WO2023060483A1 - 多肽-rbd免疫偶联物及其用途 - Google Patents

多肽-rbd免疫偶联物及其用途 Download PDF

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WO2023060483A1
WO2023060483A1 PCT/CN2021/123587 CN2021123587W WO2023060483A1 WO 2023060483 A1 WO2023060483 A1 WO 2023060483A1 CN 2021123587 W CN2021123587 W CN 2021123587W WO 2023060483 A1 WO2023060483 A1 WO 2023060483A1
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polypeptide
rbd
vaccine
immunoconjugate
present
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PCT/CN2021/123587
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English (en)
French (fr)
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刘刚
李雪媛
高军
杨俊伟
张伟
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清华大学
辽宁成大生物股份有限公司
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Priority to PCT/CN2021/123587 priority Critical patent/WO2023060483A1/zh
Publication of WO2023060483A1 publication Critical patent/WO2023060483A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum

Definitions

  • the present invention relates to the field of biomedicine, specifically, the present invention relates to a polypeptide-RBD immunoconjugate and its application, more specifically, the present invention relates to a polypeptide immunoconjugate, the use of the immunoconjugate, a Pharmaceutical compositions, vaccines, methods of preparing said vaccines, methods of preparing antibodies, and a method of stimulating the production of anti-peptide antibodies or anti-RBD antibodies in a subject.
  • Coronavirus is a positive enveloped virus with RNA, and its genome size is about 26-32kb, which is the largest RNA virus known genome. Genomic RNA and phosphorylated nucleocapsid (N) proteins are buried in the phospholipid bilayer and covered by the spike glycoprotein trimer (S), the membrane (M) protein (type III transmembrane glycoprotein) and the envelope ( E) The protein is located between the S proteins of the viral envelope.
  • Coronaviruses have a variety of hosts, including birds and mammals, especially bats. Coronavirus is a kind of virus that widely exists in nature and can cause multi-system diseases including respiratory tract, digestive tract and nervous system. Highly pathogenic coronavirus infection has become a public health problem that has attracted wide attention in the past 10 years.
  • SARS-CoV Middle East respiratory syndrome coronavirus
  • the S protein of SARS-CoV-2 is located on the outermost layer of the virus, regularly arranged in a crown structure on the membrane, and participates in the binding of the virus to the virus receptor on the surface of the host cell and mediates the process of the virus entering the cell through membrane fusion. Play an important role in the process of neutralizing antibodies.
  • the research and development of COVID-19 vaccines at home and abroad use the whole S protein or part of the S protein (receptor domain (RBD) in the S protein) as the primary target antigen.
  • RBD receptor domain
  • the molecular weight of RBD is low, and the immunogenicity of RBD monomer as antigen is poor.
  • CoV-2 mutants have RBD amino acid single-site and multiple-site mutations, which may further reduce the protection rate of vaccines targeting RBD. Therefore, how to improve the immunogenicity of RBD needs further exploration.
  • the peptide immunoconjugate is prepared into a vaccine after adding an adjuvant, which can not only improve the immunogenicity of the polypeptide, but also improve the immunogenicity of the RBD protein, and at the same time improve the broad-spectrum of the immune effect of the immunoconjugate.
  • the present invention provides a polypeptide immunoconjugate.
  • it includes: a polypeptide and the RBD of the S protein of the novel coronavirus; wherein the molar ratio of the polypeptide to the RBD of the S protein of the novel coronavirus is 1:1-15:1.
  • the polypeptide immunoconjugate formed by combining the antigenic short peptide with the carrier can increase the neutralization titer of the anti-RBD antibody.
  • the inventor creatively found that after combining the polypeptide with the RBD of the novel coronavirus S protein The obtained polypeptide immunoconjugate can significantly increase the content of anti-peptide antibody or anti-RBD antibody.
  • the polypeptide immunoconjugate produces High levels of neutralizing antibodies.
  • the polypeptide immunoconjugate may further include at least one of the following additional technical features:
  • the polypeptide is an epitope of the novel coronavirus S protein.
  • the polypeptide has the amino acid sequence shown in SEQ ID NO: 1 or has at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least Amino acid sequences with 75% identity.
  • the RBD of the novel coronavirus S protein has the amino acid sequence shown in SEQ ID NO: 2 or has at least 99%, at least 95%, at least 90%, at least 85% of SEQ ID NO: 2 , amino acid sequences of at least 80%, at least 75% identity.
  • the polypeptide is connected to the RBD through a linker.
  • the linker is sulfosuccinimide 4-(N-maleimidomethyl)cyclohexane-1-carboxylate.
  • the present invention proposes the use of the polypeptide immunoconjugate described in the first aspect in the preparation of medicaments.
  • the drug is used to prevent or treat novel coronavirus pneumonia.
  • the present invention proposes the use of the polypeptide immunoconjugate described in the first aspect in preparing a vaccine.
  • the vaccine is used to prevent or treat novel coronavirus pneumonia.
  • the vaccine provided by the invention has high neutralizing antibodies against various novel coronaviruses, and can effectively prevent or treat novel coronavirus pneumonia.
  • the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, it includes the polypeptide immunoconjugate described in the first aspect.
  • the pharmaceutical composition according to the embodiment of the present invention can significantly increase the content of anti-peptide antibody or anti-RBD antibody, and effectively prevent or treat novel coronavirus pneumonia.
  • the invention proposes a vaccine.
  • it includes the polypeptide immunoconjugate described in the first aspect; and a pharmaceutically acceptable adjuvant.
  • the vaccine according to the embodiment of the present invention has the polypeptide immunoconjugate, which can significantly increase the content of anti-peptide antibody or anti-RBD antibody. Therefore, the vaccine retention after combining the polypeptide immunoconjugate with the adjuvant It can produce high anti-peptide and anti-RBD antibodies in the human body stimulated by a variety of new coronaviruses.
  • the immunogenicity of peptides and RBD to the body is further improved, thereby improving the antibody
  • the titer can achieve the effect of effectively preventing or treating the novel coronavirus.
  • the vaccine may further include at least one of the following additional technical features:
  • the adjuvant includes at least one of aluminum hydroxide, aluminum phosphate, monophosphoryl lipid, and oligonucleotide CpG.
  • the vaccine obtained when the adjuvant according to the embodiment of the present invention is combined with the polypeptide immunoconjugate significantly improves the titer of anti-peptide antibody and anti-RBD antibody, preventing or treating new Coronavirus works better.
  • the adjuvant is aluminum hydroxide.
  • the adjuvant includes 3D-mLA with a final concentration of 100 ⁇ g/L and aluminum hydroxide with a final concentration of 1 mg/L.
  • the vaccine obtained when the adjuvant according to the embodiment of the present invention is combined with the polypeptide immunoconjugate significantly improves the titer of anti-peptide antibody and anti-RBD antibody, preventing or treating new The coronavirus works even better.
  • the content of the adjuvant in each dose of the vaccine is 1 ⁇ g-1000 ⁇ g, preferably 10 ⁇ g-500 ⁇ g, more preferably 10-100 ⁇ g, most preferably 10-50 ⁇ g per dose. According to the amount of adjuvant added in the embodiment of the present invention, the effect of the prepared vaccine in preventing or treating the novel coronavirus is better.
  • the vaccine is in any pharmaceutically acceptable dosage form.
  • the vaccine immunization method is at least one of intramuscular injection, subcutaneous injection, intradermal injection, and microneedle injection.
  • the present invention provides a method for preparing the vaccine described in the fifth aspect. According to an embodiment of the present invention, it includes the following steps: mixing the polypeptide immunoconjugate described in the first aspect and an adjuvant.
  • the vaccine according to the embodiment of the present invention has the polypeptide immunoconjugate, which can significantly increase the content of anti-peptide antibody or anti-RBD antibody. Therefore, the vaccine retention after combining the polypeptide immunoconjugate with the adjuvant It can produce high anti-peptide and anti-RBD antibodies in the human body stimulated by a variety of new coronaviruses.
  • the immunogenicity of peptides and RBD to the body is further improved, thereby improving the antibody
  • the titer can achieve the effect of effectively preventing or treating the novel coronavirus.
  • the adjuvant includes at least one of aluminum hydroxide, aluminum phosphate, monophosphoryl lipid, and oligonucleotide CpG
  • the adjuvant is aluminum hydroxide.
  • the adjuvant includes 3D-mLA with a final concentration of 100 ⁇ g/L and aluminum hydroxide with a final concentration of 1 mg/L.
  • the present invention provides a method for preparing an antibody. According to an embodiment of the present invention, it includes: using the polypeptide immunoconjugate described in the first aspect to immunize animals; collecting serum of the immunized animals; and purifying the target antibody from the serum.
  • the method for preparing the antibody proposed in the embodiment of the present invention is easy to operate, and the antibody can specifically recognize the polypeptide immunoconjugate.
  • the present invention provides a method for stimulating the production of anti-peptide antibody or anti-RBD antibody in a subject.
  • a method for stimulating the production of anti-peptide antibody or anti-RBD antibody in a subject it is achieved by at least one of the following methods: 1) administering the polypeptide immunoconjugate described in the first aspect to the subject; 2) administering the drug described in the fourth aspect to the subject a composition; and 3) administering the vaccine of the fifth aspect to a subject.
  • a higher titer of anti-peptide antibody and anti-RBD antibody can be produced in the subject, which greatly improves the body's immunity to the new coronavirus pneumonia force.
  • the present invention proposes a method for treating or preventing novel coronavirus pneumonia.
  • at least one of the following is included: 1) administering the aforementioned polypeptide immunoconjugate to the subject; 2) administering the aforementioned pharmaceutical composition to the subject; and 3) administering to the subject Subjects are administered the vaccines described above.
  • the aforementioned polypeptide immunoconjugates can stimulate the body to produce higher titers of novel coronavirus neutralizing antibodies and anti-polypeptide antibodies, while improving the broad-spectrum immune effect of the immunoconjugates Therefore, the pharmaceutical composition or vaccine containing the polypeptide immunoconjugate can also stimulate the body to produce a higher titer of anti-peptide antibody or anti-RBD antibody, which can effectively treat or prevent novel coronavirus pneumonia.
  • Fig. 1 is the SDS-PAGE electrophoresis picture obtained after the polypeptides (7#, S2#, 45# and 46#) according to the embodiment of the present invention are respectively coupled to the RBD domain of the new coronavirus S protein, where RBD represents : Only the RBD domain group in the new coronavirus S protein; RBD-SMCC means: RBD protein and sulfosuccinimide 4-(N-maleimidomethyl) cyclohexane-1-carboxylate Product group after reaction; RBD-7#, RBD-S2#, RBD-45#, RBD-46# represent RBD-polypeptide group;
  • Figure 2 is an SDS-PAGE electrophoresis gel image obtained after coupling polypeptides (7# and S2#) according to an embodiment of the present invention to the RBD domain of the new coronavirus S protein, in which the 7# polypeptide and the new coronavirus S
  • the molar ratio of the RBD domain of the protein is 10:1
  • the molar ratio of the S2# polypeptide to the RBD domain of the new coronavirus S protein is 10:1;
  • Fig. 3 is the SDS-PAGE electrophoresis gel image obtained after the polypeptides (45# and 46#) according to the embodiment of the present invention are respectively coupled to the RBD domain of the new coronavirus S protein, wherein, the 45# polypeptide and the new coronavirus S
  • the molar ratio of the RBD domain of the protein is 10:1
  • the molar ratio of the 46# polypeptide to the RBD domain of the novel coronavirus S protein is 10:1;
  • Fig. 4 is a diagram showing the detection results of the neutralizing antibody level of the South African strain novel coronavirus by the polypeptide immunoconjugates of the RBD-HA204 adjuvant group and the RBD-45#+HA204 adjuvant group according to an embodiment of the present invention.
  • new coronavirus In this article, the terms “new coronavirus”, “new coronavirus” and “COVID-19” all refer to the pathogen that causes new coronavirus pneumonia.
  • nucleotide identity in order to compare two or more nucleotide sequences, the first sequence and the The percentage of "sequence identity" between the second sequences. nucleotides in the second sequence] minus [total number of nucleotides in the first sequence], then multiplied by [100%], where each nucleotide deletion, insertion in the second nucleotide sequence , substitutions or additions - relative to the first nucleotide sequence - are considered differences in a single nucleotide (position).
  • the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings using known computer algorithms for sequence alignment, such as NCBI Blast v2.0.
  • Some other techniques, computer algorithms and arrangements for determining the degree of sequence identity are e.g. -A.
  • the percentage "sequence identity" between a first amino acid sequence and a second amino acid sequence can be determined by dividing [the number of amino acid residues in the first amino acid sequence by Identical to [the amino acid residue at the corresponding position in the second amino acid sequence] as [total number of nucleotides in the first amino acid sequence], multiplied by [100%], where each deletion, insertion, substitution or addition corresponds to A "residue" of amino acid residues in a second amino acid sequence compared to a first amino acid sequence is considered to be a difference in a single amino acid residue (position), ie, an "amino acid difference" as defined herein.
  • the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms, such as those described above for determining the degree of sequence identity of nucleotide sequences, again using standard settings.
  • amino acid sequence with the largest number of amino acid residues will be taken as the "first" amino acid sequence and the other amino acid sequence will be taken as "Second” amino acid sequence.
  • conservative amino acid substitutions can generally be described as amino acid substitutions in which an amino acid residue is replaced. Another amino acid residue with a similar chemical structure that has little or no effect on the function, activity or other biological properties of a polypeptide.
  • conservative amino acid substitutions are well known in the art, for example, from WO 04/037999, GB-A-2357768, WO 98/49185, WO 00/46383 and WO 01/09300; and WO 01/09300.
  • the type and/or combination of such substitutions can be selected and/or (preferred) based on the relevant teachings from WO 04/037999 and WO 98/49185 and from other references cited therein.
  • the present invention proposes a polypeptide immunoconjugate, comprising: a polypeptide and the RBD of the S protein of the novel coronavirus; wherein the molar ratio of the polypeptide to the RBD of the S protein of the novel coronavirus is 1:1-15:1.
  • the polypeptide immunoconjugate formed by combining the antigenic short peptide with the carrier can increase the neutralization titer of the anti-RBD antibody.
  • the inventor creatively found that after combining the polypeptide with the RBD of the novel coronavirus S protein The obtained polypeptide immunoconjugate can significantly increase the content of anti-peptide antibody or anti-RBD antibody.
  • the polypeptide immunoconjugate produces High levels of neutralizing antibodies.
  • the polypeptide is an antigenic epitope of the novel coronavirus S protein.
  • the polypeptide is not particularly limited, and any novel coronavirus S protein epitope can be used.
  • the polypeptide has the amino acid sequence shown in SEQ ID NO: 1 or has at least 99%, at least 95%, at least 90%, at least 85%, at least 80% of SEQ ID NO: 1 , an amino acid sequence of at least 75% identity.
  • the RBD of the novel coronavirus S protein has the amino acid sequence shown in SEQ ID NO: 2 or has at least 99%, at least 95%, at least 90%, at least Amino acid sequences that are 85%, at least 80%, at least 75% identical.
  • the polypeptide is connected to the RBD through a linker.
  • the linker is sulfosuccinimide 4-(N-maleimidomethyl)cyclohexane-1-carboxylate.
  • the present invention proposes the use of the aforementioned polypeptide immunoconjugate in the preparation of medicines.
  • the drug is used to prevent or treat novel coronavirus pneumonia.
  • the present invention proposes the use of the aforementioned polypeptide immunoconjugate in the preparation of vaccines.
  • the vaccine is used to prevent or treat novel coronavirus pneumonia.
  • the present invention proposes a pharmaceutical composition, including the aforementioned polypeptide immunoconjugate.
  • the pharmaceutical composition may include: a pharmaceutically acceptable adjuvant, which includes at least one of a stabilizer, a wetting agent, an emulsifier, a binding agent, and an isotonic agent;
  • the composition is at least one of tablet, granule, powder, capsule, solution, suspension and freeze-dried preparation.
  • the pharmaceutical composition can effectively prevent or treat novel coronavirus pneumonia.
  • the present invention proposes a vaccine, including the aforementioned polypeptide immunoconjugate; and a pharmaceutically acceptable adjuvant.
  • the vaccine according to a specific embodiment of the present invention contains the polypeptide immunoconjugate, which can significantly increase the content of anti-peptide antibody or anti-RBD antibody. Therefore, the vaccine after combining the polypeptide immunoconjugate with the adjuvant It retains the characteristics of being able to produce higher anti-peptide and anti-RBD antibodies in humans stimulated by a variety of new coronaviruses. At the same time, due to the addition of adjuvants, the immunogenicity of peptides and RBD to the body is further improved, thereby improving The titer of the antibody can effectively prevent or treat the novel coronavirus pneumonia.
  • the adjuvant includes at least one of aluminum hydroxide, aluminum phosphate, monophosphoryl lipid, and oligonucleotide CpG.
  • the adjuvant is aluminum hydroxide.
  • the adjuvant includes 3D-mLA with a final concentration of 100 ⁇ g/L and aluminum hydroxide with a final concentration of 1 mg/L.
  • the content of the adjuvant in each dose of the vaccine is 1 ⁇ g-1000 ⁇ g, preferably 10 ⁇ g-500 ⁇ g, more preferably 10-100 ⁇ g, most preferably 10-50 ⁇ g per dose.
  • the vaccine is in any pharmaceutically acceptable dosage form.
  • the vaccine immunization method is at least one of intramuscular injection, subcutaneous injection, intradermal injection and microneedle injection.
  • the present invention proposes a method for preparing the aforementioned vaccine, comprising the following steps: mixing the aforementioned polypeptide immune conjugate with an adjuvant.
  • the adjuvant includes at least one of aluminum hydroxide, aluminum phosphate, monophosphoryl lipid, and oligonucleotide CpG.
  • the adjuvant is aluminum hydroxide.
  • the adjuvant includes 3D-mLA with a final concentration of 100 ⁇ g/L and aluminum hydroxide with a final concentration of 1 mg/L.
  • the present invention proposes a method for preparing antibodies, comprising: using the aforementioned polypeptide immunoconjugate to immunize animals; collecting the serum of the immunized animals; and purifying the target antibody from the serum.
  • the present invention proposes a method for stimulating the production of anti-peptide antibody or anti-RBD antibody in a subject, which is achieved by at least one of the following methods: 1) administering the aforementioned polypeptide immunoconjugate to the subject 2) administering the aforementioned pharmaceutical composition to the subject; and 3) administering the aforementioned vaccine to the subject.
  • the method according to some specific embodiments of the present invention can generate higher titers of anti-peptide antibody and anti-RBD antibody in the subject, which greatly improves the body's immunity to the novel coronavirus pneumonia.
  • the present invention proposes a method for treating or preventing novel coronavirus pneumonia, including at least one of the following: 1) administering the aforementioned polypeptide immunoconjugate to the subject; 2) administering the aforementioned polypeptide immunoconjugate to the subject 3) administering the aforementioned vaccine to the subject.
  • the aforementioned polypeptide immunoconjugates, pharmaceutical compositions or vaccines can stimulate the body to produce higher titers of anti-peptide antibodies or anti-RBD antibodies, which can effectively treat or prevent the new coronavirus Viral pneumonia.
  • administering refers to introducing a predetermined amount of a substance into a patient by some suitable means.
  • the polypeptide immunoconjugate, pharmaceutical composition or vaccine in the embodiments of the present invention can be administered through any common route, as long as it can reach the intended tissue.
  • Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, dermal, oral, topical, nasal, pulmonary and rectal, but the invention is not limited to these exemplified modes of administration.
  • the active ingredient of the composition for oral administration should be coated or formulated to prevent its degradation in the stomach.
  • the compositions of the present invention are administered as injectable formulations.
  • the pharmaceutical compositions of the present invention can be administered using specific devices that deliver the active ingredient to target cells.
  • the administration frequency and dose of the polypeptide immunoconjugates, pharmaceutical compositions or vaccines in the embodiments of the present invention can be determined by a number of related factors, including the type of disease to be treated, route of administration, patient age, gender , weight and severity of the disease and the type of drug that is the active ingredient.
  • the daily dose can be divided into 1 dose, 2 doses or multiple doses in a suitable form, so as to be administered once, twice or multiple times throughout the time period, as long as the therapeutically effective amount is achieved. Can.
  • terapéuticaally effective amount refers to an amount sufficient to significantly improve certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosage regimen.
  • treatment is used to refer to obtaining a desired pharmacological and/or physiological effect.
  • Treatment encompasses administering the polypeptide immunoconjugates, pharmaceutical compositions or vaccines of the embodiments of the invention to individuals for treatment, including but not limited to administering the compounds described herein to individuals in need.
  • polypeptide immunoconjugates are the inventors of the present application Discovered and accomplished through painstaking creative labor and optimization work.
  • peptides 7#, 45#, 46# and S2 were prepared by using the conventional solid-phase peptide synthesis method, and the amino acid sequences of the peptides are shown in Table 1.
  • amino acid sequences of peptide No. 7, No. 45 and No. 46 after synthesis have the C-terminal form of -CONH 2 or COOH; the N-terminal form of NH 2 or CH 3 CONH-.
  • the polypeptide lysate is treated with strong acid, such as TFA (trifluoroacetic acid).
  • strong acid such as TFA (trifluoroacetic acid).
  • the resin obtained in step (2) was treated with TFA, water, EDT dimercaptoethanol, and phenol (volume ratio: 92.5:2.5:2.5:2.5) for 2 hours at room temperature. Then carefully collect the lysate into a glass collector, and add ice-precooled ether to collect the precipitated peptide, and continue to wash with cold ether for 5-6 times to obtain the crude peptide.
  • the crude peptide is purified, collected, and freeze-dried by HPLC (high performance liquid chromatography), and finally the purity (214nm wavelength) is greater than 85% and the correct molecular weight is checked by mass spectrometry.
  • Example 2 the synthetic peptide prepared in Example 1 is used to form a conjugate with the RBD protein through a linker.
  • the specific experimental operation is as follows:
  • RBD protein The RBD plasmid was transfected into HEK293F cells, and the cells were cultured to the logarithmic growth phase to collect the cell supernatant. After concentration, the protein is purified by Anti-flag G1Affinity resin. The crude protein is then purified by molecular sieves to obtain RBD protein.
  • RBD protein was prepared into a solution with a concentration of 1 mg/mL in PBS. Take the required amount of RBD solution, add Sulfo-SMCC (10mg/mL) according to the molar ratio of 1:2-1:40, react at room temperature for 1 hour, and centrifuge the reaction solution at 4°C and 3500rpm with an ultrafiltration concentrator tube. After the volume was concentrated to 1/10 of the reaction solution, PBS was added to the original reaction solution volume, and the centrifugal concentration was continued, and the centrifugal concentration was continued for 5 times to remove free Sulfo-SMCC. The concentration of the final RBD-SMCC stock solution obtained was 10 mg/mL.
  • Immunoconjugate a conjugate of RBD protein and the polypeptide shown in SEQ ID NO:1;
  • Adjuvant HA204 [each mL contains 3D-MLA 100 ⁇ g, aluminum hydroxide (calculated as aluminum ion) 1mg];
  • the experimental grouping of this embodiment is shown in Table 3, wherein, the molar ratio of 7# peptide to RBD is 10:1, the molar ratio of 45# peptide to RBD is 7.5:1, the molar ratio of 46# to RBD is 7:1, S2 #The molar ratio of peptide to RBD is 6:1, and the naming rule in the column of "Group” is "RBD domain of novel coronavirus and polypeptide conjugate-adjuvant", such as “RBD in "RBD-7#+HA204" -7#" indicates the conjugate of the RBD binding domain of the novel coronavirus S protein and peptide No. 7, and "HA204" indicates the adjuvant named HA204.
  • Example 2 Mix the immunogenic conjugate prepared in Example 2 with the adjuvant HA204 and PBS solution, shake it at room temperature and 30 RPM for 1 hour, and the resulting final concentration contains 100 ⁇ g/mL of polypeptide RBD protein antigen and 12.5% of the adjuvant HA204 (v/ v), which is the final sample.
  • the grouping of the immune effect experiment is shown in Table 3.
  • the samples to be tested prepared in Table 3 were used to study the immunogenicity of the mice, and the healthy BALB/c mice with a body weight of 16-18 g were randomly divided into groups, 6 in each group Mice; using the samples to be tested of each experimental group (RBD, RBD+HA204, RBD-7#+HA204, RBD-45#+HA204, RBD-46#+HA204, RBD-S2+HA204) in Table 3
  • Rats were intraperitoneally injected once on the 0th day, 14th day, and 21st day, and the volume of each administration was 200 ⁇ L/mouse; mice in the control group were injected with normal saline intraperitoneally, On the 14th day and the 21st day, each intraperitoneal injection was performed once, and the volume of each administration was 100 ⁇ L/mouse.
  • the OD 450 value of the serum antibody was measured using a microplate reader.
  • the results of the anti-RBD antibody level after the polypeptide conjugate was combined with different adjuvants are shown in Table 4. The results showed that when the No. 45 peptide was conjugated with RBD, the anti-RBD antibody titer could be significantly increased. Spend.
  • the inventor compared the level of the South African strain pseudovirus neutralizing antibody produced by the RBD-HA204 group and the RBD-45#+HA204 group mouse D21 and D28 serum.
  • the specific experimental operations are as follows:
  • mice serum obtained in Experiment 2 of this example was inactivated in a water bath at 56° C. for 30 minutes, and diluted in a 96-well cell culture plate with complete DMEM medium;
  • the levels of pseudovirus neutralizing antibodies produced by the RBD-HA204 group and the RBD-45#+HA204 group are shown in Figure 4, in which, after peptide No. 45 was conjugated with RBD, the level of neutralizing antibodies on day 21 was significantly higher than that of the RBD group, On the 28th day, the level of neutralizing antibodies produced by the RBD-45#+HA204 group was higher than that of the RBD-HA204 group, but the difference was not significant. Both groups showed good immune effects.

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Abstract

本发明提出了多肽免疫偶联物及其应用，该多肽免疫偶联物包括：多肽和新型冠状病毒S蛋白的RBD；其中，所述多肽与新型冠状病毒S蛋白的RBD的摩尔比为15：1～1：1。

Description

多肽-RBD免疫偶联物及其用途
优先权信息
无。
技术领域
本发明涉及生物医药领域，具体地，本发明涉及多肽-RBD免疫偶联物及其应用，更具体地，本发明涉及一种多肽免疫偶联物、所述免疫偶联物的用途、一种药物组合物、疫苗、制备所述疫苗的方法、制备抗体的方法以及一种受试者体内刺激抗肽抗体或抗RBD抗体生成的方法。
背景技术
冠状病毒是一种带有RNA的正向包膜病毒，其基因组大小约为26～32kb，是已知基因组最大的RNA病毒。基因组RNA和磷酸化核衣壳(N)蛋白被埋在磷脂双层中并被刺突糖蛋白三聚体(S)覆盖，膜(M)蛋白(III型跨膜糖蛋白)和包膜(E)蛋白位于病毒包膜的S蛋白之间。冠状病毒具有多种宿主，包括禽类和哺乳动物，特别是蝙蝠。冠状病毒是在自然界中广泛存在的一类病毒，可引起包括呼吸道、消化道及神经系统在内的多系统疾病，高致病性冠状病毒感染已成为近10年来广受关注的公共卫生问题。2002年11月，首例严重急性呼吸系统综合症(SARS)发生在中国佛山。2012年，中东呼吸综合征冠状病毒(MERS-CoV)是21世纪发现的第二种高致病性冠状病毒。MERS-CoV的死亡率很高，多达40％的患者死亡。2019新型冠状病毒(SARS-CoV-2)于2019年12月首次发现，其感染导致的新型冠状病毒肺炎(COVID-19)正在影响全球上百万的患者。
COVID-19患者会出现不同程度的症状，从发烧或轻度咳嗽到肺炎，更严重者甚至死亡。目前COVID-19的致死率约为2％至4％，尽管死亡率低于SARS和MERS，但新型冠状病毒(SARS-CoV-2)具有潜伏期长、传染性强和重症率较高的特点，与引发非典型肺炎的SARS病毒不同，部分病例潜伏期具有传染性，另有一些病毒携带者没有表现出任何明显症状，这增加了疫情的防控难度。因此，快速研制出能够提升群体免疫水平并阻断病毒传播的预防疫苗已成为全球当前最为迫切的重大需求。
SARS-CoV-2的S蛋白位于病毒最外层，在膜上规则排列成冠状结构，参与病毒与宿主细胞表面的病毒受体结合并介导病毒通过膜融合进入细胞的过程，在诱导宿主产生中和抗体过程中起重要作用。目前国内外COVID-19疫苗的研发是以全S蛋白或部分S蛋白(S蛋白中的受体结域(RBD))作为首要目标抗原。研究报道，同属冠状病毒的SARS-CoV的S蛋白可能诱发抗体依赖性感染增强反应(ADE)，RBD的分子量较低，以RBD单体为抗原的免疫原性较差，全球流行的部分SARS-CoV-2突变株存在RBD氨基酸单位点及多位点突变，这可能进一步降低以RBD为目标抗原疫苗的保护率，因此，如何提高RBD的免疫原性还需进一步探索。
发明内容
本本申请是基于发明人对以下事实和问题的发现和认识作出的：
研究报道，新型冠状病毒RBD的分子量较低，以RBD单体为抗原的免疫原性较差，发明人经过大量实验研究，意外地获得了一种预防新型冠状病毒肺炎COVID-19的多肽免疫偶联物；多肽免疫偶联物加入佐剂后制备成疫苗，既可提高多肽的免疫原性，又提高了RBD蛋白的免疫原性，同时提高了免疫偶联物免疫效果的广谱性。
为此，在本发明的第一方面，本发明提出了一种多肽免疫偶联物。根据本发明的实施例，包括：多肽和新型冠状病毒S蛋白的RBD；其中，所述多肽与新型冠状病毒S蛋白的RBD的摩尔比为1：1～15：1。将抗原性的短肽与载体结合形成的多肽免疫偶联物可增加抗RBD抗体的中和滴度，发明人创造性的发现，将所述多肽和所述新型冠状病毒S蛋白的RBD进行组合后获得的多肽免疫偶联物可以显著提高抗肽抗体或抗RBD抗体含量，当多肽与新型冠状病毒S蛋白的RBD的摩尔比为1：1～1：30时，所述多肽免疫偶联物产生中和抗体的含量较高。
根据本发明的实施例，所述多肽免疫偶联物还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述多肽为新型冠状病毒S蛋白抗原表位。
根据本发明的实施例，所述多肽具有SEQ ID NO：1所示的氨基酸序列或者与SEQ ID NO:1具有至少99％、至少95％、至少90％、至少85％、至少80％、至少75％同一性的氨基酸序列。
CFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLN-NH2(SEQ ID NO:1)。
根据本发明的实施例，所述新型冠状病毒S蛋白的RBD具有SEQ ID NO：2所示的氨基酸序列或者与SEQ ID NO:2具有至少99％、至少95％、至少90％、至少85％、至少80％、至少75％同一性的氨基酸序列。
MLRGLCCVLL LCGAVFVSPS QEIHARFRRG ARGRVQPTES IVRFPNITNL CPFGEVFNAT RFASVYAWNR KRISNCVADY SVLYNSASFS TFKCYGVSPT KLNDLCFTNV YADSFVIRGD EVRQIAPGQT GKIADYNYKL PDDFTGCVIA WNSNNLDSKV GGNYNYLYRL FRKSNLKPFE RDISTEIYQA GSTPCNGVEG FNCYFPLQSY GFQPTNGVGY QPYRVVVLSF ELLHAPATVC GPKKSTNLVK NKCVNFWSHP QFEKDYKDDD DK(SEQ ID NO：2)。
根据本发明的实施例，所述多肽通过连接体与所述RBD相连。
根据本发明的实施例，所述连接体为磺基琥珀酰亚胺4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯。
在本发明的第二方面，本发明提出了第一方面所述的多肽免疫偶联物在制备药物中的用途。根据本发明的实施例，所述药物用于预防或治疗新型冠状病毒肺炎。
在本发明的第三方面，本发明提出了第一方面所述的多肽免疫偶联物在制备疫苗中的用途。根据本发明的实施例，所述疫苗用于预防或治疗新型冠状病毒肺炎。本发明提供的疫苗对多种新型冠状病毒均 具有较高的中和抗体，可有效预防或治疗新型冠状病毒肺炎。
在本发明的第四方面，本发明提出了一种药物组合物。根据本发明的实施例，包括第一方面所述的多肽免疫偶联物。根据本发明实施例的药物组合物可以显著提高抗肽抗体或抗RBD抗体的含量，有效的预防或治疗新型冠状病毒肺炎。
在本发明的第五方面，本发明提出了一种疫苗。根据本发明的实施例，包括第一方面所述的多肽免疫偶联物；以及药学上可接受的佐剂。根据本发明实施例的疫苗具有所述多肽免疫偶联物，可以显著提高抗肽抗体或抗RBD抗体的含量，因此，将所述多肽免疫偶联物与所述佐剂进行结合后的疫苗保留了可以对多种新型冠状病毒刺激的人体内均能产生较高的抗肽和抗RBD抗体的特点，同时由于佐剂的加入，进一步提高了多肽和RBD对机体的免疫原性，从而提高抗体的效价，达到有效预防或治疗新型冠状病毒的效果。
根据本发明的实施例，所述疫苗还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述佐剂包括氢氧化铝、磷酸铝、单磷酰脂质、寡核苷酸CpG中的至少之一。根据本发明实施例的佐剂与所述多肽免疫偶联物进行结合时获得的疫苗相较于利用其它佐剂制备的疫苗显著提高了抗肽抗体和抗RBD抗体的效价，预防或治疗新型冠状病毒的效果较佳。
根据本发明的实施例，所述佐剂为氢氧化铝。
根据本发明的实施例，所述佐剂中包括终浓度为100μg/L的3D-mLA、终浓度为1mg/L的氢氧化铝。根据本发明实施例的佐剂与所述多肽免疫偶联物进行结合时获得的疫苗相较于利用其它佐剂制备的疫苗显著提高了抗肽抗体和抗RBD抗体的效价，预防或治疗新型冠状病毒的效果更佳。
根据本发明的实施例，每剂量所述疫苗中佐剂的含量为1μg～1000μg，优选10μg～500μg，更优选10～100μg，最优选每剂量10～50μg。根据本发明实施例的佐剂添加剂量，所制备的疫苗预防或治疗新型冠状病毒的效果更佳。
根据本发明的实施例，所述疫苗为任何药学上可接受的剂型。
根据本发明的实施例，所述疫苗免疫方式为肌肉注射、皮下注射、皮内注射、微针注射中至少之一种。
在本发明的第六方面，本发明提出了一种制备第五方面所述疫苗的方法。根据本发明的实施例，包括以下步骤：将第一方面所述的多肽免疫偶联物与佐剂进行混合处理。根据本发明实施例的疫苗具有所述多肽免疫偶联物，可以显著提高抗肽抗体或抗RBD抗体的含量，因此，将所述多肽免疫偶联物与所述佐剂进行结合后的疫苗保留了可以对多种新型冠状病毒刺激的人体内均能产生较高的抗肽和抗RBD抗体的特点，同时由于佐剂的加入，进一步提高了多肽和RBD对机体的免疫原性，从而提高抗体的效价，达到有效预防或治疗新型冠状病毒的效果。
根据本发明的实施例，所述佐剂包括氢氧化铝、磷酸铝、单磷酰脂质、寡核苷酸CpG中的至少之一
。
根据本发明的实施例，所述佐剂为氢氧化铝。
根据本发明的实施例，所述佐剂中包括终浓度为100μg/L的3D-mLA、终浓度为1mg/L的氢氧化铝。
在本发明的第七方面，本发明提出了一种制备抗体的方法。根据本发明的实施例，包括：利用第一方面所述的多肽免疫偶联物对动物进行免疫接种；采集经过免疫接种的动物的血清；以及从所述血清中纯化出目的抗体。本发明实施例所提出的制备抗体的方法，操作简便，抗体可特异性识别所述多肽免疫偶联物。
在本发明的第八方面，本发明提出了一种在受试者体内刺激抗肽抗体或抗RBD抗体生成的方法。根据本发明的实施例，是通过下列方式的至少之一实现的：1)给与受试者第一方面所述的多肽免疫偶联物；2)给予受试者第四方面所述的药物组合物；以及3)向受试者施用第五方面所述的疫苗。根据本发明实施例的体内刺激抗肽抗体或抗RBD抗体生成的方法可以在受试者体内产生较高滴度的抗肽抗体以及抗RBD抗体，极大的提高机体对新型冠状病毒肺炎的免疫力。
在本发明的第九方面，本发明提出了一种治疗或预防新型冠状病毒肺炎的方法。根据本发明的实施例，包括下列中的至少之一：1)给与受试者前面所述的多肽免疫偶联物；2)给予受试者前面所述的药物组合物；以及3)向受试者施用前面所述的疫苗。根据本发明的一些具体实施例，前面所述的多肽免疫偶联物可刺激机体产生较高滴度的新型冠状病毒中和抗体以及抗多肽抗体，同时提高了免疫偶联物免疫效果的广谱性，因此，包含所述多肽免疫偶联物的药物组合物或疫苗同样可以刺激机体产生较高滴度的抗肽抗体或抗RBD抗体，可以有效的治疗或预防新型冠状病毒肺炎。
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
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明实施例的多肽(7#、S2#、45#和46#)分别与新型冠状病毒S蛋白的RBD结构域偶联后获得的SDS-PAGE电泳胶图，其中，RBD表示：仅有新冠病毒S蛋白中的RBD结构域组；RBD-SMCC表示：RBD蛋白与磺基琥珀酰亚胺4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯反应后产物组；RBD-7#、RBD-S2#、RBD-45#、RBD-46#表示RBD-多肽组；
图2是根据本发明实施例的多肽(7#和S2#)分别与新型冠状病毒S蛋白的RBD结构域偶联后获得的SDS-PAGE电泳胶图，其中，7#多肽与新型冠状病毒S蛋白的RBD结构域的摩尔比为10：1，S2#多肽与新型冠状病毒S蛋白的RBD结构域的摩尔比为10：1；
图3是根据本发明实施例的多肽(45#和46#)分别与新型冠状病毒S蛋白的RBD结构域偶联后获得的SDS-PAGE电泳胶图，其中，45#多肽与新型冠状病毒S蛋白的RBD结构域的摩尔比为10：1，46# 多肽与新型冠状病毒S蛋白的RBD结构域的摩尔比为10：1；以及
图4是根据本发明实施例的RBD-HA204佐剂组、RBD-45#+HA204佐剂组多肽免疫偶联物对南非株新型冠状假病毒中和抗体水平检测结果图。
发明详细描述
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
术语解释
在文中，术语“新型冠状病毒”、“新冠病毒”、“COVID-19”、均指引发新型冠状病毒肺炎的病原体。
同一性，本发明，为了比较两个或更多个核苷酸序列，可以通过将[第一序列中与相应位置的核苷酸相同的核苷酸的数目相除]来计算第一序列和第二序列之间的“序列同一性”的百分比。第二个序列中的核苷酸]减去[第一个序列中核苷酸的总数]，然后乘以[100％]，其中第二个核苷酸序列中每个核苷酸的缺失，插入，取代或添加-相对于第一核苷酸序列-被认为是单个核苷酸(位置)上的差异。
或者，可以使用标准设置，使用用于序列比对的已知计算机算法，例如NCBI Blast v2.0，计算两个或多个核苷酸序列之间的序列同一性程度。
用于确定序列同一性程度的一些其他技术，计算机算法和设置例如在WO 04/037999，EP 0 967 284，EP 1 085 089，WO 00/55318，WO 00/78972，WO 98/49185和GB 2357768-A。
同一性，本发明，为了比较两个或多个氨基酸序列，第一氨基酸序列和第二氨基酸序列之间的“序列同一性”百分比可以通过将[第一氨基酸序列中的氨基酸残基数目除以与[第二个氨基酸序列中相应位置的氨基酸残基]相同，为[第一个氨基酸序列中核苷酸的总数]，然后乘以[100％]，其中每个缺失，插入，取代或添加与第一氨基酸序列相比，第二氨基酸序列中氨基酸残基的“残基”被认为是单个氨基酸残基(位置)上的差异，即，如本文所定义的“氨基酸差异”。
备选地，可以再次使用标准设置，使用已知的计算机算法来计算两个氨基酸序列之间的序列同一性程度，例如上述用于确定核苷酸序列的序列同一性程度的算法。
通常，为了根据上文概述的计算方法确定两个氨基酸序列之间的“序列同一性”的百分比，将具有最大氨基酸残基数量的氨基酸序列作为“第一”氨基酸序列，另一个氨基酸序列将作为“第二”氨基酸序列。
同样，在确定两个氨基酸序列之间的序列同一性程度时，技术人员可以考虑所谓的“保守”氨基酸取代，其通常可以描述为其中氨基酸残基被替换为的氨基酸取代。具有相似化学结构的另一个氨基酸残基，其对多肽的功能，活性或其他生物学特性几乎没有影响或基本没有影响。这样的保守氨基酸取代在本领域中是众所周知的，例如，从WO 04/037999，GB-A-2357768，WO 98/49185，WO 00/46383和WO01/09300；和WO 01/09300。可以基于来自WO 04/037999以及WO 98/49185的相关教导以及从其中引用的其他参考文献中选择和/或(优选)这种取代的类型和/或组合。
本发明提出了一种多肽免疫偶联物，包括：多肽和新型冠状病毒S蛋白的RBD；其中，所述多肽与新型冠状病毒S蛋白的RBD的摩尔比为1：1～15：1。将抗原性的短肽与载体结合形成的多肽免疫偶联物可增加抗RBD抗体的中和滴度，发明人创造性的发现，将所述多肽和所述新型冠状病毒S蛋白的RBD进行组合后获得的多肽免疫偶联物可以显著提高抗肽抗体或抗RBD抗体含量，当多肽与新型冠状病毒S蛋白的RBD的摩尔比为1：1～15：1时，所述多肽免疫偶联物产生中和抗体的含量较高。
根据本发明一个具体的实施例，所述多肽为新型冠状病毒S蛋白抗原表位。所述多肽不受特别限制，任何新型冠状病毒S蛋白抗原表位均可使用。
根据本发明一个具体的实施例，所述多肽具有SEQ ID NO：1所示的氨基酸序列或者与SEQ ID NO:1具有至少99％、至少95％、至少90％、至少85％、至少80％、至少75％同一性的氨基酸序列。
CFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLN-NH2(SEQ ID NO:1)。
根据本发明一个具体的实施例，所述新型冠状病毒S蛋白的RBD具有SEQ ID NO：2所示的氨基酸序列或者与SEQ ID NO:2具有至少99％、至少95％、至少90％、至少85％、至少80％、至少75％同一性的氨基酸序列。
MLRGLCCVLL LCGAVFVSPS QEIHARFRRG ARGRVQPTES IVRFPNITNL CPFGEVFNAT RFASVYAWNR KRISNCVADY SVLYNSASFS TFKCYGVSPT KLNDLCFTNV YADSFVIRGD EVRQIAPGQT GKIADYNYKL PDDFTGCVIA WNSNNLDSKV GGNYNYLYRL FRKSNLKPFE RDISTEIYQA GSTPCNGVEG FNCYFPLQSY GFQPTNGVGY QPYRVVVLSF ELLHAPATVC GPKKSTNLVK NKCVNFWSHP QFEKDYKDDD DK(SEQ ID NO：2)。
根据本发明一个具体的实施例，所述多肽通过连接体与所述RBD相连。
根据本发明一个具体的实施例，所述连接体为磺基琥珀酰亚胺4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯。
本发明提出了前面所述的多肽免疫偶联物在制备药物中的用途。根据本发明的具体实施例，所述药物用于预防或治疗新型冠状病毒肺炎。
本发明提出了前面所述的多肽免疫偶联物在制备疫苗中的用途。根据本发明的具体实施例，所述疫苗用于预防或治疗新型冠状病毒肺炎。
本发明提出了一种药物组合物，包括前面所述的多肽免疫偶联物。所述药物组合物可包括：药学上可接受的辅剂，所述药学上可接受的辅剂包括稳定剂、湿润剂、乳化剂、粘合剂、等渗剂的至少之一；所述药物组合物呈片剂、颗粒剂、散剂、胶囊剂、溶液剂、悬浮剂、冻干制剂的至少一种。所述药物组合物可有效预防或治疗新型冠状病毒肺炎。
本发明提出了一种疫苗，包括前面所述的多肽免疫偶联物；以及药学上可接受的佐剂。根据本发明具体实施例的疫苗包含所述多肽免疫偶联物，可以显著提高抗肽抗体或抗RBD抗体的含量，因此，将所述多肽免疫偶联物与所述佐剂进行结合后的疫苗保留了可以对多种新型冠状病毒刺激的人体内均能 产生较高的抗肽和抗RBD抗体的特点，同时由于佐剂的加入，进一步提高了多肽和RBD对机体的免疫原性，从而提高抗体的效价，达到有效预防或治疗新型冠状病毒肺炎的效果。
根据本发明一个具体的实施例，所述佐剂包括氢氧化铝、磷酸铝、单磷酰脂质、寡核苷酸CpG中的至少之一。
根据本发明一个具体的实施例，所述佐剂为氢氧化铝。
根据本发明一个具体的实施例，所述佐剂中包括终浓度为100μg/L的3D-mLA、终浓度为1mg/L的氢氧化铝。
根据本发明一个具体的实施例，每剂量所述疫苗中佐剂的含量为1μg～1000μg，优选10μg～500μg，更优选10～100μg，最优选每剂量10～50μg。
根据本发明一个具体的实施例，所述疫苗为任何药学上可接受的剂型。
根据本发明一个具体的实施例，所述疫苗免疫方式为肌肉注射、皮下注射、皮内注射、微针注射中至少之一种。
本发明提出了一种制备前面所述疫苗的方法，包括以下步骤：将前面所述的多肽免疫偶联物与佐剂进行混合处理。
根据本发明一个具体的实施例，所述佐剂包括氢氧化铝、磷酸铝、单磷酰脂质、寡核苷酸CpG中的至少之一。
根据本发明一个具体的实施例，所述佐剂为氢氧化铝。
根据本发明一个具体的实施例，所述佐剂中包括终浓度为100μg/L的3D-mLA、终浓度为1mg/L的氢氧化铝。
本发明提出了一种制备抗体的方法，包括：利用前面所述的多肽免疫偶联物对动物进行免疫接种；采集经过免疫接种的动物的血清；以及从所述血清中纯化出目的抗体。
本发明提出了一种在受试者体内刺激抗肽抗体或抗RBD抗体生成的方法，是通过下列方式的至少之一实现的：1)给与受试者前面所述的多肽免疫偶联物；2)给予受试者前面所述的药物组合物；以及3)向受试者施用前面所述的疫苗。根据本发明的一些具体实施例的方法可以在受试者体内产生较高滴度的抗肽抗体以及抗RBD抗体，极大的提高了机体对新型冠状病毒肺炎的免疫力。
本发明提出了一种治疗或预防新型冠状病毒肺炎的方法，包括下列中的至少之一：1)给与受试者前面所述的多肽免疫偶联物；2)给予受试者前面所述的药物组合物；3)向受试者施用前面所述的疫苗。根据本发明的一些具体实施例，前面所述的多肽免疫偶联物、药物组合物或疫苗均可以刺激机体产生较高滴度的抗肽抗体或抗RBD抗体，可以有效的治疗或预防新型冠状病毒肺炎。
在本文中所使用的术语“给予”指将预定量的物质通过某种适合的方式引入病人。本发明实施例中的多肽免疫偶联物、药物组合物或疫苗可以通过任何常见的途径给药，只要它可以到达预期的组织。给药的各种方式是可以预期的，包括腹膜，静脉，肌肉，皮下，皮层，口服，局部，鼻腔，肺部和直肠，但 是本发明不限于这些已举例的给药方式。然而，由于口服给药时，口服给药的组合物的活性成分应该被包被或被配制以防止其在胃部被降解。优选地，本发明的组合物可以注射制剂被给药。此外，本发明的药物组合物可以使用将活性成分传送到靶细胞的特定器械来给药。
本发明实施例中的多肽免疫偶联物、药物组合物或疫苗的给药频率和剂量可以通过多个相关因素被确定，该因素包括要被治疗的疾病类型，给药途径，病人年龄，性别，体重和疾病的严重程度以及作为活性成分的药物类型。根据本发明的一些具体实施例，日剂量可分为适宜形式的1剂、2剂或多剂，以在整个时间段内以1次、2次或多次给药，只要达到治疗有效量即可。
术语“治疗有效量”是指足以显著改善某些与疾病或病症相关的症状的量，也即为给定病症和给药方案提供治疗效果的量。术语“治疗”用于指获得期望的药理学和/或生理学效果。本文使用的“治疗”涵盖将发明实施例中的多肽免疫偶联物、药物组合物或疫苗给予个体以治疗，包括但不限于将含本文所述的给予有需要的个体。
需要说明的是，根据本发明实施例的多肽免疫偶联物、药物组合物、疫苗、制备抗体的方法、在受试者体内刺激抗肽抗体或抗RBD抗体生成的方法是本申请的发明人经过艰苦的创造性劳动和优化工作才发现和完成的。
下面将对实施例作具体介绍。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1目标多肽的合成
本实施例采用常规的固相合成多肽的方法，制备了多肽7#、45#、46#以及S2，其中多肽的氨基酸序列如表1所示。
表1：
Figure PCTCN2021123587-appb-000001
注：合成后的7号肽、45号肽及46号肽氨基酸序列C端为-CONH 2形式或者COOH形式；N端为NH 2或者CH 3CONH-形式。
制备多肽的具体的实验操作步骤如下：
(1)脱Fmoc保护
将市售的Rink Amide AM resin装入一个有过滤器且耐有机溶剂的反应管中，并用一个耐有机溶剂的盖子盖紧。用DMF(二甲基甲酰胺)洗涤1分钟后，加入过量的20％哌啶/DMF(体积比)溶液，盖 紧后轻轻摇晃反应管，使之混合均匀并保持脱除Fmoc保护15分钟，抽干后再用DMF洗涤三次。
(2)肽键缩合
将经过预活化(预活化2-3分钟后)的3倍树脂氨基摩尔量的Fmoc保护的氨基酸、3倍树脂氨基摩尔等量的活化剂HBTU、以及6倍树脂氨基摩尔等量的DIPEA(N，N-二异丙基乙基胺)加入步骤(1)中获得的反应管中，使用DMF作为溶剂，并保证以上的试剂完全溶解，可完全覆盖树脂。每隔2-3分钟摇晃混匀树脂一次，使其反应20-30分钟。
反应结束后后，于室温下向反应管内加入50倍摩尔过量的乙酸酐DMF溶液后10分钟抽干，再在室温下加入过量的20％哌啶/DMF(体积比)溶液反应30分钟。过滤掉溶液，树脂用DMF洗涤5次。
以上步骤循环反复执行，直到拟合成多肽的全部Fmoc保护氨基酸按线性方式连接到树脂上。
(3)多肽裂解
多肽裂解液选用强酸处理，比如TFA(三氟乙酸)。室温下用TFA、水、EDT二巯基乙醇、苯酚(体积比：92.5：2.5：2.5：2.5)处理步骤(2)得到的树脂2小时。然后仔细收集裂解液到一个玻璃收集器中，并加入用冰预冷的乙醚，收集沉淀多肽，并继续用冷乙醚洗涤5-6次，得到粗品肽。
(4)多肽纯化。
粗品肽经过HPLC(高效液相色谱)纯化、收集、冻干，最终再经HPLC检验纯度(214nm波长)大于85％及质谱检验正确的分子量。
按上述操作步骤后，可获得7#、45#、46#以及S2多肽，其中，SEQ ID NO：1(表1)所示的合成肽的纯度为95％。
实施例2免疫原性偶联物的制备方法
本实施例将实施例1制备的合成肽通过连接体与RBD蛋白形成偶联物，具体实验操作如下所示：
RBD蛋白的制备：将RBD质粒转染到HEK293F细胞，将细胞培养至对数生长期后收集细胞上清。浓缩后通过Anti-flag G1Affinity resin纯化蛋白。粗品蛋白再经过分子筛进行纯化得到RBD蛋白。
RBD蛋白用PBS配制成浓度为1mg/mL溶液。取需要量的RBD溶液，按照摩尔比1：2-1：40加入Sulfo-SMCC(10mg/mL)，室温反应1小时，反应液用超滤浓缩管于4℃、3500rpm条件下离心浓缩。体积浓缩至反应液的1/10后加入PBS至原反应液体积，继续离心浓缩，连续离心浓缩5次以去除游离Sulfo-SMCC。最终获得的RBD-SMCC储液浓度为10mg/mL。称取需要量的实施例1制备的合成肽，用PBS或含有10％DMSO的PBS溶液溶解，发明人对多肽与RBD蛋白的添加比例进行优化，按照多肽与RBD蛋白摩尔比1：1-15：1加入RBD-SMCC储液，其中，多肽与RBD的添加比例如表1所示；然后添加PBS至适宜的反应体积，将获得的混合液于室温条件下反 应1h，SDS-PAGE检测共缀情况，其中，蛋白上样量为2μg，SDS-PAGE检测电泳图如图1所示，图1中7#肽与蛋白摩尔比为10:1，45#肽与蛋白摩尔比为7.5:1，46#与蛋白摩尔比为7:1，S2#肽与蛋白摩尔比为6:1。图2中7#肽与蛋白摩尔比为10:1，S2#肽与蛋白摩尔比为10:1。图3中45#肽与蛋白摩尔比为10:1，46#与蛋白摩尔比为10:1。根据图1-3所示的实验结果可知，当7#肽与蛋白摩尔比为10:1，45#肽与蛋白摩尔比为7.5:1，46#与蛋白摩尔比为7:1，S2#肽与蛋白摩尔比为6:1时形成的多聚体免疫偶联物更多，合成效果较佳。
表2：
项目 摩尔比1 摩尔比2
7#-RBD - 10：1
45#-RBD 7.5：1 10：1
46#-RBD 7：1 10：1
S2#-RBD 6：1 10：1
实施例3疫苗的制备及免疫效果检测
1、疫苗制备。
(1)实验材料
免疫偶联物：RBD蛋白与SEQ ID NO:1所示多肽的偶联物；
佐剂：HA204[每mL含3D-MLA 100μg，氢氧化铝(以铝离子计算)1mg]；
(2)实验分组
本实施例的实验分组如表3所示，其中，7#肽与RBD摩尔比为10:1，45#肽与RBD摩尔比为7.5:1，46#与RBD摩尔比为7:1，S2#肽与RBD摩尔比为6:1，“组别”一栏命名规则为“新型冠状病毒的RBD结构域与多肽偶联物-佐剂”，如“RBD-7#+HA204”中“RBD-7#”表示新型冠状病毒S蛋白的RBD结合域与7号肽的偶联物、“HA204”表示命名为HA204的佐剂。
表3：
Figure PCTCN2021123587-appb-000002
(3)操作步骤
取实施例2制备的免疫原性偶联物与佐剂HA204、PBS溶液混合，于室温、30RPM摇床1小时，所得终浓度含多肽RBD蛋白抗原100μg/mL、佐剂HA204 12.5％(v/v)，即为最终样品。
2、免疫效果实验
免疫效果实验的分组如表3所示，利用表3中制备的待测样品对小鼠进行免疫原性的研究，将体重为16-18g的健康BALB/c小鼠随机分组，每组6只小鼠；利用表3中各实验组(RBD、RBD+HA204、RBD-7#+HA204、RBD-45#+HA204、RBD-46#+HA204、RBD-S2+HA204)的待测样品对小鼠进行腹腔注射，于第0天、第14天、第21天各腹腔注射1次，每次给药体积为200μL/只；对Control组小鼠进行腹腔注射生理盐水，于第0天、第14天、第21天各腹腔注射1次，每次给药体积为100μL/只，所有小鼠分别于第0天、第21天注射前及第28天各采血1次，分离血清，将血清20倍稀释后，采用酶联免疫吸附(ELISA)方法测定各组小鼠在第21天和第28天血清抗体水平，具体操作如下：
(1)利用包被液(0.05M碳酸盐-碳酸氢盐缓冲液)将RBD溶液稀释为终浓度为1μg/mL的工作液，充分混匀后按100μL/孔加入酶标板，贴上封片纸，2～8℃过夜包被。
(2)吸掉孔内的上清液，用PBST洗5遍后，加入100μL含1％BSA的PBS于室温条件下封闭1h。
(3)封闭结束后，用PBST洗5次。
(4)加样：在步骤(3)洗涤后的每孔内加入100μL稀释后的血清样品，于室温条件下孵育2h，用PBST洗5次。
(5)加酶标抗体：在步骤(4)洗涤后的每孔内加入100μL稀释后的山羊抗小鼠IgG HRP酶标二抗，于室温条件下孵育1h。
(6)孵育完成后，用PBST洗5次。
(7)加底物液显色：在步骤(6)洗涤后的每孔内加入100μL TMB显色。
(8)终止反应：在步骤(7)加底物显色液后的每孔内加入50μL 1N硫酸终止反应。
使用酶标仪测定血清抗体的OD 450值，多肽偶联物与不同佐剂组合后抗RBD抗体水平结果如表4所示，结果表明45号肽与RBD共缀时可明显增加抗RBD抗体滴度。
表4：
Figure PCTCN2021123587-appb-000003
Figure PCTCN2021123587-appb-000004
3、假病毒中和实验
根据上述结果，发明人比较RBD-HA204组与RBD-45#+HA204组小鼠D21和D28血清产生的南非株假病毒中和抗体的水平，具体实验操作如下：
(1)将本实施例实验2获得的小鼠血清于56℃水浴灭活30min，用DMEM完全培养基在96孔细胞培养板中倍比稀释；
(2)在血清中加入假病毒(650TCID 50/孔)，细胞培养箱中(37℃，5％CO 2)孵育1小时；
(3)向96孔板中每孔加100μL Huh7细胞，使每孔细胞为2×10 4个；
(4)将96孔板放入细胞培养箱中，37℃，5％CO2培养20～28小时；
(5)从细胞培养箱中取出96孔板，每孔吸弃150μL上清，然后加入100μL荧光素酶检测试剂，室温避光反应2min；
(6)反应结束后，将反应孔中的液体反复吹吸6～8次，使细胞充分裂解，从每孔中吸出150μL液体，加于对应96孔化学发光检测板中，置于化学发光检测仪中读取发光值；
RBD-HA204组与RBD-45#+HA204组产生的假病毒中和抗体水平如图4所示，其中，45号肽与RBD共缀后，第21天中和抗体水平显著高于RBD组，第28天RBD-45#+HA204组产生中和抗体水平比RBD-HA204组高，但差异不显著，两组均呈现良好的免疫效果。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例 或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (18)

  1. 一种多肽免疫偶联物，其特征在于，包括：多肽和新型冠状病毒S蛋白的RBD；
    其中，所述多肽与新型冠状病毒S蛋白的RBD的摩尔比为1：1～15：1。
  2. 根据权利要求1所述的多肽免疫偶联物，其特征在于，所述多肽为新型冠状病毒S蛋白抗原表位。
  3. 根据权利要求1或2所述的多肽免疫偶联物，其特征在于，所述多肽具有SEQ ID NO：1所示的氨基酸序列或者与SEQ ID NO:1具有至少99％、至少95％、至少90％、至少85％、至少80％、至少75％同一性的氨基酸序列。
  4. 根据权利要求1所述的多肽免疫偶联物，其特征在于，所述新型冠状病毒S蛋白的RBD具有SEQ ID NO：2所示的氨基酸序列或者与SEQ ID NO:2具有至少99％、至少95％、至少90％、至少85％、至少80％、至少75％同一性的氨基酸序列。
  5. 根据权利要求1～4任一项所述的多肽免疫偶联物，其特征在于，所述多肽通过连接体与所述RBD相连。
  6. 根据权利要求5所述的多肽免疫偶联物，其特征在于，所述连接体为磺基琥珀酰亚胺4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯。
  7. 权利要求1～6任一项所述的多肽免疫偶联物在制备药物中的用途，所述药物用于预防或治疗新型冠状病毒肺炎。
  8. 权利要求1～6任一项所述的多肽免疫偶联物在制备疫苗中的用途，所述疫苗用于预防或治疗新型冠状病毒肺炎。
  9. 一种药物组合物，其特征在于，包括权利要求1～6任一项所述的多肽免疫偶联物。
  10. 一种疫苗，其特征在于，包括权利要求1～6任一项所述的多肽免疫偶联物；以及
    药学上可接受的佐剂。
  11. 根据权利要求10所述的疫苗，其特征在于，所述佐剂包括氢氧化铝、磷酸铝、单磷酰脂质、寡核苷酸CpG中的至少之一；
    任选地，所述佐剂为氢氧化铝；
    优选的，所述佐剂中包括终浓度为100μg/L的3D-MLA、终浓度为1mg/L的氢氧化铝。
  12. 根据权利要求10或11所述的疫苗，其特征在于，每剂量所述疫苗中佐剂的含量为1μg～1000μg，优选10μg～500μg，更优选10～100μg，最优选每剂量10～50μg。
  13. 根据权利要求10～12任一项所述的疫苗，其特征在于，所述疫苗为任何药学上可接受的剂型。
  14. 根据权利要求10～13任一项所述的疫苗，其特征在于，所述疫苗免疫方式为肌肉注射、皮下注射、皮内注射、微针注射中至少之一种。
  15. 一种制备权利要求10～14所述疫苗的方法，其特征在于，包括以下步骤：将权利要求1～6任一项所述的多肽免疫偶联物与佐剂进行混合处理；
    任选地，所述佐剂包括氢氧化铝、磷酸铝、单磷酰脂质、寡核苷酸CpG中的至少之一；
    任选地，所述佐剂为氢氧化铝；
    优选地，所述佐剂中包括终浓度为100μg/L的3D-MLA、终浓度为1mg/L的氢氧化铝。
  16. 一种制备抗体的方法，其特征在于，包括：
    利用权利要求1～6任一项所述的多肽免疫偶联物对动物进行免疫接种；
    采集经过免疫接种的动物的血清；以及
    从所述血清中纯化出目的抗体。
  17. 一种在受试者体内刺激抗肽抗体或抗RBD抗体生成的方法，其特征在于，是通过下列方式的至少之一实现的：
    1)给与受试者权利要求1～6任一项所述的多肽免疫偶联物；
    2)给予受试者权利要求9所述的药物组合物；以及
    3)向受试者施用权利要求10～14任一项所述的疫苗。
  18. 一种治疗或预防新型冠状病毒肺炎的方法，其特征在于，包括下列中的至少之一：
    1)给与受试者权利要求1～6任一项所述的多肽免疫偶联物；
    2)给予受试者权利要求9所述的药物组合物；
    3)向受试者施用权利要求10～14任一项所述的疫苗。
PCT/CN2021/123587 2021-10-13 2021-10-13 多肽-rbd免疫偶联物及其用途 WO2023060483A1 (zh)

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Publication number Priority date Publication date Assignee Title
CN112608908A (zh) * 2020-12-25 2021-04-06 中国食品药品检定研究院 重组新型冠状病毒刺突蛋白受体结合区9型腺相关病毒的构建方法
WO2021159040A2 (en) * 2020-02-07 2021-08-12 Modernatx, Inc. Sars-cov-2 mrna domain vaccines
CN113769080A (zh) * 2021-09-17 2021-12-10 清华大学 多肽免疫偶联物及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021159040A2 (en) * 2020-02-07 2021-08-12 Modernatx, Inc. Sars-cov-2 mrna domain vaccines
CN112608908A (zh) * 2020-12-25 2021-04-06 中国食品药品检定研究院 重组新型冠状病毒刺突蛋白受体结合区9型腺相关病毒的构建方法
CN113769080A (zh) * 2021-09-17 2021-12-10 清华大学 多肽免疫偶联物及其应用

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