WO2021009633A1 - Vaccin à chromatine synthétique - Google Patents

Vaccin à chromatine synthétique Download PDF

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WO2021009633A1
WO2021009633A1 PCT/IB2020/056493 IB2020056493W WO2021009633A1 WO 2021009633 A1 WO2021009633 A1 WO 2021009633A1 IB 2020056493 W IB2020056493 W IB 2020056493W WO 2021009633 A1 WO2021009633 A1 WO 2021009633A1
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chromatin
scv
antigen
vaccine
immune response
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PCT/IB2020/056493
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English (en)
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Jeong Park
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Massey University
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Priority claimed from AU2019902472A external-priority patent/AU2019902472A0/en
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Publication of WO2021009633A1 publication Critical patent/WO2021009633A1/fr

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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
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001164—GTPases, e.g. Ras or Rho
    • 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
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • A61K2039/6025—Nucleotides
    • 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

Definitions

  • the present invention relates generally to a synthetic chromatin vaccine (SCV) that can be used as an immunostimulatory agent, particularly as a vaccine or adjuvant or both, in a treatment and/or prophylactic setting.
  • SCV synthetic chromatin vaccine
  • Vaccines have been produced traditionally using live or attenuated
  • microorganisms or various preparations of their components; e.g., cell wall fractions or isolated or recombinantly produced peptides.
  • immunization using whole cells of pathogens can produce long lasting immunity but carries, among other risks, major safety risks related to causing autoimmune or strong allergic responses 1 .
  • Whole protein preparations also suffer from the potential to generate autoimmunity, as well as problems associated with protein purity and stability, large scale protein expression difficulties, difficulties with the introduction of desired post-translational modification ⁇ e.g. glycosylation) into recombinant proteins and poor or undesired immune responses (inflammation, autoimmunity, etc.) 1 .
  • a chemically defined peptide vaccine has several advantages over
  • the invention relates to a synthetic chromatin vaccine
  • nucleosomes comprising an amino acid sequence of at least one histone protein fused to an amino acid sequence of at least one peptide antigen.
  • the invention in another aspect relates to a composition
  • a composition comprising a
  • the invention in another aspect relates to a method of stimulating an immune response in a subject comprising administering a synthetic chromatin vaccine as described herein or a composition comprising a synthetic chromatin vaccine as described herein to the subject.
  • the invention relates to the use of a synthetic chromatin vaccine as described herein in the manufacture of a medicament for stimulating an immune response in a subject.
  • the invention in another aspect relates to a synthetic chromatin vaccine or composition comprising a synthetic chromatin vaccine as described herein for stimulating an immune response in a subject.
  • FIG. l provides a schematic illustration of a synthetic chromatin vaccine as described herein and its characterization.
  • A. Following assembly with pUC19/16x601, individual histones were incorporated in the multiple nucleosome array at the interval of approximately 200 base pairs of DNA. Individual mononucleosomes displayed each N- terminal tail of histones outside of the core wherein fused peptides of interest are connected and exposed on the surface.
  • Ras oncogene G12V
  • Resultant histone octamers were used to prepa re the G12V peptide-tagged chromatin using plasmid DNA. Properly assembled chromatin was confirmed by micrococcal nuclease (MNase) assay and transmission electron microscopy (TEM) . The synthetic chromatin vaccine was administered to mice as described herein to measure antibody and cytokine responses.
  • MNase micrococcal nuclease
  • TEM transmission electron microscopy
  • FIG. 1 illustrates the preparation of octameric histones displaying G12V
  • FIG. 3 illustrates the analysis of assembled synthetic chromatin vaccine by micrococcal nuclease (M Nase) assay.
  • FIG. 4 shows transmission electron microscopic (TEM) images of G12V
  • FIG. 5 shows the antibody responses to the G12V peptide displayed on
  • chromatin i .e., a synthetic chromatin vaccine as described herein
  • B. Antibody response in mice administered vaccines without added adjuvant (Trial 1). Groups of mice (n 6) were vaccinated with DNA, WT chromatin, G12V histone, G12V chromatin (SCV). Antibody responses were measured in sera using histone-coated ELISA plates.
  • C. Anti-histone antibody responses in mice administered with vaccines co-formulated with adj uvants DDA and MPL (Trial 2). Groups of mice (n 6) were vaccinated with DDA/MPL alone, G12V histone mixed with DDA/M PL, G12 chromatin mixed with DDA/M PL. Antibody responses were measured in sera using histone-coated ELISA plates.
  • FIG. 6 illustrates that vaccination with G12V-tagged chromatin does not induce cytokine responses.
  • Splenocytes from the sacrificed mice in trial 2 were stimulated by addition of PBS (control) or G12V histones. Secretion of cytokines were measured by cytometric bead array.
  • Panels A to Panel F IFN-g, TNF, IL-2, IL-6, IL-10, IL- 17A) .
  • synthetic and grammatical variations thereof as used herein to define a synthetic chromatin vaccine means that the chromatin in the array, which comprises proteins and DNA, has been produced synthetically in vitro by a self-assembly process from biological components that have been isolated, purified or chemically synthesized, and not obtained as a chromatin fiber from nature or produced as isolated and purified chromatin fibers from a natural source.
  • the DNA and proteins comprised in the synthetic chromatin vaccine described herein may comprise certain features that may be found together in nature, the DNA and proteins comprised in the synthetic chromatin vaccine described herein as a whole are not found together in nature.
  • chromatin is used herein as known in the art and refers to a
  • nucleosome and nucleosome structure
  • nucleosome or nucleosome structure in the context of this disclosure includes not only multi-nucleosome units but also monomeric nucleosomes.
  • the phrase "stimulate an immune response" and grammatical variations thereof as used herein means any measurable or observable increase in an amount or level of immune stimulation as measured by an increase in antibody production and/or cytokine production by the subject that is attributable to the SCV vaccination relative to the level of immune stimulation (i.e., antibody or cytokine production) in an appropriate control subject; e.g., placebo or non-active agent.
  • a "suitable control subject” as used herein is an appropriate control subject (untreated or treated with a defined control treatment) according to art-accepted criteria for monitoring a disease or condition.
  • the stimulation of the immune system following administration of a synthetic chromatin vaccine as described herein is a statistically significant stimulation relative to an appropriate control subject (untreated or treated with a defined control treatment) according to art-accepted criteria for monitoring a disease or condition.
  • statically significant as used herein describes a mathematical measure of difference between groups. The difference is said to be statistically significant if it is greater than what might be expected to happen by chance alone.
  • antigen refers to a molecule that contains one or more epitopes (linear, overlapping, conformational or a combination of these) that, upon exposure to a subject, can induce an immune response in the subject, that is specific for that antigen.
  • epitope refers to an antigenic determinant, i.e. a point of
  • an antigen for specific antibodies or an antigenic determinant, for example, that is presented on an MHC molecule and recognized by a T-cell receptor.
  • An antigen may contain more than one antigenic determinant.
  • a peptide antigen as described herein can be any number of amino acid
  • a fusion protein comprising the amino acid sequence encoding at least one histone protein, wherein upon expression, the histone protein-peptide antigen fusion protein forms histone octamers that form nucleosomes that form chromatin.
  • a peptide antigen may be derived from a protein which is a suitable target for prophylactic or therapeutic vaccines.
  • a peptide antigen "derived from" a target protein is to be understood herein as to comprise a contiguous amino acid sequence selected from the target protein, which, while preserving its antigenic properties, may be modified by deletion or substitution of one or more amino acids, by extension at the N- and/or C- terminus with additional amino acids or functional groups.
  • cancer associated antigen and “cancer specific antigen” when used in reference to an antigen as described herein refers to a cancer antigen that is known in the art to cause or be associated with the production, by the body's immune system, of an antibody that specifically or selectively binds to at least one epitope of the cancer antigen.
  • cancer associated antigen and “cancer specific antigen” as used herein mean a synthetic peptide antigen that comprises the amino acid sequence of at least one epitope of a protein expressed from a cancer associated or cancer specific gene.
  • composition encompasses a
  • compositions are prepared by bringing the active agent into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • Said compositions are prepared according to conventional mixing, granulating, or coating methods, respectively, and contain a percentage (%) of the active ingredient and can be determined by a skilled worker in view of the art.
  • pharmaceutically acceptable excipient or “pharmaceutically acceptable carrier” it is meant that the excipient or carrier must be compatible with the other ingredients of the formulation and not harmful to the subject to whom the composition is administered.
  • Excipients include but are not limited to sterile liquids such as water and oils, including animal, vegetable, synthetic or petroleum oils, saline solutions, aqueous dextrose and glycerol solutions, starch glucose, lactose, sucrose, gelatin, sodium stearate, glycerol monostearate, sodium chloride, propylene glycol, ethanol, wetting agents, emulsifying agents, binders, dispersants, thickeners, lubricants, pH adjusters, solubilizers, softening agents, surfactants and the like.
  • the compositions of the invention can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders and sustained-release formulations. Examples of suitable
  • compositions as described herein in an amount that does not impair the activity of the SCV as described herein, or of a composition comprising a synthetic chromatin vaccine as described herein.
  • the term "effective amount” refers to a sufficient quantity of the synthetic chromatin vaccine, in a suitable composition, and in a suitable dosage form to stimulate the immune system to obtain a measurable or observable result as compared to a suitable control subject.
  • the measurable or observable result is an increase in antibody production by the subject in response to the administration of the synthetic chromatin vaccine.
  • a "therapeutically effective amount” will typically stimulate the immune system to provide at least some level of treatment and/or prophylaxis of the desired disease conditions in the subject in addition to providing for the measurable or observable result discussed above.
  • a person skilled in the art will be able to formulate a synthetic chromatin vaccines described herein as a composition, particularly a pharmaceutical composition, by determining an appropriate mode of use, application and/or administration of the composition with reference to the literature and as described herein, and then formulating the composition for such mode with reference to the literature and as described herein.
  • administration refers to placement of the composition or compound of the invention into a subject by a method appropriate to result in an immune response.
  • the dosage form is selected and used as appropriate depending on the therapeutic purpose and the subject.
  • the dose of the composition of the invention may be selected depending on the therapeutic purpose and the cha racteristics of the subject including their age, sex, general health and disease progression.
  • the compound of the invention may be administered in a dose of 0.01 to 100 mg, preferably 0.1 to 50 mg per day, per kg of body weight, either once or divided over several administrations.
  • indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, “a bout 100” means from 90 to 110 and “about six” means from 5.4 to 6.6.
  • a nucleosome is a basic repeating unit of chromatin in the euka ryotic
  • the single nucleosome pa rticle consists of DNA of 147 base pairs wrapping around a histone octameric core containing two copies of four different histones, H3, H4, H2A and H2B 6 ' 7 .
  • Each nucleosome particle is connected by a linker DNA, which results in a 10 nanometer (nm) fiber in diameter with the appearance of "beads-on-a-string”.
  • the nucleosome array shows repeated nucleosome particles at every 200 base pairs of DNA.
  • the N-terminal histone tails are rich in basic a mino acids, such as lysine and arginine, and exposed outside of the nucleosome core pa rticle 8 ' 9 .
  • the intrinsic flexibility and surface exposure from the 10 nm chromatin fiber make the histone tails subjected to efficient modifications by diverse chromatin remodeling and modifying enzymes in cells.
  • the nucleosome array is naturally stable for several months 10 . All N-terminal tails of histones, except H2B, are dispensable for the chromatin assembly, suggesting that the N- terminal tails can be significantly altered in the nucleosomal array 11 .
  • the supercoiled DNA for the nucleosomal assembly can be any type of DNA.
  • nucleosome positioning sequences derived from the artificial 601 widom repeat or 5S rRNA gene have been shown to enhance added stability and regularity to the nucleosome array 14 .
  • Recombinant histone prepa rations from E. coli inclusion bodies and chromatin self- assembly using salt gradient dialysis method have been well-established previously 1045 .
  • the use of entirely synthetic chromatins assembled in vitro has been limited to academic investigations for elucidating chromatin structure and for studying epigenetic chromatin modifications and their effects on cells.
  • the "synthetic chromatin” of the invention and as described herein comprises a peptide segment of 17 amino acids derived from ras oncogene (G12V) displayed on the surface of chromatin.
  • the ras gene was chosen because the codon 12 mutation accounts for the most frequent oncogenic ras mutations in human cancers and the mutated peptides derived from ras oncogene are known to be presented on both MHC class I and II molecules as a tumor-specific antigen 16-19 . This makes mutant ras peptides attractive antigens for a therapeutic cancer vaccine and several mutant ras peptide vaccines have been clinically tested to treat advanced cancer patients 17,19 .
  • a synthetic chromatin vaccine as described herein induces a stronger
  • a synthetic chromatin vaccine as described herein is useful for stimulating the immune system in a T-cell independent manner by engaging and activating B-cells (Fig. 7).
  • the inventors also believe that the synthetic chromatin vaccine described herein is useful for the manufacture of a medicament for stimulating the immune system, particularly in a T-cell independent manner.
  • Example 1 details the preparation of a synthetic chromatin vaccine and the use of the synthetic chromatin vaccine as an immunostimulatory agent in mice.
  • a synthetic chromatin vaccine as described herein can be designed to display a number of different peptides, including peptide antigens.
  • the choice of peptide is believed to be within the skill of those in the art when considering the use to which the synthetic chromatin vaccine as described herein is to be put.
  • the invention relates to a synthetic chromati n vaccine comprising nucleosomes comprising an amino acid sequence of at least one histone protein fused to the amino acid sequence of at least one peptide antigen .
  • the at least one histone is translationally fused to the amino acid sequence of the at least one peptide antigen.
  • the peptide antigen comprises from 2 to 100 amino acid residues, preferably from 5 to 75, from 10 to 50, from 15 to 25, preferably from 15 to 20 amino acid residues.
  • the peptide antigen comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 65, 70, 75, 80,. 85, 90, 95 or 100 amino acids.
  • the peptide antigen comprises more than 100 amino acid residues.
  • the peptide antigen comprises 10 to 30 amino acid
  • the peptide antigen comprises 12 to 25 amino acid
  • the peptide antigen comprises 15 to 20 amino acid
  • the peptide antigen comprises 16 to 18, preferably 17 amino acid residues.
  • the peptide antigen is a cancer-associated antigen.
  • the peptide antigen is a cancer-specific antigen.
  • the cancer-associated or cancer-specific antigen is a ras antigen .
  • the ras antigen comprises the amino acid sequence KLVVVGAVGVGKSALTI (SEQ ID NO: 1).
  • the ras antigen consists essentially of, or consists of SEQ ID NO: 1.
  • the at least one histone protein is H3, H4, H2A or H2B.
  • the synthetic chromatin vaccine comprises histone
  • the histone octamers comprise a mixture of H3, H4, H2A and H2B.
  • the histone octamers are hybrid octamers comprising at least one wild type histone and at least one G12V-tagged histone.
  • the wild type histones are H3, H4 or a combination of both.
  • the at least one G12V-tagged histone is G12V-tagged H2A (G12V-H2A) or G12V-tagged H2B (G12V-H2B).
  • the hybrid octamers comprise wild type histones H3 and H4 and G12V-tagged histones G12V-H2A and G12V-H2B.
  • composition comprising a
  • the composition is a pharmaceutical composition .
  • composition or pharmaceutical composition comprises the synthetic chromatin vaccine and an adjuvant.
  • the adjuvant stimulates a T cell independent immune response of the synthetic chromatin vaccine.
  • the adjuvant is diethyldioctodecylammonium bromide (DDA) or monophosphoryl lipid A (MPL).
  • DDA diethyldioctodecylammonium bromide
  • MPL monophosphoryl lipid A
  • the carrier, diluent or excipient is a pharmaceutically acceptable carrier, diluent or excipient.
  • composition or pharmaceutical composition comprises an effective amount of the synthetic chromatin, preferably a prophylactically and/or therapeutically effective amount of the synthetic chromatin .
  • composition or pharmaceutical composition consists essentially of an effective amount of the synthetic chromatin, preferably a prophylactically and/or therapeutically effective amount of the synthetic chromatin.
  • the effective amount is an a mount of the synthetic
  • chromatin vaccine that, when administered to a subject, stimulates the immune system by at least 5%, preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, preferably at least 99% as compared to a suitable control subject.
  • An acceptable carrier, diluent, or excipient, particularly a pharmaceutically acceptable carrier, diluent or excipient may be selected as known in the art, in view of a planned manner of use, application and/or administration.
  • the invention in another aspect relates to a method of stimulating an immune response in a subject comprising administering a synthetic chromatin vaccine as described herein or a composition comprising a synthetic chromatin vaccine as described herein to the subject.
  • the subject is identified as being a subject that would benefit from stimulation of an immune response.
  • the synthetic chromatin vaccine is administered in one embodiment.
  • the synthetic chromatin vaccine is administered
  • the additional immunostimulatory agent is an antigen that stimulates an immune response.
  • the antigen stimulates a specific immune response to the antigen.
  • the antigen stimulates an immune response in a subject that has a T cell immunodeficiency.
  • the cause of the T cell immunodeficiency is selected from the group consisting of treatment of the subject for cancer by chemotherapy or radiotherapy or both, acquired immune deficiency syndrome (AIDS), and Inherited T- Cell Deficiency Disorders.
  • AIDS acquired immune deficiency syndrome
  • the additional immunostimulatory agent is an adjuvant.
  • the additional immunostimulatory agent is an adjuvant that stimulates the T cell independent immune response of the synthetic chromatin vaccine.
  • the adjuvant stimulates a general or non-specific immune response.
  • the adjuvant is diethyldioctodecylammonium bromide (DDA) or monophosphoryl lipid A (MPL).
  • DDA diethyldioctodecylammonium bromide
  • MPL monophosphoryl lipid A
  • the immune response to the SCV is a T cell independent response.
  • the T cell independent response is a T cell independent antibody response.
  • the immune response to the SCV is a B cell response.
  • stimulating an immune response is stimulating a T cell independent immune response.
  • stimulating a T cell independent immune response is stimulating a T cell independent antibody response.
  • administration is systemic or local administration.
  • administration is parenteral administration.
  • parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration.
  • a particular and effective dosage regime according to a method of stimulating an immune response according to the invention will be dependent on the desired level of stimulation to be achieved during the course of treatment, and on the responsiveness of the treated subject to the course of treatment.
  • An effective treatment may last from several hours to several days to several months, or until an acceptable therapeutic outcome is affected or assured or until an acceptable stimulation of the immune response in the subject is observed.
  • An optimal dosing schedule may be calculated from drug accumulation as measured in the body of a treated subject. It is believed to be within the skill of persons in the art to be able to easily determine optimum and/or suitable dosages, dosage formulations and dosage regimes. Of course, the optimum dosages may vary depending on the relative potency of the antibacterial combination or composition as described herein, but will be estimable from an EC50s found to be effective in suitable cells in vitro and in an appropriate in vivo animal model. In general, dosage is from 0.00001 g to 99 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, but not limited thereto.
  • the subject is an animal, preferably a mammal.
  • the mammal is selected from the group consisting of canines, felines, bovines, ovines, cervines, caprines, porcines, lagomorphs, rodents, camelids and hominids.
  • the mammal is selected from the group consisting of cats, dogs, rats, stoats, ferrets, possums, guinea pigs, mice, hamsters, zebra, elephants, lions, tigers, cheetah, monkeys, apes, macaques, tarsiers, lemurs, giraffes, prairie dogs, meerkats, bears, otters, tapiers, cows, horses, pigs, sheep, goats, deer, minks, hippopotami and humans.
  • the mammal is a human .
  • the method comprises administering the synthetic compound
  • the heterologous prime-boost regimen comprises
  • the second vaccine is a different vaccine that stimulates a T cell response.
  • the synthetic chromatin vaccine is administered before the second vaccine.
  • the second vaccine is administered before the synthetic chromatin vaccine.
  • heterologous prime boost regime enhances an
  • the invention relates to the use of a synthetic chromatin vaccine as described herein in the manufacture of a medicament for stimulating the immune system in a subject.
  • the medicament comprises an effective amount of the synthetic chromatin vaccine.
  • the effective amount is a
  • the effective amount comprises an amount of a synthetic chromatin vaccine as described herein that is effective at stimulating an immune response in a subject, preferably in a T cell independent manner.
  • the medicament comprises about 100 pg/ml, preferably about 90 pg/mL, about 80 pg/mL, about 70 pg/mL, about 60 pg/mL, about 50 pg/mL, about 40 pg/mL, about 30 pg/mL, about 20 pg/mL, about 10 pg/mL, about 5 pg/mL, preferably about 1 pg/mL of the synthetic chromatin vaccine.
  • the medicament comprises at least one additional immunostimulatory agent.
  • the medicament comprises an effective amount of the additional immunostimulatory agent.
  • the effective amount of the additional immunostimulatory agent is a therapeutically effective amount.
  • the additional immunostimulatory agent is an antigen or adj uvant as described herein.
  • the medicament comprises about 100 pg/ml, preferably about 90 pg/mL, about 80 pg/mL, about 70 pg/mL, about 60 pg/mL, about 50 pg/mL, about 40 pg/mL, about 30 pg/mL, about 20 pg/mL, about 10 pg/mL, about 5 pg/mL, preferably about 1 pg/mL of the additional immunostimulatory agent.
  • the medicament consists essentially of an effective
  • the effective amount of the synthetic chromatin vaccine and of the additional immunostimulatory agent is a therapeutically effective amount.
  • the medicament is formulated for administration, or is in a form for administration, to a subject in need thereof.
  • the medicament is in a form for, or is formulated for parenteral administration.
  • pa renteral administration is selected from the g roup consisting of di rect application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration.
  • the medicament is for, is formulated for, or is in a form for administration separately, simultaneously or sequentially with the additional immunostimulatory agent.
  • the medicament comprises a synthetic chromatin vaccine as described herein and an additional antigen or adjuvant, wherein the medicament is for, is formulated for, or is in a form for separate, simultaneous or sequential administration of the components in the combination to a subject.
  • the medicament comprises a synthetic chromatin vaccine as described herein and an antigen or adjuvant, wherein the medicament is for, is formulated for, or is in a form for administration to a subject that has shown a nonresponse or reduced response to a prior treatment.
  • the invention in another aspect relates a synthetic chromatin vaccine or composition comprising a synthetic chromatin vaccine as described herein for stimulating an immune response in a subject.
  • the pET3 expression vectors to express Xenopus laevis histones have been extensively used previously for wild type histone expression and purification 20 .
  • Addition of mutant G12V ras peptide (K 5 LVVVGAV 12 GVGKSALTI 21 ) (SEQ ID NO: 1) to the second codon of histone genes was performed using PCR-based mutagenesis and confirmed by DNA sequencing.
  • the modified histone genes can be prepared by a gene synthesis (GenScript Biotech Corp) from the sequences as shown in Table 1.
  • the mutant G12V ras peptide is shown capital letters.
  • Insoluble inclusion bodies were obtained by expressing each histone gene in BL21 (DE3) CodonPlus stain (Stratagene) using twice concentrated Luria broth (sigma) supplemented with 0.5% glucose (w/v). Induction conditions of histone expression, inclusion body preparation and two-step column purification procedures using Sephacryl S200 (GE Healthcare) and SP sepharose Fast Flow (GE Healthcare) were described previously 11 " 20 ' 21 .
  • G12V-histones Purified recombinant core histones and their G12V peptide tagged variants (G12V-histones) were dissolved in the unfolding buffer (8M Guanidine- HCI, 20 mM sodium acetate, pH 5.2, 10 mM DTT) and reconstituted to histone octamers containing wild type, G12V histones or a hybrid of wild type and G12V histones. Refolding and purification of histone octamers were performed as described previously using Superdex 200 gel filtration chromatography (HighLoad 16/600, GE Heathcare) 11 ' 20 ' 21
  • DNA containing 16 repeats of widom 601 sequence was previously described 10 ' 22 .
  • Supercoiled plasmid DNA was purified using conventional CsCI gradient ultracentrifugation.
  • various histones to DNA mass ratios were screened using microscale salt dilution procedure 23 .
  • Large-scale assembly of up to 60 pg of chromatin was done in the dialysis membrane using a step-gradient dialysis as described previously 24 . The quality of this synthetic nucleosome array was assessed by Micrococcal
  • Synthetic chromatin was dialyzed against a low salt buffer (10 mM Tris-HCI, pH 7.5, 1 mM EDTA, 2.5 mM NaCI, 10 mM b-merca ptoethanol) and adjusted to a final concentration of 10 ng/mI with distilled water.
  • the chromatin fiber was fixed with 3.7% formaldehyde in 10 mM NaC03 buffer (pH 8.5) for 10 min and dialyzed against distilled water containing 10 mM b-mercaptoethanol.
  • a formvar treated, carbon coated TEM grid was impregnated with the chromatin sample for 5 min and rinsed with water. After sequential staining with 2% uranyl acetate in water and 1% phosphotungstic acid in 70% ethanol, the grid was air dried and examined by FEI Tecnai G2 Biotwin
  • mice were purchased from a commercial small animal supplier and immunized at between 6 - 8 weeks of age.
  • mice Prior to vaccination, mice were anaesthetised using sub-cutaneous injection of ketamine and medetomidine. Immediately after vaccination, the anaethesia was reversed using atipamezole.
  • DDA diethyldioctodecylammonium bromide
  • MPL monophosphoryl lipid A
  • G12V chromatin 2 pg of each DNA and hybrid histone octamer
  • mice were anaesthetised using
  • ketamine/medetomadine and blood was collected from each mouse by cardiac puncture. The blood was allowed to clot and centrifuged prior to collecting serum. Mice were euthanised and spleens removed for culture of splenocytes using methods previously reported 28 . Splenocytes were cultured at 37°C a nd 10% C02 in air and stimulated with phosphate buffered sali ne (PBS, control), Concanavlin A (positive control, Sigma), G12V peptides, or G12V histones, each with a final concentration of 5 pg/ml .
  • PBS phosphate buffered sali ne
  • Concanavlin A positive control, Sigma
  • G12V peptides or G12V histones
  • Splenocyte culture supernatants were removed after 3 days incubation and frozen at -20°C until assayed.
  • Levels of IFN-g, IL-2, IL-4, IL-6, IL-10, IL-17A and TNF were measured using a cytometric bead array (CBA; mouse Th l-Th2 cytokine kit: BD Biosciences) according to the manufacturer's instructions. Fluorescence was measured using a FACSVerse flow cytometer (BD Biosciences) and analysed using FCAP array softwa re (BD Biosciences) . Results for all cytokines were calculated as the cytokine value of the antigen-stimulated sample minus that of the PBS-stimulated sample.
  • the level of significance was set at a P value of ⁇ 0.05.
  • Reconstituted octamers containing wild type histones were purified using Superdex 200 gel filtration chromatog raphy at the elution volume, 64 ml (Fig . 2B, upper panel).
  • hybrid octamers were prepa red by mixing wild type histones (FH3 and H4) and G12V-tagged histones (G12V-FI2A and G12V- H2B) .
  • the elution volume of hybrid octamer was 70 ml, indicating that the hybrid octamer forms more compact octamers, probably resulting from hydrophobic G12V peptide sequence.
  • Purified octamers were adj usted to 1 mg/ml of final concentration and analyzed the staining intensity using Coomassie brilliant blue G250 before chromatin assembly (Fig . 2C).
  • the MNase digestion pattern with 200 base pair of DNA ladder was clearly observed from the chromatin from hybrid histone octamer of G12V-histones (Fig . 3, lanes 3 and 4), which is compa rable to the wild type chromatin (Fig . 3, lanes 1 and 2).
  • the assembled chromatins were incubated at 37°C for two weeks (Fig . 3, lanes 5 to 12) or stored at 4°C for four months (Fig . 3, lanes 13 to 16) .
  • nucleosomes 30 are nucleosomes 30 .
  • the SCV was formulated with a n adjuvant system consisting of the cationic liposome DDA and TLR4 agonist MPL to study immune responses against the G12V peptide.
  • Mice were vaccinated DDA/MPL adj uvant alone, G12V histone mixed with the adj uvants or G12V chromatin/adjuvant.
  • ELISA plate coated with the peptide was used to measure a titer of anti-G12V peptide antibodies in the serum.
  • histone and peptide-specific IgM responses in the vaccination of G12V-tagged chromatin were not statistically different to those in the DDA/M PL and G12V histone vaccine groups (data not shown).
  • splenocytes were prepared from immunized mice and stimulated in vitro with G12V peptide or histones.
  • a cytometric bead array was used to measure the responsiveness of T cells (Thl, Th2 and Thl7) as an indicator of cell-mediated immune response. No detectable cytokine release was observed upon stimulation with G12V peptide in vitro, suggesting that none of the peptide displays, either as a fusion histone or chromatin fiber are effective at stimulating peptide-specific T cell response at the vaccine concentrations used in the trial (data not shown).
  • mice vaccinated with G12V histones showed a high response for each of the cytokines including a major inflammatory cytokine (Fig. 6).
  • the increased expression of these cytokines was not observed in splenocytes from mice vaccinated with G12V chromatin as their cytokine responses were comparable to the DDA/MPL control mice.
  • a synthetic chromatin vaccine as described herein comprising a 10 nm chromatin fiber displaying a defined peptide antigen, can be used as a vaccine to elicit an efficient TI antibody response.
  • a synthetic chromatin vaccine as described herein is useful for delivering a number of immunostimulatory agents, pa rticularly peptides, of interest, that are poorly or insoluble, have lower than desired immunogenicity by themselves, or a combination of both.
  • histone H2B but not that of histone H3 or its phosphorylation, is essential for chromosome condensation.
  • acetylation switch regulates H2A.Z deposition by the SWR-C remodeling enzyme. Science 340, 195-199, doi : 10.1126/science.1229758 (2013) .
  • Cytotoxic CD4+ and CD8+ T lymphocytes generated by mutant p21-ras ( 12Val) peptide vaccination of a patient, recognize 12Val-dependent nested epitopes present within the vaccine peptide and kill autologous tumour cells carrying this mutation.
  • N ucleosome the main autoantigen in systemic l upus erythematosus, induces direct dendritic cell activation via a MyD88-independent pathway: consequences on inflammation . J Immunol 174, 3326-3334 (2005).

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Abstract

L'invention concerne des vaccins à chromatine synthétique (SCV) et des compositions, y compris des compositions pharmaceutiques comprenant de tels vaccins. En particulier, l'invention concerne un SCV comprenant des nucléosomes comprenant une séquence d'acides aminés d'au moins une protéine histone fusionnée à la séquence d'acides aminés d'au moins un antigène peptidique, l'antigène peptidique étant un antigène spécifique du cancer. L'invention concerne en outre des procédés de stimulation d'une réponse immunitaire chez un sujet, comprenant l'administration du SCV au sujet, le SCV pouvant être formulé avec un agent immunostimulant supplémentaire tel qu'un adjuvant ou un antigène.
PCT/IB2020/056493 2019-07-12 2020-07-10 Vaccin à chromatine synthétique WO2021009633A1 (fr)

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Citations (4)

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US20100119532A1 (en) * 2005-03-18 2010-05-13 Mehdi Chenik Composition comprising the N-terminal region of histone H2B of Leishmania - use thereof for inducing an immune response
US20140315314A1 (en) * 2006-03-01 2014-10-23 Aduro Biotech Engineered listeria and methods of use thereof
US20180021419A1 (en) * 2013-12-09 2018-01-25 Targovax Asa Peptide mixture

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US20020151517A1 (en) * 1996-09-12 2002-10-17 The General Hospital Corporation, A Massachusetts Corporation Nucleosome-based anti-tumor compositions
US20100119532A1 (en) * 2005-03-18 2010-05-13 Mehdi Chenik Composition comprising the N-terminal region of histone H2B of Leishmania - use thereof for inducing an immune response
US20140315314A1 (en) * 2006-03-01 2014-10-23 Aduro Biotech Engineered listeria and methods of use thereof
US20180021419A1 (en) * 2013-12-09 2018-01-25 Targovax Asa Peptide mixture

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Title
KALIYAPERUMAL ARUNAN, MICHAELS MARISSA A., DATTA SYAMAL K.: "Naturally Processed Chromatin Peptides Reveal a Major Autoepitope That Primes Pathogenic T and B Cells of Lupus", THE JOURNAL OF IMMUNOLOGY, WILLIAMS & WILKINS CO., US, vol. 168, no. 5, 1 March 2002 (2002-03-01), US, pages 2530 - 2537, XP055784002, ISSN: 0022-1767, DOI: 10.4049/jimmunol.168.5.2530 *
MORALES VIOLETTE, RICHARD-FOY HÉLÈNE: "Role of Histone N-Terminal Tails and Their Acetylation in Nucleosome Dynamics", MOLECULAR AND CELLULAR BIOLOGY, AMERICAN SOCIETY FOR PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, US, vol. 20, no. 19, 1 October 2000 (2000-10-01), US, pages 7230 - 7237, XP055784001, ISSN: 0270-7306, DOI: 10.1128/MCB.20.19.7230-7237.2000 *

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