WO2022031087A1 - Immunogenic composition comprising pneumococcal polysaccharide-cell wall-derived material conjugates - Google Patents

Immunogenic composition comprising pneumococcal polysaccharide-cell wall-derived material conjugates Download PDF

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WO2022031087A1
WO2022031087A1 PCT/KR2021/010359 KR2021010359W WO2022031087A1 WO 2022031087 A1 WO2022031087 A1 WO 2022031087A1 KR 2021010359 W KR2021010359 W KR 2021010359W WO 2022031087 A1 WO2022031087 A1 WO 2022031087A1
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capsular polysaccharide
immunogenic composition
cell wall
derived material
carrier protein
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PCT/KR2021/010359
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French (fr)
Korean (ko)
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조경민
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(주)셀트리온
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    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • 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
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • 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
    • A61P31/04—Antibacterial 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
    • A61K2039/555—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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker

Definitions

  • the present invention relates to an immunogenic composition
  • an immunogenic composition comprising a pneumococcal polysaccharide-cell wall-derived material conjugate, each conjugate comprising a cell wall-derived material bound to a pneumococcal-derived capsular polysaccharide of a specific serotype.
  • Streptococcus pneumoniae is a major causative agent of pneumonia. According to the National Statistical Office ⁇ Development of mortality by major cause of death in 2010 ⁇ , the death rate due to pneumonia in 2010 was 14.9 per 100,000 people, one of the top 10 causes of death, and it was found that it increased by 82.9% compared to 2000. In addition, according to the WHO in 2012, 476,000 HIV-negative children under the age of 5 worldwide died from infection with Streptococcus pneumoniae in 2008, and 5% of all deaths among children under the age of 5 died from diseases caused by this bacterium. did
  • Dr. A 14-valent polysaccharide vaccine was developed by Robert Austrian, which later evolved into a 23-valent polysaccharide vaccine.
  • Polyvalent pneumococcal polysaccharide vaccines have proven useful in preventing pneumococcal disease in the elderly and high-risk patients. However, infants and children do not have a good immune response to most pneumococcal polysaccharides because of the T-cell-independent immune response.
  • Heptavalent pneumococcal conjugate vaccine (Prevnar®) contains capsular polysaccharides from the 7 most common serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.
  • the pneumococcal serotype used in the pneumococcal conjugate vaccine is usually in a state containing CPS (Capsular Polysaccharide), CWPS (Cell Wall Polysaccharide), LTA (Lipoteichoic acid), PG (Peptidoglycan), etc. and carrier protein (transfer protein).
  • CPS Capsular Polysaccharide
  • CWPS Cell Wall Polysaccharide
  • LTA Lipoteichoic acid
  • PG Poridoglycan
  • carrier protein transfer protein
  • apoptotic vaccines inactivated vaccines, etc.
  • Capsular polysaccharide a major antigen, is known to have low efficacy in inducing long-term immunity and low immune response in infants and children when used alone.
  • a specific serotype for example, serotype 3, is a serotype included in Prevena 13 (PCV13), but the serotype is known as a serotype with low protection after vaccination.
  • serotype 3 including other serotypes antibody titers and OPA titers appear after PCV13 inoculation, but in the case of serotype 3, the actual protection rate is low. The incidence rate of the type is still high.
  • the cell wall of pneumococcus is composed of LTA (Lipoteichoic acid), CWPS (cell wall polysaccharide), CPS (capsular polysaccharide, PG (Peptidoglycan), and cell membrane double layer, etc.
  • LTA Lipoteichoic acid
  • CWPS cell wall polysaccharide
  • CPS capsulear polysaccharide
  • PG Peptidoglycan
  • cell membrane double layer etc.
  • the present inventors conjugated a pneumococcal cell wall-derived material to pneumococcal capsular polysaccharide to induce structural stability of the immunogenic composition while increasing the functional antibody titer (OPA) and specific antibody titer (IgG) of a specific serotype.
  • OPA functional antibody titer
  • IgG specific antibody titer
  • an object of the present invention is to provide an immunogenic composition
  • an immunogenic composition comprising a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a cell wall-derived material.
  • Another object of the present invention is to provide a pharmaceutical composition for inducing an immune response to a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the immunogenic composition.
  • Another problem to be solved by the present invention is to provide a method for preparing the immunogenic composition.
  • the present invention provides an immunogenic composition comprising a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a cell wall derived material.
  • the capsular polysaccharide is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, It may contain a capsular polysaccharide of any one or more serotypes selected from the group consisting of 23F and 35B.
  • the capsular polysaccharide is at least one selected from the group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. serotype capsular polysaccharides.
  • the immunogenic composition according to the present invention further comprises a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a carrier protein, wherein the capsular polysaccharide is serotypes 10A, 11A, 15B, It may be a capsular polysaccharide of any one or more serotypes selected from the group consisting of 22F, 23A, and 35B.
  • the immunogenic composition according to the present invention further comprises a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a carrier protein, wherein the capsular polysaccharide is serotype 1, 4, 5, It may be a capsular polysaccharide of any one or more serotypes selected from the group consisting of 7F, 9V, 14, 18C and 23F.
  • the conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and cell wall derived material may further include a carrier protein.
  • the conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and the cell wall-derived material further comprises a linker.
  • the cell wall-derived material may be isolated from Streptococcus pneumoniae.
  • the cell wall-derived material may be covalently linked to the capsular polysaccharide.
  • the cell wall-derived material is LTA (Lipoteichoic acid), CWPS (Cell Wall Polysaccharide) CPS (Capsular Polysaccharide), PG (Peptidoglycan), LPS (Lipopolysaccharide) MDP (Muramyl Dipeptide), MTP ( Muramyl tripeptide) and PC (Phosphatidyl Choline) may be any one or more selected from the group consisting of.
  • the cell wall-derived material may be Streptococcus pneumoniae-derived CWPS (Cell Wall Polysaccharide).
  • the carrier protein is diphtheria toxoid, tetanus toxoid, pertussis toxoid, cholera toxoid, E. coli-derived inactivated toxin, Pseudomonas aeruginosa-derived inactivated toxin and bacterial outer membrane protein (OMP) ) may be any one selected from the group consisting of.
  • the diphtheria toxoid may be any one selected from the group consisting of CRM 197 , CRM 173 , CRM 228 and CRM 45 .
  • the linker is composed of adipic acid dihyrazied (ADH), Beta-propionamido, nitrophenyl-ethylamine, haloalkyl halide, hexane diamine, 6-aminocaproic acid and a glycosidic bond. It may be any one selected from the group.
  • the conjugate of the Streptococcus pneumoniae -derived capsular polysaccharide and the cell wall-derived material and the carrier protein have a structure in which they are conjugated in one of the following splicing sequences. It may be an immunogenic composition comprising:
  • the cell wall-derived material may not directly bind to a carrier protein.
  • a linker may be additionally included between each component of the conjugation sequence a), b), or c).
  • the structure conjugated in the conjugation sequence b) may be either linear or non-linear.
  • the method for conjugating the conjugate of the capsular polysaccharide with the cell wall-derived material and the carrier protein is selected from the group consisting of a CDAP conjugation method, a reductive amination method, and a Thiol-Malemide method. It may be any one or more selected.
  • the a) conjugation sequence may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • b) conjugation sequence may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • b) conjugation sequence may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence c) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • an adjuvant may be further included.
  • the adjuvant may be an aluminum salt.
  • the aluminum salt may be any one selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.
  • the immunogenic composition may contain a polysaccharide in an amount of 0.1 to 100 ⁇ g.
  • the present invention provides a pharmaceutical composition for inducing an immune response to the Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the immunogenic composition.
  • the present invention provides a method for preparing the immunogenic composition.
  • the conjugate of the Streptococcus pneumoniae -derived capsular polysaccharide and the cell wall-derived material and the carrier protein may be conjugated in any one of the following conjugation sequences.
  • a linker may be additionally included between each component of the conjugation sequence a), b), or c).
  • the method of conjugating the conjugate of the capsular polysaccharide with the cell wall-derived material and the carrier protein is a CDAP conjugation method, a reductive amination method, and a thiol-malemide method. It may be any one or more selected from the group consisting of.
  • the a) conjugation sequence may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the b) conjugation sequence may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence b) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence c) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the immunogenic composition according to the present invention can induce a more increased immune response compared to the existing vaccine.
  • it is included in the existing pneumococcal conjugate vaccine, it has an excellent effect against a specific serotype that is prevalent around the world due to its low antibody titer or low OPA titer.
  • 1 is a diagram showing the manufacturing process and conjugation sequence of pneumococcal polysaccharide-cell wall-derived material conjugates A to C.
  • FIG. 2 is a diagram showing the results of ELISA of pneumococcal capsular polysaccharide 3 serotype-specific IgG concentration.
  • FIG. 3 is a diagram showing the IgG ELISA results for the 19-valent pneumococcal vaccine serotype and the 19-valent pneumococcal vaccine including the novel conjugate (3C).
  • FIGS. 4 and 5 are diagrams showing the results of serotype-specific IgG concentration ELISA and functional antibody titer (immunogenicity) OPA for a 19-valent pneumococcal vaccine serotype and a novel conjugate (3C).
  • the present invention provides an immunogenic composition comprising a conjugate of a cell wall-derived material and a capsular polysaccharide derived from Streptococcus pneumoniae .
  • the capsular polysaccharide according to the present invention can be prepared by standard techniques known to those skilled in the art. Capsular polysaccharide can be reduced in size to reduce viscosity and increase solubility of the activated capsular polysaccharide together. Pneumococcal conjugates can be prepared by separate procedures and formulated into a single dosage form. For example, each pneumococcal polysaccharide serotype can be grown in a soy-based medium, and then the individual polysaccharide can be purified by centrifugation, precipitation, ultrafiltration.
  • the capsular polysaccharide according to the present invention is a group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 35B It may contain capsular polysaccharides of any one or more serotypes selected from
  • the capsular polysaccharide according to the present invention is a capsular polysaccharide of any one or more serotypes selected from the group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. may include
  • the capsular polysaccharide according to the present invention may include capsular polysaccharide of any one or more serotypes selected from the group consisting of serotypes 3, 6A, 6B, 19A and 19F.
  • the present invention may further include a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a carrier protein, wherein the capsular polysaccharide is serotypes 10A, 11A, 15B, 22F, 23A And it may be a capsular polysaccharide of any one or more serotypes selected from the group consisting of 35B, but is not limited thereto.
  • the present invention may further include a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a carrier protein, wherein the capsular polysaccharide is serotype 1, 4, 5, 7F, 9V , 14, 18C and 23F may be a capsular polysaccharide of any one or more serotypes selected from the group consisting of, but is not limited thereto.
  • the immunogenic composition of the present invention may range from 13 different serotypes to at least 30 different serotypes in the number of conventional capsular polysaccharides, but is not limited thereto.
  • the immunogenic composition of the present invention induces a functional immune response in humans against diseases caused by pneumococcal infection.
  • the human subject is an elderly subject and the disease is pneumonia or invasive pneumococcal disease.
  • the elderly subject is at least 50 years of age. More specifically, in one embodiment, the elderly subject is at least 55 years of age. More specifically, in one embodiment, the elderly subject is at least 60 years of age.
  • the human subject is an infant and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM).
  • IPD invasive pneumococcal disease
  • AOM acute otitis media
  • the infant is between 0 and 2 years of age or between 2 and 15 months of age.
  • the human subject is between 6 weeks of age and 17 years of age and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). In certain embodiments, the human subject is between 6 weeks of age and 5 years of age. In another embodiment, the human subject is between 5 weeks of age and 17 years of age.
  • IPD invasive pneumococcal disease
  • AOM acute otitis media
  • the capsular polysaccharide of the present invention can be prepared by standard techniques known to those skilled in the art.
  • the capsular polysaccharide can be reduced in size to reduce viscosity or to increase solubility of the activated capsular polysaccharide.
  • the present invention is an immunogenic composition
  • an immunogenic composition comprising at least 14 different polysaccharide-protein conjugates with a physiologically acceptable vehicle, each conjugate comprising a capsular polysaccharide from pneumococci of a different serotype conjugated to a carrier protein.
  • the conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and the cell wall-derived material may further include a carrier protein.
  • the carrier protein according to the present invention may be a protein which is preferably non-toxic and non-reactive, obtainable in sufficient quantity and purity.
  • the carrier protein may preferably be CRM 197 .
  • CRM 197 is a non-toxic variant (ie, toxoid) of diphtheria toxin isolated from a culture of Corynebacterium diphtheria strain C7 ( ⁇ 197) grown in casamino acids and yeast extract-based medium.
  • CRM 197 is purified via ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography.
  • CRM 197 can also be prepared by genetic recombination according to US Pat. No. 5,614,382.
  • diphtheria toxoids may also be used as carrier proteins.
  • Other carrier proteins may include, in addition to diphtheria toxoid, tetanus toxoid, pertussis toxoid, cholera toxoid, inactivated toxin from E. coli and inactivated toxin from Pseudomonas aeruginosa .
  • Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), group A or group C5a peptidase from B streptococci, or Haemophilus influenzae protein D may also be used.
  • Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivatives of tuberculin (PPD) can also be used as carrier proteins.
  • Variants of diphtheria toxin such as CRM 173 , CRM 228 , CRM 45 can also be used as carrier proteins.
  • a conventionally known conjugation method may be used.
  • a reductive amination or CDAP conjugation method may be used, but the present invention is not limited thereto.
  • Purified polysaccharides can be chemically activated to produce saccharides capable of reacting with carrier proteins. Once activated, each capsular polysaccharide is conjugated one by one to a carrier protein to form a glycoconjugate. In one embodiment according to the present invention, each capsular polysaccharide may be conjugated to the same carrier protein. Chemical activation of polysaccharides and subsequent conjugation to carrier proteins may also be performed by known methods (US Pat. Nos. 4,673,574, 4,902,506, etc.).
  • the carrier protein conjugate obtained by the above method may be purified by various methods. Examples of these methods include concentration/diafiltration processes, column chromatography and multilayer filtration. Each of the purified conjugates is mixed to formulate the immunogenic composition of the present invention, which can be used as a vaccine. Formulation of the immunogenic composition of the present invention can be accomplished using art-recognized methods. For example, individual pneumococcal conjugates can be formulated with a physiologically acceptable vehicle to prepare a composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
  • a physiologically acceptable vehicle include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
  • the conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and the cell wall-derived material may further include a linker, but is not limited thereto.
  • any one selected from the group consisting of adipic acid dihyrazied (ADH), Beta-propionamido, nitrophenyl-ethylamine, haloalkyl halide, hexane diamine, 6-aminocaproic acid and a glycosidic bond It may be one, but is not limited thereto.
  • the pneumococcal capsular polysaccharide may be conjugated to a carrier protein or a cell wall-derived material through a linker, for example, a functional linker.
  • the linker can be, for example, a heterobifunctional or homobifunctional linker having a reactive amino group and a reactive carboxylic acid group, two reactive amino groups or two reactive carboxylic acid groups.
  • the linker may have, for example, 4 to 20, 4 to 12, 5 to 10 carbon atoms.
  • the cell wall-derived material may be one isolated from Streptococcus pneumoniae, and Streptococcus pneumoniae may be CWPS (Cell Wall Polysaccharide) derived from,
  • CWPS Cell Wall Polysaccharide
  • the cell wall-derived material may be covalently linked to the capsular polysaccharide, and all conjugates present in the immunogenic composition of the present invention may be prepared by any known conjugation technique. have.
  • the conjugation method may be to activate the capsular polysaccharide using CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to form a cyanate ester.
  • CDAP 1-cyano-4-dimethylamino pyridinium tetrafluoroborate
  • the cell wall-derived material according to the present invention may not be directly bound to the protein in the polysaccharide-protein conjugate, and other components may be included between the corresponding components, or a linker for indirect connection may be included. may mean, but is not limited thereto.
  • the conjugate of the Streptococcus pneumoniae -derived capsular polysaccharide and the cell wall-derived material and the carrier protein may be conjugated in any one of the following conjugation sequences. It is not limited.
  • the cell wall-derived material may not be directly bound to a carrier protein.
  • a linker may be additionally included between each component of the conjugation order a), b) or c), and the structure conjugated in the conjugation order b) may be either linear or non-linear.
  • the present invention is not limited thereto.
  • the capsular polysaccharide or cell wall-derived material according to an embodiment of the present invention may be conjugated to an amino group on a carrier protein directly or through a linker (indirect method).
  • the cyanate ester is conjugated with hexane diamine or ADH
  • the amino-derived capsular polysaccharide can be conjugated to the carrier protein via a carbodiimide (e.g., EDAC or EDC) linkage via a carboxyl group on the carrier protein. .
  • a hydroxyl group on the capsular polysaccharide (optionally an activated hydroxyl group, for example, an activated hydroxyl group to produce a cyanate ester can be linked directly or indirectly (linker) to an amino or carboxyl group on the protein.
  • the hydroxyl group on the saccharide can be connected to the amino group on the linker using, for example, CDAP conjugation.
  • One additional amino group in the linker (eg ADH) can be connected to the carrier protein using the carbodiimide mode. It can be conjugated to the carboxylic acid group on the pneumococcal capsular polysaccharide is conjugated to the linker before the linker is conjugated to the carrier protein.
  • the present inventors also used a direct conjugation method for the production of a 13-valent or 20-valent or more multivalent vaccine, but in the present specification, an indirect conjugation method was used to compare and fabricate the effects of cell wall-derived substances.
  • the linker used in the indirect conjugation method was a commonly used ADH (adipic acid dihyrazied), and the same linker was used, and the conjugation sequence and the type of cell wall-derived material were varied and evaluated. As a result, even though the same linker and the same cell wall-derived material were used, it was confirmed that the immunogenicity (antibody and functional antibody titer) differed according to the conjugation sequence.
  • the capsular polysaccharide was activated using CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate), and then a linker was conjugated.
  • CDAP 1-cyano-4-dimethylamino pyridinium tetrafluoroborate
  • CWPS cell wall polysaccharide, cell wall-derived polysaccharide
  • the primary material and the secondary material were finally bonded using a carbodiimide bonding method. (corresponding to embodiment A of the present invention, Fig. 1)
  • a carrier protein was conjugated.
  • Primary material and CWPS was activated using CDAP, followed by conjugation of a linker.
  • Secondary material the primary material and the secondary material were finally bonded using a carbodiimide bonding method.
  • CWPS was activated with CDAP
  • a linker was attached and it was conjugated with capsular polysaccharide using an EDC binding method.
  • the carrier protein was activated by CDAP to generate an amino group, and the CWPS-linker-CPS conjugate was conjugated with the carrier protein.
  • the cell wall-derived material is LTA (Lipoteichoic acid), PS (Polysaccharide) CPS (Capsular Polysaccharide), PG (Peptidoglycan), LPS (Lipopolysaccharide) MDP (Muramyl Dipeptide), MTP (Muramyl tripeptide) and Any one or more selected from the group consisting of PC (Phosphatidyl Choline) may be conjugated in the above manner, but is not limited thereto.
  • LTA Lipoteichoic acid
  • PS Polysaccharide
  • CPS Capsular Polysaccharide
  • PG Poridoglycan
  • LPS Lipopolysaccharide
  • MDP Muramyl Dipeptide
  • MTP Muramyl tripeptide
  • PC Phosphatidyl Choline
  • the method for conjugating the conjugate of the capsular polysaccharide with the cell wall-derived material and the carrier protein is selected from the group consisting of a CDAP conjugation method, a reductive amination method, and a Thiol-Malemide method. It may be any one or more selected, but is not limited thereto.
  • the conjugation sequence a) may include the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence b) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence b) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence c) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the immunogenic composition according to the present invention may further comprise an adjuvant as a component.
  • An 'adjuvant' as defined herein is a substance used to increase the immunogenicity of the immunogenic composition of the present invention.
  • adjuvants are often given to boost the immune response and are well known to those skilled in the art.
  • Suitable adjuvants to increase the effectiveness of the composition include, but are not limited to:
  • aluminum salts may be used as adjuvants.
  • the aluminum salt may be selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide, and may be an aluminum-precipitated vaccine or an aluminum-adsorbed vaccine in its form.
  • Aluminum salts include, but are not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), and the like.
  • Aluminum chloride and sodium phosphate are mixed in a ratio of 1:1, aluminum hydroxyphosphate sulfate is precipitated. It can be prepared by dialyzing with physiological saline and sterilizing the precipitate so that the size of the precipitate becomes 2-8 ⁇ m using a high shear mixer.
  • Al(OH)3 eg Alhydrogel or Superfos
  • 50-200 g of protein can be adsorbed per 1 mg of aluminum hydroxide, and this ratio is dependent on the pI of the protein and the pH of the solvent.
  • a protein with a low pI can bind strongly compared to a protein with a high pI.
  • Aluminum salt forms an antigen reservoir that releases antigens slowly for 2-3 weeks, non-specifically activating macrophages, complement, and innate immune mechanisms.
  • the immunogenic composition may contain a polysaccharide in an amount of 0.1 to 100 ⁇ g, but is not limited thereto.
  • the present invention also provides a pharmaceutical composition (eg, a vaccine formulation) for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the immunogenic composition.
  • a pharmaceutical composition eg, a vaccine formulation
  • for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate comprising an immunologically effective amount of the immunogenic composition.
  • the vaccine formulation according to the present invention can be used to protect or treat a person susceptible to pneumococcal infection by administration by a systemic or mucosal route.
  • an 'effective amount' refers to a dose required to elicit an antibody sufficient to significantly reduce the probability or severity of pneumococcal infection.
  • Administration includes injection via the intramuscular, intraperitoneal, intradermal or subcutaneous route; or mucosal administration to the oral/digestive tract, airway tract, or genitourinary tract.
  • intranasal administration is used for the treatment of pneumonia or otitis media, since it can more effectively prevent nasopharyngeal carriers of pneumococci, thereby attenuating infection at an early stage.
  • each vaccine dose is selected such that it induces an immunoprotective response without significant side effects. This amount may vary depending on the serotype of the pneumococci. In general, each dose may contain 0.1 to 100 ⁇ g, preferably 0.1 to 10 ⁇ g, more preferably 1 to 5 ⁇ g of polysaccharide, but is not limited thereto. Optimal amounts of components for a particular vaccine can be ascertained by standard studies involving observation of an adequate immune response in subjects. For example, extrapolation of animal test results can be used to determine vaccination doses in humans. Dosage can also be determined empirically.
  • the pharmaceutical composition comprises 2 ⁇ g of each saccharide, 4 ⁇ g of capsular polysaccharide serotype; about 34 ⁇ g of CRM 197 transporter protein; 2 ⁇ g of extracellular material; 0.125 mg of elemental aluminum (0.5 mg of aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffer as excipients.
  • the composition may be filled into single dose syringes without preservatives. After shaking, the vaccine is a homogeneous white suspension that can be administered immediately intramuscularly.
  • composition of the present invention may be formulated in the form of a single dose dose vial, a multiple dose dose vial or a prefilled syringe.
  • the formulation of the present invention may include a surfactant, and may include a mixture of surfactants such as Tween 80 or Span 85.
  • the present invention provides a method for preparing the immunogenic composition.
  • the conjugate of the Streptococcus pneumoniae -derived capsular polysaccharide and the cell wall-derived material and the carrier protein may be conjugated in any one of the following conjugation sequences.
  • a linker may be additionally included between each component of the conjugation sequence a), b), or c).
  • the method of conjugation between the capsular polysaccharide and the cell wall-derived material and the carrier protein is a CDAP conjugation method, a reductive amination method, and a Thiol-Malemide method. It may be any one or more selected from the group consisting of, but is not limited thereto.
  • the conjugation sequence a) may be conjugated by a method comprising the following steps, but is not limited thereto.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence b) may be conjugated by a method comprising the following steps, but is not limited thereto.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence b) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence c) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • Pneumococcal culture and purification of capsular polysaccharide were performed by methods known to those skilled in the art.
  • Each of the pneumococcal serotypes can be obtained from the ATCC Ameircan Type Culture Collection, US.
  • Pneumococci were identified by capsular, nonmotile, Gram-positive, lancet-shaped dicocci, and alpha hemolysis in blood agar media. The serotype was confirmed based on the Banlung Test using a specific antisera.
  • Example 1-1 cell bank manufacturing
  • Pneumococci with 19 different serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 35B) was obtained from the US ATCC (Ameircan Type Culture Collection, US), which is a consignment institution.
  • a single colony was isolated by spreading the pneumococcal strain on a blood agar medium. After selecting a single colony with good growth among more than 10 single colonies, inoculating it in a liquid medium that does not contain animal-derived components and culturing, a Research Cell Bank (RCB) containing synthetic glycerol was prepared.
  • RBC Research Cell Bank
  • a master cell bank was prepared by adding synthetic glycerol after taking out one vial from the cell bank for research in which the expression of polysaccharides with a unique serotype was confirmed, proliferating the cells in a liquid medium containing no animal-derived components, and adding synthetic glycerol.
  • One vial was taken out of the bank, and the cells were grown in a liquid medium not containing animal-derived components, and then synthetic glycerol was added to prepare a manufacturing cell bank.
  • the prepared cell bank was stored at -70°C or lower for use in the next step.
  • Example 1-2 Fermentation and polysaccharide separation
  • One vial of the cell bank for production was thawed and inoculated into a liquid medium containing no animal-derived components to start the species fermentation.
  • Seed culture was performed at 37 ⁇ 2 °C without agitation until a certain cell concentration (Optical Density, OD600) was reached and the endpoint of the intermediate-exponential growth phase was reached.
  • the culture medium obtained from the seed culture was inoculated into a fermenter containing a liquid medium containing no animal-derived components to start the main fermentation (Main Fermentation).
  • the main culture was carried out while adjusting the pH of the medium with a potassium hydroxide solution at 37 ⁇ 2 °C.
  • the optimal cell density and glucose concentration in the medium were measured every 2 hours. Fermentation was terminated when the glucose in the medium was depleted.
  • the supernatant was recovered by centrifugation.
  • the recovered supernatant was passed through a depth filter, and then the buffer was exchanged with concentration and phosphate buffer solution. After the buffer exchange, the sample was passed through an active carbon filter, and impurities were removed by the following two methods.
  • CTAB Cetyltrimethylammonium Bromide
  • the two serotypes 7F and 14 that do not react with CTAB were reacted by adding an aluminum phosphate solution (Algel), and then the supernatant obtained by centrifugation was used.
  • Algel aluminum phosphate solution
  • the sample was subjected to depth filter and ultrafiltration (UF/DF) process, and then the amounts of ethanol and sodium chloride were adjusted and stored in raw form.
  • UF/DF depth filter and ultrafiltration
  • Capsular polysaccharide raw material derived from each serotype was dissolved in water for injection so that the final concentration range was within the range described below and filtered through a 0.45 ⁇ m filter.
  • serotypes 1, 3 and 4 were dissolved in the range of 0.8 - 2.0 mg/ml, serotypes 5, 6B, 9V, 18C and 19F were 4 - 8 mg/ml, serotypes 6A and 19A were 8 - In the case of 12 mg/ml, serotypes 7F, 10A, 11A and 23F, it was dissolved at 2 - 4 mg/ml and filtered. In addition, serotypes 15B, 22F and 35B were dissolved in the range of 2 - 5 mg/ml and filtration was performed.
  • the solution was incubated at the pH and temperature ranges described below for each serotype. Specifically for serotypes 1, 3, 5, 6B, 7F, 10A, 11A, 14 and 23F 70 - 80 °C overnight, for serotypes 6A and 19F 70 - 80 °C for 1-4 hours, serotypes 9V and 18C In the case of , the incubation process was performed at pH 2.0, 65 - 80 °C for 1 - 3 hours using a phosphoric acid solution. For serotypes 22F, 23A and 35B, 75-85° C. overnight, and for serotypes 4, 15B and 19A, no hydrolysis was performed. Hydrolysis was then stopped by cooling to 21-24° C. and adding sodium hydroxide to a target pH of 6.0 ⁇ 1.0.
  • CRM 197 protein transporter was prepared through a conventional production process. After inoculation into a tube containing a freeze-dried and withered seed culture medium of Corynebacterium diphtheria (ATCC 39255), it was incubated at 36° C. for 48 hours. After culturing, diphtheria cells grown on the surface were gram salted to check for contamination, and the main culture was performed. After filtration of the culture medium, primary purification was performed using the TFF system, and the process of precipitation using ammonium sulfate was repeated. Thereafter, impurities were purified through Capto Q (Anion exchange chromatography), and final TFF was performed using a buffer including the formulation composition to prepare a CRM 197 protein carrier.
  • ATCC 39255 Corynebacterium diphtheria
  • a 2 M NaCl polysaccharide solution was prepared by adding sodium chloride powder to all serotypes.
  • appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • the reaction was terminated by adding 3 to 6 molar equivalents of a glycine solution to 1 molar equivalent of CDAP added for all serotypes and adjusting the pH to 9.0.
  • the conjugation solution was stirred at 21 to 24° C. for 1 hour and then stored at a low temperature of 2 to 8° C. overnight.
  • the diluted conjugation mixture was concentrated and diafiltered through an ultrafiltration filter using at least 20 volumes of buffer.
  • the buffer was maintained in a pH range of 5.5 to 6.5, and a buffer containing 0.9% sodium chloride was used.
  • the molecular weight cutoff of the ultrafiltration filter was performed using 300 kDa for all serotypes, and the permeate was discarded.
  • Example 2-5 sterilization filtration
  • the residual solution after diafiltration was diluted to less than 0.4 g/L based on the polysaccharide content concentration using a buffer, and filtered through a 0.22 ⁇ m filter.
  • the filtered product was subjected to in-process control (sugar content, residual DMAP). In-process controls were applied to the filtered retentate to determine whether further concentration, diafiltration and/or dilution was required.
  • Example 3-1 cell wall-derived substances
  • Cell wall polysaccharide (CWPS, cat No. 3549) and lipo-teichoic acid (LTA, F-antigen, Cat No. 88840) were purchased from SSI Diagnostica.
  • a 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to all serotypes.
  • appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • a 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to all serotypes.
  • appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • Example 3-4 junction termination and filtration
  • Example 4-1 cell wall-derived substances
  • Cell wall polysaccharide (CWPS, cat No. 3549) and lipo-teichoic acid (LTA, F-antigen, Cat No. 88840) were purchased from SSI Diagnostica.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to pneumococcal capsular polysaccharide serotype 3 (CPS 3).
  • CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • ADH adipic acid dihydrazide, linker
  • the CPS 3-ADH conjugate was obtained by purification in PBS buffer (pH 7.4) using a 6-8 kDa dialysis sack.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to CWPS (cell wall polysaccharide, a cell wall-derived material).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • CRM 197 was added at a weight ratio of 1 w / w % compared to CWPS after 7 minutes. .
  • 2M glycine in the same amount as the added CDAP was added, stirred for 1 hour, and filtered to obtain a CWPS-CRM 197 conjugate.
  • EDC.HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) solution (20 mg/mL in 0.1M Tris HCl pH 7.5) was added in a ratio of 1 w/w% compared to the Pn 3-ADH conjugate. After adding 0.1M HCl solution to lower the pH to 5, the mixture was stirred at room temperature for 2 hours.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to CWPS (cell wall polysaccharide, a cell wall-derived material).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • v/v acetonitrile/water for injection
  • sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, and 7 minutes later
  • ADH was added at a weight ratio of 10 w/w % of the polysaccharide.
  • the CWPS 3-ADH conjugate was obtained by purification in PBS buffer (pH 7.4) using a 6-8 kDa dialysis sack.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to pneumococcal capsular polysaccharide serotype 3 (CPS 3).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. It was dissolved in a weight ratio of 10 w/w % of CDAP to polysaccharide, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7. After 7 minutes, CRM 197 was added in a weight ratio of 1 w/w % to polysaccharide. . After stirring at room temperature for 4 hours, 2M glycine in the same amount as the added CDAP was added, stirred for 1 hour, and filtered to obtain a CPS 3-CRM 197 conjugate.
  • an EDC.HCl solution (20 mg/mL in 0.1M Tris HCl pH 7.5) was added to the CWPS-ADH conjugate 1 w /w% was added. After adding 0.1M HCl solution to lower the pH to 5, the mixture was stirred at room temperature for 2 hours. Thereafter, 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain a CWPS-ADH-CPS 3-CRM 197 conjugate.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to CWPS (cell wall polysaccharide, a cell wall-derived material).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • v/v acetonitrile/water for injection
  • the CPS 3-ADH conjugate was obtained by purification in PBS buffer (pH 7.4) using a 6-8 kDa dialysis sack.
  • EDC.HCl solution (20 mg/mL in 0.1M Tris HCl pH 7.5)
  • CWPS -ADH conjugate was added in a ratio of 1 w/w%.
  • 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain a CWPS-ADH-CPS 3 conjugate.
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. It was dissolved in a weight ratio of 10 w/w % of CDAP to polysaccharide, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7. After 7 minutes, CRM 197 was added in a weight ratio of 1 w/w % to polysaccharide. .
  • a 2M NaCl solution was prepared by adding sodium chloride powder to LTA (Lipo-teichoic acid, Cat No. 88840).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • v/v acetonitrile/water for injection
  • sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, and 7 minutes later
  • ADH was added at a weight ratio of 10 w/w % of the polysaccharide. After stirring at room temperature for 4 hours, it was purified using a 6-8 kDa dialysis sack in PBS buffer (pH 7.4) to obtain an LTA-ADH conjugate.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to pneumococcal capsular polysaccharide serotype 3 (CPS 3).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. It was dissolved in a weight ratio of 10 w/w % of CDAP to polysaccharide, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7. After 7 minutes, CRM 197 was added in a weight ratio of 1 w/w % to polysaccharide. . After stirring at room temperature for 4 hours, 2M glycine in the same amount as the added CDAP was added, stirred for 1 hour, and filtered to obtain a CPS 3-CRM 197 conjugate.
  • an EDC.HCl solution (20 mg/mL in 0.1M Tris HCl pH 7.5) was added to the CWPS-ADH conjugate 1 w /w% was added. After adding 0.1M HCl solution to lower the pH to 5, the mixture was stirred at room temperature for 2 hours. Thereafter, 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain an LTA-ADH-CPS 3-CRM 197 conjugate.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to LTA (Lipo-teichoic acid, Cat No. 88840).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • v/v acetonitrile/water for injection
  • EDC.HCl solution (20 mg/mL in 0.1M Tris HCl pH 7.5)
  • LTA -ADH conjugate was added in a ratio of 1 w/w%.
  • 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain an LTA-ADH-CPS 3 conjugate.
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. It was dissolved in a weight ratio of 10 w/w % of CDAP to polysaccharide, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7. After 7 minutes, CRM 197 was added in a weight ratio of 1 w/w % to polysaccharide. .
  • conjugates prepared above are novel, and it was confirmed as shown in Table 1 below that the finally produced conjugates A B, C, E, F had no problems in sugar content, protein content, sugar/protein ratio and concentration as a vaccine.
  • CPS 3 pneumococcal capsular polysaccharide serotype 3
  • serotype 3 for pneumococcal capsular polysaccharide.
  • Example 5-1 Serotype 3 specific IgG concentration ELISA measurement
  • Capsular polysaccharides (PnPs) for each serotype were coated at 0.5 ⁇ g/well to 1 ⁇ g/well on 96-well plates. Equal amounts of serum were sampled from each subject and pooled into groups. The serum pool was serially diluted up to 2.5 times with an antibody dilution buffer containing Tween 20 and 5 ⁇ g/mL of CWPS, and then reacted at room temperature for 30 minutes. Plates were washed 5 times with wash buffer, then 50 ⁇ l of pre-adsorbed and diluted serum was added to the coated well plates, followed by incubation at room temperature for 2 to 18 hours.
  • the well plate was washed in the same manner, and then goat anti-mouse IgG-alkaline phosphatase conjugate was added to each well and incubated for 2 hours at room temperature.
  • the plate was washed as described above, and 1 mg/mL p-nitrophenylamine buffer as a substrate was added to each well, followed by reaction at room temperature for 2 hours.
  • the reaction was quenched by addition of 50 ⁇ l of 3 M NaOH and the absorbance was measured at 405 nm and 690 nm.
  • a commercially available 13-valent vaccine (PREVNAR13) was applied in the same procedure.
  • the Pn3C (serotype 3, Example C) group had a slightly higher antibody titer specific to serotype 3 than PCV13, and in the case of Pn3A, it showed a slightly higher tendency.
  • the antibody titer was low. This means that even in the case of the same cell wall-derived material, there is a difference in immunogenicity depending on the splicing sequence and method (FIG. 2).
  • Example 5-2 Measurement of serotype 3 functional antibody titer (OPA)
  • Functional antibody titers were assessed by testing sera in an OPA assay. Streptococcus MOPA strains stored at -70° C. or lower were diluted to the corresponding final dilution so that the concentration of each strain was about 50,000 CFU/mL. Equal amounts of serum were sampled from each subject, pooled into groups, and serially diluted 2-fold so that 20 ⁇ l of serum remained in U-bottom plates. After diluting the sample, 10 ⁇ l of the strain prepared for each serotype was mixed with the diluted sample, and the mixture was reacted at room temperature for 30 minutes to mix well with Streptococcus and the antibody.
  • a mixture of pre-differentiated HL-60 cells and complement was added and incubated in a CO 2 incubator (37° C.) for 45 minutes.
  • the phagocytosis was stopped by lowering the temperature and 10 ⁇ l of the reaction solution was spotted on a pre-dried agar plate for 30 to 60 minutes and then allowed to absorb on the plate for 20 minutes until dry.
  • a 25 mg/mL TTC stock solution was added to the prepared overlay agar, and an antibody suitable for the corresponding strain was added thereto.
  • the mixture was thoroughly mixed, then about 25 mL of the mixture was added to the plate and allowed to cure for about 30 minutes.
  • the fully cured plates were incubated in a CO 2 incubator (37° C.) for 12 to 18 hours and then colonies were counted.
  • MOPA titers were expressed as the dilution at which 50% killing was observed.
  • PREVNAR13 a commercially available, 13-valent vaccine
  • serotype 3 for pneumococcal capsular polysaccharide.
  • Example 6-1 Serotype 3 specific IgG concentration ELISA measurement
  • IgG ELISA analysis for serotype 3 was performed in the same manner as in Example 5 above. As a result, as shown in FIG. 3 , a similar level of IgG titer was confirmed in the group of Pn3C (serotype 3, Example C) compared to PCV13. However, in the case of Pn3F using the same conjugation method and conjugated with another cell wall-derived material, LTA, a lower antibody titer at the full dose than in the Pn3c group was confirmed. In Example 5, using the same conjugation method as that of Pn3B, which showed a low antibody titer, Pn3F conjugated with LTA showed a relatively low antibody titer. This shows that the antibody titers differ according to the conjugation of different cell wall-derived materials despite being prepared with the same conjugation sequence and method (Fig. 3).
  • Example 7-1 Serotype-specific IgG concentration ELISA measurement
  • Example 4-1 Except for the experimental group, this experiment was performed in the same manner as in Example 4-1, and the results are shown in Table 6 below.
  • 'Prevnar13' and 'PCV19-CRM 197 (Naive)' described in the table below are specifically in the form of existing vaccines containing only the capsular polysaccharide-CRM 197 structure, and refer to 13-valent and 19-valent vaccines, respectively.
  • 'PCV19-CRM 197 (CWPS Conjugate)' has a structural difference from the existing vaccine form as capsular polysaccharide serotype 3 is linked to CWPS and carrier protein.
  • 23A and 35B are serotypes not included in PPV23, a pneumococcal polysaccharide vaccine (PPV), and serotypes not used in pneumococcal vaccines so far are one of the serotypes that have arrived in Asia and Europe after serotype replacement.
  • PCV19-CRM 197 including 23A and 35B had an excellent level of serotype-specific IgG concentration for all serotypes.
  • CWPS was conjugated to serotype 3
  • the antibody was formed at a similar level compared to PCV13 and PCV19 (naive)-CRM.
  • the serotype common to PREVNAR13 is similar to PREVNAR13, and the added serotypes 10A, 11A, 15B, 22F, and 23A, 35B each also showed superior serotype-specific IgG concentrations. (Table 6, Fig. 4)
  • Example 7-2 functional antibody titer (OPA)
  • Example 3 An experiment was performed to evaluate whether each of the pneumococcal vaccine compositions prepared in Example 3 had the ability to induce an immune response in mice. This immunogenicity was measured via functional antibody titer (OPA). Measurements were made in the same manner as in Example 4-1 except for the experimental group, and the results are shown in Table 7 below.
  • OPA functional antibody titer
  • antibiotic-resistant strains do not exist. Accordingly, antibiotic-resistant strains were prepared and used for MOPA analysis, and the production of resistant strains was carried out based on the UAB antibiotic-resistant strain production method (MOPA protocol). was confirmed.
  • PCV19 bonded with CWPS In the (3C Conjugated) group, serotype 3 was PCV13 and PCV19 (naive) showed a similar level of functional antibody titer to the group, and the interference effect by new conjugation and increase in serotype was not confirmed in the PCV19 (naive) and PCV19 (3C conjugated) groups.
  • Table 7, FIG. 5 In the monovalent experiment in which CWPS was conjugated, a significantly higher antibody titer and functional antibody titer were confirmed compared to the naive conjugated form, but a similar level of functional antibody titer was confirmed in the multivalent (PCV19) test.

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Abstract

The present invention relates to an immunogenic composition comprising pneumococcal polysaccharide-cell wall-derived material conjugates each comprising a cell wall-derived material bound to a capsular polysaccharide derived from a specific serotype of pneumococcus. The immunogenic composition according to the present invention can induce a more increased immune response compared to existing vaccines. In particular, the immunogenic composition is included in an existing pneumococcal conjugate vaccine but has a low antibody titer or OPA titer, and thus has an excellent effect against a specific serotype that is prevalent around the world.

Description

폐렴구균 다당류-세포벽 유래 물질 접합체를 포함하는 면역원성 조성물Immunogenic composition comprising pneumococcal polysaccharide-cell wall-derived material conjugate
본 발명은 폐렴구균 다당류-세포벽 유래 물질 접합체를 포함하는 면역원성 조성물에 관한 것으로, 각 접합체는 특정 혈청형의 폐렴구균 유래의 협막 다당류와 결합된 세포벽 유래 물질을 포함한다. The present invention relates to an immunogenic composition comprising a pneumococcal polysaccharide-cell wall-derived material conjugate, each conjugate comprising a cell wall-derived material bound to a pneumococcal-derived capsular polysaccharide of a specific serotype.
스트렙토코커스 뉴모니애(Streptococcus pneumoniae)는 폐렴의 주요한 원인균이다. 통계청 「2010년 주요 사망 원인별 사망률 추이」에 의하면 2010년 폐렴으로 인한 사망률은 10만 명당 14.9 명으로 10대 사망원인의 하나이며, 2000년 대비 82.9% 증가한 것으로 나타났다. 또한 2012년 WHO에 따르면 2008년도에 전세계적으로 HIV 음성인 5세 이하 어린이 476,000명이 스트렙토코커스 뉴모니애에 의한 감염으로 사망했으며, 5세 이하 어린이 전체 사망자 중 5%가 이 균에 의한 질환으로 사망하였다. Streptococcus pneumoniae is a major causative agent of pneumonia. According to the National Statistical Office 「Development of mortality by major cause of death in 2010」, the death rate due to pneumonia in 2010 was 14.9 per 100,000 people, one of the top 10 causes of death, and it was found that it increased by 82.9% compared to 2000. In addition, according to the WHO in 2012, 476,000 HIV-negative children under the age of 5 worldwide died from infection with Streptococcus pneumoniae in 2008, and 5% of all deaths among children under the age of 5 died from diseases caused by this bacterium. did
폐렴구균에 의한 질환을 예방하기 위해 1977 년 Dr. Robert Austrian에 의해 14가 다당류 백신이 개발되었고, 이후 23가 다당류 백신으로 발전하였다. 다가 폐렴구균 다당류 백신은 노인 및 고위험 환자에서 폐렴구균 질환을 예방하는데 있어서 유용한 것으로 입증되었다. 그러나 유아 및 소아는 대부분의 폐렴구균 다당류에 대해 면역 반응이 잘 일어나지 않는데 이는 T-cell 비의존적 면역반응 현상 때문이다. 7가 폐렴구균 접합체 백신(프리베나, Prevnar®)은 가장 발생 빈도가 높은 7개 혈청형 4, 6B, 9V, 14, 18C, 19F 및 23F 유래의 협막 다당류(capsular polysaccharide)를 포함한다. 2000년 미국에서 최초로 승인된 이후 유아 및 소아에서 침습성 질환 및 중이염에 대해 면역원성이 높고 효과적인 것으로 입증되었다. 이 백신은 현재 전세계 약 80여 개 국가에서 승인되어 있다. 프리베나의 도입 이후에 수년간 축적된 감시(surveillance) 데이터에서는 예상된 바와 같이, 미국에서 프리베나에 포함된 혈청형에 의한 침습성 폐렴구균 질환이 명확히 감소하였다. 하지만 일부 지역에서는 혈청형 적용 범위에 한계가 있었으며, 프리베나에 포함되지 않은 혈청형으로 인한 침습성 폐렴구균 질환은 증가하였다.To prevent diseases caused by pneumococci, in 1977, Dr. A 14-valent polysaccharide vaccine was developed by Robert Austrian, which later evolved into a 23-valent polysaccharide vaccine. Polyvalent pneumococcal polysaccharide vaccines have proven useful in preventing pneumococcal disease in the elderly and high-risk patients. However, infants and children do not have a good immune response to most pneumococcal polysaccharides because of the T-cell-independent immune response. Heptavalent pneumococcal conjugate vaccine (Prevnar®) contains capsular polysaccharides from the 7 most common serotypes 4, 6B, 9V, 14, 18C, 19F and 23F. Since it was first approved in the United States in 2000, it has been proven to be highly immunogenic and effective against invasive diseases and otitis media in infants and children. The vaccine is currently approved in about 80 countries worldwide. As expected from the surveillance data accumulated for several years after the introduction of Prevena, invasive pneumococcal disease caused by the serotypes included in Prevena in the United States was clearly reduced. However, serotype coverage was limited in some regions, and invasive pneumococcal disease caused by serotypes not included in Prevena increased.
프리베나에 포함되지 않은 혈청형으로 인한 폐렴구균 감염이 증가됨에 따라, 폐렴구균 혈청형의 추가에 대해서는 계속해서 연구가 진행되고 있었다. 하지만, 다가 주사에 접합체를 조합함으로 인해 상이한 구성성분들 사이에 경쟁(면역 간섭)이 일어날 수 있고 임의의 개별 접합체의 면역원성에 불리한 영향을 미칠 수 있기 때문에, 면역원성 조성물에 접합체를 추가하는 것에 어려움이 있어왔다.As the number of pneumococcal infections due to serotypes not included in Prevena increased, studies on the addition of pneumococcal serotypes continued to be conducted. However, adding a conjugate to an immunogenic composition is not recommended because combining the conjugate in a multivalent injection may result in competition (immune interference) between the different components and may adversely affect the immunogenicity of any individual conjugate. There have been difficulties
이러한 면역 간섭 현상은 다가 백신에 포함될 수 있는 접합체의 수를 제한할 수 있다. 따라서, 매우 수많은 혈청형에 대한 보호가 상당한 가치가 있음에도 불구하고 조성물 중 접합체의 수를 제한하면서 이를 달성하기는 매우 어려웠다.This phenomenon of immune interference can limit the number of conjugates that can be included in a multivalent vaccine. Thus, despite the great value of protection against a very large number of serotypes, it has been very difficult to achieve while limiting the number of conjugates in the composition.
대부분의 폐렴구균 혈청형들은 Wzy 생성 회로(Wzy-dependent pathway 또는 O-antigen polymerase pathway로도 명명됨)에 의해 협막다당이 생성 되지만, 3번 혈청형 폐렴구균의 경우에는 이와 다른 별도의 회로(synthase dependent pathway)를 통해 협막다당을 생성한다. 해당 기작을 통해 생성된 협막다당은 펩티도글리칸(Peptidoglycan) 층과 공유결합을 구성하고 있지 않아, 세포벽과 약하게 결합되어 있다. 이에, 3번 혈청형을 배양하게 되면, 해당 협막다당이 폐렴구균으로부터 외부(배지)로 떨어져 나가게 된다. 폐렴구균 접합 백신에 이용되는 폐렴구균 혈청형은 보통 CPS(Capsular Polysaccharide), CWPS(Cell Wall Polysaccharide), LTA(Lipoteichoic acid), PG(Peptidoglycan) 등이 포함된 상태의 물질과 운반체 단백질(전달 단백질)이 결합된 형태이나 특정 혈청형, 예를 들면 3번 혈청형의 경우, 세포벽과 공유결합이 되지 않은 상태이므로 협막다당이 폐렴구균으로부터 분리되어 있는 형태로 존재한다. 이러한 현상은 배양 중 다른 혈청형 대비 3번 혈청형이 높은 점성을 쉽게 갖는 이유이기도 하다.Most of the pneumococcal serotypes produce capsular polysaccharide by the Wzy production cycle (also called the Wzy-dependent pathway or O-antigen polymerase pathway), but in the case of pneumococcus 3 serotype, a different cycle (synthase dependent) pathway) to produce capsular polysaccharide. Capsular polysaccharide produced through this mechanism does not form a covalent bond with the peptidoglycan layer, and is weakly bound to the cell wall. Accordingly, when serotype 3 is cultured, the capsular polysaccharide is removed from the pneumococci to the outside (medium). The pneumococcal serotype used in the pneumococcal conjugate vaccine is usually in a state containing CPS (Capsular Polysaccharide), CWPS (Cell Wall Polysaccharide), LTA (Lipoteichoic acid), PG (Peptidoglycan), etc. and carrier protein (transfer protein). In this bound form or a specific serotype, for example, serotype 3, capsular polysaccharide exists in a form separated from pneumococci because it is not covalently bound to the cell wall. This phenomenon is also the reason why serotype 3 easily has high viscosity compared to other serotypes during culture.
기존의 사멸화 백신, 불활성화 백신 등은, 면역원성이 기타 백신 형태(재조합 단백질, DNA)보다 높다고 알려져 있으며, 이는 여러 항원에 의한 부스팅 효과에서 기인함을 여러 논문에서 언급하고 있으며, 페렴구균의 주요 항원인 협막다당은 단독으로 사용 시, 장기 면역 유도 효능이 낮고, 영유아에 면역반응이 낮은 것으로 알려져 있다. Existing apoptotic vaccines, inactivated vaccines, etc., are known to have higher immunogenicity than other vaccine types (recombinant protein, DNA), and it is mentioned in several papers that this is due to the boosting effect by various antigens. Capsular polysaccharide, a major antigen, is known to have low efficacy in inducing long-term immunity and low immune response in infants and children when used alone.
특정 혈청형, 예를 들면 3번 혈청형의 경우, 프리베나 13 (PCV13)에 포함되어 있는 혈청형이지만, 해당 혈청형은 백신 접종 이후, 방어율이 낮은 혈청형으로 알려져 있다. 다른 혈청형을 포함한 3번 혈청형의 경우, PCV13 접종 후, 항체가와 OPA 역가가 나타나지만, 3번 혈청형의 경우에는 실제 방어율이 낮으며, 유럽 및 미국의 발병 현황을 참고하면, 3번 혈청형의 발병율은 여전히 높게 유지되고 있다. A specific serotype, for example, serotype 3, is a serotype included in Prevena 13 (PCV13), but the serotype is known as a serotype with low protection after vaccination. In the case of serotype 3 including other serotypes, antibody titers and OPA titers appear after PCV13 inoculation, but in the case of serotype 3, the actual protection rate is low. The incidence rate of the type is still high.
그람 양성균인 폐렴구균의 세포벽은 LTA (Lipoteichoic acid)와 CWPS (cell wall polysaccharide) CPS (capsular polysaccharide, PG (Peptidoglycan), 세포 멤브레인 이중층 등으로 구성되어 있으며, 백신 접합체의 면역원성을 올리기 위한 다양한 연구의 일환으로서 해당 구성성분들을 활용한 연구가 진행되기도 하였으나, 폐렴구균에서 특정 혈청형에 대해 면역원성을 증가시키는 연구는 현재까지 극히 제한적으로 진행된 바 있다.The cell wall of pneumococcus, a gram-positive bacterium, is composed of LTA (Lipoteichoic acid), CWPS (cell wall polysaccharide), CPS (capsular polysaccharide, PG (Peptidoglycan), and cell membrane double layer, etc. As a part of this, studies using the components have been conducted, but studies on increasing immunogenicity for specific serotypes in pneumococci have been very limited so far.
이에 본 발명자들은 특정 혈청형의 기능적 항체가(OPA) 및 특이적 항체가(IgG)를 증가시키면서도 면역원성 조성물의 구조적 안정성을 유도하고자 폐렴구균의 세포벽 유래 물질을 폐렴구균 협막다당에 접합하였고, 해당 조성물의 뛰어난 효과를 최종 확인하여 본 발명을 완성하게 되었다.Accordingly, the present inventors conjugated a pneumococcal cell wall-derived material to pneumococcal capsular polysaccharide to induce structural stability of the immunogenic composition while increasing the functional antibody titer (OPA) and specific antibody titer (IgG) of a specific serotype. The present invention was completed by finally confirming the excellent effect of the composition.
따라서, 본 발명의 해결하려는 과제는 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 세포벽 유래 물질의 접합체를 포함하는 면역원성 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide an immunogenic composition comprising a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a cell wall-derived material.
또한, 본 발명은 해결하고자 하는 다른 과제는 상기 면역원성 조성물의 면역학적 유효량을 포함하는, 스트렙토코커스 뉴모니애 협막 다당류 접합체에 대한 면역 반응을 유도하기 위한 약학 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for inducing an immune response to a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the immunogenic composition.
또한, 본 발명은 해결하고자 하는 다른 과제는 상기 면역원성 조성물을 제조하는 방법을 제공하는 것이다.In addition, another problem to be solved by the present invention is to provide a method for preparing the immunogenic composition.
상기 과제를 해결하고자, 본 발명은 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 세포벽 유래 물질(cell wall derived material)의 접합체를 포함하는 면역원성 조성물을 제공한다.In order to solve the above problems, the present invention provides an immunogenic composition comprising a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a cell wall derived material.
본 발명에 따른 면역원성 조성물에 있어서, 상기 협막 다당류는 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F 및 35B 로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류를 포함할 수 있다.In the immunogenic composition according to the present invention, the capsular polysaccharide is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, It may contain a capsular polysaccharide of any one or more serotypes selected from the group consisting of 23F and 35B.
본 발명에 따른 면역원성 조성물에 있어서, 상기 협막 다당류는 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F 및 23F로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류를 포함할 수 있다.In the immunogenic composition according to the present invention, the capsular polysaccharide is at least one selected from the group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. serotype capsular polysaccharides.
본 발명에 따른 면역원성 조성물에 있어서, 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 운반체 단백질의 접합체를 추가로 포함하며, 상기 협막 다당류는 혈청형 10A, 11A, 15B, 22F, 23A 및 35B로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류일 수 있다.The immunogenic composition according to the present invention further comprises a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a carrier protein, wherein the capsular polysaccharide is serotypes 10A, 11A, 15B, It may be a capsular polysaccharide of any one or more serotypes selected from the group consisting of 22F, 23A, and 35B.
본 발명에 따른 면역원성 조성물에 있어서, 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 운반체 단백질의 접합체를 추가로 포함하며, 상기 협막 다당류는 혈청형 1, 4, 5, 7F, 9V, 14, 18C 및 23F로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류일 수 있다.The immunogenic composition according to the present invention further comprises a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a carrier protein, wherein the capsular polysaccharide is serotype 1, 4, 5, It may be a capsular polysaccharide of any one or more serotypes selected from the group consisting of 7F, 9V, 14, 18C and 23F.
본 발명에 따른 면역원성 조성물에 있어서, 상기 스트렙토코커스 뉴모니애 유래의 협막 다당류와 세포벽 유래 물질(cell wall derived material)의 접합체는 운반체 단백질을 추가로 포함할 수 있다.In the immunogenic composition according to the present invention, the conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and cell wall derived material may further include a carrier protein.
본 발명에 따른 면역원성 조성물에 있어서, 상기 스트렙토코커스 뉴모니애 유래의 협막 다당류와 세포벽 유래 물질의 접합체는 링커(linker)를 추가로 포함In the immunogenic composition according to the present invention, the conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and the cell wall-derived material further comprises a linker.
할 수 있다.can do.
본 발명에 따른 면역원성 조성물에 있어서, 상기 세포벽 유래 물질은 스트렙토코커스 뉴모니애(Streptococcuspneumoniae)로부터 분리된 것일 수 있다.In the immunogenic composition according to the present invention, the cell wall-derived material may be isolated from Streptococcus pneumoniae.
본 발명에 따른 면역원성 조성물에 있어서, 상기 세포벽 유래 물질은 상기 협막 다당류와 공유결합으로 연결된 것일 수 있다.In the immunogenic composition according to the present invention, the cell wall-derived material may be covalently linked to the capsular polysaccharide.
본 발명에 따른 면역원성 조성물에 있어서, 상기 세포벽 유래 물질은 LTA (Lipoteichoic acid), CWPS (Cell Wall Polysaccharide) CPS (Capsular Polysaccharide), PG (Peptidoglycan), LPS (Lipopolysaccharide) MDP (Muramyl Dipeptide), MTP (Muramyl tripeptide) 및 PC (Phosphatidyl Choline)로 이루어지는 군에서 선택되는 어느 하나 이상일 수 있다.In the immunogenic composition according to the present invention, the cell wall-derived material is LTA (Lipoteichoic acid), CWPS (Cell Wall Polysaccharide) CPS (Capsular Polysaccharide), PG (Peptidoglycan), LPS (Lipopolysaccharide) MDP (Muramyl Dipeptide), MTP ( Muramyl tripeptide) and PC (Phosphatidyl Choline) may be any one or more selected from the group consisting of.
본 발명에 따른 면역원성 조성물에 있어서, 상기 세포벽 유래 물질은 스트렙토코커스 뉴모니애(Streptococcuspneumoniae)유래의 CWPS (Cell Wall Polysaccharide)일 수 있다.In the immunogenic composition according to the present invention, the cell wall-derived material may be Streptococcus pneumoniae-derived CWPS (Cell Wall Polysaccharide).
본 발명에 따른 면역원성 조성물에 있어서, 상기 운반체 단백질은 디프테리아 톡소이드, 파상풍 톡소이드, 백일해 톡소이드, 콜레라 톡소이드, 대장균 유래 불화성화 독소, 슈도모나스 애루지노사(Pseudomonas aeruginosa) 유래 불활성화 독소 및 세균 외막 단백질(OMP)로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.In the immunogenic composition according to the present invention, the carrier protein is diphtheria toxoid, tetanus toxoid, pertussis toxoid, cholera toxoid, E. coli-derived inactivated toxin, Pseudomonas aeruginosa-derived inactivated toxin and bacterial outer membrane protein (OMP) ) may be any one selected from the group consisting of.
본 발명에 따른 면역원성 조성물에 있어서, 상기 디프테리아 톡소이드는 CRM197, CRM173, CRM228 및 CRM45 로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.In the immunogenic composition according to the present invention, the diphtheria toxoid may be any one selected from the group consisting of CRM 197 , CRM 173 , CRM 228 and CRM 45 .
본 발명에 따른 면역원성 조성물에 있어서, 상기 링커는 ADH (adipic acid dihyrazied), Beta-프로피온아미도, 니트로페닐-에틸아민, 할로알킬 할라이드, 헥산 디아민, 6-아미노카프론산 및 글리코시드 결합으로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.In the immunogenic composition according to the present invention, the linker is composed of adipic acid dihyrazied (ADH), Beta-propionamido, nitrophenyl-ethylamine, haloalkyl halide, hexane diamine, 6-aminocaproic acid and a glycosidic bond. It may be any one selected from the group.
본 발명에 따른 면역원성 조성물에 있어서, 상기 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 세포벽 유래 물질의 접합체와 운반체 단백질은 하기 접합 순서 중 어느 하나로 접합된 구조임을 특징으로 하는, 면역원성 조성물 일 수 있다:In the immunogenic composition according to the present invention, the conjugate of the Streptococcus pneumoniae -derived capsular polysaccharide and the cell wall-derived material and the carrier protein have a structure in which they are conjugated in one of the following splicing sequences. It may be an immunogenic composition comprising:
a) 협막 다당류-운반체 단백질-세포벽 유래 물질; a) capsular polysaccharide-carrier protein-cell wall-derived material;
b) 운반체 단백질-협막 다당류- 세포벽 유래 물질; 및 b) carrier protein-capsular polysaccharide-cell wall-derived material; and
c) 협막 다당류-세포벽 유래 물질-운반체 단백질.c) Capsular polysaccharide-cell wall-derived material-carrier protein.
본 발명에 따른 면역원성 조성물에 있어서, 상기 세포벽 유래 물질은 운반 단백질과 직접적으로 결합하지 않을 수 있다.In the immunogenic composition according to the present invention, the cell wall-derived material may not directly bind to a carrier protein.
본 발명에 따른 면역원성 조성물에 있어서, 상기 접합 순서 a), b), 또는 c)의 각 구성성분 간에 링커를 추가적으로 포함할 수 있다.In the immunogenic composition according to the present invention, a linker may be additionally included between each component of the conjugation sequence a), b), or c).
본 발명에 따른 면역원성 조성물에 있어서, 상기 접합 순서 b)로 접합된 구조는 선형(linear) 또는 비선형(Non-linear) 중 어느 하나일 수 있다.In the immunogenic composition according to the present invention, the structure conjugated in the conjugation sequence b) may be either linear or non-linear.
본 발명에 따른 면역원성 조성물에 있어서, 상기 협막 다당류와 세포벽 유래 물질의 접합체와 운반체 단백질 내 접합 방법은 CDAP 접합 방법, 리덕티브 아미네이션 방법 및 티올-말레마이드(Thiol-Malemide) 방법으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.In the immunogenic composition according to the present invention, the method for conjugating the conjugate of the capsular polysaccharide with the cell wall-derived material and the carrier protein is selected from the group consisting of a CDAP conjugation method, a reductive amination method, and a Thiol-Malemide method. It may be any one or more selected.
본 발명에 따른 면역원성 조성물에 있어서, 상기 a) 접합 순서는 하기 단계를 포함하는 방법으로 접합될 수 있다.In the immunogenic composition according to the present invention, the a) conjugation sequence may be conjugated by a method comprising the following steps.
i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 이를 활성화시키는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to activate it;
ii) 세포벽 유래 물질에 CDAP를 가하여, 운반체 단백질과 연결하는 단계; 및 ii) adding CDAP to the cell wall-derived material and linking with a carrier protein; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 운반체 단백질의 히드록실기 또는 아미노기와 연결하는 단계.iii) linking the capsular polysaccharide of i) with a hydroxyl group or an amino group of the carrier protein of ii).
본 발명에 따른 면역원성 조성물에 있어서, 상기 b) 접합 순서는 하기 단계를 포함하는 방법으로 접합될 수 있다.In the immunogenic composition according to the present invention, b) conjugation sequence may be conjugated by a method comprising the following steps.
i) 세포벽 유래 물질에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 협막 다당류와 연결하는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the cell wall-derived material to link it with capsular polysaccharide;
ii) 운반체 단백질에 CDAP를 가하여 이를 활성화시키는 단계; 및 ii) adding CDAP to the carrier protein to activate it; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 운반체 단백질의 히드록실기 또는 아미노기와 연결하는 단계.iii) linking the capsular polysaccharide of i) with a hydroxyl group or an amino group of the carrier protein of ii).
본 발명에 따른 면역원성 조성물에 있어서, 상기 b) 접합 순서는 하기 단계를 포함하는 방법으로 접합될 수 있다.In the immunogenic composition according to the present invention, b) conjugation sequence may be conjugated by a method comprising the following steps.
i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 운반체 단백질과 연결하는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to link it with a carrier protein;
ii) 세포벽 유래 물질에 CDAP를 가하여 이를 활성화시키는 단계; 및 ii) adding CDAP to the cell wall-derived material to activate it; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 세포벽 유래 물질과 연결하는 단계.iii) linking the capsular polysaccharide of i) with the cell wall-derived material of ii).
본 발명에 따른 면역원성 조성물에 있어서, 상기 접합 순서 c)는 하기 단계를 포함하는 방법으로 접합될 수 있다.In the immunogenic composition according to the present invention, the conjugation sequence c) may be conjugated by a method comprising the following steps.
i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 이를 활성화시키는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to activate it;
ii) 세포벽 유래 물질에 CDAP를 가하여, 운반체 단백질과 연결하는 단계; 및 ii) adding CDAP to the cell wall-derived material and linking with a carrier protein; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 세포벽 유래 물질과 연결하는 단계.iii) linking the capsular polysaccharide of i) with the cell wall-derived material of ii).
본 발명에 따른 면역원성 조성물에 있어서, 애주번트를 추가로 포함할 수 있다. 상기 애주번트는 알루미늄 염일 수 있다. 상기 알루미늄 염은 알루미늄 포스페이트, 알루미늄 설페이트 및 알루미늄 하이드록사이드로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.In the immunogenic composition according to the present invention, an adjuvant may be further included. The adjuvant may be an aluminum salt. The aluminum salt may be any one selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.
본 발명에 따른 면역원성 조성물에 있어서, 상기 면역원성 조성물은 0.1 내지 100 ㎍ 용량의 다당류를 포함할 수 있다.In the immunogenic composition according to the present invention, the immunogenic composition may contain a polysaccharide in an amount of 0.1 to 100 μg.
본 발명의 또 다른 과제를 해결하고자, 본 발명은 상기 면역원성 조성물의 면역학적 유효량을 포함하는, 스트렙토코커스 뉴모니애 협막 다당류 접합체에 대한 면역 반응을 유도하기 위한 약학 조성물을 제공한다.In order to solve another object of the present invention, the present invention provides a pharmaceutical composition for inducing an immune response to the Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the immunogenic composition.
아울러, 본 발명의 또 다른 과제를 해결하고자, 본 발명은 상기의 면역원성 조성물을 제조하는 방법을 제공한다.In addition, in order to solve another problem of the present invention, the present invention provides a method for preparing the immunogenic composition.
본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 세포벽 유래 물질의 접합체와 운반체 단백질은 하기 접합 순서 중 어느 하나로 접합될 수 있다.In the method for preparing the immunogenic composition according to the present invention, the conjugate of the Streptococcus pneumoniae -derived capsular polysaccharide and the cell wall-derived material and the carrier protein may be conjugated in any one of the following conjugation sequences. can
a) 협막 다당류-운반체 단백질-세포벽 유래 물질; a) capsular polysaccharide-carrier protein-cell wall-derived material;
b) 운반체 단백질-협막 다당류- 세포벽 유래 물질; 및 b) carrier protein-capsular polysaccharide-cell wall-derived material; and
c) 협막 다당류-세포벽 유래 물질-운반체 단백질.c) Capsular polysaccharide-cell wall-derived material-carrier protein.
본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 접합 순서 a), b), 또는 c)의 각 구성성분 간에 링커를 추가적으로 포함할 수 있다.In the method for preparing the immunogenic composition according to the present invention, a linker may be additionally included between each component of the conjugation sequence a), b), or c).
본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 협막 다당류와 세포벽 유래 물질의 접합체와 운반체 단백질 내 접합 방법은 CDAP 접합 방법, 리덕티브 아미네이션 방법 및 티올-말레마이드(Thiol-Malemide) 방법으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.In the method for preparing the immunogenic composition according to the present invention, the method of conjugating the conjugate of the capsular polysaccharide with the cell wall-derived material and the carrier protein is a CDAP conjugation method, a reductive amination method, and a thiol-malemide method. It may be any one or more selected from the group consisting of.
본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 a) 접합 순서는 하기 단계를 포함하는 방법으로 접합될 수 있다.In the method for preparing an immunogenic composition according to the present invention, the a) conjugation sequence may be conjugated by a method comprising the following steps.
i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 이를 활성화시키는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to activate it;
ii) 세포벽 유래 물질에 CDAP를 가하여, 운반체 단백질과 연결하는 단계; 및 ii) adding CDAP to the cell wall-derived material and linking with a carrier protein; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 운반체 단백질의 히드록실기 또는 아미노기와 연결하는 단계.iii) linking the capsular polysaccharide of i) with a hydroxyl group or an amino group of the carrier protein of ii).
본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 b) 접합 순서는 하기 단계를 포함하는 방법으로 접합될 수 있다.In the method for preparing the immunogenic composition according to the present invention, the b) conjugation sequence may be conjugated by a method comprising the following steps.
i) 세포벽 유래 물질에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 협막 다당류와 연결하는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the cell wall-derived material to link it with capsular polysaccharide;
ii) 운반체 단백질에 CDAP를 가하여 이를 활성화시키는 단계; 및 ii) adding CDAP to the carrier protein to activate it; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 운반체 단백질의 히드록실기 또는 아미노기와 연결하는 단계.iii) linking the capsular polysaccharide of i) with a hydroxyl group or an amino group of the carrier protein of ii).
또한, 본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 접합 순서 b)가 하기 단계를 포함하는 방법으로 접합될 수 있다.In addition, in the method for preparing the immunogenic composition according to the present invention, the conjugation sequence b) may be conjugated by a method comprising the following steps.
i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 운반체 단백질과 연결하는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to link it with a carrier protein;
ii) 세포벽 유래 물질에 CDAP를 가하여 이를 활성화시키는 단계; 및 ii) adding CDAP to the cell wall-derived material to activate it; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 세포벽 유래 물질과 연결하는 단계.iii) linking the capsular polysaccharide of i) with the cell wall-derived material of ii).
아울러, 본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 접합 순서 c)가 하기 단계를 포함하는 방법으로 접합될 수 있다.In addition, in the method for preparing the immunogenic composition according to the present invention, the conjugation sequence c) may be conjugated by a method comprising the following steps.
i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 이를 활성화시키는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to activate it;
ii) 세포벽 유래 물질에 CDAP를 가하여, 운반체 단백질과 연결하는 단계; 및 ii) adding CDAP to the cell wall-derived material and linking with a carrier protein; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 세포벽 유래 물질과 연결하는 단계.iii) linking the capsular polysaccharide of i) with the cell wall-derived material of ii).
본 발명에 따른 면역원성 조성물은 기존 백신 대비 더욱 증가된 면역 반응을 유도할 수 있다. 특히, 기존 폐렴구균 접합 백신에는 포함되어 있으나 항체가가 낮거나 OPA 역가가 낮아 전 세계적으로 유행하고 있는 특정 혈청형에 대해 뛰어난 효과를 가진다.The immunogenic composition according to the present invention can induce a more increased immune response compared to the existing vaccine. In particular, although it is included in the existing pneumococcal conjugate vaccine, it has an excellent effect against a specific serotype that is prevalent around the world due to its low antibody titer or low OPA titer.
도 1은 폐렴구균 다당류-세포벽 유래 물질 접합체 A 내지 C의 제조 과정 및 접합 순서를 나타낸 그림이다.1 is a diagram showing the manufacturing process and conjugation sequence of pneumococcal polysaccharide-cell wall-derived material conjugates A to C.
도 2는 폐렴구균 협막다당 3번 혈청형 특이 IgG 농도의 ELISA 결과를 나타낸 그림이다.2 is a diagram showing the results of ELISA of pneumococcal capsular polysaccharide 3 serotype-specific IgG concentration.
도 3은 19가 폐렴구균 백신 혈청형 및 신규 접합체 (3C)를 포함한 19가 폐렴구균 백신에 대한 IgG ELISA 결과를 나타낸 그림이다.3 is a diagram showing the IgG ELISA results for the 19-valent pneumococcal vaccine serotype and the 19-valent pneumococcal vaccine including the novel conjugate (3C).
도 4 및 도 5는 19가 폐렴구균 백신 혈청형 및 신규 접합체 (3C)를 포함한 19가 폐렴구균 백신에 대한 혈청형 특이 IgG 농도 ELISA 및 기능적 항체가(면역원성) OPA 결과를 나타낸 그림이다. 4 and 5 are diagrams showing the results of serotype-specific IgG concentration ELISA and functional antibody titer (immunogenicity) OPA for a 19-valent pneumococcal vaccine serotype and a novel conjugate (3C).
이하, 본 발명을 보다 구체적으로 설명한다. Hereinafter, the present invention will be described in more detail.
그러나 본 기재는 본 발명의 이해를 돕기 위한 것일 뿐, 어떤 의미로든 본 발명의 범위가 발명의 상세한 설명에 기재된 사항들에 의하여 한정되는 것은 아니다.However, this description is only for helping the understanding of the present invention, and the scope of the present invention is not limited by the matters described in the detailed description of the present invention in any sense.
본 발명은 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 세포벽 유래 물질의 접합체를 포함하는 면역원성 조성물을 제공한다.The present invention provides an immunogenic composition comprising a conjugate of a cell wall-derived material and a capsular polysaccharide derived from Streptococcus pneumoniae .
본 발명에 따른 협막 다당류는 당업자에게 공지된 표준 기술에 의해서 제조될 수 있다. 협막 다당류는 점도를 감소시키고 활성화된 협막 다당류의 용해도를 함께 증가시키기 위해서 크기를 줄일 수 있다. 폐렴구균 접합체들은 별개의 과정에 의해서 제조되고 단일 투여 제형으로 제제화될 수 있다. 예를 들면, 각 폐렴구균 다당류 혈청형을 대두-기제 배지에서 증식시키고, 이어서 개개의 다당류를 원심분리, 침전, 한외여과를 통해서 정제할 수 있다.The capsular polysaccharide according to the present invention can be prepared by standard techniques known to those skilled in the art. Capsular polysaccharide can be reduced in size to reduce viscosity and increase solubility of the activated capsular polysaccharide together. Pneumococcal conjugates can be prepared by separate procedures and formulated into a single dosage form. For example, each pneumococcal polysaccharide serotype can be grown in a soy-based medium, and then the individual polysaccharide can be purified by centrifugation, precipitation, ultrafiltration.
본 발명에 따른 상기 협막 다당류는 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F 및 35B 로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류를 포함할 수 있다. The capsular polysaccharide according to the present invention is a group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 35B It may contain capsular polysaccharides of any one or more serotypes selected from
본 발명에 따른 상기 협막 다당류는 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F 및 23F로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류를 포함할 수 있다.The capsular polysaccharide according to the present invention is a capsular polysaccharide of any one or more serotypes selected from the group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. may include
본 발명에 따른 상기 협막 다당류는 혈청형 3, 6A, 6B, 19A 및 19F로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류를 포함할 수 있다.The capsular polysaccharide according to the present invention may include capsular polysaccharide of any one or more serotypes selected from the group consisting of serotypes 3, 6A, 6B, 19A and 19F.
상기의 특정 혈청형들의 경우, 프리베나 13 (PCV13)에 포함되어 있는 혈청형이지만, 해당 혈청형은 백신 접종 이후, 방어율이 낮은 현상(면역원성이 낮음)이 보고된 바 있다. 다른 혈청형을 포함한 해당 혈청형들의 경우, PCV13 접종 후, 항체가와 OPA 역가가 나타나지만, 실제 방어율이 낮으며, 유럽 및 미국의 발병 현황을 참고하면, 해당 혈청형의 발병율은 여전히 높게 유지되는 것으로 알려져 있다. 이는 앞서 배경설명에서 기술한 바와 같이, 해당 특정 혈청형의 협막다당이 폐렴구균 세포벽과 공유결합을 형성하고 있지 않아 분리되어 있는 형태로 존재하기 때문이다.In the case of the above specific serotypes, although they are serotypes included in Prevena 13 (PCV13), the serotype has been reported to have a low protective rate (low immunogenicity) after vaccination. In the case of the serotypes, including other serotypes, after PCV13 inoculation, antibody titers and OPA titers appear, but the actual protection rate is low. is known This is because, as described in the background description, the capsular polysaccharide of the specific serotype does not form a covalent bond with the pneumococcal cell wall and exists in a separate form.
또한, 본 발명은 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 운반체 단백질의 접합체를 추가로 포함할 수 있으며, 상기 협막 다당류는 혈청형 10A, 11A, 15B, 22F, 23A 및 35B로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류일 수 있으나, 이에 한정되는 것은 아니다.In addition, the present invention may further include a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a carrier protein, wherein the capsular polysaccharide is serotypes 10A, 11A, 15B, 22F, 23A And it may be a capsular polysaccharide of any one or more serotypes selected from the group consisting of 35B, but is not limited thereto.
아울러, 본 발명은 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 운반체 단백질의 접합체를 추가로 포함할 수 있으며, 상기 협막 다당류는 혈청형 1, 4, 5, 7F, 9V, 14, 18C 및 23F로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류일 수 있으나, 이에 한정되는 것은 아니다.In addition, the present invention may further include a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a carrier protein, wherein the capsular polysaccharide is serotype 1, 4, 5, 7F, 9V , 14, 18C and 23F may be a capsular polysaccharide of any one or more serotypes selected from the group consisting of, but is not limited thereto.
본 발명의 면역원성 조성물은 통상적인 협막 다당의 수로서 13개의 상이한 혈청형 내지 30개 이상의 상이한 혈청형 범위일 수 있으나, 이에 한정되지는 않는다.The immunogenic composition of the present invention may range from 13 different serotypes to at least 30 different serotypes in the number of conventional capsular polysaccharides, but is not limited thereto.
본 발명의 면역원성 조성물은 폐렴구균 감염에 의해 유발되는 질환에 대해 사람에서 기능적 면역 반응을 유도한다. 보다 구체적으로는 일 구현예에서, 사람 대상체는 노인 대상체이며 질환은 폐렴 또는 침습성 폐렴구균 질환이다. 보다 구체적으로는, 일 구현예에서, 노인 대상체는 적어도 50세이다. 보다 구체적으로는, 일 구현예에서, 노인 대상체는 적어도 55세이다. 보다 구체적으로는, 일 구현예에서, 노인 대상체는 적어도 60세이다. The immunogenic composition of the present invention induces a functional immune response in humans against diseases caused by pneumococcal infection. More specifically, in one embodiment, the human subject is an elderly subject and the disease is pneumonia or invasive pneumococcal disease. More specifically, in one embodiment, the elderly subject is at least 50 years of age. More specifically, in one embodiment, the elderly subject is at least 55 years of age. More specifically, in one embodiment, the elderly subject is at least 60 years of age.
보다 구체적으로는, 일 구현예에서, 사람 대상체는 유아이고 질환은 폐렴, 침습성 폐렴구균 질환(IPD), 또는 급성 중이염(AOM)이다. More specifically, in one embodiment, the human subject is an infant and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM).
보다 구체적으로는, 일 구현예에서, 유아는 0 내지 2세 또는 2 내지 15개월령이다.More specifically, in one embodiment, the infant is between 0 and 2 years of age or between 2 and 15 months of age.
다른 실시형태에서, 사람 대상체는 6주령 내지 17세이며 질환은 폐렴, 침습성 폐렴구균 질환(IPD) 또는 급성 중이염(AOM)이다. 특정 실시형태에서, 사람 대상체는 6주령 내지 5세이다. 또 다른 실시형태에서, 사람 대상체는 5주령 내지 17세이다.In another embodiment, the human subject is between 6 weeks of age and 17 years of age and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). In certain embodiments, the human subject is between 6 weeks of age and 5 years of age. In another embodiment, the human subject is between 5 weeks of age and 17 years of age.
본 발명의 협막 다당류는 당업자에게 공지된 표준 기술에 의해서 제조될 수 있다. 협막 다당류는 점도를 감소시키기 위해서 또는 활성화된 협막 다당류의 용해도를 증가시키기 위해서 크기를 줄일 수 있다.The capsular polysaccharide of the present invention can be prepared by standard techniques known to those skilled in the art. The capsular polysaccharide can be reduced in size to reduce viscosity or to increase solubility of the activated capsular polysaccharide.
본 발명은 생리학적으로 허용되는 비히클과 함께 14개 이상의 상이한 다당류-단백질 접합체를 포함하는 면역원성 조성물로서, 각각의 접합체가 운반체 단백질에 접합된 상이한 혈청형의 폐렴구균 유래의 협막 다당류를 포함한다. 본 발명에 따른 면역원성 조성물에 있어서, 상기 스트렙토코커스 뉴모니애 유래의 협막 다당류와 세포벽 유래 물질의 접합체는 운반체 단백질을 추가로 포함할 수 있다.The present invention is an immunogenic composition comprising at least 14 different polysaccharide-protein conjugates with a physiologically acceptable vehicle, each conjugate comprising a capsular polysaccharide from pneumococci of a different serotype conjugated to a carrier protein. In the immunogenic composition according to the present invention, the conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and the cell wall-derived material may further include a carrier protein.
본 발명에 따른 운반체 단백질은 바람직하게는 무독성이고 비반응원성이며, 충분한 양 및 순도로 수득할 수 있는 단백질일 수 있다. 본 발명에 따른 면역원성 조성물에 있어서, 상기 운반체 단백질은 바람직하게는 CRM197일 수 있다. CRM197은 카사미노산 및 효모 추출물-기제 배지에서 증식시킨 코리네박테리움 디프테리아(Corynebacterium diphtheria) 균주 C7 (β197)의 배양물로부터 분리된 디프테리아 독소의 무독성 변이체(즉, 톡소이드)이다. CRM197은 한외여과, 암모늄 설페이트 침전 및 이온 교환 크로마토그래피를 통해서 정제된다. CRM197은 미국특허 제5,614,382호에 따라 유전자 재조합하여 제조될 수도 있다.The carrier protein according to the present invention may be a protein which is preferably non-toxic and non-reactive, obtainable in sufficient quantity and purity. In the immunogenic composition according to the present invention, the carrier protein may preferably be CRM 197 . CRM 197 is a non-toxic variant (ie, toxoid) of diphtheria toxin isolated from a culture of Corynebacterium diphtheria strain C7 (β197) grown in casamino acids and yeast extract-based medium. CRM 197 is purified via ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography. CRM 197 can also be prepared by genetic recombination according to US Pat. No. 5,614,382.
본 발명에 따른 면역원성 조성물에 있어서, 다른 디프테리아 톡소이드도 또한 운반체 단백질로서 사용될 수 있다. 다른 운반체 단백질은 디프테리아 톡소이드 외에도, 파상풍 톡소이드, 백일해 톡소이드, 콜레라 톡소이드, 대장균 유래 불화성화 독소 및 슈도모나스 애루지노사(Pseudomonas aeruginosa) 유래 불활성화 독소가 포함될 수 있다. 세균 외막 단백질, 예를 들면, 외막 복합체 c(OMPC), 포린, 트랜스페린 결합 단백질, 뉴모리신, 폐렴구균 표면 단백질 A(PspA), 폐렴구균 어드헤신(adhesin) 단백질(PsaA), 그룹 A 또는 그룹 B 연쇄구균 유래의 C5a 펩티다제, 또는 헤모필러스 인플루엔자(Haemophilus influenzae) 단백질 D도 또한 사용될 수 있다. 오브알부민, 키홀 림펫 헤모시아닌(KLH), 소 혈청 알부민(BSA) 또는 투베르쿨린의 정제된 단백질 유도체(PPD)와 같은 다른 단백질도 또한 운반체 단백질로서 사용될 수 있다. CRM173, CRM228, CRM45와 같은 디프테리아 독소의 변이체도 운반체 단백질로 사용 가능하다.In the immunogenic composition according to the invention, other diphtheria toxoids may also be used as carrier proteins. Other carrier proteins may include, in addition to diphtheria toxoid, tetanus toxoid, pertussis toxoid, cholera toxoid, inactivated toxin from E. coli and inactivated toxin from Pseudomonas aeruginosa . Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), group A or group C5a peptidase from B streptococci, or Haemophilus influenzae protein D may also be used. Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivatives of tuberculin (PPD) can also be used as carrier proteins. Variants of diphtheria toxin such as CRM 173 , CRM 228 , CRM 45 can also be used as carrier proteins.
본 발명에 따른 운반체 단백질을 다른 구성과 접합하기 위해서는 기존에 공지된 접합 방법을 사용할 수도 있다. 예시로 리덕티브 아미네이션 또는 CDAP 접합 방법을 사용할 수 있으며, 이에 한정되지는 않는다. 운반체 단백질과 반응할 수 있는 당류를 제조하기 위하여, 정제된 다당류는 화학적으로 활성화시킬 수 있다. 일단 활성화되면, 각 협막 다당류를 운반체 단백질에 하나씩 접합시켜서 당접합체(glycoconjugate)를 형성한다. 본 발명에 따른 일 구현예에서, 각 협막 다당류를 동일한 운반체 단백질에 접합시킬 수도 있다. 다당류의 화학적 활성화 및 이어지는 운반체 단백질에의 접합은 공지의 방법에 의해 수행될 수도 있다(미국특허 제4,673,574호, 제4,902,506호 등). In order to conjugate the carrier protein according to the present invention with other components, a conventionally known conjugation method may be used. As an example, a reductive amination or CDAP conjugation method may be used, but the present invention is not limited thereto. Purified polysaccharides can be chemically activated to produce saccharides capable of reacting with carrier proteins. Once activated, each capsular polysaccharide is conjugated one by one to a carrier protein to form a glycoconjugate. In one embodiment according to the present invention, each capsular polysaccharide may be conjugated to the same carrier protein. Chemical activation of polysaccharides and subsequent conjugation to carrier proteins may also be performed by known methods (US Pat. Nos. 4,673,574, 4,902,506, etc.).
상기의 방법으로 얻어진 운반체 단백질 접합체는 다양한 방법에 의해 정제할 수도 있다. 이들 방법의 예는 농축/투석여과 공정, 칼럼 크로마토그래피 및 다층 여과를 포함한다. 정제된 접합체들은 각각을 혼합하여 본 발명의 면역원성 조성물로 제제화하고, 이를 백신으로서 사용할 수 있다. 당업계에서 인정된 방법을 사용하여 본 발명의 면역원성 조성물의 제제화를 수행할 수 있다. 예를 들면, 개개의 폐렴구균 접합체를 생리학적으로 허용되는 비히클과 함께 제형화하여 조성물을 제조할 수 있다. 이러한 비히클의 예에는, 물, 완충 식염수, 폴리올(예: 글리세롤, 프로필렌 글리콜, 액체 폴리에틸렌 글리콜) 및 덱스트로스 용액이 포함되지만, 이에 제한되는 것은 아니다.The carrier protein conjugate obtained by the above method may be purified by various methods. Examples of these methods include concentration/diafiltration processes, column chromatography and multilayer filtration. Each of the purified conjugates is mixed to formulate the immunogenic composition of the present invention, which can be used as a vaccine. Formulation of the immunogenic composition of the present invention can be accomplished using art-recognized methods. For example, individual pneumococcal conjugates can be formulated with a physiologically acceptable vehicle to prepare a composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
본 발명에 따른 면역원성 조성물에 있어서, 상기 스트렙토코커스 뉴모니애 유래의 협막 다당류와 세포벽 유래 물질의 접합체는 링커(linker)를 추가로 포함할 수도 있으나, 이에 한정되는 것은 아니다.In the immunogenic composition according to the present invention, the conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and the cell wall-derived material may further include a linker, but is not limited thereto.
본 발명에 따른 상기 링커로는 ADH(adipic acid dihyrazied), Beta-프로피온아미도, 니트로페닐-에틸아민, 할로알킬 할라이드, 헥산 디아민, 6-아미노카프론산 및 글리코시드 결합으로 이루어진 군으로부터 선택되는 어느 하나일 수 있으나, 이에 한정되는 것은 아니다. 본 발명의 일 구현에 따르면, 폐렴구균 협막 다당은 링커, 예를 들어, 기능성 링커를 통해 운반체 단백질 또는 세포벽 유래 물질에 접합될 수 있다. 링커는 예를 들어, 반응성 아미노기 및 반응성 카르복실산기, 2개의 반응성 아미노기 또는 두 개의 반응성 카르복실산기를 지니는 이종이기능성 또는 동종이기능성 링커일 수 있다. 링커는 예를 들어, 4 내지 20, 4 내지 12, 5 내지 10 개의 탄소 원자를 지닐 수 있다.As the linker according to the present invention, any one selected from the group consisting of adipic acid dihyrazied (ADH), Beta-propionamido, nitrophenyl-ethylamine, haloalkyl halide, hexane diamine, 6-aminocaproic acid and a glycosidic bond It may be one, but is not limited thereto. According to one embodiment of the present invention, the pneumococcal capsular polysaccharide may be conjugated to a carrier protein or a cell wall-derived material through a linker, for example, a functional linker. The linker can be, for example, a heterobifunctional or homobifunctional linker having a reactive amino group and a reactive carboxylic acid group, two reactive amino groups or two reactive carboxylic acid groups. The linker may have, for example, 4 to 20, 4 to 12, 5 to 10 carbon atoms.
본 발명에 따른 면역원성 조성물에 있어서, 상기 세포벽 유래 물질은 스트렙토코커스 뉴모니애(Streptococcuspneumoniae)로부터 분리된 것일 수 있고, 스트렙토코커스 뉴모니애(Streptococcuspneumoniae)유래의 CWPS (Cell Wall Polysaccharide)일 수 있으나, 이에 한정되는 것은 아니다.In the immunogenic composition according to the present invention, the cell wall-derived material may be one isolated from Streptococcus pneumoniae, and Streptococcus pneumoniae may be CWPS (Cell Wall Polysaccharide) derived from, However, the present invention is not limited thereto.
본 발명에 따른 면역원성 조성물에 있어서, 상기 세포벽 유래 물질은 상기 협막 다당류와 공유결합으로 연결된 것일 수 있으며, 본 발명의 면역원성 조성물 내에 존재하는 모든 접합체는 임의의 공지된 접합 기술에 의해 제조될 수 있다. 접합 방법은 CDAP (1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)을 이용하여 협막 다당을 활성화시켜, 시아네이트 에스테르를 형성시키는 것일 수 있다. 또한, 본 발명에 따른 상기 세포벽 유래 물질은 다당류-단백질 접합체 내의 단백질과는 직접적으로 결합된 것이 아닐 수 있으며 이는 그 해당 구성성분 사이에 기타 성분이 포함될 수도 있고, 혹은 간접 연결을 위한 링커 등이 포함될 수도 있음을 의미하나, 이에 한정되는 것은 아니다. In the immunogenic composition according to the present invention, the cell wall-derived material may be covalently linked to the capsular polysaccharide, and all conjugates present in the immunogenic composition of the present invention may be prepared by any known conjugation technique. have. The conjugation method may be to activate the capsular polysaccharide using CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to form a cyanate ester. In addition, the cell wall-derived material according to the present invention may not be directly bound to the protein in the polysaccharide-protein conjugate, and other components may be included between the corresponding components, or a linker for indirect connection may be included. may mean, but is not limited thereto.
본 발명에 따른 면역원성 조성물에 있어서, 상기 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 세포벽 유래 물질의 접합체와 운반체 단백질은 하기 접합 순서 중 어느 하나로 접합될 수 있으나, 이에 한정되는 것은 아니다.In the immunogenic composition according to the present invention, the conjugate of the Streptococcus pneumoniae -derived capsular polysaccharide and the cell wall-derived material and the carrier protein may be conjugated in any one of the following conjugation sequences. It is not limited.
a) 협막 다당류-운반체 단백질-세포벽 유래 물질; a) capsular polysaccharide-carrier protein-cell wall-derived material;
b) 운반체 단백질--협막 다당류- 세포벽 유래 물질; 및 b) carrier protein--capsular polysaccharide-cell wall-derived material; and
c) 협막 다당류-세포벽 유래 물질-운반체 단백질.c) Capsular polysaccharide-cell wall-derived material-carrier protein.
본 발명에 따른 면역원성 조성물에 있어서, 상기 세포벽 유래 물질은 운반 단백질과 직접적으로 결합하지 않은 것일 수 있다.In the immunogenic composition according to the present invention, the cell wall-derived material may not be directly bound to a carrier protein.
상기 접합 순서 a), b) 또는 c)의 각 구성성분 간에 링커를 추가적으로 포함할 수 있고, 상기 접합 순서 b)로 접합된 구조는 선형(linear) 또는 비선형(Non-linear) 중 어느 하나일 수도 있으나, 이에 한정되는 것은 아니다.A linker may be additionally included between each component of the conjugation order a), b) or c), and the structure conjugated in the conjugation order b) may be either linear or non-linear. However, the present invention is not limited thereto.
본 발명의 일 구현예에 따른 협막 다당류 또는 세포벽 유래 물질은 직접적으로 또는 링커(간접방법) 등을 통해 운반체 단백질 상의 아미노기에 접합 될 수 있다. 임의로, 시아네이트 에스테르는 헥산 디아민 또는 ADH와 함께 접합되고, 아미노-유도된 협막 다당은 운반체 단백질 상의 카르복실기를 통해 카르보디이미드(예를 들어, EDAC 또는 EDC) 결합을 통해 운반체 단백질과 접합될 수 있다.The capsular polysaccharide or cell wall-derived material according to an embodiment of the present invention may be conjugated to an amino group on a carrier protein directly or through a linker (indirect method). Optionally, the cyanate ester is conjugated with hexane diamine or ADH, and the amino-derived capsular polysaccharide can be conjugated to the carrier protein via a carbodiimide (e.g., EDAC or EDC) linkage via a carboxyl group on the carrier protein. .
협막 다당 상의 히드록실기(임의로 활성화딘 히드록실기, 예를 들어, 시아네이트 에스테르를 제조하기 위해 활성화된 히드록실기는 단백질 상의 아미노기 또는 카르복실기에 직접적 또는 간접적(링커)으로 연결될 수 있다. 링커가 존재하는 경우, 임의로 당류 상의 히드록실기는 예를 들어, CDAP 접합을 이용하여 링커 상의 아미노기에 연결될 수 있다. 링커 내의 한 추가 아미노기 (예를 들어, ADH)는 카르보디이미드 방식을 이용하여 운반체 단백질 상의 카르복실산기에 접합 될 수 있다. 폐렴구균 협막 다당은 링커가 운반체 단백질에 접합되기 전에 먼저 링커에 접합이 이루어진다. A hydroxyl group on the capsular polysaccharide (optionally an activated hydroxyl group, for example, an activated hydroxyl group to produce a cyanate ester can be linked directly or indirectly (linker) to an amino or carboxyl group on the protein. If present, optionally the hydroxyl group on the saccharide can be connected to the amino group on the linker using, for example, CDAP conjugation.One additional amino group in the linker (eg ADH) can be connected to the carrier protein using the carbodiimide mode. It can be conjugated to the carboxylic acid group on the pneumococcal capsular polysaccharide is conjugated to the linker before the linker is conjugated to the carrier protein.
본 발명자들은 13가 혹은 20가 이상의 다가 백신 제작에 직접 접합 방법을 사용하기도 하였으나, 본 명세서 내에서는 세포벽 유래 물질의 영향을 비교하기 및 제작하기 위해 간접 접합 방식을 사용하였다. 간접 접합 방법에 사용된 링커는 일반적으로 사용되는 ADH (adipic acid dihyrazied)를 사용하였으며, 동일한 링커를 하여, 접합 순서와 세포벽 유래 물질의 종류를 달리 하여, 평가 하였다. 결과적으로 동일한 링커와 동일한 세포벽 유래 물질을 사용하였음에도 접합 순서에 따라 면역원성(항체가 및 기능적 항체가)에 차이를 보이는 것을 확인하였다.The present inventors also used a direct conjugation method for the production of a 13-valent or 20-valent or more multivalent vaccine, but in the present specification, an indirect conjugation method was used to compare and fabricate the effects of cell wall-derived substances. The linker used in the indirect conjugation method was a commonly used ADH (adipic acid dihyrazied), and the same linker was used, and the conjugation sequence and the type of cell wall-derived material were varied and evaluated. As a result, even though the same linker and the same cell wall-derived material were used, it was confirmed that the immunogenicity (antibody and functional antibody titer) differed according to the conjugation sequence.
본 발명에 따른 한 구현예에서, 협막다당을 CDAP (1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 사용하여 활성화 시킨 후, 링커를 접합하였다. (1차 물질) 그리고 CWPS(cell wall polysaccharide, 세포벽 유래 폴리사카라이드)를 CDAP으로 활성화 한 후 CRM197 운반체 단백질을 접합하였다. (2차 물질) 1차 물질과 2차 물질을 카르보디이미드 결합 방식을 사용하여 최종 접합하였다. (본 발명의 실시예 A에 해당함, 도 1) In one embodiment according to the present invention, the capsular polysaccharide was activated using CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate), and then a linker was conjugated. (Primary material) and CWPS (cell wall polysaccharide, cell wall-derived polysaccharide) were activated with CDAP and then the CRM 197 transporter protein was conjugated. (Secondary material) The primary material and the secondary material were finally bonded using a carbodiimide bonding method. (corresponding to embodiment A of the present invention, Fig. 1)
본 발명에 따른 한 구현예에서, 협막다당을 CDAP로 활성화 시킨 후, 운반체 단백질을 접합 시켰다. (1차 물질), 그리고 CWPS를 CDAP을 사용하여 활성화 시킨 후 링커를 접합시켰다. (2차 물질) 그 다음, 1차 물질과 2차 물질을 카르보디이미드 결합 방식을 사용하여 최종 접합하였다. (본 발명의 실시예 B에 해당함, 도 1)In one embodiment according to the present invention, after activating the capsular polysaccharide with CDAP, a carrier protein was conjugated. (Primary material), and CWPS was activated using CDAP, followed by conjugation of a linker. (Secondary material) Then, the primary material and the secondary material were finally bonded using a carbodiimide bonding method. (corresponding to embodiment B of the present invention, Fig. 1)
본 발명에 따른 한 구현예에서, CWPS을 CDAP으로 활성화시킨 후, 링커를 부착시키고, EDC 결합 방식을 사용하여 협막다당과 접합시켰다. 이후 운반체 단백질을 CDAP 활성화를 통해 아미노기를 생성하고, CWPS-링커-CPS 접합체와 운반체 단백질을 접합하였다. (본 발명의 실시예 C에 해당함, 도 1)In one embodiment according to the present invention, after CWPS was activated with CDAP, a linker was attached and it was conjugated with capsular polysaccharide using an EDC binding method. Thereafter, the carrier protein was activated by CDAP to generate an amino group, and the CWPS-linker-CPS conjugate was conjugated with the carrier protein. (corresponding to embodiment C of the present invention, Fig. 1)
본 발명에 따른 구현예에서, 상기 세포벽 유래 물질은 LTA (Lipoteichoic acid), PS (Polysaccharide) CPS (Capsular Polysaccharide), PG (Peptidoglycan), LPS (Lipopolysaccharide) MDP (Muramyl Dipeptide), MTP (Muramyl tripeptide) 및 PC (Phosphatidyl Choline)로 이루어지는 군에서 선택되는 어느 하나 이상을상기 방식으로 접합하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the embodiment according to the present invention, the cell wall-derived material is LTA (Lipoteichoic acid), PS (Polysaccharide) CPS (Capsular Polysaccharide), PG (Peptidoglycan), LPS (Lipopolysaccharide) MDP (Muramyl Dipeptide), MTP (Muramyl tripeptide) and Any one or more selected from the group consisting of PC (Phosphatidyl Choline) may be conjugated in the above manner, but is not limited thereto.
본 발명에 따른 면역원성 조성물에 있어서, 상기 협막 다당류와 세포벽 유래 물질의 접합체와 운반체 단백질 내 접합 방법은 CDAP 접합 방법, 리덕티브 아미네이션 방법 및 티올-말레마이드(Thiol-Malemide) 방법으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있으나, 이에 한정되는 것은 아니다.In the immunogenic composition according to the present invention, the method for conjugating the conjugate of the capsular polysaccharide with the cell wall-derived material and the carrier protein is selected from the group consisting of a CDAP conjugation method, a reductive amination method, and a Thiol-Malemide method. It may be any one or more selected, but is not limited thereto.
본 발명에 따른 면역원성 조성물에 있어서, 상기 접합 순서 a)는 하기 단계를 포함할 수 있다.In the immunogenic composition according to the present invention, the conjugation sequence a) may include the following steps.
i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 이를 활성화시키는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to activate it;
ii) 세포벽 유래 물질에 CDAP를 가하여, 운반체 단백질과 연결하는 단계; 및 ii) adding CDAP to the cell wall-derived material and linking with a carrier protein; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 운반체 단백질의 히드록실기 또는 아미노기와 연결하는 단계.iii) linking the capsular polysaccharide of i) with a hydroxyl group or an amino group of the carrier protein of ii).
본 발명에 따른 면역원성 조성물에 있어서, 상기 접합 순서 b)는 하기 단계를 포함하는 방법으로 접합될 수 있다.In the immunogenic composition according to the present invention, the conjugation sequence b) may be conjugated by a method comprising the following steps.
i) 세포벽 유래 물질에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 협막 다당류와 연결하는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the cell wall-derived material to link it with capsular polysaccharide;
ii) 운반체 단백질에 CDAP를 가하여 이를 활성화시키는 단계; 및 ii) adding CDAP to the carrier protein to activate it; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 운반체 단백질의 히드록실기 또는 아미노기와 연결하는 단계.iii) linking the capsular polysaccharide of i) with a hydroxyl group or an amino group of the carrier protein of ii).
또한, 본 발명에 따른 면역원성 조성물에 있어서, 상기 접합 순서 b) 가 하기 단계를 포함하는 방법으로 접합될 수 있다.In addition, in the immunogenic composition according to the present invention, the conjugation sequence b) may be conjugated by a method comprising the following steps.
i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 운반체 단백질과 연결하는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to link it with a carrier protein;
ii) 세포벽 유래 물질에 CDAP를 가하여 이를 활성화시키는 단계; 및 ii) adding CDAP to the cell wall-derived material to activate it; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 세포벽 유래 물질과 연결하는 단계.iii) linking the capsular polysaccharide of i) with the cell wall-derived material of ii).
아울러, 본 발명에 따른 면역원성 조성물에 있어서, 상기 접합 순서 c)는 하기 단계를 포함하는 방법으로 접합될 수 있다.In addition, in the immunogenic composition according to the present invention, the conjugation sequence c) may be conjugated by a method comprising the following steps.
i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 이를 활성화시키는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to activate it;
ii) 세포벽 유래 물질에 CDAP를 가하여, 운반체 단백질과 연결하는 단계; 및 ii) adding CDAP to the cell wall-derived material and linking with a carrier protein; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 세포벽 유래 물질과 연결하는 단계.iii) linking the capsular polysaccharide of i) with the cell wall-derived material of ii).
본 발명에 따른 면역원성 조성물은 구성성분으로서 애주번트를 추가로 포함할 수 있다. 본원에서 정의되는 '애주번트'는 본 발명의 면역원성 조성물의 면역원성을 증가시키는데 사용되는 물질이다. 따라서, 애주번트는 종종 면역 반응을 부스터하기 위하여 제공되고 당업자에게 잘 알려져 있다. 조성물의 유효성을 증가시키기에 적당한 애주번트는 다음을 포함하나, 이에 제한되는 것은 아니다:The immunogenic composition according to the present invention may further comprise an adjuvant as a component. An 'adjuvant' as defined herein is a substance used to increase the immunogenicity of the immunogenic composition of the present invention. Thus, adjuvants are often given to boost the immune response and are well known to those skilled in the art. Suitable adjuvants to increase the effectiveness of the composition include, but are not limited to:
본 발명에 따른 특정 구현예에서, 알루미늄 염이 애주번트로서 사용될 수있다. 상기 알루미늄 염은 알루미늄 포스페이트, 알루미늄 설페이트 및 알루미늄 하이드록사이드로 이루어진 군으로부터 선택될 수 있고, 그 형태로는 알루미늄-침전 백신(alum-precipitated vaccine)이거나 알루미늄-흡착 백신(alum-adsorbed vaccine)일 수도 있다. 알루미늄 염에는 수화된 알루미나, 알루미나 수화물, 알루미나 3수화물(ATH) 등이 포함되지만, 이에 제한되는 것은 아니다. 염화 알루미늄과 인산나트륨을 1:1의 비율로 혼합하면 알루미늄 히드록시포스페이트 설페이트가 침전된다. 믹서(High shear mixer)를 이용하여 침전물의 크기가 2~8㎛이 되도록 한 다음 생리식염수로 투석하고 멸균하여 제조할 수 있다. 본 발명에 따른 특정 일 구현예에서, 상업적으로 이용가능한 Al(OH)3(예를 들어 알하이드로겔 또는 Superfos)를 사용하여 단백질을 흡착할 수 있다. 수산화알루미늄 1 mg당 50~200g의 단백질이 흡착될 수 있으며 이 비율은 단백질의 pI와 용매의 pH에 의존적이다. 낮은 pI의 단백질은 높은 pI를 가진 단백질에 비해 강하게 결합할 수 있다. 알루미늄 염은 2~3주간 서서히 항원을 방출하는 항원 저장소를 형성하여 비특이적으로 대식세포, 보체, 선천성 면역 메커니즘을 활성화시킨다.In certain embodiments according to the invention, aluminum salts may be used as adjuvants. The aluminum salt may be selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide, and may be an aluminum-precipitated vaccine or an aluminum-adsorbed vaccine in its form. have. Aluminum salts include, but are not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), and the like. When aluminum chloride and sodium phosphate are mixed in a ratio of 1:1, aluminum hydroxyphosphate sulfate is precipitated. It can be prepared by dialyzing with physiological saline and sterilizing the precipitate so that the size of the precipitate becomes 2-8 μm using a high shear mixer. In a specific embodiment according to the present invention, commercially available Al(OH)3 (eg Alhydrogel or Superfos) can be used to adsorb proteins. 50-200 g of protein can be adsorbed per 1 mg of aluminum hydroxide, and this ratio is dependent on the pI of the protein and the pH of the solvent. A protein with a low pI can bind strongly compared to a protein with a high pI. Aluminum salt forms an antigen reservoir that releases antigens slowly for 2-3 weeks, non-specifically activating macrophages, complement, and innate immune mechanisms.
본 발명에 따른 면역원성 조성물에 있어서, 상기 면역원성 조성물은 0.1 내지 100 ㎍ 용량의 다당류를 포함할 수 있으나, 이에 한정되는 것은 아니다.In the immunogenic composition according to the present invention, the immunogenic composition may contain a polysaccharide in an amount of 0.1 to 100 μg, but is not limited thereto.
또한, 본 발명은 상기 면역원성 조성물의 면역학적 유효량을 포함하는, 스트렙토코커스 뉴모니애 협막 다당류 접합체에 대한 면역 반응을 유도하기 위한 약학 조성물(예를 들어, 백신 제제)을 제공한다. The present invention also provides a pharmaceutical composition (eg, a vaccine formulation) for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the immunogenic composition.
본 발명에 따른 백신 제제는, 전신 또는 점막 경로로 투여함으로써, 폐렴구균에 감염되기 쉬운 사람을 보호 또는 치료하는데 사용될 수 있다. 본원에서 정의되는 '유효량'이란 폐렴구균에 감염될 확률 또는 감염의 심각성을 상당히 감소시킬 수 있을 정도의 항체를 유발하는데 필요한 투여량을 말한다. 투여에는 근육내, 복강내, 피내 또는 피하 경로를 통한 주사; 또는 구강/소화관, 기도관 또는 비뇨생식관으로의 점막 투여가 포함될 수 있다. 일 구현예에서, 폐렴 또는 중이염의 치료를 위하여 비내 투여가 사용되며, 이는 폐렴구균의 비인두 보균을 보다 효과적으로 예방하여, 초기 단계에서 감염을 약화시킬 수 있기 때문이다. The vaccine formulation according to the present invention can be used to protect or treat a person susceptible to pneumococcal infection by administration by a systemic or mucosal route. As defined herein, an 'effective amount' refers to a dose required to elicit an antibody sufficient to significantly reduce the probability or severity of pneumococcal infection. Administration includes injection via the intramuscular, intraperitoneal, intradermal or subcutaneous route; or mucosal administration to the oral/digestive tract, airway tract, or genitourinary tract. In one embodiment, intranasal administration is used for the treatment of pneumonia or otitis media, since it can more effectively prevent nasopharyngeal carriers of pneumococci, thereby attenuating infection at an early stage.
각 백신 용량에서 상기 접합체의 양은, 상당한 부작용 없이 면역보호 반응을 유도하는 양으로 선택된다. 이러한 양은 폐렴구균의 혈청형에 따라 달라질 수 있다. 일반적으로, 각 용량은 0.1 내지 100 ㎍, 바람직하게는 0.1 내지 10 ㎍, 더욱 바람직하게는 1 내지 5㎍의 다당류를 포함할 수 있으나, 이에 한정되는 것은 아니다. 특정 백신에 대한 성분의 최적량은 피험자에서 적당한 면역 반응의 관찰을 포함하는 표준 연구에 의해서 확인될 수 있다. 예를 들어 동물실험 결과를 외삽하여 사람을 대상으로 한 백신접종 용량을 결정할 수 있다. 또한 경험적으로 용량을 결정할 수 있다.The amount of the conjugate in each vaccine dose is selected such that it induces an immunoprotective response without significant side effects. This amount may vary depending on the serotype of the pneumococci. In general, each dose may contain 0.1 to 100 μg, preferably 0.1 to 10 μg, more preferably 1 to 5 μg of polysaccharide, but is not limited thereto. Optimal amounts of components for a particular vaccine can be ascertained by standard studies involving observation of an adequate immune response in subjects. For example, extrapolation of animal test results can be used to determine vaccination doses in humans. Dosage can also be determined empirically.
본 발명의 일 구현예에서, 상기 약학 조성물은 2 ㎍의 각 당류, 협막다당 혈청형 4 ㎍; 약 34 ㎍의 CRM197 운반체 단백질; 세포외 유래 물질 2 ㎍; 0.125 mg의 알루미늄 원소(0.5 mg의 알루미늄 포스페이트) 애주번트; 및 부형제로서 염화나트륨 및 나트륨 석시네이트 완충액을 함유하도록 제제화된 면역원성 조성물일 수 있다. 상기 조성물은 보존제 없이 단일 용량 주사기 속에 충전될 수 있다. 진탕하고 나면, 즉시 근육 내 투여할 수 있는 균질한 백색 현탁액의 백신이 된다.In one embodiment of the present invention, the pharmaceutical composition comprises 2 μg of each saccharide, 4 μg of capsular polysaccharide serotype; about 34 μg of CRM 197 transporter protein; 2 μg of extracellular material; 0.125 mg of elemental aluminum (0.5 mg of aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffer as excipients. The composition may be filled into single dose syringes without preservatives. After shaking, the vaccine is a homogeneous white suspension that can be administered immediately intramuscularly.
본 발명의 조성물은 단회 투여 용량 바이알, 다회 투여 용량 바이알 또는 프리필드시린지 형태로 제제화할 수 있다. The composition of the present invention may be formulated in the form of a single dose dose vial, a multiple dose dose vial or a prefilled syringe.
본 발명의 제제(Formulation)은 계면활성제를 포함할 수 있으며, Tween 80 또는 Span 85와 같은 계면활성제의 혼합물을 포함할 수 있다.The formulation of the present invention may include a surfactant, and may include a mixture of surfactants such as Tween 80 or Span 85.
아울러, 본 발명은 상기의 면역원성 조성물을 제조하는 방법을 제공한다.In addition, the present invention provides a method for preparing the immunogenic composition.
본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 세포벽 유래 물질의 접합체와 운반체 단백질은 하기 접합 순서 중 어느 하나로 접합될 수 있다.In the method for preparing the immunogenic composition according to the present invention, the conjugate of the Streptococcus pneumoniae -derived capsular polysaccharide and the cell wall-derived material and the carrier protein may be conjugated in any one of the following conjugation sequences. can
a) 협막 다당류-운반체 단백질-세포벽 유래 물질; a) capsular polysaccharide-carrier protein-cell wall-derived material;
b) 운반체 단백질--협막 다당류- 세포벽 유래 물질; 및 b) carrier protein--capsular polysaccharide-cell wall-derived material; and
c) 협막 다당류-세포벽 유래 물질-운반체 단백질.c) Capsular polysaccharide-cell wall-derived material-carrier protein.
본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 접합 순서 a), b), 또는 c)의 각 구성성분 간에 링커를 추가적으로 포함할 수 있다.In the method for preparing the immunogenic composition according to the present invention, a linker may be additionally included between each component of the conjugation sequence a), b), or c).
본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 협막 다당류와 세포벽 유래 물질의 접합체와 운반체 단백질 내 접합 방법은 CDAP 접합 방법, 리덕티브 아미네이션 방법 및 티올-말레마이드(Thiol-Malemide) 방법으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있으나, 이에 한정되는 것은 아니다.In the method for preparing the immunogenic composition according to the present invention, the method of conjugation between the capsular polysaccharide and the cell wall-derived material and the carrier protein is a CDAP conjugation method, a reductive amination method, and a Thiol-Malemide method. It may be any one or more selected from the group consisting of, but is not limited thereto.
본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 접합 순서 a) 는 하기 단계를 포함하는 방법으로 접합될 수 있으나, 이에 한정되는 것은 아니다. In the method for preparing the immunogenic composition according to the present invention, the conjugation sequence a) may be conjugated by a method comprising the following steps, but is not limited thereto.
i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 이를 활성화시키는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to activate it;
ii) 세포벽 유래 물질에 CDAP를 가하여, 운반체 단백질과 연결하는 단계; 및 ii) adding CDAP to the cell wall-derived material and linking with a carrier protein; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 운반체 단백질의 히드록실기 또는 아미노기와 연결하는 단계.iii) linking the capsular polysaccharide of i) with a hydroxyl group or an amino group of the carrier protein of ii).
본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 접합 순서 b) 는 하기 단계를 포함하는 방법으로 접합될 수 있으나, 이에 한정되는 것은 아니다.In the method for preparing the immunogenic composition according to the present invention, the conjugation sequence b) may be conjugated by a method comprising the following steps, but is not limited thereto.
i) 세포벽 유래 물질에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 협막 다당류와 연결하는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the cell wall-derived material to link it with capsular polysaccharide;
ii) 운반체 단백질에 CDAP를 가하여 이를 활성화시키는 단계; 및 ii) adding CDAP to the carrier protein to activate it; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 운반체 단백질의 히드록실기 또는 아미노기와 연결하는 단계.iii) linking the capsular polysaccharide of i) with a hydroxyl group or an amino group of the carrier protein of ii).
또한, 본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 접합 순서 b) 가 하기 단계를 포함하는 방법으로도 접합될 수 있다.In addition, in the method for preparing the immunogenic composition according to the present invention, the conjugation sequence b) may be conjugated by a method comprising the following steps.
i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 운반체 단백질과 연결하는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to link it with a carrier protein;
ii) 세포벽 유래 물질에 CDAP를 가하여 이를 활성화시키는 단계; 및 ii) adding CDAP to the cell wall-derived material to activate it; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 세포벽 유래 물질과 연결하는 단계.iii) linking the capsular polysaccharide of i) with the cell wall-derived material of ii).
아울러, 본 발명에 따른 면역원성 조성물을 제조하는 방법에 있어서, 상기 접합 순서 c)는 하기 단계를 포함하는 방법으로 접합될 수 있다.In addition, in the method for preparing the immunogenic composition according to the present invention, the conjugation sequence c) may be conjugated by a method comprising the following steps.
i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 이를 활성화시키는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to activate it;
ii) 세포벽 유래 물질에 CDAP를 가하여, 운반체 단백질과 연결하는 단계; 및 ii) adding CDAP to the cell wall-derived material and linking with a carrier protein; and
iii) 상기 i)의 협막 다당류를 상기 ii)의 세포벽 유래 물질과 연결하는 단계.iii) linking the capsular polysaccharide of i) with the cell wall-derived material of ii).
본 발명에서 인용된 문헌 및 본 출원인이 기출원한 한국특허출원 제 10-2020-0089019호, PCT국제출원 PCT/KR2020/009474는 본 발명의 명세서에 참조로서 통합된다.Documents cited in the present invention, Korean Patent Application No. 10-2020-0089019 previously filed by the present applicant, and PCT International Application PCT/KR2020/009474 are incorporated herein by reference.
본원에 기재된 상기 각 특징들은 조합되어 사용될 수 있으며, 상기 각 특징들이 특허청구범위의 서로 다른 종속항에 기재된다는 사실은 이들이 조합되어 사용될 수 없음을 나타내는 것은 아니다. Each of the features described herein may be used in combination, and the fact that each of the features is recited in different dependent claims of the claims does not indicate that they cannot be used in combination.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명한다. 그러나, 하기 실시에는 본 발명의 설명을 위한 것이며, 이들 실시예에 의해 본 발명이 제한되는 것으로 해석되어서는 안 된다.Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for the purpose of illustrating the present invention, and should not be construed as limiting the present invention by these examples.
실시예 1. 폐렴구균 협막 다당류의 제조 및 정제Example 1. Preparation and purification of pneumococcal capsular polysaccharide
폐렴구균 배양과 협막 다당류의 정제는 당업자에게 공지된 방법에 의해서 수행하였다. 폐렴 구균 각 혈청형은 수탁기관(ATCC Ameircan Type Culture Collection,, US)으로부터 입수할 수 있다. 협막과 비운동성, 그람 양성, Lancet-shaped 쌍구균, 혈액한천배지에서 알파용혈현상으로 폐렴 구균을 동정하였다. 혈청형은 특정한 항혈청을 이용한 Quellung Test를 바탕으로 확인하였다.Pneumococcal culture and purification of capsular polysaccharide were performed by methods known to those skilled in the art. Each of the pneumococcal serotypes can be obtained from the ATCC Ameircan Type Culture Collection, US. Pneumococci were identified by capsular, nonmotile, Gram-positive, lancet-shaped dicocci, and alpha hemolysis in blood agar media. The serotype was confirmed based on the Quellung Test using a specific antisera.
실시예 1-1. 세포 은행 제조Example 1-1. cell bank manufacturing
19 종의 각기 다른 혈청형(1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F 및 35B)을 가진 폐렴구균을 수탁기관인 미국 ATCC(Ameircan Type Culture Collection,, US)로부터 입수하였다.Pneumococci with 19 different serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 35B) was obtained from the US ATCC (Ameircan Type Culture Collection, US), which is a consignment institution.
폐렴구균 균주를 혈액 한천배지에 도말하여 단일 콜로니를 분리하였다. 10개 이상의 단일 콜로니 중 성장이 좋은 단일 콜로니를 선정한 후 동물 유래의 성분을 포함하지 않은 액상배지에 접종하여 배양한 후 합성 글리세롤을 함유한 연구용 세포 은행(Research Cell Bank, RCB)을 제조하였다A single colony was isolated by spreading the pneumococcal strain on a blood agar medium. After selecting a single colony with good growth among more than 10 single colonies, inoculating it in a liquid medium that does not contain animal-derived components and culturing, a Research Cell Bank (RCB) containing synthetic glycerol was prepared.
고유의 혈청형을 가진 다당류 발현이 확인 된 연구용 세포 은행 중 바이알 1 개를 꺼내어 동물 유래의 성분을 포함하지 않은 액상배지에 세포를 증식시킨 후 합성 글리세롤을 추가하여 마스터 세포은행을 제조하였으며, 마스터 세포 은행 중 바이알 1 개를 꺼내어 동물 유래의 성분들을 포함하지 않은 액상배지에 세포를 증식시킨 후 합성 글리세롤을 추가하여 제조용 세포 은행을 제조하였다. 제조된 세포 은행은 다음 단계에서 사용하기 위해 -70℃ 이하에서 보관하여 사용하였다.A master cell bank was prepared by adding synthetic glycerol after taking out one vial from the cell bank for research in which the expression of polysaccharides with a unique serotype was confirmed, proliferating the cells in a liquid medium containing no animal-derived components, and adding synthetic glycerol. One vial was taken out of the bank, and the cells were grown in a liquid medium not containing animal-derived components, and then synthetic glycerol was added to prepare a manufacturing cell bank. The prepared cell bank was stored at -70°C or lower for use in the next step.
실시예 1-2. 발효 및 다당류 분리Example 1-2. Fermentation and polysaccharide separation
제조용 세포 은행 중 바이알 1 개를 해동하여 동물 유래의 성분을 포함하지 않은 액상배지에 접종하여 종 발효를 시작하였다. 일정 균체 농도(Optical Density, OD600)에 도달하여 중간-지수 증식기의 종말점에 도달할 때까지 무교반 상태로 37 ± 2℃에서 종배양을 실시 하였다. 종배양에서 얻은 배양액을 동물 유래의 성분을 포함하지 않은 액상배지를 함유하는 발효기에 접종하여 주 발효(Main Fermentation)를 시작하였다.One vial of the cell bank for production was thawed and inoculated into a liquid medium containing no animal-derived components to start the species fermentation. Seed culture was performed at 37 ± 2 °C without agitation until a certain cell concentration (Optical Density, OD600) was reached and the endpoint of the intermediate-exponential growth phase was reached. The culture medium obtained from the seed culture was inoculated into a fermenter containing a liquid medium containing no animal-derived components to start the main fermentation (Main Fermentation).
그 다음 37 ± 2℃에서 수산화 칼륨 용액으로 배지의 pH를 조절하면서 본 배양을 실시하였다. 2 시간 마다 최적의 세포 밀도와 배지에 포함된 글루코스 농도를 측정하였다. 발효는 배지 내 글루코스가 고갈되었을 때 종료하였다.Then, the main culture was carried out while adjusting the pH of the medium with a potassium hydroxide solution at 37 ± 2 °C. The optimal cell density and glucose concentration in the medium were measured every 2 hours. Fermentation was terminated when the glucose in the medium was depleted.
발효가 종료된 후, 12 % 소듐 데옥시콜레이트(Sodium Deoxycholate)를 최종 0.12 %가 되도록 배양물에 1시간동안 첨가하여 세포를 용해시키고 세포에 결합된 다당류를 유리시켰다.After the fermentation was completed, 12% sodium deoxycholate was added to the culture to a final 0.12% for 1 hour to lyse the cells and release the polysaccharide bound to the cells.
실시예 1-3. 협막 다당의 정제Examples 1-3. Purification of capsular polysaccharide
소듐 데옥시콜레이트가 처리된 샘플에 인산을 가한 뒤, 원심 분리를 통해 상층액을 회수하였다. 회수한 상층액을 Depth Filter에 통과시킨 다음, 농축 및 인산 완충용액으로 버퍼 교환을 진행하였다. 버퍼 교환 후, 샘플을 탄소활성탄 필터(Active Carbon Filter)에 통과시킨 다음, 하기와 같은 두 가지 방법으로 불순물 제거를 진행하였다.After phosphoric acid was added to the sodium deoxycholate-treated sample, the supernatant was recovered by centrifugation. The recovered supernatant was passed through a depth filter, and then the buffer was exchanged with concentration and phosphate buffer solution. After the buffer exchange, the sample was passed through an active carbon filter, and impurities were removed by the following two methods.
17 개의 혈청형 1, 3, 4, 5, 6A, 6B, 9V, 10A, 11A, 15B, 18C, 19A, 19F, 22F, 23A, 23F 및 35B의 경우, CTAB(Cetyltrimethylammonium Bromide)와 이온 결합이 가능하므로 CTAB 공정을 진행하였다. CTAB 처리, 원심 분리, 염화나트륨(NaCl) 및 요오드화 나트륨(NaI) 처리, 및 원심 분리 과정을 수행하였다.17 serotypes 1, 3, 4, 5, 6A, 6B, 9V, 10A, 11A, 15B, 18C, 19A, 19F, 22F, 23A, 23F, and 35B are capable of ionic binding with Cetyltrimethylammonium Bromide (CTAB) Therefore, the CTAB process was performed. CTAB treatment, centrifugation, sodium chloride (NaCl) and sodium iodide (NaI) treatment, and centrifugation were performed.
CTAB와 반응하지 않는 2 개의 혈청형 7F 및 14는 인산 알루미늄겔(Algel) 용액을 첨가하여 반응시킨 다음, 원심 분리를 통해 얻어진 상층액을 사용하였다.The two serotypes 7F and 14 that do not react with CTAB were reacted by adding an aluminum phosphate solution (Algel), and then the supernatant obtained by centrifugation was used.
상기 두 가지 형태의 불순물 제거 공정을 완료한 샘플은 Depth Filter 및 한외여과(UF/DF) 공정을 거친 다음, 에탄올과 염화나트륨의 양을 조절하며 원말 형태로 만들어 보관하였다.After completing the two types of impurity removal process, the sample was subjected to depth filter and ultrafiltration (UF/DF) process, and then the amounts of ethanol and sodium chloride were adjusted and stored in raw form.
실시예 1-4. 협막 다당류의 용해 및 가수분해Examples 1-4. Dissolution and hydrolysis of capsular polysaccharides
각각의 혈청형에서 유래한 협막 다당류 원말을 최종 농도 범위가 아래 기술된 범위가 되도록 주사용수에 용해하고 0.45 ㎛ 필터를 통해 여과시켰다.Capsular polysaccharide raw material derived from each serotype was dissolved in water for injection so that the final concentration range was within the range described below and filtered through a 0.45 μm filter.
세부적으로 혈청형 1, 3 및 4의 경우 0.8 - 2.0mg/㎖의 범위로 용해하였고, 혈청형 5, 6B, 9V, 18C 및 19F 의 경우 4 - 8mg/㎖, 혈청형 6A 및 19A는 8 - 12mg/㎖, 혈청형 7F, 10A, 11A 및 23F의 경우 2 - 4mg/㎖로 용해하여 여과를 진행하였다. 또한, 혈청형 15B, 22F 및 35B의 경우 2 - 5mg/㎖의 범위로 용해하여 여과를 진행하였다.Specifically, serotypes 1, 3 and 4 were dissolved in the range of 0.8 - 2.0 mg/ml, serotypes 5, 6B, 9V, 18C and 19F were 4 - 8 mg/ml, serotypes 6A and 19A were 8 - In the case of 12 mg/ml, serotypes 7F, 10A, 11A and 23F, it was dissolved at 2 - 4 mg/ml and filtered. In addition, serotypes 15B, 22F and 35B were dissolved in the range of 2 - 5 mg/ml and filtration was performed.
혈청형 별로 아래 기술된 pH 및 온도 범위에서 용액을 항온 처리하여 진행하였다. 세부적으로 혈청형 1, 3, 5, 6B, 7F, 10A, 11A, 14 및 23F의 경우 밤새 70 - 80℃, 혈청형 6A 및 19F 경우 1 - 4시간 동안 70 - 80℃, 혈청형 9V 및 18C의 경우, 인산용액을 이용하여 1 - 3 시간 동안 pH 2.0, 65 - 80℃ 에서 항온처리 과정을 수행하였다. 혈청형 22F, 23A 및 35B의 경우 밤새 75 - 85℃, 혈청형 4, 15B, 19A의 경우 가수분해를 진행하지 않았다. 그 다음 21 내지 24℃로 냉각시키고 6.0 ± 1.0의 목표 pH로 수산화 나트륨을 첨가함으로써 가수분해를 중지시켰다.The solution was incubated at the pH and temperature ranges described below for each serotype. Specifically for serotypes 1, 3, 5, 6B, 7F, 10A, 11A, 14 and 23F 70 - 80 °C overnight, for serotypes 6A and 19F 70 - 80 °C for 1-4 hours, serotypes 9V and 18C In the case of , the incubation process was performed at pH 2.0, 65 - 80 °C for 1 - 3 hours using a phosphoric acid solution. For serotypes 22F, 23A and 35B, 75-85° C. overnight, and for serotypes 4, 15B and 19A, no hydrolysis was performed. Hydrolysis was then stopped by cooling to 21-24° C. and adding sodium hydroxide to a target pH of 6.0±1.0.
실시예 2. 폐렴구균 협막 다당류와 CRM197 단백질 운반체의 접합 및 정제Example 2. Conjugation and purification of pneumococcal capsular polysaccharide and CRM197 protein transporter
실시예 2-1. CRM197 단백질 운반체의 준비Example 2-1. Preparation of CRM197 protein transporter
CRM197 단백질 운반체는 통상적인 생산 공정을 통해 준비하였다. 코리네박테리움 디프테리아 (ATCC 39255) 의 동결건조 시들 종균배지가 들어 있는 튜브에 접종한 후 36℃에서 48시간 배양 하였다. 배양 후 표면에 자란 디프테리아 균체를 그램 염샘하여 오염여부를 확인하고, 본 배양 배을 수행하였다. 배양액을 여과한 후, TFF system을 이용하여 1차 정제를 하였고, 황산암모늄을 이용하여 침전시키는 과정을 반복하였다. 이 후, Capto Q (Anion exchange chromatography)를 통해 불순물을 정제하였고, 제형 조성을 포함한 버퍼를 이용하여 final TFF를 수행하여 CRM197 단백질 운반체를 준비하였다. CRM 197 protein transporter was prepared through a conventional production process. After inoculation into a tube containing a freeze-dried and withered seed culture medium of Corynebacterium diphtheria (ATCC 39255), it was incubated at 36° C. for 48 hours. After culturing, diphtheria cells grown on the surface were gram salted to check for contamination, and the main culture was performed. After filtration of the culture medium, primary purification was performed using the TFF system, and the process of precipitation using ammonium sulfate was repeated. Thereafter, impurities were purified through Capto Q (Anion exchange chromatography), and final TFF was performed using a buffer including the formulation composition to prepare a CRM 197 protein carrier.
실시예 2-2. 협막 다당류와 CRM197의 직접 접합Example 2-2. Direct Conjugation of Capsular Polysaccharide with CRM197
모든 혈청형에 염화나트륨 분말을 첨가하여 2 M NaCl 다당류 용액을 제조하였다. 각 혈청 별로 적절한 CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate)를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 세부적으로 혈청형 6A 및 14의 경우 다당 대비 CDAP 1 w/w%를, 혈청형 4의 경우 2 w/w%를, 혈청형 1, 3, 6B, 7F 15B 및 19A의 경우 3 w/w%를, 그 외 혈청형의 경우 4 w/w%의 무게비율로 용해하고 각각의 다당류 용액에 첨가하였다. 이어, 1 내지 3 분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내지 9.7로 상승시킨 후 다당류의 하이드록실기가 CDAP에 의해 충분히 활성화될 수 있도록 3 내지 7 분 동안 교반하였다. 다당 대비 CRM197 0.5 - 1.0 w/w%를 각 혈청형 다당 용액에 첨가하여 1 시간 내지 4 시간 동안 접합반응을 진행하였으며 HPLC-SEC을 이용하여 반응 전환율을 측정하였고 필요에 따라 CDAP를 추가 투입하였다.A 2 M NaCl polysaccharide solution was prepared by adding sodium chloride powder to all serotypes. For each serum, appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. Specifically, for serotypes 6A and 14, 1 w/w% of CDAP relative to polysaccharide, for serotype 4, 2 w/w%, and for serotypes 1, 3, 6B, 7F 15B and 19A, 3 w/w% was dissolved at a weight ratio of 4 w/w% for other serotypes and added to each polysaccharide solution. Then, after 1 to 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, followed by stirring for 3 to 7 minutes so that the hydroxyl groups of the polysaccharide were sufficiently activated by CDAP. 0.5 - 1.0 w/w% of CRM197 compared to polysaccharide was added to each serotype polysaccharide solution, and the conjugation reaction was carried out for 1 to 4 hours. The reaction conversion rate was measured using HPLC-SEC. .
실시예 2-3. 접합 반응 종료Example 2-3. End of conjugation reaction
모든 혈청형에 대해 첨가한 CDAP 1 몰 당량 대비 3 내지 6 몰 당량의 글리신 용액을 첨가하고 pH를 9.0으로 조정하여 반응을 종결하였다. 접합 용액을 21 내지 24℃에서 1시간 교반한 후 2 내지 8℃ 저온에서 밤새 보관하였다.The reaction was terminated by adding 3 to 6 molar equivalents of a glycine solution to 1 molar equivalent of CDAP added for all serotypes and adjusting the pH to 9.0. The conjugation solution was stirred at 21 to 24° C. for 1 hour and then stored at a low temperature of 2 to 8° C. overnight.
실시예 2-4. 한외 여과Example 2-4. ultrafiltration
희석된 접합 혼합물을 최소 20 용적의 완충액을 사용하여 한외여과필터에 농축 및 투석 여과시켰다. 여기서 완충액은 pH 5.5 내지 6.5의 범위를 유지하며, 0.9 % 염화나트륨을 포함한 완충액을 사용하였다. 한외여과필터의 분획 분자량은 모든 혈청형에서 300 kDa을 사용하여 실시하였고, 투과액은 폐기하였다.The diluted conjugation mixture was concentrated and diafiltered through an ultrafiltration filter using at least 20 volumes of buffer. Here, the buffer was maintained in a pH range of 5.5 to 6.5, and a buffer containing 0.9% sodium chloride was used. The molecular weight cutoff of the ultrafiltration filter was performed using 300 kDa for all serotypes, and the permeate was discarded.
실시예 2-5. 제균 여과Example 2-5. sterilization filtration
투석 여과 후의 잔류액을 완충액을 이용하여 다당함량 농도 기준으로 0.4 g/L 미만이 되도록 희석하여 0.22 ㎛ 필터를 통해서 여과시켰다. 여과된 산물에 대해 제조과정 중 제어 (당류 함량, 잔류 DMAP)를 실시하였다. 여과시킨 잔류액에 대해 제조과정 중 제어를 실시하여 추가적인 농축, 투석 여과 및/또는 희석이 필요한지의 여부를 결정하였다.The residual solution after diafiltration was diluted to less than 0.4 g/L based on the polysaccharide content concentration using a buffer, and filtered through a 0.22 μm filter. The filtered product was subjected to in-process control (sugar content, residual DMAP). In-process controls were applied to the filtered retentate to determine whether further concentration, diafiltration and/or dilution was required.
실시예 3. 폐렴구균 협막 다당류, CRM197 운반체 단백질 및 세포벽 유래 물질의 접합 및 정제Example 3. Conjugation and purification of pneumococcal capsular polysaccharide, CRM197 transport protein and cell wall-derived material
실시예 3-1. 세포벽 유래 물질Example 3-1. cell wall-derived substances
Cell wall polysaccharide(CWPS, cat No. 3549)와 Lipo-teichoic acid (LTA, F-antigen, Cat No. 88840)는 SSI Diagnostica 업체로부터 구입 하였다. Cell wall polysaccharide (CWPS, cat No. 3549) and lipo-teichoic acid (LTA, F-antigen, Cat No. 88840) were purchased from SSI Diagnostica.
실시예 3-2. 협막 다당류와 CRM197의 접합Example 3-2. Conjugation of capsular polysaccharide to CRM197
모든 혈청형에 염화나트륨 분말을 첨가하여 2M NaCl 다당류 용액을 제조하였다. 각 혈청 별로 적절한 CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate)를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 세부적으로 혈청형 6A 및 14의 경우 다당 대비 CDAP 1 w/w%를, 혈청형 4의 경우 2 w/w%를, 혈청형 1, 3, 6B, 7F 15B 및 19A의 경우 3 w/w%를, 그 외 혈청형의 경우 4 w/w%의 무게비율로 용해하고 각각의 다당류 용액에 첨가하였다. 이어, 1 내지 3 분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내지 9.7로 상승시킨 후 다당류의 하이드록실기가 CDAP에 의해 충분히 활성화될 수 있도록 3 내지 7 분 동안 교반하였다. 다당 대비 CRM197 0.5 - 1.0 w/w%를 각 혈청형 다당 용액에 첨가하여 1 시간 내지 4 시간 동안 접합반응을 진행하였으며 HPLC-SEC을 이용하여 반응 전환율을 측정하였고 필요에 따라 CDAP를 추가 투입하였다.A 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to all serotypes. For each serum, appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. Specifically, for serotypes 6A and 14, 1 w/w% of CDAP relative to polysaccharide, for serotype 4, 2 w/w%, and for serotypes 1, 3, 6B, 7F 15B and 19A, 3 w/w% was dissolved at a weight ratio of 4 w/w% for other serotypes and added to each polysaccharide solution. Then, after 1 to 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, followed by stirring for 3 to 7 minutes so that the hydroxyl groups of the polysaccharide were sufficiently activated by CDAP. 0.5 - 1.0 w/w% of CRM197 compared to polysaccharide was added to each serotype polysaccharide solution, and the conjugation reaction was carried out for 1 to 4 hours. The reaction conversion rate was measured using HPLC-SEC. .
실시예 3-3. 세포벽 유래 물질의 접합Example 3-3. Conjugation of cell wall-derived substances
모든 혈청형에 염화나트륨 분말을 첨가하여 2M NaCl 다당류 용액을 제조하였다. 각 혈청 별로 적절한 CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate)를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 세부적으로 혈청형 6A 및 14의 경우 다당 대비 CDAP 1 w/w%를, 혈청형 4의 경우 2 w/w%를, 혈청형 1, 3, 6B, 7F 15B 및 19A의 경우 3 w/w%를, 그 외 혈청형의 경우 4 w/w%의 무게비율로 용해하고 각각의 다당류 용액에 첨가하였다. 이어, 1 내지 3 분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내지 9.7로 상승시킨 후 다당류의 하이드록실기가 CDAP에 의해 충분히 활성화될 수 있도록 3 내지 7 분 동안 교반하였다. 다당 대비 CRM197 0.5 - 1.0 w/w%를 각 혈청형 다당 용액에 첨가하여 1 시간 내지 4 시간 동안 접합반응을 진행하였으며 HPLC-SEC을 이용하여 반응 전환율을 측정하였고 필요에 따라 CDAP를 추가 투입하였다.A 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to all serotypes. For each serum, appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. Specifically, for serotypes 6A and 14, 1 w/w% of CDAP relative to polysaccharide, for serotype 4, 2 w/w%, and for serotypes 1, 3, 6B, 7F 15B and 19A, 3 w/w% was dissolved at a weight ratio of 4 w/w% for other serotypes and added to each polysaccharide solution. Then, after 1 to 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, followed by stirring for 3 to 7 minutes so that the hydroxyl groups of the polysaccharide were sufficiently activated by CDAP. 0.5 - 1.0 w/w% of CRM197 compared to polysaccharide was added to each serotype polysaccharide solution, and the conjugation reaction was carried out for 1 to 4 hours. The reaction conversion rate was measured using HPLC-SEC. .
실시예 3-4. 접합 종료 및 여과Example 3-4. junction termination and filtration
접합체의 접합 반응 종료 여과는 상기 직접 접합을 하는 방식과 동일하게 수행하였다. Filtration at the end of the conjugation reaction of the conjugate was performed in the same manner as in the direct conjugation.
실시예 4. 폐렴구균-운반체 단백질 접합체 및 신규한 세포벽 유래 물질 포함 접합체의 특성Example 4. Characterization of pneumococcal-carrier protein conjugates and conjugates containing novel cell wall-derived materials
실시예 4-1. 세포벽 유래 물질Example 4-1. cell wall-derived substances
Cell wall polysaccharide(CWPS, cat No. 3549)와 Lipo-teichoic acid (LTA, F-antigen, Cat No. 88840)는 SSI Diagnostica 로부터 구입을 하였다. Cell wall polysaccharide (CWPS, cat No. 3549) and lipo-teichoic acid (LTA, F-antigen, Cat No. 88840) were purchased from SSI Diagnostica.
실시예 4-2. A 형태의 접합 (CWPS-CRM197-ADH-CPS 3)Example 4-2. Conjugation of form A (CWPS-CRM197-ADH-CPS 3)
폐렴구균 협막다당 혈청형 3(CPS 3)에 염화나트륨 분말을 첨가하여 2M NaCl 용액을 제조하였다. CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate)를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 다당 대비 CDAP 10 w/w %의 무게 비율로 용해하고 3분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내기 9.7로 상승시킨 후, 7 분 후에 ADH (adipic acid dihydrazide, 링커)를 다당 대비 10 w/w %의 무게 비율로 첨가하였다. 상온에서 4시간 동안 교반 후, 6-8 kDa dialysis sack 을 사용하여 PBS buffer (pH 7.4)하에서 정제하여 CPS 3-ADH 접합체를 얻었다. A 2M NaCl solution was prepared by adding sodium chloride powder to pneumococcal capsular polysaccharide serotype 3 (CPS 3). CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. After dissolving in a weight ratio of 10 w/w % of CDAP to polysaccharide, and adding sodium hydroxide solution after 3 minutes to raise the pH to 9.4 to 9.7, ADH (adipic acid dihydrazide, linker) was added 10 w/w to polysaccharide after 7 minutes % by weight. After stirring at room temperature for 4 hours, the CPS 3-ADH conjugate was obtained by purification in PBS buffer (pH 7.4) using a 6-8 kDa dialysis sack.
CWPS(cell wall polysaccharide, 세포벽 유래물질)에 염화나트륨 분말을 첨가하여 2M NaCl 용액을 제조하였다. CDAP를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 다당 대비 CDAP 10 w/w %의 무게 비율로 용해하고 3분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내기 9.7로 상승시킨 후 7 분 후 CRM197를 CWPS 대비 1 w/w %의 무게 비율로 첨가하였다. 상온에서 4시간 동안 교반 후, 첨가한 CDAP과 같은 양의 2M glycine를 첨가하고 1시간 교반 후 필터하여 CWPS-CRM197 접합체를 얻었다. A 2M NaCl solution was prepared by adding sodium chloride powder to CWPS (cell wall polysaccharide, a cell wall-derived material). CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. After dissolving in a weight ratio of 10 w / w % of CDAP to polysaccharide, and adding sodium hydroxide solution after 3 minutes to raise the pH to 9.4 to 9.7, CRM 197 was added at a weight ratio of 1 w / w % compared to CWPS after 7 minutes. . After stirring at room temperature for 4 hours, 2M glycine in the same amount as the added CDAP was added, stirred for 1 hour, and filtered to obtain a CWPS-CRM 197 conjugate.
CPS 3-ADH 접합체에 CWPS-CRM197 접합체를 무게 대비 1.5 w/w % 비율로 첨가한 후, EDC.HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) 용액을 (20 mg/mL in 0.1M Tris HCl pH 7.5)) Pn 3-ADH 접합체 대비 1 w/w% 비율로 첨가하였다. 0.1M HCl 용액을 첨가하여 pH 5로 낮춘 후, 상온에서 2시간 교반하였다. 그 후, 1M Tris HCl 용액을 첨가하여 pH 7로 상승시킨 후 상온에서 30분 교반 후, 탄젠셜 플로우 필터레이션 시스템으로 정제하여 CWPS-CRM197-ADH-CPS 3 접합체를 얻었다. After adding CWPS-CRM 197 conjugate to CPS 3-ADH conjugate at a ratio of 1.5 w/w % by weight, EDC.HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) solution (20 mg/mL in 0.1M Tris HCl pH 7.5)) was added in a ratio of 1 w/w% compared to the Pn 3-ADH conjugate. After adding 0.1M HCl solution to lower the pH to 5, the mixture was stirred at room temperature for 2 hours. Thereafter, 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain a CWPS-CRM 197 -ADH-CPS 3 conjugate.
실시예 4-3. B 형태의 접합 (CWPS-ADH-CPS 3-CRM197)Example 4-3. Conjugation of Form B (CWPS-ADH-CPS 3-CRM197)
CWPS(cell wall polysaccharide, 세포벽 유래물질)에 염화나트륨 분말을 첨가하여 2M NaCl 용액을 제조하였다. CDAP를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 다당 대비 CDAP 10 w/w %의 무게 비율로 용해하고 3분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내기 9.7로 상승시킨 후 7 분 후 ADH를 다당 대비 10 w/w %의 무게 비율로 첨가하였다. 상온에서 4시간 동안 교반 후, 6-8 kDa dialysis sack 을 사용하여 PBS buffer (pH 7.4)하에서 정제하여 CWPS 3-ADH 접합체를 얻었다. A 2M NaCl solution was prepared by adding sodium chloride powder to CWPS (cell wall polysaccharide, a cell wall-derived material). CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. After dissolving the polysaccharide at a weight ratio of 10 w/w % of CDAP, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, and 7 minutes later, ADH was added at a weight ratio of 10 w/w % of the polysaccharide. After stirring at room temperature for 4 hours, the CWPS 3-ADH conjugate was obtained by purification in PBS buffer (pH 7.4) using a 6-8 kDa dialysis sack.
폐렴구균 협막다당 혈청형 3(CPS 3)에 염화나트륨 분말을 첨가하여 2M NaCl 용액을 제조하였다. CDAP를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 다당 대비 CDAP 10 w/w %의 무게 비율로 용해하고 3분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내기 9.7로 상승시킨 후 7 분 후 CRM197를 다당 대비 1 w/w %의 무게 비율로 첨가하였다. 상온에서 4시간 동안 교반 후, 첨가한 CDAP과 같은 양의 2M glycine를 첨가하고 1시간 교반 후 필터하여 CPS 3-CRM197 접합체를 얻었다A 2M NaCl solution was prepared by adding sodium chloride powder to pneumococcal capsular polysaccharide serotype 3 (CPS 3). CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. It was dissolved in a weight ratio of 10 w/w % of CDAP to polysaccharide, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7. After 7 minutes, CRM 197 was added in a weight ratio of 1 w/w % to polysaccharide. . After stirring at room temperature for 4 hours, 2M glycine in the same amount as the added CDAP was added, stirred for 1 hour, and filtered to obtain a CPS 3-CRM 197 conjugate.
CWPS-ADH 접합체에 CPS 3-CRM197 접합체를 무게 대비 1.5 w/w % 비율로 첨가한 후, EDC.HCl 용액(20 mg/mL in 0.1M Tris HCl pH 7.5)을 CWPS-ADH 접합체 대비 1 w/w% 비율로 첨가하였다. 0.1M HCl 용액을 첨가하여 pH 5로 낮춘 후, 상온에서 2시간 교반하였다. 그 후, 1M Tris HCl 용액을 첨가하여 pH 7로 상승시킨 후 상온에서 30분 교반 후, 탄젠셜 플로우 필터레이션 시스템으로 정제하여 CWPS-ADH-CPS 3-CRM197 접합체를 얻었다.After adding the CPS 3-CRM 197 conjugate to the CWPS-ADH conjugate at a ratio of 1.5 w/w % by weight, an EDC.HCl solution (20 mg/mL in 0.1M Tris HCl pH 7.5) was added to the CWPS-ADH conjugate 1 w /w% was added. After adding 0.1M HCl solution to lower the pH to 5, the mixture was stirred at room temperature for 2 hours. Thereafter, 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain a CWPS-ADH-CPS 3-CRM 197 conjugate.
실시예 4-4. C 형태의 접합 (CWPS-ADH-CPS 3-CRM197)Example 4-4. C-form junction (CWPS-ADH-CPS 3-CRM197)
CWPS(cell wall polysaccharide, 세포벽 유래물질)에 염화나트륨 분말을 첨가하여 2M NaCl 용액을 제조하였다. CDAP를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 다당 대비 CDAP 10 w/w %의 무게 비율로 용해하고 3분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내기 9.7로 상승시킨 후 7 분 후 ADH를 다당 대비 10 w/w %의 무게 비율로 첨가하였다. 상온에서 4시간 동안 교반 후, 6-8 kDa dialysis sack 을 사용하여 PBS buffer (pH 7.4)하에서 정제하여 CPS 3-ADH 접합체를 얻었다. CWPS-ADH 접합체에 폐렴구균 협막다당 혈청형 3(CPS 3)를 무게 대비 1.5 w/w % 비율로 첨가한 후, EDC.HCl 용액을 (20 mg/mL in 0.1M Tris HCl pH 7.5)) CWPS-ADH 접합체 대비 1 w/w% 비율로 첨가하였다. 0.1M HCl 용액을 첨가하여 pH 5로 낮춘 후, 상온에서 2시간 교반하였다. 그 후, 1M Tris HCl 용액을 첨가하여 pH 7로 상승시킨 후 상온에서 30분 교반 후, 탄젠셜 플로우 필터레이션 시스템으로 정제하여 CWPS-ADH-CPS 3 접합체를 얻었다. A 2M NaCl solution was prepared by adding sodium chloride powder to CWPS (cell wall polysaccharide, a cell wall-derived material). CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. After dissolving the polysaccharide at a weight ratio of 10 w/w % of CDAP, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, and 7 minutes later, ADH was added at a weight ratio of 10 w/w % of the polysaccharide. After stirring at room temperature for 4 hours, the CPS 3-ADH conjugate was obtained by purification in PBS buffer (pH 7.4) using a 6-8 kDa dialysis sack. After adding pneumococcal capsular polysaccharide serotype 3 (CPS 3) to the CWPS-ADH conjugate at a ratio of 1.5 w/w % by weight, EDC.HCl solution (20 mg/mL in 0.1M Tris HCl pH 7.5)) CWPS -ADH conjugate was added in a ratio of 1 w/w%. After adding 0.1M HCl solution to lower the pH to 5, the mixture was stirred at room temperature for 2 hours. Thereafter, 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain a CWPS-ADH-CPS 3 conjugate.
그 후, CDAP를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 다당 대비 CDAP 10 w/w %의 무게 비율로 용해하고 3분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내기 9.7로 상승시킨 후 7 분 후 CRM 197을 다당 대비 1 w/w %의 무게 비율로 첨가하였다. 상온에서 4시간 동안 교반 후, 첨가한 CDAP과 같은 양의 2M glycine를 첨가하고 1시간 교반 후 탄젠셜 플로우 필터레이션 시스템으로 정제하여 CWPS-ADH-CPS 3-CRM197 접합체를 얻었다. Thereafter, CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. It was dissolved in a weight ratio of 10 w/w % of CDAP to polysaccharide, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7. After 7 minutes, CRM 197 was added in a weight ratio of 1 w/w % to polysaccharide. . After stirring at room temperature for 4 hours, 2M glycine in the same amount as the added CDAP was added, and after stirring for 1 hour, the mixture was purified by a tangential flow filtration system to obtain a CWPS-ADH-CPS 3-CRM 197 conjugate.
실시예 4-5. E 형태의 접합 (LTA-ADH-CPS 3-CRM197)Example 4-5. Conjugation of Form E (LTA-ADH-CPS 3-CRM197)
LTA (Lipo-teichoic acid, Cat No. 88840)에 염화나트륨 분말을 첨가하여 2M NaCl 용액을 제조하였다. CDAP를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 다당 대비 CDAP 10 w/w %의 무게 비율로 용해하고 3분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내기 9.7로 상승시킨 후 7 분 후 ADH를 다당 대비 10 w/w %의 무게 비율로 첨가하였다. 상온에서 4시간 동안 교반 후, 6-8 kDa dialysis sack 을 사용하여 PBS buffer (pH 7.4)하에서 정제하여 LTA-ADH 접합체를 얻었다. A 2M NaCl solution was prepared by adding sodium chloride powder to LTA (Lipo-teichoic acid, Cat No. 88840). CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. After dissolving the polysaccharide at a weight ratio of 10 w/w % of CDAP, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, and 7 minutes later, ADH was added at a weight ratio of 10 w/w % of the polysaccharide. After stirring at room temperature for 4 hours, it was purified using a 6-8 kDa dialysis sack in PBS buffer (pH 7.4) to obtain an LTA-ADH conjugate.
폐렴구균 협막다당 혈청형 3(CPS 3)에 염화나트륨 분말을 첨가하여 2M NaCl 용액을 제조하였다. CDAP를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 다당 대비 CDAP 10 w/w %의 무게 비율로 용해하고 3분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내기 9.7로 상승시킨 후 7 분 후 CRM197를 다당 대비 1 w/w %의 무게 비율로 첨가하였다. 상온에서 4시간 동안 교반 후, 첨가한 CDAP과 같은 양의 2M glycine를 첨가하고 1시간 교반 후 필터하여 CPS 3-CRM197 접합체를 얻었다A 2M NaCl solution was prepared by adding sodium chloride powder to pneumococcal capsular polysaccharide serotype 3 (CPS 3). CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. It was dissolved in a weight ratio of 10 w/w % of CDAP to polysaccharide, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7. After 7 minutes, CRM 197 was added in a weight ratio of 1 w/w % to polysaccharide. . After stirring at room temperature for 4 hours, 2M glycine in the same amount as the added CDAP was added, stirred for 1 hour, and filtered to obtain a CPS 3-CRM 197 conjugate.
LTA-ADH 접합체에 CPS 3-CRM197 접합체를 무게 대비 1.5 w/w % 비율로 첨가한 후, EDC.HCl 용액(20 mg/mL in 0.1M Tris HCl pH 7.5)을 CWPS-ADH 접합체 대비 1 w/w% 비율로 첨가하였다. 0.1M HCl 용액을 첨가하여 pH 5로 낮춘 후, 상온에서 2시간 교반하였다. 그 후, 1M Tris HCl 용액을 첨가하여 pH 7로 상승시킨 후 상온에서 30분 교반 후, 탄젠셜 플로우 필터레이션 시스템으로 정제하여 LTA-ADH-CPS 3-CRM197 접합체를 얻었다.After adding the CPS 3-CRM 197 conjugate to the LTA-ADH conjugate at a ratio of 1.5 w/w % by weight, an EDC.HCl solution (20 mg/mL in 0.1M Tris HCl pH 7.5) was added to the CWPS-ADH conjugate 1 w /w% was added. After adding 0.1M HCl solution to lower the pH to 5, the mixture was stirred at room temperature for 2 hours. Thereafter, 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain an LTA-ADH-CPS 3-CRM 197 conjugate.
실시예 4-6. F 형태의 접합 (LTA-ADH-CPS 3-CRM197)Example 4-6. Conjugation of Form F (LTA-ADH-CPS 3-CRM197)
LTA (Lipo-teichoic acid, Cat No. 88840)에 염화나트륨 분말을 첨가하여 2M NaCl 용액을 제조하였다. CDAP를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 다당 대비 CDAP 10 w/w %의 무게 비율로 용해하고 3분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내기 9.7로 상승시킨 후 7 분 후 ADH를 다당 대비 10 w/w %의 무게 비율로 첨가하였다. 상온에서 4시간 동안 교반 후, 6-8 kDa dialysis sack 을 사용하여 PBS buffer (pH 7.4)하에서 정제하여 LTA-ADH 접합체를 얻었다. LTA-ADH 접합체에 폐렴구균 협막다당 혈청형 3(CPS 3)를 무게 대비 1.5 w/w % 비율로 첨가한 후, EDC.HCl 용액을 (20 mg/mL in 0.1M Tris HCl pH 7.5)) LTA-ADH 접합체 대비 1 w/w% 비율로 첨가하였다. 0.1M HCl 용액을 첨가하여 pH 5로 낮춘 후, 상온에서 2시간 교반하였다. 그 후, 1M Tris HCl 용액을 첨가하여 pH 7로 상승시킨 후 상온에서 30분 교반 후, 탄젠셜 플로우 필터레이션 시스템으로 정제하여 LTA-ADH-CPS 3 접합체를 얻었다. A 2M NaCl solution was prepared by adding sodium chloride powder to LTA (Lipo-teichoic acid, Cat No. 88840). CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. After dissolving the polysaccharide at a weight ratio of 10 w/w % of CDAP, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, and 7 minutes later, ADH was added at a weight ratio of 10 w/w % of the polysaccharide. After stirring at room temperature for 4 hours, it was purified using a 6-8 kDa dialysis sack in PBS buffer (pH 7.4) to obtain an LTA-ADH conjugate. After adding pneumococcal capsular polysaccharide serotype 3 (CPS 3) to the LTA-ADH conjugate at a ratio of 1.5 w/w % by weight, EDC.HCl solution (20 mg/mL in 0.1M Tris HCl pH 7.5)) LTA -ADH conjugate was added in a ratio of 1 w/w%. After adding 0.1M HCl solution to lower the pH to 5, the mixture was stirred at room temperature for 2 hours. Thereafter, 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain an LTA-ADH-CPS 3 conjugate.
그 후, CDAP를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 다당 대비 CDAP 10 w/w %의 무게 비율로 용해하고 3분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내기 9.7로 상승시킨 후 7 분 후 CRM 197을 다당 대비 1 w/w %의 무게 비율로 첨가하였다. 상온에서 4시간 동안 교반 후, 첨가한 CDAP과 같은 양의 2M glycine를 첨가하고 1시간 교반 후 탄젠셜 플로우 필터레이션 시스템으로 정제하여 LTA-ADH-CPS 3-CRM197 접합체를 얻었다.Thereafter, CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. It was dissolved in a weight ratio of 10 w/w % of CDAP to polysaccharide, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7. After 7 minutes, CRM 197 was added in a weight ratio of 1 w/w % to polysaccharide. . After stirring at room temperature for 4 hours, 2M glycine in the same amount as the added CDAP was added, and after stirring for 1 hour, the mixture was purified by a tangential flow filtration system to obtain an LTA-ADH-CPS 3-CRM 197 conjugate.
상기에서 제작한 접합체들은 신규한 것으로서, 최종적으로 생성된 접합체 A B,C,E,F가 백신으로서 당 함량, 단백질 함량, 당/단백질 비율 및 농도 등에 문제가 없음을 아래 표 1과 같이 확인하였다. (폐렴구균 협막다당 혈청형 3(CPS 3)을 사용하여 제작함에 따라, 3A 내지 3C와 같이 기재하였음)The conjugates prepared above are novel, and it was confirmed as shown in Table 1 below that the finally produced conjugates A B, C, E, F had no problems in sugar content, protein content, sugar/protein ratio and concentration as a vaccine. (As produced using pneumococcal capsular polysaccharide serotype 3 (CPS 3), described as 3A to 3C)
3A* 3A * 3B* 3B * 3C* 3C *
PS titer (ug/mL)PS titer (ug/mL) 432.55432.55 155.10155.10 70.9370.93
Pr titer (ug/mL)Pr titer (ug/mL) 816.13816.13 262.88262.88 161.20161.20
Ps/Pr ratioPs/Pr ratio 0.530.53 0.590.59 0.440.44
Purity (%)Purity (%) 8282 8080 8282
* 폐렴구균 협막다당 혈청형 3(CPS 3)을 사용하여 제작 하였으므로, 3A 내지 3C와 같은 방식으로 기재함* Since it was prepared using pneumococcal capsular polysaccharide serotype 3 (CPS 3), it is described in the same way as 3A to 3C.
실시예 5. 혈청형 특이적 면역원성 측정 Example 5. Measurement of serotype-specific immunogenicity
신규 세포벽 유래 물질의 접합에 따른 3번 혈청형(폐렴구균 협막다당, CPS 3)의 면역효과 확인을 위해, 하기 표 2에 나타낸 바와 같이 조성물을 달리하여 마우스 내 면역원성 유도 실험을 진행하였다. 6-8주령의 마우스에 2주간격으로 3회 면역을 하였고, 3회 면역 이후 2주 후 3번 혈청형 특이적인 IgG 농도와 기능적 항체가에 대한 OPA (Opsonophagocytic assay)를 측정하였다. In order to confirm the immune effect of serotype 3 (pneumococcal capsular polysaccharide, CPS 3) according to the conjugation of a novel cell wall-derived material, an experiment for inducing immunogenicity in mice was conducted by changing the composition as shown in Table 2 below. Mice aged 6-8 weeks were immunized 3 times at 2 week intervals, and after 3 immunizations, 2 weeks later, serotype-specific IgG concentration and OPA (Opsonophagocytic assay) for functional antibody titers were measured.
GroupGroup NameName DescriptionsDescriptions NoteNote
1One Negative controlnegative control PBSPBS N/AN/A
22 Positive control (1)Positive control (1) Prevnar Prevnar PCV13PCV13
33 Test group 1 (Pn_3)*Test group 1 (Pn_3)* Full dose (2.2 ug)Full dose (2.2 ug) CPS-CRM(기존 형태)CPS-CRM (old form)
44 Test group 1 (Pn_3)Test group 1 (Pn_3) 1/4 dose (0.55 ug)1/4 dose (0.55 ug)
55 Test group 2 (Pn_3A)Test group 2 (Pn_3A) Full dose (2.2 ug)Full dose (2.2 ug) CWPS-CRM197-ADH-CPS 3CWPS-CRM 197 -ADH-CPS 3
66 Test group 2 (Pn_3A)Test group 2 (Pn_3A) 1/4 dose (0.55 ug)1/4 dose (0.55 ug)
77 Test group 3 (Pn_3B)Test group 3 (Pn_3B) Full dose (2.2 ug)Full dose (2.2 ug) CWPS-ADH- CPS 3-CRM197 CWPS-ADH- CPS 3-CRM 197
88 Test group 3 (Pn_3B)Test group 3 (Pn_3B) 1/4 dose (0.55 ug)1/4 dose (0.55 ug)
99 Test group 4 (Pn_3C)Test group 4 (Pn_3C) Full dose (2.2 ug)Full dose (2.2 ug) CWPS-ADH-CPS 3-CRM197 CWPS-ADH-CPS 3-CRM 197
1010 Test group 4 (Pn_3C)Test group 4 (Pn_3C) 1/4 dose (0.55 ug)1/4 dose (0.55 ug)
*: 이하, 폐렴구균 협막다당 3번 혈청형을 의미함*: Hereinafter, it means serotype 3 for pneumococcal capsular polysaccharide.
실시예 5-1. 3번 혈청형 특이 IgG 농도 ELISA 측정Example 5-1. Serotype 3 specific IgG concentration ELISA measurement
각 혈청형에 대한 협막 다당류(PnPs)를 96-웰 플레이트 상에서 0.5 μg/웰(well) 내지 1 μg/웰로 피복시켰다. 등가량의 혈청을 각 대상체로부터 샘플링하고 그룹별로 모았다. 혈청 혼주물(pool)을 Tween 20 및 CWPS 5 μg/mL를 포함하는 항체 희석 완충액으로 2.5배까지 연속 희석시킨 다음 실온에서 30분 동안 반응시켰다. 플레이트를 세척 완충액으로 5회 세척한 다음 예비-흡착되고 희석된 혈청 50 μl를 피복된 웰 플레이트에 첨가한 후, 실온에서 2시간 내지 18시간 동안 배양하였다. 웰 플레이트를 동일한 방식으로 세척한 다음 염소 항-마우스 IgG-알칼리성 포스파타제 접합체를 각 웰에 가한 후 실온에서 2시간 동안 배양하였다. 플레이트를 상기한 바와 같이 세척하고 기질로서 1mg/mL p-니트로페닐아민 완충액을 각 웰에 가한 다음 실온에서 2시간 동안 반응시켰다. 50 μl의 3 M NaOH를 첨가하여 반응을 켄칭(quenching)시키고 405 nm 및 690 nm에서 흡광도를 측정하였다. 비교 실시예로서, 상업적으로 이용 가능한 13가 백신(PREVNAR13)을 동일한 과정에 적용하였다. Capsular polysaccharides (PnPs) for each serotype were coated at 0.5 μg/well to 1 μg/well on 96-well plates. Equal amounts of serum were sampled from each subject and pooled into groups. The serum pool was serially diluted up to 2.5 times with an antibody dilution buffer containing Tween 20 and 5 μg/mL of CWPS, and then reacted at room temperature for 30 minutes. Plates were washed 5 times with wash buffer, then 50 μl of pre-adsorbed and diluted serum was added to the coated well plates, followed by incubation at room temperature for 2 to 18 hours. The well plate was washed in the same manner, and then goat anti-mouse IgG-alkaline phosphatase conjugate was added to each well and incubated for 2 hours at room temperature. The plate was washed as described above, and 1 mg/mL p-nitrophenylamine buffer as a substrate was added to each well, followed by reaction at room temperature for 2 hours. The reaction was quenched by addition of 50 μl of 3 M NaOH and the absorbance was measured at 405 nm and 690 nm. As a comparative example, a commercially available 13-valent vaccine (PREVNAR13) was applied in the same procedure.
그 결과, 도 2에 나타낸 바와 같이 PCV13 대비 Pn3C(3번 혈청형, 실시예 C)의 그룹이 약간 높은 3번 혈청형 특이적인 항체가가 발생 하였으며, Pn3A의 경우, 약간 높은 경향을 보였다. 하지만 Pn3B의 경우, 낮은 항체가를 보였다. 이는 동일한 세포벽 유래 물질인 경우에도 접합 순서와 방법에 따라 면역원성의 차이를 갖는 것을 의미한다(도 2)As a result, as shown in FIG. 2, the Pn3C (serotype 3, Example C) group had a slightly higher antibody titer specific to serotype 3 than PCV13, and in the case of Pn3A, it showed a slightly higher tendency. However, in the case of Pn3B, the antibody titer was low. This means that even in the case of the same cell wall-derived material, there is a difference in immunogenicity depending on the splicing sequence and method (FIG. 2).
실시예 5-2. 3번 혈청형 기능적 항체가 측정(OPA)Example 5-2. Measurement of serotype 3 functional antibody titer (OPA)
기능적 항체가는 OPA 검정으로 혈청을 시험함으로써 평가하였다. 각 균주의 농도가 약 50,000 CFU/mL로 되도록, -70℃ 이하에서 저장된 폐렴연쇄구균 MOPA 균주를 상응하는 최종 희석도로 희석시켰다. 등가량의 혈청을 각 대상체로부터 샘플링하고, 그룹별로 모으고, 20 μl의 혈청이 U-바닥 플레이트에 남도록 2배 연속 희석시켰다. 샘플을 희석한 후, 각 혈청형에 대해 제조된 10 μl의 균주를 희석된 샘플과 혼합하고, 폐렴연쇄구균과 항체가 잘 혼합되도록 혼합물을 실온에서 30분 동안 반응시켰다. 예비-분화된 HL-60 세포와 보체의 혼합물을 첨가하고 CO2 배양기(37℃)에서 45분 동안 반응시켰다. 온도를 낮추어 식균 작용을 중단시키고 10 μl의 반응 용액을 30 내지 60분 동안 예비-건조된 한천 플레이트 상에 스폿팅(spotting)한 다음 건조될 때까지 20분 동안 플레이트 상에 흡수되도록 하였다. 25mg/mL TTC 스톡 용액을 제조된 오버레이 한천(overlay agar)에 첨가하고, 상응하는 균주에 적합한 항체를 이에 첨가하였다. 혼합물을 완전히 혼합한 다음 약 25 mL의 혼합물을 플레이트에 첨가하고 약 30분 동안 경화시켰다. 완전히 경화된 플레이트를 CO2 배양기(37℃)에서 12 내지 18시간 동안 배양한 다음 콜로니를 계수하였다. MOPA 역가는 50% 사멸이 관찰되는 희석률로 표현되었다. 비교 실시예로서, 상업적으로 이용 가능한, 13가 백신(PREVNAR13)을 동일한 과정에 적용하였다. 결과는 하기의 표 3 및 표 4에 나타내었다.Functional antibody titers were assessed by testing sera in an OPA assay. Streptococcus MOPA strains stored at -70° C. or lower were diluted to the corresponding final dilution so that the concentration of each strain was about 50,000 CFU/mL. Equal amounts of serum were sampled from each subject, pooled into groups, and serially diluted 2-fold so that 20 μl of serum remained in U-bottom plates. After diluting the sample, 10 μl of the strain prepared for each serotype was mixed with the diluted sample, and the mixture was reacted at room temperature for 30 minutes to mix well with Streptococcus and the antibody. A mixture of pre-differentiated HL-60 cells and complement was added and incubated in a CO 2 incubator (37° C.) for 45 minutes. The phagocytosis was stopped by lowering the temperature and 10 μl of the reaction solution was spotted on a pre-dried agar plate for 30 to 60 minutes and then allowed to absorb on the plate for 20 minutes until dry. A 25 mg/mL TTC stock solution was added to the prepared overlay agar, and an antibody suitable for the corresponding strain was added thereto. The mixture was thoroughly mixed, then about 25 mL of the mixture was added to the plate and allowed to cure for about 30 minutes. The fully cured plates were incubated in a CO 2 incubator (37° C.) for 12 to 18 hours and then colonies were counted. MOPA titers were expressed as the dilution at which 50% killing was observed. As a comparative example, a commercially available, 13-valent vaccine (PREVNAR13) was applied in the same procedure. The results are shown in Tables 3 and 4 below.
샘플명sample name OI titerOI titer
PBSPBS 1One
PCV13PCV13 606606
Pn3 Full dosePn3 full dose 453453
Pn3 1/4 dose Pn3 1/4 dose 277277
Pn3A Full dosePn3A full dose 15171517
Pn3A 1/4 dose Pn3A 1/4 dose 22292229
Pn3B Full dosePn3B Full dose 12881288
Pn3B 1/4 dose Pn3B 1/4 dose 3030
Pn3C Full dosePn3C Full dose 52115211
Pn3C 1/4 dose Pn3C 1/4 dose 1749617496
샘플명sample name 샘플/PCV13 OI 역가 상대 비교Sample/PCV13 OI Titer Relative Comparison
PBSPBS 00
PCV13PCV13 1.001.00
Pn3 Full dosePn3 full dose 0.750.75
Pn3 1/4 dosePn3 1/4 dose 0.460.46
Pn3A Full dosePn3A full dose 2.502.50
Pn3A 1/4 dosePn3A 1/4 dose 3.683.68
Pn3B Full dosePn3B Full dose 2.132.13
Pn3B 1/4 dosePn3B 1/4 dose 0.050.05
Pn3C Full dosePn3C Full dose 8.608.60
Pn3C 1/4 dosePn3C 1/4 dose 28.8728.87
상기 표 3 및 표 4에 나타낸 바와 같이, CWPS(세포벽 유래 물질)와 3번 혈청형을 접합한 그룹 (A-C) 모두 PCV13 대비 높은 기능적 항체가를 보였다. 특이적 항체가 (IgG) 결과와 유사하게, 접합 순서에 따라 동일한 CWPS를 접합을 하였지만 기능적 항체가에서는 차이를 보였으며, IgG와 유사하게 Pn3C 에서 PCV13 대비 8 내지 28 배 높은 기능적 항체가를 보였다. 이는 동일한 세포벽 유래 물질 의 경우에도 접합 순서와 방법에 따라 면역원성의 차이를 가짐을 기능적 항체가 실험을 통해 재 확인하였다. (표 3 및 표 4)As shown in Tables 3 and 4, both groups (A-C) conjugated with CWPS (cell wall-derived material) and serotype 3 showed higher functional antibody titers compared to PCV13. Similar to the specific antibody titer (IgG) result, although the same CWPS was conjugated according to the conjugation sequence, there was a difference in functional antibody titer. Similar to IgG, Pn3C showed a functional antibody titer that was 8 to 28 times higher than that of PCV13. It was reconfirmed through experiments that functional antibodies have differences in immunogenicity depending on the sequence and method of conjugation even in the case of the same cell wall-derived material. (Table 3 and Table 4)
실시예 6. 혈청형 특이적 면역원성 측정Example 6. Measurement of serotype-specific immunogenicity
CWPS 이외의 세포벽 유래 물질의 접합에 따른 3번 혈청형(폐렴구균 협막다당, CPS 3)의 면역효과 확인을 위해, 하기 표 5에 나타낸 바와 같이 조성물을 달리하여 마우스 내 면역원성 유도 실험을 진행하였다. 6-8주령의 마우스에 2주간격으로 3회 면역을 하였고, 3회 면역 이후 2주 후 3번 혈청형 특이적인 IgG 농도를 측정하였다. In order to confirm the immune effect of serotype 3 (pneumococcal capsular polysaccharide, CPS 3) according to the conjugation of a cell wall-derived material other than CWPS, an experiment for inducing immunogenicity in mice was conducted by changing the composition as shown in Table 5 below. . Mice aged 6-8 weeks were immunized 3 times at 2-week intervals, and after 3 immunizations, serotype-specific IgG concentrations were measured 2 weeks later.
GroupGroup NameName DescriptionsDescriptions NoteNote
1One Negative controlnegative control PBSPBS N/AN/A
22 Positive control (1)Positive control (1) Prevnar Prevnar PCV13PCV13
33 Test group 1 (Pn_3)*Test group 1 (Pn_3)* Full dose (2.2 ug)Full dose (2.2 ug) CPS-CRM(기존 형태)CPS-CRM (old form)
44 Test group 1 (Pn_3)Test group 1 (Pn_3) 1/4 dose (0.55 ug)1/4 dose (0.55 ug)
55 Test group 2 (Pn_3F)Test group 2 (Pn_3F) Full dose (2.2 ug)Full dose (2.2 ug) LTA-CRM197-ADH-CPS 3LTA-CRM 197 -ADH-CPS 3
66 Test group 2 (Pn_3F)Test group 2 (Pn_3F) 1/4 dose (0.55 ug)1/4 dose (0.55 ug)
77 Test group 3 (Pn_3E)Test group 3 (Pn_3E) Full dose (2.2 ug)Full dose (2.2 ug) LTA-ADH- CPS 3-CRM197 LTA-ADH-CPS 3-CRM 197
88 Test group 3 (Pn_3E)Test group 3 (Pn_3E) 1/4 dose (0.55 ug)1/4 dose (0.55 ug)
99 Test group 4 (Pn_3C)Test group 4 (Pn_3C) Full dose (2.2 ug)Full dose (2.2 ug) CWPS-ADH-CPS 3-CRM197 CWPS-ADH-CPS 3-CRM 197
1010 Test group 4 (Pn_3C)Test group 4 (Pn_3C) 1/4 dose (0.55 ug)1/4 dose (0.55 ug)
*: 이하, 폐렴구균 협막다당 3번 혈청형을 의미함*: Hereinafter, it means serotype 3 for pneumococcal capsular polysaccharide.
실시예 6-1. 3번 혈청형 특이 IgG 농도 ELISA 측정Example 6-1. Serotype 3 specific IgG concentration ELISA measurement
3번 혈청형에 대한 IgG ELISA 분석은 상기 실시예 5와 동일한 방법으로 진행하였다. 그 결과, 도 3에 나타낸 바와 같이 PCV13 대비 Pn3C(3번 혈청형, 실시예 C)의 그룹이 유사한 수준의 IgG 역가가 확인되었다. 하지만 동일한 접합 방법을 사용하고, 다른 세포벽 유래 물질인 LTA를 접합한 Pn3F의 경우, Pn3c 그룹보다 full dose에서 낮은 항체가를 확인되었다. 실시예 5에서 낮은 항체가를 보인 Pn3B와 동일한 접합 방법을 사용하고, LTA로 접합한 Pn3F의 경우, 상대적으로 낮은 항체가를 보였다. 이는 동일한 접합 순서 및 방법으로 제조되었음에도 불구하고 다른 세포벽 유래 물질을 접합함에 따라 항체가의 차이를 갖는 것을 보여준다.(도 3)IgG ELISA analysis for serotype 3 was performed in the same manner as in Example 5 above. As a result, as shown in FIG. 3 , a similar level of IgG titer was confirmed in the group of Pn3C (serotype 3, Example C) compared to PCV13. However, in the case of Pn3F using the same conjugation method and conjugated with another cell wall-derived material, LTA, a lower antibody titer at the full dose than in the Pn3c group was confirmed. In Example 5, using the same conjugation method as that of Pn3B, which showed a low antibody titer, Pn3F conjugated with LTA showed a relatively low antibody titer. This shows that the antibody titers differ according to the conjugation of different cell wall-derived materials despite being prepared with the same conjugation sequence and method (Fig. 3).
실시예 7. 19가 폐렴구균 백신의 면역원성Example 7. Immunogenicity of 19-valent pneumococcal vaccine
상기 실시예에서 제조한 각각의 폐렴구균 백신 조성물이 마우스 내에서 면역 반응 유도 능력을 가지는지 여부를 평가하기 위한 실험을 수행하였다. Pn3(3번 혈청형)가 포함된 PCV19 제형과 Pn3에 CWPS를 접합한 PCV19 제형을 비교하고자 하였고, 대조군으로 프리베나13을 면역하여 채취한 혈액 샘플을 같이 분석하였다.An experiment was performed to evaluate whether each of the pneumococcal vaccine compositions prepared in the above Example has the ability to induce an immune response in mice. The purpose of this study was to compare the PCV19 formulation containing Pn3 (serotype 3) with the PCV19 formulation in which Pn3 was conjugated with CWPS, and blood samples collected by immunization with Prevena 13 as a control were also analyzed.
실시예 7-1. 혈청형 특이 IgG 농도 ELISA 측정Example 7-1. Serotype-specific IgG concentration ELISA measurement
상기 실시예 3에서 제조한 각각의 폐렴구균 백신 조성물이 마우스에서 면역 반응을 유도하는지 여부를 평가하기 위한 실험을 수행하였다. 이러한 면역원성은, 항원-특이적 ELISA를 통해 혈청 IgG 농도를 측정하여 확인하였다.An experiment was performed to evaluate whether each of the pneumococcal vaccine compositions prepared in Example 3 induces an immune response in mice. This immunogenicity was confirmed by measuring the serum IgG concentration through antigen-specific ELISA.
실험군을 제외하고 실시예 4-1에서와 동일한 방식으로 본 실험을 수행하였으며, 그 결과를 아래 표 6에 나타내었다. 하기 표에 기재된 'Prevnar13' 및 'PCV19-CRM197(Naive)'은 구체적으로 협막다당-CRM197 구조만을 포함하고 있는 기존 백신의 형태로써, 각각 13가 및 19가의 백신을 의미한다. 그러나 'PCV19-CRM197(CWPS Conjugate)'는 협막다당 3번 혈청형이 CWPS 및 운반체 단백질과 연결되어 있어 상기의 기존 백신 형태와 구조적 차이를 갖는다.Except for the experimental group, this experiment was performed in the same manner as in Example 4-1, and the results are shown in Table 6 below. 'Prevnar13' and 'PCV19-CRM 197 (Naive)' described in the table below are specifically in the form of existing vaccines containing only the capsular polysaccharide-CRM 197 structure, and refer to 13-valent and 19-valent vaccines, respectively. However, 'PCV19-CRM 197 (CWPS Conjugate)' has a structural difference from the existing vaccine form as capsular polysaccharide serotype 3 is linked to CWPS and carrier protein.
혈청형serotype Prevnar13Prevnar13 PCV19-CRM197(Naive)PCV19-CRM 197 (Naive) PCV19-CRM197(3C conjugated)PCV19-CRM 197 (3C conjugated)
1One ++ ++ ++
33 ++ ++ ++
44 ++ ++ ++
55 ++ ++ ++
6A6A ++ ++ ++
6B6B ++ ++ ++
7F7F ++ ++ ++
9V9V ++ ++ ++
10A10A N/AN/A ++ ++
11A11A N/AN/A ++ ++
1414 ++ ++ ++
15B15B N/AN/A ++ ++
18C18C ++ ++ ++
19A19A ++ ++ ++
19F19F ++ ++ ++
22F22F N/AN/A ++ ++
23A23A N/AN/A ++ ++
23F23F ++ ++ ++
35B35B N/AN/A ++ ++
23A, 35B는 폐렴구균 다당 백신 (PPV, Pneumococcal polysaccharide vaccine)인 PPV23에 포함되지 않는 혈청형이며, 현재까지 폐렴백신에 사용되지 않는 혈청형으로 아시아와 유럽에 serotype replacement이후 도래한 혈청형 중 하나이다. 실험 결과, 23A, 35B가 포함된 PCV19-CRM197 모든 혈청형에 대해 우수한 수준의 혈청형 특이 IgG 농도를 갖는 것을 확인하였다. 3번 혈청형에 CWPS를 접합한 제형의 경우, PCV13 및 PCV19(naive)-CRM 대비, 유사한 수준의 항체가 형성을 확인하였다. PREVNAR13과 공통인 혈청형은 PREVNAR13과 유사하고, 추가된 혈청형 10A, 11A, 15B, 22F, 그리고 23A, 35B 각각 또한 우수한 수준의 혈청형 특이 IgG 농도를 보여주었다. (표 6, 도 4)23A and 35B are serotypes not included in PPV23, a pneumococcal polysaccharide vaccine (PPV), and serotypes not used in pneumococcal vaccines so far are one of the serotypes that have arrived in Asia and Europe after serotype replacement. As a result of the experiment, it was confirmed that PCV19-CRM 197 including 23A and 35B had an excellent level of serotype-specific IgG concentration for all serotypes. In the case of the formulation in which CWPS was conjugated to serotype 3, it was confirmed that the antibody was formed at a similar level compared to PCV13 and PCV19 (naive)-CRM. The serotype common to PREVNAR13 is similar to PREVNAR13, and the added serotypes 10A, 11A, 15B, 22F, and 23A, 35B each also showed superior serotype-specific IgG concentrations. (Table 6, Fig. 4)
실시예 7-2. 기능적 항체가 (OPA)Example 7-2. functional antibody titer (OPA)
상기 실시예 3에서 제조한 각각의 폐렴구균 백신 조성물이 마우스에서 면역 반응 유도 능력을 가지는지 여부를 평가하기 위한 실험을 수행하였다. 이러한 면역원성은 기능적 항체가 (OPA)를 통해 측정하였다. 실험군을 제외하고 실시예 4-1에서와 동일한 방식으로 측정하였으며, 그 결과는 아래 표 7에 나타내었다. An experiment was performed to evaluate whether each of the pneumococcal vaccine compositions prepared in Example 3 had the ability to induce an immune response in mice. This immunogenicity was measured via functional antibody titer (OPA). Measurements were made in the same manner as in Example 4-1 except for the experimental group, and the results are shown in Table 7 below.
혈청형serotype Prevnar13Prevnar13 PCV19-CRM197(Naive)PCV19-CRM 197 (Naive) PCV19-CRM197(3C conjugated)PCV19-CRM 197 (3C conjugated)
1One ++ ++ ++
33 ++ ++ ++
44 ++ ++ ++
55 ++ ++++ ++++
6A6A ++ ++ ++
6B6B ++ ++ ++
7F7F ++ ++ ++
9V9V ++ ++ ++
10A10A N/AN/A ++++ ++++
11A11A N/AN/A ++++ ++++
1414 ++ ++ ++
15B15B N/AN/A ++ ++
18C18C ++ ++ ++
19A19A ++ ++ ++
19F19F ++ ++ ++
22F22F N/AN/A ++++++ ++++++
23A23A N/AN/A ++++ ++++
23F23F ++ ++ ++
35B35B N/AN/A ++ ++
23A, 35B는 폐렴구균 다당 백신 (PPV, Pneumococcal polysaccharide vaccine)인 PPV23에 포함되지 않는 혈청형이므로 항생제 내성 균주가 존재하지 않는다. 이에 항생제 내성균주를 제작하여 MOPA 분석에 사용하였으며, 내성균주의 제작은 UAB항생제 내성 균주 제조방법(MOPA protocol)에 근거하여 진행하였고, 제작 후 균주 특성화 분석 및 동정을 통해 각 23A, 35B 혈청형의 특성을 확인하였다.Since 23A and 35B are serotypes not included in PPV23, a pneumococcal polysaccharide vaccine, antibiotic-resistant strains do not exist. Accordingly, antibiotic-resistant strains were prepared and used for MOPA analysis, and the production of resistant strains was carried out based on the UAB antibiotic-resistant strain production method (MOPA protocol). was confirmed.
실험 결과, 10A, 11A, 15B, 22F, 23A, 35B가 포함된 PCV19-CRM197 (naive), PCV19-CRM197 (CWPS conjugate)의 모든 혈청형에 대해 우수한 수준의 혈청형 기능적 항체가가 확인되었다. PREVNAR13에 공통인 혈청형은 PREVNAR13과 유사한 수준의 기능적 항체가를 보였으며 새로 추가된 혈청형 10A, 11A, 15B, 22F, 그리고 23A, 35B 각각 또한 우수한 수준의 기능적 항체가를 보였다. 특히 CWPS를 접합한 PCV19 (3C Conjugated) 그룹에서는 3번 혈청형이 PCV13과 PCV19 (naive) 그룹과 유사한 수준의 기능적 항체가를 보였으며, 신규 접합 및 혈청형 증가에 의한 간섭효과가 PCV19 (naive) 및 PCV19 (3C conjugated) 그룹에서 확인되지 않았다. (표 7, 도 5) CWPS를 접합한 단가 실험에서는 naive 접합 형태 비해, 월등히 높은 항체가와 기능적 항체가가 확인되었지만, 다가 (PCV19) 실험에서는 유사한 수준의 기능적 항체가가 확인되었다.As a result of the experiment, excellent serotype functional antibody titers were confirmed for all serotypes of PCV19-CRM 197 (naive) and PCV19-CRM 197 (CWPS conjugate), including 10A, 11A, 15B, 22F, 23A, and 35B. . The serotype common to PREVNAR13 showed a similar level of functional antibody titer to PREVNAR13, and each of the newly added serotypes 10A, 11A, 15B, 22F, and 23A, 35B also showed an excellent level of functional antibody titer. In particular, PCV19 bonded with CWPS In the (3C Conjugated) group, serotype 3 was PCV13 and PCV19 (naive) showed a similar level of functional antibody titer to the group, and the interference effect by new conjugation and increase in serotype was not confirmed in the PCV19 (naive) and PCV19 (3C conjugated) groups. (Table 7, FIG. 5) In the monovalent experiment in which CWPS was conjugated, a significantly higher antibody titer and functional antibody titer were confirmed compared to the naive conjugated form, but a similar level of functional antibody titer was confirmed in the multivalent (PCV19) test.

Claims (37)

  1. 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 세포벽 유래 물질의 접합체를 포함하는 면역원성 조성물.An immunogenic composition comprising a conjugate of a cell wall-derived material and a capsular polysaccharide derived from Streptococcus pneumoniae .
  2. 제1항에 있어서, According to claim 1,
    상기 협막 다당류는The capsular polysaccharide is
    혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F 및 35B 로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류를 포함함을 특징으로 하는, 면역원성 조성물.Any one or more serotypes selected from the group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 35B An immunogenic composition comprising a capsular polysaccharide of
  3. 제2항에 있어서, 3. The method of claim 2,
    상기 협막 다당류는The capsular polysaccharide is
    혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F 및 23F로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류를 포함함을 특징으로 하는, 면역원성 조성물.Immune, characterized in that it contains capsular polysaccharide of any one or more serotypes selected from the group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F original composition.
  4. 제3항에 있어서, 4. The method of claim 3,
    상기 협막 다당류는The capsular polysaccharide is
    혈청형 3, 6A, 6B, 19A 및 19F로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류를 포함함을 특징으로 하는, 면역원성 조성물.An immunogenic composition comprising capsular polysaccharides of any one or more serotypes selected from the group consisting of serotypes 3, 6A, 6B, 19A and 19F.
  5. 제3항 또는 제4항에 있어서, 5. The method according to claim 3 or 4,
    스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 운반체 단백질의 접합체를 추가로 포함하며, Streptococcus pneumoniae ( Streptococcus pneumoniae ) It further comprises a conjugate of a capsular polysaccharide ( capsular polysaccharide ) and a carrier protein,
    상기 협막 다당류는The capsular polysaccharide is
    혈청형 10A, 11A, 15B, 22F, 23A 및 35B로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류임을 특징으로 하는, 면역원성 조성물.An immunogenic composition, characterized in that it is a capsular polysaccharide of any one or more serotypes selected from the group consisting of serotypes 10A, 11A, 15B, 22F, 23A and 35B.
  6. 제4항 또는 제5항에 있어서, 6. The method according to claim 4 or 5,
    스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 운반체 단백질의 접합체를 추가로 포함하며, Streptococcus pneumoniae ( Streptococcus pneumoniae ) It further comprises a conjugate of a capsular polysaccharide (capsular polysaccharide) and a carrier protein,
    상기 협막 다당류는The capsular polysaccharide is
    혈청형 1, 4, 5, 7F, 9V, 14, 18C 및 23F로 이루어지는 군에서 선택되는 어느 하나 이상의 혈청형의 협막 다당류임을 특징으로 하는, 면역원성 조성물.An immunogenic composition, characterized in that it is a capsular polysaccharide of any one or more serotypes selected from the group consisting of serotypes 1, 4, 5, 7F, 9V, 14, 18C and 23F.
  7. 제 1항에 있어서, The method of claim 1,
    상기 스트렙토코커스 뉴모니애 유래의 협막 다당류와 세포벽 유래 물질의 접합체는 운반체 단백질을 추가로 포함함을 특징으로 하는, 면역원성 조성물.The conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and cell wall-derived material further comprises a carrier protein, the immunogenic composition.
  8. 제 1항에 있어서, The method of claim 1,
    상기 스트렙토코커스 뉴모니애 유래의 협막 다당류와 세포벽 유래 물질의 접합체는 링커(linker)를 추가로 포함함을 특징으로 하는, 면역원성 조성물.The conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and the cell wall-derived material is characterized in that it further comprises a linker (linker), the immunogenic composition.
  9. 제1항에 있어서, According to claim 1,
    상기 세포벽 유래 물질은 스트렙토코커스 뉴모니애(Streptococcus pneumoniae)로부터 분리됨을 특징으로 하는, 면역원성 조성물.The cell wall-derived material is Streptococcus pneumoniae ( Streptococcus pneumoniae ), characterized in that isolated from, the immunogenic composition.
  10. 제9항에 있어서, 10. The method of claim 9,
    상기 세포벽 유래 물질은 상기 협막 다당류와 공유결합으로 연결됨을 특징으로 하는, 면역원성 조성물.The cell wall-derived material is characterized in that it is covalently linked to the capsular polysaccharide, the immunogenic composition.
  11. 제1항에 있어서, According to claim 1,
    상기 세포벽 유래 물질은 LTA (Lipoteichoic acid), CWPS (Cell Wall Polysaccharide), CPS (Capsular Polysaccharide), PG (Peptidoglycan), LPS (Lipopolysaccharide) MDP (Muramyl Dipeptide), MTP (Muramyl tripeptide) 및 PC (Phosphatidyl Choline)로 이루어지는 군에서 선택되는 어느 하나 이상을 포함함을 특징으로 하는, 면역원성 조성물.The cell wall-derived material is LTA (Lipoteichoic acid), CWPS (Cell Wall Polysaccharide), CPS (Capsular Polysaccharide), PG (Peptidoglycan), LPS (Lipopolysaccharide) MDP (Muramyl Dipeptide), MTP (Muramyl tripeptide) and PC (Phosphatidyl Choline) An immunogenic composition comprising any one or more selected from the group consisting of.
  12. 제1항에 있어서, According to claim 1,
    상기 세포벽 유래 물질은 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 CWPS (Cell Wall Polysaccharide)임을 특징으로 하는, 면역원성 조성물.The cell wall-derived material is Streptococcus pneumoniae ( Streptococcus pneumoniae ) CWPS (Cell Wall Polysaccharide) derived from, characterized in that, immunogenic composition.
  13. 제7항에 있어서, 8. The method of claim 7,
    상기 운반체 단백질은 디프테리아 톡소이드, 파상풍 톡소이드, 백일해 톡소이드, 콜레라 톡소이드, 대장균 유래 불화성화 독소, 슈도모나스 애루지노사(Pseudomonas aeruginosa) 유래 불활성화 독소 및 세균 외막 단백질(OMP)로 이루어진 군으로부터 선택되는 어느 하나임을 특징으로 하는, 면역원성 조성물.The carrier protein is any one selected from the group consisting of diphtheria toxoid, tetanus toxoid, pertussis toxoid, cholera toxoid, E. coli-derived inactivated toxin, Pseudomonas aeruginosa -derived inactivated toxin and bacterial outer membrane protein (OMP) characterized in that the immunogenic composition.
  14. 제13항에 있어서, 14. The method of claim 13,
    상기 디프테리아 톡소이드는 CRM197, CRM173, CRM228 및 CRM45 로 이루어진 군으로부터 선택되는 어느 하나임을 특징으로 하는, 면역원성 조성물.The diphtheria toxoid is CRM 197 , CRM 173 , CRM 228 and CRM 45 characterized in that any one selected from the group consisting of, the immunogenic composition.
  15. 제8항에 있어서, 상기 링커는 ADH (adipic acid dihyrazied), Beta-프로피온아미도, 니트로페닐-에틸아민, 할로알킬 할라이드, 헥산 디아민, 6-아미노카프론산 및 글리코시드 결합으로 이루어진 군으로부터 선택되는 어느 하나임을 특징으로 하는, 면역원성 조성물. 9. The method of claim 8, wherein the linker is selected from the group consisting of adipic acid dihyrazied (ADH), Beta-propionamido, nitrophenyl-ethylamine, haloalkyl halide, hexane diamine, 6-aminocaproic acid and a glycosidic bond. Any one, characterized in that the immunogenic composition.
  16. 제7항에 있어서, 8. The method of claim 7,
    상기 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 세포벽 유래 물질의 접합체와 운반체 단백질은 하기 접합 순서 중 어느 하나로 접합된 구조임을 특징으로 하는, 면역원성 조성물:The Streptococcus pneumoniae -derived conjugate of a capsular polysaccharide and a cell wall-derived material and a carrier protein are characterized in that the structure is conjugated in any one of the following splicing sequences, immunogenic composition:
    a) 협막 다당류-운반체 단백질-세포벽 유래 물질; a) capsular polysaccharide-carrier protein-cell wall-derived material;
    b) 운반체 단백질-협막 다당류- 세포벽 유래 물질; 및 b) carrier protein-capsular polysaccharide-cell wall-derived material; and
    c) 협막 다당류-세포벽 유래 물질-운반체 단백질.c) Capsular polysaccharide-cell wall-derived material-carrier protein.
  17. 제16항에 있어서, 17. The method of claim 16,
    상기 세포벽 유래 물질은 운반 단백질과 직접적으로 결합하지 않음을 특징으로 하는, 면역원성 조성물.An immunogenic composition, characterized in that the cell wall-derived material does not directly bind to a carrier protein.
  18. 제16항에 있어서, 17. The method of claim 16,
    상기 접합 순서 a), b), 또는 c)의 각 구성성분 간에 링커를 추가적으로 포함함을 특징으로 하는, 면역원성 조성물.An immunogenic composition, characterized in that it further comprises a linker between each component of the conjugation sequence a), b), or c).
  19. 제16항에 있어서, 17. The method of claim 16,
    상기 접합 순서 b)로 접합된 구조는 선형(linear) 또는 비선형(Non-linear) 중 어느 하나임을 특징으로 하는, 면역원성 조성물The immunogenic composition, characterized in that the structure conjugated in the conjugation sequence b) is either linear or non-linear.
  20. 제16항에 있어서, 17. The method of claim 16,
    상기 협막 다당류와 세포벽 유래 물질의 접합체와 운반체 단백질 간 접합 방법은 CDAP 접합 방법, 리덕티브 아미네이션 방법 및 티올-말레마이드(Thiol-Malemide) 방법으로 이루어진 군으로부터 선택되는 어느 하나 이상임을 특징으로 하는, 면역원성 조성물.The conjugation method between the conjugate of the capsular polysaccharide and the cell wall-derived material and the carrier protein is characterized in that at least one selected from the group consisting of a CDAP conjugation method, a reductive amination method, and a Thiol-Malemide method, Immunogenic composition.
  21. 제16항에 있어서, 17. The method of claim 16,
    상기 접합 순서 a)는 하기 단계를 포함하는 방법으로 접합됨을 특징으로 하는, 면역원성 조성물:Immunogenic composition, characterized in that the conjugation sequence a) is conjugated in a way comprising the steps of:
    i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 이를 활성화시키는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to activate it;
    ii) 세포벽 유래 물질에 CDAP를 가하여, 운반체 단백질과 연결하는 단계; 및 ii) adding CDAP to the cell wall-derived material and linking with a carrier protein; and
    iii) 상기 i)의 협막 다당류를 상기 ii)의 운반체 단백질의 히드록실기 또는 아미노기와 연결하는 단계.iii) linking the capsular polysaccharide of i) with a hydroxyl group or an amino group of the carrier protein of ii).
  22. 제16항에 있어서, 17. The method of claim 16,
    상기 접합 순서 b)는 하기 단계를 포함하는 방법으로 접합됨을 특징으로 하는, 면역원성 조성물:Immunogenic composition, characterized in that the conjugation sequence b) is conjugated in a method comprising the steps of:
    i) 세포벽 유래 물질에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 협막 다당류와 연결하는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the cell wall-derived material to link it with capsular polysaccharide;
    ii) 운반체 단백질에 CDAP를 가하여 이를 활성화시키는 단계; 및 ii) adding CDAP to the carrier protein to activate it; and
    iii) 상기 i)의 협막 다당류를 상기 ii)의 운반체 단백질의 히드록실기 또는 아미노기와 연결하는 단계.iii) linking the capsular polysaccharide of i) with a hydroxyl group or an amino group of the carrier protein of ii).
  23. 제16항에 있어서, 17. The method of claim 16,
    상기 접합 순서 b)는 하기 단계를 포함하는 방법으로 접합됨을 특징으로 하는, 면역원성 조성물:Immunogenic composition, characterized in that the conjugation sequence b) is conjugated in a method comprising the steps of:
    i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 운반체 단백질과 연결하는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to link it with a carrier protein;
    ii) 세포벽 유래 물질에 CDAP를 가하여 이를 활성화시키는 단계; 및 ii) adding CDAP to the cell wall-derived material to activate it; and
    iii) 상기 i)의 협막 다당류를 상기 ii)의 세포벽 유래 물질과 연결하는 단계.iii) linking the capsular polysaccharide of i) with the cell wall-derived material of ii).
  24. 제16항에 있어서, 17. The method of claim 16,
    상기 접합 순서 c)는 하기 단계를 포함하는 방법으로 접합됨을 특징으로 하는, 면역원성 조성물:Immunogenic composition, characterized in that the conjugation sequence c) is conjugated in a method comprising the steps of:
    i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 이를 활성화시키는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to activate it;
    ii) 세포벽 유래 물질에 CDAP를 가하여, 운반체 단백질과 연결하는 단계; 및 ii) adding CDAP to the cell wall-derived material and linking with a carrier protein; and
    iii) 상기 i)의 협막 다당류를 상기 ii)의 세포벽 유래 물질과 연결하는 단계.iii) linking the capsular polysaccharide of i) with the cell wall-derived material of ii).
  25. 제1항에 있어서, According to claim 1,
    애주번트를 추가로 포함함을 특징으로 하는, 면역원성 조성물.An immunogenic composition, characterized in that it further comprises an adjuvant.
  26. 제25항에 있어서, 26. The method of claim 25,
    상기 애주번트는 알루미늄 염임을 특징으로 하는, 면역원성 조성물.The immunogenic composition, characterized in that the adjuvant is an aluminum salt.
  27. 제26항에 있어서, 27. The method of claim 26,
    상기 알루미늄 염은 알루미늄 포스페이트, 알루미늄 설페이트 및 알루미늄 하이드록사이드로 이루어진 군으로부터 선택되는 어느 하나임을 특징으로 하는, 면역원성 조성물.The aluminum salt is characterized in that any one selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide, the immunogenic composition.
  28. 제1항에 있어서, 상기 면역원성 조성물은 0.1 내지 100 ㎍ 용량의 다당류를 포함함을 특징으로 하는, 면역원성 조성물.The immunogenic composition of claim 1, wherein the immunogenic composition comprises a dose of 0.1 to 100 μg of polysaccharide.
  29. 제1항 내지 제28항 중 어느 한 항에 따른 면역원성 조성물의 면역학적 유효량을 포함하는, 스트렙토코커스 뉴모니애 협막 다당류 접합체에 대한 면역 반응을 유도하기 위한 약학 조성물.29. A pharmaceutical composition for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate comprising an immunologically effective amount of the immunogenic composition according to any one of claims 1 to 28.
  30. 제1항 내지 제28항 중 어느 한 항에 따른 면역원성 조성물을 제조하는 방법.29. A method for preparing an immunogenic composition according to any one of claims 1-28.
  31. 제30항에 있어서, 31. The method of claim 30,
    상기 스트렙토코커스 뉴모니애(Streptococcus pneumoniae) 유래의 협막 다당류(capsular polysaccharide)와 세포벽 유래 물질의 접합체와 운반체 단백질은 하기 접합 순서 중 어느 하나로 접합됨을 특징으로 하는, 면역원성 조성물을 제조하는 방법:The Streptococcus pneumoniae -derived conjugate of a capsular polysaccharide and a cell wall-derived material and a carrier protein are conjugated in one of the following conjugation sequences, characterized in that the method for preparing an immunogenic composition:
    a) 협막 다당류-운반체 단백질-세포벽 유래 물질; a) capsular polysaccharide-carrier protein-cell wall-derived material;
    b) 운반체 단백질-협막 다당류-세포벽 유래 물질; 및 b) carrier protein-capsular polysaccharide-cell wall-derived material; and
    c) 협막 다당류-세포벽 유래 물질-운반체 단백질.c) Capsular polysaccharide-cell wall-derived material-carrier protein.
  32. 제31항에 있어서, 32. The method of claim 31,
    상기 접합 순서 a), b), 또는 c)의 각 구성성분 간에 링커를 추가적으로 포함함을 특징으로 하는, 면역원성 조성물을 제조하는 방법.A method for preparing an immunogenic composition, characterized in that it further comprises a linker between each component of the conjugation sequence a), b), or c).
  33. 제31항에 있어서, 32. The method of claim 31,
    상기 협막 다당류와 세포벽 유래 물질의 접합체와 운반체 단백질 내 접합 방법은 CDAP 접합 방법, 리덕티브 아미네이션 방법 및 티올-말레마이드(Thiol-Malemide) 방법으로 이루어진 군으로부터 선택되는 어느 하나 이상임을 특징으로 하는, 면역원성 조성물을 제조하는 방법.The method for conjugating the conjugate of the capsular polysaccharide to the cell wall-derived material and the carrier protein is at least one selected from the group consisting of a CDAP conjugation method, a reductive amination method, and a Thiol-Malemide method, characterized in that A method of making an immunogenic composition.
  34. 제31항에 있어서, 32. The method of claim 31,
    상기 접합 순서 a)는 하기 단계를 포함하는 방법으로 접합됨을 특징으로 하는, 면역원성 조성물을 제조하는 방법:Method for preparing an immunogenic composition, characterized in that the conjugation sequence a) is conjugated in a method comprising the steps of:
    i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 이를 활성화시키는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to activate it;
    ii) 세포벽 유래 물질에 CDAP를 가하여, 운반체 단백질과 연결하는 단계; 및 ii) adding CDAP to the cell wall-derived material and linking with a carrier protein; and
    iii) 상기 i)의 협막 다당류를 상기 ii)의 운반체 단백질의 히드록실기 또는 아미노기와 연결하는 단계.iii) linking the capsular polysaccharide of i) with a hydroxyl group or an amino group of the carrier protein of ii).
  35. 제31항에 있어서, 32. The method of claim 31,
    상기 접합 순서 b)는 하기 단계를 포함하는 방법으로 접합됨을 특징으로 하는, 면역원성 조성물을 제조하는 방법:Method for preparing an immunogenic composition, characterized in that the conjugation sequence b) is conjugated in a method comprising the steps of:
    i) 세포벽 유래 물질에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 협막 다당류와 연결하는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the cell wall-derived material to link it with capsular polysaccharide;
    ii) 운반체 단백질에 CDAP를 가하여 이를 활성화시키는 단계; 및 ii) adding CDAP to the carrier protein to activate it; and
    iii) 상기 i)의 협막 다당류를 상기 ii)의 운반체 단백질의 히드록실기 또는 아미노기와 연결하는 단계.iii) linking the capsular polysaccharide of i) with a hydroxyl group or an amino group of the carrier protein of ii).
  36. 제31항에 있어서, 32. The method of claim 31,
    상기 접합 순서 b)는 하기 단계를 포함하는 방법으로 접합됨을 특징으로 하는, 면역원성 조성물을 제조하는 방법:Method for preparing an immunogenic composition, characterized in that the conjugation sequence b) is conjugated in a method comprising the steps of:
    i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 운반체 단백질과 연결하는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to link it with a carrier protein;
    ii) 세포벽 유래 물질에 CDAP를 가하여 이를 활성화시키는 단계; 및 ii) adding CDAP to the cell wall-derived material to activate it; and
    iii) 상기 i)의 협막 다당류를 상기 ii)의 세포벽 유래 물질과 연결하는 단계.iii) linking the capsular polysaccharide of i) with the cell wall-derived material of ii).
  37. 제31항에 있어서, 32. The method of claim 31,
    상기 접합 순서 c)는 하기 단계를 포함하는 방법으로 접합됨을 특징으로 하는, 면역원성 조성물을 제조하는 방법:A method for preparing an immunogenic composition, characterized in that the conjugation sequence c) is conjugated in a method comprising the steps of:
    i) 협막 다당류에 CDAP(1-시아노-4-디메틸아미노 피리디늄 테트라플루오로보레이트)를 가하여 이를 활성화시키는 단계;i) adding CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to the capsular polysaccharide to activate it;
    ii) 세포벽 유래 물질에 CDAP를 가하여, 운반체 단백질과 연결하는 단계; 및 ii) adding CDAP to the cell wall-derived material and linking with a carrier protein; and
    iii) 상기 i)의 협막 다당류를 상기 ii)의 세포벽 유래 물질과 연결하는 단계.iii) linking the capsular polysaccharide of i) with the cell wall-derived material of ii).
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