WO2014065229A1 - Adjuvant, et vaccin contenant celui-ci - Google Patents

Adjuvant, et vaccin contenant celui-ci Download PDF

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Publication number
WO2014065229A1
WO2014065229A1 PCT/JP2013/078435 JP2013078435W WO2014065229A1 WO 2014065229 A1 WO2014065229 A1 WO 2014065229A1 JP 2013078435 W JP2013078435 W JP 2013078435W WO 2014065229 A1 WO2014065229 A1 WO 2014065229A1
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adjuvant
irf3
phosphatidylinositol
acid
phospholipid
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PCT/JP2013/078435
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English (en)
Japanese (ja)
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審良　静男
太郎 河合
拓実 川▲崎▼
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国立大学法人大阪大学
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Priority to JP2014543277A priority Critical patent/JP6274668B2/ja
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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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to an adjuvant and a vaccine including the same.
  • Immunostimulation is the stimulation and activation of dendritic cells, which means that antibody production (humoral immunity) and immune functions by T cells are enhanced in vivo.
  • Components that exhibit such functions are called adjuvants, immunostimulators, immune enhancers, and the like.
  • adjuvants Components that exhibit such functions
  • immunostimulators Components that exhibit such functions
  • immune enhancers Components that exhibit such functions.
  • the components having the above functions are collectively referred to as “adjuvant”, and the function is referred to as “adjuvant ability”.
  • Specific adjuvants include, for example, precipitating adjuvants such as calcium phosphate, aluminum phosphate, aluminum oxide, etc .; Freund's complete adjuvant containing paraffin oil, heated Mycobacterium tuberculosis, and surfactant; Complete Freund's adjuvant to Mycobacterium tuberculosis Examples include Freund's incomplete adjuvant excluding dead bacteria.
  • adjuvants included in vaccines specifically used in clinical practice include aluminum salts contained in vaccines shown in Non-Patent Document 2 and Non-Patent Document 3, influenza vaccines sold by Novartis Pharma And MF59 (oil-in-water type adjuvant containing squalene) contained in Celura (registered trademark).
  • vaccines containing an aluminum salt as an adjuvant have anaphylactic shock-like symptoms, multiple sclerosis, acute disseminated encephalomyelitis as serious side effects. There is a risk of causing Guillain-Barre syndrome. These may be attributed to the fact that the aluminum salt produces IgE, not IgG that acts in the neutralization reaction of the pathogen.
  • aluminum salts are contraindicated for patients undergoing dialysis therapy, and long-term administration of aluminum salts is known to cause aluminum encephalopathy, aluminum osteopathy, aluminum nephropathy, anemia, etc. ing. Furthermore, it cannot be said that the aluminum salt is sufficiently strong as an ability to enhance the immunity.
  • phospholipid is an important component that operates the IRF3-TBK1 signal cascade, which is a signal cascade that is very important for immunostimulation. I found it. Furthermore, it has been found that the phospholipids are not involved in excessive production of inflammatory cytokines that are thought to cause side effects when used in the immune system, especially as an adjuvant. Based on this finding, when the phospholipid was actually administered to mice together with the antigen, it was confirmed that antibodies against the antigen were sufficiently produced in the mouse serum.
  • Item 1 Adjuvant containing phospholipid.
  • Item 2 The adjuvant according to Item 1, comprising a phospholipid (excluding 3-deacylated-4'-monophosphoryl lipid A).
  • Item 3 The adjuvant according to Item 1 or 2, wherein the phospholipid is anionic.
  • Item 4 The adjuvant according to any one of Items 1 to 3, wherein the phospholipid is glycerophospholipid, sphingophospholipid, glycerophosphonolipid, sphingophosphonolipid, amino derivatives thereof, or salts thereof.
  • Phospholipid is phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidic acid, bisphosphatidic acid, pyrophosphatidic acid, plasmalogen, ethanolamine plasmalogen, sphingomyelin, ceramide phosphocholine, ceramide Phosphoethanolamine, ceramide phosphoserine, ceramide phosphoglycerol, ceramide phosphoglycerophosphoric acid, ethylamine diacylglycerophosphonic acid, ethylamine monoacylglycerophosphonic acid, ethylamine diacylglyceroaminoethylphosphonic acid, ethylamine monoacylglyceroaminoethylphosphonic acid, ethylamine diacylglyceroaminoethylphosphonic acid
  • Item 6 The adjuvant according to any one of Items 1 to 5, wherein the phospholipid is phosphatidylinositol, a phosphatidylinositol ester derivative, or a salt thereof.
  • the phosphatidylinositol or phosphatidylinositol ester derivative is PtdIns, PtdIns (3) P, PtdIns (4) P, PtdIns (5) P, PtdIns (3,4) P 2 , PtdIns (3,5) P 2 , and PtdIns (3, 4, 5) adjuvant according to Item 6 is at least one selected from the group consisting of P 3.
  • Item 8 The adjuvant according to Item 6 or 7, wherein the phosphatidylinositol or phosphatidylinositol ester derivative is a compound represented by the following formula (1):
  • R 1 is an alkyl group which may have an amino group or a hydroxyl group, The alkyl group may further have one or more alkanoyloxy groups having 3 to 24 carbon atoms or amino acid residues or peptide residues, R 2 , R 3 , and R 4 are the same or different and are a residue of the acid obtained by a dehydration condensation reaction between a hydrogen atom or an acid and a hydroxyl group. ).
  • Item 9 The adjuvant according to any one of Items 6 to 8, wherein the phosphatidylinositol ester derivative is phosphatidylinositol phosphate.
  • Item 10 The adjuvant according to Item 9, wherein the phosphatidylinositol phosphate is a compound represented by the following formula (2);
  • Item 11 A vaccine comprising the adjuvant and antigen according to any one of Items 1 to 10 above.
  • the antigen is composed of bacteria, viruses, fungi, parasitic protozoa, parasitic helminths, cancer cells, cancer cell-specific proteins, cancer cell melts, prions, nucleic acids, renin, angiotensin, and angiotensin receptors.
  • Item 13 The vaccine according to Item 12, wherein the antigen is a virus.
  • Item 14 An immunostimulation method comprising a step of administering a phospholipid to a living body.
  • Item 15 The immunostimulation method according to Item 14, comprising a step of administering a phospholipid (excluding 3-deacylated-4′-monophosphoryl lipid A) to a living body.
  • a phospholipid excluding 3-deacylated-4′-monophosphoryl lipid A
  • Item 16 The immunostimulation method according to Item 14 or 15, wherein the phospholipid is anionic.
  • Item 17 The immunostimulation method according to any one of Items 14 to 16, wherein the phospholipid is glycerophospholipid, sphingophospholipid, glycerophosphonolipid, sphingophosphonolipid, amino derivatives thereof, or salts thereof. .
  • Phospholipid is phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidic acid, bisphosphatidic acid, pyrophosphatidic acid, plasmalogen, ethanolamine plasmalogen, sphingomyelin, ceramide phosphocholine, ceramide Phosphoethanolamine, ceramide phosphoserine, ceramide phosphoglycerol, ceramide phosphoglycerophosphoric acid, ethylamine diacylglycerophosphonic acid, ethylamine monoacylglycerophosphonic acid, ethylamine diacylglyceroaminoethylphosphonic acid, ethylamine monoacylglyceroaminoethylphosphonic acid, ethylamine diacylglyceroaminoethylphosphonic acid
  • Item 19 The immunostimulation method according to any one of Items 14 to 18, wherein the phospholipid is phosphatidylinositol, a phosphatidylinositol ester derivative, or a salt thereof.
  • the phosphatidylinositol or the phosphatidylinositol ester derivative is PtdIns, PtdIns (3) P, PtdIns (4) P, PtdIns (5) P, PtdIns (3, 4) P 2 , PtdIns (3,5) P 2 , and PtdIns (3, 4, 5) immunostimulatory method according to item 19 is at least one selected from the group consisting of P 3.
  • Item 21 The immunostimulation method according to Item 19 or 20, wherein the phosphatidylinositol or phosphatidylinositol ester derivative is a compound represented by the following formula (1):
  • R 1 is an alkyl group optionally having an amino group or a hydroxyl group, and the alkyl group further has an alkanoyloxy group having 3 to 24 carbon atoms, an amino acid residue or a peptide residue
  • R 2 , R 3 , and R 4 are the same or different and are a residue of the acid obtained by a dehydration condensation reaction between a hydrogen atom or an acid and a hydroxyl group.
  • Item 22 The immunostimulation method according to any one of Items 19 to 21, wherein the phosphatidylinositol ester derivative is phosphatidylinositol phosphate.
  • Item 23 The immunostimulation method according to Item 22, wherein the phosphatidylinositol phosphate is a compound represented by the following formula (2):
  • Item 24 The immunostimulation method according to any one of Items 14 to 23, wherein an antigen is administered together with a phospholipid.
  • the antigen is composed of bacteria, viruses, fungi, parasitic protozoa, parasitic helminths, cancer cells, cancer cell-specific proteins, cancer cell melts, prions, nucleic acids, renin, angiotensin, and angiotensin receptors.
  • Item 26 The immunostimulation method according to Item 25, wherein the antigen is a virus.
  • Item 27 The immunostimulation method according to any one of Items 14 to 26, wherein the living body is a human.
  • Item 28 A method for screening an adjuvant candidate substance comprising the following step 1 and step 2; (1) A step 1 for bringing an intracellular component into contact with IRF3 (Interferon regulatory factor 3), and (2) a step 2 for selecting the intracellular component that phosphorylates the IRF3.
  • IRF3 Interferon regulatory factor 3
  • Item 29 A method for screening an adjuvant candidate substance comprising the following step 1 and step 2; (I) contacting an intracellular component with IRF3 (Interferon regulatory factor 3) and TBK1 (TANK binding kinase 1); II) Step 2 of selecting the intracellular component that binds to the TBK1 and phosphorylates the IRF3.
  • IRF3 Interferon regulatory factor 3
  • TBK1 TANK binding kinase 1
  • Item 30 The screening method according to Item 28 or 29, wherein the intracellular component is a component contained in the water-insoluble fraction.
  • the present invention is not limited to the invention that exhibits all of the effects described below, and it is sufficient that at least one of the effects is exhibited.
  • the adjuvant according to the present invention has an effect of being easily taken up by dendritic cells, and is excellent in the effect of activating dendritic cells. That is, it is considered that the adjuvant according to the present invention has an action of inducing antibodies and increasing the response of Th1 cells through such activation of dendritic cells. Therefore, the adjuvant which concerns on this invention is useful as a component which comprises a vaccine with an antigen.
  • the adjuvant according to the present invention is not involved in the production of inflammatory cytokines and does not enhance it, it is very unlikely to cause a side effect even as a component constituting a vaccine together with an antigen.
  • the adjuvant of the present invention originally contains a compound present in the living body, unlike an adjuvant containing a foreign compound, the possibility of causing other side effects such as fever other than inflammation and vomiting is very low.
  • the adjuvant of the present invention or the vaccine comprising the same is safe for the human body and is very excellent in immunostimulatory ability.
  • the immunostimulation method by administering the adjuvant of the present invention alone or together with the antigen to the human body is safe for the human body and is useful as a method for improving the immunostimulatory ability of the living body.
  • FIG. 1 shows the experimental results showing that PtdIns (5) P enhances phosphorylation of IRF3 by TBK1 in vitro.
  • A), (B), and (D) are in vitro kinase assays with recombinant IRF3 and TBK1 using various reagents. And it is the experimental result which identified phosphorylation of IRF3 by TBK1 using pS394IRF3 antibody.
  • A shows an in vitro kinase assay using various reagents, isolated fractions of proteins, or reconstituted proteins.
  • B shows an in vitro kinase assay using lipid fractions extracted from HEK293 cells transformed and activated with the plasmids shown in the figure.
  • FIG. 2 shows the experimental results showing that PIKfive activates the IFN ⁇ promoter or ISRE promoter.
  • the reporter activity in HEK293 cells transformed with the plasmid to be expressed (bottom panel in B) is shown.
  • F Localization of RelA after isolation of nuclear fraction (top), total RelA as a control.
  • (G)-(I) shows the results of a PIKfive knockdown experiment in GM-DC by siRNA electroporation.
  • OT-II transgenic CD4-positive T cells specific for OVA are isolated and C8-PtdIns (5) P, C8-PtdIns (4,5) P 2 or poly I: C in the presence of OVA 3 shows the production of interferon ⁇ (IFN ⁇ ) measured after co-culture of GM-DC treated with the above. The production amount of IL1 ⁇ measured by ELISA of GM-DC stimulated with C8 lipid shown in the figure is shown.
  • IFN ⁇ interferon ⁇
  • FIGS. 6-1 and 6-2 are shown in FIGS. 6-1 and 6-2.
  • the adjuvant according to the present invention is also called an immunopotentiator or an immunostimulator. All such designations are normally used to mean agents used for the same application as adjuvant.
  • the adjuvant according to the present invention contains a phospholipid.
  • phospholipids include phospholipids other than 3-deacylated-4'-monophosphoryl lipid A are preferable.
  • the phospholipid may be a phospholipid that exists naturally, particularly in vivo, or may be an industrially produced phospholipid, and is not particularly limited. Further, the industrially produced phospholipid is not particularly limited to a specific technique such as a biochemical technique or a chemical technique. That is, it may be a phospholipid obtained by synthesis in a cell or a phospholipid synthesized by an in vitro chemical reaction.
  • Examples of such specific phospholipids include glycerophospholipid, sphingophospholipid, glycerophosphonolipid, sphingophosphonolipid, and derivatives thereof.
  • Glycerophospholipids and glycerophosphonolipids are phospholipids that contain a glycerol-derived constituent alcohol moiety in the phospholipid molecule, and sphingophospholipids and sphingophosphonolipids are derived from long-chain amino alcohols such as sphingosine and ceramide.
  • a phospholipid containing an alcohol moiety is phospholipids that contain a glycerol-derived constituent alcohol moiety in the phospholipid molecule.
  • sphingophospholipids and sphingophosphonolipids are derived from long-chain amino alcohols such as sphingosine and ceramide.
  • Glycerophospholipid and sphingophospholipid are phospholipids containing a C—O—P bond (phosphate ester bond) in which an oxygen atom is bonded between a carbon atom and a phosphate atom.
  • a phosphonolipid is a phospholipid containing a phosphono group containing a CP bond in which a phosphorus atom is bonded directly to a carbon atom.
  • Specific glycerophospholipids include, for example, phosphatidylinositol, phosphatidylinositol phosphate, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidic acid, bisphosphatidic acid, pyrophosphatidic acid, plasmalogen, ethanolamine plasmalogen And derivatives thereof.
  • glycerophospholipids may be lyso forms.
  • the lyso form is one of the three hydroxyl groups of glycerol other than the above-mentioned C—O—P bond formed by ester linkage with phosphoric acid, and only one of them is fatty acid and ester. It is a bonded compound.
  • these glycerophospholipids may be amino derivatives in which at least one of the two hydroxyl groups described above is ester-bonded with an amino acid or a peptide. This is called an amino derivative of glycerophospholipid in the present invention.
  • the amino acids are not only 20 amino acids based on t-RNA genetic information, but also selenocysteine, selenomethionine, N-formylmethionine, pyrrolysine, pyroglutamic acid, cystine, hydroxyproline, hydroxylysine, thyroxine, O-phosphoserine. , ⁇ -alanine, N-methylglycine, ornithine, statin, citrulline, creatine, ⁇ -aminobutyric acid and other amino acid- and carboxy-group-containing amino acids.
  • amino acids containing side chains having a charge such as lysine, arginine, histidine, glutamic acid, aspartic acid and the like are preferable.
  • the above peptide is a peptide bond of the above 2 to about 10 amino acids. Among them, a peptide containing an amino acid residue containing a side chain having the above charge is preferable.
  • sphingophospholipids include sphingomyelin, ceramide phosphocholine, ceramide phosphoethanolamine, ceramide phosphoserine, ceramide phosphoglycerol, ceramide phosphoglycerophosphate, and derivatives thereof.
  • sphingophospholipids may be lyso forms.
  • the lyso form is a compound in which the other hydroxyl group used for the above-mentioned C—O—P bond formed by the ester bond with phosphoric acid among the two hydroxyl groups of sphingosine remains as it is.
  • these sphingophospholipids may be amino derivatives in which an amino acid or a peptide is ester-bonded to the remaining hydroxyl group. This is referred to as an amino derivative of sphingophospholipid in the present invention.
  • amino acids and peptides are as described in detail for the amino derivatives of the above glycerophospholipids.
  • Specific glycerophosphonolipids include, for example, ethylamine diacylglycerophosphonic acid, ethylamine monoacylglycerophosphonic acid, ethylamine diacylglyceroaminoethylphosphonic acid, ethylamine monoacylglyceroaminoethylphosphonic acid, ethylamine diacylglycerocholinephosphonic acid, ethylamine monoacylphosphonic acid.
  • Examples include acyl glycerocholine phosphonic acid, ethylamine diacyl glyceroserine phosphonic acid, ethylamine monoacyl glyceroserine phosphonic acid, and derivatives thereof.
  • glycerophosphonolipids may be lyso forms.
  • the lyso form means that, of the three hydroxyl groups of glycerol, only one of the remaining two hydroxyl groups other than the above-mentioned CP bond formed by directly binding to phosphoric acid is an ester bond with a fatty acid. It is a compound.
  • these glycerophosphonolipids may be amino derivatives in which an amino acid or a peptide is ester-bonded to at least one of the two hydroxyl groups described above. This is referred to as an amino derivative of glycerophosphonolipid in the present invention.
  • amino acids and peptides are as described in detail for the amino derivatives of the above glycerophospholipids.
  • sphingophosphonolipids include ceramide aminoethylphosphonic acid, acylsphingosylaminoethylphosphonic acid, and derivatives thereof.
  • sphingophosphonolipids may be lyso forms.
  • the lyso form is a compound in which the other hydroxyl group used for the above-mentioned CP bond formed by directly binding to phosphoric acid among the two hydroxyl groups of sphingosine remains as it is.
  • these sphingophosphonolipids may be amino derivatives in which an amino acid or a peptide is ester-bonded to the remaining hydroxyl group. This is called an amino derivative of sphingophosphonolipid in the present invention.
  • amino acids and peptides are as described in detail for the amino derivatives of the above glycerophospholipids.
  • Examples of the above-described phospholipids include compounds represented by the structural formulas shown in Table 1 below. Phospholipids represented by these structural formulas can be purchased as a salt form from Avanti Polar Lipid (numbers in the left column indicate product numbers).
  • phosphatidylinositol or phosphatidylinositol ester derivatives are preferable.
  • phosphatidylinositol ester derivatives examples include phosphatidylinositol (3) monophosphate, phosphatidylinositol (4) monophosphate, phosphatidylinositol (5) monophosphate, phosphatidylinositol (3,4) bisphosphate, phosphatidylinositol (3,5 ) Bisphosphoric acid, phosphatidylinositol (3,4,5) triphosphoric acid, and derivatives thereof.
  • the compound shown by following formula (1) is also mentioned as a phosphatidylinositol or a phosphatidylinositol ester derivative.
  • R 1 is an alkyl group which may have an amino group or a hydroxyl group.
  • the alkyl group may further have an alkanoyloxy group having 3 to 24 carbon atoms, or one or more amino acid residues or peptide residues.
  • it is an alkyl group further having two or more alkanoyloxy groups or amino acid residues or peptide residues.
  • the upper limit of the number of alkanoyloxy groups or amino acid residues or peptide residues that the alkyl group has is not particularly limited as long as it can be retained according to the number of carbon atoms of the alkyl group.
  • the carbon number of the alkanoyloxy group is preferably about 4 or more, more preferably about 6 or more, and still more preferably about 8 or more. Further, the upper limit of the carbon number of the alkanoyloxy group is preferably about 20, more preferably about 16, further preferably about 12, and most preferably about 8.
  • the alkanoyloxy group may contain one or more double bonds in the carbon-carbon bond.
  • the upper limit number of double bonds is determined according to the number of carbon atoms of the alkanoyloxy group, and preferably about 4 carbon-carbon double bonds may be included.
  • a carbon-carbon double bond it may be a cis isomer or a trans isomer, and is not limited to any one.
  • the number of carbon atoms of the alkyl group is not particularly limited, but is usually 2 to 8 and more preferably 3 or more.
  • the upper limit of the number of carbon atoms is preferably 7, more preferably 6, and even more preferably 5.
  • the oxygen atom that is single-bonded to the terminal carbonyl carbon atom in the alkanoyloxy group may be single-bonded to any carbon atom in the alkyl group.
  • the alkyl group has two or more alkanoyloxy groups
  • the two or more alkanoyloxy groups described above may be bonded to the same carbon atom in the alkyl group, and may be bonded to different carbon atoms of the alkyl group.
  • the alkanoyloxy group mentioned above may be couple
  • the two or more alkanoyloxy groups described above may be the same or different.
  • the amino acid residues include not only 20 types of amino acids based on t-RNA genetic information, but also selenocysteine, selenomethionine, N-formylmethionine, pyrrolysine, pyroglutamic acid, cystine, hydroxyproline, hydroxylysine, thyroxine, O- A substance in which a hydrogen atom is removed from at least one carboxy group of amino acids in a broad sense such as phosphoserine, ⁇ -alanine, N-methylglycine, ornithine, statin, citrulline, creatine, ⁇ -aminobutyric acid and other compounds having an amino group and a carboxy group It is.
  • an amino acid residue obtained by removing a hydrogen atom from an amino acid containing a charged side chain such as lysine, arginine, histidine, glutamic acid, and aspartic acid is preferable.
  • the peptide residue is a peptide residue obtained by removing a hydrogen atom from at least one carboxy group of a peptide obtained by peptide bonding of the above 2 to 10 amino acids.
  • Such peptide residues preferably include the above-mentioned preferred amino acid residues.
  • the oxygen atom that is single-bonded to the carbonyl group in the above amino acid residue or peptide residue may be single-bonded to any carbon atom in the alkyl group.
  • the alkyl group has two or more amino acid residues or peptide residues, two or more amino acid residues or peptide residues described above may be bonded to the same carbon atom in the alkyl group, The amino acid residue or peptide residue described above may be bonded to different carbon atoms of the alkyl group. Two or more amino acid residues or peptide residues described above may be the same or different.
  • R 2 , R 3 and R 4 are the same or different and are the residues of the acid obtained by a dehydration condensation reaction between a hydrogen atom or an acid and a hydroxyl group.
  • the acid may be an organic acid or an inorganic acid, and is not particularly limited, but is preferably an inorganic acid.
  • inorganic acids include phosphoric acid, phosphorous acid, hypophosphorous acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, and boronic acid, and are not particularly limited, but phosphoric acid is preferred.
  • the compound represented by the above formula (1) include compounds represented by the structural formulas shown in Tables 2 to 4 below.
  • the compounds represented by these structural formulas are in the form of salts, which can be purchased from echelon bioscience for Tables 2 to 4, and from Avanti Polar Lipid for Tables 5 and 6. Indicates the catalog number.
  • phosphatidylinositol ester derivative phosphatidylinositol phosphate is preferable, and the most preferred phosphatidylinositol derivative is a compound represented by the following chemical formula (2).
  • the above-mentioned phospholipid may be in the form of a salt.
  • a salt composed of an anion obtained by removing a proton from any one or more OH groups contained in the phospholipid and a counter cation thereof can be mentioned. More preferably, a salt comprising an anion obtained by removing a proton from an OH group of an alkylphosphono group which may have a phosphono group or one or more alkanoyloxy groups contained in a phospholipid, and a counter cation thereof may be mentioned. It is done.
  • the valence of the anion is not particularly limited, but is usually about 1 to 7 valences.
  • the type of the counter cation is not particularly limited, and examples thereof include sodium ion, potassium ion, calcium ion and the like.
  • a salt composed of a cation in which a proton is bonded to any one or more amino groups contained in the phospholipid and a counter anion thereof can be mentioned.
  • the type of the counter anion is not particularly limited, and examples thereof include fluoride ions, chloride ions, bromide ions, and the like.
  • the above-described phospholipid can be produced using a known method.
  • the phospholipid is a phospholipid having a phosphonooxy group containing a phosphate ester bond as described above, it should be produced by referring to Experimental Chemistry Course 5th edition (16) “Carboxylic acid / amino acid / peptide” as appropriate. Can do. It is also possible to purchase and obtain commercially available products.
  • the phospholipid contained in the adjuvant according to the present invention may be anionic, cationic or nonionic, but is preferably an anionic phospholipid.
  • the adjuvant according to the present invention contains the above-described phospholipid, and the content thereof is usually about 0.01 to 99.99% by weight per the adjuvant. Further, the phospholipid itself may be the adjuvant according to the present invention.
  • the adjuvant according to the present invention may contain a known component usually contained in the adjuvant as long as the effect of the adjuvant according to the present invention is not impaired.
  • Such components include preservatives, buffers, preservatives, inactivating agents, isotonic agents, pH adjusters, and the like.
  • a known component that exerts an immunostimulatory action usually contained in the adjuvant is included in addition to the above-described phospholipid. It may be.
  • Such components include aluminum hydroxide, squalene, ⁇ -tocopherol, mineral oil, paraffin oil, single-stranded RNA or analog thereof, double-stranded RNA or analog thereof, flagellin, trehalose derivative, MPL, and the like.
  • the adjuvant according to the present invention is effectively used for experimental animals that are generally employed when, for example, producing antibodies for commercial use. That is, when an antigen specifically recognizing an antibody is administered to a laboratory animal, the adjuvant according to the present invention is mixed therewith and simultaneously or separately from the antigen.
  • mice mice, rats, rabbits, sheep, chickens, donkeys, horses, ostriches, camels, llamas, and alpaca.
  • the amount of the adjuvant according to the present invention to be used for these experimental animals may be appropriately set depending on the desired degree of immunostimulation, and is not particularly limited, but in terms of the amount of the phospholipid contained in the adjuvant.
  • the dose may be about 0.5 mg to 0.5 g / kg / kg of animal individual.
  • the interval may be set as appropriate, usually about 14 to 30 days.
  • the use method of the adjuvant according to the present invention when used in the above-mentioned animals is not particularly limited, and examples thereof include oral administration, intravenous administration, transdermal administration, transmucosal administration, sublingual administration, transperitoneal administration. What is necessary is just to use by administration, transmuscular administration, etc.
  • the adjuvant according to the present invention when administered to a living body together with an antigen, dendritic cells in the living body are activated and an acquired immune effect is exhibited. Therefore, the adjuvant according to the present invention is expected to exhibit a function as a vaccine when clinically used together with an antigen.
  • the vaccine according to the present invention comprises the above-mentioned adjuvant and antigen.
  • the antigen is one that is detected by the humoral immunity or cellular immunity system, and an antibody that specifically binds to the antigen is produced, or is preyed on by white blood cells, macrophages, T cells, or the like.
  • Specific antigens include bacteria, viruses, fungi, parasitic protozoa, parasitic helminths, cancer cells, cancer cell-specific proteins, cancer cell lysates, prions, nucleic acids, renin, angiotensin, angiotensin receptors, etc. There is no particular limitation.
  • the above bacteria are not particularly limited as long as they cause bacteria to humans, pets, livestock and the like.
  • streptococci such as streptococci, pneumococci, etc.
  • Staphylococcus aureus MSSA, MRSA, etc.
  • Staphylococcus epidermidis enterococci
  • bacteria belonging to the genus Listeria Listeria, hereinafter
  • Neisseria meningitidis Neisseria gonorrhoeae, Pathogenic E.
  • coli Neisseria pneumoniae, Proteus, Pertussis, Pseudomonas aeruginosa, Serratia, Citrobacter, Acinetobacter, Enterobacter, Mycoplasma, Clostridium, M. tuberculosis, Cholera Fungus, plague, diphtheria, shigella, anthrax, treponema, tetanus, leprosy, legionella, leptospira, borrelia, francisella, coccella, rickettsia, chlamydia, rhinomycosis, pylori Etc.
  • influenza virus is not particularly limited as long as it is a virus that causes disease to humans, pets, livestock and the like.
  • influenza virus papilloma virus, hepatitis virus (A type, B type, C type, D type, E type, F type, G type, TT type, etc.), smallpox virus, measles virus, rubella virus, Poliovirus, varicella-zoster virus, norovirus, norwalk virus, sapovirus, sapporovirus, mumps virus, adenovirus, enterovirus, rotavirus, AIDS virus, rabies virus, T lymphophilic virus, yellow fever virus, cytomegalo Virus, SARS virus, polyoma virus, JC virus, BK virus, herpes virus, lymphocrypt virus, roseovirus, Japanese encephalitis virus, coxsackie virus, dengue virus, West Nile virus, coronavirus, parvovirus Epstein-Barr virus, Marburg virus, Hanta virus, Lassa
  • the above fungus is not particularly limited as long as it is a fungus that causes diseases to humans, pets, livestock, and the like. Specific examples include Aspergillus, Candida, Cryptococcus, Ringworm mycosis, Histoplasma, Pneumocystis and the like.
  • the above parasitic protozoa is not particularly limited as long as it is a parasitic protozoa causing a disease to humans, pets, livestock and the like. Specifically, amoeba dysentery, malaria, toxoplasma, leishmania, cryptosporidium, trypanosoma and the like can be mentioned.
  • the above parasitic worm is not particularly limited as long as it is a parasitic worm causing a disease to humans, pets, livestock and the like. Specific examples include Echinococcus, Schistosoma japonicum, Filaria, roundworm, and broad-headed fibroids.
  • the cancer cell is not particularly limited as long as it is a cell in a cancerous lesion that affects humans, pets, livestock, and the like.
  • cancer include leukemia, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, brain tumor, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, esophageal cancer, stomach cancer, appendic cancer, colon cancer, Liver cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma, head and neck cancer, laryngeal cancer, oral cancer, gingival cancer, tongue cancer, buccal mucosa cancer, salivary gland cancer , Sinus cancer, maxillary sinus cancer, frontal sinus cancer, ethmoid sinus cancer, sphenoid sinus cancer, thyroid cancer, kidney cancer, lung cancer, small cell lung cancer, osteosarcoma, prostate
  • the above cancer cell-specific protein is not particularly limited as long as it is a protein that is specifically expressed in the above cancer cells (also referred to as a tumor-associated antigen).
  • a protein that is specifically expressed on the surface of a cancer cell is preferable.
  • a receptor is also included.
  • Such a protein may be a full-length protein or a partial fragment thereof.
  • PSA may be mentioned.
  • the cancer cell lysate is obtained by melting the cancer cells described above.
  • the method of thawing may be simply crushing the cancer cells, for example, a method of crushing using a buffer containing a surfactant, an ultrasonic crushing method, a method of crushing using glass beads, or a French press.
  • the method etc. which are crushed are mentioned, You may combine these suitably.
  • the above-mentioned prion is an infectious factor composed of a non-wild type protein, and causes a disease by accumulating in humans, pet animals, livestock and the like.
  • Such diseases are not particularly limited, and examples include spongiform encephalopathy, kuru, Creutzfeldt-Jakob disease, lethal familial insomnia, Gerstman-Streisler-Scheinker syndrome, Alzheimer's disease, etc.
  • prions include ⁇ amyloid and the like.
  • the above-mentioned renin works in the renin-angiotensin system related to blood pressure increase.
  • the angiotensin may be type I or type II, and works in the above-mentioned renin-angiotensin system.
  • angiotensin receptor is a receptor for type I angiotensin and / or type II angiotensin, and may be the full length or a partial fragment thereof.
  • bacteria, viruses, fungi, parasitic protozoa, parasitic helminths and the like may be used as antigens, or inactivated may be used as antigens.
  • a method of performing a treatment such as heating, ultraviolet irradiation, addition of an organic solvent, addition of a surfactant or the like can be mentioned.
  • a virus coat (which is also called a capsid) may be used as an antigen.
  • components such as proteins, carbohydrates, lipids, etc. possessed by bacteria, viruses, fungi, parasitic protozoa, parasitic helminths, etc. may be used as antigens, and some of these may be used as antigens.
  • Such components are preferably present on the surface of bacteria, viruses, fungi, parasitic protozoa, parasitic helminths and the like.
  • various proteins such as HA and NA present on the surface may be used as an antigen, and a part of these proteins or a sugar chain contained therein or a part thereof may be used as an antigen.
  • the nucleic acid may be ribonucleotide or deoxyribonucleotide. Moreover, such a nucleic acid is not limited to a single-stranded form, and may be a double-stranded form or more. And said nucleic acid is not limited to what contains adenine, guanine, cytosine, thymine, and uracil as a base, The nucleic acid containing these derivatives or the modified base may be sufficient.
  • the vaccine according to the present invention is usually suitably used for humans in order to prevent diseases such as infectious diseases and cancers, but in addition to the above-mentioned pet animals (for example, dogs, cats, ferrets, Birds) and the above-mentioned livestock (cow, pig, chicken, goat, ostrich, sheep, horse, etc.).
  • pet animals for example, dogs, cats, ferrets, Birds
  • livestock cow, pig, chicken, goat, ostrich, sheep, horse, etc.
  • the content of the above-mentioned adjuvant in the vaccine according to the present invention is not particularly limited, but is usually about 0.01% to 99.99% by weight with respect to 100% by weight of the vaccine.
  • the adjuvant is usually about 10 to 1000 parts by weight with respect to 1 part by weight of the antigen.
  • the administration method of the vaccine according to the present invention is not particularly limited, and examples thereof include oral administration, intravenous administration, transdermal administration, transmucosal administration, sublingual administration, and intramuscular administration.
  • the dose of the vaccine varies depending on the desired degree of immunostimulation, age, sex, etc. of the administration target, and may be set as appropriate. Although not particularly limited, for example, if converted into the amount of phospholipid In general, the dose may be about 0.5 mg to 0.5 g / dose per kg of humans, pets or livestock.
  • the vaccine may be administered multiple times to the same human, companion animal, or domestic animal according to the degree of effect exerted by the desired vaccine, the age of the administration subject, sex, etc., and usually 2 to 4 times.
  • the upper limit may be set as the upper limit.
  • the interval may be appropriately set, and it is usually about 14 to 30 days.
  • Such a vaccine not only exhibits a sufficient preventive effect on diseases (particularly infectious diseases), but also exhibits extremely low side effects caused by the administration of the vaccine.
  • the immunostimulation method according to the present invention includes a step of administering the above-described phospholipid to a living body.
  • the living body is not particularly limited as long as it is a living body that needs to enhance the immune system. Specifically, as described in detail in ⁇ Adjuvant> above, experimental animals used in the production of antibodies for commercial use, and disease (particularly infectious diseases) detailed in ⁇ Vaccine> described above are prevented. For example, humans, pets, livestock, etc., to be administered vaccines.
  • the dose of phospholipid is not particularly limited, and is appropriately set based on, for example, the amount of adjuvant used as described in detail in ⁇ Adjuvant> above. Or you may set based on the dosage of a vaccine explained in full detail in the above ⁇ vaccine>.
  • the number of multiple doses and the administration interval at the time of multiple doses may be appropriately set based on those detailed in the above ⁇ Adjuvant> or ⁇ Vaccine>.
  • the administration method may be appropriately selected from the administration methods detailed in the above ⁇ Adjuvant> or ⁇ Vaccine>.
  • the immunostimulation method according to the present invention it is preferable to administer an antigen together with the above-mentioned adjuvant according to the present invention.
  • the specific antigen may be the same as that described in detail in ⁇ Vaccine> above, and the subject to be administered, the administration method, the number of multiple doses and the interval thereof, etc. in ⁇ Vaccine> and ⁇ Adjuvant> What is necessary is just like description.
  • the screening method according to the present invention is a method for screening an adjuvant candidate, which includes the following steps 1 and 2.
  • Process 1 (1) A step of bringing an intracellular component into contact with IRF3 (Interferon regulatory factor 3)
  • Process 2 (2) A step of selecting the intracellular component that phosphorylates the IRF3.
  • the screening method according to the present invention includes a method for screening an adjuvant candidate in an embodiment including the following step 1 ′ and step 2 ′.
  • Examples of such a method include a method of crushing using a buffer containing a surfactant, an ultrasonic crushing method, a method of crushing using glass beads, a method of crushing using a French press, etc. These may be combined.
  • the above-mentioned intracellular components obtained by crushing cells by such means include those obtained by crushing, for example, in a methanol fraction (water-soluble fraction) fractionated with a methanol / chloroform mixed solvent. May be a component contained in the chloroform fraction (water-insoluble fraction), or a component contained in both of them, and is not particularly limited. It is a component contained in the sex fraction.
  • the specific cell origin is not particularly limited and may be appropriately selected.
  • the cells may be activated in advance so that IRF3 is easily phosphorylated before disruption.
  • a specific activation method for example, a method of introducing a gene expressing IPS-1, TRIF, MyD88, TIRAP or the like into the cell can be mentioned.
  • a method for introducing a gene expressing IPS-1 is preferred.
  • the intracellular component that phosphorylates IRF3 may be selected by confirming the phosphorylation of IRF3 by a known method.
  • Specific confirmation methods include Western blotting, flow cytometry, ELISA, and the like using an antibody that recognizes phosphorylated IRF3. .
  • the method of binding to TBK1 and phosphorylating IRF3 in the above steps 2 and 2 ′ is as follows.
  • an immunoprecipitation method a QCM (quartz crystal microbalance) method, an SPR (surface plasmon resonance) method, or the like may be combined.
  • HEK293 cells and MEFs (mouse fetal fibroblasts) use a DMEM medium containing 10% inactivated FCS (Life Technology) at 37 ° C. in a 5% carbon dioxide environment. Was cultured.
  • GM-DCs Bone marrow derived dendritic cells induced by GM-CSF were obtained from 10% FCS, 100 ⁇ M 2-ME, and 10 ng / ml mouse derived GM-CSF. It was prepared by culturing in RPMI 1640 medium containing (BD Bioscience) for 6 to 8 days.
  • the ISD sequence was as follows and was prepared according to a conventional method. (Sense: 5'-TACAGATCTACTAGTGATCTATGAACTGATCTGTACATGATCTACCA; SEQ ID NO: 1).
  • LPS and poly I C and poly dA: dT were purchased from Invivogen and used to stimulate cells with Lipofectamine 2000 (Life Technology) 2: 1 (2 ⁇ g: 1 ⁇ l) respectively. And stimulated with OPTI-MEM (Life Technology).
  • YM-201636 a PIKfyve inhibitor
  • Anti-PIKfyve antibody Sigma
  • anti-pIRF3 antibody Cell Signaling
  • anti-pJNK Cell Signaling
  • anti-TBK1 Anti-TBK1
  • Anti-pTBK1 BD Bioscience
  • Plasmid and reporter assay A FLAG-tagged PIKfyve expression plasmid was prepared by amplifying PIKfyve by PCR and then incorporating it into the pFLAG-CMV6 vector. And a kinase expression mutant (K1831M PIKfyve) expression plasmid was prepared using KOD (Toyobo).
  • FLAG-tagged PIP5K ⁇ , ⁇ , and ⁇ , and expression plasmids were obtained by amplifying gene fragments encoding these from HEK293 cell cDNA and incorporating them into the pFlag-CMV6 vector.
  • PI3KIII, PTEN, TMEM55 ⁇ , TMEM55 ⁇ , and PI4K2 ⁇ expression plasmids used were those incorporated into a pCMV-SPORT6 vector (Open Biosystems).
  • HEK293 cells were seeded in a 24-well plate, HEK293 cells seeded with various expression plasmids and reporter luciferase plasmids using Lipofectamine 2000 (Life Technology) were transformed and cultured for 16 hours. The cells were lysed using a passive lysis buffer (Promega).
  • luciferase activity was measured using a dual luciferase reporter assay kit (Promega).
  • the reporter plasmids IFN ⁇ , ISRE, and NF ⁇ B were known.
  • the TK promoter that operates Renilla luciferase was used as an internal control.
  • In vitro kinase assay was performed by incubating 20 ng TBK1 and 100 nmol IRF3 for 30 minutes at 30 ° C. in reaction buffer (5 mM HEPES (pH 7.2) containing 10 ⁇ M ATP, 100 mM NaCl, 2 mM MgCl 2 ). did.
  • Liposomes were prepared by sonication by mixing PC: PE: PI at 30: 8: 2, respectively. This was also used at 40 ⁇ M for in vitro kinase assay.
  • GST-tagged STING was produced using HEK293t cells and purified using 10 mM glutathione sepharose beads. Purified STING was reconstituted by forming liposomes by removing the surfactant with azolectin lipids in a dialysis environment.
  • RNAi and quantitative PCR Double- stranded stealth RNA was obtained from Life Science Technology.
  • the target sequence of PIKfive is as follows.
  • the primers used for quantitative PCR are as follows.
  • MGAPDH 5'-TGACGTGCCCCCTGGAGAAA-3 '[sense: SEQ ID NO: 7]: 5'-AGTGTAGCCCAAGATGCCCCTTCAG-3' [reverse: SEQ ID NO: 8].
  • mice IPS-1 ⁇ / ⁇ mice, IRF3 ⁇ / ⁇ / IRF7 ⁇ / ⁇ mice, and OT-II mice were prepared by known methods. All animal experiments were conducted with the approval of the Animal Experiment Committee of the Institute for Microbial Diseases, Osaka University.
  • mice C57BL / 6J mice (CLEA Japan, Inc.) were injected intraperitoneally with 100 ⁇ g of OVA (Seikagaku Corporation) and 1 mg of alum (Sigma) every week for a total of 4 immunizations. Or immunized by intramuscular injection with 100 ⁇ g OVA / 100 ⁇ g C8-PtdIns.
  • OVA Seikagaku Corporation
  • alum Sigma
  • C8-PtdIns (5) P and C8-PtdIns (4,5) P 2 was solubilized in PBS, and mixed with OVA for immunization.
  • IRF3 The activation of IRF3 was evaluated by detecting by Western blotting using an antibody against S396 phosphorylated IRF3 after an in vitro kinase reaction.
  • Purified STING which is a signal protein that functions when TBK1 and IRF3 are known immune activators DMXAA, dsDNA, high concentrations of KCl, H 2 O 2 , and dendritic cells are activated using known DNA viruses as antigens None of these were able to enhance IRF3 phosphorylation when incubated with (FIG. 1A).
  • HEK293t cells were separated into a water-soluble fraction and a water-insoluble fraction using chloroform / methanol.
  • the water-insoluble fraction (chloroform side) containing lipid or cholesterol was dried and resuspended in a reaction buffer.
  • lipids that bind to TBK1 or IRF3 were examined using a protein-lipid overlay assay. As a result, it was found that TBK1 binds to PtdIns (5) P and IRF3 binds to several anionic lipids such as PtdIns (3) P, PtdIns (4) P, and PtdIns (5) P ( FIG. 1C). The binding of these proteins to cationic lipids or any other lipid was undetectable.
  • anionic lipids have the ability to promote phosphorylation of IRF3 in vitro.
  • various synthetic phosphatidylinositols which are anionic lipids, are mixed with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to form liposomes, These were used for in vitro kinase assays.
  • the various phospholipids used in Example 1 contain a fatty acid having 16 carbon atoms, and the specific source is the following catalog number of Avanti Polar Lipid.
  • Example 1 Such a method shown in Example 1 is useful as a screening method for an adjuvant candidate.
  • Whether or not the candidate gene induces IRF3 activation is determined by a reporter plasmid (IFR stimulation response element (ISRE)) designed to directly monitor the activity of the transcription factor IRF together with the expression plasmid of the candidate gene.
  • ISRE reporter plasmid was transformed into HEK293 cells and quantified by a reporter assay using intracellular luciferase expression as an index (FIG. 2A).
  • PIKfyve Overexpression of PIKfyve in HEK293 cells increased the promoter activity of ISRE and IFN ⁇ , but these promoters were not activated by overexpression of the PIKfyve kinase negative mutant. In addition, overexpression of PIKfyve did not affect the promoter activity of NF- ⁇ B, which is a transcription factor essential for the cellular response during virus infection (FIG. 2B).
  • PIKfyve is Kainesu for synthesizing PtdIns (5) P or PtdIns (3,5) P 2 by phosphorylation of the 5-position of the inositol ring, overexpression PtdIns (5) Production of P is induced by, specifically It was thought that the promoter activity of ISRE was increased (FIG. 2C).
  • PtdIns (5) P is specifically required for the activation of the TBK1-IRF3 signal cascade caused by RNA virus infection and suppresses PIKfyve, a metabolic factor of PtdIns (5) P. It is specified by. Furthermore, the addition of PtdIns (5) P suggests the possibility of activating dendritic cells and exerting adjuvant ability through cytokine production.
  • cytokine production in GM-DC derived from IPS-1-deficient mice or IRF3 / IRF7-deficient mice was examined.
  • mice immunized with a P produced OVA and PBS or C8-PtdIns (4,5) OVA-specific IgG of high titers than mice immunized with a P 2 (FIG. 4D).
  • C8-PtdIns (5) P induces T cell activation.
  • Transgenic CD4 + T cells expressing OVA-specific T cell receptors were treated with C8-PtdIns (5) P, C8-PtdIns (4,5) P 2 , or poly I: C in the presence of OVA protein.
  • Co-cultured with GM-DC, and the amount of IFN ⁇ produced was measured as an index of T cell activation (FIG. 4E).
  • Treatment with C8-PtdIns (5) P induced IFN ⁇ production, but treatment with C8-PtdIns (4,5) P 2 did not.
  • Various phospholipids used in Example 4 are those containing a fatty acid having 8 carbon atoms, the specific origin, except for PI (3,4,5) P 3, those of Echelon Biosciences Inc. follows catalog number It is.
  • PI: P-0008a, PI (3) P: P-3008a, PI (4) P: P-4008, PI (5) P: P-5008a, PI (3,4) P 2 : Catalog number P-3408a, PI (3, 5) P 2 : Catalog number P-3508, PI (4, 5) P 2 : Catalog number P-4508a, PI (3,4,5) P 3 are those Avanti Polar Lipid's catalog number is 850176P.
  • IgG 1 increased levels of IgG 1 were seen after immunization with C8-PtdIns (5) P, but not for other subclasses such as IgG 2 , IgG 3 , IgA, or IgE.
  • IgE is produced in a significant amount, there is a high possibility of inducing inflammation, and since IgG 1 or the like plays a leading role in exerting an immune effect, C8-PtdIns (5) has side effects such as inflammation. It was revealed that it has an adjuvant ability that does not cause it.
  • C8-PtdIns (5) P has a sufficient adjuvant ability but can be a safe and useful adjuvant with no side effects due to a specific mechanism of action.

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Abstract

L'invention a pour objectif de fournir un adjuvant qui est destiné à fabriquer un vaccin agissant de manière stable et suffisante, qui possède un effet augmentant de manière suffisante des fonctions immunitaires, et qui présente d'extrêmement faibles probabilités d'effets secondaires y compris dans le cas d'une application à un corps biologique. Plus précisément, l'invention concerne un adjuvent contenant un phospholipide, et un vaccin contenant cet adjuvent et un antigène. Enfin, l'invention concerne un procédé de criblage de substances candidates pour adjuvant qui comporte les étapes (1, 2) suivantes : (1) une étape (1) au cours de laquelle un composant intracellulaire et un facteur de régulation d'interféron (3) (IRF3) sont mis en contact ; et (2) une étape (2) au cours de laquelle le composant intracellulaire oxydant le facteur de régulation d'interféron (3), est choisi.
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WO2020023628A1 (fr) * 2018-07-24 2020-01-30 Hygia Pharmaceuticals, Llc Composés, dérivés et analogues contre le cancer

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JP2002537271A (ja) * 1999-02-17 2002-11-05 シーエスエル、リミテッド 免疫原複合体およびそれに関する方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020023628A1 (fr) * 2018-07-24 2020-01-30 Hygia Pharmaceuticals, Llc Composés, dérivés et analogues contre le cancer

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