Coronavirus-targeting broad-spectrum binding-blocking protein and use thereof

Abstract

Provided are a coronavirus-targeting broad-spectrum binding-blocking protein and a use thereof. Specifically, provided is a novel coronavirus Spike protein (S protein)-targeting binding protein having ultra-high affinity, wherein the protein can bind to an RBD area of the S protein and can block binding of a novel coronavirus to an ACE2 receptor, thereby blocking the novel coronavirus from invading host cells. Also provided is a novel coronavirus S protein-targeting self-assembling trimeric protein having high affinity, wherein the protein exhibits broad-spectrum blocking protection activity against novel coronaviruses.

Description

A class of broad-spectrum blocking proteins targeting coronaviruses and their applications Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, specifically relating to a class of broad-spectrum blocking proteins that target coronaviruses and their uses. Background Technology

[0002] SARS-CoV-2 is a single-stranded RNA virus that mainly invades the body by specifically binding to the host's ACE2 receptor protein through the Spike protein (S protein) distributed on the surface of the virus.

[0003] The spike protein (S) on the surface of the coronavirus is a large class I fusion protein. The S protein forms a trimeric complex, functionally divided into two distinct subunits separated by a protease cleavage site: S1 and S2. The S1 subunit contains a receptor-binding domain (RBD), which interacts with host cell receptor proteins to trigger membrane fusion. The S2 subunit contains the membrane fusion complex, including a hydrophobic fusion peptide and an α-heptacapeptide repeat region. Coronaviruses mediate viral invasion by binding to host cell surface receptors via the S protein's receptor-binding domain (RBD). Therefore, the S protein is considered an important target for preventing and treating coronavirus infection of host cells. Currently, vaccines (recombinant vaccines and mRNA) and neutralizing antibodies against SARS-CoV-2 primarily target the S protein for vaccine development and neutralizing antibody screening. However, due to the virus's high mutation rate, neither targeted antibodies nor vaccines can effectively inhibit treatment escape caused by rapid viral mutations. How to effectively address treatment escape caused by rapid viral mutations remains a pressing problem.

[0004] In summary, there is an urgent need in this field to develop a targeted drug that can more efficiently and broadly block the invasion of coronaviruses into the host. Summary of the Invention

[0005] The purpose of this invention is to provide a class of high-affinity small proteins that target the SARS-CoV-2 S protein. These small proteins can more efficiently block the binding of the S protein to the ACE2 protein, thereby blocking the SARS-CoV-2 virus from invading host cells.

[0006] Another objective of this invention is to provide a class of fusion proteins based on ultra-high affinity small proteins targeting the SARS-CoV-2 S protein and a method for preparing the same.

[0007] In a first aspect of the present invention, a small protein targeting the SARS-CoV-2 S protein is provided. The small protein can specifically target and bind to the SARS-CoV-2 S protein, exhibiting strong affinity, and can competitively bind to the S protein with ACE2, effectively blocking the binding of SARS-CoV-2 to the ACE2 protein.

[0008] In another preferred embodiment, the small protein consists of a single peptide chain, primarily forming a secondary structure of three α-helices and one beta-sheet.

[0009] In another preferred embodiment, the amino acid sequence of the small protein is as shown in SEQ ID NO: 1, 3 or 15.

[0010] In another preferred embodiment, the amino acid sequence of the small protein is substantially the same as that shown in SEQ ID NO:1, 3 or 15 (i.e., homology ≥90%, preferably ≥95%, more preferably ≥98%), and retains binding activity with the SARS-CoV-2 virus (preferably, retaining ≥70%, more preferably ≥80% binding activity).

[0011] In another preferred embodiment, the amino acid sequence of the small protein is shown in SEQ ID NO: 1.

[0012] The present invention also provides a recombinant protein comprising two or more small proteins of the present invention targeting the S protein, which are linked together in series.

[0013] In a second aspect of the invention, a fusion protein is provided, the fusion protein comprising a first polypeptide and / or a second polypeptide;

[0014] The first polypeptide has a structure as shown in Formula I from its N-terminus to its C-terminus, and the second polypeptide has a structure as shown in Formula II from its N-terminus to its C-terminus: P-Mx-H-Fc (Formula I) P-Fc-H-Mx (Formula II)

[0015] in,

[0016] P represents the absence of a signal peptide sequence;

[0017] M is the S protein binding region (or binding element), which includes small proteins targeting the SARS-CoV-2 S protein as described in the first aspect of the present invention, or the recombinant protein of the present invention.

[0018] H represents the hinge area;

[0019] Fc is the constant region of adenovirus or immunoglobulin, or a fragment thereof;

[0020] "-" indicates a peptide bond or linking peptide that connects the above elements;

[0021] x is a positive integer between 1 and 4.

[0022] In another preferred embodiment, the amino acid sequence of P is selected from the group consisting of:

[0023] (i) A sequence as shown in SEQ ID NO:13;

[0024] (ii) Based on SEQ ID NO:13, one or more amino acid residues are replaced, deleted, altered or inserted, or 1 to 10 amino acid residues, preferably 1 to 5 amino acid residues, are added to its N-terminus or C-terminus to obtain an amino acid sequence.

[0025] In another preferred embodiment, the nucleotide sequence encoding P is shown in SEQ ID NO:13.

[0026] In another preferred embodiment, the fusion protein is a monomer or a dimer.

[0027] In another preferred embodiment, the fusion protein is a homodimer or a heterodimer.

[0028] In another preferred embodiment, the first polypeptide can form disulfide bonds with each other, the second polypeptide with each other, or the first polypeptide with each other through cysteine ​​C on their respective Fc residues.

[0029] In another preferred embodiment, the dimer is selected from the group consisting of: a homodimer formed by two first polypeptides, a homodimer formed by two second polypeptides, or a heterodimer formed by the first polypeptide and the second polypeptide.

[0030] In another preferred embodiment, the fusion protein is a homodimer formed by two first polypeptides.

[0031] In another preferred embodiment, the sequence of M is as shown in SEQ ID NO:1, 3 or 15.

[0032] In another preferred embodiment, x is 1, 2, 3 or 4, preferably 2.

[0033] In another preferred embodiment, H is the hinge region of a human immunoglobulin.

[0034] In another preferred embodiment, the human immunoglobulin is selected from the group consisting of IgG1, IgG4, or a combination thereof.

[0035] In another preferred embodiment, the human immunoglobulin is IgG1.

[0036] In another preferred embodiment, the amino acid sequence of H is selected from the group consisting of:

[0037] (i) A sequence as shown in SEQ ID NO:5;

[0038] (ii) Based on SEQ ID NO:6, one or more amino acid residues are replaced, deleted, altered or inserted, or 1 to 10 amino acid residues, preferably 1 to 5 amino acid residues, are added to its N-terminus or C-terminus to obtain an amino acid sequence.

[0039] In another preferred embodiment, the nucleotide sequence encoding said H is shown in SEQ ID NO:5.

[0040] In another preferred embodiment, the Fc is a constant region of a human immunoglobulin or a fragment thereof.

[0041] In another preferred embodiment, the Fc is a tandem sequence of the CH2 and CH3 regions of human immunoglobulin, or is only the CH3 region of human immunoglobulin.

[0042] In another preferred embodiment, the amino acid sequence of the Fc is selected from the group consisting of:

[0043] (i) A sequence as shown in SEQ ID NO:7;

[0044] (ii) Based on SEQ ID NO:7, one or more amino acid residues are replaced, deleted, altered or inserted, or 1 to 30 amino acid sequences are added to its N-terminus or C-terminus, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, thereby obtaining an amino acid sequence.

[0045] In another preferred embodiment, the nucleotide sequence encoding the Fc is shown in SEQ ID NO:7.

[0046] In another preferred embodiment, the amino acid sequence of the first polypeptide is selected from the group consisting of:

[0047] (i) A sequence as shown in SEQ ID NO:9 or 11;

[0048] (ii) Based on SEQ ID NO:9 or 11, one or more amino acid residues are replaced, deleted, altered or inserted, or 1 to 30 amino acid residues are added to its N-terminus or C-terminus, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, thereby obtaining an amino acid sequence.

[0049] In another preferred embodiment, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO:9 or 11, and the nucleotide sequence encoding the first polypeptide is as shown in SEQ ID NO:10 or 12.

[0050] In a third aspect of the invention, a polynucleotide is provided that encodes a small protein targeting the S protein in the first aspect of the invention, or a recombinant protein thereof, or a fusion protein as described in the second aspect of the invention.

[0051] In another preferred embodiment, the sequence of the polynucleotide is as shown in SEQ ID NO:2, 4, 10, 12, 14, 16, 18 or 20.

[0052] In a fourth aspect of the invention, a carrier is provided, the carrier containing the polynucleotide described in the third aspect of the invention.

[0053] In another preferred embodiment, the vector is: pET vector, pGEM-T vector, pcDNA3.1, or a combination thereof.

[0054] In a fifth aspect of the invention, a host cell is provided, the host cell containing the vector described in the fourth aspect, or the genome integrating the polynucleotides described in the third aspect.

[0055] In a sixth aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0056] (a) the small protein targeting the S protein as described in the first aspect of the invention, or a recombinant protein thereof, or the fusion protein as described in the second aspect of the invention; and

[0057] (b) The coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0058] In another preferred embodiment, the coupling portion is a drug or toxin.

[0059] In another preferred embodiment, the coupling portion is a detectable marker.

[0060] In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents.

[0061] In a seventh aspect of the invention, a pharmaceutical composition is provided comprising:

[0062] (a) The small protein targeting the S protein as described in the first aspect of the present invention, or its recombinant protein, or the fusion protein as described in the second aspect of the present invention, or its encoding gene; or the immunoconjugate as described in the sixth aspect of the present invention; and

[0063] (b) Pharmaceutically acceptable carriers.

[0064] In another preferred embodiment, the pharmaceutical composition is used for the diagnosis or treatment of SARS-CoV-2 expressing the S protein.

[0065] In another preferred embodiment, the content of component (a) is 0.1-99.9 wt%, more preferably 10-99.9 wt%, and even more preferably 70%-99.9 wt%.

[0066] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral dosage form, an injection, or a topical dosage form.

[0067] In another preferred embodiment, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral liquids, or injections.

[0068] In another preferred embodiment, the pharmaceutical composition or formulation is selected from the group consisting of suspensions, liquids, or lyophilized formulations.

[0069] In another preferred embodiment, the liquid formulation is an aqueous injection formulation.

[0070] In another preferred embodiment, the liquid preparation has a shelf life of one to three years, more preferably one to two years, and even more preferably one year.

[0071] In another preferred embodiment, the liquid preparation is stored at a temperature of 0°C-16°C, more preferably 0°C-10°C, and even more preferably 2°C-8°C.

[0072] In another preferred embodiment, the shelf life of the lyophilized preparation is six months to two years, more preferably six months to one year, and even more preferably six months.

[0073] In another preferred embodiment, the storage temperature of the lyophilized formulation is ≤42°C, more preferably ≤37°C, and even more preferably ≤30°C.

[0074] In another preferred embodiment, the pharmaceutically acceptable carrier includes surfactants, solution stabilizers, isotonic modifiers, buffer solutions, or combinations thereof.

[0075] In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of infusion carriers and / or injection carriers, preferably one or more carriers selected from the group consisting of physiological saline, glucose saline, or combinations thereof.

[0076] In another preferred embodiment, the solution stabilizer is selected from the group consisting of sugar solution stabilizers, amino acid solution stabilizers, alcohol solution stabilizers, or combinations thereof.

[0077] In another preferred embodiment, the sugar solution stabilizer is selected from the group consisting of reducing sugar solution stabilizers or non-reducing sugar solution stabilizers.

[0078] In another preferred embodiment, the amino acid solution stabilizer is selected from the group consisting of monosodium glutamate or histidine.

[0079] In another preferred embodiment, the alcohol solution stabilizer is selected from the group consisting of: triols, higher sugar alcohols, propylene glycol, polyethylene glycol, or combinations thereof.

[0080] In another preferred embodiment, the isotonic regulator is selected from the group consisting of sodium chloride or mannitol.

[0081] In another preferred embodiment, the buffer solution is selected from the group consisting of TRIS, histidine buffer, phosphate buffer, or combinations thereof.

[0082] In another preferred embodiment, the pharmaceutical composition or preparation is administered to humans or non-human animals.

[0083] In another preferred embodiment, the non-human animal includes rodents (such as rats and mice) and primates (such as monkeys).

[0084] In another preferred embodiment, the dosage of the pharmaceutical composition or preparation is 0.01-10 g / day, more preferably 0.05-5000 mg / day, and even more preferably 0.1-3000 mg / day.

[0085] In another preferred embodiment, the pharmaceutical composition or formulation is used to inhibit and / or treat viral invasion of a host, preferably for inhibiting and / or treating viral infection; more preferably for inhibiting and / or treating SARS-CoV-2 infection.

[0086] In another preferred embodiment, the suppression and / or treatment of viral infection includes delaying the development of symptoms associated with viral infection and / or reducing the severity of these symptoms.

[0087] In another preferred embodiment, the suppression and / or treatment of viral infection also includes the reduction of pre-existing symptoms of viral infection and the prevention of the occurrence of other symptoms.

[0088] In another preferred embodiment, the pharmaceutical composition or formulation may be administered in combination with other antiviral or anti-inflammatory drugs.

[0089] In another preferred embodiment, the other antiviral or anti-inflammatory drugs administered in combination are selected from the group consisting of: viral replication inhibitors, hormonal anti-inflammatory drugs, biological response modifiers, monoclonal antibodies, or combinations thereof.

[0090] In another preferred embodiment, the viral replication inhibitor includes: drugs that affect viral nucleic acid synthesis and replication, and drugs that act on nucleic acid reverse transcription.

[0091] In another preferred embodiment, the drug affecting viral nucleic acid synthesis and replication includes remdesivir.

[0092] In another preferred embodiment, the drug acting on reverse transcription of nucleic acids is selected from the group consisting of: Molnupiravir, Barosavir, Favipiravir, or combinations thereof.

[0093] In another preferred embodiment, the hormonal anti-inflammatory drug includes an anti-estrogen, an aromatase inhibitor, or an anti-androgen; preferably, the anti-estrogen is selected from the group consisting of tamoxifen, droloxifene, exemestane, or combinations thereof; the aromatase inhibitor is selected from the group consisting of ammoniaglutide, lantron, letrozole, reninide, or combinations thereof; and the anti-androgen is selected from the group consisting of flutamethasone RH-LH agonists / antagonists: novide, phenazone, or combinations thereof.

[0094] In another preferred embodiment, the biological response modifier includes: interferon, interleukin-2, thymopeptides, or combinations thereof.

[0095] In another preferred embodiment, the monoclonal antibody includes an antibody that blocks viral invasion or an anti-inflammatory antibody; preferably, the antibody that blocks viral invasion includes: LY-CoV555, LY-CoV016, REGN10933, REGN10987, AZD8895, AZD1061, VIR-7831, BRII-196, DXP-604, or combinations thereof; the anti-inflammatory antibody includes: Tocilizumab, Sarilumab, or combinations thereof.

[0096] In an eighth aspect of the invention, a method is provided for preparing a small protein or recombinant protein targeting the S protein of the first aspect of the invention, or a fusion protein as described in the third aspect of the invention, comprising the steps of:

[0097] (a) Culturing the host cells described in the fifth aspect of the invention under suitable conditions to obtain a culture containing the small protein or its recombinant or fusion protein; and

[0098] (b) The culture obtained in step (a) is purified and / or separated to obtain the small protein or recombinant protein or fusion protein targeting the S protein.

[0099] In a ninth aspect of the invention, the use of the small protein targeting the S protein described in the first aspect of the invention, or a fusion protein thereof, or an immunoconjugate thereof, for the preparation of pharmaceuticals, reagents, detection plates, or kits is provided; wherein the reagents, detection plates, or kits are used to: detect the S protein or SARS-CoV-2 in a sample; wherein the pharmaceuticals are used to treat and / or prevent SARS-CoV-2 infection.

[0100] In another preferred embodiment, the agent is used to block the invasion of the human body by the novel coronavirus.

[0101] In another preferred embodiment, the reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0102] In another preferred embodiment, the reagent for detecting the SARS-CoV-2 S protein in the sample is a contrast agent for detecting SARS-CoV-2 or the viral S protein in vivo.

[0103] In another preferred embodiment, the detection is either in vivo or in vitro.

[0104] In another preferred embodiment, the detection includes flow cytometry, immunofluorescence assay, or a combination thereof.

[0105] In another preferred embodiment, the agent is used to block the interaction between the SARS-CoV-2 S protein and the ACE2 protein.

[0106] In another preferred embodiment, the SARS-CoV-2 virus includes, but is not limited to, wild-type SARS-CoV-2, alpha mutants, beta mutants, Gamma mutants, Delta mutants, or combinations thereof.

[0107] In a tenth aspect of the invention, a method for treating and / or preventing COVID-19 infection is provided, comprising the steps of: administering to a desired subject a safe and effective amount of the small protein or recombinant protein of the S protein targeted by the first aspect of the invention, or the fusion protein of the second aspect, or the immunoconjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect.

[0108] In another preferred embodiment, the treatment and / or prevention of COVID-19 infection includes blocking the entry of the COVID-19 virus into the human body.

[0109] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0110] Figure 1 shows a simulated structural diagram of the complex of a small protein with ultra-high affinity targeting the SARS-CoV-2 S protein. A represents the protein structure of the human ACE2-S protein complex. B represents a simulated structural diagram of the complex of small proteins AE2-mm-1 and AE2-mm-7 with the S protein.

[0111] Figure 2 shows the affinity of ultra-high affinity small protein targeting BA.4 / 5S protein determined using biomembrane interferometry (BLI).

[0112] Figure 3 shows the thermal stability of the ultra-high affinity small protein targeting the S protein, as determined using the Uncle protein stability analyzer. Specifically, the BCM values ​​of AE2-mm-1 and AE2-mm-7 were observed at three temperatures: 25°C, increased to 95°C, and decreased to 25°C, to assess the changes in the protein's secondary structure before and after temperature increases.

[0113] Figure 4 shows the thermal recoverability test results of a small protein with ultra-high affinity targeting the S protein, measured by the Uncle protein stability analyzer. The BCM values ​​of the proteins (AE2-mm-1 and AE2-mm-7) were observed as the temperature gradually increased from 25°C to 95°C. The Tm value of the protein was calculated based on the protein BCM signal changes over time.

[0114] Figure 5 shows schematic diagrams of several structural combinations of small proteins with high affinity for the S protein and their fusion proteins.

[0115] Wherein, A is a short peptide chain of a small protein that targets the S protein;

[0116] B is a small protein targeting the S protein that is linked with the antibody hinge region or linker and CH2 and CH3 to form a polypeptide chain. With the help of the high affinity small protein (or fragment) targeting the S protein provided by the present invention, a single / multi-targeting fusion protein targeting the S protein is formed.

[0117] C is a small protein targeting the S protein that is linked with the antibody hinge region or linker and CH3 to form a polypeptide chain. With the help of the high affinity small protein (or fragment) targeting the S protein provided by the present invention, a single / multi-targeting fusion protein targeting the S protein can be formed.

[0118] D is a small protein targeting the S protein that is tandemly linked with the antibody hinge region or linker and CH3 to form a polypeptide chain, thereby forming a single / multi-target fusion protein targeting the S protein using the high-affinity small protein (or fragment) provided by this invention.

[0119] E is a small protein targeting the S protein. After being linked to the small protein targeting the S protein through a linker sequence, it is connected in series with the antibody hinge region or linker and CH2 and CH3 to form a polypeptide chain. With the help of the high affinity small protein (or fragment) targeting the S protein provided by the present invention, a single / multi-target fusion protein targeting the S protein is formed.

[0120] F is a small protein targeting the S protein. After being linked to the small protein targeting the S protein through a linker sequence, it is connected in series with the antibody hinge region or linker and CH3 to form a polypeptide chain. With the help of the high affinity small protein (or fragment) targeting the S protein provided by the present invention, a single / multi-target fusion protein targeting the S protein is formed.

[0121] G is a small protein targeting the S protein. After being linked to the small protein targeting the S protein through a linker sequence, it is connected in series with the antibody hinge region or linker and CH3 to form a polypeptide chain. With the help of the high affinity small protein (or fragment) targeting the S protein provided by the present invention, a single / multi-target fusion protein targeting the S protein is formed.

[0122] H is a small protein targeting the S protein that forms a polypeptide chain with a self-assembling trimer protein through a linker. The high-affinity small protein (or fragment) targeting the S protein provided by this invention is linked with a previously applied self-assembling trimer protein to form a trivalent fusion protein targeting the S protein.

[0123] Figure 6 shows the neutralizing protective activities of AE2-mm-40 and AE2-mm-40-GS16-C3-50-130-18 against major mutant strains of coronavirus pseudoviruses.

[0124] Figure 7 shows the in vivo neutralizing protective activity of AE2-mm-40 and AE2-mm-40-GS16-C3-50-130-18 against SARS-CoV-2 XBB1.16 virus 6 hours before prophylaxis or 6 hours after administration.

[0125] Figure 8 shows the inhibitory effect of AE2-mm-40 and AE2-mm-40-GS16-C3-50-130-18 on the viral titer of SARS-CoV-2 XBB1.16 in lung tissue. Detailed Implementation

[0126] Through extensive and in-depth research, the inventors have designed and obtained a class of broad-spectrum, high-affinity small proteins targeting the SARS-CoV-2 S protein, based on the structural complex of the SARS-CoV-2 S protein and ACE2 protein, targeting the interaction surface between ACE2 and S proteins. The binding site of this small protein almost completely covers the binding site of ACE2 protein on the S protein. Experiments show that the high-affinity small protein AE2-mm-1 of this invention can broadly bind to major mutant strains of SARS-CoV-2. This protein exhibits good broad-spectrum neutralizing protective activity against SARS-CoV-2 mutant strains. Compared with traditional antibodies, the small protein of this invention has a smaller molecular weight and potentially better tissue penetration and structural stability. This invention was completed based on these findings.

[0127] Specifically, the representative ultra-high affinity small protein targeting the S protein is 142 amino acids in length, with a molecular weight much smaller than conventional antibodies, and lacks the antibody Fc moiety, thus exhibiting better tissue penetration. Furthermore, the ultra-high affinity small protein targeting the S protein of this invention has even higher affinity and can serve as a potential diagnostic and in vivo tracing reagent for SARS-CoV-2.

[0128] This invention targets small proteins and fusion proteins with high affinity for the S protein.

[0129] In this invention, a class of small proteins with ultra-high affinity targeting S proteins and a fusion protein or conjugate thereof comprising the small protein are provided.

[0130] As used herein, the terms "small protein of the present invention", "small protein of the present invention with ultra-high affinity for coronavirus S protein", and "small protein of the present invention with ultra-high affinity for S protein" are used interchangeably and all refer to the small protein having the broad-spectrum blocking protective activity against the SARS-CoV-2 S protein as described in the first aspect of the present invention.

[0131] As used herein, the term "S protein" refers to the Spike protein of the SARS-CoV-2 virus. In some implementations, the S protein may be derived from the S protein of wild-type SARS-CoV-2, alpha mutants, beta mutants, Gamma mutants, Delta mutants, or other mutants.

[0132] Preferably, the small protein of the present invention has an amino acid sequence as shown in SEQ ID NO:1 or 3.

[0133] As used herein, the term "fusion protein of the present invention" refers to a fusion protein formed by the ultra-high affinity small protein targeting the S protein described in this invention and other fusion elements. For example, the small protein of the present invention can form a fusion protein with elements such as the hinge region and the Fc region. The fusion protein of the present invention can block the binding of ACE2 protein to the S protein.

[0134] Preferably, the ultra-high affinity fusion protein of the present invention can be any ultra-high affinity small protein or a partial amino acid fragment thereof (typically at least 70% of the length of the amino acid fragment) that at least contains the complete target S protein.

[0135] Typically, the fusion protein of the present invention may have the following structure:

[0136] High-affinity small proteins or fragments targeting the S protein - Y-shaped structure of Hinge-CH2-CH3;

[0137] A Y-shaped structure of a small protein or fragment with ultra-high affinity for the S protein - Hinge-CH3;

[0138] High-affinity small proteins or fragments targeting the S protein - tracer labeling;

[0139] Small proteins or fragments with extremely high affinity targeting the S protein.

[0140] It should be understood that the above structural types are merely illustrative and do not limit the present invention. Some representative structures are shown in Figure 2. Among them, the ultra-high affinity small protein or fragment thereof targeting the S protein can be single or multiple (such as two, three or four ultra-high affinity small proteins or fragments thereof in tandem, for example, Figures 5E, 5F and 5G).

[0141] As used herein, the terms "high-affinity small protein targeting the S protein" or "fusion protein" also include variants possessing both S protein-binding activity and ACE2 / S protein-blocking activity. These variants include (but are not limited to): deletions, insertions, and / or substitutions of 1-3 amino acids (typically 1-2, more preferably 1); additions or deletions of one or more amino acids (typically up to 3, preferably up to 2, more preferably up to 1) at the C-terminus and / or N-terminus; or additions of smaller amino acid side chains (such as glycine, serine, etc.) as linkers at the N-terminus or C-terminus of the small protein. For example, in the art, substitution with amino acids of similar or comparable properties typically does not alter the protein's function. Similarly, adding or deleting one or more amino acids at the C-terminus and / or N-terminus typically does not alter the protein's structure and function. Furthermore, the term also includes the polypeptides of the invention in both monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0142] The present invention also includes active fragments, derivatives, and analogs of the aforementioned small proteins or fusion proteins targeting the S protein (especially fusion proteins formed with the Fc fragment). As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the function or activity of the high-affinity small proteins or fusion proteins targeting the S protein of the present invention.

[0143] The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) polypeptides having substituent groups in one or more amino acid residues, or (iii) polypeptides formed by fusing a polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, for example, polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (fusion proteins formed by fusing with a leader sequence, secretion sequence, or tag sequence such as 6His). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0144] A preferred class of active derivatives refers to polypeptides formed by replacing up to five, more preferably up to three, and even more preferably up to one amino acid with an amino acid of similar or analogous properties, compared to the amino acid sequence of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0145] Table A

[0146] This invention also provides analogs of the fusion protein of this invention. These analogs may differ from the peptides of this invention in terms of amino acid sequence, or in the form of modifications that do not affect the sequence, or both. Analogs also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the peptides of this invention are not limited to the representative peptides exemplified above.

[0147] Furthermore, the high-affinity small proteins or fusion proteins targeting the S protein of this invention can also be modified. Modifications (typically without altering the primary structure) include chemically derived forms of the peptide, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during peptide synthesis and processing or further processing steps. This modification can be accomplished by exposing the peptide to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Modifications also include peptides modified to improve their resistance to proteolytic hydrolysis or optimize their solubility.

[0148] The term "polynucleotide of the present invention" can refer to a polynucleotide that includes a high-affinity small protein or fusion protein encoding the S protein targeted by the present invention, or it can also include a polynucleotide that includes additional coding and / or non-coding sequences.

[0149] This invention also relates to variants of the aforementioned polynucleotides that encode fragments, analogs, and derivatives of polypeptides or fusion proteins having the same amino acid sequence as those of this invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the encoded high-affinity small protein or fusion protein targeting the S protein.

[0150] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions (or strict conditions). In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%.

[0151] The ultra-high affinity small proteins or fusion proteins and polynucleotides targeting the S protein of the present invention are preferably provided in isolated form, and more preferably, purified to homogenization.

[0152] The full-length polynucleotide sequences of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0153] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0154] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0155] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0156] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the polynucleotides of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE (RACE-cDNA end amplification) method is preferred. Primers used for PCR can be appropriately selected based on the sequence information disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0157] As used in this article, “AE2-mm-40-GS16-C3-50-130-18”, “GS16C3130” and “AE2-mm-40-GS16-C3-130” have the same meaning and can be used interchangeably. They all refer to proteins with amino acid sequences as shown in SEQ ID NO:17.

[0158] As used in this article, “AE2-mm-40” and “AEMM40” have the same meaning and can be used interchangeably. Both refer to proteins with amino acid sequences as shown in SEQ ID NO:15.

[0159] expression carrier

[0160] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors of the present invention or the coding sequences of the high-affinity small proteins or fusion proteins of the present invention that target the S protein, and methods for generating the polypeptides of the present invention via recombinant technology.

[0161] Using conventional recombinant DNA techniques, the polynucleotide sequences of this invention can be used to express or produce recombinant fusion proteins. Generally, the following steps are involved:

[0162] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding the fusion protein of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0163] (2) Host cells cultured in a suitable culture medium;

[0164] (3) Isolate and purify proteins from culture media or cells.

[0165] In this invention, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0166] In the method for preparing the target coronavirus extended-spectrum blocking binding protein or its fusion protein of the present invention, any suitable vector can be used, selected from pET, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, and the expression vector includes a fusion DNA sequence linked with suitable transcription and translation regulatory sequences.

[0167] Both eukaryotic and prokaryotic host cells can be used for the expression of the ultra-high affinity small protein or its fusion protein targeting the S protein of the present invention. The eukaryotic host cells are preferably mammalian or insect host cell culture systems, preferably COS, CHO, NSO, sf9 and sf21 cells; the prokaryotic host cells are preferably one of lemo21, DH5a, BL21(DE3) and TG1.

[0168] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the fusion protein of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0169] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0170] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0171] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces, Salmonella typhimurium bacterial cells, fungal cells such as yeast, and plant cells (such as ginseng cells).

[0172] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0173] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0174] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0175] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0176] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0177] Affinity chromatography can be used to separate and purify a class of extended-spectrum blocking proteins or their fusion proteins targeting coronaviruses disclosed in this invention. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer or pH adjustment can be used to elute the ultra-high affinity small proteins or their fusion proteins targeting S proteins bound to the affinity column.

[0178] Using the above method, small proteins with high affinity for the S protein or their fusion proteins can be purified into essentially homogeneous substances, such as a single band on SDS-PAGE electrophoresis.

[0179] Pharmaceutical Composition

[0180] In this invention, a pharmaceutical composition containing a small protein or fusion protein or an immunoconjugate thereof that targets the S protein of this invention is also provided.

[0181] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the small protein or fusion protein (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or aqueous solutions containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents. The small protein or fusion protein targeting the S protein, or its immunoconjugate, can be formulated with pharmaceutically acceptable excipients to achieve a more stable therapeutic effect. These formulations ensure the structural integrity of the amino acid core sequence of the small protein or fusion protein targeting the S protein of this invention, while also protecting the protein's multifunctional groups from degradation (including but not limited to aggregation, deamination, or oxidation). The formulation can be in various forms. Generally, liquid formulations can be stably stored for at least one year at 2°C-8°C, and lyophilized formulations remain stable for at least six months at 30°C. The formulation can be a commonly used pharmaceutical preparation such as a suspension, injection, or lyophilized form, with injections or lyophilized forms being preferred.

[0182] For the pharmaceutical compositions of the present invention targeting the S protein (such as aqueous injections or lyophilized formulations), pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonic modifiers, and buffers. Surfactants include nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleic, or octadecyl sarcosine; Pluronics; MONAQUAT™, etc., added in an amount that minimizes the tendency of the protein to granulate. Solution stabilizers can be sugars, including reducing and non-reducing sugars; amino acids, including monosodium glutamate or histidine; alcohols, including one or a combination of triols, higher sugar alcohols, propylene glycol, and polyethylene glycol. The amount of solution stabilizer added should be such that the final formulation is considered by a person skilled in the art to remain stable for a stable period of time. Isotonic modifiers can be one of sodium chloride and mannitol. Buffers can be one of TRIS, histidine buffer, and phosphate buffer.

[0183] When using the pharmaceutical composition, a safe and effective amount of the small protein or fusion protein of the present invention, or its immunoconjugate, is administered to a mammal. This safe and effective amount is typically at least about 50 micrograms per kilogram of body weight, and in most cases does not exceed about 100 milligrams per kilogram of body weight; preferably, the dose is between about 100 micrograms per kilogram of body weight and about 50 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise. Typically, the total dose should not exceed a certain range, for example, an intravenous dose of 10 to 3000 mg / day / 50 kg, preferably 100 to 1000 mg / day / 50 kg.

[0184] The small protein or its fusion protein targeting the S protein of the present invention and pharmaceutical preparations containing the same can be used as antiviral drugs for viral treatment. The antiviral drugs referred to in the present invention are drugs that have the effect of inhibiting and / or treating viral infection, and may include delaying the development of viral infection-related symptoms and / or reducing the severity of these symptoms. It further includes alleviating the accompanying symptoms of viral infection and preventing the occurrence of other symptoms.

[0185] The main advantages of this invention include:

[0186] 1) The small protein targeting the SARS-CoV-2 S protein provided by this invention has a binding site that almost covers the binding site of ACE2 and the S protein.

[0187] 2) The small protein of the present invention has a small molecular weight and a length of 142 amino acids, and has better tissue penetration.

[0188] 3) The small protein of the present invention has an extremely high affinity for the SARS-CoV-2 S protein and can effectively block the SARS-CoV-2 virus from invading host cells.

[0189] 4) The small protein of the present invention has ultra-high structural stability, with a Tm value greater than 95°C.

[0190] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0191] The sequence of the present invention is shown in Table 1 below.

[0192] Table 1. Sequence of the present invention

[0193] Example 1: Synthesis of high-affinity target for SARS-CoV-2 S protein

[0194] Using a whole-genome synthesis method, high-affinity small protein genes targeting the S protein were synthesized and named AE2-mm-1, AE2-mm-7, and AE2-mm-40, respectively. Their amino acid sequences are shown in SEQ ID NO: 1, 3, or 15, and their nucleotide sequences are shown in SEQ ID NO: 2, 4, or 16, respectively. After adding a start codon to the N-terminus of the synthesized nucleotide sequences, they were inserted into the pET29b(+) expression vector at the XhoI and NedI restriction sites.

[0195] Example 2: Structural simulation of a small protein with high affinity for the SARS-CoV-2 S protein

[0196] The obtained small protein was simulated using Alphafold2, and as shown in Figure 1B, the small protein has an ααβα structure. The structure was then visualized using ChimeraX, and Figure 1A shows the structure of the SARS-CoV-2 S protein complex with ACE2. As shown in Figure 1, the binding site of the small protein to the SARS-CoV-2 S protein almost completely overlaps with the binding site of the ACE2 protein to the S protein.

[0197] Example 3: Expression and purification of ultra-high affinity small proteins

[0198] After transforming the vector into E. coli, it was cultured in LB medium at 37°C and 270 rpm until OD200. 600 =0.6. Then, 1 mM IPTG was used to induce protein expression in the bacterial culture overnight. After harvesting, Protease Inhibitor Cocktail and Nucleases were disrupted by sonication (4 min, 10 s on, 10 s off, 80% Amp), and the supernatant was collected. After purification using a Ni column, the concentrated sample was further purified by molecular sieve. Protein expression and purification were assessed using SDS-PAGE and Coomassie brilliant blue staining. Protein concentration was further determined using [a specific method / method / approach].

[0199] This method yields high-purity candidate proteins for subsequent experiments.

[0200] Example 4: Determination of the affinity of high-affinity small proteins targeting the S protein

[0201] In this embodiment, the affinity of the high-affinity blocking protein was detected using the ForteBio Octet. First, 3 μg / ml of biotin-tagged SARS-CoV-2 S protein was loaded onto the SA chip (300 s), and unbound SARS-CoV-2 S protein was eluted in PBST solution. Then, the detection probe carrying the S protein was simultaneously immersed in a 2-fold serially diluted solution of a high-affinity small protein targeting the S protein, and the binding signal was detected (180 s). Next, the probe was immersed in PBST (300 s), and the dissociation signal of the bound protein was detected. Finally, the affinity of the high-affinity blocking protein was calculated.

[0202] As shown in Figure 2, AE2-mm-1, AE2-mm-7, and AE2-mm-40 all exhibited good binding signals. The affinity of AE2-mm-1 and AE2-mm-7 was determined by binding kinetics. AE2-mm-1 and AE2-mm-7 showed strong binding activity to the S protein of the SARS-CoV-2 mutant, with affinity reaching the femtoliter level. The affinities of AE2-mm-40, AE2-mm-40-GS16-C3-50-130-18, and AE2-mm-40-GS16-C3-50-70-11 were 3.576 nM, 0.1233 pM, and femtoliter levels, respectively.

[0203] Example 5: Detection of the thermal stability of a high-affinity small protein structure targeting the S protein

[0204] Protein structural stability was assessed using a JASCO-1500 microscope. The detection range was 190 nm–260 nm. First, the circular dichroism (BDD) signal of the AE2-mm-1 or AE2-mm-7 protein at 25 °C (0.1 mg / ml) was measured. Then, the protein was heated to 95 °C, and the DCD signal was measured again. Finally, the temperature was restored to 25 °C and allowed to stand for 5 minutes before measuring the DCD signal again. The changes in the secondary structure conformation of the protein at different temperatures were obtained, thus assessing the structural stability of the bound protein.

[0205] As shown in Figure 3, AE2-mm-1 and AE2-mm-7 exhibit a high level of α-helical protein secondary structure at 25℃. When the temperature is increased to 95℃, the secondary structure of the protein undergoes some changes due to the high temperature. However, as the temperature is cooled back to 25℃, the circular dichroism signals almost completely overlap, indicating that the secondary structure of the protein has returned to its pre-heating state. This protein exhibits excellent thermal stability.

[0206] Example 6: Detection of the thermal recovery of high-affinity small protein structures targeting the S protein

[0207] Using a JASCO-1500, the circular dichroism (CDI) signals of AE2-mm-1 and AE2-mm-7 proteins at 25°C (0.1 mg / ml) were measured. The CDI signal was detected at a wavelength of 222 nm as the protein was gradually heated from 25°C to 95°C. The CDI rate was 2°C / min, with a equilibration time of 30 seconds per minute. The Tm value of the protein was then obtained.

[0208] As shown in Figure 4, although the circular dichroism (CDI) signal increases with increasing temperature, it only increases slightly at the instrument's detection limit of 95°C. Based on this signal curve, its Tm exceeds the instrument's detection temperature limit; the Tm for both AE2-mm-1 and AE2-mm-7 is greater than 95°C. This protein exhibits exceptional thermal stability.

[0209] Example 7: Expression and purification of fusion protein

[0210] In this embodiment, a fusion protein of a small protein with extremely high affinity was prepared. The structure of the prepared fusion protein is shown in Figure 5B, and the amino acid sequence is SEQ ID NO: 9 or 11. The method is as follows:

[0211] The signal peptide sequence SEQ ID NO: 14 was ligated to the coding sequence SEQ ID NO: 10 or 12 of the fusion protein, respectively, and then introduced into the multiple cloning site of the pcDNA3.1 vector. The vector was transfected into 293F cells, which were then cultured on a shaker for 6 days. The cell culture supernatant was collected, filtered, purified using a Protein A column, and further concentrated by ultrafiltration. Protein expression and purification were assessed using SDS-PAGE and Coomassie Brilliant Blue staining.

[0212] In addition, the binding of the fusion protein to the S protein was determined using the BLI method of Example 4. The results showed that the prepared fusion protein could bind to the S protein with ultra-high affinity.

[0213] Example 8: In vitro neutralizing and protective activity assay of a small protein with ultra-high affinity targeting the S protein.

[0214] In this embodiment, the in vitro pseudovirus neutralizing and protective activities of AE2-mm-40 and AE2-mm-40-GS16-C3-50-130-18 against major mutant strains of SARS-CoV-2 will be evaluated.

[0215] This embodiment employs a method similar to antibody-pseudovirus neutralization titration. The method is described as follows: In a 96-well plate, AE2-mm-40 or AE2-mm-40-GS16-C3-50-130-18 was serially diluted 12 times at a 3-fold ratio. The diluted protein was then mixed with a SARS-CoV-2 pseudovirus carrying the luciferase reporter gene to achieve an initial protein dilution of 450 μg / mL and a pseudovirus concentration of 1000 TCID. 50 / well. After incubating at 37°C for 1 hour, the cells were incubated with 293T cells stably expressing human ACE2 at 37°C for 24 hours. Lysis buffer containing luciferase reaction substrate was added and incubated for 2 minutes. The relative fluorescence luminescence value was detected using a multi-mode microplate reader, and the IC50 was calculated using the Reed-Muench method. 50 .

[0216] As shown in Figure 6, AE2-mm-40 and AE2-mm-40-GS16-C3-50-130-18 exhibited good broad-spectrum neutralizing protective activity against major mutant strains of SARS-CoV-2, SARS-1, and other species of coronaviruses.

[0217] Example 9: In vivo neutralizing and protective activity assay of a small protein with ultra-high affinity targeting the S protein.

[0218] In this embodiment, the in vitro protective activities of AE2-mm-40 and AE2-mm-40-GS16-C3-130 against the main mutant strain of SARS-CoV-2, XBB.1.16, were evaluated.

[0219] This embodiment includes treatment and prevention trials. In the treatment group, 4-5 week old male golden hamsters were first intranasally infected with the virus (50 μl / hamster, viral load 2 × 10⁻⁶). 5 TCID 50 Six hours later, 4-5 week old male golden hamsters were treated with intranasal protein (80 μl / hamster, containing 200 μg protein). In the prevention group, 4-5 week old male golden hamsters were first treated with intranasal protein (80 μl / hamster, containing 200 μg protein), and 6 hours later, they were treated with intranasal viral infection (50 μl / hamster, viral load 2 × 10⁻⁶). 5 TCID 50 On the third day of infection, golden hamsters were deeply anesthetized with tribromoethanol, and their lungs were dissected, separated, and photographed. The tissue from the upper lobe of the right lung was homogenized with Trizol, and nucleic acid was extracted to determine the viral load.

[0220] As shown in Figures 7 and 8, both AE2-mm-40 and AE2-mm-40-GS16-C3-130 proteins exhibited in vivo protective activity against the main mutant strain of SARS-CoV-2, XBB.1.16, in both treatment and prevention trials.

[0221] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A small protein targeting the S protein of the SARS-CoV-2 virus, characterized in that, The amino acid sequence of the small protein is shown in SEQ ID NO: 1, 3 or 15. The small protein contains three α-helices and one beta-sheet secondary structure, and it blocks the binding of the SARS-CoV-2 S protein to the ACE2 protein.

2. The small protein targeting the SARS-CoV-2 S protein as described in claim 1, characterized in that, The amino acid sequence of the small protein is shown in SEQ ID NO:

1.

3. A recombinant protein, characterized in that, The recombinant protein comprises two or more small proteins targeting the S protein as described in claim 1 or 2, linked together.

4. A fusion protein, characterized in that, The fusion protein includes a first polypeptide and / or a second polypeptide; The first polypeptide has a structure as shown in Formula I from its N-terminus to its C-terminus, and the second polypeptide has a structure as shown in Formula II from its N-terminus to its C-terminus. P-Mx-H-Fc (Formula I) P-Fc-H-Mx (Formula II) in, P represents the absence of a signal peptide sequence; M is the S protein binding region (or binding element), wherein the S protein binding region is the small protein targeting the S protein as described in claim 1 or 2, or the recombinant protein as described in claim 3; H represents the hinge area; Fc is the constant region of adenovirus or immunoglobulin, or a fragment thereof; "-" indicates a peptide bond or linking peptide that connects the above elements; x is a positive integer between 1 and 4.

5. A polynucleotide, characterized in that, The polynucleotide encodes a small protein targeting the S protein as described in claim 1 or 2, a recombinant protein as described in claim 3, or a fusion protein as described in claim 4.

6. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 5.

7. A host cell, characterized in that, The host cell contains the vector as described in claim 6, or the genome is integrated with the polynucleotide as described in claim 5.

8. An immunoconjugate, characterized in that, This immunoconjugate contains: (a) the small protein targeting the S protein as described in claim 1 or 2, the recombinant protein as described in claim 3, or the fusion protein as described in claim 4; and (b) The coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

9. A pharmaceutical composition, characterized in that, include: (a) the small protein targeting the S protein as described in claim 1 or 2, or the recombinant protein as described in claim 3, or the fusion protein as described in claim 3, or its encoding gene; or the immunoconjugate as described in claim 8; and (b) Pharmaceutically acceptable carriers.

10. A method for preparing a small protein targeting the S protein as described in claim 1 or 2, or a recombinant protein as described in claim 3, or a fusion protein as described in claim 4, characterized in that, Including the following steps: (a) Culturing the host cells as described in claim 7 under suitable conditions to obtain a culture containing the small protein, recombinant protein, or fusion protein; and (b) The culture obtained in step (a) is purified and / or separated to obtain the small protein, recombinant protein or fusion protein targeting the S protein.

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