Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the present invention relates to a liposome agent, an NKT cell activator, a pharmaceutical composition, and uses thereof.
• the following mechanism is known for the immune system. That is, when immature dendritic cells take up an antigen and migrate to lymph nodes, they differentiate into mature dendritic cells and present the antigen. Then, CD8-positive T cells (killer T cells) are activated by the mature dendritic cells presenting the antigen, acquire cytotoxic activity, and attack cells that have the antigen.
• CD8-positive T cells killer T cells
• the differentiation of the immature dendritic cells into mature cells is suppressed by cancer cells, so there is a problem that the attack on cancer cells using the above mechanism is inhibited. For this reason, a treatment method that activates the immune system (so-called immunotherapy) has become common for diseases such as cancer.
• NKT cells natural killer T cells
• a ligand that NKT cells specifically recognize (hereinafter also referred to as NKT cell activation ligand) is administered as a drug to cause 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.
• 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 gamma (IFN- ⁇ ), thereby enhancing immune responses.
• IL-4 interleukin 4
• IFN- ⁇ interferon gamma
• Non-Patent Document 1 ⁇ -galactosylceramide ( ⁇ -GalCer) and its analogs activate NKT cells.
• the present invention therefore aims to provide a composition that is more suitable for practical use as a ligand for activating NKT cells.
• the liposome preparation of the present invention comprises: PEGylated liposomes, the PEGylated liposome comprises an NKT cell activating ligand and a liposome-constituting lipid; the NKT cell activating ligand is ⁇ -galactosylceramide or an analog thereof, which is specifically recognized by an NKT cell receptor on an NKT cell;
• the liposome is characterized in that the lipids constituting the liposome contain a choline-containing phospholipid, a PEGylated lipid, and a cholesterol-based lipid.
• the NKT cell activator of the present invention is characterized by containing the liposome agent of the present invention.
• the pharmaceutical composition of the present invention is characterized by containing the liposome agent of the present invention.
• the method for activating NKT cells of the present invention is characterized by including a step of contacting the liposome agent of the present invention with NKT cells.
• the liposome preparation of the present invention has excellent stability during storage and can achieve the effects of activating NKT cells in the body.
• FIG. 1 is a graph showing the results of long-term storage stability of B7 liposome in Example 2.
• FIG. 2A is a graph showing IFN- ⁇ concentrations in serum and lymph node supernatants in Example 3.
• FIG. 2B is a graph showing IL-10 concentrations in serum and lymph node supernatants in Example 3.
• Fig. 3A is a histogram of NKT cells in lymph nodes, and is a graph showing the distribution of cell numbers by relative fluorescence intensity indicating CD3 positivity for NK1.1-positive and CD3-positive cells in lymph nodes.
• Fig. 3B is a graph showing the quantification of 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, and is a graph showing the distribution of the number of NK1.1-positive and CD3-positive cells in the spleen by the relative fluorescence intensity.
• Fig. 4B is a graph showing the quantification of the amount of NK1.1-positive and CD3-positive NKT cells in the spleen.
• Fig. 5A is a histogram of NKT cells in lymph nodes, and a graph showing the distribution of cell numbers by relative fluorescence intensity showing CD positivity for NK1.1-positive and CD3-positive cells in lymph nodes.
• FIG. 5B is a histogram of NK cells in lymph nodes, and a graph showing the distribution of cell numbers by relative fluorescence intensity showing NK1.1 positivity for NK1.1-positive NK cells in lymph nodes.
• Fig. 5C is a histogram of T cells in lymph nodes, and a graph showing the distribution of cell numbers by relative fluorescence intensity showing CD3 positivity for CD3-positive T cells in lymph nodes.
• Fig. 5D is a histogram of NKT cells in lymph nodes.
• Fig. 5E is a graph quantifying the amount of NK1.1-positive and CD3-positive NKT cells in lymph nodes.
• FIG. 6A is a histogram of NKT cells in the spleen, and is a graph showing the distribution of the number of NK1.1-positive and CD3-positive cells in the spleen by the relative fluorescence intensity showing CD3 positivity.
• Fig. 6B is a histogram of NK cells in the spleen, and is a graph showing the distribution of the number of NK1.1-positive NK cells in the spleen by the relative fluorescence intensity showing NK1.1 positivity.
• Fig. 6C is a histogram of T cells in the spleen, and is a graph showing the distribution of the number of CD3-positive T cells in the spleen by the relative fluorescence intensity showing CD3 positivity.
• Fig. 6A is a histogram of NKT cells in the spleen, and is a graph showing the distribution of the number of NK1.1-positive and CD3-positive cells in the spleen by the relative fluorescence intensity showing CD3 positivity.
• FIG. 6D is a histogram of NKT cells in the spleen.
• Fig. 6E is a graph quantifying the amount of NK1.1-positive and CD3-positive NKT cells in the spleen.
• Fig. 7A is a histogram of dendritic cells in lymph nodes, and a graph showing the cell number distribution by relative fluorescence intensity showing CD11c positivity for CD11c positive cells in lymph nodes.
• Fig. 7B is a histogram of dendritic cells in lymph nodes.
• Fig. 7C is a histogram of macrophages in lymph nodes, and a graph showing the cell number distribution by relative fluorescence intensity showing CD68 positivity for CD68 positive cells in lymph nodes.
• Fig. 7A is a histogram of dendritic cells in lymph nodes, and a graph showing the cell number distribution by relative fluorescence intensity showing CD68 positivity for CD68 positive cells in lymph nodes.
• FIG. 7D is a graph quantifying the amount of CD11c positive dendritic cells in lymph nodes.
• Fig. 7E is a graph quantifying the amount of CD68 positive macrophages in lymph nodes.
• Fig. 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 by relative fluorescence intensity indicating CD11c positivity.
• Fig. 8B is a histogram of dendritic cells in the spleen.
• Fig. 8C is a histogram of macrophages in lymph nodes, and a graph showing the cell number distribution of CD68-positive cells in the spleen by relative fluorescence intensity indicating CD68 positivity.
• FIG. 8D is a graph quantifying the amount of CD11c-positive dendritic cells in the spleen.
• Fig. 8E is a graph quantifying the amount of CD68-positive macrophages in the spleen.
• FIG. 9A shows images showing fluorescence in each organ of a mouse 24 hours after administration.
• FIG. 9B is a graph showing the relative fluorescence intensity in each organ of the mouse 24 hours after administration.
• FIG. 10 is a graph showing the relationship between mouse tumor volume (V) and the number of days from the start of administration.
• FIG. 11 is a graph showing the relationship between mouse survival rate and the number of days from the start of administration.
• FIG. 12 is a graph showing the relationship between mouse body weight (g) and the number of days from the start of administration.
• FIG. 9A shows images showing fluorescence in each organ of a mouse 24 hours after administration.
• FIG. 9B is a graph showing the relative fluorescence intensity in each organ of the mouse 24 hours after administration.
• FIG. 13 is a graph showing the relationship between mouse tumor volume (V) and the number of days from the start of administration.
• FIG. 14 is a graph showing the relationship between mouse survival rate and the number of days from the start of administration.
• FIG. 15 is a graph showing the relationship between mouse body weight (g) and the number of days from the start of administration.
• FIG. 16A is a graph showing the relationship between mouse tumor volume (V) and the number of days from the start of administration.
• FIG. 16B is a graph showing the relationship between mouse body weight (g) and the number of days from the start of administration.
• FIG. 17 is a graph showing the relationship between mouse tumor volume (V) and the number of days from the start of administration.
• FIG. 18A is an image showing fluorescence in a lymph node.
• 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 the cancer cells were inoculated.
• FIG. 20 is an image showing fluorescence in the liver where cancer has metastasized.
• FIG. 21A is a graph showing the distribution of cells based on the fluorescence intensity of FITC and that of Cy5.
• Figure 21B is a graph showing the distribution of cells according to FITC fluorescence intensity.
• Figure 21C is a graph showing the quantification of the proportion of CD11c positive cells.
• Figure 21D is a graph showing the distribution of CD11c positive cells according to Cy5 fluorescence intensity.
• Figure 21E is the result of quantifying the proportion of CD11c positive and Cy5 positive cells.
• FIG. 22A is a graph showing the distribution of cells based on the fluorescence intensity of FITC and that of Cy5.
• Figure 22B is a graph showing the distribution of cells based on FITC fluorescence intensity
• Figure 22C is a graph showing the quantification of the percentage of CD68-positive cells
• Figure 22D is a graph showing the distribution of CD68-positive cells based on Cy5 fluorescence intensity
• Figure 22E is the result of quantifying the percentage of CD68-positive and Cy5-positive cells.
• FIG. 23A is a graph showing the distribution of cells based on the fluorescence intensity of NK1.1 and that of Cy5.
• Figure 23B is a graph showing the distribution of cells according to FITC fluorescence intensity.
• Figure 23C is a graph quantifying the proportion of NK1.1 positive cells.
• Figure 23D is a graph showing the distribution of NK1.1 positive cells according to Cy5 fluorescence intensity.
• Figure 23E is the result of quantifying the proportion of NK1.1 positive and Cy5 positive cells.
• FIG. 24A is a graph showing the distribution of cells based on the fluorescence intensity of FITC and that of Cy5.
• Figure 24B is a graph showing the distribution of cells according to FITC fluorescence intensity
• Figure 24C is a graph showing the quantification of the proportion of CD3 positive cells
• Figure 24D is a graph showing the distribution of CD3 positive cells according to Cy5 fluorescence intensity
• Figure 24E is the result of quantifying the proportion of CD3 positive and Cy5 positive cells.
• FIG. 25A is a graph showing the distribution of CD3-positive and NK1.1-positive double positive cells (NKT cells) by FITC fluorescence intensity.
• Fig. 25B is a result of quantifying the proportion of CD3-positive and NK1.1-positive double positive cells (NKT cells).
• Fig. 25C is a graph showing the distribution of CD3-positive and NK1.1-positive double positive cells (NKT cells) by Cy5 fluorescence intensity.
• Fig. 25D is a result of quantifying the proportion of CD3-positive/NK1.1-positive/Cy5-positive cells.
• FIG. 26 shows immunohistochemical staining micrographs of mouse cancer section slides.
• FIG. 27A shows a graph in which fluorescence intensity is superimposed on a photograph of each organ of a mouse administered with a sample of B7 liposome 12 hours after administration.
• FIG. 27B is a graph quantifying the fluorescence intensity in lymph nodes.
• FIG. 28 is an electron microscope photograph of the administered sample.
• FIG. 29A is a graph showing the accumulation amount of B7 liposomes in mouse lymph nodes as fluorescence intensity.
• FIG. 29B is a graph quantifying the fluorescence intensity, showing the accumulation of B7 liposomes in mouse lymph nodes.
• FIG. 30 is a graph showing the uptake of B7 liposomes into cells based on the fluorescence intensity of Cy5 derived from B7 liposomes.
• FIG. 31 is a graph showing the uptake of B7 liposomes into dendritic cells based on the fluorescence intensity of Cy5 derived from B7 liposomes.
• FIG. 32 is a graph showing the uptake of B7 liposomes into dendritic cells based on the fluorescence intensity of Cy5 derived from B7 liposomes.
• FIG. 33 is a fluorescence microscopy image of a mouse lymph node section.
• FIG. 34A is a graph showing the relationship between mouse tumor volume (V) and the number of days from the start of administration
• FIG. 34B is a graph showing the relationship between mouse survival rate and the number of days from the start of administration
• FIG. 34C is a graph showing the relationship between mouse body weight and the number of days from the start of administration.
• FIG. 35A is a graph showing the relationship between mouse tumor volume (V) and the number of days from the date of subcutaneous inoculation of the cancer
• FIG. 35B is a graph showing the relationship between mouse survival rate and the number of days from the date of subcutaneous inoculation of the cancer
• FIG. 35C is a graph showing the relationship between mouse body weight and the number of days from the date of subcutaneous inoculation of the cancer.
• a composition comprising a PEGylated liposome, the PEGylated liposome contains an NKT cell activating ligand and a liposome-constituting lipid; the NKT cell activating ligand is ⁇ -galactosylceramide or an analog thereof, which is specifically recognized by an NKT cell receptor on an NKT cell;
• a liposome preparation characterized in that the liposome-constituting lipids include a choline-containing phospholipid, a PEGylated lipid, and a cholesterol-based lipid.
• [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.
• An NKT cell activator comprising the liposome preparation according to any one of [1] to [22].
• a pharmaceutical composition comprising the liposome preparation according to any one of [1] to [22].
• the pharmaceutical composition according to [24] which is for anticancer use.
• the pharmaceutical composition according to [25], wherein the target cancer is breast cancer, skin cancer, blood cancer, colon cancer, prostate cancer, or ovarian cancer.
• the target cancer is triple-negative breast cancer;
• the size of the PEGylated liposome is within 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.
• a method for activating NKT cells comprising the step of administering to a subject the liposome preparation according to any one of [1] to [22], or the pharmaceutical composition according to any one of [24] to [29].
• a method for treating cancer comprising the step of administering to a subject the liposome preparation according to any one of [1] to [22], or the pharmaceutical composition according to any one of [24] to [29].
• the subject is a subject with triple-negative breast cancer, The method for treating cancer according to [32], wherein the pharmaceutical composition according to [28] or [29] is administered in the administration step.
• [34] The method for treating cancer according to [32] or [33], wherein the subject is a human or a non-human animal.
• [35] The liposome preparation according to 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 the production of a pharmaceutical composition.
• a liposome is a lipid vesicle that forms a lipid bilayer.
• the liposome preparation of the present invention is characterized in that it contains a PEGylated liposome, the PEGylated liposome contains an NKT cell activating ligand and a liposome-constituting lipid, the NKT cell activating ligand is ⁇ -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 lipid, and a cholesterol-based lipid.
• the liposome preparation of the present invention will be described below with reference to examples, but the present invention is not limited to these examples. In addition, the description of other embodiments of the liposome preparation of the present invention can be used.
• NKT cell activating ligand is usually a compound that can be specifically recognized by an NKT cell-specific T cell receptor (NKT cell receptor) on an NKT cell and can specifically activate the NKT cell.
• the NKT cell activating ligand is presented, for example, on a CD1d molecule.
• the "NKT cell activating ligand" in the present invention may be, for example, a compound that can be specifically recognized by the NKT cell receptor and can specifically activate the NKT cell.
• the NKT cell activating ligand is not particularly limited, and examples thereof include glycolipids that are already known to have a specific activation function for NKT cells, i.e., ⁇ -galactosylceramide ( ⁇ -GalCer) and derivatives (analogs thereof).
• ⁇ -GalCer ⁇ -galactosylceramide
• the following provides examples of the NKT cell activating ligand, including ⁇ -GalCer and its derivatives.
• R 11 is H or OH;
• X1 is an integer from 7 to 27;
• R 21 is a substituent selected from the group consisting of the following (a) to (e) (Y1 is an integer of 5 to 17).
• R 31 to R 91 are substituents defined in i) or ii) below.
• R 41 is H, OH, NH 2 , NHCOCH 3 , or a substituent selected from the group consisting of the following groups (A) to (D):
• R 51 is OH or a substituent selected from the group consisting of the following groups (E) and (F):
• R 71 is OH or a substituent selected from the group consisting of the following groups (A) to (D):
• R 91 is H, CH 3 , CH 2 OH, or a substituent selected from the group consisting of the following groups (A') to (D').
• R 31 , R 61 and R 71 are 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):
• R 51 is OH or a substituent selected from the group consisting of the following groups (E) and (F):
• R 81 is OH or a substituent selected from the group consisting of the following groups (A) to (D):
• R 91 is H, CH 3 , CH 2 OH or a substituent selected from the group consisting of the following groups (A') to (D').
• ⁇ -GalCer ⁇ -galactosylceramide
• formula (a) The chemical name of ⁇ -GalCer is (2S,3S,4R)-1-O-( ⁇ -D-galactopyranosyl)-2-hexacosanoylamino-1,3,4-octadecanetriol.
• the compound of formula (I), its salt, or solvate may be, for example, those described in patent publications (WO94/09020, WO94/02168, WO94/24142, WO98/44928, etc.) and papers (Science, 278, p.1626-1629, 1997), and may be produced by the methods described therein. It has already been confirmed in these documents that the compound of formula (I) functions as the NKT cell activating ligand.
• R 1 represents an ⁇ -carba sugar residue
• R 2 and R 3 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms
• X represents an oxygen atom, a sulfur atom, -CH 2 - or NH-
• Y represents -CH 2 -, -CH(OH)- or CH ⁇ CH-.
• the compound of formula (II), its salt, or solvate may be, for example, those described in patent publications (WO2008/102888, U.S. Patent No. 8299223, etc.), and may be produced by the methods described therein. It has already been confirmed in these documents that the compound of formula (II) etc. has the function of the NKT cell activating ligand.
• 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 represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms
• the compound of formula (III), its salt, or solvate may be, for example, those described in patent publications (WO2009/119692, U.S. Patent No. 8,551,959, etc.), and may be produced by the methods described therein. It has already been confirmed in these documents that the compound of formula (III) etc. has the function of the NKT cell activating ligand.
• R1 represents a hydrocarbon group having 1 to 30 carbon atoms
• R2 represents a hydrocarbon group having 1 to 20 carbon atoms
• R3 represents a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms
• R4 and R5 may be the same or different and represent a hydrogen atom or a hydrocarbon group having 1 to 5 carbon atoms, or R4 and R5 may combine together to form a divalent hydrocarbon group having 1 to 5 carbon atoms and form a ring structure together with the adjacent ethylenedioxy.
• the compound of formula (IV), its salt, or solvate may be, for example, those described in patent publications (WO2010/030012, U.S. Patent No. 8580751, etc.), and may be produced by the methods described therein. It has already been confirmed in these documents that the compound of formula (IV) etc. has the function of the NKT cell activating ligand.
• 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 -
• the compound of formula (V), its salt, or solvate may be, for example, those described in patent publications (WO2011/552842, U.S. Patent No. 8853173, etc.), and may be produced by the methods described therein. It has already been confirmed in these documents that the compound of formula (V) etc. has the function of the NKT cell activating ligand.
• X represents an alkylene group or -NH-;
• R 1 and R 2 are the same or different and each represents a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group, and each may have a substituent;
• R 1 and R 2 may be joined together with the adjacent nitrogen atom to form a 5- or 6-membered ring;
• R3 represents a hydrocarbon group having 1 to 20 carbon atoms;
• R4 represents a hydrocarbon group having 1 to 30 carbon atoms.
• the compound of formula (VI), its salt, or solvate may be, for example, those described in patent publications (WO 2013/162016 A1, U.S. Patent No. 2015/152128 A1, etc.), and may be produced by the methods described therein. It has already been confirmed in these documents that the compound of formula (VI) etc. has the function of the NKT cell activating ligand.
• NKT cell activating ligands for example, from the viewpoint of more potently inducing IFN- ⁇ production by NKT cells, compounds of formula (III) or formula (VI) are preferred, more preferably A14, B16, and B7, and particularly preferably B7.
• the liposome-constituting lipids include a choline-containing phospholipid, a PEGylated lipid, and a cholesterol-based lipid.
• the liposome-constituting lipids may, for example, include only the choline-containing phospholipid, the PEGylated lipid, and the cholesterol-based lipid, or may further include other components.
• the other components may be, for example, any components and amounts that do not inhibit the formation of liposomes by the liposome-constituting lipids.
• 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 a glycerol backbone as a hydrophilic portion.
• An example of the choline-containing phospholipid is phosphatidylcholine.
• the choline-containing phospholipid may be, for example, PEGylated or not PEGylated (non-PEGylated), and is preferably a non-PEGylated choline-containing phospholipid.
• the fatty acid has a structure having a carboxyl group at the end of the carbon chain, and may be a saturated fatty acid with a saturated carbon chain or an unsaturated fatty acid with a double bond in the carbon chain.
• the two fatty acids may be, independently of each other, saturated or unsaturated fatty acids.
• the combination of the two fatty acids include a combination of saturated fatty acids, a combination of unsaturated fatty acids, and a combination of a saturated fatty acid and an unsaturated fatty acid.
• the two fatty acids may be, independently of each other, branched or unbranched fatty acids.
• the two fatty acids may have, for example, the same number of carbon atoms or different numbers of carbon atoms, and may have carbon chains (main chains) of the same length or different lengths.
• 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.
• the choline-containing phospholipid may, for example, be a combination of fatty acids as shown in the table below, with the following phosphatidylcholine being a specific example.
• 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.
• 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.
• 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.
• the average molecular weight of PEG is, for example, a lower limit of 160 Da, 162 Da, 1,800 Da, or 4,500 Da, an upper limit of 44,000 Da, 5,500 Da, or 2,200 Da, and a range 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.
• 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 shown in the table below may vary in carbon number, for example, in the range of C8 to C22.
• the cholesterol lipid may be, for example, a PEGylated cholesterol-based lipid or a non-PEGylated cholesterol-based lipid, preferably the former.
• the liposome preparation of the present invention contains PEGylated liposomes as liposomes.
• the liposomes contained in the liposome preparation of the present invention are liposomized with the liposome-constituting lipids, and the liposome-constituting lipids contain the PEGylated phospholipids, and therefore are called PEGylated liposomes.
• the liposome preparation of the present invention may contain, for example, one PEGylated liposome or two or more PEGylated liposomes.
• the PEGylated liposome may be, for example, a monodisperse liposome containing molecules of a single molecular weight, or a polydisperse liposome containing molecules of different molecular weights.
• the size and dispersion of the PEGylated liposomes are not particularly limited.
• the size and dispersion 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 represented, for example, by particle size distribution (particle diameter distribution), and the above analytical methods can be used.
• the particle size distribution can be represented, 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.
• a zeta potential measuring device for example, Zetasizer, manufactured by Malvern Instruments Ltd.
• the evaluation conditions are not particularly limited, and for example, the detection temperature is 22 to 28°C (specific example: 25°C).
• the intensity-average hydrodynamic diameter and polydispersity index can be determined, for example, according to the cumulant method.
• 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 called the median or D50), mode (also called the mode or peak), distribution width, etc.
• the PEGylated liposomes preferably have the following sizes when the 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.
• the Z-average hydrodynamic diameter (Z-Average) of the PEGylated liposomes falls within a predetermined range.
• the Z-average hydrodynamic diameter (Z-Average) has a lower limit of, for example, 10 nm or more, 30 nm or more, 50 nm or more, 80 nm or more, or 90 nm or more, and an upper limit of, 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 a range of, 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
• the Z-average hydrodynamic diameter 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 the correlation function obtained from dynamic light scattering (DLS) measurement data and the following formula.
• the size of the PEGylated liposomes for example, in the case of normal distribution, has a standard deviation within ⁇ 2STD, ⁇ 1STD.
• the size distribution of the PEGylated liposomes can be expressed, for example, by the polydispersity index (PDI).
• 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 according to the cumulant method for the distribution measured by the dynamic light scattering method.
• 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.
• 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
• the amount of the NKT ligand is not particularly limited.
• the amount of the NKT ligand per 1 mg of the total lipid of the PEGylated liposome is not particularly limited, and the lower limit is, for example, more than 0 ⁇ g, 5 ⁇ g or more, 10 ⁇ g or more, 20 ⁇ g or more, 50 ⁇ g or more, 100 ⁇ g or more, the upper limit is, for example, 1000 ⁇ g or less, 900 ⁇ g or less, 800 ⁇ g or less, 700 ⁇ g or less, 600 ⁇ g or less, 500 ⁇ g or less, and the range is, for example, more than 0 to 1000 ⁇ g, 5 to 900 ⁇ g, 10 to 900 ⁇ g, 20 to 900 ⁇ g, 20 to 800 ⁇ g, 50 to 700 ⁇ g, 50 to 600 ⁇ g, 100 to 500 ⁇ g.
• the method for preparing the PEGylated liposomes is not particularly limited, and the NKT cell activating ligand and the liposome-constituting lipids may be used as raw materials, and a general method for preparing liposomes using lipid raw materials may be adopted.
• a paper O. H. voss et al., Curr. Protoc. Immunol., 2018, vol. 44, pp. 1-21 of 120-14
• the preparation method is exemplified below, but the present invention is not limited to these descriptions.
• the raw material liquid is prepared using the NKT cell activating ligand and the liposome-constituting lipid as raw materials.
• the raw material liquid may be prepared, for example, by separately preparing a ligand liquid containing the NKT cell activating ligand and a lipid mixture liquid containing the choline-containing phospholipid, the PEGylated phospholipid, and the cholesterol-based lipid, or the ligand liquid, the liquid containing the choline-containing phospholipid, the liquid containing the PEGylated phospholipid, and the liquid containing the cholesterol-based lipid.
• the solvent of the raw material liquid is not particularly limited, and may be, for example, an organic solvent.
• organic solvent examples include alcohol such as methanol, chloroform, dichloromethane, acetonitrile, etc., and any of them may be a single type of solvent or a mixed solvent of two or more types.
• the raw material liquid may be, for example, a diluted liquid diluted with the organic solvent.
• the various raw material liquids are mixed.
• the ligand liquid and the lipid mixture liquid may be mixed, or the ligand liquid, the choline-containing phospholipid-containing liquid, the PEGylated phospholipid-containing liquid, and the cholesterol-based lipid-containing liquid may be mixed.
• 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.
• the solvent fraction is evaporated from the raw material mixture obtained by mixing the raw materials.
• the 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 the evaporation of the solvent fraction.
• This lipid membrane is a lipid membrane composed of PEGylated liposomes containing the NKT cell activating ligand.
• the lipid membrane formed on the inner surface of the container is suspended in, for example, an aqueous solvent and subjected to ultrasonic treatment.
• an aqueous solvent is not particularly limited, and examples thereof include water, saline, buffered saline, buffer solution, etc.
• the lipid membrane decomposition product (mixture of PEGylated liposomes) can be classified into liposome fractions of the desired size and distribution, for example, by filtering using a membrane with the desired pore size.
• the form of the liposome preparation of the present invention is not particularly limited and may be liquid or solid.
• the liquid for example, it is a dispersion liquid dispersed in a solvent.
• the solvent is, for example, a solvent in which the PEGylated liposome does not dissolve, and a specific example thereof is the above-mentioned aqueous solvent.
• a solid for example, it is a state in which the aqueous solvent is removed from the dispersion liquid, or a state (dried body) in which the aqueous solvent is removed and then dried.
• the concentration of the PEGylated liposome is not particularly limited.
• 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 is, for example, 0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL at the lower limit, 1 mg/mL, 5 mg/mL, 25 mg/mL at the upper limit, and 0.05 to 25 mg/mL, 0.1 to 5 mg/mL, 0.5 to 1 mg/mL at the range.
• the concentration may be, for example, the concentration of the liposome preparation of the present invention during storage or during use.
• the conditions 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.
• the liposome preparation of the present invention may contain, for example, other additives in addition to the PEGylated liposome.
• the additives in the NKT cell activator of the present invention described below can be used.
• 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.
• 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 the change in particle size distribution of the PEGylated liposome contained in the liposome preparation over time.
• the stability can be evaluated, for example, by the stability over the storage period (stability 1), the stability due to dilution after storage (stability 2), etc.
• 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. Specifically, the dispersion (D 1 ) after a certain period of time can be compared with the dispersion (D 0 ) immediately after preparation under the same conditions to determine whether aggregates have occurred, and if aggregates have occurred, the extent of the occurrence of aggregates, etc.
• the stability 2 due to dilution after storage can be evaluated, for example, by the presence or absence or amount of aggregates in a dilution obtained by diluting the dispersion after a certain period of time has elapsed.
• the dilution (d 1 ) obtained by diluting the dispersion (D 1 ) after a certain period of time has elapsed can be compared with the dilution (d 0 ) immediately after preparing and diluting the dispersion (D 0 ) under the same conditions to determine whether aggregates have occurred, and if aggregates have occurred, the extent of the occurrence of aggregates.
• the surface charge of the PEGylated liposome is not particularly limited.
• the surface charge can be, for example, ⁇ 80 mV to 5 mV, and is preferably a negative charge, as described below.
• 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 described below, the pharmaceutical composition and treatment method of the present invention, etc.
• the descriptions of each embodiment described below can be cited.
• NKT cell activator and NKT cell activation method The NKT cell activator of the present invention is characterized by comprising the liposome agent of the present invention, as described above. Also, the NKT cell activation method of the present invention is characterized by using the liposome agent of the present invention, as described above. 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. Also, the NKT cell activator and activation method of the present invention can be described with reference to the descriptions of other embodiments.
• 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, pharma- ceutically acceptable additives. Examples of pharma-ceutically acceptable additives include those known in the pharmaceutical field.
• the additives include, for example, carriers, diluents, solvents, excipients, stabilizers, isotonic agents, etc.
• the carriers include, for example, monosaccharides such as sucrose, polysaccharides such as cellulose, the aqueous solvents, etc.
• the diluents and excipients include, for example, the same as the carriers.
• the solvents include, for example, the aqueous solvents, etc.
• protein drugs such as PD-L1 (Programmed cell Death ligand 1)
• antibodies, cellular drugs, and other drugs can also be used in combination.
• the additives are, for example, hydrophilic or weakly hydrophobic.
• the NKT cell activator of the present invention may be, for example, adjusted to the concentration of the PEGylated liposome at the time of use, or may be at a higher concentration than that and diluted at the time of use.
• concentration of the PEGylated liposome in the NKT cell activator of the present invention is not particularly limited, and for example, the concentrations exemplified for the NKT cell activator of the present invention can be used.
• 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.
• the liposome agent can be read as the NKT cell activator of the present invention or the pharmaceutical composition of the present invention described below.
• the liposome preparation of the present invention may be used, for example, in vivo or in vitro.
• the method for activating NKT cells of the present invention includes a step of administering the liposome preparation to a subject.
• the subject may be, for example, a human or a non-human animal.
• the non-human animal include non-human mammals such as mice, rats, rabbits, dogs, cats, cows, horses, monkeys, pigs, goats, and sheep.
• the administration method is not particularly limited, and may be oral or parenteral administration, with parenteral administration being preferred.
• the parenteral administration may be, for example, injection, drip infusion, etc.
• 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.
• the form of the liposome agent of the present invention is not particularly limited, and may be appropriately determined depending on the administration form.
• the form may be, for example, an injection, drip infusion, etc.
• the conditions for administering the liposome agent of the present invention are not particularly limited and can be set appropriately depending on the animal species, age, sex, health condition, weight, etc. of the recipient.
• the dosage 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 number of administrations is, for example, once every 1 to 28 days, once or twice a day.
• the PEGylated liposome When the liposome preparation of the present invention is administered subcutaneously, the PEGylated liposome can be delivered efficiently to lymph nodes such as the axillary lymph nodes, the liver, the breasts, 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 (approximately 24 hours as a specific example).
• the method for activating NKT cells of the present invention includes, for example, a step of contacting the liposome preparation with NKT cells.
• composition and cancer treatment method The pharmaceutical composition of the present invention is characterized by comprising the liposome agent of the present invention, as described above. Also, the cancer treatment method of the present invention is characterized by comprising a 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. Also, the pharmaceutical composition and treatment method of the present invention can be referred to in the descriptions of other embodiments.
• the liposome agent and the pharmaceutical composition can be read as the NKT cell activator of the present invention.
• compositions contains the liposome agent of the present invention.
• the pharmaceutical composition of the present invention may contain, for example, only the liposome agent of the present invention, or may contain other additives.
• the additives for example, the description of the NKT cell activator of the present invention can be used.
• the pharmaceutical composition of the present invention can be used, for example, for diseases that can be prevented or treated by activating NKT cells.
• diseases include cancer.
• Target cancers include breast cancer, skin cancer, blood cancer, colon cancer, prostate cancer, ovarian cancer, and the like.
• Target cancers include, for example, cancers that are highly metastatic, and specific examples include blood cancers, lung cancer, breast cancer, colon cancer, prostate cancer, and other cancers that are prone to metastasis to other organs.
• 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.
• 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.
• the present invention relates to the liposome preparation for use in the treatment of diseases such as cancer.
• the present invention also relates to the liposome preparation for use in the manufacture of pharmaceutical compositions such as anticancer agents.
• the liposome preparation of the present invention is particularly preferably applied to NKT cell-targeted therapy for intractable cancer.
• examples of liposome preparations used in NKT cell-targeted therapy for intractable cancer are shown. The above examples can be used unless otherwise specified.
• the intractable cancer in this embodiment is a type of cancer that is not affected by conventional immune cancer treatments 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 the immune reaction with the 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), is all negative.
• Examples of triple-negative breast cancer include 4T1, Hs578T, BT-20, and HCC70.
• the liposomal agent in this embodiment is preferably applied to NKT cell-targeted therapy for triple-negative breast cancer, which is one of the intractable cancers.
• the liposome agent of this embodiment can be preferably used for 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. For this reason, the liposome agent of this embodiment can be particularly preferably used for patients in whom the growth of cancer has not been suppressed even when the PD-1 antibody or the PD-L1 antibody is administered, or patients in whom the growth of cancer is expected to be incapable of being suppressed even when the PD-1 antibody or the PD-L1 antibody is administered.
• the liposomal agent of this embodiment can be applied to so-called cold cancers in addition to triple-negative breast cancer.
• cold cancer refers to a tumor to which the immune system shows little reaction.
• Cold cancer is generally considered to be difficult to treat because of its weak attack by the immune system, and its main characteristics include a small 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 is a highly metastatic tumor cell line that is mainly used in mouse breast cancer research, and is generally classified as a "cold cancer".
• 4T1 tumors have a weak immune response by cells of the innate immune system and cells of the adaptive immune system, and poor infiltration of immune cells, and 4T1 tumors have an immunosuppressive microenvironment and are considered to be easy to avoid attacks by the immune system.
• 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).
• condition 1, condition 2, and condition 3 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 can activate NKT cells more effectively, making it possible to suppress cancer growth even in patients for whom PD-1 antibodies, etc., as described above, are ineffective.
• the diameter of the PEGylated liposome preferably satisfies the following conditions. 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 the present embodiment, the diameter is more preferably 100 ⁇ 40 nm or 100 ⁇ 30 nm, and particularly preferably 100 ⁇ 20 nm or 100 ⁇ 10 nm.
• the terminal of the PEG modifying the phospholipid is preferably a hydroxyl group.
• PEG having a hydroxyl group at its terminal is hereinafter also referred to as PEG-OH
• phospholipid modified with PEG-OH is hereinafter also referred to as PEG-OH lipid.
• PEG can be represented by the structural formula (1) below, where n is a positive integer.
• n is a positive integer.
• the PEG of the PEG lipid has hydrogen atoms at both ends and a hydroxyl group at the right end, as shown in the structural formula (2) below.
• the liposome particles in the liposome agent of this embodiment have a surface rich in hydroxyl groups derived from PEG-OH.
• the liposome agent of this embodiment can be more efficiently taken up by antigen-presenting cells such as dendritic cells.
• the surface charge of the PEGylated liposome is preferably negative, and the PEGylated liposome is also referred to as being negatively charged.
• 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 a range 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
• the charge of the PEG liposome is preferably a negative charge, as described above, and specifically, -80 to -5 mV is preferable, and -30 ⁇ 20 mV or -30 ⁇ 10 mV is more preferable.
• the liposome agent of this embodiment contains a plurality of the PEGylated liposomes. Therefore, in the liposome agent 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 be charged. Furthermore, the liposome agent of the present invention as a whole may be charged.
• Choline-containing phospholipids Phosphatidylcholine (1,2-diethyl-sn-glycero-3-phosphocholine, Tokyo Chemical Industry Co., Ltd.) Cholesterol-based lipids: Cholesterol (Wako Pure Chemical Industries, Ltd.) Positively charged lipids: Didodecyldimethylammonium bromide (DDAB, Sigma Aldrich)
• PEGylated phospholipids - Phospholipid modified with PEG with a hydroxyl group at the end: HO-PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol, Broadpharm) - Phospholipid modified with PEG with a methyl group at the end: METHYL-PEG-DSPE (1,2-Distearoyl-phosphatidyl Ethanolamine-Methyl-Polyethyleneglycol Conjugate, Tokyo Chemical Industry
• Porous membrane Product name: Polycarbonate membrane (Cytiva) 1: Pore diameter 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 100nm, recovered particle size 100nm 5: Pore size 50 nm, recovered particle size 50 nm RPMI-1640 medium (RPMI, Sigma Aldrich) Buffer solution: PBS (-) (Takara Bio) Fetal bovine serum (FBS, Biological Industries, Inc.) Penicillin-Streptomycin (Life Technologies) Sulfocyanine 5 succinimidyl ester (Sulfo-Cy5-NHS, Lumiprobe)
• Cells, etc. All cells 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 in a 5% CO2 atmosphere. BALB/c mice 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.
• Example 1 PEGylated liposomes were prepared. The preparation was basically carried out as follows, except for the reagents and amounts added, according to the paper (O. H. Voss et al., Curr. Protoc. Immunol., 2018).
• liquid reagents of each component were prepared.
• Phosphatidylcholine was dissolved in chloroform to 5 mg/mL.
• the PEGylated phospholipid (HO-PEG-DSPE or METHYL-PEG-DSPE) was dissolved in chloroform to 5 mg/mL.
• the positively charged lipid (DDAB) was dissolved in chloroform to 10 mg/mL.
• Cholesterol was dissolved in chloroform to 5 mg/mL.
• the liquid reagents of each component were mixed in a 20 mL flask to obtain the following composition, to prepare a total of 1 mL of a mixed solution.
• 50% and 0% refer to the mass % of the NKT cell activating ligand in the mixed solution.
• the molar ratio of components other than the NKT cell activating ligand was 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 added with the positively charged lipid (DDAB) to make the surface charge of the liposomes formed positively or neutrally charged.
• DDAB positively charged lipid
• [NKT cell activating ligand: B7] (50% B7/mixture for negatively charged PEG-terminated -OH liposomes) B7 solution 500 ⁇ L Phosphatidylcholine solution 34 ⁇ L Cholesterol solution 40 ⁇ L HO-PEG-DSPE solution 27 ⁇ L Chloroform Remaining (10% B7/mixture for negatively charged PEG-terminated -OH liposomes) B7 solution 100 ⁇ L Phosphatidylcholine solution 114 ⁇ L Cholesterol solution 40 ⁇ L HO-PEG-DSPE solution 27 ⁇ L Chloroform Remaining (2% B7/mixture for negatively charged PEG-terminated -OH liposomes) B7 solution 20 ⁇ L Phosphatidylcholine solution 130 ⁇ L Cholesterol solution 40 ⁇ L HO-PEG-DSPE solution 27 ⁇ L Chloroform Remaining part (50% B7/mixture for negatively charged PEG-terminated Me liposomes) B7
• NKT cell activating ligand (0% control/mixture for negatively charged PEG-terminated -OH liposomes) NKT ligand solution 0 ⁇ L Phosphatidylcholine solution 134 ⁇ L Cholesterol solution 40 ⁇ L HO-PEG-DSPE solution 27 ⁇ L Chloroform Remaining (0% control/mixture for negatively charged PEG-terminated Me liposomes) NKT ligand solution 0 ⁇ L Phosphatidylcholine solution 134 ⁇ L Cholesterol solution 40 ⁇ L METHYL-PEG-DSPE solution 27 ⁇ L Chloroform Residue
• the flask was rotated using an evaporator (Tokyo Rikakikai Co., Ltd.) at 30°C for 10 minutes under a vacuum of 30 hPa to completely dry out 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 for resuspension. This suspension was treated at 28 kHz for 5 minutes using an ultrasonic bath (Aiwa Medical Industry Co., Ltd.), and further treated at 20 kHz for 30 seconds using an ultrasonic homogenizer (Taitec Co., Ltd.). This resulted in the lipid film being crushed, and PEGylated liposomes broken down into small fragments were obtained.
• the decomposed PEGylated liposomes were further processed using the porous membrane and an extruder (product name AE-10, Bio-Equip) to obtain a particle size within a specified range.
• PBS was used as the solvent during processing.
• the porous membrane 1 (recovery particle size 1000 ⁇ m) was set in the extruder, and the decomposed PEGylated liposomes were passed through it three or more times.
• the porous membrane 2 (recovery 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 it three or more times.
• PEGylated liposomes with a particle size of about 400 nm were collected as PEGylated liposome fraction A.
• the porous membrane 3 (recovery 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 it three or more times.
• PEGylated liposomes with a particle size of about 200 nm were collected as PEGylated liposome fraction B.
• 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.
• PEGylated liposomes with a particle size of about 100 nm were collected as PEGylated liposome fraction C.
• 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.
• PEGylated liposomes with a particle size of about 50 nm were collected as PEGylated liposome fraction D.
• 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 confirmed.
• the particle size and PDI were determined according to the cumulant method.
• various liposome fractions were prepared as liposome samples in the same manner as using PBS, except that dye-containing PBS was used instead of PBS.
• the dye-containing PBS was prepared as follows. That is, a dye reagent (Sulfo-cyanine 5 succinimidyl ester, product name: Sulfo-Cy5 (registered trademark)-NHS, Lumiprobe) was mixed with PBS to a concentration of 0.1 mg/mL and left for 24 hours. By leaving the dye reagent in PBS, -NHS was removed from the dye reagent by hydrolysis, and dye-containing PBS containing Sulfo-Cy5 (registered trademark) at a concentration of 0.1 mg/mL was prepared. 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.
• PBS refers to PBS that does not contain a dye.
• PEGylated liposomes prepared using PBS that does not contain a dye will be referred to as liposomes
• PEGylated liposomes prepared using PBS that contains a dye will be referred to as liposomes (PBS that contains a dye).
• Example 2 The stability of the PEGylated liposomes prepared in Example 1 was confirmed.
• Example No. 1-1 The B7 liposome (sample No. 1-1), ⁇ -GC liposome (sample No. 1-2), and NKT ligand (-) liposome (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 sample 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)
• the GalCer concentration of the liposome sample prepared from the 50% ⁇ -GarCer mixture was 0.472 mg/mL (approximately 0.5 mg/mL).
• the liposome samples were stored at 4°C.
• the particle size (number average hydrodynamic diameter) and polydispersity index (PDI) were determined for the liposome samples before the start of storage (Day 0) and for the liposome samples 10 days (Day 10) and 90 days (Day 90) after the start of storage, based on particle size distribution diagrams obtained by dynamic light scattering (DLS) measurements.
• the particle size was calculated as 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.
• Table 4A shows the rate of change in particle size
• Table 4B shows the polydispersity index (PDI).
• the rate of change in particle size 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 rate of change in particle size of ⁇ -GarCer from 10 days to 90 days also remained within the range of -13.1% to +3.70% compared to Day 0 (0%).
• the rate of change in particle size of the control NKT ligand (-) liposomes from 10 days to 90 days increased over time to +0.997% to +15.9% compared to Day 0 (0%).
• the PDI of B7 liposomes from 10 days to 90 days only varied within a range of 0.153 to 0.245 relative to Day 0 (0.170), and the PDI of ⁇ -GarCer from 10 days to 90 days also only varied within a range of 0.116 to 0.364 relative to Day 0 (0.142).
• the PDI of the control NKT ligand (-) liposomes from 10 days to 90 days increased over time to a range of 0.176 to 0.420 relative to Day 0 (0.120).
• B7 liposomes and ⁇ -GC liposomes can be stabilized by using cholesterol and PEGylated phospholipid (PEG-DSPE), and are more stable than NKT ligand (-) control liposomes when stored for a long period of time, for example, up to 90 days, and can prevent the formation of aggregates of 1000 nm or more.
• PEG-DSPE cholesterol and PEGylated phospholipid
• 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 liposome, and the right graph showing the PDI of the diluted sample of B7 liposome. As shown in the figure, even when B7 liposome was diluted about 1000 times (1/2 10 ), no formation of aggregates with a particle size exceeding 1000 nm was confirmed.
• Example 3 The PEGylated liposome prepared in Example 1 was administered to confirm its function.
• the B7 liposomes (sample No. 1-1) and NKT ligand (-) liposomes (sample No. 1-11) prepared in Example 1 above 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.
• administration control samples free B7 that was not liposomized and PBS were used, respectively.
• mice (6 weeks old, female) were subcutaneously administered with the administration sample to the left armpit. 48 hours after administration, the mice were sacrificed and blood and lymph nodes were collected.
• the lymph nodes were collected from the right paraaxillary, right upper arm axillary, left paraaxillary, and left upper arm axillary.
• the collected blood was centrifuged at 2000 g for 15 minutes to collect serum.
• the right paraaxillary lymph nodes and the right upper arm axillary lymph nodes were combined to form the right axillary lymph nodes, and the left paraaxillary lymph nodes and the left upper arm axillary lymph nodes were combined to form the left axillary lymph nodes.
• the collected lymph nodes were weighed, PBS was added to the lymph nodes to make the concentration 50 ⁇ g/ ⁇ L, and the lymph nodes were homogenized using a Dounce homogenizer. The homogenized material was centrifuged at 2000 g for 15 minutes to collect the supernatant. The serum and lymph node supernatant were used as samples, and IFN- ⁇ and IL-10 in the samples were measured using an IFN- ⁇ ELISA kit (BioLegend, catalog number 430804) according to the manufacturer's instructions.
• Figure 2A is a graph showing IFN- ⁇ concentrations in serum and lymph node supernatants
• Figure 2B is a graph showing IL-10 concentrations in serum and lymph node supernatants.
• the left shows the results for left axillary lymph node supernatant
• the middle shows the results for right axillary lymph node supernatant
• the right shows the results for serum.
• the vertical axis is the IFN- ⁇ concentration (ng/mL) of the sample
• the vertical axis is the IL-10 concentration (ng/mL) of the sample.
• the bars represent, 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).
• B7 liposomes are thought to accumulate in lymph nodes about 24 hours after administration, to be activated about 48 hours after administration, and to act on cancer about 72 hours after administration. For this reason, for example, a decrease in IFN- ⁇ in lymph nodes is expected about 96 hours after administration, but in that case, it is thought that a continuous effect can be maintained by administering the B7 liposomes again 4 to 5 days after the first administration.
• the B7 liposome (sample No. 1-1) prepared in Example 1 above was adjusted to a PEGylated liposome concentration of 1 mg/mL (total lipid equivalent) and used as the administration sample.
• PBS was used to adjust the PEGylated liposome concentration.
• PBS was used as the administration control sample.
• Lymph node cells and spleen cells were prepared as follows. First, spleens and lymph nodes were harvested from BALB/c mice (7 weeks old, female). The lymph nodes were harvested from the right paraaxilla, right upper arm axilla, left paraaxilla, and left upper arm axilla. Each of the harvested lymph nodes 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 surviving lymph node cells. The spleen was similarly harvested into single cells using a cell strainer, and the cells were collected by centrifugation.
• RBC Lysis Buffer (10X, ThermoFisher) was added to PBS (containing 1% FBS) to make a 1x solution, and the single spleen cells were resuspended in this mixture and allowed to stand for 5 minutes to remove red blood cells. The fraction after removing the red blood cells was then centrifuged at 400g for 4 minutes to recover surviving spleen cells.
• DC2.4 (Sigma Aldrich), which is a mouse dendritic cell, was cultured in RPMI. Fresh RPMI and 5x10 5 of the cultured dendritic cells were seeded in each well of a 6-well plate, the B7 liposome was added, and the cells were cultured for 24 hours. The B7 liposome was added to each well so that the final total lipid amount was 0.1 mg/mL. After 24 hours of culture, 7.5x10 5 of the lymph node cells or spleen cells were added per well and co-cultured for 24 hours. After 24 hours of co-culture, all the cells were collected. The collected cell group was blocked by 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).
• NK cell and T cell labeling were performed simultaneously.
• PBS containing 1% FBS
• Anti-mouse NK1.1-InVivo Selleck
• FlexAble CoraLite registered trademark
• Plus 488 Antibody Labeling Kit for mouse IgG Proteintech
• PBS containing 1% FBS
• PE anti-mouse CD3 Antibody Biorezen
• Each labeling reagent was added simultaneously to the blocked cell group and left to stand at 4°C for 45 minutes, thereby labeling the NK cells and the T cells in the cell group at the same time.
• the labeled cell groups were washed three times with PBS (containing 1% FBS) and then measured by flow cytometry (BD FACSAriaTM III cell sorter, DB Biosciences). During labeling, the staining concentration of each antibody was determined according to the manufacturer's instructions (same below).
• Figure 3A is a histogram of NKT cells in lymph nodes, and a graph showing the distribution of cell numbers for NK1.1-positive and CD3-positive cells in lymph nodes by the relative fluorescence intensity indicating CD3 positivity.
• the X-axis is the relative fluorescence intensity of the labeling reagent to CD3, and the Y-axis is the number of events (cell number) showing that fluorescence.
• the right-hand peak indicates NK1.1-positive and CD3-positive cells, i.e., NKT cells.
• the B7 liposome group (B7 lipo) showed an increase in the amount of NK1.1-positive and CD3-positive NKT cells (the peak on the right side in Figure 3A) when dendritic cells administered with B7 liposomes were co-cultured with lymph node cells, compared to the PBS-administered group (PBS).
• PBS PBS-administered group
• Figure 4A is a histogram of NKT cells in the spleen, and a graph showing the distribution of cell numbers by the relative fluorescence intensity for NK1.1-positive and CD3-positive cells in the spleen.
• the X-axis is the relative fluorescence intensity of the labeling reagent to CD3, and the Y-axis is the number of events (cell number) showing that fluorescence.
• the right-hand peak represents NK1.1-positive and CD3-positive cells, i.e., NKT cells.
• the B7 liposome (sample No. 1-1) prepared in Example 1 above was adjusted to a PEGylated liposome concentration of 1 mg/mL (total lipid equivalent) and used as the administration sample.
• PBS was used as the administration control sample (no NKT ligand added).
• the administration sample was subcutaneously administered to the left armpit of BALB/c mice (6 weeks old, female). After a certain time (12, 24, 48, 72 hours) from administration, the mice were sacrificed, and the spleen and lymph nodes were collected as organs. The lymph nodes were collected from the right paraaxilla, right upper arm axilla, left paraaxilla, and left upper arm axilla. The size of each organ collected was observed. The collected lymph nodes were minced, placed in a cell strainer, and gently ground in RPMI containing 1% FBS to make single cells. The single cells were centrifuged at 400g for 4 minutes, and only surviving lymph node cells were collected. The spleen was also treated in the same way.
• the collected lymph node cell group and spleen cell group were subjected to blocking and simultaneous labeling of NK cells and T cells by the same method as described in (2) above.
• the labeled cells were washed and measured by flow cytometry in the same manner as described in (2) above.
• the collected lymph node cell group and spleen cell group were blocked using the same method as in (2) above, and dendritic cell labeling was then performed.
• PBS containing 1% FBS
• FITC anti-mouse CD11c Antibody Biorezen
• the reagent was added to the cell group and left to stand at 4°C for 45 minutes to label the dendritic cells in the cell group.
• macrophage labeling was further performed for each of the cell groups in which dendritic cell labeling had been performed.
• PBS containing 1% FBS
• anti-mouse CD68 Militenyi
• FlexAble CoraLite registered trademark Plus 555 Antibody Labeling Kit for rabbit IgG (Proteintech) was used as the reagent. That is, the reagent was added to the cell group and left to stand at 4°C for 45 minutes, thereby labeling the macrophages at the same time as labeling the dendritic cells.
• the labeled cells were washed and measured by flow cytometry in the same manner as in (2) above.
• FIG. Fig. 5A is a histogram of NKT cells in lymph nodes, and a graph showing the distribution of cell numbers by relative fluorescence intensity showing CD positivity for NK1.1-positive and CD3-positive cells in lymph nodes.
• the X-axis is the relative fluorescence intensity of the labeling reagent to CD3, and the Y-axis is the number of events (cell number) showing that fluorescence.
• time (h) is the treatment time after administration of the administration sample, and a histogram is shown for each administration group.
• Figure 5B is a histogram of NK cells in lymph nodes, which is a graph showing the distribution of cell numbers by relative fluorescence intensity indicating NK1.1 positivity for NK1.1 positive cells in lymph nodes.
• Figure 5B histograms of the five treatment groups are shown superimposed.
• Figure 5C is a histogram of T cells in lymph nodes, which is a graph showing the distribution of cell numbers by relative fluorescence intensity indicating CD3 positivity for CD3 positive T cells in lymph nodes.
• FIG. 5D is a histogram of NKT cells in lymph nodes, overlaying the histograms of the five treatment groups in FIG. 5A.
• the number of NKT cells in the lymph nodes increased over time at 12 and 24 hours after administration, and it was confirmed that the increased number of NKT cells was maintained at 48 and 72 hours after administration. Furthermore, as shown in Figures 5B and C, it was confirmed that the number of NK cells and T cells also increased over time after administration, peaking at 24 to 48 hours after administration, and then the number of these cells was maintained.
• FIG. Fig. 6A is a histogram of NKT cells in the spleen, and a graph showing the distribution of cell numbers by relative fluorescence intensity showing CD3 positivity for NK1.1-positive and CD3-positive cells in the spleen.
• the X-axis is the relative fluorescence intensity of the labeling reagent to CD3, and the Y-axis is the number of events (cell number) showing that fluorescence.
• time (h) is the treatment time after administration of the administration sample, and a histogram is shown for each administration group.
• Figure 6B is a histogram of NK cells in the spleen, which is a graph showing the distribution of cell numbers by relative fluorescence intensity indicating NK1.1 positivity for NK1.1 positive NK cells in the spleen.
• Figure 6B histograms of the five treatment groups are shown superimposed.
• Figure 6C is a histogram of T cells in the spleen, which is a graph showing the distribution of cell numbers by relative fluorescence intensity indicating CD3 positivity for CD3 positive T cells in the spleen.
• FIG. 6D is a histogram of NKT cells in the spleen, overlaying the histograms of the five treatment groups in FIG. 6A.
• FIG. 7A is a histogram of dendritic cells in lymph nodes, and a graph showing the cell number distribution of CD11c-positive cells in lymph nodes by relative fluorescence intensity indicating CD11c positivity.
• the X-axis shows the relative fluorescence intensity of the labeling reagent against CD11c
• the Y-axis shows the number of events (cell number) showing that fluorescence.
• FIG. 7B is a histogram of dendritic cells in lymph nodes, overlaid with the histograms of the five treatment groups in FIG. 7A.
• Figure 7C is a histogram of macrophages in lymph nodes, which is a graph showing the distribution of cell numbers by relative fluorescence intensity indicating CD68 positivity for CD68 positive cells in lymph nodes.
• the X-axis is the relative fluorescence intensity of the labeling reagent to CD68
• the Y-axis is the number of events (cell numbers) showing that fluorescence.
• the graph shows the percentage (%) of dendritic cells in lymph node cells in the PBS-administered group at the same relative fluorescence intensity as that shown for 5% of the lymph node cells in the PBS-administered group in Fig. 7B.
• the graph shows the percentage (%) of dendritic cells in lymph node cells in the PBS administration group at the same relative fluorescence intensity as that shown in Fig. 7C, based on the relative fluorescence intensity indicating that the percentage of macrophages in lymph node cells in the PBS administration group is 5%.
• FIG. 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 by relative fluorescence intensity indicating CD11c positivity.
• the X-axis shows the relative fluorescence intensity of the labeling reagent against CD11c
• the Y-axis shows the number of events (cell number) showing that fluorescence.
• FIG. 8B is a histogram of dendritic cells in the spleen, overlaying the histograms of the five treatment groups in FIG. 8A.
• Figure 8C is a histogram of splenic macrophages, which is a graph showing the distribution of cell numbers by relative fluorescence intensity indicating CD68 positivity for CD68 positive cells in the spleen.
• the X-axis is the relative fluorescence intensity of the labeling reagent to CD68
• the Y-axis is the number of events (cell numbers) showing that fluorescence.
• the graph shows the percentage (%) of dendritic cells in the spleen cells of the PBS-administered group at the same relative fluorescence intensity as that shown in Fig. 8B, relative to the percentage of 5% dendritic cells in the spleen cells of the PBS-administered group.
• the graph shows the percentage (%) of dendritic cells in the other administration groups at the same relative fluorescence intensity, based on the relative fluorescence intensity showing a percentage of macrophages in spleen cells of the PBS administration group in Fig. 8C.
• Example 4 The in vivo distribution of the PEGylated liposome prepared in Example 1 was examined.
• 30 ⁇ L of the administration sample was subcutaneously administered to the left flank of a BALB/c mouse (6 weeks old, female) with breast cancer (4T1, average tumor volume 150 mm 3 ) and a BALB/c mouse (6 weeks old, female) without breast cancer.
• the dose of each administration sample was as follows: 30 ⁇ L of the B7 liposome administration sample: 30 ⁇ L/mouse, B7 amount: 15 ⁇ g
• the control liposome-administered sample (30 ⁇ L: 30 ⁇ L/mouse) was administered. 24 hours after administration, the mice were sacrificed and the left axillary lymph node, spleen, kidney, and liver were collected.
• each of the collected tissues was visualized using an IVIS imaging system (product name IVIS Spectrum, Sumitomo Pharma International Corporation) with 650 nm/680 nm fluorescence channels.
• Figure 9A is an image (converted to black and white) showing the fluorescence in each organ of the mouse 24 hours after administration.
• the site indicated by the arrowhead is the area where the fluorescence intensity was particularly strong.
• Figure 9A is a color image showing the fluorescence displayed in black and white, so the color does not indicate the fluorescence intensity of the fluorescent area.
• Figure 9B is a graph showing the relative fluorescence intensity in each organ of the mouse 24 hours after administration.
• the relative fluorescence intensity is the value obtained by dividing the fluorescence intensity in each organ by the weight of each organ and normalizing it with the Ctrl lip value set to 1.
• Example 5 The PEGylated liposome prepared in Example 1 was confirmed to have an antitumor effect on breast cancer cells by repeated subcutaneous administration.
• the following liposome samples prepared using PBS in Example 1 were used. (1-1) 50% B7/negatively charged PEG-terminated -OH liposome 100 nm (1-11) 0% control/negatively charged PEG-terminated -OH liposome 100 nm The liposome sample 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.
• the administration sample prepared in (1-1) above was used as the B7 liposome administration sample (B7 lipo) of the example, and the administration sample prepared in (1-11) above was used as the control liposome administration sample (Ctrl lipo).
• a free B7 sample Free B7 in which non-liposomal free B7 was suspended in PBS, and PBS were each used as an administration sample.
• Triple negative breast cancer cells 4T1 were used as breast cancer cells.
• each administration sample was as follows: 30 ⁇ L of the B7 liposome administration sample: 30 ⁇ L/mouse, B7 amount 15 ⁇ g, total lipid amount 30 ⁇ g 30 ⁇ L of the control liposome-administered sample: 30 ⁇ L/mouse, total lipid amount 30 ⁇ g Free B7 sample 30 ⁇ L: 30 ⁇ L/mouse, B7 amount 15 ⁇ g PBS sample: 30 ⁇ L/mouse
• the tumor growth and mouse body weight were monitored every 2 days from the start of administration.
• the survival of the mice was monitored, and the survival rate was analyzed by the Kaplan-Meier method. Mice with tumor volumes exceeding 2000 mm3 were euthanized, and the day was recorded as the day of death of the mice.
• administering Condition A Eight days after inoculation of the 4T1 cells, the first administration (1st) of the administration sample was performed in the left flank, which was designated as Day 0. Further subcutaneous injections were performed in the left flank four times every six days under the same conditions (a total of five times).
• FIG. 10 is a graph showing the relationship between the tumor volume (V) of the mouse and the number of days from the start of administration.
• the tumor volume on Day 0 was 100 mm 3.
• the increase in tumor volume on Day 30 was about 12 to 20 times compared to Day 0.
• the increase in tumor volume on Day 30 was about 10 times compared to Day 0, and the increase in tumor was suppressed more than in the PBS administration group and the administration group (Ctrl lipo).
• B7 liposome administration group B7 lipo
• the increase in tumor volume on Day 30 was only about 5 times that on Day 0, confirming that the increase in tumor volume was significantly suppressed. From these results, it was found that the growth of breast cancer can be effectively suppressed by administering B7 as a PEGylated liposome.
• Figure 11 is a graph showing the relationship between mouse survival rate and the number of days from the start of administration.
• the survival rate of mice in the B7 liposome administration group increased significantly, and specifically, the survival period was about four times longer compared to the PBS administration group. From this result, it is believed that the administration of B7 liposome causes immune activity, which suppresses the growth of cancer, and as a result, the survival period of the mice is extended.
• FIG. 12 is a graph showing the relationship between the weight of the mice and the number of days from the start of administration.
• the group administered with the control liposome (Ctrl lipo), the group administered with free B7 (Free B7), and the group administered with the B7 liposome (B7 lipo) did not show a significant decrease in the weight of the mice. From this result, it was confirmed that, like PBS, none of the B7 liposome, the control liposome, and free B7 caused significant side effects to the mice.
• administering condition B Five days after inoculation of the 4T1 cells, the first administration (1st) to both flanks was performed on Day 0, and five days later (Day 5), a second subcutaneous injection was performed on both flanks under the same conditions (a total of two times).
• FIG. 13 is a graph showing the relationship between the tumor volume (V) of the mouse and the number of days from the start of administration.
• the tumor volume on Day 0 was 36 mm 3.
• the increase in tumor volume on Day 32 was about 30 to 40 times compared to Day 0.
• the increase in tumor volume on Day 32 was about 27 times compared to Day 0, and the tumor increase was suppressed more than in the PBS administration group and the administration group (Ctrl lipo).
• 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.
• FIG. 15 is a graph showing the relationship between the body weight of the mice and the number of days from the start of administration. As shown in FIG. 15, compared to the group administered with PBS alone, no significant decrease in the body weight of the mice was observed in the group administered with the control liposome (Ctrl lipo), the group administered with free B7 (Free B7), and the group administered with the B7 liposome (B7 lipo).
• Example 6 The PEGylated liposome prepared in Example 1 was confirmed to have an antitumor effect against skin cancer cells by repeated subcutaneous administration.
• the following administration samples were used, which were the same as those used in Example 5 above.
• B16F10 was used as the skin cancer cells.
• B16F10 cells 1.0 ⁇ 10 6 cells/mouse
• were subcutaneously inoculated into the vicinity of the second nipple from the top of the left breast of C57BL/6j mice (5 weeks old, female, n 6-7).
• 30 ⁇ L of the administration sample was administered in the same manner as in Example 5, and the antitumor effect, safety, and tolerability were confirmed.
• Figure 16A is a graph showing the relationship between the tumor volume (V) of the mouse and the number of days from the start of administration.
• Figure 16B is a graph showing the relationship between the weight (g) of the mouse and the number of days from the start of administration.
• the increase in tumor volume on Day 6 was about 40 to 50 times compared to Day 0.
• the increase in tumor volume on Day 6 was only about 15 times compared to Day 0, and it was confirmed that the increase in tumor volume was significantly suppressed.
• Example 7 The PEGylated liposome prepared in Example 1 was confirmed to have an antitumor effect on breast cancer cells by repeated subcutaneous administration.
• the following liposome samples prepared using PBS in Example 1 were used.
• the liposome sample 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.
• the administration sample prepared in (1-2) above was used as an ⁇ GalCer liposome administration sample ( ⁇ Gal lipo) of the example, and the administration sample prepared in (1-11) above was used as a control liposome administration sample (Ctrl lipo).
• a free GalCer sample Free ⁇ Gal
• free non-liposomal ⁇ -GalCer was suspended in PBS
• PBS were each used as an administration sample.
• Triple-negative breast cancer cells 4T1 were used as breast cancer cells.
• each administration sample was as follows: 30 ⁇ L of the ⁇ -GalCer liposome administration sample: 30 ⁇ L/mouse, ⁇ -GalCer amount 15 ⁇ g, total lipid amount 30 ⁇ g 30 ⁇ L of the control liposome-administered sample: 30 ⁇ L/mouse, total lipid amount 30 ⁇ g Free ⁇ -GalCer sample 30 ⁇ L: 30 ⁇ L/mouse, ⁇ -GalCer amount 15 ⁇ g PBS: 30 ⁇ L/mouse
• 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 administered under the same conditions three times every five days (a total of four times). Then, the antitumor effect, safety, and tolerability were confirmed in the same manner as in Example 5.
• V tumor volume
• PBS PBS administration group
• Ctrl lip the control liposome administration group
• the tumor volume increased significantly as the days passed.
• the increase in tumor volume was suppressed in the free GalCer administration group (Free ⁇ Gal).
• the ⁇ GalCer liposome administration group ( ⁇ Gal lipo) showed a more significant inhibition of the increase in tumor volume than the free GalCer administration group (Free ⁇ Gal), confirming a significant inhibition of cancer growth.
• Example 8 The PEGylated liposome prepared in Example 1 was administered to mice transplanted with cancer cells, and the in vivo distribution was confirmed.
• Triple-negative breast cancer cells 4T1 were used as breast cancer cells.
• 4T1 cells (5.0 ⁇ 10 5 to 1.0 ⁇ 10 6 cells/mouse) were subcutaneously inoculated into cancer-free BALB/c mice (5 weeks old, female) near the second nipple from the top of the left breast (Day 0).
• 30 ⁇ L of the administration sample was subcutaneously injected into the left armpit or both armpits of the mice.
• the dose of each administration sample was as follows: 30 ⁇ L of the B7 liposome administration sample: 30 ⁇ L/mouse, B7 amount: 15 ⁇ g 30 ⁇ L of the control liposome-administered sample: 30 ⁇ L/mouse
• 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 body. Unless otherwise specified, the same procedure was followed as in Example 4 above.
• 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 where the cancer cells were inoculated.
• Figure 20 is an image (converted to black and white) showing fluorescence in the liver where the cancer had metastasized.
• the lower figure is a photograph of the organs that were harvested, and the upper figure is a diagram in which the fluorescence distribution has been superimposed on the photograph.
• the white arrows indicate areas where the fluorescence intensity was stronger in the B7 liposome administration group (B7 lipo) compared to the control administration group (Ctrl lipo).
• the black arrow indicates areas where severe cancer metastasis has occurred.
• mice with a high incidence of cancer metastasis to the liver also showed high accumulation of B7 liposomes in the liver (top panel of Figure 20, B7 lipo/48 hours/first, second, and fifth livers from the left).
• the control liposome administration group (Ctrl lipo)
• no accumulation of the fluorescent dye was observed even in the liver where metastasis had occurred.
• the administration samples used were the same as those in (1) above, and the administration conditions for each administration sample were as follows: 30 ⁇ L of the B7 liposome administration sample: 30 ⁇ L/mouse, B7 amount: 15 ⁇ g 30 ⁇ L of the control liposome-administered sample: 30 ⁇ L/mouse
• Triple-negative breast cancer cells 4T1 were used as breast cancer cells.
• 30 ⁇ L of the administration sample was subcutaneously injected into the left armpit or both armpits of the mice. 24 hours after administration, the mice of each administration group were euthanized and the cancer was removed. The collected cancer was minced and shaken at 37° C.
• NK cell labeling and T cell labeling were performed simultaneously for the blocked cell group.
• PBS containing 1% FBS
• Anti-mouse NK1.1-InVivo Selleck
• FlexAble CoraLite registered trademark
• Plus 488 Antibody Labeling Kit for mouse IgG Proteintech
• PBS containing 1% FBS
• PE anti-mouse CD3 Antibody Biorezen
• PBS containing 1% FBS
• FITC anti-mouse CD11c Antibody Biorezen
• Biorezen FITC anti-mouse CD11c Antibody
• Figure 21 shows the results of flow cytometry showing 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, both of which can be detected by flow cytometry.
• FIG. 21A is a graph showing the distribution of cells based on the fluorescence intensity of FITC and the fluorescence intensity of Cy5, where the horizontal axis represents the fluorescence intensity of FITC and the vertical axis represents the fluorescence intensity of Cy5.
• 21B is a graph showing the distribution of cells based on the fluorescence intensity of FITC, where the horizontal axis is the fluorescence intensity of FITC and the vertical axis is the percentage of the number of cells.
• the left peak indicates CD11c negative cells and the right peak indicates CD11 positive cells (dendritic cells).
• FIG. 21C is a graph quantifying the percentage of CD11c positive cells (peak on the right) in FIG. 21B.
• Figure 21D is a graph showing the distribution of CD11c-positive cells by Cy5 fluorescence intensity, with gating on only the CD11c-positive cells (peak on the right) in Figure 21B, where the horizontal axis represents Cy5 fluorescence intensity and the vertical axis represents the percentage of cell number.
• FIG. 21E shows the results of quantifying the proportion of CD11c-positive and Cy5-positive cells in FIG. 21D.
• Figure 22 shows the results of flow cytometry showing the co-localization of macrophages and liposomes in cancer.
• Macrophages can be detected by the fluorescence intensity based on FITC staining of their CD68, and liposomes can be detected by the fluorescence intensity based on Cy5 in the dye-containing PBS, both of which can be detected by flow cytometry.
• FIG. 22A is a graph showing the distribution of cells based on the fluorescence intensity of FITC and the fluorescence intensity of Cy5, where the horizontal axis represents the fluorescence intensity of FITC and the vertical axis represents the fluorescence intensity of Cy5.
• 22B is a graph showing the distribution of cells based on the fluorescence intensity of FITC, where the horizontal axis is the fluorescence intensity of FITC and the vertical axis is the percentage of the number of cells.
• the left peak indicates CD68-negative cells
• the right peak indicates CD68-positive cells (macrophages).
• FIG. 22C is a graph quantifying the percentage of CD68 positive cells (peak on the right) in FIG. 22B.
• FIG. 22D is a graph showing the distribution of CD68-positive cells based on Cy5 fluorescence intensity, with gating on only the CD68-positive cells in FIG. 22B, where 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.
• Figure 23 shows the results of flow cytometry showing the co-localization of NK cells and liposomes in cancer.
• NK cells can be detected by flow cytometry based on the fluorescence intensity based on FITC staining of their NK1.1, and liposomes can be detected by the fluorescence intensity based on Cy5 in the dye-containing PBS.
• FIG. 23A is a graph showing the distribution of cells based on the fluorescence intensity of NK1.1 and the fluorescence intensity of Cy5, where the horizontal axis represents the fluorescence intensity of NK1.1 and the vertical axis represents the fluorescence intensity of Cy5.
• 23B is a graph showing the distribution of cells based on the fluorescence intensity of FITC, where the horizontal axis is the fluorescence intensity of NK1.1 and the vertical axis is the percentage of the number of cells. In each administration group, the left peak indicates NK1.1-negative cells and the right peak indicates NK1.1-positive cells (NK cells).
• FIG. 23C is a graph quantifying the percentage of NK1.1 positive cells (peak on the right) in FIG. 23B.
• FIG. 23D is a graph showing the distribution of NK1.1-positive cells by Cy5 fluorescence intensity, with gating on only the NK1.1-positive cells (peak on the right) in FIG. 23B, where the horizontal axis represents Cy5 fluorescence intensity and the base number represents the percentage of the cell number.
• FIG. 23E shows the results of quantifying the percentage of NK1.1-positive and Cy5-positive cells in FIG. 23D.
• FIG. 24 shows the results of flow cytometry showing the co-localization of T cells and liposomes in cancer.
• T cells can be detected by the fluorescence intensity based on PE staining of their CD3, and liposomes can be detected by the fluorescence intensity based on Cy5 in the dye-containing PBS, both of which can be detected by flow cytometry.
• FIG. 24A is a graph showing the distribution of cells based on the fluorescence intensity of FITC and the fluorescence intensity of Cy5, where the horizontal axis represents the fluorescence intensity of CD3 and the vertical axis represents the fluorescence intensity of Cy5.
• 24B is a graph showing the distribution of cells based on the fluorescence intensity of FITC, where the horizontal axis is the fluorescence intensity of FITC and the vertical axis is the percentage of the number of cells.
• the left peak indicates CD3-negative cells and the right peak indicates CD3-positive cells (T cells).
• FIG. 24C is a graph quantifying the percentage of CD3 positive cells (peak on the right) in FIG. 24B.
• 24D is a graph showing the distribution of CD3-positive cells by Cy5 fluorescence intensity, with gating on only the CD3-positive cells (peak on the right) in FIG. 24B, where the horizontal axis represents Cy5 fluorescence intensity and the vertical axis represents the percentage of cell count.
• FIG. 24E shows the results of quantifying the percentage of CD3-positive and Cy5-positive cells in FIG. 24D.
• FIG. 25 shows the results of flow cytometry showing co-localization of CD3-positive and NK1.1-positive double positive cells (NKT cells) in cancer and liposomes.
• FIG. 25A is a graph showing the distribution of CD3-positive and NK1.1-positive double positive cells (NKT cells) based on FITC fluorescence intensity, where the horizontal axis represents FITC fluorescence intensity and the vertical axis represents the percentage of cell count.
• FIG. 25B shows the results of quantifying the proportion of CD3-positive and NK1.1-positive double positive cells (NKT cells) in FIG. 25A.
• FIG. 25C is a graph showing the distribution of the CD4+ and NK1.1-positive double positive cells (NKT cells) in FIG. 25B based on Cy5 fluorescence intensity.
• FIG. 25D shows the results of quantification of the proportion of CD4+/NK1.1+/Cy5+ cells in FIG. 25C.
• 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, it was confirmed that the colocalization of dendritic cells (CD11c positive) and liposomes (Cy5 positive) was increased more in the B7 liposome-administered group (B7 lipo) than in the B7 liposome-administered group (B7 lipo) by administering B7 as B7 liposomes.
• the proportion of dendritic cells in the cancer was increased by about 20% in the B7 liposome-administered group (B7 lipo) compared to the control liposome-administered group (Ctrl lipo), whereas the number of dendritic cells containing liposomes (CD11c positive and Cy5 positive cells) increased by about 100%. From these results, it is speculated that dendritic cells carried the liposomes to the cancer. Furthermore, Figures 22A, 22B, 22C, and 22E regarding macrophages show similar results to those shown in Figure 21 regarding dendritic cells. From these results, it is speculated that antigen-presenting phagocytes such as dendritic cells and macrophages carried the liposomes to the cancer.
• T cells, NK cells, and NKT cells do not have phagocytic activity, it is unlikely that these cells directly took up the liposomes. For this reason, it is presumed that the phagocytes (dendritic cells and macrophages) described above take up the liposomes, absorb the Cy5 contained therein, and in that state interact with T cells, NK cells, and NKT cells, and Cy5 is transferred from the phagocytes to the T cells, NK cells, and NKT cells.
• the B7 liposomes when B7 liposomes are administered, the B7 liposomes are taken up into the lymph nodes, where they are phagocytosed and absorbed by dendritic cells. Then, in the lymph nodes, dendritic cells display the B7 antigen, which activates the NKT cells. At this time, the Cy5 taken up into the dendritic cells is transferred to the NKT cells. It is believed that the activated immune system (e.g., NKT cells, dendritic cells, etc.) then migrates to the cancer and exerts an anti-cancer effect.
• the activated immune system e.g., NKT cells, dendritic cells, etc.
• (2B) The cancer excised in (2A) was fixed in 20% formaldehyde for 48 hours, the fixed tissue was embedded in paraffin, and 5 ⁇ m sections were prepared.
• the paraffin embedding was performed according to the equipment and protocol provided by Abcam.
• the sections were placed on slide glasses, deparaffinized (treated in the order of xylene, ethanol, and water), and antigen retrieval was performed by treating the section slides with citrate buffer (pH 6.0) at 98° C. for 20 minutes.
• the section slides were blocked at 4°C for 30 minutes in the same manner as in (2A) above.
• primary antibody staining was performed on the blocked sections, followed by secondary antibody staining.
• PBS containing 1% FBS containing CD56 (NCAM) Antibodies (ThermoFisher)
• PBS containing 1% FBS
• IBA1 Recombinant Rabbit Monoclonal Antibody ThermoFisher
• PBS containing 1% FBS
• CD3 Antibodies ThermoFisher
• Goat anti-Rabbit IgG H+L Secondary Antibody, HRP (ThermoFisher) was used as the reagent, and the treatment conditions were incubation at room temperature for 30 minutes to 1 hour.
• AEC 3-Amino-9-ethylcarbazole
• Peroxidase substrate substrate solution Abcam
• 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 in the section slide was observed under a microscope.
• Figure 26 is a micrograph of a section slide, specifically, macrophages, NK cells, and T cells visualized by staining for the marker proteins IBA1, CD56, and CD3, respectively.
• B7 lipo the B7 liposome-administered group
• the B7 liposome-administered group had increased numbers of macrophages, NK cells, and T cells in the cancer compared to the PBS-administered group. This suggests that administration of B7 liposomes activated the entire immune system, which in turn affected the anti-cancer effect.
• Example 9 The effects of changing conditions on the in vivo distribution and immune activity of the various B7 liposomes prepared in Example 1 were examined.
• the following liposome samples prepared using the dye-containing PBS in Example 1 were used.
• (1-1) 50% B7/negatively charged PEG-terminated -OH liposome 100 nm (1-3) 50% B7/negatively charged PEG-terminated -OH liposome 50 nm (1-4) 50% B7/negatively charged PEG-terminated -OH liposome 200 nm (1-45) 50% B7/negatively charged PEG-terminated -OH liposome 400 nm
• the liposome sample was adjusted to a PEGylated liposome concentration of 1 mg/mL (total lipid equivalent) and used as an administration sample.
• the PEGylated liposome concentration was adjusted using the dye-containing PBS.
• the administration sample contained 15 ⁇ g/30 ⁇ L of B7.
• 30 ⁇ L 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.
• Figure 27 shows the fluorescence intensity superimposed on a photograph of each organ 12 hours after administration of a mouse administered a sample of B7 liposomes. Areas with particularly strong fluorescence intensity are indicated with white arrows in Figure 27A.
• Figure 27B is a graph quantifying the fluorescence intensity in the lymph nodes.
• lymph nodes showed higher fluorescence intensity than other organs other than lymph nodes.
• 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.
• the fluorescence intensity was divided by the weight of the organ and normalized to fluorescence intensity per gram, the accumulation amount in the lymph nodes was more than 10 times higher than other organs.
• the B7 liposome administration group effectively accumulated in the lymph nodes 12 hours after administration, regardless of the size of the liposomes, and liposomes of smaller size in particular accumulated in the lymph nodes more effectively. Furthermore, while there are prior art reports that particles of 50 nm or more do not accumulate in lymph nodes, this example confirmed that even liposomes of 50 nm or more (e.g., 100 nm liposomes) accumulated in lymph nodes.
• the administration 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.).
• Figure 28 is an electron microscope photograph of the administration sample (1-1). As shown in Figure 28, observation with an electron microscope confirmed many spherical B7 liposomes, with some B7 liposomes having a long, elongated structure, the length of their short sides being approximately 25 nm. The structure of the latter liposomes is thought to be due to the interaction with the edge of the grid on which the administration sample was placed, causing them to soften and elongate. From these results, it is believed that the B7 liposomes of this example were effectively taken up by lymph nodes, even if their average particle size was, for example, greater than 50 nm, because they were soft and able to deform.
• FIG. 29 shows the results of the accumulation amount of B7 liposome in lymph nodes 24 hours after administration for mice administered with an administration sample of B7 liposome.
• FIG. 29A shows the accumulation amount of B7 liposome in lymph nodes of mice 12 hours and 24 hours after administration in terms of fluorescence intensity. The increase and decrease in fluorescence intensity from 12 hours to 24 hours after administration are indicated by arrows. The arrow pointing downward to the right indicates a decrease from 12 hours to 24 hours after administration, and the arrow pointing upward to the right indicates an increase from 12 hours to 24 hours after administration.
• FIG. 29B is a graph that quantifies the fluorescence intensity showing the accumulation amount of B7 liposome in lymph nodes of mice 12 hours and 24 hours after administration based on FIG.
• the administration groups with different particle sizes of B7 liposome showed the same accumulation amount.
• the accumulation amount increased significantly from 12 hours to 24 hours after administration, and showed the highest accumulation amount among all the administration groups.
• the cells in the wells were washed with fresh medium (the RPMI) to remove excess administration sample, and a staining reagent (0.1 mg/mL Hoechst 33342) was added to the wells, followed by incubation for 15 minutes to stain the nuclei of the cells.
• a staining reagent 0.1 mg/mL Hoechst 33342
• the cells in the wells were washed with fresh medium (RPMI) and then fixed with 4% PFA (PBS solution containing 4% paraformaldehyde) 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.
• Figure 30 is a graph showing the uptake of B7 liposomes into cells by the fluorescence intensity of Cy5 derived from B7 liposomes.
• the uptake of B7 liposomes into dendritic cells increases as the average particle size approaches 200 nm, with the highest uptake being in the administered sample with an average particle size of 200 nm. From the results of (1A) and (1D) above, it was found that uptake into lymph nodes is more likely the smaller the average diameter of the liposomes, whereas uptake into dendritic cells is more likely the closer the average diameter of the liposomes is to 200 nm.
• liposomes with an average particle size of 100 nm are relatively easily taken up by lymph nodes and are further effectively taken up by dendritic cells therein, and therefore, 24 hours after administration, the amount of accumulation in dendritic cells in lymph nodes is thought to have increased by about four times compared to liposomes of other sizes.
• 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, 100 ⁇ 30 nm, and more preferably 100 ⁇ 20 nm, 100 ⁇ 10 nm, or 100 ⁇ 5 nm.
• the administration sample was added to human dendritic cell line DC2.4 cells, incubated, stained, and nucleic acid was examined under a microscope in the same manner as in (1D) above.
• Figure 31 is a graph showing the uptake of B7 liposomes into dendritic cells by the fluorescence intensity of Cy5 derived from B7 liposomes.
• the vertical axis represents the fluorescence intensity of Cy5, 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 liposomes taken up by dendritic cells.
• Figure 31 shows, from the left, the results of 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.
• the fluorescence intensity was comparable between the groups administered with the control liposome that did not contain an NKT ligand, i.e., the Ctrl lipo-OH(-) group and the Ctrl lipo-Me(-) group.
• the B7 lipo-Me(-) group in which the PEG terminus of the B7 liposome was a methyl group, also had a fluorescence intensity comparable to that of the control liposome group.
• the B7 lipo-OH(-) administration group showed significantly higher fluorescence intensity, which indicates the amount of liposomes taken up by dendritic cells, compared to the B7 lipo-Me(-) administration group, in which the PEG terminus is a methyl group.
• Figure 32 is a graph showing the uptake of B7 liposomes into dendritic cells by the fluorescence intensity of Cy5 derived from B7 liposomes.
• the vertical axis represents the fluorescence intensity of Cy5, which indicates B7 liposomes, normalized with the fluorescence intensity of HOECHST 33342, which indicates the amount of cell nuclei, and corresponds to the amount of B7 liposomes taken up by 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 (+).
• the administered samples B7 lipo-OH(-), B7 lipo-OH(n), and B7 lipo-OH(+) all used phospholipids PEGylated with hydroxyl-terminated PEG, but the B7 lipo-OH(n) and B7 lipo-OH(+) had liposomes with neutral (n) and positive (+) charges, respectively, due to the addition of the positively charged lipid (DDAB).
• the B7 lipo-OH(-) administered group in which the liposomes were negatively charged, showed higher fluorescence intensity, indicating the amount of uptake by dendritic cells, compared to the B7 lipo-OH(n) administered group and the B7 lipo-OH(+) administered group, in which the liposomes were neutral or positively charged.
• the PEGylated liposomes of the present invention preferably use phospholipids PEGylated with PEG having a hydroxyl group at the end, and that it is preferable for the liposomes to have a negative charge.
• 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 B7 liposomes was observed using a confocal laser scanning microscope (CLSM) (LSM 880, Carl Zeiss) in the same manner as in (1D) above.
• CLSM confocal laser scanning microscope
• Figure 33 shows microscopic images of the sections, with the first row from the top being a Hoechst 33342 stained image, the second row being a fluorescent image of Cy5 derived from liposomes, and the third row being an image in which the fluorescent distribution in the second row is overlaid on the stained image in the first row.
• B7 liposome B7 lipo-OH(-) which has a negatively charged surface
• 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(-).
• the B7 lipo-OH(n) and B7 lipo-OH(+) which have a neutral or positive surface charge, showed lower accumulation in lymph nodes compared to the B7 lipo-OH(-).
• the PEGylated liposome containing the NKT cell activating ligand of the present invention preferably satisfies the following conditions. That is, it is preferable that the liposome has, 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 PEG terminal on the liposome surface is a hydroxy group.
• the following liposome samples prepared using PBS in Example 1 were used. (1-1) 50% B7/negatively charged PEG-terminated -OH liposome 100 nm (B7 lipo-OH(-)) (1-8) 50% B7/negatively charged PEG-terminated Me liposome 100 nm (B7 lipo-Me(-)) (1-11) 0% control/negatively charged PEG-terminated -OH liposome 100 nm (Ctrl lipo-OH(-)) (1-12) 0% control/negatively charged PEG-terminated Me liposome 100 nm (Ctrl lipo-Me(-)) 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 ⁇ g/30 ⁇ L of B7.
• an administration sample (Free B7) of 15 ⁇ g/30 ⁇ L in which non-liposomal B7 was suspended in PBS, and an administration sample (PBS) of PBS alone were used.
• Figure 34 The results of the antitumor effect are shown in Figure 34.
• Figure 34A is a graph showing the relationship between the tumor volume (V) of the mouse and the number of days from the start of administration
• Figure 34B is a graph showing the relationship between the survival rate of the mouse and the number of days from the start of administration
• Figure 34C is a graph showing the relationship between the body weight (g) of the mouse and the number of days from the start of administration.
• the tumor volume increased about 20 to 40 times on Day 30 compared to Day 0 (30 mm 3 ), and rapid cancer growth was confirmed.
• the increase in tumor volume on Day 30 compared to Day 0 was only about 6 times, and significant inhibition of tumor volume increase was confirmed. From this result, it was found that the cancer inhibition effect can be significantly improved by converting the PEG terminal into a hydroxy group in B7 liposome.
• the B7 lipo-OH administration group maintained a significantly higher survival rate than the other administration groups. This result shows that by converting the PEG end of the B7 liposome to a hydroxyl group, the survival-prolonging effect can be significantly improved.
• the following liposome samples prepared using PBS in Example 1 were used. (1-1) 50% B7/negatively charged PEG-terminated -OH liposome 100 nm (50% B7 lipo) (1-6) 10% B7/negatively charged PEG-terminated -OH liposome 100 nm (10% B7 lipo) (1-7) 2% B7/negatively charged PEG-terminated -OH liposome 100 nm (2% B7 lipo) (1-11) 0% control/negatively charged PEG-terminated -OH liposome 100 nm (0% B7 lipo) 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 using (1-1) had a B7 content of 15 ⁇ g/30 ⁇ L
• the administration sample using (1-6) had a B7 content of 3 ⁇ g/30 ⁇ L
• the administration sample using (1-7) had a B7 content of 0.6 ⁇ g/30 ⁇ L
• the administration sample using (1-1) had a B7 content of 0 ⁇ g/30 ⁇ L.
• Figure 35A is a graph showing the relationship between the tumor volume (V) of the mouse and the number of days from the day of subcutaneous instillation of the cancer
• Figure 35B is a graph showing the relationship between the survival rate of the mouse and the number of days from the day of subcutaneous instillation of the cancer
• Figure 35C is a graph showing the relationship between the weight of the mouse and the number of days from the day of subcutaneous instillation of the cancer.
• the tumor volume of the 0% B7 lipo administration group and the 2% B7 lipo administration group increased by about 20 to 30 times on Day 36 compared to Day 0 (46 mm 3 ), and the rapid growth of the cancer was confirmed.
• the tumor volume of the 10% B7 lipo administration group increased by only about 10 times on Day 36 compared to Day 0, and excellent inhibition of the increase in tumor volume was confirmed.
• the tumor volume of the 50% B7 lipo administration group increased by only about 7 times on Day 36 compared to Day 0, and more significant inhibition of the increase in tumor volume was confirmed. From these results, it was found that the cancer growth inhibition effect can be increased with an increase in the B7 concentration of B7 liposome.
• the liposome preparation of the present invention has excellent stability during storage and can achieve the effects of activating NKT cells in the body.

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