WO2022007478A1 - 一种定点突变的载体蛋白及其在制备疫苗中的用途 - Google Patents

一种定点突变的载体蛋白及其在制备疫苗中的用途 Download PDF

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WO2022007478A1
WO2022007478A1 PCT/CN2021/090074 CN2021090074W WO2022007478A1 WO 2022007478 A1 WO2022007478 A1 WO 2022007478A1 CN 2021090074 W CN2021090074 W CN 2021090074W WO 2022007478 A1 WO2022007478 A1 WO 2022007478A1
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site
protein
group
directed
amino acids
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PCT/CN2021/090074
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French (fr)
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王浩猛
严志红
晏巧玲
邵娟
史建明
隋秀文
李军强
朱涛
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康希诺生物股份公司
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Priority to EP21837338.9A priority Critical patent/EP4180447A4/en
Priority to CA3185421A priority patent/CA3185421A1/en
Priority to KR1020227046500A priority patent/KR20230021677A/ko
Priority to JP2022581652A priority patent/JP2023532559A/ja
Publication of WO2022007478A1 publication Critical patent/WO2022007478A1/zh
Priority to US18/150,496 priority patent/US20230183298A1/en

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    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
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    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
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    • C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
    • C07C323/59—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton with acylated amino groups bound to the carbon skeleton
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    • C07C2601/02—Systems containing only non-condensed rings with a three-membered ring

Definitions

  • the present application relates to the field of biopharmaceuticals, in particular to a site-directed mutagenesis protein, a site-directed modified protein and its use in preparing vaccines, especially meningococcal multivalent vaccines.
  • Neisseria meningococcus a worldwide acute respiratory infectious disease that still seriously endangers human health, especially are children.
  • Neisseria meningitidis can be divided into 13 serogroups according to the differences in their capsular polysaccharide structure, and all serogroups are pathogenic.
  • A, B, C, Y, W135 Neisseria meningitidis pathogens account for more than 95% of Neisseria meningitidis-related diseases.
  • group B meningococcus Neisseria meningitidis serogroup B, MenB
  • group B Nm capsular polysaccharide contains epitopes that are potentially cross-immune with human anti-stress, resulting in its weak immunogenicity and can induce autoimmune diseases, so , the vaccine research using group B Nm capsular polysaccharide as immunogen has encountered serious challenges.
  • the international research on group B Nm vaccine mainly adopts two strategies, one is the outer membrane protein-based outer membrane vesicle (Outer Menbrane Vesicle OMV) vaccine, such as three representative OMV vaccines: VA-ENGOC-BC, MenBvacTM group B OMV vaccine, group B meningococcal vaccine MeNZB.
  • the other is a recombinant protein vaccine based on reverse vaccinology technology, such as 4C MenB (Bexsero), bivalent fHBp (r-fHBp), rLP2086.
  • Bivalent fHBp is a bivalent protein vaccine containing fHbp type 1 and type 3, which has been proved to have bactericidal effect in adolescents; but in the infant group, the proportion of people with side effects such as fever and local redness and swelling is obvious Therefore, further research on Bivalent fHBp (r-fHBp) vaccine is urgently needed to improve its immunogenicity and reduce side effects.
  • the present application provides site-directed mutagenic proteins, conjugates of site-directed mutagenic proteins, vaccines or immunogenic compositions and uses thereof.
  • the site-directed mutagenesis protein introduces the amber codon TAG at the specific site of the antigen protein, and then uses the orthogonal aminoacyl tRNA synthetase-tRNA to site-directly mutate the non-natural amino acid with cross-linking property to the specific site of the antigen protein .
  • the site-directed mutated protein reacts with liposomes through Click to form covalent bonds to obtain lipoproteins.
  • the site-directed modified lipoprotein obtained in this application has the same liposome length and significantly controllable quality, which can effectively avoid the disadvantage of uneven lipidation of recombinant lipoprotein during the expression process, and provides an effective method for improving the quality of lipoprotein vaccine. s method.
  • the present application relates to a site-directed mutagenesis protein, wherein the protein is selected from one or two or more muteins of the meningococcal group B fHBP protein, wherein at least one site on the protein antigen is mutated.
  • Amino acids are mutated to unnatural amino acids containing azide, alkynyl end groups, or other reactive groups.
  • meningococcal group B fHBP protein is not limited, and can be selected according to actual needs.
  • the mutated amino acid site is preferably a site that does not affect the antigenic epitope, so as to reduce the impact of amino acid mutation on the immunogenicity of the protein antigen.
  • the protein is selected from the variant proteins formed by one or more of the fHBP proteins of group B meningococcus; preferably, the protein antigen is selected from the group B meninges Variant 2, 3 in Subfamily A or variant 1 in Subfamily B of the pneumococcal fHBP protein.
  • the non-natural amino acids are phenylalanine derivatives, tyrosine derivatives, glutamine derivatives, alanine derivatives, cysteine derivatives, serine derivatives derivatives or lysine derivatives.
  • the unnatural amino acid is an azide-containing lysine derivative.
  • described unnatural amino acid is:
  • the mutation site of the protein antigen is one or more amino acids that do not affect the epitope.
  • the protein antigen is variant2 in the subfamily A of the meningococcal fHBP protein antigen of group B, and the mutation site can be one or more amino acids at any position in SEQ ID NO: 1 .
  • the mutation site of the protein antigen is positions 2-30 in SEQ ID NO: 1 or other sites that do not affect the antigenic epitope. More preferably, the mutation site is one or more amino acids on amino acids 2-10 in SEQ ID NO: 1
  • the protein antigen is variant3 in the subfamily A of the meningococcal fHBP protein antigen of group B, and the mutation site can be one or more amino acids at any position in SEQ ID NO:2 .
  • the mutation site of the protein antigen is positions 2-30 in SEQ ID NO: 2 or other sites that do not affect the antigenic epitope. More preferably, the mutation site is one or more amino acids on amino acids 2-10 in SEQ ID NO:2.
  • the protein antigen is variant 1 in the subfamily B meningococcal fHBP protein antigen
  • the mutation site of the protein antigen is one or more on any site in SEQ ID NO: 3 of amino acids.
  • the mutation site of the protein antigen is positions 2-10 in SEQ ID NO: 3 or other sites that do not affect the antigenic epitope. More preferably, the mutation site is one or more amino acids on amino acids 2-10 in SEQ ID NO:3.
  • the difference between the site-directed mutated protein and the amino acid sequence of the target protein before mutation is that the amino acid at position X of the amino acid sequence of the protein before mutation is mutated to Lys-azido, and the connection mode of the mutated amino acids is shown in the following formula :
  • the X is the mutation site
  • AA is the amino acid before and after the mutation site.
  • the present application also provides a method for preparing a site-directed mutagenesis protein, comprising the following steps: using a gene codon expansion technique to introduce an unnatural amino acid site-directed into a specific site of the protein to obtain a site-directed mutagenesis protein.
  • the present application also provides a site-directed mutagenic group B meningococcal fHBP protein antigen conjugate, which is prepared by coupling the site-directed mutagenesis protein described in the present application with a modified compound.
  • the modified compound is a compound whose terminal group contains an alkynyl group or a modified alkynyl group.
  • the modified compound is selected from a sugar, nucleic acid, amino acid, polypeptide or small molecule compound containing an alkynyl end group, or, a sugar, nucleic acid, amino acid, polypeptide or small molecule compound through the end Modified products obtained by modification of alkynyl groups.
  • the sugar, nucleic acid, amino acid, polypeptide or small molecule compound described in this application can be a modification of the terminal alkynyl group of the sugar, nucleic acid, amino acid, polypeptide or small molecule compound, and can be prepared by site-specific coupling through the catalysis of monovalent copper. Conjugates are obtained, or sugars, nucleic acids, amino acids, polypeptides or small molecular compounds are modified substances with cyclooctyne or its derivatives as modified groups, and site-specific coupling is directly realized.
  • the site-directed mutagenesis of group B meningococcal fHBP protein described in this application is prepared by Click reaction with a molecule containing or modified alkynyl end group.
  • the Click reaction may be a monovalent copper-mediated Click reaction, or a copper-free Click reaction mediated by cyclooctyne or a derivative thereof.
  • the modified compound is a lipoprotein receptor agonist.
  • the modified compound is a TLR2 receptor agonist.
  • the agonist is selected from tripalmitoyl-S-glycerocysteine, monophosphoryl lipid A, dipalmitoyl-S-glycero-cysteine, or analogs thereof.
  • amino acid at position X of the amino acid sequence of the protein is modified by mutation into the following structure:
  • X is the mutation site
  • AA is the amino acid before and after the mutation site
  • R 2 is a TLR2 receptor agonist.
  • the R 2 is tripalmitoyl-S-glycerocysteine, monophosphoryl lipid A, dipalmitoyl-S-glyceryl-cysteine or an analog thereof, more preferably tripalmitoyl Acyl-S-glycerol cysteine analogs selected from analogs of the following structural formula:
  • the R 2 is a monophosphoryl lipid A receptor agonist or a derivative thereof, preferably a monophosphoryl lipid A receptor agonist, and the structural formula is as follows:
  • the R-terminus can be coupled with the site-directed mutation of group B meningococcal fHBP protein,
  • R 3 is selected from phosphate or H
  • R 4 is selected from n is 1, 3, 5; or
  • R 5 is selected from
  • R 6 is selected from H or
  • R 7 is selected from
  • R 8 is selected from H or OH.
  • the molar ratio of group B meningococcal fHBP egg:modified compound in the conjugate is 1:1-30.
  • the meningococcal group B fHBP protein is variant2 in Subfamily A
  • the mutation site of the fHBP protein is the one on the amino acid sequence of positions 2-30 in SEQ ID NO:1 or more amino acids, preferably, the mutation site is one or more amino acids on the 2-10th amino acid of the sequence of SEQ ID NO: 1.
  • the meningococcal group B fHBP protein is variant3 in Subfamily A
  • the mutation site of the fHBP protein is one of the amino acid sequences at positions 2-30 in SEQ ID NO:2 or A plurality of amino acids, preferably, the mutation site is one or more amino acids on amino acids 2-10 of the sequence of SEQ ID NO: 2.
  • the meningococcal group B fHBP protein is variant 1 in Subfamily B
  • the mutation site of the fHBP protein can be the one on the 2-30th amino acid sequence in SEQ ID NO:3 or multiple amino acids, preferably, the mutation site is one or more amino acids on amino acids 2-10 of the sequence of SEQ ID NO: 3.
  • the present application also relates to a vaccine or immunogenic composition comprising one or more of said site-directed mutagenic protein or said conjugate.
  • the vaccine or immunogenic composition simultaneously comprises the three site-directed mutagenic proteins to form a multivalent vaccine or immunogenic composition, or simultaneously comprises the three conjugates form a multivalent vaccine or immunogenic composition.
  • the dose of the meningococcal group B fHBP site-directed mutagenesis protein or conjugate is 10-100 micrograms.
  • the vaccine or immunogenic composition comprises a pharmaceutically acceptable excipient, carrier or diluent.
  • the vaccine or immunogenic composition is further used in combination with the ACW135Y swarm meningitis conjugate vaccine.
  • each dose of the vaccine or immunogenic composition contains 5-10 micrograms of group A meningococcal polysaccharide antigens, each dose contains 5-10 micrograms of group C meningococcal polysaccharide antigens, and each dose contains 5-10 micrograms of meningococcal polysaccharide antigens.
  • Microgram W135 meningococcal polysaccharide antigen each dose contains 5-10 microgram Y group meningococcal polysaccharide antigen
  • each dose contains 10-100 microgram B group meningococcal fHBP site-directed mutation protein or conjugate.
  • the present application also relates to the use of the site-directed mutagenesis protein, the conjugate, or the immunogenic composition in the preparation of vaccines.
  • the vaccine described therein is a meningococcal vaccine.
  • the present application also relates to a method for site-directed mutagenesis and site-directed modification of protein antigens, the method comprising using gene codon expansion technology to introduce unnatural amino acids into specific sites of proteins to obtain site-directed mutagenesis proteins, and the site-directed mutagenesis proteins further Coupling with a modified compound, the modified compound is a compound whose terminal group contains an alkynyl group or a modified alkynyl group.
  • tRNA Pyl Methanococcus archaea tRNA
  • tRNA Pyl /PylRS pyrrolysine-tRNA synthetase
  • the site-directed mutagenesis of the antigen protein can be further
  • the site-directed modified raw material (the pre-modified recombinant protein fHBP of the present application has been proved to prevent the infection of group B meningococcus), the site-directed mutated antigen protein is conjugated with liposomes to obtain a site-directed modified lipid protein .
  • the protein is selected from one or more variant proteins formed from the fHBP protein of group B meningococcus; preferably, the protein antigen is selected from the group B meningococcal fHBP protein Subfamily A variant2, 3 or variant 1 in Subfamily B.
  • Described unnatural amino acid is selected from among phenylalanine derivative, tyrosine derivative, glutamine derivative, alanine derivative, cysteine derivative, serine derivative or lysine derivative at least one of.
  • the unnatural amino acid is an azide-containing lysine derivative.
  • the unnatural amino acid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the modified compound is selected from a sugar, nucleic acid, amino acid, polypeptide or small molecule compound containing an alkynyl terminal group; or a modification obtained by modifying a terminal alkynyl group of a sugar, nucleic acid, amino acid, polypeptide or small molecule compound.
  • the modified compound is a lipoprotein receptor agonist.
  • the lipid-protein receptor agonist is selected from tripalmitoyl-S-glycerocysteine, monophosphoryl lipid A, dipalmitoyl-S-glyceryl-cysteine or its analog.
  • the lipid protein receptor agonist is a tripalmitoyl-S-glycerol cysteine analog selected from the analogs of the following structural formula:
  • the present application also relates to a tripalmitoyl-S-glycerol cysteine analog for site-directed mutagenesis and site-directed modification of protein antigens, selected from the analogs of the following structural formula:
  • the site-directed mutagenesis protein provided by this application can introduce unnatural amino acids at any site of the protein, thereby creating a protein antigen that can only be specifically modified at this site;
  • the site-directed mutagenesis protein provided by this application can achieve the purpose of efficient and specific modification by utilizing the unique active group on the non-natural amino acid;
  • the conjugates provided in this application through the optimization of the modification conditions, utilize the cyclooctyne-mediated copper-free Click reaction, which can achieve efficient, harmless, and simple modification reactions to proteins;
  • conjugates provided in this application by introducing modified groups confirmed by the structure, the obtained conjugates of meningococcal group B fHBP protein have uniform components and controllable quality, which can ensure immunogenicity and can significantly reduce side reactions. degree;
  • the site-directed mutagenesis protein or conjugate provided in this application can be used in combination with the ACW135Y group meningitis conjugate vaccine to improve its protection coverage.
  • Fig. 1 The particle size diagram of MenB-V1.55 protein
  • Fig. 5 The particle size diagram of MenB-V2.16-S3-L1 protein.
  • orthogonal refers to a molecule (eg, orthogonal tRNA (O-tRNA) and/or orthogonal aminoacyl tRNA synthetase (O-RS)) that functions as an endogenous component of the cell, However, its activity is reduced compared to its corresponding endogenous molecule within the cell or translation system, or it does not function as an endogenous component of the cell.
  • O-tRNA orthogonal tRNA
  • OF-RS orthogonal aminoacyl tRNA synthetase
  • orthogonal When used for tRNA and aminoacyl-tRNA synthetases, orthogonal means that the efficiency of the orthogonal tRNA complexed with endogenous tRNA synthetase decreases compared to when endogenous tRNA and endogenous tRNA synthetase complexed, Such as drop below 20%, 10%, 5% or 1%. Orthogonal molecules lack the normal function of endogenous complementary molecules within the cell.
  • Clark reaction uses Huisgen [3+2] cyclization of azides and alkynes.
  • Natural MenB undergoes lipidation modification at its N-terminus, which does not affect the three-dimensional structure of MenB protein, but acts to anchor the protein antigen to the cell membrane.
  • Structural studies show that the first 20 amino acids of the N-terminal of MenB protein are not folded to form a secondary structure, but are in a stretched state, and their function is to expose the antigenic part through the bacterial outer membrane to the bacterial surface. Therefore, the mutated site preferentially selects the N-terminal 20 amino acids, among which the 2-10th position is preferentially selected.
  • Tables 1-3 wherein the amino acid positions respectively refer to the positions on the sequences shown in SEQ ID NOs: 1-3.
  • pET28a-MenB-V1.55-G2 pET28a-MenB-V1.55-S3, pET28a-MenB-V1.55-S4, pET28a-MenB-V1.55-G5, pET28a-MenB-V1.55-G6, pET28a-MenB-V1.55-G7, pET28a-MenB-V1.55-G8, pET28a-MenB-V1.55-S9, pET28a-MenB-V1.55-G10.
  • pET28a-MenB-V2.16-G2 pET28a-MenB-V2.16-S3, pET28a-MenB-V2.16-S4, pET28a-MenB-V2.16-G5, pET28a-MenB-V2.16-G6, pET28a-MenB-V2.16-G7, pET28a-MenB-V2.16-G8, pET28a-MenB-V2.16-V9, pET28a-MenB-V2.16-A10.
  • pET28a-MenB-V3.45-G2 pET28a-MenB-V3.45-S3, pET28a-MenB-V3.45-S4, pET28a-MenB-V3.45-G5, pET28a-MenB-V3.45-G6, pET28a-MenB-V3.45-G7, pET28a-MenB-V3.45-G8, pET28a-MenB-V3.45-V9, pET28a-MenB-V3.45-A10.
  • the expression plasmid vectors pET28a-MenB-V1.55-G2, pET28a-MenB-V2.16-S3 and pET28a-MenB-V3.45-S4 obtained in Example 1 were cultured in LB medium at 37°C for 12-16 months After 1 hour, it was amplified again until the OD value of the bacterial solution reached 0.6 to 1.0. Lys-azido was added to the final concentration of 1 mM, and the amplification was continued for 30 minutes at 37 °C. IPTG was added to the final concentration of 0.5 mM, and the final concentration of arabinose was 0.2. %, the cells were collected after inducing expression at 24°C for 12 hours.
  • the collected cells were equilibrated and resuspended with Ni-NTA-Bind-Buffer, sonicated, centrifuged to remove cell debris, subjected to Ni-NTA metal chelate affinity chromatography, washed with Ni-NTA-Wash-Buffer, and finally washed with Ni-NTA-Wash-Buffer.
  • tripalmitoyl-S-glycerol cysteine analog 8 is as follows:
  • the reaction system is as follows:
  • pET28a-MenB-V1.55-G2 protein 1 microgram per microliter tripalmitoyl-S-glycerol cysteine compound 8 20 micrograms per microliter Cu 2+ 1mM BTTES 400 micromoles PBS 0.01M (pH ⁇ 7) Small pieces of Cu wire Sufficient
  • Example 4 The same operation steps as in Example 4 were used to obtain MenB-V3.45-S4-L1, a site-directed conjugate of tripalmitoyl-S-glycerolcysteine analog and pET28a-MenB-V3.45 protein.
  • Group B meningococcal strain 440902 was used, which is ST4821 sequence type, belongs to ST4821 sequence group, and is a recent epidemic strain of group B meningococcus in China.
  • target bacteria culture Meningococcus meningitidis 440902 strain on 8-12% blood-nourishing agar plate, incubate with 6-10% carbon dioxide at 37°C for 16-18 hours, scrape the bacterial moss into normal saline, and the bacteria are turbid According to the counting method, the target bacteria were diluted to 1 ⁇ 10 6 .
  • mice serum to be tested was extinguished at 56°C for 1 hour to extinguish the inherent complement activity of the mouse serum.
  • Pel-Freez baby rabbit complement was added to the serum of the mice to be tested, and fire-extinguishing complement and complement control were set at the same time, which were fold diluted to a 96-well culture plate, and 10 microliters of freshly prepared target bacteria were added dropwise. After shaking and mixing, incubate at 37°C for 2 to 4 hours.
  • Color development Cover the soft agar containing 150-300 micrograms per milliliter of TTC on the solid impact agar comprehensive medium cultivated overnight, and develop color at an appropriate temperature and time.
  • the lipoproteins after the V1 and V2 variants of MenB protein and liposomes are site-specifically coupled, have significant bactericidal power compared with the negative control group with normal saline, and have no significant difference with the trivalent fHBP positive control group. ;
  • the lipoprotein after the V1, V2 and V3 variants of MenB protein is coupled to the liposome has a significantly improved bactericidal power
  • V3 variant lipoprotein of MenB protein has a certain cross-protection effect on V2 strains.
  • Test method NIH mice, body weight: 18-22 g/mouse, 5 rats in each group; guinea pigs, body weight: 250-350 g/mouse, 2 rats in each group;
  • Abnormal toxicity test According to the inoculation dose specified in the 2015 edition of the Chinese Pharmacopoeia, Appendix XIIF, item XIIF abnormal toxicity test method: mice, intraperitoneal injection of 0.5ml (1 human dose), guinea pig, intraperitoneal injection of 5ml (10 human doses) .
  • Acute toxicity test According to the 2015 edition of "Chinese Pharmacopoeia" three appendix XIIF abnormal toxicity test method, the dose is 5 times the inoculation dose, and the concentrated vaccine is prepared with the valence B group meningoencephalin stock solution.
  • the dose is mice, Intraperitoneal injection of 0.5 ml (5 human doses), guinea pigs intraperitoneal injection of 5 ml (50 human doses).
  • mice and guinea pigs were inoculated with the test article, they were continuously observed for 7 days. During the observation period, the animals should be all healthy and alive, and there should be no abnormal reaction.
  • the trivalent group B meningococcal protein vaccine provided by the present invention passed the abnormal toxicity and acute toxicity tests.
  • Fig. 1 is a particle size diagram of MenB-V1.55 protein, including two main particle size ranges, which are about 20 nm and 90 nm, respectively.
  • Figure 2 is the particle size diagram of MenB-V2.16 protein, including two main particle size ranges, about 30 nm and 400 nm respectively;
  • Figure 3 is the particle size diagram of MenB-V1.55-G2-L1 protein, the main particle size The range is about 50 nanometers.
  • Figure 4 is a particle size diagram of MenB-V2.16-S3-L1 protein, and the main particle size range is about 60 nm.
  • Figure 5 is a particle size diagram of MenB-V2.16-S3-L1 protein, and the main particle size range is about 50 nm.
  • the site-directed modified lipid proteins obtained in this application have the same length of liposomes and remarkably controllable quality, which can effectively avoid the disadvantage of uneven lipidation of the recombinant lipid proteins during the expression process, ensure the immunogenicity, and at the same time significantly improve the quality of the liposomes. Reduce the degree of side effects.

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Abstract

一种定点突变、定点修饰的蛋白抗原及定点突变和定点修饰蛋白抗原的方法，所述方法包括使用基因密码子扩展技术将非天然氨基酸定点引入到蛋白抗原特定位点，借助非天然氨基酸与修饰剂，所述修饰剂如三棕榈酰-S-甘油半胱氨酸、单磷酰脂质A等受体激动剂，与蛋白抗原定点修饰。定点突变、定点修饰的蛋白抗原的应用，如作为疫苗等用途。

Description

一种定点突变的载体蛋白及其在制备疫苗中的用途 技术领域
本申请涉及生物制药领域，具体地说，本申请涉及一种定点突变的蛋白、定点修饰的蛋白质及其在制备疫苗，特别是脑膜炎球菌多价疫苗中的用途。
背景技术
脑膜炎奈瑟氏菌(Neisseria meningococcus,Nm)感染所致的流行性脑脊髓膜炎(流脑)，是一种世界范围内致病的急性呼吸道传染病，至今仍严重危害着人类健康，尤其是儿童。脑膜炎奈瑟氏菌根据其荚膜多糖结构的差异可分为13个血清群，且所有血清群均可致病。A、B、C、Y、W135群脑膜炎奈瑟氏菌所致病约占脑膜炎奈瑟氏菌相关疾病的95％以上。其中，B群脑膜炎球菌(Neisseria meningitidis serogroup B,MenB)，由于其荚膜多糖包含了与人类抗压潜在交叉免疫的表位，致使其免疫原性很弱，且能诱发自身免疫疾病，因此，以B群Nm荚膜多糖为免疫原的疫苗研究遇到了严重挑战。
目前国际上针对B群Nm疫苗的研究主要采用两种策略，一种是基于外膜蛋白的外膜囊泡(Outer Menbrane Vesicle OMV)疫苗，例如三种代表性OMV疫苗：VA-ENGOC-BC、MenBvacTM B群OMV疫苗、B群脑膜炎球菌疫苗MeNZB。另一种是基于反向疫苗学技术的重组蛋白疫苗，例如4C MenB(Bexsero)、bivalent fHBp(r-fHBp)、rLP2086。尽管B群脑膜炎球菌疫苗已有多个上市或在研产品，但仍然存在诸多问题亟待解决，例如：目前没有国际通用的B群脑膜炎球菌OMV疫苗、OMV疫苗的保护覆盖范围具有局限性、MenB OMV生产工艺影响其抗原性、“广谱”的B群脑膜炎球菌疫苗难以达到广泛保护性等问题。
总体而言，回顾数十年来对B群Nm OMV蛋白疫苗的研发，临床应用效果显示，在不同地区差异较大，分析原因可能存在以下因素：
①B群Nm型别、亚型较多，彼此间交叉保护性弱；
②各地区流行菌株的抗原性具有地域特征差异及目前尚未被揭示的诸多因素影响；
③国外疫苗制备工艺的复杂性和过度纯化，改变了菌体抗原的立体天然构象，所诱导的抗体针对自然界流行菌株侵袭缺乏特异性；
④多步纯化工艺也导致了丰富抗原位点的丢失、损伤、单一，影响其抗原性。
惠氏公司的Bivalent fHBp(r-fHBp)是含有fHbp1型和3型的双价蛋白疫苗，已经在青少年人群中证明有杀菌作用；但在婴幼儿组中，有发烧、局部红肿等副作用人群比例明显增加，因此急需对Bivalent fHBp(r-fHBp)疫苗做进一步研究，以提高其免疫原性，并降低副作用。
发明内容
有鉴于此，本申请提供了定点突变的蛋白、定点突变蛋白的偶联物、疫苗或免疫原性组合物及其应用。其中，所述定点突变的蛋白在抗原蛋白的特定位点引入琥珀密码子TAG，再利用正交氨酰tRNA合成酶-tRNA将具有交联性质的非天然氨基酸定点突变到抗原蛋白的特定位点。定点突变的蛋白与脂质体经Click反应，形成共价键获得脂质蛋白。本申请获得的定点修饰脂质蛋白，脂质体长度一致，质量显著可控，可有效避免重组脂质蛋白在表达过程中脂质化不均一的缺点，为提高脂质蛋白疫苗质量提供了有效的手段。
具体的，本申请涉及一种定点突变的蛋白，其中所述的蛋白选自B群脑膜炎球菌fHBP蛋白中的一种或两种以上突变蛋白，其中所述蛋白抗原上至少1个位点的氨基酸被突变为非天然氨基酸，所述非天然氨基酸含有叠氮基、炔基端基或其他活性基团。
对于B群脑膜炎球菌fHBP蛋白的具体种类不作限定，可以根据实际需要进行选择。对于突变的氨基酸位点优选不影响抗原表位的位点，减少氨基酸突变对蛋白抗原免疫原性的影响。
在本申请一种具体实施方式中，所述的蛋白选自B群脑膜炎球菌fHBP蛋白中的一种或两种以上形成的变异体蛋白；优选的，所述的蛋白抗原选自B群脑膜炎球菌fHBP蛋白Subfamily A中variant2、3或Subfamily B中variant 1。
在本申请一种具体实施方式中，所述的非天然氨基酸为苯丙氨酸衍生物、酪氨酸衍生物、谷氨酰胺衍生物、丙氨酸衍生物、半胱氨酸衍生物、丝氨酸衍生物或赖氨酸衍生物。
优选的，所述的非天然氨基酸为含有叠氮基的赖氨酸衍生物。
更优选的，所述的非天然氨基酸为：
Figure PCTCN2021090074-appb-000001
示例性地，所述蛋白抗原的突变位点为不影响抗原表位的一个或多个氨基酸。
在本申请的一个实施方式中，所述蛋白抗原为B群脑膜炎球菌fHBP蛋白抗原Subfamily A中variant2，所述突变位点可为SEQ ID NO:1中任何位点上一个或多个的氨基酸。优选地，所述蛋白抗原的突变位点为SEQ ID NO:1中第2-30位或其他不影响抗原表位的位点。更优选地，所述突变位点为SEQ ID NO:1中第2-10位氨基酸上一个或多个的氨基酸
在本申请的一个实施方式中，所述蛋白抗原为B群脑膜炎球菌fHBP蛋白抗原Subfamily A中variant3，所述突变位点可为SEQ ID NO:2中任何位点上一个或多个的氨基酸。优选地，所述蛋白抗原的突变位点为SEQ ID NO:2中第2-30位或其他不影响抗原表位的位点。更优选地，所述突变位点为SEQ ID NO:2中第2-10位氨基酸上一个或多个的氨基酸。
在本申请的一个实施方式中，所述蛋白抗原为B群脑膜炎球菌fHBP蛋白抗原Subfamily B中variant 1，所述蛋白抗原的突变位点为SEQ ID NO:3中任何位点上一个或多个的氨基酸。优选地，所述蛋白抗原的突变位点为SEQ ID NO:3中第2-10位或其他不对影响抗原表位的位点。更优选地，所述突变位点为SEQ ID NO:3中第2-10位氨基酸上一个或多个的氨基酸。
经过突变后，所述定点突变的蛋白与突变前目的蛋白的氨基酸序列的区别在于：突变前蛋白氨基酸序列的第X位氨基酸被突变为Lys-azido，所述突变氨基酸的连接方式如下式所示：
Figure PCTCN2021090074-appb-000002
其中，所述的X为突变位点，AA为突变位点前后的氨基酸。
本申请还提供定点突变的蛋白的制备方法，包括以下步骤：使用基因密码子扩展技术将非天然氨基酸定点引入到蛋白特定位点获得定点突变的蛋白。
本申请还提供一种定点突变的B群脑膜炎球菌fHBP蛋白抗原偶联物，所述的偶联物为本申请所述的定点突变的蛋白与进一步与修饰化合物偶联制备得到。
在本申请一种实施方式中，所述修饰化合物为端基含有炔基或修饰炔基的化合物。
在本申请一种具体实施方式中，所述的修饰化合物选自含有炔基端基的糖、核酸、氨基酸、多肽或小分子化合物，或者，糖、核酸、氨基酸、多肽或小分子化合物通过末端炔基的修饰得到的修饰物。
本申请所述的糖、核酸、氨基酸、多肽或小分子化合物可为糖、核酸、氨基酸、多肽或小分子化合物的末端炔基的修饰物，通过一价铜的催化作用，实现定点偶联制备得到偶联物，或者糖、核酸、氨基酸、多肽或小分子化合物为以环辛炔或其衍生物为修饰基的修饰物，直接实现定点偶联。
本申请所述的定点突变的B群脑膜炎球菌fHBP蛋白与含有或修饰的炔基端基的分子通过Click反应制备得到。所述的Click反应可以为一价铜介导的Click反应，也可以是环辛炔或其衍生物介导的无铜Click反应。
在本申请一种具体实施方式中，所述修饰化合物为脂质蛋白类受体激动剂。优选地，所述修饰化合物为TLR2受体激动剂。
示例性地，所述激动剂为选自三棕榈酰-S-甘油半胱氨酸、单磷酰脂质A、二棕榈酰-S-甘油基-半胱氨酸或其类似物。
示例性地，所述蛋白的氨基酸序列的第X位氨基酸被突变修饰为如下结构：
Figure PCTCN2021090074-appb-000003
或者，
Figure PCTCN2021090074-appb-000004
其中，
X为突变位点，AA为突变位点前后的氨基酸，
n＝1～20，R 2为TLR2受体激动剂。
优选地，所述的R 2为三棕榈酰-S-甘油半胱氨酸、单磷酰脂质A、二棕榈酰-S-甘油基-半胱氨酸或其类似物，更优选三棕榈酰-S-甘油半胱氨酸类似物，选自如下结构式的类似物：
Figure PCTCN2021090074-appb-000005
Figure PCTCN2021090074-appb-000006
其中n，m＝1～5。
在本申请一种具体实施方式中，所述的R 2为单磷酰脂质A受体激动剂或其衍生物，优选单磷酰脂质A受体激动剂，结构式如下：
Figure PCTCN2021090074-appb-000007
，n＝1～20，R端可与定点突变的B群脑膜炎球菌fHBP蛋白偶联，
其中，R 3选自磷酸根或H；
R 4选自
Figure PCTCN2021090074-appb-000008
n为1,3,5；或
Figure PCTCN2021090074-appb-000009
R 5选自
Figure PCTCN2021090074-appb-000010
R 6选自H或
Figure PCTCN2021090074-appb-000011
R 7选自
Figure PCTCN2021090074-appb-000012
Figure PCTCN2021090074-appb-000013
R 8选自H或OH。
示例性地，所述偶联物中B群脑膜炎球菌fHBP蛋:修饰化合物的摩尔比为1:1-30。
在本申请一种具体实施方式中，所述B群脑膜炎球菌fHBP蛋白为Subfamily A中variant2，所述fHBP蛋白的的突变位点为SEQ ID NO:1中第2-30位氨基酸序列上一个或多个的氨基酸，优选地，所述突变位点为SEQ ID NO:1的序列的第2-10位氨基酸上一个或多个的氨基酸。
在本申请一种具体实施方式中，所述B群脑膜炎球菌fHBP蛋白为Subfamily A中variant3，所述fHBP蛋白的突变位点为SEQ ID NO:2中第2-30位氨基酸序列上一个或多个的氨基酸，优选地，所述突变位点为SEQ ID NO:2的序列的第2-10位氨基酸上一个或多个的氨基酸。
在本申请一种具体实施方式中，所述B群脑膜炎球菌fHBP蛋白为Subfamily B中variant 1，所述fHBP蛋白的突变位点可为SEQ ID NO:3中第2-30氨基酸序列上一个或多个的氨基酸，优选地，所述突变位点为SEQ ID NO:3的序列的第2-10位氨基酸上一个或多个的氨基酸。
本申请还涉及一种疫苗或免疫原性组合物，所述疫苗或免疫原性组合物包含所述的定点突变的蛋白或所述的偶联物中一种或多种。
在本申请一种具体实施方式中，所述疫苗或免疫原性组合物同时包含所述的三种定点突变的蛋白形成多价疫苗或免疫原性组合物，或同时包含所述三种偶联物形成多价疫苗或免疫原性组合物。
示例性地，所述B群脑膜炎球菌fHBP定点突变的蛋白或偶联物的剂量为10～100微克。
进一步，所述的疫苗或免疫原性组合物包含药学上可接受的赋形剂、载体或稀释剂。所述疫苗或免疫原性组合物进一步与ACW135Y群流脑结合疫苗联合使用。
示例性地，所述疫苗或免疫原性组合物的每剂量含5～10微克A群脑膜炎球菌多糖抗原，每剂量含5～10微克C群脑膜炎球菌多糖抗原，每剂量含5～10微克W135群脑膜炎球菌多糖抗原，每剂量含5～10微克Y群脑膜炎球菌多糖抗原，每剂量含10～100微克B群脑膜炎球菌fHBP定点突变的蛋白或偶联物。
本申请还涉及所述的定点突变的蛋白、所述的偶联物，或所述的免疫原性组合物在制备疫苗中的用途。其中所述的疫苗为脑膜炎球菌疫苗。
本申请还涉及一种定点突变和定点修饰蛋白抗原的方法，所述方法包括使用基因密码子扩展技术将非天然氨基酸定点引入到蛋白特定位点获得定点突变的蛋白，所述定点突变的蛋白进一步与修饰化合物偶联，所述修饰化合物为端基含有炔基或修饰炔基的化合物。
发明人经过对现有技术的思考和研究，利用古甲烷球菌的tRNA(tRNA Pyl)和吡咯赖氨酸-tRNA合成酶(tRNA Pyl/PylRS)的蛋白质翻译系统使非天然氨基酸定点掺入到蛋白中，从而得到定点突变的目的肽或蛋白(预修饰)，如B群脑膜炎球菌fHBP蛋白Subfamily A中variant2、3，Subfamily B中variant 1，后将所述定点突变的抗原蛋白作为可被进一步定点修饰的原料(本申请的预修饰的重组蛋白fHBP已经证明可预防B群脑膜炎球菌的侵染)，对该定点突变的抗原蛋白与脂质体进行缀合进而得到定点修饰的脂质蛋白。
具体的，所述的蛋白选自B群脑膜炎球菌fHBP蛋白中的一种或两种以上形成的变异体蛋白；优选的，所述的蛋白抗原选自B群脑膜炎球菌fHBP蛋白Subfamily A中variant2、3或Subfamily B中variant 1。所述的非天然氨基酸选自苯丙氨酸衍生物、酪氨酸衍生物、谷氨酰胺衍生物、丙氨酸衍生物、半胱氨酸衍生物、丝氨酸衍生物或赖氨酸衍生物中的至少一种。
优选地，所述的非天然氨基酸为含有叠氮基的赖氨酸衍生物。
更优选地，所述非天然氨基酸为
Figure PCTCN2021090074-appb-000014
具体的，所述修饰化合物选自含有炔基端基的糖、核酸、氨基酸、多肽或小分子化合物；或者糖、核酸、氨基酸、 多肽或小分子化合物通过末端炔基的修饰得到的修饰物。优选地，所述修饰化合物为脂质蛋白类受体激动剂。
示例性地，所述脂质蛋白类受体激动剂选自三棕榈酰-S-甘油半胱氨酸、单磷酰脂质A、二棕榈酰-S-甘油基-半胱氨酸或其类似物。
所述脂质蛋白类受体激动剂为三棕榈酰-S-甘油半胱氨酸类似物，选自如下结构式的类似物：
Figure PCTCN2021090074-appb-000015
Figure PCTCN2021090074-appb-000016
其中n，m＝1～5。
本申请还涉及一种用于定点突变和定点修饰蛋白抗原的三棕榈酰-S-甘油半胱氨酸类似物，选自如下结构式的类似物：
Figure PCTCN2021090074-appb-000017
Figure PCTCN2021090074-appb-000018
其中n，m＝1～5。
相比于其他方法，本申请的优点可体现在如下中的一个或几个：
1、本申请提供的定点突变的蛋白，可以在蛋白质任意位点引入非天然氨基酸，从而创造可以仅对该位点进行特异性修饰的蛋白抗原；
2、本申请提供的定点突变的蛋白，利用非天然氨基酸上特有的活性基团，可以实现高效，特异性地修饰目的；
3、本申请提供的偶联物，通过修饰条件的优化，利用环辛炔介导的无铜Click反应，可以实现高效，对蛋白无害，简单易行的修饰反应；
4、本申请提供的偶联物，通过引入结构确认的修饰基团，得到的B群脑膜炎球菌fHBP蛋白的偶联物，成分均一，质量可控，保证免疫原性同时可显著降低副反应程度；
5、本申请提供的定点突变的蛋白或偶联物，可以与ACW135Y群流脑结合疫苗联合使用，提高其保护覆盖度。
附图说明
图1 MenB-V1.55蛋白粒径图；
图2 MenB-V2.16蛋白粒径图；
图3 MenB-V1.55-G2-L1蛋白粒径图；
图4 MenB-V2.16-S3-L1蛋白粒径图；
图5 MenB-V2.16-S3-L1蛋白粒径图。
具体实施方式
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。
本文所用的术语“正交”是指一个分子(如正交tRNA(O-tRNA)和/或正交氨酰tRNA合成酶(O-RS))的功能与细胞的内源性组分一样，但是其活性与细胞或翻译系统内的其相应的内源性分子相比下降，或者不具有细胞内源性组分的功能。在用于tRNA和氨酰-tRNA合成酶时，正交的是指正交的tRNA与内源性tRNA合成酶配合时其效率与内源性tRNA和内源性tRNA合成酶配合时相比下降，如下降到20％、10％、5％或1％以下。正交分子缺乏细胞内内源性互补分子的正常功能。
本文所用的术语“Click反应”使用叠氮化物和炔烃的Huisgen[3+2]环化。
实施例1定点突变的MenB蛋白表达质粒的构建
1.突变位点的选择
天然MenB在其N末端发生脂化修饰，这一修饰不影响MenB蛋白的三维结构，而是起到将蛋白抗原锚定到细胞膜上的作用。结构研究显示MenB蛋白的N末端前20个氨基酸并未折叠形成二级结构，呈舒展状态，其作用是将抗原部分穿过细菌外膜暴露于细菌表面。因此突变的位点优先选择N末端20个氨基酸，其中优先选择第2-10位。具体突变位点的信息见表1-3，其中氨基酸位置分别是指在SEQ ID NO：1-3所示的序列上的位置。
SEQ ID NO.1：
Figure PCTCN2021090074-appb-000019
SEQ ID NO.2：
Figure PCTCN2021090074-appb-000020
SEQ ID NO.3：
Figure PCTCN2021090074-appb-000021
表1 V1.55突变位点
Figure PCTCN2021090074-appb-000022
表2 V2.16突变位点
Figure PCTCN2021090074-appb-000023
表3 V3.45突变位点
Figure PCTCN2021090074-appb-000024
2.表达质粒的获得
根据NCBI Gene Bank公布的MenB V.155,V2.16及V3.45基因序列(genbank序列号分别为AAR84481，AAR84445，AAR84435，分别对应SEQ ID NO：1，SEQ ID NO：2和SEQ ID NO：3)，经分别全基因合成获得基因全长DNA片段，然后分别融合构建于pET28a载体的NcoI和XhoI两个酶切位点之间，保留C末端His纯化标签，分别获得pET28a-MenB-V1.55，pET28a-MenB-V2.16，pET28a-MenB-V3.45表达质粒。
3.定点突变
使用全式金公司的Fast Mutagenesis System定点突变试剂盒，根据其使用说明，用前述pET28a-MenB-V1.55，pET28a-MenB-V2.16和pET28a-MenB-V3.45表达质粒做模版，使用表4-6中的突变引物对，完成各突变；对于突变后获得的质粒，进行测序验证工作。测序结果表明，均成功将各突变位点基因突变为TAG，得到9个定点突变的质粒。
MenB-V1.55的9个突变克隆命名为：
pET28a-MenB-V1.55-G2，pET28a-MenB-V1.55-S3，pET28a-MenB-V1.55-S4，pET28a-MenB-V1.55-G5，pET28a-MenB-V1.55-G6，pET28a-MenB-V1.55-G7，pET28a-MenB-V1.55-G8，pET28a-MenB-V1.55-S9，pET28a-MenB-V1.55-G10。
MenB-V2.16的9个突变克隆命名为：
pET28a-MenB-V2.16-G2，pET28a-MenB-V2.16-S3，pET28a-MenB-V2.16-S4，pET28a-MenB-V2.16-G5，pET28a-MenB- V2.16-G6，pET28a-MenB-V2.16-G7，pET28a-MenB-V2.16-G8，pET28a-MenB-V2.16-V9，pET28a-MenB-V2.16-A10。
MenB-V3.45的9个突变克隆命名为：
pET28a-MenB-V3.45-G2，pET28a-MenB-V3.45-S3，pET28a-MenB-V3.45-S4，pET28a-MenB-V3.45-G5，pET28a-MenB-V3.45-G6，pET28a-MenB-V3.45-G7，pET28a-MenB-V3.45-G8，pET28a-MenB-V3.45-V9，pET28a-MenB-V3.45-A10。
表4 MenB V1.55突变引物列表
Figure PCTCN2021090074-appb-000025
表5 MenB V2.16突变引物列表
Figure PCTCN2021090074-appb-000026
Figure PCTCN2021090074-appb-000027
表6 MenB V3.45突变引物列表
Figure PCTCN2021090074-appb-000028
实施例2突变蛋白的Lys-azido掺入表达及纯化
将实施例1的获得的表达质粒载体pET28a-MenB-V1.55-G2、pET28a-MenB-V2.16-S3、pET28a-MenB-V3.45-S4在LB培养基中37℃培养12～16小时后，再经二次扩增至菌液OD值到0.6～1.0时，加入Lys-azido至终浓度1mM，37℃ 继续扩增30分钟，加入IPTG至终浓度0.5mM，阿拉伯糖终浓度0.2％，24℃诱导表达12小时后收集菌体。
将收集的菌体用Ni-NTA-Bind-Buffer平衡重悬，超声破碎，离心去除细胞碎片，经过Ni-NTA金属螯合亲和层析，用Ni-NTA-Wash-Buffer充分洗涤，最后用Ni-NTA-Elute-Buffer洗脱，得到初步纯化的蛋白样品pET28a-MenB-V1.55-G2，pET28a-MenB-V2.16-S3，pET28a-MenB-V3.45-S4纯度约为90％。
V1.55、V2.16、V3.45的其他突变蛋白也分别按上述方法制备，限于篇幅，未全部记载入本申请说明书中。
实施例3三棕榈酰-S-甘油半胱氨酸类似物8的合成
三棕榈酰-S-甘油半胱氨酸类似物8合成路线如下：
1、将化合物1(5g)和丙酮叉(5g)溶于二氯甲烷中(100ml)，待溶解完全后，冰水浴中缓慢加入PTSA(0.9g)，加入完全后撤去冰浴，室温下搅拌2小时。反应结束后，减压蒸馏除去溶剂，采用硅胶层析柱纯化得化合物2。
2、将化合物2(5g)溶于DMF(100ml)中，后依次加入EDCI(5g)、HOBT(3.5g)、TEA(10g)，搅拌3～5分钟，后加入化合物a(6g)，加入完毕后，置于80℃油浴锅中反应过夜。反应结束后，减压蒸馏除去溶剂，采用硅胶层析柱纯化得化合物3。
3、将化合物3(5g)溶于二氯甲烷(100ml)中，溶解完全后，向体系中加入1N HCl甲醇溶液(20ml)，室温搅拌3小时。反应结束后，减压蒸馏除去溶剂，采用硅胶层析柱纯化得化合物4。
4、将化合物4(5g)溶于DMF(100ml)中，溶解完全后，依次向体系中依次加入三苯基氯硅烷(8g)和咪唑(1g)，40℃下搅拌过夜。反应结束后，减压蒸馏除去溶剂，采用硅胶层析柱纯化得化合物5。
5、将化合物5(5g)和化合物b(6g)溶于DMF(100ml)中，同时加入分子筛(10g)，后加入浓硫酸3～5滴，置于80℃油浴锅中反应过夜。反应结束后，过滤除去分子筛，减压蒸馏除去溶剂，采用硅胶层析柱纯化得化合物6。
6、将化合物6(5g)溶于乙酸(100ml)中，溶解完全后，120℃下回流6h。TLC法监测反应进程，待原料反应完全后，减压蒸馏除去溶剂，采用硅胶层析柱纯化得化合物7。
7、将化合物7(5g)和化合物c(6g)溶于DMF(100ml)中，同时加入分子筛(10g)，后加入浓硫酸3～5滴，置于80℃油浴锅中反应过夜。反应结束后，过滤除去分子筛，减压蒸馏除去溶剂，采用硅胶层析柱纯化得化合物8。
流程图如下：
Figure PCTCN2021090074-appb-000029
采用相同方法，可得三棕榈酰-S-甘油半胱氨酸其他类似物L1-L15：
Figure PCTCN2021090074-appb-000030
编号 L1 L2 L3 L4 L5
m 1 2 3 4 5
n 1 2 3 4 5
Figure PCTCN2021090074-appb-000031
编号 L6 L7 L8 L9 L10
m 1 2 3 4 5
n 1 2 3 4 5
Figure PCTCN2021090074-appb-000032
编号 L11 L12 L13 L14 L15
m 1 2 3 4 5
n 1 2 3 4 5
实施例4突变蛋白pET28a-MenB-V1.55-G2与三棕榈酰-S-甘油半胱氨酸类似物通过铜催化Click反应偶联
反应体系如下：
pET28a-MenB-V1.55-G2蛋白 1微克每微升
三棕榈酰-S-甘油半胱氨酸化合物8 20微克每微升
Cu 2+ 1mM
BTTES 400微摩尔
PBS 0.01M(pH≈7)
Cu丝小段 足量
注：(1,2,3-triazol-1-yl)ethanesulfonic acid，简称BTTES)
反应条件：4℃，垂直混悬30分钟，反应结束后加入EDTA至1mM终止反应，获得的最终产物是三棕榈酰-S-甘油半胱氨酸类似物与pET28a-MenB-V1.55-G2蛋白定点偶联结合物MenB-V1.55-G2-L1。
实施例5突变蛋白pET28a-MenB-V2.16-S3与三棕榈酰-S-甘油半胱氨酸类似物通过铜催化Click反应偶联
同实施例4操作步骤，得到三棕榈酰-S-甘油半胱氨酸类似物与pET28a-MenB-V2.16-S3蛋白定点偶联结合物MenB-V2.16-S3-L1。
实施例6突变蛋白pET28a-MenB-V3.45-S4与三棕榈酰-S-甘油半胱氨酸类似物通过铜催化Click反应偶联
同实施例4操作步骤，得到三棕榈酰-S-甘油半胱氨酸类似物与pET28a-MenB-V3.45蛋白定点偶联结合物MenB-V3.45-S4-L1。
采用实施例4～6的方法，同时制备了不同脂质体的蛋白定点偶联结合物，见下表：
MenB-V1.55-G2-L2
MenB-V2.16-S3-L2
MenB-V3.45-S4-L2
MenB-V1.55-G2-L11
MenB-V2.16-S3-L11
MenB-V3.45-S4-L11
MenB-V1.55-G2-L15
MenB-V2.16-S3-L15
MenB-V3.45-S4-L15
MenB-V1.55-G2-L12
MenB-V2.16-S3-L12
MenB-V3.45-S4-L12
MenB-V1.55-G2-L14
MenB-V2.16-S3-L14
MenB-V3.45-S4-L14
MenB-V1.55-S3-L1
MenB-V2.16-S3-L1
MenB-V3.45-S4-L1
MenB-V1.55-S4-L2
MenB-V2.16-S4-L2
MenB-V3.45-S4-L2
MenB-V1.55-G5-L6
MenB-V2.16-G5-L6
MenB-V3.45-S4-L6
MenB-V1.55-G6-L11
MenB-V2.16-G6-L11
MenB-V3.45-S4-L11
MenB-V1.55-G7-L15
MenB-V2.16-G7-L15
MenB-V3.45-S4-L15
MenB-V1.55-G8-L1
MenB-V2.16-G8-L1
MenB-V3.45-S4-L1
MenB-V1.55-S9-L2
MenB-V2.16-G2-L2
MenB-V3.45-S4-L1
MenB-V1.55-G10-L1
MenB-V2.16-V9-L2
MenB-V3.45-S4-L3
实施例7三价fHBP蛋白疫苗的配制
选择本申请所制备的突变蛋白修饰物中的三个突变体，将三种变异型重组B群fHBP脂化蛋白MenB-V1.55-G2- L1、MenB-V2.16-S3-L1、MenB-V3.45-S4-L1，分别以氢氧化铝佐剂吸附，4℃搅拌过夜，吸附率在95％以上。以0.15mol每升氯化钠稀释配制，至蛋白终浓度分别为240微克每毫升。铝含量终浓度为0.45～0.6毫克每毫升，pH值5.8～7.2。
实施例8流行株的杀菌力(SBA)试验
采用B群脑膜炎球菌440902菌株，该菌株为ST4821序列型，属ST4821序列群，为国内近期B群脑膜炎球菌流行菌株，fHBP分型为V2变异体。
靶菌的制备：培养流行性脑膜炎球菌440902菌株，于8～12％养血琼脂平板上，37℃6～10％二氧化碳培养16～18小时，刮取菌苔至生理盐水中，细菌比浊法计数，根据计数，将靶菌稀释至1×10 6。
将待检小鼠血清于56℃灭火1小时，以灭火小鼠血清固有补体活性。实验过程中将Pel-Freez幼兔子补体加入到待检小鼠血清中，同时设置灭火补体、补体对照，倍比稀释至96孔培养板上，并滴加新鲜配制的靶菌10微升。震荡混匀后于37℃培养2～4小时。
点样：取培养后的混合菌液，以10微升的量滴加至固定营养琼脂综合培养基上，37℃5％二氧化碳培养过夜。
显色：将含有150～300微克每毫升的TTC的软琼脂覆盖在过夜培养的固体影响琼脂综合培养基上，以适宜温度、适宜时间显色。
计数：高清拍摄显色菌落照片，采用图像扫描技术，以专有分析软件分析并计算细菌菌落个数，以杀菌力结果计算软件计算杀菌滴度，结果如下：
小鼠血清杀菌力滴度
Figure PCTCN2021090074-appb-000033
实施例9其他偶联物杀菌力(SBA)试验结果
Figure PCTCN2021090074-appb-000034
Figure PCTCN2021090074-appb-000035
以上测试的不同定点偶联物的杀菌力结果表明：
1、MenB蛋白的V1、V2变异体与脂质体定点偶联后的脂蛋白，与阴性对照组生理盐水相比具有显著的杀菌力，同时与三价fHBP阳性对照组的杀菌力无显著差异；
2、MenB蛋白的V1、V2、V3变异体与脂质体定点偶联后的脂蛋白，与未经脂质体修饰的非脂化蛋白相比，其杀菌力具有显著提升；
3、MenB蛋白的V3变异体脂蛋白对V2型菌种具有一定的交叉保护作用。
这一结果表明，本发明通过化学合成方式得到的脂质体，再与MenB蛋白经定点偶联，可得到结构明确、成分均一、质量可控且具有高度抗原活性的脂蛋白，后期可通过准确控制、调整有效剂量来改善产品有效性和安全性。
实施例10急性毒性和异常毒性试验
本试验利用不同剂量的药物急性毒性反应，将一定剂量的供试品溶液(实施例4、5和6中制备MenB-V1.55-G2-L1、MenB-V2.16-S3-L1、MenB-V3.45-S4-L1组成得到的三价B群脑膜炎球菌蛋白疫苗)，注入受试动物(小鼠、豚鼠)体内，在规定时间内观察动物出现的毒性反应症状和死亡情况，按段供试品是否复核规定的质量要求及其安全性程度。
试验方法：NIH小鼠，体重：18～22克/只，每组5只；豚鼠，体重250～350克/只，每组2只；
注射剂量及分组
异常毒性试验：按2015版《中国药典》三部附录XIIF项异常毒性检查法规定的接种剂量：小鼠，腹腔注射0.5ml(1人用剂量)，豚鼠，腹腔注射5ml(10个人用剂量)。
重复给药试验：按以上试验给药后，观察受试动物三天内未出现异常症状，继续饲养至第七天，受试动物健存、正常，且体重增加，再次重复给与以上剂量集中给药，再连续观察7天，判定结果。
急性毒性试验：按2015版《中国药典》三部附录XIIF项异常毒性检查法规定接种剂量的5倍剂量给药，用各价B群流脑蛋白原液配制浓缩疫苗，给药剂量为小鼠，腹腔注射0.5ml(5个人用剂量)，豚鼠腹腔注射5ml(50个人用剂量)。
实验结果判定
小鼠、豚鼠接种供试品后，连续观察7天，观察期内，动物应全部健康、存活，且无异常反应，到期时动物体重增加，判定供试品合格。
试验结果
3价B群脑膜炎球菌蛋白疫苗异常毒性、急性毒性试验
Figure PCTCN2021090074-appb-000036
结果分析
无论是常规剂量异常毒性、常规剂量重复给药和5倍常规剂量急性毒性试验，接种后，各组动物活动、进食均正常，无异常反应，且均健存，体重增加，试验证实，供试品具有可靠的安全性。表明3价B群脑膜炎球菌蛋白疫苗异常毒性和急性毒性试验合格。
结论：本发明提供的3价B群脑膜炎球菌蛋白疫苗异常毒性和急性毒性试验合格。
实施例11对比定点修饰脂质蛋白与野生型脂蛋白粒径分析
利用Dynamic Light Scattering(DLS)，Zetasizer Nano ZS对各个脂质蛋白MenB-V1.55-G2-L1、MenB-V2.16-S3-L1、MenB-V3.45-S4-L1大小分布进行分析，通过定点修饰偶联蛋白得到脂质蛋白的粒度分布均匀，产品均一性良好；而通过传统发酵得到的野生型脂蛋白MenB-V1.55、MenB-V2.16粒度分布不均匀、有聚集物存在。参见图1～图5，图1为MenB-V1.55蛋白粒径图，包括两个主要粒径范围，分别约为20纳米和90纳米。图2为MenB-V2.16蛋白粒径图，包括两个主要粒径范围，分别约为30纳米和400纳米；图3为MenB-V1.55-G2-L1蛋白粒径图，主要粒径范围约为50纳米。图4为MenB-V2.16-S3-L1蛋白粒径图，主要粒径范围约为60纳米。图5为MenB-V2.16-S3-L1蛋白粒径图，主要粒径范围约为50纳米。
综上，本申请获得的定点修饰脂质蛋白，脂质体长度一致，质量显著可控，可有效避免重组脂质蛋白在表达过程中脂质化不均一的缺点，保证免疫原性同时可显著降低副反应程度。
本申请虽然以较佳实施例公开如上，但并不是用来限定权利要求，任何本领域技术人员在不脱离本申请构思的前提下，都可以做出若干可能的变动和修改，因此本申请的保护范围应当以本申请权利要求所界定的范围准。
Figure PCTCN2021090074-appb-000037
Figure PCTCN2021090074-appb-000038
Figure PCTCN2021090074-appb-000039
Figure PCTCN2021090074-appb-000040
Figure PCTCN2021090074-appb-000041
Figure PCTCN2021090074-appb-000042
Figure PCTCN2021090074-appb-000043
Figure PCTCN2021090074-appb-000044
Figure PCTCN2021090074-appb-000045
Figure PCTCN2021090074-appb-000046
Figure PCTCN2021090074-appb-000047
Figure PCTCN2021090074-appb-000048
Figure PCTCN2021090074-appb-000049
Figure PCTCN2021090074-appb-000050
Figure PCTCN2021090074-appb-000051
Figure PCTCN2021090074-appb-000052

Claims (45)

  1. 一种定点突变的蛋白，其特征在于，其中所述的蛋白选自B群脑膜炎球菌fHBP蛋白中的一种或两种以上突变蛋白，其中所述蛋白抗原上至少1个位点的氨基酸被突变为非天然氨基酸，所述非天然氨基酸含有叠氮基、炔基端基或其他活性基团。
  2. 如权利要求1所述的定点突变的蛋白，其特征在于，所述的蛋白选自B群脑膜炎球菌fHBP蛋白中的一种或两种以上形成的变异体蛋白；优选的，所述的蛋白抗原选自B群脑膜炎球菌fHBP蛋白Subfamily A中variant2、3或Subfamily B中variant 1。
  3. 如权利要求1或2任意一项所述的定点突变的蛋白，其特征在于，所述的非天然氨基酸选自苯丙氨酸衍生物、酪氨酸衍生物、谷氨酰胺衍生物、丙氨酸衍生物、半胱氨酸衍生物、丝氨酸衍生物或赖氨酸衍生物中的至少一种。
  4. 如权利要求3所述的定点突变的蛋白，其特征在于，所述的非天然氨基酸为含有叠氮基的赖氨酸衍生物。
  5. 如权利要求1-4任意一项所述的定点突变的蛋白，其特征在于，所述的非天然氨基酸为
    Figure PCTCN2021090074-appb-100001
  6. 如权利要求1-5任意一项所述的定点突变的蛋白，其特征在于，所述蛋白抗原的突变位点为不影响抗原表位的一个或多个氨基酸。
  7. 如权利要求1-6任意一项所述的定点突变的蛋白，其特征在于，所述蛋白抗原为B群脑膜炎球菌fHBP蛋白SubfamilyA中variant2，所述蛋白抗原的突变位点为SEQ ID NO:1中第2-30位氨基酸序列上一个或多个的氨基酸。
  8. 如权利要求7所述的定点突变的蛋白，其特征在于，所述突变位点为SEQ ID NO:1中第2-10位氨基酸上一个或多个的氨基酸。
  9. 如权利要求1-6任意一项所述的定点突变的蛋白，其特征在于，所述蛋白抗原为B群脑膜炎球菌fHBP蛋白抗原SubfamilyA中variant3，所述蛋白抗原的突变位点为SEQ ID NO:2中第2-30位氨基酸序列上一个或多个的氨基酸。
  10. 如权利要求9所述的定点突变的蛋白，其特征在于，所述突变位点为SEQ ID NO:2中第2-10位氨基酸上一个或多个的氨基酸。
  11. 如权利要求1-6任意一项所述的定点突变的蛋白，其特征在于，所述蛋白抗原为B群脑膜炎球菌fHBP蛋白抗原Subfamily B中variant 1，所述蛋白抗原的突变位点为SEQ ID NO:3中第2-30位氨基酸序列上一个或多个的氨基酸。
  12. 如权利要求11所述的定点突变的蛋白，其特征在于，所述突变位点为SEQ ID NO:3中第2-10位氨基酸上一个或多个的氨基酸。
  13. 如权利要求1-12任意一项所述的定点突变的蛋白，其特征在于，所述蛋白抗原的氨基酸序列中第X位氨基酸被突变为Lys-azido，所述突变氨基酸的连接方式如下式所示：
    Figure PCTCN2021090074-appb-100002
    其中，所述的X为突变位点，AA为突变位点前后的氨基酸。
  14. 一种定点突变的B群脑膜炎球菌fHBP蛋白的偶联物，其特征在于，所述的偶联物为权利要求1-13任意一项所述的定点突变的蛋白进一步与修饰化合物偶联。
  15. 如权利要求14所述的偶联物，其特征在于，所述修饰化合物为端基含有炔基或修饰炔基的化合物。
  16. 如权利要求15所述的偶联物，其特征在于，所述修饰化合物选自含有炔基端基的糖、核酸、氨基酸、多肽或小分子化合物；或者糖、核酸、氨基酸、多肽或小分子化合物通过末端炔基的修饰得到的修饰物。
  17. 如权利要求14-16任一项所述的偶联物，其特征在于，所述修饰化合物为脂质蛋白类受体激动剂。
  18. 如权利要求14-16任一项所述的偶联物，其特征在于，所述修饰化合物为TLR2受体激动剂。
  19. 如权利要求18所述的偶联物，其特征在于，所述激动剂为选自三棕榈酰-S-甘油半胱氨酸、单磷酰脂质A、二棕榈酰-S-甘油基-半胱氨酸或其类似物。
  20. 如权利要求14-19任意一项所述的偶联物，其特征在于，所述B群脑膜炎球菌fHBP蛋白的氨基酸序列的第X位氨基酸被突变修饰为如下结构：
    Figure PCTCN2021090074-appb-100003
    或者，
    Figure PCTCN2021090074-appb-100004
    其中，
    X为突变位点，AA为突变位点前后的氨基酸，
    n＝1～20，R 2为TLR2受体激动剂。
  21. 如权利要求20所述的偶联物，其特征在于，所述的R 2为三棕榈酰-S-甘油半胱氨酸、单磷酰脂质A、二棕榈酰-S-甘油基-半胱氨酸或其类似物。
  22. 如权利要求20或21所述的偶联物，其特征在于，所述的R 2为三棕榈酰-S-甘油半胱氨酸类似物，选自如下结构式的类似物：
    Figure PCTCN2021090074-appb-100005
    Figure PCTCN2021090074-appb-100006
    其中n，m＝1～5。
  23. 如权利要求20或21所述的偶联物，其特征在于，所述的R 2为单磷酰脂质A受体激动剂或其衍生物；优选单磷酰脂质A受体激动剂，结构式如下：
    Figure PCTCN2021090074-appb-100007
    ，n＝1～20，R端可与定点突变的B群脑膜炎球菌fHBP蛋白偶联，
    其中，R 3选自磷酸根或H；
    R 4选自
    Figure PCTCN2021090074-appb-100008
    n为1、3、5；或
    Figure PCTCN2021090074-appb-100009
    R 5选自
    Figure PCTCN2021090074-appb-100010
    R 6选自H或
    Figure PCTCN2021090074-appb-100011
    R 7选自
    Figure PCTCN2021090074-appb-100012
    Figure PCTCN2021090074-appb-100013
    R 8选自H或OH。
  24. 如权利要求14-23任意一项所述的偶联物，其特征在于，其中所述偶联物中B群脑膜炎球菌fHBP蛋与修饰化合物的摩尔比为1:1-30。
  25. 如权利要求14-24任意一项所述的偶联物，其特征在于，所述B群脑膜炎球菌fHBP蛋白为Subfamily A中variant2，所述fHBP蛋白的突变位点为SEQ ID NO:1中第2-30位氨基酸序列上一个或多个的氨基酸；优选地，所述突变位点为SEQ ID NO:1中第2-10位氨基酸上一个或多个的氨基酸。
  26. 如权利要求14-24任意一项所述的偶联物，其特征在于，所述B群脑膜炎球菌fHBP蛋白为Subfamily A中variant3，所述fHBP蛋白的突变位点为SEQ ID NO:2中第2-30位氨基酸序列上一个或多个的氨基酸；优选地，所述突变位点为SEQ ID NO:2中第2-10位氨基酸上一个或多个的氨基酸。
  27. 如权利要求14-24任意一项所述的偶联物，其特征在于，所述B群脑膜炎球菌fHBP蛋白为Subfamily B中variant 1，所述fHBP蛋白的突变位点为SEQ ID NO:3中第2-30位氨基酸序列上一个或多个的氨基酸；优选地，所述突变位点为SEQ ID NO:3中第2-10位氨基酸上一个或多个的氨基酸。
  28. 一种疫苗或免疫原性组合物，其特征在于，所述疫苗或免疫原性组合物包含权利要求1-13任意一项所述的定点突变的蛋白或权利要求14-27任意一项所述的偶联物中一种或多种。
  29. 如权利要求28所述的疫苗或免疫原性组合物，其特征在于，其中所述疫苗或免疫原性组合物同时包含权利要求7-9中的三种定点突变的蛋白形成多价疫苗或免疫原性组合物，或同时包含权利要求18-20中的三种偶联物形成多价疫苗或免疫原性组合物。
  30. 如权利要求28或29所述的疫苗或免疫原性组合物，其特征在于，所述B群脑膜炎球菌fHBP定点突变的蛋白或偶联物的剂量为10～100微克。
  31. 如权利要求28-30任意一项所述的疫苗或免疫原性组合物，其特征在于，进一步包含药学上可接受的赋形剂、载体或稀释剂。
  32. 如权利要求28-30任意一项所述的疫苗或免疫原性组合物，其特征在于，所述疫苗或免免疫原性组合物进一步与ACW135Y群流脑结合疫苗联合使用。
  33. 如权利要求28-32任意一项所述的疫苗或免疫原性组合物，其特征在于，所述疫苗或免疫原性组合物的每剂量含5～10微克A群脑膜炎球菌多糖抗原，每剂量含5～10微克C群脑膜炎球菌多糖抗原，每剂量含5～10微克W135群脑膜炎球菌多糖抗原，每剂量含5～10微克Y群脑膜炎球菌多糖抗原，每剂量含10～100微克B群脑膜炎球菌fHBP定点突变的蛋白或偶联物。
  34. 权利要求1-13任意一项所述的定点突变的蛋白，权利要求14-27任意一项所述的偶联物，和权利要求28-33任意一项所述的免疫原性组合物在制备疫苗中的用途。
  35. 如权利要求34所述的用途，其特征在于，其中所述的疫苗为脑膜炎球菌疫苗。
  36. 一种定点突变和定点修饰蛋白抗原的方法，其特征在于，所述方法包括使用基因密码子扩展技术将非天然氨基酸定点引入到蛋白特定位点获得定点突变的蛋白，所述定点突变的蛋白进一步与修饰化合物偶联，所述修饰化合物为端基含有炔基或修饰炔基的化合物。
  37. 如权利要求36所述的定点突变和定点修饰蛋白抗原的方法，其特征在于，所述的蛋白选自B群脑膜炎球菌fHBP蛋白中的一种或两种以上形成的变异体蛋白；优选的，所述的蛋白抗原选自B群脑膜炎球菌fHBP蛋白Subfamily A中variant2、3或Subfamily B中variant 1。
  38. 如权利要求36所述的定点突变和定点修饰蛋白抗原的方法，其特征在于，所述的非天然氨基酸选自苯丙氨酸衍生物、酪氨酸衍生物、谷氨酰胺衍生物、丙氨酸衍生物、半胱氨酸衍生物、丝氨酸衍生物或赖氨酸衍生物中的至少一种。
  39. 如权利要求36-38任意一项所述的定点突变和定点修饰蛋白抗原的方法，其特征在于，所述的非天然氨基酸为含有叠氮基的赖氨酸衍生物。
  40. 如权利要求36-38任意一项所述的定点突变和定点修饰蛋白抗原的方法，其特征在于，所述非天然氨基酸为
    Figure PCTCN2021090074-appb-100014
  41. 如权利要求36-38任意一项所述的定点突变和定点修饰蛋白抗原的方法，其特征在于，所述修饰化合物选自含有炔基端基的糖、核酸、氨基酸、多肽或小分子化合物；或者糖、核酸、氨基酸、多肽或小分子化合物通过末端炔基的修饰得到的修饰物。
  42. 如权利要求41所述的定点突变和定点修饰蛋白抗原的方法，其特征在于，所述修饰化合物为脂质蛋白类受体激动剂。
  43. 如权利要求42所述的定点突变和定点修饰蛋白抗原的方法，其特征在于，所述脂质蛋白类受体激动剂选自三棕榈酰-S-甘油半胱氨酸、单磷酰脂质A、二棕榈酰-S-甘油基-半胱氨酸或其类似物。
  44. 如权利要求43所述的定点突变和定点修饰蛋白抗原的方法，其特征在于，所述脂质蛋白类受体激动剂为三棕榈酰-S-甘油半胱氨酸类似物，选自如下结构式的类似物：
    Figure PCTCN2021090074-appb-100015
    Figure PCTCN2021090074-appb-100016
    其中n，m＝1～5。
  45. 一种用于定点突变和定点修饰蛋白抗原的三棕榈酰-S-甘油半胱氨酸类似物，其特征在于，选自如下结构式的类似物：
    Figure PCTCN2021090074-appb-100017
    Figure PCTCN2021090074-appb-100018
    其中n，m＝1～5。
PCT/CN2021/090074 2020-07-07 2021-04-27 一种定点突变的载体蛋白及其在制备疫苗中的用途 WO2022007478A1 (zh)

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KR1020227046500A KR20230021677A (ko) 2020-07-07 2021-04-27 위치지정 돌연변이의 담체 단백질 및 이의 백신 제조에서의 용도
JP2022581652A JP2023532559A (ja) 2020-07-07 2021-04-27 部位特異的に突然変異されたキャリアタンパク質及びワクチンの製造におけるその使用
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101087808A (zh) * 2004-12-21 2007-12-12 法克斯因内特公司 流行性感冒病毒蛋白质的组合物及其使用方法
CN106929482A (zh) * 2015-12-31 2017-07-07 北京大学 定点突变的流感病毒、其活疫苗及其制备方法和应用
CN110845587A (zh) * 2019-10-15 2020-02-28 康希诺生物股份公司 一种定点突变的载体蛋白及其在制备疫苗中的用途
CN111533792A (zh) * 2020-07-07 2020-08-14 康希诺生物股份公司 一种定点突变的载体蛋白及其在制备疫苗中的用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2013221448B2 (en) * 2012-02-16 2017-02-23 The University Of Toledo Xenoantigen-displaying anti-cancer vaccines and method of making
GB201614687D0 (en) * 2016-08-31 2016-10-12 Univ Oxford Innovation Ltd fHbp scaffold
US11007256B2 (en) * 2016-09-26 2021-05-18 The University Of Toledo Synthetic lipopeptide vaccines and immunotherapeutics
CN118662649A (zh) * 2016-12-30 2024-09-20 Vaxcyte公司 具有非天然氨基酸的多肽-抗原缀合物

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101087808A (zh) * 2004-12-21 2007-12-12 法克斯因内特公司 流行性感冒病毒蛋白质的组合物及其使用方法
CN106929482A (zh) * 2015-12-31 2017-07-07 北京大学 定点突变的流感病毒、其活疫苗及其制备方法和应用
CN110845587A (zh) * 2019-10-15 2020-02-28 康希诺生物股份公司 一种定点突变的载体蛋白及其在制备疫苗中的用途
CN111533792A (zh) * 2020-07-07 2020-08-14 康希诺生物股份公司 一种定点突变的载体蛋白及其在制备疫苗中的用途

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Chinese Pharmacopoeia", 2015
"NCBI", Database accession no. AAR84435
JONES LYN H: "Recent advances in the molecular design of synthetic vaccines", NATURE CHEMISTRY, vol. 7, no. 12, 20 November 2015 (2015-11-20), London, pages 952 - 960, XP037114904, ISSN: 1755-4330, DOI: 10.1038/nchem.2396 *
ROBERTO SCATENA, PATRIZIA BOTTONI, AND BRUNO GIARDINA: "Retinal Degenerative Diseases: Advances in Experimental Medicine and Biology", vol. 1030, 1 January 2017, SPRINGER , US , ISSN: 0065-2598, article KOWALCZYK RENATA, HARRIS PAUL W. R., WILLIAMS GEOFFREY M., YANG SUNG-HYUN, BRIMBLE MARGARET A.: "Peptide Lipidation – A Synthetic Strategy to Afford Peptide Based Therapeutics", pages: 185 - 227, XP055886047, DOI: 10.1007/978-3-319-66095-0_9 *
See also references of EP4180447A4

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