WO2016104647A1 - Antigène vaccinal glycosylé et agent de glycosylation - Google Patents

Antigène vaccinal glycosylé et agent de glycosylation Download PDF

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Publication number
WO2016104647A1
WO2016104647A1 PCT/JP2015/086104 JP2015086104W WO2016104647A1 WO 2016104647 A1 WO2016104647 A1 WO 2016104647A1 JP 2015086104 W JP2015086104 W JP 2015086104W WO 2016104647 A1 WO2016104647 A1 WO 2016104647A1
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group
antigen
sugar chain
formula
vaccine
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PCT/JP2015/086104
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English (en)
Japanese (ja)
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三善　英知
浩一 深瀬
良幸 真鍋
真二 高松
匡弘 種村
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国立大学法人大阪大学
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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

Definitions

  • the present invention relates to a vaccine antigen to which a sugar chain containing Gal ⁇ 1-3Gal is bound, a vaccine containing the vaccine antigen, a sugar chain introducing agent capable of introducing a sugar chain containing Gal ⁇ 1-3Gal, and the sugar chain introducing agent.
  • the present invention relates to a method for introducing the sugar chain into an antigen.
  • Cancer vaccine therapy is practiced in clinical practice as the third non-surgical therapy after chemotherapy and radiation therapy.
  • cancer patients with poor general condition have a problem in that their ability to present antigens against tumor antigens is low because their immune functions are reduced.
  • pancreatic cancer has a very poor prognosis, and there are very many patients who have no indication for surgery at the time of diagnosis. For this reason, in addition to the administration of anticancer drugs for the purpose of prolonging life, vaccine therapy is also performed.
  • pancreatic cancer vaccines used for vaccine therapy peptide vaccines such as MUC1, VEGF, and WT1 are known.
  • the effectiveness of pancreatic cancer vaccines is limited due to the low antigen presenting ability against the tumor antigens.
  • Non-patent Document 1 A technique for adding an ⁇ 1-3 galactose epitope to pancreatic cancer cells by an in vitro enzymatic reaction with ⁇ 1-3 galactose transferase is also known.
  • the ⁇ 1-3 galactose epitope is a sugar chain structure made by ⁇ 1-3 galactose transferase, which is possessed by animals below the former monkeys, but in the process of evolution, ⁇ 1-3 galactose transferase is a pseudogene in humans. Therefore, it does not have this sugar chain structure. However, since this sugar chain antigen is exposed to food such as pigs and cattle, approximately 1% of human blood IgG contains natural antibodies against ⁇ 1-3 galactose, and this antibody titer is determined at the end of cancer. It is maintained even in patients.
  • the conventional method utilizes ⁇ 1-3 galactosyltransferase, the amino acid of Asn (asparagine) side chain in the consensus sequence of Asn-X-Ser / Thr (where X is any amino acid residue)
  • a pig-type sugar chain can be introduced only into the group. That is, in the method using a transferase, since only N-type sugar chains can be formed, the number of pig-type sugar chains added is within the range of the above consensus sequence number of the protein to be introduced.
  • An object of the present invention is to introduce a vaccine antigen in which a sugar chain containing galactose ⁇ 1-3 galactose (Gal ⁇ 1-3Gal) is bound via a linker, a vaccine containing the vaccine antigen, and a sugar chain containing Gal ⁇ 1-3Gal.
  • An object is to provide a sugar chain introducing agent and a method for introducing the sugar chain into an antigen using the sugar chain introducing agent.
  • a sugar chain containing Gal ⁇ 1-3Gal and a functional group possessed by an antigen for example, amino group, carboxyl group, mercapto group, hydroxyl group A functional group (reactive group or activating group) capable of forming a bond by reacting with a peptide or protein (or a functional group possessed by an amino acid constituting the peptide or protein)] such as a group via a linker.
  • an antigen for example, amino group, carboxyl group, mercapto group, hydroxyl group A functional group (reactive group or activating group) capable of forming a bond by reacting with a peptide or protein (or a functional group possessed by an amino acid constituting the peptide or protein)
  • the sugar chain can be efficiently introduced into the antigen by bringing the compound and the antigen in which both of them are bonded via such a linker into contact with the antigen; and (iii) such a sugar chain is introduced. It was found that the sugar chain functions as an excellent adjuvant
  • the present invention includes the following inventions.
  • G represents the following formula: L represents a linker containing at least a hydrocarbon unit, A represents an antigen, Z represents a bond between the linker and the antigen, and m represents an integer of 1 or more.
  • the compound represented by the formula (2) is represented by the following formula (2A) (In the formula, L 1 is a sugar unit, L 2 is a linker containing at least a hydrocarbon unit, a is 0 or 1, and G, A, Z and m are the same as those in the formula (2).)
  • a sugar chain introducing agent for introducing a sugar chain containing Gal ⁇ 1-3Gal into an antigen comprising the following formula (1) [In the formula, G represents the following formula: L represents a linker containing at least a hydrocarbon unit, and X represents a functional group capable of forming a bond by reaction with the functional group Y of the antigen.
  • the compound represented by the formula (1) is represented by the following formula (1A)
  • L 1 is a saccharide unit
  • L 2 is a linker containing at least a hydrocarbon unit
  • a is 0 or 1
  • G and X are the same as those in the formula (1).
  • the sugar chain introducing agent according to the above [6] which is a compound represented by the formula: [8]
  • R represents a hydrocarbon unit
  • E represents a unit having a hetero atom
  • k represents an integer of 1 or more.
  • [12] A method of introducing a sugar chain containing Gal ⁇ 1-3Gal into an antigen by bringing the antigen into contact with the sugar chain introducing agent according to any of [6] to [11].
  • a vaccine comprising the vaccine antigen according to any one of [1] to [5].
  • a vaccine comprising an antigen into which a sugar chain containing Gal ⁇ 1-3Gal has been introduced by the sugar chain introducing agent according to any one of [6] to [11].
  • the vaccine according to [14] which is a cancer cell, a component of cancer cell, or a cancer vaccine into which a sugar chain containing Gal ⁇ 1-3Gal is introduced.
  • the vaccine according to [14] or [15] which is a pancreatic cancer cell, a component of pancreatic cancer cell, or a pancreatic cancer vaccine into which a sugar chain containing Gal ⁇ 1-3Gal has been introduced.
  • the present invention also includes a method for administering a vaccine antigen (or a vaccine containing this vaccine antigen) to a human and treating a disease corresponding to the antigen (for example, cancer such as pancreatic cancer).
  • a vaccine antigen or a vaccine containing this vaccine antigen
  • a disease corresponding to the antigen for example, cancer such as pancreatic cancer
  • a vaccine antigen to which a sugar chain containing Gal ⁇ 1-3Gal is bound via a linker can be provided.
  • a compound (sugar chain introduction agent) in which a Gal ⁇ 1-3Gal skeleton and a reactive group for a specific functional group are bonded via a linker can be obtained by a chemical synthesis method. According to such a compound, a sugar chain containing Gal ⁇ 1-3Gal can be efficiently introduced into an antigen (for example, a cancer cell, a cancer peptide vaccine, etc.).
  • an N-type sugar chain is formed in order to utilize a reaction with a functional group possessed by an antigen (for example, an amino group, carboxyl group, mercapto group, etc. possessed by a peptide or protein constituting the antigen).
  • an antigen for example, an amino group, carboxyl group, mercapto group, etc. possessed by a peptide or protein constituting the antigen.
  • the compound of the present invention can introduce many sugar chains containing Gal ⁇ 1-3Gal to known vaccine antigens (particularly cancer vaccine antigens) already applied clinically such as MUC1, VEGF, and WT1. Therefore, a strong adjuvant effect can be obtained without causing any obvious side effects.
  • vaccine antigens particularly cancer vaccine antigens
  • MUC1, VEGF, and WT1 cancer vaccine antigens
  • WT1 WT1
  • a strong adjuvant effect can be obtained without causing any obvious side effects.
  • the Gal ⁇ 1-3Gal skeleton can be introduced via a linker, a vaccine antigen that is easily recognized by the anti-Gal ⁇ 1-3Gal antibody can be efficiently obtained.
  • FIG. 1 shows the results of detecting the anti-BSA antibody obtained in Example 13 by Western blotting.
  • FIG. 2 shows the results of detecting the anti-MUC1 antibody obtained in Example 13 by Western blotting.
  • the compound of the present invention is represented by the following formula (1).
  • Such a compound can be used as a sugar chain introducing agent for introducing a sugar chain containing Gal ⁇ 1-3Gal, which is an epitope recognized by an antibody.
  • G represents the following formula: Wherein L represents a linker (or spacer), X represents a functional group capable of forming a bond by reaction with the functional group Y. ]
  • G is Gal ⁇ 1-3Gal (or Gal ⁇ 1-3Gal unit).
  • G may be either ⁇ -form ( ⁇ -bond) or ⁇ -form ( ⁇ -bond) depending on the type of L (or L 1 or L 2 described later), and the ⁇ -form and ⁇ -form. It may be a mixture.
  • L is a linker (linking group, spacer) for linking the Gal ⁇ 1-3Gal unit and the functional group X.
  • the linker L may contain a saccharide unit (sugar residue).
  • the sugar unit is usually adjacent (or glycosidic bond) to the Gal ⁇ 1-3Gal unit.
  • Examples of the compound represented by the formula (1) which may contain such a sugar unit include a compound represented by the following formula (1A).
  • L 1 is a saccharide unit
  • L 2 is a linker (specifically, a linker other than a saccharide unit)
  • a is 0 or 1
  • G and X are the same as in the above formula (1).
  • sugar may be a monosaccharide, it may be oligosaccharides.
  • the oligosaccharide may be a homopolysaccharide or a heteropolysaccharide.
  • the sugar may be a derivative (for example, a sugar with a reducing end modified, a sugar in which a hydroxyl group constituting the sugar is substituted with an acyloxy group or the like).
  • the sugar may be a cyclic structure (cyclic acetal or cyclic ketal), or may be a ring-opened chain structure.
  • Such a chain structure may be a structure formed by derivatization of a sugar having a cyclic structure (for example, a structure opened by modification with hydrazine or oxime at the reducing end).
  • Specific sugars include, for example, pentose (eg, ribose, arabinose, xylose, deoxyribose, etc.), hexose (eg, fructose, tagatose, allose, altrose, glucose, mannose, galactose, rhamnose, glucosamine, N-acetyl) Glucosamine, glucuronic acid, galactosamine, etc.), oligosaccharides or polysaccharides to which these monosaccharides are bound (for example, disaccharides (eg, maltose, cordobiose, cellobiose, isomaltose, gentibiose, lactose, etc.), etc.).
  • pentose eg, ribose, arabinose, xylose, deoxyribose, etc.
  • hexose eg, fructose, tagatose, allose
  • the following unit in which N-acetylglucosamine (GlcNAc) is ⁇ -1,4 linked to the Gal ⁇ 1-3Gal unit, which is an epitope, is a porcine sugar chain ( ⁇ -Gal).
  • lactosamine (Gal ⁇ 1-4GlcNAc) is transferred to a porcine sugar chain (Gal ⁇ 1-3Gal ⁇ 1-4GlcNAc), and thus an N-acetylglucosamine unit is essential. Even without an acetylglucosamine unit, the Gal ⁇ 1-3Gal unit can be introduced.
  • the group GL 1 — include, for example, a group represented by the following formula (G1) (a group in which the sugar unit is an N-acetylglucosamine unit), and the reducing end is modified in such a group G1.
  • a unit for example, a group represented by the following formula (G2), a group represented by the following formula (G3), a group represented by the following formula (G4)
  • G1 a group represented by the following formula (G2), a group represented by the following formula (G3), a group represented by the following formula (G4)
  • N-acetylglucosamine The group GL 1 -etc. substituted on the sugar unit is included.
  • Ra represents a substituent such as a hydrocarbon group (for example, a hydrocarbon group described later such as an alkyl group (such as a methyl group)), and G and Ac are the same as described above.)
  • the linker (—L— or —L 2 —) is not particularly limited, but usually has at least a hydrocarbon unit (hydrocarbon unit, hydrocarbon group). There are many cases.
  • hydrocarbon unit examples include a divalent hydrocarbon group, such as an aliphatic hydrocarbon unit [for example, an alkylene (or an alkylidene group, such as methylene, ethylene, trimethylene, propylene, tetramethylene, pentamethylene, hexamethylene, etc.
  • aliphatic hydrocarbon unit for example, an alkylene (or an alkylidene group, such as methylene, ethylene, trimethylene, propylene, tetramethylene, pentamethylene, hexamethylene, etc.
  • the hydrocarbon unit includes a unit in which two or more of the above units are connected [for example, an alkylene-arylene group (for example, methylenephenylene group, ethylenephenylene group), an alkenylene-arylene group (for example, vinylenephenylene group), etc.) Is also included.
  • an alkylene-arylene group for example, methylenephenylene group, ethylenephenylene group
  • an alkenylene-arylene group for example, vinylenephenylene group
  • the hydrocarbon unit includes a unit in which a substituent is substituted on the above unit.
  • the substituent is not particularly limited, for example, a hydrocarbon group ⁇ eg, alkyl group [eg, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, alkyl groups such as t-butyl group, pentyl group, hexyl group (for example, C 1-12 alkyl group, preferably C 1-8 alkyl group)], cycloalkyl groups (for example, cyclopentyl group, cyclohexyl group, etc.) C 4-10 cycloalkyl group, preferably C 5-8 cycloalkyl group), aralkyl group (for example, C 6-10 aryl C 1-4 alkyl group such as benzyl group, phenethyl group), polycyclic aliphatic carbonization
  • the substituents may be substituted alone or in combination with two or more hydrocarbon units.
  • Typical hydrocarbon units include alkylene groups, arylene groups, and the like.
  • the linker may have one or more hydrocarbon units.
  • the linker may comprise a unit containing a heteroatom.
  • examples of the hetero atom include a nitrogen atom, an oxygen atom, and a sulfur atom.
  • the unit containing a hetero atom may have two or more hetero atoms.
  • the unit containing a hetero atom include a unit containing a nitrogen atom (for example, an imino group (—NH—), a substituted imino group (for example, —NR a — (wherein R a is the same as above), etc.) Amide group (—NHCO—), urethane group (—NCO—), urea group (—NHCONH—), azo group (—N ⁇ N—), hydrazone group (—C ⁇ N—NH—), nitrogen-containing ring Groups (for example, polyvalent groups corresponding to nitrogen-containing heterocyclic compounds such as piperidine, pyridine, indole, quinoline, carbazole, etc.) ⁇ , units containing oxygen atoms ⁇ eg, oxygen atoms (—O—), carbonyl groups ( -CO-), ester groups (oxycarbonyl group, -OCO-), oxygen-containing ring groups (for example, tetrahydrofuran, tetrahydropyran, furan
  • the unit having a hetero atom includes a unit in which a substituent is substituted on the above unit depending on the type.
  • substituents include the substituents exemplified in the section of the unit having a hydrocarbon group (for example, a hydrocarbon group, a halogen atom, etc.).
  • Typical units containing a hetero atom include an imino group, a substituted imino group (such as an alkylimino group), an amide group, an oxygen atom (ether group, —O—), a carbonyl group (—CO—), an ester group (— OCO-, -COO-), thio group (-S-), thiocarbonyl group (-CS-) and the like.
  • the linker may have a unit containing 1 or 2 or more heteroatoms.
  • the linker has a hydrocarbon unit and a unit having a heteroatom
  • the positional relationship of these units in the linker is not particularly limited, but usually the hydrocarbon unit and the unit having a heteroatom are adjacent (or bonded). There are many cases.
  • the unit which has a hetero atom between the hydrocarbon units may be located.
  • the linker is a unit in which a hydrocarbon unit and a unit having a hetero atom [for example, an oxyalkylene unit (for example, an oxyC 2-4 alkylene group such as oxyethylene group), a peptide unit, etc.] are combined as one unit.
  • the repeating unit (repeating unit) may be formed. In such a case, the number of repetitions may be 2 or more (for example, about 2 to 20, preferably about 2 to 10, and more preferably about 2 to 6).
  • linkers include, for example, groups (linkers) represented by the following formulas (L1) to (L8) (note that “-” at the left end is G or The bond to L 1 , the “ ⁇ ” at the right end represents the bond to X).
  • R represents a hydrocarbon unit
  • E represents a unit having a hetero atom
  • k represents an integer of 1 or more.
  • hydrocarbon unit R and the unit E having a hetero atom include the units exemplified above.
  • the hydrocarbon unit may be an alkylene group, and E may be an ether group (—O—) or an amide group (—NHCO—).
  • E may be an ether group (—O—) or an amide group (—NHCO—).
  • the plurality of R or E may be the same or different units.
  • More specific linkers include, for example, groups (linkers) represented by the following formulas (L1) to (L6).
  • R 1 , R 2 and R 3 are the same or different divalent hydrocarbon groups
  • R 4 is a C 2-4 alkylene group
  • R 5 is an amino acid residue
  • k is the same as above.
  • examples of the divalent hydrocarbon group and the C 2-4 alkylene group include those exemplified above.
  • R 1 to R 3 may typically be an alkylene group.
  • R 1 is a methylene group
  • R 2 and R 3 are alkylene groups having 2 or more carbon atoms (for example, C 2-10 alkylene groups such as ethylene group, trimethylene group, tetramethylene group, hexamethylene group, etc.) Also good.
  • examples of R 4 include an ethylene group and a propylene group, and an ethylene group (—CH 2 CH 2 —) may be used.
  • R 5 is an amino acid residue, that is, a group obtained by removing an amino group and a carboxyl group from an amino acid.
  • R 5 is a methylene group.
  • k is an integer of 1 or more. When k is 2 or more, the number of k (the number of repetitions) may be, for example, about 2 to 20, preferably about 2 to 10, and more preferably about 2 to 6.
  • the functional group X may be any functional group that can react with the functional group Y different from the functional group X to form a bond.
  • the functional group Y is usually a functional group of a peptide or protein (for example, a peptide or protein that constitutes an antigen) [for example, an amino acid that constitutes a peptide or protein such as a hydroxyl group, a carboxyl group, an amino group, or a mercapto group.
  • a functional group introduced into a peptide or protein for example, an azide group, an alkynylene group (such as an acetylene group)] or the like.
  • Examples of the functional group X include a carboxyl group or a derivatized (activated) carboxyl group ⁇ for example, a carboxyl group and an N-hydroxyamine (for example, N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxynorbornene- 2,3-dicarboxylic acid imide, N-hydroxypiperidine, 2-hydroxyimino-2-cyanoacetic acid alkyl ester, 2-hydroxyimino-2-cyanoacetic acid amide, N-hydroxyimidazole, N-hydroxymaleimide, etc.) or derivatives thereof (Eg, N, N, N ′, N′-tetramethyl-O— (N-succinimidyl) uronium tetrafluoroborate) ester-bonded groups, acid halide groups, acid anhydride groups, acid azide groups, etc.
  • N-hydroxyamine for example, N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxynorbornene-
  • Active ester group ⁇ , derivatized (activated) Droxyl group for example, sulfonate group (for example, tosylate group, mesylate group, triflate group, etc.)], maleimide group, halogen atom (chlorine atom, bromine atom, iodine atom, etc.), azido group, alkynyl group (for example, An ethynyl group or an acetylene group) and a group having a hexatriene- ⁇ -carbonyl skeleton (for example, a group represented by the following formula).
  • sulfonate group for example, tosylate group, mesylate group, triflate group, etc.
  • maleimide group for example, halogen atom (chlorine atom, bromine atom, iodine atom, etc.), azido group, alkynyl group (for example, An ethynyl group or an acetylene group
  • the carboxyl group or the derivatized carboxyl group is a functional group Y such as a hydroxyl group or an amino group
  • the derivatized hydroxyl group is a functional group Y such as a hydroxyl group or a carboxyl group
  • the maleimide group is a mercapto group or the like.
  • the functional group Y, the halogen atom is a mercapto group, the nitrogen atom constituting the nitrogen ring, the functional group Y such as an imino group (for example, the imino group of an imidazolyl ring constituting histidine), and the azide group is a functional group such as an alkynyl group Y and a group having a hexatriene- ⁇ -carbonyl skeleton each react with a functional group Y such as an amino group.
  • the group -LX or the group -L 2 -X bonded to G or GL 1 includes L or L 2 as exemplified above, and X as exemplified above.
  • the following table shows an example of all the combinations. In the table, symbols such as R 1 are the same as described above.
  • carbohydrate introduction agent (compound represented by Formula (1) or (1A)) can be manufactured using a well-known organic chemical method.
  • the reducing end of the sugar corresponding to G or GL 1 is oxidized by a conventional method (for example, the hydroxyl group at the reducing end of a protected sugar is oxidized after allyl etherification, the hydroxyl group at the reducing end is alkalized.
  • the linker is —CH 2 —CONH—CH 2 CH 2 —NHCO—CH 2 —
  • the functional group X is — Compounds having I can be obtained.
  • the linker is —CH 2 —CONH—CH 2 CH 2 —NHCO—CH 2 —
  • the functional group X is A compound having a tosyloxy group can be obtained.
  • the sugar chain introducing agent of the present invention (compound represented by the formula (1) or (1A)) has a Gal1-3Gal skeleton and a functional group X, and a Gal1-3Gal skeleton (Gal1-3Gal) as an antigen.
  • a sugar chain containing Gal ⁇ 1-3Gal is bound via a linker (vaccine antigen).
  • the antigen is composed of a peptide or protein, and the amino acid constituting the peptide or protein has various functional groups Y that can react with the functional group X.
  • a sugar chain introducing agent having an active ester group as the functional group X reacts with an amino acid having an amino group as the functional group Y to form a bond.
  • the functional group X of the sugar chain introducing agent and the functional group Y of the antigen are reacted to introduce a sugar chain containing Gal1-3Gal into the antigen. (Or can be linked through a linker).
  • the amino acid may have a functional group Y at the terminal of the peptide or protein, and may have a functional group as a side chain of the main chain.
  • the amino acid located at the terminal has an amino group or a carboxyl group as the functional group Y regardless of the type of amino acid, and the functional group Y does not participate in peptide bonds with amino acids such as lysine, cysteine, serine, threonine, and glutamic acid. Therefore, these amino acids have a functional group Y (for example, amino group, hydroxyl group, carboxyl group, mercapto group, etc.) as a side chain of the peptide or protein in addition to the terminal.
  • a functional group Y for example, amino group, hydroxyl group, carboxyl group, mercapto group, etc.
  • the functional group Y located at the end or side chain of the peptide or protein can be used in the antigen, and thus the present invention is not limited to the amino group possessed by Asn of the specific consensus sequence as described above.
  • the side chain functional group Y can be used, a very large number of Gal1-3Gal skeletons can be introduced.
  • the functional group Y may contain a functional group other than the amino group derived from asparagine, which Asn of a specific consensus sequence particularly has.
  • functional groups include lysine-derived amino groups, cysteine-derived mercapto groups, serine-derived hydroxyl groups, threonine-derived hydroxyl groups, aspartic acid-derived carboxyl groups, glutamic acid-derived carboxyl groups, and these amino acids.
  • a functional group Y that does not originally exist in the antigen and react with the functional group X of the sugar chain introducing agent.
  • an azide group or acetylene group may be introduced into the antigen as the functional group Y and reacted with a sugar chain introducing agent having the acetylene group or azide group as the functional group X.
  • Such a method using the reaction between the functional group X and the functional group Y can also be referred to as a click chemistry method.
  • Representative functional groups Y include hydroxyl group, carboxyl group, amino group, mercapto group, azide group, acetylene group and the like.
  • the antigen may have a combination of two or more of these functional groups.
  • the antigen is not particularly limited, and examples thereof include pathogens (for example, viruses such as influenza viruses or bacteria), cancer antigens, and the like. Moreover, it is good also considering a cancer cell (or cancer tissue) itself as an antigen.
  • the antigen may in particular be a cancer cell, a component of a cancer cell, a cancer antigen or a cancer antigen peptide.
  • the component of the cancer cell is not particularly limited as long as it is a constituent component of a cell that does not retain the cell morphology.
  • lysate for example, lysate containing a membrane component, lysate containing no membrane component, etc.
  • membrane components membrane proteins, etc.
  • the target cancer is not particularly limited.
  • pancreatic cancer has a poor prognosis and requires strong immunogenicity. Therefore, the utility of using the sugar chain introducing agent of the present invention for pancreatic cancer antigens is extremely high.
  • cancer antigen various antigens can be used depending on the type of cancer to be treated.
  • MUC1 peptide
  • VEGF peptide
  • WT1 peptide
  • the present invention also includes a vaccine antigen in which a sugar chain containing Gal ⁇ 1-3Gal is introduced by the sugar chain introducing agent.
  • Such a vaccine antigen has a structure different from that of an antigen (cancer cell) into which a porcine sugar chain has been introduced by a transferase.
  • the vaccine antigen of the present invention has a structure in which a sugar chain containing Gal ⁇ 1-3Gal is bound to the antigen via a linker that cannot be introduced by a transferase.
  • the functional group Y reacts with a functional group other than an amino group derived from asparagine having a specific consensus sequence, and a sugar chain containing Gal ⁇ 1-3Gal is introduced. For this reason, the amount of introduction of the Gal ⁇ 1-3Gal skeleton is usually larger than that when a transferase is used.
  • such a vaccine antigen can be represented by the following formula (2) or the following formula (2A).
  • the following formula (2) and the following formula (2A) correspond to the case of using the compound represented by the formula (1) and the compound represented by the formula (1A), respectively.
  • A is an antigen
  • Z is a bond between a linker and an antigen
  • m is an integer of 1 or more
  • G, L, L 1 , L 2 , and a are the same as described above.
  • Z is a bond formed by the reaction between the functional group X of the formula (1) and the functional group Y of the antigen (for example, an amino acid-derived functional group of the antigen).
  • the functional group Y may contain at least a functional group other than an amino group derived from asparagine. Examples of coupling are as illustrated in the table and the like.
  • Z is an amide bond (—NHCO—).
  • m is an integer of 1 or more. That is, a sugar chain containing one or more Gal ⁇ 1-3Gal is introduced into the antigen. Since an antigen usually has a plurality of functional groups Y that react with the functional group X, m is usually a plurality or two or more (eg, 3 to 1000, preferably 5 to 500, The number may preferably be 10 to 300). Depending on the type of antigen and the like, it is possible to bind a sugar chain containing a large number of Gal ⁇ 1-3Gal.
  • the vaccine antigen of the present invention can be used as a vaccine as described above.
  • the form of the vaccine of the present invention is not particularly limited as long as it contains the vaccine antigen of the present invention.
  • the vaccine of the present invention may be composed only of the vaccine antigen of the present invention, and may constitute a composition (pharmaceutical composition) according to the dosage form and the like.
  • the vaccine of this invention may make the vaccine antigen of this invention into a formulation (for example, solid formulation, liquid formulation etc.) suitably.
  • a formulation for example, solid formulation, liquid formulation etc.
  • conventional additives and formulation methods can be used.
  • the vaccine of the present invention may contain one or more types of adjuvant, and may not contain an adjuvant.
  • the vaccine of the present invention is highly useful in that a high adjuvant effect can be obtained without including an adjuvant.
  • the vaccine of the present invention contains an adjuvant, it can be appropriately selected from known adjuvants.
  • aluminum adjuvant for example, aluminum salt such as aluminum hydroxide, aluminum phosphate, aluminum sulfate or a combination thereof), Freund's adjuvant (complete or incomplete), TLR ligand (for example, CpG, Poly (I : C), Pam3CSK4, etc.), BAY, DC-chol, pcpp, monophosphoryl lipid A, QS-21, cholera toxin, formylmethionyl peptide and the like.
  • it is an aluminum adjuvant, a TLR ligand, or a combination thereof.
  • the present invention also includes a method of treating the corresponding disease (for example, cancer) by administering the vaccine (or sugar chain-introduced antigen or pharmaceutical composition).
  • the dose of vaccine can be appropriately selected according to the type of disease and the type of vaccine.
  • the present invention includes aspects in which the above-described configurations are variously combined within the technical scope of the present invention.
  • Ph represents a phenyl group
  • Bn and Bz represent a benzyl group
  • Troc represents a 2,2,2-trichloroethoxycarbonyl group
  • Allyl represents an allyl group
  • Ac represents an acetyl group
  • Me represents a methyl group.
  • the reaction mixture was ultrafiltered using a centrifugal filter [Microcon (registered trademark) YM-10, 50,000 cut, Millipore] (10000 rpm for 5 minutes). Distilled water was added to this. Substitution of the solution from the buffer solution to water using this ultrafiltration was performed three times to obtain BSA16 into which a sugar chain was introduced.
  • the introduction of sugar chains was confirmed by measuring the mass spectrum in BSA before and after the reaction. That is, the molecular weight of BSA before the reaction was about 66000, and the molecular weight of BSA after the reaction was about 73,000. From this result, it was confirmed that Compound 13 reacted with amino acids constituting the protein to form a bond. It was also confirmed that compound 13 can introduce a large number of sugar chains.
  • One sugar chain was introduced because 34 of the 35 cysteine units possessed by BSA were disulfide-bonded, so that one cysteine-derived mercapto group reacted with compound 14.
  • the introduction of sugar chains was confirmed by measuring the mass spectrum in BSA before and after the reaction. That is, the molecular weight of BSA before the reaction was about 66000, and the molecular weight of BSA after the reaction was about 67,000. From this result, it was confirmed that Compound 14 reacted with amino acids constituting the protein to form a bond.
  • the introduction of sugar chains was confirmed by measuring the mass spectrum in BSA before and after the reaction. That is, the molecular weight of BSA before the reaction was about 66000, and the molecular weight of BSA after the reaction was about 70000. From this result, it was confirmed that Compound 15 reacted with amino acids constituting the protein to form a bond. It was also confirmed that compound 15 can introduce a large number of sugar chains.
  • Example 7 Sugar chain introduction using compound 13 According to the following steps, a sugar chain-introduced MUC1 peptide 20 was obtained.
  • the reaction mixture was purified by injection into HPLC [column: COSMOSIL 5C 18 -AR-300, 10 ⁇ 250 nm; MeCN in H 2 O containing 0.1% TFA (0-100% linear gradient over 50 min, 1 mL / min); UV detection at 220 nm; retention time: 30 min], and MUC1 peptide 20 (1.3 mg, 67%) into which a sugar chain was introduced was obtained.
  • HRMS (ESI-LIT-Obitrap) calcd. for C 104 H 165 N 27 O 46 Na 2 [M + 2Na] 2+ 1287.5608, found 1287.5629
  • MUC1 peptide 19 was synthesized as follows.
  • MUC1 peptide 19 was synthesized according to a general Fmoc solid phase synthesis protocol. As Fmoc protected amino acids, His (Boc), Ala, Pro, Thr (tBu), Ser (tBu), Gly, Arg (Pmc), Asp (OtBu), and Val were used, and Boc-Gly was used for the N-terminus. .
  • Glycine 2-chlorotrityl resin (0.195 mmol scale, 0.78 mmol / g, 100-200 mesh, 1% DVB, Watanabe Chemical) was used, and all amino acids were condensed according to the following procedure. went. Fmoc protected amino acid (0.975 mmol) 1-hydroxybenzotriazole (HOBt, 132 mg, 0.975 mmol), diisopropylcarbodiimide (DIC, 305 ⁇ L, 1.95 mmol) is dissolved in DMF (3.0 mL) and mixed for 30 minutes. The reagent was activated with The activated reagent was added to the resin and the suspension was shaken at room temperature for 2 hours.
  • Fmoc protected amino acid (0.975 mmol) 1-hydroxybenzotriazole (HOBt, 132 mg, 0.975 mmol), diisopropylcarbodiimide (DIC, 305 ⁇ L, 1.95 mmol) is dissolved in DMF (3.0
  • Et 2 O (30 mL) was added to the resulting precipitate, and the same centrifugation operation was performed twice to obtain a crude protected amino acid (343 mg, 62%) as a precipitate.
  • Et 2 O (4 mL) was added to the supernatant and centrifuged (15,000 rpm, 5 minutes) to remove the supernatant.
  • Example 8 Sugar chain introduction using compound 13 According to the following steps, WT1 peptide 22 into which a sugar chain was introduced was obtained.
  • Peptide 21 is a peptide having the following amino acid sequence (1-182 aa, 182aa length), and a synthesized peptide was used.
  • Example 9 Sugar chain introduction using compound 13 According to the following steps, an eEF2L peptide 24 into which a sugar chain was introduced was obtained.
  • Peptide 23 is a peptide having the following amino acid sequence and was synthesized.
  • Example 10 Sugar chain introduction into lysate According to the following steps, a cell lysate of PANC1 cells into which a sugar chain was introduced was obtained.
  • the lysate of PANC1 cells was prepared as follows. About 100 conf PANC-1 cells (5-6 ⁇ 10 6 cells) were removed by treatment with 0.25% Trypsin-0.01 mM EDTA, and the cell solution resuspended in fresh medium was collected in a 15 ml tube. . After centrifugation at 100 ⁇ g, 5 min, 4 ° C., the supernatant was removed, 1 ml of PBS ( ⁇ ) was added and suspended by pipetting, and the whole cell solution was transferred to a 1.5 ml tube. After centrifugation at 600 ⁇ g, 3 min, 4 ° C., the supernatant was removed and stored at ⁇ 80 ° C. until the cell lysate was prepared.
  • the cell lysis buffer (10 mM Tris-HCl pH 7.8, 1% Nonidet P-40, 0. 15 M NaCl, 1 mM EDTA, protease inhibitor) was added, and the cells were disrupted for 7 to 8 min using a water bath type ultrasonic crusher (BioRaptor) at 15 sec ON / OFF interval under ice cooling. Then, it centrifuged at 20,000xg, 15min, 4 degreeC. The supernatant collected after centrifugation was stored as a whole cell lysate at ⁇ 80 ° C. until sugar chain introduction.
  • BioRaptor BioRaptor
  • the immobilized PANC1 cells were prepared as follows.
  • the cell pellet collected by the method described in Example 10 was treated under a fixing condition of -20 ° C methanol for 10 minutes. After this treatment, the mixture was centrifuged at 300 ⁇ g, 3 min, 4 ° C. to remove the fixative, and washed 3 times with PBS to remove the remaining fixative. Thereafter, the fixed cell pellet was resuspended in FACS buffer and stored at 4 ° C. until FACS measurement.
  • the FACS analysis method is as follows.
  • the immobilized cells 10 about five, were suspended in FACS buffer of 100 [mu] l, added M86 antibody 2 [mu] l, and stained on ice for 30 minutes with 50-fold dilution. Thereafter, the supernatant is removed by centrifugation at 300 ⁇ g for 3 minutes, 1 ⁇ l of PE-labeled anti-mouse IgM Ab is added to 100 ⁇ l of FACS buffer (2% FBS / D-PBS) (100-fold dilution), and the cells are resuspended.
  • the mixture was allowed to stand for 20 to 30 minutes on ice, centrifuged again at 300 ⁇ g for 3 minutes, and the supernatant was removed.
  • the supernatant was resuspended in 100 ⁇ l of FACS buffer, and a FACS analysis was performed using a part thereof.
  • Example 12 Identification by ELISA using M86 antibody BSA16 obtained in the above Example was identified by ELISA using M86 antibody (an antibody recognizing ⁇ -Gal). For comparison, identification was also performed for subjects into which no sugar chain was introduced (negative control, negative control) and positive control (positive control). The results are as follows. In the following table, the magnification indicates the dilution factor of the M86 antibody.
  • the M86 antibody that recognizes ⁇ -Gal was prepared by adding 50 ⁇ l of a 12.5-fold diluted M86 antibody solution to the first well in order to prepare a 2-fold dilution series. After stirring well, the work of transferring 50 ⁇ l to the next well was sequentially performed, and finally a dilution series up to 3200-fold dilution was prepared. Excess 50 ⁇ l in the last 8 rows was removed. Thereafter, the ELISA plate was wrapped in a wrap and incubated in a 37 ° C. gas phase thermostat for 2 hours. The antibody solution was discarded, each well was washed twice with 200 ⁇ l of 0.1% Tween / PBS, and then the wash solution was removed as completely as possible by tapping the plate on a paper towel.
  • the buffer composition used is as follows. Carbonate buffer: 15 mM Na 2 CO 3 , 35 mM NaHCO 3 , 3.1 mM NaN 3 (pH 9.5) Peroxidase buffer: 50 mM Na 2 HPO 4 , 60 mM citric acid (pH 5.0)
  • the anti- ⁇ -Gal antibody titer in mouse serum was determined by measuring over time using the method described in Example 10.
  • the amount of protein used in one vaccine experiment was 10 ⁇ g / head for both labeled and unlabeled substances in the case of BSA, and 2.5 ⁇ g / head for both labeled and unlabeled substances in the case of the MUC1 peptide.
  • Groups of 5 mice were prepared.
  • Serum was prepared by administering intraperitoneal administration once a week for 5 weeks, and sampling 200 to 300 ⁇ l of blood approximately every week after each administration. Each serum was dispensed into multiple microtubes and stored at ⁇ 80 ° C.
  • the reaction was performed at room temperature for 1 hour using HRP-labeled anti-mouse IgG (Promega) diluted 5000 times with skim milk / TBST. After the completion of the reaction, similarly, washing was performed with TBST for 10 minutes three times, and then the target band was detected by chemiluminescence using ECL-Prime (GE). For detection of chemiluminescence, LAS-4000mini (GE) was used.
  • Electrophoresis was performed at a constant voltage of 150 V using 7.5% polyacrylamide gel using 30 ⁇ g of cell lysate of pancreatic cancer cell line Panc1 per lane. Thereafter, the resultant was transferred to a nitrocellulose filter (Millipore) at 120 mA for 60 minutes using a semi-dry type blotting apparatus (Bio-Rad). The nitrocellulose filter after the transfer was cut and separated for each lane and subjected to overnight blocking at 4 ° C. using 5% skim milk / TBST.
  • CST anti-MUC1 antibody
  • MUC1 peptide vaccine serum MUC1 peptide vaccine serum
  • ⁇ -gal-MUC1 peptide vaccine serum MUC1 peptide vaccine serum
  • ⁇ -1,3- a negative control
  • galactosyltransferase serum Four types of galactosyltransferase serum were used as primary antibodies and reacted at room temperature for 1 hour. After completion of the reaction, the antibody solution was removed by suction, and washed with TBST for 10 minutes three times.
  • anti-MUC1 antibody and secondary antibodies against three mouse sera were reacted with HRP-labeled anti-mouse IgG (Jackson Lab) diluted 5-fold with 5% skim milk / TBST for 1 hour at room temperature. I let you. After the completion of the reaction, similarly, washing was performed with TBST for 10 minutes three times, and then the target band was detected by chemiluminescence using ECL-Prime (GE). For detection of chemiluminescence, LAS-4000mini (GE) was used.
  • FIG. 1 shows the detection result of the anti-BSA antibody
  • FIG. 2 shows the detection result of the anti-MUC1 antibody.
  • “PonceauS” is a confirmation of the BSA band.
  • the compound or sugar chain introducing agent of the present invention can easily introduce a sugar chain containing Gal ⁇ 1-3Gal into an antigen.
  • the sugar chain can be introduced regardless of the type of amino acid (or amino acid sequence), a large amount of the sugar chain can be introduced, and a very high immune reaction can be obtained.
  • the sugar chain can be introduced into a known vaccine antigen, a high adjuvant effect can be obtained. Therefore, the compound (or sugar chain introduction agent) of the present invention can also be used as an adjuvant (immunostimulatory agent, immune enhancer).
  • Such a sugar chain introduction agent and vaccine antigen of the present invention are useful for the treatment of various diseases such as cancer (malignant tumor) and infectious diseases (influenza, etc.), and in particular, cancer vaccine therapy such as pancreatic cancer. Useful in.

Abstract

L'invention concerne un antigène vaccinal auquel est lié, par l'intermédiaire d'un lieur, un glycane contenant un galactose de type α1-3 galactose (Galα1-3Gal). L'antigène vaccinal comprend un composé représenté par la formule (2) (dans la formule, G représente un groupe représenté par [Produit chimique 2], L représente un lieur comprenant au moins une unité hydrocarbonée, A représente un antigène, Z représente une liaison entre le lieur et l'antigène, et m représente un nombre entier égal ou supérieur à 1). L'antigène est de préférence une cellule cancéreuse, un composant d'une cellule cancéreuse, un onco-antigène ou un peptide d'un onco-antigène.
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JP2019526614A (ja) * 2016-09-13 2019-09-19 センタウリ セラピューティクス リミテッド 新規化合物及びその治療上の使用

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WO2008057235A2 (fr) * 2006-10-25 2008-05-15 Newlink Genetics AUGMENTATION DE L'IMMUNOGÉNICITÉ DES ANTIGÈNES ASSOCIÉS AUX TUMEURS PAR L'ADJONCTION D'ÉPITOPES αGAL

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Publication number Priority date Publication date Assignee Title
WO2008057235A2 (fr) * 2006-10-25 2008-05-15 Newlink Genetics AUGMENTATION DE L'IMMUNOGÉNICITÉ DES ANTIGÈNES ASSOCIÉS AUX TUMEURS PAR L'ADJONCTION D'ÉPITOPES αGAL

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019526614A (ja) * 2016-09-13 2019-09-19 センタウリ セラピューティクス リミテッド 新規化合物及びその治療上の使用
JP7185622B2 (ja) 2016-09-13 2022-12-07 センタウリ セラピューティクス リミテッド 新規化合物及びその治療上の使用

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