WO2018160026A1

WO2018160026A1 - Multi-domain vesicle comprising immunoactive material, production method therefor and immunomodulatory composition comprising same

Info

Publication number: WO2018160026A1
Application number: PCT/KR2018/002516
Authority: WO
Inventors: 임용택
Original assignee: 단디바이오사이언스 주식회사
Priority date: 2017-03-02
Filing date: 2018-03-02
Publication date: 2018-09-07

Abstract

The present invention relates to a multi-domain vesicle comprising an immunoactive material, a production method for the multi-domain vesicle and an immunomodulatory composition comprising the multi-domain vesicle. According to one aspect of the present invention, the multi-domain vesicle comprises: at least two liposomes making contact and connected with each other; and a multi-domain vesicle outer wall surrounding the at least two liposomes. The multi-domain vesicle is formed from an oil phase and an aqueous phase, wherein: the oil phase comprises a first immunomodulatory material and a fluid oil; the oil phase forms a membrane of the liposomes, and the multi-domain vesicle outer wall; the aqueous phase comprises a second immunomodulatory material; the aqueous phase is an internal aqueous phase of the membrane of the liposomes, and an outer aqueous phase of the membrane of the liposomes; the first immunomodulatory material is a fat-soluble immunoactive material; the second immunomodulatory material is a water-soluble immunoactive material; and the fluid oil increases the structural stability of the at least two liposomes making contact and connected with each other.

Description

Multidomain capsule containing an immunoactive substance, preparation method thereof, and immunomodulatory composition comprising the same

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.

Currently, liposome materials containing various drugs are used. However, in such a technique using a single liposome material, low loading efficiency and instability in vivo are pointed out as a big disadvantage.

Recently, various liposomes and emulsion materials (e.g., ASO1, ASO2, AS15 and Novartis MF59) equipped with 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.

In order to overcome this drawback, 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. Attempts were made to solve the low encapsulation efficiency and stability problems that had been a fundamental disadvantage.

However, since the shape of liposomes having a multilayer lamellar structure is very nonuniform, and a manufacturing process for producing a multilayer lamellar structure having a specific structure is optional, there is a disadvantage in that a vaccine composition having uniform properties cannot be obtained at every production. Because of the use of chemical crosslinking, there is a limit that can cause toxicity to the human body.

In a similar form, a drug carrier, which is conventionally called multivesicular liposome, is described by 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]. There is a bar. These multiple liposomes are composed of a mixture of substances selected from the group consisting of neutral lipids and cholesterol and triolein.

In the conventional multiple liposomes, the principle that microvesicles maintain a single micro cluster is that the double membranes do not break down and do not scatter even under the abrupt curve change of lipid membranes in which triolein material contacts between lipid membranes of individual vesicles. Because it fixed. These multiple liposomes are currently developed as drugs loaded with bupivacaine, a pain management agent, and are commercially available under the trade name EXPARELβ.

However, 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. . In addition, multiple liposome forms into which immunoactivating drugs have been introduced have not been found to date. On the other hand, in addition to 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. In particular, in order to solve the low therapeutic efficiency and side effects of the current anti-cancer immunotherapy, it is very urgent to develop a technology that can overcome the immunosuppression phenomenon in the cancer microenvironment.

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. Among these anti-cancer immunotherapy methods, 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. And 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. However, 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.

One of these reasons is due to microenvironmental factors that suppress immune function around solid cancers. In fact, cells that degrade immune cells in the tumor microenvironment (MDSC: myeoloid-derived stromal cells, Treg: regulatory T cells, TAM: tumor-assocaited macrophaphages), immunosuppressive cytokines, metabolites, etc. In other words, the activity of immune activating substances and therapeutic immune cells is drastically reduced. Therefore, in order to increase the treatment efficiency of solid cancer, it is very urgent to develop a new treatment platform technology that can control immunosuppressive factors in the solid cancer microenvironment.

Recently, research on the development of drugs that can control various immunosuppressive factors in the tumor microenvironment has been actively conducted worldwide. However, these drugs have the disadvantage of being easily degraded by various physiological environments and enzymes in vivo when injected into the body, or delivered to tissues other than the tumor site, resulting in various unwanted side effects.

In order to overcome these disadvantages, attempts have been made in the clinical field to improve the immunotherapeutic effect by repeatedly administering high-dose drugs, but due to various drug toxicity and side effects, It is causing the consequences.

Therefore, in the solid cancer microenvironment, by releasing a drug that can control immunosuppressive environmental factors that inhibit the therapeutic function of the immunotherapy agent in a sustained release form around the solid cancer, the immunosuppressive factors can be effectively targeted and the side effects caused by the drug can be minimized. There is an urgent need to develop an anticancer immunotherapy and a treatment effect improvement technology for anticancer immunotherapy using the same.

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.

However, the problem to be solved by the present application is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

According to an aspect of the present invention, 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. Wherein 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, and the aqueous phase comprises a second immune modulator, wherein the aqueous phase is the An inner aqueous solution phase of the liposome membrane and an outer aqueous solution phase of the liposome membrane, wherein the first immunomodulatory substance is a fat-soluble immunoactive substance, the second immunomodulatory substance is a water-soluble immunoactive substance, and the fluid oil is in contact with and connected to each other. Multiple, characterized in that to enhance the structural stability of two or more liposomes Domain capsules can be provided.

According to another aspect of the present invention, it is possible to provide an immunomodulatory substance comprising the multidomain capsule and an antigen.

According to another aspect of the invention, 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. It includes, wherein 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.

In addition, 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. .

In addition, 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. The advantage is that capsules can be prepared.

In addition, 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 By constructing a multi-domain capsule, a variety of anionic and / or negatively charged immunomodulators and biomaterials such as DNA, RNA can be effectively loaded into the multi-domain capsule.

In addition, since the disintegration gradually occurs from the outer wall of the multidomain capsule to the inner membrane, 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.

On the other hand, 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.

In addition, 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.

1 is a schematic diagram showing the structure of an immunomodulatory multidomain capsule (imMDV) in one embodiment of the present invention.

2 (a) to (d), in one embodiment of the present invention, a graph (c) showing the optical microscope image (a) and the size distribution of the multi-domain capsule containing squalene, and does not include squalene Is a graph (d) showing the optical microscope image (b) and size distribution of multidomain capsules (scale bar: 20 μm).

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. Thus, it is an optical microscope image of multidomain capsules without squalene (scale bar: 4 μm).

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.

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.

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).

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).

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).

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. This is the graph shown (imMDV (R837-base) sample / 1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: OVA + imMDV sample).

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. This is the graph shown (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).

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).

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. Is a graph showing humoral immune effects (IgG) against OVA (ovalbumin) cancer antigen (1: PBS, 2: OVA, 3: OVA + R837-HCl, 4: imMDV [R837-HCl: R837-base (1)). : 1) sample].

19, in an embodiment of the present invention, imMDV (R837-HCl) + OVA sample was immunized and boosted at 5th week. 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) + OVA sample).

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).

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).

Figure 22, in one embodiment of the present invention, immunization of the imMDV (R837-HCl) + OVA sample, the humoral immune effect (IgG) against the OVA (ovalbumin) cancer antigen shown in 1-4 d. Data compared with adjuvant (DMSO (R837) + OVA) of (1: OVA, 2: imMDV (R837-HCl) + OVA, 3: DMSO (R837) + OVA, 4: DMSO).

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. FIG.

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. FIG.

FIG. 26 is a graph showing humoral immune effects of immunomodulatory substances against OVA (ovalbumin) cancer antigens in one embodiment of the present invention. FIG.

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.

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.

36 shows imMDV (PF-04691502) in Example 4-4 of the present invention.

FIG. 37 shows imMDV (Azacytidine) in Example 4-5 of the present invention. FIG.

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.

Fig. 44 shows imMDV (BLZ945 / R837) in Example 5 of the present invention.

DETAILED DESCRIPTION Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art may easily implement the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. In the drawings, parts irrelevant to the description are omitted for simplicity of explanation, and like reference numerals designate like parts throughout the specification.

Throughout this specification, when a part is said to be "connected" with another part, this includes not only the "directly connected" but also the "electrically connected" between other elements in between. do.

Throughout this specification, when a member is located “on” another member, this includes not only when one member is in contact with another member but also when another member exists between the two members.

Throughout this specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding the other components unless otherwise stated. As used throughout this specification, the terms “about”, “substantially”, and the like, are used at, or in the vicinity of, numerical values when manufacturing and material tolerances inherent in the meanings indicated are given, and an understanding of the invention Accurate or absolute figures are used to help prevent unfair use by unscrupulous infringers. As used throughout this specification, the term “step of” or “step of” does not mean “step for”.

Throughout this specification, 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.

Throughout this specification, the description of “A and / or B” means “A or B, or A and B”.

Hereinafter, with reference to the accompanying drawings will be described embodiments and embodiments of the present invention; However, the present disclosure may not be limited to these embodiments, examples, and drawings.

According to an aspect of the present invention, 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. Wherein 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, and the aqueous phase comprises a second immune modulator, wherein the aqueous phase is the An inner aqueous solution phase of the liposome membrane and an outer aqueous solution phase of the liposome membrane, wherein the first immunomodulatory substance is a fat-soluble immunoactive substance, the second immunomodulatory substance is a water-soluble immunoactive substance, and the fluid oil is in contact with and connected to each other. Multiple, characterized in that to enhance the structural stability of two or more liposomes Domain capsules may be provided.

1 is a cross-sectional view showing the structure of an immunomodulatory multidomain capsule (immunomodulatory multidomain vesicle, imMDV) according to an embodiment of the present invention. As shown in FIG. 1, 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 μm to about 100 μm.

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.

In one embodiment of the present invention, as shown in Figure 1, 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. In addition, 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".

In one embodiment, the size of the multi-domain capsule is about 1 μm to about 100 μm, about 1 μm to about 80 μm, about 1 μm to about 60 μm, about 1 μm to about 40 μm, about 1 μm to about 20 μm, about 1 μm to about 10 μm, about 10 μm to about 100 μm, about 10 μm to about 80 μm, about 10 μm to about 60 μm, about 10 μm to about 40 μm, about 10 μm to About 20 μm, about 20 μm to about 100 μm, about 20 μm to about 80 μm, about 20 μm to about 60 μm, about 20 μm to about 40 μm, about 40 μm to about 100 μm, about 40 μm to about 80 μm, about 40 μm to about 60 μm, about 60 μm to about 100 μm, about 60 μm to about 80 μm, or about 80 μm to about 100 μm.

In one embodiment of the present invention, since the multi-domain capsule is gradually collapsed from the outer wall constituting the outer side of the capsule to the inner membrane containing the two or more liposomes, 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.

In one embodiment of the invention, two or more liposomes may comprise liposomes in which the envelope is in contact with each other. For example, 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.

In one embodiment of the present invention, 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. For example, 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.

In one embodiment of the present invention, the fat-soluble immunoactive substance may be easily loaded into the multi-domain capsule by the fluid oil. For example, 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.

In one embodiment of the present invention, 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.

In one embodiment of the invention, the animal oil may be to include a fish oil.

In one embodiment of the present invention, 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. In the case of fish oils including squalene or squalane, they are readily available from commercial sources or can be obtained by methods known in the art.

In one embodiment of the present invention, the animal-derived oil may include a lard, resin (tallow), or tallow.

In one embodiment of the present invention, 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. .

In one embodiment of the present invention, the tocopherol may be tocopherol containing vitamin E. There are various tocopherols (α, β, γ, δ, ε or ξ), but generally α-tocopherol can be used, for example DL-α-tocopherol can be used.

In one embodiment of the present invention, 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. For example, when squalene and oleic acid are used as the fluid oil, 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.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, 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. .

In one embodiment of the invention, 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.

In one embodiment of the invention, the toll-like receptor agonist may be a natural toll-like receptor agonist or a synthetic toll-like receptor agonist.

In one embodiment of the invention, 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.

In one embodiment of the present invention, the toll-like receptor agonist may include a TLR-2 agonist, for example, may include Pam3Cys-Lip, but may not be limited thereto.

In one embodiment of the present invention, 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.

In one embodiment of the invention, 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.

In one embodiment of the present invention, 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.

In one embodiment of the invention, the toll-like agonist may comprise a TLR-7 agonist or a TLR-8 agonist, for example, imiquimod, R837, resquimod, or R848 Such as imidazoquinoline molecules; VTX-2337; CRX642; Imidazoquinoline covalently bound to a phospholipid group or a phosphonolipid group; And one or more materials selected from the group consisting of combinations thereof, but may not be limited thereto.

In one embodiment of the invention, the toll-like agonist may include a TLR-9 agonist, for example, may include an immune stimulating oligonucleotide, but may not be limited thereto.

In one embodiment of the present invention, the immune stimulatory oligonucleotide may include one or more CpG motifs, but may not be limited thereto.

In one embodiment of the present invention, the saponin may be selected from the group consisting of QS21, QuilA, QS7, QS17, β-Eskin, Digitonin and combinations thereof, but may not be limited thereto.

In one embodiment of the present invention, the antiviral peptide may include KLK, but may not be limited thereto.

In one embodiment of the present invention, the influmersome inducer may be TDB (trehalose-6,6-dibehenate), but may not be limited thereto.

In one embodiment of the present invention, the NOD ligand may be M-TriLYS (NOD2 agonist-synthetic Muramil tripeptide) or NOD2 agonist (N-glycolylated muramyldipeptid), but may not be limited thereto.

In one embodiment of the present invention, the CDS ligand may be Poly (dA: dT), but may not be limited thereto.

In one embodiment of the present invention, the STING ligand may be cGAMP, di-AMP, or di-GMP, but may not be limited thereto.

In one embodiment of the invention, 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.

In one embodiment of the present invention, 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.

In one embodiment of the invention, 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.

In one embodiment of the present invention, 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. For example, 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.

In one embodiment of the present invention, by the cationic lipid, the electrostatic attraction with the anionic cell membrane is induced, the intracellular delivery efficiency of the immunomodulatory substance can be further improved.

According to an embodiment of the present invention, by constructing a multi-domain capsule containing a cationic lipid, 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. For example, 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. However, this may not be limited.

In one embodiment of the present invention, the cationic lipid is DC-cholesterol (3β- [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 (lipids1 , 2-dioleoyl-3-dimethylammonium-propane), and combinations thereof may be included, but may not be limited thereto.

According to one embodiment of the present invention, 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 ethoxylates such as the Tergitol ™ NP series; Polyoxyethylene patty ethers derived from lauryl, cetyl and oleyl alcohols (known as Brij surfactants) such as triethyleneglycol monolauryl ether (Brij 30); And sorbitan esters (commonly known as SPAN), such as sorbitan trioleate (Span85) and sorbitan monolaurate, alone or in combination of two or more surfactants. For example, 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%.

According to another aspect of the present invention, there is provided an immunomodulatory substance, comprising a multidomain capsule and an antigen according to the present invention .

In one embodiment of 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. For example, 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).

In one embodiment of the invention, 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. For example, the antigen may include, but is not limited to, an influenza-derived antigen or a cancer cell-derived antigen. For example, 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.

In one embodiment of the present invention, 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. In addition, after inducing the production of proteins related to intracellular stress by applying an anticancer agent or radiation in the actual cancer tissue, it may be prepared by lysing the cancer cells, but may not be limited thereto.

In one embodiment of the present invention, 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.

According to another aspect of the invention, 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. It includes, wherein 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.

In one embodiment of the present invention, 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. For example, 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.

In one embodiment of the present invention, the lipophilic immunomodulatory substance may be easily loaded into the multi-domain capsule by the flowable oil. For example, 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.

In one embodiment of the present invention, 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. For example, when squalene and oleic acid are used as the fluid oil, 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.

In one embodiment of the invention, 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. .

In one embodiment of the invention, 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.

In one embodiment of the invention, 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.

In one embodiment of the present invention, 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. For example, 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 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 ethoxylates such as the Tergitol ™ NP series; Polyoxyethylene patty ethers derived from lauryl, cetyl and oleyl alcohols (known as Brij surfactants) such as triethyleneglycol monolauryl ether (Brij 30); And sorbitan esters (commonly known as SPAN), such as sorbitan trioleate (Span85) and sorbitan monolaurate, alone or in combination of two or more surfactants.

For example, 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%.

According to another aspect of the present invention, 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. Wherein 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, and the aqueous phase comprises a second immune modulator, wherein the aqueous phase is the An inner aqueous solution phase of the liposome membrane and an outer aqueous solution phase of the liposome membrane, wherein the first immunomodulatory substance and the second immunomodulatory substance are immunosuppressive factor controlling substances, and 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.

In addition, according to another aspect of the present invention, an immunomodulatory substance comprising the multidomain capsule and the antigen may be provided.

In addition, 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. It includes, wherein 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.

In the present invention, 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.

According to an embodiment of the present invention, 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 In addition, it is possible to prepare an immunomodulatory multidomain capsule having a micro-sized capsule form having improved structural stability of a plurality of liposomes linked by the introduced fluid oil component. In addition, according to one embodiment of the present invention, 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 according to an embodiment of the present invention, 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.

In addition, 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. By loading factor control substances, the effective duration of the immunoactivator can be increased.

Multi-domain capsule according to an embodiment of the present invention, 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 according to an embodiment of the present invention, 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 In addition, it is possible to increase the effective duration of immunosuppressive factor control substances that can control the function of immunosuppressive cells and immunosuppressive agents.

In one example of the present invention, drugs capable of controlling the function of Meloidoid-Derived Suppressor Cell (MDSC), 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.

In one example of the invention, 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.

In one example of the present invention, PI3Kdelta inhibitors are AMG-319, Idelalisib, TRG-1202, INCB050465, IPI-145, Duvelisib, Acalisib, TG-1202, RV1729, RP-6530, GDC-0032. .

In one example of the present invention, the PI3Kgamma inhibitors are characterized by IPI-549, IPI-145, and the like.

In one example of the present invention, drugs capable of controlling the function of Treg (Regulatory T cell), that is, immunosuppressive factor controlling substances include Anti-CD25 antibodies (daclizumab), Basiliximab, LMB-2, Denileukin diftitox (Ontak), Bivalent IL-2 fusiontoxin, Anti-TGF-beta antibodies, fresolimumab, TGF-betaR kinase inhibitors, LY2157299, Soluble TGF-betaR I / II, Ipilimumab, Tremelimumab, Pembrolizumab, Nivolumab, TIM-3 antibodies, LAG-3 antibodies, Anti- CD39 antibodies, Anti-73 antibodies, A (2A) R inhibitors, Celecoxib, Indomethacin, Diclofenac, Ibuprofen, TNFR2 antibodies, Anti-GITR antibodies, Bevacizumab, Anti-OX40 (CD134) antibodies, soluble GITR ligand, Blockades for chemokine receptors ( CCR4, 5, 6,10), cyclophosphamide, Sunitinib, Fludarabine, PI3K p110 (delta) inhibitors, CliniMACs, Mogamulizumab, Fingolimod, Regulators for miRNA (miR-155, miR-146a, miR-181a), 5-aza-2 -deoxycytidine, paclitaxel, Imatinib, Sorafenib, Cyclosporin A, Tacrolimus, Dasatinib, Poly-G-oligonucleotide, TLR8 ligands, gemcit abine and 5-fluorouracil, but are not limited thereto.

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.

In addition, as 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.

In addition, as 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. One feature, but is not limited to such.

In addition, drugs that can inhibit the mechanism of M2 macrophage-based cancer cell growth, i.e. immunosuppressive factors, include sunitinib, sorafenib, WP1066, corosolic acid, oleanolic acid, STAT6 inhibitors and the M2 pathway (c-). Myc, PPAR-alpha / gamma, PI3K, KLF4, HIFs, Ets2, DcR3, mTOR) inhibitors and HRG, CuNG, MDXAA, Silibinin, PPZ, but is not limited thereto.

And, 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 (BLZ945, PLX3397, Emactuzumab (RG7155), AMG-820, IMC-CS4 , GW3580, PLX6134) and ligands of toll like receptor 7 (imiquimod, 852A), NF-kB inhibitors (N-acetyl-l-cystein, Vitamin C, bortezomib, aspirin, salicylates, Indolecarboxamide derivatives, quinazolineanalogues, Thalidomide, prostaglandin metabolites) , HIF-1 inhibitors (2ME2, 17-AAG, Camptothecin, Topotecan, Pleurotin, 1-methylpropyl, 2-imidazolyl dissulphide (YC-1), CXCR4 agonist (AMD3100, AMD1498), ALX40-4C, T22, T140, CGP64222, KRH-1636), but is not limited thereto. .

One example of the present invention is transforming growth factor beta (TGF-beta) inhibitors, Nitro aspirin, Cycloxygenase-2 (COX2) inhibitors, Indoleamine 2,3-dioxygenase (IDO) inhibitors, Phosphodiesterase-5 ( Multi-domain capsule-based compositions containing PDE-5) inhibitors, Anti-Interleukin 10 (IL-10)) drugs can be provided.

In one example of the present invention, TGF-beta inhibitors include, but are not limited to, SB-505124, LY-364974, and the like.

In one example of the invention Nitro aspirin includes, but is not limited to, NCX 4040 and the like.

In one example of the invention COX-2 inhibitors include, but are not limited to, Celecoxib.

In one example of the present invention, IDO inhibitors include, but are not limited to, Indoximod, NLG919, and the like.

In one example of the present invention, PDE-5 inhibitors include, but are not limited to, Tadalafil (Cialis) and the like.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, 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.

In one example of the invention 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.

In one example of the present invention Anti-TIM-3 antibody includes, but is not limited to, TSR-022.

In one example of the present invention, 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.

In one example of the invention Anti-OX40 includes, but is not limited to, RG7888 and the like.

In one example of the present invention Anti-CD137 includes, but is not limited to, Urelumab.

In one example of the invention 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. To provide.

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.

As an example of the epigenetic mechanism in the present invention, 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.

As an example of the epigenetic mechanism in the present invention, 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, Psammaplin A, Nexturastat A, RGFP966 and the like, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to examples of the present invention, but the following examples are merely illustrated to aid the understanding of the present invention, and the contents of the present invention are not limited to the following examples.

실시예 1. 면역조절물질을 포함하는 다중도메인캡슐 제조 및 특성화Example 1 Preparation and Characterization of Multiple Domain Capsules Containing Immunomodulators

In the embodiment of the present invention, a multi-domain capsule was prepared as follows.

1-1. 스쿠알렌 기반의 다중도메인캡슐(imMDV(SQ)) 제조 및 특성화1-1. Preparation and Characterization of Squalene-based Multidomain Capsules (imMDV (SQ))

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. In addition, a control group was prepared together in the same manner as in the above example, except that squalene was not included.

As a result of observing the structure of the multi-domain capsule using optical microscope and DLS (dynamic light scattering, Otsuka), 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)).

3A to 3C are optical microscope images of multidomain capsules containing squalene, and FIGS. 3D to 3F are optical microscopes of multidomain capsules not containing squalene. Image. As shown in 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.

For the post-production stability analysis, when the centrifugation (about 2500 rpm) process is performed to remove impurities or to classify the multidomain capsules by size, the structure of the multidomain capsule including squalene [FIG. a) and (c)] showed a stable structure almost unchanged before and after centrifugation, but it was confirmed that most of the structures were destroyed in the control group containing no squalene (FIGS. 4B and 4). d)].

1-2. 스쿠알렌 기반의 MPLA를 포함하는 다중도메인캡슐(imMDV-1:imMDV(MPLA)) 제조 및 특성화1-2. Preparation and Characterization of Multidomain Capsules (imMDV-1: imMDV (MPLA)) Including Squalene-based MPLA

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.

Evaluation of immune cell activity effect of prepared multidomain capsule

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-α, IL-6, IL-12) using the ELISA test method.

When treated with imMDV (SQ) in Figure 6, it was confirmed that the secretion of TNF-α and IL-6 increases in proportion to the treated concentration, and also when treated with imMDV (MPLA) in Figure 7 TNF-α , IL-6, IL-12 secretion was increased in proportion to the concentration of the multi-domain capsules treated.

Preparation and Characterization of Multiple Domain Capsules (imMDV) Containing Squalene-based Antigen (Orbalbumin)

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. In addition, a control group was prepared together in the same manner as in the above example, except that squalene was not included.

As a result of confirming the release behavior of the multidomain capsule containing ovalbumin antigen and squalene (imMDV (SQ-OVA)) and the multidomain capsule containing only ovalbumin antigen (imMDV (OVA)), in the multidomain capsule containing squalene It was confirmed that the release behavior of the ovalbumin antigen was slowly released [Fig. 8].

1-3. 스쿠알렌 기반의 DDA를 포함하는 다중도메인캡슐(imMDV-2) 제조1-3. Preparation of multidomain capsules (imMDV-2) containing squalene-based DDA

DOPC (10 mg), cholesterol (8 mg), DDA (10 mg, Avanti Polar Lipids, USA), squalene (12 mg), glycerol trioleate (12 mg) in chloroform (1 mL) It was dissolved in to prepare an oily solution. Thereafter, the supernatant was removed by sinking in a centrifuge 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.

1-4. 스쿠알렌 기반의 MPLA/TDB를 포함하는 다중도메인캡슐(imMDV-3) 제조1-4. Preparation of multidomain capsules (imMDV-3) containing squalene-based MPLA / TDB

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.

1-5. 스쿠알렌 기반의 MPLA/DDA를 포함하는 다중도메인캡슐(imMDV-4) 제조1-5. Preparation of multidomain capsules (imMDV-4) containing squalene-based MPLA / DDA

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. 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.

1-6. 스쿠알렌 기반의 MPLA/QS21을 포함하는 다중도메인캡슐(imMDV-5) 제조1-6. Preparation of multidomain capsules (imMDV-5) containing squalene-based MPLA / QS21

DOPC (10 mg), cholesterol (8 mg), MPLA (10 mg), QS21 (10 mg, Desert King, USA), squalene (12 mg), glycerol trioleate (12 mg) in chloroform (1 mL) It was dissolved 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.

1-7. 스쿠알렌 기반의 CpG를 포함하는 다중도메인캡슐(imMDV-6) 제조1-7. Preparation of multidomain capsules (imMDV-6) containing squalene-based CpG

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.

1-8. 스쿠알렌 기반의 Poly(I:C)를 포함하는 다중도메인캡슐(imMDV-7) 제조1-8. Preparation of multidomain capsules (imMDV-7) containing squalene-based Poly (I: C)

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.

1-9. 스쿠알렌 기반의 1-9. Squalene based 레스퀴모드를Resquid mode 포함하는 다중도메인캡슐( Containing multiple domain capsules ( imMDVimMDV -8) 제조-8) manufacturing

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.

1-10. 스쿠알렌 기반의 1-10. Squalene based 이미퀴모드를Imiquimod 포함하는 다중도메인캡슐( Containing multiple domain capsules ( imMDVimMDV -9) 제조 및 면역반응 유도효과 확인-9) Confirmation of the induction effect of manufacture and immune response

DOPC (10 mg), cholesterol (8 mg), squalene (12 mg), oleic acid (2 mg, Sigma-Aldrich, USA), imiquimod (base form, Sigma-Aldrich, USA) (5 mg), glycerol tree 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. 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. 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. The solution is stirred in the range of 60 to 65 degrees until all of the imiquimod is dissolved, and after all the imiquimod is dissolved, it is cooled down to 20 to 25 degrees and maintained for about 30 minutes. The remaining solution is dried in a room temperature dryer to obtain Hcl Imiquimod. The HCl-imiquimod thus prepared was dissolved in an internal aqueous solution in the imMDV manufacturing process, and then prepared through the same process. 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. In the obtained multidomain capsules (imMDV (R837-HCl)), the release behavior of imiquimod was analyzed at 37 ° C. using a transwell. 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. In addition, when imMDV (R837-HCl) samples were treated with bone marrow-derived dendritic cells (BMDCs), the secretion of representative pro-inflammatory cytokines IL-6 related to the Th1 immune response was determined by ELISA. ) Was analyzed using the experimental method. As shown in FIG. 11, it was confirmed that the secretion of IL-6 was increased in proportion to the treated concentration, and 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.

Antibody-forming effect of the multi-domain capsules [imMDV (R837-HCl), imMDV (R837-base), imMDV [R837-HCl: R837-base] containing ovalbumin model antigens prepared in Example Were validated through mouse experiments (C57BL / 6, 6-7 week old females). Increasing humoral immune response by injecting 50 μg of multi-domain capsules into mice was measured by ELISA (Enzyme linked Immunosorbent assay).

As can be seen in Figures 12a, 12b, and 12c, 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. In addition, 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). In comparison with the oil-induced adjuvants and sustained effect of the multi-domain capsule-based adjuvant compared to the oil-type adjuvant (DMSO (R837)) used in the clinical stage, it can be confirmed that it is very excellent (Fig. 22). Above all, the greatest advantage is that the inflammatory phenomenon caused by the administration of oil-type adjuvant (DMSO (R837)) does not occur at all when the multi-domain capsule-based adjuvant (imMDV (R837-HCl)) is used. (Figure 23).

1-11. 스쿠알렌 기반의 STING (cyclic DNA)을 포함하는 다중도메인캡슐(imMDV-10) 제조1-11. Preparation of multidomain capsules (imMDV-10) containing squalene-based STING (cyclic DNA)

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.

1-12. 스쿠알렌 기반의 MPLA/CpG를 포함하는 다중도메인캡슐(imMDV-11) 제조1-12. Preparation of multidomain capsules (imMDV-11) containing squalene-based MPLA / CpG

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.

1-13. 스쿠알렌 기반의 MPLA와 Poly(I:C)를 포함하는 다중도메인캡슐(imMDV-12) 제조1-13. Preparation of multidomain capsules (imMDV-12) containing squalene-based MPLA and Poly (I: C)

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.

1-14. 스쿠알렌 기반의 CpG/Poly(I:C)를 포함하는 다중도메인캡슐(imMDV-13) 제조1-14. Preparation of multidomain capsules (imMDV-13) containing squalene based CpG / Poly (I: C)

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.

1-15. 캐스터 오일 기반의 MPLA를 포함하는 다중도메인캡슐(imMDV-14) 제조1-15. Preparation of multidomain capsules (imMDV-14) containing castor oil based MPLA

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.

1-16. 미네랄 오일 기반의 MPLA를 포함하는 다중도메인캡슐(imMDV-15) 제조1-16. Preparation of multi-domain capsules (imMDV-15) containing mineral oil-based MPLA

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.

실시예 2. 바이러스 항원에 대한 다중도메인캡슐의 면역 향상 효능 평가Example 2 Evaluation of Immune Improvement Efficacy of Multi-Domain Capsules Against Viral Antigens

In order to investigate the specific immunity effect against the avian influenza virus of the multidomain capsule samples comprising the immunomodulatory substance prepared in Example 1, during antibody specific immune response, in particular by B cells involved in antibody production The effect on the humoral immune response was investigated. First, 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.

Two weeks after the first intramuscular injection (FIG. 24) and four weeks (FIG. 25), mouse serum was collected and antibody titers against HA proteins in serum were measured by an enzyme linked immunosorbent assay (ELISA). In the ELISA method, 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. After the reaction was developed by adding 100 μL of TMB (tetramethylbenzidine, BD biosciences, USA) as a substrate, the absorbance at 450 nm was measured with an ELISA reader and the results are shown in FIGS. 24 and 25.

실시예 3. 암 항원에 대한 다중도메인캡슐의 면역 향상 효능 평가Example 3 Evaluation of Immune Improvement Efficacy of Multi-Domain Capsules Against Cancer Antigens

3-1 : 다중도메인캡슐의 OVA 특이 항체 생성 확인3-1: Confirmation of OVA Specific Antibody Production of Multi-Domain Capsules

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 µg 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.

3-2: 다중도메인캡슐의 세포매개 특정 T 세포 반응 확인3-2: Confirmation of Cell Mediated Specific T Cell Response of Multi-Domain Capsules

The cellular immune response of T cells in mouse spleens by multidomain capsules containing immunomodulatory substances was investigated. Among the 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. Transfer all the contents of the petri dish to a 50 mL tube, fill with RPMI medium, centrifuge at 1,500 rpm for 5 minutes, and add 5 mL of erythrocyte elution buffer (sigma Aldrich, Germany) to the pellet from which the supernatant was removed. Red blood cells were hemolyzed by standing in a water bath for 5 to 10 minutes. The cells contained in the tube were washed with PBS, and then suspended in RPMI medium to separate splenocytes. The isolated splenocytes were plated in IFN-gamma coated plates in 96-well 5 × 10 ⁵ cells / 100 μL and treated with MHC class I-restricted OVA peptide at a concentration of 5 μg / mL for 48 hours. Then, IFN-gamma with horseradish peroxide was added. As a substrate, the reaction was developed by adding 100 μL of ACE (3-amino-9-ethyl-carbazole, BD biosciences, USA) and measured by ELSPOT (enzyme-linked immunospot) method (FIG. 27).

실시예 4. 고형암 미세환경 조절용 약물이 로딩된 다중도메인 캡슐 제조Example 4 Preparation of Multi-Domain Capsules Loaded with Drugs for Control of Solid Cancer Microenvironment

4-1. MDSC 세포 제거 물질을 함유하는 다중도메인 캡슐 제조4-1. Preparation of Multidomain Capsules Containing MDSC Cell Scavenger

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. 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 a multidomain capsule (imMDV (SQ-Gem)). Gemstabin-loaded multidomain capsules (imMDV (OA-Gem)) containing oleic acid oil instead of squalene fluid oil and gemsatabin-loaded multidomain capsules (imMDV (Gem)) containing no squalene Figure 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).

4-2. 면역학적 세포 사멸(4-2. Immunological cell death ( immunogenicimmunogenic cell death)을 유도하는 약물을 포함하는 다중도메인 캡슐 제조 Preparation of multidomain capsules containing drugs that induce cell death

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.

4-3. Treg 세포 제거 물질을 함유하는 다중도메인 캡슐 제조4-3. Preparation of Multidomain Capsules Containing Treg Cell Remover

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).

4-4. PI3K 신호전달을 제어할 수 있는 약물을 함유하는 다중도메인 캡슐 제조4-4. Preparation of Multidomain Capsules Containing Drugs That Can Control PI3K Signaling

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).

4-5. 4-5. 에피제네틱Epigenetic 기전(epigenetic machinery)을 제어할 수 있는 약물을 포함하는 다중도메인 캡슐 제조 Preparation of multidomain capsules containing drugs capable of controlling epigenetic machinery

Among the drugs capable of inducing epigenetic mechanisms, Azacytidine, Resminostat, Panobinostat and OTX015 (iBET) were selected to prepare multidomain capsules loaded with these drugs. Specifically, imMDV (Azacytidine) (FIG. 37), imMDV (Resmonostat) (FIG. 38), imMDV (Panobinostat) (FIG. 39) and imMDV (OTX015 (iBET)) using the same method as in Example 4-1 ( 40) prepared the multidomain capsules by adding each drug to the internal aqueous phase. As can be seen in Figures 38 and 39, the loaded drug was confirmed to be slowly released over two weeks.

4-6. TAM 세포 제거 물질을 함유하는 다중도메인 캡슐 제조4-6. Preparation of Multidomain Capsules Containing TAM Cell Remover

Using the same process as in Example 4-1, but dissolved in the oil phase BLZ945 (CSF-1R kinase inhibitor), a drug capable of removing TAM cells, multi-domain capsules were prepared (Fig. 41).

4-7. 종양 면역억제 사이토카인 저해제(inhibitor)를 함유하는 다중도메인 캡슐 제조4-7. Preparation of multidomain capsules containing tumor immunosuppressive cytokine inhibitors

After dissolving 5 mg of the tumor immunosuppressive cytokine inhibitor drug (Celecoxib, Sigma-Aldrich) in the oil phase in Example 4-1, multidomain capsules were prepared (FIG. 42).

실시예 5. 고형암 미세환경 면역조절물질이 조합된 다중도메인 캡슐 제조Example 5 Preparation of Multidomain Capsules Combining Solid Cancer Microenvironmental Immunomodulators

As an example of the preparation of a multi-domain capsule in which a substance modulating immune function in the solid cancer microenvironment is combined, gemcitabine (Example 4-1) 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).

In addition, a multi-domain having a stable structure containing BLZ945 (Example 4-6), 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).

The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. . Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form. The scope of the present invention is shown by the following claims rather than the above description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention. do.

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. In addition, 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.

Claims

A multidomain capsule comprising at least two liposomes in contact with and connected to each other, and a multidomain capsule outer wall surrounding the at least two liposomes,

The multi-domain capsule is composed of an organic phase and an aqueous phase,

The organic phase comprises a first immune modulator and a flowable oil, the organic phase forming a membrane of the liposome and an outer wall of the multidomain capsule,

The aqueous phase includes a second immunomodulatory substance, wherein the aqueous phase is an inner aqueous phase of the liposome membrane and an outer aqueous 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 flowable oil is characterized in that to improve the structural stability of two or more liposomes in contact with and connected to each other, multi-domain capsule.
According to claim 1, wherein the size of the multi-domain capsule is 1 ㎛ to 100 ㎛, multi-domain capsule.
The multidomain capsule of claim 1, wherein the flowable oil comprises one selected from the group consisting of animal oils, vegetable oils, tocopherols, mineral oils, castor oils, and combinations thereof.
4. The multidomain capsule of claim 3, wherein the animal oil is squalene and the vegetable oil is oleic acid.
The multidomain capsule according to claim 1, wherein the fat-soluble and water-soluble immunoactive substance comprises a substance which induces the activation of antigen presenting cells, B cells, or T cells.
The method of claim 1, wherein the fat-soluble immunoactive substance is cationic lipid, MPLA, AGP, CRX-527, PHAD, 3D-PHAD, GLA, lipid peptide, Pam3Cys, Pam3Cys-Lip, imiquimod (base form), res A multidomain capsule comprising a substance selected from the group consisting of quimod (base form), VTX-2337, CRX642, saponin (QS21), TDB, CL401, CL429, and combinations thereof.
The method of claim 1, wherein the water-soluble immunoactive material is CpG, imiquimod (HCl form), resquimod (HCl form), Poly (I: C), STING, flagellin (flagellin), saponin, KLK peptide, A multidomain capsule comprising a material selected from the group consisting of NOD agonist peptides, Poly (dA: dT), and combinations thereof.
The multidomain capsule of claim 6, wherein the cationic lipid comprises a substance selected from the group consisting of DC-cholesterol, DDA, DOTAP, DOTMA, EPC, MVL5, DODAP, and combinations thereof.
An immunomodulatory substance comprising a multidomain capsule and an antigen according to any one of claims 1 to 8.
The immunomodulatory substance of claim 9, wherein the antigen is selected from the group consisting of proteins, genes, cells, viruses, and combinations thereof.
Dissolving the first immunomodulator and fluid oil in a solvent to prepare an oily 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 water-in-oil emulsion with a second aqueous solution and evaporating the solvent; Including,

The first immunomodulatory substance is a fat-soluble immunoactive substance, and the second immunomodulatory substance, characterized in that the water-soluble immunoactive substance, method for producing a multi-domain capsule.