T cell activator containing glycolipid derivative as active ingredient

Abstract

Disclosed herein as embodiments are: a T cell activator which contains, as an active ingredient, a glycolipid represented by formula (I) (wherein each symbol is as defined in the description) and can also activate innate immunity; and others.

Description

T cell activator containing glycolipid derivative as an active ingredient

[0001] The present invention provides, as one embodiment, a T cell activator containing a glycolipid derivative as an active ingredient, which is useful, for example, as a vaccine and can also activate innate immunity, and is useful, for example, in the field of medicine.

[0002] Tuberculosis, caused by Mycobacterium tuberculosis (M. tuberculosis; M. tb), is one of the world's largest infectious diseases and has yet to be eradicated. In 2023, approximately 10 million people will develop tuberculosis annually, and approximately 1.25 million will die from the disease. Mammals are equipped with innate immunity as one of the biological defense systems against infection with Mycobacterium tuberculosis. For example, trehalose dimycolate (TDM), which is abundant in the cell wall of Mycobacterium tuberculosis, and its synthetic analog trehalose dibehenate (TDB) are known to be ligands that strongly activate the innate immune receptor Macrophage-inducible C-type lectin receptor (Mincle) (Non-Patent Document 1, Non-Patent Document 2). Mincle enhances cellular immunity and is therefore thought to be effective in protecting against infections caused by intracellular pathogens, and attempts have been made to apply compounds that efficiently activate Mincle to vaccines (Non-Patent Document 3).

[0003] On the other hand, in relation to adaptive immunity, mammals are known to possess T cells that specifically recognize lipids in the outer membrane of Mycobacterium tuberculosis. However, only a few of the characteristic lipids present in the outer membrane of Mycobacterium tuberculosis have actually been identified as T cell antigens (Non-Patent Document 4). Among these, diacylated sulfoglycolipid (Ac2SGL) and phosphatidylinositol dimannoside (PIM2) have been proposed as lipid vaccines that can directly activate both innate immunity and T cells, but they have not yet reached a clinically viable level (Non-Patent Document 5). Therefore, there has been a need for a ligand that can directly activate both innate and adaptive immunity during Mycobacterium tuberculosis infection and be used in vaccines.

[0004] Ishikawa E, J. Exp. Med. , 2009 Schoenen, J. Immunol. , 2010 Braganza, Front. Immunol. , 2018 De Libero G, Csh Perspect Med. , 2014 Larrouy-Maumus G, Vaccine. ,2017

[0005] One of the objects of the present invention is to provide, as an embodiment, a T cell activator that can also activate innate immunity.

[0006] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have unexpectedly discovered that glycolipid derivatives with specific structures that activate innate immunity are useful as dual ligands that function as T cell ligands and can also activate adaptive immunity. Based on this novel finding, further research has led to the completion of the present invention. The present invention will be described below with reference to specific embodiments, although the present invention is not limited thereto.

[0007] [1] Formula (I):

[0008]

[0009] (In the formula, R １ represents an optionally substituted hydrocarbon group having m carbon atoms; R ２[2] A T cell activator comprising, as an active ingredient, a glycolipid derivative (hereinafter also referred to as "glycolipid derivative (I)") represented by the formula (I) or a salt thereof, wherein R represents an optionally substituted hydrocarbon group having n carbon atoms, and m+n represents an integer of 25 to 100. １ But, R １ At the β-position from the carbonyl group adjacent to the carbon atom to which １－６ The T cell activator according to the above-mentioned [1], wherein R is a hydrocarbon group substituted with an alkoxy group and may be further substituted at other positions, and wherein two adjacent substituents on the hydrocarbon group may be bonded to each other to form a cyclopropane ring or an oxacyclopropane ring. １ But, R １ At the β-position from the carbonyl group adjacent to the carbon atom to which １－６ substituted with an alkoxy group, and at other positions, a hydroxyl group, C １－６ a hydrocarbon group having m carbon atoms, which may be further substituted with 1 to 10 identical or different substituents selected from an alkoxy group, a carboxy group, and an oxo group, wherein two adjacent substituents on the hydrocarbon group may be bonded to each other to form 1 to 3 identical or different cyclopropane or oxacyclopropane rings; R ２ is a hydroxyl group, C １－６ The T cell activator according to the above-mentioned [1] or [2], wherein R is a hydrocarbon group having n carbon atoms, which may be substituted with 1 to 10 identical or different substituents selected from an alkoxy group, a carboxy group, and an oxo group. [4] In the glycolipid derivative (I), R ２ [5] The T cell activator according to any one of the above [1] to [3], wherein R is a hydrocarbon group having n carbon atoms. １ [6] In the glycolipid derivative (I), R１ and R ２ [7] The T cell activator according to any one of the above-mentioned [1] to [5], wherein the hydrocarbon groups in R are each independently an alkyl group or an alkenyl group. １ and R ２ [6] The T cell activator according to [6] above, wherein the hydrocarbon group in the formula (I) is an alkyl group. [8] The T cell activator according to any of [1] to [7] above, wherein in the glycolipid derivative (I), m+n is an integer of 25 to 90 (preferably an integer of 25 to 50, an integer of 55 to 70, or an integer of 75 to 90). [9] The T cell activator according to any of [1] to [8] above, wherein in the glycolipid derivative (I), m is an integer of 15 to 70 (preferably an integer of 15 to 65), and n is an integer of 10 to 30 (preferably an integer of 10 to 25).

[10] The T cell activator according to any one of [1] to [9] above, wherein the glycolipid derivative (I) is trehalose monomycolate (hereinafter also referred to as "TMM") derived from an acid-fast bacterium (preferably R. sp4306, R. equi, R. rubber, M. smegmatis, M. bovis BCG, M. intracellulare, or M. tuberculosis).

[11] The T cell activator according to

[10] above, wherein the glycolipid derivative (I) comprises one derivative selected from glycolipid derivatives represented by the following structural formulas, or a combination of two or more derivatives:

[0010]

[0011]

[12] The T cell activator according to any one of the above-mentioned [1] to [9], wherein the glycolipid derivative (I) comprises a glycolipid derivative represented by the following structural formula (hereinafter also referred to as "TMM(C32)").

[0012]

[0013]

[13] The T cell activator according to any one of [1] to

[12] above, wherein T cell activation is due to antigen presentation of the glycolipid derivative (I) by CD1b molecules.

[14] The T cell activator according to any one of [1] to

[13] above, wherein innate immunity is also activated.

[15] The T cell activator according to any one of [1] to

[14] above, wherein the T cell activator is used as a vaccine or a functional adjuvant.

[16] The T cell activator according to

[15] above, wherein the vaccine is for the prevention or treatment of an infectious disease.

[17] The T cell activator according to

[16] above, wherein the infectious disease is caused by bacteria expressing one or more types of trehalose monomycolate (TMM) in the cell wall or intracellularly.

[18] The T cell activator according to

[17] above, wherein the bacteria is Mycobacterium tuberculosis, Mycobacterium leprae, or nontuberculous mycobacterium.

[19] Use of the glycolipid derivative (I) or a salt thereof defined in any one of the above [1] to

[12] as a T cell activator in research.

[0014]

[20] A method for activating T cells in an animal having an immune system, comprising administering an effective amount of the glycolipid derivative (I) or its salt defined in [1] above to the animal.

[21] The method for activating T cells according to

[20] above, wherein innate immunity is also activated.

[22] The method for activating T cells according to

[20] or

[21] above, wherein the administration to the animal is in the form of a vaccine or a functional adjuvant.

[23] A method for preventing or treating an infectious disease in an animal having an immune system in need thereof, comprising administering an effective amount of the glycolipid derivative (I) or its salt defined in [1] above to the animal. *) Here, for preferred embodiments of the glycolipid derivative (I) or its salt, preferred infectious disease, etc., reference can be made to the embodiments specified as the glycolipid derivative (I) or its salt, preferred infectious disease, etc. in [2] to

[18] above.

[0015]

[24] The glycolipid derivative (I) or its salt defined in [1] above, which is used as a T cell activator.

[25] The glycolipid derivative (I) or its salt defined in [1] above, which is used as a T cell activator that can also activate innate immunity.

[26] The glycolipid derivative (I) or its salt defined in [1] above, which is used as a vaccine or a functional adjuvant.

[27] The glycolipid derivative (I) or its salt defined in

[24] above, wherein the vaccine is for the prevention or treatment of an infectious disease. *) Here, for preferred embodiments of the glycolipid derivative (I) or its salt, preferred infectious disease, etc., reference can be made to the embodiments specified as the glycolipid derivative (I) or its salt, preferred infectious disease, etc. in [2] to

[18] above.

[0016]

[28] Use of the glycolipid derivative (I) or a salt thereof defined in [1] above, for producing a T cell activator.

[29] Use of the glycolipid derivative (I) or a salt thereof defined in [1] above, for producing a T cell activator that can also activate innate immunity.

[30] Use of the glycolipid derivative (I) or a salt thereof defined in [1] above, for producing a vaccine or a functional adjuvant.

[31] Use of the glycolipid derivative (I) or a salt thereof according to

[30] above, wherein the vaccine is for the prevention or treatment of an infectious disease. *) Here, for preferred embodiments of the glycolipid derivative (I) or a salt thereof, preferred infectious diseases, etc., reference can be made to the embodiments specified as the glycolipid derivative (I) or a salt thereof, preferred infectious diseases, etc. in [2] to

[18] above.

[0017] According to one embodiment of the present invention, there is provided a T cell activator containing a glycolipid derivative as an active ingredient, which is useful as, for example, a vaccine and can also activate innate immunity.

[0018] Figure 1 shows the results of thin-layer chromatography fractionation of crude lipids extracted from Mycobacterium tuberculosis (M. tb) H37Rv strain, and the results of evaluation of GFP and CD69 expression in each fraction using NFAT-GFP reporter cells expressing clone 50-TCR (see Example 1 below). Figure 2 shows the results of MALDI-TOF / MS analysis of the active fraction (Fr2) shown in Figure 1 (see Example 2 below). Figure 3 shows the results of evaluation of GFP and CD69 expression in NFAT-GFP reporter cells expressing clone 50-TCR when stimulated with Mycobacterium tuberculosis-derived trehalose monomycolate (TMM) and trehalose dimycolate (TDM) and chemically synthesized glucose monomycolate (GMM) (see Example 3 below). Figure 4 shows the results of evaluating GFP and CD69 expression when NFAT-GFP reporter cells expressing clone 50-TCR were stimulated with TMM derived from Mycobacterium tuberculosis in the presence of human monocyte-derived dendritic cells (MoDCs) and various inhibitory antibodies against human CD1 molecules (see Example 4 below). Figure 5 shows the results of evaluating GFP and CD69 expression when NFAT-GFP reporter cells expressing clone 50-TCR were stimulated with synthetic TMM in the presence of HEK293 cells overexpressing human CD1a and other genes (see Example 5 below). Figure 6 shows the results of evaluating GFP and CD69 expression when NFAT-GFP reporter cells expressing clone 50-TCR were stimulated with various TMMs of different chain lengths derived from mycobacteria in the presence of DC2.4 cells (a mouse dendritic cell line) transduced with human CD1b (see Example 6 below). Figure 7 (left panel) shows the results of evaluating GFP expression when NFAT-GFP reporter cells co-expressing human Mincle and FcRγ were stimulated with TMM derived from Mycobacterium tuberculosis (see Example 7(1) below). Figure 7 (right panel) shows the results of evaluating GFP and CD69 expression when NFAT-GFP reporter cells expressing clone 50-TCR were stimulated with TMM derived from mycobacterium equi in the presence of DC2.4 cells (a mouse dendritic cell line) transduced with human CD1b (see Example 7(2) below).

[0019] The present invention will be described below with reference to specific embodiments. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. As mentioned above, the present invention is not limited to the following embodiments.

[0020] One embodiment provided by the present invention is a T cell activator represented by the following formula (I):

[0021]

[0022] (In the formula, R １ represents an optionally substituted hydrocarbon group having m carbon atoms; R ２ represents an optionally substituted hydrocarbon group having n carbon atoms, and m+n represents an integer of 25 to 100.) A T cell activator comprising, as an active ingredient, a glycolipid derivative represented by the formula (hereinafter also referred to as "glycolipid derivative (I)") or a salt thereof." (Hereinafter, this embodiment may also be referred to as "the present T cell activator.") This embodiment is based on the novel finding that "glycolipid derivative (I) having the above structural characteristics activates T cells," and relates to a novel use of glycolipid derivative (I) as a T cell activator. This embodiment will be described in detail below.

[0023] [Regarding Glycolipid Derivative (I)] (1) Structure of Glycolipid Derivative (I) Glycolipid derivative (I) is represented by the following formula (II):

[0024]

[0025] The partial structure represented by the following formulae (IIa) and (IIb):

[0026]

[0027] (In the above formula, R １ and R ２ are as defined above), the "glycolipid derivative (I)" can have two structures represented by the following formula:

[0028] In this embodiment, glycolipid derivative (I) can be used in either its free form or its salt form (preferably, a pharmaceutically acceptable salt thereof). Those skilled in the art can carry out this embodiment by appropriately selecting either form based on the properties of the individual glycolipid derivative (I) used. Examples of pharmaceutically acceptable salts include salts with acids such as salts with inorganic acids (e.g., hydrochloride, hydrobromide, sulfate, phosphate), salts with organic acids (e.g., acetate, fumarate, oxalate, citrate, methanesulfonate, benzenesulfonate, tosylate, maleate), salts with bases (e.g., alkali metal salts (e.g., sodium salt, potassium salt), alkaline earth metal salts (e.g., calcium salt), and salts with amino acids (e.g., glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, aspartate).

[0029] Each group in the glycolipid derivative (I) will be explained below. When the notation "Ca-b" (e.g., C1-6) is used, it indicates that the number of carbon atoms constituting the group is a to b (e.g., 1 to 6). The notation "optionally substituted" indicates that the target group may be substituted with a substituent at a substitutable position in the target group. The number of the substituents is not particularly limited, but a specific embodiment includes, for example, 1 to 10.

[0030] R １ and R ２In the above, the "hydrocarbon group" refers to a saturated or unsaturated, linear or branched hydrocarbon group, and is not limited thereto. Specific examples include, but are not limited to, methyl, ethyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, 2-hexenyl, 1-hexynyl, heptyl, 1-methylhexyl, 6-heptynyl, octyl, 2-methylheptyl, 2-octen-7-ynyl, nonyl, and 3-methyloctyl; Decyl, 4-methylnonyl, undecyl, 5-methyldecyl, dodecyl, 6-methylundecyl, tridecyl, 7-methylundecyl, tetradecyl, 8-methyltridecyl, pentadecyl, 9-methyltetradecyl, hexadecyl, 10-methylpentadecyl, 12-hexadecenyl, 1-hexadecynyl, heptadecyl, 11-methylhexadecyl, 16-heptadecynyl, octadecyl, 12-methylhexadecyl, 12-octadecen-17-ynyl, nonadecyl, 13-methyloctadecyl, 14-methylnonadecyl; Eicosyl, 15-methylicosyl, henicosyl, docosyl, 16-ethylicosyl, tricosyl, 10,17-dimethylhenicosyl, tetracosyl, 10,18-dimethylhenicosyl, pentacosyl, 19-ethyltricosyl, hexacosyl, 22-hexacosenyl, 1-hexacosinyl, 10,20-dimethyltetracosyl, heptacosyl, 21-methylhexacosyl, 26-heptacosinyl, octacosyl, 10,21-dimethylhexacosyl, 22-hexacosen-27-ynyl, nonacosyl, 10-methyl-15-ethyl-22-propyltriacosyl;triacontyl, 24-methylnonacosyl, hentriacontyl, 25-methyltriacontyl, 1,3,5,7-tetramethylheptacosyl, dotriacontyl, 26-ethyltriacontyl, tritriacontyl, 20,27-dimethylhentriacontyl, tetratriacontyl, 20,28-dimethyldotriacontyl, pentatriacontyl, 29-methyltetracontyl, hexatriacontyl, 20,30-dimethyltetratriacontyl, 32-hexatriacontenyl, 1-hexatriacontinyl, heptatriacontyl, 31-methylhexatriacontyl, 36-heptacontinyl, octatriacontyl, 30,31-dimethylhexatriacontyl, 32-octatriaconten-37-ynyl, nonatriacontyl, 20-methyl-25-ethyl-32-propyltritriacontyl; Tetracontyl, 34-methylnonatriacontyl, hentetracontyl, dotetracontyl, tritetracontyl, tetratetracontyl, pentacontyl, 10-pentacontenyl, hexacontyl, heptatetracontyl, 20,30-heptatetracontane-dienyl, octatetracontyl, nanotetracontyl, 35-nonatetracontinyl; pentacontyl, 44-methylnanotetracontyl, henpentacontyl, dopentacontyl, tripentacontyl, 19,34-tripentacontan-dienyl, tetrapentacontyl, pentapentacontyl, 20-pentapentacontenyl, hexapentacontyl, heptapentacontyl, 38-methylhexapentacontyl, octapentacontyl, 20,38-dimethylhexapentacontyl, nonapentacontyl, 45-nonapentacontinyl; Hexacontyl, 55-methylnonapentacontyl, henhexacontyl, dohexacontyl, trihexacontyl, 29,34-trihexacontandienyl, tetrahexacontyl, pentahexacontyl, hexahexacontyl, heptahexacontyl, 48-methylhexahexacontyl, octahexacontyl, 30,48-dimethylhexahexacontyl, nonahexacontyl, 55-nonahexacontinyl;Examples of the "hydrocarbon group" include alkyl groups such as heptacontyl and 65-methylnonahexacontyl, alkenyl groups, and alkynyl groups. Here, the "hydrocarbon group" preferably includes an "alkyl group" and an "alkenyl group," and more preferably includes an "alkyl group."

[0031] m is R １ represents the number of carbon atoms in the hydrocarbon group, and n represents the number of carbon atoms in the hydrocarbon group ２ m represents an integer of 15 to 70, preferably an integer of 15 to 65. n represents an integer of 10 to 30, preferably an integer of 10 to 25. m+n represents the number of carbon atoms in the hydrocarbon group of R １ and R ２ and specifically represents an integer of 25 to 100. Preferred embodiments of m+n include integers of 25 to 90, and more preferred embodiments include integers of 25 to 50, integers of 55 to 70, and integers of 75 to 90, but are not limited thereto and can be appropriately determined by a person skilled in the art.

[0032] Examples of the "substituent" in the "optionally substituted hydrocarbon group having m carbon atoms" and the "optionally substituted hydrocarbon group having n carbon atoms" include a hydroxyl group, a C １－６ Examples of the alkyl group include an alkoxy group, a carboxy group, and an oxo group. １－６ Examples of the "alkoxy group" include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like. R １ In the hydrocarbon group of R １ At the β-position from the carbonyl group adjacent to the carbon atom to which １－６ It is preferably substituted with an alkoxy group (preferably a hydroxyl group). １The hydrocarbon group of the formula (I) may have, in addition to the substituent at the β-position relative to the carbonyl group, 1 to 10 identical or different substituents selected from the above-mentioned group of substituents at substitutable positions. Two adjacent substituents on the hydrocarbon group may be bonded to each other to form a cyclopropane ring or an oxacyclopropane ring (preferably 1 to 3 identical or different rings). ２ The hydrocarbon group R may have 1 to 10 identical or different substituents selected from the above substituent group at substitutable positions. １ and R ２ The hydrocarbon group is preferably an alkyl group or an alkenyl group, more preferably an alkyl group.

[0033] Preferred specific embodiments of the glycolipid derivative (I) are exemplified below. １ But, R １ At the β-position from the carbonyl group adjacent to the carbon atom to which １－６ substituted with an alkoxy group, and at other positions, a hydroxyl group, C １－６ a hydrocarbon group (preferably an alkyl group or an alkenyl group) which may be further substituted with 1 to 10 identical or different substituents selected from an alkoxy group, a carboxy group, and an oxo group, wherein two adjacent substituents on the hydrocarbon group may be bonded to each other to form 1 to 3 identical or different cyclopropane or oxacyclopropane rings, and which has m carbon atoms; R ２ is a hydrocarbon group (preferably an alkyl group or an alkenyl group) having n carbon atoms, and m+n is an integer of 25 to 100.

[0034] Glycolipid derivative (IB) R １ But, R １ is substituted with a hydroxyl group at the β-position relative to the carbonyl group adjacent to the carbon atom to which is bonded, and at other positions, １－６The glycolipid derivative (IA) is an alkyl group having m carbon atoms, which is optionally further substituted with 1 to 10 (preferably 1 to 5, more preferably 1 to 3) identical or different substituents selected from an alkoxy group, a carboxy group, and an oxo group, and wherein two adjacent substituents on the alkyl group may be bonded to each other to form 1 to 3 cyclopropane rings, which may be the same or different, wherein m is an integer of 15 to 65, n is an integer of 10 to 25, and m+n is an integer of 25 to 90 (preferably an integer of 25 to 50, an integer of 55 to 70, or an integer of 75 to 90).

[0035] Glycolipid Derivative (IC) The glycolipid derivative (IB) above, wherein the glycolipid derivative is trehalose monomycolate derived from an acid-fast bacterium (preferably R. sp4306, R. equi, R. rubber, M. smegmatis, M. bovis BCG, M. intracellulare or M. tuberculosis).

[0036] Glycolipid Derivative (ID) The glycolipid derivative (ID) is the glycolipid derivative (IC) described above, which contains one derivative selected from glycolipid derivatives represented by the following structural formulas, or a combination of two or more derivatives (or is one derivative selected from glycolipid derivatives represented by the following structural formulas, or a combination of two or more derivatives).

[0037]

[0038] Glycolipid derivative (IE) R １ But, R １ is an alkyl group having m carbon atoms and substituted with a hydroxyl group at the β-position relative to the carbonyl group adjacent to the carbon atom to which R is bonded; ２ is an alkyl group having n carbon atoms.

[0039] Glycolipid Derivative (I-F) The glycolipid derivative (I-E) above, which contains (or is) a glycolipid derivative (TMM(C32)) represented by the following structural formula:

[0040]

[0041] (2) Method for Obtaining Glycolipid Derivative (I) As the glycolipid derivative (I), a naturally occurring derivative such as trehalose monomycolate (TMM) isolated and purified from bacteria can be used, or a chemically synthesized derivative can also be used. When TMM is commercially available, it may be purchased and used.

[0042] (Naturally Derived Derivatives) Examples of the bacteria include acid-fast bacteria such as Mycobacterium tuberculosis, Mycobacterium leprae, and nontuberculous mycobacteria. Specific examples include R. sp4306, R. equi, R. rubber, M. smegmatis, M. bovis BCG, M. intracellulare, and M. tuberculosis. Isolation and purification from bacteria can be performed by methods commonly used in the art. In this way, TMM obtained by isolation and purification from bacteria may be obtained not as a single derivative but as a mixture of multiple structurally similar derivatives. For example, TMM obtained by isolation and purification from Mycobacterium tuberculosis is known to be a mixture containing three representative TMM subclasses, as shown for the TMM preparation in Example 2 below. Generally, bacterial-derived TMM is recognized by those skilled in the art as a mixture of multiple structurally similar glycolipid derivatives (subclasses) (see also the subclasses of TMM derived from each bacterium listed in Table 1 in Example 6 below). Therefore, in this specification, when the term "TMM" is used, it includes both cases where it is used as a collective term for multiple structurally similar derivatives (subclasses) and cases where it is used to refer to a single derivative. In the former case, it may be written as "TMMs." As described above, in this embodiment, glycolipid derivative (I) can be used in either an embodiment using a single TMM or an embodiment using a combination of multiple TMMs. Specific examples of the naturally-derived glycolipid derivative (I) include the TMMs described in Example 2 below and the TMMs described in Example 6 below, as described above.

[0043] (Synthesized Derivatives) The glycolipid derivative (I) can be synthesized by condensing the hydroxyl group at the 6-position of trehalose with a fatty acid corresponding to the side chain. A known raw material (e.g., mycolic acid) can be used as the fatty acid corresponding to the side chain. Alternatively, a person skilled in the art can obtain a fatty acid corresponding to the side chain from a known compound by appropriate chemical conversion and use it in the condensation reaction. For the synthesis of the side chain fatty acid and the condensation reaction with the hydroxyl group at the 6-position of trehalose, see, for example, Chem. Commun. , 2015, 51, 5100-5103; Chemistry & Biology 21, January 16 (2014), 67-85, etc. In this embodiment, the glycolipid derivative (I) can be used in either an embodiment using a single synthesized derivative or an embodiment using a combination of multiple synthesized derivatives. A specific example of the glycolipid derivative (I) synthesized above is TMM (C32) described in Example 7 below.

[0044] As described above, when glycolipid derivatives (I) are used in this embodiment, two or more derivatives selected from glycolipid derivatives (I) can be used in combination. The number of glycolipid derivatives (I) to be combined is not particularly limited, and it will be understood by those skilled in the art that this will vary depending on, for example, the type of bacteria used and the conditions for isolation and purification. One example is the use of a combination of 1 to 20 derivatives.

[0045] [Use as a T Cell Activator] (Use as a T Cell Activator) As demonstrated in Examples 1 to 7 below, which were carried out using representative derivatives of the glycolipid derivative (I), the glycolipid derivative (I) or its salt has the effect of activating T cells via antigen presentation of the glycolipid derivative (I) by CD1b molecules. T cell activation by glycolipid derivative (I), whose antigen is presented by CD1b molecules, which have almost no genetic polymorphism in primates, is applicable to an extremely wide range of subjects. Therefore, glycolipid derivative (I) or its salt can be used alone or in the form of a composition containing it as a T cell activator, and those skilled in the art can utilize it appropriately in a manner commonly used in the art.

[0046] The use of glycolipid derivative (I) or a salt thereof as a T cell activator will be described in detail below, taking as an example the case where the glycolipid derivative (I) or a salt thereof is used as a functional adjuvant or vaccine. (Use as a functional adjuvant / vaccine) The glycolipid derivative (I) is also useful as a ligand that activates Mincle. For example, the above-mentioned TMM derived from Mycobacterium tuberculosis, a representative example of glycolipid derivative (I), is known to be a Mincle ligand (see, for example, the above-mentioned Non-Patent Document 1). The above-mentioned TMM (C32) has also been reported to be a Mincle ligand (Chem. Commun., 2015, 51, 5100-5103). Furthermore, Mincle is a receptor that controls innate immunity, and Mincle ligands are known to function as adjuvants (immunostimulants) in immune responses (see, for example, the above-mentioned Non-Patent Document 3). In addition to the above-mentioned known findings, the present inventors have confirmed for the first time that glycolipid derivative (I) or a salt thereof also has the effect of activating T cells. That is, they have discovered for the first time that glycolipid derivative (I), which activates innate immunity, unexpectedly also functions as a T cell ligand and is useful as a dual ligand that can also activate adaptive immunity. Therefore, it has been revealed that glycolipid derivative (I) not only activates innate immunity through Mincle activation and functions as an adjuvant, but also functions as a T cell antigen and activates immune function (humoral immunity) itself through T cell activation. Therefore, glycolipid derivative (I) or a salt thereof is not only useful as an adjuvant, but can also be used as a "functional adjuvant" that itself functions as a vaccine. TDM (trehalose dimycolate (cord factor); TMM is a precursor in the biosynthetic pathway) which, like TMM, is abundant in the cell wall of Mycobacterium tuberculosis, is a Mincle ligand like TMM and is a structural analog of TMM that is expected to act as an adjuvant. However, as shown in Example 3 below, T cell activation was clearly confirmed with TMM, whereas no T cell activation was observed with TDM.Such a large difference in T cell activation due to the difference in the binding mode of lipids on trehalose was something that could not be predicted even by those skilled in the art.

[0047] (Specific Use Form as a Functional Adjuvant) When the glycolipid derivative (I) or a salt thereof is used as a functional adjuvant, its dosage form may be, for example, an aqueous or non-aqueous (e.g., oily, etc.) solution, suspension, emulsion, etc. These can be prepared by mixing the glycolipid derivative (I) or a salt thereof with a pharmaceutically acceptable carrier (e.g., solvent, suspending agent, etc.) and then by methods such as manual shaking, mechanical shaking, ultrasonic dispersion, dispersion using a homomixer, self-emulsification, membrane emulsification, D-phase emulsification, vacuum emulsification, ultra-high pressure emulsification, etc. (hereinafter also referred to as "the present adjuvant"). The present adjuvant may be used in combination with "other adjuvants." Other adjuvants include adjuvants used in the art, such as Freund's incomplete adjuvant, Freund's complete adjuvant, microparticles (e.g., uric acid crystals, silica, aluminum hydroxide gel, polystyrene, asbestos, titanium dioxide, black nickel oxide, etc.), lipopolysaccharides (LPS), etc.

[0048] (Specific Use as a Vaccine) The glycolipid derivative (I) or a salt thereof is useful as a vaccine. More specifically, a T cell activator containing the glycolipid derivative (I) or a salt thereof as an active ingredient is useful as a vaccine for preventing or treating infectious diseases through the activation of T cells. In this context, infectious diseases to be prevented or treated include those caused by bacteria expressing one or more types of trehalose monomycolate in the cell wall or intracellularly, and specific examples of such bacteria include Mycobacterium tuberculosis, Mycobacterium leprae, and nontuberculous mycobacteria.

[0049] The above vaccine is a vaccine containing glycolipid derivative (I) or a salt thereof (hereinafter also referred to as "this vaccine"). This vaccine can be administered by a route selected from the group consisting of oral administration, intramuscular administration, transdermal administration, intradermal administration, subcutaneous administration, intraperitoneal administration, intratracheal administration, nasal administration (intranasal administration), intraocular administration, intravaginal administration, rectal administration, intravenous administration, small intestinal administration, and inhalation administration, with subcutaneous administration and nasal administration being particularly preferred. The content of glycolipid derivative (I) or a salt thereof in this vaccine is not particularly limited and can be adjusted appropriately depending on, for example, the subject of administration, the administration form, the administration route, etc., but in the case of oral administration, intramuscular administration, transdermal administration, intradermal administration, subcutaneous administration, or intraperitoneal administration, the content of glycolipid derivative (I) is, for example, 2 μg to 1000 mg, typically 2 μg to 500 mg, preferably 2 μg to 20 mg, and more preferably 20 μg to 200 μg. In the case of intratracheal administration, nasal administration (transnasal administration), intraocular administration, intravaginal administration, rectal administration, intravenous administration, intraintestinal administration or inhalation administration, the dose is usually 0.01 μg to 1 mg, preferably 0.1 μg to 100 μg.

[0050] The present vaccine can be produced by methods conventionally used in the field of pharmaceutical formulation, such as the methods described in the Sixteenth Edition of the Japanese Pharmacopoeia. The present vaccine may contain a desired pharmaceutically acceptable carrier in addition to the glycolipid derivative (I) or a salt thereof. Various carriers conventionally used in the field of pharmaceutical formulation can be used as the "pharmaceutically acceptable carrier." Specific examples of "pharmaceutically acceptable carriers" that can be used in solid formulations include excipients (e.g., lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, etc.), lubricants (e.g., magnesium stearate, talc, colloidal silica, etc.), binders (e.g., crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethylcellulose, etc.), and disintegrants (e.g., starch, carboxymethylcellulose, calcium carboxymethylcellulose, sodium carboxymethylstarch, L-hydroxypropylcellulose, etc.). Liquid preparations may contain solvents (e.g., water for injection, isotonic saline, alcohol, propylene glycol, macrogol, sesame oil, etc.), solubilizing agents (e.g., polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, triethanolamine, sodium carbonate, sodium citrate, etc.), suspending agents (e.g., surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, glycerin monostearate, etc.; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, etc.), isotonic agents (e.g., glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol, etc.), buffers (e.g., buffer solutions such as phosphates and citrates, etc.), and soothing agents (e.g., benzyl alcohol, etc.).If necessary, formulation additives such as preservatives (e.g., parahydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, sorbic acid, etc.), antioxidants (e.g., sulfites, ascorbic acid, α-tocopherol, etc.), colorants, sweeteners, etc. may be further added. Note that the present vaccine can exert its function simply by containing glycolipid derivative (I) or a salt thereof. However, this does not prevent the inclusion of other antigens, if necessary. In this vaccine, since glycolipid derivative (I) also functions as an adjuvant, other adjuvants are not necessary in principle. However, this does not prevent the inclusion of the above-mentioned "other adjuvants" if necessary.

[0051] The recipient of the present vaccine is not particularly limited as long as it is an animal with an immune system capable of contracting the target infectious disease, but it is, for example, administered to humans. The present vaccine may be administered as a single dose or multiple consecutive doses. When the present vaccine is administered consecutively, the administration period is not particularly limited and may be appropriately determined depending on, for example, the recipient, the administration form, and the administration route. It is typically in the range of 1 to 150 days, and can also be administered for 1 to 120 days, 1 to 60 days, or 1 to 30 days, as needed. By administering the present vaccine to a subject such as a human, infectious diseases can be prevented and / or treated. (Use as a Research Reagent) As described above, the glycolipid derivative (I) or its salt has the effect of activating T cells. Therefore, it can be used as a research reagent when T cell activation is required, for example, in drug research. For example, it can be used in in vitro tests, tests using non-human animals, etc. Such use of the glycolipid derivative (I) or its salt in research is also encompassed within the scope of the present invention.

[0052] Other embodiments provided by the present invention include, for example, those shown below.

[0053] [A-1] A method for activating T cells in an animal having an immune system, comprising administering to the animal an effective amount of glycolipid derivative (I) or a salt thereof defined in the embodiment of the above-mentioned "T cell activator." [A-2] The method for activating T cells according to the above-mentioned [A-1], wherein innate immunity is also activated. [A-3] The method for activating T cells according to the above-mentioned [A-1] or [A-2], wherein administration to the animal is carried out in the form of a vaccine or a functional adjuvant. [A-4] A method for preventing or treating an infectious disease in an animal having an immune system in need thereof, comprising administering to the animal an effective amount of glycolipid derivative (I) or a salt thereof defined in the embodiment of the above-mentioned "T cell activator." Here, for preferred embodiments of glycolipid derivative (I) or a salt thereof, preferred infectious disease, etc., reference can be made to the embodiments specified as glycolipid derivative (I) or a salt thereof, preferred infectious disease, etc. in the embodiments of the above-mentioned "T cell activator."

[0054] [B-1] The glycolipid derivative (I) or a salt thereof defined in the embodiment for the above-mentioned "T cell activator" is used as a T cell activator. [B-2] The glycolipid derivative (I) or a salt thereof defined in the embodiment for the above-mentioned "T cell activator" is used as a T cell activator that can also activate innate immunity. [B-3] The glycolipid derivative (I) or a salt thereof defined in the embodiment for the above-mentioned "T cell activator" is used as a vaccine or a functional adjuvant. [B-4] The glycolipid derivative (I) or a salt thereof according to [B-3] above, wherein the vaccine is for the prevention or treatment of an infectious disease. Here, for preferred embodiments of the glycolipid derivative (I) or a salt thereof, preferred infectious diseases, etc., reference can be made to the embodiments specified as the glycolipid derivative (I) or a salt thereof, preferred infectious diseases, etc. in the embodiments for the above-mentioned "T cell activator."

[0055] [C-1] Use of the glycolipid derivative (I) or a salt thereof defined in the embodiment for "the present T cell activator" above, to produce a T cell activator. [C-2] Use of the glycolipid derivative (I) or a salt thereof defined in the embodiment for "the present T cell activator" above, to produce a T cell activator that can also activate innate immunity. [C-3] Use of the glycolipid derivative (I) or a salt thereof defined in the embodiment for "the present T cell activator" above, to produce a vaccine or a functional adjuvant. [C-4] Use of the glycolipid derivative (I) or a salt thereof according to [C-3] above, wherein the vaccine is for the prevention or treatment of an infectious disease. Here, for preferred embodiments of the glycolipid derivative (I) or a salt thereof, preferred infectious disease, etc., reference can be made to the embodiments specified as the glycolipid derivative (I) or a salt thereof, preferred infectious disease, etc. in the embodiment for "the present T cell activator" above.

[0056] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art may modify the embodiments of the present invention in various aspects without departing from the spirit of the present invention, and such modifications are also included within the scope of the present invention.

[0057] Example 1: Crude lipids extracted from Mycobacterium tuberculosis (M. tb) H37Rv strain were fractionated by thin-layer chromatography using a mixture of chloroform, methanol, and water (16 fractions) and stained with copper(II) acetate-phosphate reagent. NFAT-GFP reporter cells (Biochem. Biophys. Res. Commun. 534:680-686) expressing a TCR (also referred to as "clone 50-TCR") previously confirmed to react with crude lipids from M. tuberculosis were stimulated with the 16 fractions obtained above for 20 hours in the presence of human monocyte-derived dendritic cells (MoDCs). The activity of the reporter cells was evaluated by measuring the expression of GFP and CD69 by flow cytometry (n=2 for each). The evaluation results are shown in Figure 1. In the figure, △ indicates the start position of development, and ▲ indicates the end position of development. As shown in Figure 1, Fr2 was the fraction containing the major active ingredient.

[0058] Example 2 The active fraction (Fr2) in Figure 1 was analyzed by MALDI-TOF / MS, and the results are shown in Figure 2. The MS spectrum of Fr2 had the same pattern as the spectrum of a trehalose monomycolate (TMM) preparation purified from Mycobacterium tuberculosis (M. tb) H37Rv strain (Fujita, Microbiol. 2005), and the major MS values ​​matched the estimated molecular weight of TMM (C76-87). Therefore, the active substance in Fr2 was identified as a TMM (a mixture of subclasses). The structures of the major analogs in the TMM are shown below.

[0059]

[0060] Example 3: NFAT-GFP reporter cells expressing Clone50-TCR were stimulated in the presence of human monocyte-derived dendritic cells (MoDCs) with TMMs (same as the TMMs listed in Table 1 below), which are glycolipid derivatives (I) isolated and purified from Mycobacterium tuberculosis, trehalose dimycolate (TDM; a mixture of analogs) isolated and purified from Mycobacterium tuberculosis in the same manner as TMMs, and a synthetic product (single derivative) of glucose monomycolate (GMM) derived from Mycobacterium tuberculosis (Angew. Chem. Int. Ed. 2020, 59, 7555-7560) (0.3 nmol / well), and the expression of GFP and CD69 was evaluated (n=3 for each). The evaluation results are shown in Figure 3. As shown in Figure 3, "TMM" (glycolipid derivative (I)) exhibited the ability to activate T cells, but neither TDM nor GMM, which have different structures, exhibited the ability to activate T cells.

[0061] Example 4 NFAT-GFP reporter cells expressing Clone50-TCR were stimulated with TMMs (same as the TMMs listed in Table 1 below) (glycolipid derivative (I)) isolated and purified from Mycobacterium tuberculosis (0.3 nmol / well) in the presence of human monocyte-derived dendritic cells (MoDCs) and human CD1 molecule inhibitory antibodies (anti-CD1a, anti-CD1b, anti-CD1c, anti-CD1d, and an isotype control antibody), and the expression of GFP and CD69 was evaluated (n=3 for each). The evaluation results are shown in Figure 4. As shown in Figure 4, the T cell activation ability of "TMM" was inhibited only in the presence of anti-CD1b antibody.

[0062] Example 5: NFAT-GFP reporter cells expressing clone 50-TCR were stimulated (1 nmol / well) with TMM (C32) (Chem. Commun., 2015, 51, 5100-5103), a synthetic product described above for glycolipid derivatives (I-F), in the presence of HEK293 cells overexpressing human CD1a, 1b, 1c, or 1d. GFP and CD69 expression was evaluated (n=3 for each). The results are shown in Figure 5. As shown in Figure 5, the ability of TMM to activate T cells was confirmed only in the presence of HEK293 cells overexpressing human CD1b.

[0063] The results of Examples 4 and 5 revealed that glycolipid derivative (I) exhibits the ability to activate T cells through antigen presentation of glycolipid derivative (I) by CD1b molecules.

[0064] Example 6 NFAT-GFP reporter cells expressing Clone50-TCR were isolated and purified from various acid-fast bacteria in the presence of DC2.4 cells (a mouse dendritic cell line) transduced with human CD1b (references are listed in the "reference" column of the table below). Expression of GFP and CD69 was evaluated (n=3 for each) upon stimulation with TMMs (1 nmol / well), which are glycolipid derivatives (I) with different chain lengths as shown in Table 1 below. The evaluation results are shown in Figure 6. As shown in Figure 6, the various glycolipid derivatives (I) activated T cells.

[0065]

[0066] In the above table, the subclass indicates the structural characteristics of the structural analogues constituting the TMMs derived from each acid-fast bacterium. The notation α indicates the R １ indicates that the alkyl group corresponding to has a cyclopropane ring structure in the alkyl chain. Also, each of the notations "methoxy", "keto", and "dicarboxy" indicates that R in the glycolipid derivative (I) of the TMMs １is substituted with a methoxy group, an oxo group, and two carboxy groups, respectively. Furthermore, the notation α′ indicates that the alkyl group corresponding to R １ In the above table, the carbon-chain length indicates the chain length (total number of carbon atoms) of the lipid side chain (fatty acid side chain forming an ester) of the structural analogue constituting the TMMs. Taking the notation C34-38 as an example, in glycolipid derivative (I), "m(R １ (number of carbon atoms in the hydrocarbon group) + n(R ２ (number of carbon atoms in the hydrocarbon group) + 2(R １ and R ２ This indicates that the number of carbon atoms represented by the notation "(which corresponds to the number of carbon atoms to which the carbon atom is bonded and the number of carbon atoms constituting the adjacent carbonyl group)" is in the range of 34 to 38. The same applies to other notations.

[0067] Example 7 NFAT-GFP reporter cells (Kiyotake, J. Biol. Chem. 2015) co-expressing human Mincle and FcRγ were stimulated with TMMs (same as the TMMs listed in Table 1 below) (glycolipid derivative (I)) isolated and purified from Mycobacterium tuberculosis, and GFP expression was evaluated (n=1). The evaluation results are shown in Figure 7 (left). This test confirmed that TMM, which is glycolipid derivative (I), activates Mincle in a dose-dependent manner, as is known from public knowledge. (2) NFAT-GFP reporter cells expressing Clone50-TCR were stimulated with TMMs (same as the TMMs listed in Table 1 below) (glycolipid derivative (I)) isolated and purified from acid-fast bacteria (R. equi) in the presence of DC2.4 cells (mouse dendritic cell line) transduced with human CD1b, and the expression of GFP and CD69 was evaluated (n=2). The evaluation results are shown in Figure 7 (right panel). This test confirmed that glycolipid derivative (I), "TMM," activates T cells in a dose-dependent manner.

[0068] As described above, it has been demonstrated that glycolipid derivative (I) can directly activate T cells (e.g., CD4-positive memory-like T cells) as a ligand for T cells. It is understood that upon TCR-mediated TMM stimulation, T cells release TNF, IFNγ, and cytotoxic effector molecules to eliminate tuberculosis bacteria (Nunes-Alves C, Nat Rev Microbiol, 12, 289, 2014).

[0069] The present invention provides, as one embodiment, a T cell activator containing a glycolipid derivative as an active ingredient, which is useful, for example, as a vaccine and can also activate innate immunity, and is useful, for example, in the field of medicine. This application is based on Japanese Patent Application No. 2024-004685 (filing date: January 16, 2024), the contents of which are incorporated in full herein.

Claims

1. The following formula (I): (In the formula, R １ represents an optionally substituted hydrocarbon group having m carbon atoms, and R ２ represents an optionally substituted hydrocarbon group having n carbon atoms, and m + n represents an integer of 25 to 100.) A T cell activator containing a glycolipid derivative (hereinafter referred to as "glycolipid derivative (I)") represented by the formula or a salt thereof as an active ingredient.

2. In the glycolipid derivative (I), R １ is, when viewed from the carbonyl group adjacent to the carbon atom to which R １ is attached, a hydroxyl group or a C １－６ alkoxy group at the β-position, and at other positions, a hydrocarbon group which may be further substituted, where two substituents adjacent to each other on the hydrocarbon group may be bonded to each other to form a cyclopropane ring or an oxacyclopropane ring, and which is a hydrocarbon group having m carbon atoms. The T cell activator according to claim 1.

3. In the glycolipid derivative (I), R １ is, when viewed from the carbonyl group adjacent to the carbon atom to which R １ is attached, substituted at the β-position with a hydroxyl group or a C １－６ alkoxy group, and at other positions, is a hydrocarbon group having 1 to 10 substituents, the same or different, selected from a hydroxyl group, a C １－６ alkoxy group, a carboxyl group, and an oxo group, and may be further substituted, where two substituents adjacent to each other on the hydrocarbon group may be bonded to each other to form 1 to 3 cyclopropane rings or oxacyclopropane rings, the same or different, and is a hydrocarbon group having m carbon atoms, and R ２ is a hydrocarbon group having n carbon atoms, which may be substituted with 1 to 10 substituents, the same or different, selected from a hydroxyl group, a C １－６ alkoxy group, a carboxyl group, and an oxo group. The T cell activator according to claim 1.

4. In the glycolipid derivative (I), R ２ is a hydrocarbon group having n carbon atoms, and the T cell activator according to any one of claims 1 to 3.

5. In the glycolipid derivative (I), R １ is a hydrocarbon group having m carbon atoms, and the T cell activator according to any one of claims 1 to 3.

6. In the glycolipid derivative (I), R １ and R ２ wherein the hydrocarbon groups in are each independently an alkyl group or an alkenyl group, the T cell activator according to any one of claims 1 to 3.

7. In the glycolipid derivative (I), R １ and R ２ The T cell activator according to any one of claims 6, wherein the hydrocarbon group in is an alkyl group.

8. The T cell activator according to any one of claims 1 to 3, wherein in the glycolipid derivative (I), m + n is an integer of 25 to 90.

9. The T cell activator according to any one of claims 1 to 3, wherein in the glycolipid derivative (I), m is an integer of 15 to 70 and n is an integer of 10 to 30.

10. The T cell activator according to any one of claims 1 to 3, wherein the glycolipid derivative (I) is trehalose monomycolate derived from mycobacteria (hereinafter also referred to as "TMM").

11. The T cell activator according to claim 10, wherein the glycolipid derivative (I) comprises one derivative selected from the glycolipid derivatives represented by the following structural formulas, or a combination of two or more derivatives.

12. The T cell activator according to any one of claims 1 to 3, wherein the glycolipid derivative (I) contains a glycolipid derivative represented by the following structural formula.

13. The T cell activator according to any one of claims 1 to 3, wherein the activation of T cells is by antigen presentation of the glycolipid derivative (I) by CD1b molecules.

14. The T cell activator according to any one of claims 1 to 3, wherein innate immunity is also activated.

15. The T cell activator according to any one of claims 1 to 3, which is used as a vaccine or a functional additive adjuvant.

16. The T cell activator according to claim 15, wherein the vaccine is for the prevention or treatment of infectious diseases.

17. The T cell activator according to claim 16, wherein the infectious disease is caused by bacteria expressing one or more trehalose monomycolates (TMM) on the cell wall or intracellularly.

18. The T cell activator according to claim 17, wherein the bacteria are Mycobacterium tuberculosis, Mycobacterium leprae, or non-tuberculous mycobacteria.

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