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WO2023108666A1 - 一类靶向covid-19病毒s蛋白的超高亲和力小蛋白及用途 - Google Patents

一类靶向covid-19病毒s蛋白的超高亲和力小蛋白及用途 Download PDF

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Publication number
WO2023108666A1
WO2023108666A1 PCT/CN2021/139445 CN2021139445W WO2023108666A1 WO 2023108666 A1 WO2023108666 A1 WO 2023108666A1 CN 2021139445 W CN2021139445 W CN 2021139445W WO 2023108666 A1 WO2023108666 A1 WO 2023108666A1
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protein
small
targeting
present
new coronavirus
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PCT/CN2021/139445
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English (en)
French (fr)
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赵磊
张帆
胡毅
姚咏明
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中国人民解放军总医院
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Priority to PCT/CN2021/139445 priority Critical patent/WO2023108666A1/zh
Publication of WO2023108666A1 publication Critical patent/WO2023108666A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the invention belongs to the fields of biotechnology and medicine, and in particular relates to a small super-affinity protein targeting COVID-19 virus S protein and a fusion protein thereof.
  • the COVID-19 virus is a single-stranded RNA virus that invades the body mainly through the specific binding of the Spike protein (S protein) distributed on the surface of the virus to the host ACE2 receptor protein.
  • S protein Spike protein
  • the spike protein (S) on the surface of coronavirus which is a large class I fusion protein.
  • the S protein forms a trimeric complex that is functionally divided into two distinct subunits, S1 and S2, separated by a protease cleavage site.
  • the S1 subunit contains a receptor-binding domain (RBD) that interacts with host cell receptor proteins to trigger membrane fusion.
  • the S2 subunit contains a membrane fusion complex, including a hydrophobic fusion peptide and an ⁇ -helical heptad repeat region.
  • Coronaviruses bind to host cell surface receptors through the receptor binding domain (RBD) of the S protein to mediate virus invasion.
  • the S protein is considered to be an important target for preventing and treating coronavirus-infected host cells.
  • both vaccines recombinant vaccines and mRNA
  • neutralizing antibodies against COVID-19 use the S protein as the main target for vaccine preparation and neutralizing antibody screening.
  • the purpose of the present invention is to provide a class of ultra-high-affinity small proteins targeting the S protein of the new coronavirus, which can more efficiently block the combination of the S protein and the ACE2 protein, thereby blocking the invasion of host cells by the new coronavirus.
  • Another object of the present invention is to provide a fusion protein based on a small protein with super high affinity targeting the S protein of the new coronavirus and its preparation method.
  • a small protein targeting the S protein of the new coronavirus is provided, the small protein can specifically target and bind the S protein of the new coronavirus, exhibit super affinity, and can compete with ACE2 sexual binding to S protein, effectively blocking the binding of S protein to ACE2 protein in the wild type of the new coronavirus and its alpha, beta, gamma and Delta mutant strains.
  • the small protein consists of one peptide chain, mainly forming three ⁇ -helical secondary structures.
  • the small protein can specifically target and bind to the S protein of the new coronavirus, showing strong affinity, respectively showing strong binding to the wild type, alpha, beta, gamma and delta of the new coronavirus Activity; wherein, the small protein is composed of a peptide chain, mainly forming three ⁇ -helical secondary structures;
  • amino acid sequence of the small protein has SEQ ID No: 1 or as shown in SEQ ID NO: 1.
  • the small protein can specifically target and bind to the S protein of the new coronavirus Delta mutant strain, showing super affinity, and can compete with the ACE2 receptor for binding to the new coronavirus S protein, effectively blocking The combination of the new coronavirus S protein and the ACE2 receptor protein; and against the new coronavirus wild type, alpha, beta and gamma all show good neutralizing and protective activity; wherein, the small protein is composed of a peptide chain, mainly forming Three ⁇ -helical secondary structures;
  • amino acid sequence of the small protein has or is shown in SEQ ID NO: 3 or 5.
  • the amino acid sequence of the small protein is shown in SEQ ID NO: 1, 3 or 5.
  • the present invention also provides a recombinant protein, which includes two or more small proteins targeting the S protein of the present invention connected in series.
  • a fusion protein which includes the first polypeptide and/or the second polypeptide;
  • the first polypeptide has the structure shown in formula I from N-terminus to C-terminus
  • the second polypeptide has the structure shown in formula II from N-terminus to C-terminus
  • P is nothing or a signal peptide sequence
  • M is the S protein binding region (or binding element), the amino acid sequence of the S protein binding region is from the amino acid sequence of the small protein targeting S protein as described in the first aspect;
  • H is the hinge region
  • Fc is none or a constant region of an immunoglobulin, or a fragment thereof;
  • x is a positive integer of 1-4.
  • amino acid sequence from the small protein targeting S protein refers to the amino acid sequence of the binding region (or binding element) of the S protein and the amino acid sequence of the targeting S protein
  • the amino acid sequences of the small proteins are identical or substantially identical (that is, homology ⁇ 90%, preferably ⁇ 95%, more preferably ⁇ 98%), and the S protein binding region (or binding element) remains the same as the new crown
  • the binding activity of the S protein of the virus Delta mutant strain preferably, ⁇ 70% of the binding activity is retained, more preferably ⁇ 80% of the binding activity).
  • amino acid sequence of P is selected from the following group:
  • amino acid residues On the basis of SEQ ID NO: 17, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 10 amino acid residues are added at its N-terminal or C-terminal, more preferably 1 to 5 amino acid residues, thereby obtaining the amino acid sequence.
  • nucleotide sequence encoding the M is shown in SEQ ID NO:18.
  • the fusion protein is a monomer or a dimer.
  • the fusion protein is a homodimer or a heterodimer.
  • between the first polypeptide and the first polypeptide, between the second polypeptide and the second polypeptide, or between the first polypeptide and the second polypeptide, can pass Cysteine C on the respective Fc forms a disulfide bond.
  • 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 homodimer formed by the first polypeptide A heterodimer formed by a peptide and a second polypeptide.
  • the fusion protein is a homodimer formed by two first polypeptides.
  • sequence of M is shown in SEQ ID NO: 1, 3 or 5.
  • said x is 1, 2, 3 or 4, preferably 2.
  • the H is the hinge region of human immunoglobulin.
  • the human immunoglobulin is selected from the group consisting of IgG1, IgG4, or a combination thereof.
  • the human immunoglobulin is IgG1.
  • amino acid sequence of H is selected from the following group:
  • amino acid residues On the basis of SEQ ID NO: 7, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 10 amino acid residues are added at its N-terminal or C-terminal, more preferably 1 to 5 amino acid residues, thereby obtaining the amino acid sequence.
  • nucleotide sequence encoding the H is shown in SEQ ID NO:8.
  • the Fc is a constant region of human immunoglobulin or a fragment thereof.
  • the Fc is the tandem sequence of CH2 and CH3 regions of human immunoglobulin, or only the CH3 region of human immunoglobulin.
  • amino acid sequence of the Fc is selected from the following group:
  • amino acid residues are replaced, deleted, changed or inserted, or 1 to 30 amino acid sequences are added at its N-terminal or C-terminal, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, thereby obtaining the amino acid sequence.
  • nucleotide sequence encoding the Fc is shown in SEQ ID NO:10.
  • amino acid sequence of the first polypeptide is selected from the following group:
  • amino acid residues On the basis of SEQ ID NO: 11, 13, or 15, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 30 amino acid sequences are added at its N-terminal or C-terminal , preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, thereby obtaining the amino acid sequence.
  • amino acid sequence of the first polypeptide is shown in SEQ ID NO: 11, 13, or 15, and the nucleotide sequence encoding the first polypeptide is shown in SEQ ID NO: 12, 14 or 16.
  • a polynucleotide which encodes the small protein targeting the S protein in the first aspect of the present invention, or its recombinant protein or the fusion protein described in the second aspect of the present invention .
  • sequence of the polynucleotide is shown in SEQ ID NO: 2, 4, 6, 12, 14 or 16.
  • a vector containing the polynucleotide described in the third aspect of the present invention is provided.
  • the vector is: pET vector, pGEM-T vector, pcDNA3.1, or a combination thereof.
  • a host cell in the fifth aspect of the present invention, contains the vector described in the fourth aspect, or the polynucleotide described in the third aspect is integrated in the genome.
  • an immunoconjugate which comprises:
  • a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
  • the coupling moiety is a drug or a toxin.
  • the coupling moiety is a detectable label.
  • 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.
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition is used for diagnosing or treating a new coronavirus expressing S protein.
  • the content of the component (a) is 0.1-99.9wt%, preferably 10-99.9wt%, more preferably 70%-99.9wt%.
  • the dosage form of the pharmaceutical composition is an oral dosage form, an injection, or an external pharmaceutical dosage form.
  • the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral liquids, or injections.
  • the pharmaceutical composition or preparation is selected from the group consisting of suspension preparation, liquid preparation or freeze-dried preparation.
  • the liquid preparation is an aqueous injection preparation.
  • the shelf life of the liquid preparation is one to three years, preferably one to two years, more preferably one year.
  • the storage temperature of the liquid preparation is 0°C-16°C, preferably 0°C-10°C, more preferably 2°C-8°C.
  • the shelf life of the freeze-dried preparation is half a year to two years, preferably half a year to one year, more preferably half a year.
  • the storage temperature of the freeze-dried preparation is ⁇ 42°C, preferably ⁇ 37°C, more preferably ⁇ 30°C.
  • the pharmaceutically acceptable carrier includes: a surfactant, a solution stabilizer, an isotonic regulator, a buffer, or a combination thereof.
  • the pharmaceutically acceptable carrier is selected from the following group: infusion solution carrier and/or injection carrier, preferably, the carrier is one or more carriers selected from the following group : Physiological saline, glucose saline, or a combination thereof.
  • the solution stabilizer is selected from the group consisting of carbohydrate solution stabilizers, amino acid solution stabilizers, alcohol solution stabilizers, or combinations thereof.
  • the sugar solution stabilizer is selected from the group consisting of reducing sugar solution stabilizers or non-reducing sugar solution stabilizers.
  • the amino acid solution stabilizer is selected from the group consisting of monosodium glutamate or histidine.
  • the alcohol solution stabilizer is selected from the group consisting of trihydric alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol, or combinations thereof.
  • the isotonicity regulator is selected from the group consisting of sodium chloride or mannitol.
  • the buffer is selected from the group consisting of TRIS, histidine buffer, phosphate buffer, or a combination thereof.
  • the administration objects of the pharmaceutical composition or preparation include humans or non-human animals.
  • the non-human animals include: rodents (such as rats, mice), primates (such as monkeys).
  • the administration amount is 0.01-10 g/day, preferably 0.05-5000 mg/day, more preferably 0.1-3000 mg/day.
  • the pharmaceutical composition or preparation is used to inhibit and/or treat virus intrusion into the host, preferably for inhibiting and/or treating viral infection; more preferably for inhibiting and/or treating new coronavirus infection .
  • the suppression and/or treatment of viral infection includes delaying the development of related symptoms after viral infection and/or reducing the severity of these symptoms.
  • the suppression and/or treatment of viral infection also includes alleviation of symptoms associated with existing viral infection and prevention of other symptoms.
  • the pharmaceutical composition or preparation can be administered in combination with other antiviral or anti-inflammatory drugs.
  • the other antiviral or anti-inflammatory drugs administered in combination are selected from the group consisting of virus replication inhibitors, hormonal anti-inflammatory drugs, biological response modifiers, monoclonal antibodies, or combinations thereof.
  • the viral replication inhibitors include: drugs that affect the synthesis and replication of viral nucleic acids, and drugs that act on nucleic acid reverse transcription.
  • the drug affecting the synthesis and replication of viral nucleic acid includes remdesivir.
  • the drug acting on nucleic acid reverse transcription is selected from the group consisting of Molnupiravir, Baloxavir, Favipiravir, or a combination thereof.
  • the hormonal anti-inflammatory drugs include anti-estrogens, aromatase inhibitors or anti-androgens; preferably, the anti-estrogens are selected from the group consisting of tamoxifen, droloxifene, Exemestane, or a combination thereof; the aromatase inhibitor is selected from the group consisting of Amglutethimide, Lantron, Letrozole, Arimidex, or a combination thereof; the antiandrogen is selected from the group : Flutamide RH-LH agonist/antagonist: Noredex, Enerton, or a combination thereof.
  • the biological response modifier includes: interferon, interleukin-2, thymosin, or a combination thereof.
  • the monoclonal antibody includes antibodies that block virus invasion, or anti-inflammatory antibodies; preferably, the antibodies that block virus invasion include: LY-CoV555, LY-CoV016, REGN10933, REGN10987 , AZD8895, AZD1061, VIR-7831, BRII-196, DXP-604, or a combination thereof; the anti-inflammatory antibodies include: Tocilizumab, Sarilumab, or a combination thereof.
  • the eighth aspect of the present invention there is provided a method for preparing the small protein targeting S protein of the first aspect of the present invention or its recombinant protein or the fusion protein described in the third aspect of the present invention, comprising the steps of:
  • step (b) purifying and/or separating the culture obtained in step (a) to obtain the small protein targeting S protein or its recombinant protein or fusion protein.
  • the small protein targeting S protein described in the first aspect of the present invention or its fusion protein, or its immunoconjugate, for the preparation of medicaments, reagents, detection plates or Kit; wherein, the reagent, detection plate or kit is used for: detecting the S protein or the new coronavirus in the sample; wherein, the medicament is used for treating and/or preventing the new coronavirus infection.
  • the medicament is used to block the invasion of the human body by the new coronavirus.
  • the reagent is one or more reagents selected from the group consisting of isotopic tracers, contrast agents, flow detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles and imaging agents .
  • the reagent for detecting the SARS-CoV-2 S protein in the sample is a contrast agent (in vivo) for detecting the SARS-CoV-2 or virus S protein.
  • the detection is an in vivo detection or an in vitro detection.
  • the detection includes flow cytometry detection, cellular immunofluorescence detection, or a combination thereof.
  • the agent is used to block the interaction between the S protein and ACE2 protein of the new coronavirus.
  • the SARS-CoV-2 includes, but is not limited to: wild-type SARS-CoV-2, alpha mutant, beta mutant, Gamma mutant, Delta mutant or a combination thereof.
  • a method for treating and/or preventing novel coronavirus infection comprising the step of: administering a safe and effective amount of the small protein targeting S protein described in the first aspect of the present invention to a subject in need Or its recombinant protein or the fusion protein described in the second aspect, or the immunoconjugate described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect.
  • the treatment and/or prevention of SARS-CoV-2 infection includes blocking the invasion of SARS-CoV-2 into the human body.
  • Figure 1 shows the structural simulation diagram of the super high affinity binding small protein and S protein complex targeting the new coronavirus S protein.
  • A is the complex protein structure of human ACE2 and S protein.
  • B is the structural simulation diagram of the small protein NC_139_error_2 binding complex with S protein.
  • Figure 2 shows the binding activity of the small ultra-high affinity protein targeting the S protein of the new coronavirus Delta mutant strain detected by flow cytometry.
  • the super-high affinity small protein targeting S protein is displayed on the surface of yeast, and the yeast displaying the small protein is traced with anti-Myc tag antibody FITC (ab1394); F(ab') 2 -goat anti-human IgG is used Fc secondary antibody, PE (H10104) will be able to track yeast cells bound to S protein with Fc tag.
  • Figure 3 shows the competitive binding activity of the small ultra-high affinity protein targeting S protein and human ACE2 protein detected by flow cytometry.
  • Figure 4 shows the determination of the targeting affinity of the ultrahigh affinity small protein targeting the S protein using biofilm interferometry (BLI).
  • BBI biofilm interferometry
  • Figure 5 shows the determination of the targeting affinity of the ultrahigh affinity small protein targeting the S protein using biofilm interferometry (BLI).
  • BBI biofilm interferometry
  • Figure 6 shows the thermal stability of small ultrahigh-affinity proteins targeting S protein measured by CD spectrometer.
  • the protein circular dichroism of NC_139_error_Delta_3 was observed at three temperatures: 25°C, heating up to 95°C, and cooling down to 25°C, and then evaluated the changes in the secondary structure of the protein before and after heating up.
  • Figure 7 shows the Tm value of the ultra-high affinity small protein targeting S protein measured by CD spectrometer. Among them, observe the circular dichroism signal of the protein detected during the process of NC_139_error_Delta_3 gradually heating up from 25°C to 95°C. According to the protein circular dichroism signal changing with time, the Tm value of the protein was calculated.
  • Figure 8 shows a schematic diagram of several structural combinations of small high-affinity proteins targeting the S protein and their fusion proteins.
  • A is a small protein short peptide chain targeting S protein
  • B is a small protein targeting the S protein and the antibody hinge region (hinge) or linker (linker) and CH2, CH3 in series to form a polypeptide chain, and the small protein (or fragment) targeting the S protein with high affinity provided by the present invention forms a target Single/multi-target fusion protein to S protein;
  • C is a small protein targeting the S protein in series with the antibody hinge region (hinge) or linker (linker) and CH3 to form a polypeptide chain, with the help of the small protein (or fragment) targeting the S protein with high affinity provided by the present invention to form a targeting S protein Single/multiple targeting fusion proteins of proteins;
  • D is a small protein targeting the S protein that is connected in series with the antibody hinge region (hinge) or linker (linker) and CH3 to form a polypeptide chain, with the help of the high-affinity small protein (or fragment) provided by the present invention to form a single/multiple protein targeting the S protein targeting fusion proteins;
  • E is after the small protein targeting S protein is connected with the small protein targeting S protein through a linker sequence, and then forms a polypeptide chain in series with the antibody hinge region (hinge) or linker (linker) and CH2 and CH3.
  • a small protein (or fragment) targeting the S protein with affinity forms a single/multiple targeting fusion protein targeting the S protein;
  • F is that the small protein targeting S protein is connected with the small protein targeting S protein through a linker sequence, and then forms a polypeptide chain in series with the antibody hinge region (hinge) or linker (linker) and CH3, with the help of the high affinity target protein provided by the present invention
  • G is that the small protein targeting S protein is connected with the small protein targeting S protein through a linker sequence, and then forms a polypeptide chain in series with the antibody hinge region (hinge) or linker (linker) and CH3, with the help of the high affinity target protein provided by the present invention
  • a single/multiple targeting fusion protein targeting the S protein is formed to a small protein (or fragment) of the S protein.
  • Figure 9 shows the in vitro pseudovirus neutralizing protective activity of NC_139_error_Delta_3 on the wild type of the new coronavirus and the main mutant strains alpha, beta, Gamma and Delta respectively.
  • the inventors designed a class of small proteins with super high affinity targeting the S protein of the new coronavirus for the interaction surface of ACE2 and the S protein.
  • the binding site of this small protein can almost completely cover the binding site of ACE2 protein on S protein.
  • the small high-affinity protein NC_139_error_2 of the present invention can broadly bind to the main mutant strains of the new coronavirus alpha, beta, gamma and Delta.
  • the ultra-high affinity small proteins NC_139_error_Delta_3 and NC_139_error_Delta_9 for the S protein of the Delta mutant strain of the new coronavirus were further optimized.
  • the protein exhibits good neutralizing and protective activity against the new coronavirus Delta mutant strain, and shows good neutralizing and protective activity against the new coronavirus wild type, alpha, beta and Gamma, and has broad-spectrum neutralizing and protective activity against the new coronavirus .
  • the small protein of the present invention has a smaller molecular weight and has potentially better tissue penetration and structural stability. The present invention has been accomplished on this basis.
  • the typical ultra-high-affinity small protein targeting the S protein is less than about 60 amino acids in length, has a molecular weight much smaller than that of conventional antibodies, and has no Fc part of the antibody, so it has better tissue penetration.
  • the ultra-high-affinity small protein targeting the S protein of the present invention has higher affinity and can be used as a potential new coronavirus diagnosis and in vivo tracer reagent.
  • the invention targets the super high affinity small protein and fusion protein of S protein
  • a class of ultrahigh-affinity small protein targeting S protein and a fusion protein comprising the small protein or a conjugate thereof are provided.
  • small protein of the present invention As used herein, the terms "small protein of the present invention”, “small ultra-high affinity protein targeting the S protein of the new coronavirus of the present invention”, and “small ultra-high affinity protein targeting the S protein of the present invention” are used interchangeably , all refer to the small protein with super high affinity for the S protein of the new coronavirus described in the first aspect of the present invention.
  • S protein of the present invention includes the S protein of the novel coronavirus wild type and its alpha, beta, gamma and Delta mutant strains.
  • the small protein of the present invention has the amino acid sequence shown in SEQ ID NO: 1, 3 or 5.
  • fusion protein of the present invention refers to a fusion protein formed by the ultra-high affinity small protein targeting S protein of the present invention and other fusion elements, for example, the small protein of the present invention can be combined with the hinge region, Fc A fusion protein formed by elements such as regions.
  • the fusion protein of the present invention has super high affinity to ACE2.
  • the term "with super high affinity for ACE2" means that the affinity of the small protein or fusion protein of the present invention to the S protein is much higher than the affinity of the ACE2 protein and the S protein of the Delta mutant strain, such as the small protein or fusion protein of the present invention
  • the affinity Q1 of the protein to the S protein of the Delta mutant strain is at least 1.5, at least 2 times or more of the affinity Q0 of the ACE2 protein to the S protein of the Delta mutant strain; or, the Kd value of the small protein or fusion protein of the present invention to the S protein of the Delta mutant strain
  • the ratio (Z1/Z0) of Z1 and ACE2 protein to the Kd value Z0 of Delta mutant S protein is ⁇ 1/1.5, more preferably ⁇ 1/2 or ⁇ 1/3 or more.
  • the ultra-high-affinity fusion protein of the present invention can be any small ultra-high-affinity protein that at least contains the complete targeting S protein or a partial amino acid fragment thereof (usually at least 70% of the length of the amino acid fragment).
  • the fusion protein of the present invention can have the following structure:
  • the small ultra-high affinity protein or its fragments targeting the S protein can be single or multiple (such as 2, 3 or 4 ultra-high affinity small proteins or fragments thereof in tandem form, for example, Fig. 2E, 2F and 2G ).
  • small ultra-high affinity protein targeting S protein also includes variant forms with S protein binding activity and ACE2/S protein blocking activity. These variations include (but are not limited to): 1-3 (usually 1-2, preferably 1) amino acid deletions, insertions and/or substitutions, additions or deletions at the C-terminal and/or N-terminal One or several (usually within 3, preferably within 2, more preferably within 1) amino acids, or add an amino acid fragment with a smaller amino acid side chain at the N-terminal or C-terminal of the small protein as a linker (such as glycine, serine, etc.). For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein.
  • adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein.
  • the term also includes monomeric and multimeric forms of the polypeptides of the invention.
  • the term also includes linear as well as non-linear polypeptides (eg, cyclic peptides).
  • the present invention also includes active fragments, derivatives and analogs of the above-mentioned small protein targeting S protein or fusion protein (especially fusion protein formed with Fc fragment).
  • fragment refers to a polypeptide that substantially retains the function or activity of the S protein-targeting ultrahigh affinity small protein or fusion protein of the present invention.
  • polypeptide fragments, derivatives or analogs of the present invention can be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more A polypeptide with substituent groups in amino acid residues, or (iii) a polypeptide fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence fused A polypeptide formed from this polypeptide sequence (a fusion protein formed by fusing with a leader sequence, a secretory sequence, or a tag sequence such as 6His).
  • Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.
  • a preferred class of active derivatives refers to that compared with the amino acid sequence of the present invention, at most 5, preferably at most 3, and more preferably at most 1 amino acid are replaced by amino acids with similar or similar properties to form polypeptides. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the invention also provides analogs of the fusion proteins of the invention.
  • the difference between these analogs and the polypeptide of the present invention may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or both.
  • Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, ⁇ , ⁇ -amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
  • the super high affinity small protein or fusion protein targeting S protein of the present invention can also be modified.
  • Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of polypeptides during synthesis and processing or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.
  • polynucleotide of the present invention may be a polynucleotide that encodes an ultrahigh-affinity small protein or a fusion protein targeting the S protein of the present invention, or a polynucleotide that also includes additional coding and/or non-coding sequences .
  • the present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or fusion proteins having the same amino acid sequence as the present invention.
  • These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering its encoded target S The function of ultra-high affinity small proteins or fusion proteins of proteins.
  • the present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides hybridizable under stringent conditions (or stringent conditions) to the polynucleotides of the present invention.
  • stringent conditions refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%.
  • the ultra-high affinity small protein or fusion protein and polynucleotide targeting S protein of the present invention are preferably provided in an isolated form, more preferably, purified to homogeneity.
  • the full-length polynucleotide sequence of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.
  • recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
  • the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • the method of amplifying DNA/RNA using PCR technique is preferably used to obtain the polynucleotide of the present invention.
  • the RACE method RACE-cDNA terminal rapid amplification method
  • the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, And can be synthesized by conventional methods.
  • Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or the super high affinity small protein or fusion protein coding sequence targeting the S protein of the present invention, and recombinant technology Methods of producing the polypeptides of the invention.
  • polynucleotide sequences of the present invention can be used to express or produce recombinant fusion proteins by conventional recombinant DNA techniques. Generally speaking, there are the following steps:
  • the polynucleotide sequence encoding the fusion protein can be inserted into the recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.
  • any suitable carrier can be used, which can be selected from pET, pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+ ), one of pDHFR, the expression vector includes a fusion DNA sequence connected with appropriate transcription and translation regulatory sequences.
  • eukaryotic/prokaryotic host cells can be used for the expression of the super high affinity small protein targeting S protein of the present invention or its fusion protein
  • eukaryotic host cells are preferably mammalian or insect host cell culture systems, preferably COS, CHO, NSO, Both sf9 and sf21 cells; prokaryotic host cells are preferably one of lemo21, DH5a, BL21(DE3), and TG1.
  • Methods well known to those skilled in the art can be used to construct an expression vector containing the fusion protein coding DNA sequence of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis.
  • promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (such as ginseng cells).
  • Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl2 . Transformation can also be performed by electroporation, if desired.
  • DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional media according to the host cells used.
  • the culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.
  • the recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. 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 precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the method of affinity chromatography can be used to separate and purify a class of ultra-high affinity small protein targeting S protein or its fusion protein disclosed in the present invention.
  • conventional methods such as high salt can be used. Buffer, changing PH and other methods to elute the super high affinity small protein targeting S protein or its fusion protein bound on the affinity column.
  • the small ultra-high affinity protein targeting the S protein or its fusion protein can be purified into a substantially uniform substance, such as a single band on SDS-PAGE electrophoresis.
  • a pharmaceutical composition containing the small protein targeting the S protein or the fusion protein or the immunoconjugate thereof of the present invention is also provided.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the small protein or fusion protein (or its conjugate) of the present invention and pharmaceutically acceptable carrier or excipient.
  • a safe and effective amount such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt% of the small protein or fusion protein (or its conjugate) of the present invention and pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be made into an injection form, for example, by normal methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under ster
  • the active ingredient is administered in a therapeutically effective amount, for example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptides of the invention can also be used with other therapeutic agents.
  • the small protein or fusion protein targeting the S protein or its immunoconjugate can be combined with pharmaceutically acceptable adjuvants to form a pharmaceutical preparation so as to exert a more stable therapeutic effect. These preparations can ensure the targeting of the S protein of the present invention Structural integrity of the amino acid core sequence of a small protein or its fusion protein, while protecting the protein's multifunctional groups from degradation (including but not limited to aggregation, deamination, or oxidation).
  • the preparations can be in various forms.
  • liquid preparations for liquid preparations, they can be stored stably for at least one year at 2°C-8°C, and for freeze-dried preparations, they can be kept stable for at least six months at 30°C.
  • the preparations here can be suspension, aqueous injection, freeze-dried and other preparations commonly used in the pharmaceutical field, preferably aqueous injection or freeze-dried preparations.
  • the pharmaceutically acceptable adjuvant includes one or a combination of a surfactant, a solution stabilizer, an isotonic regulator and a buffer
  • the surfactant includes nonionic surfactants such as polyoxyethylene sorbitan fatty acid ester (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate ; tetradecyl, linoleyl or octadecyl sarcosine; Pluronics; Sexual sugars, amino acids include monosodium glutamate or histidine, alcohols include one of trihydric alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol or a combination thereof, and the addition amount of the solution stabilizer should make the final formed Those skilled in the art believe that the preparation
  • a safe and effective amount of the small protein or fusion protein or immunoconjugate thereof of the present invention is administered to the mammal, wherein the safe and effective amount is usually at least about 50 ⁇ g/kg body weight, and in most cases Preferably the dose is about 100 micrograms/kg body weight to about 50 mg/kg body weight.
  • the total dosage cannot exceed a certain range, for example, the dosage for intravenous injection is 10 to 3000 mg/day/50kg, preferably 100 to 1000 mg/day/50kg.
  • the small protein targeting S protein or its fusion protein of the present invention and pharmaceutical preparations containing it can be used as an anti-tumor drug for tumor treatment.
  • the anti-tumor drug referred to in the present invention refers to a drug that inhibits and/or treats tumors, It can include a delay in the development of symptoms associated with tumor growth and/or a reduction in the severity of these symptoms, and it further includes a reduction of existing tumor growth associated with symptoms and preventing the appearance of other symptoms, and also reducing or preventing metastasis.
  • antineoplastic drugs for combined administration include but are not limited to: 1 , Cytotoxic drugs (1) Drugs that act on the chemical structure of DNA: alkylating agents such as nitrogen mustards, nitrosouries, and methylsulfonates; platinum compounds such as cisplatin, carboplatin, and oxalplatin; Mitomycin (MMC); (2) Drugs affecting nucleic acid synthesis: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta, etc.; thymidine synthase inhibitors such as fluorouracils (5FU, FT -207, capecitabine), etc.; purine nucleoside synthase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG, etc.; nucleotide reductase inhibitors
  • Biological response modifiers mainly suppress tumor interferon through the body's immune function; interleukin-2; thymosin; 4.
  • Monoclonal antibodies MabThera (MabThera); Cetuximab (C225); Bevacizumab (Avastin); Yervoy (Ipilimumab); Nivolumab (OPDIVO); Pembrolizumab (Keytruda); Atezolizumab (Tecentriq); Retinoids; Inducers of Apoptosis.
  • the binding site of the small protein targeting the S protein of the new coronavirus provided by the present invention can almost cover the binding site between ACE2 and the S protein.
  • the small protein of the present invention has a smaller molecular weight, less than about 60 amino acids in length, and better tumor penetration.
  • the small protein of the present invention has a super high affinity for the S protein of the new coronavirus, and can effectively block the invasion of the host cell by the new coronavirus.
  • the small protein of the present invention has ultra-high structural stability, and its Tm value is greater than 95°C.
  • Candidate proteins were screened using yeast display library technology.
  • the synthesized candidate protein gene was electroporated into EBY-100 yeast cells with the ratio of 2:1 to the pETCON carrier fragment by electroporation. After culturing at 30°C for 2 days with the help of double-deficient (-Ura/-Trp) culture plates, the electroporation efficiency (greater than 1 ⁇ 10 5 ) was confirmed.
  • the electroporated yeast cells were cultured in double-deficient medium (30° C., 250 rpm) for two days.
  • the displayed proteins were induced to express in a lactose-rich induction medium at a dilution ratio of 1:100.
  • Fc-tagged S protein was used as the target protein, and F(ab') 2 -goat anti-human IgG Fc secondary antibody, PE(H10104) and anti-Myc tag antibody FITC(ab1394) were used for dual-color flow cytometry dyeing.
  • the FITC-positive cells are yeast cells displaying the protein, and the PE/FITC double positive indicates that the displayed protein can bind to the target protein S protein with affinity. According to the affinity, the PE/FITC double-positive yeast cells corresponding to the ultra-high affinity are screened out, and then the gene sequence of the candidate protein that can bind to the target protein (that is, the ultra-high affinity small protein targeting the S protein) is obtained by gene sequencing .
  • NC_139_error_2 is a broad-spectrum high-affinity small protein targeting the new coronavirus S protein, its amino acid sequence is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 2.
  • NC_139_error_Delta_3 and NC_139_error_Delta_9 are small ultra-high affinity proteins targeting the S protein of the new coronavirus Delta mutant strain.
  • amino acid sequence of NC_139_error_Delta_3 is shown in SEQ ID NO: 3
  • its nucleotide sequence is shown in SEQ ID NO: 4.
  • the amino acid sequence of NC_139_error_Delta_9 is shown in SEQ ID NO: 5
  • its nucleotide sequence is shown in SEQ ID NO: 6.
  • the obtained small protein was simulated with the help of RoseTTAFold, as shown in Figure 1B, the small protein has an ⁇ structure.
  • the structure is displayed with the help of ChimeraX, as shown in Figure 1A is the structure diagram of the complex of the new coronavirus S protein and ACE2 protein (6M0J).
  • the binding site of the small protein on the S protein of the new coronavirus almost completely overlaps the binding site of the ACE2 protein on the S protein.
  • the N-terminus of the synthesized small protein nucleotide sequence was added with a start codon, and then loaded into the pETCON vector at the XhoI and NedI restriction sites.
  • the vector loaded with the small protein gene was transferred to EBY-100 yeast cells with the help of a yeast transformation kit. After culturing at 30°C for 2 days with the help of double-deficient (-Ura/-Trp) culture plates, the electroporation efficiency (greater than 1 ⁇ 10 5 ) was confirmed.
  • the yeast cells after electroporation were cultured in double deficient medium (30° C., 225 rpm) for two days.
  • the displayed proteins were induced to express in a lactose-rich induction medium at a dilution ratio of 1:100.
  • F(ab') 2 -goat anti-human IgG Fc secondary antibody, PE(H10104) and anti-Myc tag antibody FITC(ab1394) for two-color flow staining.
  • the FITC-positive cells are yeast cells displaying the protein, and the PE/FITC double positive indicates that the displayed protein can bind to the target protein.
  • the candidate protein displayed by NC_139_error_2 on the surface of yeast cells exhibited strong binding activity at the concentration of the target protein at 500pM and 100pM, showing a PE/FITC double positive signal.
  • the target protein concentration was diluted to 24.5pM and 12.25pM, respectively, and the two proteins still showed relatively strong binding activity at the target protein concentration of 24.5pM. Strong binding activity, still has binding activity when the target protein concentration is 12.25pM, showing PE/FITC double positive signal.
  • the target protein S protein concentration was selected to be 100 pM, and the ACE2 protein concentration was respectively 200 nM and 0 nM, and incubated with the target protein S Delta -Fc for 30 minutes at room temperature. The protein incubation mixture was then incubated with yeast cells expressing the candidate protein for 45 minutes at room temperature.
  • Competitive binding activity of candidate proteins was assessed by dual-color flow cytometry. When the competing protein ACE2 is at a concentration of 2nM (supersaturated concentration), the candidate binding protein can still show good competitive protection activity.
  • the affinity detection of high-affinity blocking protein was carried out with the help of ForteBio Octet.
  • the detection probe with the S protein was simultaneously immersed in the double-diluted high-affinity small protein solution targeting the S protein, and the binding signal was detected (300s). Then immerse the probe in PBST to detect the dissociation signal of the bound protein.
  • the affinities of the high-affinity blocking binding proteins were calculated.
  • NC_139_error_Delta_3 and NC_139_error_Delta_9 showed super strong binding activity to the S protein of the new crown Delta mutant strain, and their affinities were 7.58 ⁇ 10 -10 M and 6.083 ⁇ 10 -9 M, respectively.
  • NC_139_error_2 showed good binding activity against the wild-type 2019-nCoV and the main mutant strains alpha, beta, gamma and Delta respectively. Its affinities are 9.399 ⁇ 10 -9 M, 9.022 ⁇ 10 -9 M, 8.444 ⁇ 10 -9 M, 1.799 ⁇ 10 -8 M and 1.448 ⁇ 10 -8 M, showing broad-spectrum binding to the S protein of the new coronavirus active.
  • Example 6 Structural stability detection of high-affinity small proteins targeting S protein
  • the stability of protein structure was detected by JASCO-1500. Choose to detect from the wavelength range of 190nm-260nm, first measure the circular dichroism signal of NC_139_error_Delta_3 at 25°C (0.1mg/ml) protein, then heat the protein to 95°C to detect the circular dichroism signal of the protein, and finally return the temperature to Circular dichroism signal after standing at 25°C for 5 minutes. Obtain the conformational changes of the secondary structure of the protein at different temperatures, and then evaluate the structural stability of the binding protein.
  • NC_139_error_Delta_3 showed a higher secondary structure of ⁇ -helical protein at 25°C.
  • the secondary structure of the protein changed to some extent due to the influence of high temperature. But when the temperature was lowered to 25°C again, the circular dichroism signals almost completely overlapped, indicating that the secondary structure of the protein returned to the situation before the temperature was raised.
  • the protein exhibits superior thermal stability.
  • Example 7 Determination of the Tm value of the high-affinity blocking binding protein targeting the S protein
  • the circular dichroism signal of NC_139_error_Delta_3 was measured at 25°C (0.1mg/ml).
  • the wavelength of 222nm was selected to detect the circular dichroism signal during the process of gradually heating the protein from 25°C to 95°C. Among them, 2°C/min and equilibrate for 30 seconds per minute. Then obtain the Tm value of the protein.
  • the circular dichroic signal increases as the temperature increases, the circular dichroic signal only increases to a small extent when the detection limit temperature of the instrument is 95°C. According to the signal curve, it is determined that its Tm exceeds the upper limit of the detection temperature of the instrument, and the Tm is greater than 95°C.
  • the protein exhibits superior thermal stability.
  • Example 8 Expression and purification of fusion protein
  • a fusion protein of a small ultrahigh affinity protein was prepared.
  • the structure of the prepared fusion protein is shown in B in Figure 8, and the amino acid sequence is SEQ ID NO: 11, 13, or 15. Methods as below:
  • Example 4 the BLI method of Example 4 was used to measure the binding of the fusion protein to the S protein, and the results showed that the prepared fusion protein could bind to the S protein with superhigh affinity.
  • Example 9 In vitro neutralization and protection activity test of small ultra-high affinity protein targeting S protein
  • NC_139_error_Delta_3 the in vitro pseudovirus neutralizing protective activity of NC_139_error_Delta_3 on the wild type of the new coronavirus and the main mutant strains alpha, beta, Gamma and Delta will be evaluated.
  • NC_139_error_Delta_3 After incubating NC_139_error_Delta_3 with the new coronavirus pseudovirus with luciferase reporter gene at 37°C for 1 hour at the concentration shown in Figure 9, it was incubated with 293T cells highly expressing human ACE2 in a 37°C incubator for 24 hours. After removing the culture supernatant, add 100 ⁇ l D-luciferin and incubate for 2 minutes.

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Abstract

本发明提供了一类靶向COVID-19新冠病毒突变株Delta的Spike蛋白(S蛋白)的超高亲和力小蛋白及用途。具体地，本发明提供了一类靶向新冠病毒Delta突变株Spike蛋白且具有超高亲和力的结合蛋白。本发明蛋白能结合在S蛋白RBD区域，能阻断新冠病毒与ACE2受体的结合，进而阻断新冠病毒入侵宿主细胞。本发明还提供了包括所述靶向新冠病毒Delta突变株S蛋白的超高亲和力的融合蛋白,该蛋白不仅对COVID-19病毒Delta突变株表现出较好的中和保护活性，而且针对野生型新冠病毒及其主要突变株Alpha、Beta和Gamma都表现出较好的中和保护活性，表现出针对新冠病毒的广谱阻断保护活性。

Description

一类靶向COVID-19病毒S蛋白的超高亲和力小蛋白及用途 技术领域
本发明属于生物技术和医药领域，具体涉及靶向COVID-19病毒S蛋白的超高亲和力小蛋白及其融合蛋白。
背景技术
COVID-19病毒是一种单链RNA病毒，其主要通过分布在病毒表面的Spike蛋白(S蛋白)与宿主ACE2受体蛋白特异性结合，进而入侵机体。
冠状病毒表面的刺突蛋白(S)，它是一种大型I类融合蛋白。S蛋白形成了一个三聚体复合物，在功能上可分为两个不同的由一个蛋白酶切割位点分隔开的亚基，即S1和S2。S1亚基包含受体结合结构域(RBD)，它与宿主细胞受体蛋白相互作用，触发膜融合。S2亚基包含膜融合复合物，包括疏水性融合肽和α-螺旋七肽重复区。冠状病毒通过S蛋白的受体结合区(RBD)与宿主细胞表面受体结合，介导病毒的入侵。因此，S蛋白被认为是预防与治疗冠状病毒感染宿主细胞的重要靶点。目前，针对COVID-19的疫苗(重组疫苗和mRNA)和中和性抗体都将S蛋白作为主要靶点，进行疫苗的制备与中和性抗体的筛选。
综上所述，本领域迫切需要开发出一种能够更高效地阻断新冠病毒入侵宿主的药物。
发明内容
本发明的目的就是提供了一类靶向新冠病毒S蛋白的超高亲和力小蛋白，所述小蛋白能够更加高效地阻断S蛋白与ACE2蛋白的结合，进而阻断新冠病毒入侵宿主细胞。
本发明的另一目的是提供一类基于靶向新冠病毒S蛋白的超高亲和力小蛋白的融合蛋白及其制备方法。
在本发明的第一方面，提供了一种靶向新冠病毒S蛋白的小蛋白，所述小蛋白能特异性靶向结合新冠病毒的S蛋白，表现出超强的亲和力，并且能够与ACE2竞争性结合S蛋白，有效阻断新冠病毒野生型及其alpha、beta、gamma和Delta突变株S蛋白与ACE2蛋白的结合。
在另一优选例中，所述的小蛋白有一条肽链构成，主要形成三个α-螺旋二级结构。
在另一优选例中，所述小蛋白能特异性靶向结合新冠病毒的S蛋白，表现出较强的亲和力，分别针对新冠病毒野生型、alpha、beta、gamma和delta表现出较强的 结合活性；其中，所述的小蛋白由一条肽链构成，主要形成三个α-螺旋二级结构；
并且，所述的小蛋白其氨基酸序列具有SEQ ID No:1或如SEQ ID NO：1所示。
在另一优选例中，所述小蛋白能特异性靶向结合新冠病毒Delta突变株的S蛋白，表现出超强的亲和力，并且能够与ACE2受体竞争性结合新冠病毒S蛋白，有效阻断新冠病毒S蛋白与ACE2受体蛋白的结合；并且针对新冠病毒野生型、alpha、beta和gamma都表现出较好的中和保护活性；其中，所述的小蛋白由一条肽链构成，主要形成三个α-螺旋二级结构；
并且，所述的小蛋白其氨基酸序列具有或如SEQ ID NO：3或5所示。
在另一优选例中，所述的小蛋白的氨基酸序列如SEQ ID NO：1、3或5所示。本发明还提供了一种重组蛋白，所述重组蛋白包括串联在一起的两个或多个本发明的靶向S蛋白的小蛋白。
在本发明的第二方面，提供了一种融合蛋白，所述融合蛋白包括第一多肽和/或第二多肽；
其中，所述第一多肽从N端到C端具有如式I所示的结构，所述第二多肽从N端到C端具有如式II所示的结构，
P-Mx-H-Fc       (式I)
P-Fc-H-Mx       (式II)
其中，
P为无或信号肽序列；
M为S蛋白结合区(或结合元件)，所述S蛋白结合区的氨基酸序列来自如第一方面所述的靶向S蛋白的小蛋白的氨基酸序列；
H为铰链区；
Fc为无或免疫球蛋白的恒定区，或其片段；
“-”表示连接上述元件的肽键或连接肽；
x为1-4的正整数。
在另一优选例中，所述的“来自所述的靶向S蛋白的小蛋白的氨基酸序列”指，所述S蛋白结合区(或结合元件)的氨基酸序列与所述的靶向S蛋白的小蛋白的氨基酸序列相同或基本相同(即同源性≥90％，较佳地≥95％，更佳地≥98％)，并且所述的S蛋白结合区(或结合元件)保留与新冠病毒Delta突变株S蛋白的结合活性(较佳地，保留≥70％，更佳地≥80％的结合活性)。
在另一优选例中，所述P的氨基酸序列选自下组：
(i)如SEQ ID NO:17所示的序列；
(ii)在SEQ ID NO:17的基础上，进行一个或多个氨基酸残基的替换、缺失、改变或插入，或在其N端或C端添加1至10个氨基酸残基，更佳地1至5个氨基酸 残基，从而获得的氨基酸序列。
在另一优选例中，编码所述M的核苷酸序列如SEQ ID NO:18所示。
在另一优选例中，所述融合蛋白为单体或二聚体。
在另一优选例中，所述融合蛋白是同源二聚体或异源二聚体。
在另一优选例中，所述第一多肽与第一多肽之间、所述第二多肽与第二多肽之间，或第一多肽与第二多肽之间，可通过各自Fc上的半胱氨酸C形成二硫键。
在另一优选例中，所述二聚体选自下组：两条第一多肽形成的同源二聚体、两条第二多肽形成的同源二聚体，或由第一多肽和第二多肽形成的异源二聚体。
在另一优选例中，所述融合蛋白是两条第一多肽形成的同源二聚体。
在另一优选例中，所述M的序列如SEQ ID NO:1、3或5所示。
在另一优选例中，所述x为1、2、3或4个，较佳地为2个。
在另一优选例中，所述H为人免疫球蛋白的铰链区。
在另一优选例中，所述人免疫球蛋白选自下组：IgG1、IgG4，或其组合。
在另一优选例中，所述人免疫球蛋白是IgG1。
在另一优选例中，所述H的氨基酸序列选自下组：
(i)如SEQ ID NO:7所示的序列；
(ii)在SEQ ID NO:7的基础上，进行一个或多个氨基酸残基的替换、缺失、改变或插入，或在其N端或C端添加1至10个氨基酸残基，更佳地1至5个氨基酸残基，从而获得的氨基酸序列。
在另一优选例中，编码所述H的核苷酸序列如SEQ ID NO:8所示。
在另一优选例中，所述的Fc为人免疫球蛋白的恒定区或其片段。
在另一优选例中，所述Fc是人免疫球蛋白的CH2区和CH3区的串联序列，或仅为人免疫球蛋白的CH3区。
在另一优选例中，所述Fc的氨基酸序列选自下组：
(i)如SEQ ID NO:9所示的序列；
(ii)在SEQ ID NO:9的基础上，进行一个或多个氨基酸残基的替换、缺失、改变或插入，或在其N端或C端添加1至30个氨基酸序列，较佳地1至10个氨基酸残基，更佳地1至5个氨基酸残基，从而获得的氨基酸序列。
在另一优选例中，编码所述Fc的核苷酸序列如SEQ ID NO:10所示。
在另一优选例中，所述第一多肽的氨基酸序列选自下组：
(i)如SEQ ID NO:11、13、或15所示的序列；
(ii)在SEQ ID NO:11、13、或15的基础上，进行一个或多个氨基酸残基的替换、缺失、改变或插入，或在其N端或C端添加1至30个氨基酸序列，较佳地1至10个氨基酸残基，更佳地1至5个氨基酸残基，从而获得的氨基酸序列。
在另一优选例中，所述第一多肽的氨基酸序列如SEQ ID NO:11、13、或15所 示，编码所述第一多肽的核苷酸序列如SEQ ID NO:12、14或16所示。
在本发明的第三方面，提供了一种多核苷酸，所述多核苷酸编码本发明第一方面靶向S蛋白的小蛋白、或其重组蛋白或本发明第二方面所述的融合蛋白。
在另一优选例中，所述多核苷酸的序列如SEQ ID NO:2、4、6、12、14或16所示。
在本发明的第四方面，提供了一种载体，所述载体中含有本发明第三方面所述的多核苷酸。
在另一优选例中，所述载体为：pET载体、pGEM-T载体、pcDNA3.1，或其组合。
在本发明的第五方面，提供了一种宿主细胞，所述宿主细胞中含有第四方面所述的载体，或基因组中整合有第三方面所述的多核苷酸。
在本发明的第六方面，提供了一种免疫偶联物，该免疫偶联物含有：
(a)本发明第一方面所述的靶向S蛋白的小蛋白或其重组蛋白或第二方面所述的融合蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联物选自下组：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂。
在本发明的第七方面，提供了一种药物组合物，其包括：
(a)本发明第一方面所述的靶向S蛋白的小蛋白或其重组蛋白或本发明第二方面所述的融合蛋白，或其编码基因；或本发明第六方面所述的免疫偶联物；和
(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物用于诊断或治疗表达S蛋白的新冠病毒。
在另一优选例中，所述组分(a)的含量为0.1-99.9wt％,较佳地10-99.9wt％，更佳地70％-99.9wt％。
在另一优选例中，所述的药物组合物的剂型为口服剂型、注射剂、或外用药物剂型。
在另一优选例中，所述药物组合物的剂型包括片剂、颗粒剂、胶囊、口服液、 或注射剂。
在另一优选例中，所述药物组合物或制剂选自下组：混悬制剂、液体制剂或冻干制剂。
在另一优选例中，所述液体制剂为水针制剂。
在另一优选例中，所述液体制剂的保存期限为一年至三年，较佳地一年至两年，更佳地一年。
在另一优选例中，所述液体制剂的保存温度为0℃-16℃，较佳地0℃-10℃，更佳地2℃-8℃。
在另一优选例中，所述冻干制剂的保存期限为半年至两年，较佳地半年至一年，更佳地半年。
在另一优选例中，所述冻干制剂的保存温度为≤42℃，较佳地≤37℃，更佳地≤30℃。
在另一优选例中，所述药学上可接受的载体包括：表面活性剂、溶液稳定剂、等渗调节剂、缓冲液，或其组合。
在另一优选例中，所述的药学上可接受的载体选自下组：输液剂载体和/或注射剂载体，较佳地，所述的载体是选自下组的一种或多种载体：生理盐水、葡萄糖盐水、或其组合。
在另一优选例中，所述溶液稳定剂选自下组：糖类溶液稳定剂、氨基酸类溶液稳定剂、醇类溶液稳定剂，或其组合。
在另一优选例中，所述糖类溶液稳定剂选自下组：还原性糖类溶液稳定剂或非还原性糖类溶液稳定剂。
在另一优选例中，所述氨基酸类溶液稳定剂选自下组：谷氨酸单钠或组氨酸。
在另一优选例中，所述醇类溶液稳定剂选自下组：三元醇、高级糖醇、丙二醇、聚乙二醇，或其组合。
在另一优选例中，所述等渗调节剂选自下组：氯化钠或甘露醇。
在另一优选例中，所述缓冲液选自下组：TRIS、组氨酸缓冲液、磷酸盐缓冲液，或其组合。
在另一优选例中，所述药物组合物或制剂的施用对象为包括人或非人动物。
在另一优选例中，所述非人动物包括：啮齿动物(如大鼠、小鼠)、灵长动物(如猴)。
在另一优选例中，在所述药物组合物或制剂的施用中，施用的量为0.01-10g/天，较佳地0.05-5000mg/天，更佳地0.1-3000mg/天。
在另一优选例中，所述药物组合物或制剂用于抑制和/或治疗病毒入侵宿主，优选地用于抑制和/或治疗病毒感染；更优选地用于抑制和/或治疗新冠病毒感染。
在另一优选例中，所述抑制和/或治疗病毒感染包括伴随病毒感染后相关症状发 展的延迟和/或这些症状严重程度的降低。
在另一优选例中，所述抑制和/或治疗病毒感染还包括已存在的病毒感染伴随症状的减轻并防止其他症状的出现。
在另一优选例中，对于肿瘤的治疗，所述药物组合物或制剂可以和其他的抗病毒或抗炎症药物联合给药。
在另一优选例中，所述联合给药的其他的抗病毒或抗炎症药物选自下组：病毒复制抑制剂、激素类抗炎症药物、生物反应调节剂、单克隆抗体、或其组合。
在另一优选例中，所述病毒复制抑制剂包括：影响病毒核酸合成与复制的药物、作用于核酸逆转录的药物。
在另一优选例中，所述影响病毒核酸合成与复制的药物包括：瑞德西韦。
在另一优选例中，所述作用于核酸逆转录的药物选自下组：莫纳皮拉韦(Molnupiravir)、巴罗沙韦、法匹拉韦、或其组合。
在另一优选例中，所述激素类抗炎症药物包括抗雌激素、芳香化酶抑制剂或抗雄激素；较佳地，所述抗雌激素选自下组：三苯氧胺、屈洛昔芬、依西美坦、或其组合；所述芳香化酶抑制剂选自下组：氨鲁米特、兰特隆、来曲唑、瑞宁德、或其组合；所述抗雄激素选自下组：氟它氨RH-LH激动剂/拮抗剂：诺雷德、依那通、或其组合。
在另一优选例中，所述生物反应调节剂包括：干扰素、白细胞介素-2、胸腺肽类、或其组合。
在另一优选例中，所述单克隆抗体包括阻断病毒入侵的抗体、或抗炎症抗体；较佳地，所述阻断病毒入侵的抗体包括：LY-CoV555、LY-CoV016、REGN10933、REGN10987、AZD8895、AZD1061、VIR-7831、BRII-196、DXP-604、或其组合；所述抗炎症抗体包括：Tocilizumab、Sarilumab、或其组合。
在本发明的第八方面，提供了一种制备本发明第一方面的靶向S蛋白的小蛋白或其重组蛋白或本发明的第三方面所述的融合蛋白的方法，包括步骤：
(a)在合适的条件下，培养本发明第五方面所述的宿主细胞，从而获得含所述小蛋白或其重组蛋白或融合蛋白的培养物；和
(b)对步骤(a)中得到的培养物进行纯化和/或分离，获得所述的靶向S蛋白的小蛋白或其重组蛋白或融合蛋白。
在本发明的第九方面，提供了本发明第一方面所述的靶向S蛋白的小蛋白，或其融合蛋白、或其免疫偶联物的用途，用于制备药剂、试剂、检测板或试剂盒；其中，所述试剂、检测板或试剂盒用于：检测样品中的S蛋白或新冠病毒；其中，所述药剂用于治疗和/或预防新冠病毒感染。
在另一优选例中，所述药剂用于阻断新冠病毒入侵人体。
在另一优选例中，所述的试剂为选自下组的一种或多种试剂：同位素示踪剂、造影剂、流式检测试剂、细胞免疫荧光检测试剂、纳米磁粒和显像剂。
在另一优选例中，所述检测样品中新冠病毒S蛋白的试剂为(体内)检测新冠病毒或病毒S蛋白的造影剂。
在另一优选例中，所述的检测为体内检测或体外检测。
在另一优选例中，所述的检测包括流式检测、细胞免疫荧光检测，或其组合。
在另一优选例中，所述的药剂用于阻断新冠病毒S蛋白和ACE2蛋白的相互作用。
在另一优选例中，所述的新冠病毒包括但不限于：野生型新冠病毒、alpha突变株、beta突变株、Gamma突变株、Delta突变株或其组合。
在本发明的第十方面，提供了一种治疗和/或预防新冠病毒感染的方法，包括步骤：给需要的对象施用安全有效量的本发明第一方面所述的靶向S蛋白的小蛋白或其重组蛋白或第二方面所述的融合蛋白、或第六方面所述的免疫偶联物、或第七方面所述的药物组合物。
在另一优选例中，所述治疗和/或预防新冠病毒感染包括阻断新冠病毒入侵人体。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了靶向新冠病毒S蛋白的超高亲和力结合小蛋白与S蛋白复合物结构模拟图。其中，A为人ACE2与S蛋白复合物蛋白结构。
B为小蛋白NC_139_error_②与S蛋白结合复合物结构模拟图。
图2显示了采用流式法检测的靶向新冠病毒Delta突变株S蛋白的超高亲和力小蛋白的结合活性。其中，将靶向S蛋白的超高亲和力小蛋白展示在酵母表面，用anti-Myc tag antibody FITC(ab1394)将展示小蛋白的酵母进行示踪；用F(ab') 2-羊抗人IgG Fc二抗,PE(H10104)将能够对带有Fc标签的S蛋白结合的酵母细胞进行示踪。
图3显示了采用流式法检测的靶向S蛋白的超高亲和力小蛋白与人ACE2蛋白的竞争结合活性。
其中，将不同浓度的人ACE2蛋白与带有Fc标签的Delta突变株S蛋白室温孵育后，与展示靶向S蛋白的超高亲和力小蛋白的酵母进行孵育。采用流式细胞仪， 借助anti-Myc tag antibody FITC(ab1394)和F(ab') 2-羊抗人IgG Fc二抗,PE(H10104)双染色评估靶向S蛋白的超高亲和力小蛋白与人ACE2蛋白的竞争结合活性。
图4显示了采用生物膜干涉技术(BLI)测定靶向S蛋白的超高亲和力小蛋白靶向的亲和力。其中，NC_139_error_Delta_③和NC_139_error_Delta_⑨对新冠Delta突变株的S蛋白表现出超强的结合活性，其亲和力分别为7.58×10 -10M和6.083×10 -9M。
图5显示了采用生物膜干涉技术(BLI)测定靶向S蛋白的超高亲和力小蛋白靶向的亲和力。其中，分别将带有Fc标签的野生型、alpha、beta、Gamma或Delta突变株S蛋白包被在检测探头上后，检测不同浓度的靶向S蛋白的超高亲和力小蛋白(NC_139_error_②)与S蛋白的亲和力。
图6显示了采用CD光谱仪测定靶向S蛋白的超高亲和力小蛋白的热稳定性。其中，观察NC_139_error_Delta_③在25℃、升温至95℃、降温至25℃三种温度下的蛋白圆二色性，进而评估该蛋白在升温前后，蛋白二级结构发生的改变。
图7显示了CD光谱仪测定靶向S蛋白的超高亲和力小蛋白的Tm值。其中，观察NC_139_error_Delta_③在25℃逐渐升温至95℃过程中检测蛋白的圆二色信号。根据随时间点变化的蛋白圆二色信号，计算出该蛋白的Tm值。
图8显示了靶向S蛋白高亲和力小蛋白及其融合蛋白的几种结构组合示意图。
其中，A为靶向S蛋白的小蛋白短肽链；
B为靶向S蛋白的小蛋白与抗体铰链区(hinge)或接头(linker)以及CH2、CH3串联形成多肽链，借助本发明提供的高亲和力靶向S蛋白的小蛋白(或片段)形成靶向S蛋白的单/多靶向融合蛋白；
C为靶向S蛋白的小蛋白与抗体铰链区(hinge)或接头(linker)以及CH3串联形成多肽链，借助本发明提供的高亲和力靶向S蛋白的小蛋白(或片段)形成靶向S蛋白的单/多靶向融合蛋白；
D为靶向S蛋白的小蛋白与抗体铰链区(hinge)或接头(linker)以及CH3串联形成多肽链，借助本发明提供的高亲和力小蛋白(或片段)形成靶向S蛋白的单/多靶向融合蛋白；
E为靶向S蛋白的小蛋白通过接头序列与靶向S蛋白的小蛋白连接后，与抗体铰链区(hinge)或接头(linker)以及CH2和CH3串联形成多肽链，借助本发明提供的高亲和力靶向S蛋白的小蛋白(或片段)形成靶向S蛋白的单/多靶向融合蛋白；
F为靶向S蛋白的小蛋白通过接头序列与靶向S蛋白的小蛋白连接后，与抗体铰链区(hinge)或接头(linker)以及CH3串联形成多肽链，借助本发明提供的高亲和力靶向S蛋白的小蛋白(或片段)形成靶向S蛋白的单/多靶向融合蛋白；
G为靶向S蛋白的小蛋白通过接头序列与靶向S蛋白的小蛋白连接后，与抗体铰链区(hinge)或接头(linker)以及CH3串联形成多肽链，借助本发明提供的高亲和力 靶向S蛋白的小蛋白(或片段)形成靶向S蛋白的单/多靶向融合蛋白。
图9显示NC_139_error_Delta_③分别对新冠病毒野生型和主要突变株alpha、beta、Gamma及Delta的体外假病毒中和保护活性。分别将NC_139_error_Delta_③按照如图9所示浓度分别与带有荧光素报告酶基因的新冠假病毒孵育后，采用高表达人ACE2的293T细胞确定其中和保护活性。
具体实施方式
经过广泛而深入的研究，本发明人基于新冠病毒S蛋白与ACE2蛋白结构复合物，针对ACE2与S蛋白相互作用面，设计获得了一类靶向新冠病毒S蛋白的超高亲和力小蛋白。该小蛋白的结合位点能够几乎完全覆盖ACE2蛋白在S蛋白上的结合位点。实验表明，具有本发明的高亲和力小蛋白NC_139_error_②能够广谱结合新冠病毒主要突变株alpha、beta、gamma和Delta。在此基础上，进一步优化获得了针对新冠病毒的Delta突变株S蛋白的超高亲和力小蛋白NC_139_error_Delta_③、NC_139_error_Delta_⑨。该蛋白对新冠病毒Delta突变株表现出较好的中和保护活性，对新冠病毒野生型、alpha、beta和Gamma都表现出较好的中和保护活性，具有针对新冠病毒广谱中和保护活性。本发明的小蛋白较传统抗体来讲，分子量更小，具有潜在更好的组织穿透性和结构稳定性。在此基础上完成了本发明。
具体地，代表性的靶向S蛋白的超高亲和力小蛋白，长度小于约60个氨基酸，分子量远小于常规抗体，且无抗体Fc部分，因此具有更好的组织穿透性。此外，本发明的靶向S蛋白的超高亲和力小蛋白，具有更高的亲和力，可以作为潜在的新冠病毒诊断与体内示踪试剂。
本发明靶向S蛋白的超高亲和力小蛋白以及融合蛋白
在本发明中，提供了一类靶向S蛋白的超高亲和力小蛋白以及一种包含所述小蛋白的融合蛋白或其偶联物。
如本文所用，术语“本发明的小蛋白”、“本发明的靶向新冠病毒S蛋白的超高亲和力小蛋白”、“本发明的靶向S蛋白的超高亲和力小蛋白”可互换使用，均指具有本发明第一方面中所述的对新冠病毒S蛋白具有超高亲和力的小蛋白。
如本文所用，术语“本发明的S蛋白”，包括新冠病毒野生型及其alpha、beta、gamma和Delta突变株的S蛋白。
优选地，本发明所述的小蛋白具有如SEQ ID NO:1、3或5所示的氨基酸序列。
如本文所用，术语“本发明融合蛋白”是指本发明所述的靶向S蛋白的超高亲和力小蛋白与其他融合元件形成的融合蛋白，例如，本发明的小蛋白可以与铰链区、Fc区等元件形成的融合蛋白。本发明的融合蛋白对ACE2具有超高亲和力。
如本文所用，术语“对ACE2具有超高亲和力”指，本发明的小蛋白或融合蛋白 对S蛋白的亲和力远远高于ACE2蛋白与Delta突变株S蛋白的亲和力，例如本发明小蛋白或融合蛋白对Delta突变株S蛋白的亲和力Q1是ACE2蛋白对Delta突变株S蛋白亲和力Q0的至少1.5，至少2倍或更多；或者，本发明小蛋白或融合蛋白对Delta突变株S蛋白的Kd值Z1与ACE2蛋白对Delta突变株S蛋白的Kd值Z0的比值(Z1/Z0)≤1/1.5，更佳地≤1/2或≤1/3或更多。优选地，本发明的超高亲和力的融合蛋白可以是任何至少包含完整的靶向S蛋白的超高亲和力小蛋白或其部分氨基酸片段(通常至少70％长度的氨基酸片段)。
典型地，本发明融合蛋白可具有下述结构：
靶向S蛋白的超高亲和力小蛋白或片段-Hinge-CH2-CH3的Y字形结构；
靶向S蛋白的超高亲和力小蛋白或片段-Hinge-CH3的Y字形结构；
靶向S蛋白的超高亲和力小蛋白或片段-示踪标记；
靶向S蛋白的超高亲和力小蛋白或片段。
应理解，以上结构类型仅为示例形式，并不限制本发明。一些代表性的结构如图2所示。其中，靶向S蛋白的超高亲和力小蛋白或其片段可以为单个或多个(如串联形式的2个、3个或4个超高亲和力小蛋白或其片段，例如图2E、2F和2G)。
如本文所用，术语“靶向S蛋白的超高亲和力小蛋白”或“融合蛋白”还包括具有S蛋白结合活性以及ACE2/S蛋白阻断活性的变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，更佳地1个)氨基酸的缺失、插入和/或取代，在C末端和/或N末端添加或缺失一个或数个(通常为3个以内，较佳地为2个以内，更佳地为1个以内)氨基酸，或在小蛋白N端或C端添加氨基酸侧链较小的氨基酸片段作为linker(如甘氨酸、丝氨酸等)。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式的本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
本发明还包括上述靶向S蛋白的小蛋白或融合蛋白(尤其是与Fc片段形成的融合蛋白)的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明靶向S蛋白的超高亲和力小蛋白或融合蛋白的功能或活性的多肽。
本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与本发明的氨基酸序列相比，有至多5个，较佳地至 多3个，更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供本发明融合蛋白的类似物。这些类似物与本发明的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
此外，还可以对本发明靶向S蛋白的超高亲和力小蛋白或融合蛋白进行修饰。修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
术语“本发明的多核苷酸”可以是包括编码本发明靶向S蛋白的超高亲和力小蛋白或融合蛋白的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的 多肽或融合蛋白的片段、类似物和衍生物。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的靶向S蛋白的超高亲和力小蛋白或融合蛋白的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件(或严紧条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明的靶向S蛋白的超高亲和力小蛋白或融合蛋白和多核苷酸优选以分离的形式提供，更佳地，被纯化至均质。
本发明多核苷酸全长序列通常可以通过PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的多核苷酸。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
表达载体
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明靶向S蛋白的超高亲和力小蛋白或融合蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的融合蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明融合蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，编码融合蛋白的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
在本发明的靶向S蛋白的超高亲和力小蛋白或其融合蛋白制备方法中，可以使用任何合适的载体，可选自pET、pDR1、pcDNA3.1(+)、pcDNA3.1/ZEO(+)、pDHFR之一，表达载体中包括连接有合适的转录和翻译调节序列的融合DNA序列。
真核/原核宿主细胞均可用于本发明的靶向S蛋白的超高亲和力小蛋白或其融合蛋白的表达，真核宿主细胞优选哺乳动物或昆虫宿主细胞培养系统，优选COS、CHO、NS0、sf9及sf21等细胞均；原核宿主细胞优选为lemo21、DH5a、BL21(DE3)、TG1之一。
本领域的技术人员熟知的方法能用于构建含本发明融合蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母、植物细胞(如人参细胞)。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时 将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
可以利用亲和层析的方法对本发明公开的一类靶向S蛋白的超高亲和力小蛋白或其融合蛋白进行分离纯化，根据所利用的亲和柱的特性，可以使用常规的方法例如高盐缓冲液、改变PH等方法洗脱结合在亲和柱上的靶向S蛋白的超高亲和力小蛋白或其融合蛋白。
利用上述方法，可以将靶向S蛋白的超高亲和力小蛋白或其融合蛋白纯化为基本均一的物质，例如在SDS-PAGE电泳上为单一条带。
药物组合物
在本发明中，还提供了一种含有本发明靶向S蛋白的小蛋白或融合蛋白或其免疫偶联物的药物组合物。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明的小蛋白或融合蛋白(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制 备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。所述的靶向S蛋白的小蛋白或融合蛋白或其免疫偶联物可以和药学上可以接受的辅料一起组成药物制剂从而更稳定地发挥疗效，这些制剂可以保证本发明的靶向S蛋白的小蛋白或其融合蛋白的氨基酸核心序列的结构完整性，同时还要保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。所述制剂可以是各种形态，通常情况下，对于液体制剂，通常可以在2℃-8℃条件下至少稳定保存一年，对于冻干制剂，在30℃至少六个月保持稳定。在这里制剂可为制药领域常用的混悬、水针、冻干等制剂，优选水针或冻干制剂。
对于本发明的靶向S蛋白的药物组合物(如水针或冻干制剂)，其中药学上可以接受的辅料包括表面活性剂、溶液稳定剂、等渗调节剂和缓冲液之一或其组合，其中表面活性剂包括非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或80)；poloxamer(如poloxamer 188)；Triton；十二烷基硫酸钠(SDS)；月桂硫酸钠；十四烷基、亚油基或十八烷基肌氨酸；Pluronics；MONAQUATTM等，其加入量应使蛋白的颗粒化趋势最小，溶液稳定剂可以为糖类，包括还原性糖和非还原性糖，氨基酸类包括谷氨酸单钠或组氨酸，醇类包括三元醇、高级糖醇、丙二醇、聚乙二醇之一或其组合，溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到在稳定的时间内保持稳定状态，等渗调节剂可以为氯化钠、甘露醇之一，缓冲液可以为TRIS、组氨酸缓冲液、磷酸盐缓冲液之一。
使用药物组合物时，是将安全有效量的本发明的小蛋白或融合蛋白或其免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约50微克/千克体重，而且在大多数情况下不超过约100毫克/千克体重，较佳地该剂量是约100微克/千克体重-约50毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。典型地，通常，总给药量不能超过一定范围，例如静脉注射的剂量是10至3000mg/天/50kg，较佳地100至1000mg/天/50kg。
本发明的靶向S蛋白的小蛋白或其融合蛋白和含有其的药物制剂可以作为抗肿瘤药物用于肿瘤治疗，本发明所称的抗肿瘤药物，指具有抑制和/或治疗肿瘤的药物，可以包括伴随肿瘤生长相关症状发展的延迟和/或这些症状严重程度的降低，它进一步还包括已存在的肿瘤生长伴随症状的减轻并防止其他症状的出现，还也减少或防止转移。
上述靶向S蛋白的小蛋白或其融合蛋白及其药物制剂还可以和其他的抗肿瘤药联合给药，用于肿瘤的治疗，这些用于联合给药的抗肿瘤药包括但不限于：1、细胞毒类药物(1)作用于DNA化学结构的药物：烷化剂如氮芥类、亚硝尿类、甲基磺酸酯类；铂类化合物如顺铂、卡铂和草酸铂等；丝裂霉素(MMC)；(2)影响核酸合成的药物：二氢叶酸还原酶抑制剂如甲氨喋呤(MTX)和Alimta等；胸腺核苷合成酶 抑制剂如氟尿嘧啶类(5FU、FT-207、卡培他滨)等；嘌呤核苷合成酶抑制剂如6-巯基嘌呤(6-MP)和6-TG等；核苷酸还原酶抑制剂如羟基脲(HU)等；DNA多聚酶抑制剂如阿糖胞苷(Ara-C)和健择(Gemz)等；(3)作用于核酸转录的药物：选择性作用于DNA模板，抑制DNA依赖RNA聚合酶，从而抑制RNA合成的药物如：放线菌素D、柔红霉素、阿霉素、表阿霉素、阿克拉霉素、光辉霉素等；(4)主要作用于微管蛋白合成的药物：紫杉醇、泰索帝、长春花碱、长春瑞滨、鬼臼硷类、高三尖杉酯碱；(5)其他细胞毒药：门冬酰胺酶主要抑制蛋白质的合成；2、激素类抗雌激素：三苯氧胺、屈洛昔芬、依西美坦等；芳香化酶抑制剂：氨鲁米特、兰特隆、来曲唑、瑞宁德等；抗雄激素：氟它氨RH-LH激动剂/拮抗剂：诺雷德、依那通等；3、生物反应调节剂：主要通过机体免疫功能抑制肿瘤干扰素；白细胞介素-2；胸腺肽类；4、单克隆抗体：美罗华(MabThera)；Cetuximab(C225)；赫赛汀(Trastuzumab)；Bevacizumab(Avastin)；Yervoy(Ipilimumab)；Nivolumab(OPDIVO)；Pembrolizumab(Keytruda)；Atezolizumab(Tecentriq)；5、其他括一些目前机制不明和有待进一步研究的药物；细胞分化诱导剂如维甲类；细胞凋亡诱导剂。
本发明的主要优点包括：
1)本发明所提供的靶向新冠病毒S蛋白的小蛋白，其结合位点能够几乎覆盖ACE2与S蛋白的结合位点。
2)本发明的小蛋白分子量较小，长度小于约60个氨基酸，拥有更好的肿瘤穿透性。
3)本发明的小蛋白对新冠病毒S蛋白具有超高的亲和力，能够有效阻断新冠病毒入侵宿主细胞。
4)本发明的小蛋白具有超高的结构稳定性，其Tm值大于95℃。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
本发明序列如下表1所示
表1.本发明序列
Figure PCTCN2021139445-appb-000001
Figure PCTCN2021139445-appb-000002
Figure PCTCN2021139445-appb-000003
实施例1：高亲和力靶向新冠病毒S蛋白的合成
1.1高亲和力靶向新冠病毒S蛋白的筛选
采用酵母展示文库技术对候选蛋白进行筛选。首先将合成的候选蛋白基因借助 电转法与pETCON载体片段按照2：1的比例，电转至EBY-100酵母细胞。借助双缺陷(-Ura/-Trp)培养板30℃培养2天后，确认其电转效率(大于1×10 5)。电转后的酵母细胞在双缺陷培养基(30℃，250rpm)培养两天后。按照1：100稀释比例，在富含乳糖的诱导培养基中进行展示蛋白的诱导表达。当OD600＝0.5时，采用Fc标签的S蛋白作为靶蛋白，借助F(ab') 2-羊抗人IgG Fc二抗,PE(H10104)和anti-Myc tag antibody FITC(ab1394)进行双色流式染色。其中FITC阳性细胞为展示蛋白的酵母细胞，PE/FITC双阳性表示该展示蛋白能够与靶蛋白S蛋白发生亲和结合。按亲和力大小，将对应于超高亲和力的PE/FITC双阳性酵母细胞筛选出来，进而通过基因测序获得能够与靶蛋白结合的候选蛋白(即靶向S蛋白的超高亲和力小蛋白)的基因序列。
1.2靶向S蛋白高亲和力小蛋白的合成
采用全基因合成的方法，合成靶向S蛋白的超高亲和力小蛋白基因，命名为NC_139_error_②、NC_139_error_Delta_③和NC_139_error_Delta_⑨。其中，NC_139_error_②为靶向新冠病毒S蛋白广谱高亲和力小蛋白，其氨基酸序列如SEQ ID NO：1所示，其核苷酸序列如SEQ ID NO：2所示。
NC_139_error_Delta_③和NC_139_error_Delta_⑨为靶向新冠病毒Delta突变株S蛋白的超高亲和力小蛋白。其中，NC_139_error_Delta_③的氨基酸序列如SEQ ID NO：3所示，其核苷酸序列如SEQ ID NO：4所示。NC_139_error_Delta_⑨的氨基酸序列如SEQ ID NO：5所示，其核苷酸序列如SEQ ID NO：6所示。将合成好的核苷酸序列的N端加上起始密码子后，在XhoI和NedI酶切位点处装入pET29b(+)表达载体。
1.3靶向新冠病毒S蛋白高亲和力小蛋白的结构模拟
将获得的小蛋白借助RoseTTAFold进行蛋白结构模拟，如图1B所示小蛋白为ααα结构。借助ChimeraX进行结构显示，如图1A为新冠病毒S蛋白与ACE2蛋白复合物结构图(6M0J)。借助分子对接软件将小蛋白与新冠病毒S蛋白进行分子对接。如图1所示，小蛋白在新冠病毒S蛋白的结合位点几乎与ACE2蛋白结合在S蛋白的结合位点完全重叠。
实施例2：超高亲和力小蛋白的表达纯化
将该载体转化大肠杆菌后，在LB培养基37℃，270rpm培养至OD 600＝0.6。然后采用1mM的IPTG诱导菌液蛋白表达过夜。收菌后，加入Protease Inhibitor Cocktail和
Figure PCTCN2021139445-appb-000004
核酸酶，借助超声破菌(4分钟，10s on，10s off，80％Amp)后取上清。借助Ni柱纯化后，将浓缩后的样品过分子筛进一步纯化。采用SDS-PAGE和考马斯亮蓝染色评估蛋白表达与纯化。借助法进一步确定蛋白的浓度。
通过该方法获得高纯度候选蛋白用于后续试验。
实施例3：靶向新冠病毒S蛋白高亲和力小蛋白结合活性检测
在本实施例中，将合成的小蛋白核苷酸序列N端加上起始密码子后，在XhoI和NedI酶切位点处装入pETCON载体。借助酵母转化试剂盒将装入小蛋白基因的载体转至EBY-100酵母细胞。借助双缺陷(-Ura/-Trp)培养板30℃培养2天后，确认其电转效率(大于1×10 5)。电转后的酵母细胞在双缺陷培养基(30℃，225rpm)培养两天后。按照1：100稀释比例，在富含乳糖的诱导培养基中进行展示蛋白的诱导表达。当OD600＝0.5时，采用带有Fc标签的新冠病毒Delta突变株S蛋白作为靶蛋白，按照如图所示浓度稀释后与酵母细胞室温孵育45分钟。借助F(ab') 2-羊抗人IgG Fc二抗,PE(H10104)和anti-Myc tag antibody FITC(ab1394)进行双色流式染色。其中FITC阳性细胞为展示蛋白的酵母细胞，PE/FITC双阳性表示该展示蛋白能够与靶蛋白结合。
如图2所示，NC_139_error_②展示在酵母细胞表面的候选蛋白在靶蛋白浓度在500pM和100pM浓度下表现出较强的结合活性，表现出PE/FITC双阳性信号。为了进一步明确NC_139_error_Delta_③和NC_139_error_Delta_⑨对Delta突变株S蛋白的超强结合活性，将靶蛋白浓度分别稀释到24.5pM和12.25pM情况下，这两个蛋白在靶蛋白浓度为24.5pM情况下依然表现出较强的结合活性，在靶蛋白浓度为12.25pM情况下依然具有结合活性，表现出表现出PE/FITC双阳性信号。
实施例4：靶向S蛋白的高亲和力小蛋白竞争结合活性检测
在本实施例中，为了进一步确证靶向S蛋白的高亲和力小蛋白与新冠病毒S蛋白的竞争结合活性。我们先将过不同浓度生物素标记的人ACE2蛋白与带人抗体Fc区域的新冠病毒S蛋白融合蛋白室温孵育20分钟后，然后与展示靶向S蛋白高亲和力小蛋白的酵母细胞进行孵育，然后借助F(ab')2-羊抗人IgG Fc二抗,PE(H10104)和anti-Myc tag antibody FITC(ab1394)进行双色流式评估竞争结合活性。其中FITC阳性细胞为展示蛋白的酵母细胞，PE/FITC双阳性表示展示的小蛋白能够与S蛋白结合。
如图3所示，选择靶蛋白S蛋白浓度在100pM情况下，ACE2蛋白浓度分别从200nM和0nM，与靶蛋白S Delta-Fc在室温孵育30分钟。然后将该蛋白孵育混合物与表达候选蛋白的酵母细胞进行室温孵育45分钟。通过双色流式评估候选蛋白的竞争结合活性。竞争蛋白ACE2在2nM浓度下(过饱和浓度)，候选结合蛋白依然能够表现出较好的竞争保护活性。
实施例5：靶向S蛋白的高亲和力小蛋白亲和力测定
在本实施例中，借助ForteBio Octet对高亲和力阻断蛋白进行亲和力检测。首先 将3μg/ml带有Fc标签的新冠病毒S蛋白装载到偶联Protein A的检测探头上(300s)，在PBST溶液中洗脱尚未结合的带有Fc标签的新冠病毒S蛋白。然后将带有S蛋白的检测探头同时浸没在等两倍比稀释的靶向S蛋白高亲和力小蛋白溶液中，检测结合信号(300s)。再将探头浸没PBST中，检测结合蛋白的解离信号。最终计算出高亲和力阻断结合蛋白的亲和力。
如图4所示，NC_139_error_Delta_③和NC_139_error_Delta_⑨对新冠Delta突变株的S蛋白表现出超强的结合活性，其亲和力分别为7.58×10 -10M和6.083×10 -9M。如图5所示，NC_139_error_②分别针对野生型新冠病毒和主要突变株alpha、beta、gamma和Delta都表现出较好的结合活性。其亲和力分别为9.399×10 -9M、9.022×10 -9M、8.444×10 -9M、1.799×10 -8M和1.448×10 -8M，表现出针对新冠病毒S蛋白的广谱结合活性。
实施例6：靶向S蛋白高亲和力小蛋白结构稳定性检测
借助JASCO-1500对蛋白结构稳定性进行检测。选择从190nm-260nm波长范围进行检测，首先测定NC_139_error_Delta_③在25℃(0.1mg/ml)蛋白的圆二色信号，然后将蛋白升温至95℃后检测蛋白的圆二色信号，最后将温度恢复到25℃并静置5分钟后的圆二色信号。获得该蛋白在不同温度下，蛋白二级结构构象的变化，进而评估结合蛋白的结构稳定性。
如图6所示，NC_139_error_Delta_③在25℃时呈现出较高的α螺旋蛋白二级结构。升温至95℃时，该蛋白的二级结构由于高温影响发生一定变化。但随着温度再次降温至25℃后，其圆二色信号几乎完全重叠，表明该蛋白的二级结构恢复至升温前的情况。该蛋白表现出超强的热稳定性。
实施例7：靶向S蛋白高亲和力阻断结合蛋白Tm值测定
借助JASCO-1500，测定NC_139_error_Delta_③在25℃(0.1mg/ml)蛋白的圆二色信号。选择波长为222nm进行检测蛋白从25℃开始逐渐升温至95℃过程中的圆二色信号。其中，2℃/分钟且每分钟平衡30秒。进而获得该蛋白的Tm值。
如图7所示，虽然随着温度提高其圆二色信号有所升高，但在仪器检测极限温度95℃情况下，其圆二色信号仅有较小幅度的升高。根据该信号曲线，确定其Tm超过仪器检测温度上限，Tm大于95℃。该蛋白表现出超强的热稳定性。
实施例8：融合蛋白的表达纯化
在本实施例中，制备超高亲和力小蛋白的融合蛋白。所制备的融合蛋白的结构如图8中的B所示，氨基酸序列为SEQ ID NO:11、13、或15。方法如下：
将信号肽序列SEQ ID NO：17分别与融合蛋白的编码序列SEQ ID NO:12、14、 或16连接后，分别引入pcDNA3.1载体的多克隆位点，将该载体转染293F细胞后，在细胞培养摇床培养6天。收取细胞培养上清并过滤后，借助Protein A柱纯化并将样品进一步超滤浓缩。采用SDS-PAGE和考马斯亮蓝染色评估蛋白表达与纯化。
此外，采用实施例4的BLI方法测定融合蛋白与S蛋白的结合情况，结果表明，制备的融合蛋白可以与S蛋白以超高亲和力结合。
实施例9：靶向S蛋白的超高亲和力小蛋白体外中和保护活性试验
在本实施例总，将评估NC_139_error_Delta_③分别对新冠病毒野生型和主要突变株alpha、beta、Gamma及Delta的体外假病毒中和保护活性。分别将NC_139_error_Delta_③按照如图9所示浓度分别与带有荧光素报告酶基因的新冠假病毒37℃孵育1小时后，与高表达人ACE2的293T细胞37℃孵箱培养24小时。去掉培养上清后，加入100μl D-luciferin孵育2分钟。将混合液稀释10倍后，取150μl检测荧光素酶活性，进而计算出该蛋白分别对新冠野生型和主要突变株alpha、beta、Gamma及Delta的体外假病毒中和保护活性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种靶向新冠病毒S蛋白的小蛋白，其特征在于，所述小蛋白能特异性靶向结合新冠病毒的S蛋白，表现出较强的亲和力，分别针对新冠病毒野生型、alpha、beta、gamma和delta表现出较强的结合活性；
    其中，所述的小蛋白由一条肽链构成，主要形成三个α-螺旋二级结构；
    并且，所述的小蛋白其氨基酸序列如SEQ ID NO：1所示。
  2. 一种靶向新冠病毒Delta突变株S蛋白的小蛋白，其特征在于，所述小蛋白能特异性靶向结合新冠病毒Delta突变株的S蛋白，表现出超强的亲和力，并且能够与ACE2受体竞争性结合新冠病毒S蛋白，有效阻断新冠病毒S蛋白与ACE2受体蛋白的结合；并且针对新冠病毒野生型、alpha、beta和gamma都表现出较好的中和保护活性；
    其中，所述的小蛋白由一条肽链构成，主要形成三个α-螺旋二级结构；
    并且，所述的小蛋白其氨基酸序列如SEQ ID NO：3或5所示。
  3. 一种重组蛋白，其特征在于，所述重组蛋白包括串联在一起的两个或多个权利要求1或2所述的靶向S蛋白的小蛋白。
  4. 一种融合蛋白，其特征在于，所述融合蛋白包括第一多肽和/或第二多肽；
    其中，所述第一多肽从N端到C端具有如式I所示的结构，所述第二多肽从N端到C端具有如式II所示的结构，
    P-Mx-H-Fc  (式I)
    P-Fc-H-Mx  (式II)
    其中，
    P为无或信号肽序列；
    M为S蛋白结合区(或结合元件)，所述S蛋白结合区的氨基酸序列来自如权利要求1或2所述的靶向S蛋白的小蛋白的氨基酸序列；
    H为铰链区；
    Fc为无或免疫球蛋白的恒定区，或其片段；
    “-”表示连接上述元件的肽键或连接肽；
    x为1-4的正整数。
  5. 一种多核苷酸，其特征在于，所述多核苷酸编码如权利要求1或2所述的靶向S蛋白的小蛋白、权利要求3所述的重组蛋白、或如权利要求4所述的融合蛋白。
  6. 一种载体，其特征在于，所述载体中含有如权利要求5所述的多核苷酸。
  7. 一种宿主细胞，其特征在于，所述宿主细胞中含有如权利要求6所述的载体，或基因组中整合有如权利要求5所述的多核苷酸。
  8. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)如权利要求1或2所述的靶向S蛋白的小蛋白、如权利要求3所述的重组蛋白、或如权利要求4所述的融合蛋白；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  9. 一种药物组合物，其特征在于，包括：
    (a)如权利要求1或2所述的靶向S蛋白的小蛋白、或权利要求3所述的重组蛋白、或如权利要求3所述的融合蛋白，或其编码基因；或如权利要求8所述的免疫偶联物；和
    (b)药学上可接受的载体。
  10. 一种制备如权利要求1或2所述的靶向S蛋白的小蛋白、或权利要求3所述的重组蛋白或如权利要求4所述的融合蛋白的方法，其特征在于，包括步骤：
    (a)在合适的条件下，培养如权利要求7所述的宿主细胞，从而获得含所述小蛋白或重组蛋白或融合蛋白的培养物；和
    (b)对步骤(a)中得到的培养物进行纯化和/或分离，获得所述的靶向S蛋白的小蛋白或重组蛋白或融合蛋白。
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CN111560054A (zh) * 2020-06-16 2020-08-21 哈尔滨吉象隆生物技术有限公司 抑制新型冠状病毒感染的多肽及其筛选方法
CN113264998A (zh) * 2021-01-28 2021-08-17 四川大学华西医院 抗新冠病毒SARS-CoV-2表面S1蛋白的单链抗体及其应用
CN113264990A (zh) * 2020-02-14 2021-08-17 深圳大学 抑制新型冠状病毒（sars-cov-2）的多肽和其应用
CN113336844A (zh) * 2021-05-28 2021-09-03 闽江学院 一种靶向新冠病毒n蛋白的鲨鱼单域抗体及其制备方法和应用
CN113663073A (zh) * 2021-08-19 2021-11-19 山东大学 一种靶向s蛋白棕榈酰化的多肽在制备广谱抗冠状病毒药物中的应用

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CN113264990A (zh) * 2020-02-14 2021-08-17 深圳大学 抑制新型冠状病毒（sars-cov-2）的多肽和其应用
CN111560054A (zh) * 2020-06-16 2020-08-21 哈尔滨吉象隆生物技术有限公司 抑制新型冠状病毒感染的多肽及其筛选方法
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