WO2022152204A1 - 一种人乳头瘤病毒病毒样颗粒疫苗的稳定制剂 - Google Patents

一种人乳头瘤病毒病毒样颗粒疫苗的稳定制剂 Download PDF

Info

Publication number
WO2022152204A1
WO2022152204A1 PCT/CN2022/071782 CN2022071782W WO2022152204A1 WO 2022152204 A1 WO2022152204 A1 WO 2022152204A1 CN 2022071782 W CN2022071782 W CN 2022071782W WO 2022152204 A1 WO2022152204 A1 WO 2022152204A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
hpv
type
protein
fragment
Prior art date
Application number
PCT/CN2022/071782
Other languages
English (en)
French (fr)
Inventor
刘艳
胡萍
常欣荣
Original Assignee
神州细胞工程有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 神州细胞工程有限公司 filed Critical 神州细胞工程有限公司
Priority to EP22739080.4A priority Critical patent/EP4292607A1/en
Priority to MX2023008306A priority patent/MX2023008306A/es
Priority to CN202280008056.4A priority patent/CN117083078A/zh
Priority to JP2023542604A priority patent/JP2024503452A/ja
Priority to US18/261,199 priority patent/US20240075124A1/en
Priority to AU2022207569A priority patent/AU2022207569A1/en
Priority to CA3204802A priority patent/CA3204802A1/en
Priority to KR1020237026965A priority patent/KR20230129506A/ko
Publication of WO2022152204A1 publication Critical patent/WO2022152204A1/zh

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the field of biological medicine preparations, in particular to a stable human papilloma virus virus-like particle vaccine preparation.
  • Cervical cancer is one of the most common female malignancies, with about 500,000 new patients worldwide every year, and the incidence rate ranks second among female tumors. More than 95% of cervical cancers are closely related to human papillomavirus (HPV) infection. In addition to directly causing cervical cancer, HPV is also significantly associated with bronchial, rectal, oral and skin cancers. In addition, HPV is the main pathogenic factor that causes skin and mucosal warts.
  • HPV can be divided into high-risk, suspected carcinogenic and low-risk.
  • High-risk and suspected carcinogenic types can induce cervical cancer and other cancers;
  • high-risk types include 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59;
  • suspected carcinogenic types include 26, 53, 66 , 68, 73, 82 types.
  • Low-risk types are mostly related to genital warts, condyloma acuminatum and other diseases, including types 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and 89.
  • HPV6, 11, and 16 are the subtypes with the highest detection rate in patients with genital lesions.
  • HPV vaccine is an effective way to block papillomavirus infection.
  • Virus-like particle (VLP) vaccine is the most advantageous form of vaccine.
  • VLP-based HPV vaccines are type-restricted, that is, they only have a strong protective effect on HPV of the same type of VLPs in the vaccine.
  • the development of a multivalent HPV vaccine is necessary to provide broad protection.
  • human papillomavirus vaccine formulations undergo a storage and transport process during which the antigen undergoes physical and chemical degradation. These instabilities may reduce the immunogenicity and/or safety of the antigen, so A stable formulation is needed to ensure that the antigen remains immunogenic and safe for prophylaxis prior to administration.
  • the present invention provides a stable formulation of a multivalent human papillomavirus virus-like particle vaccine for preventing HPV-related disease or infection, comprising a plurality of papillomavirus virus-like particles, which are adsorbed on an adjuvant; physiologically Acceptable concentrations of buffers, osmotic pressure regulators, and optionally surfactants.
  • human papillomavirus virus-like particles are selected from HPV types 6, 11, 16, 18, 31, 33, 45
  • HPV virus-like particles assembled from the L1 protein of types 52 and 58;
  • HPV virus-like particles assembled from L1 proteins of other pathogenic HPV types.
  • Described buffer is selected from one or more of citrate buffer, acetate buffer or histidine buffer;
  • Described osmotic pressure regulator is selected from one or more of sodium chloride, sodium phosphate or sodium sulfate;
  • the surfactant is polyethoxy ether, preferably polysorbate 80;
  • the adjuvant is selected from one or more of aluminum hydroxyphosphate (AlPO 4 ), amorphous aluminum hydroxyphosphate sulfate (AAHS) or aluminum hydroxide (Al(OH 3 )), preferably aluminum hydroxyphosphate (AlPO 4 ).
  • the concentration of the buffer is 10 mM to 26 mM, preferably 10 mM, 18 mM or 26 mM;
  • the concentration of the osmotic pressure regulator is 150mM ⁇ 320mM, preferably 150mM or 320mM;
  • the concentration of the surfactant is 0-0.02% by weight,
  • the adjuvant has a concentration of about 1.0 mg/mL
  • the pH of the formulation is 5.9-6.5, preferably 5.9, 6.2 or 6.5.
  • the concentration of any type of papillomavirus virus-like particle comprising the polyvalent papillomavirus virus-like particle is 40 ⁇ g/mL to 120 ⁇ g/mL.
  • the formulation contains a total of 0.74 mg/mL papillomavirus virus-like particles, 1.0 mg/mL aluminum phosphate adjuvant, 18 mM histidine buffer, 320 mM sodium chloride, and the pH of the formulation solution is 6.2 ;
  • polysorbate 80 at a concentration not higher than 0.3 mg/mL.
  • the one or more other pathogenic HPV types are selected from HPV types 35, 39, 51, 56 and 59.
  • the HPV virus-like particles is a chimeric HPV virus-like particle comprising a chimeric HPV L1 protein; the chimeric HPV L1 protein From its N-terminal to C-terminal direction it includes:
  • a Derived from an N-terminal fragment of the L1 protein of a first type of papillomavirus that retains the immunogenicity of the L1 protein of the first type, wherein the first type of papillomavirus is selected from HPV types 6, 11 HPV types 16, 18, 31, 33, 45, 52 and 58 and one or more other pathogenic HPV types; and
  • the second type of papillomavirus L1 protein has the characteristics of better expression and solubility than other types of L1 protein;
  • the chimeric HPV L1 protein has the immunogenicity of the first type of papillomavirus L1 protein.
  • the N-terminal fragment is a fragment obtained by truncating the C-terminal of the native sequence of the first type papillomavirus L1 protein to any amino acid site in its ⁇ 5 region, and the same A fragment having at least 98% identity; and the C-terminal fragment is obtained by truncating the N-terminus of the native sequence of the L1 protein of type 2 papillomavirus to any amino acid site within its ⁇ 5 region fragments, as well as functional variants resulting from further mutations, deletions and/or additions to the fragments.
  • the C-terminal fragment contains one or more nuclear localization sequences.
  • the first type of papilloma L1 protein is selected from the group consisting of HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, Type 56 or Type 58; preferably, its native sequence is SEQ ID No:30, SEQ ID No:31, SEQ ID No:32, SEQ ID No:33, SEQ ID No:34, SEQ ID No:35, respectively, SEQ ID No:36, SEQ ID No:37, SEQ ID No:38, SEQ ID No:39, SEQ ID No:40, or the amino acid sequence encoded by the coding gene shown in SEQ ID No:41;
  • the second type of papillomavirus L1 protein is selected from HPV type 16, type 28, type 33, type 59, or type 68 L1 protein;
  • the second type of papillomavirus L1 protein is selected from HPV type 33 or HPV type 59 L1 protein.
  • the C-terminal fragment is SEQ ID No: 1; or a fragment with a length of m1 amino acids, preferably a fragment covering amino acids 1-m1 of SEQ ID No: 1; wherein m1 is 8- An integer of 26; or the C-terminal fragment is SEQ ID No: 2; or a fragment of m2 amino acids in length, preferably a fragment covering amino acids 1-m2 of SEQ ID No: 2; wherein m2 is 13-31 the integer.
  • the C-terminal fragment is SEQ ID No: 3; or a fragment having a length of n amino acids, preferably a fragment covering amino acids 1-n of SEQ ID No: 3; wherein n is 16- An integer of 38.
  • the N-terminal fragment of the HPV type 6 L1 protein has 98% of the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 4 at any amino acid site in its ⁇ 5 region , 98.5%, 99%, 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV 11 type L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 5 to any amino acid site in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV 16 type L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 6 to any amino acid site in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV 18 type L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 7 to any amino acid site in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV 31 type L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 8 to any amino acid site in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV 35 L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 9 to any amino acid site in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV 39 type L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 10 to any amino acid site in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV 45 type L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 11 to any amino acid site in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV 51 type L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 12 to any amino acid site in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV 52 type L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 13 to any amino acid site in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV 56 type L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 14 to any amino acid site in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity;
  • N-terminal fragment of the HPV 58 type L1 protein and the fragment obtained by truncating the C-terminal of the sequence shown in SEQ ID No: 15 to any amino acid site in its ⁇ 5 region have 98%, 98.5%, 99% , 99.5%, 99% or 100% identity.
  • the C-terminus of the N-terminal fragment is linked directly or via a linker to the N-terminus of the C-terminal fragment.
  • the following contiguous amino acid sequence exists within the range of plus or minus 4 amino acid positions of the junction point: RKFL; preferably Typically, the following contiguous amino acid sequence exists within plus or minus 6 amino acid positions of the junction: LGRKFL.
  • the chimeric HPV types 6, 11, 16, 18, 31, 35, 39, 45, 51, 52, 56 and 58 chimeric HPV The L1 protein is associated with SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 19, SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 23.
  • SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26 and SEQ ID No: 27 are 98%, 98.5%, 99%, 99.5% or 100% identical; and HPV type 33 L1 protein and HPV59 type L1 proteins are 98%, 98.5%, 99%, 99.5% or 100% identical to SEQ ID No: 28 and SEQ ID No: 29, respectively.
  • the formulation comprises SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, respectively.
  • the present invention provides a method of preventing HPV-related disease or infection, comprising: administering to a subject a stable formulation of the multivalent human papillomavirus virus-like particle vaccine formulation.
  • the prevention can be thought of as treatment, and the two are used interchangeably.
  • the subject is a human.
  • the formulation is stable for at least 24 months at 2-8°C and at least 16 weeks at 25°C.
  • the present invention provides use of the human papillomavirus virus-like particle vaccine formulation in the preparation of a vaccine for preventing HPV-related disease or infection.
  • Fig. 1 is the adsorption degree detection result of each formula sample in Example 1.
  • Fig. 2 is the antigen content analysis result of each formula sample in Example 1.
  • T0 37°C, week 0; 37°C_1W: 37°C, week 1; 37°C_2W: 37°C, week 2; 37°C_4W: 37°C, week 4.
  • Fig. 3 is the adsorption degree detection result of each formula sample in Example 2.
  • Fig. 4 is the antigen content analysis result of each formula sample in Example 2.
  • T0 37°C, week 0; 37°C_1W: 37°C, week 1; 37°C_2W: 37°C, week 2; 37°C_4W: 37°C, week 4.
  • Fig. 5 is the adsorption degree detection result of each formula sample in Example 3.
  • Fig. 6 is the antigen content analysis result of each formula sample in Example 3.
  • T0 37°C, week 0; 37°C_1W: 37°C, week 1; 37°C_2W: 37°C, week 2; 37°C_4W: 37°C, week 4.
  • Fig. 7 is the adsorption degree detection result of each formula sample in Example 4.
  • FIG. 8 is the analysis result of antigen content of each formula sample in Example 4.
  • T0 37°C, week 0; 37°C_1W: 37°C, week 1; 37°C_2W: 37°C, week 2; 37°C_4W: 37°C, week 4.
  • the invention provides a stable preparation of human papillomavirus vaccine, which solves the problem of antibody stability during storage and transportation. Ensure that the pre-administration antigen still has the immunogenicity and safety required for prophylaxis.
  • formulation refers to a composition that maintains the biological activity of the active ingredient effective and that is free of other ingredients that would be unacceptably toxic to the subject. Such formulations are sterile. "Aseptic” refers to the absence of viable bacteria or the absence or substantial absence of all viable microorganisms and their spores.
  • a “stable” formulation refers to a formulation in which the active ingredient substantially retains its physical and/or chemical stability and/or biological activity upon storage.
  • the formulation substantially retains its physical and chemical stability, as well as its biological activity, upon storage.
  • patient or “subject” are used interchangeably and refer to any mammal suffering from a condition or disease according to the present invention. People are preferred.
  • physiologically acceptable refers to a buffer, excipient, or salt at a concentration or ionic strength that renders the formulation biologically compatible with the target host, eg, a human, to be immunized.
  • the stable formulation of the present invention contains human papillomavirus virus-like particles, a buffer, an osmotic pressure regulator and an aluminum adjuvant.
  • buffer refers to a buffered solution that resists changes in pH through the action of its acid-base complexing components.
  • a histidine buffer is selected, and the pH of the formulation solution is preferably about 5.9-6.5, more preferably 6.2.
  • surfactant refers to a surfactant, in one embodiment, the surfactant herein is polysorbate 80.
  • osmotic pressure regulator means a pharmaceutically acceptable osmotic pressure regulator. Suitable osmotic pressure regulators include, but are not limited to, salts, in one embodiment of the invention sodium chloride (NaCl) at a concentration of about 150 mM to 320 mM.
  • NaCl sodium chloride
  • adjuvant refers to a compound or mixture that enhances an immune response.
  • vaccines may contain adjuvants.
  • the adjuvant used in the present invention is selected from one or more of aluminum hydroxyphosphate (AlPO 4 ), amorphous aluminum hydroxyphosphate sulfate (AAHS) or aluminum hydroxide (Al(OH 3 )), preferably a hydroxyl group Aluminum Phosphate (AlPO 4 ).
  • the "stability" of a protein after storage at a selected temperature for a selected period of time can be qualitatively and/or quantitatively assessed in a number of different ways.
  • the content of active antigen that can bind to the neutralizing antibody of recombinant human papilloma virus is obtained by enzyme-linked immunosorbent assay (ELISA), the ratio of the content of active antigen to T 0 at each time point is compared, and the content of each preparation is compared.
  • the antigens not adsorbed to the aluminum phosphate adjuvant were obtained by centrifugation, and the content was analyzed, and the adsorption degree was calculated;
  • the EC50 of recombinant human papillomavirus neutralizing antibodies was calculated as the ratio of the EC50 of the vaccine formulation to the positive control to determine the relative potency of the vaccine in vitro.
  • immunogenicity refers to the ability of a substance, such as a protein or polypeptide, to stimulate an immune response, ie to stimulate the production of antibodies, especially humoral or cell-mediated responses.
  • HPV or "HPV virus” refers to papillomaviruses of the family Papillomaviridae, which are non-enveloped DNA viruses whose genome is a double-stranded, closed-circle DNA of approximately 8 kb in size and can generally be divided into three regions: 1Early region (E), contains 6 open reading frames encoding E1, E2, E4-E7 viral replication, transcription and transformation-related non-structural proteins, as well as E3 and E8 open reading frames; 2Late region (L) contains coding The reading frames of the major capsid protein L1 and the minor capsid protein L2; 3 The long regulatory region (LCR) does not encode any protein, but has the origin of replication and multiple transcription factor binding sites.
  • E E
  • L contains 6 open reading frames encoding E1, E2, E4-E7 viral replication, transcription and transformation-related non-structural proteins, as well as E3 and E8 open reading frames
  • 2Late region (L) contains coding The reading frames of the
  • HPV L1 protein and HPV L2 protein refer to proteins encoded by the late region (L) of the HPV gene and synthesized late in the HPV infection cycle.
  • the L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa.
  • the L2 protein is a minor capsid protein. Seventy-two L1 pentamers form the shell of the icosahedral HPV virion, wrapping the closed-loop double-stranded DNA minichromosome.
  • the L2 protein is located inside the L1 protein.
  • virus-like particle is a hollow particle containing one or more structural proteins of a virus, without viral nucleic acid.
  • concentration of papillomavirus virus-like particles of any type refers to the amount of papillomavirus virus-like particles of any type in the formulation, while the term “total concentration of papillomavirus virus-like particles of all types” refers to the formulation The sum of the concentrations of the individual types of papillomavirus virus-like particles contained in .
  • One embodiment of the present invention employs the human papillomavirus multivalent immunogenic composition described in the patent application PCT/CN2020/102601 filed on July 17, 2020, which is incorporated herein by reference and claims.
  • the formulation contains 0.74 mg/mL papillomavirus virus-like particles, 1.0 mg/mL aluminum phosphate adjuvant, 18 mM histidine buffer, 320 mM sodium chloride, pH 6.2 .
  • the vaccine will contain polysorbate 80 with a concentration of not higher than 0.3 mg/mL.
  • the preparation has good stability, and can be stored stably for at least 24 months at 2-8° C. and at least 16 weeks at 25° C.
  • the formulations of the present invention may be provided in liquid form or may be provided in lyophilized form.
  • the lyophilized formulation can be reconstituted by prior administration.
  • the detection method used is as follows:
  • Positive control human papillomavirus virus-like particle standard, source: Shenzhou Cell Engineering Co., Ltd., chimeric HPV types 6, 16, 18, 31, 35, 30,
  • the recombinant human papillomavirus neutralizing antibody (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., the same below) is combined with a solid-phase carrier to form a solid-phase antibody.
  • the recombinant human papillomavirus neutralizing antibody is combined with a solid phase carrier to form a solid phase antibody.
  • the sample to be tested was centrifuged, and the supernatant was taken as the test sample.
  • Adsorption degree (%) (1-antigen concentration in supernatant/antigen concentration of test sample)%.
  • the positive control 14-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) controls, Source: Beijing Shenzhou Cell Biotechnology Group Co., Ltd., the sequence is the same as the protein sequence in the test product) and the test product were completely dissolved, as the positive control and the test product to be tested.
  • Dilute recombinant human papillomavirus neutralizing antibody to a final concentration of 2 ⁇ g/mL add 100 ⁇ L/well to a 96-well plate, tap the plate to mix the samples, and coat overnight at 4°C; use washing solution at 200 ⁇ L/well Wash the plate once and dry the ELISA plate; then block the ELISA plate with 300 ⁇ L/well of blocking solution for one hour at room temperature; wash twice with 300 ⁇ L/well of washing solution, add 100 ⁇ L of treated Blank control (corresponding buffer for the test product), positive control to be tested and test product, act for 1 hour at room temperature; after washing the plate 3 times with washing solution at 200 ⁇ L/well, add diluted enzyme at 100 ⁇ L/well Labeled recombinant human papillomavirus neutralizing antibody; act for 1 hour at room temperature, wash the plate three times with 200 ⁇ L/well of washing solution, add chromogenic solution at 200 ⁇ L/well, and leave at room temperature for 20 ⁇ 5min; stop
  • the computer program Origin or the four-parameter fitting method is used for processing. Taking the concentration of the positive control or the test product as the abscissa, and the average absorbance intensity as the ordinate, the EC 50 of the test product and the positive control is obtained, and the EC 50 of the test product is obtained. 50 was divided by the EC 50 of the positive control to obtain the in vitro relative potency of the test article (M. Shank-Retzlaff, F. Wang, T. Morley et al. Correlation between Mouse Potency and In Vitro Relative Potency for Human Papillomavirus Type 16 Virus-Like Particles and Gardasil Vaccine Samples. Human Vaccines, 1:5, 191-197).
  • Preparation formula number papillomavirus virus-like particle Aluminum phosphate adjuvant Histidine buffer Sodium chloride Polysorbate 80 pH F1 0.74mg/mL 1.0mg/mL 18mM 320mM 0.00wt% 6.5 F2 0.74mg/mL 1.0mg/mL 18mM 320mM 0.02wt% 6.5
  • Adsorption degree analysis The detection principle is to obtain the antigen not adsorbed on the aluminum phosphate adjuvant by centrifugation and analyze the content to calculate the adsorption degree.
  • Antigen content analysis The detection principle is to obtain the active antigen content that can be combined with recombinant human papillomavirus neutralizing antibody by ELISA, and compare the stability of each preparation by comparing the ratio of the active antigen content at each time point to T 0 . The higher the value, the higher the active antigen content in the preparation, and the better the activity is maintained.
  • test results showed that the adsorption degrees of the two papillomavirus virus-like particle vaccine preparations were both above 99%, and there was no significant difference in the change trend of the antigen content of the papillomavirus virus-like particle vaccine in F1 and F2. Stability is comparable.
  • Preparation formula number papillomavirus virus-like particle Aluminum phosphate adjuvant Histidine buffer Sodium chloride Polysorbate 80 pH F1 0.74mg/mL 1.0mg/mL 18mM 320mM 0.00wt% 5.9 F2 0.74mg/mL 1.0mg/mL 18mM 320mM 0.00wt% 6.2 F3 0.74mg/mL 1.0mg/mL 18mM 320mM 0.00wt% 6.5
  • Adsorption degree analysis The detection principle is to obtain the antigens that are not adsorbed on the aluminum phosphate adjuvant by centrifugation, analyze the content, and calculate the adsorption degree.
  • Antigen content analysis The detection principle is to obtain the active antigen content that can be combined with recombinant human papillomavirus neutralizing antibody by ELISA, and compare the stability of each preparation by comparing the ratio of the active antigen content at each time point to T 0 . The higher the value, the higher the active antigen content in the preparation, and the better the activity is maintained.
  • the test results showed that the adsorption degrees of the three papillomavirus virus-like particle vaccine preparations were all above 99%, and the change trend of the antigen content of the papillomavirus virus-like particle vaccine in F1 and F2 was better than that in the F3 preparation, namely F1 and F2.
  • the stability is better than F3.
  • Example 3 Screening study of osmotic pressure regulator concentration
  • Preparation formula number papillomavirus virus-like particle Aluminum phosphate adjuvant Histidine buffer Sodium chloride Polysorbate 80 pH F1 0.74mg/mL 1.0mg/mL 18mM 320mM 0.00wt% 6.2 F2 0.74mg/mL 1.0mg/mL 18mM 150mM 0.00wt% 6.2
  • Adsorption degree analysis The detection principle is to obtain the antigens that are not adsorbed on the aluminum phosphate adjuvant by centrifugation, analyze the content, and calculate the adsorption degree.
  • Antigen content analysis The detection principle is to obtain the active antigen content that can be combined with recombinant human papillomavirus neutralizing antibody by ELISA, and compare the stability of each preparation by comparing the ratio of the active antigen content at each time point to T 0 . The higher the value, the higher the active antigen content in the preparation, and the better the activity is maintained.
  • test results showed that the adsorption degrees of the two papillomavirus virus-like particle vaccine preparations were both above 99%. At 37°C for 4 weeks, there was no significant difference in the changes of the active antigen content in the F1 and F2 preparations, that is, the stability of F1 and F2. Sexually quite.
  • Preparation formula number papillomavirus virus-like particle Aluminum phosphate adjuvant Histidine buffer Sodium chloride Polysorbate 80 pH F1 0.74mg/mL 1.0mg/mL 10mM 320mM 0.00wt% 6.2 F2 0.74mg/mL 1.0mg/mL 18mM 320mM 0.00wt% 6.2 F3 0.74mg/mL 1.0mg/mL 26mM 320mM 0.00wt% 6.2
  • Adsorption degree analysis The detection principle is to obtain the antigens that are not adsorbed on the aluminum phosphate adjuvant by centrifugation, analyze the content, and calculate the adsorption degree.
  • Antigen content analysis The detection principle is to obtain the active antigen content that can be combined with recombinant human papillomavirus neutralizing antibody by ELISA, and compare the stability of each preparation by comparing the ratio of the active antigen content at each time point to T 0 . The higher the value, the higher the active antigen content in the preparation, and the better the activity is maintained.
  • test results are shown in Table 11-12 and Figure 7-8.
  • the test results showed that the adsorption degrees of the three papillomavirus virus-like particle vaccine preparations were all above 99%, and the change trend of the antigen content of the papillomavirus virus-like particle vaccine in F1 and F2 was better than that in the F3 preparation, namely F1 and F2.
  • the stability is better than F3.
  • Example 5 Prescription confirmation of various types (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) papillomavirus virus-like particle vaccine formulations
  • papillomavirus virus-like particle vaccine preparations (preparation formula is 0.74mg/mL) Papilloma virus virus-like particles + 1.0 mg/mL aluminum phosphate adjuvant + 18 mM histidine buffer + 320 mM sodium chloride, pH 6.2) to conduct a stability study at 2-8 °C, where due to the process in the preparation process Residual, the vaccine will contain polysorbate 80 at a concentration of no higher than 0.3 mg/mL.
  • the investigation time points were 3m (3 months), 6m (6 months), 9m (9 months), and 12m (12 months), and the investigation items were adsorption degree and in vitro relative efficacy.
  • Adsorption degree The detection principle is to obtain the antigen that is not adsorbed on the aluminum phosphate adjuvant by centrifugation and analyze the content to calculate the adsorption degree.
  • Relative potency in vitro The detection principle of this method is to calculate the percentage of EC 50 of the test article and the positive control by detecting the EC 50 of the test article, the positive control and the recombinant human papillomavirus neutralizing antibody respectively. The higher the relative potency of the test article in vitro, the better the quality of the test article.
  • the experimental results show that the various types of papillomavirus virus-like particle vaccine preparations disclosed in the present invention have good stability, and can be stored stably for at least 12 months at 2-8°C.
  • Example 6 Preparation of 14-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) preparation and prescription verification experiment
  • Adsorption degree The detection principle is to obtain the antigen that is not adsorbed on the aluminum phosphate adjuvant by centrifugation and analyze the content to calculate the adsorption degree.
  • Relative potency in vitro The detection principle of this method is to calculate the percentage of EC 50 of the test article and the positive control by detecting the EC 50 of the test article, the positive control and the recombinant human papillomavirus neutralizing antibody respectively. The higher the relative potency of the test article in vitro, the better the quality of the test article.
  • the experimental results show that the fourteen-valent human papillomavirus vaccine (types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) formulations disclosed in the present invention have good Stability, stable storage for at least 24 months at 2-8 °C, and stable storage for at least 16 weeks at 25 °C.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Oncology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Communicable Diseases (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Biochemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种人乳头瘤病毒病毒样颗粒疫苗的稳定制剂，由人乳头瘤病毒病毒样颗粒、缓冲液、渗透压调节剂、表面活性剂及铝佐剂组成，其中疫苗成分为由HPV 6型、11型、16型、18型、31型、33型、45型、52型和58型的L1蛋白组装而成的HPV病毒样颗粒，和一种或多种由其他致病的HPV型别的L1蛋白组装而成的HPV病毒样颗粒。该制剂可增强疫苗的稳定性，延长其在含水制剂中的有效期。

Description

一种人乳头瘤病毒病毒样颗粒疫苗的稳定制剂
相关申请的交叉引用
本申请要求2021年01月14日提交的中国专利申请202110049777.7的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明涉及生物医药制剂领域，特别是涉及一种稳定的人乳头瘤病毒病毒样颗粒疫苗制剂。
背景技术
宫颈癌是最常见的女性恶性肿瘤之一，全球每年约有50万新增病人，发病率在女性肿瘤中占第二位。95％以上的宫颈癌与人乳头瘤病毒(Human Papillomavirus,HPV)感染密切相关。HPV除了直接导致宫颈癌，还与支气管肺癌、直肠癌、口腔癌和皮肤癌有重大关系。另外，HPV还是导致皮肤、黏膜疣的主要致病因子。
目前已发现100多种HPV型，不同型的HPV可引起的不同疾病。根据与宫颈癌关系的紧密程度，可将HPV分为高危型、疑似致癌型和低危型。高危型和疑似致癌型可诱发宫颈癌等癌症；高危型包括16、18、31、33、35、39、45、51、52、56、58、59型；疑似致癌型包括26、53、66、68、73、82型。低危型多与生殖器疣、尖锐湿疣等疾病有关，包括6、11、40、42、43、44、54、61、70、72、81、89型。HPV6、11、16型是生殖器病变患者检出率最高的亚型。
通过HPV疫苗是阻断乳头瘤病毒感染的有效途径。在多种疫苗形式中类病毒颗粒(Virus-like particle，VLP)疫苗为最具优势的疫苗形式。但是，基于VLP的HPV疫苗具有型别限制性，即只对疫苗内VLPs相同型别的HPV产生较强的保护效力。为提供广泛的保护，研发多价HPV疫苗十分必要。
但是在给药前，人乳头瘤病毒疫苗制剂会经过贮存和运输过程，在该过程中，抗原会发生物理和化学降解，这些不稳定性可能降低抗原的免疫原性和/或安全性，因此需要一种稳定的制剂来保证给药前抗原仍具有预防所需要的免疫原性和安全性。
发明内容
在一个方面，本发明提供了一种预防HPV相关疾病或感染的多价人乳头瘤病毒病毒样颗粒疫苗稳定制剂，其含有多种乳头瘤病毒病毒样颗粒，其吸附在佐剂上；生理上可接受的浓度的缓冲液、渗透压调节剂以及任选地表面活性剂。
其中所述人乳头瘤病毒病毒样颗粒选自由HPV 6型、11型、16型、18型、31型、33型、45
型、52型和58型的L1蛋白组装而成的HPV病毒样颗粒；和
一种或多种由其他致病的HPV型别的L1蛋白组装而成的HPV病毒样颗粒。
在一个实施方案中，
所述缓冲液选自柠檬酸缓冲液、醋酸缓冲液或组氨酸缓冲液的一种或多种；
所述渗透压调节剂选自氯化钠、磷酸钠或硫酸钠的一种或多种；
所述表面活性剂为多乙氧基醚，优选为聚山梨酯80；
所述佐剂选自羟基磷酸铝(AlPO 4)、非晶形的羟基磷酸铝硫酸盐(AAHS)或氢氧化铝(Al(OH 3))的一种或多种，优选为羟基磷酸铝(AlPO 4)。
在一个实施方案中，
(a)所有型别的乳头瘤病毒病毒样颗粒的总计浓度为40μg/mL～740μg/mL；(b)所述缓冲液的浓度为10mM～26mM，优选10mM、18mM或26mM；
(c)所述渗透压调节剂的浓度为150mM～320mM，优选150mM或320mM；
(d)所述表面活性剂的浓度为0～0.02％wt％，；
(e)所述佐剂的浓度约为1.0mg/mL；
(f)所述制剂的pH值为5.9-6.5，优选5.9、6.2或6.5。
在一个实施方案中，组成所述多价乳头瘤病毒病毒样颗粒的任一型别乳头瘤病毒病毒样颗粒的浓度为40μg/mL～120μg/mL。
在一个实施方案中，所述制剂含有总计0.74mg/mL乳头瘤病毒病毒样颗粒，1.0mg/mL磷酸铝佐剂，18mM组氨酸缓冲液，320mM氯化钠，制剂溶液的pH值为6.2；任选地，浓度不高于0.3mg/mL的聚山梨酯80。
在一个实施方式中，所述一种或多种其他致病的HPV型别选自HPV 35型、39型、51型、56型和59型。
在一个实施方式中，其中至少一种所述HPV病毒样颗粒为嵌合的HPV病毒样颗粒，所述嵌合的HPV病毒样颗粒包含一种嵌合HPV L1蛋白；所述嵌合HPV L1蛋白自其N末端至C末端方向包含：
a.衍生于第一型别乳头瘤病毒L1蛋白的N端片段，所述N端片段保持该型别L1蛋白的免疫原性，其中第一型别的乳头瘤病毒选自HPV 6型、11型、16型、18型、31型、33型、45型、52型和58型和一种或多种其他致病的HPV型别；和
b.衍生于第二型别乳头瘤病毒L1蛋白的C端片段，所述第二型别乳头状瘤病毒L1蛋白具有相较于其他型别的L1蛋白表达量和可溶性较好的特性；
其中嵌合HPV L1蛋白具有第一型别乳头瘤病毒L1蛋白的免疫原性。
在一个实施方式中，所述N端片段为将所述第一型别乳头瘤病毒L1蛋白的天然序列的C端截短 于其α5区内的任一氨基酸位点而得到的片段，以及与其具有至少98％的同一性的片段；并且所述C端片段为将第二型别乳头状瘤病毒L1蛋白的天然序列的N末端截短于其α5区内的任一氨基酸位点而得到的片段，以及该片段进一步突变、缺失和/或添加而产生的功能性变体。
在一个实施方式中，所述C端片段含有一个或多个核定位序列。
在一个实施方式中，其中所述第一型别乳头瘤L1蛋白选自HPV 6型、11型、16型、18型、31型、35型、39型、45型、51型、52型、56型或58型；优选地，其天然序列分别为SEQ ID No:30,SEQ ID No:31，SEQ ID No:32，SEQ ID No:33，SEQ ID No:34，SEQ ID No:35，SEQ ID No:36，SEQ ID No:37，SEQ ID No:38，SEQ ID No:39，SEQ ID No:40，或SEQ ID No:41所示出的编码基因编码的氨基酸序列；
所述第二型别乳头瘤病毒L1蛋白选自HPV 16型、28型、33型、59型、或68型L1蛋白；
更优选地，所述第二型别乳头瘤病毒L1蛋白选自HPV 33型或HPV 59型L1蛋白。
在一个实施方式中，所述C端片段为SEQ ID No:1；或其长度为m1个氨基酸的片段，优选涵盖SEQ ID No:1的第1-m1位氨基酸的片段；其中m1为8-26的整数；或所述C端片段为SEQ ID No:2；或其长度为m2个氨基酸的片段，优选涵盖SEQ ID No:2的第1-m2位氨基酸的片段；其中m2为13-31的整数。
在一个实施方式中，所述C端片段为SEQ ID No:3；或其长度为n个氨基酸的片段，优选涵盖SEQ ID No:3的第1-n位氨基酸的片段；其中n为16-38的整数。
在一个实施方式中，所述HPV 6型L1蛋白的N端片段与将SEQ ID No:4所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 11型L1蛋白的N端片段与将SEQ ID No:5所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 16型L1蛋白的N端片段与将SEQ ID No:6所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 18型L1蛋白的N端片段与将SEQ ID No:7所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 31型L1蛋白的N端片段与将SEQ ID No:8所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 35型L1蛋白的N端片段与将SEQ ID No:9所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 39型L1蛋白的N端片段与将SEQ ID No:10所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 45型L1蛋白的N端片段与将SEQ ID No:11所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 51型L1蛋白的N端片段与将SEQ ID No:12所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 52型L1蛋白的N端片段与将SEQ ID No:13所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
所述HPV 56型L1蛋白的N端片段与将SEQ ID No:14所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；和
所述HPV 58型L1蛋白的N端片段与将SEQ ID No:15所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性。
在一个实施方式中，所述N端片段的C末端与所述C端片段的N末端直接连接或通过接头连接。
在一个实施方式中，当所述N端片段的C末端与所述C端片段的N末端连接时，在连接点的正负4个氨基酸位点的范围内存在以下连续氨基酸序列：RKFL；优选地，在连接点的正负6个氨基酸位点的范围内存在以下连续氨基酸序列：LGRKFL。
在一个实施方式中，所述嵌合的HPV 6型、11型、16型、18型、31型、35型、39型、45型、51型、52型、56型和58型嵌合HPV L1蛋白分别与SEQ ID No:16、SEQ ID No:17、SEQ ID No:18、SEQ ID No:19、SEQ ID No:20、SEQ ID No:21、SEQ ID No:22、SEQ ID No:23、SEQ ID No:24、SEQ ID No:25、SEQ ID No:26和SEQ ID No:27具有98％、98.5％、99％、99.5％或100％的同一性；以及HPV 33型L1蛋白和HPV59型L1蛋白分别与SEQ ID No:28和SEQ ID No:29具有98％、98.5％、99％、99.5％或100％的同一性。
在一个实施方式中，所述制剂包含分别具有SEQ ID No:16、SEQ ID No:17、SEQ ID No:18、SEQ ID No:19、SEQ ID No:20、SEQ ID No:21、SEQ ID No:22、SEQ ID No:23、SEQ ID No:24、SEQ ID No:25、SEQ ID No:26和SEQ ID No:27所示氨基酸序列的HPV 6型、11型、16型、18型、31型、35型、39型、45型、51型、52型、56型和58型嵌合HPV L1蛋白；以及
分别具有SEQ ID No:28和SEQ ID No:29所示氨基酸序列的HPV 33型L1蛋白和HPV 59型L1蛋白。在一个方面，本发明提供一种预防HPV相关疾病或感染的方法，其包括：向受试者施用该多价人乳头瘤病毒病毒样颗粒疫苗制剂稳定制剂。所述预防可被认为是治疗，两者可互换使用。在一个实施方式中，受试者是人。
在一个方面，所述制剂在2～8℃条件下至少可稳定保存24个月，在25℃至少可稳定保存16个周。
在一个方面，本发明提供一种所述人乳头瘤病毒病毒样颗粒疫苗制剂在制备一种用于预防HPV相关疾病或感染的疫苗中的应用。
附图说明
图1是实施例1中各配方样品的吸附度检测结果。
图2是实施例1中各配方样品的抗原含量分析结果。T0:37℃，第0周；37℃_1W:37℃,第1周；37℃_2W:37℃,第2周；37℃_4W:37℃,第4周。
图3是实施例2中各配方样品的吸附度检测结果。
图4是实施例2中各配方样品的抗原含量分析结果。T0:37℃，第0周；37℃_1W:37℃,第1周；37℃_2W:37℃,第2周；37℃_4W:37℃,第4周。
图5是实施例3中各配方样品的吸附度检测结果。
图6是实施例3中各配方样品的抗原含量分析结果。T0:37℃，第0周；37℃_1W:37℃,第1周；37℃_2W:37℃,第2周；37℃_4W:37℃,第4周。
图7是实施例4中各配方样品的吸附度检测结果。
图8是实施例4中各配方样品的抗原含量分析结果。T0:37℃，第0周；37℃_1W:37℃,第1周；37℃_2W:37℃,第2周；37℃_4W:37℃,第4周。
发明详述
本发明提供了一种人乳头瘤病毒疫苗的稳定制剂，解决了贮存和运输过程中的抗体稳定性问题。保证给药前抗原仍具有预防所需要的免疫原性和安全性。
术语“制剂”指保持活性组分的生物学活性有效，且不含对受试者有不可接受的毒性的其他成分的组合物。此类制剂是无菌的。“无菌”指没有活菌或者没有或基本上没有所有活的微生物及其孢子。
本文中所用的“稳定”制剂指其中的活性组分在贮藏后基本上保留其物理稳定性和/或化学稳定性和/或生物学活性的制剂。优选地，制剂在贮藏后基本上保留其物理和化学稳定性，以及其生物学活性。
术语“患者”或“受试者”可以互换使用，指患有依照本发明的状况或疾病的任何哺乳动物。优选人。
本文中使用的“生理上可接受的”指一种缓冲液、赋形剂或盐的浓度或离子强度使得制剂与所免疫的目标宿主，例如人之间生物学上相容。
本发明的稳定制剂含有人乳头瘤病毒病毒样颗粒、缓冲液、渗透压调节剂和铝佐剂。
术语“包含”及“含有”指除提及的成分之外，还可以包含另外的成分。
当用于本文和所附权利要求书中时，单数形式“一”、“一种”、“另一”和“所述”包括复数指代对象，除非上下文明确地另有指示。
如本文中使用的，“缓冲液”指通过其酸-碱配合成分的作用来抵抗pH变化的缓冲溶液。本发明的一个实施方案中，选用组氨酸缓冲液，制剂溶液的pH优选5.9-6.5左右，更优选为6.2。
如本文中使用的，“表面活性剂”指表面活性作用剂，在一个实施方案中，本文中的表面活性剂为聚山梨酯80。
术语“渗透压调节剂”意指药学可接受渗透压调节剂。合适的渗透压调节剂包括但不限于盐，在本发明的一个实施方案中为氯化钠(NaCl)浓度为约150mM至320mM。
术语“佐剂”指一种增强免疫应答的化合物或混合物。特别的，疫苗可以包含佐剂。用于本发明的佐剂选自羟基磷酸铝(AlPO 4)、非晶形的羟基磷酸铝硫酸盐(AAHS)或氢氧化铝(Al(OH 3))其中的一种或多种，优选为羟基磷酸铝(AlPO 4)。
可以多种不同方式定性和/或定量评估蛋白质在选定温度下贮藏选定时间段后的“稳定性”。本发明 的实施方案中，通过酶联免疫吸附测定(ELISA)获得可结合重组人乳头瘤病毒中和抗体的活性抗原含量，对比各时间点的活性抗原含量与T 0的比值，比较各制剂的稳定性；采用酶联免疫吸附测定(ELISA)法,通过离心获得未吸附到磷酸铝佐剂上的抗原并进行含量分析，计算得到吸附度；通过分别检测人乳头瘤病毒疫苗制剂及阳性对照与重组人乳头瘤病毒中和抗体的EC 50，计算疫苗制剂与阳性对照EC 50比值，从而测定疫苗的体外相对效力。
术语“免疫原性”是指某种物质，例如蛋白质或多肽刺激免疫应答的能力，即刺激产生抗体，尤其是产生体液或者刺激细胞介导的应答的能力。
术语“HPV”或“HPV病毒”指乳头状瘤病毒科的乳头状瘤病毒，为无包膜DNA病毒，该病毒基因组为双链闭环DNA，大小约为8kb,通常可以分为三个区域：①早期区(E)，含有编码E1、E2、E4～E7病毒复制，转录及转化有关的非结构蛋白的6个开放阅读框，以及E3和E8开放阅读框；②晚期区(L)含有编码主要衣壳蛋白L1和次要衣壳蛋白L2的阅读框；③长调控区(LCR)不编码任何蛋白，但具有复制的起源以及多个转录因子结合位点。
术语“HPV L1蛋白”及“HPV L2蛋白”指由HPV基因的晚期区(L)编码，在HPV感染周期中晚期合成的蛋白。L1蛋白质是主要的衣壳蛋白并且具有55-60kDa的分子量。L2蛋白质是次要的衣壳蛋白质。72个L1五聚体构成二十面体HPV病毒粒子的外壳,包裹闭环双链DNA微染色体。L2蛋白质位于L1蛋白质内侧。
术语“病毒样颗粒”是含有某种病毒的一个或多个结构蛋白的空心颗粒，没有病毒核酸。
术语“任一型别乳头瘤病毒病毒样颗粒的浓度”指任一型别乳头瘤病毒病毒样颗粒在制剂中的含量，而术语“所有型别乳头瘤病毒病毒样颗粒的总计浓度”为制剂中包含的各个型别的乳头瘤病毒病毒样颗粒的浓度的总和。
本发明的一个实施方案中采用记载于2020年7月17日提交的专利申请PCT/CN2020/102601的人乳头瘤病毒多价免疫原性组合物，PCT/CN2020/102601因提及而引入本说明书以及权利要求书。
本发明的一个特别优选的实施方案中，制剂含有0.74mg/mL的乳头瘤病毒病毒样颗粒，1.0mg/mL磷酸铝佐剂，18mM组氨酸缓冲液，320mM氯化钠，pH值为6.2。其中由于制剂制备过程中的工艺残留，疫苗中会含有浓度不高于0.3mg/mL的聚山梨酯80。该制剂具有良好的稳定性，在2～8℃条件下至少可稳定保存24个月，在25℃至少可稳定保存16个周。
本发明的制剂可以以液体形式提供或者可以以冻干形式提供。可以通过在给药之前重建冻干制剂。
实施例
本发明通过参照以下实施例会得到更完整的理解。然而，它们不应解释为限制本发明的范围。所有文献、专利和专利申请因引用而并入本文。
以下的实施例中，所用的各型乳头瘤病毒病毒样颗粒，其制备、表征和性能鉴定记载于2020年7 月17日提交的专利申请PCT/CN2020/102601。
以下的实施例中，所用的检测方法如下所述：
1)抗原含量分析(Enzyme-linked Immunosorbent Assay,ELISA)
使用解吸附缓冲液将阳性对照(人乳头瘤病毒病毒样颗粒标准品，来源：神州细胞工程有限公司，为嵌合的HPV 6型、16型、18型、31型、35型、30型、45型、51型、52型、56型的嵌合HPV L1蛋白以及HPV33型和HPV59型L1蛋白，分别对应氨基酸序列SEQ ID NO:16-29，下文同)及供试品完全溶解，作为待检的阳性对照及供试品。
将重组人乳头瘤病毒中和抗体(来源：北京义翘神州科技股份有限公司，下文同)与固相载体结合，形成固相抗体。用样品稀释液稀释待检的阳性对照及供试品，然后与固相抗体进行结合，形成固相抗原抗体复合物；然后加酶标抗体，加底物显色，显色产物在450nm波长处进行读数。以一系列的阳性对照的浓度与相应的吸光度进行直线回归，将供试品测得的吸光值代入直线回归方程，即可求得待检样本中的抗原含量(M.Shank-Retzlaff,F.Wang,T.Morley et al.Correlation between Mouse Potency and In Vitro Relative Potency for Human Papillomavirus Type 16 Virus-Like Particles and Gardasil Vaccine Samples.Human Vaccines,1:5,191-197)。
2)吸附度分析(Enzyme-linked Immunosorbent Assay,ELISA)
将重组人乳头瘤病毒中和抗体与固相载体结合，形成固相抗体。对待检样品进行离心，取上清液作为供试品。用样品稀释液对阳性对照、供试品进行相应的稀释，然后与固相抗体进行结合，形成固相抗原抗体复合物；然后加酶标抗体，加底物显色，显色产物在450nm波长处进行读数。以一系列的阳性对照的浓度与相应的吸光度进行直线回归，将供试品测得的吸光值代入直线回归方程，即可求得上清液中的抗原浓度，然后通过下面的公式计算得到待检样本的吸附度(Michael J.Caulfield,Li Shi,Su Wang et al.Effect of Alternative Aluminum Adjuvants on the Absorptionand Immunogenicity of HPV16 L1 VLPs in Mice.Human Vaccines 3:4,139-146)。
吸附度(％)＝(1-上清液中的抗原浓度/待检样本的抗原浓度)％。
3)体外相对效力测定(In VitroRelativePotency,IVRP)
使用解吸附缓冲液将阳性对照(十四价人乳头瘤病毒疫苗(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)对照品，来源：北京神州细胞生物技术集团股份公司，序列同供试品中的蛋白序列)及供试品完全溶解，作为待检的阳性对照及供试品。将重组人乳头瘤病毒中和抗体稀释至终浓度2μg/mL,按照100μL/孔加入到96孔板中，轻拍板子使样品混匀，4℃条件下包被过夜；用洗涤液按200μL/孔洗板1次，扣干酶标板；再用封闭液按300μL/孔封闭酶标板，室温下封闭一小时；用洗涤液按300μL/孔洗涤2次，每孔加入100μL经过处理后的空白对照(供试品相应的缓冲液)、待检的阳性对照及供试品，室温下作用1小时；用洗涤液按200μL/孔洗板3次后，以100μL/孔加入稀释后的酶标记的重组人乳头瘤病毒中和抗体；室温下作用1小时，用洗涤液按200μL/孔洗板3次，以200μL/孔加入显色液，室温放置20±5min；以50μL/孔加入终止液终止反应；用酶标仪检测450nm的吸光值。采用计算机程序Origin或四参数拟合 方法进行处理，以阳性对照或供试品浓度为横坐标，以平均吸光强度为纵坐标，获得供试品和阳性对照的EC 50，以供试品的EC 50除以阳性对照的EC 50得到供试品的体外相对效力(M.Shank-Retzlaff,F.Wang,T.Morley et al.Correlation between Mouse Potency and In Vitro Relative Potency for Human Papillomavirus Type 16 Virus-Like Particles and Gardasil Vaccine Samples.Human Vaccines,1:5,191-197)。
实施例1：表面活性剂浓度的筛选研究
本实施例的乳头瘤病毒病毒样颗粒疫苗制剂的配方如下表所示：
表1 不同表面活性剂浓度的制剂
制剂配方编号 乳头瘤病毒病毒样颗粒 磷酸铝佐剂 组氨酸缓冲液 氯化钠 聚山梨酯80 pH
F1 0.74mg/mL 1.0mg/mL 18mM 320mM 0.00wt％ 6.5
F2 0.74mg/mL 1.0mg/mL 18mM 320mM 0.02wt％ 6.5
乳头瘤病毒病毒样颗粒疫苗制剂的制备方法：
取一定量的HPV 18病毒样颗粒，调整其配方，然后与磷酸铝佐剂混合，4℃吸附过夜，使乳头瘤病毒病毒样颗粒疫苗制剂中的pH、乳头瘤病毒病毒样颗粒、磷酸铝佐剂、组氨酸、氯化钠及聚山梨酯80的相应浓度符合表1的要求，无菌分装，做好相应编号后放置于37℃恒温箱中，并于第0周取出进行吸附度、抗原含量分析，于第1、2、4周取出进行抗原含量分析。
分析检测方法：
吸附度分析：其检测原理为通过离心获得未吸附到磷酸铝佐剂上的抗原并进行含量分析，计算得到吸附度。
抗原含量分析：其检测原理为通过ELISA获得可结合重组人乳头瘤病毒中和抗体的活性抗原含量，通过将各时间点的活性抗原含量与T 0的比值，比较各制剂的稳定性，该比值越高，说明制剂中活性抗原含量越高，活性维持的越好。
检测结果见表2-3和图1-2。
表2 HPV 18病毒样颗粒制剂吸附度
Num 结合于铝佐剂的％HPV18 VLPs
F1 99.8
F2 99.9
表3 HPV 18病毒样颗粒制剂抗原含量
Figure PCTCN2022071782-appb-000001
检测结果表明，两种乳头瘤病毒病毒样颗粒疫苗制剂的吸附度均在99％以上，且乳头瘤病毒病毒样颗粒疫苗在F1、F2中的抗原含量变化趋势无明显差异，即F1、F2的稳定性相当。
实施例2：pH的筛选研究
本实施例的乳头瘤病毒病毒样颗粒疫苗制剂的配方如下表所示：
表4 不同pH的制剂
制剂配方编号 乳头瘤病毒病毒样颗粒 磷酸铝佐剂 组氨酸缓冲液 氯化钠 聚山梨酯80 pH
F1 0.74mg/mL 1.0mg/mL 18mM 320mM 0.00wt％ 5.9
F2 0.74mg/mL 1.0mg/mL 18mM 320mM 0.00wt％ 6.2
F3 0.74mg/mL 1.0mg/mL 18mM 320mM 0.00wt％ 6.5
乳头瘤病毒病毒样颗粒疫苗制剂的制备方法：
取一定量的HPV 18病毒样颗粒，调整其配方，然后与磷酸铝佐剂混合，4℃吸附过夜，使乳头瘤病毒病毒样颗粒疫苗制剂中的pH、乳头瘤病毒病毒样颗粒、磷酸铝佐剂、组氨酸、氯化钠及聚山梨酯80的相应浓度符合表4的要求，无菌分装，做好相应编号后放置于37℃恒温箱中，并于第0周取出进行吸附度、抗原含量分析，于第1、2、4周取出进行抗原含量分析。
分析检测方法：
吸附度分析：其检测原理为通过离心获得未吸附到磷酸铝佐剂上的抗原并进行含量分析，计算得到吸附度。
抗原含量分析：其检测原理为通过ELISA获得可结合重组人乳头瘤病毒中和抗体的活性抗原含量，通过将各时间点的活性抗原含量与T 0的比值，比较各制剂的稳定性，该比值越高，说明制剂中活性抗原含量越高，活性维持的越好。
检测结果见表5-6和图3-4。
表5 HPV 18病毒样颗粒制剂吸附度
Num 结合于铝佐剂的％HPV18 VLPs
F1 99.4
F2 99.4
F3 99.8
表6 HPV 18病毒样颗粒制剂抗原含量
Figure PCTCN2022071782-appb-000002
检测结果表明，3种乳头瘤病毒病毒样颗粒疫苗制剂的吸附度均在99％以上，且乳头瘤病毒病毒样颗粒疫苗在F1、F2中的抗原含量变化趋势优于F3制剂，即F1、F2的稳定性优于F3。
实施例3：渗透压调节剂浓度的筛选研究
本实施例的乳头瘤病毒病毒样颗粒疫苗制剂的配方如下表所示：
表7 不同渗透压调节剂浓度的制剂
制剂配方编号 乳头瘤病毒病毒样颗粒 磷酸铝佐剂 组氨酸缓冲液 氯化钠 聚山梨酯80 pH
F1 0.74mg/mL 1.0mg/mL 18mM 320mM 0.00wt％ 6.2
F2 0.74mg/mL 1.0mg/mL 18mM 150mM 0.00wt％ 6.2
乳头瘤病毒病毒样颗粒疫苗制剂的制备方法：
取一定量的HPV 18病毒样颗粒，调整其配方，然后与磷酸铝佐剂混合，4℃吸附过夜，使乳头瘤病毒病毒样颗粒疫苗制剂中的pH、乳头瘤病毒病毒样颗粒、磷酸铝佐剂、组氨酸、氯化钠及聚山梨酯80的相应浓度符合表7的要求，无菌分装，做好相应编号后放置于37℃恒温箱中，并于第0周取出进行吸附度、抗原含量分析，于第1、2、4周取出进行抗原含量分析。
分析检测方法：
吸附度分析：其检测原理为通过离心获得未吸附到磷酸铝佐剂上的抗原并进行含量分析，计算得到吸附度。
抗原含量分析：其检测原理为通过ELISA获得可结合重组人乳头瘤病毒中和抗体的活性抗原含量，通过将各时间点的活性抗原含量与T 0的比值，比较各制剂的稳定性，该比值越高，说明制剂中活性抗原含量越高，活性维持的越好。
检测结果见表8-9和图5-6。
表8 HPV 18病毒样颗粒制剂吸附度
Num 结合于铝佐剂的％HPV18 VLPs
F1 99.4
F2 99.6
表9 HPV 18病毒样颗粒制剂抗原含量
Figure PCTCN2022071782-appb-000003
检测结果表明，两种乳头瘤病毒病毒样颗粒疫苗制剂的吸附度均在99％以上，37℃ 4周时，F1、F2制剂中的活性抗原含量变化程度无明显差异，即F1、F2的稳定性相当。
实施例4：缓冲剂浓度的筛选研究
本实施例的乳头瘤病毒病毒样颗粒疫苗制剂的配方如下表所示：
表10 不同的缓冲剂浓度的制剂
制剂配方编号 乳头瘤病毒病毒样颗粒 磷酸铝佐剂 组氨酸缓冲液 氯化钠 聚山梨酯80 pH
F1 0.74mg/mL 1.0mg/mL 10mM 320mM 0.00wt％ 6.2
F2 0.74mg/mL 1.0mg/mL 18mM 320mM 0.00wt％ 6.2
F3 0.74mg/mL 1.0mg/mL 26mM 320mM 0.00wt％ 6.2
乳头瘤病毒病毒样颗粒疫苗制剂的制备方法：
取一定量的HPV 18病毒样颗粒，调整其配方，然后与磷酸铝佐剂混合，4℃吸附过夜，使乳头瘤病毒病毒样颗粒疫苗制剂中的pH、乳头瘤病毒病毒样颗粒、磷酸铝佐剂、组氨酸、氯化钠及聚山梨酯80的相应浓度符合表10的要求，无菌分装，做好相应编号后放置于37℃恒温箱中，并于第0周取出进行吸附度、抗原含量分析，于第1、2、4周取出进行抗原含量分析。
分析检测方法：
吸附度分析：其检测原理为通过离心获得未吸附到磷酸铝佐剂上的抗原并进行含量分析，计算得到吸附度。
抗原含量分析：其检测原理为通过ELISA获得可结合重组人乳头瘤病毒中和抗体的活性抗原含量，通过将各时间点的活性抗原含量与T 0的比值，比较各制剂的稳定性，该比值越高，说明制剂中活性抗原含量越高，活性维持的越好。
检测结果见表11-12和图7-8。
表11 HPV 18病毒样颗粒制剂吸附度
Num 结合于铝佐剂的％HPV18 VLPs
F1 99.6
F2 99.4
F3 99.6
表12 HPV 18病毒样颗粒制剂抗原含量
Figure PCTCN2022071782-appb-000004
检测结果表明，3种乳头瘤病毒病毒样颗粒疫苗制剂的吸附度均在99％以上，且乳头瘤病毒病毒样颗粒疫苗在F1、F2中的抗原含量变化趋势优于F3制剂，即F1、F2的稳定性优于F3。
实施例5：各型(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)乳头瘤病毒病毒样颗粒疫苗制剂处方确认研究
分别对各型(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)乳头瘤病毒病毒样颗粒疫苗制剂(制剂配方为0.74mg/mL乳头瘤病毒病毒样颗粒+1.0mg/mL磷酸铝佐剂+18mM组氨酸缓冲液+320mM氯化钠，pH值为6.2)进行2～8℃稳定性考察，其中由于制剂制备过程中的工艺残留，疫苗中会含有浓度不高于0.3mg/mL的聚山梨酯80。考察时间点为3m(3个月)、6m(6个月)、9m(9个月)、12m(12个月)，考察项为吸附度和体外相对效力。
分析检测方法：
吸附度：其检测原理为通过离心获得未吸附到磷酸铝佐剂上的抗原并进行含量分析，计算得到吸附度。
体外相对效力：该方法的检测原理为通过分别检测供试品及阳性对照与重组人乳头瘤病毒中和抗体的EC 50，计算供试品与阳性对照EC 50的百分比，该值越高，表明供试品的体外相对效力越高，供试品质量越好。
实验结果见表13。
实验结果表明，本发明公开的各型乳头瘤病毒病毒样颗粒疫苗制剂具有良好的稳定性，2～8℃条件下至少可稳定保存12个月。
表13 十四型(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)乳头瘤病毒病毒样颗粒佐剂吸附液制剂的稳定性数据
Figure PCTCN2022071782-appb-000005
Figure PCTCN2022071782-appb-000006
实施例6：十四价人乳头瘤病毒疫苗(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)制剂制备及处方验证实验
十四价人乳头瘤病毒疫苗(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)的制备方法：
分别取各型(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)乳头瘤病毒病毒样颗粒疫苗制剂(制剂配方为0.74mg/mL乳头瘤病毒病毒样颗粒+1.0mg/mL磷酸铝佐剂+18mM组氨酸缓冲液+320mM氯化钠，pH值为6.2)，按照一定体积比(6,11,16,18,31,33,35,39,45,51,52,56,58,59型＝1.5:2:3:2:1:1:1:1:1:1:1:1:1:1)无菌混合，得到十四价人乳头瘤病毒疫苗(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)半成品，对应各型病毒样颗粒浓度分别为0.06mg/mL，0.08mg/mL，0.12mg/mL，0.08mg/mL，0.04mg/mL，0.04mg/mL，0.04mg/mL，0.04mg/mL，0.04mg/mL，0.04mg/mL，0.04mg/mL，0.04mg/mL，0.04mg/mL，0.04mg/mL，再灌装至西林瓶中，然后加塞、轧盖、贴签，制备得到重组十四价人乳头瘤病毒疫苗(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)，其中由于制剂制备过程中的工艺残留，疫苗中会含有浓度不高于0.3mg/mL的聚山梨酯80。然后进行2～8℃(时间点为3、6、9、12、18、24个月)和25±2℃(时间点为2周、4周、8周、16周)稳定性考察，考察项为体外相对效力和吸附度。
分析检测方法：
吸附度：其检测原理为通过离心获得未吸附到磷酸铝佐剂上的抗原并进行含量分析，计算得到吸附度。
体外相对效力：该方法的检测原理为通过分别检测供试品及阳性对照与重组人乳头瘤病毒中和抗体的EC 50，计算供试品与阳性对照EC 50的百分比，该值越高，表明供试品的体外相对效力越高，供试品质量越好。
实验结果见表14-17。
表14 重组十四价人乳头瘤病毒疫苗(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)的体外相对效力(25℃)
Figure PCTCN2022071782-appb-000007
表15 重组十四价人乳头瘤病毒疫苗(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)的吸附度(25℃)
Figure PCTCN2022071782-appb-000008
表16 重组十四价人乳头瘤病毒疫苗(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)的体外相对效力(4℃)
Figure PCTCN2022071782-appb-000009
表17 重组十四价人乳头瘤病毒疫苗(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)的吸附度(4℃)
Figure PCTCN2022071782-appb-000010
Figure PCTCN2022071782-appb-000011
实验结果表明，本发明公开的十四价人乳头瘤病毒疫苗(6,11,16,18,31,33,35,39,45,51,52,56,58,59型)制剂具有良好的稳定性，2～8℃条件下至少可稳定保存24个月，在25℃至少可稳定保存16周。
序列表
Figure PCTCN2022071782-appb-000012
Figure PCTCN2022071782-appb-000013
Figure PCTCN2022071782-appb-000014
Figure PCTCN2022071782-appb-000015
Figure PCTCN2022071782-appb-000016
Figure PCTCN2022071782-appb-000017
Figure PCTCN2022071782-appb-000018
Figure PCTCN2022071782-appb-000019
Figure PCTCN2022071782-appb-000020
Figure PCTCN2022071782-appb-000021
Figure PCTCN2022071782-appb-000022
Figure PCTCN2022071782-appb-000023

Claims (20)

  1. 一种预防HPV相关疾病或感染的多价人乳头瘤病毒病毒样颗粒疫苗的稳定制剂，含有
    (a)多种人乳头瘤病毒病毒样颗粒；
    (b)佐剂；
    (c)生理上可接受的浓度的缓冲液；
    (d)生理上可接受的浓度的渗透压调节剂；以及任选地
    (e)生理上可接受的浓度的表面活性剂,
    其中所述人乳头瘤病毒病毒样颗粒选自由HPV 6型、11型、16型、18型、31型、33型、45型、52型和58型的L1蛋白组装而成的HPV病毒样颗粒；和
    一种或多种由其他致病的HPV型别的L1蛋白组装而成的HPV病毒样颗粒,
    其中人乳头瘤病毒病毒样颗粒吸附在佐剂上。
  2. 根据权利要求1所述的制剂，其特征在于
    所述缓冲液选自柠檬酸缓冲液、醋酸缓冲液或组氨酸缓冲液的一种或多种；
    所述渗透压调节剂选自氯化钠、磷酸钠或硫酸钠的一种或多种；
    所述表面活性剂为多乙氧基醚，优选为聚山梨酯80；
    所述佐剂优选为铝佐剂，更优选为羟基磷酸铝(AlPO 4)、非晶形的羟基磷酸铝硫酸盐(AAHS)或氢氧化铝(Al(OH 3))的一种或多种，最优选为羟基磷酸铝(AlPO 4)。
  3. 根据权利要求1或2所述的制剂，其特征在于
    (a)所有型别的乳头瘤病毒病毒样颗粒的总计浓度为40～740μg/mL；
    (b)所述缓冲液的浓度为10mM～26mM，优选10mM、18mM或26mM；
    (c)所述渗透压调节剂的浓度为150mM～320mM，优选150mM或320mM；
    (d)所述表面活性剂的浓度为0～0.02wt％；
    (e)所述佐剂的浓度约为1.0mg/mL；
    (f)所述制剂的pH为5.9-6.5，优选5.9、6.2或6.5。
  4. 根据权利要求3所述的制剂，其特征在于组成所述多价乳头瘤病毒病毒样颗粒的任一型别乳头瘤病毒病毒样颗粒的浓度为40μg/mL～120μg/mL。
  5. 根据权利要求1-4之任一的制剂，其含有总计0.74mg/mL乳头瘤病毒病毒样颗粒，1.0mg/mL磷酸铝佐剂，18mM组氨酸缓冲液，320mM氯化钠，pH值为6.2；
    任选地，浓度不高于0.3mg/mL的聚山梨酯80。
  6. 根据权利要求5所述的制剂，其中所述一种或多种其他致病的HPV型别选自HPV 35 型、39型、51型、56型和59型。
  7. 根据权利要求6所述的制剂，其中至少一种所述HPV病毒样颗粒为嵌合的HPV病毒样颗粒，所述嵌合的HPV病毒样颗粒包含一种嵌合HPV L1蛋白；所述嵌合HPV L1蛋白自其N末端至C末端方向包含：
    a.衍生于第一型别乳头瘤病毒L1蛋白的N端片段，所述N端片段保持该型别L1蛋白的免疫原性，其中第一型别的乳头瘤病毒选自HPV 6型、11型、16型、18型、31型、33型、45型、52型和58型和一种或多种其他致病的HPV型别；和
    b.衍生于第二型别乳头瘤病毒L1蛋白的C端片段，所述第二型别乳头状瘤病毒L1蛋白具有相较于其他型别的L1蛋白表达量和可溶性较好的特性；
    其中嵌合HPV L1蛋白具有第一型别乳头瘤病毒L1蛋白的免疫原性。
  8. 根据权利要求7所述的制剂，其中
    所述N端片段为将所述第一型别乳头瘤病毒L1蛋白的天然序列的C端截短于其α5区内的任一氨基酸位点而得到的片段，以及与其具有至少98％的同一性的片段；并且
    所述C端片段为将第二型别乳头状瘤病毒L1蛋白的天然序列的N末端截短于其α5区内的任一氨基酸位点而得到的片段，以及该片段进一步突变、缺失和/或添加而产生的功能性变体。
  9. 根据权利要求8所述的制剂，其中所述C端片段含有一个或多个核定位序列。
  10. 根据权利要求7所述的制剂，
    其中所述第一型别乳头瘤L1蛋白选自HPV 6型、11型、16型、18型、31型、35型、39型、45型、51型、52型、56型或58型；优选地，其天然序列分别为为SEQ ID No:30,SEQ ID No:31，SEQ ID No:32，SEQ ID No:33，SEQ ID No:34，SEQ ID No:35，SEQ ID No:36，SEQ ID No:37，SEQ ID No:38，SEQ ID No:39，SEQ ID No:40，或SEQ ID No:41所示出的编码基因编码的氨基酸序列；
    其中所述第二型别乳头瘤病毒L1蛋白选自HPV 16型、28型、33型、59型或68型L1蛋白；
    更优选地，所述第二型别乳头瘤病毒L1蛋白选自HPV 33型或HPV 59型L1蛋白。
  11. 根据权利要求10所述的制剂，其中所述C端片段为SEQ ID No:1；或其长度为m1个氨基酸的片段，优选涵盖SEQ ID No:1的第1-m1位氨基酸的片段；其中m1为8-26的整数；或所述C端片段为SEQ ID No:2；或其长度为m2个氨基酸的片段，
    优选涵盖SEQ ID No:2的第1-m2位氨基酸的片段；其中m2为13-31的整数。
  12. 根据权利要求10所述的制剂，其中所述C端片段为SEQ ID No:3；或其长度为n个氨基酸的片段，优选涵盖SEQ ID No:3的第1-n位氨基酸的片段；其中n为16-38的整数。
  13. 根据权利要求7所述的制剂，其中
    所述HPV 6型L1蛋白的N端片段与将SEQ ID No:4所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 11型L1蛋白的N端片段与将SEQ ID No:5所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 16型L1蛋白的N端片段与将SEQ ID No:6所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 18型L1蛋白的N端片段与将SEQ ID No:7所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 31型L1蛋白的N端片段与将SEQ ID No:8所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 35型L1蛋白的N端片段与将SEQ ID No:9所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 39型L1蛋白的N端片段与将SEQ ID No:10所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 45型L1蛋白的N端片段与将SEQ ID No:11所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 51型L1蛋白的N端片段与将SEQ ID No:12所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 52型L1蛋白的N端片段与将SEQ ID No:13所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；
    所述HPV 56型L1蛋白的N端片段与将SEQ ID No:14所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性；和
    所述HPV 58型L1蛋白的N端片段与将SEQ ID No:15所示序列的C末端截短于其α5区内的任一氨基酸位点而得到的片段具有98％、98.5％、99％、99.5％、99％或100％的同一性。
  14. 根据权利要求7所述的制剂，所述N端片段的C末端与所述C端片段的N末端直接连接或通过接头连接。
  15. 根据权利要求7所述的制剂，其中当所述N端片段的C末端与所述C端片段的N末端连接时，在连接点的正负4个氨基酸位点的范围内存在以下连续氨基酸序列：RKFL；
    优选地，在连接点的正负6个氨基酸位点的范围内存在以下连续氨基酸序列：LGRKFL。
  16. 根据权利要求7所述的制剂，其中
    所述嵌合的HPV 6型、11型、16型、18型、31型、35型、39型、45型、51型、52型、56型和58型嵌合HPV L1蛋白分别与SEQ ID No:16、SEQ ID No:17、SEQ ID No:18、SEQ ID No:19、SEQ ID No:20、SEQ ID No:21、SEQ ID No:22、SEQ ID No:23、SEQ ID No:24、SEQ ID No:25、SEQ ID No:26和SEQ ID No:27具有98％、98.5％、99％、99.5％或100％的同一性；以及
    HPV 33型L1蛋白和HPV 59型L1蛋白分别与SEQ ID No:28和SEQ ID No:29具有98％、98.5％、99％、99.5％或100％的同一性。
  17. 根据权利要求16所述的制剂，其包含
    分别具有SEQ ID No:16、SEQ ID No:17、SEQ ID No:18、SEQ ID No:19、SEQ ID No:20、SEQ ID No:21、SEQ ID No:22、SEQ ID No:23、SEQ ID No:24、SEQ ID No:25、SEQ ID No:26和SEQ ID No:27所示氨基酸序列的HPV 6型、11型、16型、18型、31型、35型、39型、45型、51型、52型、56型和58型嵌合HPV L1蛋白；以及
    分别具有SEQ ID No:28和SEQ ID No:29所示氨基酸序列的HPV 33型L1蛋白和HPV 59型L1蛋白。
  18. 根据权利要求1-17所述的乳头瘤病毒疫苗的制剂，其特征在于，其在2～8℃条件下至少可稳定保存24个月，在25℃至少可稳定保存16个周。
  19. 一种预防HPV相关疾病或感染的方法，其包括：
    向受试者施用权利要求1-18中任一项所述的制剂。
  20. 权利要求1-18中任一项所述的制剂在制备一种用于预防HPV相关疾病或感染的疫苗中的应用。
PCT/CN2022/071782 2021-01-14 2022-01-13 一种人乳头瘤病毒病毒样颗粒疫苗的稳定制剂 WO2022152204A1 (zh)

Priority Applications (8)

Application Number Priority Date Filing Date Title
EP22739080.4A EP4292607A1 (en) 2021-01-14 2022-01-13 Stable preparation of human papillomavirus virus-like particle vaccine
MX2023008306A MX2023008306A (es) 2021-01-14 2022-01-13 Preparacion estable de una vacuna con particulas similares al virus de papilomavirus humano.
CN202280008056.4A CN117083078A (zh) 2021-01-14 2022-01-13 一种人乳头瘤病毒病毒样颗粒疫苗的稳定制剂
JP2023542604A JP2024503452A (ja) 2021-01-14 2022-01-13 ヒトパピローマウイルスウイルス様粒子ワクチンの安定な調製
US18/261,199 US20240075124A1 (en) 2021-01-14 2022-01-13 Stable formulation of human papillomavirus virus-like particle vaccine
AU2022207569A AU2022207569A1 (en) 2021-01-14 2022-01-13 Stable preparation of human papillomavirus virus-like particle vaccine
CA3204802A CA3204802A1 (en) 2021-01-14 2022-01-13 Stable preparation of human papillomavirus virus-like particle vaccine
KR1020237026965A KR20230129506A (ko) 2021-01-14 2022-01-13 인간 유두종 바이러스 유사 입자 백신의 안정한 제제

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110049777 2021-01-14
CN202110049777.7 2021-01-14

Publications (1)

Publication Number Publication Date
WO2022152204A1 true WO2022152204A1 (zh) 2022-07-21

Family

ID=82446926

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/071782 WO2022152204A1 (zh) 2021-01-14 2022-01-13 一种人乳头瘤病毒病毒样颗粒疫苗的稳定制剂

Country Status (9)

Country Link
US (1) US20240075124A1 (zh)
EP (1) EP4292607A1 (zh)
JP (1) JP2024503452A (zh)
KR (1) KR20230129506A (zh)
CN (1) CN117083078A (zh)
AU (1) AU2022207569A1 (zh)
CA (1) CA3204802A1 (zh)
MX (1) MX2023008306A (zh)
WO (1) WO2022152204A1 (zh)

Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001097840A1 (en) * 2000-06-21 2001-12-27 Medimmune, Inc. Chimeric human papillomavirus (hpv) l1 molecules and uses therefor
CN101245099A (zh) * 2007-02-14 2008-08-20 马润林 重组人乳头瘤病毒l1衣壳蛋白的氨基酸序列及其应用
CN102349996A (zh) * 2011-10-17 2012-02-15 沈阳三生制药有限责任公司 人乳头瘤病毒药物组合物及其应用
WO2021013079A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒56型l1蛋白
WO2021013070A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒35型l1蛋白
WO2021013071A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 人乳头瘤病毒多价免疫原性组合物
WO2021013072A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒39型l1蛋白
WO2021013062A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒31型l1蛋白
WO2021013075A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒18型l1蛋白
WO2021013063A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒16型l1蛋白
WO2021013077A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒58型l1蛋白
WO2021013073A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的乳头瘤病毒l1蛋白
WO2021013076A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒45型l1蛋白
WO2021013078A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒52型l1蛋白
WO2021013067A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒6型l1蛋白
WO2021013060A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒51型l1蛋白
WO2021013069A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒11型l1蛋白

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001097840A1 (en) * 2000-06-21 2001-12-27 Medimmune, Inc. Chimeric human papillomavirus (hpv) l1 molecules and uses therefor
CN101245099A (zh) * 2007-02-14 2008-08-20 马润林 重组人乳头瘤病毒l1衣壳蛋白的氨基酸序列及其应用
CN102349996A (zh) * 2011-10-17 2012-02-15 沈阳三生制药有限责任公司 人乳头瘤病毒药物组合物及其应用
WO2021013079A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒56型l1蛋白
WO2021013070A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒35型l1蛋白
WO2021013071A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 人乳头瘤病毒多价免疫原性组合物
WO2021013072A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒39型l1蛋白
WO2021013062A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒31型l1蛋白
WO2021013075A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒18型l1蛋白
WO2021013063A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒16型l1蛋白
WO2021013077A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒58型l1蛋白
WO2021013073A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的乳头瘤病毒l1蛋白
WO2021013076A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒45型l1蛋白
WO2021013078A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒52型l1蛋白
WO2021013067A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒6型l1蛋白
WO2021013060A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒51型l1蛋白
WO2021013069A1 (zh) * 2019-07-19 2021-01-28 神州细胞工程有限公司 嵌合的人乳头瘤病毒11型l1蛋白

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHRISTENSEN, N. D. ET AL.: "Hybrid papillomavirus L1 molecules assemble into virus-like particles that reconstitute conformational epitopes and induce neutralizing antibodies to distinct HPV types.", VIROLOGY., vol. 291, no. 2, 20 December 2001 (2001-12-20), XP002280750, DOI: 10.1006/viro.2001.1220 *
JIN SHI: "Controlled Assembly of Human Papillomavirus Capsid Protein L1in Vitro", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, 1 May 2015 (2015-05-01), pages 1 - 140, XP055775976 *
LI, Z. H. ET AL.: "Rational design of a triple-type human papillomavirus vaccine by compromising viral-type specificity.", NAT COMMUN., vol. 9, no. 1, 18 December 2018 (2018-12-18), XP055776002, DOI: 10.1038/s41467-018-07199-6 *
M. SHANK-RETZLAFFF. WANGT. MORLEY ET AL.: "Correlation between Mouse Potency and In Vitro Relative Potency for Human Papillomavirus Type 16 Virus-Like Particles and Gardasil Vaccine Samples", HUMAN VACCINES, vol. 1, no. 5, pages 191 - 197, XP055815561, DOI: 10.4161/hv.1.5.2126
MICHAEL J. CAULFIELDLI SHISU WANG ET AL.: "Effect of Alternative Aluminum Adjuvants on the Absorption and Immunogenicity of HPV16 L1 VLPs in Mice", HUMAN VACCINES, vol. 3, no. 4, pages 139 - 146
PIMIENTA ELSA, RODRÍGUEZ SANDRA, FANDO RAFAEL, SERRANO YUNIER, ORTEGA DARIÉN, PALENZUELA ARIEL, MARRERO KAREN: "Cloning and expression in Escherichia coli of the full-lengh and deletion variants of a human papillomavirus 18 L1 gene isolated from a Cuban patient", REVISTA CUBANA DE MEDICINA TROPICAL, vol. 71, no. 2, 1 January 2019 (2019-01-01), pages e301, XP055951381 *

Also Published As

Publication number Publication date
EP4292607A1 (en) 2023-12-20
AU2022207569A1 (en) 2023-08-10
CA3204802A1 (en) 2022-07-21
US20240075124A1 (en) 2024-03-07
CN117083078A (zh) 2023-11-17
KR20230129506A (ko) 2023-09-08
MX2023008306A (es) 2023-07-19
JP2024503452A (ja) 2024-01-25

Similar Documents

Publication Publication Date Title
US7709010B2 (en) Papillomavirus vaccine compositions
Tumban et al. Preclinical refinements of a broadly protective VLP-based HPV vaccine targeting the minor capsid protein, L2
Stanley et al. Immunobiology of human papillomavirus infection and vaccination-implications for second generation vaccines
WO2008098484A1 (fr) Protéines l1 recombinantes du papillomavirus et leurs utilisations
WO2023116374A1 (zh) 带状疱疹疫苗组合物
US20210077611A1 (en) Compositions, methods and uses for thermally stable human papillomavirus formulations
CN112043825A (zh) 一种基于新型冠状病毒突刺蛋白s1区域预防新型冠状病毒感染的亚单位疫苗
Su et al. Recombinant SARS-CoV-2 RBD with a built in T helper epitope induces strong neutralization antibody response
JP2616915B2 (ja) 中和糖タンパク質のペプチド
US20160000901A1 (en) Compositions and Methods for the Production of Virus-Like Particles
Basu et al. Bacteriophage Qβ virus-like particles displaying Chikungunya virus B-cell epitopes elicit high-titer E2 protein antibodies but fail to neutralize a Thailand strain of Chikungunya virus
Zhang et al. A rationally designed flagellin-L2 fusion protein induced serum and mucosal neutralizing antibodies against multiple HPV types
US11858964B2 (en) Virus-inspired compositions and methods of redirecting preexisting immune responses using the same for treatment of cancer
KR20110053340A (ko) Ｈｐｖ에 대한 백신
JPWO2006028214A1 (ja) 経口投与ワクチン
WO2022152204A1 (zh) 一种人乳头瘤病毒病毒样颗粒疫苗的稳定制剂
WO2023001259A1 (zh) 一种可诱导广谱中和活性重组多价新冠病毒三聚体蛋白疫苗的制备及应用
WO2022100459A1 (zh) 一种预防和治疗默克尔细胞癌的新型疫苗
EP4183409A1 (en) Vaccine with improved immunogenicity against mutant coronaviruses
Yu et al. A bacterially expressed triple-type chimeric vaccine against human papillomavirus types 51, 69, and 26
WO2015068101A1 (en) Papillomavirus vaccine formulations
US11806432B2 (en) Compositions, methods and uses for thermally stable multi-targeted antigens
CN118059223A (zh) 九价人乳头瘤病毒疫苗及其应用
US20210299238A1 (en) Compositions, methods and uses for thermally stable broad-spectrum human papillomavirus formulations
WO2023142283A1 (zh) 一种新型冠状病毒mRNA疫苗及其制备方法与应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22739080

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202280008056.4

Country of ref document: CN

ENP Entry into the national phase

Ref document number: 3204802

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: MX/A/2023/008306

Country of ref document: MX

Ref document number: 2023542604

Country of ref document: JP

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112023014016

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 20237026965

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1020237026965

Country of ref document: KR

ENP Entry into the national phase

Ref document number: 2022207569

Country of ref document: AU

Date of ref document: 20220113

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 112023014016

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20230712

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022739080

Country of ref document: EP

Effective date: 20230814

WWE Wipo information: entry into national phase

Ref document number: 11202305338W

Country of ref document: SG