KR20220129939A - A composition for preventing or treating cancer comprising an anticancer compound and exosomes as active ingredients - Google Patents

Abstract

The present invention relates to a composition for preventing or treating cancer comprising an anticancer compound and exosomes as active components. Specifically, the present invention relates to a composition for treating cancer comprising an immune checkpoint inhibitor and exosomes obtained from pre-treated mesenchymal stem cells as active components.

Description

Composition for preventing or treating cancer comprising anticancer compound and exosome as effective component

The present invention relates to a composition for preventing or treating cancer comprising an anticancer compound and exosomes as active ingredients.

Immunotherapy is a drug that prevents cancer cells from evading the body's immune system or allows immune cells to better recognize and attack cancer cells. Anticancer immunotherapy is a treatment that effectively responds to cancer through cancer-specific immunity formation. . However, since cancer cells have the ability to hide themselves from immune cells, even immuno-oncology drugs are only effective in some cancer patients.

Mesenchymal stem cell (MSC) is a stem cell suitable for various clinical applications such as tissue regeneration and reduction of immune rejection during transplantation. It has been reported that the effect is not in the cells themselves, but is a paracrine effect caused by a specific substance secreted by mesenchymal stem cells. Most of the secreted substances having therapeutic effects are vesicles called exosomes.

An exosome is a vesicle with a size of 30-400 nanometers (nm) derived from a cell, and a multivesicular body, a vesicle during the maturation of an endosome, fuses with the cell membrane and is discharged. . The discharged exosomes contain various proteins, nucleic acids, lipids, etc., and can be fused with other cells to deliver the contents. Exosomes have been found to perform important functions in processes such as blood coagulation, intercellular communication, and waste disposal. Various clinical studies such as diagnosis, prognosis, and treatment using exosomes are being attempted.

On the other hand, Korea Patent Application Publication No. 2019-0037163 discloses a 'new recombinant plasma membrane-based ER for cancer treatment', which relates to a recombinant protoplast-based ER containing a VSV-G mutant protein in which histidine, the 162nd amino acid, is substituted with arginine. and Korean Patent Application Laid-Open No. 2018-0035700 discloses 'a pharmaceutical composition for treating drug-resistant cancer comprising exosomes extracted from differentiated stem cells as an active ingredient', but the anticancer compound and exosomes of the present invention There is no description of a composition for preventing or treating cancer comprising as an active ingredient.

The present invention has been derived from the above needs, and the present inventors can treat the exosomes obtained from pre-treated mesenchymal stem cells with an immune checkpoint inhibitor anti-PD-1 or anti-PD-L1 antibody. In this case, the present invention was completed by confirming the significantly increased tumor growth inhibitory effect compared to treatment with the immune checkpoint inhibitor alone.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an anticancer compound and exosomes as active ingredients.

In addition, the present invention provides a method for treating cancer in mammals other than humans, comprising administering a pharmaceutically effective amount of a pharmaceutical composition for the prevention or treatment of cancer to a mammal other than a human suffering from cancer do.

The anticancer compound according to the present invention, in particular, a composition comprising an immune checkpoint inhibitor and an exosome as an active ingredient can effectively inhibit tumor growth and induce the activity of dendritic cells and T cells, so it is useful for cancer treatment could be utilized.

1 is a result of analyzing the dendritic cell (DCs) activity-inducing effect of exosomes. CTRL: control exosomes obtained from non-pre-treated mesenchymal stem cells, Exosome: exosomes obtained from pre-treated mesenchymal stem cells. Exosome unit 'x 10 ⁹ /ml' means 'x 10 ⁹ particles/ml'.
Figure 2 is a result of analyzing the dendritic cell (DCs) activity-inducing effect according to the administration route (route) of the exosome.
3 is an evaluation of the effect of exosomes on the immune checkpoint inhibition-mediated anticancer effect, and on the 7th day after tumor transplantation, immune checkpoint inhibitors (anti-PD-1 Ab, anti-PD-L1 Ab), exosomes (CTRL: Control exosomes obtained from non-pre-treated mesenchymal stem cells, exosomes (exosomes obtained from pre-treated mesenchymal stem cells) or a combination thereof, and measurement of tumor size and weight changes .
4 is a result of analysis of the activity of cancer antigen-specific T cells according to the complex treatment of anti-PD-1 Ab and exosomes obtained from pre-treated mesenchymal stem cells.
Figure 5 confirms the effect of exosomes inducing human peripheral blood dendritic cell activity.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for treating cancer comprising an anticancer compound and exosomes as active ingredients.

In the pharmaceutical composition according to the present invention, the anti-cancer compound may be an immune checkpoint inhibitor, preferably a PD-1/PD-L1 interaction inhibitor, and the PD-1/PD-L1 interaction inhibitor is more Specifically, it may be an antibody targeting PD-1 or PD-L1 or a functional fragment of the antibody or a single chain-based antibody analog.

The antibody targeting PD-1 or PD-L1 may be, but is not limited to, Pembrolizumab, Nivolumab, Atezolizumab or Avelumab, The single chain-based antibody analog may be, but is not limited to, scFv, sdAb, diabody, monobody, variable lymphocyte receptor (VLR) or nanobody.

As used herein, the term "immune checkpoint inhibitor" refers to a type of drug that blocks certain proteins produced by certain types of cells of the immune system such as T lymphocytes and some cancer cells, and these proteins are immune responses inhibits and prevents T lymphocytes from killing cancer cells. So when these proteins are blocked, the "brake" of the immune system is released and T lymphocytes are better able to kill cancer cells.

In addition, in the pharmaceutical composition according to the present invention, the exosome may be derived from pre-conditioning mesenchymal stem cells, more preferably pre-treated human adipose-derived mesenchymal stem cells It may be derived from, but is not limited thereto.

In the pharmaceutical composition according to an embodiment of the present invention, when the exosomes derived from the pre-treated mesenchymal stem cells are treated with an anti-cancer compound, excellent tumor growth inhibition compared to the exosomes alone or treated with an anti-cancer compound alone showed the effect. In addition, it was found that exosomes derived from non-pre-treated mesenchymal stem cells had no effect on the tumor growth inhibitory effect compared to treatment with the anticancer compound alone even when treated with the anticancer compound.

In the present invention, the "pre-conditioning (pre-conditioning or pre-condition)" refers to exposing cells to specific conditions during culturing of mesenchymal stem cells, and the specific conditions include compounds, biomaterials, culture temperature, oxygen concentration, It may be a change in carbon dioxide concentration, etc., but is not limited thereto, and various states and changes that may occur in mesenchymal stem cells, such as proliferation, stimulation, stress, undifferentiation, differentiation, activity, death, and increase in secreted substances, of mesenchymal stem cells Includes any substance and/or environment that can cause, inhibit, or maintain at a certain level. In the present invention, it is intended to maximize the effect of exosomes secreted from mesenchymal stem cells through the pre-treatment.

In the pharmaceutical composition according to the present invention, the "pre-treatment" is cobalt chloride, palmitate, beta-caryophyllene, deferoxamine when culturing mesenchymal stem cells. , polyI:C (Polyinosinic:polycytidylic acid), MPLA (Monophosphoryl Lipid A), Pam3CSK4, zymosan, HMGB1 (High mobility group box 1 protein) or a combination thereof may be performed by treatment, but is limited thereto. doesn't happen

In the pharmaceutical composition according to an embodiment of the present invention, the pre-treatment may be preferably cobalt chloride treatment, more preferably 50 to 150 μM cobalt chloride treatment, even more preferably 100 It may be cobalt chloride treatment of μM, but is not limited thereto.

Cobalt chloride is an inorganic compound of cobalt and chlorine having the chemical formulas CoCl ₂ , CoCl ₃ . It is readily soluble in water and soluble in alcohol and acetone. Chloride of cobalt chloride is also used to form a hypoxia-like environment in stem cell culture.

Palmitate is a saturated fatty acid salt consisting of 16 carbons, a salt or ester of palmitic acid, and is a form of palmitic acid observed at physiological pH 7.4. Palmitic acid (or hexadecanoic acid) is the most common saturated fatty acid found in animals, plants, and microorganisms, and contains 16 carbons, and the chemical formula is CH ₃ (CH ₂ ) ₁₄ COOH, It has a molecular weight of 256.43 g/mol, a melting point of 62.65°C, and a boiling point of 351.5°C. It is insoluble in water, but soluble in alcohol and ether. It is widely distributed in the animal and plant kingdom along with stearic acid and oleic acid, and is contained in most oils and fats, especially in large amounts in wood wax or palm kernel oil.

Beta-caryophyllene is a sesquiterpene contained in essential oils of many spices and plant foods. It has the chemical formula C ₁₅ H ₂₄ with 15 carbon atoms and 24 hydrogen atoms. . Beta-caryophyllene is known to have an anti-inflammatory effect, and its mechanism of action is reported to be a selective agonist of the cannabinoid receptor CB2 [Proc Nat Acad Sci USA., (2008) 105(26), 9099- 9104]. In experiments using human monocytes, beta-caryophyllene binds to CB2 and inhibits adenylate cyclase, causes intracellular calcium transients, and mitogen-activated kinases ), Erk1/2 and p38 were weakly activated.

Deferoxamine is a polyhydroxamate iron chelator useful for reducing iron concentrations in human plasma. The chemical name for deferoxamine is N'-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N-(5 -Aminopentyl)-N-hydroxy-butanediamide. Deferoxamine possesses the desirable properties of a very low affinity for calcium (Ka = 10 ² ) with a high affinity for ferric iron (Ka = 10 ³¹ ). In addition, deferoxamine is used to induce a hypoxia-like environment in mesenchymal stem cells.

polyI:C (Polyinosinic:polycytidylic acid) is known to interact with toll-like receptor 3 (TLR3) expressed in the membranes of B cells, macrophages and dendritic cells. polyI:C is structurally similar to double-stranded RNA, which is present in some viruses and is a natural stimulator of TLR3. Therefore, Poly I:C can be regarded as a synthetic analogue of double-stranded RNA, and is used as a general tool for the study of the immune system.

Monophosphoryl Lipid A (MPLA) is an attenuated derivative of the lipid A component of bacterial lipopolysaccharide (LPS). LPS and Lipid A are potent immunostimulants that induce humoral antibody responses and cell-mediated immune responses in patients administered with the respective compounds, but Lipid A and LPS are also pyrogenic and local Schwarzman responses. toxic side effects such as MPLA is a lipid A-like molecule modified to attenuate the toxicity of LPS. Monophosphoryl lipid A, a derivative of attenuated lipid A, is used as an immune adjuvant in prophylactic vaccines against infectious diseases and therapeutic vaccines for the treatment of cancer tumors and chronic infections.

Pam3CSK4 is a synthetic triacylated lipopeptide (LP) with an analogue of the acylated amino terminus of a bacterial lipopeptide (LP). Pam3CSK4 is a potent activator of the proinflammatory transcription factor NF-kB, whose activation is mediated by an interaction between TLR2 and TLR1, which recognizes LPs with three fatty acids, a structural feature of bacterial LPs. Several TLR2 agonists, particularly lipopeptides, have been evaluated as vaccine adjuvants. Pam3CSK4 has proven to be a potent adjuvant to a variety of vaccines, including sublingual allergy vaccines, flu vaccines and leishmaniasis vaccines. In preclinical studies, Pam3CSK4, unlike other TLR ligands, has been reported to increase the antibody response to influenza antigens. It has a strong local response and has been shown to enhance IgG2a and IgG1 titers and upregulate proinflammatory and Th1 cytokine genes.

Zymosan is a glucan with repeating glucose units linked by β-1,3-glycosidic bonds and binds to TLR 2 and Dectin-1 (CLEC7A). Zymosan is a ligand found on the surface of fungi such as yeast. Zymosan is produced in the yeast cell wall and consists of a protein-carbohydrate complex and is used to induce experimental sterile inflammation. In macrophages, the responses induced by zymosan include induction of proinflammatory cytokines, arachidonate recruitment, protein phosphorylation and inositol phosphate formation. Zymosan A is also known to play a role in macrophage activation in addition to proliferation by raising cyclin D2 levels.

High mobility group box 1 protein (HMGB1) belongs to a family of high mobility group (HMG) proteins. HMGB1 protein, called HMG because of its high electrophoretic mobility in polyacrylamide gels, is the most ubiquitous non-histone protein associated with chromatin isolated from eukaryotic cells. Because this protein twists, bends, or modifies DNA structure and forms complexes with transcription factors or histones, it is commonly referred to as "constitutive" in DNA bending, looping, folding and wrapping. architectural)" role. HMGB1 protein is typically a nuclear factor (nuclear factor), in particular, a transcriptional regulatory molecule that induces DNA bending and promotes the binding of several transcriptional complexes. Extracellularly secreted HMGB1 acts as a potent cytokine and a very potent macrophage-stimulating factor. HMGB1 binds directly to the cell membrane, induces signaling and chemotaxis, has a chemokine-like function, and acts indirectly by upregulating the expression and secretion of pro-inflammatory cytokines. . Thereby, the extracellular HMGB1 protein becomes a potent chemotactic and immunomodulatory protein that promotes an effective inflammatory immune response.

In the pharmaceutical composition according to the present invention, the cancer is colorectal cancer, laryngeal cancer, lung cancer, esophageal cancer, pancreatic cancer, liver cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, breast cancer, cervical cancer, ovarian cancer, kidney cancer, gallbladder cancer, oral cancer, It may be one selected from the group consisting of colon cancer, liver cancer, and bladder cancer, preferably colon cancer, but is not limited thereto.

The pharmaceutical composition according to the present invention may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively. have.

Specifically, in the case of formulation, it may be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, etc. normally used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient for the anticancer compound and exosomes, for example, starch, calcium carbonate, It may be prepared by mixing sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally. When administered parenterally, it is possible to administer it through various routes such as intravenous injection, intranasal inhalation, intramuscular administration, intraperitoneal administration, and transdermal absorption.

In addition, the pharmaceutical composition of the present invention may be administered in combination with other anticancer agents, and may be administered sequentially or simultaneously with conventional anticancer agents.

The present invention also provides a method for treating cancer in a mammal other than a human, comprising administering a pharmaceutically effective amount of a pharmaceutical composition for the treatment of cancer to a mammal other than a human suffering from cancer.

In the method for treating cancer in the present invention, the pharmaceutical composition for treating cancer includes an anti-cancer compound and an exosome as active ingredients, and the anti-cancer compound and the exosome are the same as described above.

In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level refers to the type and severity of the subject, age, sex, and activity of the drug. , sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered in a dose of 0.1 mg/kg to 1 g/kg, and more preferably in a dose of 1 mg/kg to 500 mg/kg. Meanwhile, the dosage may be appropriately adjusted according to the age, sex and condition of the patient.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. Preparation of stem cell-derived exosomes

Human adipose-derived mesenchymal stem cells were purchased from ATCC (American Type Culture Collection). The mesenchymal stem cells were periodically subcultured and maintained in an incubator maintained at 37° C. and 5% carbon dioxide using DMEM (Gibco ^® ) medium containing 10% FBS (fetal bovine albumin) and antibiotics. In order to obtain exosomes, the mesenchymal stem cells were aliquoted in a 100 mm culture dish at a concentration of 5x10 ⁹ cells and cultured for 2 days, and then cobalt (II) chloride hexahydrate (CoCl ₂ ) at a concentration of 100 μM in the medium. ·6H ₂ O) was added and cultured for 24 hours, and then replaced with 10 ml of DMEM medium containing no serum and cultured for 1 day. Thereafter, the culture medium was recovered and centrifuged at 300x g for 10 minutes to remove the residue, and exosomes were obtained using Total Exosome Isolation (from cell culture media) reagent (Invitrogen, cat no.4478359). Exosomes cultured and obtained under the same conditions without treatment with the cobalt(II) chloride·hexahydrate were used as control exosomes (CTRL). The concentration of the obtained exosomes was confirmed through a nanoparticle tracking analysis.

2. Cancer Cell Lines and Experimental Animals

BLAB/c mice were purchased from Orient Bio (Korea). Purchased mice were raised under sterile conditions of 22-24°C and 50-60% relative humidity. This experiment complied with the guidelines made by the Institutional Animal Care and Use committee of Yeungnam University, and was performed with the permission of the Ethics for Animal Experiments committee of Yeungnam University (2020-018) . In all animal experiments, efforts were made to minimize the pain of mice using euthanasia by inhalation of carbon dioxide. The mouse cancer cell line CT26 (ATCC, CRL-2638) is an RPMI containing 10% FBS, 100 μg/ml streptomycin, 2 mM glutamine, 100 U/ml penicillin, and 1 M HEPES. Incubated in an incubator maintained at -1640 with 5% carbon dioxide and 37°C.

3. Antibodies

Isotype control Abs (IgG1, IgG2a, and IgG2b), CD3 (17A2), CD8α (53-6.7), CD4 (GK1.5), CD11c (N418), CD40 (3/23), CD86 (GL-1), CD80 (16-10A1) and anti-IgG (Poly4060) were purchased from BioLegend (USA). Anti-IFN-γ (B27), anti-MHC class I (28-8-6), and anti-MHC class II (M5/114.15.2) were purchased from eBioscience (USA).

4. Flow cytometry analysis

In order to remove the non-specific background prior to primary antibody staining, the cells were treated with unlabeled isotype control and Fc-block antibodies (BioLegend) and reacted for 15 minutes. After that, the cells were washed with PBS, and then Fluorescently conjugated antibodies were treated and incubated on ice for 20 min. After washing the remaining antibody with PBS and performing DAPI (4',6-diamidino-2-phenylindole; Sigma-Aldrich, USA) staining to indicate dead cells, FACS Aria Ⅱ (Becton Dickinson) and NovoCyte flow Cells were analyzed using a cytometer (ACEA Biosciences Inc., USA).

5. Mouse dendritic cell (DC) analysis

After homogenizing the spleen removed from the experimental animal, it was treated with collagenase for 20 minutes at room temperature. Cells were washed and resuspended in 5 ml Histopaque-1077 (Sigma-Aldrich) medium. The cell suspension was then carefully transferred to 5 ml of fresh Histopaque-1077 plus 1 ml of FBS. Thereafter, centrifugation was performed at 1,700 x g for 10 minutes, and a leukocyte fraction (<1.077 g/cm ³ ) was obtained and stained with fluorescently labeled monoclonal antibodies (mAbs) for 30 minutes. DCs are defined as lineage-CD11c ⁺ cells. Lineage staining was performed with anti-B220 (RA3-6B2), anti-CD49b (DX5), anti-CD3 (17A2), anti-Gr1 (RB68C5), anti-TER-119 (TER-119) and anti-Thy1.1 ( OX-7).

6. Human peripheral blood mononuclear cell (PBDC) analysis

PBMCs isolated from blood were treated with FITC-conjugated lineage antibodies anti-CD3 (HIT3a), anti-CD14 (63D3), anti-CD16 (3G8), anti-CD19 (HIB19), anti-CD20 (2H7) and anti- Stained with CD56 (5.1H11). Lineage-CD11c ⁺ cells were sorted as PBDCs using flow cytometry.

7. Ex vivo T cell stimulation and intracellular cytokine staining

Single cell suspensions obtained from lymph nodes were stimulated in vitro with 50 ng/ml of PMA (phorbol 12-myristate 13-acetate; Merck, USA) and 1 μM of ionomycin (Merck) for 4 h in vitro, followed by monensin solution (BioLegend). ) was added for the last 2 hours to react. For intracellular cytokine staining, first, surface molecules were stained and fixed, and then the cells were permeabilized using Cytofix/Cytoperm buffer (eBioscience), and reacted for 30 minutes using anti-cytokine Abs in Perm/Wash buffer (eBioscience). did it Staining of isotype control IgGs was performed in all experiments.

8. Enzyme-linked immunosorbent assay (ELISA)

IL-6, IL-12 and TNF-α ELISA kits for mice were purchased from eBioscience. Human IFN-γ ELISA kit was purchased from BioLegend. The concentration of IFN-γ was measured in triplicate using an ELISA kit.

9. Anti-cancer activity assay

1x10 ⁶ cells/100 μl of CT-26 cells were injected subcutaneously into BALB/c mice. On the 7th day after the injection of CT-26 cells, intraperitoneally PBS, 10 mg/kg anti-PD-1 Ab, 10 mg/kg anti-PD-L1 Ab, 5x10 ⁹ particles/ml CTRL (cobalt chloride) Exosomes obtained from mesenchymal stem cells not treated with compounds), 5x10 ⁹ particles/ml Exosome (exosomes obtained from mesenchymal stem cells treated with cobalt chloride compound), and a combination of the antibody and exosomes every 2 treated daily. The anti-PD-1 Ab and anti-PD-L1 Ab were purchased from BioXCell Korea and used. The extent of tumor growth was monitored by using a caliper until the 19th day after tumor injection, and on the final 21st day, mice in each experimental group were sacrificed to remove the tumor and photographed.

Example 1. Effect of inducing dendritic cell activity of exosomes

The immune-stimulating effect of exosomes produced by mesenchymal stem cells (MSCs) in mice was evaluated. Dendritic cells of the spleen in BALB/c mice were defined as CD11c ⁺ lineage-cells in splenocytes ( FIG. 1A ). In BALB/c mice, injection of exosomes obtained from pre-treated mesenchymal stem cells intraperitoneally (hereinafter referred to as Exosome) was confirmed to have a concentration-dependent tendency, and Exosome treatment was a costimulatory, Class It promoted up-regulation of I and II major histocompatibility complex (MHC) expression ( FIG. 1B ). In addition, the concentrations of IL-6 (interleukin-6), IL-12 and TNF-α (tumor necrosis factor-alpha) in the serum were significantly increased by treatment with Exosome when compared to treatment with PBS or control exosome (CTRL). became (Fig. 1C). In addition, the activation effect of splenic dendritic cells by exosomes obtained from pre-treated mesenchymal stem cells was almost similar to the level induced by LPS (lipopolysaccharide), a positive control.

In addition, exosome injection routes were evaluated for induction of dendritic cell activation. Although subcutaneous injection of exosomes failed to upregulate class I and II MHC, costimulatory molecules were injected with exosomes into intraperitoneal (i.p.), subcutaneous (s.c.) or intravenous (i.v.) splenic dendritic cells. was dramatically increased by (Fig. 2A). Additionally, serum concentrations of pro-inflammatory cytokines were significantly increased by other injection routes of exosomes when compared to the control group or the PBS-treated group ( FIG. 2B ). These results showed that exosomes produced by mesenchymal stem cells could induce dendritic cell-mediated immune activity in mice.

Example 2. Effect of exosomes on immune checkpoint inhibition-mediated anticancer activity

After confirming the result that the exosome obtained from the pre-treated mesenchymal stem cells induces the activity of dendritic cells in the spleen, the present inventors conducted an experiment on the induction of the anticancer immune effect of the exosome using experimental animals. carried out. BLALB/c mice were subcutaneously implanted with CT-26 cancer cells. Seven days after tumor injection, mice were treated with anti-PD-1 Ab, anti-PD-L1 Ab, control exosomes obtained from mesenchymal stem cells without pre-conditioning (hereinafter, CTRL), Exosomes obtained from pre-treated mesenchymal stem cells (hereinafter, Exosome) and a combination of each Ab and CTRL or Exosome were intraperitoneally administered every 2 days. Thereafter, changes in tumor size were observed.

As a result of the analysis, as shown in Figure 3, it can be confirmed that the immune checkpoint inhibitor (anti-PD-1 Ab, anti-PD-L1 Ab) treatment group inhibited tumor growth compared to the untreated control (PBS), CTRL-treated group and Exosome-treated group. there was. In particular, it was confirmed that the tumor growth inhibitory effect was further improved by the combined treatment of an immune checkpoint inhibitor and Exosome. However, even when CTRL was combined with an immune checkpoint inhibitor, it did not show an improved anticancer effect compared to the single treatment of the immune checkpoint inhibitor. Similar to the results of tumor size analysis, it was confirmed that the weight of the tumor was also significantly reduced in the combined treatment of exosome and immune checkpoint inhibitors (anti-PD-1 Ab, anti-PD-L1 Ab) than in other treatment groups (Fig. 3C). Through these results, it was found that the exosome obtained from the pre-treated mesenchymal stem cells of the present invention can enhance the immune checkpoint inhibition-mediated anticancer effect in mice.

Example 3. Effect on the activity of cancer antigen-specific T cell immunity

Since the complex treatment of anti-PD-1 Ab and exosomes obtained from pre-treated mesenchymal stem cells (hereinafter, Exosome) effectively suppressed the size of tumors in mice, the present inventors anti-PD-1 Ab and The activity of cancer antigen-specific T cells according to the complex treatment of exosomes was investigated. On day 24 after tumor injection, spleens were harvested and the activity of T cells promoted with CT-26 antigen was analyzed. Splenic CD4 ⁺ and CD8 ⁺ T cells were defined as shown in Fig. 4A, and CD4 ⁺ and CD8 ⁺ T cells in mice treated with the combination of anti-PD-1 Ab and Exosome were detected in response to CT-26 antigen. IFN-γ was effectively produced ( FIGS. 4B and C ). In particular, it was found that treatment with anti-PD-1 Ab or Exosome alone did not enhance the production of IFN-γ in CD4 ⁺ and CD8 ⁺ T cells. Moreover, the concentrations of IFN-γ and TNF-α measured in the culture medium were higher in splenocytes treated with the combination of anti-PD-1 Ab and Exosome than in splenocytes treated with PBS, anti-PD-1 Ab, or Exosome alone. significantly higher (Fig. 4D). These results suggested that the combination of anti-PD-1 Ab and Exosome could elicit cancer antigen-specific T cell immunity.

Example 4. Effect of exosomes on human peripheral blood dendritic cell activity induction

Since the exosomes were derived from human mesenchymal stem cells, the present inventors looked at the effects of exosomes on the induction of human dendritic cell activity. In human peripheral blood, CD11c ⁺ -lineage cells were defined as PBDCs (Fig. 5A). Co-stimulatory agents and MHC molecules were significantly increased in a concentration-dependent manner in PBDCs ( FIG. 5B ). The above results suggested that exosomes can be used as immunostimulatory molecules in humans.

Claims (7)

A pharmaceutical composition for preventing or treating cancer comprising an anticancer compound and exosomes as active ingredients. The pharmaceutical composition according to claim 1, wherein the anticancer compound is an immune checkpoint inhibitor. The pharmaceutical composition for treating cancer according to claim 2, wherein the immune checkpoint inhibitor is a PD-1/PD-L1 interaction inhibitor. The cancer according to claim 3, wherein the PD-1/PD-L1 interaction inhibitor is an antibody targeting PD-1 or PD-L1 or a functional fragment of the antibody or a single chain-based antibody analog. A therapeutic pharmaceutical composition. The pharmaceutical composition for treating cancer according to claim 1, wherein the exosomes are derived from pre-conditioning mesenchymal stem cells. The method of claim 5, wherein the pre-treatment is cobalt chloride, palmitate, beta-caryophyllene, deferoxamine, polyI:C (Polyinosinic:polycytidylic acid), MPLA (Monophosphoryl Lipid A), Pam3CSK4, zymosan (zymosan), HMGB1 (High mobility group box 1 protein), or a pharmaceutical composition for the treatment of cancer is carried out by treating a combination thereof. A method for treating cancer in a mammal other than a human, comprising administering a pharmaceutically effective amount of the pharmaceutical composition for cancer treatment of any one of claims 1 to 6 to a mammalian subject other than a human suffering from cancer .

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