WO2014178195A1 - 糖鎖抗原の免疫誘導剤 - Google Patents
糖鎖抗原の免疫誘導剤 Download PDFInfo
- Publication number
- WO2014178195A1 WO2014178195A1 PCT/JP2014/002401 JP2014002401W WO2014178195A1 WO 2014178195 A1 WO2014178195 A1 WO 2014178195A1 JP 2014002401 W JP2014002401 W JP 2014002401W WO 2014178195 A1 WO2014178195 A1 WO 2014178195A1
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- WIPO (PCT)
- Prior art keywords
- fucα1
- siaα2
- acid
- glcnac
- antigen
- Prior art date
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
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- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
- C07H15/06—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical being a hydroxyalkyl group esterified by a fatty acid
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
- C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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- A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Definitions
- the present invention relates to a method for enhancing the immunogenicity of a sugar chain antigen and a carrier compound therefor.
- the present invention also relates to a novel glycolipid compound obtained by binding a sugar chain antigen and a carrier compound, an immune enhancement method using the glycolipid compound, a method for producing an antibody that recognizes a sugar chain antigen, and particularly a method for producing a monoclonal antibody.
- Mammalian cells express sugar chains with characteristic structures, reflecting cell types, development / differentiation stages, pathological conditions, and the like.
- Sugar chains are present in cell surface layers and serum in the form of glycoconjugates such as glycoproteins and glycolipids, and effective use as biomarkers is being studied in the same manner as proteins and nucleic acids because of their properties.
- glycoconjugates such as glycoproteins and glycolipids
- biomarkers is being studied in the same manner as proteins and nucleic acids because of their properties.
- sugar chains have an important function in vivo, and research on the function is also being advanced.
- the technology related to sugar chain structure identification and detection has not been sufficiently established compared to proteins and nucleic acids, functional analysis is difficult to proceed.
- a typical example is an N-linked sugar chain of ⁇ -fetoprotein (AFP).
- AFP ⁇ -fetoprotein
- An increase in the serum level of AFP has been used for a long time as a diagnostic index for liver cancer.
- it since it also increases in hepatitis and cirrhosis, it is a drawback that only liver cancer cannot be accurately diagnosed.
- Non-patent Document 2 Non-patent Document 2
- AFP-L3 containing such a sugar chain structure is highly accurate. It is attracting attention as a diagnostic marker for liver cancer.
- Non-patent Document 3 N-linked sugar chains are useful as a marker for liver fibrosis (a pre-stage of cirrhosis), which is a symptom of cirrhosis.
- the N-linked sugar chain of ⁇ 1-acid glycoprotein (AGP) present in serum has a characteristic structure that correlates with the onset of liver fibrosis, and LecT-Hepa was developed as a marker to diagnose this. (Non-Patent Document 4).
- a peptide when used as an immunogen, there is a technique for enhancing its immunogenicity using an immune induction system based on antigen presentation using MHC molecules.
- an immune inducer in which any peptide is bound to a carrier protein such as KLH or BSA, the interaction with the MHC molecule through the carrier protein is enhanced, and the target antigen is made a thymus-dependent antigen. It is possible. If it can be a thymus-dependent antigen, B cells, which are antibody-producing cells, can interact with helper T cells to undergo specificity such as class switching, affinity maturation, and antibody production memory. It is possible to produce useful antibodies with high applicability.
- oligosaccharides are immunogens, conjugates (artificial glycoproteins) with carrier proteins such as KLH and BSA used for these peptides are prepared and immunized, and the target carbohydrate antigen is There is a report that the IgG antibody which recognizes was produced (nonpatent literatures 9 and 10).
- the oligosaccharide chain in this report refers to a study using ⁇ 1,6 glucose polymer (derived from isomaltose) prepared from dextran derived from microorganisms, and is derived from mammalian complex carbohydrate-derived oligosaccharide chains.
- the affinity of the obtained monoclonal antibody is as low as about 10 2 M ⁇ 1 to 10 5 M ⁇ 1 (Non-patent Document 11) in the binding constant. It was not enough as a manufacturing method. This result also suggests that in the case of oligosaccharide chains derived from mammalian cells, it is unlikely to function as a thymus-dependent antigen even if the same conjugation method as in the case of peptide immunization is applied as it is. Since it is unlikely that antibodies exhibiting sex / specificity can be obtained, this method could not be a practical option for those skilled in the art. Therefore, research into carrier compounds that can be used in this way has become active.
- the immunity induction method is also selected for any sugar chain. It is not a general-purpose technique for inducing antibodies.
- the reductive amination method of cleaving the reducing end sugar of the oligosaccharide chain is used as a production method when conjugating the carrier compound and the oligosaccharide chain, the oligosaccharide chain The immunity-inducing agent cannot be synthesized while maintaining the structure.
- a spacer is essential, and there is a disadvantage that a complicated synthesis step of previously binding a spacer sugar or hydroxybenzaldehyde to an oligosaccharide chain is required.
- a method using magnetic metal nanoparticles in which a sugar chain ligand is combined with an adjuvant via a linker has been proposed (Patent Document 1).
- Patent Document 1 the preparation of nanoparticles itself is not easy, and a sugar chain ligand and an adjuvant are not easily prepared. The process of binding the bands to the nanoparticles at a constant rate is not easy.
- a carrier protein that is a diphtheria toxin cross-reactive substance is bound to the reducing side of the Globo-H sugar chain via an aminoalkyl and p-nitrophenyl ester to Although it is proposed to use a vaccine (Patent Document 2), it is not a general-purpose technique that can be applied to sugar chain antigens in general.
- an anti-sugar having an immunopotentiating effect on an arbitrary sugar chain antigen, particularly an oligosaccharide chain antigen contained in an N-linked sugar chain of a glycoprotein, and having high specificity and affinity for a target sugar chain antigen
- a carrier compound capable of providing a chain antigen monoclonal antibody is provided.
- the present invention provides a method for producing an immune inducer that simply conjugates a carrier compound and an oligosaccharide chain while maintaining the structure of the target oligosaccharide chain antigen.
- the present invention searches for carrier compounds capable of binding to various target oligosaccharide antigens, particularly oligosaccharide antigens contained in N-linked sugar chains of glycoproteins, and enhancing the immunogenicity of the sugar antigens.
- various target oligosaccharide antigens particularly oligosaccharide antigens contained in N-linked sugar chains of glycoproteins
- enhancing the immunogenicity of the sugar antigens attention was paid to the lipid structure possessed by sponge-derived ⁇ -galactosylceramide (Non-patent Document 13), which is known to have an immunostimulatory action, and various glycosphingolipids that are analogs thereof.
- Non-patent Document 13 sponge-derived ⁇ -galactosylceramide
- various glycosphingolipids that are analogs thereof.
- These various glycosphingolipids are presented as antigens via CD1 molecules on dendritic cells, and are known to activate NKT cells.
- Patent Documents 2 and 3 In addition to immunotherapeutic agents, Although widely used as an adjuvant (Patent Documents 2 and 3), there is no example of using the lipid portion of ⁇ -galactosylceramide or an analog thereof as a carrier compound for the purpose of enhancing the immunogenicity of a sugar chain antigen.
- the present inventors have conceived that the lipid structure of ⁇ -galactosylceramide, which serves as a binding site for the CD1 molecule, is bound to a sugar chain antigen.
- a carrier compound (artificial lipid) having a lipid structure of the general formula (1) having a saturated alkyl group in which no alkyl group such as —OH or an unsaturated bond group is present in the alkyl group on the amino group side is most immunized.
- XY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3 ...
- General formula (1) wherein X represents —H, —OH, —SH, —NH 2 , halogen, hydrazide group, etc., Y represents — (CH 2 ) m — (m is 1 to 30, preferably 1 to 3).
- Arbitrary oligosaccharide chains targeted in the present invention can be synthesized by organic synthesis or enzymatic synthesis, and can be reacted with a carrier compound of formula (1) while protecting the hydroxyl groups other than the reducing end of the oligosaccharide chain. For example, it can be conjugated while maintaining the oligosaccharide chain structure.
- the resulting oligosaccharide chain antigen / carrier compound conjugate can be represented by the following general formula (2).
- RZY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3 ...
- R is an oligosaccharide serving as a sugar chain antigen
- Z represents a single bond, or O, S, NH, or the like
- Y, n1, and n2 are defined in the same manner as in general formula (1)).
- a preferred carrier compound was examined using the serum antibody titer against the target oligosaccharide chain (CDw75) as an index, and the carrier compound (HOCH 2 CH (NH-CO- (CH 2 ) Monoclonal antibody was produced by a conventional method using a conjugate (artificial glycolipid) (a3-1 / CDw75-C12L) of 22- CH 3 )-(CH 2 ) 9 -CH 3 : C12L). It was found that the carrier compound (C12L) has a significant immunogenicity enhancing action and also has an activity as a thymus-dependent antigen (ability to induce production of IgG antibody).
- the carrier compound of the general formula (1) can be used as an immune inducer conjugated with any oligosaccharide antigen, including oligosaccharide antigens derived from N-linked sugar chains of glycoproteins. It has been demonstrated that the oligosaccharide chain antigens can enhance the immunogenicity and can confer activity as a thymus-dependent antigen. Furthermore, by selecting an appropriate spacer sequence, further increase in activity and simplification of binding between the oligosaccharide chain and the carrier compound can be expected.
- the carrier compound of the general formula (1) it is found that the immunity induction ability tends to increase as the alkyl chain length of the fatty acid site increases, and conversely, if the chain length is too long, problems such as solubility occur and enzyme
- the discovery of a reduction in synthesis efficiency by the method also identified a range of carrier compounds of optimal size. By obtaining the above knowledge, the present invention was completed.
- the present invention includes the following.
- the present invention relates to an invention relating to the following immunity induction method.
- a mammal comprising a step of immunizing a mammal with an immunity-inducing agent containing an artificial glycolipid represented by the general formula (2) containing the target oligosaccharide chain antigen R or a salt thereof as an active ingredient
- An antigen-specific immunity induction method for the target oligosaccharide chain antigen R ;
- RZY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3
- R represents a linear or branched oligosaccharide composed of one or more kinds of monosaccharides 2 to 30.
- Z represents a single bond, or O, S or NH, or thiomethyl;
- Linked linker, aminomethylated sugar alcohol, Y represents — (CH 2 ) m —, n1 is an integer of 2 to 40, n2 is an integer of 1 to 27, and m is an integer of 1 to 30 Represents.
- the target oligosaccharide chain antigen R may be any target oligosaccharide chain antigen, but is preferably a mammal-derived oligosaccharide chain antigen, and more preferably a glycoprotein antigen. It is preferably an oligosaccharide chain antigen contained in an N-linked sugar chain. Examples of the oligosaccharide chain antigen contained in an N-linked sugar chain include sialylated sugar chain antigens, asialo sugar chains (non-sialylated sugar chains). ) An antigen or a fucosylated sugar chain antigen.
- Sialylated sugar chain antigens include “Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc: CDw75”, “Sia ⁇ 2,3Gal ⁇ 1,4GlcNAc: 3′-Sialyl-LacNAc”, “Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc: Sialyl-Lewis X ”,“ Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc: Sialyl-Lewis a ”,“ Sia ⁇ 2,3Gal ⁇ 1,3 (Fuc ⁇ 1,4) (Sia ⁇ 2,6) GlcNAc: Disialyl-Lewis a ”,“ Sia ⁇ 2,3Gal ⁇ 1, 4 (Fuc ⁇ 1,3) GlcNAc (6SO 4 ): 6-sulfo-Sialyl-Lewis X ”,“ Sia ⁇ 2,3Gal (6SO 4 ) ⁇ 1,4 (Fuc ⁇ 1,3)
- R in the general formula (2) when R in the general formula (2) is preferable, it can be expressed as an oligosaccharide chain antigen contained in an N-linked sugar chain of a glycoprotein derived from a mammal.
- preferable examples of R in the general formula (2) are “Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc: CDw75”, “Sia ⁇ 2,3Gal ⁇ 1,4GlcNAc: 3′-Sialyl-LacNAc”, “Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3 )
- GlcNAc Sialyl-Lewis X "," Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3)
- GlcNAc Sialyl-Lewis a "," Sia ⁇ 2,3Gal ⁇ 1,3 (Fuc ⁇ 1,4) (Sia ⁇ 2,6)
- Z is preferably a single bond or O, S or NH, but may be a linker bonded to thiomethyl or an aminomethylated sugar alcohol.
- linkers to be bonded to thiomethyl commercially available linkers BMPH (N- ⁇ -Maleimidopropionic acid hydrazide-TFA), KMUH (N- ⁇ -Maleimidoundecanoic acid hydrazide-TFA), EMCH (N- [ ⁇ -Maleimidocaproic acid] hydrazide-TFA) ), MPBH (4- [4-N-Maleimidophenyl] butyric acid hydrazide-HCl), and PDPH (3- [2-Pyridyldithio] propionyl hydrazide) (manufactured by Thermo Fisher scientific), preferably aminomethyl
- the sugar alcohol to be converted is preferably selected from N-acetylglucosaminitol, N-acetylgalact
- n1 examples include 14-39, 16-31, 17-28, and 20-40, and more preferred embodiments are 20-28, 21-27, and most preferably 22-24.
- n2 is an integer of 1 to 27.
- Preferred embodiments include 2 to 15, 2 to 13, and 3 to 13. More preferred embodiments are 5 to 13 and 6 to 12, and most preferably 7 to 11.
- m is an integer of 1 to 30, preferably 1 to 10, more preferably an integer of 1 to 3.
- a carrier compound represented by the general formula (1) or a salt thereof for a target oligosaccharide chain antigen R composed of a linear or branched oligosaccharide composed of one or more kinds of monosaccharides 2 to 30
- a target oligosaccharide chain antigen comprising a step of synthesizing an artificial glycolipid represented by the general formula (2) conjugated with and immunizing a mammal with an immune inducer containing the glycolipid or a salt thereof as an active ingredient
- Antigen-specific immunity induction method for R ;
- General formula (1) XY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3
- X represents —H, —OH, —SH, —NH 2 , halogen or hydrazide group
- Y represents — (CH 2 ) m —
- n1 represents an integer
- Z represents a single bond, or O, S or NH, or thiomethyl;
- Linked linker, aminomethylated sugar alcohol Y represents — (CH 2 ) m —, n1 is an integer of 2 to 40, n2 is an integer of 1 to 27, and m is an integer of 1 to 30 Represents.
- preferred cases of the target oligosaccharide chain antigens R and Z and n1, n2 and m in the formula are the same as those described in [1-2] and [1-3] above.
- An immune inducer comprising, as an active ingredient, an artificial glycolipid represented by the general formula (2) or a salt thereof conjugated with a target oligosaccharide chain antigen R and a carrier compound represented by the general formula (1) or a salt thereof
- An antigen-specific immunity induction method for a target oligosaccharide chain antigen R in a mammal comprising a step of administering to the mammal;
- General formula (1) XY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3 (In the formula, X represents —H, —OH, —SH, —NH 2 , halogen or hydrazide group, Y represents — (CH 2 ) m —, n1 represents an integer of 2 to 40, and n2 represents 1 to (An integer of 27, m represents an integer of 1 to 30)
- Z represents a single bond, or O, S or NH, or thiomethyl;
- Linked linker, aminomethylated sugar alcohol Y represents — (CH 2 ) m —, n1 is an integer of 2 to 40, n2 is an integer of 1 to 27, and m is an integer of 1 to 30 Represents.
- preferred cases of the target oligosaccharide chain antigens R and Z and n1, n2 and m in the formula are the same as those described in [1-2] and [1-3] above.
- the present invention also relates to an invention relating to the following immunity-inducing agent.
- An immunity-inducing agent represented by the following general formula (2) and containing an artificial glycolipid containing the target oligosaccharide chain antigen R or a salt thereof as an active ingredient;
- General formula (2) RZY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3 (In the formula, R represents a linear or branched oligosaccharide composed of one or more kinds of monosaccharides 2 to 30.
- Z represents a single bond, or O, S or NH, or thiomethyl;
- Linked linker, aminomethylated sugar alcohol Y represents — (CH 2 ) m —, n1 is an integer of 2 to 40, n2 is an integer of 1 to 27, and m is an integer of 1 to 30 Represents.
- preferred cases of the target oligosaccharide chain antigens R and Z and n1, n2 and m in the formula are the same as those described in [1-2] and [1-3] above.
- the invention relating to the immunity-inducing agent of the present invention can also be expressed as follows.
- An artificial glycolipid containing a target oligosaccharide chain antigen R represented by the following general formula (2) or a salt thereof for use in an antigen-specific immunity induction method for the target oligosaccharide chain antigen R Artificial glycolipids or salts thereof; General formula (2) RZY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3 (In the formula, R represents a linear or branched oligosaccharide composed of one or more kinds of monosaccharides 2 to 30.
- Z represents a single bond, or O, S or NH, or thiomethyl;
- Linked linker, aminomethylated sugar alcohol Y represents — (CH 2 ) m —, n1 is an integer of 2 to 40, n2 is an integer of 1 to 27, and m is an integer of 1 to 30 Represents.
- preferred cases of the target oligosaccharide chain antigens R and Z and n1, n2 and m in the formula are the same as those described in [1-2] and [1-3] above.
- [2-3] Use of an artificial glycolipid containing a target oligosaccharide chain antigen R represented by the following general formula (2) or a salt thereof, and an artificial for producing an immunity-inducing agent against the target oligosaccharide chain antigen R Use of glycolipids or salts thereof;
- General formula (2) RZY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3 (In the formula, R represents a linear or branched oligosaccharide composed of one or more kinds of monosaccharides 2 to 30.
- Z represents a single bond, or O, S or NH, or thiomethyl;
- Linked linker, aminomethylated sugar alcohol Y represents — (CH 2 ) m —, n1 is an integer of 2 to 40, n2 is an integer of 1 to 27, and m is an integer of 1 to 30 Represents.
- preferred cases of the target oligosaccharide chain antigens R and Z and n1, n2 and m in the formula are the same as those described in [1-2] and [1-3] above.
- a vaccine represented by the following general formula (2) and containing an artificial glycolipid containing the target oligosaccharide chain antigen R or a salt thereof as an active ingredient;
- General formula (2) RZY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3 (In the formula, R represents a linear or branched oligosaccharide composed of one or more kinds of monosaccharides 2 to 30.
- the vaccine is a vaccine having an effect of causing antigen-specific immunity induction against the target oligosaccharide chain antigen R, and when the target oligosaccharide chain antigen R, Z and n1, n2 and m in the formula are preferred, This is the same as described in [1-2] and [1-3] above.
- the invention according to the vaccine of the present invention can also be expressed as follows.
- An artificial glycolipid containing a target oligosaccharide chain antigen R represented by the following general formula (2) or a salt thereof, and used for a vaccination method against the target oligosaccharide chain antigen R Or a salt thereof;
- General formula (2) RZY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3 (In the formula, R represents a linear or branched oligosaccharide composed of one or more kinds of monosaccharides 2 to 30.
- Z represents a single bond, or O, S or NH, or thiomethyl;
- Linked linker, aminomethylated sugar alcohol Y represents — (CH 2 ) m —, n1 is an integer of 2 to 40, n2 is an integer of 1 to 27, and m is an integer of 1 to 30 Represents.
- preferred cases of the target oligosaccharide chain antigens R and Z and n1, n2 and m in the formula are the same as those described in [1-2] and [1-3] above.
- Z represents a single bond, or O, S or NH, or thiomethyl;
- Linked linker, aminomethylated sugar alcohol Y represents — (CH 2 ) m —, n1 is an integer of 2 to 40, n2 is an integer of 1 to 27, and m is an integer of 1 to 30 Represents.
- preferred cases of the target oligosaccharide chain antigens R and Z and n1, n2 and m in the formula are the same as those described in [1-2] and [1-3] above.
- the present invention relates to an artificial conjugated conjugate of a target oligosaccharide chain antigen R and a carrier compound used in the invention related to the immunity induction method of [1], the immunity induction agent of [2] or the vaccine of [3].
- the present invention also relates to an invention related to a novel artificial glycolipid or salt thereof itself represented by the following general formula (2).
- General formula (2) RZY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3 (In the formula, R represents a linear or branched oligosaccharide composed of one or more kinds of monosaccharides 2 to 30.
- n1 in the formula corresponds to a fatty acid having 22 or more carbon atoms classified as a super long chain fatty acid, and n1 is an integer of 20 to 40, preferably 20 to 28, more preferably This is preferably 21 to 27, and most preferably 22 to 24.
- the preferred cases of the target oligosaccharide chain antigens R and Z, and n2 and m are the same as those described in [1-2] and [1-3] above.
- the invention relating to the novel artificial glycolipid of the present invention or a salt thereof itself is “the artificial glycolipid or a salt thereof according to the above [4] having immunity-inducing activity against the target oligosaccharide chain antigen R”. It can also be expressed as “the artificial glycolipid or salt thereof as described in [4] above for induction of immunity against oligosaccharide R”.
- the present invention has the activity of causing immunity-inducing activity against the target oligosaccharide chain antigen R when used in a state conjugated to the target oligosaccharide chain antigen R in the immunity induction method according to [1].
- the present invention also relates to an invention relating to a carrier compound itself composed of a novel artificial lipid represented by the following general formula (1) or a salt thereof. That is, the invention relating to the novel artificial lipid or salt thereof of the present invention can be expressed as follows.
- An artificial lipid represented by the following general formula (1) or a salt thereof;
- General formula (1) XY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3 (Wherein X represents —H, —OH, —SH, —NH 2 , halogen or hydrazide group, Y represents — (CH 2 ) m —, n1 represents an integer of 20 to 40, and n2 represents 1 to 27 represents an integer, m represents an integer of 1 to 30).
- R and Z, and n2 and m are preferred, they are the same as those described in [1-2] and [1-3], and n1 is the same as in [4] ].
- the invention related to the artificial lipid of the present invention or a salt thereof is characterized for a specific use, and “a carrier compound comprising an artificial lipid represented by the following general formula (1) or a salt thereof: ", Which consists of an artificial lipid represented by the following general formula (1) or a salt thereof, and binds to the reducing end side of the target oligosaccharide chain antigen R to cause immunity induction activity against the target oligosaccharide chain antigen R.
- a carrier compound having activity is characterized for a specific use, and “a carrier compound comprising an artificial lipid represented by the following general formula (1) or a salt thereof: ", Which consists of an artificial lipid represented by the following general formula (1) or a salt thereof, and binds to the reducing end side of the target oligosaccharide chain antigen R to cause immunity induction activity against the target oligosaccharide chain antigen R.
- a carrier compound having activity is characterized for a specific use, and “a carrier compound comprising an artificial lipid represented
- the carrier compound of the present invention By using the carrier compound of the present invention, it is possible to remarkably enhance the immunogenicity of oligosaccharide antigens contained in N-linked sugar chains of glycoproteins that are difficult to induce immunity. .
- the serum antibody titer against the oligosaccharide chain antigen of the immunogen is about 12 as compared with the case of simple lipid modification (a2).
- the serum antibody titer against glycoprotein (Fetuin) containing the target sugar chain is about 8 times as potent as immunogenicity (FIG. 3).
- Non-patent Document 8 a monoclonal antibody that reacts with CDw75 is produced by immunization with an immunity-inducing agent containing a CDw75 oligosaccharide chain, but a specific anti-CDw75 antibody in a strict sense is obtained.
- various experimental data on the CDw75-reactive antibody (241-5-2 antibody) described in this document were compared with the data obtained when the anti-CDw75 antibody was obtained in the present invention.
- the appearance rate of positive hybridoma cells producing the target antibody later was about 13 times higher.
- the affinity of the finally obtained monoclonal antibody for the epitope was about 3,000 times as high.
- the immunogenicity enhancing effect of the carrier compound of the present invention has enabled the development of monoclonal antibodies having high specificity and affinity for the target sugar chain epitope and hybridoma cells producing the same from the host animal after immunization.
- the immunity-inducing agent of the present invention has been confirmed to have the ability to induce the production of IgG class antibodies produced by class switching in mice immunized with the immunity-inducing agent, and has activity as a thymus-dependent antigen. . This activity has not been confirmed by conventional techniques (Non-Patent Documents 7 and 8) using an immunity-inducing agent using phosphatidylethanolamine as a carrier compound.
- Non-patent Document 9 Reported that when antibody production against microorganism-derived oligosaccharide chains was attempted, IgG antibodies were produced with low affinity by immunization with conjugates such as KLH, BSA, and biotinavidin (Non-patent Document 9) 10 and 12), however, it is a mammal-derived oligosaccharide chain and induces immunity with class switching in the oligosaccharide chain antigen contained in the N-linked sugar chain of glycoprotein, and has high affinity It is the first time that the present invention has been able to produce antibodies.
- the immunity-inducing agent of the present invention is recognized by the immune system as a thymus-dependent antigen, it can undergo antibody specificity, such as class switching and affinity maturation, and an affinity enhancement process. An antibody having high specificity and affinity can be obtained.
- the conjugation method of the carrier compound and the target oligosaccharide chain in the present invention it is possible to produce an immune inducer by binding with maintaining the structure of the oligosaccharide chain.
- the carrier compound used in the prior art Non-Patent Documents 7 to 12
- the oligosaccharide chain of interest is generally conjugated by a reductive amination method
- the sugar chain is combined with the carrier compound.
- the binding site (sugar on the reducing side) is cleaved, and an immune inducer cannot be synthesized while maintaining the structure of the target oligosaccharide chain.
- a carrier compound that can enhance the immunogenicity of any oligosaccharide chain provided by the present invention, it is possible to efficiently construct an “oligosaccharide chain-carrier compound” conjugate as an immunity-inducing agent. Development of monoclonal antibodies that recognize epitopes, vaccines that target sugar chains, and the like is facilitated.
- Serum antibody titer against immunogen after immunization Serum antibody titer against immunogen was measured by ELISA after immunization of mice with each of the a1, b1, and c1 compounds as immunity inducers.
- the white circles indicate the antibody sera in non-immunized (D0) mice
- the gray circles indicate the antibody titers in the mouse serum on the third day after the final immunization (D3)
- N 6-12. It was confirmed by Student's test that the serum antibody titers of D3 and D7 were significantly increased compared to the serum antibody titers of unimmunized mice.
- Serum antibody titer against glycoprotein after immunity induction Serum antibody with the antigen of glycoprotein (Fetuin) having the same oligosaccharide chain structure as the immunogen after immunization of mice with each of the a1, b1, and c1 compounds as immunity inducers The titer was measured by ELISA. For c1, Fetuin-a (Table 1) from which sialic acid of all binding modes was removed by sialidase treatment was used as an antigen.
- the white circles indicate the antibody sera in non-immunized (D0) mice
- the gray circles indicate the antibody titers in the mouse serum on the third day after the final immunization (D3)
- the black circles on the seventh day after the final immunization (D7).
- N 6-12). It was confirmed by Student's test that the serum antibody titers of D3 and D7 were significantly increased compared to the serum antibody titers of unimmunized mice.
- Serum antibody titer after immunity induction (IgM subclass): After immunizing mice with each of the a1 to a3-3 compounds as an immunity inducing agent, the antibody titer against the target sugar chain epitope for the immunoglobulin in the serum (IgM subclass) is ELISA It was evaluated by the law.
- Serum antibody titer after immunity induction (IgG subclass): After immunizing mice with each of the a1 to a3-3 compounds as an immunity inducer, the antibody titer against the target sugar chain epitope for immunoglobulin (IgG subclass) in the serum is determined by ELISA. It was evaluated by the law.
- Serum antibody titer after immunity induction by Globoside Serum antibody titer of mice when immunized with glycolipid (sphingoglycolipid: Globoside) containing natural ceramide is shown as a comparison target.
- the antibody titers of IgM subclass (left figure) and IgG subclass (right figure) in mouse serum after immunization were evaluated by ELISA as reactivity to the target sugar chain epitope.
- the white circles indicate the antibody sera in non-immunized (D0) mice
- the gray circles indicate the antibody titers in the mouse serum on the third day after the final immunization (D3)
- the black circles on the seventh day after the final immunization (D7).
- Affinity to epitope of developed monoclonal antibody FR9 A: Regarding affinity to epitope of FR9 antibody, ELISA method and Scatchard plot of dissociation constant (Kd value) using glycoprotein (Fetuin) containing CDw75 sugar chain as antigen Determined.
- the x-axis is the amount of antigen-antibody complex (Ag-Ab) divided by the total amount of antibody (Abt) (Ag-Ab / Abt), and the y-axis is (Ag-Ab / Abt) as the total amount of free antigen (Agf).
- the divided value (Ag-Ab / Abt ⁇ Agf) is plotted.
- the Kd value (8.86 ⁇ 10 ⁇ 7 M) was calculated from the value.
- B About the affinity with respect to the epitope of FR9 antibody, the detection sensitivity was measured by ELISA which used the glycoprotein (Fetuin: white circle) containing CDw75 sugar chain and the immunogen (CDw75-C12L) as an antigen.
- Specificity analysis of the developed monoclonal antibody FR9 by ELISA The antigen recognition specificity of the FR9 antibody was analyzed by ELISA using various sugar chain antigen compounds shown in Table 1.
- sLe X -C12L and Le X -C12L by immune induction after serum antibody titer sLe X -C12L and Le X -C12L compounds each after immunizing mice as immunity-inducing agent, each subclass of immunoglobulin in serum (IgM , IgG) antibody titer was evaluated by ELISA.
- A, B are sLe X -C12L mice sera immunized with, C, D serum antibody titers of mice immunized with Le X -C12.
- A, C Evaluation by ELISA method using immunogen as antigen.
- B Evaluation by ELISA method using a cell extract (HL60) containing a glycoprotein containing the target sugar chain epitope as an antigen.
- D Evaluation by ELISA using a glycoprotein (AGP) containing the target sugar chain epitope as an antigen.
- D0 serum antibody titer before immunization
- D7 serum antibody titer on the seventh day after final immunization, the antibody titer in each mouse serum is shown in a graph. It was confirmed by Student's t test that the serum antibody titers of D3 and D7 were significantly increased compared to the unimmunized mouse serum antibody titers.
- Detection of CDw75 expressed on the surface of cancer cells (B cell tumor cells): Using FR9 antibody, CDw75 expressed on the cell surface of B cell tumor cells (Burkitt lymphoma cell line: Raji cell) was detected. 1: Negative control, 2: FR9 antibody.
- oligosaccharide chain antigen or simply “sugar chain antigen” refers to an antibody against the oligosaccharide chain itself or a modified product thereof.
- the immunity induction method of the present invention can enhance immunogenicity for any oligosaccharide antigen, but typical “oligosaccharide antigens” targeted in the present invention are secreted glycoproteins of various organisms.
- oligosaccharide antigen (R) is “a sugar chain structure contained in a complex carbohydrate, and is a linear or branched chain composed of one or more types of monosaccharides 2 to 30”. It is composed of a “shaped oligosaccharide”.
- the glycan antigen (R) that is the object of the present invention is sialic acid (N-acetylneuraminic acid, N-glycolylneuraminic acid, ⁇ -ketodeoxynononic acid), galactose, glucose, N-acetylglucosamine , N-acetylgalactosamine, glucosamine, galactosamine, mannose, fucose, xylose, glucuronic acid, iduronic acid, inositol, erythrose, erythronic acid, erythrulonic acid, erythralic acid, erythritol, threose, threonic acid, treuronic acid, treallic acid, threitol , Ribose
- complex sugar chains derived from glycoproteins and glycolipids constituting the cell membrane are covered with various complex sugar chains derived from glycoproteins and glycolipids constituting the cell membrane.
- these complex sugar chains serve as identification markers for each cell as oligosaccharide chain antigens.
- a pathogen is a biomarker for diagnosis and drug targeting.
- eubacterial O antigen is known.
- mammalian cells a complex having a complex sugar chain with a characteristic structure on the cell surface, particularly reflecting the pathology of cancer cells and serious diseases. Numerous sugars have been reported to be expressed, and these complex sugar chains also serve as diagnostic markers and biomarkers for drug targets.
- CA19-9 Sialyl-Lewis a
- the present invention is mainly directed to oligosaccharide chain antigens contained in complex carbohydrates derived from mammalian cells.
- Typical sugar chain structures of such known oligosaccharide chain antigens derived from mammalian cells are classified into the following (A) to (D).
- glycolipid type oligosaccharide chain has a distinctive feature that it contains a lactose structure (Gal ⁇ 1,4Glc) at the reducing end except for the GM4 type oligosaccharide chain (Sia ⁇ 2,3Gal).
- a lactose structure Gal ⁇ 1,4Glc
- GM4 type oligosaccharide chain Sia ⁇ 2,3Gal
- the immunity induction method of the present invention can be applied to any oligosaccharide antigen contained in an N-linked sugar chain, an O-linked sugar chain, a proteoglycan-type sugar chain and a glycolipid-type sugar chain. Although it exhibits a potentiating action, in the present invention, despite its attention as a diagnostic marker for serious diseases such as highly malignant cancer, it is low in immunogenicity and cannot provide an effective antibody.
- the target is “oligosaccharide antigens contained in N-linked sugar chains”.
- oligosaccharide antigen contained in an N-linked sugar chain is an oligosaccharide antigen consisting of an N-linked sugar chain of a glycoprotein derived from a mammal or a partial structure thereof.
- Such “oligosaccharide antigens contained in N-linked sugar chains” are roughly classified into the following three types, sialylated sugar chains (a), asialo sugar chains (b), fucosylated sugar chains (c (Essentials of Glycobiology, Cold Spring Harbor Laboratory Press, 1999).
- N-linked sugar chains those classified as sialylated sugar chains Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc, Sia ⁇ 2,3Gal ⁇ 1,4GlcNAc, Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc, Sia ⁇ 2,3Gal ⁇ 1,3 (Fuc ⁇ 1,4) GlcNAc, Sia ⁇ 2,3Gal ⁇ 1,3GlcNAc, Sia ⁇ 2,3Gal ⁇ 1,3 (Sia ⁇ 2,6) GlcNAc, Sia ⁇ 2,3Gal ⁇ 1,3 (Fuc ⁇ 1,4) (Sia ⁇ 2,6) GlcNAc, Sia ⁇ 2,3Gal ⁇ 1,4GlcNAc ⁇ 1,2Man Sia ⁇ 2,3Gal ⁇ 1,4GlcNAc ⁇ 1,4Man, Sia ⁇ 2,3Gal ⁇ 1,4GlcNAc ⁇ 1,6Man, Sia ⁇ 2,3Gal ⁇ 1,4GlcNAc ⁇ 1,6Man, Sia ⁇ 2,3Gal ⁇ 1,4Glc
- sialylated sugar chains include “Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc: CDw75”, “Sia ⁇ 2,3Gal ⁇ 1,4GlcNAc: 3′-Sialyl-LacNAc”, “Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1 , 3) GlcNAc: Sialyl-Lewis X "," Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc: Sialyl-Lewis a "," Sia ⁇ 2,3Gal ⁇ 1,3 (Fuc ⁇ 1,4) (Sia ⁇ 2,6) GlcNAc: Disialyl- Lewis a ”,“ Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc (6SO 4 ): 6-sulfo-Sialyl-Lewis X ”,“ Sia ⁇ 2,3Gal (6SO 4 ) ⁇ 1,4 (Fuc ⁇ 1 ),“ Sia
- N-linked sugar chains those classified as asialo sugar chains (non-sialylated sugar chains) Gal ⁇ 1,4GlcNAc, Gal ⁇ 1,3GlcNAc, Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc, Gal ⁇ 1,3 (Fuc ⁇ 1 , 4) GlcNAc, Fuc ⁇ 1,2Gal ⁇ 1,3 (Fuc ⁇ 1,4) GlcNAc, Fuc ⁇ 1,2Gal ⁇ 1,4GlcNAc, Fuc ⁇ 1,2Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc, Fuc ⁇ 1,6GlcNAc, GlcNAc ⁇ 1,4 (Fuc ⁇ 1,6) GlcNAc , 4GlcNAc ⁇ 1,4 (Fuc ⁇ 1,6) GlcNAc, Man ⁇ 1,6 (Man ⁇ 1,3) Man ⁇ 1,4GlcNAc ⁇ 1,4 (Fuc ⁇ 1,6) GlcNAc, Man ⁇ 1,6 (GlcNAc ⁇ 1,4) (
- Gal ⁇ 1,4GlcNAc LacNAc
- GlcNAc ⁇ 1,4Man “GlcNAc ⁇ 1,6Man”
- Gal ⁇ 1,4GlcNAc ⁇ 1,6Man “Gal ⁇ 1,4GlcNAc ⁇ 1,6Man”
- GalNAc ⁇ 1,4GlcNAc “GalNAc ⁇ 1,4GlcNAc ⁇ 1,2Man”
- GalNAc (4SO 4 ) ⁇ 1,4GlcNAc “Gal ⁇ 1,4 (Fuc ⁇ 1,3)
- GlcNAc Lewis X ”, “Gal ⁇ 1,3 (Fuc ⁇ 1,4)
- GlcNAc Lewis a ”,“ Fuc ⁇ 1,2Gal ⁇ 1,3 (Fuc ⁇ 1,4)
- GlcNAc Lewis b
- GlcNAc Lewis y ”,“ Fuc ⁇ 1,6GlcNAc ”,
- fucosylated sugar chain antigens include “Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc: Lewis X ”, “Gal ⁇ 1,3 (Fuc ⁇ 1,4) GlcNAc: Lewis a ”, “Fuc ⁇ 1 , 2Gal ⁇ 1,3 (Fuc ⁇ 1,4) GlcNAc: Lewis b ”,“ Fuc ⁇ 1,2Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc: Lewis y ”,“ Fuc ⁇ 1,6GlcNAc ”,“ GlcNAc ⁇ 1,4 (Fuc ⁇ 1,6) GlcNAc ”, “Man ⁇ 1,4GlcNAc ⁇ 1,4 (Fuc ⁇ 1,6) GlcNAc”, “Man ⁇ 1,6 (Man ⁇ 1,3) Man ⁇ 1,4GlcNAc ⁇ 1,4 (Fuc ⁇ 1,6) GlcNAc”, “Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc : Sialyl
- the immunity induction method using the carrier compound of the present invention is based on these known diagnosis and various complex sugar chains that are biomarkers for drug targets. Rather, it is a technology that can acquire a monoclonal antibody that recognizes with specific and high affinity for any newly discovered complex sugar chain.
- These arbitrary oligosaccharide chains may be purified from cell membrane fractions derived from them by conventional methods, and if the sugar chain structure is known, they can be synthesized by applying known organic synthesis methods or enzymatic synthesis methods. is there.
- a plurality of representative oligosaccharide chain antigens are selected from three types of oligosaccharide chain antigen groups contained in a typical N-linked sugar chain, and the present invention is selected.
- this immunity induction method was applied, it was confirmed that antigen-specific immunity induction was caused when any oligosaccharide antigen was used.
- the immunity induction method of the present invention can be applied to the entire "oligosaccharide chain antigens contained in N-linked sugar chains".
- any oligosaccharide chain antigen, including other oligosaccharide chain antigens that are more likely to induce immunity in view of the fact that "oligosaccharide chain antigens contained in N-linked sugar chains" can enhance immunity. It can also be seen that this is a technology that can be applied.
- CDw75 is a typical sialylated glycan that exists as a non-reducing terminal structure of N-linked glycan produced by mammalian cells, and is a tumor marker that serves as a diagnostic index for determining malignancy of gastric cancer and colon cancer (CDw75) has attracted attention as a molecular target for malignant tumor treatment (Non-patent Document 14). And there is also a report as a human influenza virus infection receptor (Non-patent Document 15), which is also expected as a target for influenza treatment.
- CDw75 Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc
- CDw75 is a carbohydrate antigen that is rarely expressed in normal human stomach and colon tissues, whereas when these tissues become cancerous, strong expression is observed.
- Non-Patent Document 14 On the other hand, CDw75 is found as a major N-linked sugar chain structure in glycoproteins present in mammalian serum. Since it is present in the mouse as well, it is very difficult to induce immunity compared to microbial-derived sugar chains. In particular, antibodies that recognize "glycan antigens contained in N-linked sugar chains of glycoproteins" Production is considered difficult.
- 3′-Sialyl-LacNAc is also a typical sialylated sugar chain that exists as a non-reducing terminal structure of an N-linked protein produced by mammalian cells.
- influenza viruses it is known that a type that infects birds (avian influenza virus) uses this sugar chain structure as an infection receptor (Non-patent Document 15).
- “LacNAc” is similar to “3'-Sialyl-LacNAc” and forms the non-reducing terminal structure and internal structure of N-linked proteins, O-linked sugar chains and oligosaccharide chains expressed in mammalian cells. Is a typical asialo sugar chain (non-sialylated sugar chain). It becomes a precursor structure of various sugar chain structures.
- Sialyl-Lewis X Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3 ) GlcNAc
- oligosaccharide chain antigen for the "" Sialyl-Lewis X Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3 ) GlcNAc Is represented by the following general formula (6).
- Sialyl-Lewis X (Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc) is a structure found in N-linked sugar chains and O-linked sugar chains of glycoproteins produced by mammalian cells. This sugar chain is a ligand of an adhesion molecule protein (E-selectin) expressed at an inflammatory site (Non-patent Document 16). In healthy individuals, lymphocytes are recruited to the site of inflammation by cell adhesion mediated by Sialyl-Lewis X and E-selectin. On the other hand, some metastatic cancer cells express Sialyl-Lewis X and utilize this cell adhesion mechanism when metastasized to other organs.
- E-selectin adhesion molecule protein
- This sugar chain antigen is a sugar chain antigen contained in a glycoprotein specifically expressed in large amounts in human promyelocytic leukemia cell line (HL60) of human leukemia cells (Kobzdej MM., Et al., Blood, 100, 4485-4494, 2002). Sialyl-Lewis X- containing glycoprotein can be easily prepared from HL60 cells.
- Lewis X (Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc) is a sugar chain antigen known as CD15, SSEA-1. It is a structure found in N-linked sugar chains and O-linked sugar chains of glycoproteins such as ⁇ 1-acid glycoprotein (AGP) produced by mammalian cells, and is a precursor of the above Sialyl-Lewis X. SSEA-1 is famous as an antigen widely used as a marker for mouse stem cells / iPS cells (Non-patent Documents 17 and 18). Application as a tumor marker is also expected as a marker for bladder cancer and cancer stem cells (Non-Patent Documents 16 and 19).
- the carrier compound of the present invention is a novel compound of the following general formula (1), which is similar to the lipid structure of ⁇ -galactosylceramide that serves as a binding site with CD1 molecule known to have an NKT cell activation effect. It is a lipid molecule.
- the lipid structure of natural glycosphingolipids such as A-galactosylceramide is a saturated alkyl in which one chain bonded to the carbonyl group side of the binding structure "-CH (NH-CO)-" with sugar is not substituted.
- the alkyl group on the amino group side is a saturated alkyl group, whereas in the case of natural glycosphingolipid, it is bonded to the amino group side.
- the alkyl group is always modified with a group containing oxygen such as —OH group and ⁇ O group.
- a glycosphingolipid derived from a mammal containing an oligosaccharide chain of two or more sugars there is a difference that an alkyl group bonded to the amino group side always includes an unsaturated bond as well as an —OH group.
- the carrier compound of the present invention is an “artificial lipid or a salt thereof” similar to natural lipids.
- XY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3 ...
- X is —H, —OH, —SH, —NH 2 , or a functional group capable of reacting with a reducing terminal hydroxyl group of a sugar chain antigen, such as halogen (—Br, —I, —Cl, —F ), A hydrazide group and the like.
- Y is a spacer sequence.
- the spacer sequence Y in the present invention is represented by “— (CH 2 ) m —”.
- m is an integer of 1 to 30, preferably 1 to 10, more preferably 1 to 3. Longer methylene chains are easier to bind to ligands (receptor proteins and antibodies on lymphocytes) and can be expected to increase immunogenicity.
- a preferable numerical range was set as an easy to handle range.
- a spacer sequence can also be introduced through the functional group of X.
- n1 is an integer of 2 to 40, and preferred embodiments include 14 to 39, 16 to 31, 17 to 28, and 20 to 40. More preferred embodiments are 20 to 28, 21 to 27, and most preferably 22 to 24. It is.
- n1 20 or more
- ultra-long-chain fatty acids fatty acids having 22 or more carbon atoms
- the properties of fatty acids are known to be different from normal long-chain fatty acids.
- activation of cell membrane signaling in immune cells Korean A., J. Biochem., 152, 387-395, 2012.
- the upper limit value is set within a preferable numerical range, but it is a numerical value that is temporarily set from the viewpoint of ease of synthesis, difficulty in handling, and the like, not the viewpoint of immunity induction ability. That is, if n1 is 20 or more, an extremely high immunogenicity can be ensured. Therefore, preferable numerical ranges including numerical ranges that are easy to synthesize and handle are preferably 20 to 28, 21 to 27, and most preferably 22 to 24.
- n2 is an integer of 1 to 27.
- Preferred embodiments include 2 to 15, 2 to 13, and 3 to 13. More preferred embodiments are 5 to 13 and 6 to 12, and most preferably 7 to 11. If n2 is less than 5, the immunity induction ability tends to decrease.If n2 exceeds 9, the solubility in an aqueous solvent decreases and the synthesis efficiency by the enzymatic method is reduced. There is no problem.
- the compound of the general formula (1) can be a pharmaceutically acceptable non-toxic salt.
- the salt of the compound of the general formula (1) includes an acid addition salt, for example, a salt with an inorganic acid (for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid), or an organic acid (for example, acetic acid, propionic acid, maleic acid, olein).
- an inorganic acid for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid
- organic acid for example, acetic acid, propionic acid, maleic acid, olein.
- Immunity-inducing agent comprising conjugate (artificial glycolipid) of target sugar chain antigen and carrier compound, and production method thereof (3-1) Immunity-inducing agent of the present invention
- the conjugate can be represented by the following general formula (2).
- R is an arbitrary oligosaccharide that becomes a sugar chain antigen, and is sialic acid (N-acetylneuraminic acid, N-glycolylneuraminic acid, ⁇ -ketodeoxynononic acid), galactose, glucose , N-acetylglucosamine, N-acetylgalactosamine, glucosamine, galactosamine, mannose, fucose, xylose, glucuronic acid, iduronic acid, inositol, erythrose, erythronic acid, erythrulonic acid, erythralic acid, erythritol, threose, threonic acid, treuronic acid, Treareal acid, threitol, ribose, ribbon acid, ribonic acid, ribivalic acid, ribitol, arabinose, arabinonic acid, arabinuronic acid,
- sugar chain antigens in the present invention, 1. As described in (1), it is mainly directed to oligosaccharide antigens derived from mammalian cells.
- oligosaccharide antigens contained in N-linked sugar chains found in glycoproteins of mammalian cells. Is preferred.
- oligosaccharide antigens contained in N-linked sugar chains include “Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc: CDw75”, “Sia ⁇ 2,3Gal ⁇ 1,4GlcNAc: 3 ′” -Sialyl-LacNAc "Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc: Sialyl-Lewis X ", "Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc: Sialyl-Lewis a ", "Sia ⁇ 2,3Gal ⁇ 1,3 ( Fuc ⁇ 1,4) (Sia ⁇ 2,6) GlcNAc: Disialyl-Lewis a ”,“ Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc (6SO 4 ): 6-sulfo-Sialyl-Lewis X ”,“ Sia ⁇ 2,3Gal (6SO 4 ):
- Z represents a single bond, or O, S or NH.
- Z becomes S or NH, but even if the carrier compound contains S or NH, It does not affect immunity induction (Non-patent Documents 7 and 12).
- bonded with thiomethyl or the sugar alcohol by which aminomethylation was carried out may be sufficient.
- linkers to be bonded to thiomethyl commercially available linkers BMPH (N- ⁇ -Maleimidopropionic acid hydrazide-TFA), KMUH (N- ⁇ -Maleimidoundecanoic acid hydrazide-TFA), EMCH (N- [ ⁇ -Maleimidocaproic acid] hydrazide-TFA) ), MPBH (4- [4-N-Maleimidophenyl] butyric acid hydrazide-HCl), and PDPH (3- [2-Pyridyldithio] propionyl hydrazide) (manufactured by Thermo Fisher scientific).
- the carrier compound is conjugated with the target oligosaccharide chain antigen R to produce a conjugate (artificial glycolipid) that acts as an immunity-inducing agent against the target oligosaccharide chain antigen R.
- a conjugate artificial glycolipid
- it is cleaved to contain a sugar alcohol.
- N-acetylglucosaminitol N-acetylgalactosaminitol, mannitol, galactitol and other sugar alcohols
- aminomethylated sugar alcohols include N-acetylglucosaminitol, It is preferably selected from N-acetylgalactosaminitol, mannitol, galactitol.
- Z or a compound containing a sugar is not included in Z.
- Y is a spacer sequence and is defined as "-(CH 2 ) m- (m is an integer of 1 to 30, preferably 1 to 10, more preferably 1 to 3)" as in the general formula (1). Is done. Longer methylene chains are easier to bind to ligands (receptor proteins and antibodies on lymphocytes) and can be expected to increase immunogenicity. A preferable numerical range was set as an easy to handle range.
- a spacer sequence can also be introduced through the functional group of X.
- linkers BMPH N- ⁇ -Maleimidopropionic acid hydrazide-TFA
- KMUH N- ⁇ -Maleimidoundecanoic acid hydrazide-TFA
- EMCH N- [ ⁇ -Maleimidocaproic acid] hydrazide-TFA
- MPBH 4- [4-N-Maleimidophenyl] butyric acid hydrazide-HCl
- PDPH 3- [2-Pyridyldithio] propionyl hydrazide
- n1 and n2 are also defined similarly to the general formula (1).
- n1 corresponding to the alkyl chain length of the fatty acid moiety is an integer of 2 to 40, and preferred embodiments include 14 to 39, 16 to 31, 17 to 28, and 20 to 40, and more preferred embodiments are 20 to 28, 21 to 27, most preferably 22 to 24.
- the immunity induction ability tends to increase, and when the number of n1 is 20 or more (in the case of a very long chain fatty acid having 22 or more carbon atoms), the immunity induction ability is remarkably increased. .
- the upper limit value of the preferable numerical value range is a numerical value provided temporarily from the viewpoint of ease of synthesis, difficulty in handling, and the like. That is, n1 is an integer of 20 to 40, which is a preferable numerical range that can ensure high immunogenicity, is easy to synthesize, and is easy to handle, and further preferably 20 to 28, 21 to 27, most preferably 22 to 24 It is. N2 corresponding to the alkyl chain length on the amino group side is an integer of 1 to 27. Preferred embodiments include 2 to 15, 2 to 13, and 3 to 13, and more preferred embodiments are 5 to 13 and 6 to 12. And most preferably 7-11.
- n2 is less than 5, the immunity induction ability tends to decrease. If n2 exceeds 9, the solubility in an aqueous solvent decreases and the synthesis efficiency by the enzymatic method is reduced. There is no problem.
- a novel artificial glycolipid represented by the following general formula (2) or a salt thereof It also relates to an invention relating to the salt itself.
- RZY-CH (NH-CO- (CH 2 ) n1 -CH 3 )-(CH 2 ) n2 -CH 3
- R represents a linear or branched oligosaccharide composed of one or more kinds of monosaccharides 2 to 30.
- Z represents a single bond, or O, S or NH, or thiomethyl;
- Linked linker, aminomethylated sugar alcohol Y represents — (CH 2 ) m —, n1 is an integer of 2 to 40, n2 is an integer of 1 to 27, and m is an integer of 1 to 30 Represents.
- Z is preferably a single bond or O, S or NH, but may be a linker bonded to thiomethyl or an aminomethylated sugar alcohol.
- linkers to be bonded to thiomethyl commercially available linkers BMPH (N- ⁇ -Maleimidopropionic acid hydrazide-TFA), KMUH (N- ⁇ -Maleimidoundecanoic acid hydrazide-TFA), EMCH (N- [ ⁇ -Maleimidocaproic acid] hydrazide-TFA) ), MPBH (4- [4-N-Maleimidophenyl] butyric acid hydrazide-HCl), and PDPH (3- [2-Pyridyldithio] propionyl hydrazide) (manufactured by Thermo Fisher scientific), preferably aminomethyl
- the sugar alcohol to be converted is preferably selected from N-acetylglucosaminitol, N-acetylgalactosaminitol, mannitol, and galactitol.
- the compound of the general formula (2) can be a pharmaceutically acceptable non-toxic salt.
- the salt of the compound of the general formula (2) include acid addition salts such as salts with inorganic acids (for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid), or organic acids (for example, acetic acid, propionic acid, maleic acid, olein). Acid, palmitic acid, citric acid, succinic acid, tartaric acid, fumaric acid, glutamic acid, pantothenic acid, laurylsulfonic acid, methanesulfonic acid and phthalic acid). It may be a solvate (for example, hydrate).
- the conjugate of the general formula (2) (artificial glycolipid) in the present invention alone has an action as an immunity-inducing agent, but when actually immunizing experimental animals, Lipid is used as an adjuvant.
- Lipid is used as an adjuvant.
- -A, LPS, acid-treated Salmonella minnesota strain R595, heat-killed bacteria, etc. are preferably used in combination.
- Complete Freund's adjuvant a mixture of paraffin oil, Aracel A, heat-killed Mycobacterium tuberculosis
- Complete Freund's adjuvant a mixture of paraffin oil, Aracel A, heat-killed Mycobacterium tuberculosis
- liposomes prepared by dissolving in lipids such as cholesterol, phospholipid, diacylglycerol, monoacylglycerol, glycerol, fatty acid, glycocholic acid, taurocholic acid, glycolipid without an adjuvant or with an adjuvant are intravenously administered.
- the liposome method for injection (Brodin et al .; Eur J Immunol., 16, 951-956, 1986) is preferably used.
- the conjugate of the general formula (2) may be dissolved alone in a cell culture solution for lymphocyte culture and allowed to act on immune cells.
- the above-mentioned method of Brodin et al. Can be dissolved in a high concentration in the cell culture medium after treatment such as adsorption to the lipid binding protein. Therefore, it is possible to effectively act on immune cells. Therefore, when referring to an immunity inducer in the present invention, the compound itself of the general formula (2), a pharmaceutically acceptable non-toxic salt thereof, or the compound of the general formula (2) is used as an active ingredient, and Lipid-A or the like A composition having an ability to induce immunity including an adjuvant, and a liposome composition dissolved in lipid are also included.
- (3-2) Production method (3-2-1) Method of binding a carrier compound (artificial lipid) to an oligosaccharide chain antigen directly or via a spacer sequence Purified from a natural product that is an arbitrary oligosaccharide chain as a target
- the carrier compound represented by the general formula (1) of the present invention can be directly bound to the prepared oligosaccharide chain or the oligosaccharide chain obtained by organic synthesis.
- a sugar in which a hydroxyl group other than the reducing end of the oligosaccharide chain is protected and a leaving group for example, an acetyl group, a trichloroacetimidate group, a hydroxyl group, a thioalkyl group, a halogen, etc.
- a donor is prepared, and this is conjugated with a carrier compound of the general formula (1), or a part of the prepared sugar donor and a carrier substance of the general formula (1) is conjugated, and then an enzyme reaction or chemistry is performed.
- Conjugation can be performed while maintaining the structure of the oligosaccharide chain by extending the sugar chain or constructing the carrier part by reaction or the like. According to this method, even when the sugar chain structure of the oligosaccharide antigen of interest is unknown, it can bind to the carrier compound of the present invention.
- the method of protecting hydroxyl groups other than the reducing end in the oligosaccharide chain at that time is as follows.
- a protection step of reacting an arbitrary oligosaccharide chain with a protecting reagent to protect a hydroxyl group other than the reducing end with a protecting group (i) a substitution step of reacting the product of the above step with a leaving group introduction reagent to replace the hydroxyl group at the 1-position of the reducing end with a leaving group; (iii) reacting the product of the above step with a reaction reagent in the presence of an activating agent to convert the reducing end; (iv) A deprotection step of reacting the product of the above step with a deprotection reagent to deprotect the protecting group to obtain a product in which the reducing end of any oligosaccharide chain is converted.
- Examples of the protecting reagent used in the protecting step include acetic anhydride / pyridine, benzyl chloride / sodium hydride and the like.
- Examples of the activator used in the conversion step include AgOTf when the leaving group is halogen, and Lewis acids such as MeOTf or BF 3 when the leaving group is a thioalkyl group.
- reaction reagent used in the step examples include 12-azidododecyl-1-ol, 3-azidopropyl-1-ol, p-methoxyphenol and the like.
- bases such as sodium methoxide and DBU are used for acyl protecting groups, and metal catalysts such as Pd and Pt are used for benzyl protecting groups.
- catalytic reduction reaction with sodium and birch reduction reaction with sodium-ammonia examples of the reaction reagent used in the step.
- fatty acids examples include Butyric acid, Valeric acid, Hexanoic acid, Heptanoic acid, Octanoic acid, Nonanoic acid, Decanoic acid, Undecanoic acid, Dodecanoic acid, Tridecanoic acid, Myristic acid, Pentadecanoic acid, Palmitic acid, Heptadecanoic acid, Steric acid, Nonadecanoic acid, Arachidic acid, Heneicosanoic acid, Behenic acid, Tricosanoic acid, Lignoceric acid, Pentacosanoic acid, Hexacosanoic acid, Heptacosanoic acid, Octacosanoic acid, Nonacosanoic acid, Melissicic acid, Hetriacontanoic acid, Racceroicano acid Hexatriacontanoic acid, Heptariacontanoic acid, Octatriacontanoic acid, Nonatriaconta
- alkyl diol examples include 1,2-Pentanediol, 1,2-Hexanediol, 1,2-Heptanediol, 1,2-Octanediol, 1,2-Nonanediol, 1,2-Decanediol, 1,2-Undecanediol, 1, 2-Dodecandiol, 1,2-Tridecanediol, 1,2-Tetradecanediol, 1,2-Pentadecanediol, 1,2-Hexadecanediol can be used.
- 1,3-nonanediol, 1,4-Heptanediol and the like can be used.
- the oligosaccharide chain can be introduced by a reductive amination method.
- an oligosaccharide containing an extra sugar serving as a spacer is used to maintain the structure of the target oligosaccharide chain, or hydroxybenzaldehyde is used according to the method described in Non-Patent Document 8. It is introduced after binding to the oligosaccharide chain.
- the oligosaccharide chain can be introduced through a spacer having a functional group that reacts with the thiol group among the above-mentioned commercially available linkers.
- the oligosaccharide chain can be introduced by reacting the oligosaccharide chain with a solvent such as PBS.
- a solvent such as PBS.
- halogen group it can be introduced by reacting in a solvent after protecting the hydroxyl group other than the reducing end of the oligosaccharide chain.
- a method for producing a conjugate as an active ingredient of the immunity-inducing agent of the present invention containing the sugar chain structure “Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc (CDw75)” as a model is shown below.
- Sugar donors derived from N-acetyl-glucosamine eg, 3,4,6-tri-O-acetyl-2-deoxy-2- (4,5-dichlorophthalimide) -D-glucopyranosyl bromide
- 2-Azidoalkyl alcohol CH 3 (CH 2 ) n2 CH (N 3 ) CH 2 OH is coupled by glycosylation, followed by hydroxy group deprotection, side chain azide group reduction, etc.
- a glucosamine derivative GlcNAc ⁇ 1-CH 2 CH (NH 2 ) (CH 2 ) n 2 CH 3 having an amino group was synthesized.
- This glucosamine derivative intermediate is subjected to an enzymatic reaction using ⁇ 1,4-galactosyltransferase and ⁇ 2,6-sialyltransferase to extend the sugar chain, and Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc ⁇ 1-CH 2 CH (NH 2 ) (CH 2) converted to n2 CH 3.
- Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc ⁇ 1-CH 2 CH (NH 2 ) (CH 2 ) n2 CH 3 and a fatty acid are condensed by an amidation reaction, and Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc ⁇ 1-CH 2 CH (NH-CO - (CH 2) n1 -CH 3 ) - (CH 2) synthesizing n2 -CH 3.
- Fatty acids were condensed using N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, converted to succinimide ester and isolated. This fatty acid active ester was subjected to coupling with an alkyl glycoside.
- Immunization method (4-1) Immunization method As a method for producing the monoclonal antibody of the present invention, conventional methods well known in the art can be applied (for example, Shepherd P. and Dean C., Monoclonal Antibodies, Oxford University Press, 2000, etc.). Specifically, using a conjugate (general formula (2)) in which a carrier compound (general formula (1)) developed by the present inventors is bound to a target oligosaccharide chain, For example, rats, mice, rabbits, etc., preferably mice are immunized as usual. For immunization, the liposome method (Brodin et al. Method; Eur. J. Immunol., 16, 951- 956, 1986) is preferably used.
- the conjugate (general formula (2)) is absorbed into acid-treated Salmonella minnesota strain R595 and injected intraperitoneally and intravenously (Galanos et al .; Eur. J. Biochem., 24, 116-122). , 1971).
- the dose of fused sugar chain antigen per animal is 0.05 to 0.2 mg using an adjuvant.
- Salmonella minnesota strain R595 treated with acid, complete Freund's adjuvant, or the like can be used, but a liposome method containing Lipid-A is preferred.
- booster immunization can also be performed. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally.
- the immunization interval is not particularly limited, and immunization is carried out 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks. Then, antibody-producing cells are collected 1 to 60 days, preferably 1 to 14 days after the last immunization day.
- antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred.
- Spleen cells are fused with myeloma cells by a conventional method, incubated in a medium together with thymic feeder cells in the presence of IL-6, and then selected by HAT in IMDM medium. Then, the supernatant of the growing clone is screened using the target oligosaccharide chain.
- immunohistochemical analysis or the like may be used, but an enzyme immunoassay (such as ELISA) or a simple screening method by Western blot can be applied to the target oligosaccharide chain immobilized on the substrate.
- the ELISA method or the Western blot method can be applied to the selection of the target oligosaccharide chain antibody-producing hybridoma.
- the antibody titer in the culture supernatant is preferably evaluated by an ELISA method, and hybridomas are selected using the antibody titer as an index.
- the antibody titer was evaluated, for example, as the activity of peroxidase, which is a labeling enzyme of the anti-mouse immunoglobulin antibody used as the secondary antibody.
- TMB is used as the chromogenic substrate for peroxidase, and the intensity of absorbance at 450 nm after adding 2N sulfuric acid after the reaction is evaluated.
- a monoclonal antibody-producing hybridoma cell that recognizes a glycoprotein that contains the glycolipid antigen and target oligosaccharide chain structure as an immunogen is selected, and then reacts with a sugar chain structure that approximates the target sugar chain structure.
- a monoclonal antibody-producing hybridoma having no sex is selected.
- Test Method for Characteristics of Monoclonal Antibody Recognizing Target Oligosaccharide Chain of the Present Invention 5-1 Specificity Test ⁇ ELISA Method> A protein (or lipid) having a similar oligosaccharide chain is immobilized on a substrate (ELISA plate) by evaporating to dryness together with a protein (or lipid) having an oligosaccharide chain antigen as a target of the present invention, and an antibody is obtained by ELISA. Evaluation is made as a value (intensity of absorbance at 450 nm). A commercially available sugar chain array may be used.
- ⁇ Western blot analysis> The sample is separated by SDS-polyacrylamide gel electrophoresis and electrically transferred to a membrane such as PVDF using a blotting apparatus.
- the membrane after transfer is blocked with 1% skim milk-PBST or the like, and then the primary antibody diluted appropriately with PBST is infiltrated into the membrane and reacted. After reacting at room temperature for 1 to 3 hours, the membrane is washed by shaking with a washing buffer such as PBST, and reacted with an HRP-labeled secondary antibody. After washing with PBST, the chemiluminescent substrate is reacted, and the antibody reaction is detected with an image analyzer.
- ⁇ Glycan array method An antibody appropriately diluted with PBST or the like is dropped onto a “sugar chain array” in which sugar chains are immobilized on a substrate. Cover the substrate surface with a cover glass to infiltrate the antibody solution and incubate at room temperature for about 2 hours. After gently stirring and washing with PBST or the like, the fluorescently labeled secondary antibody is reacted. After washing, the slide is dried, and fluorescence on the slide is detected using a microarray scanner.
- the concentration at which the competitive agent inhibits the antigen-antibody reaction is evaluated.
- Cell fluorescence staining method After the cells expressing the target oligosaccharide chain antigen are incubated with the antibody, the antibody reaction is labeled with a fluorescently labeled secondary antibody and detected with a fluorescence detection device.
- the antibody titer was evaluated as the activity of peroxidase which is a labeling enzyme of the secondary antibody.
- TMB was used as the chromogenic substrate for peroxidase, and the intensity of absorbance at 450 nm after adding 2N sulfuric acid after the reaction was evaluated.
- 1 to 3 show antibody titers of IgM subclass
- FIG. 4 shows antibody titers of IgG subclass.
- the pharmaceutical composition or vaccine for enhancing immunity containing the immunity-inducing agent of the present invention can be used without an adjuvant or with aluminum phosphate, aluminum chloride, Together with adjuvants such as aluminum salts, aluminum hydroxide, aluminum sulfate, Montanide ISA51, Montanide ISA720VG, MF59, AS03, cholesterol, phospholipids, diacylglycerol, monoacylglycerol, glycerol, fatty acids, glycocholic acid, taurocholic acid,
- adjuvants such as aluminum salts, aluminum hydroxide, aluminum sulfate, Montanide ISA51, Montanide ISA720VG, MF59, AS03, cholesterol, phospholipids, diacylglycerol, monoacylglycerol, glycerol, fatty acids, glycocholic acid, taurocholic acid,
- avian influenza Mainly intended for humans, but not limited to humans, domestic animals such as dogs, monkeys, cats, rabbits, domestic animals such as cattle, horses, pigs, and laboratory animals such as mice and rats
- it can be administered to birds in advance as a vaccine for avian influenza that may infect humans.
- the “Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc (CDw75)” antigen is used as an active ingredient of a pharmaceutical composition for treating malignant tumors or a pharmaceutical composition for preventing or treating influenza.
- the compound of the general formula (2) or a salt thereof is an appropriate dosage form determined by the treatment method, administration method, and administration purpose, specifically, injection, suspension, emulsifier, ointment, cream tablet, capsule , Granules, powders, pills, fine granules, troches, rectal administration, oily suppositories, water-soluble suppositories and the like.
- a solvent for example, water, physiological saline
- a bulking agent and a filler for example, lactose, tempun, crystalline cellulose, mannitol, maltose, calcium hydrogen phosphate, soft anhydrous silicic acid, Calcium carbonate
- solubilizers for example, ethanol, polysorbate
- binders for example, starch, polyvinylpyrrolidone, hydroxypropylcellulose, ethylcellulose, carboxymethylcellulose, gum arabic
- disintegrants for example, Starch, carboxymethylcellulose calcium
- lubricant eg, magnesium stearate, talc, hydrogenated oil
- stabilizer eg, lactose, mannitol, maltose, polysorbates, macrogol, polyoxyethylene hydrogenated castor oil
- Tonicity agent wet Agents, lubricants, dispersing agents, buffering agents, and the like can be used solubilizer.
- Additives as necessary include antioxidants, preservatives, flavoring agents, soothing agents, stabilizers, colorants, sweeteners and the like.
- glycerin, dimethylacetamide, 70% sodium lactate, surfactants, basic substances eg, ethylenediamine, ethanolamine, sodium carbonate, arginine, meglumine, trisaminomethane
- the administration route of the compound of the general formula (2) or a salt thereof is intravenous administration, local administration by injection, subcutaneous administration, intramuscular administration, sublingual administration, percutaneous absorption or rectal administration. Can do. Intravenous administration is most preferred.
- each active ingredient in the therapeutic agent according to the present invention can be administered continuously or intermittently depending on individual circumstances.
- the specific dose varies depending on the disease to be treated and its severity, as well as the administration method and various conditions of the patient, such as age, weight, sex, sensitivity, administration time, concomitant drugs, and the like.
- the dose of the compound of the general formula (2) is, for example, about 0.001 to 10 mg, preferably 0.01 to 1 mg per day for a human adult in intravenous administration.
- the compound of the general formula (2) is preferably made into a lyophilized preparation, and is preferably dissolved in distilled water for injection immediately before administration and administered to a patient.
- immunization of an animal such as a mouse with the immunity-inducing agent of the present invention to produce target sugar chain antigen-specific antisera or monoclonal antibodies can It can be used as an active ingredient of a pharmaceutical composition for preventing or treating a disease.
- a1, b1, and c1 are sphingosaccharide ceramide derivatives that are conjugates of the oligosaccharide antigens shown in the general formulas (3), (4), and (5) and a ceramide analog (CerA), respectively. It is.
- CerA represents a ceramide analog (HOCH 2 CH (NH—CO— (CH 2 ) 16 —CH 3 ) — (CH 2 ) 9 —CH 3 ), which is a typical example of the carrier compound of the present invention, and has a hydroxyl group. It is linked to the reducing end of each sugar chain via Cer is a ceramide (natural body), and LacCer, GM3, GM1, GD1a, and Globoside are natural glycolipids each having a sugar chain bound to ceramide.
- R represents the basic structure of Fetuin (Fetuin sugar chain and core protein part constituting the reducing side of the sugar chain structure shown in the table).
- Fetuin-a and Fetuin-b were prepared by selectively degrading the sugar chain structure of Fetuin with sialidase or ⁇ 2,3-sialidase, respectively.
- HL60 indicates a glycoprotein fraction extracted from HL60 cells, and Table 1 shows the main sugar chain structure.
- the backbone structure is indicated by R2.
- Glycoprotein AGP also showed the main sugar chain structure, and its basic structure was indicated by R3.
- the main sugar chain structure of HL60 is Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc (Sialyl-Lewis X ), and the main sugar chain structure of AGP is Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc (CDw75 ) And Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc (Lewis X ).
- Example 1 Production of Immune Inducing Agent of the Present Invention (1-1) Production of Immune Inducing Agent Having “6′-Sialyl-LacNAc (CDw75)” Sugar Chain Structure
- a mammal "6'-” is a type of sialylated sugar chain that is expressed in cells and is attracting attention as a tumor marker (CDw75) that serves as a diagnostic index for determining the malignancy of gastric cancer and colorectal cancer, and as a molecular target for the treatment of malignant tumors.
- Synthesis was performed according to the following reaction formula (4). Specifically, a sugar donor derived from N-acetyl-glucosamine (eg, 3,4,6-tri-O-acetyl-2-deoxy-2- (4,5-dichlorophthalimide) -D-glucopyranosyl bromide (Shimizu H., et al., Biosci. Biotech. Biochem., 60, 73-76, 1996)) and 2-azidododecanol CH 3 (CH 2 ) 9 CH ( After coupling N 3 ) CH 2 OH by a glycosylation reaction, a hydroxy group deprotection reaction, a side chain azide group reduction reaction, etc.
- N-acetyl-glucosamine eg, 3,4,6-tri-O-acetyl-2-deoxy-2- (4,5-dichlorophthalimide) -D-glucopyranosyl bromide (Shimizu H., et al.,
- the obtained intermediate compound was subjected to an enzymatic reaction using ⁇ 1,4-galactosyltransferase according to the following reaction formula (5), and the sugar chain was elongated to produce LacNAc-CH 2 CH (NH 2 ) (CH 2 ) 9 This was converted to CH 3 and further subjected to an enzymatic reaction using ⁇ 2,6-sialyltransferase to induce sialyl trisaccharide.
- A2 (S form) was synthesized in the same manner as the a1 compound except that acetic acid was used in place of stearic acid as the fatty acid to be reacted with the side chain amino group.
- the a2 (S) compound was purified by normal phase silica gel column chromatography and subjected to mass spectrometry by 1 H-NMR spectrum and MALDI-TOF to confirm the obtained compound.
- the a3-1 (S) isomer was synthesized by the same method as the a1 compound except that lignoceric acid was used in place of stearic acid as the fatty acid to be reacted with the side chain amino group.
- the ⁇ 1,4-galactosyltransferase reaction is followed by an enzymatic reaction using ⁇ 1,3 fucosyltransferase and ⁇ 2,3-sialyltransferase, and the sugar chain is elongated to sLe X —CH 2 CH (NH 2) (CH 2) except that was converted to 9 CH 3 synthesized by a method similar to the above a3-1 compound.
- the sLe X -C12L compound was purified by normal phase thin layer silica gel column chromatography and subjected to mass spectrometry by MALDI-TOF to confirm the obtained compound.
- MALDI-TOFMS C 67 H 123 N 3 O 24 [M + Na] + calcd. (M / z) 1376.84, found. (M / z) 1376.91
- Example 2 Evaluation of immunity induction ability by various immunity inducers (a1, b1, c1)
- various immunity synthesized in Examples (1-1), (1-2) and (1-3) respectively.
- the racemate of the inducer (a1, b1, c1) is dissolved in a lipid containing cholesterol and phospholipid together with adjuvant (Lipid-A), and the liposome is introduced into a mouse (C3H / HeN strain), which is a general-purpose host for monoclonal antibodies.
- the immunity was evaluated by the method (Eur. J. Immunol., 16, 951-956, 1986), and the immunity induction ability was evaluated.
- Each immunity-inducing agent was immunized subcutaneously on the first day and boosted intraperitoneally two weeks later. Blood was collected 3 days and 7 days after booster immunization, and serum was prepared. The antibody titer in the obtained serum was evaluated by ELISA.
- the ELISA method uses two types of antigens: immunogen and 6'-Sialyl-LacNAc, 3'-Sialyl-LacNAc, and glycoprotein (Fetuin) containing LacNAc structure. The antibody titer was evaluated.
- the secondary antibody was evaluated using an antibody specific for IgM and IgG.
- the antibody titer was evaluated as the activity of peroxidase which is a labeling enzyme of the secondary antibody.
- TMB was used as the chromogenic substrate for peroxidase, and the intensity of absorbance at 450 nm after adding 2N sulfuric acid after the reaction was evaluated.
- FIG. 1 to (FIG. 2) show antibody titers of IgM subclasses. All of the compounds (a1, b1, c1) used were significantly significant at p ⁇ 0.05, p ⁇ 0.01, and p ⁇ 0.001, respectively, by ELISA using immunogen (FIG. 1) and glycoprotein (FIG. 2) as antigens. An increase in antibody titer was confirmed.
- Example 3 Evaluation of immunity induction ability by structural change of carrier compound (3-1) Regarding immunity inducer having "6'-Sialyl-LacNAc ⁇ 1 (CDw75)" sugar chain structure
- a more active carrier compound Are selected from five candidate compounds (a1, a2, a3-1, a3-2, a3-3) in which the structure of the carrier compound is changed using the compound a1 synthesized in Example (1-1) as a basic structure. ), Its immunity induction ability was evaluated in the same manner as described above.
- Each immunity inducer was immunized subcutaneously on the first day, and boosted intraperitoneally two weeks after the carrier compound. Blood was collected 3 days and 7 days after booster immunization, and serum was prepared. The antibody titer in the obtained serum was evaluated by ELISA.
- ELISA In the ELISA method, two kinds of antigens, ie, an immunogen, 6'-Sialyl-LacNAc, and a glycoprotein (Fetuin) containing a structure, were evaluated as an antibody titer to the immunogen and an antibody titer to the target sugar chain, respectively.
- the secondary antibody was evaluated using an antibody specific for IgM and IgG.
- the antibody titer was evaluated as the activity of peroxidase, which is a secondary antibody labeling enzyme. TMB was used as the chromogenic substrate for peroxidase, and the intensity of absorbance at 450 nm after adding 2N sulfuric acid after the reaction was evaluated.
- FIG. 3 shows the antibody titer of the IgM subclass. In the compounds (a1, a2, a3-1, a3-2), a significant increase in antibody titer was confirmed by ELISA using immunogen and fetuin as antigens.
- the globoside used in the study is a purified product derived from human erythrocytes, and a ceramide containing a very long chain fatty acid (lignoceric acid) is bound to the P antigen sugar chain of human P blood group.
- FIG. 5 shows the serum antibody titers of mice when globoside was immunized by the liposome method.
- IgM subclass FIG. 5, left panel
- Example 4 An immune inducer having a sugar chain structure of "Sialyl-Lewis X (Sia ⁇ 2,3Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc)" and "Lewis X (Gal ⁇ 1,4 (Fuc ⁇ 1,3) GlcNAc)" Evaluation of (Example 3)
- (Example 1) (1 -4), typical fucosylated sugar chains, Sialyl Lewis X (sLe X ), and representative asialo sugar chains, Lewis X (Le X ), manufactured in (1-5)
- the ability to induce immunity against the target oligosaccharide chain antigen in the conjugates with oligosaccharide chains was verified.
- FIG. 9 shows the serum antibody titers of the mice when each compound was immunized.
- IgM subclass in the sera of mice immunized with sLe X -C12L (FIG. 9, A)
- significant increase of antibody to the glycoprotein (HL60) containing the immunogen and the desired oligosaccharide chain antigen in the sera after 3 and 7 days after immunization was confirmed.
- IgG subclass FIG. 9
- the carrier compound (C12L) developed in the present invention is a typical oligosaccharide antigen classified into a fucosylated sugar chain as well as a sialylated sugar chain and an asialo (non-sialylated) sugar chain. It was demonstrated that a strong immunity-inducing activity can be imparted to any of these.
- the carrier compound of the present invention includes sialylated sugar chains, asialo (non-sialylated) sugar chains, and fucosylated sugars, which are oligosaccharide antigens contained in N-linked sugar chains present in mammalian glycoproteins. It was confirmed that this technique can be applied to all of the chains.
- Example 5 Production of monoclonal antibody Using a3-1 (CDw75-C12L), which was confirmed to have the strongest immunogenicity in (Example 3), a specific antibody of the target sugar chain epitope was actually produced. It was confirmed. Specifically, a monoclonal antibody was prepared as follows. (5-1) Production of anti-CDw75 monoclonal antibody-producing hybridoma 100 ⁇ g of glycolipid antigen “CDw75-C12L” was prepared according to the liposome method (Brodin et al .; Eur. J. Immunol., 16, 951-956, 1986).
- Lipids, cholesterol and Lipid-A were mixed in methanol solution, evaporated, and then dissolved in 50 ° C PBS buffer to form liposomes. This was used as an immune inducer for mice (C3H / HeN strain ).
- the immunity-inducing agent was immunized subcutaneously 4 times at 2-week intervals, and immunized intraperitoneally 2 weeks later, and then spleen cells were collected 3 days later, and hybridoma cells were prepared by cell fusion with the myeloma cell Sp1 strain.
- the antibody titer in the culture supernatant was evaluated by ELISA, and hybridomas were selected using an absorbance at 450 nm of 0.1 or more as an index.
- the antibody titer (absorbance) was evaluated as the activity of peroxidase, which is a labeling enzyme of the anti-mouse immunoglobulin antibody used as the secondary antibody.
- TMB was used as the chromogenic substrate for peroxidase, and the intensity of absorbance at 450 nm after adding 2N sulfuric acid after the reaction was evaluated.
- 0.05 ml of the culture supernatant of hybridoma cells cultured on 8 96-well plates so that the number of colonies is 1 / well (total of 768 wells) is collected, and the antibody titer is measured using an absorbance of 0.1 nm or higher at 450 nm by ELISA as an index.
- Non-patent Document 8 phospholipid (phosphatidylethanolamine) is used as a carrier compound, and an antibody having the CDw75 sugar chain as an epitope is similarly induced. The appearance rate was only 8 wells (appearance rate 1.3%) out of 581 wells.
- glycolipid antigen “CDw75-C12L” as the immunogen and the precursor “Gal ⁇ 1,4GlcNAc” in general formula (5) and the sugar chain present in the mammalian body are the closest in structure to the target oligosaccharide chain “Sia ⁇ 2,3Gal ⁇ 1,4GlcNAc” glycolipid having a sugar chain structure of the general formula (4) (b1, c1 in Table 1) and a monoclonal antibody-producing hybridoma cell not reactive to glycoprotein (Fetuin-a) And finally isolated two cell lines as high affinity clones.
- the monoclonal antibodies obtained from these hybridomas are all anti-CDw75 monoclonal antibodies having high specificity for the “CDw75” sugar chain structure, and are the anti-CDw75 monoclonal antibodies of the present invention.
- “Hybridoma FR9” which produces the anti-CDw75 monoclonal antibody with the highest antibody titer, specificity and affinity, was deposited with the NITE Patent Microorganism Deposit Center on January 21, 2013. . After the “Accession Number: NITE P-1516” was granted on March 13, 2013, it was transferred to the International Deposit as “NITE BP-01516” on April 15, 2014.
- the FR9 antibody was able to detect a trace amount of Fetuin (about 15 ng) (FIG. 6B).
- the antibody induced by the prior art (Non-patent Document 8) requires 50 ⁇ g or more of Fetuin for detection, the FR9 antibody is 3000 times higher than the antibody prepared by a known technique. It can be seen that it has detection sensitivity.
- the antigen recognition specificity of the FR9 antibody was evaluated by ELISA (FIG. 7). Table 1 shows the sugar chain structure of the antigen used.
- the FR9 antibody was developed because it strictly identifies epitopes only of glycolipids and glycoproteins with CDw75 structure as antigen, and does not react to the 3'-Sialyl-LacNAc structure or the precursor LacNAc structure.
- the antibody has high specificity for the CDw75 sugar chain.
- a fetuin glycoprotein containing the 6'-Sialyl-LacNAc structure as an antigen was treated with two types of sialidases, and the specificity analysis was performed in parallel.
- the FR9 antibody detected the fetuin glycoprotein.
- the immunity-inducing agent developed according to the present invention produces a specific antibody against the target sugar chain epitope, and that the produced antibody has a high activity and a performance that can be used for various applications.
- the anti-CDw75 antibody (FR9 antibody) of the present invention has the same reactivity as that of Fetuin even when the CDw75 sugar chain is contained in a glycoprotein other than Fetuin glycoprotein, and the reactivity was confirmed by Western blot analysis using AGP glycoprotein (FIG. 11).
- AGP glycoprotein contains “3w-Sialyl-LacNAc” (Sia ⁇ 2,3Gal ⁇ 1,4GlcNAc) as well as “CDw75 (Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc)” as a sugar chain structure like Fetuin glycoprotein.
- the observed antibody titer is absent from AGP (AGP-a) from which sialic acid has been removed, and selectively from AGP (AGP-b) from which “3'-Sialyl-LacNAc” structure has been removed. Confirmed that the antibody titer did not change.
- Table 2 shows the main sugar chain structures of AGP, AGP-a, and AGP-b.
- R represents the basic structure of AGP (AGP sugar chain and core protein part constituting the reducing side of the sugar chain structure shown in the table).
- CDw75 sugar chains are expressed as cell surface antigens in the form of glycoproteins and glycolipids, and cell surface antigens are molecular targets for cell diagnosis and malignant tumors. It becomes. It was confirmed by cell fluorescence staining that the FR9 antibody reacted with CDw75 on the cell surface. After incubating high-grade B-cell tumor cells (Burkitt lymphoma Raji cells) expressing CDw75 with FR9 antibody, the antibody reaction was labeled with a fluorescently labeled secondary antibody and detected with a fluorescence detector (FIG. 12). ). A clear increase in fluorescence was detected as compared with the negative control to which no FR9 antibody was added, indicating that the FR9 antibody reacts with CDw75 on the surface of Raji cells.
- the FR9 antibody of the present invention is an antibody that recognizes even the fine structure of the CDw75 sugar chain, including not only the Sia ⁇ 2,6 structure but also the GlcNAc structure at the reducing end, including “6'-Sialyl-LacNAc (Sia ⁇ 2,6Gal ⁇ 1,4GlcNAc ) "Is the epitope. It has a strict epitope recognition specificity that GlcNAc does not react with Glc (FIG. 10), and it can be said that extremely high specificity was achieved compared to existing antibodies.
- the FR9 antibody of the present invention can be detected even with a trace amount of Fetuin (about 15 ng) (FIG. 6B). In view of this, it can be said that the FR9 antibody of the present invention achieved a detection sensitivity 3000 times higher than that of the 241-2-5-2 antibody with respect to the affinity for the CDw75 sugar chain antigen.
- the monoclonal antibody obtained by applying the immunity induction method of the present invention is a monoclonal antibody having high specificity and affinity for the target oligosaccharide chain antigen.
- An immunity-inducing agent for sugar chains can be used for the development of polyclonal and monoclonal antibodies that recognize specific sugar chain structures.
- the developed antibody can be applied to basic research related to sugar chain function and research and development for industrial application, and demand as a research reagent is expected.
- molecular species that are specifically expressed in pathological cells such as cancer cells are expected to be applied as diagnostics and antibody drugs by developing specific antibodies.
- the developed immunity-inducing agent is also expected to be used as a vaccine in various diseases such as cancer and viral infections related to sugar chains.
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Abstract
Description
しかし、糖鎖の構造同定や検出に関する技術が、タンパク質や核酸と比べて十分に確立されていないため、機能解析がなかなか進まない。複合糖質の糖鎖構造を特徴づける糖鎖の部分構造を識別し検出できる抗体が容易に開発できるようになれば、タンパク質解析に汎用されるELISA法やウエスタンブロット法等による糖鎖解析が可能となり、糖鎖の機能解明や産業応用に関する研究開発が加速すると考えられる。
複合糖質の糖鎖構造のうちでも、特に糖タンパク質に含まれるN-結合型糖鎖構造の場合は各種の癌診断マーカーや、重篤な疾患の診断マーカーとなる場合が多いため、N-結合型糖鎖構造を認識できる抗体の開発への期待はきわめて大きい。
例えば、以前より肝臓がんにおける糖タンパク質のN-結合型糖鎖構造の変化ががん診断マーカーとして利用できることが知られている(非特許文献1)。代表例として、α-フェトプロテイン(AFP)のN-結合型糖鎖がある。AFPの血清値の上昇は、肝臓がんの診断指標として以前より利用されてきたが、肝炎や肝硬変でも上昇するため、肝臓がんのみを正確に診断できないことが欠点であった。最近になり、AFPタンパク質のN-結合型糖鎖の構造が肝臓がんに特異的に変化することがわかり(非特許文献2)、このような糖鎖構造を含むAFP-L3が、高精度な肝臓がん診断マーカーとして注目されている。卵巣がんについても、コアフコース型、sLeX型のN-結合糖鎖を含むイムノグロブリン、急性期タンパク質が新規なマーカー候補として見いだされている(非特許文献3)。
また、肝硬変の前症状である肝線維症(肝硬変の前段階)のマーカーとしてもN-結合型糖鎖が有用であることがわかってきている。血清中に存在するα1-酸性糖タンパク質(AGP)のN-結合型糖鎖は、肝線維症の発症と相関して特徴的な構造をとり、これを診断するマーカーとしてLecT-Hepaが開発された(非特許文献4)。さらには、より高感度かつ簡便に検出できるよう改良されたFastLec-Hepaでは、血液中のM2BP糖タンパク質のN-結合型糖鎖に見られる肝線維症特異的な変化を検出する(非特許文献5)。
その他、糖尿病などでも膵臓に発現するグルコーストランスポータータンパク質のN-結合型糖鎖の構造異常が病態発症の原因となることが提唱されている(非特許文献6)。
これまでのところ、ビオチン化アミノピリジンのアミノ基にオリゴ糖鎖を還元的に結合させ、続けてビオチンをリガンドであるアビジンに多価に結合させたものを免疫原として免疫を誘導する方法(非特許文献12)が知られているが、上述のイソマルトース派生産物(7糖体)をオリゴ糖鎖として用いて、免疫誘導はなされたものの免疫増強能が低く、標的のオリゴ糖鎖に対する有効なモノクローナル抗体の開発には至っていない。
また、典型的な糖タンパク質のN-結合型糖鎖中のオリゴ糖鎖抗原である「Galβ1,4GlcNAcβ1,2Man」及び「非還元末端のβ-GlcNAc残基」、並びに「Siaα2,6Galβ1,4GlcNAc(CDw75)」については、ホスファチジルエタノールアミンをキャリア化合物として、そのアミノ基にオリゴ糖鎖を還元的に結合させた化合物を免疫原とし免疫を誘導する方法(非特許文献7、8)が報告されている。しかし、これらの免疫誘導方法により得られたモノクローナル抗体の親和性、特異性は十分ではないため、実用的な抗体が提供できたとはいえず、当該免疫誘導方法も任意の糖鎖に対して選択的に抗体を誘導する汎用的な技術ではない。しかも、これらの免疫誘導方法では、キャリア化合物とオリゴ糖鎖とをコンジュゲートする際の製造方法としてオリゴ糖鎖の還元末端糖を開裂させる還元的アミノ化法を利用しているため、オリゴ糖鎖の構造を維持したままで免疫誘導剤が合成できない。そのため、開裂を防ぐためにはスペーサーが必須であり、あらかじめスペーサー用の糖やヒドロキシベンズアルデヒドをオリゴ糖鎖に結合させるという煩雑な合成工程が必要となるという欠点もある。
また、糖鎖リガンドをアジュバントと共にリンカーを介して結合させた磁性の金属ナノ粒子を用いる方法(特許文献1)が提案されているが、ナノ粒子の調製自体が容易ではなく、糖鎖リガンドとアジュバンドを一定の割合でナノ粒子へと結合させる工程も簡単ではない。乳癌特異的なGlobo-H糖鎖に対しては、Globo-H糖鎖の還元側に、アミノアルキル及びp-ニトロフェニルエステルを介し、ジフテリア毒素交差反応物質であるキャリアタンパク質を結合させて、癌ワクチンとすることが提案されている(特許文献2)が、糖鎖抗原一般に適用できる汎用的技術ではない。
本発明者らは、糖鎖抗原にCD1分子との結合部位となるα-ガラクトシルセラミドの脂質構造を結合させることを思いつき、種々の脂質構造を検討した結果、天然には存在しない脂質構造である、アミノ基側のアルキル基中に-OHなど酸素原子を含む基も不飽和結合基も存在しない飽和アルキル基を有する一般式(1)の脂質構造を有するキャリア化合物(人工脂質)が、最も免疫原性増強作用があることを見いだした。しかもその免疫原性増強作用は、特定の糖鎖抗原のみに留まらず、N-結合型糖鎖由来に含まれる各種オリゴ糖鎖抗原を含め、どのような糖鎖抗原に対しても適用できるという極めて高い汎用性が高いことも実証できた。
X-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
・・・一般式(1)
(式中、Xは-H、-OH、-SH、-NH2、ハロゲン、又はヒドラジド基などを表し、Yは-(CH2)m-(mは1~30、好ましくは1~3の整数)などのスペーサー配列を表す。n1は2~40 の整数で、n2は1~27、好ましくは2~13の整数を表す。)
本発明で対象とする任意のオリゴ糖鎖は、有機合成でも酵素合成法でも合成可能であり、オリゴ糖鎖の還元末端以外の水酸基を保護して、式(1)のキャリア化合物を反応させれば、オリゴ糖鎖構造を維持したままコンジュゲートできる。
得られるオリゴ糖鎖抗原とキャリア化合物のコンジュゲート(人工糖脂質)は、下記の一般式(2)で表すことができる。
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
・・・一般式(2)
(式中、Rは糖鎖抗原となるオリゴ糖であり、Zは、単結合、又はO、S、もしくはNHなどを表し、Y、n1及びn2は一般式(1)と同様に定義される。)
このことから、一般式(1)のキャリア化合物は、糖タンパク質のN-結合型糖鎖由来のオリゴ糖鎖抗原を含め、どのようなオリゴ糖鎖抗原とコンジュゲートした免疫誘導剤としても、標的のオリゴ糖鎖抗原の免疫原性を増強し、かつ胸腺依存性抗原としての活性を付与することができることが実証された。さらに、スペーサー配列を適宜選ぶことで、活性のさらなる上昇やオリゴ糖鎖とキャリア化合物との結合の簡便化も期待できる。
また、一般式(1)のキャリア化合物において、脂肪酸部位のアルキル鎖長が伸長するほど免疫誘導能が増強する傾向を見出し、反対に鎖長が長すぎると溶解性等の問題が発生して酵素法による合成効率が低減することも併せて発見したことで、最適なサイズのキャリア化合物の範囲を同定した。
以上の知見を得たことで、本発明を完成した。
〔1〕本発明は、以下の免疫誘導方法に係る発明に関する。
〔1-1〕標的オリゴ糖鎖抗原Rを含む一般式(2)で表される人工糖脂質又はその塩を有効成分とする免疫誘導剤を用いて哺乳動物を免疫する工程を含む、哺乳動物において標的オリゴ糖鎖抗原Rに対する抗原特異的な免疫誘導方法;
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
シアリル化糖鎖抗原としては、「Siaα2,6Galβ1,4GlcNAc:CDw75」、「Siaα2,3Galβ1,4GlcNAc:3’-Sialyl-LacNAc」、「Siaα2,3Galβ1,4(Fucα1,3)GlcNAc:Sialyl-LewisX 」、「Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc:Sialyl-Lewisa」、「Siaα2,3Galβ1,3 (Fucα1,4) (Siaα2,6)GlcNAc:Disialyl-Lewisa」、「Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc(6SO4):6-sulfo-Sialyl-LewisX」、「Siaα2,3Gal (6SO4)β1,4 (Fucα1,3)GlcNAc:6’-sulfo-Sialyl-LewisX」、「Siaα2,3Galβ1,4GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc:VIM-2 antigen」、「Siaα2,6GalNAcβ1,4GlcNAc」、「Siaα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc:Sialyl-LewisX-i」、「Siaα2,3Galβ1,4GlcNAcβ1,3(Siaα2,3Galβ1,4GlcNAcβ1,6)Gal:Sialyl-I」であることが好ましく、
アシアロ糖鎖(非シアリル化糖鎖)抗原としては、「Galβ1,4GlcNAc:LacNAc」、「GlcNAcβ1,4Man」、「GlcNAcβ1,6Man」、「Galβ1,4GlcNAcβ1,6Man」、「GalNAcβ1,4GlcNAc」、「GalNAcβ1,4GlcNAcβ1,2Man」、「GalNAc(4SO4)β1,4GlcNAc」、「Galβ1,4 (Fucα1,3)GlcNAc:LewisX」、「Galβ1,3 (Fucα1,4)GlcNAc:Lewisa」、「Fucα1,2Galβ1,3(Fucα1,4)GlcNAc:Lewisb」、「Fucα1,2Galβ1,4(Fucα1,3)GlcNAc:Lewisy」、「Fucα1,6GlcNAc」、「GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Galβ1,4GlcNAcβ1,3(Galβ1,4GlcNAcβ1,6)Gal:I-antigen」であることが好ましく、そして、
フコシル化糖鎖抗原としては、「Galβ1,4 (Fucα1,3)GlcNAc:LewisX」、「Galβ1,3 (Fucα1,4)GlcNAc:Lewisa」、「Fucα1,2Galβ1,3(Fucα1,4)GlcNAc:Lewisb」、「Fucα1,2Galβ1,4(Fucα1,3)GlcNAc:Lewisy」、「Fucα1,6GlcNAc」、「GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Siaα2,3Galβ1,4(Fucα1,3)GlcNAc:Sialyl-LewisX 」、「Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc:Sialyl-Lewisa」、「Siaα2,3Galβ1,3 (Fucα1,4) (Siaα2,6)GlcNAc:Disialyl-Lewisa」、「Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc(6SO4):6-sulfo-Sialyl-LewisX」、「Siaα2,3Gal (6SO4)β1,4 (Fucα1,3)GlcNAc:6’-sulfo-Sialyl-LewisX」、「Siaα2,3Galβ1,4GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc:VIM-2 antigen」、「Siaα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc:Sialyl-LewisX-i」であることが特に好ましい。
また、一般式(2)中のRの好ましい場合を、「Siaα2,6Galβ1,4GlcNAc:CDw75」、「Siaα2,3Galβ1,4GlcNAc:3’-Sialyl-LacNAc」、「Siaα2,3Galβ1,4(Fucα1,3)GlcNAc:Sialyl-LewisX 」、「Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc:Sialyl-Lewisa」、「Siaα2,3Galβ1,3 (Fucα1,4) (Siaα2,6)GlcNAc:Disialyl-Lewisa」、「Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc(6SO4):6-sulfo-Sialyl-LewisX」、「Siaα2,3Gal (6SO4)β1,4 (Fucα1,3)GlcNAc:6’-sulfo-Sialyl-LewisX」、「Siaα2,3Galβ1,4GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc:VIM-2 antigen」、「Siaα2,6GalNAcβ1,4GlcNAc」、「Siaα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc:Sialyl-LewisX-i」、「Siaα2,3Galβ1,4GlcNAcβ1,3(Siaα2,3Galβ1,4GlcNAcβ1,6)Gal:Sialyl-I」からなるシアリル化糖鎖抗原、「Galβ1,4GlcNAc:LacNAc」、「GlcNAcβ1,4Man」、「GlcNAcβ1,6Man」、「Galβ1,4GlcNAcβ1,6Man」、「GalNAcβ1,4GlcNAc」、「GalNAcβ1,4GlcNAcβ1,2Man」、「GalNAc(4SO4)β1,4GlcNAc」、「Galβ1,4 (Fucα1,3)GlcNAc:LewisX」、「Galβ1,3 (Fucα1,4)GlcNAc:Lewisa」、「Fucα1,2Galβ1,3(Fucα1,4)GlcNAc:Lewisb」、「Fucα1,2Galβ1,4(Fucα1,3)GlcNAc:Lewisy」、「Fucα1,6GlcNAc」、「GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Galβ1,4GlcNAcβ1,3(Galβ1,4GlcNAcβ1,6)Gal:I-antigen」からなるアシアロ糖鎖(非シアリル化糖鎖)抗原、及び「Galβ1,4 (Fucα1,3)GlcNAc:LewisX」、「Galβ1,3 (Fucα1,4)GlcNAc:Lewisa」、「Fucα1,2Galβ1,3(Fucα1,4)GlcNAc:Lewisb」、「Fucα1,2Galβ1,4(Fucα1,3)GlcNAc:Lewisy」、「Fucα1,6GlcNAc」、「GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Siaα2,3Galβ1,4(Fucα1,3)GlcNAc:Sialyl-LewisX 」、「Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc:Sialyl-Lewisa」、「Siaα2,3Galβ1,3 (Fucα1,4) (Siaα2,6)GlcNAc:Disialyl-Lewisa」、「Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc(6SO4):6-sulfo-Sialyl-LewisX」、「Siaα2,3Gal (6SO4)β1,4 (Fucα1,3)GlcNAc:6’-sulfo-Sialyl-LewisX」、「Siaα2,3Galβ1,4GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc:VIM-2 antigen」、「Siaα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc:Sialyl-LewisX-i」からなるフコシル化糖鎖抗原から選択されたオリゴ糖鎖抗原である、と表現することもできる。
チオメチルと結合させるリンカーとしては、市販リンカーのBMPH(N-β-Maleimidopropionic acid hydrazide-TFA)、KMUH(N-κ-Maleimidoundecanoic acid hydrazide-TFA)、EMCH(N-[ε-Maleimidocaproic acid]hydrazide- TFA)、MPBH (4-[4-N-Maleimidophenyl]butyric acid hydrazide-HCl)、及びPDPH (3-[2-Pyridyldithio]propionyl hydrazide)(Thermo Fisher scientific社製)から選択されることが好ましく、アミノメチル化される糖アルコールとしては、N-アセチルグルコサミニトール、N-アセチルガラクトサミニトール、マンニトール、ガラクチトールから選択されることが好ましい。
n1の好ましい態様として14~39、16~31、17~28、及び20~40が挙げられ、より好ましい態様は、20~28、21~27であり、最も好ましくは22~24である。
n2は1~27の整数で、好ましい態様として2~15、2~13、3~13が挙げられ、より好ましい態様は5~13、6~12であり、最も好ましくは7~11である。
mは1~30の整数で、好ましくは1~10、より好ましくは1~3の整数である。
1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖からなる標的オリゴ糖鎖抗原Rに対して、一般式(1)で表されるキャリア化合物又はその塩がコンジュゲートされた一般式(2)で表される人工糖脂質を合成し、当該糖脂質又はその塩を有効成分とする免疫誘導剤により哺乳動物を免疫する工程を含む、標的オリゴ糖鎖抗原Rに対する抗原特異的な免疫誘導方法;
一般式(1)
X-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Xは-H、-OH、-SH、-NH2、ハロゲン又はヒドラジド基を表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
ここで、式中の標的オリゴ糖鎖抗原R、Z並びにn1、n2及びmの好ましい場合は、前記〔1-2〕及び〔1-3〕で述べた場合と同様である。
標的オリゴ糖鎖抗原Rと一般式(1)で表されるキャリア化合物又はその塩とをコンジュゲートした一般式(2)で表される人工糖脂質又はその塩を有効成分とする免疫誘導剤を哺乳動物に投与する工程を含むことを特徴とする、哺乳動物において標的オリゴ糖鎖抗原Rに対する抗原特異的な免疫誘導方法;
一般式(1)
X-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Xは-H、-OH、-SH、-NH2、ハロゲン又はヒドラジド基を表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
ここで、式中の標的オリゴ糖鎖抗原R、Z並びにn1、n2及びmの好ましい場合は、前記〔1-2〕及び〔1-3〕で述べた場合と同様である。
〔2-1〕下記一般式(2)に示され、標的オリゴ糖鎖抗原Rを含む人工糖脂質又はその塩を有効成分として含む免疫誘導剤;
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
ここで、式中の標的オリゴ糖鎖抗原R、Z並びにn1、n2及びmの好ましい場合は、前記〔1-2〕及び〔1-3〕で述べた場合と同様である。
また、本発明の免疫誘導剤に係る発明は、以下のように表現することもできる。
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
ここで、式中の標的オリゴ糖鎖抗原R、Z並びにn1、n2及びmの好ましい場合は、前記〔1-2〕及び〔1-3〕で述べた場合と同様である。
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
ここで、式中の標的オリゴ糖鎖抗原R、Z並びにn1、n2及びmの好ましい場合は、前記〔1-2〕及び〔1-3〕で述べた場合と同様である。
〔3-1〕下記一般式(2)に示され、標的オリゴ糖鎖抗原Rを含む人工糖脂質又はその塩を有効成分として含むワクチン;
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
ここで、ワクチンは、標的オリゴ糖鎖抗原Rに対する抗原特異的な免疫誘導を引き起こす作用を有するワクチンであり、式中の標的オリゴ糖鎖抗原R、Z並びにn1、n2及びmの好ましい場合は、前記〔1-2〕及び〔1-3〕で述べた場合と同様である。また、本発明のワクチンに係る発明は、以下のように表現することもできる。
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
ここで、式中の標的オリゴ糖鎖抗原R、Z並びにn1、n2及びmの好ましい場合は、前記〔1-2〕及び〔1-3〕で述べた場合と同様である。
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
ここで、式中の標的オリゴ糖鎖抗原R、Z並びにn1、n2及びmの好ましい場合は、前記〔1-2〕及び〔1-3〕で述べた場合と同様である。
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は20~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
ここで、式中のn1については、超長鎖脂肪酸に分類される炭素数22以上の脂肪酸に対応させた、n1が20以上の整数の20~40であって、好ましくは20~28、よりこのましくは21~27であり、最も好ましくは22~24である。
また、標的オリゴ糖鎖抗原R及びZ、並びにn2及びmの好ましい場合は、前記〔1-2〕及び〔1-3〕で述べた場合と同様である。
すなわち、本発明の新規な人工脂質又はその塩に係る発明は、以下のように表現することができる。
下記一般式(1)に示された人工脂質又はその塩;
一般式(1)
X-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Xは-H、-OH、-SH、-NH2、ハロゲン又はヒドラジド基を表し、Yは-(CH2)m-を表す。n1は20~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
ここで、標的となるオリゴ糖鎖抗原R及びZ、並びにn2及びm好ましい場合は、前記〔1-2〕及び〔1-3〕で述べた場合と同様であり、n1については、前記〔4〕の場合と同様である。
先行技術(非特許文献8)においてもCDw75オリゴ糖鎖を含む免疫誘導剤で免疫し、CDw75と反応するモノクローナル抗体が産生されているが、厳密な意味での特異的抗CDw75抗体は得られておらず、当該文献中に記載されたCDw75反応性の抗体(241-5-2抗体)についての各種実験データを、本発明で得られた抗CDw75抗体作製の際のデータと比較すると、免疫誘導後の目的抗体を産生する陽性ハイブリドーマ細胞の出現率が約13倍も高かった。また、最終的に得られたモノクローナル抗体のエピトープに対する親和性は約3,000倍もの高さであった。
本発明のキャリア化合物による免疫原性増強効果により、免疫後の宿主動物より、目的糖鎖エピトープに対する特異性及び親和性の高いモノクローナル抗体及びその産生ハイブリドーマ細胞の開発が可能となった。
また本発明の免疫誘導剤は、当該免疫誘導剤を免疫したマウスにおいて、クラススイッチにより産生されるIgGクラスの抗体の産生を惹起できる能力が確認されており、胸腺依存性抗原としての活性を有する。当該活性は、ホスファチジルエタノールアミンをキャリア化合物とする免疫誘導剤を用いた従来技術(非特許文献7、8)では確認されていない。
微生物由来オリゴ糖鎖に対する抗体産生を試みた場合にはKLHやBSA、ビオチンアビジンなどのキャリア化合物とするコンジュゲートによる免疫で、親和性は低いながらIgG抗体を産生させたという報告(非特許文献9、10、12)はあったものの、哺乳動物由来オリゴ糖鎖であり、かつ糖タンパク質のN-結合型糖鎖に含まれるオリゴ糖鎖抗原においてクラススイッチを伴う免疫を誘導し、親和性の高い抗体を産生できたのは本発明がはじめてである。
このように、本発明の免疫誘導剤は、胸腺依存性抗原として免疫システムに認識されるため、クラススイッチや親和性成熟などの抗体の特異性、親和性の増強過程を経ることができるため、特異性、親和性の高い抗体を得ることが可能となる。
従来技術(非特許文献7~12)で用いられたキャリア化合物の場合、一般的には目的とするオリゴ糖鎖を還元的アミノ化法によりコンジュゲートすることになるため、糖鎖はキャリア化合物との結合部位(還元側の糖)が開裂し、目的とするオリゴ糖鎖の構造を維持したまま免疫誘導剤を合成することはできない。
本発明により提供された、任意のオリゴ糖鎖の免疫原性を増強できるキャリア化合物の場合、免疫誘導剤としての「オリゴ糖鎖-キャリア化合物」コンジュゲートの効率的な構築が可能となり、糖鎖エピトープを認識するモノクローナル抗体や糖鎖を標的とするワクチン等の開発が容易となる。
(1-1)標的オリゴ糖鎖抗原について
本発明において「オリゴ糖鎖抗原」または単に「糖鎖抗原」というとき、当該オリゴ糖鎖自身またはその修飾物に抗体産生細胞の刺激活性が備わっているオリゴ糖鎖をいう。本発明の免疫誘導法は、任意のオリゴ糖鎖抗原を対象として免疫原性を増強可能であるが、本発明において標的とする典型的な「オリゴ糖鎖抗原」は、各種生物の分泌糖タンパク質、細胞膜を構成する糖タンパク質又は糖脂質などの複合糖質に含まれる糖鎖領域の全部もしくは一部の糖鎖構造として存在している、2~30、好ましくは2~16、より好ましくは2~6個の糖から構成されるオリゴ糖鎖抗原である。つまり、本発明の「オリゴ糖鎖抗原(R)」は、「複合糖質に含まれる糖鎖構造であって、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖」により構成される。とりわけ、哺乳動物細胞由来の糖タンパク質に見られるN-結合型糖鎖に含まれる糖鎖構造からなるオリゴ糖鎖抗原が好ましい。
すなわち、本発明において対象となる糖鎖抗原(R)は、シアル酸(N-アセチルノイラミン酸、N-グリコリルノイラミン酸、αケトデオキシノノン酸)、ガラクトース、グルコース、N-アセチルグルコサミン、N-アセチルガラクトサミン、グルコサミン、ガラクトサミン、マンノース、フコース、キシロース、グルクロン酸、イズロン酸、イノシトール、エリトロース、エリトロン酸、エリトルロン酸、エリトラル酸、エリトリトール、トレオース、トレオン酸、トレウロン酸、トレアル酸、トレイトール、リボース、リボン酸、リブロン酸、リバル酸、リビトール、アラビノース、アラビノン酸、アラビヌロン酸、アラビナル酸、アラビニトール、キシロン酸、キシルロン酸、キシラル酸、キシリトール、リキソース、リキソン酸、リキスロン酸、アロース、アロン酸、アルロン酸、アラル酸、アリトール、アルトロース、アルトロン酸、アルトルロン酸、アルトラル酸、アルトリトール、グルコン酸、グルカル酸、グルシトール、マンノン酸、マンヌロン酸、マンナル酸、マンニトール、グロース、グロン酸、グルロン酸、イドース、イドン酸、イダル酸、イジトール、ガラクトン酸、ガラクツロン酸、ガラクタル酸、ガラクチトール、タロース、タロン酸、タルロン酸、ジヒドロキシアセトン、エリトルロース、リブロース、プシコース、フルクトース、ソルボース、タガトース、セドヘプツロース、コリオース、デオキシリボース、ラムノース、フクロース、アロメチロース、キノボース、アンチアロース、タロメチロース、ジギタロース、ジギトキソース、シマロース、チベロース、アベコース、パラトース、コリトース、アスカリロース、及びこれらが硫酸化、アセチル化、リン酸化、もしくはメチル化された糖、好ましくは、シアル酸、ガラクトース、グルコース、N-アセチルグルコサミン、N-アセチルガラクトサミン、グルコサミン、ガラクトサミン、マンノース、フコース、キシロース、グルクロン酸、イズロン酸、イノシトール、及びこれらが硫酸化、アセチル化、もしくはメチル化された糖から選択される1又は複数種の単糖を構成要素とする直鎖状又は分枝状の分子であり、Rを構成する単糖の数は2~30好ましくは2~16、より好ましくは2~6個の糖からなる、と表現することができる。
哺乳動物細胞においては、特に癌細胞や重篤な疾患など病態などを反映して細胞表面に特徴的な構造の複合糖鎖を有する複合糖質が発現してくることが多数報告されており、これら複合糖鎖も診断マーカー、及び薬剤標的のためのバイオマーカーとなる。例えば、消化器癌のマーカーとしてCA19-9(Sialyl-Lewisa)が知られている。
したがって、本発明では、主として哺乳動物細胞由来の複合糖質に含まれるオリゴ糖鎖抗原を対象とする。
このような哺乳動物細胞由来の既知のオリゴ糖鎖抗原の代表的な糖鎖構造は、以下の(A)~(D)に分類される。
Siaα2,6Galβ1,4GlcNAc、Siaα2,3Galβ1,4GlcNAc、Galβ1,4GlcNAc、Galβ1,3GlcNAc、Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc、Galβ1,4 (Fucα1,3)GlcNAc、Siaα2,3Galβ1,3 (Fucα1,4)GlcNAc、Siaα2,3Galβ1,3GlcNAc、Galβ1,3 (Fucα1,4)GlcNAc、Siaα2,3Galβ1,3 (Siaα2,6)GlcNAc、Siaα2,3Galβ1,3 (Fucα1,4)(Siaα2,6)GlcNAc、Fucα1,2Galβ1,3(Fucα1,4)GlcNAc、Fucα1,2Galβ1,4GlcNAc、Fucα1,2Galβ1,4(Fucα1,3)GlcNAc、Fucα1,6GlcNAc、GlcNAcβ1,4 (Fucα1,6)GlcNAc、Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc、Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc、Manα1,6 (GlcNAcβ1,4) (Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc、Manα1,6 (GlcNAcβ1,4) (Manα1,3)Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc、GlcNAcβ1,4GlcNAc、Manβ1,4GlcNAcβ1,4 GlcNAc、GlcNAcβ1,2Man、GlcNAcβ1,4Man、GlcNAcβ1,6Man、Galβ1,3GlcNAcβ1,2Man、Galβ1,3GlcNAcβ1,4Man、Galβ1,3GlcNAcβ1,6Man、Galβ1,4GlcNAcβ1,2Man、Galβ1,4GlcNAcβ1,4Man、Galβ1,4GlcNAcβ1,6Man、Siaα2,3Galβ1,4GlcNAcβ1,2Man、Siaα2,3Galβ1,4GlcNAcβ1,4Man、Siaα2,3Galβ1,4GlcNAcβ1,6Man、Siaα2,3Galβ1,3GlcNAcβ1,2Man、Siaα2,3Galβ1,3GlcNAcβ1,4Man、Siaα2,3Galβ1,3GlcNAcβ1,6Man、Siaα2,6Galβ1,4GlcNAcβ1,2Man、Siaα2,6Galβ1,4GlcNAcβ1,4Man、Siaα2,6Galβ1,4GlcNAcβ1,6Man、GalNAcβ1,4GlcNAc、GalNAcβ1,4GlcNAcβ1,2Man、GalNAc(4SO4)β1,4GlcNAc、GalNAc(4SO4)β1,4GlcNAcβ1,2Man、GalNAc(4SO4)β1,4GlcNAcβ1,4Man、GalNAc(4SO4)β1,4GlcNAcβ1,6Man、GlcAβ1,3Galβ1,4GlcNAc、GlcA(3SO4)β1,3Galβ1,4GlcNAc、Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc(6SO4)、Siaα2,3Galβ1,4 GlcNAc(6SO4)、Galβ1,4 GlcNAc(6SO4)、Galβ1,4 (Fucα1,3) GlcNAc(6SO4)、Siaα2,3Gal (6SO4)β1,4 (Fucα1,3)GlcNAc、Siaα2,3Gal (6SO4)β1,4GlcNAc、Gal(6SO4)β1,4 GlcNAc、Gal(6SO4)β1,4 GlcNAc(6SO4)、Galβ1,4GlcNAcβ1,3Gal、GlcNAcβ1,3Galβ1,4GlcNAc、Galβ1,4GlcNAc(6SO4)β1,3Gal、GlcNAc(6SO4)β1,3Galβ1,4GlcNAc、Gal(6SO4)β1,4GlcNAc(6SO4)β1,3Gal、GlcNAc(6SO4)β1,3Gal(6SO4)β1,4GlcNAc、Siaα2,3Galβ1,4GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc、Fucα1,2Galβ1,3GlcNAc、Galα1,3(Fucα1,2)Galβ1,3GlcNAc、Galα1,3(Fucα1,2)Galβ1,4GlcNAc、GalNAcα1,3(Fucα1,2)Galβ1,3GlcNAc、GalNAcα1,3(Fucα1,2)Galβ1,4GlcNAc、Fucα1,2Galβ1,4(Fucα1,3)GlcNAcβ1,3Gal、Gal(3SO4)β1,4GlcNAc、Gal(3SO4)β1,4(Fucα1,3)GlcNAc、Gal(3SO4)β1,3GlcNAc、Gal(3SO4)β1,3(Fucα1,4)GlcNAc、Gal(3SO4)β1,4GlcNAc(6SO4)、Gal(3SO4)β1,4(Fucα1,3)GlcNAc(6SO4)、Siaα2,8Siaα2,8Sia、Siaα2,6GalNAcβ1,4GlcNAc、Siaα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc、Galβ1,4GlcNAcβ1,3(Galβ1,4GlcNAcβ1,6)Gal、Siaα2,3Galβ1,4GlcNAcβ1,3(Siaα2,3Galβ1,4GlcNAcβ1,6)Gal、などが挙げられる。
Siaα2,3Galβ1,3GalNAc、Galβ1,3GalNAc、Siaα2,3Galβ1,3(GlcNAcβ1,6)GalNAc、GalNAcα1,3GalNAc、GlcNAcβ1,3(Siaα2,6)GalNAc、GalNAcα1,6GalNAc、GlcNAcβ1,6GalNAc、Fucα1,2GalNAc、Galβ1,3(GlcNAcβ1,6)GalNAc、Siaα2,6GalNAc、Siaα2,6(GlcNAcβ1,6)GalNAc、Siaα2,3Galβ1,3(Siaα2,6)GalNAc、Galβ1,4GalNAc、Siaα2,3Galβ1,4GalNAc、GlcNAcα1,4Gal、GlcNAcα1,4Galβ1,4GlcNAc、GlcNAcα1,4Galβ1,4GalNAc、GlcNAcα1,4Galβ1,4(GlcNAcα1,4Galβ1,4GlcNAcβ1,6)GalNAc、Siaα2,3Galβ1,4(Fucα1,3)GlcNAc(6SO4)β1,6GalNAc、Gal(3SO4)β1,4GlcNAcβ1,6(Siaα2,3Galβ1,3)GalNAc、Siaα2,3Galβ1,4(Fucα1,3)GlcNAc(6SO4)β1,3Galβ1,3GalNAc、Siaα2,3Galβ1,4(Fucα1,3)GlcNAc(6SO4)β1,3Galβ1,3(Siaα2,3Galβ1,4(Fucα1,3)GlcNAc(6SO4)β1,6)GalNAc、Siaα2,3Gal(6SO4) β1,4GlcNAcβ1,3Galβ1,3GalNAc、Gal(3SO4)β1,3GalNAc(6SO4)、Siaα2,3Galβ1,3GalNAc(6SO4)、Gal(3SO4)β1,3(Siaα2,6)GalNAc、Gal(3SO4)β1,3GalNAc、などが挙げられる。
Galβ1,4Xyl、Galβ1,3Galβ1,4Xyl、GlcAβ1,3Galβ1,3Galβ1,4Xyl、GalNAcβ1,4IdoAα1,3Galβ1,3Galβ1,4Xyl、GalNAcβ1,4GlcAβ1,3Galβ1,3Galβ1,4Xyl、GlcNAcα1,4GlcAβ1,3Galβ1,3Galβ1,4Xyl、GlcAβ1,3GalNAcβ1,4GlcA、GalNAcβ1,4GlcAβ1,3GalNAc、GlcAβ1,3GalNAc(4SO4)β1,4GlcA、GalNAc(4SO4)β1,4 GlcAβ1,3GalNAc、GlcAβ1,3GalNAc(6SO4)β1,4GlcA、GalNAc(6SO4)β1,4 GlcAβ1,3GalNAc、GlcA(2SO4)β1,3(6SO4)GalNAcβ1,4GlcA、GalNAc(6SO4)β1,4GlcA(2SO4)β1,3GalNAc、GlcAβ1,3GalNAc(4,6SO4)β1,4GlcA、GalNAc(4,6SO4)β1,4GlcAβ1,3GalNAc、GalNAc(4,6SO4)β1,4GlcA(2SO4)β1,3GalNAc(6SO4)、IdoAα1,3GalNAcβ1,4IdoA、GalNAcβ1,4IdoAα1,3GalNAc、IdoAα1,3GalNAc(4SO4)β1,4IdoA、GalNAc(4SO4)β1,4IdoAα1,3GalNAc、GlcAβ1,4GlcNAcα1,4GlcA、GlcNAcα1,4GlcAβ1,4GlcNAc、GlcA(2SO4)β1,4GlcNAcα1,4GlcA、GlcNAcα1,4GlcA(2SO4)β1,4GlcNAc、IdoA(2SO4)α1,4GlcNAcα1,4GlcA、GlcNAcα1,4IdoA(2SO4)α1,4GlcNAc、GlcA(2SO4)β1,4GlcNSO3α1,4GlcA、GlcNSO3α1,4GlcA(2SO4)β1,4GlcNAc、IdoA(2SO4)α1,4GlcNSO3Hα1,4GlcA、GlcNSO3α1,4IdoA(2SO4)α1,4GlcNAc、GlcAβ1,4GlcNα1,4GlcA、GlcAβ1,4GlcNSO3α1,4GlcA、GlcNSO3α1,4GlcAβ1,4GlcNAc、IdoAα1,4GlcNSO3α1,4GlcA、GlcNSO3α1,4IdoAα1,4GlcNAc、GlcAβ1,4GlcNSO3(6SO4)α1,4GlcA、GlcNSO3(6SO4)α1,4GlcAβ1,4GlcNAc、IdoAα1,4GlcNSO3(6SO4)α1,4GlcA、GlcNSO3(6SO4)α1,4IdoAα1,4GlcNAc、IdoA(2SO4)α1,4GlcNSO3(6SO4)α1,4GlcA、GlcNSO3(6SO4)α1,4IdoA(2SO4)α1,4GlcNAc、IdoAα1,4 GlcNSO3α1,4IdoA、GlcAβ1,3GlcNAcβ1,4GlcA、GlcNAcβ1,4GlcAβ1,3GlcNAc、IdoAα1,4GlcNSO3、IdoAα1,4GlcNSO3(6SO4)、IdoA(2SO4)α1,4GlcNSO3、IdoA(2SO4)α1,4GlcNSO3(6SO4)、IdoA(2SO4)α1,4GlcNSO3(3SO4)、GlcAβ1,4GlcNSO3、GlcAβ1,4GlcNSO3(6SO4)、GlcA(2SO4)β1,4GlcNAc、GlcA(2SO4)β1,4GlcNSO3(3SO4)、GlcAβ1,4GlcNSO3(3,6SO4)、GlcAβ1,4GlcNSO3(3SO4)、
Galα1,3Galβ1,4Glc、GalNAcβ1,3Galα1,3Galβ1,4Glc、Galβ1,3GalNAcβ1,3Galα1,3Galβ1,4Glc、Galα1,4Galβ1,4Glc、GalNAcβ1,3Galα1,4Galβ1,4Glc、GalNAcα1,3GalNAcβ1,3Galα1,4Galβ1,4Glc、Galβ1,3GalNAcβ1,3Galα1,4Galβ1,4Glc、Galα1,4Galβ1,4GlcNAcβ1,3Galβ1,4Glc、GlcNAcβ1,3Galβ1,4Glc、Galβ1,4GlcNAcβ1,3Galβ1,4Glc、Galβ1,4Glc、Siaα2,3Galβ1,4Glc、Siaα2,8Siaα2,3Galβ1,4Glc、Siaα2,8Siaα2,8Siaα2,3 Galβ1,4Glc、GalNAcβ1,4Galβ1,4Glc、GalNAcβ1,4 (Siaα2,3) Galβ1,4Glc、Galβ1,3GalNAcβ1,4Galβ1,4Glc、Galβ1,3GalNAcβ1,4 (Siaα2,3) Galβ1,4Glc、Siaα2,3Galβ1,3GalNAcβ1,4Galβ1,4Glc、Siaα2,3Galβ1,3GalNAcβ1,4 (Siaα2,3) Galβ1,4Glc、Siaα2,3Galβ1,3(Siaα2,6)GalNAcβ1,4Galβ1,4Glc、Siaα2,3Galβ1,3(Siaα2,6)GalNAcβ1,4 (Siaα2,3) Galβ1,4Glc、Siaα2,3Galβ1,3GalNAcβ1,4 (Siaα2,8Siaα2,3) Galβ1,4Glc、Siaα2,3Galβ1,3(Siaα2,6)GalNAcβ1,4 (Siaα2,8Siaα2,3) Galβ1,4Glc、Siaα2,8Siaα2,3Galβ1,3GalNAcβ1,4 (Siaα2,8Siaα2,3) Galβ1,4Glc、Siaα2,3Galβ1,3GalNAcβ1,3Galα1,4Galβ1,4Glc、Siaα2,3Galβ1,3(Siaα2,6)GalNAcβ1,3Galα1,4Galβ1,4Glc、Siaα2,3Galβ1,3GlcNAcβ1,3Galβ1,4Glc、Siaα2,3Galβ1,3(Siaα2,6)GlcNAcβ1,3Galβ1,4Glc、Galβ1,3GlcNAcβ1,3Galβ1,4Glc、Siaα2,3Galβ1,4GlcNAcβ1,3Galβ1,4Glc、Siaα2,8Siaα2,3Galβ1,4GlcNAcβ1,3Galβ1,4Glc、Siaα2,3Galβ1,3(Fucα1,4)GlcNAcβ1,3Galβ1,4Glc、Gal(3SO4)β1,4Glc、GalNAcβ1,4Gal(3SO4)β1,4Glc、GalNAc(3SO4)β1,4Galβ1,4Glc、GalNAc(3SO4)β1,4Gal(3SO4)β1,4Glc、Gal(3SO4)β1,3GalNAcβ1,4Gal(3SO4)β1,4Glc、Gal(3SO4)β1,3GalNAcβ1,4(Siaα2,3)Galβ1,4Glc、GalNAc(3SO4)β1,3Galα1,4Galβ1,4Glc、Gal(3SO4)β1,3GalNAcβ1,3Galα1,4Galβ1,4Glc、GalNAc(3SO4)β1,3Galα1,3Galβ1,4Glc、Gal(3SO4)β1,3GalNAcβ1,3Galα1,3Galβ1,4Glc、Siaα2,3Galβ1,4 (Fucα1,3) GlcNAcβ1,3 Galβ1,4 (Fucα1,3)GlcNAcβ1,3Galβ1,4Glc、Siaα2,3 Galβ1,4 (Fucα1,3) GlcNAcβ1,3 Galβ1,4 GlcNAcβ1,3 Galβ1,4Glc、Fucα1,2 Galβ1,3 GalNAcβ1,3Galα1,4Galβ1,4Glc、Siaα2,3 Gal、などが挙げられる。
本発明の免疫誘導方法は、N-結合型糖鎖、O-結合型糖鎖、プロテオグリカン型糖鎖及び糖脂質型糖鎖に含まれるいずれのオリゴ糖鎖抗原に対しても適用でき、免疫原増強作用を示すが、本発明においては、主として、悪性の高い癌などの重篤な疾患の診断マーカーとして注目されているにもかかわらず、免疫原性が低く、有効な抗体が提供できていない「N-結合型糖鎖に含まれるオリゴ糖鎖抗原」を対象としている。なお、本発明において「N-結合型糖鎖に含まれるオリゴ糖鎖抗原」というときには、哺乳動物由来の糖タンパク質のN-結合型糖鎖またはその部分構造からなるオリゴ糖鎖抗原である。
そのような「N-結合型糖鎖に含まれるオリゴ糖鎖抗原」としては、大きく分けて以下の3種類、シアリル化糖鎖(a)、アシアロ糖鎖(b)、フコシル化糖鎖(c)に分類される(Essentials of Glycobiology, Cold Spring Harbor Laboratory Press, 1999)。
(a)N-結合型糖鎖のうち、シアリル化糖鎖に分類されるもの
Siaα2,6Galβ1,4GlcNAc、Siaα2,3Galβ1,4GlcNAc、Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc、Siaα2,3Galβ1,3 (Fucα1,4)GlcNAc、Siaα2,3Galβ1,3GlcNAc、Siaα2,3Galβ1,3 (Siaα2,6)GlcNAc、Siaα2,3Galβ1,3 (Fucα1,4)(Siaα2,6)GlcNAc、Siaα2,3Galβ1,4GlcNAcβ1,2Man、Siaα2,3Galβ1,4GlcNAcβ1,4Man、Siaα2,3Galβ1,4GlcNAcβ1,6Man、Siaα2,3Galβ1,3GlcNAcβ1,2Man、Siaα2,3Galβ1,3GlcNAcβ1,4Man、Siaα2,3Galβ1,3GlcNAcβ1,6Man、Siaα2,6Galβ1,4GlcNAcβ1,2Man、Siaα2,6Galβ1,4GlcNAcβ1,4Man、Siaα2,6Galβ1,4GlcNAcβ1,6Man、Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc(6SO4)、Siaα2,3Galβ1,4 GlcNAc(6SO4)、Siaα2,3Gal (6SO4)β1,4GlcNAc、Siaα2,3Gal (6SO4)β1,4 (Fucα1,3)GlcNAc、Siaα2,3Galβ1,4GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc、Siaα2,8Siaα2,8Sia、Siaα2,6GalNAcβ1,4GlcNAc、Siaα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc、Siaα2,3Galβ1,4GlcNAcβ1,3(Siaα2,3Galβ1,4GlcNAcβ1,6)Gal、などが挙げられる。
これらのうち、特に典型的で有用なシアリル化糖鎖としては、「Siaα2,6Galβ1,4GlcNAc:CDw75」、「Siaα2,3Galβ1,4GlcNAc:3’-Sialyl-LacNAc」、「Siaα2,3Galβ1,4(Fucα1,3)GlcNAc:Sialyl-LewisX 」、「Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc:Sialyl-Lewisa」、「Siaα2,3Galβ1,3 (Fucα1,4) (Siaα2,6)GlcNAc:Disialyl-Lewisa」、「Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc(6SO4):6-sulfo-Sialyl-LewisX」、「Siaα2,3Gal (6SO4)β1,4 (Fucα1,3)GlcNAc:6’-sulfo-Sialyl-LewisX」、「Siaα2,3Galβ1,4GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc:VIM-2 antigen」、「Siaα2,6GalNAcβ1,4GlcNAc」、「Siaα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc:Sialyl-LewisX-i」、「Siaα2,3Galβ1,4GlcNAcβ1,3(Siaα2,3Galβ1,4GlcNAcβ1,6)Gal:Sialyl-I」、が挙げられる。
Galβ1,4GlcNAc、Galβ1,3GlcNAc、Galβ1,4 (Fucα1,3)GlcNAc、Galβ1,3 (Fucα1,4)GlcNAc、Fucα1,2Galβ1,3(Fucα1,4)GlcNAc、Fucα1,2Galβ1,4GlcNAc、Fucα1,2Galβ1,4(Fucα1,3)GlcNAc、Fucα1,6GlcNAc、GlcNAcβ1,4 (Fucα1,6)GlcNAc、Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc、Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc、Manα1,6 (GlcNAcβ1,4) (Manα1,3)Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc、Manα1,6 (GlcNAcβ1,4) (Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc、GlcNAcβ1,4GlcNAc、Manβ1,4GlcNAcβ1,4 GlcNAc、GlcNAcβ1,2Man、GlcNAcβ1,4Man、GlcNAcβ1,6Man、Galβ1,3GlcNAcβ1,2Man、Galβ1,3GlcNAcβ1,4Man、Galβ1,3GlcNAcβ1,6Man、Galβ1,4GlcNAcβ1,2Man、Galβ1,4GlcNAcβ1,4Man、Galβ1,4GlcNAcβ1,6Man、GalNAcβ1,4GlcNAc、GalNAcβ1,4GlcNAcβ1,2Man、GalNAc(4SO4)β1,4GlcNAc、GalNAc(4SO4)β1,4GlcNAcβ1,2Man、GalNAc(4SO4)β1,4GlcNAcβ1,4Man、GalNAc(4SO4)β1,4GlcNAcβ1,6Man、GlcAβ1,3Galβ1,4GlcNAc、GlcA(3SO4)β1,3Galβ1,4GlcNAc、Galβ1,4 GlcNAc(6SO4)、Galβ1,4 (Fucα1,3) GlcNAc(6SO4)、Gal(6SO4)β1,4 GlcNAc、Gal(6SO4)β1,4 GlcNAc(6SO4)、Galβ1,4GlcNAcβ1,3Gal、GlcNAcβ1,3Galβ1,4GlcNAc、Galβ1,4GlcNAc(6SO4)β1,3Gal、GlcNAc(6SO4)β1,3Galβ1,4GlcNAc、Gal(6SO4)β1,4GlcNAc(6SO4)β1,3Gal、GlcNAc(6SO4)β1,3Gal(6SO4)β1,4GlcNAc、Fucα1,2Galβ1,3GlcNAc、Galα1,3(Fucα1,2)Galβ1,3GlcNAc、Galα1,3(Fucα1,2)Galβ1,4GlcNAc、GalNAcα1,3(Fucα1,2)Galβ1,3GlcNAc、GalNAcα1,3(Fucα1,2)Galβ1,4GlcNAc、Fucα1,2Galβ1,4(Fucα1,3)GlcNAcβ1,3Gal、Gal(3SO4)β1,4GlcNAc、Gal(3SO4)β1,4(Fucα1,3)GlcNAc、Gal(3SO4)β1,3GlcNAc、Gal(3SO4)β1,3(Fucα1,4)GlcNAc、Gal(3SO4)β1,4GlcNAc(6SO4)、Gal(3SO4)β1,4(Fucα1,3)GlcNAc(6SO4)、Galβ1,4GlcNAcβ1,3(Galβ1,4GlcNAcβ1,6)Gal、などが挙げられる。
これらのうち、特に典型的で有用なアシアロ糖鎖(非シアリル化糖鎖)としては、「Galβ1,4GlcNAc:LacNAc」、「GlcNAcβ1,4Man」、「GlcNAcβ1,6Man」、「Galβ1,4GlcNAcβ1,6Man」、「GalNAcβ1,4GlcNAc」、「GalNAcβ1,4GlcNAcβ1,2Man」、「GalNAc(4SO4)β1,4GlcNAc」、「Galβ1,4 (Fucα1,3)GlcNAc:LewisX」、「Galβ1,3 (Fucα1,4)GlcNAc:Lewisa」、「Fucα1,2Galβ1,3(Fucα1,4)GlcNAc:Lewisb」、「Fucα1,2Galβ1,4(Fucα1,3)GlcNAc:Lewisy」、「Fucα1,6GlcNAc」、「GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Galβ1,4GlcNAcβ1,3(Galβ1,4GlcNAcβ1,6)Gal:I-antigen」が挙げられる。
Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc、Galβ1,4 (Fucα1,3)GlcNAc、Siaα2,3Galβ1,3 (Fucα1,4)GlcNAc、Galβ1,3 (Fucα1,4)GlcNAc、Siaα2,3Galβ1,3 (Fucα1,4)(Siaα2,6)GlcNAc、Fucα1,2Galβ1,3(Fucα1,4)GlcNAc、Fucα1,2Galβ1,4GlcNAc、Fucα1,2Galβ1,4(Fucα1,3)GlcNAc、Fucα1,6GlcNAc、GlcNAcβ1,4 (Fucα1,6)GlcNAc、Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc、Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc、Manα1,6 (GlcNAcβ1,4) (Manα1,3)Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc、Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc(6SO4)、Galβ1,4 (Fucα1,3) GlcNAc(6SO4)、Siaα2,3Gal (6SO4)β1,4 (Fucα1,3)GlcNAc、Siaα2,3Galβ1,4GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc、Fucα1,2Galβ1,3GlcNAc、Galα1,3(Fucα1,2)Galβ1,3GlcNAc、Galα1,3(Fucα1,2)Galβ1,4GlcNAc、GalNAcα1,3(Fucα1,2)Galβ1,3GlcNAc、GalNAcα1,3(Fucα1,2)Galβ1,4GlcNAc、Fucα1,2Galβ1,4(Fucα1,3)GlcNAcβ1,3Gal、Gal(3SO4)β1,4(Fucα1,3)GlcNAc、Gal(3SO4)β1,3(Fucα1,4)GlcNAc、Gal(3SO4)β1,4(Fucα1,3)GlcNAc(6SO4)、などが挙げられる。
これらのうち、特に典型的で有用なフコシル化糖鎖抗原としては、「Galβ1,4 (Fucα1,3)GlcNAc:LewisX」、「Galβ1,3 (Fucα1,4)GlcNAc:Lewisa」、「Fucα1,2Galβ1,3(Fucα1,4)GlcNAc:Lewisb」、「Fucα1,2Galβ1,4(Fucα1,3)GlcNAc:Lewisy」、「Fucα1,6GlcNAc」、「GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Manα1,6(Manα1,3)Manβ1,4GlcNAcβ1,4 (Fucα1,6)GlcNAc」、「Siaα2,3Galβ1,4(Fucα1,3)GlcNAc:Sialyl-LewisX 」、「Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc:Sialyl-Lewisa」、「Siaα2,3Galβ1,3 (Fucα1,4) (Siaα2,6)GlcNAc:Disialyl-Lewisa」、「Siaα2,3Galβ1,4 (Fucα1,3)GlcNAc(6SO4):6-sulfo-Sialyl-LewisX」、「Siaα2,3Gal (6SO4)β1,4 (Fucα1,3)GlcNAc:6’-sulfo-Sialyl-LewisX」、「Siaα2,3Galβ1,4GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc:VIM-2 antigen」、「Siaα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,3Galβ1,4 (Fucα1,3)GlcNAc:Sialyl-LewisX-i」、が挙げられる。
本発明のキャリア化合物を用いた免疫誘導方法は、これらの既知の診断、及び薬剤標的のためのバイオマーカーである各種複合糖鎖のみならず、新たに発見されるいかなる複合糖鎖に対しても、特異的かつ高い親和性で認識するモノクローナル抗体を取得できる技術である。
これら任意のオリゴ糖鎖は、由来の細胞膜画分から常法により精製してもよく、また、糖鎖構造がわかれば、既知の有機合成法または酵素合成法を適用して合成することも可能である。
以下、本発明の実施の態様では、典型的なN-結合型糖鎖に含まれる3種類のオリゴ糖鎖抗原群から、それぞれの代表的なオリゴ糖鎖抗原を複数種選択して、本発明の免疫誘導方法を適用したところ、いずれのオリゴ糖鎖抗原を対象とした場合も抗原特異的な免疫誘導を引き起こすことが確認できた。具体的には、代表的なシアリル化糖鎖である、「6’-Sialyl-LacNAc(Siaα2,6Galβ1,4GlcNAc):CDw75」、「3’-Sialyl-LacNAc(Siaα2,3Galβ1,4GlcNAc)」、「Sialyl-LewisX(Siaα2,3Galβ1,4(Fucα1,3)GlcNAc)」、代表的なアシアロ糖鎖である、「LacNAc(Galβ1,4GlcNAc)」「LewisX(Galβ1,4(Fucα1,3)GlcNAc)」、並びに代表的なフコシル化糖鎖である「Sialyl-LewisX(Siaα2,3Galβ1,4(Fucα1,3)GlcNAc)」、及び「LewisX(Galβ1,4(Fucα1,3)GlcNAc)」の糖鎖構造を、本発明の対象のオリゴ糖鎖抗原(R)とした。このことから、本発明の免疫誘導法は、「N-結合型糖鎖に含まれるオリゴ糖鎖抗原」全般に亘って適用可能であることがいえる。さらに「N-結合型糖鎖に含まれるオリゴ糖鎖抗原」でも免疫増強ができたことからみて、より免疫誘導されやすいとされる他のオリゴ糖鎖抗原も含めた、任意のオリゴ糖鎖抗原に適用できる技術であることもわかる。
典型的なオリゴ糖鎖抗原「CDw75」の糖鎖構造は、
「6’-Sialyl-LacNAc(Siaα2,6Galβ1,4GlcNAc)」(CDw75)の下記一般式(3)で表される。
CDw75(Siaα2,6Galβ1,4GlcNAc)は、人の正常な胃・大腸組織ではほとんど発現していない糖鎖抗原であるのに対して、これらの組織が癌化した場合、強い発現が認められるようになることから、がんの診断マーカーとしての応用が検討されている(非特許文献14)。
一方で、哺乳動物の血清中に存在する糖タンパク質では、CDw75が主要なN-結合型糖鎖構造として見られる。マウス体内でも同様に存在していることから、微生物由来糖鎖と比較してきわめて免疫誘導されにくく、とりわけ、「糖タンパク質のN-結合型糖鎖に含まれる糖鎖抗原」を認識する抗体の産生は難しいと考えられている。
典型的なオリゴ糖鎖抗原の「3’-Sialyl-LacNAc(Siaα2,3Galβ1,4GlcNAc)」の糖鎖構造は、下記一般式(4)で表される。
オリゴ糖鎖抗原の「LacNAc(Galβ1,4GlcNAc)」の糖鎖構造は、下記一般式(5)で表される。
オリゴ糖鎖抗原「「Sialyl-LewisX(Siaα2,3Galβ1,4(Fucα1,3)GlcNAc)」の糖鎖構造は、下記一般式(6)で表される。
オリゴ糖鎖抗原「LewisX(Galβ1,4(Fucα1,3)GlcNAc)」の糖鎖構造は、下記一般式(7)で表される。
本発明のキャリア化合物は、NKT細胞の活性化作用が知られているCD1分子との結合部位となるα-ガラクトシルセラミドの脂質構造に類似した下記一般式(1)の新規な脂質分子である。A-ガラクトシルセラミドなど天然のスフィンゴ糖脂質の脂質構造とは、糖との結合構造「-CH(NH-CO)-」のカルボニル基側に結合している片方の鎖が置換されていない飽和アルキル基である点は共通しているが、本発明のキャリア化合物ではアミノ基側のアルキル基が飽和アルキル基であるのに対して、天然のスフィンゴ糖脂質の場合は、アミノ基側に結合しているアルキル基に必ず-OH基、=O基など酸素を含む基で修飾されている点で明確に区別できる。さらに2糖以上のオリゴ糖鎖を含む哺乳動物由来のスフィンゴ糖脂質の場合は、アミノ基側に結合しているアルキル基に-OH基と共に不飽和結合も必ず含むという相違点もある。したがって、本発明のキャリア化合物は、天然脂質と類似した「人工脂質又はその塩」であるということもできる。
X-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
・・・一般式(1)
ここで、式中、Xは-H、-OH、-SH、-NH2、又は糖鎖抗原の還元末端水酸基と反応し得る官能基、例えばハロゲン(-Br、-I、-Cl、-F)、ヒドラジド基などを表す。
Yはスペーサー配列である。
本発明におけるスペーサー配列Yは、「-(CH2)m-」で表される。mは1~30、好ましくは1~10、より好ましくは1~3の整数である。メチレン鎖を長くしたほうがリガンド(リンパ球上の受容体タンパク質や抗体)と結合しやすく、免疫原性を高める効果が期待できるが、長すぎると極性溶媒への可溶性が低くなり扱いにくくなるため、扱いやすい範囲として好ましい数値範囲を設定した。Xの官能基を介してスペーサー配列を導入することもできる。
n1は2~40の整数で、好ましい態様として14~39、16~31、17~28及び20~40が挙げられ、より好ましい態様は、20~28、21~27、最も好ましくは22~24である。n1の数が大きいほど、すなわち脂肪酸部位のアルキル鎖長が伸長するほど免疫誘導能が増強する傾向があるが、2~10であっても免疫増強効果は得られる。また、長鎖脂肪酸のうちでも炭素数22以上(n1=20以上)の脂肪酸は超長鎖脂肪酸と呼ばれ、脂肪酸としての性質もそれより短い通常の長鎖脂肪酸とは性質が異なることが知られており、免疫細胞においても細胞膜シグナル伝達を活性化しやすい差異がある(Kihara A., J. Biochem., 152, 387-395, 2012)。本発明のキャリア化合物の脂肪酸部位においても、炭素数22以上(n1=20以上)の超長鎖脂肪酸の場合に、免疫誘導能が格段に高まることが確認されている。本発明では好ましい数値範囲で上限値を設けているが、免疫誘導能の観点ではなく、合成のしやすさ、扱いにくさなどの観点で一応設けられている数値である。すなわち、n1が20以上であれば、極めて高い免疫原性が確保できるため、合成しやすく、扱いやすい数値範囲も含めた好ましい数値範囲は上述の20~28、21~27、最も好ましくは22~24である。
n2は1~27の整数で、好ましい態様として2~15、2~13、3~13が挙げられ、より好ましい態様は5~13、6~12であり、最も好ましくは7~11である。n2が5未満の場合は免疫誘導能が低下する傾向にあり、n2が9を超えた場合は水性溶媒中への溶解度が低下し、酵素法による合成効率が下がる欠点があるものの、免疫誘導能には問題はない。
また、一般式(1)の化合物は、薬学上許容される非毒性塩であることができる。一般式(1)の化合物の塩としては、酸付加塩、例えば、無機酸(例えば塩酸、硫酸、硝酸、リン酸)との塩、あるいは有機酸(例えば、酢酸、プロピオン酸、マレイン酸、オレイン酸、パルミチン酸、クエン酸、コハク酸、酒石酸、フマル酸、グルタミン酸、パントテン酸、ラウリルスルホン酸、メタンスルホン酸およびフタル酸)との塩が挙げられる。溶媒和物(例えば、水和物)であってもよい。
(3-1)本発明の免疫誘導剤
本発明の標的オリゴ糖鎖抗原とキャリア化合物とのコンジュゲートは、下記の一般式(2)で表すことができる。
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
・・・一般式(2)
ここで、式中、Rは糖鎖抗原となる任意のオリゴ糖であって、シアル酸(N-アセチルノイラミン酸、N-グリコリルノイラミン酸、αケトデオキシノノン酸)、ガラクトース、グルコース、N-アセチルグルコサミン、N-アセチルガラクトサミン、グルコサミン、ガラクトサミン、マンノース、フコース、キシロース、グルクロン酸、イズロン酸、イノシトール、エリトロース、エリトロン酸、エリトルロン酸、エリトラル酸、エリトリトール、トレオース、トレオン酸、トレウロン酸、トレアル酸、トレイトール、リボース、リボン酸、リブロン酸、リバル酸、リビトール、アラビノース、アラビノン酸、アラビヌロン酸、アラビナル酸、アラビニトール、キシロース、キシロン酸、キシルロン酸、キシラル酸、キシリトール、リキソース、リキソン酸、リキスロン酸、アロース、アロン酸、アルロン酸、アラル酸、アリトール、アルトロース、アルトロン酸、アルトルロン酸、アルトラル酸、アルトリトール、グルコン酸、グルカル酸、グルシトール、マンノン酸、マンヌロン酸、マンナル酸、マンニトール、グロース、グロン酸、グルロン酸、イドース、イドン酸、イダル酸、イジトール、ガラクトン酸、ガラクツロン酸、ガラクタル酸、ガラクチトール、タロース、タロン酸、タルロン酸、アルトラル酸、アルトリトール、ジヒドロキシアセトン、エリトルロース、リブロース、プシコース、フルクトース、ソルボース、タガトース、セドヘプツロース、コリオース、デオキシリボース、ラムノース、フクロース、アロメチロース、キノボース、アンチアロース、タロメチロース、ジギタロース、ジギトキソース、シマロース、チベロース、アベコース、パラトース、コリトース、アスカリロース、及びこれらが硫酸化、アセチル化、リン酸化、もしくはメチル化された糖、好ましくは、シアル酸、ガラクトース、グルコース、N-アセチルグルコサミン、N-アセチルガラクトサミン、グルコサミン、ガラクトサミン、マンノース、フコース、キシロース、グルクロン酸、イズロン酸、イノシトール、及びこれらが硫酸化、アセチル化、もしくはメチル化された糖から選択される1又は複数種の単糖を構成要素とする直鎖状又は分枝状の分子であり、Rを構成する単糖の数は2~30好ましくは2~16、より好ましくは2~6個の糖からなる。
チオメチルと結合させるリンカーとしては、市販リンカーのBMPH(N-β-Maleimidopropionic acid hydrazide-TFA)、KMUH(N-κ-Maleimidoundecanoic acid hydrazide-TFA)、EMCH(N-[ε-Maleimidocaproic acid]hydrazide- TFA)、MPBH (4-[4-N-Maleimidophenyl]butyric acid hydrazide-HCl)、及びPDPH (3-[2-Pyridyldithio]propionyl hydrazide)(Thermo Fisher scientific社製)から選択されることができる。このような市販リンカーを用いる場合には極性基を多く含むため、長いメチレン鎖を有していても扱いやすい。
後述のように、本発明において、当該キャリア化合物を標的オリゴ糖鎖抗原Rとコンジュゲートさせて、標的オリゴ糖鎖抗原Rに対する免疫誘導剤として働くコンジュゲート(人工糖脂質)を製造させる方法として、周知の還元的アミノ化法を用いる場合は、開裂されて糖アルコールを含む。その場合の糖アルコールとしては、N-アセチルグルコサミニトール、N-アセチルガラクトサミニトール、マンニトール、ガラクチトールなどの糖アルコール、また、アミノメチル化される糖アルコールとしては、N-アセチルグルコサミニトール、N-アセチルガラクトサミニトール、マンニトール、ガラクチトールから選択されることが好ましい。
なお、糖又は糖を含む化合物は、Zには含まれない。特に2糖以上を含む場合、標的オリゴ糖鎖抗原の糖鎖を伸ばすことになり、新たな抗原性を付与するなど標的オリゴ糖鎖抗原自体の抗原性を損なうことになるため、好ましくないからである。
Yはスペーサー配列であり、一般式(1)の場合と同様に「-(CH2)m-(mは1~30、好ましくは1~10、より好ましくは1~3の整数)」と定義される。メチレン鎖を長くしたほうがリガンド(リンパ球上の受容体タンパク質や抗体)と結合しやすく、免疫原性を高める効果が期待できるが、長すぎると極性溶媒への可溶性が低くなり扱いにくくなるため、扱いやすい範囲として好ましい数値範囲を設定した。Xの官能基を介してスペーサー配列を導入することもできる。この場合、例えば市販リンカーのBMPH(N-β-Maleimidopropionic acid hydrazide-TFA)、KMUH(N-κ-Maleimidoundecanoic acid hydrazide-TFA)、EMCH(N-[ε-Maleimidocaproic acid]hydrazide- TFA)、MPBH (4-[4-N-Maleimidophenyl]butyric acid hydrazide-HCl)、PDPH (3-[2-Pyridyldithio]propionyl hydrazide)(Thermo Fisher scientific社製)を用いることができる。これらは極性基を多く含むため、長いメチレン鎖を有していても扱いやすい。
具体的には、脂肪酸部位のアルキル鎖長に対応するn1は2~40の整数で、好ましい態様として14~39、16~31、17~28及び20~40が挙げられ、より好ましい態様は、20~28、21~27、最も好ましくは22~24である。ここで、n1の数が伸長するほど免疫誘導能が増強する傾向があり、n1の数が20以上の場合(炭素数22以上の超長鎖脂肪酸の場合)に、免疫誘導能が格段に高まる。一方、好ましい数値範囲の上限値は、合成のしやすさ、扱いにくさなどの観点で一応設けられている数値である。すなわち、n1が20~40の整数の場合が高い免疫原性が確保でき、かつ合成しやすく、扱いやすい好ましい数値範囲であり、さらに20~28、21~27が好ましく、最も好ましくは22~24である。
アミノ基側のアルキル鎖長に対応するn2は1~27の整数で、好ましい態様として2~15、2~13、3~13が挙げられ、より好ましい態様は5~13、6~12であり、最も好ましくは7~11である。n2が5未満の場合は免疫誘導能が低下する傾向にあり、n2が9を超えた場合は水性溶媒中への溶解度が低下し、酵素法による合成効率が下がる欠点があるものの、免疫誘導能には問題はない。
ここで、本発明の免疫誘導剤として用いられる上記一般式(2)からなる人工糖脂質又はその塩に含まれる化合物のうち、RがSiaα2,3GalβGlcであり、Z=O、Y=-(CH2)-であって、かつn1=16、n2=9の化合物は上皮細胞成長因子レセプター(EGFR)の阻害剤として抗癌作用が期待されているGM3類似体として、既知物質(特許文献5)である。しかし、本発明の免疫誘導剤のうちの好ましい態様であるn1=20~40の場合の人工糖脂質については、その免疫誘導能の点で、超長鎖脂肪酸とは呼べないn1=16の場合のGM3類似体とは明らかに区別ができる。
すなわち、本発明の免疫誘導剤として用いられる標的オリゴ糖鎖抗原Rとキャリア化合物がコンジュゲートした人工糖脂質又はその塩のうち、下記の一般式(2)に示される新規な人工糖脂質又はその塩自体に係る発明にも関する。
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
Zは、単結合又はO、SもしくはNHが好ましいが、チオメチルと結合させたリンカー、又はアミノメチル化された糖アルコールであってもよい。
チオメチルと結合させるリンカーとしては、市販リンカーのBMPH(N-β-Maleimidopropionic acid hydrazide-TFA)、KMUH(N-κ-Maleimidoundecanoic acid hydrazide-TFA)、EMCH(N-[ε-Maleimidocaproic acid]hydrazide- TFA)、MPBH (4-[4-N-Maleimidophenyl]butyric acid hydrazide-HCl)、及びPDPH (3-[2-Pyridyldithio]propionyl hydrazide)(Thermo Fisher scientific社製)から選択されることが好ましく、アミノメチル化される糖アルコールとしては、N-アセチルグルコサミニトール、N-アセチルガラクトサミニトール、マンニトール、ガラクチトールから選択されることが好ましい。
そして、免疫に際しては、アジュバントなしで、もしくはアジュバントと共にコレステロール、リン脂質、ジアシルグリセロール、モノアシルグリセロール、グリセロール、脂肪酸、グリココール酸、タウロコール酸、糖脂質などの脂質に溶解して作製したリポソームを静脈注射するリポソーム法(Brodinらの方法;Eur J Immunol., 16, 951-956, 1986)などが好適に用いられる。
その他、本発明のコンジュゲートの免疫誘導能は強力であるため、in vitro免疫に供することも可能である。この際、リンパ球培養用の細胞培養液に一般式(2)のコンジュゲートを単独で溶解し、免疫細胞に作用させても良いが、上述のBrodinらの方法によりリポソームとする、あるいはBSAなどの脂質結合タンパク質に吸着させる等の処理後により、細胞培養液に高濃度に溶解することができる。よって、免疫細胞に効果的に作用させることが可能となる。
したがって、本発明において免疫誘導剤というとき、一般式(2)の化合物自体、薬学的に許容されるその非毒性塩、または一般式(2)の化合物を有効成分とし、さらにLipid-Aなどのアジュバントを含む免疫誘導能を有する組成物、さらに、脂質に溶解したリポソーム組成物も包含される。
(3-2-1)オリゴ糖鎖抗原にキャリア化合物(人工脂質)を直接もしくはスペーサー配列を介して結合させる方法
標的となる任意のオリゴ糖鎖である天然物から精製されたオリゴ糖鎖、又は有機合成にて得られるオリゴ糖鎖に対して、本発明の一般式(1)で表されるキャリア化合物を直接結合させることができる。方法としては、例えばオリゴ糖鎖の還元末端以外の水酸基を保護して、還元末端水酸基に脱離基(例えば、アセチル基、トリクロロアセトイミデート基、水酸基、チオアルキル基、ハロゲンなど)を導入した糖供与体を作製し、これと一般式(1)のキャリア化合物をコンジュゲートする、あるいは作成した糖供与体と一般式(1)のキャリア物質の一部をコンジュゲートし、その後、酵素反応や化学反応等で糖鎖の伸長やキャリア部の構築をおこなうことで、オリゴ糖鎖の構造を維持したままコンジュゲートできる。
当該方法であれば、対象となるオリゴ糖鎖抗原の糖鎖構造が不明な場合でも、本発明のキャリア化合物と結合できる。
なお、その際のオリゴ糖鎖における還元末端以外の水酸基を保護する方法は以下の通りである。
(i)任意のオリゴ糖鎖と保護化試薬を反応させて、還元末端以外のヒドロキシル基以外を保護基で保護する保護化工程;
(ii)前記工程の生成物と脱離基導入試薬を反応させて、還元末端1-位のヒドロキシル基を脱離基に置換する置換工程;
(iii)前記工程の生成物と反応試薬を活性化剤の存在下で反応させて、還元末端を変換する工程;
(iv)前記工程の生成物と脱保護試薬を反応させて保護基を脱保護して、任意のオリゴ糖鎖の還元末端が変換された生成物を得る脱保護工程。
ここで保護化工程に使用される保護化試薬としては、無水酢酸/ピリジン、ベンジルクロリド/水素化ナトリウム等を挙げることができる。置換工程に使用される脱離基導入試薬としては、無水臭化水素酸、アルキルチオール(EtSH、PhSH等)/ルイス酸(TMSOTf、BF3等)、無水酢酸/ピリジンや、トリクロロアセトイミデート等を挙げることができる。変換工程に使用される活性化剤としては、脱離基がハロゲンの場合はAgOTf、チオアルキル基の場合はMeOTf若しくはBF3等のルイス酸を挙げることが出来る。また、同工程に使用される反応試薬としては、12-アジドドデシル-1-オール、3-アジドプロピル-1-オール、p-メトキシフェノール等を挙げることが出来る。さらに、脱保護工程に使用される脱保護試薬としては、アシル系保護基に対してはナトリウムメトキシド、DBU等の塩基類を、ベンジル保護基に対してはPd、Pt等の金属触媒存在下による接触還元反応や、ナトリウム-アンモニアによるバーチ還元反応等を挙げることが出来る。
<セラミド類似体の製造方法>
(i)2-アジドアルキルアルコールの合成法
アルキルジオール1位の水酸基(第一級アルコール)をベンジルクロリド等の保護化試薬で保護し、続けて側鎖水酸基(第三級アルコール)をトシルクロリドによりトシル基とした後、アジ化ナトリウムを作用させてアジド基に置換する。続けて脂肪酸を導入する場合は、側鎖アジド基の還元反応を行い、脂肪酸をアミド化反応により縮合する。
脂肪酸としては、例えばButyric acid、Valeric acid、Hexanoic acid、Heptanoic acid、Octanoic acid、Nonanoic acid、Decanoic acid、Undecanoic acid、Dodecanoic acid、Tridecanoic acid、Myristic acid、Pentadecanoic acid、Palmitic acid、Heptadecanoic acid、Stearic acid、Nonadecanoic acid、Arachidic acid、Heneicosanoic acid、Behenic acid、Tricosanoic acid、Lignoceric acid、Pentacosanoic acid、Hexacosanoic acid、Heptacosanoic acid、Octacosanoic acid、Nonacosanoic acid、Melissic acid、Henatriacontanoic acid、Lacceroic acid、Tritriacontanoic acid、Tetratriacontanoic acid、Pentatriacontanoic acid、Hexatriacontanoic acid、Heptariacontanoic acid、Octatriacontanoic acid、Nonatriacontanoic acid 、Tetracontanoic acid、Hentetracontanoic acid、Dotetracontanoic acidを用いることができる。
アルキルジオールとしては、例えば1,2-Pentanediol、1,2-Hexanediol、1,2-Heptanediol、1,2-Octanediol、1,2-Nonanediol、1,2-Decanediol、1,2-Undecanediol、1,2-Dodecandiol、1,2-Tridecanediol、1,2-Tetradecanediol、1,2-Pentadecanediol、1,2-Hexadecanediolを用いることができる。
また、Y(スペーサー配列)としてm=1以上のメチレン鎖を導入する場合、1,3-Nonanediol、1,4-Heptanediolなどを用いることができる。
1位の水酸基をトシルクロリドによりトシル基とした後、アジ化ナトリウムを作用させてアジド基に置換し、還元剤によりアミノ基へと変換する。
(iii)Xへのチオール基の導入
1位の水酸基を三臭化リンにてブロモ基に置換し、アルカリ存在下にて硫化水素と反応させることでチオール基を導入する。
(iv)Xへのヒドラジド基の導入
第一級アルコールに酸化剤(過マンガン酸カリウム等)を作用させてカルボン酸に変換し、続けて塩化チオニルを作用させてとカルボン酸塩化物とした後、ヒドラジンを作用さることでヒドラジドに変換する。
(v)Xへのハロゲン基の導入
第一級アルコールにハロゲン化剤(三臭化リン、ヨウ化水素、塩化チオニル、DAST等)を作用させて変換する。
(vi)Xにアミノ基を有する場合、オリゴ糖鎖は還元的アミノ化法にて導入できる。この場合、還元末端の糖が開裂するため、標的オリゴ糖鎖の構造を維持するためにスペーサーとなる糖を余分に含むオリゴ糖を用いるか、あるいはヒドロキシベンズアルデヒドを非特許文献8に記載の方法でオリゴ糖鎖に結合させた後に導入する。
(vii)Xにチオール基を有する場合、オリゴ糖鎖の導入は上記市販リンカーのうち、チオール基と反応する官能基を有するスペーサーを介して導入することができる。
(viii)ヒドラジド基の有する場合、オリゴ糖鎖とPBS等の溶媒中で反応させることで導入できる。
(viii)ハロゲン基を有する場合、オリゴ糖鎖の還元末端以外の水酸基を保護した後、溶媒中で反応させることで導入できる。
まず、オリゴ糖鎖構造のうちの還元末端側の単糖のみと2-アジドアルキルアルコールとのカップリング反応を行い、次いで、単糖の非還元末端側に糖鎖を酵素反応により伸長させて目的のオリゴ糖鎖の基本となる糖鎖構造を合成した後に、側鎖アミノ基に対して脂肪酸を縮合し、セラミドミミックな糖脂質誘導体を製造する。
具体的には、モデルとして糖鎖構造「Siaα2,6Galβ1,4GlcNAc (CDw75)」を含む本発明の免疫誘導剤の有効成分となるコンジュゲートの製造方法を以下に示す。
N-アセチル-グルコサミンから誘導される糖供与体(例えば、3,4,6-トリ-O-アセチル-2-デオキシ-2-(4,5-ジクロロフタルイミド)-D-グルコピラノシル ブロミド)と受容体となる2-アジドアルキルアルコールCH3(CH2)n2CH(N3)CH2OHをグリコシル化反応によりカップリングさせた後、ヒドロキシ基の脱保護反応、側鎖アジド基の還元反応などを行い、アミノ基を有したグルコサミン誘導体GlcNAcβ1-CH2CH(NH2)(CH2)n2CH3を合成した。
(4-1)免疫方法
本発明のモノクローナル抗体の製造法は当該分野で周知の常法が適用できる(例えばShepherd P. and Dean C., Monoclonal Antibodies, Oxford University Press, 2000など)。具体的には、標的オリゴ糖鎖に対して、本発明者らが開発したキャリア化合物(一般式(1))を結合したコンジュゲート(一般式(2))を用いて、非ヒト哺乳動物、例えばラット、マウス、ウサギなど、好ましくはマウスに常法通り免疫する。
免疫に際しては、アジュバント(Lipid-A)と共にコレステロール、リン脂質などの脂質に溶解して作製したリポソームを腹腔及び静脈注射するリポソーム法(Brodinらの方法;Eur. J. Immunol., 16, 951-956, 1986)などが好適に用いられる。他に酸で処理したSalmonella minnesota strain R595にコンジュゲート(一般式(2))を吸収させ、これを腹腔及び静脈注射する方法(Galanosらの方法;Eur. J. Biochem., 24, 116-122, 1971)でも良い。
融合糖鎖抗原の動物1匹当たりの投与量は、アジュバント利用下にて0.05~0.2 mgである。アジュバントとしては、酸で処理したSalmonella minnesota strain R595、完全フロイントアジュバンド等を用いることができるが、Lipid-Aを含むリポソーム法が好ましい。その際に、追加免疫を行うこともできる。免疫は、主として静脈内、皮下、腹腔内に注入することにより行われる。また、免疫の間隔は特に限定されず、数日から数週間間隔、好ましくは2~5週間間隔で、1~10回、好ましくは2~5回免疫を行う。そして、最終の免疫日から1~60日後、好ましくは1~14日後に抗体産生細胞を採集する。抗体産生細胞としては、脾臓細胞、リンパ節細胞、末梢血細胞等が挙げられるが、脾臓細胞又は局所リンパ節細胞が好ましい。
常法により、脾臓細胞をミエローマ細胞と融合し、IL-6の存在下で胸腺フィーダー細胞と一緒に培地中でインキュベートした後、IMDM培地のHATにより選択する。次いで、増殖するクローンの上清を標的オリゴ糖鎖を用いてスクリーニングする。その際に、免疫組織化学分析などを用いてもよいが、基板上に固定した標的オリゴ糖鎖に対して、酵素免疫測定法(ELISAなど)又はウエスタンブロットによる簡便なスクリーニング法が適用できる。ここで、標的オリゴ糖鎖抗体産生ハイブリドーマの選択に、ELISA法又はウエスタンブロット法が適用できることも本発明のメリットである。
本発明では、好ましくは培養上清中の抗体価はELISA法により評価し、抗体価を指標としてハイブリドーマを選別する。抗体価は、例えば、二次抗体として用いた抗マウスイムノグロブリン抗体の標識酵素であるペルオキシダーゼの活性として評価した。ペルオキシダーゼの発色基質にはTMBを用い、反応後に2規定の硫酸を加えた後の450nmの吸光度の強度を評価する。
まず、免疫原とした糖脂質抗原及び標的オリゴ糖鎖構造を含有する糖タンパク質を認識するモノクローナル抗体産生ハイブリドーマ細胞を選別し、次いで、当該標的糖鎖構造と近似する糖鎖構造に対しては反応性を有さないモノクローナル抗体産生ハイブリドーマを選別する。
(5-1)特異性試験
<ELISA法>
本発明の標的となるオリゴ糖鎖抗原を有するタンパク質(又は脂質)と共に、類似のオリゴ糖鎖を有するタンパク質(又は脂質)を基板(ELISAプレート)に蒸発乾固によって固定化し、ELISA法により、抗体価(450nmの吸光度の強度)として評価する。
市販の糖鎖アレイを用いてもよい。
<ウエスタンブロット解析>
試料をSDS-ポリアクリルアミドゲル電気泳動にて分離し、ブロッティング装置を用いて電気的にPVDF等のメンブレンに転写する。転写後のメンブレンを、1%スキムミルク-PBSTなどでブロッキングした後、PBSTにて適宜希釈した一次抗体をメンブレンに浸潤させ、反応させる。
室温にて1~3時間反応させた後、メンブレンをPBST等の洗浄バッファーにて振盪洗浄し、HRP標識二次抗体を反応させる。PBSTにて洗浄後、化学発光基質を反応させ、抗体反応をイメージアナライザーにて検出する。
<糖鎖アレイ法>
基盤上に糖鎖を固定化した「糖鎖アレイ」にPBST等で適宜希釈した抗体を滴下する。
カバーガラス等により被うことで基盤表面に抗体溶液を浸潤させ、室温にて2時間程度インキュベートする。PBST等にて穏やかに撹拌洗浄した後、蛍光標識二次抗体を反応させる。洗浄後スライドを乾燥させた後、マイクロアレイ用スキャナを用いてスライド上の蛍光を検出する。
<競合阻害アッセイ>
上記ELISA法にて、あらかじめ競合剤となるオリゴ糖鎖を抗体とインキュベートした後に、抗体価を評価する。加える競合剤の量を変化させることで、競合剤が抗原抗体反応を阻害する濃度を評価する。
<細胞蛍光染色法>
標的となるオリゴ糖鎖抗原を発現する細胞を抗体とインキュベートした後、抗体反応を蛍光標識二次抗体にて標識し、蛍光検出装置で検出する。
標的糖鎖への抗体の親和性は、ELISA法による算出法(Friguet B., et al., J. Immunol. Methods, 77, 305-319, 1985)にて決定した。抗体を1×10-7 Mの濃度にPBS等にて希釈調製し、25~1.56×10-7 Mの濃度になるように段階希釈した抗原と混和・インキュベーションすることで、抗原抗体複合体を形成させた。この混和液中のFreeの抗体量を、抗原固相化プレートを用いたELISAにより算出し、得られた値からScatchard プロットによってKd値(解離定数)を決定した(図6A)。また、Fetuin及びCDw75-C12Lを段階希釈して固相化したプレートを用いたELISA法にて、作製した抗体がどの程度の量の抗原まで検出できるかを測定した(図6B)。
各免疫原を初日に皮下に免疫し、2週間後に腹腔内に追加免疫した。追加免疫後、3日後及び7日後の血液を採取し、血清を調製した。得られた血清中の抗体価について、ELISA法により評価した。ELISA法には免疫原及び目的糖鎖を含有する糖タンパク質を固相化抗原として用い、それぞれ免疫原に対する抗体価及び目的糖鎖への抗体価として評価した(図1~4)。
産生されるイムノグロブリンのサブクラス(IgM及びIgG)をそれぞれ評価するため、二次抗体にIgM及びIgGに特異的な抗体を用いて評価した。抗体価は、二次抗体の標識酵素であるペルオキシダーゼの活性として評価した。ペルオキシダーゼの発色基質にはTMBを用い、反応後に2規定の硫酸を加えた後の450nmの吸光度の強度を評価した。図1~図3に、IgMサブクラスの抗体価を、図4にIgGサブクラスの抗体価を示す。
上記(5-3)にて、抗原に対するIgGクラス抗体の抗体価の上昇を、免疫原(本発明の免疫誘導剤)胸腺依存性活性の指標として評価した。
また、本発明の免疫誘導剤となる一般式(2)の化合物又はその塩は、アジュバントなしで、又はリン酸アルミニウム、塩化アルミニウム、アルミニウム塩、水酸化アルミニウム、硫酸アルミニウム、Montanide ISA51、Montanide ISA720VG、MF59、AS03などのアジュバントと共に、さらには、コレステロール、リン脂質、ジアシルグリセロール、モノアシルグリセロール、グリセロール、脂肪酸、グリココール酸、タウロコール酸、糖脂質などの脂質に溶解して作製したリポソームの状態で各種の悪性腫瘍や悪性疾患の罹患者体内での、標的糖鎖抗原特異的なIgG抗体産生能増強効果を引き起こすことのできる免疫増強用医薬組成物の有効成分として用いることができる。主として、ヒトを対象とするが、ヒトに限られることはなく、イヌ、サル、ネコ、ウサギなど愛玩動物、ウシ、ウマ、ブタ、などの家畜動物、マウス、ラットなどの実験動物などの哺乳類の他、ヒトに感染する恐れのある鳥インフルエンザ用ワクチンとして、鳥類にあらかじめ投与しておくこともできる。
例えば、「Siaα2,6Galβ1,4GlcNAc(CDw75)」抗原に対するコンジュゲート化合物の場合は悪性腫瘍治療用医薬組成物又はインフルエンザの予防もしくは治療用医薬組成物の有効成分として、「Siaα2,3Galβ1,4GlcNAc」抗原に対するコンジュゲート化合物の場合は鳥インフルエンザの予防もしくは治療用医薬組成物の有効成分として用いることができる。
これらの各種製剤は、薬学上許容される担体等を用いて常法により製造することができる。具体的には、賦形剤として、溶剤(例えば、水、生理食塩水)、増量剤および充てん剤(例えば、乳糖、テンプン、結晶セルロース、マンニトール、マルトース、リン酸水素カルシウム、軟質無水ケイ酸、炭酸カルシウム)などが、補助剤として、可溶化剤(例えば、エタノール、ポリソルベート剤)、結合剤(例えば、デンプン、ポリビニルピロリドン、ヒドロキシプロピルセルロース、エチルセルロース、カルボキシメチルセルロース、アラビアゴム)、崩壊剤(例えば、デンプン、カルボキシメチルセルロースカルシウム)、滑沢剤(例えば、ステアリン酸マグネシウム、タルク、硬化油)、安定剤(例えば、乳糖、マンニトール、マルトース、ポリソルベート類、マクロゴール類、ポリオキシエチレン硬化ヒマシ油)、等張化剤、湿潤化剤、潤滑剤、分散剤、緩衝剤、溶解補助剤などを用いることができる。必要に応じ添加剤として、抗酸化剤、保存剤、矯味矯臭剤、無痛化剤、安定化剤、着色剤、および甘味剤などが含まれる。
また、各種製剤には、必要に応じて、グリセリン、ジメチルアセトアミド、70%乳酸ナトリウム、界面活性剤、塩基性物質(例えば、エチレンジアミン、エタノールアミン、炭酸ナトリウム、アルギニン、メグルミン、トリスアミノメタン)を添加することもできる。
本発明において、一般式(2)の化合物又はその塩の投与経路は、静脈内投与、注射による局所投与、皮下投与、筋肉内投与、舌下投与、経皮吸収または直腸内投与により投与することができる。静脈内投与がもっとも好ましい。
また、本発明の免疫誘導剤をマウスなどの動物に免疫して、標的糖鎖抗原特異的な抗血清又はモノクローナル抗体を製造して、これら標的糖鎖抗原特異的抗血清又はモノクローナル抗体を、各種疾患用の予防又は治療用の医薬組成物の有効成分とすることができる。
本発明におけるその他の用語や概念は、当該分野において慣用的に使用される用語の意味に基づくものであり、本発明を実施するために使用する様々な技術は、特にその出典を明示した技術を除いては、公知の文献等に基づいて当業者であれば容易かつ確実に実施可能である。また、各種の分析などは、使用した分析機器又は試薬、キットの取り扱い説明書、カタログなどに記載の方法を準用して行った。
なお、本明細書中に引用した技術文献、特許公報及び特許出願明細書中の記載内容は、本発明の記載内容として参照されるものとする。
ELISAによる血清抗体価の評価に用いた化合物の糖鎖構造を下記(表1)に示す。
表中、a1、b1、c1は、それぞれ前記一般式(3)、(4)、(5)に示したオリゴ糖鎖抗原と、セラミド類似体(CerA)とのコンジュゲートであるスフィンゴ糖セラミド誘導体である。
CerAは、本発明のキャリア化合物の典型的な例であるセラミド類似体(HOCH2CH(NH-CO-(CH2)16-CH3)-(CH2)9-CH3)を示し、水酸基を介してそれぞれの糖鎖の還元末端と結合させてある。Cerはセラミド(天然体)であり、LacCer, GM3, GM1, GD1a, Globosideはそれぞれセラミドに糖鎖が結合した天然体の糖脂質である。RはFetuinの基幹構造(表に示した糖鎖構造の還元側を構成するFetuin糖鎖及びコアタンパク質部分)を示す。Fetuin-a、Fetuin-b は、それぞれFetuinの糖鎖構造をシアリダーゼ又はα2,3-シアリダーゼによって選択的に分解することで調製した。
表1中、HL60はHL60細胞から抽出した糖タンパク質画分を指し、表1ではその主要な糖鎖構造を示した。また、その基幹構造をR2で示した。糖タンパク質AGPについても主要な糖鎖構造を示し、その基幹構造をR3で示した。HL60が有している主要な糖鎖構造は、Siaα2,3Galβ1,4(Fucα1,3)GlcNAc(Sialyl-LewisX )であり、AGPの有する主要な糖鎖構造は、Siaα2,6Galβ1,4GlcNAc(CDw75)及び Galβ1,4(Fucα1,3)GlcNAc(LewisX)である。
(1-1)「6’-Sialyl-LacNAc(CDw75)」糖鎖構造を有する免疫誘導剤の製造
本発明の標的オリゴ糖鎖として、哺乳動物細胞で発現するシアリル化糖鎖の1種であり、胃癌、大腸癌の悪性度判定の診断指標となる腫瘍マーカー(CDw75)として、また悪性腫瘍治療の分子標的として注目されている「6’-Sialyl-LacNAc(CDw75)」に対して、本発明のキャリア化合物X-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3(Y=-CH2-の場合)を融合させたコンジュゲート(免疫誘導剤)を製造した。
a1化合物(一般式1,n1=16,n2=9の場合)の製造
(S)体 : 1H NMR (400MHz, CD3OD) : δ 4.46 (1H, d, J = 8.0 Hz, H-1 Gal or Glc), 4.32 (1H, d, J = 7.2 Hz, H-1 Gal or Glc), 4.04 (1H, bt, J = 8.6 Hz, H-5 Gal), 2.77 (1H, dd, J = 4.0, 12.4 Hz, H-3eq Sia (NeuAc)); MALDI-TOFMS : C55H101N3O20 [M+Na]+ calcd. (m/z) 1146.69, found. (m/z) 1146.49
(R)体 : 1H NMR (400MHz, CD3OD) : δ 4.53 (1H, d, J = 8.4 Hz, H-1 Gal or Glc), 4.32 (1H, d, J = 7.2 Hz, H-1 Gal or Glc), 4.05 (1H, bt, J = 8.8 Hz, H-5 Gal), 2.77 (1H, dd, J = 4.0, 12.0 Hz, H-3eq Sia (NeuAc)); MALDI-TOFMS : C55H101N3O20 [M+Na]+ calcd. (m/z) 1146.69, found. (m/z) 1146.55
a2(S)化合物は順相シリカゲルカラムクロマトグラフィーにより精製し、1H-NMRスペクトルおよびMALDI-TOFによる質量分析を行ない、得られた化合物を確認した。
(S)体 : 1H NMR (400MHz, CD3OD) : δ 4.46 (1H, d, J = 8.4 Hz, H-1 Gal or Glc), 4.33 (1H, d, J = 7.2 Hz, H-1 Gal or Glc), 4.04 (1H, bt, J = 8.8 Hz, H-5 Gal), 2.77 (1H, dd, J = 4.2, 12.2 Hz, H-3eq Sia (NeuAc)), 2.00, 1.99 and 1.94 (3H × 3, 3s, -NHC(=O)CH3); MALDI-TOFMS : C39H69N3O20 [M+Na]+ calcd. (m/z) 922.44, found. (m/z) 922.17
a3-1(S)化合物を順相シリカゲルカラムクロマトグラフィー(酢酸エチル:エタノール:水=6:2:1)により精製し、1H-NMRスペクトルおよびMALDI-TOFによる質量分析を行ない、得られた化合物を確認した。
1H NMR (400MHz, CD3OD:CDCl3 = 4:1) : δ 4.44 (1H, d, J = 8.0 Hz, H-1 Gal or Glc), 4.33 (1H, d, J = 7.2 Hz, H-1 Gal or Glc), 2.77 (1H, dd, J = 4.6, 12.2 Hz, H-3eq Sia (NeuAc)), 2.01 and 2.00 (3H × 2, 2s, -NHC(=O)CH3)
MALDI-TOFMS : C61H113N3O20 [M+Na]+ calcd. (m/z) 1230.78, found. (m/z) 1230.48
MALDI-TOFMS : C57H105N3O20 [M+Na]+ calcd. (m/z) 1174.72, found. (m/z) 1174.78
a3-3化合物を逆相カラムを用いた高速液体クロマトグラフィーにより精製し、MALDI-TOFによる質量分析を行ない、得られた化合物を確認した。
MALDI-TOFMS : C65H121N3O20 [M+Na]+ calcd. (m/z) 1286.84, found. (m/z) 1287.26
b1化合物(一般式2,n1=16,n2=9の場合)の製造
b1化合物を順相シリカゲルカラムクロマトグラフィー(酢酸エチル:エタノール:水=7:2:1)により精製し、MALDI-TOFによる質量分析を行ない、得られた化合物を確認した。
(S)体 : MALDI-TOFMS : C55H101N3O20 [M+Na]+ calcd. (m/z) 1146.69, found. (m/z) 1146.40
(R)体 : MALDI-TOFMS : C55H101N3O20 [M+Na]+ calcd. (m/z) 1146.69, found. (m/z) 1145.42
c1化合物(一般式3,n1=16,n2=9の場合)の製造
c1化合物を順相薄層シリカゲルカラムクロマトグラフィー(クロロホルム:メタノール:水=5:4:1)により精製し、MALDI-TOFによる質量分析を行ない、得られた化合物を確認した。
(S)体 : MALDI-TOFMS : C44H84N2O12 [M+Na]+ calcd. (m/z) 855.59, found. (m/z) 855.24
(R)体 : MALDI-TOFMS : C44H84N2O12 [M+Na]+ calcd. (m/z) 855.59, found. (m/z) 855.13
sLeX-C12L 化合物(Sialyl-LewisX(Siaα2,3Galβ1,4(Fucα1,3)GlcNAc)-C12L)の製造
sLeX-C12L化合物を順相薄層シリカゲルカラムクロマトグラフィーにより精製し、MALDI-TOFによる質量分析を行ない、得られた化合物を確認した。
MALDI-TOFMS : C67H123N3O24 [M+Na]+ calcd. (m/z) 1376.84, found. (m/z) 1376.91
LeX-C12L(Galβ1,4(Fucα1,3)GlcNAc-C12L)の製造
MALDI-TOFMS : C56H106N2O16 [M+Na]+ calcd. (m/z) 1085.74, found. (m/z) 1085.96
次に、実施例(1-1)、(1-2)及び(1-3)でそれぞれ合成した各種免疫誘導剤(a1,b1,c1)のラセミ体を、アジュバント(Lipid-A)と共にコレステロール、リン脂質を含む脂質に溶解し、モノクローナル抗体の汎用作製宿主であるマウス(C3H/HeN strain)に、リポソーム法(Eur. J. Immunol., 16, 951-956, 1986)にて免疫し、その免疫誘導能を評価した。
また、産生されるイムノグロブリンのサブクラス(IgM及びIgG)を評価するため、二次抗体にIgM及びIgGに特異的な抗体を用いて評価した。抗体価は、二次抗体の標識酵素であるペルオキシダーゼの活性として評価した。ペルオキシダーゼの発色基質にはTMBを用い、反応後に2規定の硫酸を加えた後の450nmの吸光度の強度を評価した。(図1)~(図2)に、IgMサブクラスの抗体価を示す。
用いた化合物(a1,b1,c1)全てにおいて、免疫原(図1)及び糖タンパク質(図2)を抗原としたELISA法にて、それぞれp<0.05、p<0.01及びp<0.001にて有意な抗体価の上昇が確認された。一方で、a1,b1,c1の免疫誘導剤ではIgGサブクラスにおける有意な抗体価は見られなかった(図示せず)。また、それぞれ(S)体と(R)体の間に免疫増強能の著明な差は確認されなかった(図示せず)。
(3-1)「6’-Sialyl-LacNAcβ1(CDw75)」糖鎖構造を有する免疫誘導剤について
次に、より活性の強いキャリア化合物を選別するため、実施例(1-1)で合成した化合物a1を基本構造としてキャリア化合物の構造を変化させた5種類の候補化合物(a1, a2, a3-1, a3-2, a3-3)に対して、上記同様にその免疫誘導能を評価した。
上述の検討の結果、長鎖長の脂肪酸(超長鎖脂肪酸)を含有するセラミド類似体に強い免疫原性増強作用を見いだした。天然体のセラミドの場合は、アミノ基側のアルキル基に水酸基を有し不飽和結合も有している点で本発明のセラミド類似体である免疫誘導剤とは構造的には異なるが、天然体のセラミドの場合でも、このような超長鎖脂肪酸を含有する場合がある。そこで、天然体の超長鎖脂肪酸含有セラミド自身も高い免疫原性を有している可能性があると考え、その免疫原性の程度を検討した。検討した天然体セラミドは、炭素数24(n1=22に相当)の脂肪酸を有する下記のGlobosideである。
globosideをリポソーム法にて免疫した際の、マウスの血清抗体価を図5に示す。免疫誘導後のマウス血清中のIgMサブクラス(図5,左図)では、免疫後3日後および7日後の血清において、免疫原(globoside)に対する抗体価の有意な上昇が確認された。一方で、IgGサブクラス(図5,右図)では、抗体価の上昇は確認されなかった。
この結果は、天然体のセラミドが結合した糖脂質化合物には免疫原性はあるが、胸腺依存性抗原としての十分な活性までは超長鎖脂肪酸を含有する分子種であっても発揮できないことを示唆している。
前記(実施例3)(3-1)での検討の結果、最も強い免疫原性を確認したC12L化合物のフコシル化糖鎖への汎用性を検討するべく、(実施例1)(1-4)で製造した、代表的なフコシル化糖鎖である、Sialyl LewisX(sLeX)及び同(1-5)で製造した、代表的なアシアロ糖鎖である、LewisX(LeX)オリゴ糖鎖とのコンジュゲート(sLeX-C12L、LeX-C12L)における、目的のオリゴ糖鎖抗原に対する免疫誘導能を検証した。sLeX、LeXは前述のように腫瘍マーカーや幹細胞マーカーとして有用なオリゴ糖鎖抗原である。
それぞれの化合物を免疫した際の、マウスの血清抗体価を図9に示す。sLeX-C12Lを免疫したマウス血清中のIgMサブクラス(図9,A)では、免疫3日後及び7日後の血清において免疫原及び目的オリゴ糖鎖抗原を含む糖タンパク質(HL60)に対する抗体化の有意な上昇が確認された。IgGサブクラス(図9,B)の場合でも、一次追加免疫のみにもかかわらず、十分なIgGクラススイッチが起こっており、免疫7日後にて免疫原に対する抗体価の上昇傾向が確認され、また糖タンパク質(HL60)に対しても、抗体価の有意な上昇が確認された。
LeX-C12Lを免疫したマウス血清中のIgMサブクラス(図9,C)の場合も、免疫3日後及び7日後の血清において免疫原及び目的オリゴ糖鎖抗原を含む糖タンパク質(AGP)に対する優位な抗体価の上昇が確認された。IgGサブクラスの場合(図9,D)は、免疫原に対する抗体価の上昇傾向が免疫7日後に確認され、糖タンパク質(AGP)に対しても、免疫3日後に抗体価の有意な上昇が確認された。
以上の結果から、本発明で開発されたキャリア化合物(C12L)は、シアリル化糖鎖及びアシアロ(非シアリル化)糖鎖と共に、フコシル化糖鎖のそれぞれに分類される代表的なオリゴ糖鎖抗原のいずれに対しても、強力な免疫誘導活性を付与できることが実証された。すなわち、本発明のキャリア化合物は、哺乳動物の糖タンパク質に存在するN-結合型糖鎖に含まれるオリゴ糖鎖抗原である、シアリル化糖鎖、アシアロ(非シアリル化)糖鎖及びフコシル化糖鎖の全てに適用可能な技術であることが確認できた。
前記(実施例3)で最も強い免疫原性を確認したa3-1(CDw75-C12L)を用いて実際に目的糖鎖エピトープの特異抗体が産生されていることを確認した。具体的には、以下のようにモノクローナル抗体を作製した。
(5-1)抗CDw75モノクローナル抗体産生ハイブリドーマの製造
糖脂質抗原「CDw75-C12L」100μgを、リポソーム法(Brodinらの方法;Eur. J. Immunol., 16, 951-956, 1986)に従い、リン脂質、コレステロール、Lipid-Aとのメタノール溶液中にて混合し、蒸発させた後に50℃のPBS緩衝液中に溶解することでリポソームを形成させ、これを免疫誘導剤としてマウス(C3H/HeN strain)を免疫した。
免疫誘導剤は2週間間隔で4回皮下に免疫し、2週間後に腹腔内に免疫した後、3日後に脾臓細胞を採取し、ミエローマ細胞Sp1株と細胞融合することでハイブリドーマ細胞を作製した。
培養上清中の抗体価はELISA法により評価し、450nmの吸光度0.1以上を指標としてハイブリドーマを選別した。抗体価(吸光度)は、二次抗体として用いた抗マウスイムノグロブリン抗体の標識酵素であるペルオキシダーゼの活性として評価した。ペルオキシダーゼの発色基質にはTMBを用い、反応後に2規定の硫酸を加えた後の450nmの吸光度の強度を評価した。
まず、96ウェルプレート8枚にコロニー数1/ウェルになるよう(計768ウェル)培養したハイブリドーマ細胞の培養上清を0.05 mlずつ採取し、抗体価としてELISA法による450nmの吸光度0.1以上を指標として、6’-Sialyl-LacNAc(CDw75)糖鎖構造を含有する糖タンパク質(Fetuin)を認識するモノクローナル抗体産生ハイブリドーマ細胞を選別したところ、131ウェルにて陽性クローンを得た(出現率17.1%)。
一方、先行技術(非特許文献8)では、リン脂質(フォスファチジルエタノールアミン)をキャリア化合物として用い、同様にCDw75糖鎖をエピトープとする抗体を誘導しているが、この段階における陽性クローンの出現率は、581ウェル選別したうちのわずか8ウェル(出現率1.3%)であった。
これらのハイブリドーマから得られるモノクローナル抗体は、いずれも「CDw75」糖鎖構造に対して特異性の高い抗CDw75モノクローナル抗体であり、本発明の抗CDw75モノクローナル抗体である。
これら2個のハイブリドーマ中で、最も抗体価が高く、特異性、親和性が優れた抗CDw75モノクローナル抗体を産生する「ハイブリドーマFR9」を、2013年1月21日付でNITE特許微生物寄託センターに寄託した。2013年3月13日付で「受託番号:NITE P-1516」が付与されたあと、2014年4月15日付で「NITE BP-01516」として国際寄託へ移管されている。
FR9細胞の培養上清中に含まれるモノクローナル抗体(以下「FR9抗体」ともいう。)のCDw75に対する親和性について、CDw75を含有するFetuinを抗原とした解離定数(Kd値)を決定することで評価したところ、そのKd値は8.86×10-7 M であった。糖鎖に結合する抗体やレクチンのKd値は、一般的に1×10-3 ~1×10-6 Mであることから、開発した抗体はCDw75糖鎖に対する強い親和性を有する(図6A)。
ELISAによるFetuin及びCDw75-C12Lの検出限界の測定結果では、FR9抗体は、極微量なFetuin(約15 ng)を検出可能であった(図6B)。 一方、先行技術(非特許文献8)にて誘導された抗体は、50μg以上のFetuinが検出に必要であるため、FR9抗体が既知の技術にて作製された抗体よりも、3000倍以上の高い検出感度を有することがわかる。
次に、FR9抗体の抗原認識特異性についてELISA法により評価した(図7)。用いた抗原の糖鎖構造を表1に示す。FR9抗体は、抗原としたCDw75構造を有する糖脂質、糖タンパク質のみを厳密にエピトープ識別し、構造が近似する3’-Sialyl-LacNAc構造や前駆体であるLacNAc構造には反応しないため、開発した抗体はCDw75糖鎖に対する高い特異性を有する。
続けて、FR9抗体が特定のタンパク質検出に汎用されるウエスタンブロット法に適用可能かどうかを検討した(図8)。
抗原とした6’-Sialyl-LacNAc構造を含むFetuin 糖タンパク質を、2種類のシアリダーゼにて処理したものを調製し、特異性の解析も平行しておこなったところ、FR9抗体はFetuin糖タンパク質を検出し、CDw75糖鎖を酵素反応にて消失させたものには反応せず、一方で3’-Sialyl-LacNAc糖鎖を選択的に消失させたFetuin糖タンパク質には未処理のFetuin同等に反応した。
よって本発明により開発した免疫誘導剤が、目的とする糖鎖エピトープに対する特異抗体を産生させること、また産生された抗体が高活性かつ様々な用途に利用できる性能を有することが証明された。一方、比較のために市販されている抗CDw75抗体(LN-1)に対しても同様の手法で特異性、親和性の検討をしたがELISA法での検出限界以下であり、CDw75糖鎖そのものに対する特異性、親和性が非常に低いことが確認された。
CDw75糖鎖は糖タンパク質や糖脂質の形態で細胞表層抗原として発現しており、細胞表層抗原は細胞診断や悪性腫瘍の分子標的となる。FR9抗体が細胞表層のCDw75と反応することを細胞蛍光染色法で確認した。CDw75を発現する高悪性度B細胞性腫瘍細胞(バーキットリンパ腫のRaji細胞)をFR9抗体とインキュベートした後、抗体反応を蛍光標識二次抗体にて標識し、蛍光検出装置で検出した(図12)。FR9抗体を加えないネガティブコントロールと比較して、明らかな蛍光の増大が検出されたことから、FR9抗体がRaji細胞表層のCDw75に反応することがわかる。
先行技術文献(非特許文献8)においても、CDw75糖鎖抗原を認識するモノクローナル抗体(241-5-2抗体)が得られているので、その特異性及び親和性を本発明の抗CDw75抗体(FR9抗体)の結果と比較する。
非特許文献8中の記載によれば、得られた241-5-2抗体は、CDw75糖鎖を含むFetuin糖タンパク質と反応するものの、当該241-5-2抗体が認識するエピトープは、CDw75糖鎖「6’-Sialyl-LacNAc(Siaα2,6Galβ1,4GlcNAc)」中の部分構造である「Siaα2,6構造」であると推測されている(同第303頁)。つまり、241-5-2抗体はCDw75糖鎖に特異的な抗体とはいえず、「Siaα2,6構造」を有している糖鎖であれば反応する。
一方、本発明のFR9抗体はCDw75糖鎖の微細構造まで認識する抗体であり、Siaα2,6構造のみでなく還元末端のGlcNAc構造までも含めた「6’-Sialyl-LacNAc(Siaα2,6Galβ1,4GlcNAc)」の全長がエピトープである。GlcNAcがGlcであると反応しないという厳密なエピトープの認識特異性を有しており(図10)、既存の抗体と比較して極めて高い特異性を達成できたといえる。
また、非特許文献8における、241-5-2抗体についてのELISAによる測定結果(同第302頁)によると、Fetuin検出のために50μg以上のFetuinを必要としている。一方、本発明のFR9抗体は、極微量なFetuin(約15 ng)であっても検出可能である(図6B)。このことからみて、本発明のFR9抗体は、CDw75糖鎖抗原に対する親和性に関しても、241-5-2抗体と比較して、3000倍以上の高い検出感度が達成できたといえる。
このように、本発明の免疫誘導法を適用して得られるモノクローナル抗体は、目的のオリゴ糖鎖抗原に対する高い特異性及び親和性を有するモノクローナル抗体である。
Claims (9)
- 標的オリゴ糖鎖抗原Rを含む一般式(2)で表される人工糖脂質又はその塩を有効成分とする免疫誘導剤を用いて哺乳動物を免疫する工程を含む、哺乳動物において標的オリゴ糖鎖抗原Rに対する抗原特異的な免疫誘導方法;
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。 - 標的オリゴ糖鎖抗原Rが、哺乳動物由来の糖タンパク質のN-結合型糖鎖に含まれるオリゴ糖鎖抗原である、請求項1に記載の免疫誘導方法。
- 下記一般式(2)に示され、標的オリゴ糖鎖抗原Rを含む人工糖脂質又はその塩を有効成分として含む免疫誘導剤;
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。 - 標的オリゴ糖鎖抗原Rが、哺乳動物由来の糖タンパク質のN-結合型糖鎖に含まれるオリゴ糖鎖抗原である、請求項3に記載の免疫誘導剤。
- 下記一般式(2)に示され、標的オリゴ糖鎖抗原Rを含む人工糖脂質又はその塩を有効成分として含むワクチン;
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は2~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。 - 標的オリゴ糖鎖抗原Rが、哺乳動物由来の糖タンパク質のN-結合型糖鎖に含まれるオリゴ糖鎖抗原である、請求項5に記載のワクチン。
- 下記一般式(2)に示される人工糖脂質又はその塩;
一般式(2)
R-Z-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Rは、1又は複数種の単糖2~30から構成される直鎖状又は分枝状のオリゴ糖を表す。Zは、単結合、又はO、SもしくはNH、又はチオメチルと結合させたリンカー、アミノメチル化された糖アルコールを表し、Yは-(CH2)m-を表す。n1は20~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。 - 式中Rが、哺乳動物由来の糖タンパク質のN-結合型糖鎖に含まれるオリゴ糖鎖抗原である、請求項7に記載の糖脂質又はその塩。
- 下記一般式(1)に示される人工脂質又はその塩;
一般式(1)
X-Y-CH(NH-CO-(CH2)n1-CH3)-(CH2)n2-CH3
(式中、Xは-H、-OH、-SH、-NH2、ハロゲン又はヒドラジド基を表し、Yは-(CH2)m-を表す。n1は20~40の整数、n2は1~27の整数、mは1~30の整数を表す。)。
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JP2016216387A (ja) * | 2015-05-19 | 2016-12-22 | 公立大学法人名古屋市立大学 | 未分化細胞のアポトーシス誘導剤 |
EP3344806A4 (en) * | 2015-09-04 | 2019-03-20 | OBI Pharma, Inc. | GLYCAN NETWORKS AND METHODS OF USE |
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US11642400B2 (en) | 2016-07-27 | 2023-05-09 | Obi Pharma, Inc. | Immunogenic/therapeutic glycan compositions and uses thereof |
US11643456B2 (en) | 2016-07-29 | 2023-05-09 | Obi Pharma, Inc. | Human antibodies, pharmaceutical compositions and methods |
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EP2993182A1 (en) | 2016-03-09 |
EP2993182B1 (en) | 2020-10-14 |
US20160074522A1 (en) | 2016-03-17 |
US10307471B2 (en) | 2019-06-04 |
JPWO2014178195A1 (ja) | 2017-02-23 |
EP2993182A4 (en) | 2017-01-04 |
JP6143240B2 (ja) | 2017-06-07 |
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