CN116617388A

CN116617388A - Use of antibodies targeting coronaviruses for preventing, treating or ameliorating covd-19

Info

Publication number: CN116617388A
Application number: CN202310087475.8A
Authority: CN
Inventors: 黄贤明; 杨晓蕾; 张慧; 李胜峰
Original assignee: Bio Thera Solutions Ltd
Current assignee: Bio Thera Solutions Ltd
Priority date: 2022-01-26
Filing date: 2023-01-19
Publication date: 2023-08-22
Also published as: WO2023143407A1

Abstract

The present invention discloses the use of an antibody or antigen binding fragment targeting coronavirus for the prevention, treatment or amelioration of covd-19, the method of treatment comprising administering to a patient in need thereof an effective amount of an antibody or antigen binding fragment targeting coronavirus.

Description

Use of antibodies targeting coronaviruses for preventing, treating or ameliorating covd-19

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of an antibody targeting coronavirus in preventing, treating or improving COVID-19.

Background

Coronaviruses are non-segmented single-stranded positive-strand RNA viruses, and are classified into four genera, α, β, γ and δ according to the serotype and genome characteristics, and are named as corolla due to the presence of protrusions extending around the viral envelope. Novel coronaviruses (SARS-CoV-2 or 2019-nCoV) belong to the genus beta and are enveloped, and the particles are round or oval, often polymorphic, with diameters of 60-140nm.

The novel coronavirus infection, covd-19, is transmitted primarily through respiratory tract infections, which may also be transmitted by contact. The people are generally susceptible, the old and the people with basic diseases have serious illness after being infected, and children and infants also have illness. The main clinical symptoms of the infected person are fever, hypodynamia, dry cough, and the symptoms of the upper respiratory tract such as nasal obstruction, nasal discharge and the like are rare. In early stages of onset, the total number of leukocytes in the patient is normal or decreased, or the number of lymphocytes is decreased, and part of the patients show increased liver enzymes, myoenzymes and myoglobin. Chest images show that patients present multiple small patches and interstitial changes in early stages, and are evident in pulmonary external zones; further, it develops double lung multiple abrasion glass shadow and infiltration shadow, and severe cases may develop excessive lung changes and gradually develop dyspnea, and severe cases develop Acute Respiratory Distress Syndrome (ARDS), shock, and various tissue injuries and dysfunction of lung tissue, heart and kidney. Most patients with mild infections have a good prognosis, and severe patients often suffer from critical illness or even die.

Disclosure of Invention

The present invention provides methods or uses of antibodies targeting coronaviruses for preventing, treating or ameliorating a disease caused by a novel coronavirus (covd-19). In some embodiments, the antibody that targets coronavirus is an antibody having high affinity for the spike protein of SARS-CoV-2. In some embodiments, the antibody that targets the coronavirus is a bispecific antibody, a single domain antibody, or a heavy chain antibody or antigen binding fragment.

The present invention provides bispecific antibodies or antigen binding fragments having high affinity for the spike protein of SARS-CoV-2. Bispecific antibodies or antigen binding fragments can bind spike proteins, prevent viral particle and cell binding, and can mediate immune cell phagocytosis and clearance of viral particles. Bispecific antibodies or antigen binding fragments are useful for preventing, treating or ameliorating a covd-19 such as a novel coronavirus infection, and for diagnosing covd-19.

Through binding of its surface spike protein (S protein or spike protein) to a type of surface of lung epithelial cells called angiotensin converting enzyme 2 (ACE 2), SARS-CoV-2 enters the cell and utilizes the cell for its synthesis of new viral particles; the new viral particles are released outside the cell and the virus infects surrounding normal cells in the same manner. The bispecific antibody or antigen binding fragment of the targeted spike protein can block the combination of the spike protein and ACE2, thereby blocking the virus from entering cells and playing an antiviral role. Bispecific antibodies or antigen-binding fragments of the invention may also mediate immune cell phagocytosis and viral clearance.

Some embodiments provide a bispecific antibody targeting a coronavirus comprising a first binding moiety that binds a spike protein and a second binding moiety that binds a spike protein linked by a linker L1. First binding moiety that binds to spike protein

In some embodiments, the first binding moiety that binds to a spike protein comprises:

(a) HCDR1 comprising an amino acid sequence as shown in SEQ ID No. 1 or 2, or a variant thereof having single or multiple site substitutions, deletions or insertions; (b) HCDR2 comprising an amino acid sequence as shown in SEQ ID No. 3 or 4, or a variant thereof having single or multiple site substitutions, deletions or insertions; and/or (c) HCDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NOs 5-42, or a variant thereof having single or multiple site substitutions, deletions or insertions.

In some embodiments, the first binding moiety comprises:

(a) HCDR1 comprising an amino acid sequence as shown in SEQ ID No. 1 or 2, or a variant thereof having single or multiple site substitutions, deletions or insertions; (b) HCDR2 comprising an amino acid sequence as shown in SEQ ID No. 3 or 4, or a variant thereof having single or multiple site substitutions, deletions or insertions; and (c) HCDR3, which comprises an amino acid sequence as set forth in any one of SEQ ID NOs 5-42, or a variant thereof having single or multiple site substitutions, deletions or insertions.

In some embodiments, the first binding moiety comprises:

(a) HCDR1 comprising an amino acid sequence as set forth in SEQ ID NO. 1 or 2; (b) HCDR2 comprising an amino acid sequence as shown in SEQ ID No. 3 or 4; and (c) HCDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NOs 5-42.

In some embodiments, the first binding moiety comprises:

(a) HCDR1 comprising an amino acid sequence as shown in SEQ ID No. 1 or 2, or a variant thereof having single or multiple site substitutions, deletions or insertions; (b) HCDR2 comprising an amino acid sequence as shown in SEQ ID No. 3 or 4, or a variant thereof having single or multiple site substitutions, deletions or insertions; (c) HCDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NOs 5-42 or a variant thereof having single or multiple site substitutions, deletions or insertions; (d) LCDR1 comprising an amino acid sequence as set forth in SEQ ID NO. 43 or 44, or a variant thereof having single or multiple site substitutions, deletions or insertions; (e) LCDR2 comprising an amino acid sequence as set forth in SEQ ID NO. 45 or 46, or a variant thereof having single or multiple site substitutions, deletions or insertions; and/or (f) LCDR3 comprising an amino acid sequence as set forth in SEQ ID NO. 47 or 48, or a variant thereof having single or multiple site substitutions, deletions or insertions.

In some embodiments, the first binding moiety comprises:

(a) HCDR1 comprising an amino acid sequence as shown in SEQ ID No. 1 or 2, or a variant thereof having single or multiple site substitutions, deletions or insertions; (b) HCDR2 comprising an amino acid sequence as shown in SEQ ID No. 3 or 4, or a variant thereof having single or multiple site substitutions, deletions or insertions; (c) HCDR3 comprising an amino acid sequence as set forth in any one of SEQ ID NOs 5-42 or a variant thereof having single or multiple site substitutions, deletions or insertions; (d) LCDR1 comprising an amino acid sequence as set forth in SEQ ID NO. 43 or 44, or a variant thereof having single or multiple site substitutions, deletions or insertions; (e) LCDR2 comprising an amino acid sequence as set forth in SEQ ID NO. 45 or 46, or a variant thereof having single or multiple site substitutions, deletions or insertions; and (f) LCDR3 comprising an amino acid sequence as set forth in SEQ ID NO. 47 or 48, or a variant thereof having single or multiple site substitutions, deletions or insertions.

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

In some embodiments, the first binding moiety comprises one, two, three, four, five or all of HCDR1 as set forth in SEQ ID NO:1 or 2, HCDR2 as set forth in SEQ ID NO:3 or 4, HCDR3 as set forth in any one of SEQ ID NO:5-42, LCDR1 as set forth in SEQ ID NO:43 or 44, LCDR2 as set forth in SEQ ID NO:45 or 46, and LCDR3 as set forth in SEQ ID NO:47 or 48.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 5, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 6, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 7, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 8, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 9, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 10, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 11, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 12, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 13, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 14, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 15, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 16, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 17, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 18, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 19, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 20, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 21, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 22, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 23, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 24, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 25, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 26, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 27, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 28, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 29, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 30, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 31, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 32, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 33, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 34, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 35, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 36, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 37, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 38, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 39, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 40, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 41, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45, and LCDR3 as shown in SEQ ID NO. 47.

In some embodiments, the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 2, HCDR2 as shown in SEQ ID NO. 4, HCDR3 as shown in SEQ ID NO. 42, LCDR1 as shown in SEQ ID NO. 44, LCDR2 as shown in SEQ ID NO. 46, and LCDR3 as shown in SEQ ID NO. 48.

In some embodiments, the first binding moiety comprises a heavy chain variable region and/or a light chain variable region.

In some embodiments, the framework region of the first binding moiety heavy chain variable region comprises heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4; the heavy chain FR1 comprises a sequence as set forth in SEQ ID NO. 49 or 50, or a sequence having at least 90% identity to the sequence as set forth in SEQ ID NO. 49 or 50, 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. 49 or 50; and/or

The heavy chain FR2 comprises a sequence as set forth in SEQ ID NO. 51 or 52, or a sequence having at least 90% identity to the sequence as set forth in SEQ ID NO. 51 or 52, 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. 51 or 52; and/or

The heavy chain FR3 comprises a sequence as set forth in SEQ ID NO. 53 or 54, or a sequence having at least 90% identity to a sequence as set forth in SEQ ID NO. 53 or 54, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence as set forth in SEQ ID NO. 53 or 54; and/or

The heavy chain FR4 comprises the sequence shown as SEQ ID NO. 55, or a sequence having at least 90% identity to the sequence shown as SEQ ID NO. 55, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown as SEQ ID NO. 55.

In some embodiments, the heavy chain FR1 comprises a sequence set forth in SEQ ID NO. 49 or 50, or a sequence having at least 90% identity to a sequence set forth in SEQ ID NO. 49 or 50, or an amino acid sequence having one or more conservative amino acid substitutions compared to a sequence set forth in SEQ ID NO. 49 or 50; the heavy chain FR2 comprises a sequence as set forth in SEQ ID NO. 51 or 52, or a sequence having at least 90% identity to the sequence as set forth in SEQ ID NO. 51 or 52, 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. 51 or 52; the heavy chain FR3 comprises a sequence as set forth in SEQ ID NO. 53 or 54, or a sequence having at least 90% identity to a sequence as set forth in SEQ ID NO. 53 or 54, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence as set forth in SEQ ID NO. 53 or 54; the heavy chain FR4 comprises the sequence shown as SEQ ID NO. 55, or a sequence having at least 90% identity to the sequence shown as SEQ ID NO. 55, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown as SEQ ID NO. 55. 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. 49, the heavy chain FR2 comprises the sequence shown in SEQ ID NO. 51, the heavy chain FR3 comprises the sequence shown in SEQ ID NO. 53 and the heavy chain FR4 comprises the sequence shown in SEQ ID NO. 55. In some embodiments, the heavy chain FR1 comprises the sequence shown in SEQ ID NO. 50, the heavy chain FR2 comprises the sequence shown in SEQ ID NO. 52, the heavy chain FR3 comprises the sequence shown in SEQ ID NO. 54 and the heavy chain FR4 comprises the sequence shown in SEQ ID NO. 55. In some embodiments, the heavy chain variable region comprises the structure FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4.

In some embodiments, the first binding moiety heavy chain variable region comprises FR1 shown as SEQ ID NO. 49, HCDR1 shown as SEQ ID NO. 1, FR2 shown as SEQ ID NO. 51, HCDR2 shown as SEQ ID NO. 3, FR3 shown as SEQ ID NO. 53, HCDR3 shown as any one of SEQ ID NO. 5-41 and FR4 shown as SEQ ID NO. 55.

In some embodiments, the first binding moiety heavy chain variable region comprises FR1 shown as SEQ ID NO. 50, HCDR1 shown as SEQ ID NO. 2, FR2 shown as SEQ ID NO. 52, HCDR2 shown as SEQ ID NO. 4, FR3 shown as SEQ ID NO. 54, HCDR3 shown as SEQ ID NO. 42 and FR4 shown as SEQ ID NO. 55.

In some embodiments, the first binding moiety heavy chain variable region comprises a sequence set forth in any one of SEQ ID NOS: 56, 57, 83-105, a sequence having at least 80% identity to a sequence set forth in any one of SEQ ID NOS: 56, 57, 83-105, 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: 56, 57, 83-105.

In some embodiments, the first binding moiety light chain variable region comprises a sequence set forth in SEQ ID NO. 58 or 59, a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 58 or 59, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 58 or 59.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 56 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 57 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 59.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 83 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 84 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 85 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 86 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 87 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 88 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 89 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 90 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 91 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 92 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 93 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 94 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 95 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO:96 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO: 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 97 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 98 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 99 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 100 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 101 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 102 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 103 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 104 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 105 and the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO. 58.

In some embodiments, the first binding moiety 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 first binding moiety is one of IgG, igM, igA, igE or IgD isoforms, or a fragment thereof. In some embodiments, the isotype is IgG1, igG2, igG3, or IgG4, or a fragment thereof. In some embodiments, the C-terminal end of the heavy chain constant region in the first binding moiety is truncated. In some embodiments, the C-terminal end of the heavy chain constant region in the first binding moiety of IgG1 or IgG4 type lacks amino acid residues G and K. Without limitation, the first binding moiety is a chimeric, humanized or fully human antibody. In a certain aspect, the first binding moiety is a fully humanized antibody.

In some embodiments, the Fc is a variant Fc region. In some embodiments, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions, or insertions, relative to the parent Fc region. In some embodiments, amino acid modifications of the Fc region alter effector function activity relative to parent Fc region activity. In some embodiments, the variant Fc region may have altered (i.e., increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding. In some embodiments, the Fc region amino acid modification may alter the affinity of the variant Fc region for fcγr (fcγreceptor) relative to the parent Fc region. In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc region mutation is N297A. In some embodiments, the Fc region mutation is N297A, L234A or L235A (Eu numbering). In some embodiments, the Fc region mutation is E345R or S440Y (Eu numbering).

In some embodiments, the heavy chain constant region comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 60 or 61, a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 60 or 61, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 60 or 61; and/or

The light chain constant region comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 62, a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 62, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 62.

In some embodiments, the heavy chain constant region comprises a sequence having the amino acid sequence set forth in SEQ ID NO. 60 or 61 and/or the light chain constant region comprises a sequence having the amino acid sequence set forth in SEQ ID NO. 62. In some embodiments, the heavy chain constant region comprises a sequence having the amino acid sequence set forth in SEQ ID NO. 60 and the light chain constant region comprises a sequence having the amino acid sequence set forth in SEQ ID NO. 62. In some embodiments, the heavy chain constant region comprises a sequence having the amino acid sequence set forth in SEQ ID NO. 61 and the light chain constant region comprises a sequence having the amino acid sequence set forth in SEQ ID NO. 62.

In some embodiments, the heavy chain constant region comprises an amino acid sequence as set forth in amino acid 1 to amino acid 328 of SEQ ID NO. 60 or 61, or a sequence having at least 80% identity to the sequence set forth in amino acid 1 to amino acid 328 of SEQ ID NO. 60 or 61, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in amino acid 1 to amino acid 328 of SEQ ID NO. 60 or 61; and/or

The light chain constant region comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 62, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 62, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 62.

In some embodiments, the heavy chain constant region comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 60 from amino acid 1 to amino acid 328, and the light chain constant region comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 62. In some embodiments, the heavy chain constant region comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 61 from amino acid 1 to amino acid 328, and the light chain constant region comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety comprises a heavy chain and/or a light chain.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in any one of SEQ ID NOS: 56, 57, 83-105 and a heavy chain constant region as set forth in SEQ ID NOS: 60 or 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 or 59 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 83 and a heavy chain constant region as shown in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 84 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 85 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 86 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 87 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 88 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 89 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 90 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 91 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 92 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 93 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 94 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 95 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 96 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 97 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 98 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 99 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 100 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 101 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 102 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 103 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 104 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 105 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 56 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 57 and a heavy chain constant region as set forth in SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 59 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in any one of SEQ ID NOS: 56, 57, 83-105 and a heavy chain constant region as set forth in amino acids 1 to 328 of SEQ ID NOS 60 or 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 or 59 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 83 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 84 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 85 and a heavy chain constant region as set forth in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 86 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 87 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 88 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 89 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 90 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 91 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 92 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 93 and a heavy chain constant region as set forth in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 94 and a heavy chain constant region as set forth in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 95 and a heavy chain constant region as set forth in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 96 and a heavy chain constant region as set forth in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 97 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 98 and a heavy chain constant region as set forth in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as set forth in SEQ ID NO. 99 and a heavy chain constant region as set forth in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 100 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 101 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 102 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 103 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 104 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 105 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 56 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 58 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain comprises a heavy chain variable region as shown in SEQ ID NO. 57 and a heavy chain constant region as shown in amino acids 1 to 328 of SEQ ID NO. 61; the first binding moiety light chain comprises a light chain variable region as set forth in SEQ ID NO. 59 and a light chain constant region as set forth in SEQ ID NO. 62.

In some embodiments, the heavy chain comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 71 or 72, a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 71 or 72, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 71 or 72; and/or

The light chain comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 73 or 74, a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 73 or 74, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 73 or 74.

In some embodiments, the heavy chain of the first binding moiety comprises a sequence having the amino acid sequence shown in SEQ ID NO. 71 or 72, and/or the light chain of the first binding moiety comprises a sequence having the amino acid sequence shown in SEQ ID NO. 73 or 74.

In some embodiments, the heavy chain of the first binding moiety comprises a sequence having the amino acid sequence shown in SEQ ID NO. 71 and the light chain of the first binding moiety comprises a sequence having the amino acid sequence shown in SEQ ID NO. 73. In some embodiments, the heavy chain of the first binding moiety comprises a sequence having the amino acid sequence shown in SEQ ID NO. 72 and the light chain of the first binding moiety comprises a sequence having the amino acid sequence shown in SEQ ID NO. 74.

In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% identity to the sequence shown as amino acid 1 to amino acid 450 in SEQ ID NO:71 or amino acid 1 to amino acid 451 in SEQ ID NO:72, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown as amino acid 1 to amino acid 450 in SEQ ID NO:71 or amino acid 1 to amino acid 451 in SEQ ID NO: 72; and/or

In some embodiments, the heavy chain of the first binding moiety comprises a sequence as set forth in amino acid sequence 1 to 450 in SEQ ID NO:71 or amino acid 1 to 451 in SEQ ID NO:72, and/or the light chain of the first binding moiety comprises a sequence as set forth in SEQ ID NO:73 or 74.

In some embodiments, the heavy chain of the first binding moiety comprises a sequence having an amino acid sequence as set forth in SEQ ID NO:71 from amino acid 1 to amino acid 450, and the light chain of the first binding moiety comprises a sequence having an amino acid sequence as set forth in SEQ ID NO: 73. In some embodiments, the heavy chain of the first binding moiety comprises a sequence of amino acids set forth in amino acids 1 to 451 of SEQ ID NO. 72, and the light chain of the first binding moiety comprises a sequence of amino acids set forth in SEQ ID NO. 74.

In some embodiments, the first binding moiety comprises 2 heavy chains that are identical in sequence and 2 light chains that are identical in sequence.

Connector L1

In some embodiments, the linker L1 is a polypeptide comprising glycine and serine.

In some embodiments, the sequence of linker L1 is (G _m S) _n Wherein each m is independently 2, 3, 4 or 5 and n is independently 1, 2, 3, 4 or 5. In some embodiments, the sequence of linker L1 is (GGGGS) _n And n is independently 1, 2, 3, 4 or 5. In some embodiments, the linker L1 is GGGGS. In some embodiments, the linker L1 is (GGGGS) ₂ As shown in SEQ ID NO. 65. In some embodiments, the linker L1 is (GGGGS) ₃ . In some embodiments, the linker L1 is (GGGGS) ₄ As shown in SEQ ID NO. 63. In some embodiments, the linker L1 is (GGGGS) ₅ As shown in SEQ ID NO. 64.

Second binding moiety that binds to spike protein

In some embodiments, the second binding moiety is a single domain antibody. In some embodiments, the single domain antibody is a VHH.

In some embodiments, the second binding moiety is a single domain antibody and comprises:

(a) HCDR1 comprising the amino acid sequence shown as SEQ ID No. 66 or a variant thereof having single or multiple site substitutions, deletions or insertions; (b) HCDR2 comprising the amino acid sequence shown as SEQ ID No. 67 or a variant thereof having single or multiple site substitutions, deletions or insertions; and/or (c) HCDR3, comprising the amino acid sequence set forth in SEQ ID NO. 68, or a variant thereof having single or multiple site substitutions, deletions or insertions.

(a) HCDR1 comprising the amino acid sequence shown as SEQ ID No. 66 or a variant thereof having single or multiple site substitutions, deletions or insertions; (b) HCDR2 comprising the amino acid sequence shown as SEQ ID No. 67 or a variant thereof having single or multiple site substitutions, deletions or insertions; and (c) HCDR3, which comprises the amino acid sequence set forth in SEQ ID NO. 68, or a variant thereof having single or multiple site substitutions, deletions or insertions.

(a) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO. 66; (b) HCDR2 comprising the amino acid sequence set forth in SEQ ID NO. 67; and (c) HCDR3, comprising the amino acid sequence set forth in SEQ ID NO. 68.

In some embodiments, the second binding moiety is a single domain antibody and comprises the amino acid sequence shown as SEQ ID NO. 69.

Bispecific antibodies

In some embodiments, the bispecific antibody comprises a first binding moiety that binds a spike protein as described above and a second binding moiety that binds a spike protein, linked by a linker L1 as described above.

In some embodiments, the bispecific antibody comprises the following features:

the first binding moiety comprises at least one, two, three, four, five or all of HCDR1 as shown in SEQ ID NO. 2, HCDR2 as shown in SEQ ID NO. 4, HCDR3 as shown in SEQ ID NO. 42, LCDR1 as shown in SEQ ID NO. 44, LCDR2 as shown in SEQ ID NO. 46 and LCDR3 as shown in SEQ ID NO. 48; and/or

The second binding moiety is a VHH and comprises at least one, two or three of HCDR1 shown as SEQ ID NO. 66, HCDR2 shown as SEQ ID NO. 67 and HCDR3 shown as SEQ ID NO. 68; and/or

The C-terminal end of the first binding moiety is linked to the N-terminal end of the second binding moiety via a linker L1, the C-terminal end of the first binding moiety being the C-terminal end of the heavy chain or the C-terminal end of the light chain in the first binding moiety; and/or

The amino acid sequence of the linker L1 is (GGGGS) _n And n is independently 1, 2, 3, 4 or 5.

In some embodiments, the bispecific antibody comprises a first binding moiety that binds a spike protein and a second binding moiety that binds a spike protein, the bispecific antibody comprising the following features:

the first binding moiety comprises HCDR1 as shown in SEQ ID NO. 2, HCDR2 as shown in SEQ ID NO. 4, HCDR3 as shown in SEQ ID NO. 42, LCDR1 as shown in SEQ ID NO. 44, LCDR2 as shown in SEQ ID NO. 46 and LCDR3 as shown in SEQ ID NO. 48; and/or

The second binding moiety is a VHH and comprises HCDR1 as shown in SEQ ID NO. 66, HCDR2 as shown in SEQ ID NO. 67, and HCDR3 as shown in SEQ ID NO. 68; and/or

The sequence of the linker L1 is (GGGGS) _n And n is independently 1, 2, 3, 4 or 5.

In some embodiments, the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO. 57, a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 57, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 57.

In some embodiments, the first binding moiety light chain variable region comprises a sequence set forth in SEQ ID NO. 59, a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 59, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 59.

In some embodiments, the first binding moiety heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 57 and the first binding moiety light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 59.

In some embodiments, the first binding moiety 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 first binding moiety is IgG, igM, igA, igE or IgD type, or a fragment thereof. In some embodiments, the first binding moiety is of the IgG1, igG2, igG3, or IgG4 type, or a fragment thereof. In some embodiments, the C-terminal end of the heavy chain constant region in the first binding moiety is truncated. In some embodiments, the C-terminal end of the heavy chain constant region in the first binding moiety of IgG1 or IgG4 type lacks amino acid residues G and K. In some embodiments, the Fc is a variant Fc region. In some embodiments, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions, or insertions, relative to the parent Fc region. In some embodiments, the first binding moiety is scFV, fab, fab', F (ab) ₂ Or F (ab) ₂ ’。

In some embodiments, the first binding moiety and/or the second binding moiety is a chimeric, humanized or fully human antibody.

In some embodiments, the first binding moiety heavy chain constant region comprises an amino acid sequence as shown in amino acid 1 to amino acid 328 of SEQ ID NO. 60 or 61, or a sequence having at least 80% identity to the sequence shown in amino acid 1 to amino acid 328 of SEQ ID NO. 60 or 61, or an amino acid sequence having one or more conservative amino acid substitutions to the sequence shown in amino acid 1 to amino acid 328 of SEQ ID NO. 60 or 61; and/or

The first binding moiety light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO. 62, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 62, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 62.

In some embodiments, the heavy chain constant region of the first binding moiety comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 60 from amino acid 1 to amino acid 328, and the light chain constant region of the first binding moiety comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 62. In some embodiments, the heavy chain constant region of the first binding moiety comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 61 from amino acid 1 to amino acid 328, and the light chain constant region of the first binding moiety comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID No. 60 or 61, or a sequence having at least 80% identity to a sequence set forth in SEQ ID No. 60 or 61, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence set forth in SEQ ID No. 60 or 61; and/or

The first binding moiety light chain constant region comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 62, a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 62, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 62.

In some embodiments, the first binding moiety heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 60 and the first binding moiety light chain constant region comprises the amino acid sequence set forth in SEQ ID NO. 62. In some embodiments, the first binding moiety heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO. 61 and the first binding moiety light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO. 62.

In some embodiments, the second binding moiety is a VHH. In some embodiments, the second binding moiety comprises a sequence as set forth in SEQ ID NO:69, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:69, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO: 69.

In some embodiments, a bispecific antibody is provided, comprising a first binding moiety and a single domain antibody, and comprising the following features:

the first binding moiety comprises a heavy chain and a light chain; the heavy chain of the first binding moiety comprises an amino acid sequence as shown in amino acids 1 to 451 of SEQ ID NO. 72, or a sequence having at least 80% identity to the sequence shown in amino acids 1 to 451 of SEQ ID NO. 72, or an amino acid sequence having one or more conservative amino acid substitutions to the sequence shown in amino acids 1 to 451 of SEQ ID NO. 72; and/or

The light chain of the first binding moiety comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 74, a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 74, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 74; and/or

The C-terminal end of the heavy chain of the first binding moiety is covalently linked to the single domain antibody via a linker L1 as shown in SEQ ID NO. 63; and/or

The single domain antibody comprises a sequence as set forth in SEQ ID NO. 69, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 69, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 69.

In some embodiments, the heavy chain of the first binding moiety comprises a sequence having an amino acid sequence as set forth in amino acid 1 to amino acid 451 of SEQ ID NO. 72, and the light chain of the first binding moiety comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 74; the C-terminus (i.e., CH3 terminus) of the heavy chain of the first binding moiety is covalently linked by a linker L1 as shown in SEQ ID NO:63 to a single domain antibody comprising the sequence shown in SEQ ID NO: 69.

In some embodiments, bispecific antibodies are provided that comprise a first binding moiety and a single domain antibody, and comprise the following features:

the first binding moiety comprises a heavy chain and a light chain; the heavy chain of the first binding moiety comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 72, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 72, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 72; and/or

The light chain of the first binding moiety comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 74, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 74, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 74; and/or

The C-terminal end of the light chain of the first binding moiety is covalently linked to the single domain antibody via a linker L1 as shown in SEQ ID NO. 64; and/or

In some embodiments, the heavy chain of the first binding moiety comprises a sequence having the amino acid sequence shown in SEQ ID NO. 72 and the light chain of the first binding moiety comprises a sequence having the amino acid sequence shown in SEQ ID NO. 74; the C-terminus (i.e., CL terminus) of the light chain of the first binding moiety is covalently linked by a linker L1 as shown in SEQ ID NO:64 to a single domain antibody comprising the sequence shown in SEQ ID NO: 69.

In some embodiments, the bispecific antibody comprises a first polypeptide and a second polypeptide. In some embodiments, the bispecific antibody comprises 2 first polypeptides that are identical in sequence and 2 second polypeptides that are identical in sequence.

In some embodiments, the first polypeptide comprises, or consists of, the heavy chain of the first binding moiety, linker L1, single domain antibody, in order from the N-terminus to the C-terminus; the second polypeptide comprises or consists of a light chain.

In some embodiments, the first polypeptide comprises or consists of a sequence having an amino acid sequence as set forth in SEQ ID NO. 77, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 77, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 77; and/or

The second polypeptide comprises or consists of a sequence having an amino acid sequence as set forth in SEQ ID NO. 74, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 74, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 74.

In some embodiments, the first polypeptide comprises a sequence having the amino acid sequence set forth in SEQ ID NO:77 and the second polypeptide comprises a sequence having the amino acid sequence set forth in SEQ ID NO: 74.

In some embodiments, the first polypeptide comprises, or consists of, a heavy chain; the second polypeptide comprises or consists of a light chain, a linker L1, a single domain antibody of the first binding moiety in order from the N-terminus to the C-terminus.

In some embodiments, the first polypeptide comprises or consists of a sequence having an amino acid sequence as set forth in SEQ ID NO. 72, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 72, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 72; and/or

The second polypeptide comprises or consists of a sequence having an amino acid sequence as shown in SEQ ID NO. 78, or a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 78, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 78.

In some embodiments, the first polypeptide comprises a sequence having an amino acid sequence set forth in SEQ ID NO. 72 and the second polypeptide comprises a sequence having an amino acid sequence set forth in SEQ ID NO. 78.

In some embodiments, the bispecific antibody is an isolated bispecific antibody. In some embodiments, the bispecific antibody is a bispecific monoclonal antibody. In some embodiments, the isolated bispecific antibody is a monoclonal antibody.

In some embodiments, the bispecific antibody first binding moiety specifically binds to a spike protein. In some embodiments, the second binding portion of the bispecific antibody specifically binds to a spike protein.

Single domain antibodies

The invention also provides single domain antibodies with high affinity for the spike protein of SARS-CoV-2. The single domain antibodies can bind to spike proteins, prevent viral particles from binding to cells, and can mediate immune cell phagocytosis and clearance of viral particles. The single domain antibodies can be used to prevent, treat or ameliorate COVID-19, and can also be used to diagnose COVID-19.

Some embodiments provide a single domain antibody targeting a coronavirus, the single domain antibody comprising:

(a) HCDR1 comprising the amino acid sequence shown as SEQ ID No. 66 or a variant thereof having a single site substitution, deletion or insertion; (b) HCDR2 comprising the amino acid sequence shown as SEQ ID No. 67 or a variant thereof having a single site substitution, deletion or insertion; and/or (c) HCDR3, comprising an amino acid sequence as set forth in SEQ ID NO. 68, or a variant thereof having a single site substitution, deletion, or insertion.

In some embodiments, the single domain antibody comprises:

(a) HCDR1 comprising the amino acid sequence shown as SEQ ID No. 66 or a variant thereof having a single site substitution, deletion or insertion; (b) HCDR2 comprising the amino acid sequence shown as SEQ ID No. 67 or a variant thereof having a single site substitution, deletion or insertion; and (c) HCDR3, which comprises the amino acid sequence set forth in SEQ ID NO. 68, or a variant thereof having a single site substitution, deletion, or insertion.

In some embodiments, the single domain antibody comprises HCDR1 as set forth in SEQ ID NO:66, HCDR2 as set forth in SEQ ID NO:67, and HCDR3 as set forth in SEQ ID NO: 68.

In some embodiments, the single domain antibody is a VHH. In some embodiments, the single domain antibody comprises a sequence as set forth in SEQ ID NO. 69, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 69, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 69.

In some embodiments, the single domain antibody comprises or consists of the sequence set forth in SEQ ID NO. 69.

In some embodiments, the single domain antibody is an isolated antibody. In some embodiments, the single domain antibody is an isolated monoclonal antibody.

Heavy chain antibodies

The invention also provides heavy chain antibodies comprising a single domain antibody that can bind to a spike protein, as described herein. The heavy chain antibodies prevent viral particles from binding to cells and can mediate immune cell phagocytosis and clearance of viral particles. The heavy chain antibodies can be used to prevent, treat or ameliorate COVID-19, and can also be used to diagnose COVID-19.

Some embodiments provide a heavy chain antibody targeting a coronavirus, the variable region of the heavy chain antibody comprising:

In some embodiments, the heavy chain antibody comprises:

In some embodiments, the variable region of the heavy chain antibody comprises HCDR1 as shown in SEQ ID NO:66, HCDR2 as shown in SEQ ID NO:67, and HCDR3 as shown in SEQ ID NO: 68.

In some embodiments, the variable region of the heavy chain antibody comprises or consists of a sequence as set forth in SEQ ID NO. 69, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 69, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 69.

In some embodiments, the variable region of the heavy chain antibody comprises or consists of the sequence set forth in SEQ ID NO. 69.

In some embodiments, the heavy chain antibody comprises a variable region, a linker L2, and an Fc region.

In some embodiments, the linker L2 is a polypeptide comprising glycine and serine. In some embodiments, the sequence of linker L2 is (G _m S) _n Wherein each m is independently 2, 3, 4 or 5 and n is independently 1, 2, 3, 4 or 5. In some embodiments, the sequence of linker L2 is (GGGGS) _n And n is independently 1, 2, 3, 4 or 5. In some embodiments, the linker L2 is GGGGS. In some embodiments, the linker L2 is (GGGGS) ₂ As shown in SEQ ID NO. 65. In some embodiments, the linker L2 is (GGGGS) ₃ . In some embodiments, the linker L2 is (GGGGS) ₄ As shown in SEQ ID NO. 63. In some embodiments, the linker L2 is (GGGGS) ₅ As shown in SEQ ID NO. 64.

In some embodiments, the Fc region comprises a sequence having an amino acid sequence as set forth in SEQ ID NO. 70, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 70, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 70.

In some embodiments, the heavy chain antibody comprises an amino acid sequence as set forth in SEQ ID NO. 79, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 79, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 79.

In some embodiments, the heavy chain antibody comprises a sequence having the amino acid sequence set forth in SEQ ID NO. 79.

In some embodiments, the antibody specifically binds to a spike protein.

In some embodiments, the antibody or antigen-binding fragment thereof is an isolated antibody or antigen-binding fragment.

Nucleic acid and cell for expressing antibody

The invention also provides nucleic acids encoding such antibodies, including bispecific antibodies, single domain antibodies, and heavy chain antibodies. In some embodiments, the nucleic acid is an isolated nucleic acid. In some embodiments, the nucleic acid is as shown in tables 5 and 7.

The invention also provides a vector containing the nucleic acid. In some embodiments, the carrier is an isolated carrier. In some embodiments, the vector comprising the nucleic acid is a nucleic acid fragment, a plasmid, a phage, or a virus. In some embodiments, the vector is an isolated plasmid.

The invention also provides a host cell comprising said nucleic acid or said vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, HEK293 cell, cos1 cell, cos7 cell, CV1 cell, or murine L cell.

Application of

In some embodiments, the invention provides the use of the antibody (e.g., bispecific antibody, single domain antibody, heavy chain antibody, etc.) or antigen binding fragment or pharmaceutical composition comprising the same in the prevention, treatment, or amelioration of covd-19.

In some embodiments, the invention provides a method of preventing, treating, or ameliorating a covd-19 comprising administering to a patient in need thereof an effective amount of about 10mg to 2000mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or an antigen-binding fragment described herein. In some embodiments, the patient has a disease caused by a novel coronavirus. In some embodiments, the patient is a mild, moderate, heavy or critical infected patient with a novel coronavirus pneumonia. In some embodiments, the subject is in need of ICU therapy prior to treatment by the present method. In some embodiments, the subject is in need of in vitro ECMO and/or IMV treatment prior to treatment by the present methods. In some embodiments, the subject is in need of oxygen therapy prior to treatment by the present methods. In some embodiments, the subject is in need of NIV/HFNC treatment prior to treatment by the present method. In some embodiments, the method reduces patient status score by at least 1 grade. In some embodiments, the method reduces patient status score by at least 2 orders. In some embodiments, the present methods result in recovery of the patient.

In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment is administered in an amount of about 1mg/kg to 35mg/kg. In some embodiments, the dosage schedule and mode of administration depend on risk assessment of benefit and general clinical practice guidelines for antibodies (e.g., bispecific antibodies, single domain antibodies, or heavy chain antibodies) or antigen binding fragments (or formulations) in certain patient populations. In some embodiments, the antibody is a combination of any one or more of antibodies 1-23. In some embodiments, the antibody is antibody 2F8. In some embodiments, the antibody is a single domain antibody VHH18. In some embodiments, the antibody is the heavy chain antibody VHH18-Fc. In some embodiments, the antibody is a bispecific antibody 2F8-VH-VHH18 or 2F8-VL-VHH18.

In some embodiments, antibodies (e.g., bispecific antibodies, single domain antibodies, or heavy chain antibodies) or antigen binding fragments may be formulated into pharmaceutical compositions and administered to a patient in a variety of forms suitable for the chosen route of administration, such as oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, topical (e.g., by powder, ointment, drops, or transdermal patch), buccal, or by oral or nasal spray. In some embodiments, antibodies (e.g., bispecific antibodies, single domain antibodies, or heavy chain antibodies) or antigen binding fragments may be administered intravenously or subcutaneously or intramuscularly.

In some embodiments, about 1mg/kg to 35mg/kg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment or a formulation comprising such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment is administered to a patient in need thereof. In some embodiments, the amount of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment administered is about 1mg/kg, about 1.5mg/kg, about 2mg/kg, about 3mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 13mg/kg, about 15mg/kg, about 17mg/kg, about 21mg/kg, about 23mg/kg, about 25mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 35mg/kg, or a range between any two of these values (inclusive), or a formulation containing such a dose of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment. In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment is administered in a single dose.

In some embodiments, about 100mg to 1500mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment is administered to a patient in need thereof. In some embodiments, the amount of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment administered is about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1500mg, or a range between any two of these values (inclusive) or any value therein, or a formulation containing such a dose of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment. In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment is administered in a single dose.

In some embodiments, about 100mg to 1000mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment is administered to a patient in need thereof. In some embodiments, the amount of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment administered is about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, or a range between any two of these values (inclusive) or any value therein, or a formulation containing such a dose of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment. In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment is administered in a single dose.

In some embodiments, about 100mg to 600mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment is administered to a patient in need thereof. In some embodiments, the amount of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment administered is about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, or a range between any two of these values (inclusive), or any value therein, or a formulation containing such a dose of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment. In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment is administered in a single dose.

In some embodiments, about 100mg to 300mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment is administered to a patient in need thereof. In some embodiments, the amount of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment administered is about 100mg, about 200mg, about 300mg, or a range between any two of these values (inclusive), or any value therein, or a formulation containing such an amount of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment. In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment is administered in a single dose.

In some embodiments, about 300mg to 1500mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment is administered to a patient in need thereof. In some embodiments, the amount of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment administered is about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1200mg, about 1300mg, about 1500mg, or a range between any two of these values (inclusive), or any value therein, or a formulation containing such a dose of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment. In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment is administered in a single dose.

In some embodiments, about 300mg to 1000mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment is administered to a patient in need thereof. In some embodiments, the amount of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment administered is about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, or a range between any two of these values (inclusive) or any value therein, or a formulation containing such amount of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment. In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment is administered in a single dose.

In some embodiments, about 300mg to 600mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment is administered to a patient in need thereof. In some embodiments, the amount of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment administered is about 300mg, about 400mg, about 500mg, about 600mg, or a range between any two of these values (inclusive) or any value therein, or a formulation containing such a dose of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment. In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment is administered in a single dose.

In some embodiments, about 600mg to 1000mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment is administered to a patient in need thereof. In some embodiments, the amount of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment administered is about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, or a range between any two of these values (inclusive), or any value therein, or a formulation containing such a dose of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment. In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment is administered in a single dose.

In some embodiments, about 80mg to 150mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, or a formulation comprising such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, is administered to a patient in need thereof; such as about 100mg administered in a single administration to a patient in need thereof. In some embodiments, about 100mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, is administered in a single dose to a patient in need thereof.

In some embodiments, about 180mg to 320mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, or a formulation comprising such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, is administered to a patient in need thereof; such as about 300mg administered in a single dose to a patient in need thereof. In some embodiments, about 300mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, is administered in a single dose to a patient in need thereof.

In some embodiments, about 580mg to 620mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, or a formulation comprising such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, is administered to a patient in need thereof; such as about 600mg administered in a single administration to a patient in need thereof. In some embodiments, about 600mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, is administered in a single dose to a patient in need thereof.

In some embodiments, about 980mg to 1200mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen-binding fragment, or a formulation comprising such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen-binding fragment, is administered to a patient in need thereof; such as about 1000mg administered in a single dose to a patient in need thereof. In some embodiments, about 1000mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, is administered in a single dose to a patient in need thereof.

In some embodiments, about 1450mg to 1550mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, is administered to a patient in need thereof; such as about 1500mg administered in a single dose to a patient in need thereof. In some embodiments, about 1500mg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, or a formulation containing such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, is administered in a single dose to a patient in need thereof.

In some embodiments, about 1mg/kg to 35mg/kg of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, or a formulation comprising such a dose of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, is administered to a patient in need thereof. In some embodiments, the amount of antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment administered is about 1mg/kg to 25mg/kg, about 1.7mg/kg to 16.7mg/kg, about 1.7mg/kg to 5mg/kg, about 5mg/kg to 25mg/kg, about 5mg/kg to 16.7mg/kg, about 16.7mg/kg to 25mg/kg, about 1.4mg/kg to 21.4mg/kg, about 1.4mg/kg to 14.3mg/kg, about 1.4mg/kg to 4.3mg/kg, about 4.3mg/kg to 21.4mg/kg, about 4.3mg/kg to 14.3mg/kg, about 14.3mg/kg to 21.4mg/kg. In some embodiments, the amount of antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment administered is about 1mg/kg, about 1.2mg/kg, about 1.4mg/kg, about 1.5mg/kg, about 1.7mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 4mg/kg, about 4.3mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14.3mg/kg, about 15mg/kg, about 16mg/kg, about 16.7mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 21.4mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 27mg, about 32mg, about 26mg, about 32mg, or any of the antibody, or any fragment therebetween, including a range therebetween, or a specific antibody, or a range therebetween. In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment is administered in a single dose.

In some embodiments, the symptoms of the patient are alleviated after a single administration of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment. In some embodiments, after a single administration of an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment, the patient's symptoms are not expected to be alleviated prior to administration to the patient.

In some embodiments, the antibody is selected from the group consisting of antibodies 1-24, antibody 2F8.

In some embodiments, the antibody is a single domain antibody VHH18.

In some embodiments, the antibody is the heavy chain antibody VHH18-Fc.

In some embodiments, the antibody is the bispecific antibody 2F8-VH-VHH18.

In some embodiments, the antibody is the bispecific antibody 2F8-VL-VHH18.

In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment (or formulation) is administered by subcutaneous (s.c.) injection, intraperitoneal (i.p.) injection, parenteral injection, intraarterial injection, intramuscular Injection (IM), or intravenous (i.v.) injection, among others. In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment (or formulation) is administered by infusion. In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment (or formulation) is administered as a bolus.

In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment (or formulation) is administered by intravenous (i.v.) infusion. In some embodiments, the intravenous infusion duration is about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, or a range between any two of these values (inclusive) or any value therein. In some embodiments, the intravenous infusion is greater than or equal to 60 minutes in duration. In some embodiments, the intravenous infusion duration is less than or equal to 60 minutes. In some embodiments, the intravenous infusion is greater than or equal to 30 minutes in duration.

In some embodiments, the antibody (e.g., bispecific antibody, single domain antibody, or heavy chain antibody) or antigen binding fragment (or formulation) is used in combination with other therapeutic methods for preventing, treating, or ameliorating covd-19, e.g., hormone, in vitro ECMO, IMV, NIV, HFNC, and/or oxygen therapy.

In another aspect, the invention also discloses the use of said antibody (e.g. bispecific, single domain or heavy chain antibody) or antigen binding fragment in the manufacture of a medicament for the prevention, treatment or amelioration of covd-19. In some embodiments, the agent for preventing, treating, or ameliorating covd-19 comprises an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or an antigen binding fragment.

In another aspect, the invention also discloses a kit comprising an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or an antigen binding fragment (or formulation) and instructions for administering the antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment (or formulation) to a patient in need thereof.

Diagnostic methods and uses are also provided. In some embodiments, methods are provided for detecting SARS-CoV-2 expression in a sample by contacting the sample with an antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment described herein, such that the antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment binds to a spike protein, and detecting its binding, i.e., the amount of spike protein in the sample. In some embodiments, there is provided the use of the antibody (e.g., bispecific, single domain, or heavy chain antibody) or antigen binding fragment in the preparation of a kit for diagnosing covd-19. In some embodiments, a diagnostic kit comprising the antibody (e.g., a bispecific antibody, a single domain antibody, or a heavy chain antibody) or antigen binding fragment is provided.

Pharmaceutical composition

In another aspect, the invention also discloses pharmaceutical compositions comprising the antibodies or antigen-binding fragments described herein and pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is a pharmaceutical composition suitable for injection, such as a bolus-type pharmaceutical composition or an infusion (instillation) type pharmaceutical composition or a spray-type pharmaceutical composition. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (herein water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, dextrose injection, sterile water or Phosphate Buffered Saline (PBS), ethanol, solvents or dispersion media for polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycols, and the like), and suitable mixtures thereof. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier may comprise an antibacterial and/or antifungal agent, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, benzyl alcohol, and the like. In some embodiments, the pharmaceutically acceptable carrier may comprise isotonic agents, such as sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride. In some embodiments, the pharmaceutical composition comprises at least 0.1% antibodies (e.g., bispecific antibodies, single domain antibodies, or heavy chain antibodies) or antigen binding fragments. The percentage of antibody may vary and is between about 2% to about 90% of the weight of a given dosage form. The amount of antibody (e.g., bispecific, single domain, or heavy chain antibody) or antigen binding fragment in such a therapeutically useful pharmaceutical composition can be an effective amount for administration.

On the other hand, the invention also discloses a preparation method of the pharmaceutical composition, which comprises the following steps: the antibodies (e.g., bispecific, single domain, or heavy chain antibodies) or antigen binding fragments described herein are admixed with a pharmaceutically acceptable carrier (e.g., water for injection, physiological saline, dextrose injection, or the like). Methods of mixing the above-described antibodies (e.g., bispecific antibodies, single domain antibodies, or heavy chain antibodies) or antigen binding fragments with a pharmaceutically acceptable carrier are generally known in the art.

The invention provides a bispecific antibody of targeting coronavirus and application thereof, wherein a first binding part and a second binding part in the bispecific antibody cooperate to prevent SARS-CoV-2 virus particles from infecting cells, mediate immune cells to phagocytose and remove virus particles, and prevent, treat or improve COVID-19; the bispecific antibodies of the invention can also be used for diagnostic detection of whether a patient is infected with SARS-CoV-2.

Drawings

FIG. 1 is a graph of inhibition of SARS-CoV-2 binding to ACE2 in ELISA experiments using a portion of the anti-spike protein antibodies of the invention. In fig. 1, the abscissa represents the concentration, and the ordinate represents the OD value; 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.

FIG. 2 shows that antibodies block binding of spike RBD to ACE 2; wherein no antibody was added to the ACE2 control group.

FIG. 3 shows the inhibition curves of the antibody 2F8-VH-VHH18 against pseudoviruses of different strains of the novel coronavirus.

FIG. 4 shows that antibodies block virus infection in mice; in the figure, the ordinate indicates pulmonary virus titer.

FIG. 5 shows the effect of antibodies on mouse body weight; in the figure, the ordinate indicates the percentage of the weight of the mice, and the abscissa indicates the number of days.

Terminology

Unless otherwise indicated, each term below shall have the meaning described below.

Definition of the definition

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

The terms "comprising" or "including" as used herein mean that the compositions and methods, etc., include the recited elements, e.g., components or steps, but do not exclude the others. By "consisting essentially of … …" it is meant that the compositions and methods exclude other elements that have a fundamental impact on the characteristics of the combination, but do not exclude elements that have no essential impact on the compositions or methods. "consisting of … …" means that elements not specifically recited are excluded.

The term "polypeptide" is intended to encompass both the singular and the plural of "polypeptides" and refers to molecules formed from amino acid monomers that are linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any single chain or multiple chains comprising two or more amino acids, and does not refer to a particular length of product. Thus, the definition of "polypeptide" includes 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 addition to, any of the terms described above. The term "polypeptide" is also intended to refer to products of modification of the polypeptide after expression, 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 the 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 directly or in precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides, a so-called codon or base triplet. Each amino acid is encoded by at least one codon. The same amino acid is encoded by different codons called "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 that contains a side chain (R group) that is similar in chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will 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 chains: 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 number of conservative amino acid substitutions of "VL, VH, VHH" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15 conservative amino acid substitutions, or a range between any two of these values (inclusive) or any value therein. The number of amino acids of a "conservative amino acid substitution of a heavy chain constant region, a light chain constant region, a heavy chain or light chain, a fusion protein first polypeptide or a second polypeptide" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values (including the endpoints) or any value therein.

The term "isolated" as used herein with respect to cells, nucleic acids, polypeptides, antibodies, etc., e.g., "isolated" DNA, RNA, polypeptides, antibodies, etc., refers to molecules that are separated by one or more of the other components of the cell's natural environment, such as DNA or RNA, respectively. 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 medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not exist in a natural state and do not exist in a natural state. The term "isolated" is also used herein to refer to cells or polypeptides isolated from other cellular proteins or tissues. An isolated polypeptide is 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, the purity of the isolated nucleic acid, polypeptide, antibody, etc., is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values (inclusive) or any value therein.

The term "recombinant" refers to a polypeptide or polynucleotide, meaning a form of the polypeptide or polynucleotide that does not exist in nature, and non-limiting examples can be combined to produce a polynucleotide or polypeptide that does not normally exist.

"homology" or "identity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology or identity 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 or identical at that position. The degree of homology between sequences is a function of the number of matched or homologous positions shared by the sequences.

"at least 80% identical" is about 80% identical, about 81% identical, about 82% identical, about 83% identical, about 85% identical, about 86% identical, about 87% identical, about 88% identical, about 90% identical, about 91% identical, about 92% identical, about 94% identical, about 95% identical, about 98% identical, about 99% identical, or a range between any two of these values (inclusive) or any value therein.

"at least 90% identical" is about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 94% identical, about 95% identical, about 92% identical, about 96% identical, about 99% identical, or a range between any two of these values (inclusive of the endpoints) or any value therein. A nucleic acid 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 when the sequences are aligned, the percentage of bases (or amino acids) in the two sequences that are compared are identical. The percentage of alignment identity or sequence identity may be determined using visual inspection or software programs known in the art, such as the software program described in Ausubel et al eds. (2007) in Current Protocols in Molecular Biology. Preferably, the alignment is performed using default parameters. One such alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both of which use the following default parameters: genetics code = standard; filter = none; strand = both; cutoff = 60; expect=10; matrix = BLOSUM62; descriptive = 50sequences; sortby=highscore; databases = non-redundants; genbank+embl+ddbj+pdb+genbank cdstransplations+swi ssprotein+spldate+pir. Biologically equivalent polynucleotides are those that have the indicated percent identity and encode polypeptides having the same or similar biological activity.

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

The terms "polynucleotide," "nucleic acid," 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 may have any three-dimensional structure and may perform any function, known or unknown. The following are examples of non-limiting polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, structural modification of the nucleotide may be performed 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 indicated 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 constitute the double stranded form.

The term "encoding" when applied to a polynucleotide refers to a polynucleotide referred to as "encoding" a polypeptide, which polypeptide and/or fragment thereof may be produced by transcription and/or translation in its native state or when manipulated by methods well known to those skilled in the art.

An "antibody" refers to a polypeptide or complex of polypeptides that specifically recognizes and binds an antigen. The antibody may be an intact antibody, any antigen-binding fragment thereof, or a single chain thereof. The term "antibody" thus includes any protein or peptide comprising at least a portion of an immunoglobulin molecule having biological activity for binding to an antigen in a molecule. Antibodies and antigen-binding fragments include, but are not limited to, 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 (F R), or any portion thereof, or at least a portion of a binding protein, as described in the examples of heavy or light chains or ligand-binding portions thereof. The CDR regions include the CDR regions of the light chain (LCDR 1-3) and the heavy chain (HCDR 1-3). The variable region may comprise the structure FR1-CDR1-FR2-CDR2-FR3-CD R3-FR4.

The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F (ab') ₂ 、F(ab) ₂ Fab', fab, fv, scFv, etc. Regardless of its structure, the antibody fragment binds to the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, stereoisomers, and diabodies. The term "antigen binding fragment" also includes antibodies that are raised by binding to a specific antigen to form a complexAny synthetic or genetically engineered protein that acts on the body.

"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 short-linked peptides of 10 to about 25 amino acids. The linker may be glycine-rich to increase flexibility, 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 a linker introduced, it retains the original immunoglobulin specificity. ScFv molecules are generally known in the art and are described, for example, in U.S. Pat. No. 5,892,019.

The term "single domain antibody" or "sdAb" refers to a single antigen binding polypeptide having three Complementarity Determining Regions (CDRs). The sdAb alone is capable of binding to an antigen, but does not pair with the corresponding CDR-containing polypeptide. In some cases, sdabs are engineered from camelid hcabs, and their heavy chain variable domains are referred to herein as "VHHs. Camelsdabs are among the smallest known antigen-binding antibody fragments (see, e.g., hamers-Casterman et al, nature363:446-8 (1993); greenberg et al, nature 374:168-73 (1995); hassazadeh-Ghasssaboeh et al, nanomedicine (Lond), 8:1013-26 (2013)).

The term "heavy chain antibody" or "HcAb" refers to a functional antibody that comprises heavy chains (VHH, CH2, and CH 3), but lacks the light chains typically found in antibodies. Camelids (e.g. camels, llamas or alpacas) are known to produce hcabs.

In some embodiments, the first binding moiety in the bispecific antibody is an antibody that targets the spike protein, either as an intact antibody or an antigen-binding fragment. In some embodiments, the first binding moiety is an IgG-type antibody. In some embodiments, the first binding moiety is an IgG-type antibody, and the C-terminus of its heavy chain is truncated.

In some embodiments, the second binding moiety in the bispecific antibody is an antibody that targets the spike protein, either as an intact antibody or an antigen-binding fragment. In some embodiments, the second binding moiety in the bispecific antibody is a single domain antibody.

The term "antibody" includes a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that the heavy chain classes include gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), some of which are also subclasses (e.g., γ1- γ4). The nature of this chain determines the "class" of antibody as IgG, igM, igA, igG or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., igG1, igG2, igG3, igG4, igG5, etc., have been well characterized and the functional specificities conferred are 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, wherein the light chain starts at the "Y" mouth and continues through the variable region surrounding the heavy chain.

Antibodies, antigen binding fragments, or derivatives of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric/single chain antibodies, epitope-binding fragments such as Fab, fab ', and F (ab') ₂ Fd, fvs, single chain Fvs (scFv), disulfide linked Fvs (sdFv), fragments comprising a VK or VH domain, or fragments generated from a Fab expression library, and an anti-idiotype (anti-Id) antibody. The immunoglobulins 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 IgA 2) or subclass of immunoglobulin.

Light chains can be classified as kappa (kappa) or lambda (lambda). 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 thereof are bound by covalent bonds, and the "tail" portions of the two heavy chains are bound 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. Immunoglobulin kappa light chain variable region vkappa; immunoglobulin lambda light chain variable region 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 terms of function. The light chain variable region (VL) and the heavy chain variable region (VH) 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, and the like. Conventionally, the numbering of constant regions increases as they become farther from the antigen binding site or amino terminus of an antibody. The N-terminal portion is a variable region and the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chains, respectively.

As described above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. In particular, the VL domain of an antibody and a subset of VH domains or Complementarity Determining Regions (CDRs) combine to form a variable region that defines a three-dimensional antigen binding site. The antibody quaternary structure forms an antigen binding site present at each arm end of Y. More specifically, the antigen binding site is defined by three CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR 3) in each of the VH and VL chains. In some cases, for example some immunoglobulin molecules derived from or engineered based on camelid immunoglobulins, the complete immunoglobulin molecule may consist of heavy chains only, without light chains. See, for example, hamers-Casterman et al, nature 363:446-448 (1993).

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, provided that the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining other amino acids in the antigen binding domain, known as the "framework" region, exhibit less intermolecular variability. The framework regions largely adopt a β -sheet conformation, with the CDRs forming a loop structure linked thereto, or in some cases forming part of a β -sheet structure. Thus, the framework regions form scaffolds to position the CDRs in the correct orientation by non-covalent interactions between the chains. An antigen binding domain with CDRs at specific positions forms a surface complementary to an epitope on an antigen that facilitates non-covalent binding of the antibody to its epitope. For a given heavy or light chain variable region, one of ordinary skill in the art can identify amino acids comprising CDRs and framework regions by known methods (see Kabat, e., et al, U.S. device ofHealth and Human Services, sequences ofProteins of Immunological Interest, (1983) and Chothia and Lesk, j. Mol. Biol.,196:901-917 (1987)).

Where there are two or more definitions of terms used and/or accepted in the art, the definitions of terms used herein include 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 overlapping 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. One skilled in the art can generally determine which specific residues a CDR comprises based on the variable region amino acid sequence of an antibody.

Kabat et al also define a numbering system for variable region sequences suitable for use with any antibody. The "Kabat numbering" system can be applied to any variable region sequence by one of ordinary skill in the art independent of other experimental data than the sequence itself. "Kabat numbering" refers to the numbering system set forth by Kabat et al, U.S. Dept. Ofhealth and Human Services at "Sequence ofProteins ofImmunological Interest" (1983). Antibodies may also use the EU numbering system.

The antibodies or antigen binding fragments 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 a cartilage class (condricchoice) source (e.g., from shark).

"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, an antibody or antigen binding fragment of the disclosure comprises a CH1 domain; comprises a CH1 domain, at least a portion of a hinge region, and a CH2 domain; comprising 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, a CH2 domain, and a CH3 domain. In another embodiment, an antibody or antigen binding fragment of the disclosure 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 can be modified such that the amino acid sequence of their naturally occurring immunoglobulin molecules is altered.

The heavy chain constant regions of antibodies may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may comprise a polypeptide derived from an IgG ₁ CH1 domain of a molecule and derived from IgG ₃ Hinge region of the molecule. In another embodiment, the heavy chain constant region may comprise a portion derived from an IgG ₁ Molecules and moieties derived from IgG ₃ Hinge region of the molecule. In another embodiment, a portion of the heavy chain may comprise a portion derived from IgG ₁ Molecules and moieties derived from IgG ₄ Chimeric hinge region of the molecule.

"light chain constant region" includes amino acid sequences 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 the collection of light and heavy chains that can form dimers through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

As mentioned above, the subunit structure and three-dimensional configuration of the constant regions of various immunoglobulin classes are well known. The "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain and the "CH1 domain" includes the first (most amino terminal) constant region of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is the amino terminus of the immunoglobulin heavy chain molecule hinge region. 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. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule, approximately comprising 108 residues. The "hinge region" includes a portion of the heavy chain region connecting 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 (rouxet al, j.immunol 161:4083 (1998)).

"disulfide" refers to a covalent bond formed between two sulfur atoms. The thiol group of cysteine may form a disulfide bond or bridge with the 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 region is obtained or derived from a first species, while its constant region (which may be intact, partial or modified) is 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.

"specifically binding" or "specific for … …" generally refers to the complementary binding of an antibody or antigen binding fragment to a particular antigen through its antigen binding domain to an epitope to form a relatively stable complex. "specificity" may be expressed in terms of the relative affinity of an antibody or antigen binding fragment to bind to a particular antigen or epitope. For example, if antibody "a" has a greater relative affinity for the same antigen than antibody "B", antibody "a" may be considered to have a higher specificity for that antigen than antibody "B". Specific binding can be described by equilibrium dissociation constants (KD), a smaller KD meaning a tighter binding. Methods for 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. Antibodies that "specifically bind" a spike protein include 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. A monospecific antibody may specifically bind one antigen or one epitope, whereas a bispecific antibody may specifically bind two different antigens or two different epitopes.

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures, with the object of preventing, slowing, ameliorating or stopping an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the disease state, delay or slowing of disease progression, amelioration, palliation or disappearance (whether partial or total), prolongation of life span expected when not receiving treatment, and the like. Patients in need of treatment include those already with the condition or disorder, those prone to the condition or disorder, or those in need of prophylaxis of the condition or disorder, for whom administration of the disclosed antibodies or pharmaceutical compositions for detection, diagnostic procedures, and/or treatment would be expected to benefit.

The term "administering" as used herein refers to administering a substance (e.g., bispecific antibody 2F8-VH-VHH 18) for therapeutic purposes (e.g., treatment of COVID-19).

As used herein, the term "in need of" means that the patient has been identified as in need of a particular method or treatment. In some embodiments, the identification may be performed by any diagnostic means. In any of the methods and treatments described herein, the patient may need.

An "effective amount" refers to an amount of an active compound or agent that is capable of eliciting a biological or medical response in a tissue, system, animal, individual or human; an effective amount is sought by a researcher, veterinarian, medical doctor or other clinician.

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

"about" refers to a conventional error range of corresponding numerical values as readily known to one of ordinary skill in the relevant art. In some embodiments, references herein to "about" refer to the values described and ranges thereof of ± 10%, ± 5% or ± 1%.

"ECMO" refers to extracorporeal membrane oxygenation (Extracorporeal Membrane Oxygenation, ECMO), a medical emergency technical device, primarily used to provide sustained in vitro respiration and circulation to a patient suffering from severe cardiopulmonary failure to maintain the patient's life.

The ICU refers to a intensive care unit (Intensive Care Unit), and can synchronously perform treatment, nursing and rehabilitation, provide isolation places and equipment for patients suffering from serious illness or coma, and provide services such as optimal nursing, comprehensive treatment, medical and nursing combination, early postoperative rehabilitation, joint nursing exercise treatment and the like.

"IMV" refers to intermittent commanded ventilation (intermittent mandatory ventilation) that is the implementation of periodic volume or pressure ventilation according to a preset time interval, i.e., time trigger. This period allows the patient to breathe spontaneously at any set base pressure level during the commanded ventilation. In spontaneous breathing, the patient may breathe spontaneously with continuous airflow support, or the machine will valve open on demand to allow spontaneous breathing. Most ventilators can provide pressure support during spontaneous breathing.

"HFNC", i.e., nasal High flow oxygen therapy (High-flow nasal cannula oxygen therapy), is an oxygen therapy that delivers a High flow of air-mixed oxygen gas of a certain oxygen concentration directly to a patient through a nasal obstruction tube without sealing, as a form of noninvasive respiratory support, which can rapidly improve oxygenation. The traditional Chinese medicine composition can be applied to patients with acute hypoxia respiratory failure, patients after surgical operation, patients without tracheal intubation for respiratory failure, patients with immunosuppression, patients with cardiac insufficiency and the like.

"NIV" refers to Non-invasive ventilation (Non-invasine Ventilation) and refers to atraumatic mechanical ventilation other than tracheal intubation and tracheotomy.

“EC ₅₀ "half maximum effect concentration (concentration for 50%ofmaximal effect,EC) ₅₀ ) Refers to the concentration that causes 50% of the maximum effect.

“IC ₅₀ "means a 50% inhibition concentration, i.e., the concentration of drug or inhibitor required to inhibit half of a given biological process.

The "parent Fc region" in the present invention may be a naturally occurring Fc region, and the gene encoding the Fc region may be from a human, mouse, rabbit, camel, monkey, preferably human and mouse; for example, the parent Fc region is the Fc region of SEQ ID NO. 60, SEQ ID NO. 61 or SEQ ID NO. 66.

The relevant descriptions of the patents and publications mentioned herein are incorporated by reference in their entirety.

Bispecific, single domain antibodies and heavy chain antibodies

The present invention provides antibodies, including bispecific antibodies, single domain antibodies, and heavy chain antibodies, having high affinity for spike proteins. Bispecific antibodies, single domain antibodies, and heavy chain antibodies exhibit potent binding activity and are useful for therapeutic and diagnostic applications. For example, these antibodies can prevent fusion of SARS-CoV-2 viral particles and cell membranes, as well as mediate immune cell phagocytosis and clearance of viral particles.

Some embodiments provide bispecific antibodies in which the C-terminus (i.e., CH3 terminus) of the heavy chain of the first binding moiety is covalently linked to a single domain antibody via a linker L1. In some embodiments, the bispecific antibody comprises 2 first polypeptides that are identical in sequence and 2 second polypeptides that are identical in sequence; the amino acid sequence of the first polypeptide is shown as SEQ ID NO. 77, and the amino acid sequence of the second polypeptide is shown as SEQ ID NO. 74.

Some embodiments provide bispecific antibodies in which the C-terminus of the light chain of the first binding moiety (i.e., the CL terminus) is covalently linked to a single domain antibody via a linker L1. In some embodiments, the bispecific antibody comprises 2 first polypeptides that are identical in sequence and 2 second polypeptides that are identical in sequence; the amino acid sequence of the first polypeptide is shown as SEQ ID NO. 72, and the amino acid sequence of the second polypeptide is shown as SEQ ID NO. 78.

Some embodiments provide a single domain antibody having the amino acid sequence shown in SEQ ID NO. 69.

Some embodiments provide a heavy chain antibody comprising 2 heavy chains of identical sequence, the heavy chain sequence of which is shown in SEQ ID NO. 79.

In some embodiments, a bispecific antibody, a single domain antibody, or a heavy chain antibody may also be linked to an amino acid sequence or one or more modifying groups. For example, a bispecific, single domain or heavy chain antibody of the present disclosure may comprise a malleable linker sequence, or may be modified to add a functional group (e.g., PEG, drug, toxin, or tag).

The bispecific, single domain or heavy chain antibodies disclosed herein also comprise modified derivatives, i.e. modified by covalent attachment of any type of molecule to the antibody, wherein the covalent attachment does not prevent the binding of the antibody to an epitope. Including but not limited to the following examples, the antibodies may be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized with 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 the prior art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

In some embodiments, the bispecific antibody, single domain antibody, or heavy chain antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, agent, or PEG.

Bispecific antibodies, single domain antibodies, or heavy chain antibodies may be conjugated or fused to therapeutic agents, which may include detectable labels such as radiolabels, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic agents, diagnostic agents, cytotoxic agents, ultrasound enhancers, non-radioactive labels and combinations thereof, and other such agents known in the art.

Bispecific antibodies, single domain antibodies, or heavy chain antibodies can be detectably labeled by coupling them to chemiluminescent compounds. The presence of the chemiluminescent-tagged antibody is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts and oxalic esters.

Also disclosed are polynucleotides or nucleic acid molecules encoding the bispecific antibodies, single domain antibodies and heavy chain antibodies of the invention. The polynucleotides disclosed herein may encode a heavy chain, a light chain, a heavy chain variable region, a light chain variable region, an Fc region, a portion of a heavy chain variable region, or a portion of a light chain variable region, a bispecific, single domain antibody, or a heavy chain antibody. Methods of making antibodies are well known in the art and are described in the present invention. In certain embodiments, the variable and constant regions of the antibodies disclosed herein are all human in 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 directed against a particular antigen can be prepared by administering the antigen to transgenic animals that have been modified to produce fully human antibodies in response to antigen challenge. Exemplary techniques that can be used to prepare such antibodies are 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 detrimental immune response in the animal (e.g., human) to be treated. In one embodiment, the antibodies (including bispecific antibodies, single domain antibodies, or heavy chain antibodies) disclosed herein are modified to reduce their immunogenicity using art-recognized techniques. For example, the antibodies may be humanized, primatized, deimmunized or chimeric antibodies 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 accomplished by a variety of methods, including (a) grafting the entire non-human variable region to a human constant region to produce a chimeric antibody; (b) Transplanting at least a portion of one or more non-human Complementarity Determining Regions (CDRs) into framework and constant regions of human origin, with or without the retention of critical framework residues; or (c) transplanting the entire non-human variable regions, but "hiding" them by replacing surface residues with human-like moieties. Typically the framework residues in the human framework region will be replaced 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 interactions of CDRs and framework residues to identify framework residues that play an important role in antigen binding and by sequence alignment to identify aberrant framework residues at specific positions. (see U.S. Pat. No. 5,585,089; 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 (EP 592,106; EP519,596; and chain rearrangement (U.S. Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.

Deimmunized can also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunizing" includes altering antibodies to modify T cell epitopes (see, e.g., WO/9852976A1 and WO/0034317A 2). For example, heavy and light chain variable region sequences from the starting antibody are analyzed and a "map" of human T cell epitopes from each variable region is generated showing the positions of the epitopes relative to the Complementarity Determining Regions (CDRs) and other key residues within the sequence. Single T cell epitopes from the T cell epitope map were 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 a combination of amino acid substitutions are designed and subsequently incorporated into a series of binding polypeptides. The genes for the complete heavy and light chains, comprising the modified variable and human constant regions, are then cloned into expression vectors, and the plasmids are subsequently transferred into cell lines to produce the complete antibodies. The antibodies are then compared using appropriate biochemical and biological experiments to identify the best antibodies.

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

Preparation of scFv can be seen in the art of producing single chain units (U.S. Pat. No. 4,694,778). The heavy and light chain fragments of the Fv region are bridged by amino acids to form single chain units, resulting in a single chain fusion peptide. Techniques for assembling functional Fv fragments in E.coli can also be used (Skerra et al Science 242:1038-1041 (1988)).

Examples of techniques that can 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. 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 a variable region of murine monoclonal antibodies and a constant region of human immunoglobulins. Methods of producing chimeric antibodies are known in the art, see U.S. Pat. nos. 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated herein by reference.

Naturally occurring VHH domains directed against a specific antigen or target may be obtained from a (native or immune) library of camelidae VHH sequences. Such libraries and techniques are described, for example, in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, a modified synthetic or semisynthetic library derived from a (native or immune) VHH library may be used, e.g. a VHH library obtained from a (native or immune) VHH library by techniques such as random mutagenesis and/or CDR shuffling, e.g. as described in WO 00/43507.

In addition, another efficient method for producing recombinant antibodies is disclosed in Newman, biotechnology 10:1455-1460 (1992), which in particular enables the production of primate antibodies comprising monkey variable and human constant region sequences, the entire content of which is incorporated herein by reference. In addition, this technology is also mentioned in commonly assigned U.S. Pat. nos. 5,658,570, 5,693,780 and 5,756,096, the entire contents of each of which are incorporated herein by reference.

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

In another embodiment, the DNA encoding the desired monoclonal antibody may be isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of a murine antibody). Isolated and subcloned hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into a prokaryotic or eukaryotic host cell, such as an e.coli cell, simian COS cell, chinese Hamster Ovary (CHO) cell, or myeloma cell that does not produce other immunoglobulins. Isolated DNA (which may be synthetic as described herein) may also be used to prepare sequences of constant and variable regions of antibodies, as described in U.S. patent 5,658,570, which is incorporated herein by reference in its entirety. The method extracts RNA from selected cells and converts it into 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.

Furthermore, one or more CDRs of an antibody of the invention can be inserted into a framework region, e.g., into a human framework region, using conventional recombinant DNA techniques to construct a humanized non-fully human antibody. The framework regions may be naturally occurring or consensus framework regions, preferably human framework regions (see Chothia et al, J. Mol. Biol.278:457-479 (1998), which lists a range of human framework regions). Some polynucleotides may encode antibodies that bind specifically to at least one epitope of an antigen of interest produced by the combination of framework regions and CDRs. One or more amino acid substitutions may be made within the framework region, and amino acid substitutions may be selected that improve binding of the antibody to its antigen. In addition, substitution or deletion of cysteine residues in one or more of the variable regions involved in interchain disulfide formation may be performed in this manner, thereby producing an antibody molecule lacking one or more interchain disulfide bonds. Other variations on polynucleotides within the skill of the art are also encompassed by the present invention.

In some embodiments, DNA encoding an antibody may be synthesized according to the amino acid sequence design of the antibodies described herein, placed into an expression vector, and then transfected into a host cell, and the transfected host cell cultured in culture medium to produce the antibody. In some embodiments, the expression vector includes at least one promoter element, an antibody, antigen binding fragment, or fusion protein coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, kozak sequences, and donor and acceptor sites for RNA splicing flanking the insertion. Efficient transcription can be obtained by the early and late promoters of SV40, the early promoters from the long terminal repeats of retroviruses such as RSV, HTLV1, HIVI and Cytomegalovirus (CMV), and promoters of other cells such as actin promoters may 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, pSV dhfr, pBC12MI, pCS2 or pCHO1.0, etc. Commonly used mammalian cells include chinese hamster ovary cells (CHO cells) (e.g., CHO-K1 cells) or CHO-S, CHO-dhfr-, CHO/DG44 or expi CHO, NSO myeloma cells, COS1 cells, COS7 cells, SP2 cells, CV1 cells, murine L cells, human embryonic kidney cells HEK293 or HEK293T, HEK293F or HEK293E cells modified from HEK293 cells, etc.

In some embodiments, the insert should contain a selectable marker, common selectable markers including dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, and the like, to facilitate selection and isolation of transfected cells. The constructed plasmid is transfected into host cells without the genes, and the transfected cells grow in a large quantity after being cultured by a selective medium to generate target proteins to be obtained.

Antibody-producing cell lines may 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, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L.Hacker, F.M.Wurm, in Reference Module in Life Sciences,2017, the entire contents of which, including the supplementary contents, are incorporated by reference in their entirety.

For recombinant expression of the antibodies of the invention, the host cell may be co-transfected with two recombinant expression vectors, a first encoding the antibody heavy chain and a second encoding the antibody light chain. The two recombinant expression vectors may contain the same selectable marker, or they may each contain a different selectable marker. Alternatively, host cells can be transfected with recombinant expression vectors that co-encode the heavy and light chains of the antibody.

Antibodies of the invention may also be produced by chemical synthesis (e.g., by the method described in SolidPhase Peptide Synthesis, 2 nd edition, 1984The Pierce Chemical Co, rockford, ill.). Variant antibodies can also be generated using a cell-free platform (see, e.g., chu et al, biochem No.2, 2001 (Roche Molecular Biologicals) and Murray et al, 2013,Current Opinion in Chemical Biology,17:420-426).

Antibodies produced by recombinant expression may be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity and fractionation column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. For example, affinity chromatography using protein A or protein G. Alternatively, specific antigens or epitopes thereof targeted by immunoglobulins can be immobilized on the column to purify the immunospecific antibodies by immunoaffinity chromatography. The antibodies of the invention may be fused to heterologous polypeptide sequences known in the art to facilitate purification. Purification of immunoglobulins can be found in The article d.wilkinson (The scientific, inc., philiadelphia Pa., vol.14, 8 (4 months 17 of 2000), pages 25-28).

In addition, mutations may be introduced into the nucleotide sequences encoding the antibodies of the invention using standard techniques known to those skilled in the art, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis that result in amino acid substitutions. Variants (including derivatives) encode substitutions of less than 50 amino acids, less than 40 amino acids, less than 30 amino acids, less than 25 amino acids, less than 20 amino acids, less than 15 amino acids, less than 10 amino acids, less than 5 amino acids, less than 4 amino acids, less than 3 amino acids, or less than 2 amino acids relative to the original heavy chain variable regions HCDR1, HCDR2, HCDR3, and light chain variable regions LCDR1, LCDR2, or LCDR 3. Alternatively, mutations may be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants may be screened for biological activity to identify mutants that retain activity. In some embodiments, the substitution may be a conservative amino acid substitution.

Therapeutic method

The invention also provides methods of treatment and uses. In some embodiments, methods for preventing, treating, or ameliorating covd-19 are provided that include administering to a patient an effective dose of an antibody (including a bispecific antibody, a single domain antibody, or a heavy chain antibody). In some embodiments, there is provided the use of the antibodies (including bispecific antibodies, single domain antibodies, or heavy chain antibodies) in the prevention, treatment, or amelioration of covd-19. In some embodiments, there is provided the use of the antibody (including a bispecific antibody, a single domain antibody or a heavy chain antibody) in the manufacture of a medicament for the prevention, treatment or amelioration of covd-19. In some embodiments, the patient is a patient suspected of being infected with SARS-CoV-2 virus. In some embodiments, the patient is a patient in contact with a carrier of SARS-CoV-2 virus. In some embodiments, the patient is a patient diagnosed with infection with SARS-CoV-2 virus. In some embodiments, the patient is a patient with mild symptoms. In some embodiments, the patient is a patient with severe symptoms. In some embodiments, the patient has fever, cough, hypotension, hypoxia, and/or Acute Respiratory Distress Syndrome (ARDS).

The specific dosage and treatment regimen for any particular patient will depend on a variety of factors including the antibody (including bispecific antibody, single domain antibody or heavy chain antibody) used, the age and weight of the patient, the general health, sex and diet, and the time of administration, frequency of excretion, drug combination, and the severity of the particular disease undergoing therapy. These factors are judged by medical care personnel included within the scope of one of ordinary skill in the art. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound used, the severity of the disease and the desired effect. The dosages used can be determined by pharmacological and pharmacokinetic principles well known in the art.

Methods of antibody (including bispecific, single domain, or heavy chain antibodies) administration 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, by absorption through epithelial or skin mucosa (e.g. oral mucosa, rectal and intestinal mucosa, etc.), and may be co-administered with other bioactive agents. Thus, the pharmaceutical compositions containing the antibodies of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by powders, ointments, drops, or transdermal patches), bucally, or by oral or nasal spray.

The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intranasal, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion. The mode of administration may be systemic or local.

In some embodiments, the compositions of the invention comprise a nucleic acid or polynucleotide encoding a protein, which can be administered in vivo by constructing it as part of a suitable nucleic acid expression vector to facilitate expression of the protein it encodes, and then administering the part of the vector to become an intracellular part by, for example, 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 liposomes or cell surface receptors or transfection reagents, or by ligation with a homeobox-like peptide 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 antibodies of the invention, including bispecific antibodies, single domain antibodies, or heavy chain antibodies, are administered to a patient at a dose of 10mg to 2000mg, or 1mg/kg to 35mg/kg of patient body weight. The dosing frequency may be a single dose. Alternatively, each administration may be at least 1 to 3 days apart; or at least one week. Alternatively, the dosing frequency may be 2 times per week, 1 time per 2 weeks, 1 time per 3 weeks, 1 time per 4 weeks, 1 time per 5 weeks, 1 time per 6 weeks, 1 time per 7 weeks, 1 time per 8 weeks, or 1 time per 12 weeks. The dosage and frequency of administration of the antibodies of the invention may be reduced by modification, e.g., lipidation, to enhance the uptake and tissue penetration capabilities of the antibodies (e.g., into the brain).

Various known delivery systems may be used to administer the bispecific, single domain or heavy chain antibodies 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. 262:4429-4432), construction of nucleic acids as part of retroviruses or other vectors, and the like.

Combination therapy

In some embodiments, the antibodies of the invention, including bispecific antibodies, single domain antibodies, or heavy chain antibodies, can be combined with other therapeutic or prophylactic regimens, including administration of one or more antibodies of the invention, together with one or more other therapeutic agents or methods, or in combination. For combination therapy, the antibodies of the invention may be administered simultaneously or separately with other therapeutic agents. When administered separately, the antibodies of the invention may be administered before or after administration of another other therapeutic agent.

Some patients with severe or critical coronavirus pneumonia have cytokine storm phenomenon, and the antibody of the invention can be combined with adalimumab (adalimumab, for exampleAnd biological analogues thereof, e.g. Abrilada ^TM (adalimumab-afzb)，Amjevita(adalimumab-att)，Cyltezo ^TM (adalimumab-adbm)，Hyrimoz ^TM (adalimumab-adaz)，Hulio ^TM ，/>(/>BAT 1406)) or tobalizumab (tobalizumab, e.g. +. >And biological analogs thereof, such as BAT 1806) are useful in combination therapy, which may slow down inflammatory responses resulting from upregulation of TNF- α expression. In some embodiments, the patient treated by the present methods is diagnosed with a novel coronavirus infection and has an increase in one or more cytokines, including tumor necrosis factor alpha (TNF-alpha), IFN-gamma, IL-1β, 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 (MCP 1), macrophage inflammatory protein 1α (MIP 1A). In some embodiments, the patient treated by the present methods has an increase in TNF- α. In some embodiments, the one or more cytokines are at least 50% above normal. In some embodiments, the one or more cytokines are at least 2-fold, 3-fold, or 4-fold higher than normal levels. In some embodiments, the subject has fever, hypotension, hypoxia, and/or Acute Respiratory Distress Syndrome (ARDS) prior to treatment by the present method. In some embodiments, the patient has his/her lungs filled with an inflammatory fluid (i.e., the so-called "white lung") prior to treatment by the present method.In some embodiments, the subject has cytokine storm-induced cytokine release syndrome (Cytokine Release Syndrome, CRS) prior to treatment by the present method.

In some embodiments, the antibodies of the invention are used in combination with ICU therapy. In some embodiments, the antibodies of the invention bind to in vitro ECMO and/or IMV treatment. In some embodiments, the antibodies of the invention bind to oxygen therapy. In some embodiments, the antibodies of the invention bind NIV/HFNC therapy. In some embodiments, the patient's one or more cytokines is reduced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% after treatment compared to before treatment. In some embodiments, the present methods result in recovery of the patient.

Diagnostic method

In some samples, spike protein positivity was observed, or patients infected with SARS-CoV-2 virus may respond to treatment with the antibodies of the invention. Thus, the antibodies of the invention may also be used for detection and diagnosis.

The sample may be obtained from a patient. After the sample is optionally pre-treated, the sample may be incubated with the antibodies of the invention under conditions that allow the antibodies to interact with spike proteins that may be present in the sample. Antibodies can be used to detect the presence of spike protein in a sample using methods such as ELISA.

The presence (e.g., amount or concentration) of spike protein in a sample may be used to diagnose a disease of interest, as an indication that a patient is being treated with an antibody, or as an indication that a patient has (or has not) responded to a treatment for a disorder. For prognostic methods, one, two or more tests can 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 antibody as described herein and pharmaceutically acceptable excipients.

In some embodiments, the term "pharmaceutically acceptable" refers to substances listed in the pharmacopoeia approved by a regulatory agency of the government or otherwise generally recognized for use in animals, and particularly in humans. In addition, "pharmaceutically acceptable excipients" generally refer to any type of nontoxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation aid, or the like.

The term "adjuvant" 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 vegetable origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can 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 agents, emulsifying agents, or pH buffering agents, such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parahydroxybenzoate, antioxidants such as ascorbic acid or sodium bisulphite, chelating agents such as ethylenediamine tetraacetic 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 using 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, 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 a suitable 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 packaged in ampules, disposable syringes or multiple dose vials made of glass or plastic.

In some embodiments, the composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous injection into the human body. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffers. The pharmaceutical composition may also contain a solubilizing agent and a local anesthetic such as lidocaine, thereby alleviating pain at the injection site. In general, the active ingredients are supplied individually or in admixture in unit dosage form, such as in dry lyophilized powder or dry concentrate form, in sealed containers (e.g., ampules, penicillin bottles or sachets) that are indicative of the amount of active agent. In the case of administering the composition by infusion, the composition may be dispensed using an infusion bottle or bag containing sterile pharmaceutical grade water, physiological saline, or dextrose injection. In the case of administering the composition by injection, an ampoule bottle or a penicillin bottle of sterile water for injection or physiological saline or dextrose injection may be used so that the active ingredients may be mixed prior to administration.

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

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.

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

Example 1 preparation of anti-spike protein antibodies

Antibodies (including monospecific antibodies, bispecific antibodies, single domain antibodies, and heavy chain antibodies) can be prepared by the following methods or other known methods: sequence optimization is carried out according to the characteristic of CHO codon preference of the host cell, and DNA sequences are obtained from amino acid sequences. Cloning the optimized and synthesized sequences into vectors respectively, and then extracting a large number of plasmids respectively to construct stable expression cell strains: uniformly mixing the linearized expression vector with CHO cells, and then adding a 0.4cm electric rotating cup for electric rotating; after the electrotransformation is finished, spreading 1200 cells on a 96-well cell culture plate every well, selecting a high-expression-level mother clone to perform cell expansion culture and expression level detection from 96-well to 24-well to 6-well to shake flask after about 2-3 weeks, selecting a high-shake flask-expression-level clone to perform subcloning, performing subcloning expansion culture and expression identification on the same mother clone, selecting a monoclonal stable cell strain according to the expression level and the stability of the cell strain, harvesting a supernatant after about 12 days of suspension culture, performing protein A affinity capture, and performing anion and cation chromatography to obtain the antibody with the purity of more than 95%.

The variable regions of exemplary antibodies 1-24 and 2F8 are shown in Table 1, the framework and CDR regions of the heavy chain variable region and the light chain variable region are shown in Table 2, VH and CH (shown as SEQ ID NO: 61) make up the heavy chain of the antibody, and VL and CL (shown as SEQ ID NO: 62) make up the light chain of the antibody; the sequences of the bispecific antibody, the single domain antibody, the heavy chain antibody and the monospecific antibody are shown in tables 3-8; wherein, the antibody 2F8 (monospecific antibody) contains two heavy chains with the same sequence (shown as SEQ ID NO: 72) and two light chains with the same sequence (shown as SEQ ID NO: 74); the amino acid sequence of the single domain antibody VHH18 is shown as SEQ ID NO. 69; the heavy chain antibody VHH18-Fc contains 2 heavy chains (shown as SEQ ID NO: 79) with the same sequence, and the heavy chains consist of a single domain antibody, a linker and Fc; the bispecific antibody 2F8-VH-VHH18 comprises two first polypeptides (shown as SEQ ID NO: 77) with the same sequence and two second polypeptides (shown as SEQ ID NO: 74) with the same sequence, wherein the first polypeptides consist of a heavy chain, a linker and a single domain antibody, and the second polypeptides consist of a light chain; bispecific antibody 2F8-VL-VHH18 comprises 2 first polypeptides of identical sequence (as shown in SEQ ID NO: 72) consisting of a heavy chain and 2 second polypeptides of identical sequence (as shown in SEQ ID NO: 78) consisting of a light chain, a linker and a single domain antibody. The Fc regions of the heavy chains in tables 3 and 5 are underlined.

The sequence of the purified antibody was confirmed by sequencing as described above.

TABLE 1 variable and constant regions of anti-spike protein antibodies (monospecific antibodies)

Antibody numbering	VH SEQ ID NO	VL SEQ ID NO
			1	83	58
2	84	58
			3	85	58
4	86	58
			5	87	58
6	88	58
			7	89	58
8	90	58
			9	91	58
10	92	58
			11	93	58
12	94	58
			13	95	58
14	96	58
			15	97	58
16	98	58
			17	99	58
18	100	58
			19	101	58
20	102	58
			21	103	58
22	104	58
			23	105	58
24	56	58
			2F8	57	59

TABLE 2 heavy chain variable region and light chain variable region of anti-spike protein antibodies (monospecific antibodies)

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TABLE 3 amino acid sequences

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TABLE 4 amino acid sequences of Single-Domain antibodies and heavy chain antibodies

TABLE 5 amino acid sequences of monospecific antibodies

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TABLE 6 nucleic acid sequence of antibody 2F8

TABLE 7 amino acid sequences of bispecific antibodies and heavy chain antibodies

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TABLE 8 nucleic acid sequences of bispecific antibodies and heavy chain antibodies

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EXAMPLE 2 detection of antibody binding Activity to SARS-CoV-2spike protein

The Elisa detection is carried out on the antibody, and the detection method comprises the following steps: 96-well plates (Corning, 9018) were coated with spike-RBD-mFC (sinobiologicals), sealed with tape and stored; plates were washed 3 times in wash buffer PBST (PBS containing 0.05% tween 20), followed by addition of blocking solution (200 μl per well of 10mg/ml BSA, solvent wash buffer); after incubation (1 hour (h), 37 ℃) the plates were washed 3 times with wash buffer, and then 100 μl of the gradient diluted sample was added per well; after incubation (1.5 h,37 ℃), the plates were washed with wash buffer, then anti-human kappa light chain antibody-peroxidase conjugate (diluted to 1:2000 in blocking solution, 100 μl/well) was added; the plates were washed with wash buffer and the test samples were incubated (1 h,37 ℃) before 100. Mu.L TMB (tetramethylzidine, biopanda TMB-S-001) substrate/well was added; after the color development was carried out for 10 minutes, 100. Mu.L/well of 0.1MH was added ₂ SO ₄ The reaction was terminated and then 96-well plates were measured at absorbance of 450 nm.

Calculation of EC by absorbance ₅₀ EC of various monospecific antibodies binding to SARS-CoV-2spike protein ₅₀ The values are shown in Table 9.

TABLE 9 EC of binding of monospecific antibodies to SARS-CoV-2spike protein ₅₀ Value of

EXAMPLE 3 antibody blocking the binding of SARS-CoV-2spike protein to angiotensin converting enzyme 2

1) The ability of certain antibodies described above to block binding of spike protein to angiotensin converting enzyme 2 (ACE 2) was tested against the competition Elisa. The detection method comprises the following steps: 96-well plates (Corning, 9018) were coated with spike-RBD-mFC (sino biologicals), sealed with tape and stored overnight at 4 ℃; plates were washed 3 times in wash buffer (PBS containing 0.05% Tween 20), followed by addition of blocking solution (200. Mu.L per well of 10mg/ml BSA, solvent wash buffer); after incubation (2 h,37 ℃), the plates were washed 3 times in wash buffer, antibody samples of different concentrations were added, followed by biotinylated angiotensin converting enzyme 2 (50 ng/ml); after incubation (1 h,37 ℃), the plates were washed 3 times with wash buffer, then 100 μl of streptavidin peroxidase conjugate (diluted 1:10,000 in blocking solution) was added per well, after incubation (1 h,37 ℃), the plates were washed with wash buffer, and 100 μl/well of TMB substrate was added; after 10 minutes of development, 50. Mu.L/well 0.1. 0.1M H was added ₂ SO ₄ The reaction was terminated and absorbance was measured at an absorbance of 450 nm.

The graph of the various antibodies blocking binding of the covd-19 spike protein to ACE2 is shown in figure 1.

2) Detection was performed using SPR (surface plasmon resonance) technique: the antibody-captured probe was incubated with 100 nMAE 2 (paraxial organisms, C419) protein by first binding 100nM biotinylated spike RBD (Acrobiosystems, SPD-C82E 9) protein to the streptavidine probe, incubating the biotinylated spike RBD-bound probe with 100nM antibody solution, and detecting whether the antibody-bound spike RBD protein can also bind to ACE2 in solution.

As shown in fig. 2, antibodies 2F8 and 2F8-VH-VHH18 may block ACE2 binding to the spike RBD, and antibody VHH18-Fc may partially block ACE2 binding to the RBD.

Example 4 determination of affinity of antibodies to spike protein

BiaCore T200 (GE Healthcare) (biomolecular interaction analysis) was tested at 25 ℃): detection was performed using a ProteinA chip, and the antibody was diluted with 1 XHBSEP+ (0.1M HEPES,1.5MNaCl,0.03M EDTA, supplemented with 0.005% surfactant P20) and captured through the experimental flow paths (Fc 2, fc 4) at a flow rate of 10. Mu.l/min; then, the flow rate was adjusted to 30. Mu.l/min, dilutions (0 nM, 3.125nM, 6.25nM, 12.5nM, 25nM and 50nM, diluted with 1 XHBSEP+) of Spike S1 RBD and mutants were analyzed sequentially, and simultaneously, binding and dissociation were performed by flowing over the surfaces of the experimental (Fc 2, fc 4) and reference (Fc 1, fc 3) channels, and finally, the chips were regenerated with a pH1.5glycine buffer and then put into the next cycle. The kinetic constant spike primer (Acrobiosystems, SPN-C52H 8) (ka is the binding rate, kD is the dissociation rate, and kD is the binding dissociation equilibrium constant) was calculated on BiaCore DataAnalysis software using a 1:1Langmuir binding model.

As shown in tables 10 to 12, antibodies 2F8, 2F8-VL-VHH18, 2F8-VH-VHH18 and VHH18-Fc bind well to Spike S1 RBD and mutants thereof.

TABLE 10 affinity constants of antibodies with Spike S1 RBD (Acrobiosystems, SPD-C52H 3)

Antibodies to	K _a (1/Ms)	K _d (1/s)	K _D (M)
				2F8	4.7E+07	1.23E-03	2.61E-11
2F8-VL-VHH18	2.37e+07	5.39e-04	2.27e-11
				VHH18-Fc	8.17e+04	4.14e-04	5.07e-11

TABLE 11 affinity constants of antibodies to trimeric spike trimers (Acrobiosystems, SPN-C52H 8)

Antibodies to	K _a (1/Ms)	K _d (1/s)	K _D (M)
				2F8	4.44E+07	2.45E-06	5.51E-14
2F8-VL-VHH18	1.36e+06	6.41e-07	4.78e-13
				VHH18-Fc	4.78e+05	3.75e-04	7.85e-10

TABLE 12 affinity constant for binding of antibody 2F8-VH-VHH18 to antigen (NA represents non-mutant, WT RBD monomer, spike S1 RBD)

Example 5 detection of binding Activity of antibodies

1) The spike RBD protein (Acrobiosytems, SPD-C52H 3), the P.1. Mutant S1 protein (Yiqiaoshen, 40591-V08H 14), the B.1.351 mutant S1 protein (Yiqiaoshen, 40591-V08H 15), the B.1.617 mutant (Yiqiaoshen, 40592-V08H 88), the B.1.1.7 mutant (Yiqiaoshen, 40591-V08H 8), the D614G mutant (offshore organism, DRA 57), omicron BA.1RBD, omicron BA.2RBD were diluted to 2 μg/ml and 100 μl per well was placed in 96 well plates (Corning, 9018) and coated overnight at 4deg.C; the 96-well plate was washed 3 times in wash buffer PBST (PBS buffer containing 0.05% Tween-20), followed by addition of blocking solution (200. Mu.L 3mg/ml BSA per well, solvent wash buffer), and incubation at 37℃for 2h; then, the 96-well plate was washed 3 times with a washing buffer, 100. Mu.L of a gradient diluted antibody solution was added to each well, and incubated at 37℃for 1.5 hours; the 96-well plate was washed 5 times with wash buffer and 100. Mu.L of anti-human kappa light chain antibody-peroxidation was added The physical enzyme conjugate (diluted to 1:2000 in blocking solution) was incubated at 37℃for 1h; the 96-well plate was washed 8 times with a washing buffer, and 100. Mu.L of TMB (tetramethylene, biopanda TMB-S-001) substrate was added thereto for color development; after color development for 10-15min, 50. Mu.L of 0.1. 0.1M H is added ₂ SO ₄ The reaction was terminated, and the absorbance of the 96-well plate was measured at an absorbance of 450 nm.

As shown in Table 13, antibodies VHH18-Fc and 2F8-VH-VHH18 were able to bind the spike RBD protein and the mutant protein; the results show that antibody 2F8-VH-VHH18 binds to the EC of the spike RBD ₅₀ The value was about 10ng/ml.

TABLE 13 binding Activity of antibodies EC ₅₀ (ng/ml)

"-" indicates no detection.

2) The binding activity of antibody 2F8-VH-VHH18 to the spike RBD or variants thereof was tested using the method described above and the results are shown in Table 14: the antibody 2F8-VH-VHH18 can bind to the spike RBD protein and various mutant proteins.

TABLE 14 binding Activity of antibodies EC ₅₀ (pM)

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⁺ EXAMPLE 6 bispecific antibody and heavy chain antibody inhibit pseudovirus infection of ACE2293 cells

1) The experiment is to evaluate from vitro that antibodies inhibit the infection of ACE 2-expressing cells (namely ACE 2) by spike-pseudotyping pseudovirus (gemini organisms) ⁺ 293 cells). Using ACE2 ⁺ 293 cells detect the ability of the antibody to inhibit infection of cells by SARS-CoV-2 pseudovirus with luciferase gene. The main principle is as follows: using ACE2 ⁺ 293F cells as easily infected cells, different concentrationsIncubation of the antibody with SARS-CoV-2-Fluc pseudovirus system; when the antibody is combined with pseudovirus by incubation, the virus infection is blocked from entering ACE2 ⁺ 293 cells; pseudoviruses cannot effectively infect cells, and the luciferase reporter gene on the genome cannot be expressed in the cells and generates a fluorescent signal; since the signal value of the fluorescent signal is inversely related to the concentration of the added antibody, the ability of the antibody to inhibit viral infection in vitro can be detected. Wherein wild-type pseudovirus strain WT has a accession number of GM-0220PV07 (Ji-quan), mutant 1 (E484K) has a accession number of GM-0220PV35 (Ji-quan), mutant 2 (W436R) has a accession number of GM-0220PV26 (Ji-quan), mutant 3 (B.1.1.7/VUI-202012/01del 145Y) has a accession number of GM-0220PV33 (Ji-quan), mutant 4 (B.1.1.7/VUI-202012/01 del 144Y/145Y) has a accession number of GM-0220PV34 (Ji-quan), mutant 5 (B.1.351/501Y.V2, beta) has a accession number of GM-0220PV32-96T (Ji-quan), mutant 6 (D614G) has a accession number of GM-0220PV14 (Ji-quan), mutant 7 (D614G, D936Y) (GM-0220 PV19, gibby organism), mutant 8 (D839Y) (GM-0220 PV6, gibby organism), mutant 9 (V483A) (GM-0220 PV17, gibby organism), mutant 10 (D614G, A831V) (GM-0220 PV24, gibby organism), mutant 11 (W436R) (GM-0220 PV26, gibby organism), mutant 12 (E484 K+K417 N+N501Y) (GM-0220 PV31, gibby organism), mutant 13SARS-COV-2Spike (B.1.1.529, gibby organism) (GM-0220 PV84, gibby organism), mutant 14 (K417N) (GM-0220 PV30, gibby organism), mutant 15 (N501Y, D614G) (GM-0220 PV29, gibby organism), mutant 16 (N354D, D364Y) (GM-0220 PV13, gibby organism).

The method for detecting the pseudovirus inhibition capacity comprises the following steps: diluting the antibody to 4 mug/ml, performing gradient dilution by 4 times, and transferring the antibody to a 96-well detection plate according to the volume of 50 mu l of each well for later use; respectively diluting the pseudovirus stock solutions of different mutant strains with DMEM culture medium containing 10% FBS, transferring the diluted pseudovirus solution into the 96-well plate containing the antibody according to 25 mu l of each well, uniformly mixing, and standing at room temperature for 1h; ACE2 ⁺ 293 cells were digested with 0.25% Trypsin-EDTA (Gibco, 25200-072) and counted to adjust the cell density to 4X 10 ⁵ cells/ml, 50. Mu.l per well, were added to the 96-well assay plate and incubated in an incubator at 37℃for 48 hours; 50 μl Bi was added per wello-Lite luciferase assay system (Norvezan, DD 1201-03) detection reagent, was left to stand for 3 minutes and read, and the inhibition was calculated from the reading: inhibition ratio = [1- (sample group-blank control group)/(negative control group-blank control group)]X 100%; wherein, the negative control group is added with the pseudovirus solution and no antibody is added, and the blank control group is not added with the pseudovirus solution.

ACE2 ⁺ The construction method of 293 cells comprises the following steps: HEK293 cells (ACS-4500 TM, ATCC) were cultured in DMEM complete medium containing 10% FBS, transfected with ACE2 expression plasmid (HG 10108-M, yiqiao Shenzhou) using lipofectamine 2000transfection reagent (Thermo Fisher, 11668019), followed by pressurized screening with hygromycin (200. Mu.g/ml) and flow sorting (Anti-Human IgG-Fc coupled with 10. Mu.g/ml Anti-ACE2 and PE) and cells were further expanded to select PE positive rate >90% of the monoclonal cells were amplified in the next step and HEK293 cells expressing ACE2, i.e.ACE2, were selected ⁺ 293 cells.

The results show that antibody VHH18-Fc was IC for mutant 2 ₅₀ 327.4ng/ml; as shown in Table 15, 2F8-VH-VHH18 and 2F8-VL-VHH18 were effective in inhibiting pseudovirus-infected cells.

TABLE 15 antibody to pseudovirus IC ₅₀ (ng/ml)

"-" indicates no detection.

2) The inhibition of the infection activity of pseudoviruses by antibody 2F8-VH-VHH18 was detected by the method described above and the results are shown in Table 16: the 2F8-VH-VHH18 can effectively inhibit various pseudovirus infection cells.

TABLE 16 antibody to pseudovirus IC ₅₀ (pM)

3) Inhibition ability of antibody 2F8-VH-VHH18 on pseudovirus-infected cells

The operation steps are as follows: 1) Preparing a dilution medium: 5mL of heat-inactivated FBS is added into 45mL of DMEM Basic (1X) culture medium (Thermo, product number: C11995500 CP) and mixed for later use, and the dosage is adjusted according to the actual needs in proportion. 2) The antibody 2F 8-VHH 18 was diluted with a dilution medium to an antibody concentration of 1. Mu.g/mL, 18. Mu.g/mL, 20. Mu.g/mL, respectively, and 2-fold or 3-fold gradient dilution was performed depending on the pseudovirus species (wherein the initial concentration of the corresponding antibody was 1. Mu.g/mL for wild strain (Gibby organism, GM-0220PV 07), omicron BA.1 (Gibby organism, GM-0220PV 84), and Omicron BA.2.76 (Nannuo vone Biotech Co., ltd., DD 1782-03); the initial concentration of the antibodies corresponding to Omicron BF.7 (Nanjinopran Biotechnology Co., ltd., DD 1789-03) and Omicron BQ.1.1 (Nanjinopran Biotechnology Co., ltd., DD 1792-03) was 18. Mu.g/mL, and 3-fold gradient dilution was performed; the initial concentration of the corresponding antibody to Omicron BA.4/5 (Gibby organism, GM-0220PV 90) was 20. Mu.g/mL, 2-fold gradient dilution was performed, 10 total dilution gradients, and then added to 96 Kong Baiban (Costar, cat: 3917 In (3) a step of setting a value of a parameter; 3) The pseudovirus is taken out from a refrigerator at-80 ℃ and placed on ice for slow dissolution, and diluted to 4 multiplied by 10 by using a dilution culture medium ⁵ TU/mL, pseudovirus dilutions were added to 96 Kong Baiban, and 25. Mu.L/well of virus dilutions were added to each other except for the CC control group. 96 Kong Baiban was placed in an incubator at 37℃for 1h of incubation. 4) ACE2 ⁺ 293 cells were cultured to a confluency of about 90%, centrifuged to remove supernatant, resuspended, and then counted, and the cells were diluted to 4X 10 with dilution medium ⁵ cells/mL, 96 Kong Baiban after 1h incubation was removed and cell suspension (2X 10) was added at 50. Mu.L/well ⁴ cells/well), the edges of 96 Kong Baiban were gently tapped to disperse the cells evenly, and 96 Kong Baiban was placed in an incubator for culture. Taking out 96 Kong Baiban after 48 hours, adding 50 mu L of Bio-LiteTM Luciferase Assay System solution into each hole after the solution is restored to room temperature, carrying out light-proof reaction at room temperature for 2 minutes, reading a Luminescence signal by using a ELISA reader Luminecence detection module, and calculating the inhibition rate according to the reading: inhibition ratio = [1- (sample group-CC control group)/(VC control group-CC control group)]×100％。

The experimental results of the neutralization virus show (see fig. 3): the antibody 2F8-VH-VHH18 can effectively inhibit wild strains (Wildtype), omicron BA.1 and BA.2.76, and can effectively inhibit currently popular strain pseudoviruses, such as: omicron BA.4/5, BF.7, BQ.1.1.

EXAMPLE 7 antibody inhibition of eukaryotic viruses

Gradient dilution (initial concentration 60nM, 3-fold gradient dilution) of the antibody was performed in BSL-3 laboratory, and the antibody dilution was mixed with a 200PFU SARS-CoV-2 wild-type novel coronavirus particle (strain number: 2019-nCoV/IQTC01/human/2020/Guangzhou, genBank is MT 123290.1) or SARS-CoV-2delta (from Guangdong disease prevention control center) in equal volumes while setting up an antibody-free virus control group and a virus-free cell control group; setting 3 compound holes in each experimental group, and standing at 37 ℃ for 1h; the supernatant of Vero E6 cells (ATCC CRL-1587) of the African green monkey kidney cell line in a 96-well plate is sucked and removed, 50 μl of the incubated virus antibody mixture is transferred to a Vero E6 cell plate, and the plates are placed in a cell incubator at 37 ℃ for incubation for 1 hour; the supernatant of the Vero E6 cell plate was aspirated, 100. Mu.l of a 37℃pre-heated DMEM medium (containing 1.6% CMC (carboxymethylcellulose)) was added, and the mixture was placed in a 37℃cell incubator for cultivation for 24 hours; after 24h, the cell plates were removed, 200 μl of 4% pfa (paraformaldehyde solution) was added, and the immobilized inactivated virus and cells were incubated overnight at 4 ℃; the next day, after supernatant was aspirated, fresh 4% PFA was replaced and 96 well cell plates were brought into BSL-2 laboratory for subsequent experiments; detection was performed using the FRNT50 (plaque/focus reduction neutralization test (P/FRNT)) experiment: after membrane rupture and closure, SARS-CoV-2N rabbit polyclonal antibody (product number 40143-T62, beijing Yiqiao Shenzhou) and HRP coat-anti-rabit IgG (Jackson, product number 111-035-003) are used as primary antibody and secondary antibody respectively, and incubation is carried out sequentially; after washing, developing by using True blue; and (5) taking an on-machine reading result of the immune ELISAPOT, and carrying out data analysis by using Graphpad software.

The results show that antibodies VHH18-Fc, 2F8-VH-VHH18 and 2F8-VL-VHH18 can effectively inhibit SARS-CoV-2 wild-type eukaryotic infection cells: IC corresponding to antibody VHH18-Fc ₅₀ A value of 23.63nIC corresponding to M, antibody 2F8-VH-VHH18 ₅₀ IC corresponding to antibody 2F8-VL-VHH18 with a value of 0.03nM ₅₀ A value of 0.033nM; antibody 2F8-VH-VHH18 can effectively inhibit SARS-CoV-2delta eukaryotic virus infection cells, and the corresponding IC of antibody 2F8-VH-VHH18 ₅₀ The value was 0.09nM.

Example 8 in vivo blocking of real Virus efficacy test in mice

A virus infection test was performed in BSL-3 laboratory using Balb/c female mice (Hunan Stokes laboratory animals Co., ltd.) transfected with hACE2 (Ad 5-hACE 2), and the animals were divided into 5 groups of 12 animals each; group G1: nasal drop infection of mice 10 ⁵ PBS was administered as a control after the administration of the new coronavirus (strain number: 2019-nCoV/IQTC 01/human/2020/Guangzhou, genBank MT 123290.1); group G2: mice were intraperitoneally injected with 1mg of antibody 2F8-VH-VHH18, and nasal drops 10 were made 24 hours (h) ⁵ Individual infection with a new coronavirus; group G3: nasal drop infection of mice 10 ⁵ The new coronavirus was given by intraperitoneal injection of 1mg of antibody 2F8-VH-VHH18 after 18 hours; group G4: nasal drop infection 10 was performed 24 hours after nasal drop administration of 1mg of antibody 2F8-VH-VHH18 to mice ⁵ New coronaviruses. On day 3 after infection with the new coronavirus, lung tissues of 4 mice were homogenized for each group, and the FRNT method was used to detect the viable lung virus titer of mice. Each group of mice was subjected to weight measurement daily for 14 days after infection with virus and injection of antibody.

1) As shown in fig. 4, groups G2, G3 and G4 each block infection of lung tissue by new coronaviruses.

2) As shown in fig. 5, the body weight of the G2 group and the G4 group mice did not significantly fluctuate, and the body weight of the G3 group mice significantly decreased in the first 4 days, and then returned to the normal level.

EXAMPLE 9 neutralizing Effect of antibodies on SARS-CoV-2

Neutralization levels of 2F8-VH-VHH18 on SARS-CoV-2 wild-type strain, alpha, beta, gamma, delta and Omicron mutant strain were determined by a microdilution neutralization method.

Experimental results show that 2F8-VH-VHH18 has significant neutralization titers on 6 strains of SARS-CoV-2 wild-type strain, alpha, beta, gamma, delta and Omacron mutant strains detected.

EXAMPLE 10 pharmacokinetic Studies

Since 2F8-VH-VHH18 targets the SARS-CoV-2 novel coronavirus spike protein RBD domain and no related species are present, 2F8-VH-VHH18 project selection performed single-dose pharmacokinetic experiments in SD rats.

After single intravenous infusion of 2F8-VH-VHH18 to SD rats, blood samples were collected, the concentration of 2F8-VH-VHH18 in the blood samples was determined by ELISA, and the main pharmacokinetic parameters were calculated. A total of 30 SD rats (male and female halves) were used in the study, randomized into 3 groups of 5 females and males each. Each group of animals was given 20, 50 and 150mg/kg of 2F8-VH-VHH18, respectively, without a fasted single intravenous bolus. Blood samples were collected to Day 14 after dosing. The results of the study showed that there was no significant sex difference in the systemic exposure of 2F 8-VHH 18 in serum over the dose range of 20-150mg/kg and that the systemic exposure in serum was positively correlated with the dose. No abnormalities were found by clinical detailed observation of all animals throughout the experiment.

Male SD rats were given a single intravenous bolus of 20, 50, 150mg/kg of 2F8-VH-VHH18 followed by t of 2F8-VH-VHH18 in serum _1/2 413.08h, 244.19h and 260.91h respectively, peak times Tmax of 2h, 2h and 3h respectively, peak concentrations Cmax of 469754ng/mL, 1306101ng/mL and 3600117ng/mL respectively, and system exposure AUC _(0-t) 64683258h ng/mL, 177004803h ng/mL and 495684682 h ng/mL, respectively. After single intravenous bolus administration of 20, 50, 150mg/kg of 2F8-VH-VHH18 to female SD rats, t of 2F8-VH-VHH18 in serum _1/2 258.78h, 277.74h and 240.82h respectively, peak time Tmax of 2h, peak concentration Cmax of 518253ng/mL, 1421953ng/mL and 3794472ng/mL respectively, and system exposure AUC _(0-t) 6876660 h ng/mL, 214527346h ng/mL and 476887380h ng/mL, respectively.

EXAMPLE 11 hemolysis assay and tissue Cross-reaction

Hemolysis was performed using New Zealand rabbit red blood cells. When 2F8-VH-VHH18 (50 mg/mL) was diluted more than 10-fold for injection, 2F8-VH-VHH18 was found not to cause hemolysis or agglutination of rabbit erythrocytes in vitro.

Cross-reaction tests of 2F8-VH-VHH18 and normal human and SD rat tissues from 3 different individuals were performed, and the results were analyzed and compared, and after 2F8-VH-VHH18 was added dropwise at a concentration of 2 and 10 μg/mL, no positive staining was seen in all 35 human tissues and 36 SD rat tissues tested.

Example 12 clinical trial

The study is a single-center, open, dose escalation assay for the evaluation of pharmacokinetics, safety, tolerability and pharmacodynamics of 2F8-VH-VHH18 in healthy humans.

Each group was enrolled in 8 healthy subjects, 2 of which were treated with placebo, at 3:1 proportion random distribution received single intravenous 2F8-VH-VHH18 injection or 5% glucose injection. Each group will be administered in the manner of a sentinel administration.

Wherein the first administration of each group was performed simultaneously with 2 subjects observing a full 24 hours, and required to include 1 placebo subject. After the first 2 subjects in the group can be dosed with the remaining 6 subjects.

Classification of safety events in this trial for healthy subjects will be published using CDE 2019: guidelines for adverse event grading criteria for clinical trials of prophylactic vaccines. Where the standard > = grade 3 event is defined as Dose Limiting Toxicity (DLT).

After all subjects in a single dose group have completed dosing observation for 7 days, it is determined that, if Dose Limiting Toxicity (DLT) as defined by the present protocol of > or=50% does not occur, dosing treatment for the next dose group is entered until either MTD (maximum tolerated dose) is explored or termination study criteria are met. Based on the data collected by the test, the pharmacokinetic profile, safety, tolerability and pharmacodynamic activity of the drug were analyzed and studied. Wherein the pharmacodynamic activity assay will utilize SARS-CoV-2 pseudovirus neutralization assay to determine the neutralization capacity of a subject's blood to a novel coronavirus (key mutant pseudovirus). HEK293F cells over-expressing ACE2 are infected with a novel coronavirus carrying a luciferase reporter system, and the blocking capacity of the novel coronavirus on the virus is measured by detecting the signal intensity of luciferase.

Dose group climbing is performed by four groups of dose increment of 100mg,300mg,1000mg and 1500mg, and safety discussion of medicines is performed after the 1500mg group to determine whether to continue climbing.

Subjects were required to study pharmacokinetics by leaving a PK blood sample at fixed time points to detect the concentration of drug in serum: study drug pre-administration (predose); at the end of drug administration, the study was completed at 0.5h, 2h, 8h, 12h, 24h, 36h, 48h, 72h, 96h, day 7, 9, 12, 15, 18, 22, 28, 42, 56 days. Meanwhile, ADA samples are collected on 7 days, 15 days, 28 days, 42 days and 56 days before study drug administration (predose); PD samples were collected 12h, 24h, 48h, 72h, 96h, 7 th day, 15 days, 28 days, 56 days after the end of study drug administration (predose).

During the entire study, researchers will perform safety assessments of vital signs, physical examinations, injection site reactions, electrocardiography, clinical laboratory examinations, and adverse events. The immunogenicity (ADA, ADA titer and nAb) was evaluated simultaneously.

Frequency of administration and route of administration: single administration, for 60 minutes intravenous infusion mode. Safety data for infusion reactions can be reviewed to determine whether infusion time can be adjusted to 30 minutes.

Pharmacokinetic evaluation index: the study will collect a blood sample from a subject to detect the concentration of 2F8-VH-VHH18 in its serum for calculation of pharmacokinetic parameters.

Pharmacodynamic index: the study will collect a blood sample from a subject to test its serum for its neutralizing capacity against SARS-CoV-2 pseudovirus (key mutant virus).

Safety evaluation index: vital signs, physical examination, electrocardiographic parameters, clinical laboratory examination (blood routine, blood biochemistry, urine routine, blood clotting routine), adverse events, and injection site reactions.

Immunogenicity evaluation index: anti-drug antibody (ADA) positive rate, ADA titer and neutralizing activity of ADA.

EXAMPLE 13 results of antibody 2F8-VH-VHH18 clinical phase I trial

The study is a single-center, randomized, double-blind, placebo-controlled, dose-escalated clinical trial evaluating the pharmacokinetics, safety, tolerability, immunogenicity, and pharmacodynamics of the injectable antibody 2F8-VH-VHH18 in healthy subjects. The main objective of this study was to evaluate the safety and tolerability of antibody 2F8-VH-VHH18 in healthy humans.

Each group was planned into 8 healthy subjects, 6 of which used study medication, 2 of which used placebo, test groups, dose escalation ratios, number of persons, etc., as shown in table 17. There are 4 fixed dose groups, namely 100mg, 300mg, 1000mg and 1500mg dose groups. The Dose Limiting Toxicity (DLT) observation period was 7 days post-administration. Each dose group will have a first 2 subjects, with 1 placebo subject. After the first two observations of the dose group are completed, the dosing and observations of the remaining 6 subjects of the subsequent group can be completed.

Table 17 design of antibody 2F8-VH-VHH18 clinical phase I dose escalation protocol

Group of	Dosage (mg)	Increasing ratio of	The number of people
				A1	100	Is not suitable for	6+2
A2	300	300％	6+2
				A3	1000	333％	6+2
A4	1500	150％	6+2

Security results:

the severity of the observed adverse events (Treatment EmergentAdverse Event, TEAE) during the treatment period in this clinical study were both grade 1 and grade 2, no TEAE was occurring above grade 3, no serious adverse events, no TEAE leading to withdrawal or death, and no Dose Limiting Toxicity (DLT) event. From the safety point of view, injection of antibody 2F8-VH-VHH18100 mg, 300mg, 1000mg and 1500mg in healthy subjects was better in safety and tolerability.

Pharmacokinetic results:

the antibody 2F8-VH-VHH18 was absorbed faster in vivo after a single dose of 100-1500 mg in the climbing dose, and the detailed pharmacokinetic results are shown in Table 18.

TABLE 18 pharmacokinetic results

Remarks: except T _max Other PK parameters besides Median (Min, max) are Geoman (% CV)

Pharmacodynamic results:

according to clinical protocols, serum from subjects was obtained at different time points and antibody 2F8-VH-VHH18 pairs were detectedNeutralization ability (IC) of Omicron BA.1 pseudovirus ₅₀ ). The pharmacodynamics results are shown in Table 19, and the IC of antibody 2F8-VH-VHH18 for inhibiting SARS-CoV-2 pseudovirus at climbing dosage of 100-1500 mg ₅₀ The average value of the values is 16.850-20.510 ng/mL, and the result shows that the antibody 2F8-VH-VHH18 can effectively neutralize the key variant strain of SARS-CoV-2 pseudovirus.

TABLE 19 pharmacodynamic results

Remarks: IC (integrated circuit) ₅₀ Values are expressed in Mean (SD).

In conclusion, injection of antibody 2F8-VH-VHH18100 mg, 300mg, 1000mg and 1500mg in healthy subjects was safe and well tolerated, and antibody 2F8-VH-VHH18 was able to effectively neutralize the critical variant strain of SARS-CoV-2 pseudovirus.

Claims

1. A method for preventing, treating or ameliorating covd-19, the method comprising: administering 10mg to 2000mg of the antibody or antigen-binding fragment to a patient in need thereof;

the antibody is a coronavirus-targeting bispecific antibody comprising a first binding moiety that binds a spike protein and a second binding moiety that binds a spike protein, wherein

The first binding moiety comprises one or more of HCDR1 as shown in SEQ ID NO. 1 or 2, HCDR2 as shown in SEQ ID NO. 3 or 4, HCDR3 as shown in any one of SEQ ID NO. 5-42, LCDR1 as shown in SEQ ID NO. 43 or 44, LCDR2 as shown in SEQ ID NO. 45 or 46, and LCDR3 as shown in SEQ ID NO. 47 or 48;

the C-terminal or N-terminal end of the first binding moiety is linked to the second binding moiety via a linker L1.

2. The method of claim 1, wherein the linker L1 is a polypeptide comprising glycine and serine; alternatively, the sequence of the linker L1 is (G _m S) _n Wherein each m is independently 2, 3, 4 or5, n is independently 1, 2, 3, 4 or 5; alternatively, the linker L1 has the sequence (GGGGS) _n And n is independently 1, 2, 3, 4 or 5.

3. The method of claim 1 or 2, wherein the first binding moiety comprises HCDR1 as shown in SEQ ID No. 1 or 2, HCDR2 as shown in SEQ ID No. 3 or 4, HCDR3 as shown in any one of SEQ ID nos. 5-42, LCDR1 as shown in SEQ ID No. 43 or 44, LCDR2 as shown in SEQ ID No. 45 or 46, and LCDR3 as shown in SEQ ID No. 47 or 48; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 6, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 9, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 15, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 16, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 18, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 19, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 20, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 21, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 22, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 23, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 24, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 25, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 28, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 29, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 30, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 32, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 33, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 34, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 35, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 36, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 37, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 39, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 3, HCDR3 as shown in SEQ ID NO. 40, LCDR1 as shown in SEQ ID NO. 43, LCDR2 as shown in SEQ ID NO. 45 and LCDR3 as shown in SEQ ID NO. 47; or (b)

The first binding moiety comprises HCDR1 as shown in SEQ ID NO. 2, HCDR2 as shown in SEQ ID NO. 4, HCDR3 as shown in SEQ ID NO. 42, LCDR1 as shown in SEQ ID NO. 44, LCDR2 as shown in SEQ ID NO. 46 and LCDR3 as shown in SEQ ID NO. 48.

4. A method according to any one of claims 1 to 3, wherein the first binding moiety comprises a heavy chain variable region comprising the sequence set forth in any one of SEQ ID NOs 56, 57, 83 to 105, or a sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NOs 56, 57, 83 to 105, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOs 56, 57, 83 to 105; and/or

The light chain variable region comprises the sequence set forth in SEQ ID NO. 58 or 59, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 58 or 59, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 58 or 59.

5. The method of any one of claims 1-4, wherein the second binding moiety comprises one, two, or three of HCDR1 as shown in SEQ ID No. 66, HCDR2 as shown in SEQ ID No. 67, and HCDR3 as shown in SEQ ID No. 68; alternatively, the second binding moiety comprises HCDR1 as shown in SEQ ID NO. 66, HCDR2 as shown in SEQ ID NO. 67, and HCDR3 as shown in SEQ ID NO. 68; alternatively, the second binding moiety is a single domain antibody or a heavy chain antibody; alternatively, the second binding moiety comprises a sequence as shown in SEQ ID NO. 69 or 79.

6. The method of any one of claims 1-5, wherein the bispecific antibody comprises a first polypeptide and a second polypeptide; wherein the method comprises the steps of

The first polypeptide comprises a sequence as set forth in SEQ ID NO. 72 or 77, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 72 or 77, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 72 or 77; and/or

The second polypeptide comprises a sequence as set forth in SEQ ID NO. 74 or 78, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO. 74 or 78, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO. 74 or 78.

7. A method for preventing, treating or ameliorating covd-19, the method comprising: administering 10mg to 2000mg of the antibody or antigen-binding fragment to a patient in need thereof;

the antibody or antigen binding fragment comprises one, two or three of HCDR1 as shown in SEQ ID NO. 66, HCDR2 as shown in SEQ ID NO. 67 and HCDR3 as shown in SEQ ID NO. 68; alternatively, the antibody or antigen binding fragment comprises HCDR1 as shown in SEQ ID NO. 66, HCDR2 as shown in SEQ ID NO. 67, and HCDR3 as shown in SEQ ID NO. 68; alternatively, the antibody or antigen binding fragment is a single domain antibody or a heavy chain antibody; alternatively, the antibody or antigen binding fragment comprises the sequence shown as SEQ ID NO 69 or 79.

8. The method of any one of claims 1-7, wherein the patient has a disease caused by a novel coronavirus; alternatively, the patient is a mild, moderate, severe or critical infected patient with a novel coronavirus infection.

9. The method of any one of claims 1-8, wherein the antibody or antigen-binding fragment is administered in an amount of 1mg/kg to 35mg/kg.

10. The method of any one of claims 1-9, wherein the dosing frequency of the dosing is a single dosing; or at a frequency of 2 times per week, 1 time per 2 weeks, 1 time per 3 weeks, 1 time per 4 weeks, 1 time per 5 weeks, 1 time per 6 weeks, 1 time per 7 weeks, 1 time per 8 weeks, or 1 time per 12 weeks; alternatively, the administration is injection; alternatively, the mode of administration is intravenous, subcutaneous, or intramuscular; alternatively, the mode of administration is intravenous infusion.