CN113637083A

CN113637083A - Fusion protein for treating coronavirus and application thereof

Info

Publication number: CN113637083A
Application number: CN202010344084.6A
Authority: CN
Inventors: 张慧; 汪志炜; 陈俊有; 陈振埕; 李闯; 晏庆威; 郑丹丹; 杨少伟; 李嘉萍; 秦超; 黄贤明; 李胜峰; 黄皓晖; 苏紫琪
Original assignee: Bio Thera Solutions Ltd
Current assignee: Bio Thera Solutions Ltd
Priority date: 2020-04-27
Filing date: 2020-04-27
Publication date: 2021-11-12

Abstract

The invention provides a fusion protein for treating coronavirus and application thereof. The fusion protein of the invention can prevent the fusion of SARS-CoV or SARS-CoV-2 virus particle and cell by the cooperation of the polypeptide and the antibody part or antigen binding fragment, and mediate the phagocytosis of immune cell and eliminate virus particle.

Description

Fusion protein for treating coronavirus and application thereof

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a fusion protein for treating coronavirus and application thereof.

Background

Coronaviruses are nonsegmented single-stranded positive-strand RNA viruses, which are classified into four genera, alpha, beta, gamma and delta, according to the serotype and the genomic characteristics, and are named because the virus envelope has protrusions extending around, and is shaped like a corolla. The novel coronavirus (SARS-CoV-2 or 2019-nCoV) discovered in 2019 belongs to a novel coronavirus of beta genus, and has an envelope, a round or oval particle, usually polymorphism and a diameter of 60-140 nm. Current studies show that SARS-CoV-2 has a high homology with SARS-CoV.

The novel coronavirus pneumonia COVID-19 is mainly transmitted through respiratory tract, and may also be transmitted by contact. The population is common and susceptible, the disease of the old and the people with basic diseases is serious after infection, and children and infants also have diseases. Based on current epidemiological investigations, the latency of the new coronaviruses is generally 1-14 days, mostly 3-7 days. The main clinical symptoms of the infected patients are fever, hypodynamia and dry cough, and the symptoms of upper respiratory tract such as nasal obstruction, watery nasal discharge and the like are rare. In the early stage of onset, the total white blood cell count of a patient is normal or reduced, or the number of lymphocytes is reduced, and liver enzyme, myozyme and myoglobin are increased in part of the patients. The chest image shows that the patient presents multiple small spot images and interstitial changes in early stage, and the extrapulmonary zone is obvious; further, the lung is developed into double lungs with multiple wear and wear of vitreous shadows and infiltrations, serious patients can have lung consolidation and gradually have dyspnea, and serious patients have Acute Respiratory Distress Syndrome (ARDS), shock and various tissue injuries and dysfunctions of lung tissues, heart and kidney. The prognosis of most patients with mild infection is good, and severe patients are often critically ill or even die.

Recently, fundamental, clinical and epidemiological studies on COVID-19 have been published or published, but there is still a lack of effective therapeutic drugs.

Disclosure of Invention

The invention provides a fusion protein. The antibody part or antigen binding fragment in the fusion protein has high affinity to spike protein of SARS-CoV and SARS-CoV-2, and these antibodies can specifically bind to the spike protein; the polypeptide in the fusion protein may interfere with the formation of the "six-helix bundle (6-HB)". The fusion protein can prevent the fusion of virus particles and cell membranes and mediate immune cells to phagocytize and eliminate virus particles. These fusion proteins can be used for treating or improving SARS and COVID-19, and can also be used for diagnosing SARS and COVID-19.

The virus particles first bind to the surface of the lung epithelial cells, a so-called angiotensin converting enzyme 2(ACE2), via the S1 subunit of spike protein (S protein or spike protein) on their surface. After the virus binds to the receptor and is hydrolyzed by proteases, the S2 subunit located at the N-terminus of the S protein is exposed and embedded into the serous membrane or endocytotic membrane. The HR2 (head predicted 2) structural domain in the S2 subunit is combined with the HR1 (head predicted 1) structural domain in the S2 subunit to form a 6-HB fusion core, so that the viral outer shell is fused with the cell membrane, SARS-CoV or SARS-CoV-2 enters into the cell and synthesizes new viral particles for the cell; the new viral particles are released outside the cell and infect the surrounding normal cells in the same way. The antibody in the fusion protein can block the combination of spike protein and ACE2, and the polypeptide part prevents the fusion of virus shell and cell membrane, so as to block the virus from entering into cell and play the role of antivirus; antibody moieties in fusion proteins may also mediate phagocytosis by immune cells and clearance of the virus.

Some embodiments provide a fusion protein comprising an antibody or antigen-binding fragment that specifically binds to a spike protein and a polypeptide, and comprising: a) VH CDR1 comprising the amino acid sequence shown as SEQ ID NO. 1, or a single site substitution, deletion or insertion variant thereof; (b) VH CDR2 comprising the amino acid sequence shown in SEQ ID NO. 2, or a single site substitution, deletion or insertion variant thereof; and/or (c) a VH CDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NOs 3-38 or 105, or a variant thereof having a single site substitution, deletion or insertion;

the C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to the polypeptide by a linker. In some embodiments, the linker comprises glycine and serine.

Some embodiments provide a fusion protein comprising an antibody or antigen-binding fragment that specifically binds to a spike protein and a polypeptide, and comprising: (a) VH CDR1 comprising the amino acid sequence shown as SEQ ID NO. 1, or a single site substitution, deletion or insertion variant thereof; (b) VH CDR2 comprising the amino acid sequence shown in SEQ ID NO. 2, or a single site substitution, deletion or insertion variant thereof; and (c) a VH CDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NOs 3-38 or 105, or a variant thereof having a single site substitution, deletion or insertion;

Some embodiments provide a fusion protein comprising an antibody or antigen-binding fragment that specifically binds to a spike protein and a polypeptide, and comprising:

(a) VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO 1; (b) VH CDR2 comprising the amino acid sequence shown as SEQ ID NO. 2; and (c) a VH CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs 3-38 or 105;

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

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

In some embodiments, the substitution variants are conservative amino acid substitution variants.

In some embodiments, the linker has the sequence (G)_mS)_nWherein each m is independently 2, 3,4 or 5 and n is 1, 2, 3,4 or 5. In some embodiments, the linker has the sequence (GGGGS)_nAnd n is 1, 2, 3,4 or 5. In some embodiments, the linker is GGGGS. In some embodiments, the linker is (GGGGS)₂. In some embodiments, the linker is (GGGGS)₃. In some embodiments, the linker is (GGGGS)₄Shown as SEQ ID NO. 59. In some embodiments, the linker is (GGGGS)₅。

In some embodiments, the polypeptide comprises a sequence as set forth in SEQ ID No. 58, a sequence having at least 90% identity to a sequence set forth in SEQ ID No. 58, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence set forth in SEQ ID No. 58. In some embodiments, at least 90% identity is at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or a range between any two of these values (including endpoints), or any value therein. In some embodiments, the one or more conservative amino acid substitutions is about 1, about 2, or about 3 conservative amino acid substitutions. In some embodiments, the polypeptide comprises the sequence set forth as SEQ ID NO: 58.

In some embodiments, the fusion protein comprises an antibody or antigen-binding fragment and a polypeptide, the fusion protein comprising the following features:

the antibody or antigen-binding fragment comprises at least one, two, three, four, five or all of a VH CDR1 shown in SEQ ID NO. 1, a VH CDR2 shown in SEQ ID NO. 2, a VH CDR3 shown in SEQ ID NO. 3-38 or 105, a VL CDR1 shown in SEQ ID NO. 39, a VL CDR2 shown in SEQ ID NO. 40 and a VL CDR3 shown in SEQ ID NO. 41; and/or

The C-terminus of the antibody or antigen-binding fragment is linked to the polypeptide by a linker, the C-terminus of the antibody or antigen-binding fragment being the C-terminus of the heavy chain portion or the C-terminus of the light chain portion of the antibody or antigen-binding fragment; and/or

The sequence of the linker is (GGGGS)_nN is 1, 2, 3,4 or 5; and/or

The polypeptide comprises a sequence shown as SEQ ID NO. 58, a sequence with at least 90% identity with the sequence shown as SEQ ID NO. 58, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 58.

the antibody or antigen-binding fragment comprises at least a VH CDR1 shown in SEQ ID NO. 1, a VH CDR2 shown in SEQ ID NO. 2, a VH CDR3 shown in any one of SEQ ID NO. 3-38 or 105, a VL CDR1 shown in SEQ ID NO. 39, a VL CDR2 shown in SEQ ID NO. 40, and a VL CDR3 shown in SEQ ID NO. 41; and/or

The sequence of the linker is (GGGGS)_nN is 1, 2, 3,4 or 5; and/or

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In some embodiments, the heavy chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID No. 42, a sequence having at least 90% identity to the sequence set forth in SEQ ID No. 42, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 42; and/or

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 to the sequence shown in SEQ ID NO. 43, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 43; and/or

The heavy chain FR3 of the antibody or antigen-binding fragment comprises the sequence shown as SEQ ID NO. 44, a sequence having at least 90% identity to the sequence shown as SEQ ID NO. 44, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown as SEQ ID NO. 44; and/or

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 to the sequence shown in SEQ ID NO. 45, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 45;

the heavy chain FR1, heavy chain FR2, heavy chain FR3 and heavy chain FR4 are framework regions of the heavy chain variable region.

In some embodiments, the heavy chain FR1 comprises the sequence set forth in SEQ ID No. 42, a sequence having at least 90% identity to the sequence set forth in SEQ ID No. 42, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 42; the heavy chain FR2 comprises the sequence shown as SEQ ID NO. 43, a sequence with at least 90% identity to the sequence shown as SEQ ID NO. 43, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown as SEQ ID NO. 43; the heavy chain FR3 comprises the sequence shown as SEQ ID NO. 44, a sequence with at least 90% identity with the sequence shown as SEQ ID NO. 44, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 44; the heavy chain FR4 comprises the sequence shown as SEQ ID NO. 45, a sequence having at least 90% identity with the sequence shown as SEQ ID NO. 45, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown as SEQ ID NO. 45. In some embodiments, at least 90% identity is at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or a range between any two of these values (including endpoints), or any value therein. In some embodiments, the one or more conservative amino acid substitutions is about 1, about 2, or about 3 conservative amino acid substitutions.

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 heavy chain variable region comprises the structure FR1-VH CDR1-FR2-VH CDR2-FR3-VH CDR3-FR 4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises a sequence set forth in any one of SEQ ID NOs 46-49 or 107, a sequence having at least 80% identity to a sequence set forth in any one of SEQ ID NOs 46-49 or 107, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence set forth in any one of SEQ ID NOs 46-49 or 107.

In some embodiments, at least 80% identity is at least about 80% identity, at least about 81% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least about 86% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or a range between any two of these values (including endpoints), or any value therein.

In some embodiments, the one or more conservative amino acid substitutions is about 1, about 2, about 3, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 13, about 14, about 15, about 17, about 18, about 19, about 20 conservative amino acid substitutions, or a range between any two of these values (including endpoints), or any value therein.

In some embodiments, the light chain variable region of the antibody or antigen binding fragment comprises the sequence set forth in SEQ ID No. 50, a sequence having at least 80% identity to the sequence set forth in SEQ ID No. 50, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 50.

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

(a) VH CDR1 comprising the amino acid sequence shown as SEQ ID NO: 60; (b) VH CDR2 comprising the amino acid sequence shown as SEQ ID NO: 61; and (c) a VH CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs 62-97 or 106;

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

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

The sequence of the linker is (GGGGS)_nN is 1, 2, 3,4 or 5; and/or

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

The sequence of the linker is (GGGGS)_nN is 1, 2, 3,4 or 5; and/or

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

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

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

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

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

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

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

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

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

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

In some embodiments, the antibody or antigen-binding fragment comprises at least a VH CDR1 shown in SEQ ID NO:60, a VH CDR2 shown in SEQ ID NO:61, a VH CDR3 shown in SEQ ID NO71, a VL CDR1 shown in SEQ ID NO:98, a VL CDR2 shown in SEQ ID NO:99, and a VL CDR3 shown in SEQ ID NO: 100.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In some embodiments, the heavy chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID No. 101, a sequence having at least 90% identity to the sequence set forth in SEQ ID No. 101, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 101; and/or

The heavy chain FR2 of the antibody or antigen-binding fragment comprises the sequence shown as SEQ ID NO. 102, a sequence having at least 90% identity to the sequence shown as SEQ ID NO. 102, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown as SEQ ID NO. 102; and/or

The heavy chain FR3 of the antibody or antigen-binding fragment comprises the sequence shown as SEQ ID NO. 103, a sequence having at least 90% identity to the sequence shown as SEQ ID NO. 103, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown as SEQ ID NO. 103; and/or

The heavy chain FR4 of the antibody or antigen-binding fragment comprises the sequence shown as SEQ ID NO. 104, a sequence having at least 90% identity to the sequence shown as SEQ ID NO. 104, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown as SEQ ID NO. 104;

In some embodiments, the heavy chain FR1 comprises the sequence shown in SEQ ID No. 101, the heavy chain FR2 comprises the sequence shown in SEQ ID No. 102, the heavy chain FR3 comprises the sequence shown in SEQ ID No. 103, and the heavy chain FR4 comprises the sequence shown in SEQ ID No. 104.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs 46-49 or 107, and/or the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO 50.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO. 46 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO. 50.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO. 47 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO. 50.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO. 48 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth 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 comprises the sequence set forth in SEQ ID NO. 107 and the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO. 50.

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 kappa or lambda chain constant region. In some embodiments, the antibody or fragment thereof is of one of the isotypes IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG 4. Without limitation, the antibody or antigen-binding fragment is a chimeric antibody, a humanized antibody, or a fully human antibody. In a certain aspect, the antibody or antigen-binding fragment is a humanized antibody.

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 a scFV, Fab, F (ab)₂Or IgG. In some embodiments, the antibody or antigen binding fragment is a monoclonal antibody.

In some embodiments, the heavy chain constant region of the antibody or antigen binding fragment comprises a sequence having an amino acid sequence as set forth in SEQ ID No. 51, a sequence having at least 80% identity to the sequence set forth in SEQ ID No. 51, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 51; and/or

The light chain constant region of the antibody or antigen-binding fragment comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 52, a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 52, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 52.

In some embodiments, at least 80% identity is at least about 80% identity, at least about 81% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least about 86% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or a range between any two of these values (including endpoints), or any value therein. In some embodiments, the one or more conservative amino acid substitutions is about 1, about 2, about 3, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 13, about 14, about 15, about 17, about 18, about 19, about 20, about 22, about 24, about 25, about 27, about 30, about 32, about 33, about 36 conservative amino acid substitutions, or a range between any two of these values (including endpoints) or any value therein.

In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises a sequence having an amino acid sequence set forth in SEQ ID NO. 51 and/or the light chain constant region of the antibody or antigen-binding fragment comprises a sequence having an amino acid sequence set forth in SEQ ID NO. 52.

In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises a sequence having an amino acid sequence set forth in SEQ ID NO. 51 and the light chain constant region of the antibody or antigen-binding fragment comprises a sequence having an amino acid sequence set forth in SEQ ID NO. 52.

In some embodiments there is provided a fusion protein comprising the following features:

the heavy chain of the antibody comprises a sequence having an amino acid sequence as set forth in any one of SEQ ID NOs 53-56 or 108, a sequence having at least 80% identity to a sequence set forth in any one of SEQ ID NOs 53-56 or 108, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence set forth in any one of SEQ ID NOs 53-56 or 108; and/or

The light chain of the antibody comprises a sequence with an amino acid sequence shown as SEQ ID NO. 57, a sequence with at least 80% identity with the sequence shown as SEQ ID NO. 57, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 57; and/or

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

In some embodiments, the fusion protein comprises an antibody having a heavy chain comprising a sequence having an amino acid sequence set forth in SEQ ID NO. 53 and a light chain comprising a sequence having an amino acid sequence set forth in SEQ ID NO. 57; the C-terminus of the heavy chain (i.e., the CH3 terminus) or the C-terminus of the light chain (i.e., the CL terminus) of the antibody is covalently linked through a linker as set forth in SEQ ID NO:59 to a polypeptide comprising the sequence set forth in SEQ ID NO: 58.

In some embodiments, the fusion protein comprises an antibody having a heavy chain comprising a sequence having an amino acid sequence set forth in SEQ ID NO:54 and a light chain comprising a sequence having an amino acid sequence set forth in SEQ ID NO: 57; the C-terminus of the heavy chain (i.e., the CH3 terminus) or the C-terminus of the light chain (i.e., the CL terminus) of the antibody is covalently linked through a linker as set forth in SEQ ID NO:59 to a polypeptide comprising the sequence set forth in SEQ ID NO: 58.

In some embodiments, the fusion protein comprises an antibody having a heavy chain comprising a sequence having an amino acid sequence set forth in SEQ ID NO. 55 and a light chain comprising a sequence having an amino acid sequence set forth in SEQ ID NO. 57; the C-terminus of the heavy chain (i.e., the CH3 terminus) or the C-terminus of the light chain (i.e., the CL terminus) of the antibody is covalently linked through a linker as set forth in SEQ ID NO:59 to a polypeptide comprising the sequence set forth in SEQ ID NO: 58.

In some embodiments, the fusion protein comprises an antibody having a heavy chain comprising a sequence having an amino acid sequence set forth in SEQ ID NO:56 and a light chain comprising a sequence having an amino acid sequence set forth in SEQ ID NO: 57; the C-terminus of the heavy chain (i.e., the CH3 terminus) or the C-terminus of the light chain (i.e., the CL terminus) of the antibody is covalently linked through a linker as set forth in SEQ ID NO:59 to a polypeptide comprising the sequence set forth in SEQ ID NO: 58.

In some embodiments, the fusion protein comprises an antibody having a heavy chain comprising a sequence having an amino acid sequence set forth in SEQ ID NO:108 and a light chain comprising a sequence having an amino acid sequence set forth in SEQ ID NO: 57; the C-terminus of the heavy chain (i.e., the CH3 terminus) or the C-terminus of the light chain (i.e., the CL terminus) of the antibody is covalently linked through a linker as set forth in SEQ ID NO:59 to a polypeptide comprising the sequence set forth in SEQ ID NO: 58.

In some embodiments, the fusion protein comprises 2 identical heavy chains and 2 identical light chains, the C-termini of the heavy chains being covalently linked to the polypeptide by a linker. In some embodiments, the fusion protein comprises 2 identical heavy chains and 2 identical light chains, the C-terminus of which is covalently linked to the polypeptide by a linker.

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

The invention also provides a nucleic acid molecule for encoding the fusion protein. In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

The invention also provides a vector comprising the nucleic acid molecule. In some embodiments, the vector is an isolated vector.

The invention also provides a host cell comprising the nucleic acid molecule. In some embodiments, the host cell is an isolated host cell.

The invention also provides a host cell containing the vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, 293 cell, Cos1 cell, Cos7 cell, CV1 cell, murine L cell.

The invention also provides a pharmaceutical composition, which comprises the fusion protein and a pharmaceutically acceptable carrier.

The invention also provides methods of treatment and uses. In some embodiments, methods are provided for treating or ameliorating SARS or COVID-19, the methods comprising administering to a patient an effective dose of the fusion protein. In some embodiments, the use of the fusion protein in treating or ameliorating SARS or COVID-19 is provided. In some embodiments, there is provided a use of the fusion protein in the preparation of a medicament for treating or ameliorating SARS or COVID-19.

The invention also provides diagnostic methods and uses. In some embodiments, methods are provided for detecting the expression of SARS-CoV or SARS-CoV-2 in a sample by contacting the sample with the fusion protein such that the antibody or antigen-binding fragment in the fusion protein binds to the spike protein and detecting its binding, i.e., the amount of spike protein in the sample. In some embodiments, there is provided use in the manufacture of a kit for diagnosing SARS or COVID-19. In some embodiments, a diagnostic kit comprising the fusion protein is provided.

The invention provides a fusion protein for treating coronavirus, wherein the polypeptide in the fusion protein and an antibody part or an antigen binding fragment cooperate to prevent SARS-CoV or SARS-CoV-2 virus particles from fusing with cells, mediate immune cell phagocytosis, remove virus particles and treat SARS or COVID-19; the antibody or antigen binding fragment in the fusion protein of the invention can also be used for diagnosing and detecting whether a patient is infected with SARS-CoV or SARS-CoV-2.

Drawings

FIG. 1 is a graph of the inhibition of SARS-CoV-2 binding to ACE2 by a portion of an anti-spike protein antibody of the invention in an ELISA assay, wherein the abscissa represents concentration and the ordinate represents OD; wherein 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.

Term(s) for

Unless otherwise specified, each of the following terms shall have the meaning set forth below.

Definition of

It should be noted that the term "an" entity refers to one or more of the entities, e.g., "an antibody" should be understood as one or more antibodies, and thus the terms "a" (or "an"), "one or more" and "at least one" may be used interchangeably herein.

The term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptide" and refers to a molecule composed of monomers of amino acids linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single chain or multiple chains of two or more amino acids and does not refer to a particular length of the product. Thus, included within the definition of "polypeptide" are peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to two or more amino acid chains, and the term "polypeptide" may be used in place of, or in alternation with, any of the above terms. The term "polypeptide" is also intended to refer to the product of post-expression modification of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modification. The polypeptide may be derived from a natural biological source or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, and it may be produced in any manner, including chemical synthesis.

"amino acid" refers to an organic compound containing both amino and carboxyl groups, such as an alpha-amino acid, which may be encoded by a nucleic acid, either directly or in the form of a precursor. 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 same amino acid is encoded by different codons and is referred to as "degeneracy of the genetic code". Amino acids include natural amino acids and unnatural 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).

"conservative amino acid substitution" refers to the substitution of one amino acid residue with another amino acid residue having a side chain (R group) of similar chemical nature (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Examples of classes of amino acids containing chemically similar side chains include: 1) aliphatic side chain: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chain: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) basic side chain: lysine, arginine and histidine; 6) acidic side chain: aspartic acid and glutamic acid.

The term "isolated" as used herein with respect to a cell, nucleic acid, polypeptide, antibody, etc., e.g., "isolated" DNA, RNA, polypeptide, antibody, refers to a molecule that is separated from one or more of the other components, e.g., DNA or RNA, respectively, in the natural environment of the cell. The term "isolated" as used herein also refers to nucleic acids or peptides that are substantially free of cellular material, viral material, or cell culture media when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that do not occur in nature, and which do not occur in nature. The term "isolated" is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to include both purified and recombinant polypeptides. Isolated polypeptides, antibodies, and the like are typically prepared by at least one purification step. In some embodiments, an isolated nucleic acid, polypeptide, antibody, etc., is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% pure, or ranges between any two of these values (including endpoints) or any value therein.

The term "recombinant" refers to a polypeptide or polynucleotide, and means a form of a polypeptide or polynucleotide that does not occur in nature, and non-limiting examples may include combinations that produce polynucleotides or polypeptides that do not normally occur.

"homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "identity" or "sequence identity" with another sequence, meaning that the percentage of bases (or amino acids) in the two sequences being compared are the same when the sequences are aligned. The percent identity or sequence identity of the alignment can be determined using visual inspection or software programs known in the art, such as the software program described in Current Protocols in Molecular Biology, Ausubel et al. Preferably, the alignment is performed using default parameters. One alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both using the following default parameters: geneticcode ═ standard; filter is none; strand ═ booth; cutoff is 60; expect is 10; matrix ═ BLOSUM 62; descriptors is 50 sequences; sortby ═ HIGHSCORE; databases is non-redundant; GenBank + EMBL + DDBJ + PDB + GenBank CDStranslations + SwissProtein + Spupdate + PIR. A biologically equivalent polynucleotide is a polynucleotide having the above specified percentage of identity and encoding a polypeptide having the same or similar biological activity.

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

The terms "polynucleotide" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. The polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are examples of non-limiting polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), an exon, an intron, messenger RNA (mrna), transfer RNA, ribosomal RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, structural modifications to the nucleotide can be made before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, embodiments of any polynucleotide of the present disclosure include a double-stranded form and each of two complementary single-stranded forms known or predicted to comprise the double-stranded form.

The term "encoding" as applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide and/or fragments thereof that can be produced by transcription and/or translation in its native state or when manipulated by methods well known to those skilled in the art.

"antibody," "antigen-binding fragment," refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. The antibody may be a whole antibody and any antigen binding fragment thereof or a single chain thereof. The term "antibody" thus includes any protein or peptide in a molecule that contains at least a portion of an immunoglobulin molecule having biological activity that binds to an antigen. Antibodies and antigen-binding fragments include, but are not limited to, the Complementarity Determining Regions (CDRs), heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions (CH), light chain constant regions (CL), Framework Regions (FR), or any portion thereof, or at least a portion of a binding protein, of a heavy or light chain, or ligand-binding portion thereof, as described in the examples. The CDR regions include the CDR regions of the light chain (VL CDR1-3) and the CDR regions of the heavy chain (VH CDR 1-3). The variable region may comprise the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, e.g., F (ab')₂、F(ab)₂Fab', Fab, Fv, scFv, etc. Regardless of its structure, an antibody fragment binds to the same antigen that is recognized by an intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by forming a complex with a particular antigen.

"Single chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some aspects, these regions are linked to a short linker peptide of 10 to about 25 amino acids. The linker may be glycine rich to increase flexibility and serine or threonine rich to increase solubility and may link the N-terminus of VH and the C-terminus of VL, or vice versa. Although the protein has the constant region removed and the linker introduced, it retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art and are described in, for example, U.S. Pat. No. 5,892,019.

The term "antibody" includes a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that the class of heavy chains includes gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ 1- γ 4). The nature of this chain determines the "class" of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgG5, and the like have been well characterized and the functional specificity conferred is also known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is an IgG class. IgG typically comprises two identical light chain polypeptides having a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides having a molecular weight of about 53,000-70,000. The four chains are linked by disulfide bonds in a "Y" configuration, in which the light chain begins at the "Y" opening and continues through the variable region surrounding the heavy chain.

The antibodies, antigen binding fragments, or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies, epitope binding fragments such as Fab, Fab ', and F (ab')₂Fd, Fvs, single chain Fvs (scFv), disulfide linked Fvs (sdFv), fragments comprising VK or VH domains, or fragments produced from Fab expression libraries and anti-idiotypic (anti-Id) antibodies. The immunoglobulin or antibody molecules disclosed herein may be of 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 immunoglobulin.

Light chains can be classified as kappa (. kappa.) or lambda (. lamda.). Each heavy chain may be associated with a kappa or lambda light chain. In general, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, the light and heavy chains are joined by covalent bonds and the "tail" portions of the two heavy chains are joined by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the forked end of the Y configuration to the C-terminus of the bottom of each chain. The immunoglobulin kappa light chain variable region is Vkappa; immunoglobulin lambda light chain variable region is V_λ。

Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in accordance with function. The variable regions of the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. The constant regions of the light and heavy chains confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement fixation, etc. By convention, the numbering of constant regions increases as they become further away from the antigen binding site or amino terminus of the antibody. The N-terminal part is a variable region and the C-terminal part is a constant region; the CH3 and CL domains actually comprise the carboxy-termini of the heavy and light chains, respectively.

As described above, the variable regions enable the antibody to selectively recognize and specifically bind to an epitope on an antigen. In particular, the VL domain and VH domain of an antibody, or a subset of Complementarity Determining Regions (CDRs), combine to form variable regions that define a three-dimensional antigen binding site. The antibody quaternary structure forms an antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs (i.e., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) in each of the VH and VL chains. In certain cases, such as certain camelid derived or engineered immunoglobulin molecules based on camelid immunoglobulins, the intact immunoglobulin molecule may consist of only heavy chains, without light chains. See, for example, Hamers-Casterman et al, Nature, 363: 446-.

In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous amino acid sequences that form the antigen binding domain that specifically bind to an antigen, assuming the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining other amino acids in the antigen binding domain, referred to as the "framework" region, show less intermolecular variability. The framework regions largely adopt a β -sheet conformation with the CDRs forming a loop structure attached to, or in some cases forming part of, the β -sheet structure. Thus, the framework regions allow the CDRs to be positioned in the correct orientation by forming a scaffold via interchain non-covalent interactions. The antigen binding domain with the CDRs at a particular location forms a surface complementary to an epitope on an antigen that facilitates non-covalent binding of an antibody to its antigenic epitope. Amino acids comprising CDRs and framework regions can be identified by known methods for a given heavy or light chain variable region by one of ordinary skill in the art (see Kabat, E., et al, U.S. department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J.mol.biol.,196:901-917 (1987)).

Where two or more definitions are provided for a term used and/or accepted in the art, the definition of the term as used herein includes all such meanings unless explicitly stated to the contrary. One specific example is the use of the term "complementarity determining regions" ("CDRs") to describe non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. This particular region is described in Kabat et al, U.S. Dept. of Health and Human services,,, Sequences of Proteins of Immunological Interest (1983) and Chothia et al, J.mol.biol.196:901-917(1987), which are incorporated herein by reference in their entirety.

CDRs defined according to Kabat and Chothia include overlaps or subsets of amino acid residues when compared to each other. Nevertheless, it is within the scope of the invention to apply either definition to refer to the CDRs of an antibody or variant thereof. The exact residue number comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which specific residues a CDR contains based on the amino acid sequence of the variable region of an antibody.

Kabat et al also define a numbering system for the variable region sequences applicable to any antibody. One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence without relying on other experimental data beyond the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al, U.S. Dept. of Health and Human Services in "sequence of proteins of Immunological Interest" (1983). Antibodies can also be used with the EU numbering system.

The antibodies disclosed herein may be derived from any animal, including birds and mammals. Preferably, the antibody is of human, murine, donkey, rabbit, goat, camel, llama, horse or chicken origin. In another embodiment, the variable region may be of chondrocyclic (condricthoid) origin (e.g. from sharks).

"heavy chain constant region" includes amino acid sequences derived from immunoglobulin heavy chains. The polypeptide comprising a heavy chain constant region comprises at least one of a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. For example, the antibodies or antigen binding fragments disclosed herein comprise a CH1 domain; comprising a CH1 domain, at least a portion of a hinge region, and a CH2 domain; comprises a CH1 domain and a CH3 domain; comprising a CH1 domain and at least a portion of a hinge region and a CH3 domain; or comprises a CH1 domain, at least a portion of a hinge region, and a CH2 domain and a CH3 domain. In another embodiment, the disclosed antibody or antigen binding fragment comprises a CH3 domain. Furthermore, the antibodies or antigen-binding fragments used in the present invention may lack part or all of the CH2 domain. As described above, it will be appreciated by those of ordinary skill in the art that the heavy chain constant regions may be modified such that the amino acid sequence of their naturally occurring immunoglobulin molecules is altered.

The heavy chain constant region of an antibody may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may comprise a heavy chain constant region derived from an IgG₁CH1 domain of molecule and derived from IgG₃The hinge region of the molecule. In another embodiment, the heavy chain constant region may comprise a portion derived from an IgG₁Molecules and moieties derived from IgG₃The hinge region of the molecule. In another embodiment, a portion of the heavy chain may comprise a portion derived from IgG₁Molecules and moieties derived from IgG₄A chimeric hinge region of the molecule.

"light chain constant region" includes the amino acid sequence from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. "light chain-heavy chain pair" refers to a collection of light and heavy chains that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

As mentioned above, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. The "VH domain" comprises the amino-terminal variable domain of an immunoglobulin heavy chain and the "CH 1 domain" comprises the first (largely amino-terminal) constant region of an immunoglobulin heavy chain. The CH1 domain is contiguous with the VH domain and is the amino terminus of the hinge region of an immunoglobulin heavy chain molecule. The CH2 domain is not tightly paired with other domains, but rather two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. It is also documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues. "hinge region" includes that portion of the heavy chain region that connects the CH1 domain and the CH2 domain. The hinge region comprises about 25 residues and is flexible, thereby enabling independent movement of the two N-terminal antigen-binding regions. The hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains (rouxetal, j.immunol161:4083 (1998)).

"disulfide bond" refers to a covalent bond formed between two sulfur atoms. The thiol group of cysteine may 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 corresponding positions 239 and 242 in the Kabat numbering system (EU numbering system positions 226 and 229).

"chimeric antibody" refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be intact, partial, or modified) are derived from a second species. In certain embodiments, the variable region is from a non-human source (e.g., mouse or primate) and the constant region is from a human source.

"specific binding" or "specific for … …" generally refers to the formation of a relatively stable complex of an antibody or antigen-binding fragment and a particular antigen through complementary binding of its antigen-binding domain to an epitope. "specificity" can be expressed in terms of the relative affinity of an antibody or antigen-binding fragment for binding to a particular antigen or epitope. For example, an antibody "a" can be considered to have a higher specificity for the same antigen than an antibody "B" if antibody "a" has a greater relative affinity for the antigen than antibody "B". Specific binding can be described by the equilibrium dissociation constant (KD), with a smaller KD implying tighter binding. Methods of determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, biofilm layer optical interferometry, and the like. An antibody that "specifically binds" a spike protein includes an antibody that has an equilibrium dissociation constant KD for the spike protein of less than or equal to about 100nM, less than or equal to about 10nM, less than or equal to about 5nM, less than or equal to about 1nM, or less than or equal to about 0.5 nM.

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures, with the object of preventing, slowing, ameliorating, or halting undesirable physiological changes or disorders, such as the progression of a disease, including, but not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration, palliation, alleviation or abolition (whether partial or total) of disease state, extending the expected life span when not treated, and the like, whether detectable or undetectable. Patients in need of treatment include patients already with a condition or disorder, patients susceptible to a condition or disorder, or patients in need of prevention of the condition or disorder, patients who may or are expected to benefit from administration of the antibodies or pharmaceutical compositions disclosed herein for detection, diagnostic procedures, and/or treatment.

"patient" refers to any mammal in need of diagnosis, prognosis or treatment, including humans, dogs, cats, rabbits, rats, mice, horses, cattle, etc.

Fusion proteins

The present invention provides fusion proteins. The antibody part or antigen binding fragment in the fusion protein has high affinity to spike protein of SARS-CoV and SARS-CoV-2, and these antibodies can specifically bind to the spike protein; the polypeptide in the fusion protein may interfere with the formation of the "six-helix bundle (6-HB)". The antibodies tested exhibit potent binding activity and are useful for therapeutic and diagnostic purposes. For example, these antibodies or antigen-binding fragments can prevent the fusion of SARS-CoV and SARS-CoV-2 viral particles with cell membranes, and mediate phagocytosis of immune cells, clearance of viral particles.

Accordingly, one embodiment of the present disclosure provides a fusion protein. Fusion proteins include antibodies or antigen-binding fragments against spike proteins, as well as linkers, polypeptides.

The sequences of the fusion proteins disclosed in the embodiments of the present invention are shown in Table 1, wherein the sequences include CDR regions, FR regions, heavy and light chain constant regions, heavy and light chain variable regions, linkers, polypeptides, etc., of the antibody or antigen binding fragment; the CDR, FR region partitions for antibodies or antigen binding fragments may also be divided according to the Kabat definition, as shown in table 2.

In some embodiments, the fusion proteins disclosed herein comprise a polypeptide and an antibody or antigen-binding fragment, the C-terminus of the heavy chain or the C-terminus of the light chain of the antibody being covalently linked to the polypeptide by a linker (as set forth in SEQ ID NO: 59), the polypeptide comprising a sequence as set forth in SEQ ID NO:58, a sequence having at least 90% identity to the sequence as set forth in SEQ ID NO:58, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence as set forth in SEQ ID NO: 58.

In some embodiments, the fusion proteins disclosed herein include a polypeptide and an antibody or antigen-binding fragment comprising a VH set forth as SEQ ID NO 46, 47, 48, 49, or 107, a VL set forth as SEQ ID NO 50, or a biological equivalent of each. A biological equivalent of a VH or VL is a sequence that comprises a particular amino acid, i.e., the sequence as a whole has about 80%, about 85%, about 90%, about 95%, about 98%, about 99% sequence identity, or a range between any two of these values (including endpoints), or any value therein.

In some embodiments, the C-terminal (i.e., CH3 terminal) linker of the heavy chain of the antibody is covalently linked to the polypeptide. In some embodiments, the C-terminus of the light chain of the antibody (i.e., the CL terminus) is covalently linked to the polypeptide through a linker.

In some embodiments, the C-terminus of the heavy chain of the antibody (i.e., the CH3 terminus) is covalently linked through a linker as set forth in SEQ ID NO:59 to a polypeptide comprising the sequence set forth in SEQ ID NO: 58. In some embodiments, the C-terminus of the light chain of the antibody (i.e., the CL terminus) is covalently linked through a linker as set forth in SEQ ID NO:59 to a polypeptide comprising a sequence set forth in SEQ ID NO: 58.

In some embodiments, a fusion protein, e.g., wherein the antibody may further be linked to an amino acid sequence or one or more modifying groups. For example, fusion proteins are disclosed wherein, for example, the antibody may comprise a flexible linker sequence, or may be modified to add a functional group (e.g., PEG, drug, toxin, or tag).

Fusion proteins are disclosed, for example, wherein the antibody, antigen-binding fragment, further comprises a modified derivative, i.e., modified by covalent attachment of any type of molecule to the antibody, wherein the covalent attachment does not prevent the antibody from binding to the epitope. Including, but not limited to, examples where antibodies can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be made by existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

In some embodiments, the fusion protein, for example, wherein the antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, agent, or PEG.

Fusion proteins, for example, wherein the antibody can be conjugated or fused to a therapeutic agent, which can include detectable labels, such as radioactive labels, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic agents, diagnostic agents, cytotoxic agents, ultrasound enhancing agents, nonradioactive labels, and combinations thereof, and other such agents known in the art.

Fusion proteins, for example, wherein the antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the luminescence that occurs during the course of the chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

Polynucleotides encoding antibodies and methods of making antibodies

Also disclosed are polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof of the invention. The polynucleotides disclosed herein may encode a heavy chain, a light chain, a variable region of a heavy chain, a variable region of a light chain, an Fc region, a portion of a variable region of a heavy chain, or a portion of a variable region of a light chain. Methods of making antibodies are well known in the art and are described herein. In certain embodiments, the variable and constant regions of the antibodies, antigen-binding fragments disclosed herein are all of human origin. Fully human antibodies and antigen-binding fragments can be prepared using techniques disclosed in the art and described herein. For example, fully human antibodies to a particular antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce fully human antibodies in response to antigen challenge. Exemplary techniques that can be used to prepare such antibodies are described in U.S. patent 6,458,592; 6,420,140, the entire contents of which are incorporated herein by reference.

In certain embodiments, the antibodies produced do not elicit a deleterious immune response in the animal (e.g., human) to be treated. In one embodiment, the antibodies, antigen-binding fragments, or derivatives disclosed herein are modified to reduce their immunogenicity using art-recognized techniques. For example, the antibody may be humanized, primatized, deimmunized or a chimeric antibody may 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 are less immunogenic in humans. This can be achieved by a variety of methods, including (a) grafting the entire variable region of non-human origin to a constant region of human origin to produce a chimeric antibody; (b) grafting at least a portion of one or more non-human Complementarity Determining Regions (CDRs) into a framework and constant region of human origin, with or without retention of critical framework residues; or (c) transplanting the entire variable regions of non-human origin, but "hiding" them by replacing surface residues with portions of human-like origin. Typically, framework residues in the human framework region will be substituted with corresponding residues from the CDR donor antibody, such as residues capable of improving antigen binding. These framework substitutions can be identified by methods well known in the art, for example, by modeling the interaction of the CDRs with framework residues to identify framework residues that play a significant role in antigen binding and by sequence alignment to identify framework residues that are aberrant at particular positions. (see U.S. Pat. No. 5,585,089; Riechmann et al, Nature 332:323 (1988); incorporated herein by reference in its entirety). Antibodies can be humanized using a variety of techniques well known in the art, such as CDR grafting (EP 239,400; WO 91/09967; U.S. Pat. Nos. 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); studnika 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. Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.

Deimmunization may also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunization" includes the alteration of antibodies to modify T cell epitopes (see, e.g., WO/9852976A1 and WO/0034317A 2). For example, the heavy and light chain variable region sequences from the starting antibody are analyzed and a human T cell epitope "map" is generated from each variable region, showing the position of the epitope relative to the 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 lower risk of altering antibody activity. A series of alternative heavy chain variable region sequences and light chain variable region sequences comprising combinations of amino acid substitutions are designed and subsequently incorporated into a series of binding polypeptides. The genes comprising the modified variable regions and the complete heavy and light chains of the human constant regions are then cloned into expression vectors, and the plasmids are subsequently transferred into cell lines to produce complete antibodies. The antibodies are then compared in appropriate biochemical and biological experiments to identify the best antibody.

The binding specificity of the antibodies and antigen binding fragments in the fusion proteins disclosed herein can be detected by in vitro assays, such as co-immunoprecipitation, Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

Alternatively, techniques for producing single stranded cells (U.S. Pat. No. 4,694,778; Bird, Science 242: 423-. The heavy and light chain fragments that bridge the Fv region by amino acids form a single chain unit, resulting in a single chain fusion peptide. Techniques for assembling functional Fv fragments in E.coli can also be used (Skerra et al, Science 242: 1038-.

Examples of techniques that may be used to produce single chain fv (scFv) and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498, and Huston et al, Methods in Enzymology 203:46-88(1991), Shu et al, Proc. Natl.Sci.USA 90: 1995-. For certain uses, including the use of antibodies in humans and in vitro detection assays, chimeric, humanized or fully human antibodies may be used. Chimeric antibodies are a class of molecules in which different portions of the antibody are derived from different animal species, such as antibodies having the variable regions of murine monoclonal antibodies and human immunoglobulin constant regions. Methods for producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); oi et al, BioTechniques 4:214 (1986); gillies 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, which are incorporated herein by reference in their entirety.

In addition, another efficient method for producing recombinant antibodies is disclosed in Newman, Biotechnology 10: 1455-. In addition, this technique is also mentioned in commonly assigned U.S. Pat. Nos. 5,658,570, 5,693,780, and 5,756,096, each of which is incorporated herein by reference in its entirety.

Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries from immunoglobulin sequences. Reference may also be made to U.S. Pat. Nos. 4,444,887 and 4,716,111, as well as PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, the entire contents of each of which are incorporated herein by reference.

Fully human antibodies are particularly desirable for treating human patients. Fully human antibodies can be prepared by a variety of methods known in the art, for example, human antibodies can be produced by transgenic mice that do not express functional endogenous immunoglobulins but express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, in addition to human heavy and light chain genes, human variable, constant and diversity regions can be introduced into mouse embryonic stem cells. Immunoglobulin genes of mouse heavy and light chains can be disabled by homologous recombination separately or simultaneously by introduction of human immunoglobulin loci. For example, homozygous deletion of the JH region can prevent the production of endogenous antibodies. The modified embryonic stem cells were expanded and microinjected into blastocysts to generate chimeric mice. Chimeric mice were then bred to produce homozygous progeny expressing the human antibody. Transgenic mice are immunized in a conventional manner with selected antigens, e.g., all or part of the target polypeptide target. Antigen-targeting monoclonal antibodies can be obtained from immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation, followed by class switching and somatic mutation. Accordingly, IgG, IgA, IgM, and IgE antibodies useful for therapy can be produced using this technique. For a related 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 this technique 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.

Fully human antibodies recognizing selective epitopes can also be produced using a technique known as "guided selection". In this method, a selected non-human monoclonal antibody (e.g., a mouse antibody) is used to direct the screening of fully human antibodies that recognize the same epitope (see U.S. Pat. No. 5,565,332, the entire contents of which are incorporated herein by reference).

In another embodiment, DNA encoding the desired monoclonal antibody can be isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Isolated and subcloned hybridoma cells can serve as a source of such DNA. Once isolated, the DNA may be placed into 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. Isolated DNA (which may be synthetic as described herein) may also be used to prepare the sequences of the constant and variable regions of antibodies, as described in U.S. Pat. No. 5,658,570, which is incorporated herein by reference in its entirety. This method extracts RNA from selected cells and converts it to cDNA, which is then amplified by PCR techniques using Ig-specific primers. Suitable probes for this purpose are also mentioned in U.S. Pat. No. 5,658,570.

In addition, using conventional recombinant DNA techniques, one or more CDRs of an antibody of the invention can be inserted into a framework region, e.g., into a human framework region, to construct a humanized non-fully human antibody. The framework regions may be naturally occurring or shared framework regions, preferably human framework regions (see Chothia et al, J.mol.biol.278: 457-. Some polynucleotides may encode antibodies produced by the framework region and CDR combination that specifically bind to at least one epitope of an antigen of interest. One or more amino acid substitutions may be made within the framework regions, and amino acid substitutions may be selected which improve binding of the antibody to its antigen. Alternatively, substitution or deletion of cysteine residues in one or more of the variable regions involved in interchain disulfide bond formation can be performed in this manner, thereby producing an antibody molecule lacking one or more interchain disulfide bonds. Other variations of polynucleotides within the skill of the art are also encompassed by the present invention.

Antibody-producing cell lines 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 as being suitable for use with the present invention are referenced to Current Protocols in Immunology, Coligan 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 by reference in their entirety.

In some embodiments, the expression antibody vector comprises at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences and donor and acceptor sites for RNA splicing on both sides of the insert. High transcription efficiency can be achieved by the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI and the early promoters of cytomegalovirus, and other cellular promoters such as actin can also be used. 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, pCS2, and the like. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, murine L cells, CHO cells, and the like.

In some embodiments, the inserted gene fragment contains a selection marker, and common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, and the like, so as to facilitate the selection and isolation of cells that are transfected successfully. The constructed plasmid is transfected to host cells without the genes, and the cells successfully transfected grow in large quantities through selective culture medium culture to produce the target protein to be obtained.

In some embodiments, the plasmid is pBAT, which is derived from engineered optimized pcDNA3.1(-), which contains the glutamine synthetase gene under the control of the SV40 early promoter. Cells successfully transfected can be selectively cultured in a glutamine-free medium added with MSX (L-amino sulfoxide methionine), cells with weak endogenous glutamine synthesis capacity such as CHO and the like cannot grow, and cells successfully transfected and containing the target gene grow normally. The screening system has the advantages of high yield, fast screening process, high stability, easy success of expanding culture and the like, and is widely used.

In some embodiments, plasmid pBAT contains a strong promoter for RSV (rous sarcoma virus) for expression of the gene of interest and an enhancer segment from human Cytomegalovirus (CMV). The plasmid has restriction enzyme recognition sites such as HindIII, EcoRI, BsiWI, pvuI and NotI, which are convenient for inserting a target gene. This plasmid contains the 3' intron and polyadenylation and termination signals of the rat preproinsulin gene after these cloning sites. Other high efficiency promoters may also be used for expression, for example the human β -agonist promoter, the SV40 early or late promoter or the long terminal repeat strong promoter from other retroviruses such as HIV and HTLVI. For polyadenylation of mRNA, signals from other genes such as the human growth hormone or globin genes may also be used. Stable cell lines carrying the gene of interest integrated into the chromosome may also be selected by using a selectable marker such as G418 or a resistance gene to hygromycin. At the outset, more than one selectable marker, such as G418 plus methotrexate, may be used.

In addition, standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequences encoding the antibodies of the present invention, including but not limited to site-directed mutations resulting in amino acid substitutions and PCR-mediated mutations. Variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions relative to the original heavy chain variable region VH CDR1, VH CDR2, VH CDR3 and light chain variable region VL CDR1, VL CDR2 or VL CDR 3. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

In some embodiments, the substitutions described herein are conservative amino acid substitutions.

Method of treatment

The invention also provides methods of treatment and uses. In some embodiments, methods are provided for treating or ameliorating SARS or COVID-19, the methods comprising administering to a patient an effective dose of the fusion protein. In some embodiments, the use of the fusion protein in treating or ameliorating SARS or COVID-19 is provided. In some embodiments, there is provided a use of the fusion protein in the preparation of a medicament for treating or ameliorating SARS or COVID-19. 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 in contact with SARS-CoV or a SARS-CoV-2 virus carrier. In some embodiments, the patient is diagnosed with SARS-CoV or SARS-CoV-2 virus. In some embodiments, the patient is a mildly symptomatic patient. In some embodiments, the patient is a severely symptomatic patient. In some embodiments, the patient has fever, cough, hypotension, hypoxia, and/or Acute Respiratory Distress Syndrome (ARDS).

The specific dose and treatment regimen for any particular patient will depend upon a variety of factors including the particular fusion protein or derivative used, the age and weight of the patient, general health, sex and diet, and the time of administration, frequency of excretion, drug combination, and the severity of the particular disease being treated. These factors are judged by a medical caregiver who is within the purview of one of ordinary skill in the art. The dosage will also depend upon the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound employed, the severity of the disease and the effect desired. The dosage employed can be determined by pharmacological and pharmacokinetic principles well known in the art.

Methods of administration of the fusion protein or derivative include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral injection. The pharmaceutical compositions may be administered by any convenient route, for example by infusion or bolus injection, absorbed through epithelial or cutaneous mucosa (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be co-administered with other biologically active agents. Thus, a pharmaceutical composition comprising a fusion protein of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drop, or transdermal patch), buccally, or by oral or nasal spray.

The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The mode of administration may be systemic or local. Furthermore, it may be desirable to introduce the antibodies of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be assisted by an intraventricular catheter connected to, for example, a reservoir (which may be an Ommaya reservoir). Pulmonary administration is also possible, for example by using an inhaler or nebulizer, and also by using a nebulized formulation.

The fusion proteins of the invention may be administered topically to the area in need of treatment; the following may be used, but not limited to: local infusion during surgery, for example in combination with a post-operative wound dressing, is achieved by injection, through a catheter, by means of a suppository or by means of an implant, which is a porous, non-porous or gelatinous material, including membranes (e.g. silicone rubber membranes) or fibres. Preferably, when administering the proteins of the invention (including antibodies), care must be taken to use materials that do not absorb the protein.

In some embodiments, the compositions of the invention comprise a nucleic acid or polynucleotide encoding a protein, which can be administered in vivo to facilitate expression of the protein encoded by it by constructing it as part of a suitable nucleic acid expression vector, which can then be made intracellular by administering such parts of the vector, for example, by using a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by using microprojectile bombardment (e.g., gene gun; Biolistic, Dupont), or coated with lipids or cell surface receptors or transfection reagents, or by ligation with homeobox-like peptides known to enter the nucleus (see, e.g., Joliot et al, 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), and the like. Alternatively, the nucleic acid may be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression.

In some embodiments, the fusion protein of the invention is administered to a patient at a dose of 0.01mg/kg to 100mg/kg of patient body weight, or 0.1mg/kg to 20mg/kg of patient body weight. A second or more doses of the antibody or antigen-binding fragment may be administered subsequently after the initial dose, at about the same or less dose as the initial dose, wherein the subsequent doses may be separated by at least 1 to 3 days; or at least one week. The dosage and frequency of administration of the antibodies of the invention can be reduced by enhancing the uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as lipidation.

Methods for treating diseases are generally performed in vitro assays comprising administering a fusion protein or derivative of the invention, then testing for the desired therapeutic or prophylactic activity in vivo in an acceptable animal model, and finally administering to a human. Suitable animal models, including transgenic animals, are well known to those of ordinary skill in the art. For example, in vitro assays for demonstrating therapeutic use of the antibodies, antigen-binding fragments, fusion proteins of the invention include the effect of the antibody on a cell line or patient tissue sample. The effect of the antibodies, fusion proteins on cell lines and/or tissue samples can be detected using techniques known to those skilled in the art, such as those disclosed elsewhere herein. In accordance with the teachings of the present invention, in vitro assay experiments useful for determining whether to administer a specific antibody, fusion protein, include in vitro cell culture experiments in which a patient tissue sample is cultured in culture and exposed to or otherwise administered a compound, and the effect of such compound on the tissue sample is observed.

Various known delivery systems may be used to administer the fusion proteins or derivatives of the invention or polynucleotides encoding them, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endocytosis (see, e.g., Wu and Wu,1987, J.biol. chem.262: 4429. about. 4432), construction of nucleic acids as part of a retrovirus or other vector, and the like.

Combination therapy

In some embodiments, the fusion proteins of the invention may be used together or in combination with other therapeutic or prophylactic regimens, including the administration of one or more fusion proteins of the invention and one or more other therapeutic agents or methods. For combination therapy, the fusion protein may be administered simultaneously or separately with the other therapeutic agent. When administered separately, the fusion protein of the invention may be administered before or after administration of another additional therapeutic agent.

In some embodiments, the therapeutic agent administered in combination with the fusion protein of the invention is at least one of: HIV drugs, antimalarial drugs, RNA polymerase inhibitors, antiviral drugs, and monoclonal drugs. In some embodiments, the HIV drug comprises lopinavir/ritonavir, ASC 09/ritonavir, and darunavir. In some embodiments, the antimalarial comprises chloroquine, hydroxychloroquine, and chloroquine phosphate. In some embodiments, the RNA polymerase inhibitor comprises reicepivir. In some embodiments, the antiviral drug comprises abidol, favipiravir. In some embodiments, the monoclonal antibody comprises BDB-001. In some embodiments, the antiviral drug is an antibody or antigen-binding fragment of a fusion protein of the invention.

Some patients with severe or critical coronavirus pneumonia have cytokine storm phenomenon, and the fusion protein of the invention can be combined with adalimumab (for example

And biological analogues thereof, e.g. Abrilada^TM(adalimumab-afzb)，Amjevita(adalimumab-att)，Cyltezo^TM(adalimumab-adbm)，Hyrimoz^TM(adalimumab-adaz)，Hulio^TM，

(

BAT1406)) or toslizumab (tocilizumab, e.g.

And its biological analogs, such as BAT1806) for treating inflammation caused by up-regulation of TNF-alpha expression. In some embodiments, patients treated by the present methods are diagnosed as infected with the novel coronavirus and have an increased level of one or more cytokines including tumor necrosis factor alpha (TNF-alpha), IFN-gamma, IL-1 beta, IL-2, IL-4, IL-7, IL-8, IL-10, IL-12p70, IL-13, granulocyte colony stimulating factor (GSCF), Interferon inducible protein-10 (IP-10), monocyte chemotactic protein-1 (MCP1), macrophage inflammatory protein 1 alpha (MIP 1A). In some embodiments, the subject treated by the present methods has increased TNF- α. In some embodiments, the one or more cytokines are at least 50% higher than normal levels. In some embodiments, the one or more cytokines are at least 2-fold, 3-fold, or 4-fold of the normal level. In some embodiments, the subject is suffering from fever, hypotension, hypoxia, and/or Acute Respiratory Distress Syndrome (ARDS) prior to treatment with the present method. In some embodiments, the patient has lungs filled with inflammatory fluid (so-called "white lungs") prior to treatment by the present method. In some embodiments, the patient has Cytokine storm induced Cytokine Release Syndrome (CRS) prior to treatment by the method.

In some embodiments, the fusion proteins of the invention are used in therapy in conjunction with ICU. In some embodiments, the fusion proteins of the invention are combined with in vitro ECMO and/or IMV therapy. In some embodiments, the fusion protein of the invention is combined with oxygen therapy. In some embodiments, the antibodies or antigen binding fragments of the invention bind NIV/HFNC therapy. In some embodiments, after treatment, the patient has at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction in one or more cytokines over before treatment. In some embodiments, the method results in the patient recovering.

Diagnostic method

The expression of spike proteins is observed in certain samples, and patients with spike protein expressing cells may respond to treatment with the fusion proteins of the invention. Thus, the fusion proteins of the invention may also be used for diagnosis and prognosis.

A sample comprising cells may be obtained from a patient. After the sample is optionally pretreated, the sample can be incubated with the fusion protein of the invention under conditions that allow the antibody to interact with spike proteins that may be present in the sample. The presence of spike protein in a sample can be detected using an antibody in the fusion protein using methods such as ELISA.

The presence (e.g., amount or concentration) of spike protein in a sample can be used to diagnose a related disease, as an indication that the patient is eligible for fusion protein therapy, or as an indication that the patient has (or has not) responded to treatment of the disorder. For prognostic methods, one, two or more tests may be performed at a particular stage at the beginning of disease treatment to indicate the progress of the treatment.

Pharmaceutical composition

The invention also provides a pharmaceutical composition. Such compositions comprise an effective amount of the fusion protein and a pharmaceutically acceptable carrier.

In some embodiments, the term "pharmaceutically acceptable" refers to substances for animals (particularly for humans) that are approved by a governmental regulatory agency or other generally recognized pharmacopoeia. Furthermore, a "pharmaceutically acceptable carrier" is generally intended to mean any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid, and the like.

The term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which the active ingredient may be administered to a patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. If desired, the pharmaceutical compositions may also contain minor amounts of wetting, emulsifying, or pH buffering agents 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 ethylenediaminetetraacetic acid, and tonicity adjusting agents such as sodium chloride or dextrose are also contemplated. These pharmaceutical compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The pharmaceutical compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable Pharmaceutical carriers are described in Remington's Pharmaceutical Sciences of e.w. martin, which is incorporated herein by reference. Such compositions will contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, together with an appropriate amount of carrier to provide a form of administration suitable for the patient. The formulation should be suitable for the mode of administration. The formulations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In some embodiments, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous injection into a human. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. The pharmaceutical composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the site of injection. Generally, the active ingredients are provided in unit dosage forms, either separately or in admixture, e.g., as a dry lyophilized powder or as an anhydrous concentrate, in a sealed container (e.g., ampoule or sachet) which is indicative of the serving size of the active agent. In the case of administration of the composition by infusion, the composition may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water or saline for injection may be used so that the effective ingredients may be mixed before administration.

The compounds of the present invention may be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

"about" refers to the conventional error range for corresponding numerical values as would be readily understood by one of ordinary skill in the relevant art. In some embodiments, reference herein to "about" refers to the numerical values recited and ranges of ± 10%, ± 5%, or ± 1% thereof.

"ECMO" refers to the Extracorporeal Membrane Oxygenation (ECMO), which is a medical emergency technology device mainly used to provide continuous Extracorporeal respiration and circulation to patients with severe cardiopulmonary failure to maintain the life of the patients.

The ICU refers to Intensive Care Unit (Intensive Care Unit), and can be used for synchronously carrying out treatment, nursing and rehabilitation, providing isolation places and equipment for patients with severe or coma, providing optimal nursing, comprehensive treatment, medical and nursing combination, early rehabilitation after operation, joint nursing exercise treatment and other services.

"IMV" refers to intermittent commanded ventilation (intermittent ventilation), which is the implementation of periodic volume or pressure ventilation based on a preset time interval, i.e., time trigger. During which the patient is allowed to breathe spontaneously at any set basal pressure level during the commanded ventilation. In spontaneous breathing, the patient may breathe spontaneously with continuous airflow support, or the machine will open an on-demand valve to allow spontaneous breathing. Most ventilators can provide pressure support during spontaneous breathing.

"HFNC", a nasal High-flow nasal oxygen therapy (High-flow nasal oxygenation therapy), is an oxygen therapy modality that delivers directly air-oxygen mixed High-flow gas of a certain oxygen concentration to a patient through a nasal prong catheter that does not need to be sealed, as a form of noninvasive respiratory support, which can rapidly improve oxygenation. Can be applied to patients with acute hypoxic respiratory failure, patients after surgical operation, patients with respiratory failure without trachea cannula, patients with immunosuppression, patients with cardiac insufficiency, and the like.

"NIV" refers to Non-invasive Ventilation (Non-invasive Ventilation) and refers to Non-invasive mechanical Ventilation other than intubation of trachea tubes and tracheotomy.

"EC 50" or half maximal effect concentration (EC 50) refers to the concentration that causes 50% of the maximal effect.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

scFV against spike protein and antibody preparation

Constructing a phage library, and screening out scFv combined with spike protein; methods for preparing and screening such libraries are known in the art. The amino acid sequence of the scFv antibody is shown in Table 1 after sequencing. The screened VH and CH form the heavy chain of the antibody, and the screened VL and CL form the light chain of the antibody; the sequence of CH is shown as SEQ ID NO. 51, and the sequence of CL is shown as SEQ ID NO. 52. The antibody can be prepared by the following method or other known methods: sequence optimization is carried out according to the codon preference characteristics of CHO of host cells, and a DNA sequence is obtained from the amino acid sequence. Cloning the optimized and synthesized sequence clones into vectors respectively, then extracting a large number of plasmids respectively, and performing transient expression: uniformly mixing the linearized expression vector and CHO cells, and adding a 0.4cm electric rotating cup for electric rotation; after the electrotransfer is completed, 1200 cells are paved on a 96-hole cell culture plate per hole, mother clones with high expression level are selected after about 2-3 weeks to perform cell amplification culture and expression level detection from 96 holes to 24 holes to 6 holes to a shake flask, clones with high expression level in the shake flask are selected to perform subcloning, subcloning amplification culture and expression identification on the same mother clone, a monoclonal stable cell strain is selected according to the expression level condition and the stability of the cell strain, supernatant is harvested after suspension culture for about 12 days, proA affinity capture is performed, and an antibody with purity of more than 95% is obtained by anion and cation chromatography. Wherein: the VH of antibody 1 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 4, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 2 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 7, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 3 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 13, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 4 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 14, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 5 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 16, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 6 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 17, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 7 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 18, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 8 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 19, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 9 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 20, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 10 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 21, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 11 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 22, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 12 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 23, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 13 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 26, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 14 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 27, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 15 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 28, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 16 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 30, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 17 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 31, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 18 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 32, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 19 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 33, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 20 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 34, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 21 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 35, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 22 comprises FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 37, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50;

the VH of antibody 23 includes FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 38, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50.

The VH of antibody 24 includes FR1 shown in SEQ ID No. 42, VH CDR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 43, VH CDR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 44, VH CDR3 shown in SEQ ID No. 105, FR4 shown in SEQ ID No. 45, and VL of antibody 1 is shown in SEQ ID No. 50

TABLE 1 amino acid sequence

TABLE 2

Preparation of fusion proteins

Fusion proteins can be prepared by the following methods or other known methods, depending on the sequence of the fusion protein described herein. Sequence optimization is carried out according to the codon preference characteristics of CHO of host cells, and a DNA sequence is obtained from the amino acid sequence. Cloning the optimized and synthesized sequence clones into vectors respectively, then extracting a large number of plasmids respectively, and performing transient expression: uniformly mixing the linearized expression vector and CHO cells, and adding a 0.4cm electric rotating cup for electric rotation; after the electrotransfer is completed, 1200 cells are paved on a 96-hole cell culture plate per hole, mother clones with high expression level are selected after about 2-3 weeks to perform cell amplification culture and expression level detection from 96 holes to 24 holes to 6 holes to a shake flask, clones with high expression level in the shake flask are selected to perform subcloning, subcloning amplification culture and expression identification on the same mother clone, a monoclonal stable cell strain is selected according to the expression level condition and the stability of the cell strain, supernatant is harvested after suspension culture for about 12 days, proA affinity capture is performed, and an antibody with purity of more than 95% is obtained by anion and cation chromatography.

Detection of binding Activity of antibody or fusion protein to SARS-CoV-2 spike protein

Carrying out Elisa detection on the antibody, wherein the detection method comprises the following steps: coating a 96-well plate (Corning,9018) with spike-RBD-mFC (sine biologicals), sealing with tape and storing; the plates were washed 3 times in wash buffer (PBS, 0.05% Tween 20) before the addition of blocking solution (200. mu.L per well of 10mg/ml BSA, solvent as wash buffer); after incubation (1h, 37 ℃), the plates were washed 3 times with washing buffer, and then 100 μ Ι _ of sample was added per well; after incubation (1.5h, 37 ℃), the plates were washed with wash buffer and then anti-human kappa light chain antibody-peroxidase conjugate (diluted to 1:2000, 100. mu.L/well in blocking solution) was added; the plates were washed with wash buffer and the test samples were incubated (1h, 37 ℃) before adding 100 μ L of TMB (tetramethyllbenzidine, biopnda TMB-S-001) substrate/well; after 10 minutes of development, 100. mu.L/well of 0.1M H was added₂SO₄The reaction was terminated, and then 96-well plates were measured at an absorbance of 450 nm.

EC50 was calculated from the absorbance values, and the EC50 values for binding of various antibodies to SARS-CoV-2 spike protein are shown in Table 3.

The binding activity of the fusion protein to the SARS-CoV-2 spike protein can be determined by the same or similar method. It is expected that the fusion proteins of the invention will be capable of specifically binding to the SARS-CoV-2 spike protein.

TABLE 3

Activity detection of antibody or fusion protein for blocking SARS-CoV-2 spike protein and angiotensin converting enzyme 2 combination

Competition for Elisa was performed against some of the antibodies or fusion proteins described above to test their ability to block the binding of spike protein to angiotensin converting enzyme 2(ACE 2). The detection method comprises the following steps: 96-well plates (Corning,9018) were coated with spike-RBD-mFC (sine biologicals), sealed with tape and stored overnight at 4 ℃; the plates were washed 3 times in wash buffer (PBS, 0.05% Tween 20) before the addition of blocking solution (200. mu.L per well of 10mg/ml BSA, solvent as wash buffer); after incubation (2h, 22 ℃), the plates were washed 3 times in washing buffer, samples of antibody or fusion protein at different concentrations were added, followed by the addition of biotinylated angiotensin-converting enzyme 2(50 ng/ml); after incubation (1h, 22 ℃) the plates were washed 3 times with washing buffer, then 100. mu.L of streptavidin peroxidase conjugate (diluted 1:10,000 in blocking solution) was added per well, after incubation (1.0h, 22 ℃), the plates were washed with washing buffer, 100. mu.L/well of TMB substrate was added; after development for 10 minutes, the reaction was stopped by adding 50. mu.L/well of 0.1M H2SO4 and absorbance was measured at 450 nm.

A graph of the binding of the COVID-19 spike protein to ACE2 blocked by the various antibodies described above is shown in FIG. 1.

The activity of the fusion protein to block the binding of SARS-CoV-2 spike protein to angiotensin-converting enzyme 2 can be determined by the same or similar method. It is expected that the fusion protein of the present invention will block the binding of SARS-CoV-2 spike protein to angiotensin converting enzyme 2.

Binding experiments of anti-spike protein antibodies or fusion proteins to spike proteins on cells

The binding of anti-spike protein antibodies, fusion proteins, to 293T cells expressing spike protein can be analyzed by flow cytometry.

293T cells were transiently transfected to express the spike protein of Covid-19 and used for FACS analysis 48-72 hours later.

For binding analysis, 293T cells expressing Spike protein were incubated with various concentrations of antibody or fusion protein for 30 minutes at 4 ℃. The samples were washed twice, then incubated with the PE-conjugated goat anti-human Fc detection antibody for 30 minutes at 4 ℃ and the samples were washed twice. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter). The results were analyzed by FlowJo. It is expected that the antibodies, fusion proteins of the invention will bind to cells expressing SARS-CoV-2 spike.

Activity detection of anti-spike protein antibody or fusion protein for blocking SARS-CoV-2 spike protein and angiotensin converting enzyme 2 combination on cell

The blockade of the binding of spike protein to human ACE2 on cells by the antibody or fusion protein can be analyzed by FACS.

Mu.g/ml of biotin-Covid-19 spike protein was preincubated with various concentrations of anti-spike protein antibody or fusion protein on ice for 1 hour. The above mixture was then incubated with human ACE2 expressing cells on ice for 1 hour, the samples were washed twice, and then incubated with avidin-PE conjugate on ice for 30 minutes and the samples were washed twice.

Flow cytometry analysis of cells was performed using the CytoFLEX flow cytometer. The results were analyzed by FlowJo. The antibody, fusion protein of the invention is expected to block binding of cells expressing SARS-CoV-2 spike protein to angiotensin converting enzyme 2.

Animal model

The following animal experiments may test the efficacy of the anti-spike protein antibodies or fusion proteins described herein to treat COVID-19. 21 macaques expressing angiotensin converting enzyme 2 on the surface of lung epithelial cells were infected with SARS-CoV-2 virus (10000 TCID). On day 3 post-infection, macaques were randomized into three groups and received treatment by intravenous injection (vehicle, 3mg/kg anti-S protein antibody or 10mg anti-S protein antibody, 3mg/kg fusion protein or 10mg fusion protein), respectively. Symptoms of monkeys, such as body temperature, lung function and oxygen content, were recorded daily from the time of infection with the virus. Blood samples were drawn before infection, on day 1, day 3 after infection (before and after treatment), on

days

5,7, 10, 14, 20, and plasma viral loads, antibody concentrations, levels of cytokines (e.g., tumor necrosis factor alpha (TNF-. alpha.), IFN-. gamma., IL-1. beta., IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, granulocyte colony stimulating factor (GSCF), interferon inducible protein 10(IP-10), monocyte chemotactic protein-1 (MCP1), macrophage inflammatory protein 1. alpha. (MIP1A)) were determined.

It is expected that this experiment will show that animals receiving the antibodies described herein have fewer symptoms, lower viral load and more normal cytokine levels, and that the effects of the antibodies are dose-dependent compared to animals receiving the vehicle.

It is expected that this experiment will show that animals receiving the fusion proteins described herein have less symptoms, lower viral load and more normal cytokine levels, and that the effect of the fusion proteins is dose-dependent compared to animals receiving the vehicle.

Sequence listing

<110> Baiotai biopharmaceutical GmbH

<120> fusion protein for treating coronavirus and application thereof

<160> 108

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 2

<211> 10

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<213> Artificial Sequence (Artificial Sequence)

<400> 2

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr

1 5 10

<210> 3

<211> 11

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<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ala Asp Tyr Ala Ser Thr Val Tyr Asp Tyr Arg

1 5 10

<210> 4

<211> 11

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<400> 4

Asp Arg Asp Pro Ser Ala Ser Tyr Tyr Leu Asp

1 5 10

<210> 5

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<400> 5

Gly Pro Leu Gly Phe Ser Ala Gly Gly Leu Pro

1 5 10

<210> 6

<211> 11

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<400> 6

Gly Pro Phe Gly Ile Tyr Asp Gly Tyr Leu Pro

1 5 10

<210> 7

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Gly Pro Tyr Gly Tyr Ser Pro Gly Tyr Leu Pro

1 5 10

<210> 8

<211> 11

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<400> 8

Gly Pro Phe Gly Phe Ser Asp Gly Tyr Leu Pro

1 5 10

<210> 9

<211> 11

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<400> 9

His Asp Asp Ala Ser Pro Pro Gly Pro Asp Ser

1 5 10

<210> 10

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Pro Phe Gly Tyr Ser Ala Gly Tyr Leu Pro

1 5 10

<210> 11

<211> 11

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<400> 11

Ala Asp Tyr Ala Ser Thr Val Tyr Asp Gly Arg

1 5 10

<210> 12

<211> 11

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<400> 12

His Leu Gly Pro Thr Tyr Gly Gly Tyr Ala Arg

1 5 10

<210> 13

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gly Leu Tyr Gly Tyr Asp Leu Tyr Leu Asp Arg

1 5 10

<210> 14

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Glu Thr Ser Tyr Asp Gly Tyr Val Thr Leu Ala

1 5 10

<210> 15

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

His Ala Glu Asp Ser Pro Pro Gly Pro Asp Ser

1 5 10

<210> 16

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gly Ser Tyr Gly Tyr Asp Leu Tyr Leu Asp Arg

1 5 10

<210> 17

<211> 11

<212> PRT

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<400> 17

Gly Pro Phe Gly Tyr Ser Asp Gly Tyr Leu Pro

1 5 10

<210> 18

<211> 11

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<400> 18

Asp Thr Gly Tyr Phe Tyr Ala Thr Thr Leu Asp

1 5 10

<210> 19

<211> 11

<212> PRT

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<400> 19

Asp Leu Asp Pro Asp Ala Phe Tyr Tyr Leu Asp

1 5 10

<210> 20

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Asp Arg Asp Pro Ser Ala Pro Tyr Tyr Leu Asp

1 5 10

<210> 21

<211> 11

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<213> Artificial Sequence (Artificial Sequence)

<400> 21

Asp Arg Asp Pro Ala Ala Tyr Tyr Tyr Leu Asp

1 5 10

<210> 22

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Asp Arg Asp Pro Thr Ala Val Tyr Tyr Leu Asp

1 5 10

<210> 23

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Glu Ser Ser Tyr Asp Gly Tyr Val Asp Leu Ala

1 5 10

<210> 24

<211> 11

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<213> Artificial Sequence (Artificial Sequence)

<400> 24

Asp Arg Asp Pro Ser Ala Tyr Tyr Tyr Leu Asp

1 5 10

<210> 25

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Gly Pro Phe Gly Val Ser Asp Gly Tyr Leu Pro

1 5 10

<210> 26

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

His Pro Asp Glu Ser Pro Pro Leu Pro Asp Arg

1 5 10

<210> 27

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Gly Pro Tyr Gly Tyr Ser Ala Gly Tyr Leu Pro

1 5 10

<210> 28

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

His Pro Thr Arg Tyr Pro Ala Tyr Thr Asp Pro

1 5 10

<210> 29

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Gly Pro Phe Gly Tyr Ser Ser Gly Tyr Leu Pro

1 5 10

<210> 30

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Glu Leu Tyr Pro Gly Arg Phe Phe Pro Leu Tyr

1 5 10

<210> 31

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

His Arg Tyr Pro Arg Ser Tyr Ser Pro Leu Ser

1 5 10

<210> 32

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Asp Arg Asp Pro Ser Ala Thr Tyr Tyr Leu Asp

1 5 10

<210> 33

<211> 11

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<213> Artificial Sequence (Artificial Sequence)

<400> 33

Asp Thr Ala Tyr Tyr Tyr Tyr Thr Thr Leu Asp

1 5 10

<210> 34

<211> 11

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<213> Artificial Sequence (Artificial Sequence)

<400> 34

Arg Val Asp Leu Gly Leu Tyr Tyr Leu Asp Ala

1 5 10

<210> 35

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Asp Thr Arg Tyr Tyr Tyr Leu Ser Thr Leu Asp

1 5 10

<210> 36

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Val Pro Thr Arg Gly Ile Gly Arg Tyr Asp Phe Leu

1 5 10

<210> 37

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Asp Arg Ala Arg Tyr Asn Ser Tyr Leu Val His

1 5 10

<210> 38

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

Gly Pro Phe Gly Ser Ser Pro Gly Tyr Leu Pro

1 5 10

<210> 39

<211> 6

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<213> Artificial Sequence (Artificial Sequence)

<400> 39

Gln Gly Ile Ser Ser Tyr

1 5

<210> 40

<211> 3

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<400> 40

Ala Ala Ser

1

<210> 41

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 41

Gln Gln His Tyr Thr Thr Pro

1 5

<210> 42

<211> 28

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 42

Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly

20 25

<210> 43

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 43

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

1 5 10 15

<210> 44

<211> 39

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 44

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Arg

35

<210> 45

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 45

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 46

<211> 124

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 46

Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Asp Pro Ser Ala Ser Tyr Tyr Leu Asp Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 47

<211> 124

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 47

Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Gly Pro Phe Gly Tyr Ser Ala Gly Tyr Leu Pro Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 48

<211> 124

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 48

Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg His Leu Gly Pro Thr Tyr Gly Gly Tyr Ala Arg Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 49

<211> 124

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 49

Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Glu Thr Ser Tyr Asp Gly Tyr Val Thr Leu Ala Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 50

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 50

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 51

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 51

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 52

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 52

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 53

<211> 454

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 53

Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Asp Pro Ser Ala Ser Tyr Tyr Leu Asp Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 54

<211> 454

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 54

Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Gly Pro Phe Gly Tyr Ser Ala Gly Tyr Leu Pro Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 55

<211> 454

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 55

Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg His Leu Gly Pro Thr Tyr Gly Gly Tyr Ala Arg Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 56

<211> 454

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 56

Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser

20 25 30

Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Glu Thr Ser Tyr Asp Gly Tyr Val Thr Leu Ala Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 57

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 57

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 58

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 58

Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile

1 5 10 15

Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp

20 25 30

Leu Gln Glu Leu

35

<210> 59

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 59

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 60

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 60

Ser Tyr Ala Met Ser

1 5

<210> 61

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 61

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 62

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 62

Ala Asp Tyr Ala Ser Thr Val Tyr Asp Tyr Arg Asp Tyr

1 5 10

<210> 63

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 63

Asp Arg Asp Pro Ser Ala Ser Tyr Tyr Leu Asp Asp Tyr

1 5 10

<210> 64

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 64

Gly Pro Leu Gly Phe Ser Ala Gly Gly Leu Pro Asp Tyr

1 5 10

<210> 65

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 65

Gly Pro Phe Gly Ile Tyr Asp Gly Tyr Leu Pro Asp Tyr

1 5 10

<210> 66

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 66

Gly Pro Tyr Gly Tyr Ser Pro Gly Tyr Leu Pro Asp Tyr

1 5 10

<210> 67

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 67

Gly Pro Phe Gly Phe Ser Asp Gly Tyr Leu Pro Asp Tyr

1 5 10

<210> 68

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 68

His Asp Asp Ala Ser Pro Pro Gly Pro Asp Ser Asp Tyr

1 5 10

<210> 69

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 69

Gly Pro Phe Gly Tyr Ser Ala Gly Tyr Leu Pro Asp Tyr

1 5 10

<210> 70

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 70

Ala Asp Tyr Ala Ser Thr Val Tyr Asp Gly Arg Asp Tyr

1 5 10

<210> 71

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 71

His Leu Gly Pro Thr Tyr Gly Gly Tyr Ala Arg Asp Tyr

1 5 10

<210> 72

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 72

Gly Leu Tyr Gly Tyr Asp Leu Tyr Leu Asp Arg Asp Tyr

1 5 10

<210> 73

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 73

Glu Thr Ser Tyr Asp Gly Tyr Val Thr Leu Ala Asp Tyr

1 5 10

<210> 74

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 74

His Ala Glu Asp Ser Pro Pro Gly Pro Asp Ser Asp Tyr

1 5 10

<210> 75

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 75

Gly Ser Tyr Gly Tyr Asp Leu Tyr Leu Asp Arg Asp Tyr

1 5 10

<210> 76

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 76

Gly Pro Phe Gly Tyr Ser Asp Gly Tyr Leu Pro Asp Tyr

1 5 10

<210> 77

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 77

Asp Thr Gly Tyr Phe Tyr Ala Thr Thr Leu Asp Asp Tyr

1 5 10

<210> 78

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 78

Asp Leu Asp Pro Asp Ala Phe Tyr Tyr Leu Asp Asp Tyr

1 5 10

<210> 79

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 79

Asp Arg Asp Pro Ser Ala Pro Tyr Tyr Leu Asp Asp Tyr

1 5 10

<210> 80

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 80

Asp Arg Asp Pro Ala Ala Tyr Tyr Tyr Leu Asp Asp Tyr

1 5 10

<210> 81

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 81

Asp Arg Asp Pro Thr Ala Val Tyr Tyr Leu Asp Asp Tyr

1 5 10

<210> 82

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 82

Glu Ser Ser Tyr Asp Gly Tyr Val Asp Leu Ala Asp Tyr

1 5 10

<210> 83

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 83

Asp Arg Asp Pro Ser Ala Tyr Tyr Tyr Leu Asp Asp Tyr

1 5 10

<210> 84

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 84

Gly Pro Phe Gly Val Ser Asp Gly Tyr Leu Pro Asp Tyr

1 5 10

<210> 85

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 85

His Pro Asp Glu Ser Pro Pro Leu Pro Asp Arg Asp Tyr

1 5 10

<210> 86

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 86

Gly Pro Tyr Gly Tyr Ser Ala Gly Tyr Leu Pro Asp Tyr

1 5 10

<210> 87

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 87

His Pro Thr Arg Tyr Pro Ala Tyr Thr Asp Pro Asp Tyr

1 5 10

<210> 88

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 88

Gly Pro Phe Gly Tyr Ser Ser Gly Tyr Leu Pro Asp Tyr

1 5 10

<210> 89

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 89

Glu Leu Tyr Pro Gly Arg Phe Phe Pro Leu Tyr Asp Tyr

1 5 10

<210> 90

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 90

His Arg Tyr Pro Arg Ser Tyr Ser Pro Leu Ser Asp Tyr

1 5 10

<210> 91

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 91

Asp Arg Asp Pro Ser Ala Thr Tyr Tyr Leu Asp Asp Tyr

1 5 10

<210> 92

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 92

Asp Thr Ala Tyr Tyr Tyr Tyr Thr Thr Leu Asp Asp Tyr

1 5 10

<210> 93

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 93

Arg Val Asp Leu Gly Leu Tyr Tyr Leu Asp Ala Asp Tyr

1 5 10

<210> 94

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 94

Asp Thr Arg Tyr Tyr Tyr Leu Ser Thr Leu Asp Asp Tyr

1 5 10

<210> 95

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 95

Val Pro Thr Arg Gly Ile Gly Arg Tyr Asp Phe Leu Asp Tyr

1 5 10

<210> 96

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 96

Asp Arg Ala Arg Tyr Asn Ser Tyr Leu Val His Asp Tyr

1 5 10

<210> 97

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 97

Gly Pro Phe Gly Ser Ser Pro Gly Tyr Leu Pro Asp Tyr

1 5 10

<210> 98

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 98

Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala

1 5 10

<210> 99

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 99

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 100

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 100

Gln Gln His Tyr Thr Thr Pro Pro Thr

1 5

<210> 101

<211> 30

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 101

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser

20 25 30

<210> 102

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 102

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

1 5 10

<210> 103

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 103

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 104

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 104

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 105

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 105

Gly Pro Phe Gly Ile Asp Asp Ala Tyr Leu Pro

1 5 10

<210> 106

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 106

Gly Pro Phe Gly Ile Asp Asp Ala Tyr Leu Pro Asp Tyr

1 5 10

<210> 107

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 107

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Phe Gly Ile Asp Asp Ala Tyr Leu Pro Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 108

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 108

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Pro Phe Gly Ile Asp Asp Ala Tyr Leu Pro Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

Claims

1. A fusion protein comprising an antibody or antigen-binding fragment that specifically binds to SARS-CoV-2 or a spike protein of SARS-CoV and a polypeptide, the antibody or antigen-binding fragment comprising at least one or more of VH CDR1 shown in SEQ ID NO:1, VH CDR2 shown in SEQ ID NO:2, VH CDR3 shown in any one of SEQ ID NO:3-38, VL CDR1 shown in SEQ ID NO:39, VL CDR2 shown in SEQ ID NO:40, and VL CDR3 shown in SEQ ID NO: 41;

the C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to the polypeptide by a linker comprising glycine and serine.

2. The fusion protein of claim 1, wherein the fusion protein comprises the following features:

the antibody or antigen-binding fragment comprises at least a VH CDR1 shown in SEQ ID NO. 1, a VH CDR2 shown in SEQ ID NO. 2, a VH CDR3 shown in any one of SEQ ID NO. 3-38, a VL CDR1 shown in SEQ ID NO. 39, a VL CDR2 shown in SEQ ID NO. 40, and a VL CDR3 shown in SEQ ID NO. 41; and/or

The sequence of the linker is (GGGGS)_nN is 1, 2, 3,4 or 5; and/or

3. The fusion protein of claim 2, wherein the antibody or antigen-binding fragment comprises at least a VH CDR1 as set forth in SEQ ID No. 1, a VH CDR2 as set forth in SEQ ID No. 2, a VH CDR3 as set forth in SEQ ID No. 4, a VL CDR1 as set forth in SEQ ID No. 39, a VL CDR2 as set forth in SEQ ID No. 40, and a VL CDR3 as set forth in SEQ ID No. 41; or

The antibody or antigen binding fragment comprises at least a VH CDR1 shown in SEQ ID NO. 1, a VH CDR2 shown in SEQ ID NO. 2, a VH CDR3 shown in SEQ ID NO. 10, a VL CDR1 shown in SEQ ID NO. 39, a VL CDR2 shown in SEQ ID NO. 40, and a VL CDR3 shown in SEQ ID NO. 41; or

The antibody or antigen binding fragment comprises at least a VH CDR1 shown in SEQ ID NO. 1, a VH CDR2 shown in SEQ ID NO. 2, a VH CDR3 shown in SEQ ID NO. 12, a VL CDR1 shown in SEQ ID NO. 39, a VL CDR2 shown in SEQ ID NO. 40, and a VL CDR3 shown in SEQ ID NO. 41; or

The antibody or antigen binding fragment comprises at least a VH CDR1 shown in SEQ ID NO. 1, a VH CDR2 shown in SEQ ID NO. 2, a VH CDR3 shown in SEQ ID NO. 14, a VL CDR1 shown in SEQ ID NO. 39, a VL CDR2 shown in SEQ ID NO. 40, and a VL CDR3 shown in SEQ ID NO. 41.

4. The fusion protein of any one of claims 1-3, wherein the heavy chain FR1 of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO. 42, a sequence having at least 90% identity to the sequence set forth in SEQ ID NO. 42, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 42; and/or

5. The fusion protein of claim 4, wherein the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in any one of SEQ ID NOs 46-49, a sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NOs 46-49, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in any one of SEQ ID NOs 46-49; and/or

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 to the sequence shown in SEQ ID NO. 50, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 50.

6. The fusion protein of claim 5, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence set forth in any one of SEQ ID NOs 46-49 and/or a light chain variable region having an amino acid sequence set forth in SEQ ID NO 50.

7. The fusion protein of any one of claims 1-6, wherein the heavy chain constant region of the antibody or antigen-binding fragment comprises an amino acid sequence set forth in SEQ ID No. 51, a sequence having at least 80% identity to the sequence set forth in SEQ ID No. 51, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 51; and/or

8. The fusion protein of any one of claims 1-6, wherein the fusion protein comprises the following features:

the heavy chain of the antibody comprises a sequence having an amino acid sequence as set forth in any one of SEQ ID NOs 53-56, a sequence having at least 80% identity to a sequence set forth in any one of SEQ ID NOs 53-56, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence set forth in any one of SEQ ID NOs 53-56; and/or

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

9. A nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1-8.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-8 and a pharmaceutically acceptable carrier.