhibit virus-mediated fusion with target cells. In some embodiments, the fusion is reduced by at least about 10%, for example, 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 fusion reduction is approximately 10-99%, for example, approximately: 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%.

[0977] In some embodiments, the effective dose is sufficient to interfere with conformational changes in viral envelope proteins required for cellular infectivity.

[0978] The therapeutic agents described herein can be administered via a variety of routes, including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intra-arterial, intravenous, intramuscular, subcutaneous, intradermal injection), intravenous infusion, and inhalation (e.g., intrabronchial, intranasal, or oral inhalation, nasal drops), depending on the compound and the specific disease to be treated. As indicated, administration can be local or systemic. Preferred methods of administration may vary depending on the specific compound selected.

[0979] In some embodiments, the peptides, compositions, or pharmaceutical compositions disclosed herein are administered to a subject as a single therapy.

[0980] In some embodiments, the 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 another 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 been treated with one or more therapeutic agents prior to administration of the polypeptide, composition, or pharmaceutical composition disclosed herein. In some embodiments, the methods disclosed herein include administering a therapeutically effective amount of one or more additional therapeutic agents to the subject simultaneously or after administration of the polypeptide, composition, or pharmaceutical composition disclosed herein. In some embodiments, the methods disclosed herein include administering a therapeutically effective amount of one or more prophylactic agents to the subject before, simultaneously, or after administration of the polypeptide, composition, or pharmaceutical composition disclosed herein. In some embodiments, the subject has previously received a therapeutic agent or prophylactic agent. In some embodiments, the subject has previously been infected with a β-coronavirus, such as SARS-CoV-2.

[0981] Other non-limiting examples of therapeutic agents include antibiotics (e.g., azithromycin), antibodies or their antigen-binding fragments (e.g., other SARS-CoV-2 binding antibodies or antigen-binding fragments), antimalarial agents (e.g., chloroquine or hydroxychloroquine), antiviral agents (e.g., monoupiravi (LAGEVRIO, Merck), PF-07817883 (Pfizer), STI-1558 (Sorrento Therapeutics), PBI-0451 (Pardes Biosciences)). 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), and renin-angiotensin-aldosterone system inhibitors (e.g., ACE2 inhibitors or angiotensin receptor blockers (ARBs)).

[0982] In some embodiments, the antiviral agent is selected from the group consisting of: monoprevir (LAGEVRIO, Merck), PF-07817883 (Pfizer), STI-1558 (Sorrento Medical Technologies), PBI-0451 (Peribio Biosciences, Inc.), EDP-235 (Inanda Pharmaceuticals, Inc.), 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.

[0983] In some embodiments, the antiviral agent is selected from the group consisting of: monoprevir (LAGEVRIO, Merck), PF-07817883 (Pfizer), STI-1558 (Sorrento Medical Technologies), PBI-0451 (Pyribio Biosciences, Inc.), EDP-235 (Enanda Pharmaceuticals, Inc.), and combinations thereof.

[0984] In some embodiments, additional anti-SARS-CoV-2 antibodies target the S1 domain of the SARS-CoV-2 spike protein. In some embodiments, additional anti-SARS-CoV-2 antibodies target the class 4 region of the S1 domain. In some embodiments, additional anti-SARS-CoV-2 monoclonal antibodies target the RBD (e.g., RBD class 1, 2, 3, or 4 epitopes) of the S1 domain of SARS-CoV-2. In some embodiments, additional anti-SARS-CoV-2 antibodies target the class 3 region of the RBD domain. In some embodiments, additional anti-SARS-CoV-2 antibodies target the N-terminal domain (NTD) – non-supersite region of the S1 domain. In some embodiments, additional anti-SARS-CoV-2 antibodies target the SD1 region of the S1 domain.

[0985] In some embodiments, additional anti-SARS-CoV-2 monoclonal antibodies target (e.g., bind to) the S2 domain of the spike protein of SARS-CoV-2. In some embodiments, additional anti-SARS-CoV-2 monoclonal antibodies are neutralizing monoclonal antibodies (e.g., as determined by a neutralization assay described herein or known in the art). Non-limiting examples of anti-SARS-CoV-2 monoclonal antibodies include barnivirumab (LY-CoV555 or LY3819253), ethesvirumab (LY-CoV016 or LY3832479), betelvirumab (LY-CoV1404, LY3853113), camrelizumab (REGN10933), edemizumab (REGN10987), cigavirumab, tesaxagvirumab, AZD7442 / Entasca (tesaxagvirumab-cigaribumab), rimantavirumab, sotopimab (Vir Biotechnology, Inc.), ADG20 (Adagio Therapeutics, Inc.), enveloperi (MP0420) (DARPin, Novartis), and P2G3 (Aerium). Tx) and S2X259 (Tortorici MA et al., neutralization of broad sarbecovirus by a human monoclonal antibody. Nature. Sep 2021; 597(7874):103-108. doi:10.1038 / s41586-021-03817-4. e-version 19 July 2021 PMID:34280951). Other examples of anti-SARS-CoV-2 antibodies include those described in U.S. Patent Nos. 11,168,128, 11,192,940, WO 2022 / 010912 A1, WO 2022 / 010921 A1, WO 2022 / 047033 A1, WO 2021 / 173753 A1, WO 2021 / 158521 A1, WO 2021 / 203053 A1, WO 2021 / 211775 A1, and WO 2021 / 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.

[0986] In some embodiments, the subject is further treated (previously, concurrently, or sequentially) with (e.g., an effective amount) of one or more RBD class 4 antibodies (or their antigen-binding fragments), such as S2X259 or a variant thereof. Further examples of SARS-CoV-2 RBD class 4 antibodies include, for example, the 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... Four types of mAb-3d, having the VH and VL sequences listed in Tables 10 and 11 herein, and those described in U.S. Patent Applications No. 63 / 424,947 (filed November 13, 2022), No. 63 / 383,699 (filed November 14, 2022), No. 63 / 480,919 (filed January 20, 2023), and No. 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.

[0987] In some embodiments, the subject is further treated (previously, concurrently, or sequentially) with one or more SARS-CoV-2 RBD class 3 antibodies (or their antigen-binding fragments), such as sotopemilab, betelvirumab, AZD1061, P2G3, and endostatin. In some embodiments, the subject is treated with (e.g., an effective amount) betelvirumab. In some embodiments, the subject is treated with (e.g., an effective amount) endostatin. In some embodiments, the subject is treated with (e.g., an effective amount) tesaxagvirumab. In some embodiments, the subject is treated with (e.g., an effective amount) cigavirumab.

[0988] In some embodiments, subjects are further treated (previously, concurrently, or sequentially) with one or more SARS-CoV-2 N-terminal domain (NTD)-nonsupersite antibodies (or their antigen-binding fragments) (such as C1520 and C1717).

[0989] In some embodiments, subjects are further treated (previously, concurrently, or sequentially) with one or more SARS-CoV-2SD1 antibodies (or their antigen-binding fragments) (such as S3H3 and P008_60).

[0990] In some embodiments, the ACE2 inhibitor is selected from the group consisting of: RNAi targeting ACE2, siRNA targeting 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 viral vaccine. In some embodiments, the vaccine is mRNA-1273, BNT162, INO-4800, AZD1222, Ad5-nCoV, PiCoVacc, NVX-CoV2373, JNJ-78436735, or combinations thereof.

[0991] Administration of two or more therapeutic agents encompasses the co-administration of the therapeutic agents in a substantially synchronous manner (e.g., in combination of drugs). In some embodiments, such administration encompasses the co-administration of each therapeutic agent in multiple containers or separate containers (e.g., capsules, powders, and liquids). Such administration also encompasses the sequential use of each type of therapeutic agent at approximately simultaneous or at different times. The compositions and second therapeutic agents described herein may be administered via the same route of administration or via different routes of administration.

[0992] In some embodiments, this disclosure provides a method for preventing SARS-CoV-2 infection in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and wherein the active ingredient is either a polypeptide or a fusion protein described herein.

[0993] In some embodiments, this disclosure provides a method for treating a subject with SARS-CoV-2 infection, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and wherein the active ingredient is any polypeptide or fusion protein described herein.

[0994] In some embodiments, this disclosure provides a method for reducing the viral load of SARS-CoV-2 in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable load, and wherein the active ingredient is any polypeptide or fusion protein described herein.

[0995] In some embodiments, this disclosure provides a method for inhibiting the binding of SARS-CoV-2 to target cells, the method comprising contacting the target cells with an effective amount of any polypeptide or fusion protein described herein.

[0996] In some embodiments, this disclosure provides a method for inhibiting the binding of SARS-CoV-2 to target proteins on target cells, the method comprising contacting the target cells with an effective amount of any polypeptide or fusion protein described herein.

[0997] In some embodiments, this disclosure provides a method for inhibiting virus-mediated fusion with target cells, the method comprising contacting the target cells with an effective amount of any polypeptide or fusion protein described herein.

[0998] Unless otherwise defined, all technical terms, symbols, and other scientific terms or proprietary terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. In some instances, for clarity and / or ease of reference, terms having a commonly understood meaning are defined herein, and the inclusion of such definitions herein should not necessarily be construed as representing a material difference from the meaning commonly understood in the art. It should be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning as otherwise defined in the relevant field and / or herein.

[0999] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[1000] As used herein, unless the context clearly indicates otherwise, the indefinite articles “a / an” and “the” should be understood to include plural pronouns.

[1001] Throughout this specification and the appended claims, unless the context otherwise requires, the word “comprise” and its variations (e.g., “comprises” or “comprising”) should be understood to imply inclusion of, for example, the stated integers or steps or groups of integers or steps, but not to exclude any other integers or steps or groups of integers or steps. When used herein, the term “comprising” may be replaced by the terms “containing” or “including”.

[1002] As used herein, "consisting of..." excludes any element, step, or component not specified in the elements of the claims. When used herein, "consisting substantially of..." does not exclude materials or steps that do not substantially affect the essential and novel characteristics of the claims. Any of the terms "comprising," "containing," "including," and "having," whenever used in the context of one aspect or embodiment disclosed herein, may in some embodiments be replaced by the terms "consisting of..." or "substantially of..." to change the scope of the disclosure herein.

[1003] As used herein, the connecting term "and / or" between multiple elements is understood to encompass both individual options and combined options. For example, in the case where two elements are connected by "and / or", the first option refers to the applicability of the first element in the absence of the second element. The second option refers to the applicability of the second element in the absence of the first element. The third option refers to the applicability of the first and second elements together. Any of these options is understood to fall within this meaning and therefore satisfies the requirement of the term "and / or" as used herein. The concurrent applicability of more than one option is also understood to fall within this meaning and therefore satisfies the requirement of the term "and / or".

[1004] When a list is presented, unless otherwise stated, it should be understood that each individual element of the list and each combination of the list will be a separate embodiment. For example, a list of embodiments presented as "A, B, or C" will be interpreted as including embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C". For all numerical ranges of certain parameters described in this application, such as "about", "at least", "less than", "less than", and "more than", the description must also cover any range defined by the referenced value. Thus, for example, the description "at least 1, 2, 3, 4, or 5" also specifically describes the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, etc.

[1005] The headings used in this application are for convenience only and do not affect the interpretation of this application.

[1006] Preferred features of each aspect or embodiment provided by the present invention, with necessary modifications, are applicable to all other aspects or embodiments of the invention, and are not limited to those exemplified by the dependent claims, but also cover combinations and arrangements of various features (e.g., elements, including numerical ranges and exemplary embodiments) of specific embodiments and aspects of the invention (including working examples). For example, specific experimental parameters exemplified in working examples can be applied individually to the claimed invention without departing from the invention. For example, for the disclosed materials, although specific references to each of the various individual and collective combinations and arrangements of these compounds may not be explicitly disclosed, each is specifically considered 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 examples of combinations of elements AD are disclosed, then each is considered individually and uniformly, even if not individually enumerated. Thus, in this example, each combination of combinations of AE, AF, BD, BE, BF, CD, CE, and CF is specifically considered and should be considered as disclosed from the disclosures of A, B, and C; D, E, and F; and the example combination AD. Similarly, any subset or combination of these combinations is also specifically considered and disclosed. Therefore, for example, subgroups of AE, BF, and CE are specifically considered and should be considered as disclosed in the disclosures of A, B, and C; D, E, and F; and the example combination AD. This concept applies to all aspects of this application, including the elements of the material composition and the steps of methods for preparing or using the composition.

[1007] As will be recognized by those skilled in the art from the teachings of the specification, the foregoing aspects of the invention may be claimed in any combination or arrangement, provided that they are novel and non-obvious relative to the prior art—therefore, within the scope of the elements described in one or more references known to those skilled in the art, they may be excluded from the claimed invention by means of negative appendices or disclaimers regarding features or combinations thereof.

[1008] Example

[1009] 1. A polypeptide that specifically binds to the S2 domain of the β-coronavirus spike glycoprotein, wherein the polypeptide has one or more properties selected from:

[1010] Broad neutralizing activity against a variety of known and predicted beta coronaviruses;

[1011] Binding affinity for highly conserved S2 domain epitopes across multiple β-coronaviruses; and

[1012] Inhibitory activity against potential emerging β-coronavirus escape variants.

[1013] 2. A polypeptide that specifically binds to the spike glycoprotein of SARS-CoV-2, said polypeptide comprising a complementary site substantially similar to the complementary site of an antibody, said antibody comprising an amino acid sequence selected from:

[1014] SEQ ID NO:4 and SEQ ID NO:51(AB-1);

[1015] SEQ ID NO:5 and SEQ ID NO:52(AB-2);

[1016] SEQ ID NO:6 and SEQ ID NO:53(AB-3);

[1017] SEQ ID NO:7 and SEQ ID NO:54(AB-4);

[1018] SEQ ID NO:8 and SEQ ID NO:51(AB-5);

[1019] SEQ ID NO:9 and SEQ ID NO:55(AB-6);

[1020] SEQ ID NO:10 and SEQ ID NO:56(AB-7);

[1021] SEQ ID NO:11 and SEQ ID NO:57(AB-8);

[1022] SEQ ID NO:12 and SEQ ID NO:58(AB-9);

[1023] SEQ ID NO:13 and SEQ ID NO:59(AB-10);

[1024] SEQ ID NO:14 and SEQ ID NO:60(AB-11);

[1025] SEQ ID NO:15 and SEQ ID NO:56(AB-12);

[1026] SEQ ID NO:16 and SEQ ID NO:51(AB-13);

[1027] SEQ ID NO:10 and SEQ ID NO:50 (AB-14);

[1028] SEQ ID NO:17 and SEQ ID NO:61(AB-15);

[1029] SEQ ID NO:18 and SEQ ID NO:62(AB-16);

[1030] SEQ ID NO:6 and SEQ ID NO:63(AB-17);

[1031] SEQ ID NO:19 and SEQ ID NO:64(AB-18);

[1032] SEQ ID NO:4 and SEQ ID NO:61(AB-19);

[1033] SEQ ID NO:20 and SEQ ID NO:61(AB-20);

[1034] SEQ ID NO:21 and SEQ ID NO:65(AB-21);

[1035] SEQ ID NO:22 and SEQ ID NO:66(AB-22);

[1036] SEQ ID NO:4 and SEQ ID NO:67(AB-23);

[1037] SEQ ID NO:23 and SEQ ID NO:56(AB-24);

[1038] SEQ ID NO:24 and SEQ ID NO:68(AB-25);

[1039] SEQ ID NO:25 and SEQ ID NO:51(AB-26);

[1040] SEQ ID NO:26 and SEQ ID NO:56(AB-27);

[1041] SEQ ID NO:27 and SEQ ID NO:61(AB-28);

[1042] SEQ ID NO:28 and SEQ ID NO:56(AB-29);

[1043] SEQ ID NO:28 and SEQ ID NO:69(AB-30);

[1044] SEQ ID NO:29 and SEQ ID NO:70(AB-31);

[1045] SEQ ID NO:30 and SEQ ID NO:71(AB-32);

[1046] SEQ ID NO:31 and SEQ ID NO:72(AB-33);

[1047] SEQ ID NO:32 and SEQ ID NO:67(AB-34);

[1048] SEQ ID NO:33 and SEQ ID NO:56(AB-35);

[1049] SEQ ID NO:34 and SEQ ID NO:73(AB-36);

[1050] SEQ ID NO:35 and SEQ ID NO:51(AB-37);

[1051] SEQ ID NO:36 and SEQ ID NO:56(AB-38);

[1052] SEQ ID NO:37 and SEQ ID NO:63(AB-39);

[1053] SEQ ID NO:38 and SEQ ID NO:69 (AB-40);

[1054] SEQ ID NO:39 and SEQ ID NO:74(AB-41);

[1055] SEQ ID NO:40 and SEQ ID NO:52 (AB-42);

[1056] SEQ ID NO:41 and SEQ ID NO:51(AB-43);

[1057] SEQ ID NO:42 and SEQ ID NO:75(AB-44);

[1058] SEQ ID NO:43 and SEQ ID NO:56(AB-45);

[1059] SEQ ID NO:44 and SEQ ID NO:51(AB-46);

[1060] SEQ ID NO:45 and SEQ ID NO:75(AB-47);

[1061] SEQ ID NO:46 and SEQ ID NO:53 (AB-48);

[1062] SEQ ID NO:47 and SEQ ID NO:52 (AB-49);

[1063] SEQ ID NO:48 and SEQ ID NO:76 (AB-50); or

[1064] SEQ ID NO:3 and SEQ ID NO:56(AB-51), or

[1065] Any combination thereof.

[1066] 3. The polypeptide according to Example 1 or 2, comprising an immunoglobulin heavy chain variable domain (V H ) and immunoglobulin light chain variable domain (V L ).

[1067] 4. A polypeptide that specifically binds to the spike glycoprotein of SARS-CoV-2, said polypeptide comprising:

[1068] a) Variable domains of immunoglobulin heavy chain (V H ) amino acid sequence, the V H The amino acid sequences include HCDR1, HCDR2, and HCDR3, which are substantially similar to the heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of the amino acid sequences of any of SEQ ID NO:4-48, respectively; and

[1069] b) Variable domains of immunoglobulin light chains (V L ) amino acid sequence, the V L The amino acid sequences include LCDR1, LCDR2 and LCDR3, which are substantially similar to the light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) of the amino acid sequences of any of SEQ ID NO:51-76, respectively.

[1070] 5. A polypeptide according to any one of Examples 1-4, comprising the antibody HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, wherein the antibody comprises an amino acid sequence selected from:

[1071] SEQ ID NO:4 and SEQ ID NO:51(AB-1);

[1072] SEQ ID NO:5 and SEQ ID NO:52(AB-2);

[1073] SEQ ID NO:6 and SEQ ID NO:53(AB-3);

[1074] SEQ ID NO:7 and SEQ ID NO:54(AB-4);

[1075] SEQ ID NO:8 and SEQ ID NO:51(AB-5);

[1076] SEQ ID NO:9 and SEQ ID NO:55(AB-6);

[1077] SEQ ID NO:10 and SEQ ID NO:56(AB-7);

[1078] SEQ ID NO:11 and SEQ ID NO:57(AB-8);

[1079] SEQ ID NO:12 and SEQ ID NO:58(AB-9);

[1080] SEQ ID NO:13 and SEQ ID NO:59(AB-10);

[1081] SEQ ID NO:14 and SEQ ID NO:60(AB-11);

[1082] SEQ ID NO:15 and SEQ ID NO:56(AB-12);

[1083] SEQ ID NO:16 and SEQ ID NO:51(AB-13);

[1084] SEQ ID NO:10 and SEQ ID NO:50 (AB-14);

[1085] SEQ ID NO:17 and SEQ ID NO:61(AB-15);

[1086] SEQ ID NO:18 and SEQ ID NO:62(AB-16);

[1087] SEQ ID NO:6 and SEQ ID NO:63(AB-17);

[1088] SEQ ID NO:19 and SEQ ID NO:64(AB-18);

[1089] SEQ ID NO:4 and SEQ ID NO:61(AB-19);

[1090] SEQ ID NO:20 and SEQ ID NO:61(AB-20);

[1091] SEQ ID NO:21 and SEQ ID NO:65(AB-21);

[1092] SEQ ID NO:22 and SEQ ID NO:66(AB-22);

[1093] SEQ ID NO:4 and SEQ ID NO:67(AB-23);

[1094] SEQ ID NO:23 and SEQ ID NO:56(AB-24);

[1095] SEQ ID NO:24 and SEQ ID NO:68(AB-25);

[1096] SEQ ID NO:25 and SEQ ID NO:51(AB-26);

[1097] SEQ ID NO:26 and SEQ ID NO:56(AB-27);

[1098] SEQ ID NO:27 and SEQ ID NO:61(AB-28);

[1099] SEQ ID NO:28 and SEQ ID NO:56(AB-29);

[1100] SEQ ID NO:28 and SEQ ID NO:69(AB-30);

[1101] SEQ ID NO:29 and SEQ ID NO:70(AB-31);

[1102] SEQ ID NO:30 and SEQ ID NO:71(AB-32);

[1103] SEQ ID NO:31 and SEQ ID NO:72(AB-33);

[1104] SEQ ID NO:32 and SEQ ID NO:67(AB-34);

[1105] SEQ ID NO:33 and SEQ ID NO:56(AB-35);

[1106] SEQ ID NO:34 and SEQ ID NO:73(AB-36);

[1107] SEQ ID NO:35 and SEQ ID NO:51(AB-37);

[1108] SEQ ID NO:36 and SEQ ID NO:56(AB-38);

[1109] SEQ ID NO:37 and SEQ ID NO:63(AB-39);

[1110] SEQ ID NO:38 and SEQ ID NO:69 (AB-40);

[1111] SEQ ID NO:39 and SEQ ID NO:74(AB-41);

[1112] SEQ ID NO:40 and SEQ ID NO:52 (AB-42);

[1113] SEQ ID NO:41 and SEQ ID NO:51(AB-43);

[1114] SEQ ID NO:42 and SEQ ID NO:75(AB-44);

[1115] SEQ ID NO:43 and SEQ ID NO:56(AB-45);

[1116] SEQ ID NO:44 and SEQ ID NO:51(AB-46);

[1117] SEQ ID NO:45 and SEQ ID NO:75(AB-47);

[1118] SEQ ID NO:46 and SEQ ID NO:53 (AB-48);

[1119] SEQ ID NO:47 and SEQ ID NO:52 (AB-49);

[1120] SEQ ID NO:48 and SEQ ID NO:76 (AB-50); or

[1121] SEQ ID NO:3 and SEQ ID NO:56(AB-51).

[1122] 6. The polypeptide according to Example 4 or 5, comprising a complementary site identical to the complementary site of the antibody, wherein the antibody comprises an amino acid sequence selected from:

[1123] SEQ ID NO:4 and SEQ ID NO:51(AB-1);

[1124] SEQ ID NO:5 and SEQ ID NO:52(AB-2);

[1125] SEQ ID NO:6 and SEQ ID NO:53(AB-3);

[1126] SEQ ID NO:7 and SEQ ID NO:54(AB-4);

[1127] SEQ ID NO:8 and SEQ ID NO:51(AB-5);

[1128] SEQ ID NO:9 and SEQ ID NO:55(AB-6);

[1129] SEQ ID NO:10 and SEQ ID NO:56(AB-7);

[1130] SEQ ID NO:11 and SEQ ID NO:57(AB-8);

[1131] SEQ ID NO:12 and SEQ ID NO:58(AB-9);

[1132] SEQ ID NO:13 and SEQ ID NO:59(AB-10);

[1133] SEQ ID NO:14 and SEQ ID NO:60(AB-11);

[1134] SEQ ID NO:15 and SEQ ID NO:56(AB-12);

[1135] SEQ ID NO:16 and SEQ ID NO:51(AB-13);

[1136] SEQ ID NO:10 and SEQ ID NO:50 (AB-14);

[1137] SEQ ID NO:17 and SEQ ID NO:61(AB-15);

[1138] SEQ ID NO:18 and SEQ ID NO:62(AB-16);

[1139] SEQ ID NO:6 and SEQ ID NO:63(AB-17);

[1140] SEQ ID NO:19 and SEQ ID NO:64(AB-18);

[1141] SEQ ID NO:4 and SEQ ID NO:61(AB-19);

[1142] SEQ ID NO:20 and SEQ ID NO:61(AB-20);

[1143] SEQ ID NO:21 and SEQ ID NO:65(AB-21);

[1144] SEQ ID NO:22 and SEQ ID NO:66(AB-22);

[1145] SEQ ID NO:4 and SEQ ID NO:67(AB-23);

[1146] SEQ ID NO:23 and SEQ ID NO:56(AB-24);

[1147] SEQ ID NO:24 and SEQ ID NO:68(AB-25);

[1148] SEQ ID NO:25 and SEQ ID NO:51(AB-26);

[1149] SEQ ID NO:26 and SEQ ID NO:56(AB-27);

[1150] SEQ ID NO:27 and SEQ ID NO:61(AB-28);

[1151] SEQ ID NO:28 and SEQ ID NO:56(AB-29);

[1152] SEQ ID NO:28 and SEQ ID NO:69(AB-30);

[1153] SEQ ID NO:29 and SEQ ID NO:70(AB-31);

[1154] SEQ ID NO:30 and SEQ ID NO:71(AB-32);

[1155] SEQ ID NO:31 and SEQ ID NO:72(AB-33);

[1156] SEQ ID NO:32 and SEQ ID NO:67(AB-34);

[1157] SEQ ID NO:33 and SEQ ID NO:56(AB-35);

[1158] SEQ ID NO:34 and SEQ ID NO:73(AB-36);

[1159] SEQ ID NO:35 and SEQ ID NO:51(AB-37);

[1160] SEQ ID NO:36 and SEQ ID NO:56(AB-38);

[1161] SEQ ID NO:37 and SEQ ID NO:63(AB-39);

[1162] SEQ ID NO:38 and SEQ ID NO:69 (AB-40);

[1163] SEQ ID NO:39 and SEQ ID NO:74(AB-41);

[1164] SEQ ID NO:40 and SEQ ID NO:52 (AB-42);

[1165] SEQ ID NO:41 and SEQ ID NO:51(AB-43);

[1166] SEQ ID NO:42 and SEQ ID NO:75(AB-44);

[1167] SEQ ID NO:43 and SEQ ID NO:56(AB-45);

[1168] SEQ ID NO:44 and SEQ ID NO:51(AB-46);

[1169] SEQ ID NO:45 and SEQ ID NO:75(AB-47);

[1170] SEQ ID NO:46 and SEQ ID NO:53 (AB-48);

[1171] SEQ ID NO:47 and SEQ ID NO:52 (AB-49);

[1172] SEQ ID NO:48 and SEQ ID NO:76 (AB-50); or

[1173] SEQ ID NO:3 and SEQ ID NO:56(AB-51).

[1174] 7. A polypeptide that specifically binds to the spike glycoprotein of SARS-CoV-2, said polypeptide comprising an immunoglobulin heavy chain variable domain (V... H ), the V HThe amino acid sequence containing SEQ ID NO:2, wherein:

[1175] X1 is not S;

[1176] X2 is not D;

[1177] X3 is not T;

[1178] X4 is not L;

[1179] X5 is not S;

[1180] X6 is not N;

[1181] X7 is not G;

[1182] X8 is not V; or

[1183] X9 is not Q.

[1184] Or any combination thereof.

[1185] 8. The polypeptide according to Example 7, comprising an immunoglobulin light chain variable domain (V L ), the V L The amino acid sequence containing SEQ ID NO:49 includes:

[1186] X 10 Not Q;

[1187] X 11 Not G;

[1188] X 12 Not S;

[1189] X 13 Not S;

[1190] X 14 Not N;

[1191] X 15 Not S;

[1192] X 16 Not F; or

[1193] X 17 Not Y,

[1194] Or any combination thereof.

[1195] 9. A polypeptide that specifically binds to the spike glycoprotein of SARS-CoV-2, said polypeptide comprising an immunoglobulin heavy chain variable domain (V... H ), the V H The amino acid sequence containing SEQ ID NO:2, wherein:

[1196] X1 is S, N, A, R, L, or F;

[1197] X2 is either D or E;

[1198] X3 is either T or V;

[1199] X4 is either L or V;

[1200] X5 is S, Q, R, K, Y, D, or E;

[1201] X6 is N, K, A, S, R, or E;

[1202] X7 is G, N, or L;

[1203] X8 is V, I, S, or K; or

[1204] X9 is Q, Y, K, F, or H.

[1205] Or any combination thereof.

[1206] 10. The polypeptide according to Example 9, wherein:

[1207] X1 is N, A, R, L, or F;

[1208] X2 is E;

[1209] X3 is V;

[1210] X4 is V;

[1211] X5 is Q, R, K, Y, D, or E;

[1212] X6 is K, A, S, R, or E;

[1213] X7 is either N or L;

[1214] X8 is I, S, or K; or

[1215] X9 is Y, K, F, or H.

[1216] Or any combination thereof.

[1217] 11. The polypeptide according to Example 9 or 10, comprising an immunoglobulin light chain variable domain (V L ), the V L The amino acid sequence containing SEQ ID NO:49 includes:

[1218] X 10 It is Q, K, or I;

[1219] X 11 Is it G or S;

[1220] X12 Is it S, R, or V;

[1221] X 13 It is S or N;

[1222] X 14 It is N, H, D, Y, or S;

[1223] X 15 Is it S or Q?

[1224] X 16 It is F, Y, L, V, T, or D; or

[1225] X 17 Is it Y or L?

[1226] Or any combination thereof.

[1227] 12. The polypeptide according to Example 11, wherein:

[1228] X 10 It is K or I;

[1229] X 11 It is S;

[1230] X 12 It is R or V;

[1231] X 13 It is N;

[1232] X 14 It is H, D, Y, or S;

[1233] X 15 It's Q;

[1234] X 16 It is Y, L, V, T, or D; or

[1235] X 17 It is L.

[1236] Or any combination thereof.

[1237] 13. The polypeptide according to any one of Examples 1 to 4 and 6 to 12, wherein the V H The HCDR1, HCDR2 and HCDR3 contain the same heavy chain complementarity determination region 1 (HCDR1), heavy chain complementarity determination region 2 (HCDR2) and heavy chain complementarity determination region 3 (HCDR3) as any of the SEQ ID NO:4-48 respectively.

[1238] 14. The polypeptide according to any one of Examples 1 to 4 and 6 to 13, wherein the V LLCDR1, LCDR2 and LCDR3 contain the same light chain complementarity determination region 1 (LCDR1), light chain complementarity determination region 2 (LCDR2) and light chain complementarity determination region 3 (LCDR3) as any of the amino acid sequences in SEQ ID NO:51-76.

[1239] 15. The polypeptide according to any one of Examples 1 to 5 and 7 to 14, wherein the polypeptide comprises V with an amino acid sequence selected from the following H / V LComplementary positions with the same complementary positions in combination: 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 IDNO: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).

[1240] 16. The polypeptide according to any one of Examples 1 to 15, wherein V H It has at least 85% sequence identity with any or more of the amino acid sequences in SEQ ID NO:4-48.

[1241] 17. The polypeptide according to any one of Examples 1 to 16, wherein the V amino acid sequence is relative to any one or more of SEQ ID NO:4-48 H It contains approximately 1-10 amino acid substitutions.

[1242] 18. The polypeptide according to any one of Examples 1 to 17, wherein V L It has at least 85% sequence identity with any one or more of the amino acid sequences in SEQ ID NO:51-76.

[1243] 19. The polypeptide according to any one of Examples 1 to 18, wherein the V amino acid sequence is relative to any one or more of SEQ ID NO: 51-76 L It contains approximately 1-10 amino acid substitutions.

[1244] 20. The polypeptide according to Example 17 or 19, wherein the amino acid substitution is a conservative substitution.

[1245] 21. The polypeptide according to Example 20, wherein the amino acid substitution is a highly conserved substitution.

[1246] 22. The polypeptide according to any one of Examples 1 to 20, wherein:

[1247] a) The V H Contains the amino acid sequence of SEQ ID NO:4; and

[1248] b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-1).

[1249] 23. The polypeptide according to any one of Examples 1 to 20, wherein:

[1250] a) The V H Contains the amino acid sequence of SEQ ID NO:5; and

[1251] b) The V L The amino acid sequence containing SEQ ID NO:52 (AB-2).

[1252] 24. The polypeptide according to any one of Examples 1 to 20, wherein:

[1253] a) The V H Contains the amino acid sequence of SEQ ID NO:6; and

[1254] b) The V L The amino acid sequence containing SEQ ID NO:53 (AB-3).

[1255] 25. The polypeptide according to any one of Examples 1 to 20, wherein:

[1256] a) The V H Contains the amino acid sequence of SEQ ID NO:7; and

[1257] b) The V L The amino acid sequence containing SEQ ID NO:54 (AB-4).

[1258] 26. The polypeptide according to any one of Examples 1 to 20, wherein:

[1259] a) The V H Contains the amino acid sequence of SEQ ID NO:8; and

[1260] b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-5).

[1261] 27. The polypeptide according to any one of Examples 1 to 20, wherein:

[1262] a) The V H Contains the amino acid sequence of SEQ ID NO:9; and

[1263] b) The V L The amino acid sequence containing SEQ ID NO:55 (AB-6).

[1264] 28. The polypeptide according to any one of Examples 1 to 20, wherein:

[1265] a) The V H Contains the amino acid sequence of SEQ ID NO:10; and

[1266] b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-7).

[1267] 29. The polypeptide according to any one of Examples 1 to 20, wherein:

[1268] a) The V H Contains the amino acid sequence of SEQ ID NO:11; and

[1269] b) The V L The amino acid sequence containing SEQ ID NO:57 (AB-8).

[1270] 30. The polypeptide according to any one of Examples 1 to 20, wherein:

[1271] a) The V H Contains the amino acid sequence of SEQ ID NO:12; and

[1272] b) The V L The amino acid sequence containing SEQ ID NO:30 (AB-9).

[1273] 31. The polypeptide according to any one of Examples 1 to 20, wherein:

[1274] a) The V H Contains the amino acid sequence of SEQ ID NO:13; and

[1275] b) The V L The amino acid sequence containing SEQ ID NO:59 (AB-10).

[1276] 32. The polypeptide according to any one of Examples 1 to 20, wherein:

[1277] a) The V H Contains the amino acid sequence of SEQ ID NO:14; and

[1278] b) The V L The amino acid sequence containing SEQ ID NO:60 (AB-11).

[1279] 33. The polypeptide according to any one of Examples 1 to 20, wherein:

[1280] a) The V H Contains the amino acid sequence of SEQ ID NO:15; and

[1281] b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-12).

[1282] 34. The polypeptide according to any one of Examples 1 to 20, wherein:

[1283] a) The V H Contains the amino acid sequence of SEQ ID NO:16; and

[1284] b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-13).

[1285] 35. The polypeptide according to any one of Examples 1 to 20, wherein:

[1286] a) The V H Contains the amino acid sequence of SEQ ID NO:10; and

[1287] b) The V L The amino acid sequence containing SEQ ID NO:50 (AB-14).

[1288] 36. The polypeptide according to any one of Examples 1 to 20, wherein:

[1289] a) The V HContains the amino acid sequence of SEQ ID NO:17; and

[1290] b) The V L The amino acid sequence containing SEQ ID NO:61 (AB-15).

[1291] 37. The polypeptide according to any one of Examples 1 to 20, wherein:

[1292] a) The V H Contains the amino acid sequence of SEQ ID NO:18; and

[1293] b) The V L The amino acid sequence containing SEQ ID NO:62 (AB-16).

[1294] 38. The polypeptide according to any one of Examples 1 to 20, wherein:

[1295] a) The V H Contains the amino acid sequence of SEQ ID NO:6; and

[1296] b) The V L The amino acid sequence containing SEQ ID NO:63 (AB-17).

[1297] 39. The polypeptide according to any one of Examples 1 to 20, wherein:

[1298] a) The V H Contains the amino acid sequence of SEQ ID NO:19; and

[1299] b) The V L The amino acid sequence containing SEQ ID NO:64 (AB-18).

[1300] 40. The polypeptide according to any one of Examples 1 to 20, wherein:

[1301] a) The V H Contains the amino acid sequence of SEQ ID NO:4; and

[1302] b) The V L The amino acid sequence containing SEQ ID NO:61 (AB-19).

[1303] 41. The polypeptide according to any one of Examples 1 to 20, wherein:

[1304] a) The V H Contains the amino acid sequence of SEQ ID NO:20; and

[1305] b) The V L The amino acid sequence containing SEQ ID NO:61 (AB-20).

[1306] 42. The polypeptide according to any one of Examples 1 to 20, wherein:

[1307] a) The V H Contains the amino acid sequence of SEQ ID NO:21; and

[1308] b) The V L The amino acid sequence containing SEQ ID NO:65 (AB-21).

[1309] 43. The polypeptide according to any one of Examples 1 to 20, wherein:

[1310] a) The V H Contains the amino acid sequence of SEQ ID NO:22; and

[1311] b) The V L The amino acid sequence (AB-22) containing SEQ ID NO:66.

[1312] 44. The polypeptide according to any one of Examples 1 to 20, wherein:

[1313] a) The V H Contains the amino acid sequence of SEQ ID NO:4; and

[1314] b) The V L The amino acid sequence containing SEQ ID NO:67 (AB-23).

[1315] 45. The polypeptide according to any one of Examples 1 to 20, wherein:

[1316] a) The V H Contains the amino acid sequence of SEQ ID NO:23; and

[1317] b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-24).

[1318] 46. ​​The polypeptide according to any one of Examples 1 to 20, wherein:

[1319] a) The V H Contains the amino acid sequence of SEQ ID NO:24; and

[1320] b) The V L The amino acid sequence containing SEQ ID NO:68 (AB-25).

[1321] 47. The polypeptide according to any one of Examples 1 to 20, wherein:

[1322] a) The V H Contains the amino acid sequence of SEQ ID NO:25; and

[1323] b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-26).

[1324] 48. The polypeptide according to any one of Examples 1 to 20, wherein:

[1325] a) The V H Contains the amino acid sequence of SEQ ID NO:26; and

[1326] b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-27).

[1327] 49. The polypeptide according to any one of Examples 1 to 20, wherein:

[1328] a) The V H Contains the amino acid sequence of SEQ ID NO:27; and

[1329] b) The V L The amino acid sequence (AB-28) containing SEQ ID NO:61.

[1330] 50. The polypeptide according to any one of Examples 1 to 20, wherein:

[1331] a) The V H Contains the amino acid sequence of SEQ ID NO:28; and

[1332] b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-29).

[1333] 51. The polypeptide according to any one of Examples 1 to 20, wherein:

[1334] a) The V H Contains the amino acid sequence of SEQ ID NO:28; and

[1335] b) The V L The amino acid sequence containing SEQ ID NO:69 (AB-30).

[1336] 52. The polypeptide according to any one of Examples 1 to 20, wherein:

[1337] a) The V H Contains the amino acid sequence of SEQ ID NO:29; and

[1338] b) The V L The amino acid sequence containing SEQ ID NO:70 (AB-31).

[1339] 53. The polypeptide according to any one of Examples 1 to 20, wherein:

[1340] a) The V H Contains the amino acid sequence of SEQ ID NO:30; and

[1341] b) The V L The amino acid sequence (AB-32) containing SEQ ID NO:71.

[1342] 54. The polypeptide according to any one of Examples 1 to 20, wherein:

[1343] a) The V H Contains the amino acid sequence of SEQ ID NO:31; and

[1344] b) The V L The amino acid sequence containing SEQ ID NO:72 (AB-33).

[1345] 55. The polypeptide according to any one of Examples 1 to 20, wherein:

[1346] a) The V H Contains the amino acid sequence of SEQ ID NO:32; and

[1347] b) The V L The amino acid sequence containing SEQ ID NO:67 (AB-34).

[1348] 56. The polypeptide according to any one of Examples 1 to 20, wherein:

[1349] a) The V H The amino acid sequence containing SEQ ID NO:33; and

[1350] b) The V L The amino acid sequence (AB-35) containing SEQ ID NO:56.

[1351] 57. The polypeptide according to any one of Examples 1 to 20, wherein:

[1352] a) The V HThe amino acid sequence containing SEQ ID NO:34; and

[1353] b) The V L The amino acid sequence containing SEQ ID NO:73 (AB-36).

[1354] 58. The polypeptide according to any one of Examples 1 to 20, wherein:

[1355] a) The V H Contains the amino acid sequence of SEQ ID NO:35; and

[1356] b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-37).

[1357] 59. The polypeptide according to any one of Examples 1 to 20, wherein:

[1358] a) The V H Contains the amino acid sequence of SEQ ID NO:36; and

[1359] b) The V L The amino acid sequence (AB-38) containing SEQ ID NO:56.

[1360] 60. The polypeptide according to any one of Examples 1 to 20, wherein:

[1361] a) The V H The amino acid sequence containing SEQ ID NO:37; and

[1362] b) The V L The amino acid sequence containing SEQ ID NO:63 (AB-39).

[1363] 61. The polypeptide according to any one of Examples 1 to 20, wherein:

[1364] a) The V H The amino acid sequence containing SEQ ID NO:38; and

[1365] b) The V L The amino acid sequence containing SEQ ID NO:69 (AB-40).

[1366] 62. The polypeptide according to any one of Examples 1 to 20, wherein:

[1367] a) The V H Contains the amino acid sequence of SEQ ID NO:39; and

[1368] b) The V L The amino acid sequence containing SEQ ID NO:74 (AB-41).

[1369] 63. The polypeptide according to any one of Examples 1 to 20, wherein:

[1370] a) The V H Contains the amino acid sequence of SEQ ID NO:40; and

[1371] b) The V L The amino acid sequence containing SEQ ID NO:52 (AB-42).

[1372] 64. The polypeptide according to any one of Examples 1 to 20, wherein:

[1373] a) The V H Contains the amino acid sequence of SEQ ID NO:41; and

[1374] b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-43).

[1375] 65. The polypeptide according to any one of Examples 1 to 20, wherein:

[1376] a) The V H Contains the amino acid sequence of SEQ ID NO:42; and

[1377] b) The V L The amino acid sequence containing SEQ ID NO:75 (AB-44).

[1378] 66. The polypeptide according to any one of Examples 1 to 20, wherein:

[1379] a) The V H Contains the amino acid sequence of SEQ ID NO:43; and

[1380] b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-45).

[1381] 67. The polypeptide according to any one of Examples 1 to 20, wherein:

[1382] a) The V H Contains the amino acid sequence of SEQ ID NO:44; and

[1383] b) The V L The amino acid sequence (AB-46) containing SEQ ID NO:51.

[1384] 68. The polypeptide according to any one of Examples 1 to 20, wherein:

[1385] a) The V H Contains the amino acid sequence of SEQ ID NO:45; and

[1386] b) The V L The amino acid sequence containing SEQ ID NO:75 (AB-47).

[1387] 69. The polypeptide according to any one of Examples 1 to 20, wherein:

[1388] a) The V H Contains the amino acid sequence of SEQ ID NO:46; and

[1389] b) The V L The amino acid sequence containing SEQ ID NO:53 (AB-48).

[1390] 70. The polypeptide according to any one of Examples 1 to 20, wherein:

[1391] a) The V H The amino acid sequence containing SEQ ID NO:47; and

[1392] b) The V L The amino acid sequence containing SEQ ID NO:52 (AB-49).

[1393] 71. The polypeptide according to any one of Examples 1 to 20, wherein:

[1394] a) The V H The amino acid sequence containing SEQ ID NO:48; and

[1395] b) The V L The amino acid sequence containing SEQ ID NO:76 (AB-50).

[1396] 72. The polypeptide according to any one of Examples 1 to 20, wherein:

[1397] a) The V H Contains the amino acid sequence of SEQ ID NO:3; and

[1398] b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-51).

[1399] 73. The polypeptide according to any one of Examples 3 to 72, wherein the VH and the V L It is humanized and contains human frame regions or combinations thereof.

[1400] 74. The polypeptide according to any one of Examples 1 to 73, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.

[1401] 75. The polypeptide according to Example 74, wherein the antigen-binding fragment is selected from Fab, F(ab')2, Fab', scFv or Fv.

[1402] 76. The polypeptide according to Example 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.

[1403] 77. The polypeptide according to Example 76, wherein the antibody heavy chain constant domain is selected from the group consisting of: IgA constant domain, IgD constant domain, IgE constant domain, IgG constant domain and IgM constant domain.

[1404] 78. The polypeptide according to Example 77, wherein the antibody heavy chain constant domain is the IgG1 heavy chain constant domain.

[1405] 79. The polypeptide according to any one of Examples 76 to 78, comprising an antibody light chain constant domain selected from the group consisting of a κ constant domain or a λ constant domain.

[1406] 80. The polypeptide according to Example 79, wherein the antibody light chain constant domain is a κ light chain constant domain.

[1407] 81. The polypeptide according to any one of Examples 1 to 80, wherein the polypeptide is conjugated with a heterologous portion.

[1408] 82. The polypeptide according to Example 81, wherein the heterologous portion is a therapeutic agent, a diagnostic agent, or a combination thereof.

[1409] 83. The polypeptide according to Example 81, wherein the heterologous portion is selected from the group consisting of: polyethylene glycol (PEG), hexadecanoic acid, hydrogel, lipid nanoparticles, polymer nanoparticles and heterologous polypeptide sequences or combinations thereof.

[1410] 84. The polypeptide according to Example 83, wherein the polymer nanoparticles comprise poly(lactic-co-glycolic acid) (PLGA).

[1411] 85. The polypeptide according to Example 81, wherein the heterologous polypeptide sequence comprises a carrier polypeptide.

[1412] 86. The polypeptide according to Example 85, wherein the carrier polypeptide is albumin or Fc polypeptide.

[1413] 87. The polypeptide according to any one of Examples 1 to 86, wherein the polypeptide:

[1414] a) with 1 μM or less K D Binds to SARS-CoV-2;

[1415] b) with an IC50 concentration of approximately 25,000 ng / mL or less. 50 Neutralizes SARS-CoV-2 infection in human host cells;

[1416] c) Reduce the infectivity of SARS-CoV-2 in human cells.

[1417] Or any combination thereof.

[1418] 88. The polypeptide according to Example 87, wherein the SARS-CoV-2 is a variant comprising T19R, 156del, 157del, R158G, L452R, T478K, D614G, P681R and D950N and optionally comprising G142D.

[1419] 89. The polypeptide according to Example 87 or 88, wherein the polypeptide is in a K+ of 100 nM or less. D It binds to SARS-CoV-2.

[1420] 90. The polypeptide according to Example 87, wherein the polypeptide is expressed at an IC50 concentration of about 25,000 ng / mL or less. 50 Neutralizes SARS-CoV-2 infection in human host cells.

[1421] 91. The polypeptide according to Example 87, wherein the polypeptide reduces the infectivity of SARS-CoV-2 in human cells by at least about 30%.

[1422] 92. A fusion protein comprising a polypeptide according to any one of Examples 1 to 91.

[1423] 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 Examples 1 to 80 or a fusion protein according to Example 92.

[1424] 94. A vector (e.g., an expression vector, including a viral delivery vector) comprising the polynucleotides described in Example 93.

[1425] 95. A host cell comprising the polynucleotide described in Example 93 or the vector described in Example 94.

[1426] 96. A composition comprising a polypeptide according to any one of Examples 1 to 91, a fusion protein according to Example 92, or a polynucleotide according to Example 93.

[1427] 97. The composition according to Example 96, comprising one or more pharmaceutical excipients, diluents or carriers.

[1428] 98. A method of treating a subject in need, the method comprising administering to the subject an effective amount of the composition according to Example 96 or 97.

[1429] 99. A method for reducing the infectivity of β-coronaviruses such as SARS-CoV-2 in cells of a subject, the method comprising contacting the cells with an effective amount of the composition according to Example 96 or 97.

[1430] 100. The method according to Example 98 or 99, wherein the subject has (e.g., confirmed by testing, such as by PCR or rapid testing) or is suspected of having COVID-19.

[1431] 101. The method according to Example 98 or 99, wherein the subject is at risk of developing COVID-19.

[1432] 102. The method according to any one of Examples 98 to 101, wherein the subject is a human.

[1433] 103. The method according to any one of Examples 98 to 102, wherein the subject suffers from heart disease.

[1434] 104. The method according to Example 103, wherein the heart 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.

[1435] 105. The method according to Example 104, wherein the heart failure is congestive heart failure.

[1436] 106. The method according to Example 104, wherein the inflammatory heart disease is selected from the group consisting of endocarditis, cardiac hypertrophy, myocarditis, and combinations thereof.

[1437] 107. The method according to any one of Examples 98 to 106, wherein the subject suffers from diabetes.

[1438] 108. The method according to any one of Examples 98 to 107, wherein the subject suffers from lung disease.

[1439] 109. The method according to Example 108, wherein the lung disease is selected from the group consisting of: acute respiratory distress syndrome, asthma, bronchitis, COPD, emphysema, lung tumor, pleural cavity disease, pulmonary vascular disease, respiratory tract infection, and combinations thereof.

[1440] 110. The method according to Example 109, wherein:

[1441] a) The respiratory infection mentioned is an upper respiratory tract infection, a lower respiratory tract infection, or pneumonia;

[1442] b) The pleural cavity disease mentioned is pleural mesothelioma or tension pneumothorax;

[1443] c) The pulmonary vascular disease is embolism, edema, hypertension, or hemorrhage; or

[1444] d) Its combination.

[1445] 111. The method according to any one of Examples 98 to 110, wherein the subject is a smoker.

[1446] 112. The method according to any one of Examples 98 to 111, wherein the subject is immune-impaired.

[1447] 113. The method according to Example 112, wherein the subject is receiving immunosuppressive therapy.

[1448] 114. The method according to any one of Examples 98 to 113, wherein the subject is 40 years of age or older.

[1449] 115. The method according to any one of Examples 98 to 114, wherein the method comprises administering to the subject a therapeutically effective amount of an additional therapeutic or preventative agent.

[1450] 116. The method according to Example 115, wherein 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.

[1451] 117. The method according to embodiment 116, wherein:

[1452] a) The additional SARS-CoV-2 spike-binding antibodies are selected from the group consisting of: bamlanivimab, etesevimab, bebtelovimab, casirivimab, imdevimab, cilgavimab, tixagevimab, AZD7442 (tixagevimab-cigarette), Regdanvimab, sotrovimab, and combinations thereof;

[1453] b) The antiviral agents are selected from the group consisting of: monoprevir (LAGEVRIO, Merck), PF-07817883 (Pfizer), STI-1558 (Sorrento Therapeutics), PBI-0451 (PyribioSciences, Inc.), EDP-235 (Enanda Pharmaceuticals, Inc.), oseltamivir (Tamiflu), favipiravir, amantadine, remdesivir, rimantadine, proconazole, antisense RNA against SARS-CoV-2, siRNA against SARS-CoV-2, and combinations thereof;

[1454] c) The ACE2 inhibitor is selected from the group consisting of: RNAi targeting ACE2, siRNA targeting ACE2, CRISPR-based inhibitors of ACE2, soluble ACE2, soluble ACE2 variants, anti-ACE2 antibodies, and combinations thereof.

[1455] d) The antibiotics mentioned include azithromycin;

[1456] e) The antimalarial agent mentioned contains chloroquine;

[1457] f) The vaccine is a nucleic acid vaccine or an inactivated viral vaccine; or

[1458] g) Its combination.

[1459] 118. The method according to Example 117, wherein the vaccine is mrna-1273, BNT162, INO-4800, AZD1222, Ad5-nCoV, PiCoVacc, NVX-CoV2373, JNJ-78436735 or a combination thereof.

[1460] 119. The method according to any one of Examples 98 to 118, wherein the subject is further treated (previously, simultaneously, or sequentially) with one or more RBD class 4 antibodies (or antigen-binding fragments thereof).

[1461] 120. The method according to Example 119, wherein the one or more RBD class 4 antibodies are selected from the group consisting of: S2X259, RBD class 4 mAb-1a having a VH containing SEQ ID NO:174 and a VL containing SEQ ID NO:178, RBD class 4 mAb-1b having a VH containing SEQ ID NO:174 and a VL containing SEQ ID NO:179, RBD class 4 mAb-1c having a VH containing SEQ ID NO:174 and a VL containing SEQ ID NO:180, RBD class 4 mAb-1d having a VH containing SEQ ID NO:174 and a VL containing SEQ ID NO:181, RBD class 4 mAb-2a having a VH containing SEQ ID NO:175 and a VL containing SEQ ID NO:182, and RBD class 4 mAb-2a having a VH containing SEQ ID NO:175 and a VL containing SEQ ID NO:183. Class 4 mAb-2b, having an RBD containing a VH of SEQ ID NO:175 and a VL of SEQ ID NO:184; Class 4 mAb-2c, having an RBD containing a VH of SEQ ID NO:175 and a VL of SEQ ID NO:185; Class 4 mAb-2d, having an RBD containing a VH of SEQ ID NO:176 and a VL of SEQ ID NO:186; Class 4 mAb-3a, having an RBD containing a VH of SEQ ID NO:176 and a VL of SEQ ID NO:187; Class 4 mAb-3b, having an RBD containing a VH of SEQ ID NO:176 and a VL of SEQ ID NO:188; Class 4 mAb-3c, having an RBD containing a VH of SEQ ID NO:176 and a VL of SEQ ID NO:189; Class 4 mAb-3d and having a VH of SEQ ID NO:177 and a VL of SEQ ID NO:189. NO:190 VL RBD 4 class mAb-4a, any of the variants of the foregoing, or any combination of the foregoing.

[1462] 121. The method according to any one of Examples 98 to 120, wherein the subject has previously received a therapeutic agent or a preventative agent.

[1463] 122. The method according to any one of Examples 98 to 121, wherein the subject has previously been infected with a β coronavirus, such as SARS-CoV-2.

[1464] 123. The method according to any one of Examples 98 to 122, the method comprising administering to the subject an effective amount of a combination of AB-1 and sotopimab.

[1465] 124. The method according to any one of Examples 98 to 122, the method comprising administering to the subject an effective amount of a combination of AB-1 and betelvirumab.

[1466] 125. The method according to any one of Examples 98 to 122, the method comprising administering an effective amount of a combination of AB-1 and Enbrel to the subject.

[1467] 126. The method according to any one of Examples 98 to 122, wherein the method comprises administering an effective amount of a combination of AB-1 and AZD-1061 to the subject.

[1468] 127. The method according to any one of Examples 98 to 122, wherein the method comprises administering an effective amount of a combination of AB-1 and P2G3 to the subject.

[1469] 128. The method according to any one of Examples 98 to 122, the method comprising administering to the subject an effective amount of a combination of AB-1 and RBD 4 type mAb-1a.

[1470] Example

[1471] Example 1. Materials and Methods

[1472] DELFIA (Determination of Alternative Pathogen Immunization)

[1473] Spike protein diluted with 5 μg / ml phosphate-buffered saline (PBS) was coated onto polystyrene MaxiSorp plates (Thermo Fisher Scientific, Waltham, MA, catalog number 460372) and incubated overnight at 4°C. Tris-buffered saline was then used to coat the plates. The plate was washed with 20% buffer (TBS-T) (Thermo Fisher Scientific, Waltham, MA, Catalog No. 28360) and blocked for 1 hour at room temperature with assay diluent (BioLegend, San Diego, CA, Catalog No. 421205). Antibody was serially diluted (1:4) in PBS / bovine serum albumin (BSA) starting at 4.5 μg / ml. After one wash with TBS-T, the serially diluted antibody was transferred to a pre-coated plate and incubated for 1 hour at room temperature. Next, the plate was washed three times with TBS-T and europium-labeled secondary antibody (PerkinElmer, Waltham, MA, Catalog No. 1244-330) was added for 30 minutes at room temperature. After incubation, the plate was washed three times with TBS-T and enhancement solution (PerkinElmer, Waltham, MA, Catalog No. 4001-0010) was applied. Time-resolved fluorescence was read at 615 nm using an EnVision plate reader (PerkinElmer, Waltham, Massachusetts).

[1474] pseudovirus neutralization assay

[1475] To neutralize the pseudovirus, Vero-TMPRSS2 cells were charged at 3.5 × 10⁻⁶. 3Cells / well were plated at a density of 20 μl in 384-well tissue culture-treated plates (Thermo Fisher Scientific, Waltham, MA, Catalogue No. 164610). The plates were briefly rotated downwards at <50 g and incubated at 37°C, 5% CO2 for 2–4 hours. To generate 5-point, 10-point, or 12-point titration profiles, antibodies were serially diluted 3, 4, or 6-fold in PBS / 0.2% BSA / 1-fold penicillin-strep (Pen-Strep) buffer, starting at 72 μg / ml or 18 μg / ml (4-fold final concentration), in 96-well round-bottom plates (Thermo Fisher Scientific, Waltham, MA, Catalogue No. 268200). The antibody was mixed with an equal volume of diluted SARS-CoV-2 pseudovirus (lentivirus pseudotyped with SARS-CoV-2δ, BA.1, SARS-CoV-1, or WIV1 spike proteins, and VSV-dG pseudotyped with SARS-CoV-2D614G, δ, BA.2, BA.2.12.1, BA.4 / 5, BA.4 / 5+K444T, BQ.1, BQ.1.1, XBB.1.5, or SARS-CoV-1 or WIV1 spike proteins). The antibody-virus mixture was incubated at 37°C, 5% CO2 for 30–60 minutes. Then, 20 μl of the antibody-virus mixture was transferred to a 384-well plate pre-inoculated with Vero-TMPRSS2 cells. The 384-well plate was briefly rotated downwards at <50 g and incubated at 37°C, 5% CO2 for 24 hours (VSV-dG) or 72 hours (lentivirus). At the end of the incubation, add 40 μl of luciferase detection buffer (BPS Bioscience ONE-Step). TM A luciferase assay system (BPS Bioscience, San Diego, CA, catalog number 60690-3) was added to each well of a cell culture plate. The plate was centrifuged at 50 g for <5 seconds and incubated with gentle agitation for 15 minutes. The luminescence signal was recorded using an Envision plate reader. Results were expressed as a percentage of neutralization and analyzed using Prism 9. Curves were generated by fitting the data using the following equation: logarithm of response relative to normalization (inhibitor) - variable slope (four parameters).

[1476] SARS-CoV-2 infection in the body

[1477] In vivo experiments were conducted at Bioqual. Male hamsters were intraperitoneally injected with the indicated antibody (variable region [VH / VL], expressing human IgG1 [huIgG1] or hamster IgG2a [hamIgG2a]) one day prior to intranasal inoculation with SARS-CoV-2δ. Body weight was recorded daily until day 7 and reported as a percentage change relative to day 0. Hamsters were euthanized on day 7, their lungs were collected, and their body weight was recorded. Viral titers in the nostrils and lungs were recorded (day 4 post-infection), and lung tissue pathology was performed (day 7 post-infection).

[1478] Live virus neutralization assay

[1479] Live virus neutralization assays were performed at Virology Research Services. Live SARS-CoV-2δ, BA.1, or BA.5 virus was incubated for 1 hour with 3- or 4-fold serial dilutions of each antibody, and then the mixture was added to Vero cells. Antiviral activity was determined using an immunofluorescence-based assay after 6 hours. Results are expressed as a percentage of neutralization and analyzed using Prism 9. Curves were generated by fitting the data using the following equation: logarithm of response relative to normalization (inhibitor) - variable slope (four parameters).

[1480] Example 2. Generation and characterization of S2-binding peptides

[1481] The S2 domain of the SARS-CoV-2 spike protein contains a fusion mechanism and includes a conserved sequence across SARS-CoV-2 variants and the entire subgenus *Sabeclovirus*. One of these sequences is a stem-helix peptide targeted by antibodies with neutralizing activity; however, these antibodies exhibit low neutralizing potency in vitro and show 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 probe the impact of sequence variations and binding postures on antibody function.

[1482] The activity selected a human IgG1 antibody isolated from a recovered donor and targeted a stem-helical peptide targeted by a reference antibody. The target epitopes of the reference antibody are highly conserved across the sequenced SARS-CoV-2 genome to date, with mutations occurring at a very low frequency (the most common mutations over the past 3 months were P1162L and P1162S, which were detected in only 0.4% and 0.2% of the SARS-CoV-2 genome sequences, respectively). A variant set of 182 antibodies was generated, and the following datasets were obtained: (1) exploitability: PSR, AC-SINS, SEC; (2) spike protein binding: SARS-CoV-2 related viruses (δ, BA.1, BA.2), SARS-CoV-1 related viruses (SARS-CoV-1, WIV1); and (3) neutralization of pseudotyped viruses: SARS-CoV-2 related viruses (δ, BA.2), SARS-CoV-1 related viruses (SARS-CoV-1, WIV1).

[1483] Many of the antibodies produced have high affinity for the spike proteins of SARS-CoV-1 and SARS-CoV-2 related viruses. Figures 4A-4B ) combine, and some of them also neutralize SARS-CoV-2δ, BA.2 ( Figures 5A-5B XBB.1.5 and BQ.1.1 Figure 21 Compared to the clinical-stage molecule, the sotopimab used in these experiments did not have the LS mutation and had three additional amino acid mutations due to differential use of the IgG allele. The ADG20 used in these experiments had the LS mutation in the Fc region, while the clinical-stage molecule had the LA mutation.

[1484] Combined and neutralized data were incorporated into a comprehensive suitability score to rank the top 20 screening hits. Then, 12 molecules with acceptable developability parameters (AC-SINS < 20, SEC > 90%, PSR < 10) were further selected as seeds for the next round of project learning. Notably, the reference antibody expressed 6 sequence biases (4 if we disregard the 2 cysteines, as they form bonds), posing a low to moderate risk to process development, and some of the seeds showed a reduced number of biases. As part of the project learning activities, the aim was to improve antibody function and further reduce the number of sequence biases.

[1485] A few seeds also showed promising properties as lead molecules. AB-1 effectively neutralized past (SARS-CoV-1), present (SARS-CoV-2δ, BA.2), and potential (WIV1) sabecloviruses. Figures 6A-6BIt has four mutations from the reference antibody and only one sequence bias besides two cysteine ​​residues. AB-2 is less potent than AB-1. Figures 7A-7B However, it has 7 mutations away from the reference antibody, has the best AC-SINS (<10), and has no bias other than 2 cysteines.

[1486] Seed molecules identified by screening this variant set (as indicated in Figures 6 and 7) were used to generate a new variant set of 364 antibodies, and the following datasets were obtained as part of the screening activity: (1) Developability: PSR, AC-SINS, SEC; (2) Spike protein binding: SARS-CoV-2 related viruses (δ, BA.1, BA.2), SARS-CoV-1 related viruses (SARS-CoV-1, WIV1); and (3) Neutralization of pseudotyped viruses: SARS-CoV-2 related viruses (δ, BA.2). Antibodies were ranked based on a comprehensive neutralization score, and the top 20 were selected. Figures 8A-8B Among them, based on the optimal binding, neutralization, and exploitability curves, three antibodies (AB-17, AB-15, and AB-1) were further selected. Figures 9A-9B And Table 4). These antibodies (SARS-CoV-2 BA.2, BA.2.12.1, and BA.4 / 5VSV dG; SARS-CoV-2 δ, SARS-CoV-1, and WIV1 pseudotyped lentiviruses) were then tested in additional neutralization assays, and benchmarks were performed against a reference antibody (reference Ab) and sotopimumab (cinnigs used as an isotype control). Figures 9A-9B (See Table 4). In all neutralization assays, AB-17, AB-15, and AB-1 demonstrated increased neutralizing potency and efficacy compared to the reference antibody. They also showed greater neutralizing activity than (SARS-CoV-2BA.2, BA.2.12.1, and BA.4 / 5VSV-dG) or comparable to (SARS-CoV-2δ, SARS-CoV-1, and WIV1) sotopimumab. In all assays, the isotype control was inactive.

[1487] Example 3. Developing candidate selection and characterization

[1488] Monoclonal antibodies (mAbs) targeting the spike protein of SARS-CoV-2 have proven effective for the prevention and treatment of COVID-19. To date, several mAbs targeting different regions of the spike protein (receptor-binding domain [RBD], N-terminal domain [NTD], S2) have been characterized, and several anti-RBD mAbs have received emergency use authorization in 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 accumulate several mutations in the RBD, leading to escape from many mAbs. Therefore, there is a need to develop mAbs targeting conserved regions of the spike protein that exhibit robust neutralizing activity against multiple SARS-CoV-2 variants.

[1489] The S2 domain contains highly conserved epitopes, namely fusion peptides and stem-helical peptides. Antibodies targeting these epitopes have been isolated from convalescent / vaccinated donors and immunized animals and have shown neutralizing activity, albeit with low potency. However, the discovery of anti-S2 neutralizing antibodies with sufficient potency could be potentially very valuable due to the broad conservatism of the S2 epitope across coronaviruses. The reference antibody is a human IgG1 anti-S2 antibody isolated from convalescent donors and showing promising neutralizing potency against coronaviruses of the subgenus Sabeclovirus (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):110210(2021)). The binding mode is known from the co-crystal structure with the stem-helical peptide. Furthermore, the target epitopes of the reference antibody are highly conserved across the sequenced SARS-CoV-2 genome to date, with only two mutations occurring at a very low frequency at position P1162 (P1162L and P1162S, representing 0.4% and 0.2% of the SARS-CoV-2 genome sequences over the past 3 months, respectively). It is hypothesized that by exploring the antibody sequence space compatible with this binding pattern, it may be possible to identify sequences with improved neutralizing potency compared to the reference antibody.

[1490] Novel anti-S2 stem-helix binding agents were generated using an in-house computational protein design scheme. Starting with the co-crystal structure of a reference antibody that binds to the S2 stem-helix peptide, in-house machine learning (ML) models were used to predict sequence patterns compatible with the binding conformation (Ingraham et al., Generative models for graph-based protein design, 33rd Conference on Neural Information Processing Systems (NeurIPS2019), Vancouver, Canada; Zhou et al., A general-purpose protein design framework based on mining sequence–structure relationships in known protein structures, Proceedings of the National Academy of Sciences 117(2):1059-68(2020)), of which 182 different sequences were sampled for experimental testing.

[1491] To test these designs, antibody variants were generated in human IgG1 format and screened for their function (pseudovirus neutralization), affinity (binding to the SARS-CoV-2 spike protein estimated by DELFIA), and developability properties (self-association tendency [AC-SINS], monomericity [aSEC], and multispecific reactivity [PSR]). Pseudovirus neutralization of SARS-CoV-2 variants δ, BA.1, BA.2, and SARS-CoV-1 and WIV1 was measured at five different titration concentrations. Affinity to the spike protein of SARS-CoV-1, WIV1, and SARS-CoV-2 variants δ, BA.1, and BA.2 was measured at eight different titration concentrations. Based on these experiments, a subset of antibodies with promising neutralization and binding curves was identified.

[1492] To further improve the neutralizing power of the candidate sequences, a second round of computational design was performed. Here, experimental measurements were used to train a model to predict power and affinity from the sequences. Using the resulting model, the predicted power and affinity were then co-optimized within the context of the sequence patterns described above to produce a set of 364 second-round sequences.

[1493] After experimental characterization of all antibody variants, lead antibody sequences were selected based on functional, binding, and exploitability properties measured among 533 unique sequences generated by two rounds of sequence design.

[1494] Based on screening data, three lead molecules with optimal functional curves were selected: 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 group of pseudoviruses representing SARS-CoV-2 variants (δ, oBA.1, BA.2, BA.2.12.1, and BA.4 / 5) and other sabecloviruses (SARS-CoV-1, WIV1), along with isotype controls (Sinagesi), reference antibodies, and clinical baselines (IgG1 molecules expressing the variable region of sotopemumab and two molecules constituting endostatin [AZD8895, AZD1061]). Figures 10A-10B Compared to the reference antibody, the three lead molecules exhibited comparable neutralization curves and improved overall neutralizing potency in all pseudoviruses (e.g., via IC50). 50 (Measured) and / or potency (maximum neutralization). Interestingly, compared with sotopimumab, the three lead molecules showed improved neutralizing potency and efficacy against the BA.2 sublineage (BA.2, BA.2.12.1, BA.4 / 5), and the BA.4 / 5 neutralization curve was comparable to that of AZD1061. Figures 10A-10B (Tables 5 and 6). Preliminary experiments using live virus also confirmed the neutralization of SARS-CoV-2δ and oBA.1 by the three lead molecules. Figure 11 (Table 7).

[1495] The FDA clinical-phase antibody prescribing information states that neutralization curves are considered comparable when the ratio between neutralizing efficiencies against two viruses is <5. To compare neutralizing efficiencies across experiments, the neutralizing efficiencies of the three lead molecules against each virus were expressed as a ratio to the neutralizing efficiency of sotopemumab against SARS-CoV-2δ. A ratio <5 was interpreted as comparable neutralization to sotopemumab neutralization against SARS-CoV-2δ. This is likely a relevant metric because sotopemumab has demonstrated clinical efficacy against SARS-CoV-2δ, and therefore a ratio <5 may indicate the human efficacy of the lead molecules at clinical doses (500 mg, IV) of sotopemumab. Notably, the calculated ratios for all three lead molecules were <5 across all viruses, confirming comparable neutralizing efficiencies to sotopemumab neutralization against δ (Table 8).

[1496] Overall, the three lead candidates demonstrated robustness and fairly consistent neutralization curves against a range of coronaviruses. To select a development candidate from the three lead candidates, developmental parameters were evaluated. AB-1 exhibited the best PSR and acceptable AC-SISN, and was therefore selected as the development candidate. Figure 12 ).

[1497] The neutralizing activity of AB-1 was further validated in a SARS-CoV-2δ-induced hamster model. For these in vivo experiments, the variable region (V) of AB-1, isotype (sinakis), and clinical-stage (sotovizumab) controls was [not specified]. H / V L ) is expressed as human IgG1 (huIgG1, Figures 15A-15D ) and hamster IgG2a (hamIgG2a, Figures 15E-15H To explain the interaction with the hamster Fcγ receptor. Hamsters were injected with the indicated antibody one day before infection with SARS-CoV-2δ. Body weight was recorded daily and expressed as a percentage change from day 0 (pre-infection). Hamsters were sacrificed on day 7, their lungs were collected, and their body weight was recorded (as a representative of lung inflammation). AB-1 expressing human IgG1 or hamster IgG2a prevented weight loss in a dose-dependent manner. Figure 15A -B and EF), and these results were associated with a reduction in lung weight ( Figure 15C and G).

[1498] The neutralizing activity of AB-1 was further validated in a SARS-CoV-2oBA.2-induced hamster model. For these in vivo experiments, the variable region (V) of AB-1, isotype (sinakis), and clinical-stage (sotovizumab) controls was measured. H / V L ) is expressed as hamster IgG2a (hamIgG2a, Figures 16A-16D To explain the interaction with the hamster Fcγ receptor. Hamsters were injected with the indicated antibody one day before infection with SARS-CoV-2oBA.2. Body weight was recorded daily and expressed as a percentage change from day 0 (pre-infection). Hamsters were sacrificed on day 7, their lungs were collected, and their body weight was recorded (as a representative of lung inflammation). AB-1 expressed as hamster IgG2a prevented weight loss in a dose-dependent manner. Figures 16A-16B ), and these results were associated with a decrease in lung weight. Figure 16C Therefore, further in vivo characterization of AB-1 confirmed its protection against weight loss in the oBA.2 infection model, and the protection achieved by measuring weight loss was not associated with a reduction in viral load in the lungs. Figure 15D (and H and 16D).

[1499] Figures 17A-17D Figures 18A-18C and 19 demonstrate that AB-1, alone or in combination with another antibody (e.g., a class 4 anti-RBD antibody), exhibits significant neutralizing potency and efficacy against multiple variants (such as oBQ.1.1, oBA.5, and δ).

[1500] Overall, these results indicate that AB-1 has robust neutralizing activity against SARS-CoV-2 variants both in vitro and in vivo.

[1501] Recently, many SARS-CoV-2 variants have exhibited significant clinical-stage antibody escape. Some of these variants show signs of convergent evolution, such as mutations at position K444. Therefore, the neutralization of AB-1 and clinical-stage antibodies sotopemumab and betronidazole against BA.4 / 5 pseudoviruses (BA.4 / 5+K444T) with and without the K444T mutation was evaluated. Sotopemumab showed poor neutralization against both pseudoviruses, while betronidazole showed significant impaired neutralization against BA.4 / 5+K444T pseudoviruses, as indicated by IC50. 50 The measured neutralizing power of AB-1 neutralizes BA.4 / 5 and BA.4 / 5+K444T pseudoviruses (Table 9).

[1502] AB-1 targets the S2 stem-helix epitope, which does not overlap with the epitope targeted by anti-RBD antibodies. In a neutralization assay, AB-1 was tested in combination with clinical-stage class 3 anti-RBD antibodies (sotovizumab, betelvirumab) or class 4 anti-RBD antibodies (anti-RBD4) using the BA.4 / 5+K444T pseudovirus (which impairs the neutralizing activity of sotopizumab and betelvirumab). As assessed by AUC (area under the curve) and efficacy (neutralization % at maximum concentration), all combinations showed enhanced neutralization curves compared to AB-1 alone. Figures 13A-13B ).

[1503] Example 4. Neutralization assay for pseudoviruses

[1504] Vero-TMPRSS2 cells were loaded at 3.5 × 10⁻⁶. 3Cells / well were plated at a density of 20 μl in 384-well tissue culture-treated plates (Thermo Fisher Scientific, Waltham, MA, Catalogue No. 164610). The plates were briefly rotated downwards at 50 g for <5 seconds and incubated at 37°C, 5% CO2 for 2–4 hours. To generate a 12-point titration profile, antibodies (alone or in combination) were serially diluted 4-fold in PBS / 0.2% BSA / 1-fold penicillin-strep (Pen-Strep) buffer, starting at 72 μg / ml (4-fold final concentration), in 96-well round-bottom plates (Thermo Fisher Scientific, Waltham, MA, Catalogue No. 268200). The antibodies were mixed with an equal volume of diluted SARS-CoV-2 pseudovirus (VSV-dG pseudotyped with SARS-CoV-2BQ.1.1 spike pseudotype). The antibody-virus mixture was incubated at 37°C, 5% CO2 for 30–60 minutes. Then, 20 μl of the antibody-virus mixture was transferred to a 384-well plate pre-inoculated with Vero-TMPRSS2 cells. The 384-well plate was briefly rotated downwards 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) was added. TM A luciferase assay system (BPS Biosciences, San Diego, CA, catalog number 60690-3) was added to each well of a cell culture plate. The plate was centrifuged at 50 g for <5 seconds and incubated with gentle agitation for 15 minutes. The luminescence signal was recorded using an Envision plate reader. Results are expressed as a percentage of neutralization and analyzed using Prism 9. Curves were generated by fitting the data using the following equation: logarithm of response relative to normalization (inhibitor) - variable slope (four parameters).

[1505] like Figure 19 As shown, AB-1 exhibits therapeutically significant in vitro neutralizing efficacy across multiple variants, suggesting that it can be used to target different variants by binding to the low immunogenicity and highly conserved region of SARS-CoV-2.

[1506] Pseudoviruses (VSV-dG pseudotyped with spike protein) representing SARS-CoV-2 variants tracked by the CDC or variants with increasing global prevalence will be generated, such as δ, 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, XBB, and non-SARS-CoV-2 sabecloviruses SARS-CoV-1 and WIV1. The pseudotyped system will be used to evaluate the neutralizing activity of the peptides disclosed herein (e.g., AB-1) against potentially emerging current and novel SARS-CoV-2 variants. These results will be confirmed with live virus. Pseudoviruses carrying mutations in the spike epitopes that may impair the neutralizing activity of the peptides (e.g., AB-1) will be developed and evaluated. These mutations will be selected based on structural and / or biochemical assessments of binding epitopes, analysis of mutations in selected sequences reported in public databases, and escape experiments with replicating VSVs, or a combination of the foregoing. Finally, the neutralizing activity of a peptide (e.g., AB-1) against a pseudovirus carrying a mutation that results in impaired neutralizing activity against a clinical-stage monoclonal antibody will be tested. For mutations already represented in the O sublineage, neutralizing activity against the O sublineage will be tested, rather than against pseudoviruses generated with selected escape mutations.

[1507] Example 5. Biochemical characterization of the AB-1 epitope

[1508] The monoclonal antibody (mAb) AB-1 was developed using an in-house machine learning model and leveraging the co-crystal structures of human reference antibodies (Hurlburt 2022, Jennewein 2021, Ullah 2021, Li 2022). AB-1 is a human IgG1 mAb that targets the spike S2 ​​stem-helical peptide of SARS-CoV-2, with an LS mutation in the Fc region to prolong its half-life and promote translocation to mucosal tissues. AB-1 exhibits robust neutralization against all major SARS-CoV-2 variants and other sabecloviruses associated with previous epidemics (SARS-CoV-1) or potential pandemics (WIV1, bat SARS-like coronaviruses). It is hypothesized that AB-1, as a single agent and potentially in combination with one or more other anti-spike mAbs, would be effective in preventing COVID-19 in high-risk groups across all current sublineages and future relevant variants.

[1509] Example 5 aimed 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). Dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) and surface plasmon resonance (SPR) were used to assess the binding of AB-1 and the reference antibody to the biotinylated peptides reported in Hurlburt 2022 (partially overlapping peptides spanning the reference antibody target epitope (aa 1133-1162), peptides representing the C-terminus of the stem-helix (aa 1149-1167), and a control 15-mer peptide derived from the HIV-1 Env protein). An additional biotinylated peptide was designed to cover the SARS-CoV-2 spike S2 ​​stem-helix (aa 1143-1162).

[1510] A. Biochemical characterization of the AB-1 epitope of DELFIA

[1511] • Biotinylated peptides: SARS-CoV-2 spike S2 ​​peptide 1133-1147 (Biosynth, Gardner, MA, lot number LP10933), 1137-1151 (Biosynth, LP10934), 1141-1155 (Biosynth, LP10935), 1145-1159 (Biosynth, LP10936), 1149- 1163 (Biosynth, batch number LP10945), 1153-1167 (Biosynth, batch number LP10938), 1157-1171 (Biosynth, batch number LP10939), 1149-1167 (Biosynth, batch number LP10937), 1143-1162 (Biosynth, batch number BU17943); HIV-1 Env peptide (Biosynth, batch number LP10940). See, for example, Table 36.

[1512] • Antibodies: AB-1 (Lonza, lot number 1100-130922-01), reference antibody (GenScript USA, Inc., Piscataway, NJ, lot number U737NHI220, a reference antibody variable region expressing human IgG1 with LS mutation in the Fc region), and isotype (GenScript USA, Inc., lot number U799WHJ270-3, palizumab variable region targeting RSV F protein and expressing human IgG1 with LS mutation in the Fc region).

[1513] • Neutral avidin: Neutral avidin protein (Thermo Fisher Scientific, Waltham, MA, catalog number 31000).

[1514] • Dilution plate: 96-well round bottom, untreated, polypropylene (Corning, Corning, NY, catalog number 3365; Greiner, Kremsmünster, Austria, catalog number 650201).

[1515] DELFIA plate: 384-well plate, white, MaxiSorp, polystyrene (Thermo Fisher Scientific, catalog number 460372).

[1516] • TBS-T: 20X TBS Tween-20 (Thermo Fisher Scientific, catalog number 28360) diluted 1:20 in ddH2O.

[1517] • Blocking solution: ELISA assay diluent B (5X) (Baijin Biotech, San Diego, California, catalog number 421205) was diluted 1:5 in PBS.

[1518] Secondary antibody: DELFIA Eu-N1 anti-human IgG (PerkinElmer, Waltham, Massachusetts, catalog number 1244-330).

[1519] • Enhancement solution: DELFIA enhancement solution (PerkinElmer, catalog number 4001-0010).

[1520] DELFIA is a time-resolved fluorescence (TRF) intensity technique used to determine the binding of AB-1 to different SARS-CoV-2 spike S2 ​​stem-helical peptides. The DELFIA assay is designed to detect the presence of the analyte of interest using reagents labeled with lanthanide (e.g., europium [Eu]) chelates. Upon completion of the immunoreaction, Eu ions dissociate from the labeled immunohistochemicals bound to the solid phase by the addition of an enhancement solution. Eu fluorescence is then measured by TRF.

[1521] 384-well plates were coated overnight at room temperature with neutral avidin (2 μg / mL). The plates were washed and coated with biotinylated peptide (50 nM) for 1 hour at room temperature. The plates were then washed and incubated with blocking solution for 2 hours at room temperature, followed by washing again and incubation with antibodies (AB-1, reference antibody, and allotype control) for 1 hour at room temperature. Antibody binding was assessed using a 12-point titration curve prepared in triplicate (1:4 serial dilutions prepared in blocking solution, starting at 18 μg / mL). Subsequently, the plates were washed and incubated at room temperature with Eu-labeled anti-human IgG secondary antibody (0.1 μg / mL) for 30 minutes. Finally, the plates were washed and incubated with DELFIA enhancement solution for 15 minutes. TRF was recorded at 615 nm using a PerkinElmer EnVision plate reader.

[1522] Statistical analysis was performed using Prism 9.5.0 software with data from two independent replicas. The area under the curve (AUC) and 95% confidence intervals for the log-transformed antibody titration curves were derived using the trapezoidal rule. The AUC values ​​of AB-1 and the reference antibody were statistically compared using a two-dimensional ANOVA corrected for multiple comparisons, as reported in the software documentation (https: / / www.graphpad.com / support / faqid / 2031 / ).

[1523] B. Biochemical characterization of the AB-1 epitope of SPR

[1524] • Biacore 8K+ (Cytiva, Marlborough, MA, serial number 2873569).

[1525] • Biacore 8K control software (Stopfan Company, version 4.0.8.19879).

[1526] • Biacore Insight assessment software (Stopfan, version 4.0.8.19879).

[1527] ·GraphPad Prism (version 9.5.0).

[1528] • Biotinylated peptides: SARS-CoV-2 spike S2 ​​peptide 1133-1147 (Biosynth, batch number LP10933), 1137-1151 (Biosynth, batch number LP10934), 1141-1155 (Biosynth, batch number LP10935), 1145-1159 (Biosynth, batch number LP10936), 1149-1163 (Biosynth) (Batch numbers LP10945), 1153-1167 (Biosynth, batch number LP10938), 1157-1171 (Biosynth, batch number LP10939), 1149-1167 (Biosynth, batch number LP10937), 1143-1162 (Biosynth, batch number BU17943); HIV-1 Env peptide (Biosynth, batch number LP10940). See, for example, Table 36.

[1529] •Fab: AB-1 (in-house prepared), reference antibody (in-house prepared).

[1530] • Antibody: AB-1 (internal generation), reference antibody variable domain (GenScript Biotech, lot number U3767HH180-11, expressed in the Fc region as the reference antibody variable domain of human IgG1 with LS mutation).

[1531] • The S-series Biotin Capture kit (Stopfan, catalog number 28920234) contains biotin capture reagent at 50 μg / mL in HBS-EP, with regeneration stock solution 1 (8M guanidine hydrochloride) and regeneration stock solution 2 (1M sodium hydroxide).

[1532] • 20X HBS-EP+, pH 7.6 (Teknova, Hollister, CA, catalog number H8022), composed of 0.2M HEPES (pH 7.4), 3M NaCl, 60mM EDTA, and 1% surfactant P20, and prepared at 1X.

[1533] • Microplate, 96W, deep well, U-bottom, PP, 2mL (Porvair, Ashland, VA, catalog number 219020).

[1534] · Microporous foil 96W (Stopfan Company, catalog number 28975816).

[1535] 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 through an iterative process that finds the best fit to a set of equations describing the interactions. The association rate constant k... a (M -1 s -1 The dissociation rate constant k controls the rate of complex formation. d (s -1 The equilibrium dissociation constant K controls the rate of complex dissociation. D (M) describes the strength of the interaction.

[1536] To evaluate the response units (RUs) of AB-1 and reference antibodies to peptides binding across the S2 stem-helix region of the SARS-CoV-2 spike protein, a multi-cycle kinetic approach was implemented using a Biacore 8K+. Biotinylated peptides were captured using a Biotin-Capture kit. The reaction was carried out at 2 μL / min. -1 Inject biotin-CAPture reagent at a flow rate of 10 μL / min for 180 seconds. -1 Inject biotinylated peptides (called ligands) at a flow rate of 10 seconds to achieve a capture level of approximately 10 to 20 RU. At a rate of 30 μL / min... -1 AB-1 and reference antibody (referred to as analyte) were injected at a flow rate of 100 nM onto the surface for 180 seconds, and 1X HBS-EP+ was used as the run buffer to allow the complex to dissociate for 900 seconds. The flow rate was measured at 10 μL / min. -1 The surface was regenerated by injecting a solution of 3 portions of regeneration stock solution 1 and 1 portion of regeneration stock solution 2 at a flow rate of 120 seconds. Measurements were performed at 25°C. A capture level reporting point was obtained 25 seconds after ligand injection and a binding response reporting point was obtained 5 seconds before the end of analyte injection. The binding response was normalized relative to the ligand capture level using GraphPad Prism and reported as a normalized binding response.

[1537] To evaluate the kinetics of AB-1 and reference antibody binding to peptides spanning the S2 stem-helix region of the SARS-CoV-2 spike protein, a single-cycle kinetic approach was implemented using a Biacore 8K+. Biotinylated peptides were captured using a Biotin-Capture kit. The loading was 2 μL / min. -1 Inject biotin-CAPture reagent at a flow rate of 10 μL / min for 180 seconds. -1 Inject biotinylated peptides (called ligands) at a flow rate of 10 seconds to achieve a capture level of approximately 10 to 20 RU. At a rate of 30 μL / min... -1The flow rate was used to sequentially inject AB-1 and the reference antibody (referred to as the analyte) onto the surface at increasing analyte concentrations for 180 seconds, and to allow the complex to dissociate for 900 seconds using 1X HBS-EP+ as the run buffer. The concentration series consisted of 3-fold serial dilutions starting at 300 nM, for a total of six concentrations. The concentrations were measured at 10 μL / min. -1 The surface was regenerated by injecting solutions of 3 portions of regeneration stock solution 1 and 1 portion of regeneration stock solution 2 at a flow rate of 120 seconds. Assays were performed at 25°C. Kinetic parameters for the concentration series were obtained by globally fitting the data to a 1:1 binding model with mass transfer limitations using dual references and Biacore Insight evaluation software. The kinetics of binding of AB-1 and the reference antibody Fab to the SARS-CoV-2 spike S2 ​​peptide (aa 1149-1167) representing the C-terminus of the stem-helix are reported, and the resulting sensor plots are shown.

[1538] C. Result

[1539] DELFIA and SPR data show the binding curves of AB-1 to the SARS-CoV-2 spike S2 ​​peptide. Figure 21 , Figure 22 (Table 13). The specificity of these results was confirmed by the lack of binding to an allotype control in the DELFIA assay and the lack of binding to the HIV-1 Env peptide, used as a negative control, in both the DELFIA and SPR assays by AB-1 and the reference antibody. Notably, AB-1Fab bound with a higher affinity to the SARS-CoV-2 spike S2 ​​peptide (aa 1149-1167) representing the C-terminus of the stem-helix (K) compared to the reference antibody. D Values: AB-1, 0.9 nM; Reference antibody, 5.4 nM (Table 14, Figures 23A-23D )).

[1540] In summary, Example 5 demonstrates that AB-1 binds to the SARS-CoV-2 spike S2 ​​stem-helix of the reference antibody with higher affinity.

[1541] Example 6. Structural characterization of the AB-1 epitope

[1542] The purpose of Example 6 was to collect structural information on AB-1 complexed with the SARS-CoV-2 BA.1 spike trimer and the spike S2 ​​(aa 1149-1167) stem-helical peptide and to determine its binding site.

[1543] X-ray crystallography was used to determine the structure of AB-1Fab complexed with the spike S2 ​​(aa 1149-1167) stem-helical peptide at high resolution. Cryo-electron microscopy (Cryo-EM) was used to determine the structure of AB-1Fab complexed with the SARS-CoV-2BA.1 spike trimer. As observed in previous structural determination attempts, the helical coiled S2 stem-helical peptide of the SARS-CoV-2BA.1 spike trimer is highly flexible and may be extremely challenging to resolve by Cryo-EM. To stabilize the movement in this region, the Fab region of a monoclonal antibody (R-AB-3a) targeting a class 4 epitope of the spike receptor-binding domain (RBD) was used to complex with the SARS-CoV-2BA.1 spike trimer bound to AB-1Fab.

[1544] A. Structural characterization using the AB-1 epitope of Cryo-EM

[1545] • Antigen: SARS-CoV-2BA.1 spike trimer (produced internally)

[1546] • Fab: AB-1 (internal production), R-AB-3a (internal production)

[1547] ·Expi293F TM Cells (Thermo Fisher Scientific, Catalog No. A14527)

[1548] Gibco Expi293 TM Expression medium (Thermo Fisher Scientific, catalog number A14351-01)

[1549] ·ExpiFectamine TM 293 Transfection Kit (Thermo Fisher Scientific, Catalog No. A14525)

[1550] • Nickel Sepharose Excel (Sitefan Company, Catalog No. 17371201, Batch No. 10313877)

[1551] LambdaFabSelect (Catalog No. 17548201, Batch No. 10302825)

[1552] ·Capto TM L (Situofan Company, Catalog No. 17547802, Batch No. 10305645)

[1553] • Quantifoil Gold R1.2 / 3 grid (Electron Microscope Sciences, catalog number 261655, lot number Q82801)

[1554] instrument:

[1555] • Kuhner oscillator (Kuhner, model: ISF1-ZC Peltier)

[1556] · Pure (Sitefan Company)

[1557] • SRT-C SEC-500 (Separ, PN: 235500-4630)

[1558] Superose 6 Increase 10 / 300GL (Stopfan Company, Catalog No. 29091596, Lot No. 10325571)

[1559] Nanodrop One (Thermo Fisher Scientific, Nanodrop One model)

[1560] • HPLC 1260 Infinity II (Agilent Technologies, TT No.: 1581T8)

[1561] • Pelco easiGLOW (Pelco, model: 91000)

[1562] • Vitrobot Mark IV (Thermo Fisher Scientific, serial number 220301059)

[1563] Glacios (Thermo Fisher Scientific, serial number 9956936)

[1564] software:

[1565] EPU (Thermo Fisher Scientific, v3.2)

[1566] • cryoSPARC (Structura Biotechnology, v4.1.1)

[1567] ChimeraX (UCSF RVBI, v1.5)

[1568] a. Expression and purification of SARS-CoV-2BA.1 spike trimer

[1569] According to GibcoTM Expi293 TM The expression system scheme, in Expi293 TM The SARS-CoV-2BA.1 spike trimer was expressed in cells. Briefly, three million cells were transfected with approximately 1 mg of plasmid DNA. Cells were incubated at 37°C, 80% relative humidity, and 8% CO2 on an orbital oscillator at 150 RPM. Four days post-transfection, cells were harvested and granulated at 3,900 x g for 30 min at 4°C. The supernatant was decanted into a 0.22 μm filter unit and stored at 4°C until purification. The SARS-CoV-2BA.1 spike trimer was purified using Nickel Sepharose Excel resin. The supernatant was incubated overnight with 2 mL of Nickel Sepharose Excel resin at 4°C and purified by gravity flow.

[1570] b. Expression and purification of AB-1 and R-AB-3a Fab

[1571] Following the same protocol used against the SARS-CoV-2 BA.1 spike trimer, AB-1 and R-AB-3a Fab were used in Expi293. TM Expression in cells. The supernatants of the two Fabs were mixed with Capto... TM Incubate overnight at 4°C with either L-resin (AB-1Fab) or LambdaFabSelect resin (R-AB-3a Fab). Load the mixture of supernatant and resin onto a 10 mL disposable column equilibrated in 1X PBS (pH 7.4). Wash the column with 10 CV of 1X PBS (pH 7.4). Elute AB-1 and R-AB-3a with 50 mM glycine (pH 2.5). Immediately neutralize the protein with 1 M Tris-HCl (pH 8.0). Exchange each protein with PD-10 desalting column buffer and elute with 1X PBS (pH 7.4). Concentrate the purified protein and maintain at 4°C until it complexes with the SARS-CoV-2BA.1 spike trimer.

[1572] c. Complexation of SARS-CoV-2BA.1 spike trimer with AB-1 and R-AB-3a Fab

[1573] The SARS-CoV-2 BA.1 spike trimer was gently mixed with AB-1 and R-AB-3a Fab at a molar ratio of 1:2:2 and incubated overnight at 4°C. The complex was purified by size exclusion chromatography on a Superose 6 Increase 10 / 300GL column equilibrated in 50 mM HEPES (pH 8.0) and 150 mM NaCl. Prior to injection, the incubated sample was rotated at 1699 x g. 1 mL of the complex was injected onto the column through a 1 mL loop; the flow rate for the entire run was 0.5 mL / min. Each elution peak was analyzed by SDS-PAGE to identify which fraction contained the complex between the SARS-CoV-2 BA.1 spike trimer and AB-1 and R-AB-3a Fab. These fractions were further analyzed by aSEC on an SRT-CSEC-500 column equilibrated in 50 mM HEPES (pH 8.0) and 150 mM NaCl.

[1574] d. Cryo-EM sample preparation

[1575] Four microliters of sample were applied to a Quantifoil gold grid, which was glow-discharged for 30 seconds at a negative electrode plasma current of 0.15 mA.

[1576] e.Cryo-EM Data Collection

[1577] 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 zero-loss slit width of 10 eV. 5160 images were collected at 130,000x magnification, with a pixel size of [missing information]. The total dose per frame is 51.3 electrons / square angstrom. The target defocus range is 0.5–2.4 μm.

[1578] f. Cryo-EM data processing

[1579] All computational steps were performed using the cryoSPARC (v4.1.1) software suite and ChimeraX (v1.5) molecular visualization software. EER format films were imported and segmented into 40 frames, sampled at physical pixel size. Beam-induced motion correction, per-frame dose weighting, and CTF estimation were performed using patch motion correction and patch CTF jobs in cryoSPARC (v4.1.1). Exposures were selected semi-automatically using an interactive exposure management tool. After setting strict cutoff values ​​for CTF fitting resolution, defocus, and relative ice thickness, 2,921 images were selected for further processing.

[1580] Low-pass filter was applied to SARS-CoV-2BA1 spike trimer. Template-based particle acquisition was performed using projections of 3D images. 466,074 particles were extracted from 2,921 micrographs. 2D classification across 200 categories was used to identify incorrectly acquired or broken particles, and 87,564 particles were retained for further processing.

[1581] Three classes of ab initio reconstructions were performed, yielding initial 3D maps of SARS-CoV-2BA.1 spike trimers with the expected size and shape from a subset of 51,996 particles. These particles were then subjected to non-uniform 3D refinement in cryoSPARC, and the desired 3D model was obtained according to the gold standard FSC criteria. The 'shared graph' of resolution.

[1582] g. Key classifications and model fitting

[1583] Using the 'Fit Plot' tool in ChimeraX (v1.5), the atomic model calculations of the SARS-CoV-2BA.1 spike trimer without Fab binding were aligned with the common plot. The plot and density fit very well, except for a few uncalculated densities in the RBD and S2 stem-helix region of the plot. Gaussian low-pass filtering of the plot revealed that these densities are dumbbell-shaped with a central pore, as expected for Fab molecules. The 'Segment Plot' tool in ChimeraX and the volume tool in cryoSPARC were used to generate a focused mask around the S2 stem-helix, enclosing the assumed Fab density in a propeller shape. A matchless, focused 3D classification of ten categories in cryoSPARC was performed to identify a subset of 9,822 particles with a high putative S2 stem-helix binding Fab density. This subset was refined using non-uniform refinement without applying symmetry by the gold standard FSC criteria. The resolution.

[1584] Local resolution estimations in cryoSPARC show that the local resolution of the S2 stem-helix binding Fab is lower than that of the core of the SARS-CoV-2BA.1 spike trimer. The local resolution of the CDR-containing domains in the Fab is approximately... The local resolution of the Fab's flexible associative framework domain is approximately Therefore, a width of [missing information] was applied in ChimeraX (v1.5). The Gaussian low-pass filter. One Fab is significantly weaker than the other two. However, these densities are sufficient to definitively align three copies of the AB-1Fab-S2 stem-helix complex crystal structure (described below) to each of the three S2 stem-helix Fab densities using ChimeraX's 'fitting plot' tool. The RBD-bonded R-AB-3a Fab structure also aligns to a dumbbell-shaped density at the spiked RBD.

[1585] B. Structural characterization of the AB-1 epitope via X-ray crystallography

[1586] •Fab: AB-1 (internal preparation).

[1587] • Fabalactica Midispin Fab Digestion Kit (Genovis, catalog number A2-AFK-100).

[1588] • Peptide: SARS-CoV-2 spike S2 ​​peptide 1149-1167 (Biosynth, lot number LP10941).

[1589] • MCSG-3 crystallization sieve (Anatrace, part number MCSG-3).

[1590] · 16 / 900 Superdex 200pg column (custom).

[1591] a. Production of AB-1Fab and SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptides

[1592] AB-1 was produced by Lonza, and the monomer content was evaluated by SE-HPLC. Fab was digested and purified from IgG using the Genovese 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 at Biosynth and its quality was verified by HPLC, mass spectrometry, and amino acid analysis. The binding of AB-1 to the SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide was evaluated in AB-1-001-PD.

[1593] b. AB-1Fab: Co-crystallization assay of SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptides:

[1594] AB-1Fab and SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptides were added at a 1:1 molar ratio, incubated on ice for 20 min, concentrated, and injected onto a 16 / 900 Superdex 200 pg column (buffer: 25 mM HEPES, pH 7.5, 150 mM NaCl). Peak fractions were analyzed by SDS-PAGE, and the fractions were pooled and concentrated to 10 mg / mL. An ARI Crystal Gryphon droplet setting robot was used with 96-3 Intelliplates to set the crystallization conditions at 10 mg / mL with three ratios of the complex (1:1, 2:1, and 3:1). 96-well MCSG-3 commercial sieves were set and incubated at 4 and 20 °C.

[1595] c. AB-1Fab: Data collection and structural determination of SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptides

[1596] Crystals were acquired from the MCSG-2A2 (1:1 ratio) at 4°C and cryoprotected in 20% glycerol. The crystals were then fed to the NSLS2 synchrotron, and X-ray datasets were collected on the AMX beamline using an Eiger X 9M detector. The data were processed using DIALS and XDS. After processing, the dataset was phased using the AlphaFold models of Phaser_MR and AB-1Fab, along with reference antibodies for peptides, through molecular substitution. Several rounds of refinement were performed using Phenix and Coot.

[1597] C. Result

[1598] Structural characterization of the AB-1Fab:SARS-CoV-2BA.1 spike trimer complex via Cryo-EM and the AB-1Fab:SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide complex via X-ray crystallography revealed the binding of AB-1 to the S2 peptide. Figures 24A-24C In addition, the results showed that three copies of AB-1Fab could bind to the SARS-CoV-2BA.1 spike trimer.

[1599] In summary, structural characterization of AB-1Fab, which is complexed with the SARS-CoV-2 BA.1 spike trimer and the SARS-CoV-2 spike S2 ​​(aa 1149-1167) peptide, indicates that AB-1 binds to the SARS-CoV-2 spike S2 ​​stem-helical peptide.

[1600] Example 7. Mutation analysis of AB-1 epitope and adjacent sequences

[1601] The purpose of Example 7 is to assess the sequence conservation of regions of the SARS-CoV-2 spike protein that may affect the neutralizing efficacy of AB-1 by defining the relative mutation frequencies of the AB-1 epitope, adjacent epitope regions (within 5 Å of the epitope in the protein structure), heptapeptide repeat 1 (HR1), and heptapeptide repeat 2 (HR2) in publicly reported SARS-CoV-2 spike protein sequences during the pandemic (January 6, 2020 to March 1, 2023).

[1602] A. Definition of the area of ​​interest

[1603] In this study, the four regions of interest for the SARS-CoV-2 spike protein were: the AB-1 epitope (referred to as "epitope"), the residues adjacent to the epitope (referred to as "adjacent epitopes"), HR1, and HR2. Using the epitope residues and the publicly available structure of the SARS-CoV-2 spike protein (PDB ID: 6VSB), "adjacent epitope" residues were defined as residues containing any atom within 5 angstroms of any epitope residue atom. The residues corresponding to HR1 and HR2 are defined in the NCBI entry for the SARS-CoV-2 surface glycoprotein YP_009724390.1. Table 15 summarizes the location of each of these four regions in the SARS-CoV-2 spike sequence. Because the epitope intersects with HR2, to avoid double-counting residues falling into both regions, the intersecting region was defined as part of the "epitope" category and excluded from the HR2 category throughout the analysis. Similarly, the adjacent epitope location also intersects with HR2; this intersection was defined as part of HR2 and excluded from the adjacent epitope category for analysis.

[1604] B. Identification of relevant mutations in AB-1 epitopes, adjacent regions, and heptapeptide repeat sequences

[1605] The covSPECTRUM API (Application Programming Interface) is used to query mutations occurring in each of the four regions of interest in the GenBank database (Chen 2021). For each mutation, the relative frequency of the mutation was calculated for the end of each month from January 6, 2020 to March 1, 2023, using three different backtracking periods for each month: 1 month, 3 months, and all time (since January 6, 2020). The 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.

[1606] Mutations that meet the following criteria have been identified:

[1607] • Falls within “table position”, “adjacent table position”, “HR1” or “HR2”.

[1608] • Satisfies a), b), or both:

[1609] a) Relative frequency over all time: The relative frequency of all stored sequences is at least 0.001 from the start of data availability (January 6, 2020) to March 1, 2023.

[1610] b) Recent relative frequency: The relative frequency is at least 0.01 among all sequences stored from January 1, 2023 to March 1, 2023, and has been observed at least 100 times during this period.

[1611] Relative frequency data were plotted throughout the pandemic to see whether the prevalence of the mutation is currently increasing or decreasing.

[1612] C. Identification of the most prevalent lineages in sequences carrying mutations

[1613] To identify the most prevalent strains carrying each mutation of interest, covSPECTRUM was queried to obtain a set of sequences with each mutation observed from January 6, 2020, when data availability began, to March 1, 2023. For each mutation, all sequences carrying the mutation were lineage-assigned (nextcladePangoLineage assignment reported by covSPECTRUM), and lineages were ranked based on absolute counts. For each mutation and lineage, to convert the counts to the relative frequency of strains among all sequences with the mutation, the count of the lineage sequences with the mutation was divided by the total number of sequences with the mutation observed from January 6, 2020, to March 1, 2023. In this analysis, the four most common lineages associated with each mutation are reported.

[1614] D. The relative frequency of mutations of interest in the most common lineages

[1615] Five of the most prevalent lineages were identified by querying the frequency of the lineages observed in each of the three time periods up to March 1, 2023 (1 month, 3 months, and since January 6, 2020) in ...

Claims

1. A polypeptide that specifically binds to the spike glycoprotein of SARS-CoV-2, said polypeptide comprising: a) Variable domains of immunoglobulin heavy chain (V H ) amino acid sequence, the V H The amino acid sequence comprises HCDR1, HCDR2, and HCDR3, which are substantially similar to the heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of the amino acid sequences of any of SEQ ID NO: 4-48, respectively; and b) Variable domains of immunoglobulin light chains (V L ) amino acid sequence, the V L The amino acid sequences comprise LCDR1, LCDR2, and LCDR3, which are substantially similar to the light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) of the amino acid sequences of any of SEQ ID NO:51-76, respectively.

2. The polypeptide of claim 1, comprising the antibody HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, wherein the antibody comprises an amino acid sequence selected from: 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 ... 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).

3. The polypeptide according to claim 1 or 2, wherein V H It has at least 85% sequence identity with any or more of the amino acid sequences in SEQ ID NO:4-48.

4. The polypeptide according to any one of claims 1 to 3, wherein V L It has at least 85% sequence identity with any one or more of the amino acid sequences in SEQ ID NO:51-76.

5. The polypeptide according to any one of claims 1 to 4, comprising the antibody HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, wherein the antibody comprises the amino acid sequences (AB-1) of SEQ ID NO:4 and SEQ ID NO:

51.

6. The polypeptide according to claim 5, comprising the amino acid sequences of HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 of SEQ ID NO:77, 80, 91, 133, 141 and 144, respectively.

7. The polypeptide according to any one of claims 1 to 6, wherein the V H It has at least 85% sequence identity with the amino acid sequence of SEQ ID NO:

4.

8. The polypeptide according to any one of claims 1 to 7, wherein the V L It has at least 85% sequence identity with the amino acid sequence of SEQ ID NO:

51.

9. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:4; and b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-1).

10. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:5; and b) The V L The amino acid sequence containing SEQ ID NO:52 (AB-2).

11. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:6; and b) The V L The amino acid sequence containing SEQ ID NO:53 (AB-3).

12. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:7; and b) The V L The amino acid sequence containing SEQ ID NO:54 (AB-4).

13. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:8; and b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-5).

14. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:9; and b) The V L The amino acid sequence containing SEQ ID NO:55 (AB-6).

15. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:10; and b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-7).

16. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:11; and b) The V L The amino acid sequence containing SEQ ID NO:57 (AB-8).

17. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:12; and b) The V L The amino acid sequence containing SEQ ID NO:30 (AB-9).

18. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:13; and b) The V L The amino acid sequence containing SEQ ID NO:59 (AB-10).

19. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:14; and b) The V L The amino acid sequence containing SEQ ID NO:60 (AB-11).

20. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:15; and b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-12).

21. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:16; and b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-13).

22. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:10; and b) The V L The amino acid sequence containing SEQ ID NO:50 (AB-14).

23. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:17; and b) The V L The amino acid sequence containing SEQ ID NO:61 (AB-15).

24. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:18; and b) The V L The amino acid sequence containing SEQ ID NO:62 (AB-16).

25. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:6; and b) The V L The amino acid sequence containing SEQ ID NO:63 (AB-17).

26. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:19; and b) The V L The amino acid sequence containing SEQ ID NO:64 (AB-18).

27. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:4; and b) The V L The amino acid sequence containing SEQ ID NO:61 (AB-19).

28. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:20; and b) The V L The amino acid sequence containing SEQ ID NO:61 (AB-20).

29. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:21; and b) The V L The amino acid sequence containing SEQ ID NO:65 (AB-21).

30. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:22; and b) The V L The amino acid sequence (AB-22) containing SEQ ID NO:

66.

31. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:4; and b) The V L The amino acid sequence containing SEQ ID NO:67 (AB-23).

32. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:23; and b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-24).

33. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:24; and b) The V L The amino acid sequence containing SEQ ID NO:68 (AB-25).

34. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:25; and b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-26).

35. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:26; and b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-27).

36. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:27; and b) The V L The amino acid sequence (AB-28) containing SEQ ID NO:

61.

37. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:28; and b) The V L The amino acid sequence containing SEQ ID NO:56 (AB-29).

38. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:28; and b) The V L The amino acid sequence containing SEQ ID NO:69 (AB-30).

39. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:29; and b) The V L The amino acid sequence containing SEQ ID NO:70 (AB-31).

40. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:30; and b) The V L The amino acid sequence (AB-32) containing SEQ ID NO:

71.

41. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:31; and b) The V L The amino acid sequence containing SEQ ID NO:72 (AB-33).

42. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:32; and b) The V L The amino acid sequence containing SEQ ID NO:67 (AB-34).

43. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H The amino acid sequence containing SEQ ID NO:33; and b) The V L The amino acid sequence (AB-35) containing SEQ ID NO:

56.

44. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H The amino acid sequence containing SEQ ID NO:34; and b) The V L The amino acid sequence containing SEQ ID NO:73 (AB-36).

45. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:35; and b) The V L The amino acid sequence containing SEQ ID NO:51 (AB-37).

46. ​​The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:36; and b) The V L The amino acid sequence (AB-38) containing SEQ ID NO:

56.

47. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H The amino acid sequence containing SEQ ID NO:37; and b) The V L The amino acid sequence containing SEQ ID NO:63 (AB-39).

48. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H The amino acid sequence containing SEQ ID NO:38; and b) The V L The amino acid sequence containing SEQ ID NO:69 (AB-40).

49. The polypeptide according to any one of claims 1 to 8, wherein: a) The V H Contains the amino acid sequence of SEQ ID NO:39; and b) The V L The amino acid sequence containing SEQ ID NO:74 (AB-41).

50. The polypeptide according to any one of claims 1 to 8, wherein: c) The V H Contains the amino acid sequence of SEQ ID NO:40; and d) The V L The amino acid sequence containing SEQ ID NO:52 (AB-42).

51. The polypeptide according to any one of claims 1 to 8, wherein: e) The V H Contains the amino acid sequence of SEQ ID NO:41; and f) The V L The amino acid sequence containing SEQ ID NO:51 (AB-43).

52. The polypeptide according to any one of claims 1 to 8, wherein: g) The V H Contains the amino acid sequence of SEQ ID NO:42; and h) The V L The amino acid sequence containing SEQ ID NO:75 (AB-44).

53. The polypeptide according to any one of claims 1 to 8, wherein: i) The V H Contains the amino acid sequence of SEQ ID NO:43; and j) The V L The amino acid sequence containing SEQ ID NO:56 (AB-45).

54. The polypeptide according to any one of claims 1 to 8, wherein: k) V H Contains the amino acid sequence of SEQ ID NO:44; and l) The V L The amino acid sequence (AB-46) containing SEQ ID NO:

51.

55. The polypeptide according to any one of claims 1 to 8, wherein: m) V H Contains the amino acid sequence of SEQ ID NO:45; and n) The V L The amino acid sequence containing SEQ ID NO:75 (AB-47).

56. The polypeptide according to any one of claims 1 to 8, wherein: o) The V H Contains the amino acid sequence of SEQ ID NO:46; and p) The V L The amino acid sequence containing SEQ ID NO:53 (AB-48).

57. The polypeptide according to any one of claims 1 to 8, wherein: q) The V H The amino acid sequence containing SEQ ID NO:47; and r) the V L The amino acid sequence containing SEQ ID NO:52 (AB-49).

58. The polypeptide according to any one of claims 1 to 8, wherein: s) The V H The amino acid sequence containing SEQ ID NO:48; and t) The V L The amino acid sequence containing SEQ ID NO:76 (AB-50).

59. The polypeptide according to any one of claims 1 to 8, wherein: u) The V H Contains the amino acid sequence of SEQ ID NO:3; and v) The V L The amino acid sequence containing SEQ ID NO:56 (AB-51).

60. A polypeptide that specifically binds to the spike glycoprotein of SARS-CoV-2, said polypeptide comprising an immunoglobulin heavy chain variable domain (V... H ), the V H The amino acid sequence containing SEQ ID NO:2, wherein: X1 is not S; X2 is not D; X3 is not T; X4 is not L; X5 is not S; X6 is not N; X7 is not G; X8 is not V; or X9 is not Q. Or any combination thereof.

61. The polypeptide of claim 60, comprising an immunoglobulin light chain variable domain (V... L ), the V L The amino acid sequence containing SEQ ID NO:49 includes: X 10 Not Q; X 11 Not G; X 12 Not S; X 13 Not S; X 14 Not N; X 15 Not S; X 16 Not F; or X 17 Not Y, Or any combination thereof.

62. A polypeptide that specifically binds to the spike glycoprotein of SARS-CoV-2, said polypeptide comprising an immunoglobulin heavy chain variable domain (V... H ), the V H The amino acid sequence containing SEQ ID NO:2, wherein: X1 is S, N, A, R, L, or F; X2 is either D or E; X3 is either T or V; X4 is either 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 thereof.

63. The polypeptide according to claim 62, wherein: 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 either N or L; X8 is I, S, or K; or X9 is Y, K, F, or H. Or any combination thereof.

64. The polypeptide according to claim 62 or 63, comprising an immunoglobulin light chain variable domain (V... L ), the V L The amino acid sequence containing SEQ ID NO:49 includes: X 10 It is Q, K, or I; X 11 Is it G or S; X 12 Is it S, R, or V; X 13 It is S or N; X 14 It is N, H, D, Y, or S; X 15 Is it S or Q? X 16 It is F, Y, L, V, T, or D; or X 17 Is it Y or L? Or any combination thereof.

65. The polypeptide according to claim 64, wherein: X 10 It is K or I; X 11 It is S; X 12 It is R or V; X 13 It is N; X 14 It is H, D, Y, or S; X 15 It's Q; X 16 It is Y, L, V, T, or D; or X 17 It is L. Or any combination thereof.

66. The polypeptide according to any one of claims 1 to 65, wherein the V H and the V L It is humanized and contains human frame regions or combinations thereof.

67. The polypeptide according to any one of claims 1 to 66, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.

68. The polypeptide of claim 67, wherein the antigen-binding fragment is selected from Fab, F(ab')2, Fab', scFv or Fv.

69. The polypeptide of claim 67, 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.

70. The polypeptide of claim 69, wherein the antibody heavy chain constant domain is selected from the group consisting of: IgA constant domain, IgD constant domain, IgE constant domain, IgG constant domain and IgM constant domain.

71. The polypeptide of claim 70, wherein the antibody heavy chain constant domain is an IgG1 heavy chain constant domain.

72. The polypeptide according to any one of claims 69 to 71, comprising an antibody light chain constant domain selected from the group consisting of a κ constant domain or a λ constant domain.

73. The polypeptide of claim 72, wherein the antibody light chain constant domain is a κ light chain constant domain.

74. The polypeptide according to any one of claims 1 to 73, wherein the polypeptide is conjugated with a heterologous portion.

75. The polypeptide of claim 74, wherein the heterologous portion is a therapeutic agent, a diagnostic agent, or a combination thereof.

76. The polypeptide of claim 74, wherein the heterologous portion is selected from the group consisting of: polyethylene glycol (PEG), hexadecanoic acid, hydrogel, lipid nanoparticles, polymer nanoparticles and heterologous polypeptide sequences or combinations thereof.

77. The polypeptide of claim 76, wherein the polymer nanoparticles comprise poly(lactic-co-glycolic acid) (PLGA).

78. The polypeptide of claim 74, wherein the heterologous polypeptide sequence comprises a carrier polypeptide.

79. The polypeptide of claim 78, wherein the carrier polypeptide is albumin or an Fc polypeptide.

80. The polypeptide according to any one of claims 1 to 79, wherein the polypeptide: a) with 1 μM or less K D Binds to SARS-CoV-2; b) with an IC50 concentration of approximately 25,000 ng / mL or less. 50 Neutralizes SARS-CoV-2 infection in human host cells; c) Reduce the infectivity of SARS-CoV-2 in human cells. Or any combination thereof.

81. The polypeptide of claim 80, wherein the SARS-CoV-2 is a variant comprising T19R, 156del, 157del, R158G, L452R, T478K, D614G, P681R and D950N and optionally comprising G142D.

82. The polypeptide according to claim 80 or 81, wherein the polypeptide is in a K+ of 100 nM or less. D It binds to SARS-CoV-2.

83. The polypeptide of claim 80, wherein the polypeptide is expressed at an IC50 concentration of about 25,000 ng / mL or less. 50 Neutralizes SARS-CoV-2 infection in human host cells.

84. The polypeptide of claim 80, wherein the polypeptide reduces the infectivity of SARS-CoV-2 in human cells by at least about 30%.

85. A composition comprising a polypeptide according to any one of claims 1 to 84.

86. The composition of claim 85, comprising one or more pharmaceutical excipients, diluents, or carriers.

87. A method of treating a subject who has or is suspected of having COVID-19, the method comprising administering to the subject an effective amount of the composition according to claim 85 or 86.

88. A method for reducing the infectivity of β-coronaviruses such as SARS-CoV-2 in cells of a subject, the method comprising contacting the cells with an effective amount of the composition according to claim 85 or 86.

89. The method according to claim 87 or 88, wherein the method comprises administering to the subject a therapeutically effective amount of an additional therapeutic or preventative agent.

90. The method of claim 89, wherein 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.

91. The method according to claim 90, wherein: a) The additional SARS-CoV-2 spike-binding antibodies are selected from the group consisting of: bamlanivimab, etesevimab, bebtelovimab, casirivimab, imdevimab, cilgavimab, tixagevimab, AZD7442 (tixagevimab-cigarette), Regdanvimab, sotrovimab, and combinations thereof; b) The antiviral agents are selected from the group consisting of: Molnupivir (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 targeting ACE2, siRNA targeting ACE2, CRISPR-based inhibitors of ACE2, soluble ACE2, soluble ACE2 variants, anti-ACE2 antibodies, and combinations thereof. d) The antibiotic mentioned includes azithromycin; e) The antimalarial agent contains chloroquine; f) The vaccine is a nucleic acid vaccine or an inactivated viral vaccine; or g) Its combination.

92. The method according to any one of claims 87 to 91, wherein the subject is further treated with at least one antibody or antigen-binding fragment thereof that binds to the receptor-binding domain (RBD) epitope of the SARS-CoV-2 spike protein.

93. A fusion protein comprising a polypeptide according to any one of claims 1 to 84.

94. 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 claims 1 to 84 or a fusion protein according to claim 93.

95. A vector (e.g., an expression vector, including a viral delivery vector) comprising the polynucleotide according to claim 94.

96. A host cell comprising the polynucleotide of claim 94 or the vector of claim 95.

97. A composition comprising a polypeptide according to any one of claims 1 to 84, a fusion protein according to claim 93, or a polynucleotide according to claim 94.

98. The composition of claim 97, comprising one or more pharmaceutical excipients, diluents, or carriers.

99. A polypeptide that specifically binds to the S2 domain of the β-coronavirus spike glycoprotein, wherein the polypeptide has one or more properties selected from: Broad neutralizing activity against a variety of known and predicted beta coronaviruses; Binding affinity for highly conserved S2 domain epitopes across multiple β-coronaviruses; and Inhibitory activity against potential emerging β-coronavirus escape variants.

100. A polypeptide that specifically binds to the spike glycoprotein of SARS-CoV-2, said polypeptide comprising a complementary site substantially similar to the complementary site of an antibody, said antibody comprising an amino acid sequence selected from: 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 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:3 ... 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 thereof.

101. The polypeptide of claim 100, comprising an immunoglobulin heavy chain variable domain (V... H ) and immunoglobulin light chain variable domain (V L ).

102. The polypeptide according to claim 100 or 101, comprising a complementary site identical to the complementary site of the antibody, said antibody comprising an amino acid sequence selected from: 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 ... 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).

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