Novel compositions comprising terpenoids and polymers, and uses thereof

Abstract

Provided herein are novel compositions comprising oil@HA derivative particles with high potential for use in stimulating immune responses. Also provided are methods of producing such compositions and methods of their use in treating or preventing diseases or disorders.

Description

[Technical Field]

[0001] The present invention relates to a composition comprising an oil such as a terpenoid and a polymer, and uses thereof, and more particularly to an oil-in-water composition comprising an oil such as a terpenoid and a polymer, and uses thereof. [Background technology]

[0002] Vaccination is the most effective strategy for preventing or limiting the severity of diseases such as infection-related syndromes and cancer. Due to safety concerns, new generation vaccine candidates usually employ highly purified portions of target cells (such as portions of target pathogens or target tumor cells) as antigens; however, these components often lack immunogenicity and therefore require adjuvants to promote the induction of adaptive immunity. Currently, the most common salts for mass vaccination are aluminum-based mineral salts, which primarily function as reservoirs for promoting humoral immunity; however, they are usually limited by weak stimulation of cell-mediated immunity.

[0003] Several developmental adjuvants have been approved or licensed for use in prophylactic human vaccines to enhance cell-mediated immunity, including MF59. However, squalene-based adjuvants such as MF59 have been unsuitable for use in cancer vaccines due to poor cytotoxic T lymphocyte (CTL) responses. Thus, there is an unmet need to develop efficient adjuvants that can stimulate immune responses, such as T lymphocyte responses. Summary of the Invention

[0004] It is therefore an object of the present invention to provide an efficient adjuvant suitable for stimulating an immune response in a host animal, such as in a mammal.

[0005] It is another object of the present invention to provide an efficient adjuvant suitable for promoting humoral and / or cell-mediated immunity.

[0006] It is a further object of the present invention to provide useful vaccine compositions.

[0007] It is a further object of the present invention to provide an efficient adjuvant suitable for use in cancer vaccines.

[0008] It is a further object of the present invention to provide methods for stimulating an immune response or treating cancer, tumors, infectious diseases, and other diseases in a subject.

[0009] 1. A composition comprising an oil and a polymer; the oil and the polymer together form a plurality of particles; and the polymer at least one first unit, the first unit being a first disaccharide; and at least one second unit, the second unit being a conjugation of a second disaccharide and a hydrophobic compound; Including, Compositions are provided herein, wherein at least one first unit and at least one second unit are linked together to form a polymer.

[0010] In some embodiments, the oil is a terpenoid.

[0011] In some embodiments, the first disaccharide is composed of D-glucuronic acid and N-acetylglucosamine.

[0012] In some embodiments, the second disaccharide is composed of D-glucuronic acid and N-acetylglucosamine; in the second unit, the hydrophobic compound is attached to carbon 6 of the D-glucuronic acid.

[0013] In some embodiments, the D-glucuronic acid and N-acetylglucosamine in the first disaccharide are linked by a β-(1,3) bond.

[0014] In some embodiments, the N-acetylglucosamine and D-glucuronic acid in the second disaccharide are linked by a β-(1,3) bond.

[0015] In some embodiments, at least one first unit and at least one second unit are linked to each other by a β-(1,4) bond to form the polymer.

[0016] In some embodiments, the hydrophobic compound is selected from the group consisting of cholesterol derivatives, octadecylamine, octadecylamine derivatives, steroids, steroid derivatives, saturated or unsaturated long-chain fatty amines, and saturated or unsaturated long-chain fatty amine derivatives, or any combination thereof. As used herein, the term "derivative" refers to a product obtained by a chemical reaction that modifies an original compound (such as cholesterol, octadecylamine, steroids, saturated or unsaturated long-chain fatty amines) into a derivative having a primary amine group. In some embodiments, examples of cholesterol derivatives include, but are not limited to, cholesterol-derived amines, cholesterol-amino acid conjugates, or any combination thereof. In some embodiments, the amino acid in the cholesterol-amino acid conjugate is selected from the group consisting of amino acids having a main carbon chain of 2 to 6 carbon atoms, such as glycine, alanine, valine, leucine, and isoleucine.

[0017] In some embodiments, the oil is selected from the group consisting of squalene, squalane, ocimene, farnesene, and paraffin oil, or any combination thereof.

[0018] In some embodiments, the weight ratio of oil to polymer in the composition is in the range of 1:100 to 80:1.

[0019] In some embodiments, the weight percentage of oil is equal to or greater than 0.01% to 40% based on the total weight of the composition.

[0020] In some embodiments, the weight percentage of the polymer is equal to or greater than 0.001% to 3% based on the total weight of the composition.

[0021] In some embodiments, at least 50% (w / w) of the oil in the composition is encapsulated within the particles, based on 100% total oil content in the composition by weight.

[0022] In some embodiments, the composition comprises at least 1% (v / v) oil, based on the total volume of the composition.

[0023] In some embodiments, the polymer is prepared by a method comprising mixing 0.0001 to 2 equivalents of a hydrophobic compound with 1 equivalent of hyaluronic acid.

[0024] In some embodiments, the composition has an average molar ratio of first units to second units per polymer in the range of 99:1 to 1:1.

[0025] 1. A composition comprising a terpenoid and a polymer; the terpenoid and the polymer together form a plurality of particles; and the polymer at least one first unit, the first unit being a disaccharide in which D-glucuronic acid is linked to N-acetylglucosamine; and at least one second unit, the second unit being a disaccharide derivative in which D-glucuronic acid is linked to N-acetylglucosamine and a hydrophobic compound is attached to carbon 6 of the D-glucuronic acid; Including, Also provided herein are compositions wherein the at least one first unit and the at least one second unit are linked together to form a polymer.

[0026] In some embodiments, the D-glucuronic acid and N-acetylglucosamine in the disaccharide are linked by a β-(1,3) bond.

[0027] In some embodiments, the N-acetylglucosamine and D-glucuronic acid attached by the hydrophobic compound in the disaccharide derivative are linked by a β-(1,3) bond.

[0028] In some embodiments, at least one first unit and at least one second unit are linked to each other by a β-(1,4) bond to form the polymer.

[0029] In some embodiments, the macromolecule is a polymer.

[0030] In some embodiments, the polymer is a hyaluronic acid derivative.

[0031] In some embodiments, the polymer is a hyaluronic acid-hydrophobic compound conjugate.

[0032] In some embodiments, the polymer has formula (I):

[0033] [ka]

[0034] where the A group is R—NH— or R—X—NH—, where the R group is a hydrophobic group derived from a hydrophobic compound, and X is a heteroatom (such as O, S, or N), a carbonyl group, or —O—C—O—C 1~5represents alkylene-, where the alkylene can be optionally substituted with one or more substituents selected from the group consisting of alkyl, aromatic, antiaromatic, cycloalkyl, acetyl, amino, hydroxyl, and thiol groups, or an alkane or alkene group whose main carbon chain has from 1 to 10 carbon atoms, and such an alkane or alkene group can be optionally substituted with one or more substituents selected from the group consisting of alkyl, aromatic, antiaromatic, cyclic groups, carbonyl, acetyl, keto, amino, hydroxyl, and heteroatoms (such as O, S, or N); or, the -X-NH- group can be represented as an amino acid whose main chain has from 2 to 6 carbon atoms, such as glycine, alanine, valine, leucine, isoleucine, etc.; and x and y each independently represent an integer value, x≧1, y≧x, and x+y≧3, e.g., x+y≧10. is a compound of

[0035] In some embodiments, the particles are detectable by a particle size analyzer, a transmission electron microscope, or a scanning electron microscope.

[0036] In some embodiments, the composition is an aqueous composition or an oil-in-water composition.

[0037] In some embodiments, the polymer is an amphiphilic compound, and the polymer has a hydrophilic-lipophilic balance value (HLB value) greater than 7.

[0038] In some embodiments, the polymer is an amphiphilic compound, and the polymer has a hydrophilic-lipophilic balance value (HLB value) of greater than 10.

[0039] In some embodiments, the polymer is an amphiphilic compound, and the polymer has a hydrophilic-lipophilic balance value (HLB value) in the range of 7-25.

[0040] In some embodiments, the polymer is an amphiphilic compound, and the polymer has a hydrophilic-lipophilic balance value (HLB value) of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0041] In some embodiments, the hydrophobic compound is an organic compound and has between 8 and 50 carbon atoms.

[0042] In some embodiments, the hydrophobic compound is an organic compound and has 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 carbons.

[0043] In some embodiments, the hydrophobic compound is selected from the group consisting of a steroid, a steroid derivative, a saturated or unsaturated long chain fatty amine, and a saturated or unsaturated long chain fatty amine derivative, or any combination thereof.

[0044] In some embodiments, the hydrophobic compound is selected from the group consisting of cholesterol derivatives, octadecylamine, and octadecylamine derivatives, or any combination thereof.

[0045] In some embodiments, the polymer has formula (II):

[0046] [ka]

[0047] wherein x and y each independently represent integer values, x≧1, y≧x, and x+y≧3, e.g., x+y≧10.

[0048] In some embodiments, the macromolecule is a hyaluronic acid-cholesterol compound conjugate.

[0049] In some embodiments, the polymer has formula (III):

[0050] [ka]

[0051] wherein x and y each independently represent integer values, x≧1, y≧x, and x+y≧3, e.g., x+y≧10.

[0052] In some embodiments, the polymer is a hyaluronic acid-octadecylamine conjugate.

[0053] In some embodiments, the terpenoid is a liquid or solid at room temperature.

[0054] In some embodiments, the terpenoid is a cyclic or acyclic compound.

[0055] In some embodiments, the carbon skeleton of the terpenoid comprises (or consists of) 1 to 20 units of an isoprene carbon skeleton.

[0056] In some embodiments, the carbon skeleton of the terpenoid comprises (or consists of) at least 21 units of an isoprene carbon skeleton.

[0057] In some embodiments, the carbon skeleton of the terpenoid comprises (or consists of) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 units of an isoprene carbon skeleton.

[0058] In some embodiments, the carbon skeleton of the terpenoid comprises (or consists of) 1 to 10 units of a terpene carbon skeleton.

[0059] In some embodiments, the carbon skeleton of the terpenoid comprises (or consists of) at least 11 units of a terpene carbon skeleton.

[0060] In some embodiments, the carbon skeleton of the terpenoid comprises (or consists of) 2, 3, 4, 5, 6, 7, 8, or 9 units of a terpene carbon skeleton.

[0061] In some embodiments, the terpenoid is an unsaturated terpenoid or a saturated terpenoid.

[0062] In some embodiments, the terpenoid is squalene, squalane, ocimene, or farnesene.

[0063] In some embodiments, the polymer has an average molecular weight of at least 10 kDa and less than or equal to 1000 kDa (10k-1000 kDa). In some embodiments, the macromolecule is 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 105 kDa, 110 kDa, 115 kDa, 120 kDa, 125 kDa, 130 kDa, 135 kDa, 140 kDa, 145 kDa, 150 kDa, 155 kDa, 160 kDa, 165 kDa, 170 kDa, 175 kDa, 180 kDa, 185 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kD kDa, 185kDa, 190kDa, 195kDa, 200kDa, 205kDa, 210kDa, 215kDa, 220kDa, 225kDa, 230kDa, 235kDa, 240kDa, 245kDa, 250kDa, 255kDa, 260kDa, 265kDa, 270kDa, 275kDa, 280kDa, 285kDa, 290kDa, 295kDa, 300kDa, 305kDa, 310kDa, 315kDa, 320kDa, 325kDa, 330kDa, 335kDa, 340kDa, 345k Da, 350kDa, 355kDa, 360kDa, 365kDa, 370kDa, 375kDa, 380kDa, 385kDa, 390kDa, 395kDa, 400kDa, 405kDa, 410kDa, 415kDa, 420kDa, 425kDa, 4 30kDa, 435kDa, 440kDa, 445kDa, 450kDa, 455kDa, 460kDa, 465kDa, 470kDa, 475kDa, 480kDa, 485kDa, 490kDa, 495kDa, 500kDa, 505kDa, 510kD a, 515kDa, 520kDa, 525kDa, 530kDa, 535kDa, 540kDa, 545kDa, 550kDa, 555kDa, 560kDa, 565kDa, 570kDa, 575kDa, 580kDa, 585kDa, 590kDa, 59 5kDa, 600kDa, 605kDa, 610kDa, 615kDa, 620kDa, 625kDa, 630kDa, 635kDa, 640kDa, 645kDa, 650kDa, 655kDa, 660kDa, 665kDa, 670kDa, 675kDa,680kDa, 685kDa, 690kDa, 695kDa, 700kDa, 705kDa, 710kDa, 715kDa, 720kDa, 725kDa, 730kDa, 735kDa, 740kDa, 745kDa, 750kDa, 755kDa, 760 kDa, 765kDa, 770kDa, 775kDa, 780kDa, 785kDa, 790kDa, 795kDa, 800kDa, 805kDa, 810kDa, 815kDa, 820kDa, 825kDa, 830kDa, 835kDa, 840kDa , 845 kDa, 850 kDa, 855 kDa, 860 kDa, 865 kDa, 870 kDa, 875 kDa, 880 kDa, 885 kDa, 890 kDa, 895 kDa, 900 kDa, 905 kDa, 910 kDa, 915 kDa, 920 kDa, 925 kDa, 930 kDa, 935 kDa, 940 kDa, 945 kDa, 950 kDa, 955 kDa, 960 kDa, 965 kDa, 970 kDa, 975 kDa, 980 kDa, 985 kDa, 990 kDa, or 995 kDa. In some embodiments, the average molecular weight is a number average molecular weight as determined by gel filtration chromatography (GFC). In some embodiments, the average molecular weight is the weight molecular weight determined by GFC (see Bernice Yeung and Dale Marecak, "Molecular weight determination of hyaluronic acid by gel filtration chromatography coupled to matrix-assisted laser desorption ionization mass spectrometry." Journal of Chromatography A 85.2 (1999): 573-581).

[0064] In some embodiments, the composition has, on average, at least 70% by molar ratio of units per polymer as the first unit.

[0065] In some embodiments, the weight ratio of terpenoid to polymer in the composition is in the range of 1:100 to 80:1.

[0066] In some embodiments, the weight ratio of terpenoid to polymer in the composition is 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, or 80:1.

[0067] In some embodiments, the weight percentages of the terpenoid and polymer in the composition are between 0.01% and 40% and between 0.001% and 3.0%, respectively, based on the total weight of the composition.

[0068] In some embodiments, the weight percentage of the terpenoid in the composition is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, or 39%.

[0069] In some embodiments, the weight percentage of the polymer in the composition is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.50%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.60%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.80%, 0.81%, 0.82%, 0.83 .35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.50%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.60%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76% ,0.77%,0.78%,0.79%,0.80%,0.81%,0.82%,0.83%,0.84%,0.85%,0.86%,0.87%,0.88%,0.89%,0.90%,0.91%,0.92%,0.93%,0.94%,0.95%,0.96%,0.97 %,0.98%,0.99%,1.00%,1.01%,1.02%,1.03%,1.04%,1.05%,1.06%,1.07%,1.08%,1.09%,1.10%,1.11%,1.12%,1.13%,1.14%,1.15%,1.16%,1.17%,1.1 8%, 1.19%, 1.20%, 1.21%, 1.22%, 1.23%, 1.24%, 1.25%, 1.26%, 1.27%, 1.28%, 1.29%, 1.30%, 1.31%, 1.32%, 1.33%, 1.34%, 1.35%, 1.36%, 1.37%, 1.38%, 1. 39%, 1.40%, 1.41%, 1.42%, 1.43%, 1.44%, 1.45%, 1.46%, 1.47%, 1.48%, 1.49%, 1.50%, 1.51%, 1.52%, 1.53%, 1.54%, 1.55%, 1.56%, 1.57%, 1.58%, 1.59%, 1.60%, 1.61%, 1.62%, 1.63%, 1.64%, 1.65%, 1.66%, 1.67%, 1.68%, 1.69%, 1.70%, 1.71%, 1.72%, 1.73%, 1.74%, 1.75%, 1.76%, 1.77%, 1.78%, 1.79%, 1.80%, 1.81%, 1.82%, 1.83%, 1.84%, 1.85%, 1.86%, 1.87%, 1.88%, 1.89%, 1.90%, 1.91%, 1.92%, 1.93%, 1.94%, 1.95% 5%, 1.96%, 1.97%, 1.98%, 1.99%, 2.00%, 2.01%, 2.02%, 2.03%, 2.04%, 2.05%, 2.06%, 2.07%, 2.08%, 2.09%, 2.10%, 2.11%, 2.12%, 2.13%, 2.14%, 2.15%, 2.16%, 2.17%, 2.18%, 2.19%, 2.20%, 2.21%, 2.22%, 2.23%, 2.24%, 2.25%, 2.26%, 2.27%, 2.28%, 2.29%, 2.30 %, 2.31%, 2.32%, 2.33%, 2.34%, 2.35%, 2.36%, 2.37%, 2.38%, 2.39%, 2.40%, 2.41%, 2.42%, 2.43%, 2.44%, 2.45%, 2.46%, 2.47%, 2.48%, 2.49%, 2.50%, 2.51%, 2.52%, 2.53%, 2.54%, 2.55%, 2.56%, 2.57%, 2.58%, 2.59%, 2.60%, 2.61%, 2.62%, 2.63%, 2.64%, 2.65% , 2.66%, 2.67%, 2.68%, 2.69%, 2.70%, 2.71%, 2.72%, 2.73%, 2.74%, 2.75%, 2.76%, 2.77%, 2.78%, 2.79%, 2.80%, 2.81%, 2.82%, 2.83%, 2.84%, 2.85%, 2.86%, 2.87%, 2.88%, 2.89%, 2.90%, 2.91%, 2.92%, 2.93%, 2.94%, 2.95%, 2.96%, 2.97%, 2.98%, or 2.99%.

[0070] In some embodiments, at least 50% (w / w) of the terpenoids in the composition are encapsulated within the particles, based on 100% total terpenoid content in the composition by weight. In some embodiments, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% (w / w) of the terpenoids in the composition are encapsulated within the particles, based on 100% total terpenoid content in the composition by weight.

[0071] In some embodiments, the composition comprises at least 1% (v / v) terpenoid based on the total volume of the composition. In some embodiments, the composition comprises 1% to 40% (v / v) terpenoid based on the total volume of the composition. In some embodiments, the composition comprises 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, or 39% (v / v) terpenoid based on the total volume of the composition.

[0072] In some embodiments, the composition further comprises a solvent. In some embodiments, the solvent is water, saline, pure water, electrolyte water, glucose water, or other aqueous solutions that can be used in pharmaceutical injections. In some embodiments, the weight percentage of the solvent in the composition is 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 7%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, or 82% based on the total weight of the composition. ,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,93.5%,93.6%,93.7%,93.8%,93.9%,94.0%,94.1%,94.2%,94.3%,94.4%,94.5%,94.6%,94.7%,94.8%,94.9%,95.0%,95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97. 6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.In some embodiments, the volume percentage of solvent in the composition is 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 7%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, or 83%, based on the total volume of the composition. ,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,93.5%,93.6%,93.7%,93.8%,93.9%,94.0%,94.1%,94.2%,94.3%,94.4%,94.5%,94.6%,94.7%,94.8%,94.9%,95.0%,95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97. 6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

[0073] In some embodiments, the solvent is water.

[0074] In some embodiments, the polymer is prepared by a method comprising mixing 0.0001 to 2 equivalents of a hydrophobic compound with 1 equivalent of hyaluronic acid.

[0075] In some embodiments, the polymer is prepared by a method comprising combining 0.0001, 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 equivalents of a hydrophobic compound with 1 equivalent of hyaluronic acid.

[0076] In some embodiments, the composition has an average molar ratio of first units to second units per polymer in the range of 99:1 to 1:1.

[0077] In some embodiments, the molar ratio of first units to second units per polymer in the composition is on average 98:1, 97:1, 96:1, 95:1, 94:1, 93:1, 92:1, 91:1, 90:1, 89:1, 88:1, 87:1, 86:1, 85:1, 84:1, 83:1, 82:1, 81:1, 80:1, 79:1, 78:1, 77:1, 76:1, 75:1, 74:1, 73:1, 72:1, 71:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1 , 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0078] In some embodiments, the particles have a diameter of 3 to 1000 nm.

[0079] In some embodiments, the particle diameter is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 150, 160, 180, 200, 220, 240, 150, 260, 280, 300, 320, 340, 350, 360, 380, 400 , 420, 440, 450, 460, 480, 500, 520, 540, 550, 560, 580, 600, 620, 640, 650, 660, 680, 700, 720, 740, 750, 760, 780, 800, 820, 840, 850, 860, 880, 900, 920, 940, 950, 960, or 980 nm.

[0080] In some embodiments, the particles have a polydispersity index value (PDI) of less than 0.4 to about 0.01.

[0081] In some embodiments, the particles have a polydispersity index value (PDI) of less than 0.375, 0.35, 0.325, 0.3, 0.275, 0.25, 0.225, 0.2, 0.175, 0.15, 0.125, 0.1, 0.075, 0.05, or 0.025.

[0082] In some embodiments, provided herein are pharmaceutical compositions comprising the compositions provided herein.

[0083] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0084] Also provided herein is the use of a composition or pharmaceutical composition provided herein in stimulating an immune response in a subject.

[0085] Also provided herein is the use of a composition or pharmaceutical composition provided herein in the treatment or prevention of a disease or disorder.

[0086] Also provided herein are methods of stimulating an immune response in a subject in need thereof, comprising administering to the subject a composition or pharmaceutical composition provided herein.

[0087] Also provided herein are methods for treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a composition or pharmaceutical composition provided herein.

[0088] In some embodiments, the pharmaceutical composition is a vaccine.

[0089] In some embodiments, the disease or disorder is a tumor or cancer.

[0090] In some embodiments, the tumor or cancer is breast cancer.

[0091] In some embodiments, the disease or disorder is a pathogenic disease, an HIV or other viral infection, a fungal infection, a protozoal infection, or a bacterial infection.

[0092] In some embodiments, the subject is a mammal, such as a human.

[0093] Also provided herein are methods for increasing the immune response elicited by an antigen component, comprising administering a composition or pharmaceutical composition provided herein having the antigen component.

[0094] Also provided herein are kits (or vaccines) that include the compositions or pharmaceutical compositions provided herein having an antigen component.

[0095] In some embodiments, the antigen component is a target pathogen, a target tumor cell, a portion of a target pathogen, a portion of a target tumor cell, a pathogen antigen, a cancer antigen, or any combination thereof.

[0096] In some embodiments, the antigen component is selected from HER2, HER3, EGFR, VEGF, VEGFR2, CA-125, MUC series, p53, MAGE, NY-ESO-1, GAGE, BAGE, KRAS, NRAS, BCR-ABL translocation, ETV6, NPM / ALK, ALK, EBV LMP-1 / LMP-2A, HPV E6 / E7, HTLV-1 Tax, MelanA / MART-1, gp100, tyrosinase, PSA, CEA, hTERT, p53, survivin, WT1, cyclin B, globo H, and SSEA series, or any combination thereof. [Brief explanation of the drawings]

[0097] [Figure 1A] FIG. 1 provides the results of a structural identification performed on (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-((tert-butoxycarbonyl)amino)acetate, analyzed by H NMR (CDCl, 400 MHz) and mass spectrometry (ESI-MS). [Figure 1B] FIG. 1 provides the results of a structural identification performed on (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate, analyzed by H NMR (CDCl, 400 MHz) and mass spectrometry (ESI-MS). [Figure 1C] FIG. 1 provides the results of the structural identification performed on HACH20, analyzed by 1H NMR spectroscopy (D2O / d6-DMSO, 400 MHz). [Figure 2] FIG. 1 provides visual appearances and TEM images of HA and SQ@HA derivatives in the present invention. [Figure 3A] Figure 3 provides results showing the effect of Oil@HACH on antigen-presenting cell (APC) recruitment. Figure 3A: Dendritic cell recruitment; Figure 3B: Macrophage recruitment. [Figure 3B] Figure 3 provides results showing the effect of Oil@HACH on antigen-presenting cell (APC) recruitment. Figure 3A: Dendritic cell recruitment; Figure 3B: Macrophage recruitment. [Figure 4A] Figure 4 provides results showing the effects of reactants and products in the squalene@HACH synthesis process on antigen-presenting cell (APC) recruitment. Figure 4A: Dendritic cell recruitment; Figure 4B: Macrophage recruitment. Note that the symbol "*" indicates that an animal in this group died during the experiment. [Figure 4B] Figure 4 provides results showing the effects of reactants and products in the squalene@HACH synthesis process on antigen-presenting cell (APC) recruitment. Figure 4A: Dendritic cell recruitment; Figure 4B: Macrophage recruitment. Note that the symbol "*" indicates that an animal in this group died during the experiment. [Figure 5A] Figure 5 provides results showing the effect of a mixture containing squalene@HACH and a target antigen on antigen-presenting cell (APC) recruitment. Figure 5A: Dendritic cell recruitment; Figure 5B: Macrophage recruitment. [Figure 5B] Figure 5 provides results showing the effect of a mixture containing squalene@HACH and a target antigen on antigen-presenting cell (APC) recruitment. Figure 5A: Dendritic cell recruitment; Figure 5B: Macrophage recruitment. [Figure 6] FIG. 1 provides results showing the effect of a mixture containing squalene@HACH and a target antigen on T cell-associated cytokine production. [Figure 7A] Figure 7 provides results from a mouse model study demonstrating the antitumor activity of WT-1 + squalene@HACH. Figure 7A provides the tumor-free rate curve, and Figure 7B provides the survival rate curve. [Figure 7B] Figure 7 provides results from a mouse model study demonstrating the antitumor activity of WT-1 + squalene@HACH. Figure 7A provides the tumor-free rate curve, and Figure 7B provides the survival rate curve. [Figure 8A] Figure 8 provides results showing the effects of reactants and products in the squalene@HAODA synthesis process on antigen-presenting cell (APC) recruitment: Figure 8A: dendritic cell recruitment; Figure 8B: macrophage recruitment. [Figure 8B] Figure 8 provides results showing the effects of reactants and products in the squalene@HAODA synthesis process on antigen-presenting cell (APC) recruitment: Figure 8A: dendritic cell recruitment; Figure 8B: macrophage recruitment. DETAILED DESCRIPTION OF THE INVENTION

[0098] PEGylated formulations have been widely applied in food, cosmetics, medicine, and many other related fields, leading to the ubiquitous presence of PEG antibodies in the modern human body; however, these pre-existing anti-PEG antibodies can bind to PEGylated formulations, activate the complement system, and release anaphylatoxins (C3a or C5a), which can further stimulate mast cells, basophils, and tissue macrophages to release secondary mediators that can cause CARPA. (See Mohamed, Marwa et al., "PEGylated liposomes: immunological responses." Science and Technology of Advanced Materials 20.1 (2019): 710-724.)

[0099] Compositions such as SQ@HACH are provided herein. Compared with the approved squalene-based adjuvant MF59 containing a PEG derivative, the APC recruitment ability of the present invention and MF59 is comparable; however, the present invention does not stimulate the production of anti-PEG antibodies, meaning that it does not induce additional immunogenic responses. This makes the present invention potentially a less allergenic alternative. Because vaccines are injected into a large number of healthy people rather than patients, greater consideration should be given to adjuvants in terms of health hazards.

[0100] Provided herein is an adjuvant composition comprising a terpenoid and a polymer; the terpenoid and polymer together form a plurality of particles; the polymer comprises: (1) at least one first unit, the first unit being a disaccharide in which D-glucuronic acid is linked to N-acetylglucosamine; and (2) at least one second unit, the second unit being a disaccharide derivative in which D-glucuronic acid is linked to N-acetylglucosamine and a hydrophobic compound is attached to carbon 6 of the D-glucuronic acid; and the at least one first unit and the at least one second unit are linked to each other to form the polymer.

[0101] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments specified herein, and that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

[0102] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature used in connection with, and techniques of, organic chemistry, cell and tissue culture, molecular biology, immunology, microbiology, and chemistry and hybridization described herein are those well known and commonly used in the art.

[0103] 1.1 Composition The present invention provides a composition comprising an oil and a polymer; the oil and the polymer together form a plurality of particles; the polymer comprises (1) at least one first unit, the first unit being a first disaccharide; and (2) at least one second unit, the second unit being a conjugation of the second disaccharide and a hydrophobic compound; the at least one first unit and the at least one second unit are linked together to form a polymer. In some embodiments, the oil can be any plant oil, animal oil, mineral oil, or synthetically prepared oil that can be metabolized by the body of the subject to which the adjuvant will be administered. In some embodiments, the oil component of the present invention can be any long-chain alkane, alkene, or alkyne, or its acid or alcohol derivative, either as a free acid, a salt thereof, or an ester, such as a mono-, di-, or tri-ester, for example, a triglyceride, and an ester of 1,2-propanediol or a similar polyhydroxy alcohol.

[0104] Provided herein is a composition comprising a terpenoid and a polymer; the terpenoid and polymer together form a plurality of particles; the polymer comprises: (1) at least one first unit, the first unit being a disaccharide in which D-glucuronic acid is linked to N-acetylglucosamine; and (2) at least one second unit, the second unit being a disaccharide derivative in which D-glucuronic acid is linked to N-acetylglucosamine and a hydrophobic compound is attached to carbon 6 of the D-glucuronic acid; and the at least one first unit and the at least one second unit are linked to each other to form the polymer.

[0105] The term "terpenoid" as used herein refers to "an organic compound comprising a monoterpene, sesquiterpene, diterpene, or triterpene and composed of linked isoprene units" or "a carbohydrate consisting of a terpene attached to an oxygen-containing group" or "an organic compound comprising a monoterpene, sesquiterpene, diterpene, triterpene, tetraterpene, or polyterpene" or "an organic compound whose carbon skeleton is composed of at least one unit of an isoprene carbon skeleton" or "an organic compound consisting of a terpene attached to an oxygen-containing group."

[0106] In some embodiments, the terpenoid is a liquid or solid at room temperature. Preferably, in some embodiments, the terpenoid is a liquid at room temperature. In some embodiments, the terpenoid is a cyclic or acyclic compound.

[0107] In some embodiments, the terpenoid comprises (or consists of) 1 to 10 terpenes; or the terpenoid comprises (or consists of) at least 11 terpenes. Preferably, in some embodiments, the terpenoid comprises (or consists of) 1 to 10 terpenes.

[0108] In some embodiments, the terpenoid is squalene, squalane, ocimene, or farnesene, other terpenoids, or any combination thereof.

[0109] As used herein, the term "polymer" refers to a molecule containing at least 100 atoms.

[0110] In some embodiments, the macromolecule is a polymer, hi some embodiments, the macromolecule is a hyaluronic acid derivative.

[0111] In some embodiments, the polymer is a hyaluronic acid-hydrophobic compound conjugate.

[0112] In some embodiments, the polymer is a compound of formula (I). In some embodiments, the polymer is a compound of formula (II) or (III).

[0113] In some embodiments, the terpenoid is an oil and the polymer is an emulsifier; the composition is an oil-in-water composition.

[0114] In some embodiments, the polymer has a hydrophilic-lipophilic balance (HLB) value greater than 7. In some embodiments, the polymer has a hydrophilic-lipophilic balance (HLB) value greater than 10. In some embodiments, the polymer has a hydrophilic-lipophilic balance (HLB) value in the range of 7-20.

[0115] In some embodiments, the polymer has a hydrophilic-lipophilic balance value (HLB value) of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

[0116] In some embodiments, the particles comprise (a) a microstructure formed by a polymer, and (b) a terpenoid encapsulated within the microstructure.

[0117] In some embodiments, the particles are detectable by a particle size analyzer, a transmission electron microscope, or a scanning electron microscope.

[0118] In some embodiments, the particles have a diameter of 3 to 1000 nm.

[0119] In some embodiments, the particle diameter is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 150, 160, 180, 200, 220, 240, 150, 260, 280, 300, 320, 340, 350, 360, 380, 400 , 420, 440, 450, 460, 480, 500, 520, 540, 550, 560, 580, 600, 620, 640, 650, 660, 680, 700, 720, 740, 750, 760, 780, 800, 820, 840, 850, 860, 880, 900, 920, 940, 950, 960, or 980 nm.

[0120] In some embodiments, the particles have a polydispersity index value (PDI) of less than 0.4 to about 0.01.

[0121] In some embodiments, the particles have a polydispersity index value (PDI) of less than 0.375, 0.35, 0.325, 0.3, 0.275, 0.25, 0.225, 0.2, 0.175, 0.15, 0.125, 0.1, 0.075, 0.05, or 0.025.

[0122] As used herein, the term "first unit" refers to a disaccharide composed of D-glucuronic acid and N-acetylglucosamine, wherein the D-glucuronic acid is linked to the N-acetylglucosamine.

[0123] In some embodiments, the D-glucuronic acid and N-acetylglucosamine in the first unit are linked by a β-(1,3) bond.

[0124] As used herein, the term "second unit" refers to a disaccharide derivative composed of D-glucuronic acid, N-acetylglucosamine, and a hydrophobic compound; the D-glucuronic acid is linked to the N-acetylglucosamine, and the hydrophobic compound is attached to carbon 6 of the D-glucuronic acid.

[0125] In some embodiments, the D-glucuronic acid and N-acetylglucosamine in the second unit are linked by a β-(1,3) bond.

[0126] In some embodiments, the hydrophobic compound is selected from the group consisting of a steroid, a steroid derivative, a saturated or unsaturated long chain fatty amine, and a saturated or unsaturated long chain fatty amine derivative, or any combination thereof.

[0127] In some embodiments, the hydrophobic compound is selected from the group consisting of cholesterol derivatives, octadecylamine, and octadecylamine derivatives, or any combination thereof.

[0128] In some embodiments, the polymer is composed of one first unit and at least two second units, which are linked together to form the polymer. In some embodiments, the polymer is composed of at least two first units and one second unit, which are linked together to form the polymer. In some embodiments, the polymer is composed of at least two first units and at least two second units, which are linked together to form the polymer.

[0129] In some embodiments, two first units located in a portion of a polymer are linked to each other to form a portion of the polymer. In some embodiments, two second units located in a portion of the polymer are linked to each other to form a portion of the polymer. In some embodiments, a first unit and a second unit located in a portion of the polymer are linked to each other to form a portion of the polymer.

[0130] In some embodiments, the units (first unit and / or second unit) are linked together by β-(1,4) bonds to form a polymer.

[0131] In some embodiments, the polymer is prepared by a method comprising mixing 0.0001 to 2 equivalents of a hydrophobic compound with 1 equivalent of hyaluronic acid. For example, in some embodiments, to prepare HACH10, 0.1 equivalents of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate (a hydrophobic compound) are mixed with 1 equivalent of hyaluronic acid. To prepare HACH20, 0.2 equivalents of cholesterol-glycine-NH2 and 1 equivalent of hyaluronic acid were mixed; to prepare HACH30, 0.3 equivalents of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthate were mixed. For the preparation of HACH5, 0.05 equivalents of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate were mixed with 1 equivalent of hyaluronic acid; for the preparation of HACH5, 0.05 equivalents of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate were mixed. and 1 equivalent of hyaluronic acid are mixed; for the preparation of HACH25, 0.25 equivalents of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate are mixed with 1 equivalent of hyaluronic acid. In some embodiments, to prepare HAODA5, 0.05 equivalents of octadecylamine (a hydrophobic compound) are mixed with 1 equivalent of hyaluronic acid; to prepare HAODA15, 0.15 equivalents of octadecylamine are mixed with 1 equivalent of hyaluronic acid; to prepare HAODA25, 0.25 equivalents of octadecylamine are mixed with 1 equivalent of hyaluronic acid.

[0132] In the composition, the molar ratio of the first unit to the second unit per polymer is in the range of 99:1 to 1:1 on average. In some embodiments, the molar ratio of first units to second units per polymer in the composition is on average 98:1, 97:1, 96:1, 95:1, 94:1, 93:1, 92:1, 91:1, 90:1, 89:1, 88:1, 87:1, 86:1, 85:1, 84:1, 83:1, 82:1, 81:1, 80:1, 79:1, 78:1, 77:1, 76:1, 75:1, 74:1, 73:1, 72:1, 71:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 5:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1 , 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0133] 1.2 Pharmaceutical Compositions In some embodiments, provided herein are pharmaceutical compositions comprising the compositions provided herein.

[0134] In some embodiments, the pharmaceutical composition is a vaccine. In some embodiments, the pharmaceutical composition is a natural vaccine, a synthetic vaccine, or a recombinant vaccine. In some embodiments, the pharmaceutical composition is a DNA vaccine or an RNA vaccine. In some embodiments, the pharmaceutical composition is an inactivated vaccine, a live attenuated vaccine, a messenger RNA (mRNA) vaccine, a subunit, recombinant, polysaccharide, and conjugate vaccine, a toxoid vaccine, or a viral vector vaccine.

[0135] In some embodiments, the compositions or pharmaceutical compositions provided herein further comprise an active ingredient or target antigen (or antigen, or antigen component). In some embodiments, the active ingredient or target antigen (or antigen, or antigen component) is for treating a disease or disorder. In some embodiments, the active ingredient or target antigen (or antigen, or antigen component) is for treating cancer, and the active ingredient or target antigen (or antigen, or antigen component) is WT1 protein, OVA, or a combination thereof.

[0136] As would be understood by one of ordinary skill in the art, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a material that is suitable for drug administration to an individual in conjunction with, for example, an active agent or target molecule or antigen, without causing undesired biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition.

[0137] In some embodiments, the pharmaceutical composition is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multi-phase materials. The term "aqueous formulation" is defined as a formulation that contains at least 50% w / w water.

[0138] 1.3 How to use In some embodiments, provided herein is a use of a composition or pharmaceutical composition provided herein in the treatment or prevention of a disease or disorder.

[0139] In some embodiments, provided herein is a method of stimulating an immune response in a subject, comprising administering to the subject a composition or pharmaceutical composition provided herein.

[0140] In some embodiments, the immune response is an antibody response (humoral immune response), an antigen-specific T cell response (cell-mediated immune response), an innate immune response, or any combination thereof. In some embodiments, the antigen-specific T cell response is a CD8 + T cell response and / or CD4 + In some embodiments, the immune response is a T-cell mediated immune response. In some embodiments, the immune response is a T-helper cell immune response. In some embodiments, the T-helper cell immune response is a T-helper 1 response, or a T-helper 2 response, or a T-helper 17 response, or any of them. In some embodiments, the immune response is a dendritic cell (CD11c + ) or macrophages (CD11b + In some embodiments, the immune response is the recruitment of T cell immunity, CD4 + T cell response, antigen-specific T cell immunity, or Th1 / Th2 / Th17-associated cytokine production.

[0141] The term "treating," as used herein in reference to a disease or condition, or a subject having a disease or condition, refers to the act of suppressing, eliminating, reducing, and / or ameliorating the symptoms, symptom severity, and / or symptom frequency associated with the disease or disorder being treated. For example, when used in reference to a cancer or tumor, the term "treating" refers to the act of reducing the severity of the cancer or tumor or slowing or slowing the progression of the cancer or tumor, including (a) inhibiting the growth or halting the progression of the cancer or tumor, (b) causing the regression of the cancer or tumor, or (c) delaying, ameliorating, or minimizing one or more symptoms associated with the presence of the cancer or tumor.

[0142] As used herein, the term "administering" refers to the act of delivering or causing to be delivered a therapeutic or pharmaceutical composition to the body of a subject by methods described herein or otherwise known in the art. A therapeutic agent can be a compound, a polypeptide, an antibody, a cell, or a population of cells. Administering a therapeutic or pharmaceutical composition includes formulating the therapeutic or pharmaceutical composition to be delivered to the body of a subject.

[0143] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the administration of an agent to a subject, either alone or as part of a pharmaceutical composition, and in a single dose or as part of a series of doses, in an amount that, when administered to a subject, can have any detectable positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition. A therapeutically effective amount can be ascertained by measuring the relevant physiological effect. The exact amount required will vary from subject to subject, depending on the age, weight, and general condition of the subject, the severity of the condition being treated, the judgment of the clinician, and the like. An appropriate "effective amount" in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

[0144] As used herein, the term "subject" refers to any animal (e.g., a vertebrate). A subject includes, but is not limited to, humans, non-human primates, apes, canines, felines, rodents, etc., which may be recipients of a particular treatment. A subject may be a human. A subject may be a mammal. A subject may be a farm animal. A subject may be a pet. A subject may have a particular disease or condition.

[0145] The patient or subject to be treated can be a human patient with the disease or disorder described herein.In some embodiments, the subject is a cancer patient.In some embodiments, the subject is a viral infection patient, a bacterial infection patient, a fungal infection patient, or a protozoan infection patient.In some embodiments, the subject has cancer or tumor and / or is being treated for it.

[0146] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges within that range as well as individual numerical values. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, etc. This applies regardless of the broadness of the range.

[0147] 1.4 Method for preparing the composition 1.4.1 Polymer Preparation Method Scheme (a) provides a synthetic route to some of the polymers of the present invention (referring to amphiphilic HA derivatives or amphiphilic HA-hydrophobic compound conjugates).

[0148] [ka]

[0149] In formula (a), the compound AH is a hydrophobic compound, and the A group is R-NH- or R-X-NH-, which conjugates with the carboxylic acid group of hyaluronic acid to form a polymer comprising a first unit and a second unit in some embodiments of the present invention (referred to as an amphiphilic HA derivative or an amphiphilic HA-hydrophobic compound conjugate); wherein the R group is a hydrophobic group derived from the hydrophobic compound, and X is a heteroatom (such as O, S, or N), a carbonyl group, or -O-CO-C 1~5represents alkylene-, where the alkylene can be optionally substituted with one or more substituents selected from the group consisting of alkyl, aromatic, antiaromatic, cycloalkyl, acetyl, amino, hydroxyl, and thiol groups, or an alkane or alkene group whose main carbon chain has 1 to 10 carbon atoms, and such an alkane or alkene group can be optionally substituted with one or more substituents selected from the group consisting of alkyl, aromatic, antiaromatic, cyclic groups, carbonyl, acetyl, keto, amino, hydroxyl, and heteroatoms (such as O, S, or N); or the -X-NH- group can be represented as an amino acid whose main chain has 2 to 6 carbon atoms, such as glycine, alanine, valine, leucine, isoleucine, etc.; and x and y each independently represent an integer value, n=x+y, x≧1, y≧x, and x+y≧3, e.g., x+y≧10.

[0150] The inventors of the present invention further provide a method for the synthesis of these polymers (referring to amphiphilic HA derivatives or amphiphilic HA-hydrophobic compound conjugates), comprising the steps of: First, 1.0 equivalents of hyaluronic acid is dissolved in a co-solvent, a mixture of water and an organic solvent (e.g., DMSO). A mixture containing 1.1 equivalents of ethyl cyanohydroxyiminoacetate and an appropriate equivalent (e.g., depending on the expected reactivity of the substituted compound) of RX-NH2 is dissolved in an organic solvent (e.g., DMSO), and the resulting solution is then added to the hyaluronic acid solution (HA solution). 2.0 equivalents of N,N'-diisopropylcarbodiimide are slowly added to the mixed solution and stirred for 24 hours. The resulting solution is transferred to an appropriate molecular weight cut-off (MWCO) dialysis bag (e.g., a 3500 Da MWCO dialysis bag) and purified by sequential dialysis against the co-solvent (50 / 50, v / v), 0.3 M NaCl aqueous solution, and purified water. Finally, water is removed from the dialyzed product solution by freeze-drying to obtain a polymer having a molecular weight higher than the MWCO (such as a polymer having a molecular weight higher than 3500 Da; note that hyaluronic acid, which consists of 10 disaccharide units, has a molecular weight of approximately 3800 Da).

[0151] In formula (a), the hydrophobic compound R is (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate; ,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminopropanoate, or (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17 -((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-aminopropanoate, or (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4 ,7,8,9,10,11,12,13,14,15,16,17-Tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-aminobutanoate, or (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl amino acid ester, or octan-1-amine, or nonan-1-amine, or decan-1-amine, or undecane-1-amine, or dodecane-1-amine, or tridecane-1-amine, or tetradecane-1-amine, or pentadecane-1-amine, or hexadecan-1-amine, or heptadecane-1-amine, or octadecane-1-amine, or a saturated long-chain fatty amine having more than 19 carbon atoms, or an unsaturated octan-1-amine, or an unsaturated nonan-1-amine or unsaturated decan-1-amine, or unsaturated undecane-1-amine, or unsaturated dodecane-1-amine, or unsaturated tridecane-1-amine, or unsaturated tetradecane-1-amine, or unsaturated pentadecane-1-amine, or unsaturated hexadecan-1-amine, or unsaturated heptadecane-1-amine, or unsaturated octadecane-1-amine, or an unsaturated long-chain fatty amine having more than 19 carbon atoms, or a saturated long-chain fatty amine, or a saturated long-chain fatty amine derivative, or an unsaturated long-chain fatty amine derivative, or any combination thereof.

[0152] 1.4.2 Preparation method of oil@polymer particles In some embodiments, oil and a polymer prepared by the method described in Section 1.4.1 are added to an aqueous solution (such as a sodium citrate solution) to obtain a resulting solution that is a composition comprising oil@polymer particles.

[0153] In some embodiments, oil and a polymer prepared by the method described in Section 1.4.1 are added to an aqueous solution (such as a sodium citrate solution) to obtain a resulting solution, which is premixed in a test tube rotor and then homogenized with a high-pressure microfluidizer to obtain a resulting solution that is a composition comprising oil@polymer particles.

[0154] In some embodiments, the oil is a terpenoid and the oil@polymer particle is a terpenoid@polymer particle.

[0155] To evaluate the particle size and stability of the oil@polymer particles, the particle size and polydispersity index (PDI) of the oil@polymer particles in the composition were measured by dynamic light scattering or a particle size analyzer. The oil@polymer particles were stained with PTA negative stain and observed by transmission electron microscopy.

[0156] experiment The examples provided below are for illustrative purposes only and, unless otherwise specified, are not intended to be limiting. Thus, the present invention should in no way be construed as limited to the following examples, but rather as encompassing any and all variations that become evident as a result of the teachings provided herein.

[0157] Experiment 1: Compositions containing oil@HACH particles Experiment 1-1 Preparation of a composition containing oil@HACH particles In this embodiment, a hyaluronic acid-cholesterol conjugate (HACH), which is a polymer of the present invention, was used.

[0158] Schemes (b) to (d) provide a synthetic route to the hyaluronic acid-cholesterol conjugate (HACH) of the present invention.

[0159] Synthetic route for formula (b) (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-((tert-butoxycarbonyl)amino)acetate; (Cholesterol modification):

[0160] [ka]

[0161] Synthetic route for formula (c) (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate; ((8S,9S,1 Deprotection of 0R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-((tert-butoxycarbonyl)amino)acetate:

[0162] [ka]

[0163] Formula (d) Synthesis route of HACH; (Conjugation of HA with (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate):

[0164] [ka]

[0165] where n, x, and y each independently represent integer values, n=x+y, x≧1, y≧x, and x+y≧3, e.g., x+y≧10.

[0166] Experiment 1-1-1: Synthetic route to (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-((tert-butoxycarbonyl)amino)acetate; (Cholesterol modification) Referring to formula (b), in the first step for the preparation of HACH, cholesterol was modified with Boc-Gly-OH by DCC / DMAP esterification to give (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-((tert-butoxycarbonyl)amino)acetate. The detailed process in this first step includes the following: 1000 mg (2.59 mmol) of cholesterol and 480 mg (2.74 mmol) of Boc-Gly-OH were dissolved in 125 mL of DCM, and then 1070 mg (5.18 mmol) of DCC and 372 mg (3.04 mmol) of DMAP were added to the mixture and reacted at room temperature under a N2 atmosphere for 12 hours. The reaction was monitored by thin layer chromatography (TLC) to confirm that the coupling reaction was complete. After removing the solvent by rotary evaporation, the residue was purified by silica gel chromatography using a mobile phase mixture of 3 / 1 hexane / acetone. The structure of the product was as follows: 1The product of this first step was identified as (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-((tert-butoxycarbonyl)amino)acetate by H-NMR spectroscopy (Agilent Technologies 400 MHz NMR, Santa Clara, CA, USA) and mass spectrometry (TSQ Altis™ triple quadrupole mass spectrometer, Thermo Fisher Scientific, Waltham, MA, USA). 1 H-NMR(400MHz,CDCl3)δ5.37(d,J=4.4Hz,1H),4.99(s,1H),4.68(m,1H),3.87(d,J =5.3Hz,2H),2.33(d,J=7.8Hz,2H),2.01(m,2H),1.95(t,J=4.5Hz,1H),1.90-1.77 (m,3H),1.63-1.41(m,9H),1.45(s,9H),1.40-1.23(m,5H),1.20-1.03(m,8H),1.0 1(s,3H),0.91(d,J=6.6Hz,3H),0.86(dd,J=6.6,1.6Hz,6H),0.67(s,3H);ESI-MS:C 34 H 58 No. 4 + [M+H] + 543.9 m / z. Experiment 1-1-2: Synthetic route of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-aminoacetate ((8S,9S,1 Deprotection of (0R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-((tert-butoxycarbonyl)amino)acetate See formula (c), in the second step for the preparation of HACH, the Boc group was removed by trifluoroacetic acid (TFA) under acidic conditions to produce (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate. The detailed process in this second step includes the following: 500 mg (0.92 mmol) of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-((tert-butoxycarbonyl)amino)acetate was dissolved in 2 mL of DCM in an ice bath, and then 2 mL of trifluoroacetic acid (TFA) was added to the solution, which was stirred at room temperature under a N atmosphere for 3 h. The reaction was monitored by TLC to check for the completion of deprotection of the Boc group. After deprotection, the mixture was neutralized with saturated aqueous NaHCO3, and the precipitate was filtered and dried in vacuo to give a white powder of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate. The structure of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate is 1 Identification was carried out by 1 H-NMR spectroscopy (Agilent Technologies 400 MHz NMR) and mass spectrometry (TSQ Altis™ triple quadrupole mass spectrometer). 1 H-NMR (400MHz, CDCl3) δ5.37 (wide width s, 1H), 4.68 (m, 1H), 3.79 (wide width s, 2H), 2.33 (m, 2H), 2.01 (m, 2H), 1.95 (t, J=4.5Hz, 1H), 1.90-1.77 (m, 3H), 1 .63-1.41(m,7H),1.40-1.23(m,6H),1.20-1.03(m,7H),1.01(s,3H),0.91(d,J=6.5Hz,3H),0.86(d,J=6.5Hz,6H),0.67(s,3H);ESI-MS:C 29 H 50 NO2 + [M+H]+ 444.2 m / z. Experiment 1-1-3: Synthesis of HACH (Conjugation of HA with (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-aminoacetate) See formula (d), in the final step for the preparation of HACH, (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate was conjugated to the carboxylic acid group of HA by DIC / oxime activation to form HACH. In this step, HACH with a glycine linker was synthesized by grafting (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate onto HA in a DMSO / HO cosolvent system using DIC / Oxima as an amide coupling agent. After dialysis and lyophilization, a cotton-like HACH product was obtained. The detailed process for this final step includes the following: First, 500 mg (1.25 mmol, 1.0 equiv.) of hyaluronic acid (100 kDa) was dissolved in a mixture of 70 mL of water and 90 mL of DMSO. A mixture containing 250 mg (1.11 mmol) of oxymer and 113 mg (0.25 mmol, 0.2 equiv.) of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate was dissolved in 10 mL of DMSO, and the resulting solution was then added to the hyaluronic acid solution (HA solution). To the mixture, 405 μL (2.58 mmol) of DIC was slowly added and stirred for 24 hours. The resulting solution was transferred to a 3500-MWCO dialysis bag (e.g., a 3500 Da molecular weight cutoff dialysis bag) and purified by sequential dialysis against DMSO / water (50 / 50, v / v), 0.3 M NaCl aqueous solution, and purified water. Finally, the dialyzed product solution was freeze-dried to remove water, yielding HACH20. (The resulting HACH20 will have a molecular weight higher than the molecular weight cutoff (MWCO), i.e., higher than 3500 Da; note that the molecular weight of hyaluronic acid, consisting of 10 disaccharide units, is approximately 3800 Da.) HACH with various DS% was synthesized by adding the corresponding equivalent of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-aminoacetate; HACH 10 and HACH30 refers to 0.1 and 0.3 equivalents of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate, respectively, with respect to HA conjugation.The conjugation ratios (DS%) of HACH are shown in Table 1 and were determined by elemental analysis (Elementarvario EL cube, Langenselbold, Hesse, Germany).

[0167] See Figures 1A-1C. Figures 1A-1B show 1 (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-((tert-butoxycarbonyl)-2-methylpropional)- ... The structural identification results were obtained for (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate (Figure 1A) and (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate (Figure 1B); Figure 1C shows the structural identification results for (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate (Figure 1B). 1 1 shows the results of structural identification performed on HACH20 analyzed by H NMR spectroscopy (D2O / d6-DMSO, 400 MHz).

[0168] As shown in Figure 1 , the characteristic peaks of cholesterol (methyl groups at 0.67, 0.86, 0.91, and 1.01 ppm) and tert-butyl groups (peak h, 1.45 ppm, singlet, 9H) were detected in (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-((tert-butoxycarbonyl)amino)acetate (Figure 1 ). 1A), and after trifluoroacetic acid (TFA) incubation, the tert-butyl group of the Boc (tert-butyloxycarbonyl) peak at 1.45 ppm disappeared (Figure 1B), indicating the successful deprotection process to give (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate. The molecular weight of (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-aminoacetate was also confirmed by mass spectrometry.

[0169] The DS% of HACH was analyzed by elemental analyzer (EA) and calculated using the following formula (IV):

[0170]

number

[0171] In the formula, R y is the C / N ratio of HACH, and R0 and R 100represent the C / N ratios of unmodified HA and theoretically fully modified HACH (100%), respectively. Actual analytical methods will be known or apparent to those skilled in the art.

[0172] See Table 1. The theoretical carbon-to-nitrogen (C / N) ratios of HA alone or HACH with a 100% degree of substitution (DS) were 14 and 21.5, respectively. To determine the DS% of cholesterol in the HACH examples in this study, the inventors employed EA to examine the C / N ratios of free HA and HACH, and then calculated the DS ratio of HACH using formula (IV). As shown in Table 1, the actual C / N of HA measured by EA was 14.01, which was consistent with the theoretical C / N ratio of HA, demonstrating the reliability of EA analysis in determining the C / N ratio of HA or HA derivatives. The C / N of HACH5 analyzed by EA was 14.36, and the DS ratio was calculated to be 4.8%, close to the estimated DS ratio of 5%. HACH10 revealed a C / N ratio of 14.66 and a DS ratio of 8.8%, even though the DS ratio of HACH was estimated to be 10% due to the conjugation process. When the estimated DS ratio increased to 20%, 25%, and 30%, the measured C / N ratio increased to 14.97, 15.27, and 15.56, respectively, and the DS ratios were 12.9%, 16.9%, and 20.8%, respectively.

[0173] [Table 1]

[0174] Furthermore, the hydrophilic-lipophilic balance (HLB) value of the amphiphilic HA derivatives was calculated according to the Griffin method and expressed as the following formula (V): HLB HA誘導体 =20×(1-DS) See Table 2. As shown in Table 2, the HLB values ​​of the HACH examples in this experiment are in the range of 15 to 20.

[0175] [Table 2]

[0176] Experiment 1-1-4 Preparation of a composition containing oil@HACH particles In this experiment, various types of oils were used to prepare compositions containing oil@HACH particles. These oils were as follows: (1) Paraffin oil, a mineral oil. (2) Squalane, a saturated terpenoid consisting of three terpenes (triterpenes) and classified as a saturated triterpene. (3) Squalene is an unsaturated terpenoid consisting of three terpenes (triterpenes) and is classified as an unsaturated triterpene. (4) Ocimene is a terpenoid consisting of one terpene (monoterpene) and is classified as a monoterpene. (5) Farnesene is a terpenoid consisting of 1.5 terpenes (sesquiterpenes) and is classified as a sesquiterpene.

[0177] 100 mg of HACH20 (final concentration in the composition is 1% (w / v)) was dissolved in 9.5 mL of sodium citrate solution (pH = 6.5, 10 mM), and then 500 μL of oil (final concentration in the composition is 5% (v / v)) was added to the HACH solution. The resulting solution was premixed in a high-shear mixer for 10 minutes, and then homogenized using a high-pressure homogenizer. (A) Composition containing paraffin oil@HACH particles (paraffin oil@HACH emulsion); (B) composition containing squalane@HACH particles (squalane@HACH emulsion); (C) composition containing squalene@HACH particles (squalene@HACH emulsion); (D) a composition comprising ocimene@HACH particles (ocimene@HACH emulsion); and (E) Composition containing farnesene@HACH particles (farnesene@HACH emulsion) Various oil@HACH particle-containing compositions were obtained, including:

[0178] To evaluate the particle size and stability of the oil@HACH particles, aliquots of the oil@HACH particle-containing compositions were placed in 1.5-mL Eppendorf tubes and stored separately at 4°C to mimic typical vaccine storage conditions. The appearance and particle size of the compositions were recorded at predetermined time points. The particle size and polydispersity index (PDI) of HACH20 and squalane@HACH (SQ@HACH) in the aqueous solutions were measured by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS90, Malvern, UK). HACH20 and squalane@HACH (SQ@HACH) were stained with PTA negative stain, and their morphology was observed by transmission electron microscopy (TEM, JEOL JEM-1400 Electron Microscopy, Tokyo, Japan).

[0179] See Table 3. As shown in Table 3, among the various oil@HACH particles, paraffin oil@HACH particles were the largest, reaching approximately 1 μm after the first homogenization using a high-shear mixer, likely due to the fluidity or viscosity characteristics of paraffin oil (an example of mineral oil). In contrast, the other terpenoid oil@HACH particles were stable nanoscale particles suitable for injection. Furthermore, squalene@HACH particles were the smallest, reaching approximately 190 nm after the second homogenization using a high-pressure homogenizer. It is noted that particle size is highly correlated with immunostimulatory potency, suggesting that terpenoid oil@HA derivative particles may have superior immunostimulatory efficacy than mineral oil-based particles. Additionally, paraffin oil@HACH particles exhibited significant phase separation after 24 h of storage in a 4°C refrigerator; paraffin oil@HACH has been demonstrated to be unsuitable for constructing vaccines with antigens requiring cryogenic storage.

[0180] [Table 3]

[0181] See Figure 2. Figure 2 shows the visual appearance and TEM images of the HA derivatives and SQ@HA derivatives of the present invention.

[0182] The resulting HA derivative product was turbid and well dispersed in aqueous solution, as shown in Figure 2. In TEM images, we found that the HA derivative could self-associate to form particles that were approximately 200 nm.

[0183] To evaluate the potential of amphiphilic HA derivatives as emulsifiers for stabilizing oil / water interfaces, the HA derivatives were mixed with 5% squalene and then passed through a high-pressure homogenizer. As shown in Figure 2, after homogenization of the squalene / HA derivative / citrate buffer solution, an isotropic emulsion formulation (named squalane@HA derivative or SQ@HA derivative) was obtained. TEM images reveal that the SQ@HA derivative spherical particles consisted of several squalene droplets (bright core) surrounded by the amphiphilic HA derivative (dark shell). DLS results for the SQ@HA derivatives indicate a uniform size of approximately 190 ± 2 nm and a polydispersity of 0.136 ± 0.027. Experiment 1-2: Analysis of oil distribution in compositions containing oil@HACH particles The composition containing squalene@HACH particles was divided into two equal parts. The first part was mixed with an equal volume of ethyl acetate to dissolve the squalene in the first part (both the squalene inside the squalene@HACH particles and the squalene outside the squalene@HACH particles) in the ethyl acetate phase. A sample of the ethyl acetate phase was collected and analyzed by GC-MS to determine the total squalene content in the first part.

[0184] The second portion was centrifuged to obtain squalene@HACH particles. The resulting squalene@HACH particles were mixed with an equal volume of ethyl acetate to dissolve the squalene inside the squalene@HACH particles in the ethyl acetate phase. A sample of the ethyl acetate phase was collected and analyzed by GC-MS to determine the total squalene content inside the squalene@HACH particles obtained from the second portion.

[0185] The percentage of squalene content inside the squalene@HACH particles relative to the total squalene content in the entire composition was calculated: Squalene @ Percentage of squalene inside HACH particles = Total squalene content inside HACH particles ÷ Total squalene content in the composition The results of this experiment indicate that at least 95% of the squalene in the composition is encapsulated within the squalene@HACH particles, based on a total squalene content of 100% by weight in the composition.

[0186] Experiment 1-3: Conditions suitable for self-assembly of squalene@HACH particles In this experiment, the conditions suitable for the self-assembly of squalene@HACH particles were assessed and shown in the ternary diagram.

[0187] There were 28 groups in this experiment, namely, Groups 1 to 28. The preparation procedure for the composition in each group was almost the same as the experimental procedure for the composition containing squalene@HACH particles described in Experiment 1-1-4. The only differences were the weight of squalene, the weight of HACH, and the weight of the sodium citrate solution (aqueous buffer solution).

[0188] The squalene@HACH particles in the composition were detected and analyzed by dynamic light scattering, particle size analyzer, or transmission electron microscope. Furthermore, it should be noted that if self-assembly of the squalene@HACH particles occurs in the composition, the composition will be a milky mixture solution (a milky suspension). Conversely, if self-assembly of the squalene@HACH particles does not occur in the composition, oil and aqueous phase separation (oil / water phase separation) will occur in the composition. When the composition contains squalene@HACH particles, a shaded area in the ternary diagram will be created, indicating that the composition could be successfully prepared using the material ratio in the composition.

[0189] See Table 4. Table 4 shows the results of 28 compositions containing various amounts of squalene, HACH, and aqueous buffer solution in this experiment, indicating the ratio of squalene, HACH, and aqueous buffer solution suitable for self-assembly of squalene@HACH particles. The results indicate that squalene@HACH forms particles when the amount of squalene in the composition is less than 40% w / w (less than 40% w / w in the HACH aqueous buffer solution). Furthermore, the inventors of the present invention found that the solubility of HACH is approximately 3% w / w in aqueous buffer solution. It is noted that by forming squalene@HACH particles together, 80 weight units of squalene can be coated with 1 weight unit of HACH. For example, by forming squalene@HACH particles together, 40% w / w of squalene can be coated with 0.5% w / w of HACH.

[0190] [Table 4]

[0191] Experiment 1-4: Effect of oil type on antigen-presenting cell (APC) recruitment In this experiment, (A) Composition containing paraffin oil@HACH particles (paraffin oil@HACH emulsion); (B) composition containing squalane@HACH particles (squalane@HACH emulsion); (C) composition containing squalene@HACH particles (squalene@HACH emulsion); (D) a composition comprising ocimene@HACH particles (ocimene@HACH emulsion); and (E) Composition containing farnesene@HACH particles (farnesene@HACH emulsion) There were five compositions, including:

[0192] The procedure for preparing the composition in this experiment was the same as the experimental procedure for the composition containing oil@HACH particles described in Experiment 1-1-4.

[0193] Fifteen BALB / c mice were randomly assigned to five groups (three mice per group): paraffin oil@HACH group, squalane@HACH group, squalene@HACH group, ocimene@HACH group, and farnesene@HACH group.

[0194] 50 μL of a composition containing paraffin oil@HACH particles, a composition containing squalane@HACH particles, a composition containing squalene@HACH particles, a composition containing ocimene@HACH particles, or a composition containing farnesene@HACH particles was intramuscularly injected into the right quadriceps of mice in the paraffin oil@HACH group, the squalane@HACH group, the squalene@HACH group, the ocimene@HACH group, and the farnesene@HACH group, respectively; 50 μL of sodium citrate buffer (pH = 6.5) (vehicle control in this experiment) was intramuscularly injected into the left quadriceps of mice in these groups, and the effects induced by the injection in each mouse were monitored.

[0195] The mice in each group were sacrificed on the fifth day after injection.

[0196] The left and right quadriceps muscles of each mouse were harvested. The tendons were removed, and the muscles were digested with 3 mL of phosphate-buffered saline (PBS) containing 0.05% (w / v) type II collagenase, 10 μg / mL DNase I, and 0.5% BSA at 37°C for 40 minutes. Muscle digestion was stopped by adding excess medium; the cell suspension was then collected by centrifugation at 1200 rpm for 5 minutes, resuspended in PBS, and filtered through a 70-μm nylon mesh (BD Biosciences).

[0197] Cell suspensions obtained from the left and right quadriceps muscles of each mouse were mixed with medium containing titrated fluorescent conjugates of the monoclonal antibodies CD11b-FITC (BD Pharmingen) and CD11c-PE (Thermo Fisher Scientific), respectively. The cells were incubated in the dark at 4°C for 40 minutes. Finally, the stained cells were washed with staining buffer and analyzed using a BD Accuri™ C6 flow cytometer.

[0198] Dendritic cells (CD11c + ) The degree of mobilization was assessed according to the following formula (VI): Dendritic cells (CD11c + ) Degree of recruitment = total CD11c in the right quadriceps + Cells ÷ Total CD11c in left quadriceps + cell.

[0199] Macrophage cells (CD11b + ) The degree of mobilization was assessed according to the following formula (VII): Macrophage cells (CD11b + ) Degree of recruitment = total CD11b in the right quadriceps + Cells ÷ Total CD11b in left quadriceps + cell.

[0200] See Figures 3A and 3B. Figure 3A shows the effect of oil@HACH on dendritic cell recruitment. Figure 3B shows the effect of oil@HACH on macrophage recruitment. Note that the symbol "*" indicates that the group is significantly different from the squalene@HACH group.

[0201] As shown in Figure 3A, these compositions containing oil@HACH particles stimulated the proliferation of dendritic cells (CD11c + ), and the composition containing squalene@HACH particles recruited more dendritic cells (CD11c) than the compositions containing other oil@HACH particles. + As shown in Figure 3B, these compositions containing oil@HACH particles recruit macrophages (CD11b + ), and compositions containing squalene@HACH particles or farnesene@HACH particles were able to recruit more macrophages (CD11b) than compositions containing other oil@HACH particles. + ) will be mobilized.

[0202] These results suggested that oils classified as unsaturated terpenoids (such as squalene), terpenoids consisting of less than or equal to three terpene carbon skeletons (or less than or equal to six isoprene carbon skeletons, such as squalane, squalene, ocimene, and farnesene), and / or paraffin oils are suitable for preparing oil@HACH particles for use in stimulating an immune response. Preferably, oils classified as unsaturated terpenoids (such as squalene) and / or terpenoids consisting of less than three terpene carbon skeletons (or less than six isoprene carbon skeletons, such as ocimene and farnesene) are more suitable for preparing oil@HACH particles for use in stimulating an immune response.

[0203] Experiment 1-5: Effect of reactants in the oil@HACH synthesis process on antigen-presenting cell (APC) recruitment Twelve BALB / c mice were randomly assigned to four groups (three mice per group): cholesterol group, squalene group, hyaluronic acid group, and squalene@HACH group.

[0204] The experimental procedures were essentially the same as those described in Experiments 1-4, with the only difference being the composition administered to the mice.

[0205] Cholesterol group: 50 μL of N-methyl-2-pyrrolidone (NMP) solution containing cholesterol (0.25 mg / mL) (test sample in this group) was intramuscularly injected into the right quadriceps of the mice; 50 μL of N-methyl-2-pyrrolidone solution (vehicle control in this group) was intramuscularly injected into the left quadriceps of the mice, and the effects induced by the injection in each mouse were monitored.

[0206] Squalene group: 50 μL of N-methyl-2-pyrrolidone (NMP) solution (test sample in this group) containing squalene (50 μL / mL) was intramuscularly injected into the right quadriceps of the mice; 50 μL of N-methyl-2-pyrrolidone solution (vehicle control in this group) was intramuscularly injected into the left quadriceps of the mice, and the effects induced by the injection in each mouse were monitored.

[0207] Hyaluronic acid group: 50 μL of sodium citrate solution (test sample in this group) containing hyaluronic acid (8.73 mg / mL) and glycine (0.25 mg / mL) was intramuscularly injected into the right quadriceps of mice; 50 μL of sodium citrate buffer solution (pH=6.5) (vehicle control in this group) was intramuscularly injected into the left quadriceps of mice, and the effect induced by each injection was monitored.

[0208] Squalene@HACH group: 50 μL of a composition (test sample in this group) containing squalene@HACH particles (cholesterol content: 0.0589 mg; squalene content: 2.5 μL; hyaluronic acid content: 0.4325 mg) prepared by the method described in Experiment 1-1-4 was intramuscularly injected into the right quadriceps of the mice; 50 μL of sodium citrate buffer (pH=6.5) (vehicle control in this group) was intramuscularly injected into the left quadriceps of the mice in these groups, and the effects induced by the injection in each mouse were monitored.

[0209] The mice in each group were sacrificed on the fifth day after injection.

[0210] See Figures 4A and 4B. Figure 4A shows the effects of reactants and products in the synthesis of squalene@HACH on dendritic cell recruitment. Figure 4B shows the effects of reactants and products in the synthesis of squalene@HACH on macrophage recruitment. Note that the symbol "*" indicates that an animal in this group died during the experiment, and "#" indicates that the group had a significant difference compared to the other three groups.

[0211] As shown in Figures 4A and 4B, all of the reactants in the synthesis of squalene@HACH (cholesterol, squalene, and hyaluronic acid) lack the ability to recruit dendritic cells (CD11c+) and macrophages (CD11b+). Unexpectedly, squalene@HACH particles significantly increased the recruitment of dendritic cells (CD11c+) by more than 35-fold. + ) and 25-fold more than macrophages (CD11b + ) was mobilized.

[0212] As shown in Figure 4A , a comparison between the dendritic cell recruitment potency of the cholesterol group, squalene group, hyaluronic acid group, and squalene@HACH group (SQ@HACH group) demonstrated that cholesterol, squalene, and hyaluronic acid in squalene@HACH particles exhibited a synergistic effect in dendritic cell recruitment potency.

[0213] As shown in Figure 4B , a comparison between the macrophage recruitment potency of the cholesterol group, squalene group, hyaluronic acid group, and squalene@HACH group (SQ@HACH group) demonstrated that cholesterol, squalene, and hyaluronic acid in squalene@HACH particles exhibited a synergistic effect in macrophage recruitment potency.

[0214] Experiment 1-6: Effect of a mixture containing squalene@HACH and target antigen on antigen-presenting cell (APC) recruitment Thirty-six BALB / c mice were randomly assigned to three groups (12 mice per group): WT1 group, WT1 + squalene@HACH group, and WT1 + Addvax group. Twelve mice in each group were randomly assigned to four subgroups (3 mice per subgroup) for different treatment periods (1, 3, 5, or 7 days).

[0215] A composition containing squalene@HACH particles (squalene@HACH emulsion) was prepared by the method described in Experiment 1-1-4. The experimental procedure was almost the same as that described in Experiment 1-4. The only difference was the composition administered to the mice.

[0216] WT1 group: 50 μL of sodium citrate solution containing 25 μL of WT1 protein (test sample in this group) was intramuscularly injected into the right quadriceps of 12 mice; 50 μL of sodium citrate buffer (pH=6.5) (vehicle control in this group) was intramuscularly injected into the left quadriceps of 12 mice, and the effects induced by the injection in each mouse were monitored.

[0217] WT1 + squalene@HACH group: 50 μL of a composition containing 25 μL of WT1 protein and 25 μL of a composition containing squalene@HACH particles (squalene@HACH emulsion) (the test sample in this group) was intramuscularly injected into the right quadriceps of 12 mice; 50 μL of sodium citrate buffer (pH = 6.5) (the vehicle control in this group) was intramuscularly injected into the left quadriceps of 12 mice, and the effects induced by the injection in each mouse were monitored.

[0218] WT1+Addvax group: 50 μL of a composition (test sample in this group) containing 25 μL of WT1 protein and 25 μL of Addvax™ (MF59-like squalene adjuvant for vaccine research, purchased from Invivogen, San Diego, LA, USA) was intramuscularly injected into the right quadriceps of 12 mice; 50 μL of sodium citrate buffer (pH=6.5) (vehicle control in this group) was intramuscularly injected into the left quadriceps of 12 mice, and the effects induced by the injection in each mouse were monitored.

[0219] Mice in each group were sacrificed on days 1, 3, 5, or 7 after injection.

[0220] See Figures 5A and 5B. Figure 5A shows the effect of a mixture containing squalene@HACH and a target antigen on dendritic cell recruitment. Figure 5B shows the effect of a mixture containing squalene@HACH and a target antigen on macrophage recruitment.

[0221] As shown in Figures 5A and 5B, WT1 alone lacks the ability to recruit dendritic cells (CD11c+) and macrophages (CD11b+). Unexpectedly, the mixture containing WT1 and squalene@HACH (WT1+squalene@HACH group, also known herein as WT-1+SQ@HACH group) recruited over 35-fold more dendritic cells (CD11c+) and macrophages (CD11b+) on day 5 after injection. + ) and 25-fold more than macrophages (CD11b + ) was mobilized.

[0222] Therefore, the oil@HACH particles of the present invention can stimulate an immune response, such as recruiting dendritic cells and / or recruiting macrophages, and thus can be mixed with an active ingredient or target antigen (or antigens) to treat or prevent a disease or disorder. Furthermore, the oil@HACH particles of the present invention can stimulate an immune response and are therefore efficient adjuvants suitable for vaccine use (such as for cancer vaccine use or other disease or disorder vaccine use).

[0223] Experiment 1-7: Effect of a mixture containing squalene@HACH and target antigen on T cell immunity Twelve BALB / c mice were randomly assigned to four groups (three mice per group): PBS group, WT1 group, WT1 + IFN-alpha-based adjuvant group, and WT1 + squalene@HACH group.

[0224] Squalene@HACH particles were prepared by the method described in Experiment 1-1-4.

[0225] PBS group: 50 μL of phosphate buffered saline was injected intramuscularly into both the left and right quadriceps muscles of the mice on days 0 and 14.

[0226] WT1 group: 50 μL of sodium citrate solution containing 25 μL of WT1 protein was intramuscularly injected into both the left and right quadriceps of mice on days 0 and 14.

[0227] WT1 + IFN-alpha-based adjuvant group: 50 μL of sodium citrate solution containing 25 μL of WT1 protein and 25 μL of IFN-alpha-based adjuvant (supplied by Latham Medical Institution, Japan; the IFN-alpha-based adjuvant was used as a positive control) was injected intramuscularly into both the left and right quadriceps muscles of mice on days 0 and 14.

[0228] WT1 + squalene@HACH group: 50 μL of sodium citrate solution containing 25 μL of WT1 protein and 25 μL of a composition containing squalene@HACH particles (squalene@HACH emulsion) was intramuscularly injected into both the left and right quadriceps muscles of mice on days 0 and 14.

[0229] The mice in each group were sacrificed on the 28th day.

[0230] Spleens were harvested and temporarily pooled in sterile lymphocyte culture medium (LCM) to avoid cell death. Lymphocyte culture medium contained 900 mL of RPMI 1640, 100 mL of FBS, 25 mL of HEPES (25 mM), and 50 μL of β-mercaptoethanol (150 μM).

[0231] Splenocytes were isolated by the following procedure: the spleen was gently pressed onto a 70-μm cell strainer using the plunger seal of a 5-mL syringe into a 50-mL tube, and the cell strainer was rinsed with RPMI 1640 containing 10% (v / v) FBS. The wash buffer was then removed by centrifugation at 1200 rpm for 5 minutes at 4 °C. The cell pellet was resuspended in 5 mL of red blood cell lysis buffer (diluted from RBC lysis buffer (10x), BioLegend, lot: B166991) and incubated in an ice bath. 30 mL of chilled PBS was then added to stop the lysis, and the supernatant was removed by centrifugation at 1200 rpm for 5 minutes at 4 °C. Finally, the cell pellet was gently resuspended in LCM. The cell solution was counted and diluted to the desired cell concentration.

[0232] Cell suspensions obtained from the spleens of mice in each group were mixed with medium containing titrated fluorescent conjugates of monoclonal antibody CD8a-Alexa Fluor 647 (BD Pharmingen) and CD4-FITC (Thermo Fisher Scientific). The cells were incubated in the dark at 4°C for 40 minutes. Finally, the stained cells were washed with staining buffer and analyzed using a BD Accuri™ C6 flow cytometer.

[0233] The results were as follows: WT-1 + squalene @ HACH group (CD4 + T cells: 37.9%; CD8 + T cells: 33.0%, indicating that the highest amount of T cells was in the PBS group (CD4 + :29.2%;CD8 + :24.7%), WT-1 group (CD4 + :34.1%;CD8 + : 27.5%), and WT1 + IFN-alpha-based adjuvant group (CD4 + :33.0%;CD8 + These results indicate that the T cell immunity induced by squalene@HACH particles was higher than that induced by the common clinical adjuvant IFN-alpha.

[0234] Therefore, the oil@HACH particles can successfully stimulate an immune response, such as inducing T cell immunity, and thus can be mixed with an active ingredient or target antigen (or antigen, or antigen component) to treat or prevent a disease or disorder. Furthermore, the oil@HACH particles of the present invention can stimulate an immune response and are therefore an efficient adjuvant suitable for vaccine use (such as for cancer vaccine use or other disease or disorder vaccine use).

[0235] Experiment 1-8: Effect of a mixture containing squalene@HACH and target antigen on antigen-specific T cell responses Splenocytes obtained from the spleens of mice in the WT1 group, the WT1 + IFN-alpha-based adjuvant group, and the WT1 + squalene@HACH group described in Experiments 1-7 were used in the WT1 group, the WT1 + IFN-alpha-based adjuvant group, and the WT1 + squalene@HACH group in this experiment, respectively.

[0236] Splenocytes obtained from the spleens of mice in the same groups described in Experiments 1-7 were pooled together. 2 x 106 Splenocytes were suspended in 1 mL of LCM and added to a 24-well plate. Splenocytes in each group were restimulated with 10 μL of WT1 antigen at 37°C for 72 hours. Supernatants were collected for multiplex cytokine assays (IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α) using a cytometric bead array (CBA) mouse Th1 / Th2 / Th17 cytokine kit according to the manufacturer's instructions.

[0237] See Figure 6. Figure 6 shows the effect of a mixture containing squalene@HACH and a target antigen on T cell-associated cytokine production.

[0238] As shown in Figure 6, the highest amount of T cell-related cytokines (IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-alpha) was produced in the WT1 + squalene@HACH group (WT-1 + SQ@HACH group), which was higher than the amounts of T cell-related cytokines produced in the WT-1 group and the WT1 + IFN-alpha-based adjuvant group. These results indicate that the antigen-specific T cell immunity induced by squalene@HACH particles was higher than that induced by the common clinical adjuvant IFN-alpha.

[0239] These results indicate that oil@HACH particles successfully stimulate antigen-specific T cell immunity and induce the production of Th1 / Th2 / Th17-associated cytokines after antigen restimulation.

[0240] Therefore, the oil@HACH particles successfully stimulate immune responses, such as inducing antigen-specific T cell immunity and Th1 / Th2 / Th17-associated cytokine production. That is, the oil@HACH particles of the present invention can be mixed with active ingredients or target antigens (or antigens, or antigen components) to treat or prevent diseases or disorders. Furthermore, the oil@HACH particles of the present invention can stimulate immune responses and are therefore efficient adjuvants suitable for vaccine use (such as cancer vaccine use or other disease or disorder vaccine use).

[0241] Experiment 1-9 Antitumor activity of mixtures containing oil@HACH and target antigens Experiment 1-9-1 Ability of a mixture containing oil@HACH and a target antigen to treat and prevent breast cancer Eighteen BALB / c mice were randomly assigned to three groups (6 mice per group): PBS group, WT1 group, and WT1+squalene@HACH group.

[0242] A composition containing squalene@HACH particles was prepared by the method described in Experiment 1-1-4.

[0243] PBS group: 50 μL of phosphate buffered saline was injected intramuscularly into both the left and right quadriceps muscles of the mice on days −14 and 0.

[0244] WT1 group: 50 μL of sodium citrate solution containing 25 μL of WT1 protein was intramuscularly injected into both the left and right quadriceps of mice on days −14 and 0.

[0245] WT1 + squalene@HACH group: 50 μL of sodium citrate solution containing 25 μL of WT1 protein and 25 μL of a composition containing squalene@HACH particles was intramuscularly injected into both the left and right quadriceps muscles of mice on days −14 and 0.

[0246] On day 0, after the injection course described above was completed, 4T1 cells (2 × 10 6Mice in each group were subcutaneously injected with 0.05 mL of phosphate-buffered saline (PBS) containing 4T1 cells / mL (4T1 cells are a breast cancer cell line) into the abdominal mammary fat pad. On this day (day 0), the tumor volume and survival status of the mice were first monitored. Tumor volume was calculated by 1 / 2 * (4 * 3.14 / 3) * (length / 2) * (width / 2) * height * 1000, and mice were defined as either dead or paralyzed, or when the tumor volume of the mice reached 1000 mm. 3 When the tumor volume and survival status were exceeded, observations were discontinued.

[0247] See Table 5 and Figures 7A and 7B, which provide results from a mouse model study demonstrating the antitumor activity of WT1+squalene@HACH.

[0248] As shown in Table 5, all six mice in the PBS group developed tumors (tumors first appeared between days 18 and 32); similarly, all six mice in the WT1 group developed tumors (tumors first appeared between days 18 and 25). Unexpectedly, a significant delay in tumor development was observed for four mice in the WT1 + squalene@HACH group (tumors first appeared between days 25 and 46); more surprisingly, no tumors were developed in the remaining two mice in this WT1 + squalene@HACH group (WT1 + SQ@HACH group). These results demonstrated that the oil@HACH particles of the present invention can successfully prevent tumor formation and tumor development.

[0249] Furthermore, as shown in Table 5, all six mice in the PBS group died on day 53; similarly, all six mice in the WT group died on day 53. Unexpectedly, five mice in the WT1 + squalene@HACH group were still alive on day 53 (only one mouse in this WT1 + squalene@HACH group died on day 53, but this mouse died from "attack by other mice" rather than "tumor"). These results demonstrated that the oil@HACH particles of the present invention can successfully extend the lifespan of subjects with cancer or tumor diseases.

[0250] [Table 5]

[0251] Therefore, the oil@HACH particles mixed with an active ingredient or target antigen (or antigens) can stimulate an immune response to successfully prevent or treat a disease or disorder, such as cancer or tumor. Furthermore, the oil@HACH particles of the present invention can stimulate an immune response and are therefore efficient adjuvants suitable for vaccine use (such as for cancer vaccine use or other disease or disorder vaccine use). Experiment 1-9-2 Ability of a mixture containing oil@HACH and an antigen to treat and prevent lymphoma Nine BALB / c mice were randomly assigned to three groups (three mice per group): PBS group, OVA group, and OVA+squalene@HACH group.

[0252] A composition containing squalene@HACH particles was prepared by the method described in Experiment 1-1-4.

[0253] PBS group: 50 μL of phosphate buffered saline was injected intramuscularly into both the left and right quadriceps muscles of the mice on days −14 and 0.

[0254] OVA group: 50 μL of sodium citrate solution containing 25 μg of OVA protein (ovalbumin, a well-known antigen commonly used as a model antigen in vaccine formulations) was injected intramuscularly into both the left and right quadriceps muscles of mice on days −14 and 0.

[0255] WT1 + squalene@HACH group: 50 μL of sodium citrate solution containing 20 μg of OVA protein and 25 μL of a composition containing squalene@HACH particles was intramuscularly injected into both the left and right quadriceps muscles of mice on days −14 and 0.

[0256] On day 0, after the injection course described above was completed, E.G7-OVA cells (3 × 10 5 Mice in each group were subcutaneously injected with 0.1 mL of phosphate-buffered saline (PBS) containing 1000 cells / mL (e.g., G7-OVA cells are a lymphoma cell line) into the abdominal mammary fat pad. On this day (day 0), the tumor volume and survival status of the mice were first monitored. Tumor volume was calculated by 1 / 2 * (4 * 3.14 / 3) * (length / 2) * (width / 2) * height * 1000. Mice were defined as either dead or paralyzed, or their tumor volume reached 1500 mm. 3 When the tumor volume and survival status were exceeded, observations were discontinued.

[0257] The inventors of the present invention have found that the oil@HACH particles of the present invention can be successful in preventing tumor formation and preventing tumor development, as well as in extending the lifespan of subjects with cancer or tumor diseases.

[0258] Experiments 1-10 Ability of mixtures containing oil@HACH and target antigens to treat and prevent infectious diseases Nine BALB / c mice were randomly assigned to three groups (three mice per group): PBS group, split-H7N9 group, and split-H7N9+squalene@HACH group.

[0259] A composition containing squalene@HACH particles was prepared by the method described in Experiment 1-1-4.

[0260] PBS group: 50 μL of phosphate buffered saline was injected intramuscularly into both the left and right quadriceps muscles of mice on days −28 and −14.

[0261] Split-H7N9 group: 50 μL of sodium citrate solution containing 0.5 μg of split-H7N9 protein (influenza H7N9 vaccine antigen, purchased from ADIMMUNE Corporation, Taiwan, China) was injected intramuscularly into both the left and right quadriceps of mice on days −28 and −14.

[0262] Split-H7N9 + squalene@HACH group: 50 μL of sodium citrate solution containing 0.5 μg of split-H7N9 protein and 25 μL of a composition containing squalene@HACH particles was intramuscularly injected into both the left and right quadriceps muscles of mice on days −28 and −14.

[0263] On day 0, serum samples from immunized mice in each group were collected by drawing blood from the submandibular glands at predetermined times, which were centrifuged at 7500 rpm for 15 minutes.

[0264] The hemagglutination inhibition (HI) test for influenza virus was used in this experiment to quantify the relative concentration of influenza virus. The actual analytical method will be known or obvious to those skilled in the art (De et al., 2003). In the hemagglutination inhibition (HI) test described here, serial dilutions of serum from immunized mice are incubated with virus (influenza H7N9 vaccine antigen, provided by ADIMMUNE Corporation, Taiwan, China), and red blood cells are added. After incubation, the HI titer is read as the highest dilution of serum that inhibits hemagglutination.

[0265] Preliminary data from this experiment suggest that a composition containing split-H7N9 and squalene@HACH particles can induce a geometric mean titer (GMT) of 127 at week 2, which means that the oil@HACH particles of the present invention can induce neutralizing antibodies sufficient to prevent H7N9 virus infection.

[0266] Thus, the oil@HACH particles of the present invention, mixed with an active ingredient or target antigen (or antigens), can be used to stimulate an immune response to prevent or treat a disease or disorder, such as an infectious disease (viral, fungal, protozoal, or bacterial infection). Furthermore, the oil@HACH particles of the present invention can stimulate an immune response and are therefore suitable and efficient adjuvants for vaccine use (such as for cancer vaccine use, infectious disease vaccine use, or other disease or disorder vaccine use).

[0267] Experiment 2: Composition containing oil@HAODA particles Experiment 2-1 Preparation of a composition containing oil@HAODA particles In this embodiment, a hyaluronic acid-octadecylamine conjugate (HAODA), which is a polymer of the present invention, was used.

[0268] Scheme (e) provides a synthetic route to the hyaluronic acid-octadecylamine conjugate (HAODA) of the present invention.

[0269] [ka]

[0270] where n, x, and y each independently represent integer values, n=x+y, x≧1, y≧x, and x+y≧3, e.g., x+y≧10.

[0271] Experiment 2-1-1 Synthetic route of HAODA (conjugation of HA with octadecylamine) See formula (e), in this step for the preparation of HAODA, octadecylamine was conjugated to the carboxylic acid group of HA by DIC / oxymer activation to form HAODA. The detailed process for this final step includes: First, 200 mg (0.5 mmol, 1.0 equiv.) of hyaluronic acid (36 kDa or 360 kDa) was dissolved in a mixture of 20 mL of water and 80 mL of acetone. A mixture containing 50 mg (0.55 mmol, 1.1 equiv.) of oxymer and 20 mg (0.15 equiv.) of octadecylamine was dissolved in 32 mL of acetone, and the resulting solution was then added to the hyaluronic acid solution (HA solution). 162 μL (1.0 mmol, 2.0 equiv.) of DIC was slowly added to the mixture and stirred for 24 hours. The resulting solution was transferred to a 3500-MWCO dialysis bag and purified by sequential dialysis against acetone / water (50 / 50, v / v), 0.3 M NaCl aqueous solution, and pure water. Finally, water was removed from the dialyzed product solution by freeze-drying to obtain HAODA15. HAODA with various degrees of substitution (DS) were synthesized by adding the corresponding equivalent amount of octadecylamine; HAODA5, HAODA15, and HAODA25 represent 0.05, 0.15, and 0.25 equivalents of octadecylamine, respectively, in terms of HA conjugation. The conjugation ratios (DS%) of HAODA are shown in Table 6. 1 Determined by H-NMR spectroscopy (Agilent Technologies 400 MHz NMR).

[0272] HAODA's DS, 1 The OH-NMR spectroscopy was analyzed and calculated using the following formula (VIII):

[0273]

number

[0274] In the formula, A ODA and A HA represent the integrated area of ​​the terminal methyl group of octadecylamine (δ 0.8-0.9, spread s, 3H) and the integrated area of ​​the N-acetyl group of glucosamine in HA (δ 1.8-2.2, spread s, 3H), respectively. The actual analytical methods will be known or apparent to those skilled in the art.

[0275] See Table 6. The DS% of HAODA with various molecular weights (MW) and their estimated DS ratios are listed. The DS% of 36k-HAODA5, 36k-HAODA15, and 36k-HAODA25 were 5.21%, 15.61%, and 23.36%, respectively. The DS% of 360k-HAODA5, 360k-HAODA15, and 360k-HAODA25 (HAODAs with higher MW) were 5.01, 15.42, and 26.52, respectively. These DS ratios were close to the corresponding estimated DS ratios.

[0276] [Table 6]

[0277] Furthermore, the hydrophilic-lipophilic balance values ​​(HLB values) of the amphiphilic HA derivatives were calculated according to the Griffin method and expressed as formula (V) described above: See Table 7. As shown in Table 7, the HLB value of HAODA in this experiment is in the range of 14 to 20.

[0278] [Table 7]

[0279] Experiment 2-1-2 Preparation of a composition containing oil@HAODA particles In this experiment, various types of oils were used to prepare compositions containing oil@HAODA particles. These oils were as follows: (1) Paraffin oil, a mineral oil. (2) Squalane, a saturated terpenoid consisting of three terpenes (triterpenes) and classified as a saturated triterpene. (3) Squalene is an unsaturated terpenoid consisting of three terpenes (triterpenes) and is classified as an unsaturated triterpene. (4) Ocimene is a terpenoid consisting of one terpene (monoterpene) and is classified as a monoterpene. (5) Farnesene is a terpenoid consisting of 1.5 terpenes (sesquiterpenes) and is classified as a sesquiterpene.

[0280] 100 mg of HAODA (final concentration in the composition is 1% (w / v)) was dissolved in 9.5 mL of sodium citrate solution (pH = 6.5, 10 mM), and then 500 μL of oil (final concentration in the composition is 5% (v / v)) was added to the HAODA solution. The resulting solution was premixed in a high-shear mixer for 10 minutes and then homogenized with a high-pressure homogenizer to obtain compositions containing various oil@HACH particles, including: (A) Composition containing paraffin oil@HAODA particles (paraffin oil@HAODA emulsion); (B) composition containing squalane@HAODA particles (squalane@HAODA emulsion); (C) composition containing squalene@HAODA particles (squalene@HAODA emulsion); (D) a composition containing ocimene@HAODA particles (ocimene@HAODA emulsion); and (E) Composition containing farnesene@HAODA particles (farnesene@HAODA emulsion).

[0281] To evaluate the particle size and stability of the oil@HAODA particles, aliquots of the oil@HAODA particle-containing compositions were placed in 1.5-mL Eppendorf tubes and stored separately at 4°C and 37°C. The appearance and particle size of the compositions were recorded at predetermined time points. The particle size and polydispersity index (PDI) of HAODA and oil@HAODA in the aqueous solutions were measured by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS90, Malvern, UK). HAODA and oil@HAODA were stained with PTA negative stain, and their morphology was observed by transmission electron microscopy (TEM, JEOL JEM-1400 Electron Microscopy, Tokyo, Japan).

[0282] Among the various oil@HAODA particles, the paraffin oil@HAODA particles were larger, likely due to the fluidity or viscosity characteristics of paraffin oil (an example of mineral oil). In contrast, the other terpenoid oil@HAODA particles were stable nanoscale particles suitable for injection. Furthermore, particle size was highly correlated with immune stimulatory potency, suggesting that terpenoid oil@HA derivative particles may have superior immune stimulatory potency than mineral oil particles. Experiment 2-1-3 Effect of reactants in the synthesis of squalene@HAODA on antigen-presenting cell (APC) mobilization Nine BALB / c mice were randomly assigned to three groups (three mice per group): hyaluronic acid group, ODA group, and squalene@HAODA group.

[0283] The experimental procedures were essentially the same as those described in Experiments 1-5, with the only difference being the composition administered to the mice.

[0284] Hyaluronic acid group: 50 μL of sodium citrate solution (test sample in this group) containing hyaluronic acid (8.73 mg / mL) was intramuscularly injected into the right quadriceps of mice; 50 μL of sodium citrate buffer solution (pH=6.5) (vehicle control in this group) was intramuscularly injected into the left quadriceps of mice, and the effect induced by each injection was monitored.

[0285] ODA group: 50 μL of the mixture solution (test sample in this group) containing 0.036 mg of octadecane-1-amine (octadecylamine) dissolved in 25 μL of N-methyl-2-pyrrolidone (NMP) and 25 μL of PBS buffer solution was intramuscularly injected into the right quadriceps of the mice; 50 μL of the mixture solution (vehicle control in this group) containing 25 μL of N-methyl-2-pyrrolidone (NMP) and 25 μL of PBS buffer solution was intramuscularly injected into the left quadriceps of the mice, and the effects induced by the injection of each mouse were monitored.

[0286] Squalene@HAODA group: 50 μL of a composition (test sample in this group) containing squalene@HAODA particles (octadecane-1-amine content: 0.036 mg; squalene content: 2.5 μL; hyaluronic acid content: 0.4325 mg) prepared by the method described in Experiment 2-1-2 was intramuscularly injected into the right quadriceps of the mice; 50 μL of sodium citrate buffer (pH = 6.5) (vehicle control in this group) was intramuscularly injected into the left quadriceps of the mice in these groups, and the effects induced by the injection in each mouse were monitored.

[0287] The mice in each group were sacrificed on the fifth day after injection.

[0288] See Figures 8A and 8B. Figure 8A shows the effects of reactants and products in the synthesis of squalene@HAODA on dendritic cell recruitment. Figure 8B shows the effects of reactants and products in the synthesis of squalene@HAODA on macrophage recruitment. Note that the symbol "#" indicates that the group is significantly different from the other three groups.

[0289] As shown in Figures 8A and 8B, all of the reactants and products (ODA and hyaluronic acid) in the squalene@HAODA synthesis process lack the ability to recruit dendritic cells (CD11c+) and macrophages (CD11b+). Unexpectedly, squalene@HAODA particles recruited over 30-fold fewer dendritic cells (CD11c+) and macrophages (CD11b+). + ) and 15-fold more than macrophages (CD11b + ) was mobilized.

[0290] As shown in Figure 8A , a comparison between the dendritic cell recruitment potency of the ODA group, hyaluronic acid group, and squalene@HAODA group demonstrated that octadecane-1-amine and hyaluronic acid in squalene@HAODA particles exhibited a synergistic effect in dendritic cell recruitment potency.

[0291] As shown in Figure 8B , a comparison between the macrophage recruitment potency of the ODA group, the hyaluronic acid group, and the squalene@HAODA group demonstrated that octadecane-1-amine and hyaluronic acid in the squalene@HAODA particles exhibited a synergistic effect in macrophage recruitment potency.

[0292] The above description is merely a preferred embodiment of the present invention, and is not intended to limit the scope of the patent application of the present invention. Therefore, any changes or modifications that do not deviate from the spirit disclosed herein should be included in the scope of the patent application of the present invention.

[0293] All publications mentioned herein are hereby incorporated by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies disclosed herein, which may be used in connection with the presently described invention. The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the inventors described herein are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.

[0294] References: 1. De Jong, JC, Palache, AM, Beyer, WE, Rimmelzwaan, GF, Boon, AC, & Osterhaus, AD (2003). Haemagglutination-inhibiting antibody to influenza virus. Developments in biologicals, 115, 63-73.

Claims

1. 1. A composition comprising an oil and a polymer; the oil and polymer together form a plurality of particles; and the polymer at least one first unit, the first unit being a first disaccharide; and at least one second unit, the second unit being a conjugation of a second disaccharide with a hydrophobic compound; Including, The composition, wherein the at least one first unit and the at least one second unit are linked to each other to form the polymer.

2. the oil is a terpenoid; or the first disaccharide is composed of D-glucuronic acid and N-acetylglucosamine; or the second disaccharide is composed of D-glucuronic acid and N-acetylglucosamine; and in the second unit, the hydrophobic compound is attached to carbon 6 of the D-glucuronic acid. The composition of claim 1.

3. the D-glucuronic acid and N-acetylglucosamine in the first disaccharide are linked by a β-(1,3) bond; or the N-acetylglucosamine and the D-glucuronic acid in the second disaccharide are linked by a β-(1,3) bond; or the at least first unit and the at least second unit are linked to each other by a β-(1,4) bond to form the polymer; The composition of claim 2.

4. The polymer has the formula (I): 【Chemical 1】 (wherein the A group is R—NH— or R—X—NH—, where the R group is a hydrophobic group derived from the hydrophobic compound, and X is a heteroatom (such as O, S, or N), a carbonyl group, or —O—CO—C １～５ alkylene-, wherein the alkylene is optionally substituted with one or more substituents selected from the group consisting of alkyl, aromatic, antiaromatic, cycloalkyl, acetyl, amino, hydroxyl, and thiol groups, or an alkane or alkene group whose main carbon chain has from 1 to 10 carbon atoms, and such alkane or alkene group is optionally substituted with one or more substituents selected from the group consisting of alkyl, aromatic, antiaromatic, cyclic groups, carbonyl, acetyl, keto, amino, hydroxyl, and heteroatoms (such as O, S, or N); or the -X-NH- group may be represented as an amino acid whose main chain has from 2 to 6 carbon atoms, such as glycine, alanine, valine, leucine, or isoleucine; and x and y are each independently integer values, where x≧1, y≧x, and x+y≧3. or a compound of The particles are detectable by a particle size analyzer, a transmission electron microscope, or a scanning electron microscope. The composition according to any one of claims 1 to 3.

5. an aqueous composition or an oil-in-water composition; or the polymer is an amphiphilic compound, and the hydrophilic-lipophilic balance value (HLB value) of the polymer is greater than 7; The composition according to any one of claims 1 to 4.

6. 6. The composition of any one of claims 1 to 5, wherein the hydrophobic compound is selected from the group consisting of cholesterol derivatives, octadecylamine, octadecylamine derivatives, steroids, steroid derivatives, saturated or unsaturated long-chain fatty amines, and saturated or unsaturated long-chain fatty amine derivatives, or any combination thereof.

7. the terpenoid is a liquid or solid at room temperature; or The terpenoid is a cyclic or acyclic compound; or the terpenoid consists of 1 to 20 sesquiterpenes; or The terpenoid is an unsaturated terpenoid. The composition according to any one of claims 2 to 6.

8. The composition of any one of claims 1 to 7, wherein the oil is selected from the group consisting of squalene, squalane, ocimene, farnesene, and paraffin oil, or any combination thereof.

9. the polymer has an average molecular weight of 10 kDa or more and 1000 kDa or less; or In the composition, on average, at least 70% by molar ratio of the units per polymer are the first units. The composition according to any one of claims 1 to 8.

10. In the composition, the weight ratio of the oil to the polymer ranges from 1:100 to 80:1; or the weight percentages of the oil and polymer are equal to or greater than 0.01% to 40% and equal to or greater than 0.001% to 3%, respectively, based on the total weight of the composition; or At least 50% (w / w) of the oil in the composition is encapsulated within the particles, based on 100% by weight of the total oil content in the composition; or The composition contains at least 1% (v / v) oil based on the total volume of the composition; The composition according to any one of claims 1 to 9.

11. The composition of any one of claims 1 to 10, wherein the polymer is prepared by a method comprising mixing 0.0001 to 2 equivalents of the hydrophobic compound with 1 equivalent of hyaluronic acid; or wherein in the composition, the molar ratio of the first unit and the second unit per polymer is, on average, in the range of 99:1 to 1:

1.

12. A pharmaceutical composition comprising the composition of any one of claims 1 to 11.

13. Use of the composition or pharmaceutical composition according to any one of claims 1 to 12 in stimulating an immune response in a subject.

14. Use of a composition or pharmaceutical composition according to any one of claims 1 to 12 in the treatment or prevention of a disease or disorder.

15. A method of stimulating an immune response in a subject, comprising administering to the subject a composition or pharmaceutical composition according to any one of claims 1 to 12.

16. A method for treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject the composition or pharmaceutical composition of any one of claims 1 to 12.

17. 17. The use or method of claim 14 or 16, wherein the disease or disorder is a tumor or cancer.

18. 18. The use or method of claim 17, wherein the tumor or cancer is breast cancer or other solid tumor.

19. 17. The use or method of claim 14 or 16, wherein the disease or disorder is a pathogenic disease, an HIV or other viral infection, a fungal infection, a protozoal infection, or a bacterial infection.

20. The use or method of any one of claims 13 to 19, wherein the pharmaceutical composition is a vaccine; or the subject is a human.

Images