WO2018160026A1 - Vésicule à domaines multiples comprenant un matériau immunoactif, procédé de production associé et composition immunomodulatrice la comprenant - Google Patents

Vésicule à domaines multiples comprenant un matériau immunoactif, procédé de production associé et composition immunomodulatrice la comprenant Download PDF

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WO2018160026A1
WO2018160026A1 PCT/KR2018/002516 KR2018002516W WO2018160026A1 WO 2018160026 A1 WO2018160026 A1 WO 2018160026A1 KR 2018002516 W KR2018002516 W KR 2018002516W WO 2018160026 A1 WO2018160026 A1 WO 2018160026A1
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capsule
multidomain
substance
oil
present
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PCT/KR2018/002516
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English (en)
Korean (ko)
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임용택
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단디바이오사이언스 주식회사
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Priority claimed from KR1020180024900A external-priority patent/KR102069670B1/ko
Application filed by 단디바이오사이언스 주식회사 filed Critical 단디바이오사이언스 주식회사
Priority to AU2018229137A priority Critical patent/AU2018229137A1/en
Priority to US16/489,781 priority patent/US20190380960A1/en
Priority to RU2019130877A priority patent/RU2736639C1/ru
Priority to CN201880029326.3A priority patent/CN110582275A/zh
Priority to CA3055067A priority patent/CA3055067A1/fr
Priority to JP2019547392A priority patent/JP2020510663A/ja
Priority to EP18761258.5A priority patent/EP3590508A4/fr
Publication of WO2018160026A1 publication Critical patent/WO2018160026A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals

Definitions

  • the present invention relates to a multi-domain capsule containing an immunoactive substance, a method for preparing the multi-domain capsule, and an immunomodulatory composition comprising the multi-domain capsule.
  • various liposomes and emulsion materials e.g., ASO1, ASO2, AS15 and Novartis MF59
  • immunoactivating substances for activating immune cells have been immunoactivated to prevent or treat various infectious diseases and cancers. It is used as a substance.
  • the single liposome-based materials are infectious disease prevention vaccine compositions, but are currently in clinical trials, but due to the low duration of antigens and immunoactivating substances, these substances may be used for a period of time in order to effectively activate immune cells in vivo. There was a disadvantage that additional injection three times.
  • the Irvive Darrel group of MIT recently developed an immunoactivated cancer vaccine having a multilamellar liposome structure (Nature Materials, 10, 243-251, 2011).
  • the cancer vaccine is loaded with an antigen and an immunoactivating material in a liposome having a multi-lamellar structure, and then each lipid layer is made of a crosslinking structure using polyvalent metal ions or a chemical linker, thereby forming a single liposome material.
  • a drug carrier which is conventionally called multivesicular liposome
  • multivesicular liposome a drug carrier, which is conventionally called multivesicular liposome
  • Kim Shin-il and colleagues of the University of California, USA [Biochimica Biophysica Acta 1983 Mar. 9 728 (3) 339-348, Mantripragada's team in 2002 [Progress of Lipids Research 41 (2002) 392-406], and Wafa's team in 2007 [International Journal of Pharmaceutics 331 (2007) 182-185].
  • These multiple liposomes are composed of a mixture of substances selected from the group consisting of neutral lipids and cholesterol and triolein.
  • the multi-liposomes prepared in this way have a problem in that the structure stabilization efficiency due to triolein is very low, and microclusters are collapsed during preparation (for example, centrifugation and temperature change), resulting in uneven size or shape. .
  • multiple liposome forms into which immunoactivating drugs have been introduced have not been found to date.
  • the immunostimulation (immunostimulation) technology the important thing in the regulation of immune function is the development of a technology that can control the immunosuppression (immunosuppression) in the body.
  • Anti-cancer immunotherapy methods for treating cancer using the body's immune system have the advantage of minimizing side effects compared to conventional chemotherapy or radiation therapy.
  • these anti-cancer immunotherapy methods include cell therapy methods that inject T cells (including CAR-T), dendritic cells, natural killer cells, etc., which are therapeutic immune cells, and inject them directly into the body after activation in vitro.
  • anticancer vaccines for increasing anticancer efficacy by directly activating immune cells present in the body by injecting cancer antigens and immune activating substances into the body, and the like.
  • these cell therapies or anticancer vaccines are mainly used for blood cancer-related diseases, and in solid cancers, most of them have the disadvantage that their therapeutic efficacy is very low.
  • the present invention provides a multi-domain capsule containing an immunoactive substance, a method for preparing the multi-domain capsule, and an immunomodulatory composition comprising the multi-domain capsule.
  • a multi-domain capsule comprising two or more liposomes in contact with and connected to each other, and a multi-domain capsule outer wall surrounding the two or more liposomes, wherein the multi-domain capsule is composed of an organic phase and an aqueous phase.
  • the organic phase comprises a first immune modulator and a fluid oil
  • the organic phase forms a membrane of the liposome, and the outer wall of the multidomain capsule
  • the aqueous phase comprises a second immune modulator
  • the aqueous phase is the An inner aqueous solution phase of the liposome membrane and an outer aqueous solution phase of the liposome membrane
  • the first immunomodulatory substance is a fat-soluble immunoactive substance
  • the second immunomodulatory substance is a water-soluble immunoactive substance
  • the fluid oil is in contact with and connected to each other.
  • an immunomodulatory substance comprising the multidomain capsule and an antigen.
  • the step of dissolving the first immune modulator and the fluid oil in a solvent to prepare an oil phase solution Dispersing a first aqueous phase comprising a second immunomodulatory substance in the oil phase solution to produce a water-in-water (W / O) emulsion; And mixing the oil-in-water emulsion with a second aqueous solution and evaporating the solvent.
  • the first immunomodulatory substance is a fat-soluble immunoactive substance
  • the second immunomodulatory substance can be provided, characterized in that the method for producing a multi-domain capsule.
  • the present invention provides an immunomodulatory multiple-modal capsule having a micro-sized capsule form with improved structural stability of a plurality of liposomes linked to each other while forming respective domains based on an immunomodulator, and having a flowable oil component introduced therein. Domain capsules can be provided.
  • the immunomodulatory composition according to the present invention has the advantage of overcoming the disadvantages of low encapsulation efficiency and short effective duration of a single liposome material that is used as various pharmaceutical compositions and increasing the effective duration of the immunomodulatory effect. .
  • the method for preparing a multi-domain capsule according to the present invention by introducing a fluid oil such as squalene, instead of the triolein that was conventionally introduced to maintain the structural stability of the multi-liposomes, stability and The storage stability can be improved, and the introduction of the flowable oil facilitates solubilization of representative poorly soluble immunomodulatory substances that are insoluble in common organic solvents, and thus include multiple domains containing the various poorly soluble immunomodulatory substances.
  • a fluid oil such as squalene
  • the multi-domain capsule according to the present invention by tuning the surface charge of the multi-domain capsule, can increase the loading efficiency and effective duration of the antigen and immunomodulatory substance having the opposite charge characteristics, and includes cationic lipids
  • the multi-domain capsule by tuning the surface charge of the multi-domain capsule, can increase the loading efficiency and effective duration of the antigen and immunomodulatory substance having the opposite charge characteristics, and includes cationic lipids
  • a variety of anionic and / or negatively charged immunomodulators and biomaterials such as DNA, RNA can be effectively loaded into the multi-domain capsule.
  • the antigens and / or immunomodulators loaded on the outer wall and the inside of the multidomain capsule are released, thereby increasing the effective duration of the antigens and the immunomodulators. That has the advantage.
  • the multi-domain capsule according to the present invention by loading various immunomodulators having lipophilic properties on the membrane of the liposome and / or the outer wall of the multi-domain capsule, it is possible to increase the effective duration of the immunomodulators, By loading various immunomodulators with hydrophilic properties inside the liposomes, the effective duration of the immunomodulators can be increased, and various immunomodulators with hydrophilic properties inside the liposomes, membranes of liposomes and / or the capsules. By simultaneously loading a lipophilic immunomodulatory substance on the outer wall of the can, the effective duration of the immunomodulatory substance can be increased.
  • the multi-domain capsule according to the present invention may be a surfactant is coated on the outside of the multi-domain capsule so that the multi-domain capsule can be stably dispersed in the aqueous solution.
  • FIG. 1 is a schematic diagram showing the structure of an immunomodulatory multidomain capsule (imMDV) in one embodiment of the present invention.
  • 3 (a) to 3 (c) are optical microscopic images of multidomain capsules containing squalene in one embodiment of the present invention, and (d) to (f) are one embodiment of the present invention.
  • it is an optical microscope image of multidomain capsules without squalene (scale bar: 4 ⁇ m).
  • Figure 4 (a) to (d), in an embodiment of the present invention as a result of the stability analysis of the multi-domain capsule, before centrifugation (a) and after the centrifugation of the multi-domain capsule containing squalene (c) Microscopy images of) and before (b) and after centrifugation (d) of multidomain capsules that do not contain squalene.
  • FIG. 5 is an optical microscope image of multidomain capsules (imMDV (MPLA)) comprising squalene-based MPLA in one embodiment of the present invention.
  • imMDV multidomain capsules
  • Figure 6 shows the expression levels of cytokines secreted when imMDV (SQ) is treated with BMDC in one embodiment of the present invention (a: TNF-alpha, b: IL-6).
  • Figure 7 shows the expression levels of cytokines secreted when imMDV (MPLA) is treated with BMDC in one embodiment of the present invention (a: TNF-alpha, b: IL-6, c: IL-12p70) ).
  • FIG. 8 is a graph showing the release behavior of OVA according to whether squalene is included in a multi-domain capsule loaded with protein antigen (OVA, ovalbumin) according to one embodiment of the present invention.
  • Figure 9 in one embodiment of the present invention, it shows a multi-domain capsule for immune function regulation loaded with imiquimod (acid and base structure), an immunoactivating material (a: imMDV (R837-HCl) sample, b : imMDV (R837-base) sample, c: imMDV [R837-HCl: R837-base (1: 1) sample].
  • Figure 10 in one embodiment of the present invention, shows the release behavior of R837 over time in the immunomodulatory multi-domain capsule (imMDV (R837-HCl) loaded with imiquimod.
  • imMDV immunomodulatory multi-domain capsule
  • FIG. 11 illustrates the expression level of IL-6 cytokines secreted when imiquimod-loaded multidomain capsules (imMDV (R837-HCl)) are treated at different concentrations in BMDC. Indicates.
  • FIG. 12A is a graph showing humoral immune effects (IgG, 1 week after injection) against an OVA (ovalbumin) cancer antigen in a multidomain capsule loaded with imiquimod according to one embodiment of the present invention (FIG. imMDV (R837-HCl) sample / 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV sample).
  • FIG. 12B is a graph showing humoral immune effects (IgG, 1 week after injection) against an OVA (ovalbumin) cancer antigen against multidomain capsules loaded with imiquimod in one embodiment of the present invention (FIG. imMDV (R837-base) sample / 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV sample).
  • FIG. 12C is a graph showing humoral immune effects (IgG, 1 week after injection) against an OVA (ovalbumin) cancer antigen in a multidomain capsule loaded with imiquimod according to one embodiment of the present invention.
  • imMDV R837-HCl: R837-base (1: 1) / 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV sample).
  • FIG. 13A is a graph showing humoral immune effects (IgG, 3 week after injection) against an OVA (ovalbumin) cancer antigen in a multidomain capsule loaded with imiquimod according to one embodiment of the present invention (FIG. imMDV (R837-HCl) sample / 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV sample).
  • FIG. 13B is a graph showing humoral immune effects (IgG, 3 week after injection) against an OVA (ovalbumin) cancer antigen in a multidomain capsule loaded with imiquimod according to one embodiment of the present invention.
  • imMDV R837-base sample / 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV sample).
  • FIG. 13C is a graph showing humoral immune effects (IgG, 3 week after injection) against an OVA (ovalbumin) cancer antigen in a multidomain capsule loaded with imiquimod according to one embodiment of the present invention.
  • imMDV R837-HCl: R837-base (1: 1) sample / 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV sample).
  • FIG. 14A is a graph showing humoral immune effects (IgG, 5 week after injection) against OVA cancer antigens for imiquimod-loaded multidomain capsules according to one embodiment of the present invention (imMDV (R837) -HCl) samples, 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV).
  • imMDV R837 -HCl
  • FIG. 14B is a graph showing humoral immune effects (IgG, 5 week after injection) against OVA cancer antigens, for multidomain capsules loaded with imiquimod according to one embodiment of the present invention (imMDV (R837) -base) sample, 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV).
  • imMDV R837 -base
  • FIG. 14C is a graph showing humoral immune effects (IgG, 5 week after injection) against OVA cancer antigens against imiquimod-loaded multidomain capsules according to one embodiment of the present invention (imMDV [R837] -HCl: R837-base (1: 1) sample, 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV).
  • imMDV [R837] -HCl R837-base (1: 1) sample, 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV).
  • FIG. 15A illustrates humoral immune effects (IgG, 1 week after boosting of 5 weeks mice) on an OVA (ovalbumin) cancer antigen against multidomain capsules loaded with imiquimod in one embodiment of the present invention.
  • This is the graph shown (imMDV (R837-HCl) sample / 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV sample).
  • FIG. 15B illustrates a humoral immune effect (IgG, 1 week after boosting of 5 weeks mice) on an OVA (ovalbumin) cancer antigen against multi-domain capsules loaded with imiquimod according to one embodiment of the present invention.
  • IgG immunoglobulin
  • OVA ovalpha-associated antigen
  • FIG. 15B illustrates a humoral immune effect (IgG, 1 week after boosting of 5 weeks mice) on an OVA (ovalbumin) cancer antigen against multi-domain capsules loaded with imiquimod according to one embodiment of the present invention.
  • IgG humoral immune effect
  • FIG. 15C illustrates humoral immune effects (IgG, 1 week after boosting of 5 weeks mice) on OVA (ovalbumin) cancer antigens in a multidomain capsule loaded with imiquimod according to one embodiment of the present invention.
  • IgG humoral immune effects
  • OVA ovalalbumin
  • FIG. 15C illustrates humoral immune effects (IgG, 1 week after boosting of 5 weeks mice) on OVA (ovalbumin) cancer antigens in a multidomain capsule loaded with imiquimod according to one embodiment of the present invention.
  • imMDV [R837-HCl: R837-base (1: 1) sample / 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV sample)).
  • FIG. 16 shows the body fluids for OVA (ovalbumin) cancer antigens in mice boosted at week 5 and mice not boosted after immunization of imMDV (R837-HCl) + OVA sample according to one embodiment of the present invention. It is a graph showing the sexual immune effect (IgG) (1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: imMDV (R837-HCl) + OVA).
  • IgG sexual immune effect
  • FIG. 17 shows the body fluids for OVA (ovalbumin) cancer antigens in mice boosted at week 5 and mice not boosted after immunization of imMDV (R837-base) + OVA sample according to one embodiment of the present invention. It is a graph showing the sexual immune effect (IgG) (1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: imMDV (R837-base) + OVA).
  • IgG sexual immune effect
  • FIG. 18 shows an example of an imMDV [R837-HCl: R837-base (1: 1) sample] + OVA sample, which is boosted at 5 weeks and not boosted, according to one embodiment of the present invention.
  • imMDV (R837-HCl) + OVA sample was immunized and boosted at 5th week.
  • FIG. 20 illustrates that imMDV (R837-base) + OVA samples are immunized and boosted at 5th week in an embodiment of the present invention.
  • This is a graph showing humoral immune effects (IgG) against OVA (ovalbumin) cancer antigens, which are continuously observed at weeks 1, 2 and 6 after boosting (1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: imMDV (R837-base) + OVA sample).
  • IgG humoral immune effects against OVA (ovalbumin) cancer antigens
  • FIG. 21 illustrates that imMDV [R837-HCl: R837-base (1: 1) sample] + OVA sample was immunized and boosted at 5th week in one embodiment of the present invention.
  • This is a graph showing humoral immune effects (IgG) against OVA (ovalbumin) cancer antigens, which are continuously observed at weeks 1, 2 and 6 after boosting (1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: imMDV [R837-HCl: R837-base (1: 1) sample).
  • FIG. 23 compares the effects of inflammatory response after immunization of two vaccines [imMDV (R837-HCl) + OVA and DMSO (R837) + OVA] in mice in an embodiment of the present invention.
  • FIG. 24 is a graph showing humoral immune effects (two weeks after muscle injection) of immunomodulatory substances against HA (hemagglutinin) virus antigen in one embodiment of the present invention.
  • HA hemagglutinin
  • FIG. 25 is a graph showing humoral immune effects (4 weeks after muscle injection) of immunomodulatory substances against HA (hemagglutinin) virus antigen in one embodiment of the present invention.
  • HA hemagglutinin
  • FIG. 26 is a graph showing humoral immune effects of immunomodulatory substances against OVA (ovalbumin) cancer antigens in one embodiment of the present invention.
  • OVA ovalalbumin
  • FIG. 27 is a graph showing the cellular immune inducing effect of immunomodulatory substances against OVA (ovalbumin) cancer antigen in one embodiment of the present invention.
  • FIG. 28 shows optical microscope images of multi-domain capsule imMDV (SQ-Gem), imMDV (OA-Gem), and imMDV (Gem) samples according to one embodiment of the present invention.
  • FIG. 29 shows that, in one embodiment of the present invention, loaded gemcitabine is gradually released in a multidomain capsule containing squalene, while most of the drugs loaded in 24 hours are released in a multidomain capsule not containing squalene. It is a graph confirming that
  • FIG. 30 shows that in one embodiment of the present invention, when oleic acid vegetable oil is used instead of an animal oil such as squalene, the sustained release behavior of loaded gemcitabine may have a plateau shape for 24-72 hours.
  • the graph shows the linear behavior after 72 hours.
  • FIG. 31 is a graph showing imMDV (paclitaxel) and drug release behavior thereof in Example 4-2 of the present invention.
  • FIG. 31 is a graph showing imMDV (paclitaxel) and drug release behavior thereof in Example 4-2 of the present invention.
  • Fig. 32 shows imMDV (doxorubicin) in Example 4-2 of the present invention.
  • Figure 33 shows imMDV (methotrexate) in Example 4-2 of the present invention.
  • Fig. 34 shows imMDV (oxaliplatin) in Example 4-2 of the present invention.
  • Fig. 35 shows imMDV (MK-2206) in Example 4-3 of the present invention.
  • FIG. 37 shows imMDV (Azacytidine) in Example 4-5 of the present invention.
  • Example 38 is a graph showing imMDV (Resmonostat) and drug release behavior thereof in Example 4-5 of the present invention.
  • Fig. 39 is a graph showing imMDV (Panobinostat) and drug release behavior thereof in Example 4-5 of the present invention.
  • Fig. 40 shows imMDV (OTX015 (iBET)) in Example 4-5 of the present invention.
  • Fig. 41 shows imMDV (BLZ945) in Example 4-6 of the present invention.
  • Fig. 42 shows imMDV (Celecoxib) in Example 4-7 of the present invention.
  • FIG. 43 shows imMDV (GEM / R837) in Example 5 of the present invention.
  • Fig. 44 shows imMDV (BLZ945 / R837) in Example 5 of the present invention.
  • the term "combination (s) thereof" included in the expression of a makushi form refers to one or more mixtures or combinations selected from the group consisting of components described in the expression of makushi form, It means to include one or more selected from the group consisting of the above components.
  • a multi-domain capsule comprising two or more liposomes in contact with and connected to each other, and a multi-domain capsule outer wall surrounding the two or more liposomes, wherein the multi-domain capsule is composed of an organic phase and an aqueous phase.
  • the organic phase comprises a first immune modulator and a fluid oil
  • the organic phase forms a membrane of the liposome, and the outer wall of the multidomain capsule
  • the aqueous phase comprises a second immune modulator
  • the aqueous phase is the An inner aqueous solution phase of the liposome membrane and an outer aqueous solution phase of the liposome membrane
  • the first immunomodulatory substance is a fat-soluble immunoactive substance
  • the second immunomodulatory substance is a water-soluble immunoactive substance
  • the fluid oil is in contact with and connected to each other.
  • the multidomain capsule includes an outer wall of the multidomain capsule including a fat-soluble immunoactive substance, and inside the outer wall of the multidomain capsule surrounding the two or more liposomes, two or more liposomes each form a domain. Forming a capsule structure of about 1 ⁇ m to about 100 ⁇ m.
  • Multi-domain capsules containing two or more liposomes may be improved in duration, immune cell activation efficacy, encapsulation efficiency, or physiological stability of immune cell activating substances, as compared to conventional single liposomes and single emulsions.
  • the inside of the liposome membrane means the inner aqueous solution phase
  • the outside of the liposome membrane means the outer aqueous solution phase
  • the inner aqueous solution phase and the outer aqueous solution phase All means "first aqueous phase”.
  • the outer aqueous solution phase that is outside of the liposome membrane means a space between the liposome membrane and the outer wall of the multidomain capsule.
  • the multi-domain capsule may be dispersed in a solvent, wherein the dispersed phase in which the multi-domain capsule is dispersed, that is, the outside of the multi-domain capsule means "second aqueous solution phase".
  • the size of the multi-domain capsule is about 1 ⁇ m to about 100 ⁇ m, about 1 ⁇ m to about 80 ⁇ m, about 1 ⁇ m to about 60 ⁇ m, about 1 ⁇ m to about 40 ⁇ m, about 1 ⁇ m to about 20 ⁇ m, about 1 ⁇ m to about 10 ⁇ m, about 10 ⁇ m to about 100 ⁇ m, about 10 ⁇ m to about 80 ⁇ m, about 10 ⁇ m to about 60 ⁇ m, about 10 ⁇ m to about 40 ⁇ m, about 10 ⁇ m to About 20 ⁇ m, about 20 ⁇ m to about 100 ⁇ m, about 20 ⁇ m to about 80 ⁇ m, about 20 ⁇ m to about 60 ⁇ m, about 20 ⁇ m to about 40 ⁇ m, about 40 ⁇ m to about 100 ⁇ m, about 40 ⁇ m to about 80 ⁇ m, about 40 ⁇ m to about 60 ⁇ m, about 60 ⁇ m to about 100 ⁇ m, about 60 ⁇ m to about 80 ⁇ m, or about
  • the antigen loaded in the capsule and And / or the immunomodulatory substance may extend the release time compared to a single liposome or a single emulsion and consequently regulate the function of immune cells in vivo over a long time.
  • two or more liposomes may comprise liposomes in which the envelope is in contact with each other.
  • the liposomes of the multidomain capsule are interfacial contact between the outer skin, and thus the liposomes are not easily broken as compared to the multiple liposomes in which the outer skin is separated from each other, thereby improving the structural stability and sustained release effect of the multidomain capsule. Can be.
  • the fluid oil may improve the stability of the multi-domain capsule by acting as a glue (glue) between the domain consisting of each liposome.
  • the multi-domain capsule may be to improve the stability of the multi-domain capsule by introducing a fluid oil to the outer wall of the domain capsule, the contact of the outer wall of the liposomes, accordingly, the sustained-release effect and structural stability May be increased.
  • the fat-soluble immunoactive substance may be easily loaded into the multi-domain capsule by the fluid oil.
  • the lubricating oil such as a poorly soluble immunomodulating substance, which is difficult to solubilize in a general organic solvent, is easily solubilized, and thus, multi-domain capsules with the fluent fluid in the space between the liposomes and the liposomes. Can be loaded into.
  • the flowable oil may serve as an adjuvant to assist the activation of immune cells, for example animal oil, vegetable oil, tocopherol, mineral oil, castor oil, and combinations thereof It may include selected from the group consisting of.
  • the animal oil may be to include a fish oil.
  • the fish oil may be used without limitation as long as it is a metabolizable oil, for example, may include cod liver oil, shark liver oil, whale oil and the like.
  • the shark liver oil contains squalene, a molecule known as 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, unsaturated terpene, Saturation analogues may also be included.
  • fish oils including squalene or squalane they are readily available from commercial sources or can be obtained by methods known in the art.
  • the animal-derived oil may include a lard, resin (tallow), or tallow.
  • the vegetable-derived oil may be an oil derived from nuts, seeds, grains, and the like, and may include, for example, peanut oil, soybean oil, coconut oil, olive oil, and the like. .
  • the tocopherol may be tocopherol containing vitamin E.
  • various tocopherols ⁇ , ⁇ , ⁇ , ⁇ , ⁇ or ⁇
  • ⁇ -tocopherol can be used, for example DL- ⁇ -tocopherol can be used.
  • the fluid oil by introducing the fluid oil into the multi-domain capsule, it is possible to easily solubilize the immunomodulatory substance, it is possible to enhance the structural stability of the multi-domain capsule.
  • lipophilic or poorly soluble immunomodulators can be easily solubilized, and synergistic effects with the immunomodulation can be obtained by the immunoactivating effect of squalene and oleic acid itself. It may increase the structural stability of the multi-domain capsule, but may not be limited thereto.
  • the fat-soluble and water-soluble immunoactive substance may be an immunomodulatory substance expressed in stressed cancer cells, for example, a heat-shock protein, or activation of T cells. It may be a substance for inducing.
  • the fat-soluble and water-soluble immunoactive substance is a toll-like receptor agonist, saponin, antiviral peptide, inflammasome inducer, NOD ligand (NOD ligand), CDS ligand (cytosolic DNA sensor ligand), stimulator of interferon genes (STING) ligand, and combinations thereof may include one or more materials selected from, but may not be limited thereto. .
  • the toll-like receptor agonist as a direct ligand or indirect ligand means a component that can cause a signaling response through the TLT signaling pathway through the production of endogenous or exogenous ligands It may be.
  • the toll-like receptor agonist may be a natural toll-like receptor agonist or a synthetic toll-like receptor agonist.
  • the toll-like receptor agonist may be one that can cause a signaling response through TLR-1, for example, tri-acylated lipopeptide (LP); Phenol-soluble modulins; Cobacterium tuberculosis (Mycobacterium tuberculosis) lipopeptide; S- (2,3-bis (palmitoyloxy)-(2-RS) -propyl) -N-palmitoyl- (R) -Cys- (S) -Ser- (S) -Lys (4) -OH ; Bacterial lipopeptides from Borrelia burgdorfei; Trihydrochloride (Pam3Cys) lipopeptides that mimic the acetylated amino termini of OspA lipopeptides; And one or more materials selected from the group consisting of combinations thereof, but may not be limited thereto.
  • LP tri-acylated lipopeptide
  • Phenol-soluble modulins Cobacterium tub
  • the toll-like receptor agonist may include a TLR-2 agonist, for example, may include Pam3Cys-Lip, but may not be limited thereto.
  • the toll-like agonist may include a TLR-3 agonist, for example, Poly (I: C), Poly (ICLC), Poly ( IC12U), ampligen, and the like, but may not be limited thereto.
  • TLR-3 agonist for example, Poly (I: C), Poly (ICLC), Poly ( IC12U), ampligen, and the like, but may not be limited thereto.
  • the toll-like agonist may comprise a TLR-4 agonist, for example, Shigella flexineri (Shigella flexineri) outer membrane protein preparation, AGP, CRX-527, MPLA , PHAD, 3D-PHAD, GLA, and combinations thereof may be one or more materials selected from the group consisting of, but may not be limited thereto.
  • Shigella flexineri Shigella flexineri (Shigella flexineri) outer membrane protein preparation
  • AGP CRX-527
  • MPLA CRX-527
  • MPLA PHAD
  • 3D-PHAD 3D-PHAD
  • GLA GLA
  • the toll-like agonist may include a TLR-5 agonist, for example, may include, but is not limited to, flagellin or a fragment thereof. have.
  • the toll-like agonist may comprise a TLR-7 agonist or a TLR-8 agonist, for example, imiquimod, R837, resquimod, or R848
  • a TLR-7 agonist for example, imiquimod, R837, resquimod, or R848
  • imidazoquinoline molecules VTX-2337; CRX642; Imidazoquinoline covalently bound to a phospholipid group or a phosphonolipid group;
  • the toll-like agonist may include a TLR-9 agonist, for example, may include an immune stimulating oligonucleotide, but may not be limited thereto.
  • the immune stimulatory oligonucleotide may include one or more CpG motifs, but may not be limited thereto.
  • the saponin may be selected from the group consisting of QS21, QuilA, QS7, QS17, ⁇ -Eskin, Digitonin and combinations thereof, but may not be limited thereto.
  • the antiviral peptide may include KLK, but may not be limited thereto.
  • the influmersome inducer may be TDB (trehalose-6,6-dibehenate), but may not be limited thereto.
  • the NOD ligand may be M-TriLYS (NOD2 agonist-synthetic Muramil tripeptide) or NOD2 agonist (N-glycolylated muramyldipeptid), but may not be limited thereto.
  • the CDS ligand may be Poly (dA: dT), but may not be limited thereto.
  • the STING ligand may be cGAMP, di-AMP, or di-GMP, but may not be limited thereto.
  • the immunomodulatory substance may comprise a combination of one or more toll-like receptor agonists, for example, CL401 (dual TLR2 and TLR7 agonists) or CL429 (dual TLR2 And NOD2 agonist), but may not be limited thereto.
  • CL401 dual TLR2 and TLR7 agonists
  • CL429 dual TLR2 And NOD2 agonist
  • the immunomodulatory substance included in the multi-domain capsule is, for example, Pam3Cys-Lip, polycysi, CRX-527, MPLA, flagellin, imiquimod, resquimod, CpG , QS21, M-TriLys (MurNAc-Ala-D-isoGln-Lys), trehalose-6,6-dibehenate (TDB), 8837, Poly (dA: dT), cGAMP, and combinations thereof It may be, but may not be limited thereto.
  • the fat-soluble immunoactive substance is, for example, cationic lipid, MPLA, AGP, CRX-527, PHAD, 3D-PHAD, GLA, lipid peptide, Pam3Cys, Pam3Cys-Lip, DDA , A substance selected from the group consisting of imiquimod (base form), resquimod (base form), VTX-2337, CRX642, saponin (QS21), TDB, CL401, CL429, and combinations thereof Can be.
  • the hydrophilic immunoactive material is, for example, CpG, imiquimod (HCl form), resquimod (HCl form), Poly (I: C), STING, flagellin ( flagellin), saponins, KLK peptides, NOD agonist peptides, Poly (dA: dT), and combinations thereof.
  • the hydrophilic material may be conjugated to the outer wall of the multidomain capsule through the chemical bonding group of the end group, but may not be limited thereto.
  • the intracellular delivery efficiency of the immunomodulatory substance can be further improved.
  • an anionic and / or negatively charged various immunoregulatory substances and biomaterials such as DNA, RNA are effectively loaded into the multi-domain capsule Can be.
  • anionic or negatively charged biomaterials and / or DNA, RNA amino acid based immunomodulatory substances may be loaded via electrostatic bonding to the membrane of the outer wall or inner liposome of the multidomain capsules exhibiting cationic properties.
  • this may not be limited.
  • the cationic lipid is DC-cholesterol (3 ⁇ - [N- (N '(N', N'-dimethylaminoethane) -carbamoyl] cholesterol hydrochloride), DDA (dimethyldioctadecylammonium), DOTAP (1,2- dioleoyl-3-trimethylammonium-propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), EPC (1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine), MVL5 (N1- [2 -((1S) -1-[(3-aminopropyl) amino] -4- [di (3-amino-propyl) amino] butylcarboxamido) ethyl] -3,4-di [oleyloxy] -benzamide), DODAP (lipid
  • a surfactant is coated on the outside of the multi-domain capsule, so that the multi-domain capsule can be stably dispersed in the aqueous solution.
  • the surfactant is coated on the outside of the multidomain capsule so that the multidomain capsule can be dispersed in an aqueous solution, for example, polyoxyethylene sorbitan ester surfactant (commonly called Tween), in particular polysorb Bait 20 and polysorbate 80; Copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO); Octosinol (eg, Triton X-100, or t-octylphenoxypolyethoxyethanol); (Octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); As phospholipid (phospholipid component), phosphatidylcholine (lecithin) phosphatidylethanolaniline, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin; Nonylphenol eth
  • the surfactant may be a mixture of these surfactants, such as a Tween 80 / Span 85 mixture. Combinations of polyoxyethylene sorbitan esters and octosinol may also be used. Other useful combinations may include laureth 9, polyoxyethylene sorbitan esters and / or octosinol.
  • the surfactant may be used in an amount of 0.001 to 20% by weight based on the total weight of the multidomain capsule, for example, 0.01 to 1%, 0.001 to 0.1%, and 0.005 to 0.02%; It may be used at a weight of 0.1 to 20%, 0.1 to 10%, 0.1 to 1% or about 0.5%.
  • an immunomodulatory substance comprising a multidomain capsule and an antigen according to the present invention .
  • the antigen may include one selected from the group consisting of proteins, genes, cells, viruses, and combinations thereof, but may not be limited thereto.
  • the protein may include overalbumin, recombinant protein, subunit, split protein antigen, and the cell may include, for example, dendritic cells, T cells,
  • the virus may include, but may not be limited to, for example, influenza, hepatitis B virus (HBV), hepatitis A virus (HAV), and human papilloma virus (HPV).
  • the antigen is attenuated live complete microorganisms, inert microorganisms, ruptured microorganisms, proteins of pathogens, recombinant proteins, glycoproteins, peptides, polysaccharides, lipopolysaccharides, lipopeptides, polynucleotides, cells, Virus, and combinations thereof, but may not be limited thereto.
  • the antigen may include, but is not limited to, an influenza-derived antigen or a cancer cell-derived antigen.
  • the immunomodulatory substance for intradermal administration may induce multiple immune responses in the body by including one or more antigens, but may not be limited thereto.
  • the cancer cells may be obtained by using a cancer cell line (cell line), or may be separated from cancer tissue (tumor tissue) existing in the body.
  • a cancer cell line cell line
  • cancer tissue tumor tissue
  • the cancer cells may include cancer cells of lung, colon, central nervous system, skin, ovary, kidney, breast, stomach, or colon, but may not be limited thereto.
  • the step of dissolving the first immunomodulator and fluid oil in a solvent to prepare an oil phase solution Dispersing a first aqueous phase comprising a second immunomodulatory substance in the oil phase solution to produce a water-in-water (W / O) emulsion; And mixing the oil-in-water emulsion with a second aqueous solution and evaporating the solvent.
  • the first immunomodulatory substance is a fat-soluble immunoactive substance
  • the second immunomodulatory substance is characterized in that the water-soluble immunoactive substance, a method for producing a multi-domain capsule.
  • the antigen loaded in the capsule and And / or the immunomodulatory substance may extend the release time compared to a single liposome or a single emulsion and consequently regulate the function of immune cells in vivo over a long time.
  • two or more liposomes may comprise liposomes in which the envelope is in contact with each other.
  • the liposomes of the multidomain capsule are interfacial contact between the outer skin, and thus the liposomes are not easily broken as compared to the multiple liposomes in which the outer skin is separated from each other, thereby improving the structural stability and sustained release effect of the multidomain capsule. Can be.
  • the fluid oil is characterized in that the role of glue (glue) between the domain consisting of each liposome, the stability of the multi-domain capsule is improved.
  • the multi-domain capsule may be to improve the stability of the multi-domain capsule by introducing a fluid oil to the outer wall of the domain capsule, the contact of the outer wall of the liposomes, accordingly, the sustained-release effect and structural stability May be increased.
  • the lipophilic immunomodulatory substance may be easily loaded into the multi-domain capsule by the flowable oil.
  • the lubricating oil such as a poorly soluble immunomodulating substance, which is difficult to solubilize in a general organic solvent, is easily solubilized, and thus, multi-domain capsules with the fluent fluid in the space between the liposomes and the liposomes. Can be loaded into.
  • the flowable oil may serve as an adjuvant to assist the activation of immune cells, for example animal oil, vegetable oil, tocopherol, mineral oil, castor oil, and combinations thereof It may include selected from the group consisting of.
  • the animal oil may be to include a fish oil.
  • the fish oil may be used without limitation as long as it is a metabolizable oil, for example, may include cod liver oil, shark liver oil, whale oil and the like.
  • the shark liver oil contains squalene, a molecule known as 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, unsaturated terpene, Saturation analogues may also be included.
  • fish oils including squalene or squalane they are readily available from commercial sources or can be obtained by methods known in the art.
  • the animal-derived oil may include a lard, resin (tallow), or tallow.
  • the vegetable-derived oil may be an oil derived from nuts, seeds, grains, and the like, and may include, for example, peanut oil, soybean oil, coconut oil, olive oil, and the like. .
  • the tocopherol may be tocopherol containing vitamin E.
  • various tocopherols ⁇ , ⁇ , ⁇ , ⁇ , ⁇ or ⁇
  • ⁇ -tocopherol can be used, for example DL- ⁇ -tocopherol can be used.
  • the fluid oil by introducing the fluid oil into the multi-domain capsule, it is possible to easily solubilize the immunomodulatory substance, it is possible to enhance the structural stability of the multi-domain capsule.
  • lipophilic or poorly soluble immunomodulators can be easily solubilized, and synergistic effects with the immunomodulators can be achieved by the immunoactivating effect of squalene and oleic acid itself. It may represent, but may increase the structural stability of the multi-domain capsule, but may not be limited thereto.
  • the fat-soluble and water-soluble immunoactive substance may be an immunomodulatory substance expressed in stressed cancer cells, for example, a heat-shock protein, or activation of T cells. It may be a substance for inducing.
  • the fat-soluble and water-soluble immunoactive material is a toll-like receptor agonist, saponin, antiviral peptide, inflammasome inducer, NOD ligand (NOD ligand), CDS ligand (cytosolic DNA sensor ligand), stimulator of interferon genes (STING) ligand, and combinations thereof may include one or more materials selected from, but may not be limited thereto. .
  • the toll-like receptor agonist as a direct ligand or indirect ligand means a component that can cause a signaling response through the TLT signaling pathway through the production of endogenous or exogenous ligands It may be.
  • the toll-like receptor agonist may be a natural toll-like receptor agonist or a synthetic toll-like receptor agonist.
  • the toll-like receptor agonist may be one that can cause a signaling response through TLR-1, for example, tri-acylated lipopeptide (LP); Phenol-soluble modulins; Cobacterium tuberculosis (Mycobacterium tuberculosis) lipopeptide; S- (2,3-bis (palmitoyloxy)-(2-RS) -propyl) -N-palmitoyl- (R) -Cys- (S) -Ser- (S) -Lys (4) -OH ; Bacterial lipopeptides from Borrelia burgdorfei; Trihydrochloride (Pam3Cys) lipopeptides that mimic the acetylated amino termini of OspA lipopeptides; And one or more materials selected from the group consisting of combinations thereof, but may not be limited thereto.
  • LP tri-acylated lipopeptide
  • Phenol-soluble modulins Cobacterium tub
  • the toll-like receptor agonist may include a TLR-2 agonist, for example, may include Pam3Cys-Lip, but may not be limited thereto.
  • the toll-like agonist may include a TLR-3 agonist, for example, Poly (I: C), Poly (ICLC), Poly ( IC12U), ampligen, and the like, but may not be limited thereto.
  • TLR-3 agonist for example, Poly (I: C), Poly (ICLC), Poly ( IC12U), ampligen, and the like, but may not be limited thereto.
  • the toll-like agonist may comprise a TLR-4 agonist, for example, Shigella flexineri (Shigella flexineri) outer membrane protein preparation, AGP, CRX-527, MPLA , PHAD, 3D-PHAD, GLA, and combinations thereof may be one or more materials selected from the group consisting of, but may not be limited thereto.
  • Shigella flexineri Shigella flexineri (Shigella flexineri) outer membrane protein preparation
  • AGP CRX-527
  • MPLA CRX-527
  • MPLA PHAD
  • 3D-PHAD 3D-PHAD
  • GLA GLA
  • the toll-like agonist may include a TLR-5 agonist, for example, may include, but is not limited to, flagellin or a fragment thereof. have.
  • the toll-like agonist may comprise a TLR-7 agonist or a TLR-8 agonist, for example, imiquimod, R837, resquimod, or R848
  • a TLR-7 agonist for example, imiquimod, R837, resquimod, or R848
  • imidazoquinoline molecules VTX-2337; CRX642; Imidazoquinoline covalently bound to a phospholipid group or a phosphonolipid group;
  • the toll-like agonist may include a TLR-9 agonist, for example, may include an immune stimulating oligonucleotide, but may not be limited thereto.
  • the immune stimulatory oligonucleotide may include one or more CpG motifs, but may not be limited thereto.
  • the saponin may be selected from the group consisting of QS21, QuilA, QS7, QS17, ⁇ -Eskin, Digitonin and combinations thereof, but may not be limited thereto.
  • the antiviral peptide may include KLK, but may not be limited thereto.
  • the influmersome inducer may be TDB (trehalose-6,6-dibehenate), but may not be limited thereto.
  • the NOD ligand may be M-TriLYS (NOD2 agonist-synthetic Muramil tripeptide) or NOD2 agonist (N-glycolylated muramyldipeptid), but may not be limited thereto.
  • the CDS ligand may be Poly (dA: dT), but may not be limited thereto.
  • the STING ligand may be cGAMP, di-AMP, or di-GMP, but may not be limited thereto.
  • the immunomodulatory substance may comprise a combination of one or more toll-like receptor agonists, eg, CL401 (dual TLR2 and TLR7 agonists) or CL429 (dual TLR2 And NOD2 agonist), but may not be limited thereto.
  • toll-like receptor agonists eg, CL401 (dual TLR2 and TLR7 agonists) or CL429 (dual TLR2 And NOD2 agonist
  • the immunomodulatory substance included in the multi-domain capsule is, for example, Pam3Cys-Lip, polycysi, CRX-527, MPLA, flagellin, imiquimod, resquimod, CpG , QS21, M-TriLys (MurNAc-Ala-D-isoGln-Lys), trehalose-6,6-dibehenate (TDB), 8837, Poly (dA: dT), cGAMP, and combinations thereof It may be, but may not be limited thereto.
  • the fat-soluble immunomodulatory substance is, for example, cationic lipid, MPLA, AGP, CRX-527, PHAD, 3D-PHAD, GLA, lipid peptide, Pam3Cys, Pam3Cys-Lip, DDA , A substance selected from the group consisting of imiquimod (base form), resquimod (base form), VTX-2337, CRX642, saponin (QS21), TDB, CL401, CL429, and combinations thereof Can be.
  • the hydrophilic immunomodulatory substance is, for example, CpG, imiquimod (HCl form), resquimod (HCl form), Poly (I: C), STING, flagellin ( flagellin), saponins, KLK peptides, NOD agonist peptides, Poly (dA: dT), and combinations thereof.
  • the hydrophilic material may be conjugated to the outer wall of the multidomain capsule through the chemical bonding group of the end group, but may not be limited thereto.
  • the intracellular delivery efficiency of the immunomodulatory substance can be further improved.
  • an anionic and / or negatively charged various immunoregulatory substances and biomaterials such as DNA, RNA are effectively loaded into the multi-domain capsule Can be.
  • anionic or negatively charged biomaterials and / or DNA, RNA amino acid based immunomodulatory substances may be loaded via electrostatic bonding to the membrane of the outer wall or inner liposome of the multidomain capsules exhibiting cationic properties.
  • this may not be limited.
  • the cationic lipid is DC-cholesterol (3 ⁇ - [N- (N '(N', N'-dimethylaminoethane) -carbamoyl] cholesterol hydrochloride), DDA (dimethyldioctadecylammonium), DOTAP (1,2- dioleoyl-3-trimethylammonium-propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), EPC (1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine), MVL5 (N1- [2 -((1S) -1-[(3-aminopropyl) amino] -4- [di (3-amino-propyl) amino] butylcarboxamido) ethyl] -3,4-di [oleyloxy] -benzamide), DODAP (lipid
  • a surfactant is coated on the outside of the multi-domain capsule, so that the multi-domain capsule can be stably dispersed in the aqueous solution.
  • the surfactant is coated on the outside of the multidomain capsule so that the multidomain capsule can be dispersed in an aqueous solution, for example, polyoxyethylene sorbitan ester surfactant (commonly called Tween), in particular polysorb Bait 20 and polysorbate 80; Copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO); Octosinol (eg, Triton X-100, or t-octylphenoxypolyethoxyethanol); (Octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); As phospholipid (phospholipid component), phosphatidylcholine (lecithin) phosphatidylethanolaniline, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin; Nonylphenol eth
  • the surfactant may be a mixture of these surfactants, such as a Tween 80 / Span 85 mixture. Combinations of polyoxyethylene sorbitan esters and octosinol may also be used. Other useful combinations may include laureth 9, polyoxyethylene sorbitan esters and / or octosinol.
  • the surfactant may be used in an amount of 0.001 to 20% by weight based on the total weight of the multidomain capsule, for example, 0.01 to 1%, 0.001 to 0.1%, and 0.005 to 0.02%; It may be used at a weight of 0.1 to 20%, 0.1 to 10%, 0.1 to 1% or about 0.5%.
  • a multi-domain capsule comprising two or more liposomes in contact with and connected to each other, and a multi-domain capsule outer wall surrounding the two or more liposomes, wherein the multi-domain capsule is composed of an organic phase and an aqueous phase.
  • the organic phase comprises a first immune modulator and a fluid oil
  • the organic phase forms a membrane of the liposome, and the outer wall of the multidomain capsule
  • the aqueous phase comprises a second immune modulator
  • the aqueous phase is the An inner aqueous solution phase of the liposome membrane and an outer aqueous solution phase of the liposome membrane
  • the first immunomodulatory substance and the second immunomodulatory substance are immunosuppressive factor controlling substances
  • the fluid oil is composed of two or more liposomes in contact with and connected to each other.
  • Multidomain capsules can be provided, characterized by improving structural stability. The.
  • the first immunomodulatory substance and the second immunomodulatory substance may further include the above-described immunoactive substance. That is, the first immunomodulatory substance and the second immunomodulatory substance may include an immunosuppressive factor controlling substance together with an immunoactive substance.
  • an immunomodulatory substance comprising the multidomain capsule and the antigen may be provided.
  • dissolving the first immune modulator and the fluid oil in a solvent to prepare an oil phase solution; Dispersing a first aqueous phase comprising a second immunomodulatory substance in the oil phase solution to produce a water-in-water (W / O) emulsion; And mixing the oil-in-water emulsion with a second aqueous solution and evaporating the solvent.
  • the first immunomodulatory substance, and the second immunomodulatory substance is characterized in that the immunosuppressive factor controlling substance, may be provided a method for producing a multi-domain capsule.
  • the multi-domain capsule-based solid cancer microenvironmental control composition is a new type of immunomodulatory composition for regulating the microenvironment of cancer, in addition to the substances for activating immune cells mentioned above, immunosuppressive cells appearing in the solid cancer microenvironment. And it is characterized in that it comprises a drug (immunosuppressive factor controlling substance) that can control the function of the immunosuppressive substance.
  • a plurality of liposomes are linked to each other while forming respective domains based on an immunosuppressive factor, that is, an immunosuppressive factor control material that can control the functions of the immunosuppressive cells and the immunosuppressive material
  • an immunosuppressive factor control material that can control the functions of the immunosuppressive cells and the immunosuppressive material
  • a new multiple that can overcome the shortcomings of the low encapsulation efficiency and short effective duration of a single liposome material that is used as a variety of pharmaceutical compositions, and increase the effective duration of the immunomodulatory effect Domain capsule-based anticancer immunotherapy composition can be prepared.
  • Multi-domain capsule while slowly decaying from the outer wall of the capsule to the inner membrane, the immune that can control the function of the immunosuppressive cells and immunosuppressive material loaded into the outer wall and the inside of the capsule Since inhibitor inhibitors are released, the effective duration of immune mechanism modulators can be increased.
  • the multi-domain capsule according to an embodiment of the present invention, immunosuppression capable of controlling the functions of various immunosuppressive cells and immunosuppressive substances having lipophilic properties on the membrane of the liposome and / or the outer wall of the multidomain capsule.
  • immunosuppression capable of controlling the functions of various immunosuppressive cells and immunosuppressive substances having lipophilic properties on the membrane of the liposome and / or the outer wall of the multidomain capsule.
  • Multi-domain capsule by loading the immunosuppressive factor control material that can control the function of various immunosuppressive cells and immunosuppressive agents having hydrophilic properties inside the liposome, immunosuppressive factor control material It can increase the effective duration of.
  • Multi-domain capsule by simultaneously loading a lipophilic immunosuppressive factor control material on the outer wall of the various immunosuppressive control agents, hydrophobic membranes and / or the capsule having hydrophilic properties inside the liposomes
  • a lipophilic immunosuppressive factor control material on the outer wall of the various immunosuppressive control agents, hydrophobic membranes and / or the capsule having hydrophilic properties inside the liposomes
  • drugs capable of controlling the function of Meloidoid-Derived Suppressor Cell that is, immunosuppressive factor controlling substances, include Tadalafil, Sildenafil, L-AME, Nitroaspirin, Celecoxib, NOHA, Bardoxolone methyl, D, L-1-methyl-tryptophan, 5-Fluorouracil, Gemcitabine, 17-DMAG, Peptide-Fc fusionproteins, ATRA, Vitamin A, Vitamin D3, Vitamin E, GR1 antibodies, Zoledronic acid, Sunitinib, Axitinib, Decetaxel, Sorafenib, CucurbitacinB, JSI-124, Anti IL-17 antibodies, Anti-glycan antibodies, Anti-VEGF antibodies, Bevacizumab, Antracycline, Tasquinimod, Imatinib, cyclophosphamide, but are not limited thereto.
  • MDSC Meloidoid-Derived Suppressor Cell
  • PI3K inhibitors include PX-866, Wortmannin, PI-103, Pictilisib, GDC-0980, PF-04691502, BEZ235, XL765, XL147, BAY80-6946, GSK-2126458, Buparlisib, BYL719, AZD8186, GSK-2636771, CH5132799, INK-1117 and the like.
  • PI3Kdelta inhibitors are AMG-319, Idelalisib, TRG-1202, INCB050465, IPI-145, Duvelisib, Acalisib, TG-1202, RV1729, RP-6530, GDC-0032. .
  • the PI3Kgamma inhibitors are characterized by IPI-549, IPI-145, and the like.
  • One example of the present invention is a drug capable of coordinating the function of a TAM (tumor associated macrophage), that is, a drug capable of inhibiting the recruitment of macrophage as an immunosuppressive factor controlling agent, CCL2 / CCR2 inhibitors (Yondeli, RS102895), M-CSF or M-CSFR inhibitors (anti-M-CSF antibodies, JNJ-28312141, GW2580), chemoattractants (CCL5, CXCL-12, VEGF) and their inhibitors and HIFs inhibitors. It is not limited to this.
  • a drug that can inhibit the survival of TAM that is, immunosuppressant control agent
  • it is a drug that can induce the expression of Bisphosphonates, Clodronate, Dasatinib, anti-FRbeta antibodies, Shigella flexneri, Legumain and CD1d It is not limited to this.
  • drugs capable of improving the properties of the M1 macrophage that is, immunosuppressive factor controlling substances, NF-kB agonists TLR agonists, Anti-CD40 antibodies, Thiazolidinediones, Tasquinimod, Anti-IL-10R antibodies, Anti-IL -10 antibodies, oligonucleotides (Anti-IL-10R Anti-IL-10), STAT1 agonists, interferons, SHIP and M1 pathway-inducing SHIP, GM-CSF, IL-12, Thymosin alpha1, etc.
  • immunosuppressive factor controlling substances that is, immunosuppressive factor controlling substances, NF-kB agonists TLR agonists, Anti-CD40 antibodies, Thiazolidinediones, Tasquinimod, Anti-IL-10R antibodies, Anti-IL -10 antibodies, oligonucleotides (Anti-IL-10R Anti-IL-10), STAT1 agonists, interferons, SHIP and M1 pathway-inducing SHIP, GM-
  • drugs that can inhibit the mechanism of M2 macrophage-based cancer cell growth include sunitinib, sorafenib, WP1066, corosolic acid, oleanolic acid, STAT6 inhibitors and the M2 pathway (c-).
  • the target miRNA that can control the function of macrophage under tumor microenvironment is miR-155, miR-511-3p, miR-26a).
  • Targeting drugs that can enhance anticancer efficacy by targeting Macrophage under tumor microenvironment include Paclitaxel, Docetaxel, 5-Flurouracil, Alendronate, Doxorubicin, Simvastatin, Hydrazinocurcumin, Amphotericin B, Ciprofloxacin, Rifabutin, Rifampicin, Efavirenz, Cilatin Theophyline, Pseudomonas exotoxin A, Zoledronic acid, Trabectedin, Siltuximab (Anti-IL-6 antibodies), Dasatinib, CpG-ODN, Interferon-alpha, beta, gamma, GM-CSF, IL-12, Thymosin alpha-1, Sunitinib, 5,6-Dimethylxanthenone-4-acetic acid, Silibinin, CCL2-CCR2 inhibitors (PF-04136309, Trabectedin, Carlumab), CSF1-CSF1R signaling blocker (
  • TGF-beta transforming growth factor beta
  • COX2 Cycloxygenase-2
  • IDO Indoleamine 2,3-dioxygenase
  • Phosphodiesterase-5 Multi-domain capsule-based compositions containing PDE-5) inhibitors
  • Anti-Interleukin 10 (IL-10)) drugs can be provided.
  • TGF-beta inhibitors include, but are not limited to, SB-505124, LY-364974, and the like.
  • Nitro aspirin includes, but is not limited to, NCX 4040 and the like.
  • COX-2 inhibitors include, but are not limited to, Celecoxib.
  • IDO inhibitors include, but are not limited to, Indoximod, NLG919, and the like.
  • PDE-5 inhibitors include, but are not limited to, Tadalafil (Cialis) and the like.
  • the solid cancer microenvironment immunosuppressive factor controlling agent contained in the multi-domain capsule may be composed of two or more combinations of the above drugs.
  • natural killer cells and T cells having a therapeutic ability to find and kill cancer cells present in the body directly survive effectively in the body, and include a multi-domain capsule capable of improving therapeutic efficacy. It may be an immunomodulatory substance.
  • One example of the present invention is an antibody that acts as an inhibitor of immune checkpoints (PD-1, PDL-1 CTLA-4, LAG-3, TIM-3, CEACAM1) as a method of T cell activation through direct binding in a solid cancer microenvironment. It can provide a multi-domain capsule-based composition comprising them.
  • PD-1 immune checkpoints
  • PDL-1 CTLA-4 LAG-3
  • TIM-3 TIM-3
  • CEACAM1 CEACAM1
  • Anti-CTLA-4 antibody includes, but is not limited to, Ipilimumab.
  • Anti-PD1-antibody in one example of the present invention includes, but is not limited to Nivolumab.
  • Anti-PDL1 antibody in one embodiment of the present invention includes, but is not limited to, Atezolizumab.
  • Anti-LAG-3 antibody in one embodiment of the present invention includes, but is not limited to, BMS-986016.
  • Anti-TIM-3 antibody includes, but is not limited to, TSR-022.
  • Anti-CEACAM1 antibody includes, but is not limited to, CM-24.
  • One example of the present invention provides a multi-domain capsule-based composition comprising a coactivator (OX40, CD137, CD27, CD40) and the like as a T cell activation method through direct binding in a solid cancer microenvironment.
  • a coactivator OX40, CD137, CD27, CD40
  • Anti-OX40 includes, but is not limited to, RG7888 and the like.
  • Anti-CD137 includes, but is not limited to, Urelumab.
  • Anti-CD27 includes, but is not limited to, Varlilumab.
  • Anti-CD40 in one example of the present invention includes, but is not limited to, BMS-986090 and the like.
  • One example of the present invention is a multidomain capsule-based composition containing a drug capable of inhibiting immunosuppressive factors (Treg, MDSC, TAM, IDO, PD-L1) by T cell activation through indirect binding in a solid cancer microenvironment.
  • a drug capable of inhibiting immunosuppressive factors Treg, MDSC, TAM, IDO, PD-L1
  • One example of the present invention may provide a multi-domain capsule-based composition comprising an anticancer agent that increases the efficacy of immune cells through induction of immunogenic cell death through chemotherapy.
  • One example of the present invention provides a multi-domain capsule-based composition comprising a drug capable of killing cancer cells or controlling the tumor microenvironment through epigenetic machinery.
  • the DNA methyltransferase inhibitor (DNMTi) substance is 5-Azacytidine, 5-Aza-2-deoxycytidine, Decitabine, SGI-110, Zebularine, CP-4200, Cladribine, Fludarabine, Clofarabine, Procainamide, Procaine, Hydralazine, Disulfiram, RG108, Nanaomycin A, Genistein, Equol, Curcumin, EEGG, Resveratrol, Parthenolide and the like, but is not limited thereto.
  • DNMTi DNA methyltransferase inhibitor
  • the histone deacetylase inhibitor (HDACi) material is Vorinostat, Abexinostat, Suberoylanilide, Hydroxamic acid, Belinostat, Panobinostat, Romidepsin, Valproic acid, Entinostat, Givinostat, Resminostat, Quisinostat, Pracinostat, Dacinostat, Pyroxamide, CHR-3996, CBHA, Trichostatin A, Oxamflatin, MC1568, Tubacin, PCI-30451, Tacedinaline, Mocetinostat, Chidamide, BML-210, M344, Butyrate, Sodium butyrate, Trapoxin A, Apicidin, Nicotinamide, Splitomicin, EX -527, Dihydrocoumarin, Tenovin-D3, AGK2, AEM1, AEM2, Cambinol, Sirtinol, Salermide, Tenovin-6, TMP-269, Psamm
  • a multi-domain capsule was prepared as follows.
  • DOPC (10 mg), cholesterol (8 mg), squalene (12 mg) and glycerol trioleate (12 mg) were dissolved in chloroform (1 mL) to prepare an oily solution.
  • the prepared oil phase solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, this formed double emulsion was dispersed in dichloromethane solution. The dichloromethane was removed using a vacuum distillation and the temperature was raised to 37 ° C. to remove residual solvent.
  • the solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • a control group was prepared together in the same manner as in the above example, except that squalene was not included.
  • the multi-domain capsule containing squalene was uniform in size compared to the control without squalene, At the interface of the interface showed a clear boundary (Fig. 2 (a) and (b)). On the other hand, it was found that the control group containing no squalene maintains non-uniform size and shape (FIG. 2 (c) and (d)).
  • FIGS. 3D to 3F are optical microscopes of multidomain capsules not containing squalene. Image.
  • Figure 3 by applying the rhodamine fluorescent dye in the oil phase solution, it was possible to clearly observe the structure of the multi-domain capsule, the case of the multi-domain capsule containing squalene has a distinct boundary point, dispersed in an aqueous solution I could see that.
  • DOPC (10 mg), cholesterol (8 mg), MPLA [monophosphoryl lipid A, 10 mg, Avanti Polar Lipids, USA], squalene (12 mg), glycerol trioleate, 12 mg ) was dissolved in chloroform (1 mL) to prepare an oily solution.
  • the prepared oil phase solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, this formed double emulsion was dispersed in dichloromethane solution.
  • the dichloromethane was removed using a vacuum distillation and the temperature was raised to 37 ° C. to remove residual solvent.
  • the solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • 5 is an optical microscope image of multidomain capsules (imMDV-1: imMDV (MPLA)) comprising squalene-based MPLA.
  • Samples of imMDV (SQ) and imMDV (MPLA) prepared in Examples 1-1 and 1-2 were obtained from bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophage (BMMCs). The effect on the activation was measured by the secretion amount of pro-inflammatory cytokines (TNF- ⁇ , IL-6, IL-12) using the ELISA test method.
  • BMDCs bone marrow-derived dendritic cells
  • BMMCs bone marrow-derived macrophage
  • DOPC (10 mg), cholesterol (8 mg), squalene (12 mg) and glycerol trioleate (12 mg) were dissolved in chloroform (1 mL) to prepare an oily solution.
  • the prepared oily solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose) containing ovalbumin (5 mg, Sigma Aldrich, USA). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, this formed double emulsion was dispersed in a dichloromethane solution.
  • the dichloromethane was removed using a reduced pressure distillation and the temperature was raised to 37 ° C. to remove residual solvent.
  • the solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • a control group was prepared together in the same manner as in the above example, except that squalene was not included.
  • a homogenizer 20,000 X g
  • the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent.
  • the solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • DOPC (10 mg), cholesterol (8 mg), MPLA (10 mg), squalene (12 mg), TDB (10 mg, Avanti Polar Lipids, USA), glycerol trioleate (12 mg) was dissolved in chloroform (1 mL) to prepare an oily solution.
  • the prepared oil phase solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent. The solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • DOPC (10 mg), cholesterol (8 mg), MPLA (10 mg), DDA (10 mg), squalene (12 mg) and glycerol trioleate (12 mg) were dissolved in chloroform (1 mL) to form an oily solution.
  • the prepared oil phase solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent. The solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • the prepared oil phase solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent. The solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • An oily solution was prepared by dissolving DOPC (10 mg), cholesterol (8 mg), squalene (12 mg), and glycerol trioleate (12 mg) in chloroform (1 mL).
  • the prepared oil phase solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose, CpG 1 mg, Bioneer, Korea). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent. The solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • An oily solution was prepared by dissolving DOPC (10 mg), cholesterol (8 mg), squalene (12 mg), and glycerol trioleate (12 mg) in chloroform (1 mL).
  • the prepared oil phase solution was used for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose, Poly (I: C) (Sigma-Aldrich, USA) 1 mg). Dispersed. Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent. The solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • An oily solution was prepared by dissolving DOPC (10 mg), cholesterol (8 mg), squalene (12 mg), and glycerol trioleate (12 mg) in chloroform (1 mL). Dispersion of the prepared oil phase solution in 1 mL of an internal aqueous phase (5% sucrose, 5 mg of Resquimod (Sigma-Aldrich, USA)) using a homogenizer (20,000 X g) for 10 minutes I was. Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent. The solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • An oleate (12 mg) was dissolved in chloroform (1 mL) to prepare an oily solution.
  • the prepared oil phase solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds.
  • HCl-type imiquimod dissolved in an aqueous solution was prepared by the following process. Dissolve 400 g of imiquimod in 2000 ml of distilled water and 900 ml of n-butanol (or 1-butanol). Stirring and add 150ml of 37% HCl solution at the same time.
  • FIG. 9 shows an optical microscope image of a multi-domain capsule loaded with imiquimod in HCl, imiquimod in base, and imiquimod in two forms simultaneously.
  • imMDV R837-HCl
  • the amount of drug released was quantified using a UV-Vis spectrometer (FIG. 10). As shown in FIG. 10, about 70% of the loaded drug was released over 8 days.
  • imMDV R837-HCl
  • BMDCs bone marrow-derived dendritic cells
  • the secretion of representative pro-inflammatory cytokines IL-6 related to the Th1 immune response was determined by ELISA.
  • IL-6 was increased in proportion to the treated concentration
  • R837-HCl encapsulated in the multi-domain capsule was confirmed to show a similar behavior to that of R837-HCl used as a control. It can be seen that it is released to activate immune cells.
  • a humoral immune effect (IgG, 1 week after injection) against an OVA (ovalbumin) model antigen is shown in the multidomain capsule sample (12a: imMDV (R837-) loaded with imiquimod. HCl) sample, 12b: imMDV (R837-base) sample, 12c: imMDV [R837-HCl: R837-base (1: 1) sample]) administration group was significantly increased.
  • the increased humoral immunity effect can be confirmed to persist even after 3 weeks (Fig. 13a, 13b and 13c) and 5 weeks (Fig. 14a, 14b and 14c) after injection.
  • Humoral immunopotentiation effect is significantly increased when additional boosting once 5 weeks after the first injection (Figs. 15a, 15b, 15c, 16, 17, and 18). This increased humoral immune effect can be confirmed that it is maintained continuously in weeks 1, 2, 6 after boosting 5 weeks (Figs. 19, 20, and 21).
  • DMSO oil-type adjuvant
  • An oily solution was prepared by dissolving DOPC (10 mg), cholesterol (8 mg), squalene (12 mg), and glycerol trioleate (12 mg) in chloroform (1 mL).
  • the prepared oily solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose, 1 mg of STING (InvivoGen, USA)). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent. The solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • An oily solution was prepared by dissolving DOPC (10 mg), cholesterol (8 mg), squalene (12 mg), MPLA (10 mg) and glycerol trioleate (12 mg) in chloroform (1 mL).
  • the prepared oil phase solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose, CpG 1 mg). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent. The solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • An oily solution was prepared by dissolving DOPC (10 mg), cholesterol (8 mg), squalene (12 mg), MPLA (10 mg) and glycerol trioleate (12 mg) in chloroform (1 mL).
  • the prepared oil phase solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose, 1 g of Poly (I: C)). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent. The solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • An oily solution was prepared by dissolving DOPC (10 mg), cholesterol (8 mg), squalene (12 mg), and glycerol trioleate (12 mg) in chloroform (1 mL).
  • the prepared oil phase solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase [5% sucrose, 1 mg of CpG, 1 mg of Poly (I: C)]. Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent. The solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • DOPC (10 mg), cholesterol (8 mg), MPLA (10 mg), castor oil (12 mg, Sigma-Aldrich, USA) and glycerol trioleate (12 mg) in chloroform (1 mL) was prepared.
  • the prepared oil phase solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent. The solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • Oily solution by dissolving DOPC (10 mg), cholesterol (8 mg), MPLA (10 mg), mineral oil (12 mg, Sigma-Aldrich, USA), glycerol trioleate (12 mg) in chloroform (1 mL) was prepared.
  • the prepared oil phase solution was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL of an internal aqueous phase (5% sucrose). Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds. Finally, the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent. The solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain liposomes.
  • mice Female BALB / c and C57BL / 6 mice (5-6 weeks old) were purchased from KOATECH (Pyeongtaek, Korea). All experiments with mice were conducted in accordance with Korean NIH guidelines for the care and use of laboratory study animals.
  • mouse serum was collected and antibody titers against HA proteins in serum were measured by an enzyme linked immunosorbent assay (ELISA).
  • ELISA enzyme linked immunosorbent assay
  • the plate coated with HA protein was blocked using PBS / 3% bovine serum albumin (BSA), and the control group serum was incubated at various serial dilutions. Then, mouse IgG attached with horseradish peroxidase was added. All the incubations were performed at 37 ° C. for 1 hour, and after each step mentioned, washed three times with PBS / 0.05% Tween 20.
  • BSA bovine serum albumin
  • the effect of the cancer prevention vaccine of the multidomain capsule containing the immunomodulatory substance prepared in Example 1 was verified through a mouse experiment (C57BL / 6, 6 to 7 week old female).
  • Increasing humoral immune response by injecting 50 ⁇ g of an immunomodulatory substance (cancer preventive vaccine) containing a multi-domain capsule into mice was measured by ELISA (Enzyme linked Immunosorbent assay) method, and the results are shown in FIG. 26. (IgG production measurement).
  • the humoral immune response was confirmed by performing ophthalmic blood collection in mice after vaccination, and comparing the production of immunoglobulin IgG with the control group.
  • mice inoculated in Example 3-1 three mice of the OVA and OVA-multiple-capsule group were selected, and after 2 weeks, spleens were extracted from each mouse, and then the spleen tissues were sterilized in a petri dish. The spleens were ground using a cell strainer to separate cells from the tissue coating.
  • the isolated splenocytes were plated in IFN-gamma coated plates in 96-well 5 ⁇ 10 5 cells / 100 ⁇ L and treated with MHC class I-restricted OVA peptide at a concentration of 5 ⁇ g / mL for 48 hours. Then, IFN-gamma with horseradish peroxide was added. As a substrate, the reaction was developed by adding 100 ⁇ L of ACE (3-amino-9-ethyl-carbazole, BD biosciences, USA) and measured by ELSPOT (enzyme-linked immunospot) method (FIG. 27).
  • ACE enzyme-linked immunospot
  • An oily solution was prepared by dissolving DOPC (10 mg), cholesterol (8 mg), squalene (12 mg), and glycerol trioleate (12 mg) in chloroform (1 mL).
  • the prepared oily solution was homogenizer (20,000) in 1 mL of internal aqueous phase (5% sucrose, gemcitabine: Gemzar® (Eli Lilly and Company, Indianapolis, Ind., USA), 5 mg).
  • X g was used to disperse for 10 minutes. Thereafter, the mixed solution was vortexed in 3 mL of an external aqueous phase (7.5% glucose, 40 mM lysine) for 10 seconds.
  • the formed double emulsion was removed using the vacuum distillation apparatus to remove the chloroform and the temperature was raised to 37 ° C. to remove residual solvent.
  • the solvent-free multidomain capsule dispersion was precipitated at low temperature, or it was settled by centrifugation to remove the supernatant to obtain a multidomain capsule (imMDV (SQ-Gem)).
  • FIG. 28 shows an optical microscope image of the three samples prepared in this way: In a multidomain capsule containing squalene, the loaded gemcitabine is slowly released, while it does not contain squalene. In the multi-domain capsule, it can be seen that most of the loaded drugs are released within 24 hours (FIG. 29) Also, when oleic acid vegetable oil is used instead of animal oil such as squalene, the sustained release behavior of loaded gemcitabine is 24. It was shown that the plateau shape for -72 hours and then linear behavior after 72 hours. It means that by using the liquid oil, can tune the release behavior of drug-loaded (Fig. 30).
  • paclitaxel By inducing the death of cancer cells, paclitaxel, doxorubicin, metotrexate and oxaliplatin are selected from anticancer agents that play a role in enabling antigen-presenting cells to recognize cancer antigens effectively.
  • Multidomain capsules loaded with these drugs were prepared. Prepared using the same method as in Example 4-1, but imMDV (paclitaxel) (Fig. 31) was used to add paclitaxel drug to the oily solution, imMDV (doxorubicin) (Fig. 32), imMDV (methotrexate) ( 33) and imMDV (oxaliplatin) (FIG. 34) prepared the multidomain capsules by adding the respective drugs on an internal aqueous solution. As can be seen in Figure 31, it was confirmed that the loaded drug is slowly released over two weeks.
  • An oily solution was prepared by dissolving DOPC (10 mg), cholesterol (8 mg), squalene (12 mg), and glycerol trioleate (12 mg) in chloroform (1 mL).
  • the prepared oil phase solution was prepared by internal aqueous phase (5% sucrose) in which MK-2206 (an Akt inhibitor, SelleckChem, 5 mg) was dissolved (Imatinib: Gleevec® (Novartis Pharmaceuticals Corp, East Hanover, NJ, USA) 5 mg) was dispersed for 10 minutes using a homogenizer (20,000 X g). Subsequently, a multi-domain capsule was prepared through the same process as in Example 4-1 (FIG. 35).
  • An oily solution was prepared by dissolving DOPC (10 mg), cholesterol (8 mg), squalene (12 mg), and glycerol trioleate (12 mg) in chloroform (1 mL).
  • the prepared oil phase solution was PF-04691502 (PI3K inhbitor, SelleckChem, 5 mg) in an internal aqueous phase (5% sucrose, (Imatinib: Gleevec® (Novartis Pharmaceuticals Corp, East Hanover, NJ, USA)) 5 mg) was dispersed for 10 minutes using a homogenizer (20,000 X g) in 1 mL. Thereafter, a multi-domain capsule was prepared by the same process as in Example 4-1 (FIG. 36).
  • Azacytidine, Resminostat, Panobinostat and OTX015 were selected to prepare multidomain capsules loaded with these drugs.
  • imMDV Azacytidine
  • imMDV Resmonostat
  • imMDV Panobinostat
  • imMDV OTX015
  • iBET imMDV
  • gemcitabine capable of killing MDSC and cancer cells and a toll-like receptor that acts on immune cell activation Multidomain capsules (imMDV (GEM / R837)) having a stable structure while simultaneously containing quimod (Examples 1-9) were prepared (FIG. 43).
  • BLZ945 which is a drug capable of removing TAM cells, and imiquimod (Example 1-9), a toll-like receptor that acts on immune cell activation Capsules (imMDV (BLZ945 / R837)) were prepared (FIG. 44).
  • Multi-domain capsule according to the present invention by loading a variety of immunomodulators having hydrophilic properties inside the liposomes, the membrane of the liposomes and / or the outer wall of the capsule at the same time, the effective maintenance of the immunomodulators You can increase the time.
  • the manufacturing method of the multi-domain capsule according to the present invention by introducing a fluid oil such as squalene, it is possible to improve the stability and storage stability in the manufacturing process of the multi-domain capsule, due to the introduction of the fluid oil, general Representative poorly soluble immunomodulatory substances that are not soluble in organic solvents can be easily solubilized, and thus, there are advantages in that multiple domain capsules containing the various poorly soluble immunomodulatory substances can be prepared.

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Abstract

La présente invention concerne une vésicule à domaines multiples comprenant un matériau immunoactif, un procédé de production de la vésicule à domaines multiples et une composition immunomodulatrice comprenant la vésicule à domaines multiples. Selon un aspect de la présente invention, la vésicule à domaines multiples comprend : deux liposomes ou plus qui entrent en contact et sont reliés l'un à l'autre ; et une paroi externe de vésicule à plusieurs domaines entourant les deux liposomes ou plus. La vésicule à domaines multiples est formée à partir d'une phase huileuse et d'une phase aqueuse, la phase huileuse comprenant un premier matériau immunomodulateur et une huile fluide ; la phase huileuse forme une membrane des liposomes et la paroi externe de la vésicule à domaines multiples ; la phase aqueuse comprend un second matériau immunomodulateur ; la phase aqueuse est une phase aqueuse interne de la membrane des liposomes et une phase aqueuse externe de la membrane des liposomes ; le premier matériau immunomodulateur est un matériau immunoactif liposoluble ; le second matériau immunomodulateur est un matériau immunoactif soluble dans l'eau ; et l'huile fluide augmente la stabilité structurelle des deux liposomes ou plus qui entrent en contact et sont reliés l'un à l'autre.
PCT/KR2018/002516 2017-03-02 2018-03-02 Vésicule à domaines multiples comprenant un matériau immunoactif, procédé de production associé et composition immunomodulatrice la comprenant WO2018160026A1 (fr)

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AU2018229137A AU2018229137A1 (en) 2017-03-02 2018-03-02 Multi-domain vesicle comprising immunoactive material, production method therefor and immunomodulatory composition comprising same
US16/489,781 US20190380960A1 (en) 2017-03-02 2018-03-02 Multi-domain vesicle comprising immunoactive material, production method therefor and immunomodulatory composition comprising same
RU2019130877A RU2736639C1 (ru) 2017-03-02 2018-03-02 Мультидоменная везикула, содержащая иммуностимулирующий материал, способ её производства и содержащая её иммуномодулирующая композиция
CN201880029326.3A CN110582275A (zh) 2017-03-02 2018-03-02 包含免疫刺激物质的多域囊泡、其制备方法和包含多域囊泡的免疫调节组合物
CA3055067A CA3055067A1 (fr) 2017-03-02 2018-03-02 Vesicule a domaines multiples comprenant un materiau immunoactif, procede de production associe et composition immunomodulatrice la comprenant
JP2019547392A JP2020510663A (ja) 2017-03-02 2018-03-02 免疫活性物質を含む多重ドメインカプセル、その製造方法、及びこれを含む免疫調節組成物
EP18761258.5A EP3590508A4 (fr) 2017-03-02 2018-03-02 Vésicule à domaines multiples comprenant un matériau immunoactif, procédé de production associé et composition immunomodulatrice la comprenant

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114617876A (zh) * 2022-01-28 2022-06-14 四川大学华西医院 一种抗肿瘤联合用药物

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6071534A (en) * 1988-02-18 2000-06-06 Skyepharma Inc. Multivesicular liposomes with controlled release of active agents encapsulated in the presence of a hydrochloride
US20020039596A1 (en) * 1997-11-14 2002-04-04 Hartoun Hartounian Production of multivesicular liposomes
JP2014058469A (ja) * 2012-09-18 2014-04-03 Kyoto Univ 複数の被内包リポソームを内包するリポソーム及びその製造方法
US20140112979A1 (en) * 2011-07-04 2014-04-24 Statens Serum Institut Methods for producing liposomes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6071534A (en) * 1988-02-18 2000-06-06 Skyepharma Inc. Multivesicular liposomes with controlled release of active agents encapsulated in the presence of a hydrochloride
US20020039596A1 (en) * 1997-11-14 2002-04-04 Hartoun Hartounian Production of multivesicular liposomes
US20140112979A1 (en) * 2011-07-04 2014-04-24 Statens Serum Institut Methods for producing liposomes
JP2014058469A (ja) * 2012-09-18 2014-04-03 Kyoto Univ 複数の被内包リポソームを内包するリポソーム及びその製造方法

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIOCHIMICA BIOPHYSICA ACTA, vol. 728, no. 3, 9 March 1983 (1983-03-09), pages 339 - 348
INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 331, 2007, pages 182 - 185
JAIN, SANJAY K.: "Design and development of multivesicular liposomal depot delivery system for controlled systemic delivery of acyclovir sodium", AAPS PHARMSCITECH, vol. 6 1, 2005, pages E35 - E41, XP055214963, DOI: doi:10.1208/pt060108 *
NATURE MATERIALS, vol. 10, 2011, pages 243 - 251
PROGRESS OF LIPIDS RESEARCH, vol. 41, 2002, pages 392 - 406
See also references of EP3590508A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114617876A (zh) * 2022-01-28 2022-06-14 四川大学华西医院 一种抗肿瘤联合用药物
CN114617876B (zh) * 2022-01-28 2023-04-07 四川大学华西医院 一种抗肿瘤联合用药物

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