WO2021170113A1 - Method for treating coronavirus by using ace-2-fc fusion protein - Google Patents

Abstract

A method for treating coronavirus by using an ACE-2-Fc fusion protein, relating to the field of virus treatment. A recombinant fusion protein, wherein the protein comprises an angiotensin-converting enzyme 2 (ACE2) region connected to an Fc region of immunoglobulin, and the fusion protein can bind to a coronavirus spike protein, thereby blocking the binding of ACE2 to the coronavirus spike protein. A method for preventing, treating or relieving coronavirus infections. The method comprises administrating a protein comprising an ACE2 extracellular domain to a subject infected or suspected to be infected with coronavirus, the coronavirus being SARS-Cov-2, and the ACE2 extracellular domain comprising an amino acid sequence represented by SEQ ID NO: 1, wherein the protein exists in the form of a dimer.

Description

ACE-2-Fc fusion protein treatment method of coronavirus Technical field

The invention belongs to the field of virus therapy, and relates to a method and a pharmaceutical composition for treating SARS-CoV-2 and related coronaviruses caused by an ACE2-Fc fusion protein.

Background technique

The pneumonia caused by the new coronavirus SARS-CoV-2 (also known as SARS-CoV-2) has infected more than 67,000 people worldwide, and more than 1,000 people have died and have not been effectively controlled. Our understanding of the SARS-CoV-2 virus transmission route and other transmission dynamics, clinical disease manifestations, and pathology (such as the length of the incubation period) is still very limited.

This virus is closely related to the virus that caused severe acute respiratory syndrome (SARS-CoV) in 2003. SARS-CoV infected more than 8,000 people and caused a 10% death rate. SARS-CoV is said to originate from Chinese chrysanthemum bats and is spread to humans through intermediate hosts such as civet cats and raccoons. Comparative genome analysis shows that SARS-CoV-2 and MERS-CoV, SARS-like bat Cov and SARS-CoV belong to the same β-coronavirus branch, but in terms of the whole genome sequence, compared to SARS-CoV, it is similar to SARS-like bats. CoV is closer, and even less related to MERS-CoV. (DOI: https://doi.org/10.1016/j.chom.2020.02.001 ).

SARS-CoV-2 enters host cells through the combination of spike glycoprotein (S) and receptor angiotensin-converting enzyme 2 (ACE2). Studies have shown that ACE2 is a metalloprotease with a total length of 805 amino acids, including an N-terminal signal peptide sequence composed of 17 amino acids and a C-terminal membrane anchoring region. Its role is believed to be involved in the regulation of cardiovascular function and may have a protective effect in acute lung injury. The protein drug can be synthesized through gene recombination technology (US 9,561,263) and has been clinically tested in two indications of type lung injury and pulmonary hypertension, and has entered phase II. As the receptor for SARS-CoV-2 to enter the cell, ACE2 exists as a dimer on the cell membrane and has two conformations: "open" and "closed". The conversion between the two conformations is achieved by the rotation of the protease domain (PD) on ACE2, and PD is the direct binding site of the coronavirus S protein, which is the entrance for the virus to infect the human body. It has been previously reported that ACE2 can form dimers (U.S. 8,586,319). However, the dimer formed by ACE-2 used as a drug to prevent SARS-CoV-2 virus from entering the host cell to prevent virus infection has not been verified and tried.

Summary of the invention

The present invention provides a specific and effective recombinant fusion protein and a method for preventing or treating SARS-CoV-2 infection by using the protein. The method of the present invention is effective against multiple virus strains of SARS-CoV-2 and other close relative strains of the coronavirus. The invention can also be used to detect or diagnose SARS-CoV-2 and the diseases caused by it.

The present invention found that the ACE2 dimer can bind with SARS-CoV-2 S protein with high affinity, and its binding force is higher than that with SARS-CoV-2 S protein. The present invention further discovered that the neutralizing ability of ACE-2 dimer for SARS-CoV-2 is 10 times that of SARS virus. The present invention suggests that ACE-2 dimer is a novel therapeutic agent for SARS-CoV-2.

In one embodiment, ACE2 can form a dimer by adding an Fc segment to the C-terminus, and bind to SARS and SARS-CoV-2 S proteins and have similar binding capabilities. The present invention further found that even though the ACE-2-Fc binding ability is equivalent, the neutralizing ability of ACE-2-Fc to SARS-CoV-2 is 10 times that of SARS virus. The present invention suggests that the dimer formed by ACE-2-Fc is a novel therapeutic agent for SARS-CoV-2.

One aspect of the present invention provides a recombinant fusion protein, including angiotensin converting enzyme 2 (ACE2) region and an immunoglobulin Fc region. In some embodiments, the ACE2 region is connected to the Fc region of the immunoglobulin, and the fusion protein can bind to the spike protein of the coronavirus, thereby blocking the binding of ACE2 to the spike protein of the coronavirus.

In some embodiments, the ACE2 region is the extracellular domain of ACE2. In a preferred embodiment, the ACE2 region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the sequence shown in SEQ ID NO:1. In a specific embodiment, the ACE2 region sequence is shown in SEQ ID NO:1.

In some embodiments, the Fc region of the immunoglobulin comprises the Fc region of IgG1, the Fc region of IgG4, or a mutant of the IgG4 Fc region. In some preferred embodiments, the Fc region of the immunoglobulin comprises the Fc region of human IgG1. In other preferred embodiments, the Fc region of the immunoglobulin comprises the Fc region of human IgG4 or a variant thereof. In other embodiments, the Fc region of the immunoglobulin comprises the Fc region of IgG2. In some preferred embodiments, the Fc region of the immunoglobulin comprises the Fc region of murine IgG2. In a specific embodiment, the immunoglobulin Fc region is the Fc region of IgG1. In another specific embodiment, the Fc region of the immunoglobulin is the Fc region of IgG2. In a specific embodiment, the immunoglobulin Fc region is the Fc region of IgG4.

In some embodiments, the ACE2 region is directly connected to the immunoglobulin Fc region or through an alternative linker. In some preferred embodiments, the ACE2 region and the immunoglobulin Fc region are directly connected by forming an amide bond. In other preferred embodiments, the ACE2 region and the immunoglobulin Fc region are connected by a linker. The linker is a polypeptide linker, such as a GS linker. In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:6. In a specific embodiment, the amino acid sequence of the linker is shown in SEQ ID NO: 6.

In some embodiments, the coronavirus is selected from SARS-CoV, MERS-CoV or SARS-CoV-2, preferably SARS-CoV-2.

In some embodiments, the recombinant fusion protein comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the sequence shown in SEQ ID NO: 2, 3 or 4. Sexual amino acid sequence. In a specific embodiment, the recombinant fusion protein comprises the amino acid sequence shown in SEQ ID NO: 2, 3 or 4. In another specific embodiment, the recombinant fusion protein comprises the amino acid sequence shown in SEQ ID NO: 2 or 3. In a specific embodiment, the amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO: 2, 3 or 4.

Another aspect of the present invention provides a dimer formed by the above-mentioned recombinant fusion protein. Two recombinant fusion proteins forming the dimer are connected by any linker or automatically form a dimer.

Another aspect of the present invention provides a recombinant protein dimer, including any form of dimer formed by the ACE2 region. In some embodiments, two of the ACE2 regions are joined by any linker to form or automatically form a dimer.

In some embodiments, the linker is selected from polypeptides or disulfide bonds. In other embodiments, the linker is a chemical linker. In a specific embodiment, the linker comprises an immunoglobulin Fc region. In a specific embodiment, the ACE2 region is connected to the Fc region of an immunoglobulin to form a dimer through one or more disulfide bonds in the Fc region. In another specific embodiment, the ACE2 region is connected to the immunoglobulin Fc region by comprising the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the Fc region of the immunoglobulin comprises the Fc region of IgG1, the Fc region of IgG2, the Fc region of IgG4, or a variant of the IgG4 Fc region.

In some embodiments, the ACE2 region is the extracellular domain of ACE2. In some preferred embodiments, the ACE2 region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the sequence shown in SEQ ID NO:1. In a specific embodiment, the ACE2 region sequence is shown in SEQ ID NO:1.

The present invention also provides a polynucleotide encoding the above-mentioned recombinant fusion protein or the dimer of the recombinant fusion protein or the dimer of the recombinant protein, further comprising an expression vector of the polynucleotide, and further comprising an expression vector of the expression vector Host cell.

Another aspect of the present invention provides a pharmaceutical composition, which comprises the above-mentioned recombinant fusion protein or the dimer of the above-mentioned recombinant fusion protein or the recombinant protein dimer, and a pharmaceutically acceptable carrier.

The present invention provides a method for preventing, treating or alleviating coronavirus infection, the method comprising administering the above-mentioned recombinant fusion protein, the dimer of the above-mentioned recombinant fusion protein, or the dimerization of the above-mentioned recombinant fusion protein to a subject who is infected or suspected of being infected with the coronavirus Body, or the above-mentioned pharmaceutical composition.

Another aspect of the present invention provides a method for detecting coronavirus in a sample, the method comprising: a. contacting the above-mentioned recombinant fusion protein, the above-mentioned recombinant fusion protein dimer or the recombinant protein dimer with the sample; b. It is determined whether the recombinant fusion protein or the dimer of the recombinant fusion protein or the recombinant protein dimer specifically binds to the molecules in the sample.

The method for preventing, treating or alleviating coronavirus infection or the method for detecting coronavirus in a sample according to the present invention, wherein the coronavirus is selected from SARS-CoV-2.

The sample of the present invention is derived from serum, whole blood, sputum, oral/nasopharyngeal secretions or lotions, urine, feces, pleural effusion, cerebrospinal fluid, tissues that are infected or suspected of being infected with SARS-CoV-2 virus Specimen or non-biological samples such as water, beverages.

The present invention also provides a method for preventing, treating or alleviating coronavirus infection, the method comprising administering a protein containing the extracellular domain of ACE2 to a subject infected or suspected of being infected with a coronavirus, and the coronavirus is SARS-CoV-2 The ACE2 extracellular domain includes the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the amino acid sequence of the extracellular domain of ACE2 is shown in SEQ ID NO:1. In some embodiments, wherein the protein exists as a dimer.

The present invention also provides the application of the protein containing the ACE2 extracellular domain in the preparation of drugs for preventing, treating or alleviating SARS-CoV-2 coronavirus infection, wherein the ACE2 extracellular domain comprises the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the amino acid sequence of the extracellular domain of ACE2 is shown in SEQ ID NO:1. In some embodiments, the protein exists as a dimer.

Detailed description of the invention

Unless otherwise specified, the technical and scientific terms used in the present invention have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

The term "new coronavirus" (SARS-CoV-2), also known as 2019-nCoV, belongs to the β-coronavirus, has an envelope, and the particles are round or elliptical, often pleomorphic, with a diameter of 60-140nm . Its genetic characteristics are obviously different from SARS-CoV and MERS-CoV. Studies have shown that it has more than 85% homology with bat SARS-like coronavirus (bat-SL-CoVZC45). When isolated and cultured in vitro, SARS-CoV-2 can be found in human respiratory epithelial cells in about 96 hours, while it takes about 6 days to isolate and culture in Vero E6 and Huh-7 cell lines.

The term "Fc region of immunoglobulins" refers to the fragment crystallizable (Fc) of immunoglobulins, in which immunoglobulins are generally composed of two identical light chains and two identical heavy chains. Peptide chain structure connected by disulfide bonds. It also refers to antibodies, which can be divided into five categories, namely immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) and immunoglobulin E (IgE) , Is composed of two parts, the antigen-binding fragment Fab and the crystallizable fragment Fc, where Fab can bind to antigen and Fc can bind to Fc receptors. The Fc region of an immunoglobulin may be IgG Fc, such as IgG1 Fc, IgG2 Fc, IgG4 Fc or variants thereof. The Fc of IgG can be of human or murine origin. The immunoglobulin Fc region may be the Fc of human IgG1, the Fc of human IgG4, or the Fc of murine IgG2. In some embodiments, the Fc region of the immunoglobulin may be the Fc of human IgG4 or a variant thereof. In some embodiments, the Fc of human IgG4 comprises the amino acid sequence shown in SEQ ID NO:7. Specifically, the amino acid sequence of the Fc region of human IgG4 is shown in SEQ ID NO:7. A variant of the Fc region of human IgG4 refers to a sequence of the Fc region of human IgG4 that contains one or more amino acid substitutions, deletions or insertions. If the Fc region of human IgG4 contains substitutions at positions 228 and 235, the substitutions can be S228P and/or L235E. In some embodiments, the human IgG4Fc variant comprises the amino acid sequence shown in SEQ ID NO: 8 or 9. Specifically, the amino acid sequence of the human IgG4Fc variant is shown in SEQ ID NO: 8 or 9.

The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersants, coatings, antibacterial and antifungal agents, isotonic and sustained release agents, and the like that are compatible with drug administration. Suitable carriers are described in the standard reference documents in the latest edition of Remington’s Pharmaceutical Sciences, which are incorporated herein by reference in their entirety. Examples of suitable carriers or diluents include, but are not limited to, water, saline solution, ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and hydrophobic-aqueous media such as fixed oils can also be used. The use of media and agents for pharmaceutically active substances is well known in the art. Except for those conventional media or reagents that are incompatible with the active ingredients, its use in the ingredients can achieve the desired effect.

The "percent (%) amino acid sequence identity" of a peptide or polypeptide sequence is defined as comparing the sequences and introducing gaps when necessary to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Candidates The percentage of amino acid residues in the sequence that are identical to the amino acid residues in the specific peptide or polypeptide sequence. Sequence comparisons can be performed in a variety of ways within the skill of the art to determine percent amino acid sequence identity, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required to obtain the maximum comparison over the entire length of the sequence being compared.

When "administering" and "treatment" are used to refer to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, it means to combine exogenous drugs, therapeutic agents, diagnostic agents or compositions with animals, humans, and recipients. Contact with the person being treated, cells, tissues, organs or biological fluids. "Administration" and "treatment" can refer to, for example, treatment methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting the reagent with the cell and contacting the reagent with a fluid, where the fluid is in contact with the cell. "Administration" and "treatment" also mean the treatment of cells in vitro and ex vivo, for example, by reagents, diagnostic agents, binding compositions, or by other cells.

The term "subject" as used herein refers to an animal in need of alleviation, prevention and/or treatment of a disease or condition such as a viral infection, preferably a mammal, more preferably a human. The term includes human subjects who have a coronavirus such as SARS-CoV-2 infection or are at risk of having a coronavirus such as SARS-CoV-2 infection.

"Pharmaceutically acceptable carrier" refers to a carrier for administration, including various excipients, diluents and buffers, etc. These substances are suitable for human and/or animal administration without excessive adverse side effects, and at the same time It is suitable for maintaining the vitality of the drug or active agent located therein.

Unless specifically stated otherwise, the use of the singular includes the plural. Unless specifically stated otherwise, the words "a" or "an" mean "at least one." The use of "or" means "and/or" unless stated otherwise. The meaning of the phrase "at least one" is equivalent to the meaning of the phrase "one or more". In addition, the use of the term "including" and other forms such as "includes" and "included" is not limiting. In addition, unless specifically stated otherwise, terms such as "element" or "component" include elements or components that include one unit as well as elements and components that include more than one unit.

Recombinant Fusion Protein

The recombinant fusion protein of the present invention includes two parts: angiotensin converting enzyme 2 (ACE2) region and immunoglobulin Fc region.

In some embodiments, the recombinant fusion protein includes an angiotensin-converting enzyme 2 (ACE2) region, which is connected to the Fc region of an immunoglobulin, and the fusion protein can bind to the spike protein of the coronavirus, thereby blocking ACE2 Binding with the coronavirus spike protein. In the present invention, the ACE2 region is the extracellular domain of ACE2. In a preferred embodiment, the ACE2 region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the sequence shown in SEQ ID NO:1. In a specific embodiment, the ACE2 region sequence is shown in SEQ ID NO:1.

The present invention can use any Fc fragment of immunoglobulin. Immunoglobulins include IgG, IgA, IgM, IgD and IgA, of which IgG is the most abundant and relatively stable. The Fc fragment of IgG, such as the Fc fragment of IgG1, the Fc fragment of IgG2, the Fc fragment of human IgG1 or the Fc fragment of mouse IgG2, is preferred, because these Fc fragments show the highest binding to staphylococcus protein A (staphylococcus Protein A) It is easy to be purified.

In some embodiments, the Fc region of the immunoglobulin comprises the Fc region of IgG1. In some preferred embodiments, the Fc region of the immunoglobulin comprises the Fc region of human IgG1. In other embodiments, the Fc region of the immunoglobulin comprises the Fc region of IgG2. In some preferred embodiments, the Fc region of the immunoglobulin comprises the Fc region of murine IgG2a.

In some embodiments, the recombinant fusion protein includes at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the sequence shown in SEQ ID NO: 2, 3 or 4. Sexual amino acid sequence. In some embodiments, the recombinant fusion protein includes at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the sequence shown in SEQ ID NO: 2 or 3. Amino acid sequence. In a specific embodiment, the recombinant fusion protein includes the amino acid sequence shown in SEQ ID NO: 2, 3 or 4. In another specific embodiment, the recombinant fusion protein includes the amino acid sequence shown in SEQ ID NO: 2. In a specific embodiment, the recombinant fusion protein includes the amino acid sequence shown in SEQ ID NO: 3. In another specific embodiment, the recombinant fusion protein includes the amino acid sequence shown in SEQ ID NO:4.

Another aspect of the present invention provides a dimer formed by the above-mentioned recombinant fusion protein. Two recombinant fusion proteins forming the dimer are connected by any linker or automatically form a dimer. In some embodiments, a homodimer of a recombinant fusion protein is provided, wherein the homodimer includes two fusion protein molecules connected by one or more disulfide bonds.

The length of the Fc fragment in the present invention can be 232 amino acids, including one cysteine in the hinge region, two cysteines in the CH2 region, and two cysteines in the CH3 region. The cysteine in the hinge region is used to form a disulfide bond between the two monomers, thereby producing a dimer. The cystine in the CH2 region and the CH3 region can form an intra-bond disulfide bond to stabilize the recombinant fusion A homodimer of protein.

Recombinant protein dimer

Another aspect of the present invention provides a recombinant protein dimer, including any form of dimer formed by the ACE2 region. In some embodiments, two of the ACE2 regions are connected by any linker to form or automatically form a dimer. The linker includes any chemical linker that can connect the ACE2 protein. In some embodiments, the linker is selected from polypeptides or disulfide bonds. In a specific embodiment, the linker is an immunoglobulin Fc region.

The present invention can use any Fc fragment of immunoglobulin. Immunoglobulins include IgG, IgA, IgM, IgD and IgA, of which IgG is the most abundant and relatively stable. The Fc fragment of IgG, such as the Fc fragment of IgG1, the Fc fragment of IgG2, the Fc fragment of human IgG1 or the Fc fragment of mouse IgG2, is preferred, because these Fc fragments show the highest level of staphylococcus protein A (staphylococcus Protein A). It is easy to be purified because of its binding properties.

The present invention includes any ACE2 dimer, ACE2-linker-ACE2, or ACE-Fc that can automatically form a dimer after expression.

In some embodiments provided by the present invention, the recombinant protein dimer is that the ACE region is connected to the immunoglobulin Fc region, and the dimer is formed through one or more disulfide bonds in the Fc region. In some embodiments, the Fc region of the immunoglobulin comprises the Fc region of IgG1 or the Fc region of IgG2. In other embodiments, the ACE2 region is the extracellular domain of ACE2. Preferably, the ACE2 region contains at least 80%, at least 85%, at least 90%, at least 95%, and the sequence shown in SEQ ID NO:1. An amino acid sequence with at least 97% or at least 99% identity. In a specific embodiment, the ACE2 region sequence is shown in SEQ ID NO:1.

The method for preparing the recombinant protein of the present invention includes: (1) providing a polynucleotide molecule for encoding; (2) constructing an expression vector containing the polynucleotide molecule described in (1); (3) ) Transfecting or transforming a suitable host cell with the expression vector described in (2), and culturing in the host cell to express the protein; and (4) Purifying the protein. The preparation can be carried out by techniques known to those skilled in the art. The dimer of the recombinant fusion protein in the present invention is spontaneously connected by one or more disulfide bonds in the Fc region through the expressed recombinant fusion protein to form a dimer of two recombinant fusion proteins.

At the same time, the present invention provides a polynucleotide molecule encoding a recombinant fusion protein or a recombinant protein dimer, and an expression vector for expressing the recombinant fusion protein. Examples of the vectors include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), transformable artificial chromosomes (TAC), mammalian artificial chromosomes (MAC) and artificial additional chromosomes (HAEC) .

The present invention provides a host cell including the above-mentioned expression vector. Host cells can be transformed or transfected with expression vectors. Suitable host cells include E. coli, yeast and other eukaryotes. Preferably, use of E. coli, yeast or mammalian cell lines (such as COS or CHO). In some embodiments, the host cell is a CHO cell.

Pharmaceutical composition

Another aspect of the present invention provides a pharmaceutical composition, which comprises the above-mentioned recombinant fusion protein, the above-mentioned recombinant fusion protein dimer or the above-mentioned recombinant protein dimer, and a pharmaceutically acceptable carrier.

In the pharmaceutical composition of the present invention, the recombinant fusion protein includes an angiotensin-converting enzyme 2 (ACE2) region, which is connected to the Fc region of immunoglobulin, and the fusion protein can bind to the spike protein of the coronavirus, thereby Blocks the binding of ACE2 to the coronavirus spike protein. In the pharmaceutical composition of the present invention, the dimer of the recombinant fusion protein includes two of the fusion protein molecules connected by one or more disulfide bonds. In the pharmaceutical composition of the present invention, the recombinant protein dimer includes any form of dimer formed by the ACE2 region.

The pharmaceutical composition may be added with a pharmaceutically acceptable carrier as required, and the carrier includes diluents, excipients, swelling agents, binding agents, wetting agents, disintegrants, absorption enhancers, Surfactants, adsorption carriers, lubricants, etc.

In some embodiments, the pharmaceutical composition is administered subcutaneously, intravenously, intracutaneously, intraperitoneally, orally, intramuscularly, or intracranially. Wherein the administration generally refers to injection, and these injectable preparations can be prepared by publicly known methods. For example, injectable preparations can be prepared by dissolving, suspending or emulsifying the above-mentioned antibody or salt thereof in a sterile aqueous medium or oily medium conventionally used for injection. As an aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be combined with appropriate solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol) , Non-ionic surfactants [for example, polysorbate 80, hydrogenated castor oil HCO-50 (polyoxyethylene (50 mol) adduct)], etc. are used in combination. As the oily medium, there are used, for example, sesame oil, soybean oil, etc., which can be used in combination with a solubilizer such as benzyl benzoate, benzyl alcohol, and the like. Therefore, the prepared injection is preferably filled in an appropriate ampoule. The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes.

In some embodiments, the pharmaceutical composition is administered to a subject who is infected with a coronavirus or is at risk of infection, and the coronavirus is selected from SARS-CoV, MERS-CoV or SARS-CoV-2, preferably SARS-CoV -2.

In another aspect of the present invention, the pharmaceutical composition further includes a second therapeutic agent. In one embodiment of the present invention, the second therapeutic agent is selected from the group consisting of anti-inflammatory drugs, antiviral drugs, other antibodies against coronavirus spike protein, vaccines against coronavirus or antibiotics. In some embodiments, the antiviral drug is selected from the group consisting of lopinavir, ribavirin, ritonavir, and remdesivir.

The recombinant fusion protein or recombinant protein dimer of the present invention can also be combined with non-polypeptide molecules to obtain desired properties, such as reducing degradation and/or increasing half-life, reducing toxicity, reducing immunogenicity and/or improving Biological activity. Examples of such molecules include, but are not limited to: polymers, such as polyethylene glycol (PEG), polylysine, dextran; blood lipids; cholesterol groups (such as hormones); carbohydrates, or oligosaccharide molecules.

Prevention, treatment or mitigation methods

In yet another aspect of the present invention, there is provided a method for preventing, treating or alleviating coronavirus infection, the method comprising administering the above-mentioned recombinant fusion protein, the dimer of the above-mentioned recombinant fusion protein, or Recombinant protein dimer, or the above-mentioned pharmaceutical composition. In some embodiments, the coronavirus to be treated is selected from SARS-CoV, MERS-CoV or SARS-CoV-2, preferably SARS-CoV-2.

The present invention also provides a method for preventing, treating or alleviating coronavirus infection, the method comprising administering a protein containing the extracellular domain of ACE2 to a subject infected or suspected of being infected with a coronavirus, and the coronavirus is SARS-CoV-2 The amino acid sequence of the extracellular domain of ACE2 is shown in SEQ ID NO:1. In some embodiments, wherein the protein exists as a homodimer.

The present invention also provides the application of the protein containing the extracellular domain of ACE2 in the preparation of drugs for preventing, treating or alleviating SARS-CoV-2 coronavirus infection, wherein the amino acid sequence of the extracellular domain of ACE2 is shown in SEQ ID NO:1. In some embodiments, the protein exists as a homodimer.

In some embodiments, the present invention provides a method for preventing, treating or alleviating at least one symptom of SARS-CoV-2 infection, the method comprising adding a therapeutically effective amount of the aforementioned recombinant fusion protein, the same type of the aforementioned recombinant fusion protein The dimer or pharmaceutical composition is used for subjects in need thereof. In some embodiments, the present invention provides that by administering the recombinant fusion protein or the homodimer of the recombinant fusion protein of the present invention, the severity of at least one symptom or indication of SARS-CoV-2 infection in a subject can be alleviated or reduced. Sexual method, wherein the at least one symptom or indication is selected from the group consisting of lung inflammation, alveolar injury, multiple ground-glass shadows or infiltration shadows in the lungs, small patches and interstitial changes outside the lungs, fever, Cough, shortness of breath, diarrhea, organ failure, septic shock, and death. In some embodiments, the present invention provides a method for reducing viral load in a subject, which comprises administering to the subject an effective amount of the aforementioned recombinant fusion protein, homodimer or pharmaceutical composition of the aforementioned recombinant fusion protein, said The recombinant fusion protein or the homodimer of the recombinant fusion protein can bind to the spike protein of SARS-CoV-2 and block the binding of SARS-CoV-2 to host cell receptors.

In some embodiments, wherein the pharmaceutical composition is administered prophylactically to a subject selected from the group consisting of immunocompromised individuals, elderly people (greater than 65 years old), medical staff, people with a history of medical problems, and People who have come into contact with people with confirmed or suspected coronavirus infections. The subject of the preventive administration is also a subject at risk of infection.

Diagnosis and detection methods

Another aspect of the present invention provides a method for detecting coronavirus in a sample, the method comprising: a. contacting the above-mentioned recombinant fusion protein or the homodimer of the above-mentioned recombinant fusion protein with the sample; b. determining the recombinant fusion Whether the homodimer of the protein or recombinant fusion protein specifically binds to the molecules in the sample.

The method for preventing, treating or alleviating coronavirus infection or the method for detecting coronavirus in a sample according to the present invention, wherein the coronavirus is selected from SARS-CoV-2.

The sample of the present invention is derived from serum, whole blood, sputum, oral/nasopharyngeal secretions or lotions, urine, feces, pleural effusion, cerebrospinal fluid, tissues that are infected or suspected of being infected with SARS-CoV-2 virus Specimen or non-biological samples such as water, beverages. The subject of the (potential) infection can be a human, but animals suspected of carrying a coronavirus such as SARS-CoV-2 can also use the composition or pharmaceutical composition to test the presence of the coronavirus. The sample can be processed first to make it more suitable for the detection method. Processing refers to the processing of samples suspected of containing and/or containing coronaviruses, whereby the coronaviruses are broken down into antigenic components such as proteins, (poly) peptides or other antigenic fragments. Preferably, the composition or the pharmaceutical composition and the sample are in such a way that the binding molecules in the composition or the pharmaceutical composition and the coronavirus or its antigenic components that may be present in the sample form a binding complex. Contact under conditions. The formation of the binding complex indicates the presence of coronavirus in the sample, which is then detected and determined by appropriate means. These methods include homogeneous or heterogeneous binding immunoassays, such as radioimmunoassay (RIA), ELISA, immunofluorescence, immunohistochemistry, FACS, BIACORE, and Western blot analysis.

Description of the drawings

Figure 1A-1C shows the binding of ACE-2-mFc to SARS S protein or SARS-Cov-2 S protein by ELISA, Figure 1A shows the binding of ACE-2-mFc to the RBD domain of SARS S protein, and Figure 1B shows ACE-2 -mFc binds to the SARS-CoV2 RBD (His tag) domain. Figure 1C shows the binding of ACE-2-mFc to the extracellular domain of SARS-CoV2.

Figure 2A shows the measurement of the binding of ACE-2-mFc to SARS S protein or SARS-Cov-2 S protein by flow cytometry. The left panel of Figure 2A shows the binding of ACE-2-mFc to SARS S protein, and the right panel of Figure 2A shows ACE. -2-mFc binds to SARS-Cov-2 S protein; Figure 2B shows the measurement of ACE-2-hFc binding to SARS S protein or SARS-Cov-2 S protein by flow cytometry. The left picture of Figure 2B shows ACE-2 -hFc binds to SARS S protein. The right picture of Figure 2B shows the binding of ACE-2-hFc to SARS-Cov-2 S protein.

Figure 3 shows the ability of ACE-2-mFc to neutralize SARS pseudovirus and SARS-Cov-2 S pseudovirus. The NCP group is a pseudovirus constructed by SARS-Cov-2 envelope S protein.

Figure 4 is the SEC-HPLC chart of ACE-2-hFc.

Figures 5A-5B show the binding of ACE-2-hFc to SARS-CoV-2 S protein by ELISA. Figure 5A shows the binding of ACE-2-hFc to the RBD domain of SARS-CoV-2 S protein. Figure 5B shows the binding of ACE-2 to SARS-CoV-2 S protein. -mFc binds to the extracellular domain of SARS-CoV2 S protein.

Figure 6 shows the binding of ACE-2-hFc to SARS-CoV-2 S protein measured by Biacore. Figure 6A shows the binding of ACE-2-hFc to SARS-CoV-2 RBD (His tag) domain. Figure 6B shows the binding of ACE-2-hFc to SARS-CoV-2 RBD (His tag) domain. hFc binds to the extracellular domain of SARS-CoV-2 S protein.

Figure 7 shows the ability of ACE2-hFc to neutralize SARS-Cov-2 true virus.

Figure 8 is the experimental diagram of SARS-CoV-2 live virus challenge treatment and protection, Figure 8A is the body weight change curve of SARS-CoV-2 infected mice after injection of BSA and ACE2-hFc within 10 days, and Figure 8B is the SARS-CoV infection Fig. 8C shows the HE staining picture of lung slices of SARS-CoV-2 infected mice after injection of BSA and ACE2-hFc;

Figure 9 is the experimental diagram of the prevention and protection of SARS-CoV-2 live virus challenge. Figure 9A is the body weight change curve of mice injected with BSA and ACE2-hFc within 10 days after the SARS-CoV-2 live virus is infected; Figure 9B is the BSA injection Figure 9C shows the lung virus infection titers of mice infected with SARS-CoV-2 live virus in mice infected with SARS-CoV-2 and ACE2-hFc. Figure 9C shows mice infected with SARS-CoV-2 injected with BSA and mice injected with ACE2-hFc infected with SARS-CoV -2 HE stained picture of lung section after live virus

Detailed ways

Example 1 Plasmid construction of coronavirus S protein

Construction method of plasmid p3XFLAG-CMV14-2019nCoV-S expressing NCP coronavirus S protein and plasmid p3XFLAG-CMV14-SARS-S expressing SARS coronavirus S protein

NCP coronavirus S protein ORF (see SEQ ID NO: 5 for the sequence) or SARS virus S protein DNA sequence after gene synthesis is digested with restriction DNA endonucleases HindIII and XbaI, and the same restriction endonucleases are used at the same time Plasmid vector p3XFLAG-CMV14 (Sigma, catalog number E4901), after digestion, the S protein ORF with sticky ends and plasmid vector fragments were ligated with T4 ligase to transform E. coli competent cells to obtain plasmid p3XFLAG-CMV14-2019nCoV- S and p3XFLAG-CMV14-SARS-S (DOI: https://doi.org/10.1371/journal.pone.0076469 ).

Example 2 Production and purification of ACE-2-Fc dimer

2.1 Cell culture and transient transfection

CHO-3E7 cells were grown in serum-free FreeStyleTM CHO expression medium (Life Technologies, Carlsbad, California, USA). The cells were kept in an Erlenmeyer flask (Corning Inc., Acton, MA) on an orbital shaker (VWR Scientific, Chester, PA) at 37°C and 5% CO2. On the day of transfection, the DNA encoding the ACE2-Fc fusion protein of SEQ ID NO: 2 and PEI (Polysciences, Eppelheim, Germany) were mixed at a ratio of 1:2, and then added to the flask together with the cells to be transfected. About 1 ml of supernatant collected on the 5th day was used for expression level detection. The supernatant collected on day 6 was used for further purification.

2.2 Purification and analysis

Centrifuge the cell culture fluid and then filter. The filtered supernatant was loaded onto a 5ml Protein A CIP chromatographic column (GenScript, catalog number L00433) at a rate of 3.0 ml/min. After washing and eluting with an appropriate buffer, the eluted fractions are combined and the buffer is replaced with PBS with pH 7.2. Based on the SEC-HPLC analysis of DEF-33, the second step of gel filtration purification was carried out. The DEF-33 elution fraction after purification in the first step was loaded onto HiLoad 26/60 Superdex 200 pg 320 ml (GE, catalog number 28-9893-36) at a rate of 2.0 ml/min. The purified antibodies were analyzed by SDS-PAGE, Western blotting, endotoxin and SEC-HPLC, using standard procedures for molecular weight, yield and purity measurements. The results are shown in Table 1. As shown in Figure 4, the purity of the dimer of ACE2-hFc is about 95%.

Table 1 Characterization of ACE2-hFc

With reference to the above method, the DNA encoded by SEQ ID NO: 3 and SEQ ID NO: 4 were used to replace the DNA encoded by SEQ ID NO: 2 to prepare another ACE2-hFc fusion protein and ACE2-mFc.

Example 3 Detection of the binding of ACE2-Fc to the coronavirus spike (S) protein on the cell surface

3.1 Transient transfection of HEK293FT cells to express coronavirus spike S protein

Inoculate HEK293FT cells (Thermo Fisher Scientific, Catalog No. R70007) in a 6-well cell culture plate at a density of 7×10 ⁵ per well and 3 ml of DMEM complete medium per well. After 16 hours, 3ml of DMEM complete medium was aspirated, and 2ml of fresh DMEM complete medium was added. After 2 hours, 5μg of the plasmid p3XFLAG-CMV14-2019nCoV-S expressing the NCP coronavirus S protein and the plasmid p3XFLAG-CMV14-SARS-S expressing the SARS coronavirus S protein were added to 100μl OptiMEM serum-free medium, and 10μl PEI was added to 100μl OptiMEM serum-free medium, and left to stand at room temperature for 5 minutes. Mix 100 μl of OptiMEM serum-free medium containing PEI with 100 μl of OptiMEM serum-free medium containing plasmids, and let stand at room temperature for 8 minutes, and add 200 μl of the mixture to one well of a 6-well plate to transfect HEK293FT cells. 16 hours after transfection, the medium containing the transfection mixture was replaced with 2 ml of fresh DMEM complete medium. After 6 hours, the transfected HEK293FT cells were trypsinized and centrifuged, and resuspended in pre-cooled FACS buffer (PBS buffer containing 1% FBS), and the final cell density was 1×10 ⁶ cells/ml.

3.2 Measure the binding of ACE2-Fc to SARS S protein or SARS-CoV-2 S protein by ELISA

Add 100ul of PBS buffer containing 0.5μg SARS S protein (SinoBio, catalog number 40634-V08B1) or SARS-Cov-2 S protein (SinoBio, catalog number 40589-V08B1) into each well of a 96-well ELISA test plate, and incubate at 37°C1 Hour. Discard the PBS buffer containing the protein, add 100 μl of blocking buffer (PBS buffer containing 5% BSA), and block at 37°C for 1 hour. Discard the blocking buffer, add 100 μl of ACE2-Fc protein solution gradiently diluted in PBS buffer to the corresponding wells, incubate for 1 hour at room temperature, discard the solution, wash with 200 μl PBS buffer, and repeat three times. Add 100μl of 0.5ug/ml HRP-labeled anti-mouse IgG (Jackson Lab catalog number 115-035-071) or anti-human IgG secondary antibody (Rockland catalog number 609-103-123) PBS buffer, incubate at room temperature for 30 minutes, discard the antibody Solution, rinse with 200μl PBS buffer, repeat three times. Add 100μl OPD substrate solution, react at room temperature for 15 minutes, add 2M sulfuric acid solution to stop the reaction, and read on the microplate reader.

In order to verify whether ACE2-mFc was successfully produced and bound to SARS S protein, ACE2-mFc was incubated with the RBD domain in SARS S protein by ELISA. As shown in Figure 1A, ACE2-mFc binds to the RBD domain of SARS S protein (EC50=87.5ng/ml), which proves that the candidate drug binds correctly and recognizes the expected binding epitope.

Secondly, in order to verify whether ACE-2-mFc binds to SARS-Cov-2 S protein, ACE-2-mFC and the RBD domain in SARS-Cov-2 S protein (SinoBio, catalog number 40592-V08B) (Figure 1B) and The extracellular domain of S protein (SinoBio, catalog number 40589-V08B1) (Figure 1C) was incubated together, and the results showed that ACE-2-mFc and SARS-Cov-2 S protein RBD domain (EC50 = 108.4ng/ml) and S protein The extracellular region (EC50=135ng/ml) of the combined.

Finally, in order to verify whether ACE-2-hFc binds to SARS-Cov-2 S protein, ACE-2-hFc is combined with the RBD domain of SARS-CoV-2 S protein (Figure 5A) and the extracellular region of S protein (Figure 5A). 5B) Co-incubation, the results show that ACE-2-mFc binds to the RBD domain (EC50=0.06nM) of the SARS-CoV-2 S protein and the extracellular domain (EC50=1.73nM) of the S protein.

3.3 Measure the binding of ACE-2-Fc to SARS S protein or SARS-CoV-2 S protein by flow cytometry (FACS) method

In order to further verify whether ACE-2-Fc binds to the trimeric protein in the natural conformation expressed on the cell membrane surface, the plasmid expressing the SARS S protein and the plasmid expressing the SARS-CoV-2 S protein were transfected into HEK-293T cells. The S protein of the virus is expressed on the cell surface, and ACE-2-Fc is further incubated with the cells expressing the S protein, and flow cytometry is used to detect whether ACE-2-Fc can bind to the S protein on the cell surface.

Add 100μl of cell suspension to a 96-well V-bottomed plate, centrifuge at 300g at 4°C for 5 minutes, and use 100μl of FACS buffer containing 1μg/ml ACE-2-mFc or ACE-2-hFc (containing 1% FBS (Thermo Fisher Scientific, Cat.No: 26140079) PBS buffer) after resuspension, incubate at 4°C for 1 hour, add 100μl FACS buffer to wash the cells, centrifuge at 300g at 4°C for 5 minutes, and wash again, add 1μg/ml Alexa647-labeled Anti-mouse (Jackson Lab, Cat. No. 715-605-151) or human IgG secondary antibody (Jackson Lab, Cat. No. 109-605-044), incubate at 4°C for 40 minutes, and repeat the above cell washing step twice. The cells were resuspended in 200μl FACS buffer, and the data was collected by the BD Canto2 flow cytometer, and the data was analyzed by FlowJo software.

As shown in the left panel of Figure 2A and the left panel of Figure 2B, either ACE-2-mFc or ACE-2-hFc can bind to the SARS S protein, suggesting that the candidate molecule can correctly recognize the conformation of the target epitope. As shown in the right panels of Figure 2A and Figure 2B, either ACE-2-mFc or ACE-2-hFc can bind to SARS-Cov-2 S protein.

3.4 Measure the binding of ACE-2-Fc to SARS-Cov-2 S protein by Biacore

After activating the surface of CM5 chip (GE Healthcare, article number BR-1005-30) with a mixture of 50mmol/LNHS and 200mmol/L EDC for 200s, dilute the SARS-CoV-2 S protein RBD or ECD at 10mmol/L sodium acetate (pH 4.5) In the buffer solution to 2ug/ml, flow into the activated chip surface at 25°C at 10ul/min to react for 200s, and then flow into 1mol/L ethanolamine solution to stop the reaction for 200s. After diluting ACE-2-hFc in HBS-EP buffer (GE Healthcare, article number BR100188) to the corresponding concentration (molar concentrations are 1.14nM, 2.28nM, 4.56nM, 9.12nM, 18.25nM, 36.5nM, 73nM), Flow 30ul/min into the surface of the chip coated with SARS-CoV-2 S protein RBD or ECD, detect and record the binding curve for 180s, flow 30ul/min into HBS-EP buffer, detect and record the dissociation curve for 600s. The binding and dissociation curves were fitted with exponential functions to obtain the binding constant K _on =4.11x10 ⁴ Ms ^-1 , and the dissociation constant K _off =1.79x10 ^-4 s ^{-1 of the ACE-2-hFc and SARS-CoV-2 S protein RBD.} , The equilibrium constant K _D =4.36x10 ^-9 M (Figure 6A), the binding constant of ACE-2-hFc and SARS-CoV-2 S protein ECD K _on =5.51x10 ⁴ Ms ^-1 , the dissociation constant K _off =4.03x10 ^{- 4} s ^-1, the equilibrium constant K _D = 7.32x10 ^-9 M (FIG. 6B).

Example 4 Pseudovirus production and neutralization experiment

4.1 Fake virus production

^{Inoculate 4x10 6} HEK293FT cells in a 10 cm cell culture dish, aspirate the original medium on the second day after inoculation, and add 10 ml of fresh DMEM complete medium. After 2 hours, 5 μg of the plasmid p3XFLAG-CMV14-2019nCoV-S expressing the NCP coronavirus S protein and the plasmid p3XFLAG-CMV14-SARS-S expressing the SARS coronavirus S protein were mixed with 20 μg HIV-Luc plasmids, and 500 μl OptiMEM was added. In the serum medium, add 50 μl PEI to 500 μl OptiMEM serum-free medium at the same time, and let stand at room temperature for 5 minutes. The OptiMEM serum-free medium containing PEI was mixed with 500 μl of the OptiMEM serum-free medium containing plasmids, and the mixture was allowed to stand at room temperature for 8 minutes, and 1 ml of the mixture was added to HEK293FT cells. 24 hours after transfection, the medium containing the transfection mixture was replaced with 10 ml of fresh DMEM complete medium. The culture supernatant containing the pseudovirus was harvested 48 hours after transfection, filtered with a 0.45 μm pore filter, and frozen at -80°C.

4.2 Test of the ability of constructed pseudovirus to infect target cells

DNA sequence of human ACE2 protein (sequence information can be found in UniProtKB, Q9BYF1). After gene synthesis, the plasmid vector pLVX-Puro (Takara CatNo: 632164) was digested with the same restriction enzymes, and the ORF of human ACE2 protein was obtained after digestion. DNA fragments and plasmid vector fragments with sticky ends were ligated using CloneEZ (Genscript) and transformed into competent E. coli cells to obtain plasmid pLV-Puro-ACE2.

Inoculate HEK293FT cells (Thermo Fisher Scientific, Catalog No. R70007) in a 6-well cell culture plate at a density of 7×10 ⁵ per well and 3 ml of DMEM complete medium per well. After 16 hours, 3ml of DMEM complete medium was aspirated, and 2ml of fresh DMEM complete medium was added. After 2 hours, 5 μg of plasmid pLVX-Puro-ACE2 expressing human ACE2 protein was added to 100 μl of OptiMEM serum-free medium, while 10 μl of PEI were added to 100 μl of OptiMEM serum-free medium, and left standing at room temperature for 5 minutes. Mix 100 μl of OptiMEM serum-free medium containing PEI with 100 μl of OptiMEM serum-free medium containing plasmids, and let stand at room temperature for 8 minutes, and add 200 μl of the mixture to one well of a 6-well plate to transfect HEK293FT cells. 16 hours after transfection, the medium containing the transfection mixture was replaced with 2 ml of fresh DMEM complete medium to obtain HEK293FT-ACE2 cells.

Inoculate HEK293FT-ACE2 cells in a 96-well flat-bottom cell culture plate at a seeding density of 5000 cells per well and 100μl DMEM complete medium per well. On the second day after inoculation, 25 μl of pseudovirus suspension was added to the 96-well plate for culturing HEK293FT-ACE2 cells. After 16 hours of infection, the culture supernatant of the cells was aspirated, 200 μl of fresh DMEM complete medium was added, and the culture was continued. 48 hours after infection, aspirate the cell culture supernatant, add 200μl PBS buffer to wash the cells, aspirate the PBS, add 50μl cell lysate, place at -80℃, freeze-thaw once, aspirate 30μl cell lysate, and transfer to Luciferase detection 96-well plate, add 30μl Luciferase reaction substrate, put into Luciferase fluorescence microplate reader (ThermoFisher) to read the data.

4.3 Pseudovirus neutralization experiment

Inoculate HEK293FT-ACE2 cells in a 96-well flat-bottom cell culture plate at a density of 5000 cells per well and 100 μl DMEM complete medium per well. On the second day after inoculation, dilute ACE2-Fc with serum-free OptiMEM to a specific concentration (100ug/ml, 20ug/ml, 4ug/ml, 0.8ug/ml), mix 25μl ACE2-Fc dilution with 25μl pseudovirus suspension, After standing at room temperature for 1 hour, 100 μl of the suspension containing 30,000 HEK293FT-ACE2 cells was added to the antibody and virus mixture 96-well plate. 48 hours after infection, aspirate the cell culture supernatant, add 50μl cell lysate and place at -80°C. After freezing and thawing once, aspirate 30μl cell lysate, transfer to Luciferase detection 96-well plate, add 30μl Luciferase reaction substrate , Put into Luciferase fluorescence microplate reader (ThermoFisher) to read the data. Figure 3 shows the results, ACE2-mFC having false virus neutralizing capacity, and neutralizing IC SARS-CoV-2 of _{50 (10.2nM)} ratio of SARS-CoV and the IC ₅₀ (107.4nM) is about 10 times lower , Suggesting that ACE-2-mFC is more effective for SARS-CoV-2.

Example 6 Live virus neutralization experiment and challenge protection experiment

6.1 SARS-CoV-2 live virus neutralization experiment

Inoculate Vero-E6 cells in a 24-well flat-bottom cell culture plate with a seeding density of 160,000 cells per well and 1ml DMEM complete medium per well. On the second day after inoculation, dilute ACE2-hFc with PBS to a specific concentration (100ul/ml, 50ug/ml, 16.67ug/ml, 5.56ug/ml, 1.85ug/ml, 0.62ug/ml, 0.21ug/ml), Mix 50μl ACE2-hFc dilution with 50ul SARS-CoV-2 live virus suspension containing 150FFU (concentrated forming unit) (provided by the P3 laboratory of Guangzhou Medical University and commissioned to perform the following experiments). After standing at room temperature for 1 hour, 100μl of antibody and virus mixture was added to Vero-E6 cells. 1 hour after infection, aspirate the cell culture supernatant and add methyl cellulose gel. After 24 hours of infection, remove the gel layer to stain the cells with crystal violet, count the stained positive cell clusters, and calculate the infection inhibition rate. Infection inhibition rate = 1-(average number of positive spots in the no antibody group-number of positive spots in the antibody test group)/average number of positive spots in the no antibody group x 100%. The results in Figure 7 show that ACE2-hFc has the ability to neutralize live viruses, and the IC ₅₀ for neutralizing live SARS-CoV-2 viruses is 4.1 nM.

6.2 SARS-CoV-2 live virus challenge treatment protection experiment

8 Balb/c mice were inoculated intranasally with adenovirus Ad5-hACE2 (source can be found in Cell.2020Aug 6; 182(3):734-743.e5.) overexpressing human ACE2 protein in the lungs. ^{Five days later, 10 5} TCID units of SARS-CoV-2 live virus were inoculated by intranasal drip, and ACE2-hFc or BSA was injected intraperitoneally on the second day after virus inoculation, and the injection dose was 50 mg/kg. After SARS-CoV-2 live virus inoculation, mice were weighed every day to the 10th day after inoculation. After 3 days of inoculation, the lungs of 3 mice were homogenized to detect the SARS-CoV-2 live virus infection titer. One mouse was anesthetized and perfused with PBS, and the lung tissue was fixed with 4% formaldehyde solution and embedded Cut into 4 micron thick tissue sections in paraffin and stain with hematoxylin/eosin. The results in Fig. 8A show that, compared with injection of BSA, injection of ACE2-hFc can significantly alleviate the weight loss of mice infected with SARS-CoV-2. The results in Fig. 8B show that the live SARS-CoV-2 virus infection titer in the lungs of mice injected with ACE2-hFc was significantly reduced. The results in Fig. 8C showed that the lungs of mice injected with ACE2-hFc did not show pulmonary fibrosis and immune cell infiltration caused by virus infection.

6.3 SARS-CoV-2 live virus challenge prevention and protection experiment

Eleven Balb/c mice were inoculated intranasally with adenovirus Ad5-hACE2 to overexpress human ACE2 protein in the lungs. Four days later, ACE2-hFc or BSA was injected intraperitoneally at a dose of 50 mg/kg. On the 5th day, 10 ⁵ TCID units of SARS-CoV-2 live virus were inoculated by intranasal drip (provided by P3 Laboratory of Guangzhou Medical University) . After SARS-CoV-2 live virus inoculation, mice were weighed every day to the 10th day after SARS-CoV-2 live virus infection. After 1 and 3 days of infection, the lungs of 3 mice were homogenized to detect the titers of live SARS-CoV-2 virus. After 3 days of infection, one mouse was anesthetized and perfused with PBS. The lung tissue was taken and fixed with 4% formaldehyde solution, embedded in paraffin, and sliced into tissue sections with a thickness of 4 microns, and stained with hematoxylin/eosin. The result of Fig. 9A shows Compared with BSA injection, ACE2-hFc injection can significantly alleviate the weight loss of mice infected with SARS-CoV-2. The results in Fig. 9B show that the SARS-CoV-2 live virus infection titer in the lungs of mice injected with ACE2-hFc was significantly reduced. The results in Fig. 9C showed that the lungs of mice injected with ACE2-hFc did not show pulmonary fibrosis and immune cell infiltration caused by virus infection.

Sequence information:

SEQ ID NO:1 ACE2 ECD

SEQ ID NO: 2 ACE2 ECD-human IgG1 Fc

SEQ ID NO: 3 ACE2 ECD-linker-human IgG1 Fc

SEQ ID NO: 4 ACE2 ECD-mouse IgG2a Fc

SEQ ID NO: 5 (DNA sequence of NCP coronavirus S protein ORF)

SEQ ID NO: 6 linker

SEQ ID NO: 7 Human IgG4 Fc sequence

SEQ ID NO: 8 Human IgG4 Fc (S228P, L235E) sequence

SEQ ID NO: 9 Human IgG4 Fc (S228P) sequence

Claims (31)

1. A recombinant fusion protein including angiotensin converting enzyme 2 (ACE2) region and immunoglobulin Fc region.
2. The recombinant fusion protein of claim 1, wherein the ACE2 region is connected to the Fc region of the immunoglobulin, and the recombinant fusion protein can bind to the spike protein of the coronavirus, thereby blocking the ACE2 and the coronavirus spike Protein binding.
3. The recombinant fusion protein of claim 1 or 2, wherein the ACE2 region is the extracellular domain of ACE2.
4. The recombinant fusion protein of claim 3, wherein the ACE2 region comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence shown in SEQ ID NO:1 The amino acid sequence.
5. The recombinant fusion protein according to any one of claims 1 to 4, wherein the Fc region of the immunoglobulin comprises IgG1 Fc region, IgG4 Fc region or IgG4 Fc region mutants, preferably human IgG1 or IgG4.
6. The recombinant fusion protein according to any one of claims 1 to 4, wherein the Fc region of the immunoglobulin comprises an Fc region of IgG2, preferably murine IgG2.
7. The recombinant fusion protein according to any one of claims 1 to 6, wherein the ACE2 region is directly connected to the immunoglobulin Fc region or is connected through an alternative linker.
8. The recombinant fusion protein according to any one of claims 1-6, the coronavirus is selected from SARS-CoV, MERS-CoV or SARS-CoV-2, preferably SARS-CoV-2.
9. The recombinant fusion protein according to any one of claims 1-7, comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or the sequence shown in SEQ ID NO: 2, 3 or 4 Amino acid sequence with at least 99% identity.
10. The dimer formed by the recombinant fusion protein of any one of claims 1-9, and the two recombinant fusion proteins forming the dimer are connected by any linker or automatically form a dimer.
11. A recombinant protein dimer, including any form of dimer formed by the ACE2 region.
12. The recombinant protein dimer according to claim 11, wherein the two ACE2 regions are connected by any linker to form a dimer or automatically form a dimer.
13. The recombinant protein dimer according to claim 12, wherein the linker is selected from a polypeptide or a disulfide bond.
14. The recombinant protein dimer according to claim 12 or 13, wherein the linker comprises an immunoglobulin Fc region.
15. The recombinant protein dimer according to any one of claims 11-14, wherein the ACE region is connected to the immunoglobulin Fc region, and the dimer is formed through one or more disulfide bonds in the Fc region.
16. The recombinant protein dimer according to claim 14 or 15, wherein the Fc region of the immunoglobulin comprises IgG1 Fc region, IgG2 Fc region, IgG4 Fc region or IgG4 Fc region mutants.
17. The recombinant fusion protein dimer according to any one of claims 11-16, wherein the ACE2 region is the extracellular domain of ACE2.
18. According to claim 16, the ACE2 region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the sequence shown in SEQ ID NO:1.
19. A polynucleotide encoding the recombinant fusion protein of any one of claims 1-9 or the recombinant white dimer of any one of claims 11-18.
20. An expression vector comprising the polynucleotide of claim 19.
21. A host cell comprising the expression vector of claim 20.
22. A pharmaceutical composition comprising the recombinant fusion protein of any one of claims 1-9 or the dimer of the recombinant fusion protein of claim 10 or the recombinant protein dimer of any of claims 11-18 , And a pharmaceutically acceptable carrier.
23. A method for preventing, treating or alleviating coronavirus infection, said method comprising administering the recombinant fusion protein according to claim 1-9, the recombinant fusion protein according to claim 10 to a subject infected or suspected of being infected with coronavirus Dimer, the recombinant protein dimer of any one of claims 11-18, or the pharmaceutical composition of claim 22.
24. The method of claim 23, wherein the coronavirus is selected from SARS-CoV-2.
25. A method for detecting coronavirus in a sample, the method comprising: a. Combining the recombinant fusion protein of any one of claims 1-9, the dimer of the recombinant fusion protein of claim 10, or claims 11-18 Contacting the recombinant protein dimer of any one with a sample; b. Determine whether the recombinant protein specifically binds to a molecule in the sample.
26. The method according to claim 25, wherein the coronavirus is selected from SARS-CoV-2.
27. The method according to claim 25 or 26, wherein the sample is derived from an infected or suspected SARS-CoV-2 infection

    Virus serum, whole blood, sputum, oral/nasopharyngeal secretions or lotions, urine, stool, pleural effusion, cerebrospinal fluid, tissue specimens or non-biological samples such as water, beverages.
28. A method for preventing, treating or alleviating coronavirus infection, the method comprising administering a protein containing ACE2 extracellular domain to a subject infected or suspected of being infected with SARS-CoV-2 coronavirus, said ACE2 extracellular domain comprising SEQ ID NO: The amino acid sequence shown in 1.
29. The method of claim 28, wherein the protein exists as a dimer.
30. The application of the protein containing the ACE2 extracellular domain in the preparation of drugs for preventing, treating or alleviating SARS-CoV-2 coronavirus infection, wherein the ACE2 extracellular domain comprises the amino acid sequence shown in SEQ ID NO:1.
31. The use of claim 30, wherein the protein exists in the form of a dimer.

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