Antibodies, fusion proteins for treating coronavirus and uses thereof

Abstract

The present application provides antibodies, fusion proteins and their uses for treating coronavirus. The 6-HB interfering polypeptide in the fusion protein of the present application cooperates with the antibody portion or antigen binding fragment to prevent SARS-CoV or SARS-CoV-2 virus particles from fusing with cells, and to mediate phagocytosis of immune cells, clearing virus particles.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to antibodies, fusion proteins and their applications for treating coronaviruses.

[0002] background

[0003] Coronaviruses are non-segmented, single-stranded, positive-sense RNA viruses. Based on serotype and genomic characteristics, the Coronaviridae subfamily is divided into four genera: α, β, γ, and δ. They are named for the flower-like shape of the protrusions extending outwards from their viral envelope. The novel coronavirus discovered in 2019 (SARS-CoV-2 or 2019-nCoV) belongs to the β genus. It is enveloped, and its particles are round or oval, often pleomorphic, with a diameter of 60-140 nm. Current research shows that SARS-CoV-2 and SARS-CoV share high homology.

[0004] COVID-19 is primarily transmitted through the respiratory tract, but can also spread through contact. The general population is susceptible, with the elderly and those with underlying medical conditions experiencing more severe illness. Children and infants can also develop the disease. Based on current epidemiological investigations, the incubation period for COVID-19 is generally 1-14 days, with most cases occurring between 3-7 days. The main clinical symptoms are fever, fatigue, and dry cough, while upper respiratory symptoms such as nasal congestion and runny nose are less common. In the early stages of the disease, patients may have a normal or decreased total white blood cell count, or a decreased number of lymphocytes. Some patients may exhibit elevated liver enzymes, muscle enzymes, and myoglobin. Chest imaging initially shows multiple small patchy opacities and interstitial changes, more pronounced in the peripheral lung fields; this progresses to multiple ground-glass opacities and infiltrates in both lungs. In severe cases, pulmonary consolidation may occur, gradually leading to dyspnea, and in severe cases, acute respiratory distress syndrome (ARDS), shock, and damage and dysfunction of multiple tissues including the lungs, heart, and kidneys. Most patients with mild infection have a good prognosis, while severe cases are often critically ill and may even result in death.

[0005] Recently, numerous basic, clinical, and epidemiological studies on COVID-19 have been published or released, yet effective therapeutic drugs remain lacking. Summary of the Invention

[0006] The present invention provides an antibody or antigen-binding fragment having high affinity for the spike protein of SARS-CoV and SARS-CoV-2, comprising a fusion protein thereof.

[0007] This invention provides an antibody or antigen-binding fragment with high affinity for the spike protein of SARS-CoV and SARS-CoV-2. These antibodies can specifically bind to the spike protein, preventing viral particles from binding to cells and mediating phagocytosis and clearance of viral particles by immune cells. These antibodies can be used to treat or improve SARS and COVID-19, and can also be used for the diagnosis of SARS and COVID-19.

[0008] SARS-CoV or SARS-CoV-2 enters lung epithelial cells by binding to angiotensin-converting enzyme 2 (ACE2) on its surface via its spike protein (S protein or spike spike protein). The cell then synthesizes new viral particles. These new viral particles are released outside the cell and infect surrounding normal cells in the same way. Antibodies against the spike protein can block the binding of the spike protein to ACE2, thereby blocking viral entry into cells and exerting an antiviral effect. The antibodies of this invention can also mediate the phagocytosis and clearance of viruses by immune cells.

[0009] Some embodiments provide an antibody or antigen-binding fragment that specifically binds to the spike protein and comprises:

[0010] (a) VH CDR1, comprising the amino acid sequence shown in SEQ ID NO:1, or a variant thereof with a single-site substitution, deletion, or insertion; (b) VH CDR2, comprising the amino acid sequence shown in SEQ ID NO:2, or a variant thereof with a single-site substitution, deletion, or insertion; and / or (c) VH CDR3, comprising the amino acid sequence shown in any one of SEQ ID NO:3-38 or 105, or a variant thereof with a single-site substitution, deletion, or insertion.

[0011] Some embodiments provide an antibody or antigen-binding fragment that specifically binds to the spike protein and comprises:

[0012] (a) VH CDR1, comprising the amino acid sequence shown in SEQ ID NO:1, or a variant thereof with a single-site substitution, deletion, or insertion; (b) VH CDR2, comprising the amino acid sequence shown in SEQ ID NO:2, or a variant thereof with a single-site substitution, deletion, or insertion; and (c) VH CDR3, comprising the amino acid sequence shown in any one of SEQ ID NO:3-38 or 105, or a variant thereof with a single-site substitution, deletion, or insertion.

[0013] Some embodiments provide an antibody or antigen-binding fragment that specifically binds to the spike protein and comprises:

[0014] (a) VH CDR1, which contains the amino acid sequence shown in SEQ ID NO:1; (b) VH CDR2, which contains the amino acid sequence shown in SEQ ID NO:2; and (c) VH CDR3, which contains the amino acid sequence shown in any one of SEQ ID NO:3-38 or 105.

[0015] Some embodiments provide an antibody or antigen-binding fragment that specifically binds to the spike protein and comprises:

[0016] (a) VH CDR1, comprising the amino acid sequence shown in SEQ ID NO:1, or a variant thereof with a single-site substitution, deletion, or insertion; (b) VH CDR2, comprising the amino acid sequence shown in SEQ ID NO:2, or a variant thereof with a single-site substitution, deletion, or insertion; (c) VH CDR3, comprising the amino acid sequence shown in any one of SEQ ID NO:3-38 or 105, or a variant thereof with a single-site substitution, deletion, or insertion; (d) VL CDR1, comprising the amino acid sequence shown in SEQ ID NO:39, or a variant thereof with a single-site substitution, deletion, or insertion; (e) VL CDR2, comprising the amino acid sequence shown in SEQ ID NO:40, or a variant thereof with a single-site substitution, deletion, or insertion; and / or (f) VL CDR3, comprising the amino acid sequence shown in SEQ ID NO:41, or a variant thereof with a single-site substitution, deletion, or insertion.

[0017] In some embodiments, the antibody or antigen-binding fragment specifically binds to the spike protein and comprises:

[0018] (a) VH CDR1, comprising the amino acid sequence shown in SEQ ID NO:1, or a variant thereof with a single-site substitution, deletion, or insertion; (b) VH CDR2, comprising the amino acid sequence shown in SEQ ID NO:2, or a variant thereof with a single-site substitution, deletion, or insertion; (c) VH CDR3, comprising the amino acid sequence shown in any one of SEQ ID NO:3-38 or 105, or a variant thereof with a single-site substitution, deletion, or insertion; (d) VL CDR1, comprising the amino acid sequence shown in SEQ ID NO:39, or a variant thereof with a single-site substitution, deletion, or insertion; (e) VL CDR2, comprising the amino acid sequence shown in SEQ ID NO:40, or a variant thereof with a single-site substitution, deletion, or insertion; and (f) VL CDR3, comprising the amino acid sequence shown in SEQ ID NO:41, or a variant thereof with a single-site substitution, deletion, or insertion.

[0019] In some embodiments, the substitution variant is a conserved amino acid substitution variant.

[0020] In some embodiments, the antibody or antigen-binding fragment specifically binds to the spike protein, and the antibody or antigen-binding fragment comprises at least one, two, three, four, five, or all of the following: VH CDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in any one of SEQ ID NO:3-38 or 105, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0021] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in any one of SEQ ID NO:3-38 or 105, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VLCDR3 as shown in SEQ ID NO:41.

[0022] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:3, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0023] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:4, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0024] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:5, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0025] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:6, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0026] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:7, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0027] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:8, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0028] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:9, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0029] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:10, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0030] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:11, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0031] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:12, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0032] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:13, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0033] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:14, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0034] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:15, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0035] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:16, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0036] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:17, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0037] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:18, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0038] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:19, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0039] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:20, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0040] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:21, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0041] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:22, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0042] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:23, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0043] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:24, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0044] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:25, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0045] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:26, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0046] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:27, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0047] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:28, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0048] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:29, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0049] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:30, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0050] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:31, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0051] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:32, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0052] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:33, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0053] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:34, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0054] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:35, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0055] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:36, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0056] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:37, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0057] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:38, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0058] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:105, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0059] In some embodiments, the frame region of the heavy chain variable region of the antibody or antigen-binding fragment includes heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4, wherein the heavy chain FR1 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:42, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:42, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:42; and / or

[0060] The heavy chain FR2 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:43, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:43, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:43; and / or

[0061] The heavy chain FR3 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:44, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:44, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:44; and / or

[0062] The heavy chain FR4 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:45, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:45, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:45.

[0063] In some embodiments, the heavy chain FR1 comprises the sequence shown in SEQ ID NO:42, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:42, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:42; the heavy chain FR2 comprises the sequence shown in SEQ ID NO:43, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:43, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:43; the heavy chain FR3 comprises the sequence shown in SEQ ID NO:44, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:44, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:44; and the heavy chain FR4 comprises the sequence shown in SEQ ID NO:45, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:45, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:45. In some implementations, one or more conserved amino acid substitutions are made by about 1, about 2, or about 3 conserved amino acid substitutions.

[0064] In some embodiments, the heavy chain FR1 comprises the sequence shown in SEQ ID NO:42, the heavy chain FR2 comprises the sequence shown in SEQ ID NO:43, the heavy chain FR3 comprises the sequence shown in SEQ ID NO:44, and the heavy chain FR4 comprises the sequence shown in SEQ ID NO:45. In some embodiments, the variable region of the heavy chain comprises the structure FR1-VH CDR1-FR2-VH CDR2-FR3-VH CDR3-FR4.

[0065] Some embodiments provide an antibody or antigen-binding fragment that specifically binds to the spike protein and comprises: (a) VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO:60, or a variant thereof with a single-site substitution, deletion, or insertion; (b) VH CDR2, which comprises the amino acid sequence shown in SEQ ID NO:61, or a variant thereof with a single-site substitution, deletion, or insertion; and / or (c) VH CDR3, which comprises the amino acid sequence shown in any one of SEQ ID NO:62-97 or 106, or a variant thereof with a single-site substitution, deletion, or insertion.

[0066] Some embodiments provide an antibody or antigen-binding fragment that specifically binds to the spike protein and comprises:

[0067] (a) VH CDR1, which contains the amino acid sequence shown in SEQ ID NO:60; (b) VH CDR2, which contains the amino acid sequence shown in SEQ ID NO:61; and (c) VH CDR3, which contains the amino acid sequence shown in any one of SEQ ID NO:62-97 or 106.

[0068] Some embodiments provide an antibody or antigen-binding fragment that specifically binds to a spike protein and comprises: (a) VH CDR1, comprising the amino acid sequence shown in SEQ ID NO:60, or a variant thereof with a single-site substitution, deletion, or insertion; (b) VH CDR2, comprising the amino acid sequence shown in SEQ ID NO:61, or a variant thereof with a single-site substitution, deletion, or insertion; (c) VH CDR3, comprising the amino acid sequence shown in any one of SEQ ID NO:62-97 or 106, or a variant thereof with a single-site substitution, deletion, or insertion; (d) VL CDR1, comprising the amino acid sequence shown in SEQ ID NO:98, or a variant thereof with a single-site substitution, deletion, or insertion; (e) VLCDR2, comprising the amino acid sequence shown in SEQ ID NO:99, or a variant thereof with a single-site substitution, deletion, or insertion; and / or (f) VL CDR3, comprising the amino acid sequence shown in SEQ ID NO:100, or a variant thereof with a single-site substitution, deletion, or insertion. In some embodiments, the substitution variant is a conserved amino acid substitution variant.

[0069] In some embodiments, the antibody or antigen-binding fragment comprises at least one, two, three, four, five, or all of the following: VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in any of SEQ ID NO:62-97 or 106, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0070] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in any one of SEQ ID NO:62-97 or 106, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0071] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:62, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0072] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:63, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0073] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:64, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0074] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:65, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0075] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:66, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0076] In some embodiments, the antibody or antigen-binding fragment includes at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:67, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0077] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:68, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0078] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:69, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0079] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:70, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0080] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:71, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0081] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:72, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0082] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:73, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0083] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:74, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0084] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:75, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0085] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:76, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0086] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:77, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0087] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:78, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0088] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:79, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0089] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:80, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0090] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:81, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0091] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:82, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0092] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:83, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0093] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:84, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0094] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:85, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0095] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:86, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0096] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:87, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0097] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:88, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0098] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:89, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0099] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:90, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0100] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:91, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0101] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:92, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0102] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:93, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0103] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:94, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0104] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:95, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0105] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:96, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0106] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:97, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0107] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:106, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0108] In some embodiments, the heavy chain FR1 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:101, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:101, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:101; and / or the heavy chain FR2 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:102, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:102, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:102; and / or

[0109] The heavy chain FR3 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:103, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:103, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:103; and / or

[0110] The heavy chain FR4 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:104, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:104, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:104.

[0111] In some embodiments, the heavy chain FR1 contains the sequence shown in SEQ ID NO:101, the heavy chain FR2 contains the sequence shown in SEQ ID NO:102, the heavy chain FR3 contains the sequence shown in SEQ ID NO:103, and the heavy chain FR4 contains the sequence shown in SEQ ID NO:104.

[0112] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in any one of SEQ ID NO:46-49 or 107, a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NO:46-49 or 107, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in any one of SEQ ID NO:46-49 or 107.

[0113] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:50, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:50, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:50.

[0114] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in any one of SEQ ID NO:46-49 or 107, and / or the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:50. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:46, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:50. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:47, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:50. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:48, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:50. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:49, and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:50. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes the sequence shown in SEQ ID NO:107, and the light chain variable region of the antibody or antigen-binding fragment includes the sequence shown in SEQ ID NO:50.

[0115] In some embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a κ or λ chain constant region. In some embodiments, the antibody or fragment thereof is an isotype of IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4. Without limitation, the antibody or antigen-binding fragment is a chimeric antibody, a humanized antibody, or a fully human antibody. In one aspect, the antibody or antigen-binding fragment is a humanized antibody.

[0116] In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is scFV, Fab, F(ab)2, or IgG. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.

[0117] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:51, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:51, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:51; and / or

[0118] The light chain constant region of the antibody or antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:52, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:52, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:52.

[0119] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment contains the amino acid sequence as shown in SEQ ID NO:51, and / or the light chain constant region of the antibody or antigen-binding fragment contains the amino acid sequence as shown in SEQ ID NO:52. In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment contains the amino acid sequence as shown in SEQ ID NO:51, and the light chain constant region of the antibody or antigen-binding fragment contains the amino acid sequence as shown in SEQ ID NO:52.

[0120] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO:53-56 or 108, a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NO:53-56 or 108, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in any one of SEQ ID NO:53-56 or 108; and / or

[0121] The light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:57, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:57.

[0122] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO:53-56 or 108, and / or the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57.

[0123] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:53, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:57. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:57. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:55, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:57. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:56, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:57. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:108, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:57.

[0124] In some embodiments, the antibody comprises two identical heavy chains and two identical light chains. In some embodiments, the antibody or antigen-binding fragment can specifically bind to the spike protein and block the binding of SARS-CoV or SARS-CoV-2 viral particles to cells, as well as mediate phagocytosis and clearance of viral particles by immune cells. In some embodiments, the antibody or antigen-binding fragment can specifically bind to the S1 subunit of the spike protein.

[0125] This invention also provides a fusion protein. The antibody portion or antigen-binding fragment in the fusion protein has high affinity for the spike proteins of SARS-CoV and SARS-CoV-2, and these antibodies can specifically bind to the spike proteins; the 6-HB interfering polypeptide in the fusion protein can interfere with the formation of the "six-helix bundle (6-HB)". The fusion protein of this invention can prevent the fusion of viral particles and cell membranes and can mediate the phagocytosis and clearance of viral particles by immune cells. These fusion proteins can be used to treat or improve SARS and COVID-19, and can also be used for the diagnosis of SARS and COVID-19.

[0126] Viral particles first bind to angiotensin-converting enzyme 2 (ACE2) on the surface of lung epithelial cells via the receptor-binding domain (RBD) of the S1 subunit of the spike protein (S protein). After binding to the receptor and being hydrolyzed by the protease, the S2 subunit located at the N-terminus of the S protein is exposed and embedded in the plasma membrane or endosome membrane. The HR2 (heptad repeated 2) domain of the S2 subunit binds to the HR1 (heptad repeated 1) domain of the S2 subunit, forming a 6-HB fusion core. This leads to the fusion of the viral capsid with the cell membrane, allowing SARS-CoV or SARS-CoV-2 to enter the cell and utilize the cell to synthesize new viral particles. These new viral particles are released extracellularly and infect surrounding normal cells in the same way. Antibodies in the fusion protein can block the binding of the spike protein to ACE2, and the 6-HB interfering polypeptide portion prevents the fusion of the viral capsid with the cell membrane, thereby blocking viral entry into the cell and exerting an antiviral effect. The antibody portion of the fusion protein can also mediate phagocytosis and clearance of the virus by immune cells.

[0127] Some embodiments provide a fusion protein comprising an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, the antibody or antigen-binding fragment specifically binding to a spike protein and comprising: (a) VH CDR1, comprising the amino acid sequence shown in SEQ ID NO:1, or a variant thereof having a single-site substitution, deletion, or insertion; (b) VHCDR2, comprising the amino acid sequence shown in SEQ ID NO:2, or a variant thereof having a single-site substitution, deletion, or insertion; and / or (c) VH CDR3, comprising the amino acid sequence shown in any one of SEQ ID NO:3-38 or 105, or a variant thereof having a single-site substitution, deletion, or insertion;

[0128] The C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to a 6-HB interfering polypeptide via a linker. In some embodiments, the linker is a polypeptide containing glycine and serine.

[0129] Some embodiments provide a fusion protein comprising an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, the antibody or antigen-binding fragment specifically binding to a spike protein and comprising: (a) VH CDR1, comprising the amino acid sequence shown in SEQ ID NO:1, or a variant thereof having a single-site substitution, deletion, or insertion; (b) VHCDR2, comprising the amino acid sequence shown in SEQ ID NO:2, or a variant thereof having a single-site substitution, deletion, or insertion; and (c) VH CDR3, comprising the amino acid sequence shown in any one of SEQ ID NO:3-38 or 105, or a variant thereof having a single-site substitution, deletion, or insertion.

[0130] The C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to a 6-HB interfering polypeptide via a linker. In some embodiments, the linker is a polypeptide containing glycine and serine.

[0131] Some embodiments provide a fusion protein comprising an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, wherein the antibody or antigen-binding fragment specifically binds to the spike protein and comprises:

[0132] (a) VH CDR1, which contains the amino acid sequence shown in SEQ ID NO:1; (b) VH CDR2, which contains the amino acid sequence shown in SEQ ID NO:2; and (c) VH CDR3, which contains the amino acid sequence shown in any one of SEQ ID NO:3-38 or 105;

[0133] The C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to a 6-HB interfering polypeptide via a linker. In some embodiments, the linker is a polypeptide containing glycine and serine.

[0134] Some embodiments provide a fusion protein comprising an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, the antibody or antigen-binding fragment specifically binding to a spike protein and comprising: (a) VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:1, or a variant thereof having a single-site substitution, deletion, or insertion; (b) VHCDR2 comprising the amino acid sequence shown in SEQ ID NO:2, or a variant thereof having a single-site substitution, deletion, or insertion; (c) VH CDR3 comprising the amino acid sequence shown in any one of SEQ ID NO:3-38 or 105, or a variant thereof having a single-site substitution, deletion, or insertion; (d) VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:39, or a variant thereof having a single-site substitution, deletion, or insertion; (e) VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:40, or a variant thereof having a single-site substitution, deletion, or insertion; and / or (f) VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:1 ... (c) VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:1, or a variant thereof having a single-site substitution, deletion, or insertion; and (d) VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:39, or a variant thereof having a single-site substitution, deletion, or insertion; and (e) VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:40, or a variant thereof having a single-site substitution, deletion, or insertion; and / or (f) VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in NO:41, or a variant thereof with a single site substitution, deletion, or insertion;

[0135] The C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to a 6-HB interfering polypeptide via a linker. In some embodiments, the linker is a polypeptide containing glycine and serine.

[0136] In some embodiments, the fusion protein comprises an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, the antibody or antigen-binding fragment specifically binding to the spike protein and comprising: (a) VH CDR1, comprising the amino acid sequence shown in SEQ ID NO:1, or a variant thereof having a single-site substitution, deletion, or insertion; (b) VH CDR2, comprising the amino acid sequence shown in SEQ ID NO:2, or a variant thereof having a single-site substitution, deletion, or insertion; (c) VH CDR3, comprising the amino acid sequence shown in any one of SEQ ID NO:3-38 or 105, or a variant thereof having a single-site substitution, deletion, or insertion; (d) VL CDR1, comprising the amino acid sequence shown in SEQ ID NO:39, or a variant thereof having a single-site substitution, deletion, or insertion; (e) VL CDR2, comprising the amino acid sequence shown in SEQ ID NO:40, or a variant thereof having a single-site substitution, deletion, or insertion; and (f) VL CDR3, comprising the amino acid sequence shown in SEQ ID NO:1 ...c) VH CDR3, comprising the amino acid sequence shown in SEQ ID NO:1, or a variant thereof having a single-site substitution, deletion, or insertion; and (d) VL CDR1, comprising the amino acid sequence shown in SEQ ID NO:39, or a variant thereof having a single-site substitution, deletion, or insertion; and (e) VL CDR2, comprising the amino acid sequence shown in SEQ ID NO:40, or a variant thereof having a single-site substitution, deletion, or insertion; and (f) VL CDR3, comprising the amino acid sequence shown in SEQ ID NO:1, or a variant thereof having a single-site substitution, deletion, or insertion; and (f) VL CDR3, comprising the amino acid sequence shown The amino acid sequence shown in NO:41, or a variant thereof with a single site substitution, deletion, or insertion;

[0137] The C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to a 6-HB interfering polypeptide via a linker. In some embodiments, the linker is a polypeptide containing glycine and serine. In some embodiments, the substitution variant is a conserved amino acid substitution variant.

[0138] In some implementations, the sequence of the connector is (G m S) n Each m is independently 2, 3, 4, or 5, and n is 1, 2, 3, 4, or 5. In some embodiments, the sequence of the connectors is (GGGGS). n The n is 1, 2, 3, 4, or 5. In some embodiments, the connector is GGGGS. In some embodiments, the connector is (GGGGS)2. In some embodiments, the connector is (GGGGS)3. In some embodiments, the connector is (GGGGS)4, as shown in SEQ ID NO:59. In some embodiments, the connector is (GGGGS)5.

[0139] In some embodiments, the 6-HB interfering polypeptide comprises the sequence shown in SEQ ID NO:58, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:58, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:58.

[0140] In some embodiments, the fusion protein comprises an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, and the fusion protein includes the following characteristics:

[0141] The antibody or antigen-binding fragment comprises at least one, two, three, four, five, or all of the following: VH CDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:3-38 or 105, VLCDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41; and / or

[0142] The C-terminus of the antibody or antigen-binding fragment is linked to a 6-HB interfering peptide via a linker, wherein the C-terminus of the antibody or antigen-binding fragment is the C-terminus of the heavy chain portion or the C-terminus of the light chain portion; and / or

[0143] The sequence of the connector is (GGGGS) n The n is 1, 2, 3, 4, or 5; and / or

[0144] The 6-HB interfering polypeptide comprises the sequence shown in SEQ ID NO:58, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:58, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:58.

[0145] In some embodiments, the fusion protein comprises an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, and the fusion protein includes the following characteristics:

[0146] The antibody or antigen-binding fragment comprises at least VH CDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in any one of SEQ ID NO:3-38 or 105, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41; and / or

[0147] The C-terminus of the antibody or antigen-binding fragment is linked to a 6-HB interfering peptide via a linker, wherein the C-terminus of the antibody or antigen-binding fragment is the C-terminus of the heavy chain portion or the C-terminus of the light chain portion; and / or

[0148] The sequence of the connector is (GGGGS) n The n is 1, 2, 3, 4, or 5; and / or

[0149] The 6-HB interfering polypeptide comprises the sequence shown in SEQ ID NO:58, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:58, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:58.

[0150] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:3, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0151] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:4, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0152] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:5, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0153] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:6, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0154] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:7, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0155] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:8, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0156] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:9, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0157] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:10, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0158] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:11, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0159] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:12, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0160] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:13, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0161] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:14, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0162] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:15, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0163] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:16, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0164] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:17, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0165] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:18, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0166] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:19, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0167] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:20, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0168] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:21, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0169] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:22, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0170] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:23, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0171] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:24, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0172] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:25, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0173] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:26, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0174] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:27, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0175] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:28, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0176] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:29, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0177] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:30, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0178] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:31, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0179] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:32, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0180] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:33, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0181] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:34, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0182] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:35, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0183] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:36, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0184] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:37, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0185] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:38, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0186] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:105, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:41.

[0187] In some embodiments, the frame region of the heavy chain variable region of the antibody or antigen-binding fragment includes heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4, wherein the heavy chain FR1 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:42, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:42, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:42; and / or

[0188] The heavy chain FR2 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:43, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:43, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:43; and / or

[0189] The heavy chain FR3 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:44, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:44, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:44; and / or

[0190] The heavy chain FR4 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:45, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:45, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:45.

[0191] In some embodiments, the heavy chain FR1 comprises the sequence shown in SEQ ID NO:42, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:42, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:42; the heavy chain FR2 comprises the sequence shown in SEQ ID NO:43, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:43, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:43; the heavy chain FR3 comprises the sequence shown in SEQ ID NO:44, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:44, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:44; and the heavy chain FR4 comprises the sequence shown in SEQ ID NO:45, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:45, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:45.

[0192] In some embodiments, the heavy chain FR1 comprises the sequence shown in SEQ ID NO:42, the heavy chain FR2 comprises the sequence shown in SEQ ID NO:43, the heavy chain FR3 comprises the sequence shown in SEQ ID NO:44, and the heavy chain FR4 comprises the sequence shown in SEQ ID NO:45.

[0193] Some embodiments provide a fusion protein comprising an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, the antibody or antigen-binding fragment specifically binding to a spike protein and comprising: (a) VH CDR1, comprising the amino acid sequence shown in SEQ ID NO:60, or a variant thereof having a single-site substitution, deletion, or insertion; (b) VH CDR2, comprising the amino acid sequence shown in SEQ ID NO:61, or a variant thereof having a single-site substitution, deletion, or insertion; and / or (c) VH CDR3, comprising the amino acid sequence shown in any one of SEQ ID NO:62-97 or 106, or a variant thereof having a single-site substitution, deletion, or insertion;

[0194] The C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to a 6-HB interfering polypeptide via a linker. In some embodiments, the linker is a polypeptide containing glycine and serine.

[0195] Some embodiments provide a fusion protein comprising an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, wherein the antibody or antigen-binding fragment specifically binds to the spike protein and comprises:

[0196] (a) VH CDR1, which contains the amino acid sequence shown in SEQ ID NO:60; (b) VH CDR2, which contains the amino acid sequence shown in SEQ ID NO:61; and (c) VH CDR3, which contains the amino acid sequence shown in any one of SEQ ID NO:62-97 or 106;

[0197] The C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to a 6-HB interfering polypeptide via a linker. In some embodiments, the linker is a polypeptide containing glycine and serine.

[0198] Some embodiments provide a fusion protein comprising an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, the antibody or antigen-binding fragment specifically binding to a spike protein and comprising: (a) VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:60, or a variant thereof having a single-site substitution, deletion, or insertion; (b) VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:61, or a variant thereof having a single-site substitution, deletion, or insertion; (c) VH CDR3 comprising the amino acid sequence shown in any one of SEQ ID NO:62-97 or 106, or a variant thereof having a single-site substitution, deletion, or insertion; (d) VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:98, or a variant thereof having a single-site substitution, deletion, or insertion; (e) VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:99, or a variant thereof having a single-site substitution, deletion, or insertion; and / or (f) VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:60 ... (c) VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:60, or a variant thereof having a single-site substitution, deletion, or insertion; and (d) VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:60, or a variant thereof having a single-site substitution, deletion, or insertion; and (e) VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:60, or a variant thereof having a single-site substitution, deletion, or insertion; and (f) VL CDR3 comprising the amino acid sequence shown The amino acid sequence shown in NO:100, or a variant thereof with a single site substitution, deletion, or insertion;

[0199] The C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to a 6-HB interfering polypeptide via a linker. In some embodiments, the linker is a polypeptide containing glycine and serine.

[0200] In some implementations, the sequence of the connector is (G m S) n Each m is independently 2, 3, 4, or 5, and n is 1, 2, 3, 4, or 5. In some embodiments, the sequence of the connectors is (GGGGS). nThe n is 1, 2, 3, 4, or 5. In some embodiments, the connector is GGGGS. In some embodiments, the connector is (GGGGS)2. In some embodiments, the connector is (GGGGS)3. In some embodiments, the connector is (GGGGS)4, as shown in SEQ ID NO:59. In some embodiments, the connector is (GGGGS)5.

[0201] In some embodiments, the 6-HB interfering polypeptide comprises the sequence shown in SEQ ID NO:58, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:58, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:58.

[0202] In some embodiments, the fusion protein comprises an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, and the fusion protein includes the following characteristics:

[0203] The antibody or antigen-binding fragment comprises at least one, two, three, four, five, or all of the following: VH CDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in any one of SEQ ID NO:62-97 or 106, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100; and / or

[0204] The C-terminus of the antibody or antigen-binding fragment is linked to a 6-HB interfering peptide via a linker, wherein the C-terminus of the antibody or antigen-binding fragment is the C-terminus of the heavy chain portion or the C-terminus of the light chain portion; and / or

[0205] The sequence of the connector is (GGGGS) n The n is 1, 2, 3, 4, or 5; and / or

[0206] The 6-HB interfering polypeptide comprises the sequence shown in SEQ ID NO:58, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:58, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:58.

[0207] In some embodiments, the fusion protein comprises an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, and the fusion protein includes the following characteristics:

[0208] The antibody or antigen-binding fragment comprises at least VH CDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in any one of SEQ ID NO:62-97 or 106, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100; and / or

[0209] The C-terminus of the antibody or antigen-binding fragment is linked to a 6-HB interfering peptide via a linker, wherein the C-terminus of the antibody or antigen-binding fragment is the C-terminus of the heavy chain portion or the C-terminus of the light chain portion; and / or

[0210] The sequence of the connector is (GGGGS) n The n is 1, 2, 3, 4, or 5; and / or

[0211] The 6-HB interfering polypeptide comprises the sequence shown in SEQ ID NO:58, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:58, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:58.

[0212] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in any one of SEQ ID NO:62-97 or 106, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0213] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:62, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0214] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:63, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0215] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:64, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0216] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:65, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0217] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:66, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0218] In some embodiments, the antibody or antigen-binding fragment includes at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:67, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0219] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:68, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0220] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:69, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0221] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:70, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0222] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:71, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0223] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:72, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0224] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:73, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0225] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:74, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0226] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:75, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0227] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:76, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0228] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:77, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0229] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:78, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0230] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:79, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0231] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:80, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0232] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:81, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0233] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:82, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0234] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:83, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0235] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:84, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0236] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:85, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0237] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:86, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0238] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:87, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0239] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:88, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0240] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:89, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0241] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:90, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0242] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:91, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0243] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:92, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0244] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:93, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0245] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:94, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0246] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:95, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0247] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:96, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0248] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:97, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0249] In some embodiments, the antibody or antigen-binding fragment comprises at least VHCDR1 as shown in SEQ ID NO:60, VH CDR2 as shown in SEQ ID NO:61, VH CDR3 as shown in SEQ ID NO:106, VL CDR1 as shown in SEQ ID NO:98, VL CDR2 as shown in SEQ ID NO:99, and VL CDR3 as shown in SEQ ID NO:100.

[0250] In some embodiments, the heavy chain FR1 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:101, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:101, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:101; and / or

[0251] The heavy chain FR2 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:102, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:102, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:102; and / or

[0252] The heavy chain FR3 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:103, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:103, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:103; and / or

[0253] The heavy chain FR4 of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:104, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:104, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:104.

[0254] In some embodiments, the heavy chain FR1 contains the sequence shown in SEQ ID NO:101, the heavy chain FR2 contains the sequence shown in SEQ ID NO:102, the heavy chain FR3 contains the sequence shown in SEQ ID NO:103, and the heavy chain FR4 contains the sequence shown in SEQ ID NO:104.

[0255] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment contains the sequence shown in any one of SEQ ID NO:46-49 or 107, and / or the light chain variable region of the antibody or antigen-binding fragment contains the sequence shown in SEQ ID NO:50.

[0256] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes the sequence shown in SEQ ID NO:46, and the light chain variable region of the antibody or antigen-binding fragment includes the sequence shown in SEQ ID NO:50. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes the sequence shown in SEQ ID NO:47, and the light chain variable region of the antibody or antigen-binding fragment includes the sequence shown in SEQ ID NO:50. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes the sequence shown in SEQ ID NO:48, and the light chain variable region of the antibody or antigen-binding fragment includes the sequence shown in SEQ ID NO:50. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes the sequence shown in SEQ ID NO:49, and the light chain variable region of the antibody or antigen-binding fragment includes the sequence shown in SEQ ID NO:50. In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment includes the sequence shown in SEQ ID NO:107, and the light chain variable region of the antibody or antigen-binding fragment includes the sequence shown in SEQ ID NO:50.

[0257] In some embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a κ or λ chain constant region. In some embodiments, the antibody or fragment thereof is an isotype of IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4. Without limitation, the antibody or antigen-binding fragment is a chimeric antibody, a humanized antibody, or a fully human antibody. In one aspect, the antibody or antigen-binding fragment is a humanized antibody.

[0258] In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is scFV, Fab, F(ab)2, or IgG. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.

[0259] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:51 or the sequence shown from amino acid position 1 to amino acid position 328 in SEQ ID NO:51, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO:51 or the sequence shown from amino acid position 1 to amino acid position 328 in SEQ ID NO:51, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:51 or the sequence shown from amino acid position 1 to amino acid position 328 in SEQ ID NO:51; and / or

[0260] The light chain constant region of the antibody or antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:52, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:52, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:52.

[0261] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:51, and the light chain constant region of the antibody or antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:52.

[0262] In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment contains an amino acid sequence as shown in amino acids 1 to 328 of SEQ ID NO:51, and the light chain constant region of the antibody or antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:52.

[0263] In some embodiments, a fusion protein is provided, the fusion protein comprising the following characteristics:

[0264] The heavy chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO:53-56, 108, or the sequence shown in any one of the amino acids from position 1 to position 450 in SEQ ID NO:53-56, 108, or a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NO:53-56, 108, or the sequence shown in any one of the amino acids from position 1 to position 450 in SEQ ID NO:53-56, 108, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in any one of SEQ ID NO:53-56, 108, or the sequence shown in any one of the amino acids from position 1 to position 450 in SEQ ID NO:53-56, 108; and / or

[0265] The light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:57, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:57; and / or

[0266] The C-terminus of the heavy chain or the C-terminus of the light chain of the antibody is covalently linked to a 6-HB interfering polypeptide via a linker as shown in SEQ ID NO:59, the 6-HB interfering polypeptide comprising the sequence shown in SEQ ID NO:58, a sequence having at least 90% or at least 95% identity with the sequence shown in SEQ ID NO:58, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:58.

[0267] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:53, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0268] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in amino acids 1 to 450 of SEQ ID NO:53, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0269] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:54, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0270] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in amino acids 1 to 450 of SEQ ID NO:54, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0271] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:55, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0272] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in amino acids 1 to 450 of SEQ ID NO:55, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0273] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:56, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0274] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in amino acids 1 to 450 of SEQ ID NO:56, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0275] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:108, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0276] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in amino acids 1 to 450 of SEQ ID NO:108, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0277] In some embodiments, the fusion protein comprises two identical heavy chains (or heavy chain fragments) and two identical light chains, with the C-terminus of each heavy chain covalently linked to the 6-HB interfering peptide via a linker. In some embodiments, the fusion protein comprises two identical heavy chains and two identical light chains, with the C-terminus of the light chains covalently linked to the 6-HB interfering peptide via a linker.

[0278] In some embodiments, the antibody portion or antigen-binding fragment in the fusion protein can specifically bind to the spike protein and prevent SARS-CoV or SARS-CoV-2 viral particles from binding to cells, as well as mediate phagocytosis and clearance of viral particles by immune cells. In some embodiments, the antibody or antigen-binding fragment in the fusion protein can specifically bind to the S1 subunit of the spike protein.

[0279] The present invention also provides a nucleic acid molecule encoding the aforementioned antibody, its antigen-binding fragment, or a fusion protein thereof. In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

[0280] The present invention also provides a vector comprising the aforementioned nucleic acid molecule. In some embodiments, the vector is a separate vector.

[0281] The present invention also provides a host cell comprising the aforementioned nucleic acid molecules. In some embodiments, the host cell is an isolated host cell.

[0282] The present invention also provides a host cell comprising the aforementioned vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, a 293 cell, a Cos1 cell, a Cos7 cell, a CV1 cell, or a mouse L cell.

[0283] The present invention also provides a pharmaceutical composition comprising the antibody, its antigen-binding fragment or fusion protein thereof, and a pharmaceutically acceptable carrier.

[0284] The present invention also provides treatment methods and uses. In some embodiments, a method for treating or improving SARS or COVID-19 is provided, the method comprising administering to a patient an effective dose of the said antibody, its antigen-binding fragment, or fusion protein. In some embodiments, the use of the said antibody, its antigen-binding fragment, or fusion protein in treating or improving SARS or COVID-19 is provided. In some embodiments, the use of the said antibody, its antigen-binding fragment, or fusion protein in the preparation of a medicament for treating or improving SARS or COVID-19 is provided.

[0285] This invention also provides diagnostic methods and uses. In some embodiments, a method is provided for detecting SARS-CoV or SARS-CoV-2 expression in a sample by contacting the sample with the stated antibody, its antigen-binding fragment, or fusion protein, such that the antibody, its antigen-binding fragment, or fusion protein binds to the spike protein, and the binding is detected, i.e., the amount of spike protein in the sample. In some embodiments, its use in the preparation of kits for diagnosing SARS or COVID-19 is provided. In some embodiments, a diagnostic kit comprising the stated antibody, its antigen-binding fragment, or fusion protein is provided.

[0286] This invention provides antibodies, antigen-binding fragments thereof, or fusion proteins for treating coronaviruses. The 6-HB interfering polypeptide in the fusion protein synergistically prevents the fusion of SARS-CoV or SARS-CoV-2 virus particles with cells, and mediates the phagocytosis and clearance of virus particles by immune cells, thereby treating SARS or COVID-19. The antibodies, antigen-binding fragments thereof, or the antibodies or antigen-binding fragments in the fusion proteins of this invention can also be used to diagnose whether a patient is infected with SARS-CoV or SARS-CoV-2. Attached Figure Description

[0287] Figure 1 The graph shows the inhibition of SARS-CoV-2 binding to ACE2 by some of the anti-spike protein antibodies of this invention in ELISA experiments. The horizontal axis represents the concentration and the vertical axis represents the OD value. In the graph, 1 represents antibody 1, 7 represents antibody 7, 8 represents antibody 8, 9 represents antibody 9, 12 represents antibody 12, 18 represents antibody 18, 19 represents antibody 19, 20 represents antibody 20, 21 represents antibody 21, and 22 represents antibody 22.

[0288] Figure 2 shows the binding of the antibody, fusion protein A, and spike S1 RBD; among them... Figure 2A The Spike S1RBD is wild-type. Figure 2B The Spike S1 RBD includes the wild type and its variants.

[0289] Figure 3 The fusion protein A was shown to block the binding of spike S1 RBD to ACE2.

[0290] Figure 4 The figure shows the binding of fusion protein A to CHO cells overexpressing spike protein; the vertical axis represents the average fluorescence intensity, and the horizontal axis represents the concentration.

[0291] Figure 5The figure shows that fusion protein A inhibits pseudovirus infection of Vero cells; the vertical axis represents the inhibition rate and the horizontal axis represents the concentration; sACE2 in the figure represents soluble ACE2 as a control.

[0292] the term

[0293] Unless otherwise stated, each of the following terms shall have the meaning described below.

[0294] definition

[0295] It should be noted that the term “a” entity refers to one or more of the same entity. For example, “an antibody” should be understood as one or more antibodies. Therefore, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably in this document.

[0296] The term "polypeptide" is intended to encompass both the singular and plural forms of "polypeptide" and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids and does not imply a specific length of the product. Therefore, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to two or more amino acid chains, and the term "polypeptide" can be used in place of any of the foregoing terms or interchangeably with any of the foregoing terms. The term "polypeptide" is also intended to refer to products modified after polypeptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. Polypeptides can be derived from natural biological sources or produced through recombinant technologies, but they do not necessarily have to be translated from a specified nucleic acid sequence; they can be produced in any manner, including chemical synthesis.

[0297] An amino acid is an organic compound containing both an amino group and a carboxyl group, such as an α-amino acid, which can be encoded by nucleic acids directly or in its precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code." Amino acids include both natural and non-natural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0298] "Conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) with similar chemical properties (such as charge or hydrophobicity). Generally, conservative amino acid substitution does not substantially change the functional properties of a protein. Examples of amino acid classes containing chemically similar side chains include: 1) Aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) Aliphatic hydroxyl side chains: serine and threonine; 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) Basic side chains: lysine, arginine, and histidine; 6) Acidic side chains: aspartic acid and glutamic acid.

[0299] The number of conserved amino acid substitutions in “VL, VH” is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, or any range between any two of these values ​​(including the endpoints) or any of these values. The number of amino acids in "conserved amino acid substitutions in the heavy chain constant region, light chain constant region, heavy chain or light chain, or the first or second polypeptide of the fusion protein" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, or about 45 conserved amino acid substitutions, or a range (including endpoints) of any two of these values, or any value therein. The term "isolated," as used in this invention with respect to cells, nucleic acids, polypeptides, antibodies, etc., such as "isolated" DNA, RNA, polypeptides, and antibodies, refers to molecules isolated from one or more other components such as DNA or RNA in the cellular native environment. The term "isolated" as used in this invention also refers to nucleic acids or peptides that are substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA technology, or chemical precursors or other chemicals used in chemical synthesis. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring and will not be naturally occurring. The term "isolated" is also used in this invention to refer to cells or polypeptides isolated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc., are typically prepared by at least one purification step. In some embodiments, the purity of the isolated nucleic acids, polypeptides, antibodies, etc., is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values ​​(including the endpoint) or any value therein.

[0300] The term “recombination” refers to polypeptides or polynucleotides, meaning forms of polypeptides or polynucleotides that do not exist naturally. Unrestricted embodiments can be created by combining polynucleotides or polypeptides that do not normally exist.

[0301] "Homology," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing comparable positions in each sequence. Molecules are homologous at those positions when the positions in the compared sequences are occupied by the same bases or amino acids. The degree of homology between sequences is a function of the number of shared matching or homologous positions.

[0302] "At least 80% identity" means approximately 80% identity, approximately 81% identity, approximately 82% identity, approximately 83% identity, approximately 85% identity, approximately 86% identity, approximately 87% identity, approximately 88% identity, approximately 90% identity, approximately 91% identity, approximately 92% identity, approximately 94% identity, approximately 95% identity, approximately 98% identity, approximately 99% identity, or a range (including endpoints) between any two of these values ​​or any value therein.

[0303] "At least 90% identity" means approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 92%, approximately 96%, approximately 99%, or a range (including endpoints) of any two of these values, or any value within that range. "Identity" or "sequence identity" of a polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) with another sequence at a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that, when sequence alignment is performed, that percentage of bases (or amino acids) are identical in the two sequences being compared. This alignment identity percentage or sequence identity can be determined visually or using software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Alignment is preferably performed using default parameters. One alignment procedure is BLAST using default parameters, such as BLASTN and BLASTP, which use the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that have the above-specified percentages of identity and encode polypeptides with the same or similar biological activities.

[0304] Polynucleotides are specific sequences of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T), or, when the polynucleotide is RNA, thymine replaced by uracil (U). The "polynucleotide sequence" can be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, such as functional genomics and homology searches.

[0305] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Examples of unrestricted polynucleotides include: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonucleases, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modification is present, structural modifications to the nucleotides can be made before or after assembly of the polynucleotide. The sequence of the nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeled components. This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiments of the polynucleotides disclosed herein include double-stranded forms and each of two complementary single-stranded forms known or predicted to constitute a double-stranded form.

[0306] When the term “encoding” is applied to polynucleotides, it refers to a polynucleotide called the “encoding” polypeptide that, in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce the polypeptide and / or fragments thereof.

[0307] "Antibody" and "antigen-binding fragment" refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody, any antigen-binding fragment, or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen. Antibody and antigen-binding fragments include, but are not limited to, the complementarity-determining region (CDR), heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), framework region (FR), or any portion thereof of the heavy chain or light chain or its ligand-binding moiety as described in the examples, or at least a portion of the binding protein. The CDR region includes the light chain CDR region (VL CDR1-3) and the heavy chain CDR region (VHCDR1-3). The variable region may contain the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0308] The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror isoforms, and bivalent antibodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0309] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some respects, these regions are linked to short linker peptides of 10 to approximately 25 amino acids. The linker may be enriched with glycine to increase flexibility, and with serine or threonine to increase solubility, and may link the N-terminus of the VH to the C-terminus of the VL, and vice versa. Although the constant region of the protein has been removed and the linker has been introduced, it retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art, for example, as described in U.S. Patent 5,892,019.

[0310] The term "antibody" encompasses a wide range of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chain classes include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with further subclasses (e.g., γ1-γ4). The properties of this chain determine the "type" of the antibody, such as IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and their assigned functional specificities are known. All immunoglobulin types are within the scope of protection disclosed in this invention. In some embodiments, the immunoglobulin molecule is of the IgG type. IgG typically comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000 Daltons. These four chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain begins at the "Y" port and continues to surround the heavy chain through a variable region.

[0311] The antibodies, antigen-binding fragments, or derivatives disclosed in this invention include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primate-like, and chimeric antibodies; single-chain antibodies; epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv); fragments containing VK or VH domains; fragments generated from Fab expression libraries; and anti-idiotypic (anti-Id) antibodies. The immunoglobulin or antibody molecules disclosed in this invention can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulins.

[0312] Light chains can be divided into kappa (κ) or lambda (λ). Each heavy chain can bind to either a κ or λ light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are covalently bonded, and the "tail" portions of the two heavy chains are linked by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration forked end to the C-terminus at the bottom of each chain. The variable region of the immunoglobulin κ light chain is Vκ; the variable region of the immunoglobulin λ light chain is V... λ .

[0313] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used according to function. The variable regions of the light chain (VL) and heavy chain (VH) determine antigen recognition and specificity. The constant regions of the light and heavy chains confer important biological properties such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. By convention, the numbering of constant regions increases as they move further away from the antibody's antigen-binding site or N-terminus. The N-terminal region is the variable region, and the C-terminal region is the constant region; the CH3 and CL domains actually contain the carboxyl termini of the heavy and light chains, respectively.

[0314] As described above, the variable region enables antibodies to selectively recognize and specifically bind to epitopes on antigens. Specifically, a subset of the antibody's VL domain and VH domain, or complementarity-determining region (CDR), binds to form a variable region that defines a three-dimensional antigen-binding site. This antibody quaternary structure forms antigen-binding sites at the ends of each arm of the Y-chain. More specifically, the antigen-binding site is defined by three CDRs (i.e., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and VL CDR3) in each of the VH and VL chains. In some cases, such as certain immunoglobulin molecules derived from camels or modified immunoglobulins based on camel immunoglobulins, the complete immunoglobulin molecule may consist only of the heavy chain, without the light chain. See, for example, Hamers-Casterman et al., Nature, 363:446-448 (1993).

[0315] In naturally occurring antibodies, assuming the antibody presents its three-dimensional conformation in an aqueous environment, the six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are short, discontinuous amino acid sequences that bind specifically to the antigen, forming the antigen-binding domain. The remaining amino acids in the framework region, known as the "framework region," exhibit less intermolecular variability. The framework region mostly adopts a β-sheet conformation, with CDRs forming linked loop structures, or in some cases, forming part of a β-sheet structure. Thus, the framework region positions the CDRs in the correct orientation by forming a scaffold through non-covalent interchain interactions. The antigen-binding domain with a CDR at a specific location forms a surface complementary to the epitope on the antigen, which facilitates the non-covalent binding of the antibody to its antigenic epitope. For a given heavy or light chain variable region, those skilled in the art can identify the amino acids containing the CDR and framework regions by known methods (see Kabat, E., et al., USDepartment of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J.Mol.Biol., 196:901-917 (1987)).

[0316] Where a term used and / or accepted in this field has two or more definitions, the definition of the term used herein includes all of those meanings unless explicitly stated otherwise. A concrete example is the use of the term “complementarity-determining region” (“CDR”) to describe a discontinuous antigen-binding site found within the variable region of heavy and light chain polypeptides. This specific region is described in Kabat et al., USDept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al. in J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entirety.

[0317] According to the definitions of Kabat and Chothia, a CDR includes overlaps or subsets of amino acid residues when compared with each other. Nevertheless, the application of either definition to refer to a CDR of an antibody or a variant thereof is within the scope of this invention. The exact residue numbering containing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can typically determine which specific residues a CDR contains based on the amino acid sequence of the variable region of the antibody.

[0318] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can apply this "Kabat numbering" system to any variable region sequence without relying on experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., USDept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also use the EU numbering system.

[0319] The antibodies disclosed in this invention can be derived from any animal, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, camel, llama, horse, or chicken-derived antibodies. In another embodiment, the variable region can be of condricthoid origin (e.g., from sharks).

[0320] "Heavy chain constant regions" comprise amino acid sequences derived from the immunoglobulin heavy chain. Polypeptides containing heavy chain constant regions include at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge regions), a CH2 domain, a CH3 domain, or variants or fragments thereof. For example, the antibody or antigen-binding fragments disclosed in this invention contain a CH1 domain; contain a CH1 domain, at least a portion of the hinge region, and a CH2 domain; contain both a CH1 domain and a CH3 domain; contain a CH1 domain and at least a portion of the hinge region and a CH3 domain; or contain a CH1 domain, at least a portion of the hinge region, and both a CH2 and CH3 domain. In another embodiment, the antibody or antigen-binding fragments disclosed in this invention contain a CH3 domain. Furthermore, the antibody or antigen-binding fragments used in this invention may lack some or all of the CH2 domain. As described above, those skilled in the art will understand that heavy chain constant regions can be modified to alter the amino acid sequence of naturally occurring immunoglobulin molecules.

[0321] The heavy chain constant region of an antibody can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another embodiment, the heavy chain constant region may include a hinge region partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In yet another embodiment, a portion of the heavy chain may include a chimeric hinge region partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0322] The “light chain constant region” comprises the amino acid sequence from the antibody light chain. Preferably, the light chain constant region contains at least one of a constant κ domain or a constant λ domain. A “light chain-heavy chain pair” refers to a set of light and heavy chains that can form dimers via disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0323] As described above, the subunit structures and three-dimensional conformations of the constant regions of various immunoglobulin species are well known. The “VH domain” comprises the N-terminal variable domain of the immunoglobulin heavy chain, and the “CH1 domain” comprises the first (mostly N-terminal) constant region of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is the N-terminus of the hinge region of the immunoglobulin heavy chain molecule. The CH2 domain does not pair tightly with other domains but inserts two N-linked branched carbohydrate chains between the two CH2 domains of the intact native IgG molecule. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues. The “hinge region” comprises a portion of the heavy chain connecting the CH1 and CH2 domains. The hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0324] A disulfide bond is a covalent bond formed between two sulfur atoms. The thiol group of cysteine ​​can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions 239 and 242 in the Kabat numbering system (positions 226 and 229 in the EU numbering system).

[0325] A "chimeric antibody" is any antibody whose variable region is derived from or derived from a first species, while its constant region (which may be complete, partial, or modified) is derived from a second species. In some embodiments, the variable region is derived from a non-human source (e.g., a mouse or primate), while the constant region is derived from a human source.

[0326] "Specific binding" or "specific to..." generally refers to the formation of a relatively stable complex by which an antibody or antigen-binding fragment binds to a specific antigen through the complementarity of its antigen-binding domain with the epitope. "Specificity" can be expressed as the relative affinity of the antibody or antigen-binding fragment to a specific antigen or epitope. For example, if antibody "A" has a greater relative affinity to the same antigen than antibody "B," antibody "A" can be considered to have higher specificity for that antigen than antibody "B." Specific binding can be described by the equilibrium dissociation constant (KD), with a smaller KD indicating a tighter binding. Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and optical interferometry of biofilm layers. Antibodies that "specifically bind" to spike proteins include antibodies with equilibrium dissociation constants (KD) less than or equal to about 100 nM, less than or equal to about 10 nM, less than or equal to about 5 nM, less than or equal to about 1 nM, or less than or equal to about 0.5 nM.

[0327] "Treatment" refers to therapeutic treatments and preventative or preventative measures aimed at preventing, mitigating, improving, or stopping adverse physiological changes or disorders, such as disease progression, including but not limited to the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, mitigation, reduction, or disappearance of the disease state (whether partial or complete), and prolongation of expected survival without treatment. Patients requiring treatment include those already suffering from the condition or disorder, those susceptible to the condition or disorder, or those needing prevention of the condition or disorder, as well as those who can or are expected to benefit from the application of the antibody or pharmaceutical composition disclosed in this invention for detection, diagnostic procedures, and / or treatment.

[0328] "Patient" refers to any mammal that requires diagnosis, prognosis, or treatment, including humans, dogs, cats, rabbits, rats, mice, horses, cattle, etc.

[0329] Antibodies and fusion proteins

[0330] This invention provides antibodies or antigens with high affinity for the spike protein, and their binding fragments. These antibody or antigen binding fragments exhibit effective binding activity and can be used for therapeutic and diagnostic purposes. For example, these antibody or antigen binding fragments can prevent the fusion of SARS-CoV and SARS-CoV-2 viral particles with the cell membrane, and mediate the phagocytosis and clearance of viral particles by immune cells.

[0331] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO:53-56 or 108, and / or the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:53, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:54, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:55, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:56, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:108, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57.

[0332] This invention also provides fusion proteins. The antibody portion or antigen-binding fragment in the fusion protein has high affinity for the spike proteins of SARS-CoV and SARS-CoV-2, and these antibodies can specifically bind to the spike proteins; the 6-HB interfering polypeptide in the fusion protein can interfere with the formation of the "six-helix bundle (6-HB)". The fusion protein exhibits effective binding activity and can be used for therapeutic and diagnostic purposes. For example, these antibody or antigen-binding fragments in the fusion protein can prevent the fusion of SARS-CoV and SARS-CoV-2 viral particles with the cell membrane, and mediate the phagocytosis and clearance of viral particles by immune cells.

[0333] In some embodiments, the C-terminus (i.e., the CH3-terminus) of the heavy chain of the antibody described herein is covalently linked to a 6-HB interfering peptide via a linker. In some embodiments, the C-terminus (i.e., the CL-terminus) of the light chain of the antibody described herein is covalently linked to a 6-HB interfering peptide via a linker.

[0334] The sequences of the antibodies, antigen-binding fragments, and fusion proteins disclosed in the embodiments of this invention are shown in Tables 1-3. The sequences include the CDR region, FR region, constant regions of the heavy and light chains, variable regions of the heavy and light chains of the antibody or antigen-binding fragment, as well as linkers and 6-HB interfering peptides, etc. The division of the CDR region and FR region of the antibody or antigen-binding fragment can also be based on the Kabat definition, as shown in Table 2.

[0335] In some embodiments, the fusion protein disclosed in this invention comprises a 6-HB interfering polypeptide and an antibody or antigen-binding fragment, wherein the C-terminus of the heavy chain (or heavy chain fragment) or the C-terminus of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide via a linker (as shown in SEQ ID NO:59), the 6-HB interfering polypeptide comprising the sequence shown in SEQ ID NO:58, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:58, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:58.

[0336] In some embodiments, the C-terminus (i.e., the CH3-terminus) of the antibody's heavy chain is covalently linked to a 6-HB interfering peptide via a linker as shown in SEQ ID NO:59, the 6-HB interfering peptide comprising the sequence shown in SEQ ID NO:58. In some embodiments, the C-terminus (i.e., the CL-terminus) of the antibody's light chain is covalently linked to a 6-HB interfering peptide via a linker as shown in SEQ ID NO:59, the 6-HB interfering peptide comprising the sequence shown in SEQ ID NO:58.

[0337] In some embodiments, the fusion protein disclosed in this invention comprises a 6-HB interfering polypeptide and an antibody or antigen-binding fragment, said antibody or antigen-binding fragment comprising VH as shown in SEQ ID NO:46, 47, 48, 49, or 107, VL as shown in SEQ ID NO:50, or their respective biological equivalents. The biological equivalent of VH or VL is a sequence containing specific amino acids, i.e., the sequence having approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 98%, or approximately 99% sequence identity overall, or a range (including the endpoint) of any two of these values, or any value therein.

[0338] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in amino acids 1 to 450 of SEQ ID NO:53, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0339] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in amino acids 1 to 450 of SEQ ID NO:54, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0340] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in amino acids 1 to 450 of SEQ ID NO:55, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0341] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in amino acids 1 to 450 of SEQ ID NO:56, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0342] In some embodiments, the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in amino acids 1 to 450 of SEQ ID NO:108, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:57; the C-terminus (i.e., CH3-terminus) of the heavy chain or the C-terminus (i.e., CL-terminus) of the light chain of the antibody is covalently linked to the 6-HB interfering polypeptide by a linker as shown in SEQ ID NO:59, wherein the 6-HB interfering polypeptide comprises a sequence as shown in SEQ ID NO:58.

[0343] In some embodiments, the antibody, or fusion protein, may also be linked to an amino acid sequence or one or more modifying groups. For example, the antibody or fusion protein disclosed in this invention may contain a resilient linker sequence or may be modified to add functional groups (e.g., PEG, pharmaceuticals, toxins, or tags).

[0344] The antibodies or fusion proteins disclosed in this invention, such as antibodies or antigen-binding fragments, further comprise modified derivatives, i.e., modified by covalent linking any type of molecule to the antibody, wherein the covalent linking does not prevent the antibody from binding to the epitope. Examples include, but are not limited to, glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization via known protecting / blocking groups, proteolytic cleavage, and linking to cellular ligands or other proteins. Any of these numerous chemical modifications can be performed using existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin.

[0345] In some implementations, the antibody, or fusion protein, may be conjugated to a therapeutic agent, drug precursor, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

[0346] Antibodies, or fusion proteins, such as those conjugated or fused with therapeutic agents, may include detectable markers, such as radioactive markers, immunomodulators, hormones, enzymes, oligonucleotides, photosensitizing agents, diagnostic agents, cytotoxic agents, ultrasound enhancers, non-radioactive markers, and combinations thereof, and other such agents known in the art.

[0347] Antibodies, or fusion proteins, can be detectably labeled by conjugating them to a chemiluminescent compound. The presence of the chemiluminescently labeled antibody is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of chemiluminescently labeled compounds include luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalates.

[0348] Polynucleotides encoding antibodies and fusion proteins and methods for preparing antibodies

[0349] This invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, fusion proteins, and derivatives thereof described herein. The polynucleotides disclosed herein can encode heavy chains, light chains, heavy chain variable regions, light chain variable regions, Fc regions, partially heavy chain variable regions, partially light chain variable regions, or fusion proteins. Methods for preparing antibodies and fusion proteins are well known in the art and are described herein. In some embodiments, the variable and constant regions of the antibodies, antigen-binding fragments, and antigen-binding fragments disclosed herein are fully human. Fully human antibodies and antigen-binding fragments can be prepared using techniques disclosed in the art and techniques described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to transgenic animals that have been modified to produce fully human antibodies in response to antigen challenge. Exemplary techniques that can be used to prepare such antibodies are found in U.S. Patents 6,458,592; 6,420,140, ​​the entire contents of which are incorporated herein by reference.

[0350] In some embodiments, the prepared antibodies and fusion proteins do not induce a harmful immune response in the animal to be treated (e.g., human). In one embodiment, the antibodies, antigen-binding fragments, fusion proteins, or derivatives disclosed in this invention are modified using techniques recognized in the art to reduce their immunogenicity. For example, antibodies can be humanized, primate-like, deimmunized, or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, typically murine or primate antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody but have lower immunogenicity in humans. This can be achieved by a variety of methods, including (a) transplanting the entire non-human variable region into a human constant region to produce a chimeric antibody; (b) transplanting at least a portion of one or more non-human complementarity-determining regions (CDRs) into human framework and constant regions, retaining or not retaining key framework residues; or (c) transplanting the entire non-human variable region but “hiding” them by replacing surface residues with human-like portions. Typically, framework residues in the human framework region are replaced by corresponding residues from the CDR donor antibody, such as residues that improve antigen binding. These framework substitutions can be identified using methods well-known in the art, such as simulating the interaction between CDRs and framework residues to identify framework residues that play an important role in antigen binding and using sequence comparison to identify aberrant framework residues at specific positions. (Refer to U.S. Patent 5,585,089; Riechmann et al., Nature 332:323 (1988); the entire contents of which are incorporated herein by reference). Antibodies can be humanized using a variety of techniques known in the art, such as CDR transplantation (EP 239,400; WO 91 / 09967; U.S. Patents 5,225,539,5,530,101 and 5,585,089), repair or surface rearrangement (EP592,106; EP519,596; Padlan, et al., Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska, et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and chain rearrangement (U.S. Patent 5,565,332), the entire contents of which are incorporated herein by reference.

[0351] Deimmunization can also be used to reduce the immunogenicity of antibodies. In this invention, the term "deimmunization" includes altering antibodies to modify T-cell epitopes (see, for example, WO / 9852976A1 and WO / 0034317A2). For example, the heavy chain variable region sequence and light chain variable region sequence from the starting antibody are analyzed, and a "map" of human T-cell epitopes from each variable region is generated, showing the position of the epitope relative to complementarity-determining regions (CDRs) and other key residues within the sequence. Individual T-cell epitopes from the T-cell epitope map are analyzed to identify alternative amino acid substitutions with a lower risk of altering antibody activity. A series of alternative heavy chain variable region sequences and light chain variable region sequences containing combinations of amino acid substitutions are designed and subsequently incorporated into a series of binding peptides. Genes containing the modified variable regions and human constant regions of the complete heavy and light chains are then cloned into expression vectors, and plasmids are subsequently transformed into cell lines to produce complete antibodies. The antibodies are then compared using appropriate biochemical and biological experiments to identify the optimal antibody.

[0352] The binding specificity of the antibody or antigen-binding fragment in the fusion protein disclosed in this invention can be detected by in vitro experiments, such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

[0353] Alternatively, techniques for producing single-chain units (US Patent 4,694,778; Bird, Science 242:423-442 (1988), Huston et al., Proc. Natl. Acad. Sci. USA 55:5879-5883 (1988), and Ward et al., Nature 334:544-554 (1989)) can be applied to produce the single-chain units disclosed in this invention. Single-chain units are formed by bridging the heavy and light chain fragments of the Fv region with amino acids, resulting in single-chain fusion peptides. Techniques for assembling functional Fv fragments in E. coli (Skerra et al., Science 242:1038-1041 (1988)) can also be used.

[0354] Examples of techniques that can be used to produce single-chain Fv (scFv) and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498, as well as Huston et al., Methods in Enzymology 203:46-88 (1991), Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993), and Skerra et al., Science 240:1038-1040 (1988). For certain applications, including the use of antibodies in humans and in vitro assays, chimeric antibodies, humanized antibodies, or fully human antibodies may be used. Chimeric antibodies are a class of molecules whose different portions are derived from different animal species, such as antibodies containing the variable region of a murine monoclonal antibody and the constant region of a human immunoglobulin. Methods for producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gilles et al., J. Immunol. Methods 125:191-202 (1989); Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature 314:452-454 (1985); and U.S. Patents 5,807,715, 4,816,567 and 4,816,397, the entire contents of which are incorporated herein by reference.

[0355] Furthermore, another efficient method for producing recombinant antibodies is disclosed in Newman, Biotechnology 10:1455-1460 (1992), specifically, this technique produces primate antibodies containing monkey variable region and human constant region sequences, the entire contents of which are incorporated herein by reference. This technique is also mentioned in commonly assigned U.S. Patents 5,658,570, 5,693,780, and 5,756,096, the entire contents of which are incorporated herein by reference.

[0356] Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from immunoglobulin sequences. Reference can also be made to U.S. Patents 4,444,887 and 4,716,111, and PCT publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO91 / 10741, the entire contents of which are incorporated herein by reference.

[0357] Fully human antibodies are particularly desirable for treating human patients. Fully human antibodies can be prepared using various methods known in the art, such as transgenic mice that do not express functional endogenous immunoglobulins but do express human immunoglobulin genes. For example, human heavy chain and light chain immunoglobulin gene complexes can be randomly introduced or introduced via homologous recombination into mouse embryonic stem cells. Alternatively, in addition to human heavy chain and light chain genes, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy chain and light chain immunoglobulin genes can be defunctionalized via homologous recombination, either separately or simultaneously, by introducing human immunoglobulin gene loci. For example, homozygous deletion of the JH region can prevent the production of endogenous antibodies. Modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. These chimeric mice are then cultured to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized in a conventional manner with selected antigens, such as all or part of the target peptide. Monoclonal antibodies targeting the antigen can be obtained from the immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgenes carried by transgenic mice undergo rearrangement during B cell differentiation, followed by class switching and somatic mutations. Therefore, this technology can be used to produce therapeutically applicable IgG, IgA, IgM, and IgE antibodies. For a review of this technology for producing fully human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of the technology for producing fully human antibodies and human monoclonal antibodies, and the steps for producing such antibodies, see PCT publications WO 98 / 24893, WO 96 / 34096, WO 96 / 33735, and U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, the entire contents of which are incorporated herein by reference.

[0358] A technique known as “guided selection” can also be used to produce fully human antibodies that recognize selective epitopes. In this method, a selected non-human monoclonal antibody (e.g., a mouse antibody) is used to guide the screening of fully human antibodies that recognize the same epitope (see U.S. Patent 5,565,332, the entire contents of which are incorporated herein by reference).

[0359] In another embodiment, DNA encoding the desired monoclonal antibody can be isolated and sequenced using conventional methods, such as oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies. Isolated and subclonal hybridoma cells can serve as sources of this DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into prokaryotic or eukaryotic host cells such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins. The isolated DNA (which may be synthetic, as described herein) can also be used to prepare sequences of the constant and variable regions of the antibody, as described in U.S. Patent 5,658,570, the entire contents of which are incorporated herein by reference. This method involves extracting RNA from selected cells and converting it into cDNA, which is then amplified using Ig-specific primers via PCR. Suitable probes for this purpose are also mentioned in U.S. Patent 5,658,570.

[0360] Furthermore, using conventional recombinant DNA techniques, one or more CDRs of the antibodies of this invention can be inserted into the frame region, for example, into the human frame region, to construct humanized non-fully human antibodies. The frame region can be a naturally occurring or common frame region, preferably a human frame region (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a series of human frame regions). Some polynucleotides can encode antibodies that specifically bind to at least one epitope of the target antigen, resulting from the combination of the frame region and the CDR. One or more amino acid substitutions can be made within the frame region, selectively choosing amino acid substitutions that improve the binding of the antibody to its antigen. Additionally, this method can be used to substitute or delete cysteine ​​residues in one or more variable regions involved in the formation of interchain disulfide bonds, thereby producing antibody molecules lacking one or more interchain disulfide bonds. Other modifications to polynucleotides within the scope of the art are also covered in this invention.

[0361] Cell lines for antibody production can be selected, constructed, and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications. In this regard, the techniques described below suitable for use in this invention are referenced in Current Protocols in Immunology, Colligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), the entire contents of which, including supplements, are incorporated herein by reference.

[0362] In some embodiments, DNA encoding antibodies, antigen-binding fragments, or fusion proteins can be designed and synthesized using conventional methods based on the amino acid sequences of the antibodies, antigen-binding fragments, or fusion proteins described herein. This DNA is then placed into an expression vector, transfected into host cells, and cultured in a culture medium to produce antibodies, antigen-binding fragments, or fusion proteins. In some embodiments, the expression vector includes at least one promoter element, the antibody, antigen-binding fragment, or fusion protein coding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, and donor and acceptor sites for RNA splicing flanking the insert sequence. Efficient transcription can be achieved using early and late promoters of SV40, early promoters from long terminal repeat sequences of retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus, or other cellular promoters such as the actin promoter. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, etc. Commonly used mammalian cell lines include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells, etc.

[0363] In some implementations, the inserted gene fragment needs to contain selection markers. Common selection markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, and hygromycin resistance genes to facilitate the selection and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking the aforementioned genes, and after being cultured in a selective medium, the successfully transfected cells grow in large numbers, producing the desired target protein.

[0364] Furthermore, standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the antibody, antigen-binding fragment, or fusion protein described in this invention, including but not limited to site-directed mutagenesis leading to amino acid substitutions and PCR-mediated mutations. Variants (including derivatives) encode substitutions of fewer than 50 amino acids, fewer than 40 amino acids, fewer than 30 amino acids, fewer than 25 amino acids, fewer than 20 amino acids, fewer than 15 amino acids, fewer than 10 amino acids, fewer than 5 amino acids, fewer than 4 amino acids, fewer than 3 amino acids, or fewer than 2 amino acids relative to the original heavy chain variable regions VH CDR1, VH CDR2, VH CDR3 and light chain variable regions VL CDR1, VL CDR2, or VL CDR3. Alternatively, mutations can be randomly introduced along all or part of the coding sequence, for example, through saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

[0365] In some implementations, the substitutions described herein are conserved amino acid substitutions.

[0366] Treatment

[0367] This invention also provides treatment methods and uses. In some embodiments, a method for treating or improving SARS or COVID-19 is provided, the method comprising administering an effective dose of the antibody, antigen-binding fragment, or fusion protein to a patient. In some embodiments, the use of the antibody or fusion protein in treating or improving SARS or COVID-19 is provided. In some embodiments, the use of the antibody or fusion protein in the preparation of a medicament for treating or improving SARS or COVID-19 is provided. In some embodiments, the patient is a patient suspected of being infected with SARS-CoV or SARS-CoV-2 virus. In some embodiments, the patient is a patient who has had contact with a carrier of SARS-CoV or SARS-CoV-2 virus. In some embodiments, the patient is a patient confirmed to be infected with SARS-CoV or SARS-CoV-2 virus. In some embodiments, the patient is a patient with mild symptoms. In some embodiments, the patient is a patient with severe symptoms. In some embodiments, the patient has fever, cough, hypotension, hypoxia, and / or acute respiratory distress syndrome (ARDS).

[0368] The specific dosage and treatment regimen for any given patient will depend on a variety of factors, including the specific antibody, antigen-binding fragment, or fusion protein or derivative used, the patient's age and weight, general health condition, sex, and diet, as well as the timing of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. These factors will be determined by a healthcare professional, including those skilled in the art. The dosage will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined using principles of pharmacology and pharmacokinetics well known in the art.

[0369] Methods of administration of antibodies, antigen-binding fragments, fusion proteins, or derivatives include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral injection. Pharmaceutical compositions can be administered via any convenient route, such as by infusion or bolus, absorption through epithelial or mucosal membranes (e.g., oral mucosa, rectal and intestinal mucosa), and can be co-administered with other bioactive agents. Therefore, pharmaceutical compositions containing the antibodies, antigen-binding fragments, or fusion proteins of the present invention can be administered orally, rectally, parenterally, intracerebrospinally, vaginally, intraperitoneally, topically (e.g., via powder, ointment, drops, or transdermal patch), orally, or via oral or nasal spray.

[0370] The term "parenteral" as used in this invention refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injection and infusion. Administration methods can be systemic or local.

[0371] In some embodiments, the compositions of the present invention comprise nucleic acids or polynucleotides encoding proteins, which can be administered in vivo to promote the expression of the proteins encoded therein by constructing them as part of a suitable nucleic acid expression vector, and then the aforementioned partial vector is administered to make it an intracellular part, for example by using a retroviral vector (see U.S. Patent 4,980,286), or by direct injection, or by using microparticle bombardment (e.g., gene gun; Biolistic, DuPont), or by coating with lipids or cell surface receptors or transfection reagents, or by administration by linking to a homeobox peptide known to enter the cell nucleus (see, for example, Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc. Optionally, the nucleic acid can be introduced into the cell and integrated into the host cell DNA for expression via homologous recombination.

[0372] In some embodiments, the antibody, antigen-binding fragment, or fusion protein of the present invention is administered to a patient at a dose of 0.01 mg / kg to 100 mg / kg of patient body weight, or 0.1 mg / kg to 20 mg / kg of patient body weight. A second or more doses of the antibody, antigen-binding fragment, or fusion protein may be subsequently administered after the initial dose, at a dose substantially the same as or less than the initial dose, wherein the subsequent doses may be spaced at least 1 to 3 days; or at least one week apart. The uptake and tissue penetration (e.g., into the brain) of the antibody or fusion protein can be enhanced by modifications such as lipolysis, thereby reducing the dose and frequency of administration of the antibody or fusion protein of the present invention.

[0373] Various known delivery systems can be used to administer the antibodies, antigen-binding fragments, or fusion proteins or derivatives of the present invention, or their encoded polynucleotides, for example, encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), and the construction of nucleic acids as part of retroviruses or other vectors.

[0374] combination therapy

[0375] In some embodiments, the antibodies, antigen-binding fragments, or fusion proteins of the present invention can be combined with other treatment or preventative regimens, including the administration of one or more of the antibodies, antigen-binding fragments, or fusion proteins of the present invention together with one or more other therapeutic agents or methods. For combination therapy, the antibodies, antigen-binding fragments, or fusion proteins can be administered simultaneously or separately from other therapeutic agents. When administered separately, the antibodies, antigen-binding fragments, or fusion proteins of the present invention can be administered before or after the administration of another therapeutic agent.

[0376] In some embodiments, the therapeutic agent used in combination with the antibody, antigen-binding fragment, or fusion protein of the present invention is at least one of the following: HIV drugs, antimalarial drugs, RNA polymerase inhibitors, antiviral drugs, and monoclonal antibody drugs. In some embodiments, HIV drugs include lopinavir / ritonavir, ASC09 / ritonavir, and darunavir. In some embodiments, antimalarial drugs include chloroquine, hydroxychloroquine, and chloroquine phosphate. In some embodiments, RNA polymerase inhibitors include remdesivir. In some embodiments, antiviral drugs include arbidol and favipiravir. In some embodiments, monoclonal antibody drugs include BDB-001. In some embodiments, the antiviral drug is an antibody or antigen-binding fragment in the fusion protein of the present invention.

[0377] Some patients with severe or critical COVID-19 pneumonia exhibit cytokine storms. The antibodies, antigen-binding fragments, or fusion proteins of this invention can bind to adalimumab (e.g.,...). and its biosimilars, such as Abrilada TM (adalimumab-afzb), Amjevita (adalimumab-att), Cyltezo TM (adalimumab-adbm), Hyrimoz TM (adalimumab-adaz), Hulio TM , ( BAT1406) or tocilizumab (e.g., BAT1406) or tocilizumab (e.g., tochilizumab) In combination with its biosimilars, such as BAT1806, this method can alleviate the inflammatory response caused by upregulation of TNF-α expression. In some embodiments, patients treated with this method are diagnosed with COVID-19 and have elevated levels of one or more cytokines, including tumor necrosis factor-α (TNF-α), IFN-γ, IL-1β, IL-2, IL-4, IL-7, IL-8, IL-10, IL-12p70, IL-13, granulocyte colony-stimulating factor (GSCF), interferon-induced protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP1), and macrophage inflammatory protein 1α (MIP1A). In some embodiments, patients treated with this method have elevated TNF-α. In some embodiments, one or more cytokines are at least 50% above normal levels. In some embodiments, one or more cytokines are at least 2, 3, or 4 times the normal level. In some embodiments, patients prior to treatment with this method have fever, hypotension, hypoxia, and / or acute respiratory distress syndrome (ARDS). In some implementations, patients prior to treatment had lungs filled with inflammatory fluid (i.e., so-called "white lung"). In some implementations, patients prior to treatment had cytokine release syndrome (CRS) caused by a cytokine storm.

[0378] In some embodiments, the antibodies, antigen-binding fragments, or fusion proteins of the present invention are used in conjunction with ICU treatment. In some embodiments, the antibodies, antigen-binding fragments, or fusion proteins of the present invention are used in conjunction with in vitro ECMO and / or IMV treatment. In some embodiments, the antibodies, antigen-binding fragments, or fusion proteins of the present invention are used in conjunction with oxygen therapy. In some embodiments, the antibodies, antigen-binding fragments, or fusion proteins of the present invention are used in conjunction with NIV / HFNC treatment. In some embodiments, after treatment, one or more cytokines in the patient are reduced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to before treatment. In some embodiments, this method leads to patient recovery.

[0379] Diagnostic methods

[0380] Spike protein expression has been observed in certain samples, and patients with spike protein-expressing cells may respond to treatment using the antibodies, antigen-binding fragments, or fusion proteins of the present invention. Therefore, the antibodies, antigen-binding fragments, or fusion proteins of the present invention can also be used for diagnosis and prognosis.

[0381] Samples containing cells can be obtained from a patient. After selective pretreatment of the sample, the sample can be incubated with the fusion protein (or antibody) of the present invention under conditions that allow the antibody (or fusion protein) to interact with the spike protein that may be present in the sample. The presence of the spike protein in the sample can be detected using methods such as ELISA, utilizing the antibody or the antibody in the fusion protein.

[0382] The presence (e.g., amount or concentration) of spike protein in a sample can be used to diagnose related diseases, as an indicator of whether a patient is suitable for antibody or fusion protein therapy, or as an indicator that a patient has (or has not) responded to disease treatment. For prognostic methods, one, two, or more tests can be performed at specific stages at the start of disease treatment to indicate treatment progress.

[0383] Pharmaceutical Composition

[0384] The present invention also provides pharmaceutical compositions. Such compositions comprise an effective dose of an antibody or fusion protein and a pharmaceutically acceptable carrier.

[0385] In some implementations, the term "pharmaceuticalally acceptable" refers to a substance approved by a government regulatory agency or listed in other recognized pharmacopoeias for use in animals (especially for humans). Furthermore, "pharmaceuticalally acceptable carrier" generally refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0386] The term "carrier" refers to a diluent, adjuvant, excipient, or carrier that can be administered to a patient along with the active ingredient. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline, glucose, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the drug composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers such as acetates, citrates, or phosphates. Antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as EDTA, and tonic agents such as sodium chloride or dextran are also foreseeable. These pharmaceutical compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The pharmaceutical composition can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which are incorporated herein by reference. Such compositions will contain a clinically effective dose of an antibody or antigen-binding fragment, preferably in a purified form, along with a suitable number of carriers to provide a dosage form suitable for the patient. The formulation should be suitable for the mode of administration. The formulation can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0387] In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection into the human body according to conventional procedures. Compositions for intravenous administration are typically solutions in sterile isotonic buffer solutions. The pharmaceutical composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the active ingredient is supplied individually or in combination in unit doses, such as as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water or saline for injection can be used, allowing the active ingredient to be mixed before administration.

[0388] The compounds of the present invention can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include those derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0389] "Approximately" refers to a typical error range for a given value that is readily known to those skilled in the art. In some embodiments, "approximately" as used herein refers to the described value and its range of ±10%, ±5%, or ±1%.

[0390] "ECMO" refers to Extracorporeal Membrane Oxygenation, a medical emergency device mainly used to provide continuous extracorporeal breathing and circulation for patients with severe cardiopulmonary failure in order to maintain their lives.

[0391] "ICU" refers to Intensive Care Unit, where treatment, nursing care, and rehabilitation can be carried out simultaneously. It provides isolation facilities and equipment for critically ill or comatose patients, offering optimal care, comprehensive treatment, integrated medical and elderly care, as well as services such as early postoperative rehabilitation, joint care, and exercise therapy.

[0392] "IMV" stands for Intermittent Mandatory Ventilation, which involves periodic volume or pressure ventilation based on pre-set time intervals (time triggers). During mandatory ventilation, the patient is allowed to breathe spontaneously at any set baseline pressure level. During spontaneous breathing, the patient can breathe independently with continuous airflow support, or the machine can open valves on demand to allow spontaneous breathing. Most ventilators provide pressure support during spontaneous breathing.

[0393] "HFNC" stands for High-flow nasal cannula oxygen therapy. It's a non-invasive form of respiratory support that delivers a high-flow-rate mixture of oxygen and air to the patient directly through a non-sealed nasal cannula. It rapidly improves oxygenation and is currently used for patients with acute hypoxic respiratory failure, post-surgical patients, patients with respiratory failure who haven't undergone intubation, immunosuppressed patients, and patients with heart failure.

[0394] “NIV” stands for Non-invasine Ventilation, which refers to non-invasive mechanical ventilation other than endotracheal intubation and tracheotomy.

[0395] "EC50" refers to the concentration for 50% of the maximal effect, which is the concentration that can cause 50% of the maximum effect.

[0396] "IC50" represents the 50% inhibition concentration, which is the concentration of a drug or inhibitor required to inhibit a specified biological process by half.

[0397] "TICD50" stands for 50% tissue culture infection dosing method, which is a standard method for determining viral titers. Detailed Implementation

[0398] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0399] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0400] Example 1: Preparation of Anti-spike Protein Antibody

[0401] The amino acid sequences of the antibodies are shown in Table 1. VH and CH constitute the heavy chain of the antibody, and VL and CL constitute the light chain. The sequence of CH is shown in SEQ ID NO:51, and the sequence of CL is shown in SEQ ID NO:52. Antibodies can be prepared by the following methods or other known methods: sequence optimization based on the CHO codon preference characteristics of the host cell, yielding the DNA sequence from the amino acid sequence. The optimized and synthesized sequence clones were cloned into vectors, and then a large number of plasmids were extracted for transient expression: the linearized expression vector was mixed with CHO cells and added to a 0.4 cm electroporation cuvette for electroporation; after electroporation, 1200 cells per well were seeded into 96-well cell culture plates. After about 2-3 weeks, the parent clone with high expression was selected for cell expansion culture in 96-well, 24-well, 6-well, and shake flasks, and the expression level was detected. The clone with high expression in the shake flasks was selected for subcloning. The subclone expansion culture and expression identification were the same as the parent clone. Based on the expression level and cell line stability, a single-clone stable cell line was selected. After about 12 days of suspension culture, the supernatant was harvested and proA affinity capture was performed. Anion and cation chromatography were used to obtain antibodies with a purity greater than 95%. Wherein: the VH of antibody 1 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:4, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 1 is shown in SEQ ID.NO:50.

[0402] The VH of antibody 2 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:7, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 2 is shown in SEQ ID.NO:50.

[0403] The VH of antibody 3 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:13, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 3 is shown in SEQ ID.NO:50.

[0404] The VH of antibody 4 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:14, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 4 is shown in SEQ ID.NO:50.

[0405] The VH of antibody 5 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:16, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 5 is shown in SEQ ID.NO:50.

[0406] The VH of antibody 6 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:17, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 6 is shown in SEQ ID.NO:50.

[0407] The VH of antibody 7 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:18, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 7 is shown in SEQ ID.NO:50.

[0408] The VH of antibody 8 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:19, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 8 is shown in SEQ ID.NO:50.

[0409] The VH of antibody 9 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:20, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 9 is shown in SEQ ID.NO:50.

[0410] The VH of antibody 10 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:21, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 10 is shown in SEQ ID.NO:50.

[0411] The VH of antibody 11 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:22, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 11 is shown in SEQ ID.NO:50.

[0412] The VH of antibody 12 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:23, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 12 is shown in SEQ ID.NO:50.

[0413] The VH of antibody 13 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:26, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 13 is shown in SEQ ID.NO:50.

[0414] The VH of antibody 14 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:27, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 14 is shown in SEQ ID.NO:50.

[0415] The VH of antibody 15 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:28, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 15 is shown in SEQ ID.NO:50.

[0416] The VH of antibody 16 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:30, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 16 is shown in SEQ ID.NO:50.

[0417] The VH of antibody 17 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:31, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 17 is shown in SEQ ID.NO:50.

[0418] The VH of antibody 18 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:32, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 18 is shown in SEQ ID.NO:50.

[0419] The VH of antibody 19 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:33, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 19 is shown in SEQ ID.NO:50.

[0420] The VH of antibody 20 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:34, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 20 is shown in SEQ ID.NO:50.

[0421] The VH of antibody 21 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:35, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 21 is shown in SEQ ID.NO:50.

[0422] The VH of antibody 22 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:37, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 22 is shown in SEQ ID.NO:50.

[0423] The VH of antibody 23 includes FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:38, and FR4 as shown in SEQ ID.NO:45; the VL of antibody 23 is shown in SEQ ID.NO:50.

[0424] The VHs of antibody 24 include FR1 as shown in SEQ ID.NO:42, VH CDR1 as shown in SEQ ID.NO:1, FR2 as shown in SEQ ID.NO:43, VH CDR2 as shown in SEQ ID.NO:2, FR3 as shown in SEQ ID.NO:44, VH CDR3 as shown in SEQ ID.NO:105, and FR4 as shown in SEQ ID.NO:45. The VLs of antibody 24 are shown in SEQ ID.NO:50.

[0425] Table 1 Amino acid sequences

[0426]

[0427]

[0428]

[0429]

[0430] Table 2 Amino acid sequences

[0431]

[0432]

[0433] Example 2: Preparation of fusion protein

[0434] Based on the sequence of the fusion protein described herein, the fusion protein can be prepared by the following methods or other known methods. The fusion protein comprises a first polypeptide and a third polypeptide with identical sequences, as well as a second polypeptide and a fourth polypeptide with identical sequences; the first polypeptide contains a heavy chain (or heavy chain fragment), a linker, and a 6-HB interfering polypeptide, and the second polypeptide contains a light chain. Sequence optimization is performed according to the CHO codon preference characteristics of the host cell, and the DNA sequence is obtained from the amino acid sequence. Taking fusion protein A as an example, the amino acid sequence of the first polypeptide of fusion protein A is shown in SEQ ID NO:111, and the nucleic acid sequence is shown in SEQ ID NO:109; the amino acid sequence of the second polypeptide of fusion protein A is shown in SEQ ID NO:112, and the nucleic acid sequence is shown in SEQ ID NO:110. The optimized and synthesized sequence clones were cloned into vectors, and then large amounts of plasmids were extracted for transient expression. The linearized expression vector was mixed with CHO cells and electroporated in a 0.4 cm electroporation cuvette. After electroporation, 1200 cells were seeded into 96-well cell culture plates. After approximately 2-3 weeks, the parent clone with high expression levels was selected for cell expansion culture in 96-well, 24-well, 6-well, and shake flasks for expression level detection. Clones with high expression levels in shake flasks were selected for subcloning. Subclone expansion culture and expression identification were performed similarly to the parent clone. Single-clone stable cell lines were selected based on expression levels and cell line stability. After approximately 12 days of suspension culture, the supernatant was harvested for proA affinity capture. Anion and cation chromatography were used to obtain antibodies with a purity greater than 95%. Sequencing confirmed that the fusion protein A sequence was as described above.

[0435] Table 3. Nucleic acid and amino acid sequences of fusion protein A

[0436]

[0437]

[0438] Example 3: Detection of antibody binding activity to SARS-CoV-2 spike protein

[0439] ELISA detection was performed on the above antibodies. The detection method was as follows: A 96-well plate (Corning, 9018) was coated with spike-RBD-mFC (Sino Biologicals), sealed with tape, and stored. The plate was washed three times with washing buffer PBST (PBS, 0.05% Tween 20), followed by the addition of blocking solution (200 μL of 10 mg / ml BSA per well, using washing buffer as the solvent). After incubation (1 h, 37°C), the plate was washed three times with washing buffer, and then 100 μL of serially diluted sample was added to each well. After incubation (1.5 h, 37°C), the plate was washed with washing buffer, and then 100 μL of anti-human κ light chain antibody-peroxidase conjugate (diluted to 1:2000 in blocking solution, 100 μL / well) was added. The plate was washed with washing buffer, and then 100 μL of TMB (Tetramethylbenzidine, Biopanda) was added. Before adding the TMB-S-001 substrate / well, incubate the test sample (1 h, 37 °C); after 10 minutes of color development, add 100 μL / well of 0.1 M H2SO4 to terminate the reaction, and then measure the absorbance of the 96-well plate at 450 nm.

[0440] The EC50 values ​​of various antibodies binding to the SARS-CoV-2 spike protein were calculated using absorbance values, and are shown in Table 4.

[0441] Table 4

[0442]

[0443] Example 4: Detection of the binding activity of fusion protein A with SARS-CoV-2 spike protein and mutants

[0444] 1) ELISA method: Add 100 μl / well of 1 μg / ml spike-RBD to a 96-well plate and pass at 4°C; wash twice with 300 μl / well of PBST; block with 300 μl / well of blocking buffer (PBST containing 3% BSA) and incubate at 37°C for 2 hours; add 10 three-fold diluted fusion protein A to the blocked 96-well plate sequentially, with the highest concentration being 5 μg / ml, and incubate at 37°C for 1 hour, repeating each concentration in duplicate; add 100 μl / well of 1:10,000 diluted HRP-goat anti-human IgG-Fc antibody (Abcam, Ab97225) and incubate at 37°C for 1 hour; wash 8 times with 300 μl / well of PBST; add 100 μl / well of TMB and incubate at 37°C for 15-25 minutes; stop the reaction with 50 μl / well of 0.2M H2SO4 and read the values ​​within 30 minutes. Among them, spike-RBD is divided into wild-type spike S1 RBD (AcroBiosystems, SPD-C52H3), variant S protein RBD (V367F) (Acrosystems, SPD-S52H4), variant S protein RBD (N354D, D364Y) (Acrosystems, SPD-S52H3), variant S protein RBD (W436R) (Acrosystems, SPD-S52H7), and variant S1 protein (D614G) (Novoprotein, DRA57).

[0445] like Figure 2A and Figure 2B As shown, fusion protein A binds to wild-type Spike S1 RBD with high affinity. Fusion protein A binds to wild-type Spike S1 RBD and its variants with high affinity: the EC50 value of fusion protein A binding to wild-type Spike S1 RBD is 0.15 nM; the binding activity of fusion protein A to Spike S1 RBD variants (N354D / D364Y; V37F; W436R) is similar to that of wild-type. Fusion protein A can bind to wild-type virus and viral mutants with high affinity.

[0446] 2) BiaCore (Biomolecular Interaction Analysis): Fusion protein A was coated onto a chip at a concentration of 10 μg / mL; binding kinetics were measured using BiaCore at 25°C; probe equilibration was performed; recombinant spike S1RBD protein or trimer spike trimer (Acrobiosystems, SPN-C52H8) was passed through the chip coated with fusion protein A at concentrations of 10-400 nM; background and non-specific binding signals were subtracted using only flow-through buffer (PBS containing 0.05% Tween-20) as a control; kinetic constants were calculated using a 1:1 Langmuir binding model on BiaCore Data Analysis software.

[0447] According to BiaCore's assessment, the EC50 value of fusion protein A binding to wild-type Spike S1 RBD is 4.54 nM, and the EC50 value of fusion protein A binding to natural trimer spike trimer is 0.773 nM.

[0448] Example 5: Detection of the activity of antibody or fusion protein A in blocking the binding of SARS-CoV-2 spike protein to angiotensin-converting enzyme 2.

[0449] 1) Competitive ELISA was performed against some of the above-mentioned antibodies to detect their ability to block the binding of spike protein to angiotensin-converting enzyme 2 (ACE2). The detection method was as follows: 96-well plates (Corning, 9018) were coated with spike-RBD-mFC (sinobiologicals), sealed with tape, and stored at 4°C overnight; the plates were washed three times in washing buffer (PBS, 0.05% Tween 20), and then blocking solution (200 μL per well, 10 mg / ml) was added. BSA (using washing buffer as solvent); after incubation (2 h, 37 °C), the plate was washed three times with washing buffer, and different concentrations of antibody or fusion protein samples were added, followed by the addition of biotinylated angiotensin-converting enzyme 2 (50 ng / ml); after incubation (1 h, 37 °C), the plate was washed three times with washing buffer, and then 100 μL of streptavidin peroxidase conjugate (diluted 1:10,000 in blocking solution) was added to each well. After incubation (1.0 h, 37 °C), the plate was washed with washing buffer, and 100 μL / well of TMB substrate was added; after 10 minutes of color development, 50 μL / well of 0.1 M H2SO4 was added to terminate the reaction, and the absorbance was measured at 450 nm.

[0450] The curves showing the blocking effects of various antibodies on the binding of COVID-19 spike protein to ACE2 are shown below. Figure 1 .

[0451] 2) The same or similar methods can be used to determine the activity of fusion protein A in blocking the binding of SARS-CoV-2 spike protein to angiotensin-converting enzyme 2.

[0452] A competitive ELISA was performed on the aforementioned fusion protein A to detect its ability to block the binding of spike protein to angiotensin-converting enzyme 2 (ACE2). The assay method was as follows: 96-well plates (Costar, 9018) were coated with 100 μl / well of 1 μg / mL spike S1 RBD (AcroBiosystems, SPD-C82E9), sealed with tape, and stored overnight at 4°C. The plates were washed three times with 300 μl / well of washing buffer PBST (PBS containing 0.05% Tween 20), followed by 300 μl / well of blocking solution (PBST containing 3% BSA). After incubation (2 h, 37°C), nine 3-fold diluted fusion protein A solutions were sequentially added to the sealed 96-well plates, with a maximum concentration of 7.5 μg / mL. The plates were incubated at 37°C for 1 h, with each concentration repeated in duplicate. Then, 25 ng / mL of biotinylated angiotensin-converting enzyme 2 (soluble ACE2, AcroBiosystems) was added. AC2-H5257), 100 μl / well; incubate (1 h, 37 °C), then wash 8 times with 300 μl / well of PBST; then add 100 μL of blocking solution to each well to dilute streptavidin-peroxidase conjugate (Jackson, 016-030-084) 10,000 times; wash 8 times with 300 μl / well of PBST; add 100 μL / well of TMB substrate, incubate at 37 °C for 15-25 minutes; stop the reaction by adding 50 μL / well of 0.2 M H2SO4, and measure the absorbance at 450 nm.

[0453] like Figure 3 As shown, fusion protein A can bind to spike S1 RBD and effectively block the binding of RBD to ACE2.

[0454] Example 6: Binding experiment of fusion protein A to spike protein on cells

[0455] Cells were collected by centrifugation and washed with 1x flow cytometry buffer (PBS containing 0.1% sodium azide and 5% FBS) (cells were CHO cells expressing SARS-CoV-2 spike or negative cells); Sample preparation: fusion protein A was diluted 3-fold, with an initial concentration of 8.33 μg / mL, and a total of 8 dilutions were prepared; only a 100 nM concentration was used to detect spike-negative cells (i.e., negative cells do not express spike protein); diluted fusion protein A was added to the cells, 100 μl / well, and incubated on ice for 1 hour; washed with flow cytometry buffer: 300 μl / well, 3 washes; diluted PE-goat anti-human IgG-Fc antibody (Thermo Fisher, 12-4998-82) was added, 100 μl / well, and incubated on ice for 1 hour; washed with flow cytometry buffer: 300 μL / well, 3 washes; cells were resuspended in flow cytometry buffer: 200 μL / well; data were analyzed on a CytoFlex using flowjo 10.1.

[0456] CHO cells expressing SARS-CoV-2 spike: CHO-K1 cells were transfected with the expression vector pCMV3-untagged (Beijing Yiqiao Shenzhou Technology Co., Ltd., VG40589) containing the full-length spike protein gene via electroporation. Then, CHO cells expressing SARS-CoV-2 spike were selected by hygromycin pressure screening and flow cytometry sorting (using biotinylated ACE2 labeling and SA-PE (phycoerythrin-labeled streptavidin) labeling).

[0457] like Figure 4 As shown, fusion protein A binds with high affinity to CHO cell lines overexpressing spike, with an EC50 value of 0.5 nM. Furthermore, the experiment verified that fusion protein A does not bind to cells that do not express spike (such as CHO cells, Raji cells, Jurkat cells, TF1 cells, H929 cells, and RPMI8226). This indicates that the binding of the fusion protein to the SARS-CoV-2 spike protein is specific and does not exhibit non-specific binding to spike-negative cells; therefore, it should not produce off-target effects in vivo.

[0458] Example 7: Fusion protein A used to neutralize pseudoviruses

[0459] This experiment evaluated the ability of fusion protein A to inhibit spike-pseudotyped pseudovirus infection of ACE2-expressing cells in vitro. Vero cells were used to detect the ability of fusion protein A to inhibit SARS-CoV-2 pseudovirus infection of cells carrying the luciferase gene. The main principle is as follows: Vero, an African green monkey kidney cell line expressing an ACE2 receptor similar to that of humans, was used as a susceptible cell line; different concentrations of fusion protein A were incubated with the SARS-CoV-2-Fluc pseudovirus system; when fusion protein A binds to the pseudovirus, it blocks viral infection into Vero cells; since the pseudovirus cannot effectively infect cells, its luciferase reporter gene cannot be expressed intracellularly and will not produce a fluorescent signal; since the fluorescence signal value is negatively correlated with the concentration of added fusion protein A, the ability of fusion protein A to inhibit viral infection in vitro can be detected.

[0460] Experimental method: On the first day of infection, fusion protein A was diluted twice in a 96-well plate, and an equal volume of Pseudovirus stock (2×10⁻⁶) was added. 4 TICD 50 Add the diluted fusion protein A to the solution, mix well, and incubate at 37°C for 1 hour. For negative control wells, do not add Pseudovirus stock (Beijing Tiantan Pharmaceutical Biotechnology Development Co., Ltd., A01007424). For positive control wells, add the virus but do not add fusion protein A. During incubation, harvest Vero cells (Chinese Academy of Sciences Cell Bank, GN017) by trypsin digestion, wash once with PBS, and then add 50 μl of a solution containing 2 × 10⁻⁶ FU / mL of fusion protein A. 4 The cells were transferred from DMEM medium to the pre-incubated fusion protein A-pseudovirus mixture; incubated at 37°C for 24-28 hours; 100 μL of luciferase substrate (Novozymes, DD1201) was added; readings were taken after two minutes. The inhibition rate was calculated using the formula: Inhibition rate % = (1 - (sample group – negative control group) / (positive control group – negative control group) × 100).

[0461] like Figure 5 As shown, soluble ACE2 exhibits very limited inhibitory ability at the same concentration. Fusion protein A effectively inhibited SARS-CoV-2 spike pseudovirus infection of Vero cells, with an IC50 value of 0.5 nM.

Claims

1. A fusion protein, characterized in that, The invention includes an antibody or antigen-binding fragment and a six-helix bundle interference polypeptide for interfering with the fusion of the viral capsid with the cell membrane. The antibody or antigen-binding fragment specifically binds to the spike protein of SARS-CoV-2. The antibody or antigen-binding fragment comprises VH CDR1 as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, VH CDR3 as shown in SEQ ID NO:105, VL CDR1 as shown in SEQ ID NO:39, VL CDR2 as shown in SEQ ID NO:40, and VL CDR3 as shown in SEQ ID NO:

41. The C-terminus of the heavy chain of the antibody or antigen-binding fragment is linked to a six-helix bundle interference polypeptide via a linker, wherein the linker is a polypeptide containing glycine and serine. The antibody or antigen-binding fragment comprises two identical heavy chains and two identical light chains.

2. The fusion protein as described in claim 1, characterized in that, The sequence of the connector is (GGGGS) n The n is 1, 2, 3, 4 or 5.

3. The fusion protein as described in claim 1, characterized in that, The six-helix beam interference polypeptide contains the sequence shown in SEQ ID NO:

58.

4. The fusion protein as described in claim 2, characterized in that, The six-helix beam interference polypeptide contains the sequence shown in SEQ ID NO:

58.

5. The fusion protein as described in claim 2, characterized in that, The frame region of the heavy chain variable region of the antibody or antigen-binding fragment includes heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4, and the heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4 are selected from one or more of the following group: (a) Heavy chain FR1 containing the sequence shown in SEQ ID NO:42; (b) Heavy chain FR2 containing the sequence shown in SEQ ID NO:43; (c) Heavy chain FR3 containing the sequence shown in SEQ ID NO:44; (d) Heavy chain FR4 containing the sequence shown in SEQ ID NO:

45.

6. The fusion protein as described in claim 3, characterized in that, The frame region of the heavy chain variable region of the antibody or antigen-binding fragment includes heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4, and the heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4 are selected from one or more of the following group: (a) Heavy chain FR1 containing the sequence shown in SEQ ID NO:42; (b) Heavy chain FR2 containing the sequence shown in SEQ ID NO:43; (c) Heavy chain FR3 containing the sequence shown in SEQ ID NO:44; (d) Heavy chain FR4 containing the sequence shown in SEQ ID NO:

45.

7. The fusion protein as described in claim 4, characterized in that, The frame region of the heavy chain variable region of the antibody or antigen-binding fragment includes heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4, and the heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4 are selected from one or more of the following group: (a) Heavy chain FR1 containing the sequence shown in SEQ ID NO:42; (b) Heavy chain FR2 containing the sequence shown in SEQ ID NO:43; (c) Heavy chain FR3 containing the sequence shown in SEQ ID NO:44; (d) Heavy chain FR4 containing the sequence shown in SEQ ID NO:

45.

8. The fusion protein as described in claim 5, characterized in that, The heavy chain variable region of the antibody or antigen-binding fragment contains the sequence shown in SEQ ID NO:

107.

9. The fusion protein as described in claim 6, characterized in that, The heavy chain variable region of the antibody or antigen-binding fragment contains the sequence shown in SEQ ID NO:

107.

10. The fusion protein as described in claim 7, characterized in that, The heavy chain variable region of the antibody or antigen-binding fragment contains the sequence shown in SEQ ID NO:

107.

11. The fusion protein as described in claim 5, characterized in that, The light chain variable region of the antibody or antigen-binding fragment contains the sequence shown in SEQ ID NO:

50.

12. The fusion protein as described in claim 6, characterized in that, The light chain variable region of the antibody or antigen-binding fragment contains the sequence shown in SEQ ID NO:

50.

13. The fusion protein as described in claim 7, characterized in that, The light chain variable region of the antibody or antigen-binding fragment contains the sequence shown in SEQ ID NO:

50.

14. The fusion protein as described in claim 8, characterized in that, The light chain variable region of the antibody or antigen-binding fragment contains the sequence shown in SEQ ID NO:

50.

15. The fusion protein as described in claim 9, characterized in that, The light chain variable region of the antibody or antigen-binding fragment contains the sequence shown in SEQ ID NO:

50.

16. The fusion protein of claim 10, characterized in that, The light chain variable region of the antibody or antigen-binding fragment contains the sequence shown in SEQ ID NO:

50.

17. The fusion protein according to any one of claims 1-16, characterized in that, The heavy chain constant region of the antibody or antigen-binding fragment contains an amino acid sequence as shown in amino acid positions 1 to 328 of SEQ ID NO:

51.

18. The fusion protein according to any one of claims 1-16, characterized in that, The light chain constant region of the antibody or antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:

52.

19. The fusion protein of claim 17, characterized in that, The light chain constant region of the antibody or antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:

52.

20. The fusion protein according to any one of claims 1-16, characterized in that, The fusion protein includes the following characteristics: The heavy chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:108, from amino acid 1 to amino acid 450.

21. The fusion protein of claim 17, characterized in that, The fusion protein includes the following characteristics: The heavy chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:108, from amino acid 1 to amino acid 450.

22. The fusion protein of claim 18, characterized in that, The fusion protein includes the following characteristics: The heavy chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:108, from amino acid 1 to amino acid 450.

23. The fusion protein as described in claim 19, characterized in that, The fusion protein includes the following characteristics: The heavy chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:108, from amino acid 1 to amino acid 450.

24. The fusion protein according to any one of claims 1-16, characterized in that, The fusion protein includes the following characteristics: The light chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:

57.

25. The fusion protein of claim 17, characterized in that, The fusion protein includes the following characteristics: The light chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:

57.

26. The fusion protein as described in claim 18, characterized in that, The fusion protein includes the following characteristics: The light chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:

57.

27. The fusion protein of claim 19, characterized in that, The fusion protein includes the following characteristics: The light chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:

57.

28. The fusion protein of claim 20, characterized in that, The fusion protein includes the following characteristics: The light chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:

57.

29. The fusion protein as described in claim 21, characterized in that, The fusion protein includes the following characteristics: The light chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:

57.

30. The fusion protein of claim 22, characterized in that, The fusion protein includes the following characteristics: The light chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:

57.

31. The fusion protein as described in claim 23, characterized in that, The fusion protein includes the following characteristics: The light chain of the antibody contains an amino acid sequence as shown in SEQ ID NO:

57.

32. A nucleic acid molecule encoding the fusion protein according to any one of claims 1-31.

33. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fusion protein as described in any one of claims 1-31 and a pharmaceutically acceptable carrier.

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