Liposome agent, NKT cell activator, pharmaceutical composition, and uses of these

Abstract

The purpose of the present invention is to provide a composition that is superior for practical use with regard to a ligand for activating NKT cells. A liposome agent according to the present invention is characterized by containing a PEGylated liposome, and is also characterized in that: the PEGylated liposome contains an NKT cell ligand and a liposome-constituting lipid; the NKT cell ligand is an α-galactosylceramide, or an analog thereof, that is specifically recognized by an NKT cell receptor on an NKT cell; and the liposome-constituting lipid contains a choline-containing phospholipid, a PEGylated phospholipid, and a cholesterol-based lipid.

Description

Liposome agent, NKT cell activator, pharmaceutical composition, and uses thereof

[0001] The present invention relates to a liposome agent, an NKT cell activator, a pharmaceutical composition, and uses thereof.

[0002] The immune system is known to have the following mechanism: Immature dendritic cells take up antigens, migrate to lymph nodes, differentiate into mature dendritic cells, and present the antigens. CD8-positive T cells (killer T cells) are then activated by the mature dendritic cells presenting the antigens, acquire cytotoxic activity, and attack cells bearing the antigens. However, in living organisms that have developed cancer, the differentiation of immature dendritic cells into mature cells is suppressed by cancer cells, resulting in the problem of inhibiting the attack on cancer cells using the mechanism described above. For this reason, treatment methods that activate the immune system (so-called immunotherapy) have become common for diseases such as cancer.

[0003] In recent years, attempts have been made to activate natural killer T cells (hereinafter referred to as NKT cells) present in the body of patients as a way to activate the immune system. In this method, for example, a ligand specifically recognized by NKT cells (hereinafter also referred to as NKT cell-activating ligand) is administered as a drug, causing antigen-presenting cells in the body to present the ligand via CD1d. NKT cells are activated by specifically recognizing and binding to the presented ligand. Activation of NKT cells induces differentiation of immature dendritic cells, the differentiation of which is suppressed by cancer cells, into mature dendritic cells. As a result, cancer antigens are presented by mature dendritic cells, enabling killer T cells with cytotoxic activity to attack cancer cells. Furthermore, activated NKTs can activate other immune system cells, such as killer T cells and natural killer cells (hereinafter referred to as NK cells), by releasing cytokines such as interleukin-4 (IL-4) and interferon-γ (IFN-γ), thereby enhancing immune responses. Immunotherapy targeting NKT cells in this way is also called, for example, NKT cell-targeted therapy.

[0004] Regarding the NKT cell-activating ligands, various compounds have already been studied. For example, it has been reported that α-galactosylceramide (α-GalCer) and its analogs activate NKT cells (Patent Documents 1 to 4, Non-Patent Document 1).

[0005] On the other hand, when it comes to drugs, not only efficacy but also stability and effective delivery are important for practical application.

[0006] WO94 / 09020 WO94 / 02168 WO94 / 24142 WO98 / 44928

[0007] Science, 278, p. 1626-1629, 1997

[0008] Therefore, an object of the present invention is to provide a composition that is more suitable for practical use as a ligand that activates NKT cells.

[0009] In order to achieve the above object, the liposome agent of the present invention comprises a PEGylated liposome, wherein the PEGylated liposome comprises an NKT cell-activating ligand and a liposome-constituting lipid, wherein the NKT cell-activating ligand is α-galactosylceramide or an analog thereof that is specifically recognized by an NKT cell receptor on an NKT cell, and wherein the liposome-constituting lipid comprises a choline-containing phospholipid, a PEGylated lipid, and a cholesterol-based lipid.

[0010] The NKT cell activator of the present invention is characterized by comprising the liposome agent of the present invention.

[0011] The pharmaceutical composition of the present invention is characterized by containing the liposome agent of the present invention.

[0012] The method for activating NKT cells of the present invention is characterized by comprising the step of contacting the liposome agent of the present invention with NKT cells.

[0013] The liposome preparation of the present invention has, for example, excellent stability during storage, and can obtain the effect of activating NKT cells in the body.

[0014] FIG. 1 is a graph showing the results of long-term storage stability of B7 liposomes in Example 2. FIG. 2A is a graph showing IFN-γ concentrations in serum and lymph node supernatant in Example 3. FIG. 2B is a graph showing IL-10 concentrations in serum and lymph node supernatant in Example 3. FIG. 3A is a histogram of NKT cells in lymph nodes, showing the distribution of cell numbers for NK1.1-positive and CD3-positive cells in lymph nodes based on the relative fluorescence intensity indicating CD3 positivity. FIG. 3B is a graph quantifying the amount of NK1.1-positive and CD3-positive NKT cells in lymph nodes. FIG. 4A is a histogram of NKT cells in the spleen, showing the distribution of cell numbers for NK1.1-positive and CD3-positive cells in the spleen based on the relative fluorescence intensity. FIG. 4B is a graph quantifying the amount of NK1.1-positive and CD3-positive NKT cells in the spleen. Figure 5A is a histogram of NKT cells in lymph nodes, and is a graph showing the distribution of cell numbers for NK1.1-positive and CD3-positive cells in lymph nodes based on relative fluorescence intensity indicating CD3 positivity. Figure 5B is a histogram of NK cells in lymph nodes, and is a graph showing the distribution of cell numbers for NK1.1-positive NK cells in lymph nodes based on relative fluorescence intensity indicating NK1.1 positivity. Figure 5C is a histogram of T cells in lymph nodes, and is a graph showing the distribution of cell numbers for CD3-positive T cells in lymph nodes based on relative fluorescence intensity indicating CD3 positivity. Figure 5D is a histogram of NKT cells in lymph nodes. Figure 5E is a graph quantifying the amount of NK1.1-positive and CD3-positive NKT cells in lymph nodes. Figure 6A is a histogram of NKT cells in the spleen, which is a graph showing the distribution of cell numbers for NK1.1-positive and CD3-positive cells in the spleen based on the relative fluorescence intensity indicating CD3 positivity. Figure 6B is a histogram of NK cells in the spleen, which is a graph showing the distribution of cell numbers for NK1.1-positive NK cells in the spleen based on the relative fluorescence intensity indicating NK1.1 positivity.Figure 6C is a histogram of T cells in the spleen, showing the distribution of cell numbers of CD3-positive T cells in the spleen based on relative fluorescence intensity indicating CD3 positivity. Figure 6D is a histogram of NKT cells in the spleen. Figure 6E is a graph quantifying the amount of NK1.1-positive and CD3-positive NKT cells in the spleen. Figure 7A is a histogram of dendritic cells in the lymph node, showing the distribution of cell numbers of CD11c-positive cells in the lymph node based on relative fluorescence intensity indicating CD11c positivity. Figure 7B is a histogram of dendritic cells in the lymph node. Figure 7C is a histogram of macrophages in the lymph node, showing the distribution of cell numbers of CD68-positive cells in the lymph node based on relative fluorescence intensity indicating CD68 positivity. Figure 7D is a graph quantifying the amount of CD11c-positive dendritic cells in the lymph node. Figure 7E is a graph quantifying the amount of CD68-positive macrophages in the lymph node. Figure 8A is a histogram of dendritic cells in the spleen, and a graph showing the cell number distribution of CD11c-positive cells in the spleen based on the relative fluorescence intensity indicating CD11c positivity. Figure 8B is a histogram of dendritic cells in the spleen. Figure 8C is a histogram of macrophages in lymph nodes, and a graph showing the cell number distribution of CD68-positive cells in the spleen based on the relative fluorescence intensity indicating CD68 positivity. Figure 8D is a graph quantifying the amount of CD11c-positive dendritic cells in the spleen. Figure 8E is a graph quantifying the amount of CD68-positive macrophages in the spleen. Figure 9A is an image showing fluorescence in each organ of a mouse 24 hours after administration. Figure 9B is a graph showing relative fluorescence intensity in each organ of a mouse 24 hours after administration. Figure 10 is a graph showing the relationship between tumor volume (V) in mice and the number of days since the start of administration. Figure 11 is a graph showing the relationship between mouse survival rate and the number of days since the start of administration. Figure 12 is a graph showing the relationship between mouse body weight (g) and the number of days since the start of administration. Figure 13 is a graph showing the relationship between mouse tumor volume (V) and the number of days since the start of administration. Figure 14 is a graph showing the relationship between mouse survival rate and the number of days since the start of administration.FIG. 15 is a graph showing the relationship between mouse body weight (g) and the number of days since the start of administration. FIG. 16A is a graph showing the relationship between mouse tumor volume (V) and the number of days since the start of administration. FIG. 16B is a graph showing the relationship between mouse body weight (g) and the number of days since the start of administration. FIG. 17 is a graph showing the relationship between mouse tumor volume (V) and the number of days since the start of administration. FIG. 18A is an image showing fluorescence in lymph nodes. FIG. 18B is a graph showing the fluorescence intensity in all excised lymph nodes. FIG. 19 is an image showing fluorescence in the cancer and spleen at the site where cancer cells were inoculated. FIG. 20 is an image showing fluorescence in the liver to which cancer has metastasized. FIG. 21A is a graph showing the distribution of cells based on the fluorescence intensity of FITC and Cy5. FIG. 21B is a graph showing the distribution of cells based on the fluorescence intensity of FITC. FIG. 21C is a graph quantifying the proportion of CD11c-positive cells. FIG. 21D is a graph showing the distribution of CD11c-positive cells based on Cy5 fluorescence intensity. FIG. 21E is a graph showing the results of quantifying the proportion of CD11c-positive and Cy5-positive cells. FIG. 22A is a graph showing the distribution of cells based on FITC fluorescence intensity and Cy5 fluorescence intensity. FIG. 22B is a graph showing the distribution of cells based on FITC fluorescence intensity. FIG. 22C is a graph showing the quantification of the proportion of CD68-positive cells. FIG. 22D is a graph showing the distribution of CD68-positive cells based on Cy5 fluorescence intensity. FIG. 22E is a graph showing the quantification of the proportion of CD68-positive and Cy5-positive cells. FIG. 23A is a graph showing the distribution of cells based on NK1.1 fluorescence intensity and Cy5 fluorescence intensity. FIG. 23B is a graph showing the distribution of cells based on FITC fluorescence intensity. FIG. 23C is a graph showing the quantification of the proportion of NK1.1-positive cells. Figure 23D is a graph showing the distribution of NK1.1-positive cells based on Cy5 fluorescence intensity. Figure 23E shows the results of quantifying the proportion of NK1.1-positive and Cy5-positive cells. Figure 24A is a graph showing the distribution of cells based on FITC fluorescence intensity and Cy5 fluorescence intensity. Figure 24B is a graph showing the distribution of cells based on FITC fluorescence intensity. Figure 24C is a graph quantifying the proportion of CD3-positive cells. Figure 24D is a graph showing the distribution of CD3-positive cells based on Cy5 fluorescence intensity.Figure 24E shows the results of quantifying the proportion of CD3-positive and Cy5-positive cells. Figure 25A is a graph showing the distribution of CD3-positive and NK1.1-positive double-positive cells (NKT cells) by FITC fluorescence intensity. Figure 25B shows the results of quantifying the proportion of CD3-positive and NK1.1-positive double-positive cells (NKT cells). Figure 25C is a graph showing the distribution of CD3-positive and NK1.1-positive double-positive cells (NKT cells) by Cy5 fluorescence intensity. Figure 25D shows the results of quantifying the proportion of CD3-positive / NK1.1-positive / Cy5-positive cells. Figure 26 shows immunohistochemically stained microscopic photographs of mouse cancer section slides. Figure 27A shows a photograph of each organ 12 hours after administration of a mouse administered a B7 liposome sample, with fluorescence intensity superimposed. Figure 27B shows a graph quantifying fluorescence intensity in lymph nodes. Figure 28 is an electron microscope photograph of an administered sample. Figure 29A is a diagram showing the amount of B7 liposome accumulation in mouse lymph nodes as fluorescence intensity. Figure 29B is a graph quantifying the fluorescence intensity indicating the amount of B7 liposome accumulation in mouse lymph nodes. Figure 30 is a graph showing the uptake of B7 liposomes into cells as measured by the fluorescence intensity of Cy5 derived from B7 liposomes. Figure 31 is a graph showing the uptake of B7 liposomes into dendritic cells as measured by the fluorescence intensity of Cy5 derived from B7 liposomes. Figure 32 is a graph showing the uptake of B7 liposomes into dendritic cells as measured by the fluorescence intensity of Cy5 derived from B7 liposomes. Figure 33 is a fluorescence microscope image of a mouse lymph node section. Figure 34A is a graph showing the relationship between mouse tumor volume (V) and the number of days since the start of administration, Figure 34B is a graph showing the relationship between mouse survival rate and the number of days since the start of administration, Figure 34C is a graph showing the relationship between mouse body weight and the number of days since the start of administration, Figure 35A is a graph showing the relationship between mouse tumor volume (V) and the number of days since the day of subcutaneous instillation of the cancer, Figure 35B is a graph showing the relationship between mouse survival rate and the number of days since the day of subcutaneous instillation of the cancer, and Figure 35C is a graph showing the relationship between mouse body weight and the number of days since the day of subcutaneous instillation of the cancer.

[0015] [1] A liposome preparation comprising a PEGylated liposome, the PEGylated liposome comprising an NKT cell activating ligand and a liposome-constituting lipid, the NKT cell activating ligand being α-galactosylceramide or an analog thereof that is specifically recognized by an NKT cell receptor on an NKT cell, and the liposome-constituting lipids comprising a choline-containing phospholipid, a PEGylated lipid, and a cholesterol-based lipid. [2] The liposome preparation according to [1], wherein the choline-containing phospholipid is phosphatidylcholine. [3] The liposome preparation according to [2], wherein two fatty acids in the phosphatidylcholine are each independently a saturated fatty acid or an unsaturated fatty acid. [4] The liposome preparation according to [2] or [3], wherein two fatty acids in the phosphatidylcholine are each independently a fatty acid having 8 to 27 carbon atoms. [5] The liposome preparation according to any one of [2] to [4], wherein the phosphatidylcholine is at least one selected from the group consisting of dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, palmitoyl oleoyl phosphatidylcholine, and stearoyl oleoyl phosphatidylcholine. [6] The liposome preparation according to any one of [1] to [5], wherein the fatty acid in the PEGylated lipid is a saturated fatty acid or an unsaturated fatty acid. [7] The liposome preparation according to any one of [1] to [6], wherein the fatty acid in the PEGylated lipid is a fatty acid having 8 to 70 carbon atoms. [8] The liposome preparation according to any one of [1] to [7], wherein the PEGylated lipid is at least one selected from the group consisting of DSPE-PEG, DMG-PEG, DPPE-PEG, DSG-PEG, DOPE-PEG, ceramide-PEG, and stearic acid-PEG. [9] The liposome preparation according to any one of [1] to [8], wherein the average molecular weight of PEG in the PEGylated lipid is 160 to 44,000 Da.

[10] The liposome preparation according to any one of [1] to [9], wherein the terminal of PEG in the PEGylated lipid is a hydroxyl group.

[11] The liposome preparation according to any one of [1] to

[10] , wherein the cholesterol-based lipid is cholesterol.

[12] The liposome preparation according to any one of [1] to

[11] , wherein the size of the PEGylated liposome is in the range of 10 to 500 nm in average particle size.

[13] The liposome preparation according to any one of [1] to

[12] , wherein in the PEGylated liposome, the mol % ratio of the choline-containing phospholipid (X), the PEGylated lipid (Y), and the cholesterol-based lipid (Z) is X:Y:Z=1:(0.03 to 0.176):(0.34 to 1.35).

[14] The liposome preparation according to any one of [1] to

[13] , wherein the α-galactosylceramide and analog thereof is a compound represented by formula (I), a salt thereof, or a solvate thereof.

[15] The liposome preparation according to any one of [1] to

[13] , wherein the α-galactosylceramide analog is a compound represented by formula (II), a salt thereof, or a solvate thereof:

[16] The liposome preparation according to any one of [1] to

[13] , wherein the α-galactosylceramide analog is a compound represented by formula (III), a salt thereof, or a solvate thereof:

[17] The liposome preparation according to any one of [1] to

[13] , wherein the α-galactosylceramide analog is a compound represented by formula (IV), a salt thereof, or a solvate thereof:

[18] The liposome preparation according to any one of [1] to

[13] , wherein the α-galactosylceramide analog is a compound represented by formula (V), a salt thereof, or a solvate thereof:

[19] The liposome preparation according to any one of [1] to

[13] , wherein the α-galactosylceramide analog is a compound represented by formula (VI), a salt thereof, or a solvate thereof:

[20] The liposome preparation according to

[19] , wherein the α-galactosylceramide analog is a compound represented by formula (VI-1), a salt thereof, or a solvate thereof:

[21] The liposome preparation according to any one of [1] to

[20] , wherein the PEGylated liposome satisfies conditions 1, 2, and 3. (Condition 1) The size of the PEGylated liposome has an average particle size in the range of 60 to 140 nm. (Condition 2) In the PEGylated lipid, the terminal of PEG is a hydroxyl group. (Condition 3) The surface charge of the PEGylated liposome is negative.

[22] The liposome preparation according to

[21] , wherein in condition 3, the surface charge of the PEGylated liposome is −80 to 5 mV.

[0016]

[23] An NKT cell activator comprising the liposome agent according to any one of [1] to

[22] .

[24] A pharmaceutical composition comprising the liposome agent according to any one of [1] to

[22] .

[25] The pharmaceutical composition according to

[24] , which is for anticancer use.

[26] The pharmaceutical composition according to

[25] , wherein the target cancer is breast cancer, skin cancer, blood cancer, colon cancer, prostate cancer, or ovarian cancer.

[27] The pharmaceutical composition according to any one of

[24] to

[26] , which is an injection.

[28] The pharmaceutical composition according to any one of

[24] to

[27] , which is for anticancer use, wherein the target cancer is triple-negative breast cancer, and wherein the PEGylated liposome contained in the liposome agent satisfies conditions 1, 2, and 3. (Condition 1) The size of the PEGylated liposome has an average particle size in the range of 60 to 140 nm. (Condition 2) In the PEGylated lipid, the terminal of PEG is a hydroxyl group. (Condition 3) The surface charge of the PEGylated liposome is negative.

[29] The pharmaceutical composition according to

[28] , wherein, in the condition 3, the surface charge of the PEGylated liposome is −80 to 5 mV.

[0017]

[30] A method for activating NKT cells, comprising the step of administering to a subject the liposome agent described in any one of [1] to

[22] or the pharmaceutical composition described in any one of

[24] to

[29] .

[31] The method for activating NKT cells described in

[30] , wherein the subject is a human or a non-human animal.

[32] A method for treating cancer, comprising the step of administering to a subject the liposome agent described in any one of [1] to

[22] or the pharmaceutical composition described in any one of

[24] to

[29] .

[33] The method for treating cancer described in

[32] , wherein the subject is a subject with triple-negative breast cancer, and wherein the pharmaceutical composition described in

[28] or

[29] is administered in the administering step.

[34] The method for treating cancer described in

[32] or

[33] , wherein the subject is a human or a non-human animal.

[35] The liposome agent described in any one of [1] to

[22] , for use in activating NKT cells.

[36] The liposome preparation according to any one of [1] to

[22] , for use in producing a pharmaceutical composition.

[0018] Unless otherwise specified, terms used in this specification can be used in the sense commonly used in the art.

[0019] Liposomes are lipid vesicles that form a lipid bilayer.

[0020] 1. Liposome Preparation As described above, the liposome preparation of the present invention comprises a PEGylated liposome, the PEGylated liposome comprising an NKT cell-activating ligand and liposome-constituting lipids, the NKT cell-activating ligand being α-galactosylceramide or an analog thereof that is specifically recognized by the NKT cell receptor on an NKT cell, and the liposome-constituting lipids comprising a choline-containing phospholipid, a PEGylated lipid, and a cholesterol-based lipid. The liposome preparation of the present invention will be described below using examples, but the present invention is not limited to these examples. Furthermore, the description of other embodiments can be used for the liposome preparation of the present invention.

[0021] (1) NKT cell-activating ligand The NKT cell-activating ligand is typically a compound that is specifically recognized by an NKT cell-specific T cell receptor (NKT cell receptor) on NKT cells and can specifically activate NKT cells. The NKT cell-activating ligand is presented, for example, on a CD1d molecule. The "NKT cell-activating ligand" of the present invention may be, for example, a compound that is specifically recognized by the NKT cell receptor and can specifically activate NKT cells. In the present invention, the NKT cell-activating ligand is not particularly limited, and examples include glycolipids that are already known to have a specific activation function for NKT cells, namely, α-galactosylceramide (α-GalCer) and derivatives (analogs thereof). α-GalCer and its derivatives are exemplified below as examples of the NKT cell-activating ligand.

[0022] (1-1) A compound of formula (I), a salt thereof, or a solvate thereof.

[0023] In formula (I), R １１ is H or OH, X １ is an integer from 7 to 27, and R ２１ is a substituent selected from the group consisting of the following (a) to (e) (Y1 is an integer of 5 to 17): (a) —CH ２ (CH ２ ) Ｙ１ CH ３ (b) -CH(OH)(CH ２ ) Ｙ１ CH ３ (c) -CH(OH)(CH ２ ) Ｙ１ CH (CH ３ ) ２ (d) -CH=CH(CH ２ ) Ｙ１ CH ３ (e) -CH(OH)(CH ２ ) Ｙ１ CH (CH ３ ) CH ２ CH ３

[0024] In formula (I), R ３１ ~R９１ is a substituent defined in i) or ii) below. ３１ , R ６１ , and R ８１ When is H, R ４１ is H, OH, NH ２ , NHCOCH ３ or a substituent selected from the group consisting of the following groups (A) to (D): ５１ is OH or a substituent selected from the group consisting of the following groups (E) and (F), ７１ is OH or a substituent selected from the group consisting of the following groups (A) to (D), ９１ is H, CH ３ , C.H. ２ OH or a substituent selected from the group consisting of the following groups (A') to (D'): ３１ , R ６１ and R ７１ When is H, R ４１ is H, OH, NH ２ , NHCOCH ３ or a substituent selected from the group consisting of the following groups (A) to (D): ５１ is OH or a substituent selected from the group consisting of the following groups (E) and (F), ８１ is OH or a substituent selected from the group consisting of the following groups (A) to (D), ９１ is H, CH ３ , C.H. ２ It is OH or a substituent selected from the group consisting of the following groups (A') to (D').

[0025]

[0026] A preferred example of the compound of formula (I) is α-GalCer (α-galactosylceramide) shown in the following formula (a): α-GalCer has the chemical name (2S,3S,4R)-1-O-(α-D-galactopyranosyl)-2-hexacosanoylamino-1,3,4-octadecanetriol.

[0027]

[0028] The compound of formula (I), its salt, or solvate can be, for example, those described in patent publications (WO94 / 09020, WO94 / 02168, WO94 / 24142, WO98 / 44928, etc.) and papers (Science, 278, pp. 1626-1629, 1997), and can be produced by the methods described therein. It has already been confirmed in these documents that the compound of formula (I) has the function of the NKT cell-activating ligand.

[0029] (1-2) A compound of formula (II), a salt thereof, or a solvate thereof.

[0030] In formula (II), R １ indicates an α-carba sugar residue, and R ２ and R ３ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and X represents an oxygen atom, a sulfur atom, or —CH ２ - or NH-, and Y is -CH ２ -, -CH(OH)- or CH=CH-.

[0031] Of the compounds of formula (II), the following compounds are preferred. [1] (2S,3S,4R)-1-(5a-carba-α-D-galactopyranosyloxy)-2-(hexacosanoylamino)-3,4-octadecanediol [2] (2S,3S,4R)-1-(5a-carba-α-D-galactopyranosylthio)-2-(hexacosanoylamino)-3,4-octadecanediol [3] (2S,3S,4R)-1-(5a-carba-α-D-glucopyranosyloxy)-2-(hexacosanoylamino)-3,4-octadecanediol [4] (2S,3S,4R)-1-(5a-carba-α-D-glucopyranosylthio)-2-(hexacosanoylamino)-3,4-octadecanediol [5] (2S,3S,4R)-1-(5a-carba-α-D-fucopyranosyloxy)-2-(hexacosanoylamino)-3,4-octadecanediol

[0032] The compound of formula (II), its salt, or solvate may be, for example, those described in patent publications (WO 2008 / 102888, U.S. Patent No. 8,299,223, etc.), and may be produced by the methods described therein. It has already been confirmed in these publications that the compound of formula (II), etc., has the function of the NKT cell-activating ligand.

[0033] (1-3) A compound of formula (III), a salt thereof, or a solvate thereof.

[0034] In formula (III), R １ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom; R ２ and R ３ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents —CH ２ -, -CH(OH)- or CH=CH-, where R １ is a hydrogen atom, R ２ represents a substituted or unsubstituted hydrocarbon group having 24 to 28 carbon atoms.

[0035] Of the compounds of formula (III), the following compounds A12, A13, A14, and A15 are particularly preferred.

[0036] A12

[0037] A13

[0038] A14

[0039] A15

[0040] The compound of formula (III), its salt, or solvate may be, for example, those described in patent publications (WO 2009 / 119692, U.S. Patent No. 8,551,959, etc.), and may be produced by the methods described therein. It has already been confirmed in these publications that the compound of formula (III), etc., has the function of the NKT cell-activating ligand.

[0041] (1-4) A compound of formula (IV), a salt thereof, or a solvate thereof.

[0042] In formula (IV), R １ represents a hydrocarbon group having 1 to 30 carbon atoms, and R ２ represents a hydrocarbon group having 1 to 20 carbon atoms, and R ３ represents a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms. ４ and R ５ may be the same or different and represent a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms, or R ４ and R ５ may combine with the adjacent ethylenedioxy to form a divalent hydrocarbon group having 1 to 5 carbon atoms, and may form a ring structure with the adjacent ethylenedioxy.

[0043] The compound of formula (IV), its salt, or solvate may be, for example, those described in patent publications (WO 2010 / 030012, U.S. Patent No. 8,580,751, etc.), and may be produced by the methods described therein. It has already been confirmed in these publications that the compound of formula (IV) and the like have the function of the NKT cell-activating ligand.

[0044] (1-5) A compound of formula (V), a salt thereof, or a solvate thereof.

[0045] In formula (V), R １ represents an aldopyranose residue, the hydroxyl group at position 6 may be alkylated, R ２ represents a hydrocarbon group having 1 to 26 carbon atoms, which may have a substituent; R ３ represents a hydrocarbon group having 1 to 26 carbon atoms, which may have a hydrogen atom or a substituent; R ４ represents a hydrocarbon group having 1 to 21 carbon atoms, which may have a substituent; X represents an oxygen atom or CH ２ -, and Y is -CH ２ -, -CH(OH)- or CH=CH-.

[0046] Of the compounds of formula (V), the following compounds are particularly preferred: [1] (2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-(tetracosanylureido)-3,4-octadecanediol [2] (2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-(hexadecanylureido)-3,4-octadecanediol [3] (2S,3S,4R)-1-(6-O-methyl-α-D-galactopyranosyloxy)-2-(tetracosanylureido)-3,4-octadecanediol

[0047] The compound of formula (V), its salt, or solvate can be, for example, those described in patent publications (WO 2011 / 552842, U.S. Patent No. 8,853,173, etc.), and can be produced by the methods described therein. It has already been confirmed in these publications that the compound of formula (V) and the like have the function of the NKT cell-activating ligand.

[0048] (1-6) A compound of formula (VI), a salt thereof, or a solvate thereof.

[0049] In formula (VI), X represents an alkylene group or —NH—; １ and R ２ are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group, and may have a substituent; R １ and R ２ may form a 5- or 6-membered ring together with the adjacent nitrogen atom, R ３ represents a hydrocarbon group having 1 to 20 carbon atoms, R ４ represents a hydrocarbon group having 1 to 30 carbon atoms.

[0050] Of the compounds of formula (VI), the following compounds are preferred:

[0051] B7

[0052] B16

[0053] The compound of formula (VI), its salt, or solvate can be, for example, those described in patent publications (WO 2013 / 162016 A1, U.S. Patent No. 2015 / 152128 A1, etc.), and can be produced by the methods described therein. It has already been confirmed in these publications that the compound of formula (VI) and the like have the function of the NKT cell-activating ligand.

[0054] Among the NKT cell-activating ligands, for example, from the viewpoint of more potently inducing IFN-γ production by NKT cells, the compounds of formula (III) or (VI) are preferred, more preferably A14, B16, and B7, and particularly preferably B7.

[0055] (2) Lipids Constituting Liposomes The lipids constituting liposomes include a choline-containing phospholipid, a PEGylated lipid, and a cholesterol-based lipid. The lipids constituting liposomes may contain, for example, only the choline-containing phospholipid, the PEGylated lipid, and the cholesterol-based lipid, or may further contain other components. The other components may be any components and in any amounts that do not inhibit liposome formation by the lipids constituting liposomes.

[0056] (2-1) Choline-containing phospholipid The choline-containing phospholipid has a structure in which two fatty acids are ester-bonded to a glycerol backbone as a hydrophobic portion, and choline is phosphate-bonded to the glycerol backbone as a hydrophilic portion. An example of the choline-containing phospholipid is phosphatidylcholine. The choline-containing phospholipid may be PEGylated or not PEGylated (non-PEGylated), and is preferably a non-PEGylated choline-containing phospholipid.

[0057] The fatty acid has, for example, a structure having a carboxyl group at the end of the carbon chain, and may be a saturated fatty acid whose carbon chain is saturated, or an unsaturated fatty acid whose carbon chain has a double bond.In the choline-containing phospholipid, the two fatty acids may each independently be saturated or unsaturated fatty acids.The combination of the two fatty acids may, for example, be a combination of saturated fatty acids, a combination of unsaturated fatty acids, or a combination of saturated and unsaturated fatty acids.The two fatty acids may, for example, each independently be branched or unbranched fatty acids.The two fatty acids may, for example, have the same number of carbon atoms or different numbers of carbon atoms, or may have the same length of carbon chain (main chain) or different lengths of carbon chain (main chain).

[0058] The number of carbon atoms in the fatty acid is, for example, 8 to 70, 24 to 70, or 42 to 58. The number of carbon atoms in the main chain of the fatty acid is, for example, 4 to 35, 12 to 35, or 21 to 29.

[0059] The choline-containing phospholipids include, for example, combinations of fatty acids as shown in the table below, and specific examples include the following phosphatidylcholines.

[0060]

[0061] (2-2) PEGylated Lipid The PEGylated lipid is a phospholipid modified with PEG. The PEGylated lipid has a structure in which two fatty acids are ester-bonded to a glycerol backbone as a hydrophobic portion, and PEG is ester-bonded to the glycerol backbone as a hydrophilic portion.

[0062] The fatty acid may be, for example, a saturated fatty acid or an unsaturated fatty acid. The fatty acid may be, for example, a branched fatty acid or an unbranched fatty acid.

[0063] The number of carbon atoms in the fatty acid is, for example, 8 to 70, 24 to 70, or 42 to 58. The number of carbon atoms in the main chain of the fatty acid is, for example, 4 to 35, 12 to 35, or 21 to 29.

[0064] In the PEGylated lipid, the average molecular weight of PEG (polyethylene glycol) is, for example, 160 Da, 162 Da, 1,800 Da, or 4,500 Da at the lower limit and 44,000 Da, 5,500 Da, or 2,200 Da at the upper limit, and ranges of 160 to 44,000 Da, 162 to 44,000 Da, 1,800 to 5,500 Da, or 1,800 to 2,200 Da. The average molecular weight is, for example, a number average molecular weight, and can be measured by gel permeation chromatography (GPC), NMR, or the like.

[0065] Specific examples of the PEGylated lipid are shown in the table below. The table below also shows examples of lipids in the PEGylated lipid and their fatty acids. The lipid may be, for example, a phospholipid. Examples of the phospholipid include phosphatidylcholine and phosphoethanolamine-containing phospholipids. The fatty acids exemplified in the table below may vary in carbon number, for example, within the range of C8 to C22.

[0066]

[0067] (2-3) Cholesterol-Based Lipids As the cholesterol-based lipids, for example, reference can be made to articles by Konrad B. et al. (Science, 1965, Vol. 150, p. 3692) and Nsairat H. et al. (J. Liposome Res., 2023), and specific examples that can be used include cholesterol, β-sitosterol, sodium cholate, and sodium dodecyl sulfate.

[0068] The cholesterol lipid may be, for example, a PEGylated cholesterol-based lipid or a non-PEGylated cholesterol-based lipid, with the former being preferred.

[0069] (3) PEGylated Liposomes As described above, the liposome preparation of the present invention contains PEGylated liposomes as liposomes. As described above, the liposomes contained in the liposome preparation of the present invention are liposomalized with the liposome-constituting lipids, and the liposome-constituting lipids contain the PEGylated phospholipids, and therefore are called PEGylated liposomes.

[0070] The liposome preparation of the present invention may contain, for example, one or two or more of the PEGylated liposomes. In the latter case, the PEGylated liposomes may be, for example, monodisperse liposomes containing molecules of a single molecular weight, or polydisperse liposomes containing molecules of different molecular weights.

[0071] The size and distribution of the PEGylated liposomes are not particularly limited. The size and distribution of the PEGylated liposomes can be measured by analytical methods such as dynamic light scattering (DLS), laser diffraction, SEM, and TEM. The size of the PEGylated liposomes contained in the liposomal agent of the present invention can be expressed, for example, by particle size distribution (particle diameter distribution), and the above-mentioned analytical methods can be used. The particle size distribution can be expressed, for example, by a frequency distribution showing the relationship between frequency (%) and particle diameter, or by an integrated distribution showing the relationship between cumulative (%) and particle diameter.

[0072] When evaluating size by dynamic light scattering, for example, a zeta potential measuring device (for example, Zetasizer, product name, manufactured by Malvern Instruments Ltd.) equipped with a diode laser (for example, λ = 532 nm) can be used. The evaluation conditions are not particularly limited, and for example, the detection temperature is 22 to 28°C (specifically, 25°C). The intensity-averaged hydrodynamic diameter and polydispersity index can be determined, for example, according to the cumulant method.

[0073] The size of the PEGylated liposomes contained in the liposome preparation of the present invention can be expressed, for example, by the average value (average particle size), median value (also referred to as the median or D50), mode (also referred to as the mode or peak), distribution width, etc. As specific examples, the PEGylated liposomes preferably have the following sizes when their size is confirmed, for example, by dynamic light scattering. Note that, in the present invention, the method for analyzing the size of the PEGylated liposomes is not limited to dynamic light scattering.

[0074] The PEGylated liposome preferably has a Z-average hydrodynamic diameter (Z-Average) measured by, for example, a dynamic scattering method using the zeta potential measuring device, within a predetermined range. The lower limit of the Z-average hydrodynamic diameter (Z-Average) is, for example, 10 nm or more, 30 nm or more, 50 nm or more, 80 nm or more, or 90 nm or more, and the upper limit is, for example, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, or 110 nm or less, and the range is, for example, 10 to 500 nm, 30 to 500 nm, 10 to 400 nm, 30 to 400 nm, 30 to 300 nm, 50 to 200 nm, 80 to 120 nm, or 100±50 nm, 100±40 nm, 100±30 nm, 100±20 nm, or 100±10 nm.

[0075] The Z-average hydrodynamic diameter (Z-Average) is a hydrodynamic diameter based on the ISO standard and can be determined by the cumulants method. The Z-Average can be calculated, for example, using a correlation function obtained from measurement data by dynamic light scattering (DLS) and the following formula:

[0076]

[0077] For example, in the case of a normal distribution, the standard deviation of the size of the PEGylated liposome is within ±2 STD, or within ±1 STD.

[0078] The size distribution of the PEGylated liposomes can be expressed, for example, by the polydispersity index (PDI). The PDI is an index for evaluating the width of particle size distribution. The PDI of the PEGylated liposomes is, for example, 1, or 0.1 to 0.5, 0.15 to 0.35, or 0.2 to 0.3. The PDI can be determined, for example, from fitting analysis of the distribution measured by the dynamic light scattering method using the cumulant method.

[0079] In the PEGylated liposome, the ratio (X:Y:Z) of the choline-containing phospholipid (X), the PEGylated lipid (Y), and the cholesterol-based lipid (Z) is not particularly limited. Specific examples, in terms of mol%, can be given as follows: X:Y:Z=1:0.03-0.176:0.34-1.35, 1:0.05-0.176:0.34-0.675, 1:0.06-0.088:0.5-0.675. Furthermore, the ratio (X+Y+Z:L) of the NKT ligand (L) to these liposome-constituting lipids (X+Y+Z) is not particularly limited. Specific examples, in terms of mol%, can be given as follows: (X+Y+Z):L=1:(0.05-2), 1:(0.2-1), 1:(0.5-1).

[0080] In the PEGylated liposome, the amount of the NKT ligand is not particularly limited. When the amount of the PEGylated liposome is converted into total lipid, the amount of the NKT ligand per 1 mg of total lipid in the PEGylated liposome is not particularly limited, and the lower limit is, for example, more than 0 μg, 5 μg or more, 10 μg or more, 20 μg or more, 50 μg or more, or 100 μg or more, and the upper limit is, for example, 1000 μg or less, 900 μg or less, 800 μg or less, 700 μg or less, 600 μg or less, or 500 μg or less, and the range is, for example, more than 0 to 1000 μg, 5 to 900 μg, 10 to 900 μg, 20 to 900 μg, 20 to 800 μg, 50 to 700 μg, 50 to 600 μg, or 100 to 500 μg.

[0081] (4) Method for Preparing PEGylated Liposomes The method for preparing the PEGylated liposomes is not particularly limited, and any general liposome preparation method using lipid raw materials can be employed, as long as the NKT cell-activating ligand and the liposome-constituting lipids are used as raw materials. For liposome preparation methods, reference can be made to, for example, a paper (O. H. Voss et al., Curr. Protoc. Immunol., 2018, vol. 120-14, pp. 1-21) or the like. Examples of preparation methods are shown below, but the present invention is not limited to these descriptions.

[0082] A raw material solution is prepared using the NKT cell activating ligand and the liposome-constituting lipids as raw materials. The raw material solution may be, for example, a ligand solution containing the NKT cell activating ligand and a lipid mixture solution containing the choline-containing phospholipid, the PEGylated phospholipid, and the cholesterol-based lipid, each prepared separately. Alternatively, the ligand solution, the choline-containing phospholipid-containing solution, the PEGylated phospholipid-containing solution, and the cholesterol-based lipid-containing solution may each be prepared separately. The solvent for the raw material solution is not particularly limited, and examples thereof include organic solvents. Examples of organic solvents include alcohols such as methanol, chloroform, dichloromethane, and acetonitrile, and any of these may be used alone or in combination. The raw material solution may be, for example, a diluted solution diluted with the organic solvent.

[0083] Next, the various raw material solutions are mixed. For example, the ligand solution and the lipid mixture solution may be mixed, or the ligand solution, the choline-containing phospholipid-containing solution, the PEGylated phospholipid-containing solution, and the cholesterol-based lipid-containing solution may be mixed. In this case, the mixing ratio (X:Y:Z) of the choline-containing phospholipid (X), the PEGylated lipid (Y), and the cholesterol-based lipid (Z), and the mixing ratio (X+Y+Z:L) of the NKT ligand (L) to these liposome-constituting lipids (X+Y+Z) are not particularly limited, and the mol% ratios described above can be exemplified.

[0084] Then, the solvent fraction is evaporated from the raw material mixture obtained by mixing the raw materials. Evaporation of the solvent fraction can be performed, for example, by placing the raw material mixture in a container such as a flask and subjecting it to an evaporator. A lipid membrane can be formed on the inner surface of the container by evaporation of the solvent fraction. This lipid membrane is a lipid membrane composed of PEGylated liposomes containing the NKT cell-activating ligand.

[0085] The lipid membrane formed on the inner surface of the container is then suspended in, for example, an aqueous solvent and subjected to ultrasonic treatment. By disrupting the lipid membrane by ultrasonic treatment, the lipid membrane can be decomposed into PEGylated liposomes containing the NKT cell-activating ligand. The aqueous solvent is not particularly limited, and examples thereof include water, saline, buffered saline, and buffer solutions.

[0086] The lipid membrane disintegrate (a mixture of PEGylated liposomes) can be classified into liposome fractions of a desired size and distribution, for example, by filtration using a membrane of a desired pore size.

[0087] (5) Form of liposome preparation The form of the liposome preparation of the present invention is not particularly limited and may be liquid or solid. In the case of a liquid, for example, it is a dispersion in a solvent. The solvent is, for example, a solvent in which the PEGylated liposome does not dissolve, and specific examples include the above-mentioned aqueous solvents. In the case of a solid, for example, it is a state in which the aqueous solvent is removed from the dispersion, or a state (dried body) in which the aqueous solvent is removed and then dried.

[0088] When the liposome preparation of the present invention is a liquid, the concentration of the PEGylated liposome is not particularly limited. In the liposome preparation of the present invention, the concentration of the PEGylated liposome can be expressed, for example, using the lipid amount of the PEGylated liposome. The concentration of the PEGylated liposome preparation in the liposome preparation of the present invention, converted into the lipid amount of the PEGylated liposome, can have a lower limit of 0.05 mg / mL, 0.1 mg / mL, or 0.5 mg / mL, an upper limit of 1 mg / mL, 5 mg / mL, or 25 mg / mL, or a range of 0.05 to 25 mg / mL, 0.1 to 5 mg / mL, or 0.5 to 1 mg / mL. The concentration may be, for example, the concentration of the liposome preparation of the present invention during storage or during use.

[0089] The conditions for storing the liposome preparation of the present invention are not particularly limited. The storage temperature is, for example, 2 to 15°C or 3 to 5°C, and the storage period is, for example, 90 days or less, 30 days or less, or 14 days or less.

[0090] The liposome preparation of the present invention may contain, for example, other additives in addition to the PEGylated liposome. For example, the additives in the NKT cell activator of the present invention described below can be used. For example, the descriptions of the NKT cell activator of the present invention and the pharmaceutical composition of the present invention described below can be used for the liposome preparation of the present invention.

[0091] (6) Properties of the liposome preparation The liposome preparation of the present invention is, for example, excellent in stability. The stability of the liposome preparation of the present invention can be expressed, for example, by a change in the particle size distribution of the PEGylated liposomes contained in the liposome preparation over time. The stability can be evaluated, for example, by stability over a storage period (stability 1) or stability upon dilution after storage (stability 2).

[0092] The storage stability 1 can be evaluated, for example, by the presence or absence or amount of aggregates in the dispersion after a certain period of time has elapsed. １ ) was immediately prepared under the same conditions. ０) and can be compared to determine whether or not aggregates have occurred, and if so, the extent of the occurrence of aggregates, etc.

[0093] The stability 2 due to dilution after storage can be evaluated, for example, by the presence or absence or amount of aggregates in a diluted solution obtained by diluting the dispersion after a certain period of time has elapsed. １ ) diluted with dilution solution (d １ ) under the same conditions, the dispersion (D ０ ) was prepared and diluted immediately after dilution (d ０ ) and can be compared to determine whether or not aggregates have occurred, and if so, the extent of the occurrence of aggregates, etc.

[0094] In the liposome preparation of the present invention, the surface charge of the PEGylated liposome is not particularly limited. The surface charge may be, for example, −80 mV to 5 mV, and is preferably a negative charge, as exemplified below.

[0095] (7) Use of Liposome Preparation The liposome preparation of the present invention can be used, for example, in the NKT cell activator and method for activating NKT cells of the present invention, the pharmaceutical composition and treatment method of the present invention, etc., which will be described later. The description of each embodiment described later can be used for the method of using the liposome preparation of the present invention.

[0096] 2. NKT cell activator and method for activating NKT cells As described above, the NKT cell activator of the present invention is characterized by comprising the liposome agent of the present invention. Furthermore, as described above, the method for activating NKT cells of the present invention is characterized by using the liposome agent of the present invention. The NKT cell activator and activation method of the present invention are characterized by using the liposome agent of the present invention, and other conditions are not particularly limited. Furthermore, the descriptions of other embodiments can be used for the NKT cell activator and activation method of the present invention.

[0097] (1) NKT cell activator The NKT cell activator of the present invention comprises the liposome agent of the present invention. The NKT cell activator of the present invention may, for example, comprise only the liposome agent of the present invention, or may comprise other additives. The additives are preferably, for example, pharmaceutically acceptable additives. Examples of pharmaceutically acceptable additives include those known in the pharmaceutical field.

[0098] Examples of the additives include carriers, diluents, solvents, excipients, stabilizers, isotonic agents, etc. Examples of the carriers include monosaccharides such as sucrose, polysaccharides such as cellulose, and the aqueous solvents mentioned above. Examples of the diluents and excipients include those similar to the carriers mentioned above. Examples of the solvents include the aqueous solvents mentioned above. Furthermore, drugs such as protein drugs, such as PD-L1 (Programmed Cell Death Ligand 1) and antibodies, and cellular drugs can also be used in combination as the additives. It is preferable that the additives be hydrophilic, weakly hydrophobic, etc.

[0099] In the NKT cell activator of the present invention, for example, the PEGylated liposome may be adjusted to a concentration at the time of use, or may be at a higher concentration than that and be diluted at the time of use. The concentration of the PEGylated liposome in the NKT cell activator of the present invention is not particularly limited, and the concentrations exemplified for the NKT cell activator of the present invention can be used.

[0100] (2) Method for Activating NKT Cells The method for activating NKT cells of the present invention is a method for activating NKT cells by using the liposome agent of the present invention. In the present invention, the liposome agent can be interpreted as the NKT cell activator of the present invention or the pharmaceutical composition of the present invention described below.

[0101] In the present invention, the liposome agent of the present invention may be used, for example, in vivo or in vitro.

[0102] When the liposome preparation of the present invention is used in vivo, the method for activating NKT cells of the present invention comprises the step of administering the liposome preparation to a subject.

[0103] The subject may be, for example, a human or a non-human animal, such as a mouse, rat, rabbit, dog, cat, cow, horse, monkey, pig, goat, or sheep.

[0104] The administration method is not particularly limited and may be oral or parenteral administration, with parenteral administration being preferred. Examples of parenteral administration include injection and infusion. The administration site may be, for example, subcutaneous, intravenous, intraarterial, intramucosal, intralymph node, or local. The local site may be, for example, the target affected tissue, or a site where the liposome agent can be delivered to the affected area. As mentioned above, the form of the liposome agent of the present invention is not particularly limited and can be appropriately determined depending on the administration form. Examples of the form include injection and infusion.

[0105] The conditions for administering the liposome agent of the present invention are not particularly limited and can be appropriately determined depending on the animal species, age, sex, health condition, body weight, etc. of the recipient. When the recipient is an adult human male, for example, the dose of the PEGylated liposome per administration is 0.005 to 50 mg / kg body weight, 0.15 to 15 mg / kg body weight, or 1 to 15 mg / kg body weight. The frequency of administration is, for example, once or twice a day, every 1 to 28 days.

[0106] When the liposome preparation of the present invention is administered subcutaneously, the PEGylated liposome can be efficiently delivered to, for example, lymph nodes such as axillary lymph nodes, the liver, the breast, etc. Furthermore, when the liposome preparation of the present invention is administered subcutaneously in the flank, the PEGylated liposome can be accumulated in the axillary lymph nodes, for example, within 12 to 48 hours (specifically, about 24 hours).

[0107] When the liposome preparation of the present invention is used in vivo, the method for activating NKT cells of the present invention comprises, for example, the step of contacting the liposome preparation with NTK cells.

[0108] 3. Pharmaceutical Composition and Cancer Treatment Method As described above, the pharmaceutical composition of the present invention is characterized by comprising the liposome agent of the present invention. Furthermore, the cancer treatment method of the present invention is characterized by comprising the step of administering the liposome agent of the present invention or the pharmaceutical composition of the present invention to a subject, as described above. The pharmaceutical composition and treatment method of the present invention are characterized by using the liposome agent of the present invention, and other conditions are not particularly limited. Furthermore, the descriptions of other embodiments can be used for the pharmaceutical composition and treatment method of the present invention. In the present invention, the liposome agent and the pharmaceutical composition can be read as the NKT cell activator of the present invention.

[0109] (1) Pharmaceutical Composition The pharmaceutical composition of the present invention comprises the liposome agent of the present invention. The pharmaceutical composition of the present invention may, for example, contain only the liposome agent of the present invention, or may contain other additives. For example, the same descriptions as those for the NKT cell activator of the present invention can be used for the additives.

[0110] The pharmaceutical composition of the present invention can be used for, for example, diseases that can be prevented or treated by activating NKT cells. Examples of such diseases include cancer. Target cancers include breast cancer, skin cancer, blood cancer, colon cancer, prostate cancer, and ovarian cancer. Target cancers include, for example, cancers with high metastatic potential, such as blood cancer, lung cancer, breast cancer, colon cancer, and prostate cancer, which are prone to metastasize to other organs.

[0111] (2) Cancer Treatment Method The cancer treatment method of the present invention is characterized by comprising a step of administering the liposome agent or the pharmaceutical composition of the present invention to a subject. The subject is not particularly limited and may be a human or a non-human animal as described above.

[0112] 4. Uses The present invention relates to the liposome preparation for use in activating NKT cells. The present invention also relates to the liposome preparation for use in producing an NKT cell activator.

[0113] The present invention relates to the liposome preparation for use in the treatment of diseases such as cancer, etc. The present invention also relates to the liposome preparation for use in the production of a pharmaceutical composition such as an anticancer agent.

[0114] [Embodiment 1] The liposome preparation of the present invention is preferably applied to NKT cell-targeted therapy for intractable cancers in particular. In this embodiment, examples of liposome preparations to be used in NKT cell-targeted therapy for intractable cancers are shown. The above examples can be used unless otherwise specified.

[0115] In this embodiment, the intractable cancer is a cancer type that is resistant to conventional immunotherapeutic agents such as PD-1 antibody (anti-PD1) and PD-L1 antibody (anti-PDL1), and a specific example is triple-negative breast cancer. Triple-negative breast cancer refers to breast cancer in which immune responses to all hormone receptors expressed on the surface of breast cancer cells—estrogen receptor (ER), progesterone receptor (PgR), and HER2 (human epidermal growth factor receptor type 2)—are negative. Examples of triple-negative breast cancer include 4T1, Hs578T, BT-20, and HCC70. The liposomal preparation of this embodiment is preferably applied to NKT cell-targeted therapy for triple-negative breast cancer, which is one of the intractable cancers.

[0116] The liposome preparation of this embodiment can be preferably used for, for example, patients with triple-negative breast cancer, as described above. Patients with triple-negative breast cancer, for example, are less likely to respond to immunotherapy, and are less likely to achieve the therapeutic effects of the PD-1 antibody or the PD-L1 antibody described above. Therefore, the liposome preparation of this embodiment can be particularly preferably used for, for example, patients in whom the administration of the PD-1 antibody or the PD-L1 antibody has not been successful in suppressing cancer growth, or patients in whom the administration of the PD-1 antibody or the PD-L1 antibody is expected to be unable to suppress cancer growth.

[0117] The liposomal agent of this embodiment can be applied to not only triple-negative breast cancer but also so-called "cold tumors." In cancer immunology, "cold tumors" refer to tumors to which the immune system shows little or no response. Cold tumors are generally considered difficult to treat due to their weak immune system attack. Their main characteristics include a low number of immune cells (especially T cells) in the tumor, low immune cell infiltration, lack of antigen presentation, and an immunosuppressive environment. The 4T1 cell line, a highly metastatic tumor cell line primarily used in mouse breast cancer research, is generally classified as a "cold tumor." This is due to the fact that 4T1 tumors are characterized by weak immune responses by cells of the innate and adaptive immune systems and poor immune cell infiltration. 4T1 tumors have an immunosuppressive microenvironment, which makes them more likely to evade immune system attack.

[0118] The liposome agent of this embodiment preferably satisfies the following conditions. Specifically, the liposome agent of this embodiment preferably satisfies all of the following conditions (condition 1, condition 2, and condition 3). By satisfying these conditions, for example, the liposome agent can be more efficiently accumulated in lymph nodes, and the liposome agent can be more efficiently taken up by antigen-presenting cells such as dendritic cells. This enables NKT cells to be activated more effectively, and therefore cancer growth can be suppressed even in patients for whom the aforementioned PD-1 antibodies and the like are ineffective.

[0119] (Condition 1) In the liposome preparation of this embodiment, the diameter of the PEGylated liposome preferably satisfies the following condition. That is, when expressed as a Z-average hydrodynamic diameter (Z-average), the lower limit is, for example, 10 nm or more, 50 nm or more, 60 nm, 80 nm or more, or 90 nm or more, and the upper limit is, for example, 400 nm or less, 200 nm or less, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, or 110 nm or less, and the range is, for example, 10 to 400 nm, 50 to 200 nm, 60 to 140 nm, 60 to 130 nm, or 80 to 120 nm. In this embodiment, the diameter is more preferably 100±40 nm or 100±30 nm, and particularly preferably 100±20 nm or 100±10 nm.

[0120] (Condition 2) In the liposome preparation of this embodiment, in the PEGylated lipid (2-2) contained as the liposome-constituting lipid, it is preferable that the terminal of the PEG modifying the phospholipid is a hydroxyl group. PEG with a terminal hydroxyl group will hereinafter be referred to as PEG-OH, and a phospholipid modified with PEG-OH will hereinafter be referred to as PEG-OH-modified lipid.

[0121] PEG can be represented by the following structural formula (1), where n is a positive integer. In this embodiment, the PEG of the PEG lipid preferably has hydrogen atoms at both ends and a hydroxyl group at the right end, as shown in the following structural formula (2). When a PEG-OH-modified phospholipid modified with PEG-OH of the following formula (2) is used, the liposome particles in the liposome preparation of this embodiment have a surface rich in hydroxyl groups derived from PEG-OH.

[0122]

[0123] By satisfying the above-mentioned condition 2, the liposome preparation of this embodiment can be more efficiently taken up by antigen-presenting cells such as dendritic cells, for example.

[0124] (Condition 3) In the liposome preparation of the present embodiment, the surface charge of the PEGylated liposome is preferably negative, and it is also said that the PEGylated liposome is negatively charged.

[0125] The charge (unit: coulomb) of the PEGylated liposome has a lower limit of, for example, −80 mV or more, −60 mV or more, −45 mV or more, −40 mV or more, or −30 mV or more, and an upper limit of, for example, 5 mV or less, 0 mV or less, less than 0 mV, −5 mV or less, −10 mV or less, or −20 mV or less, and ranges of, for example, −80 to 5 mV, −80 to 0 mV, −60 to 0 mV, −80 to less than 0 mV, −60 to less than 0 mV, −80 to −5 mV, −45 to −5 mV, −40 to −5 mV, −40 to −10 mV, −40 mV to −20 mV, −30±20 mV, −30±10 mV, or −20±10 mV. Furthermore, as described above, the charge of the PEGylated liposome is preferably a negative charge, specifically, preferably -80 to -5 mV, more preferably -30±20 mV or -30±10 mV. The liposome preparation of this embodiment contains a plurality of the PEGylated liposomes. Therefore, in the liposome preparation of this embodiment, for example, all of the PEGylated liposomes may have the same charge or different charges. Furthermore, for example, 50% or more, 80% or more, or 95% or more of all of the PEGylated liposomes may satisfy the charge. Furthermore, the liposome preparation of the present invention as a whole may satisfy the charge.

[0126] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

[0127] [Reagents, etc.] Choline-containing phospholipid: Phosphatidylcholine (1,2-Diercoyl-sn-glycero-3-phosphocholine, Tokyo Chemical Industry Co., Ltd.) Cholesterol-based lipid: Cholesterol (Wako Pure Chemical Industries, Ltd.) Positively charged lipid: Didodecyldimethylammonium bromide (DDAB, Sigma-Aldrich) PEGylated phospholipid: Phospholipid modified with PEG having a hydroxyl group at the end HO-PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol, Broadpharm) Phospholipid modified with PEG having a methyl group at the end METHYL-PEG-DSPE (1,2-Distearoyl-phosphatidyl Ethanolamine-Methyl-Polyethyleneglycol Conjugate, Tokyo Chemical Industry Co., Ltd.) NKT cell activating ligand: B7 (NERD Chemicals, Inc.) α-GalCer (NERD Chemicals Co., Ltd.)

[0128] Porous membrane: Trade name: Polycarbonate membrane (Cytiva) 1: Pore size 1000 nm, recovered particle size 1000 nm 2: Pore size 400 nm, recovered particle size 400 3: Pore size 200 nm, recovered particle size 200 nm 4: Pore size 100 nm, recovered particle size 100 nm 5: Pore size 50 nm, recovered particle size 50 nm RPMI-1640 medium (RPMI, Sigma Aldrich) Buffer solution: PBS (-) (Takara Bio Inc.) Fetal bovine serum (FBS, Biological Industries Co., Ltd.) Penicillin-streptomycin (Life Technologies Inc.) Sulfocyanine 5 succinimidyl ester (Sulfo-Cy5-NHS, Lumiprobe)

[0129] [Cells, etc.] All cells used in the examples were obtained from the RIKEN BioResource Center. Each cell was cultured in RPMI medium containing 10% FBS and 1x penicillin-streptomycin at 37°C and 5% CO ２BALB / c and C57BL / 6j mice were purchased from Charles River Japan. Animal experimental procedures were performed in accordance with the guidelines for the care and use of laboratory animals at the University of Tokyo.

[0130] Example 1: PEGylated liposomes were prepared. The preparation was carried out as follows, essentially according to the paper (OH voss et al., Curr. Protoc. Immunol., 2018), except for the reagents used and the amounts added.

[0131] First, liquid reagents of each component were prepared. The NKT ligand (B7 or α-GalCer) was dissolved in a mixed solvent (MeOH:chloroform = 4:6, volume ratio) to a concentration of 1 mg / mL. α-GalCer was dissolved in a mixed solvent (MeOH:chloroform = 4:6, volume ratio) to a concentration of 1 mg / mL. Phosphatidylcholine was dissolved in chloroform to a concentration of 5 mg / mL. The PEGylated phospholipid (HO-PEG-DSPE or METHYL-PEG-DSPE) was dissolved in chloroform to a concentration of 5 mg / mL. The positively charged lipid (DDAB) was dissolved in chloroform to a concentration of 10 mg / mL. Cholesterol was dissolved in chloroform to a concentration of 5 mg / mL.

[0132] The liquid reagent components described above were mixed in a 20 mL flask to obtain the following composition, to prepare a total of 1 mL of a mixed solution. In the following mixed solutions, 50% and 0% refer to the mass % of the NKT cell-activating ligand in the mixed solution. The molar ratios of components other than the NKT cell-activating ligand were kept constant among the mixed solutions, and the total lipid concentration in each mixed solution was set to 1 mg / mL. The following 50% B7 / neutral PEG-terminated-OH liposome mixed solution and 50% B7 / positively charged PEG-terminated-OH liposome mixed solution were supplemented with the positively charged lipid (DDAB) to make the surface charge of the liposomes formed positively or neutrally charged.

[0133] [NKT cell activating ligand: B7] (50% B7 / mixture for negatively charged PEG-terminated -OH liposomes) B7 solution 500 μL Phosphatidylcholine solution 34 μL Cholesterol solution 40 μL HO-PEG-DSPE solution 27 μL Chloroform Remainder (10% B7 / mixture for negatively charged PEG-terminated -OH liposomes) B7 solution 100 μL Phosphatidylcholine solution 114 μL Cholesterol solution 40 μL HO-PEG-DSPE solution 27 μL Chloroform Remainder (2% B7 / mixture for negatively charged PEG-terminated -OH liposomes) B7 solution 20 μL Phosphatidylcholine solution 130 μL Cholesterol solution 40 μL HO-PEG-DSPE solution 27 μL Chloroform Remainder (50% B7 / mixture for negatively charged PEG-terminated Me liposomes) B7 solution 500 μL Phosphatidylcholine solution 34 μL Cholesterol solution 40 μL METHYL-PEG-DSPE 27 μL Chloroform Remainder (50% B7 / mixture for neutral PEG-terminated OH liposomes) B7 solution 500 μL Phosphatidylcholine solution 23.4 μL Cholesterol solution 40 μL HO-PEG-DSPE solution 27 μL DDAB solution 5.3 μL Chloroform Remainder (50% B7 / mixture for positively charged PEG-terminated OH liposomes) B7 solution 500 μL Phosphatidylcholine solution 12.8 μL Cholesterol solution 40 μL HO-PEG-DSPE solution 27 μL DDAB solution 10.6 μL chloroform remainder

[0134] [NKT cell activating ligand: α-GalCer] (50% α-GalCer / mixture for negatively charged PEG-terminated -OH liposomes) α-GalCer solution 500 μL Phosphatidylcholine solution 32.7 μL Cholesterol solution 41 μL HO-PEG-DSPE solution 28 μL Chloroform Remainder

[0135] [NKT cell activating ligand: No addition] (0% control / mixture for negatively charged PEG-terminated -OH liposomes) NKT ligand solution 0 μL Phosphatidylcholine solution 134 μL Cholesterol solution 40 μL HO-PEG-DSPE solution 27 μL Chloroform Remainder (0% control / mixture for negatively charged PEG-terminated -Me liposomes) NKT ligand solution 0 μL Phosphatidylcholine solution 134 μL Cholesterol solution 40 μL METHYL-PEG-DSPE solution 27 μL Chloroform Remainder

[0136] The flask was rotated using an evaporator (Tokyo Rikakikai Co., Ltd.) under a vacuum of 30 hPa at 30°C for 10 minutes to completely dry the solvent in the mixture. As a result, a thin lipid film was formed on the inner surface of the flask. 1 mL of PBS was then added to the flask and the mixture was resuspended. This suspension was treated at 28 kHz for 5 minutes using an ultrasonic bath (Aiwa Medical Industry Co., Ltd.) and then at 20 kHz for 30 seconds using an ultrasonic homogenizer (Taitec Co., Ltd.). This disrupted the lipid film, yielding PEGylated liposomes broken down into small fragments.

[0137] The degraded PEGylated liposomes were further processed using the porous membrane and an extruder (product name AE-10, Bio-Equip) to reduce the particle size to a predetermined range. PBS was used as the solvent during processing. Specifically, first, the porous membrane 1 (recovered particle size: 1000 μm) was set in the extruder, and the degraded PEGylated liposomes were passed through the extruder three or more times. Next, the porous membrane 2 (recovered particle size: 400 nm) was set in the extruder, and the PEGylated liposome fraction that had passed through the porous membrane 1 was passed through the extruder three or more times. As a result, PEGylated liposomes with a particle size of approximately 400 nm were recovered as PEGylated liposome fraction A. Next, the porous membrane 3 (recovered particle size: 200 nm) was set in the extruder, and the PEGylated liposome fraction that had passed through the porous membrane 2 was passed through the extruder three or more times. As a result, PEGylated liposomes with a particle size of approximately 200 nm were recovered as PEGylated liposome fraction B. Subsequently, two of the porous membranes 4 (recovered particle size 100 nm) were stacked and set in the extruder, and the PEGylated liposome fraction that had passed through the porous membrane 3 was passed through three or more times. As a result, PEGylated liposomes with a particle size of approximately 100 nm were recovered as PEGylated liposome fraction C. Furthermore, the porous membrane 5 (recovered particle size 50 nm) was set in the extruder, and the PEGylated liposomes that had passed through the porous membrane 4 were passed through 15 or more times. As a result, PEGylated liposomes with a particle size of approximately 50 nm were recovered as PEGylated liposome fraction D.

[0138] The collected PEGylated liposome fractions (hereinafter also referred to as liposome samples) were each suspended in PBS, and the particle size (Z-average hydrodynamic diameter (Z-average)), polydispersity index (PDI), and zeta potential of the PEGylated liposomes were determined.

[0139] Specifically, the particle size (Z-average hydrodynamic diameter (Z-average)), PDI, and zeta potential of the PEGylated liposomes were evaluated by dynamic light scattering (DLS) at 25°C and a detection angle of 173° using a zeta potential measurement device (trade name Zetasizer, Malvern Instruments Ltd.) equipped with a diode laser (λ = 532 nm). The particle size and PDI were determined according to the cumulant method.

[0140] The results of the liposome particle size (Z-average hydrodynamic diameter (Z-average)), PDI, and zeta potential for the liposome sample (the PEGylated liposome fraction) are shown in the table below. (1-1) 50% B7 / negatively charged PEG-terminated -OH liposomes Fraction C (particle size: approximately 100 nm) (1-2) 50% α-GalCer / negatively charged PEG-terminated -OH liposomes Fraction C (particle size: approximately 100 nm) (1-3) 50% B7 / negatively charged PEG-terminated -OH liposomes Fraction D (particle size: approximately 50 nm) (1-4) 50% B7 / negatively charged PEG-terminated -OH liposomes Fraction B (particle size: approximately 200 nm) (1-5) 50% B7 / negatively charged PEG-terminated -OH liposomes Fraction A (particle size: approximately 400 nm) (1-6) 10% B7 / negatively charged PEG-terminated -OH liposomes Fraction C (particle size: approximately 100 nm) (1-7) 2% B7 / negatively charged PEG-terminated -OH liposomes Fraction C (particle size: approx. 100 nm) (1-8) 50% B7 / negatively charged PEG-terminated-Me liposomes Fraction C (particle size: approx. 100 nm) (1-9) 50% B7 / neutral PEG-terminated-OH liposomes Fraction C (particle size: approx. 100 nm) (1-10) 50% B7 / positively charged PEG-terminated-OH liposomes Fraction C (particle size: approx. 100 nm) (1-11) 0% control / negatively charged PEG-terminated-OH liposomes Fraction C (particle size: approx. 100 nm) (1-12) 0% control / negatively charged PEG-terminated-Me liposomes Fraction C (particle size: approx. 100 nm)

[0141]

[0142] In addition, various liposome fractions were prepared as liposome samples using the same method as that using PBS, except that dye-containing PBS was used instead of PBS. The dye-containing PBS was prepared as follows. Specifically, a dye reagent (Sulfo-cyanine 5 succinimidyl ester, trade name: Sulfo-Cy5 (registered trademark)-NHS, Lumiprobe) was mixed with PBS to a concentration of 0.1 mg / mL and allowed to stand for 24 hours. By leaving the dye reagent in PBS, the -NHS was removed from the dye reagent by hydrolysis, preparing dye-containing PBS containing Sulfo-Cy5 (registered trademark) at a concentration of 0.1 mg / mL. It has been confirmed that each PEGylated liposome prepared using the dye-containing PBS has the same particle size and PDI as the PEGylated liposome prepared using dye-free PBS.

[0143] Hereinafter, unless otherwise specified, PBS refers to dye-free PBS. Furthermore, PEGylated liposomes prepared using dye-free PBS will be referred to as liposomes, and PEGylated liposomes prepared using dye-containing PBS will be referred to as liposomes (dye-containing PBS).

[0144] Example 2 The stability of the PEGylated liposomes prepared in Example 1 was confirmed.

[0145] (1) Long-Term Storage Stability The B7 liposomes (Sample No. 1-1), α-GC liposomes (Sample No. 1-2), and NKT ligand (-) liposomes (Sample No. 1-11) prepared in Example 1 were adjusted to a PEGylated liposome concentration of 1 mg / mL (converted to lipid amount, the same applies below) and used as liposome samples. PBS was used to adjust the PEGylated liposome concentration. The NKT ligand concentration in the liposome samples was measured using pyrene-1-boronic acid (Sigma-Aldrich) by a conventional glycolipid detection method using fluorescently modified boronic acid (https: / / trace.tennessee.edu / utk_graddiss / 5634 / ). The B7 concentration of the liposome sample prepared from the 50% B7 mixture (PEGylated liposome concentration: 1 mg / mL) was 0.471 mg / mL (approximately 0.5 mg / mL), and the GalCer concentration of the liposome sample prepared from the 50% α-GalCer mixture (PEGylated liposome concentration: 1 mg / mL) was 0.472 mg / mL (approximately 0.5 mg / mL).

[0146] The liposome samples were stored at 4°C. The particle size (number average hydrodynamic diameter) and polydispersity index (PDI) of the liposome samples before the start of storage (Day 0) and those after 10 days (Day 10) and 90 days (Day 90) of storage were determined based on particle size distribution diagrams obtained by dynamic light scattering (DLS) measurements. The particle size was calculated by calculating the percentage change in particle size after storage, based on the particle size before the start of storage (Day 0). Storage stability was evaluated from the percentage change in particle size and PDI.

[0147] The results of the long-term storage stability of the B7 liposomes and the control liposomes are shown in the following tables: Table 4A shows the percent change in particle size, and Table 4B shows the polydispersity index (PDI).

[0148]

[0149] As shown in the table, the particle size change rate of B7 liposomes from 10 days to 90 days remained within the range of -1.01% to -6.87% compared to Day 0 (0%), and the particle size change rate of α-GarCer from 10 days to 90 days also remained within the range of -13.1% to +3.70% compared to Day 0 (0%). On the other hand, the particle size change rate of the control NKT ligand (-) liposomes from 10 days to 90 days increased over time from +0.997% to +15.9% compared to Day 0 (0%). Furthermore, as shown in the table above, the PDI of B7 liposomes from 10 days to 90 days fluctuated only within the range of 0.153 to 0.245 relative to Day 0 (0.170), and the PDI of α-GarCer from 10 days to 90 days also fluctuated only within the range of 0.116 to 0.364 relative to Day 0 (0.142). In contrast, the PDI of the control NKT ligand (-) liposomes from 10 days to 90 days increased over time to the range of 0.176 to 0.420 relative to Day 0 (0.120). These results show that B7 liposomes and α-GC liposomes can be stabilized by using cholesterol and PEGylated phospholipid (PEG-DSPE), and are more stable than NKT ligand (-) control liposomes, for example, after long-term storage for 90 days, and that the formation of aggregates of 1000 nm or larger can be prevented.

[0150] (2) Stability after Dilution The B7 liposome sample (Day 14) stored in (1) above was diluted with PBS at various dilutions (2-fold to 1024-fold) to prepare multiple diluted samples. The stability of the undiluted sample after storage and the diluted samples was evaluated based on the particle size (Z-average hydrodynamic diameter (Z-average)) and polydispersity index (PDI).

[0151] The results for B7 liposomes are shown in Figure 1. Figure 1 is a graph showing the results of dilution stability, with the left graph showing the particle size of the diluted sample of B7 liposomes and the right graph showing the PDI of the diluted sample of B7 liposomes. As shown in the figure, B7 liposomes, for example, have a high PDI of about 1000 times (1 / 2 １０), no formation of aggregates with particle sizes exceeding 1000 nm was observed.

[0152] Example 3 The PEGylated liposome prepared in Example 1 was administered to confirm its function.

[0153] (1) Immunostimulatory Activity The immunostimulatory activity of the PEGylated liposomes was confirmed.

[0154] The B7 liposomes (sample No. 1-1) and NKT ligand (-) liposomes (sample No. 1-11) prepared in Example 1 were adjusted to a PEGylated liposome concentration of 1 mg / mL (total lipid equivalent) and used as administration samples. PBS was used to adjust the PEGylated liposome concentration. As control samples for administration, free B7 that was not liposomal and PBS were used, respectively.

[0155] The test sample was administered subcutaneously to the left flank of 6-week-old female BALB / c mice. 48 hours after administration, the mice were sacrificed, and blood and lymph nodes were collected. Lymph nodes were collected from the right paraxilla, right upper arm axilla, left paraxilla, and left upper arm axilla. The collected blood was centrifuged at 2000 g for 15 minutes to recover serum. The right paraxilla and right upper arm axilla lymph nodes were combined to form the right axillary lymph nodes, and the left paraxilla and left upper arm axilla lymph nodes were combined to form the left axillary lymph nodes. The collected lymph nodes were weighed, and PBS was added to a concentration of 50 μg / μL. The homogenized material was then centrifuged at 2000 g for 15 minutes, and the supernatant was collected. The serum and lymph node supernatant were used as samples, and IFN-γ and IL-10 in the samples were measured using an IFN-γ ELISA kit (BioLegend, catalog number 430804) according to the manufacturer's instructions.

[0156] The results are shown in Figure 2A. Figure 2A is a graph showing the IFN-γ concentrations in serum and lymph node supernatant, and Figure 2B is a graph showing the IL-10 concentrations in serum and lymph node supernatant. In Figures 2A and 2B, the left shows the results for left axillary lymph node supernatant, the middle shows the results for right axillary lymph node supernatant, and the right shows the results for serum. In each graph in Figure 2A, the vertical axis represents the IFN-γ concentration (ng / mL) of the sample, and in each graph in Figure 2B, the vertical axis represents the IL-10 concentration (ng / mL) of the sample. In Figures 2A and 2B, the bars indicate, from the left, the results of administration of PBS, NKT ligand (-) liposome (0% Ctrl lipo-OH(-) 100 nm), free B7, and B7 liposome (50% B7 lipo-OH(-) 100 nm).

[0157] As shown in Figure 2A, administration of B7 liposomes resulted in a significant increase in IFN-γ in the left axillary lymph node supernatant 48 hours after administration. In contrast, administration of free B7 resulted in levels comparable to those of controls such as PBS, and no immune activation was observed. Furthermore, an increase in IFN-γ was observed in the right axillary lymph node supernatant, although this was lower than in the left axillary lymph node. This indicates that B7 liposomes can activate lymph nodes throughout the body, not just the lymph nodes in the left armpit where the administration occurred, suggesting the potential for systemic therapeutic effects. Furthermore, B7 liposomes are thought to accumulate in lymph nodes approximately 24 hours after administration, become activated approximately 48 hours after administration, and act on cancer approximately 72 hours after administration. Therefore, for example, a decrease in IFN-γ in lymph nodes is expected approximately 96 hours after administration. In such cases, a continuous effect can be maintained by administering the B7 liposomes again 4-5 days after the initial administration.

[0158] Furthermore, as shown in Figure 2A, the immune activation in serum after B7 liposome administration was lower than that in lymph nodes, indicating that B7 liposome administration significantly enhanced immune activation in lymph nodes, for example, in the spleen and other immune organs.

[0159] It is known that repeated administration of α-GalCer produces an analgic effect and induces immune regulation (Sci. Rep., 2018, Vol. 8, p. 8225). Furthermore, it is known that such immune regulation is associated with IL-10 secretion (JCI, 2014, Vol. 124(9), p. 3725). Measuring the amount of IL-10 can determine whether repeated administration of an immune activator is appropriate. Therefore, when IL-10 was examined, as shown in Figure 2(B), no increase in IL-10 was observed 48 hours after subcutaneous administration of B7 liposomes.

[0160] As described above, subcutaneous administration of B7 liposomes did not result in an increase in IL-10 secretion, demonstrating that B7 liposomes can suppress the analgic effect. Furthermore, these results suggest that the problem of repeated administration of conventional NKT activating ligands, namely, the reduction in efficacy due to repeated administration, can be avoided.

[0161] (2) In Vitro Immune Activity After adding PEGylated liposomes to dendritic cells, the dendritic cells were co-cultured with lymph node cells or spleen cells, and changes in the number of NKT cells were examined.

[0162] The B7 liposome (sample No. 1-1) prepared in Example 1 was adjusted to a PEGylated liposome concentration of 1 mg / mL (total lipid equivalent) and used as an administration sample. PBS was used to adjust the PEGylated liposome concentration. PBS was used as a control sample for administration.

[0163] Lymph node cells and spleen cells were prepared as follows. First, spleens and lymph nodes were collected from BALB / c mice (7 weeks old, female). The lymph nodes were collected from the right paraxilla, right upper arm axilla, left paraxilla, and left upper arm axilla. Each collected lymph node was minced, placed in a cell strainer, and gently ground in RPMI containing 1% FBS to obtain single cells. The single cells were centrifuged at 400 g for 4 minutes to collect only viable lymph node cells. The spleen was similarly separated into single cells using a cell strainer, and the cells were collected by centrifugation. 10% RBC Lysis Buffer (10X, ThermoFisher) was added to PBS (containing 1% FBS) to make a 1X solution, and the spleen single cells were resuspended in this mixture and allowed to stand for 5 minutes to remove red blood cells. The fraction after removal of red blood cells was then centrifuged at 400 g for 4 minutes to recover viable spleen cells.

[0164] Mouse dendritic cells, DC2.4 (Sigma-Aldrich), were cultured in RPMI. Fresh RPMI and 5 x 10 of the cultured dendritic cells were added to each well of a 6-well plate. ５ The B7 liposomes were added to each well so that the final total lipid concentration in each well was 0.1 mg / mL. After culturing for 24 hours, 7.5 x 10 cells / well were added. ５ 100% of the lymph node cells or spleen cells were added and co-cultured for 24 hours. After 24 hours of co-culture, all cells were collected. The collected cells were blocked using a blocking reagent at 4°C for 45 minutes. The blocking reagent used was Blocking One (Nacalai Tesque) containing CD16 / CD32 Monoclonal Antibody (ThermoFisher).

[0165] Next, the blocked cell population was subjected to labeling to simultaneously label NK cells and T cells. For the NK cell labeling, PBS (containing 1% FBS) containing Anti-mouse NK1.1-InVivo (Selleck) and FlexAble CoraLite® Plus 488 Antibody Labeling Kit for mouse IgG (Proteintech) was used as the reagents. For the T cell labeling, PBS (containing 1% FBS) containing PE anti-mouse CD3 Antibody (Biorezen) was used as the reagent. Each labeling reagent was simultaneously added to the blocked cell population and allowed to stand at 4°C for 45 minutes, thereby simultaneously labeling the NK cells and the T cells in the cell population. The labeled cells were washed three times with PBS (containing 1% FBS) and then analyzed by flow cytometry (BD FACSAria™ III Cell Sorter, DB Biosciences). The staining concentration of each antibody during labeling was determined according to the manufacturer's instructions (the same applies below).

[0166] Co-culture and measurement were carried out in the same manner, except that PBS (containing 1% FBS) was added per well in an amount of 1 / 10 (volume ratio) of the culture solution instead of the B7 liposome.

[0167] First, the results for lymph nodes are shown in Figure 3. Figure 3A is a histogram of NKT cells in lymph nodes, and is a graph showing the distribution of cell numbers for NK1.1-positive and CD3-positive cells in lymph nodes by relative fluorescence intensity indicating CD3 positivity. In Figure 3A, the X-axis represents the relative fluorescence intensity of the labeling reagent relative to CD3, and the Y-axis represents the number of events (cell number) showing that fluorescence. Of the two peaks in Figure 3A, the right-hand peak represents NK1.1-positive and CD3-positive cells, i.e., NKT cells. Figure 3B is a graph quantified, based on Figure 3A, for the amount of NK1.1-positive and CD3-positive NKT cells in lymph nodes for the three administration groups (n = 4 mice).

[0168] As shown in Figure 3A, the B7 liposome group (B7 lipo) showed an increased amount of NK1.1-positive and CD3-positive NKT cells (the peak on the right in Figure 3A) by co-culturing B7 liposome-administered dendritic cells with lymph node cells, compared to the PBS-administered group (PBS).

[0169] Next, the results for the spleen are shown in Figure 4. Figure 4A is a histogram of NKT cells in the spleen, and is a graph showing the distribution of cell numbers for NK1.1-positive and CD3-positive cells in the spleen based on the relative fluorescence intensity. In Figure 4A, the X-axis represents the relative fluorescence intensity of the labeling reagent to CD3, and the Y-axis represents the number of events (cell number) showing that fluorescence. Of the two peaks in Figure 4A, the right-hand peak represents NK1.1-positive and CD3-positive cells, i.e., NKT cells. Figure 4B is a graph quantifying the amount of NK1.1-positive and CD3-positive NKT cells in the spleen (n = 4 mice).

[0170] As shown in FIG. 4A, in the spleen, similar to the lymph nodes, the amount of NK1.1-positive and CD3-positive NKT cells (the peak on the right in FIG. 4A) was increased in the B7 liposome (B7 lipo) group compared to the PBS-administered group (PBS).

[0171] (3) Changes in the Number of Immune Cells The changes in the number of immune cells in the body after administration of PEGylated liposomes were examined.

[0172] The B7 liposome (sample No. 1-1) prepared in Example 1 was adjusted to a PEGylated liposome concentration of 1 mg / mL (total lipid equivalent) and used as an administration sample. PBS (no NKT ligand added) was used as a control sample for administration.

[0173] The test sample was administered subcutaneously to the left flank of 6-week-old female BALB / c mice. After the specified time (12, 24, 48, and 72 hours) following administration, the mice were sacrificed, and the spleen and lymph nodes were collected. Lymph nodes were collected from the right paraxilla, right upper arm axilla, left paraxilla, and left upper arm axilla. The size of each collected organ was observed. Each collected lymph node was minced, placed in a cell strainer, and gently ground in RPMI containing 1% FBS to obtain single cells. The single cells were centrifuged at 400 g for 4 minutes to collect only viable lymph node cells. The spleen was also treated in the same manner. Single cells were collected using a cell strainer and then centrifuged. 10% RBC Lysis Buffer (10X, ThermoFisher) was added to PBS (containing 1% FBS) to make a 1X solution, and the spleen cells were resuspended in this mixture and allowed to stand for 5 minutes to remove red blood cells. The fraction after removal of red blood cells was centrifuged at 400 g for 4 minutes to recover viable spleen cells.

[0174] The collected lymph node cells and spleen cells were blocked and simultaneously labeled with NK cells and T cells using the same method as described in (2) above. The labeled cells were washed and analyzed by flow cytometry in the same manner as described in (2) above.

[0175] The collected lymph node and spleen cell populations were then blocked using the same method as in (2) above, followed by dendritic cell labeling. Dendritic cell labeling was performed using PBS (containing 1% FBS) containing FITC anti-mouse CD11c antibody (Biorezend) as a reagent. Dendritic cells in the cell populations were labeled by adding the reagent to the cells and incubating at 4°C for 45 minutes. At the same time, macrophage labeling was performed on each of the cell populations that had undergone dendritic cell labeling. Macrophage labeling was performed using PBS (containing 1% FBS) containing anti-mouse CD68 (Militenyi) and FlexAble CoraLite® Plus 555 Antibody Labeling Kit for rabbit IgG (Proteintech) as reagents. That is, the reagent was added to the cell population and the mixture was left to stand at 4° C. for 45 minutes, thereby labeling macrophages simultaneously with dendritic cells. The labeled cells were washed and measured by flow cytometry in the same manner as in (2) above.

[0176] First, Figure 5 shows the changes in the proportions of T cells, NK cells, and NKT cells in lymph nodes. Figure 5A is a histogram of NKT cells in lymph nodes, a graph showing the distribution of cell numbers for NK1.1-positive and CD3-positive cells in lymph nodes based on the relative fluorescence intensity indicating CD3 positivity. In Figure 5A, the X-axis represents the relative fluorescence intensity of the labeling reagent relative to CD3, and the Y-axis represents the number of events (cell counts) showing that fluorescence. Also, in Figure 5A, time (h) represents the treatment time after administration of the administration sample, and histograms are shown for each administration group. Figure 5B is a histogram of NK cells in lymph nodes, a graph showing the distribution of cell numbers for NK1.1-positive NK cells in lymph nodes based on the relative fluorescence intensity indicating NK1.1 positivity. Figure 5B shows histograms for five administration groups superimposed. Figure 5C is a histogram of T cells in lymph nodes, showing the distribution of cell numbers of CD3-positive T cells in lymph nodes by relative fluorescence intensity indicating CD3 positivity. Figure 5C shows histograms of five administration groups superimposed. Figure 5D is a histogram of NKT cells in lymph nodes, showing the histograms of the five administration groups in Figure 5A superimposed. The darker the lines in Figures 5B, 5C, and 5D, the longer the treatment time after administration of the administration sample. Figure 5E is a graph quantified, based on Figure 5A, for the amount of NK1.1-positive and CD3-positive NKT cells in lymph nodes for five administration groups (n = 4 mice). Specifically, the graph shows the percentage (%) of NKT cells in lymph node cells in the PBS-administered group at the same relative fluorescence intensity as in Figure 5A, where the percentage of NKT cells in lymph node cells in the PBS-administered group is 5%.

[0177] As shown in Figures 5A, D, and E, in the B7 liposome group, the number of NKT cells in the lymph nodes increased over time at 12 and 24 hours after administration, and the increased number of NKT cells was maintained at 48 and 72 hours after administration. Furthermore, as shown in Figures 5B and C, the number of NK cells and T cells also increased over time after administration, peaking at 24 to 48 hours after administration, and then maintained at that level. These results demonstrate that in vivo, administration of B7 liposomes gradually induces immune activity between 12 and 24 hours after administration, and this activity is maintained up to 72 hours after administration.

[0178] Next, Figure 6 shows the changes in the proportions of T cells, NK cells, and NKT cells in the spleen. Figure 6A is a histogram of NKT cells in the spleen, and is a graph showing the distribution of cell numbers for NK1.1-positive and CD3-positive cells in the spleen based on the relative fluorescence intensity indicating CD3 positivity. In Figure 6A, the X-axis represents the relative fluorescence intensity of the labeling reagent relative to CD3, and the Y-axis represents the number of events (cell counts) showing that fluorescence. Also in Figure 6A, time (h) represents the treatment time after administration of the administration sample, and histograms are shown for each administration group. Figure 6B is a histogram of NK cells in the spleen, and is a graph showing the distribution of cell numbers for NK1.1-positive NK cells in the spleen based on the relative fluorescence intensity indicating NK1.1 positivity. Figure 6B shows histograms for five administration groups superimposed. Figure 6C is a histogram of T cells in the spleen, showing the distribution of cell numbers of CD3-positive T cells in the spleen by the relative fluorescence intensity indicating CD3 positivity. Figure 6C shows histograms of five administration groups superimposed. Figure 6D is a histogram of NKT cells in the spleen, showing the histograms of the five administration groups in Figure 6A superimposed. The darker the lines in Figures 6B, 6C, and 6D, the longer the treatment time after administration of the administration sample. Figure 6E is a graph quantifying the amount of NK1.1-positive and CD3-positive NKT cells in the spleen for the five administration groups (n=4 mice) based on Figure 6A. Specifically, the graph shows the percentage (%) of NKT cells in the spleen cells of the PBS-administered group at the same relative fluorescence intensity as that of the other administration groups in Figure 6A.

[0179] As shown in Figures 6A, 6D, and 6E, the number of NKT cells in the spleen in the B7 liposome group was confirmed to increase over time up to 72 hours after administration. Furthermore, as shown in Figure 6E, an increase in NKT cells was confirmed in the B7 liposome group compared to the PBS group 12 hours after administration, but the difference was not significant. The number of NKT cells in the B7 liposome group continued to increase up to 72 hours after administration, suggesting that immune activity in the spleen occurs more slowly and continues for a longer period of time than in lymph nodes. This is thought to be due, for example, to the fact that a drug administered subcutaneously is first absorbed into the adjacent lymph nodes and then migrates to the spleen.

[0180] As shown in Figure 6B, it was confirmed that the number of NK cells, like that of NKT cells, increased over time after administration. Meanwhile, as shown in Figure 6C, with regard to the number of T cells, CD3-positive cells (peak on the right) decreased over time, while CD3-negative cells (peak on the left) increased over time, reaching a peak 48 hours after administration. This indicates that the proportion of T cells decreased along with immune activity in the spleen. This is thought to be because the immune activity induced by the initial administration of B7 liposomes was innate immune activity, primarily resulting in an increase in NK cells and NKT cells.

[0181] Next, Figure 7 shows the changes in the proportions of dendritic cells and macrophages in lymph nodes. Figure 7A is a histogram of dendritic cells in lymph nodes, and is a graph showing the cell number distribution of CD11c-positive cells in lymph nodes based on the relative fluorescence intensity indicating CD11c positivity. In Figure 7A, the X-axis represents the relative fluorescence intensity of the labeling reagent relative to CD11c, and the Y-axis represents the number of events (cell number) exhibiting that fluorescence. Figure 7B is a histogram of dendritic cells in lymph nodes, and is an overlay of the histograms of the five administration groups in Figure 7A. Figure 7C is a histogram of macrophages in lymph nodes, and is a graph showing the cell number distribution of CD68-positive cells in lymph nodes based on the relative fluorescence intensity indicating CD68 positivity. In Figure 7C, the X-axis represents the relative fluorescence intensity of the labeling reagent relative to CD68, and the Y-axis represents the number of events (cell number) exhibiting that fluorescence. Figure 7D is a graph quantified based on Figure 7B for the amount of CD11c-positive dendritic cells in lymph nodes for five treatment groups (n=4 mice). Specifically, the graph shows the percentage (%) of dendritic cells in other treatment groups at the same relative fluorescence intensity, relative to the relative fluorescence intensity representing a 5% proportion of dendritic cells among lymph node cells in the PBS-treated group in Figure 7B. Figure 7E is a graph quantified based on Figure 7C for the amount of CD68-positive macrophages in lymph nodes for five treatment groups (n=4 mice). Specifically, the graph shows the percentage (%) of dendritic cells in other treatment groups at the same relative fluorescence intensity, relative to the relative fluorescence intensity representing a 5% proportion of macrophages among lymph node cells in the PBS-treated group in Figure 7C.

[0182] As shown in Figures 7A, 7B, and 7D, in the B7 liposome-administered group, the proportion of dendritic cells in lymph nodes gradually increased until 48 hours after administration, reaching approximately twice that of the PBS-administered group, and then slightly decreased at 72 hours after administration. On the other hand, as shown in Figures 7C and 7E, in the B7 liposome-administered group, the proportion of macrophages slightly increased after B7 liposome administration, but there was no significant difference compared to the PBS-administered group.

[0183] Next, Figure 8 shows the changes in the proportions of dendritic cells and macrophages in the spleen. Figure 8A is a histogram of dendritic cells in the spleen, and is a graph showing the cell number distribution of CD11c-positive cells in the spleen based on the relative fluorescence intensity indicating CD11c positivity. In Figure 8A, the X-axis represents the relative fluorescence intensity of the labeling reagent relative to CD11c, and the Y-axis represents the number of events (cell number) exhibiting that fluorescence. Figure 8B is a histogram of dendritic cells in the spleen, and is an overlay of the histograms of the five administration groups in Figure 8A. Figure 8C is a histogram of macrophages in the spleen, and is a graph showing the cell number distribution of CD68-positive cells in the spleen based on the relative fluorescence intensity indicating CD68 positivity. In Figure 8C, the X-axis represents the relative fluorescence intensity of the labeling reagent relative to CD68, and the Y-axis represents the number of events (cell number) exhibiting that fluorescence. Figure 8D is a graph quantified based on Figure 8B for the amount of CD11c-positive dendritic cells in the spleen for five administration groups (n=4 mice). Specifically, the graph shows the percentage (%) of dendritic cells in the other administration groups at the same relative fluorescence intensity, relative to the relative fluorescence intensity indicating a 5% proportion of dendritic cells among spleen cells in the PBS-administered group in Figure 8B. Figure 8E is a graph quantified based on Figure 8C for the amount of CD68-positive macrophages in the spleen for five administration groups (n=4 mice). Specifically, the graph shows the percentage (%) of dendritic cells in the other administration groups at the same relative fluorescence intensity, relative to the relative fluorescence intensity indicating a 5% proportion of macrophages among spleen cells in the PBS-administered group in Figure 8C.

[0184] As shown in Figures 8A, 8B, and 8D, in the B7 liposome-administered group, the proportion of dendritic cells in the spleen, as well as in the lymph nodes, increased to approximately twice that of the PBS-administered group. On the other hand, as shown in Figures 8C and 8E, in the B7 liposome-administered group, the proportion of macrophages did not change as significantly as in the dendritic cells. Thus, the increase in dendritic cells in spleen cells was more pronounced than in lymph nodes.

[0185] Example 4 The biodistribution of the PEGylated liposomes prepared in Example 1 was confirmed.

[0186] (1) Biodistribution of B7 Liposomes The following liposome samples prepared using the dye-containing PBS in Example 1 were used. (1-1) 50% B7 / negatively charged PEG-terminated -OH liposomes 100 nm (1-11) 0% control / negatively charged PEG-terminated -OH liposomes 100 nm The liposome samples were adjusted to a PEGylated liposome concentration of 1 mg / mL (total lipid equivalent) and used as administration samples. The dye-containing PBS was used to adjust the PEGylated liposome concentration. The administration sample prepared in (1-1) above was used as the B7 liposome administration sample (B7 lipo) of this example, and the administration sample prepared in (1-11) above was used as the control liposome administration sample (Ctrl lipo).

[0187] Breast cancer (4T1, average tumor volume 150mm ３ 30 μL of the administration sample was subcutaneously administered to the left flank of BALB / c mice (6 weeks old, female) with breast cancer and BALB / c mice (6 weeks old, female) without breast cancer. The dose of each administration sample was as follows: 30 μL of the B7 liposome administration sample: 30 μL / mouse, 15 μg of B7; 30 μL of the control liposome administration sample: 30 μL / mouse. 24 hours after administration, the mice were sacrificed, and the left axillary lymph nodes, spleen, kidney, and liver were collected.

[0188] Then, each of the collected tissues was visualized using an IVIS imaging system (product name: IVIS Spectrum, Sumitomo Pharma International Corporation) having fluorescence channels of 650 nm / 680 nm.

[0189] The results are shown in Figure 9. Figure 9A is an image (converted to black and white) showing fluorescence in each organ of a mouse 24 hours after administration. In Figure 9A, the area indicated by the arrowhead is the region where the fluorescence intensity was particularly strong. Note that Figure 9A is a color image showing fluorescence displayed in black and white, so the color does not represent the fluorescence intensity of the fluorescent region. Figure 9B is a graph showing the relative fluorescence intensity in each organ of a mouse 24 hours after administration. In Figure 9B, the relative fluorescence intensity is the value obtained by dividing the fluorescence intensity in each organ by the weight of each organ and normalizing the Ctrl+Lip value by 1.

[0190] As shown in Figure 9A, in the B7 liposome administration group (B7 lipo), strong fluorescence was detected in the left axillary lymph node 24 hours after administration, confirming the accumulation of a large amount of B7 liposomes. Fluorescence was also detected in the liver, confirming the accumulation of some B7 liposomes. On the other hand, as shown in Figure 9A, strong fluorescence was not detected in the spleen, kidneys, or other organs (thymus, lungs, heart, not shown). These results demonstrate that B7 liposomes enable selective delivery, particularly to lymph nodes. On the other hand, in the control liposome administration group (Ctrl lipo), fluorescence was detected in the liver, but almost no fluorescence was detected in other organs. Also, in Figure 9B, the relative fluorescence intensity in the lymph nodes was higher in the B7 liposome administration group (B7 lipo) than in the control liposome administration group (Ctrl lipo), confirming that the B7 liposome effectively accumulates in the lymph nodes.

[0191] Example 5 The PEGylated liposomes prepared in Example 1 were subjected to repeated subcutaneous administration to confirm their antitumor effect on breast cancer cells.

[0192] The following liposome samples prepared using PBS in Example 1 were used. (1-1) 50% B7 / negatively charged PEG-terminated -OH liposomes 100 nm (1-11) 0% control / negatively charged PEG-terminated -OH liposomes 100 nm The liposome samples were adjusted to a PEGylated liposome concentration of 1 mg / mL (total lipid equivalent) and used as administration samples. PBS was used to adjust the PEGylated liposome concentration. The administration sample prepared in (1-1) above was used as the B7 liposome administration sample (B7 lipo) of this example, and the administration sample prepared in (1-11) above was used as the control liposome administration sample (Ctrl lipo). In addition, a free B7 sample (Free B7) prepared by suspending unliposomal free B7 in PBS, and PBS, were also used as administration samples.

[0193] (1) Antitumor Effect Triple-negative breast cancer cells 4T1 were used as breast cancer cells. 4T1 cells (5.0 × 10 ５ ~1.0 x 10 ６ The average tumor volume was 30-180 mm ３ When the mice reached 100 mg / kg, the mice were randomly divided into groups. Then, the mice were subcutaneously injected with the administration samples under the left armpit or both armpits under administration condition A or administration condition B described below (Day 0). The doses of each administration sample were as follows: B7 liposome administration sample 30 μL: 30 μL / mouse, B7 amount 15 μg, total lipid amount 30 μg Control liposome administration sample 30 μL: 30 μL / mouse, total lipid amount 30 μg Free B7 sample 30 μL: 30 μL / mouse, B7 amount 15 μg PBS sample: 30 μL / mouse

[0194] Tumor growth and body weight in mice were monitored every two days from the start of administration. Tumors were measured with calipers, and tumor volume V was calculated as follows: V = π / 6 × L × W, where L is the length of the tumor and W is the width of the tumor. ２The survival of the mice was monitored, and the survival rate was analyzed by the Kaplan-Meier method. ３ Mice exceeding this age were euthanized and the date was recorded as the day of death of the mouse.

[0195] (Administration Condition A) Eight days after the inoculation of the 4T1 cells, the administration sample was administered for the first time (1st) to the left flank, which was designated as Day 0. Further subcutaneous injections to the left flank were administered four times every six days under the same conditions (a total of five times).

[0196] (A1) Antitumor Effect The results of the antitumor effect are shown in Figure 10. Figure 10 is a graph showing the relationship between the tumor volume (V) of the mice and the number of days from the start of administration. As shown in Figure 10, the tumor volume on Day 0 was 100 mm ３ As shown in FIG. 10 , the PBS-only administration group and the control liposome administration group (Ctrl lipo) showed an increase in tumor volume by approximately 12 to 20 times on Day 30 compared to Day 0. Furthermore, the free B7 administration group (Free B7) showed an increase in tumor volume by approximately 10 times on Day 30 compared to Day 0, demonstrating a greater suppression of tumor growth than the PBS administration group and the Ctrl lipo administration group. In contrast, the B7 liposome administration group (B7 lipo) showed only an increase in tumor volume by approximately 5 times on Day 30 compared to Day 0, demonstrating a significant suppression of tumor volume growth. These results demonstrate that administration of B7 as PEGylated liposomes can effectively suppress breast cancer growth.

[0197] Next, the results of the survival rate are shown in Figure 11. Figure 11 is a graph showing the relationship between the mouse survival rate and the number of days from the start of administration. As shown in Figure 11, the survival rate of mice in the B7 liposome administration group was significantly increased, and specifically, the survival period was about four times longer than that of the PBS administration group. From these results, it is believed that the administration of B7 liposome induced immune activation, which suppressed cancer growth and, as a result, extended the survival period of the mice.

[0198] (A2) Tolerability / Safety The results of tolerability and safety are shown in Figure 12. Figure 12 is a graph showing the relationship between mouse body weight and the number of days from the start of administration. As shown in Figure 12, compared to the group administered with PBS alone, no significant decrease in mouse body weight was observed in the group administered with the control liposome (Ctrl lipo), the group administered with free B7 (Free B7), or the group administered with the B7 liposome (B7 lipo). From these results, it was confirmed that, like PBS, none of the B7 liposome, the control liposome, or free B7 caused significant side effects in mice.

[0199] (Administration Condition B) Five days after the inoculation of the 4T1 cells, the first administration (1st) of the administration sample into both flanks was designated as Day 0, and five days later (Day 5), a second subcutaneous injection into both flanks was performed under the same conditions (a total of two times).

[0200] (B1) Antitumor Effect The results of the antitumor effect are shown in Figure 13. Figure 13 is a graph showing the relationship between the tumor volume (V) of the mice and the number of days from the start of administration. As shown in Figure 13, the tumor volume on Day 0 was 36 mm ３ As shown in Figure 13, the PBS-only administration group and the control liposome administration group (Ctrl lipo) showed an increase in tumor volume by approximately 30 to 40 times on Day 32 compared to Day 0. Furthermore, the free B7 administration group (Free B7) showed an increase in tumor volume by approximately 27 times on Day 32 compared to Day 0, demonstrating a greater suppression of tumor growth than the PBS administration group and the B7 liposome administration group (Ctrl lipo). In contrast, the B7 liposome administration group (B7 lipo) showed only an increase in tumor volume by approximately 7 times on Day 32 compared to Day 0, demonstrating a significant suppression of tumor volume growth. These results demonstrate that breast cancer growth can be effectively suppressed by administering B7 as PEGylated liposomes.

[0201] Next, the results of the survival rate are shown in Figure 14. Figure 14 is a graph showing the relationship between the survival rate of mice and the number of days from the start of administration. As shown in Figure 14, the survival rate of mice in the B7 liposome administration group was significantly maintained compared to the other administration groups.

[0202] Similar results were obtained with subcutaneous administration under the above-mentioned administration condition A and subcutaneous administration under this administration condition B. This indicates that B7 liposomes activate the immune system by subcutaneous administration, regardless of the differences in these administration conditions, thereby exhibiting excellent antitumor and life-prolonging effects.

[0203] (B2) Tolerability / Safety The results of tolerability and safety are shown in Figure 15. Figure 15 is a graph showing the relationship between mouse body weight and the number of days from the start of administration. Furthermore, as shown in Figure 15, compared to the group administered with PBS alone, no significant decrease in mouse body weight was observed in the group administered with the control liposome (Ctrl lipo), the group administered with free B7 (Free B7), or the group administered with the B7 liposome (B7 lipo).

[0204] Example 6 The PEGylated liposomes prepared in Example 1 were subjected to repeated subcutaneous administration to confirm their antitumor effect on skin cancer cells.

[0205] The same administration samples as in Example 5 were used: B7 liposome administration sample (B7 lipo) 30 μL: 30 μL / mouse, PEGylated liposome concentration 1 mg / mL (total lipid equivalent), B7 amount 15 μg; control liposome administration sample (Ctrl lipo) 30 μL: 30 μL / mouse, PEGylated liposome concentration 1 mg / mL (total lipid equivalent); free B7 sample 30 μL: 30 μL / mouse, B7 amount 15 μg; PBS sample: 30 μL / mouse

[0206] B16F10 cells were used as skin cancer cells. B16F10 cells (1.0 × 10 ６Then, in the same manner as in Example 5, 30 μL of the administration sample was administered to confirm the antitumor effect, safety, and tolerability.

[0207] The results of the antitumor effect are shown in Figure 16. Figure 16A is a graph showing the relationship between mouse tumor volume (V) and the number of days since the start of administration. Figure 16B is a graph showing the relationship between mouse body weight (g) and the number of days since the start of administration. As shown in Figure 16A, in the PBS-administered group (black circles) and the free B7-administered group (black squares), the increase in tumor volume on Day 6 compared to Day 0 was approximately 40-50 times. In contrast, in the B7 liposome-administered group (white squares), the increase in tumor volume on Day 6 compared to Day 0 was only approximately 15 times, confirming that tumor volume increase was significantly suppressed. These results demonstrate that administration of B7 as PEGylated liposomes can effectively suppress the growth of skin cancer. Furthermore, as shown in Figure 16B, no significant weight loss was observed in any of the administration groups from Day 0 to Day 10. These results confirm that B7 liposomes, like free B7, do not exhibit significant toxicity.

[0208] Example 7 The PEGylated liposomes prepared in Example 1 were subjected to repeated subcutaneous administration to confirm their antitumor effect on breast cancer cells.

[0209] The following liposome samples prepared using PBS in Example 1 were used: (1-2) 50% α-GalCer / negatively charged PEG-terminated -OH liposomes 100 nm; (1-11) 0% control / negatively charged PEG-terminated -OH liposomes 100 nm. The liposome samples were adjusted to a PEGylated liposome concentration of 1 mg / mL (total lipid equivalent) and used as administration samples. PBS was used to adjust the PEGylated liposome concentration. The administration sample prepared in (1-2) above was used as the α-GalCer liposome administration sample (αGal lipo) of this example, and the administration sample prepared in (1-11) above was used as the control liposome administration sample (Ctrl lipo). In addition, a free GalCer sample (Free αGal) prepared by suspending non-liposomal free α-GalCer in PBS, and PBS, were also used as administration samples.

[0210] Triple-negative breast cancer cells 4T1 were used as breast cancer cells. 4T1 cells (5.0 × 10 ５ ~1.0 x 10 ６ The average tumor volume was 40-100 mm ３ At this point, the mice were randomly divided into groups. Then, the mice were subcutaneously injected with the administration samples into the left armpit (Day 0). The doses of each administration sample were as follows: α-GalCer liposome administration sample 30 μL: 30 μL / mouse, α-GalCer amount 15 μg, total lipid amount 30 μg α-GalCer liposome administration sample 30 μL: 30 μL / mouse, total lipid amount 30 μg α-GalCer free sample 30 μL: 30 μL / mouse, α-GalCer amount 15 μg PBS: 30 μL / mouse

[0211] The first administration (1st) of the administration sample to the left flank was designated as Day 0, and further subcutaneous injections to the left flank were performed three times every five days under the same conditions (a total of four times).The antitumor effect, safety, and tolerability were then confirmed in the same manner as in Example 5.

[0212] The results of the antitumor effect are shown in Figure 17. Figure 17 is a graph showing the relationship between tumor volume (V) and the number of days from the start of administration for mice in each administration group (n = 2 for each group). As shown in Figure 17, tumor volume significantly increased over time in the PBS-administered group (PBS) and the control liposome-administered group (Ctrl lip). Compared to these administration groups, the increase in tumor volume was suppressed in the free GalCer-administered group (Free αGal). On the other hand, the αGalCer liposome-administered group (αGal lipo) showed a more significant suppression of tumor volume increase than the free GalCer-administered group (Free αGal), confirming significant suppression of cancer growth. These results demonstrate that administration of α-GalCer as PEGylated liposomes can effectively suppress breast cancer growth. Note that Figure 17 shows results for n = 2 for each group, but similar behavior was observed in the other mice in each group.

[0213] Example 8 The PEGylated liposomes prepared in Example 1 were administered to mice transplanted with cancer cells, and the biodistribution was confirmed.

[0214] (1) Biodistribution after Administration of B7 Liposomes in Tumor-Bearing Mice The following liposome samples prepared using the dye-containing PBS in Example 1 were used. (1-1) 50% B7 / negatively charged PEG-terminated -OH liposomes 100 nm (1-11) 0% control / negatively charged PEG-terminated -OH liposomes 100 nm The liposome samples were adjusted to a PEGylated liposome concentration of 1 mg / mL (total lipid equivalent) and used as administration samples. The dye-containing PBS was used to adjust the PEGylated liposome concentration. The administration sample prepared in (1-1) above was used as the B7 liposome administration sample (B7 lipo) of this example, and the administration sample prepared in (1-11) above was used as the control liposome administration sample (Ctrl lipo).

[0215] Triple-negative breast cancer cells 4T1 were used as breast cancer cells. 4T1 cells (5.0 × 10 cells) were injected into the left breast of a non-cancer-bearing BALB / c mouse (5-week-old, female) near the second nipple from the top. ５~1.0 x 10 ６ The average tumor volume was 100 mm ３ On day 8, 30 μL of the administration sample was subcutaneously injected into the left armpit or both armpits of the mice. The doses of each administration sample were as follows: 30 μL of the B7 liposome administration sample: 30 μL / mouse, 15 μg of B7; 30 μL of the control liposome administration sample: 30 μL / mouse

[0216] 12 hours, 24 hours, and 48 hours after administration, the mice in each administration group were euthanized, and the lymph nodes, cancer cells, liver, and spleen were removed to confirm the distribution of B7 liposomes in the living body. Unless otherwise specified, the same procedures as in Example 4 were carried out.

[0217] These results are shown in Figures 18 to 20. Figure 18A is an image (converted to black and white) showing fluorescence in lymph nodes, where AL is the proper axillary lymph node and BL is the accessory axillary lymph node. Figure 18B is a graph showing the fluorescence intensity in all excised lymph nodes. Figure 19 is an image (converted to black and white) showing fluorescence in the cancer and spleen at the site of cancer cell inoculation. Figure 20 is an image (converted to black and white) showing fluorescence in the liver to which cancer had metastasized. In Figure 20, the bottom image is a photograph of the excised organ, and the top image is a graph overlaying the fluorescence distribution on the photograph. In the fluorescence images in Figures 18 to 20, white arrows indicate areas with stronger fluorescence intensity in the B7 liposome-administered group (B7 lipo) compared to the control-administered group (Ctrl lipo). In addition, in the liver photograph in the bottom image of Figure 20, black arrows indicate areas with severe cancer metastasis.

[0218] As shown in Figures 18 and 19, in the control liposome-administered group (Ctrl lipo), no fluorescence was observed in the lymph nodes or tumor, and liposome localization was not observed. In contrast, in the B7 liposome-administered group (B7 lipo), fluorescence was observed and B7 liposome localization was observed, as shown in Figures 18 (lymph nodes) and 19 (cancer). Furthermore, in the B7 liposome-administered group, B7 liposome distribution in the lymph nodes was confirmed 12 to 24 hours after administration (Figure 18), and B7 liposome distribution in the tumor was confirmed 24 to 48 hours after administration (Figures 19 and 20). Furthermore, in the B7 liposome-administered group, mice with a high incidence of cancer metastasis to the liver also showed high accumulation of B7 liposome in the liver (top panel of Figure 20, B7 lipo / 48 hours / first, second, and fifth livers from the left). On the other hand, in the control liposome group (Ctrl lipo), no accumulation of the fluorescent dye was observed even in the liver where metastasis had occurred. These results suggest that the incorporation of B7 into liposomes induced a stronger selective interaction than nonspecific accumulation, such as the EPR (Enhanced Permeability and Retention) effect, effectively accumulating the substances in the liposomes (i.e., B7 and the fluorescent dye) in the cancer. The administered B7 liposomes were then taken up by lymph nodes and further taken up by dendritic cells, which then migrated to the cancer.

[0219] (2) Distribution of B7 liposomes within cancer cells and changes in the number of immune cells within cancer cells in tumor-bearing mice

[0220] (2A) To investigate what cells delivered B7 liposomes to cancer cells after administration, the colocalization of fluorescently labeled liposomes with cancer cells was measured using flow cytometry.

[0221] The same administration samples as those in (1) above were used, and the administration conditions for each administration sample were as follows: 30 μL of the B7 liposome administration sample: 30 μL / mouse, 15 μg of B7; 30 μL of the control liposome administration sample: 30 μL / mouse

[0222] Triple-negative breast cancer cells 4T1 were used as breast cancer cells. 4T1 cells (5.0 × 10 cells) were injected into the area of ​​the second nipple from the top of the left breast of BALB / c mice (5-week-old, female, n = 4) without cancer. ５ ~1.0 x 10 ６ The average tumor volume was 100 mm ３ On day 8, mice were injected subcutaneously with 30 μL of the treatment sample under the left or both armpits. Twenty-four hours after administration, mice from each treatment group were euthanized and tumors were removed. The tumors were minced and shaken at 37°C and 500 RPM in RPMI containing 0.2% collagenase (Fujifilm Wako Co., Ltd.) for 30 minutes. The mixture was then placed in a cell strainer and gently crushed in RPMI containing 1% FBS to obtain single cells. The single cells were centrifuged at 400 g for 4 minutes to collect viable cells.

[0223] The collected cell population was blocked using the same method as in Example 3(3). Next, the blocked cell population was simultaneously labeled with NK cells and T cells. For the NK cell labeling, PBS (containing 1% FBS) containing Anti-mouse NK1.1-InVivo (Selleck) and FlexAble CoraLite® Plus 488 Antibody Labeling Kit for mouse IgG (Proteintech) was used as the reagent. For the T cell labeling, PBS (containing 1% FBS) containing PE anti-mouse CD3 Antibody (Biorezen) was used as the reagent. Each labeling reagent was simultaneously added to the blocked cell population and allowed to stand at 4°C for 45 minutes, thereby simultaneously labeling the NK cells and the T cells in the cell population.

[0224] Dendritic cells were labeled by adding PBS (1% FBS) containing FITC anti-mouse CD11c antibody (Biorezen) to the collected cells and incubating at 4°C for 45 minutes. Macrophages were labeled by adding PBS (1% FBS) containing anti-mouse CD68 (Militenyi) and FlexAble CoraLite® Plus 488 Antibody Labeling Kit for rabbit IgG (Proteintech) to the cells and incubating at 4°C for 45 minutes.

[0225] The labeled cells were washed three times with PBS (containing 1% FBS) and then analyzed by flow cytometry (BD FACSAria™ III Cell Sorter, DB Biosciences). The results are shown in Figures 21-25 (n=4).

[0226] 21 shows the results of flow cytometry demonstrating the co-localization of dendritic cells and liposomes in cancer. Dendritic cells can be detected by the fluorescence intensity based on FITC staining of their CD11c, and liposomes can be detected by the fluorescence intensity based on Cy5 in the dye-containing PBS.

[0227] Figure 21A is a graph showing the distribution of cells based on FITC fluorescence intensity and Cy5 fluorescence intensity, with the horizontal axis representing FITC fluorescence intensity and the vertical axis representing Cy5 fluorescence intensity. Figure 21B is a graph showing the distribution of cells based on FITC fluorescence intensity, with the horizontal axis representing FITC fluorescence intensity and the vertical axis representing the percentage of cell count. In each administration group, the left peak represents CD11c-negative cells, and the right peak represents CD11-positive cells (dendritic cells). Figure 21C is a graph quantifying the percentage of CD11c-positive cells (right peak) in Figure 21B. Figure 21D is a graph showing the distribution of CD11c-positive cells based on Cy5 fluorescence intensity, with only the CD11c-positive cells (right peak) in Figure 21B gated. The horizontal axis represents Cy5 fluorescence intensity and the vertical axis represents the percentage of cell count. FIG. 21E shows the results of quantifying the percentage of CD11c-positive and Cy5-positive cells in FIG. 21D.

[0228] 22 shows the results of flow cytometry demonstrating the co-localization of macrophages and liposomes in cancer. Macrophages can be detected by fluorescence intensity based on FITC staining of their CD68, and liposomes can be detected by fluorescence intensity based on Cy5 in the dye-containing PBS.

[0229] Figure 22A is a graph showing the distribution of cells based on FITC fluorescence intensity and Cy5 fluorescence intensity, with the horizontal axis representing FITC fluorescence intensity and the vertical axis representing Cy5 fluorescence intensity. Figure 22B is a graph showing the distribution of cells based on FITC fluorescence intensity, with the horizontal axis representing FITC fluorescence intensity and the vertical axis representing the percentage of cell count. In each administration group, the left peak represents CD68-negative cells, and the right peak represents CD68-positive cells (macrophages). Figure 22C is a graph quantifying the percentage of CD68-positive cells (right peak) in Figure 22B. Figure 22D is a graph showing the distribution of CD68-positive cells based on Cy5 fluorescence intensity, with only the CD68-positive cells in Figure 22B gated. The horizontal axis represents Cy5 fluorescence intensity and the vertical axis represents the percentage of cell count. FIG. 22E shows the results of quantifying the percentage of CD68-positive and Cy5-positive cells in FIG. 22D.

[0230] 23 shows the results of flow cytometry demonstrating the co-localization of NK cells and liposomes in cancer. NK cells can be detected by flow cytometry based on the fluorescence intensity of their NK1.1 staining with FITC, and liposomes can be detected by the fluorescence intensity of Cy5 from the dye-containing PBS.

[0231] Figure 23A is a graph showing the distribution of cells based on NK1.1 fluorescence intensity and Cy5 fluorescence intensity, with the horizontal axis representing NK1.1 fluorescence intensity and the vertical axis representing Cy5 fluorescence intensity. Figure 23B is a graph showing the distribution of cells based on FITC fluorescence intensity, with the horizontal axis representing NK1.1 fluorescence intensity and the vertical axis representing the percentage of cell count. In each administration group, the left peak represents NK1.1-negative cells, and the right peak represents NK1.1-positive cells (NK cells). Figure 23C is a graph quantifying the percentage of NK1.1-positive cells (right peak) in Figure 23B. Figure 23D is a graph showing the distribution of NK1.1-positive cells based on Cy5 fluorescence intensity, with only the NK1.1-positive cells (right peak) in Figure 23B gated. The horizontal axis represents Cy5 fluorescence intensity, and the vertical axis represents the percentage of cell count. FIG. 23E shows the results of quantifying the percentage of NK1.1-positive and Cy5-positive cells in FIG. 23D.

[0232] 24 shows the results of flow cytometry demonstrating the co-localization of T cells and liposomes in cancer. T cells can be detected by fluorescence intensity based on PE staining of their CD3, and liposomes can be detected by fluorescence intensity based on Cy5 in the dye-containing PBS.

[0233] Figure 24A is a graph showing the distribution of cells based on FITC fluorescence intensity and Cy5 fluorescence intensity, with the horizontal axis representing CD3 fluorescence intensity and the vertical axis representing Cy5 fluorescence intensity. Figure 24B is a graph showing the distribution of cells based on FITC fluorescence intensity, with the horizontal axis representing FITC fluorescence intensity and the vertical axis representing the percentage of cell count. In each administration group, the left peak represents CD3-negative cells and the right peak represents CD3-positive cells (T cells). Figure 24C is a graph quantifying the percentage of CD3-positive cells (right peak) in Figure 24B. Figure 24D is a graph showing the distribution of CD3-positive cells based on Cy5 fluorescence intensity, with only the CD3-positive cells (right peak) in Figure 24B gated. The horizontal axis represents Cy5 fluorescence intensity and the vertical axis represents the percentage of cell count. Figure 24E shows the results of quantifying the percentage of CD3-positive and Cy5-positive cells in Figure 24D.

[0234] Figure 25 shows the results of flow cytometry demonstrating the colocalization of CD3-positive and NK1.1-positive double-positive cells (NKT cells) and liposomes in cancer. Figure 25A is a graph showing the distribution of CD3-positive and NK1.1-positive double-positive cells (NKT cells) by FITC fluorescence intensity, with the horizontal axis representing FITC fluorescence intensity and the vertical axis representing the percentage of cell count. Figure 25B shows the results of quantifying the percentage of CD3-positive and NK1.1-positive double-positive cells (NKT cells) in Figure 25A. Figure 25C is a graph showing the distribution of CD3-positive and NK1.1-positive double-positive cells (NKT cells) in Figure 25B by Cy5 fluorescence intensity. Figure 25D shows the results of quantifying the percentage of CD3-positive / NK1.1-positive / Cy5-positive cells in Figure 25C.

[0235] First, as shown in Figures 21A, 21B, and 21C regarding dendritic cells, the number of dendritic cells in the cancer was increased in the B7 liposome-administered group (B7 lipo) compared to the control liposome-administered group (Ctrl lipo). Furthermore, as shown in Figures 21D and 21E, the B7 liposome-administered group (B7 lipo) demonstrated increased colocalization of dendritic cells (CD11c-positive) and liposomes (Cy5-positive) compared to the B7 liposome-administered group (B7 lipo). In particular, the B7 liposome-administered group (B7 lipo) showed an approximately 20% increase in the number of dendritic cells in the cancer compared to the control liposome-administered group (Ctrl lipo), while the number of liposome-administered dendritic cells (CD11c-positive and Cy5-positive cells) increased by approximately 100%. From these results, it is speculated that dendritic cells delivered the liposomes to the cancer. Furthermore, the results of Figures 22A, 22B, 22C, and 22E for macrophages were similar to those of Figure 21 for dendritic cells. From these results, it is speculated that antigen-presenting phagocytes such as dendritic cells and macrophages delivered the liposomes to the cancer.

[0236] Similarly, as shown in Figures 23 (23A-23E), 24 (24A-24E), and 25 (25A-25E) for NK cells, T cells, and NKT cells, increases in the number of cancer cells and the number of liposome-containing cells (Cy5-positive cells) were confirmed for these cells as well. In particular, for NKT cells shown in Figure 25, the B7 liposome-administered group (B7 lipo) showed a 200% increase in the number of liposome-containing cells (Cy5-positive cells) in the cancer compared to the control liposome-administered group (Ctrl lipo), demonstrating a significantly higher increase rate. However, because T cells, NK cells, and NKT cells do not have phagocytic activity, it is unlikely that these cells directly ingested liposomes. Therefore, it is speculated that the aforementioned phagocytes (dendritic cells and macrophages) ingested the liposomes, absorbed the Cy5 contained therein, and then interacted with T cells, NK cells, and NKT cells, transferring Cy5 from the phagocytes to the T cells, NK cells, and NKT cells. In particular, given the increase in fluorescence intensity in the lymph nodes shown in Figure 9 and the increase in dendritic cells and NKT cells in the lymph nodes 12 to 24 hours after administration shown in Figures 5-8, but no increase in macrophages, the following hypothesis can be further proposed: When B7 liposomes are administered, they are taken up by the lymph nodes and phagocytosed and absorbed by dendritic cells. Then, in the lymph nodes, dendritic cells display B7 as an antigen, which activates NKT cells. During this process, the Cy5 taken up by the dendritic cells is transferred to the NKT cells. It is thought that the activated immune system (e.g., NKT cells, dendritic cells, etc.) then migrates to the cancer and exerts an anti-cancer effect.

[0237] (2B) The tumor tissue excised in (2A) was fixed in 20% formaldehyde for 48 hours, embedded in paraffin, and cut into 5 μm sections. The paraffin embedding was performed according to the equipment and protocol provided by Abcam. The sections were placed on slides and deparaffinized (treated in the order of xylene, ethanol, and water). The section slides were then treated with citrate buffer (pH 6.0) at 98°C for 20 minutes for antigen retrieval.

[0238] The section slides were blocked at 4°C for 30 minutes as described in (2A). The blocked sections were then stained with a primary antibody, followed by secondary antibody staining. Primary antibody staining was performed using PBS (containing 1% FBS) containing CD56 (NCAM) Antibodies (ThermoFisher), PBS (containing 1% FBS) containing IBA1 Recombinant Rabbit Monoclonal Antibody (ThermoFisher), or PBS (containing 1% FBS) containing CD3 Antibodies (ThermoFisher), with incubation at 25°C for 45 minutes. Secondary antibody staining was performed using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (ThermoFisher), with incubation at room temperature for 30 minutes to 1 hour. AEC ((3-Amino-9-ethylcarbazole), Peroxidase substrate) substrate solution (Abcam) was then added to the section slide and allowed to stand for 5 minutes to stain the target protein red. Distilled water was then added to the section slide to stop the staining reaction, and the section slide was further stained with hematoxylin to stain the nuclei blue. The stained tissue on the section slide was observed under a microscope.

[0239] These results are shown in Figure 26. Figure 26 is a micrograph of a section slide, specifically, a photograph in which macrophages, NK cells, and T cells were visualized by staining for the marker proteins IBA1, CD56, and CD3, respectively. As shown in Figure 26, the B7 liposome-administered group (B7 lipo) showed increased numbers of macrophages, NK cells, and T cells in the cancer compared with the PBS-administered group. This suggests that administration of B7 liposomes activated the entire immune system, which in turn affected the anti-cancer effect.

[0240] Example 9 The effects of changing conditions on the biodistribution and immune activity of the various B7 liposomes prepared in Example 1 were examined.

[0241] (1) Effect of size change on B7 liposome uptake by dendritic cells in lymph nodes

[0242] (1A) The following liposome samples prepared using the dye-containing PBS in Example 1 were used. (1-1) 50% B7 / negatively charged PEG-terminated -OH liposomes 100 nm (1-3) 50% B7 / negatively charged PEG-terminated -OH liposomes 50 nm (1-4) 50% B7 / negatively charged PEG-terminated -OH liposomes 200 nm (1-5) 50% B7 / negatively charged PEG-terminated -OH liposomes 400 nm The liposome samples were adjusted to a PEGylated liposome concentration of 1 mg / mL (total lipid equivalent) and used as administration samples. The dye-containing PBS was used to adjust the PEGylated liposome concentration. The administration sample contained 15 μg / 30 μL of B7.

[0243] 30 μL of the administration sample was subcutaneously injected into the left flank of a cancer-free BALB / c mouse (5 weeks old, female). 12 and 24 hours after administration, the mouse was euthanized, and the lymph nodes, spleen, kidneys, lungs, heart, thymus, and liver were removed.

[0244] The distribution of B7 liposomes in each of the excised organs was confirmed in the same manner as in Example 4, unless otherwise specified.

[0245] These results are shown in Figure 27. Figure 27A shows a photograph of each organ of a mouse administered with a B7 liposome sample 12 hours after administration, with fluorescence intensity superimposed on the image. Areas with particularly strong fluorescence intensity are indicated by white arrows in Figure 27A. Figure 27B is a graph quantifying the fluorescence intensity in the lymph nodes.

[0246] As shown in Figure 27, lymph nodes exhibited higher fluorescence intensity than other organs. For example, the fluorescence intensity of the lungs at 50 nm, which had the highest fluorescence intensity among other organs, was equal to or lower than the fluorescence intensity of the lymph nodes at 200 nm. Furthermore, when the fluorescence intensity was divided by the organ weight and normalized to fluorescence intensity per gram, the accumulation in lymph nodes was more than 10 times higher than that of other organs.

[0247] As shown in Figure 27B, the B7 liposome-administered group effectively accumulated in lymph nodes 12 hours after administration, regardless of the size of the liposomes, with smaller liposomes being particularly effective. While prior art reports have shown that particles of 50 nm or larger do not accumulate in lymph nodes, this example confirmed that liposomes of 50 nm or larger (e.g., 100 nm liposomes) also accumulated in lymph nodes.

[0248] (1B) Next, the administered sample (1-1, 100 nm) was used as a cryo-sample and observed using a transmission electron microscope (CryoTEM). The cryoTEM sample was prepared using a LEICA EM GP (Leica Microsystems) according to the protocol provided by Leica Microsystems. The cryoTEM was measured using a JEM2100 (JEOL Ltd.).

[0249] The results are shown in Figure 28. Figure 28 is an electron microscope photograph of the administration sample (1-1). As shown in Figure 28, observation with the electron microscope confirmed many spherical B7 liposomes, and some B7 liposomes with an elongated structure were also present, with the short side length being approximately 25 nm. The structure of the latter liposomes is thought to be due to softening and elongation caused by interaction with the edge of the grit on which the administration sample was placed. From these results, it is believed that the B7 liposomes of this example were effectively taken up by lymph nodes, even though their average particle size was, for example, greater than 50 nm, due to their softness and ability to deform.

[0250] (1C) Next, Figure 29 shows the results of the accumulation of B7 liposomes in lymph nodes 24 hours after administration for mice administered with B7 liposome samples. Figure 29A shows the accumulation of B7 liposomes in lymph nodes of mice 12 hours and 24 hours after administration, expressed as fluorescence intensity. The arrows indicate the increase or decrease in fluorescence intensity from 12 hours to 24 hours after administration. The downward-sloping arrow indicates a decrease from 12 hours to 24 hours after administration, while the upward-sloping arrow indicates an increase from 12 hours to 24 hours after administration. Figure 29B is a graph quantifying the fluorescence intensity showing the accumulation of B7 liposomes in lymph nodes of mice 12 hours and 24 hours after administration, based on Figure 29A. As shown in Figure 29, 12 hours after administration, the administration groups with different B7 liposome particle sizes showed similar accumulation levels. In the administration group of the administration sample (1-1) (average diameter 100 nm), the accumulation amount increased significantly from 12 hours to 24 hours after administration, and showed the highest accumulation amount among all the administration groups.

[0251] (1D) Next, human dendritic cell line DC2.4 cells were seeded into 8-well glass-bottom chambers at 20,000 cells / well and incubated overnight at 37°C in RPMI containing 10% v / v FBS and 1% v / v penicillin-streptomycin. The same administration sample (B7 concentration: 15 μg / 30 μL) as in (1A) above was then added to the wells to a final B7 concentration of 0.05 mg / mL, followed by incubation for 6 hours. After incubation, the cells in the wells were washed with fresh medium (the RPMI) to remove excess administration sample. A staining reagent (0.1 mg / mL Hoechst 33342) was added to the wells, and the cell nuclei were stained by incubation for 15 minutes. Next, the cells in the wells were washed with fresh medium (the above-mentioned RPMI) and then fixed with 4% PFA (4% paraformaldehyde in PBS) at room temperature for 15 minutes. The fixed cell samples were observed with a confocal laser scanning microscope (CLSM) (LSM 880, Carl Zeiss). Each fluorescence was observed using different excitation (ex.) and emission (em.) filters as follows: Hoechst 33342: ex. 405 nm / em. 410-485 nm Sulfo-Cy5: ex. 633 nm / em. 638-746 nm

[0252] These results are shown in Figure 30. Figure 30 is a graph showing the uptake of B7 liposomes into cells based on the fluorescence intensity of Cy5 derived from B7 liposomes. As shown in Figure 30, the uptake of B7 liposomes into dendritic cells increased as the average particle size approached 200 nm, with the highest uptake being in the administered sample with an average particle size of 200 nm. The results of (1A) and (1D) above showed that uptake into lymph nodes was more likely the smaller the average diameter of the liposomes, whereas uptake into dendritic cells was more likely the closer the average diameter of the liposomes was to 200 nm.

[0253] Furthermore, based on the results of (1A) to (1D) above, the following is considered, but the present invention is not limited to these findings. That is, liposomes with a small average particle size of 50 nm effectively and quickly accumulate in lymph nodes, but are difficult to be taken up by dendritic cells therein and are therefore likely to be eliminated over time. Furthermore, liposomes with large average particle sizes of 200 nm and 400 nm are difficult to be taken up by lymph nodes and therefore are unlikely to accumulate in dendritic cells therein. In contrast, liposomes with an average particle size of 100 nm are relatively easily taken up by lymph nodes and further effectively taken up by dendritic cells therein, and therefore, 24 hours after administration, the amount of liposomes accumulated in dendritic cells in lymph nodes is thought to be approximately four times higher than that of liposomes of other sizes.

[0254] Based on the results of (1A) to (1D) above, it was found that the liposome sample (100 nm) of (1-1) above is preferable from the viewpoint of accumulation in lymph nodes and dendritic cells. From this, it can be said that the average particle size of the PEGylated liposome containing the NKT cell-activating ligand of the present invention is preferably 100 nm±40 nm or 100±30 nm, and more preferably 100±20 nm, 100±10 nm, or 100±5 nm.

[0255] (2) Effect of changes in the PEG terminal and surface charge of B7 liposomes on the accumulation of dendritic cells in lymph nodes

[0256] (2A) The following liposome samples were used, which were prepared using the dye-containing PBS in Example 1. The abbreviations for each sample indicate the charge on the liposome surface, with (-) meaning negative charge, (+) meaning positive charge, and (n) meaning neutral. (1-1) 50% B7 / negatively charged PEG-terminated -OH liposomes 100 nm (B7 lipo-OH(-)) (1-8) 50% B7 / negatively charged PEG-terminated -Me liposomes 100 nm (B7 lipo-Me(-)) (1-11) 0% control / negatively charged PEG-terminated -OH liposomes 100 nm (Ctrl lipo-OH(-)) (1-12) 0% control / negatively charged PEG-terminated -Me liposomes 100 nm (Ctrl lipo-Me(-)) (1-9) 50% B7 / neutral PEG-terminated -OH liposomes 100 nm (B7 lipo-OH(n)) (1-10) 50% B7 / positively charged PEG-terminated -OH liposomes 100 nm (B7 The liposome sample was adjusted to a PEGylated liposome concentration of 1 mg / mL (total lipid equivalent) and used as an administration sample. The administration sample contained 15 μg / 30 μL of B7.

[0257] Except for using the above-mentioned administration sample, the administration sample was added to human dendritic cell line DC2.4 cells, followed by incubation, staining, and nucleic acid analysis under a microscope in the same manner as in (1D) above.

[0258] These results are shown in Figures 31 and 32. Figure 31 is a graph showing the uptake of B7 liposomes into dendritic cells as measured by the fluorescence intensity of Cy5 derived from B7 liposomes. In Figure 31, the vertical axis represents the Cy5 fluorescence intensity, which indicates B7 liposomes, normalized by the fluorescence intensity of HOECHST 33342, which indicates the amount of cell nuclei, and corresponds to the amount of B7 liposome uptake in dendritic cells. Figure 31 shows, from left to right, the results for the Ctrl lipo-OH(-) administration group, the Ctrl lipo-Me(-) administration group, the B7 lipo-OH(-) administration group, and the B7 lipo-Me(-) administration group.

[0259] As shown in Figure 31, the fluorescence intensity was comparable between the groups administered with control liposomes not containing an NKT ligand, i.e., the Ctrl lipo-OH(-) administration group and the Ctrl lipo-Me(-) administration group. Furthermore, the B7 lipo-Me(-) administration group, in which the PEG terminal of the B7 liposome was a methyl group, also had a fluorescence intensity comparable to that of the control liposome administration group. Thus, in the case of the control liposome Ctrl lipo-OH(-), which does not contain an NKT cell-activating ligand, the use of a phospholipid PEGylated with a hydroxyl-terminated PEG did not improve uptake into dendritic cells. In contrast, the B7 lipo-OH(-) administration group, which contained B7 as a ligand and had a hydroxyl-terminated PEG, showed significantly higher fluorescence intensity, indicating the amount of liposome uptake into dendritic cells, compared to the control liposome administration group. Furthermore, the B7 lipo-OH(-) administration group showed significantly higher fluorescence intensity, which indicates the amount of liposome uptake by dendritic cells, compared to the B7 lipo-Me(-) administration group in which the PEG has a methyl group at the end.

[0260] Figure 32 is a graph showing the uptake of B7 liposomes into dendritic cells as measured by the fluorescence intensity of Cy5 derived from B7 liposomes. In Figure 32, the vertical axis represents the Cy5 fluorescence intensity, which indicates B7 liposomes, normalized by the fluorescence intensity of HOECHST 33342, which indicates the amount of cell nuclei, and corresponds to the amount of B7 liposome uptake in dendritic cells. Figure 32 shows the results for the B7 lipo-OH(-) administration group, in which the surface charge of B7 liposomes is negative (-), the B7 lipo-OH(n) administration group, in which the surface charge of B7 liposomes is neutral (n), and the B7 lipo-OH(+) administration group, in which the surface charge of B7 liposomes is positive (+).

[0261] The B7 lipo-OH(-), B7 lipo-OH(n), and B7 lipo-OH(+) administration samples all used phospholipids PEGylated with hydroxyl-terminated PEGs. However, the B7 lipo-OH(n) and B7 lipo-OH(+) liposomes were neutrally (n) and positively (+) charged by the addition of the positively charged lipid (DDAB). As a result, as shown in Figure 32, the B7 lipo-OH(-) administration group, which had negatively charged liposomes, exhibited higher fluorescence intensity, indicating the amount of dendritic cell uptake, compared with the B7 lipo-OH(n) administration group and the B7 lipo-OH(+) administration group, which had neutrally or positively charged liposomes.

[0262] From these results, it was found that the PEGylated liposome of the present invention preferably uses a phospholipid PEGylated with PEG having a terminal hydroxyl group, and that the liposome preferably has a negative charge.

[0263] (2B) In the same manner as in (1A), except that 30 μL of the same administration sample as in (2A) was used, the administration sample was subcutaneously injected into the left armpit of a cancer-free BALB / c mouse (5-week-old, female), and lymph nodes were excised 24 hours after administration. The B7 lipo-Me(-), the B7 lipo-OH(-), the B7 lipo-OH(n), and the B7 lipo-OH(+).

[0264] The excised lymph nodes were thinly sliced, and the cell nuclei in the resulting slices were stained with the staining reagent (0.1 mg / mL Hoechst 33342). The stained slices were then used to confirm the distribution of B7 liposomes within the lymph nodes. The distribution of the B7 liposomes was observed using a confocal laser scanning microscope (CLSM) (LSM 880, Carl Zeiss) in the same manner as in (1D) above.

[0265] These results are shown in Figure 33. Figure 33 shows microscopic images of the section, with the first row from the top being a Hoechst 33342 stained image, the second row being a liposome-derived Cy5 fluorescence image, and the third row being an image in which the fluorescence distribution in the second row is superimposed on the stained image in the first row. As shown in Figure 33, it was confirmed that B7 liposome B7 lipo-OH(-), which has a negatively charged surface, is most easily absorbed into lymph nodes. In contrast, B7 lipo-Me(-), which has a negatively charged surface but a methyl group at the end of the PEG, showed significantly reduced uptake into lymph nodes compared to B7 liposome B7 lipo-OH(-). Furthermore, the B7 lipo-OH(n) and B7 lipo-OH(+), which have a neutral or positive surface charge, showed lower accumulation in lymph nodes than the B7 lipo-OH(-).

[0266] Based on the results of (1A) to (2B) above, in order to accumulate in lymph nodes and most efficiently target dendritic cells in the lymph nodes, the PEGylated liposomes of the present invention containing an NKT cell-activating ligand preferably satisfy the following conditions: That is, it is preferable that the liposomes have, for example, an average particle size of 100 nm±40 nm, 100±30 nm, 100±20 nm, 100±10 nm, or 100±5 nm, a negative surface charge, and a hydroxyl group at the PEG terminal on the liposome surface.

[0267] (3) Effect of Changes in the PEG End of the B7 Liposome Surface on Anticancer Effect Next, the effect of changes in the surface properties of the liposome on immune activity was examined.

[0268] The following liposome samples prepared using PBS in Example 1 were used. (1-1) 50% B7 / negatively charged PEG-terminated -OH liposomes 100 nm (B7 lipo-OH(-)) (1-8) 50% B7 / negatively charged PEG-terminated -Me liposomes 100 nm (B7 lipo-Me(-)) (1-11) 0% control / negatively charged PEG-terminated -OH liposomes 100 nm (Ctrl lipo-OH(-)) (1-12) 0% control / negatively charged PEG-terminated -Me liposomes 100 nm (Ctrl lipo-Me(-)) The liposome samples were adjusted to a PEGylated liposome concentration of 1 mg / mL (total lipid equivalent) and used as administration samples. The administration sample contained 15 μg / 30 μL of B7. In addition, a 15 μg / 30 μL administration sample (Free B7) in which non-liposomal B7 was suspended in PBS, and a PBS administration sample (PBS) alone were used.

[0269] In the same manner as in Example 5(1) [Antitumor effect], 4T1 cells (5.0 × 10 ５ ~1.0 x 10 ６ The average tumor volume was 30 mm ３ At the time when the tumor volume reached 100 μL, the mice were randomly divided into groups. Then, the administration sample was subcutaneously injected into both armpits of the mice (Day 0) under the administration condition B of Example 5(1) above, except that 30 μL of the administration sample was used. Then, tumor growth, mouse weight, and survival were monitored in the same manner as in Example 5(1) above.

[0270] The results of the antitumor effect are shown in Figure 34. Figure 34A is a graph showing the relationship between mouse tumor volume (V) and the number of days since the start of administration, Figure 34B is a graph showing the relationship between mouse survival rate and the number of days since the start of administration, and Figure 34C is a graph showing the relationship between mouse body weight (g) and the number of days since the start of administration.

[0271] As shown in FIG. 34A, the PBS administration group (PBS), Ctrl lipo-OH administration group, Free B7 administration group, B7 lipo-Me administration group, and Ctrl lipo-Me administration group showed a significant difference in the number of mice treated with PBS on Day 0 (30 mm ３ On Day 30, tumor volume increased approximately 20-40 times compared to Day 0, confirming rapid cancer growth. In contrast, in the B7 lipo-OH administration group, tumor volume increased only approximately 6 times on Day 30 compared to Day 0, confirming significant suppression of tumor volume increase. These results demonstrate that the cancer suppression effect of B7 liposomes can be significantly improved by converting the PEG terminal to a hydroxy group.

[0272] As shown in Figure 34B, the survival rate of mice in the B7 lipo-OH administration group was significantly maintained compared to the other administration groups. This result indicates that the survival effect of B7 liposomes can be significantly improved by converting the PEG terminal to a hydroxy group.

[0273] As shown in Figure 34C, no significant decrease in body weight was observed in any of the administration groups. From these results, it is considered that the B7 lipo-OH has no problems with tolerability and safety, and has no toxic effects.

[0274] (4) Effect of Change in B7 Concentration in B7 Liposome on Immune Activity We investigated whether immune activity changes depending on the B7 concentration in the B7 liposome.

[0275] The following liposome samples prepared using PBS in Example 1 were used: (1-1) 50% B7 / negatively charged PEG-terminated -OH liposomes 100 nm (50% B7 lipo) (1-6) 10% B7 / negatively charged PEG-terminated -OH liposomes 100 nm (10% B7 lipo) (1-7) 2% B7 / negatively charged PEG-terminated -OH liposomes 100 nm (2% B7 lipo) (1-11) 0% control / negatively charged PEG-terminated -OH liposomes 100 nm (0% B7 lipo) The liposome samples were adjusted to a PEGylated liposome concentration of 1 mg / mL (equivalent to total lipids) and used as administration samples. The administration sample using (1-1) had a B7 amount of 15 μg / 30 μL, the administration sample using (1-6) had a B7 amount of 3 μg / 30 μL, the administration sample using (1-7) had a B7 amount of 0.6 μg / 30 μL, and the administration sample using (1-1) had a B7 amount of 0 μg / 30 μL.

[0276] In the same manner as in Example 5(1) [Antitumor effect], 4T1 cells (5.0 × 10 ５ ~1.0 x 10 ６ The average tumor volume was 46 mm ３ At the time when the tumor volume reached 100 μL, the mice were randomly divided into groups. Then, the administration sample was subcutaneously injected into the left armpit of the mice (Day 4) under the administration condition A in Example 5(1) above, except that 30 μL of the administration sample was used. Then, tumor growth, mouse weight, and survival were monitored in the same manner as in Example 5(1) above.

[0277] The results of the antitumor effect are shown in Figure 35. Figure 35A is a graph showing the relationship between mouse tumor volume (V) and the number of days since the day of subcutaneous administration of the cancer, Figure 35B is a graph showing the relationship between mouse survival rate and the number of days since the day of subcutaneous administration of the cancer, and Figure 35C is a graph showing the relationship between mouse body weight and the number of days since the day of subcutaneous administration of the cancer.

[0278] As shown in FIG. 35A, the 0% B7 lipo-administered group and the 2% B7 lipo-administered group showed a significant decrease in the number of sera on Day 0 (46 mm３ On Day 36, tumor volume increased approximately 20-30 fold compared to Day 0, confirming rapid cancer growth. In contrast, in the 10% B7 lipo-administered group, tumor volume increased only approximately 10-fold compared to Day 0, confirming excellent suppression of tumor volume increase. Furthermore, in the 50% B7 lipo-administered group, tumor volume increased only approximately 7-fold compared to Day 0, confirming even more significant suppression of tumor volume increase. These results demonstrate that the cancer growth inhibitory effect can be increased with increasing B7 concentration in B7 liposomes.

[0279] As shown in Figure 35B, it was confirmed that the life-extending effect increased with increasing B7 concentration in B7 liposomes. This result suggests that the higher the B7 concentration contained in B7 liposomes, the more effective the immune activation, resulting in antitumor and life-extending effects.

[0280] As shown in Figure 35C, no significant decrease in body weight was observed in any of the administration groups. From this result, it is believed that the B7 concentration in the B7 liposomes does not affect toxicity.

[0281] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above-described embodiments. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0282] This application claims priority based on Japanese Patent Application No. 2023-204574, filed December 4, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0283] The liposome preparation of the present invention has, for example, excellent stability during storage, and can obtain the effect of activating NKT cells in the body.

Claims

1. A liposome preparation comprising a PEGylated liposome, the PEGylated liposome comprising an NKT cell activating ligand and a liposome-constituting lipid, the NKT cell activating ligand being α-galactosylceramide or an analog thereof which is specifically recognized by an NKT cell receptor on an NKT cell, and the liposome-constituting lipid comprising a choline-containing phospholipid, a PEGylated lipid, and a cholesterol-based lipid.

2. The liposome preparation according to claim 1, wherein the choline-containing phospholipid is a phosphatidylcholine.

3. The liposome preparation according to claim 2, wherein the two fatty acids in the phosphatidylcholine are each independently a saturated fatty acid or an unsaturated fatty acid.

4. The liposome preparation according to claim 2 or 3, wherein the two fatty acids in the phosphatidylcholine are each independently a fatty acid having 8 to 27 carbon atoms.

5. The liposome preparation according to any one of claims 2 to 4, wherein the phosphatidylcholine is at least one selected from the group consisting of dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, palmitoyl oleoyl phosphatidylcholine, and stearoyl oleoyl phosphatidylcholine.

6. The liposome preparation according to any one of claims 1 to 5, wherein the fatty acid in the PEGylated lipid is a saturated fatty acid or an unsaturated fatty acid.

7. The liposome preparation according to any one of claims 1 to 6, wherein the fatty acid in the PEGylated lipid is a fatty acid having 8 to 70 carbon atoms.

8. The liposome preparation according to any one of claims 1 to 7, wherein the PEGylated lipid is at least one selected from the group consisting of DSPE-PEG, DMG-PEG, DPPE-PEG, DSG-PEG, DOPE-PEG, ceramide-PEG, and stearate-PEG.

9. The liposome preparation according to any one of claims 1 to 8, wherein the average molecular weight of PEG in the PEGylated lipid is 160 to 44,000 Da.

10. The liposome preparation according to any one of claims 1 to 9, wherein in the PEGylated lipid, the terminus of PEG is a hydroxyl group.

11. The liposome preparation according to any one of claims 1 to 10, wherein the cholesterol-based lipid is cholesterol.

12. The liposome preparation according to any one of claims 1 to 11, wherein the size of the PEGylated liposomes is in the range of 10 to 500 nm in average particle size.

13. The liposome preparation according to any one of claims 1 to 12, wherein in the PEGylated liposome, the mol % ratio of the choline-containing phospholipid (X), the PEGylated lipid (Y), and the cholesterol-based lipid (Z) is X:Y:Z=1:(0.03-0.176):(0.34-1.35).

14. The liposome preparation according to any one of claims 1 to 13, wherein the α-galactosylceramide and analogues thereof are compounds represented by formula (I), salts thereof, or solvates thereof.

15. The liposome preparation according to any one of claims 1 to 13, wherein the α-galactosylceramide analog is a compound represented by formula (II), a salt thereof, or a solvate thereof.

16. The liposome preparation according to any one of claims 1 to 13, wherein the α-galactosylceramide analog is a compound represented by formula (III), a salt thereof, or a solvate thereof.

17. The liposome preparation according to any one of claims 1 to 13, wherein the α-galactosylceramide analog is a compound represented by formula (IV), a salt thereof, or a solvate thereof.

18. The liposome preparation according to any one of claims 1 to 13, wherein the α-galactosylceramide analog is a compound represented by formula (V), a salt thereof, or a solvate thereof:

19. The liposome preparation according to any one of claims 1 to 13, wherein the α-galactosylceramide analog is a compound represented by formula (VI), a salt thereof, or a solvate thereof.

20. The liposome preparation according to claim 19, wherein the α-galactosylceramide analog is a compound represented by the following formula (VI-1), a salt thereof, or a solvate thereof:

21. The liposome preparation according to any one of claims 1 to 20, wherein the PEGylated liposome satisfies conditions 1, 2 and 3. (Condition 1) The size of the PEGylated liposome is in the range of 60 to 140 nm in average particle size. (Condition 2) In the PEGylated lipid, the terminal of PEG is a hydroxyl group. (Condition 3) The surface charge of the PEGylated liposome is negative.

22. The liposome preparation according to claim 21, wherein under condition 3, the surface charge of the PEGylated liposome is −80 to 5 mV.

23. An NKT cell activator comprising the liposome agent according to any one of claims 1 to 22.

24. A pharmaceutical composition comprising a liposomal agent according to any one of claims 1 to 22.

25. The pharmaceutical composition according to claim 24, for use in anticancer purposes.

26. The pharmaceutical composition of claim 25, wherein the target cancer is breast cancer, skin cancer, blood cancer, colon cancer, prostate cancer, or ovarian cancer.

27. A pharmaceutical composition according to any one of claims 24 to 26, which is an injectable preparation.

28. The pharmaceutical composition according to any one of claims 24 to 27, for use in anticancer, the target cancer being triple-negative breast cancer, and the PEGylated liposome contained in the liposome agent satisfies conditions 1, 2, and 3. (Condition 1) The size of the PEGylated liposome is in the range of an average particle size of 60 to 140 nm, (Condition 2) In the PEGylated lipid, the terminal of PEG is a hydroxyl group, and (Condition 3) The surface charge of the PEGylated liposome is negative.

29. The pharmaceutical composition according to claim 28, wherein under condition 3, the surface charge of the PEGylated liposome is −80 to 5 mV.

30. A method for activating NKT cells, comprising the step of administering to a subject a liposome agent described in any one of claims 1 to 22, or a pharmaceutical composition described in any one of claims 24 to 29.

31. The method for activating NKT cells according to claim 30, wherein the subject is a human or a non-human animal.

32. A method for treating cancer, comprising the step of administering to a subject a liposomal agent according to any one of claims 1 to 22, or a pharmaceutical composition according to any one of claims 24 to 29.

33. The method for treating cancer according to claim 30, wherein the subject is a subject with triple-negative breast cancer, and the pharmaceutical composition according to claim 28 or 29 is administered in the administration step.

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