KR20140021153A - Pharmaceutical composition for treating immune-related disease comprising human blood derived hematosphere - Google Patents

Abstract

The present invention relates to a technology in which only trace amounts of adult stem cells and progenitor cells present in the blood are effectively cultured and proliferated by using blood mononuclear cells, thereby differentiating inflammatory mononuclear cells into anti-inflammatory mononuclear cells and efficiently securing masses of anti-inflammatory Type 2 helper lymphocytes and regulatory lymphocytes, and a method for treating immune-related diseases using the technology. The anti-inflammatory mononuclear cells, Type 2 helper lymphocytes, and regulatory lymphocytes according to the present invention are immune cells derived from patients, and thus can greatly contribute to the development of cell therapeutic agents for autoimmune diseases.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for treating an immune-related disease comprising hematopoietic cells derived from human blood,

The present invention effectively cultivates and proliferates anti-inflammatory mononuclear cells, type 2 helper lymphocytes, or regulatory lymphocytes, which are present only in trace amounts in blood, using hematopoietic cells derived from human blood, thereby efficiently securing immune cells that are difficult to obtain outside the body And a method for treating immune-related diseases using the same.

A stem cell is a cell that can differentiate into various cells constituting a biological tissue, and is collectively referred to as undifferentiated cells before differentiation, which can be obtained from each tissue of the embryo, fetus, and adult body. Among adult stem cells, adult stem cells are stem cells derived from bone marrow. They refer to multipotent stem cells that can differentiate into all organs of the human body and blood.

Bone marrow-derived stem cells are considered the ultimate therapeutic means of diseases such as blood cancer, lymphoma, and bone marrow failure. However, since it is difficult to directly collect bone marrow, recently, a method of mobilizing bone marrow stem cells into blood through G-CSF injection has been used. However, this method has potential for side effects of G-CSF drug itself .

On the other hand, the stem cells contained in the cord blood are also actively stored and stored in a cord blood bank, but the number is limited. Development of stem cell technology in vitro to obtain a sufficient number of adult adult stem cells in the above situation is one of the important challenges facing the industry.

Most of the existing extracorporeal stem cell stem cell amplification techniques have been used to artificially create artificial marrow environment using artificial materials, and have been partially successful (Peerani R, Zandstra PW, Enabling stem cell therapies through synthetic stem cell-niche engineering. Journal of Clinical Investigation . 2010; 120: 60-70). Since the bone marrow environment of the adult adult stem cells includes contact with various supporting cells, cytokines provided by these cells, and extracellular matrix, these artificial substances sufficiently provide an environment for promoting the proliferation of adult stem cells It is hard to do.

Meanwhile, in the study on existing adult tissue-derived stem cells, several researchers have successfully developed a method of amplifying the number of stem cells or maintaining pluripotency by culturing cells derived from brain tissue or cardiac tissue in a spherical form (Caldwell MA, He X, Wilkie N, et al . Growth factors regulate the survival and fate of cells derived from human neurospheres. Nat Biotech . 2001; 19: 475-479 and Messina E, De Angelis L, Frati G, et al . Isolation and expansion of adult cardiac stem cells from human and murine heart. Circ Res . 2004; 95: 911-921).

As described above, bone marrow-derived stem cells can differentiate into various cells that form organs of our body, and can be differentiated into immune cells capable of protecting our host cells from pathogens or viruses that can be infected in vitro have. One of them, anti-inflammatory mononuclear cells, type 2 tidal lymphocyte (Th2) and regulatory lymphocyte (Treg) play a very important role in our body's immune system, and it is an important lymphocyte indispensable especially in the adaptive immune system. These cells are known to directly attack and kill infected host cells through viruses and pathogens, and to allow other immune cells to exert their functions. In addition, they are crucial for the production of antibodies by B lymphocytes (B cells) to amplify and activate cytotoxic T cells, and these cells are crucial for ensuring that the phagocytic cells and macrophages are capable of sterilization. The importance of these can be confirmed by human immunodeficiency virus. The particular virus infects and destroys the type 2 CD4 T lymphocytes, and the infected host eventually gets acquired immunodeficiency syndrome. Another lymphocyte, regulatory lymphocyte, inhibits the immune system in our body, inhibits the immune response that can be induced by the self-antigen, and tolerates the immune responses that occur in our bodies. In addition, many studies are now being conducted on their role in autoimmune disease and cancer research because they help down regulation of autoimmune diseases.

On the other hand, since it is very difficult to study type II helper lymphocytes and regulatory lymphocytes in humans, researchers are studying their functions only mainly through rodents.

In order to solve the problems of the prior art as described above, the present inventors have found that human blood-derived mononuclear cells are cultured in a high-density three-dimensional culture medium for anti-inflammatory monocytes, type 2 helper lymphocytes and regulated lymphocytes recruited and amplified only at specific sites and conditions in vivo The present inventors have completed the present invention as a result of intensive efforts to develop a culturing method capable of amplifying in vivo through the method and finally studying the function of these in humans rather than rodents.

Therefore, the present invention solves the problem that the anti-inflammatory mononuclear cells, type 2 helper lymphocytes and regulatory lymphocytes can not be obtained by sacrificing rodents for cell therapy or research, and can not be applied to the final target person, Anti-inflammatory mononuclear cells, type 2 tidal lymphocytes and regulatory lymphocytes provide a new manufacturing method by which researchers can study their function very easily and easily in humans. Ultimately, the present invention provides a method for treating an immune-related disease using a hematopoietic cell derived from human blood.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for the treatment of immune-related diseases comprising a hematopoietic cell derived from human blood, which is formed through three-dimensional coagulation culture after separating mononuclear cells from human blood.

In one embodiment of the present invention, the pharmaceutical composition may further include a single cell that does not form a cell mass when the three-dimensional coagulation culture is performed.

In another embodiment of the present invention, the cell mass or single cell may comprise a mononuclear cell, an anti-inflammatory type 2 helper lymphocyte, and a regulatory lymphocyte cell.

In another embodiment of the present invention, the immune related disease may include an autoimmune disease and a chronic inflammatory disease.

The present invention also provides a method of treating an immune-related disease by administering to a subject a pharmaceutically effective amount of the above pharmaceutical composition.

According to the present invention, when monocyte cells present in blood are coagulated and cultured through a three-dimensional method to form a cell mass, anti-inflammatory mononuclear cells, type 2 helper lymphocytes, and regulatory lymphocytes can be formed and amplified intracellularly or externally. It is derived from a non-rodent person and can be produced in vitro. Therefore, it can solve the problems and hassles that have not been able to be directly linked to a disease model so far, and also has the advantage of solving ethical problems . In addition, according to the present invention, it is possible to solve the conventional problems in which only a trace amount of anti-inflammatory monocyte cells, type 2 helper lymphocytes, and regulatory lymphocytes derived from human blood can be collected by administering specific cytokines and other biochemical substances Since the anti-inflammatory mononuclear cells, type II helicotropic lymphocytes, and regulatory lymphocytes according to the present invention are patient-derived immune cells, they can be effectively used for treatment of various immune-related disease cells including autoimmune diseases. Ultimately, the human blood-derived hematopoietic cell of the present invention, which contains a large amount of anti-inflammatory mononuclear cells, type 2 helper lymphocytes or regulatory lymphocytes, is expected to be used in the development of cell therapy for various immune related diseases including cancer.

FIG. 1 is a schematic diagram showing a process of culturing and producing human blood-derived blood cell mass (BBHS) after high-density coagulation culture of a mononuclear cell isolated from a human blood using a three-dimensional culture technique.
FIG. 2 is a graph showing changes in anti-inflammatory mononuclear cells (FACS) measured in human peripheral blood mononuclear cells (PBMC) and human blood-derived blood cell mass.
FIG. 3 shows the results of RT-PCR to examine the expression of anti-inflammatory genes in human blood-derived hematopoietic cells.
Fig. 4 compares the expression of anti-inflammatory and inflammatory cytokines when three-dimensional culture of human blood-derived blood cell mass and two-dimensional culture of human peripheral blood mononuclear cells (PBMC) are performed.
FIG. 5 is a schematic view of a human blood-derived hemocyte cell mass. When a separated mononuclear cell is cultured using a three-dimensional culture technique, human blood hematopoietic cells and non-BBHS cells isolated therefrom are formed.
FIG. 6 shows changes in type 1, type 2, and type 17 helper lymphocytes and regulatory lymphocytes in comparison with three-dimensional culture of human peripheral blood mononuclear cells and human blood-derived blood cell mass.
FIG. 7 is a graph showing the absolute counts of type 1, type 2 and type 17 helper lymphocytes and regulatory lymphocytes when human blood-derived hemocyte cell cultures were cultured for 3 days and 5 days.
FIG. 8 shows the results of confirming a control lymphocyte (CD4 (+) / Foxp3 (+)) using immunofluorescence staining for single cell (non-BBHS) isolated from human blood-derived hemocyte cell mass.
FIG. 9 shows the results of analysis of anti-inflammatory and inflammatory cytokines related to assisted and regulated lymphocytes by culturing human blood-derived hemocyte cell mass using an enzyme-linked immunosorbent assay (ELISA) as an enzyme immunoassay.

The present invention provides a novel method for amplifying anti-inflammatory monocytes, type 2 helper lymphocytes, or regulatory lymphocytes outside the body using human blood-derived cell mass, and a method for treating immune-related diseases using the human blood- .

The term " blood-born hematopoietic cell (BBHS) " used in the present invention refers to an aggregate of mononuclear cells existing in blood and specific cells having stem cells, and forms a three-dimensional structure It means that they form spheres like inner cell masses of blastocyst.

As used herein, the term 'anti-inflammatory mononuclear cells' refers to myeloid-derived suppressor cells (MDSCs), which are not originally immunocompetent monocyte cells but are now known by immunologists, ), Which have anti-inflammatory properties. The mononuclear cells originally mean anti-inflammatory mononuclear cells capable of promoting angiogenesis, not monocyte cells having strong immunity.

The term " Type 2 T lymphocytes (Th2) " or " Regulatory T lymphocytes (Treg) " used in the present invention is an important lymphocyte that can not be excluded from an adaptive immune system. They act indirectly on the immune system, destroying pathogens or virus-infected host cells and helping them to multiply.

The present invention provides a pharmaceutical composition for the treatment of immune-related diseases comprising hematopoietic cells derived from human blood. The cell mass may comprise anti-inflammatory mononuclear cells, type 2 helper lymphocytes, or regulatory lymphocytes. The immune related diseases include various autoimmune diseases and chronic inflammatory diseases.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include, but is not limited to, physiological saline, polyethylene glycol, ethanol, vegetable oil, and / or isopropyl myristate.

The present invention also provides a method of treating an immune disorder by administering to said individual a pharmaceutically effective amount of said pharmaceutical composition. In the present invention, 'individual' means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, and cow Means such mammals. In addition, in the present invention, the 'pharmaceutical effective amount' may be adjusted in various ways depending on the weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease of the patient. It is obvious to

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route, and the period, but can be appropriately selected by those skilled in the art. However, it is preferably administered at a daily dose of 0.001 to 100 mg / kg body weight, more preferably 0.01 to 30 mg / kg body weight. The administration can be carried out once a day or divided into several times.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. The method of administration is not limited and can be administered, for example, orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, or intra-cerebroventricular injection.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1. Human hematopoietic stem cells Cell mass  ( blood - born hematosphere , BBHS )

(1) Mononuclear cell separation step in peripheral blood

Peripheral blood was obtained by applying Heparin (approximately 100 ul) to a 50 ml syringe. 10 ml of peripheral blood was poured into 50 ml tubes, and 30 ml of PBS (phosphate-buffered saline) was added and carefully mixed. 10 ml of Ficoll used for the density gradient in the diluted blood was slowly poured into the bottom of the tube using a pipette to separate the clear Ficoll layer from the red blood layer and then minimized the stopping speed at 25 ° C for 30 minutes at 2,500 rpm And centrifuged. After the yellow serum layer, the white mononuclear layer, the red erythrocytes and the polynuclear morphology layer were separated at the upper part and the lower part of the transparent monocyte layer, the yellow mononuclear layer was inhaled and the white mononuclear layer was carefully transferred to a new tube. The transferred mononuclear layer was divided into two tubes, filled with PBS, and centrifuged at 1800 rpm for 10 minutes at 4 ° C. The cell pellet was vortexed and unfolded into single cells, then filled to the end with PBS and centrifuged at 1,700 rpm for 10 min at 4 ° C. The above rinse procedure was repeated three times to remove the substances used in the density gradient and the residues in the blood. Cell counts were measured with a hemocytometer prior to the final centrifugation.

(2) Three-dimensional culture step

The rinsed cells were suspended in an ultra-low-attach culture dish at a density of 10 6 / ml or higher at a density of 10 6 / ml or higher using a culture medium supplemented with 5% FBS in Endothelial basal medium-2 (EBM-2) The cells were incubated at 37 ° C in a 5% CO ₂ -concentrated incubator and the same medium was added for the first two days after incubation.

FIG. 1 is a schematic diagram showing a process of culturing a blood-born hematosphere (BBHS) for 5 days after the separation of human peripheral blood mononuclear cells (PBMC).

(3) Cell mass  Disassembly step into single cell

The cell mass obtained through the culturing process was separated into single cells for use in characterization and treatment. The suspended cells were collected by centrifugation at 4 ° C for 10 minutes at 1,700 rpm. After the cells were collected by centrifugation, the cells were collected by centrifugation. The cell pellet was lightly loosened with 1 ml of Accutase, a cell disassembly solution, and cultured in a 37 ° C incubator for 2 to 3 minutes. After incubation, 1 ml of the cell culture was added, followed by several pipetting, followed by centrifugation at 1,700 rpm at 4 ° C for 10 minutes.

In the following, an experiment was carried out to confirm that the type 2 hematopoietic cell (BBHS) from human blood-derived cell mass (BBHS) is changed from inflammatory mononuclear cells to mostly anti-inflammatory mononuclear cells and that type II helper cells or regulatory lymphocytes increase Respectively.

Example  2. Identification of anti-inflammatory mononuclear cell increase

After culturing human peripheral blood mononuclear cells (PBMC) and human hematopoietic cell mass (BBHS) developed by the present invention for 3 days and 5 days, only the cell mass was selected and the change of anti-inflammatory mononuclear cells Respectively. It is known that CD14 (+) CD16 (-) is a marker of inflammatory monocyte (M1) and CD14 (+) CD16 (+) is a marker of antiinflammatory monocyte (M2) The mononuclear cell (M2) was analyzed and the results are shown in Fig.

As shown in FIG. 2, when BBHS was cultured for 3 days and 5 days, the number of inflammatory mononuclear cells (M2) was abruptly increased to about 5% in the case of human peripheral blood mononuclear cells And the number of anti-inflammatory monocytes increased. In addition, it can be seen that at the initial stage (D3), the growth rapidly increased to about 88% (see FIG. 2a), and similar results were obtained from all the other eight applicants (see FIG.

Example  3. Expression of anti-inflammatory genes and confirmation of increased cytokine secretion

In FIG. 3, RNA was extracted from human peripheral blood mononuclear cells (PBMC) and the cell mass obtained in Example 1 (BBHS), and RT-PCR was performed to examine the expression of anti-inflammatory genes. The results are shown in FIG.

As a result, as shown in FIG. 3, IL-4, IL-6, IL-10, IL-13 and the transforming growth factor beta 1 (TGF- -1 expression was significantly increased in the cell mass (BBHS) compared with human peripheral blood mononuclear cells (PBMC, OD), and the cell mass was further increased when cultured for 5 days (5D) than the 3 days culture (3D).

In addition, the cell mass obtained in Example 1 (BBHS) and the PBMC-attached group were cultured for 5 days. Specifically, the supernatant obtained from Suspension (3D) culture and Attached (2D) culture was obtained, and the secretion of anti-inflammatory and inflammatory cytokines was compared and shown in FIG.

As shown in Fig. 4, when the cell mass (BBHS) was formed, the anti-inflammatory cytokine IL-8 was increased and the inflammatory cytokine TNF-a was decreased compared to the PBMC-attached group (Attach in FIG. 4) Respectively.

Example  4. Analysis of Type 1 T helper lymphocytes, type II helper lymphocytes and regulatory lymphocytes

Human blood hematopoietic cell (BBHS) is formed by three-dimensional culture of human peripheral blood mononuclear cells (hPBMC), and cell mass (BBHS) is formed and maintained as a single cell (Sinlge cell, Non-BBHS) Group. The following two groups of cells were analyzed (see FIG. 5a).

As a result, as shown in FIG. 5B, the group forming the cell mass (BBHS) is mostly monocyte, which is a CD14 (+) cell, and the non-BBHS group is mostly a CD3 (+) cell, (Lymphocyte).

Specifically, human peripheral blood mononuclear cells (hPBMCs) and cell mass (BBHS) were cultured for 3 days and 5 days, and then the cell mass was removed, and lymphocytes were analyzed in single cells (Non-BBHS, Non-incorporated BBHS) . For analysis, each cell was treated with phorobol 12-myristate 13-acetate (PMA) (Sigma Aldrich) at 50 ng / ml and Ionomycin (Sigma Aldrich) at 25 ng / ml and stimulated for 4 hours. Monensin (BD biosciences ). (CD4 FITC (BD biosciences), IFN-? PE (R & D systems), STAT6 APC (R & D systems), and the like were analyzed using the following antibodies: Type 1 helper lymphocyte, type 2 helper lymphocyte, IL-4 Pe-Cy7 (BD biosciences), CD25 APC-Cy7 (BD biosciences), Foxp3 APC (eBioscience), IL-17A FITC (eBioscience).

As shown in FIG. 6, cell debris (BBHS) was cultured for 3 days and 5 days compared to immediately after the isolation of human PBMC (hPBMC) (0 day PBMC) CD4 (+) IL-4 (+) STAT6 (+), which is important for anti-inflammatory activity, when cultured three-dimensionally than fresh PBMCs. ) CD25 (+) FoxP3 (+) regulatory lymphocytes. Th1, which is an inflammatory CD4 (+) IFN-gamma (+), and Th17, which is CD4 (+) IL-17A (+), did not change significantly.

FIG. 7 is a graph showing the results of cell growth (BBHS) cultured for 3 days and 5 days immediately after separation of human peripheral blood mononuclear cells (hPBMC) (0 day PBMC) , CD4 (+) CD25 (+) FoxP3 (+) regulatory lymphocytes, CD4 (+) IFN-Gamma (+) And CD4 (+) IL-17A (+), respectively, and the number of absolute lymphocytes was measured in a 17-type helper lymphocyte. As shown in the graph, the number of cells in the anti-inflammatory type 2 tidal lymphocytes and regulatory lymphocytes increased. However, the number of cells in the inflammatory type 1-assisted lymphocyte and type 17-assisted lymphocyte was not changed.

(BBHS) was cultured for 5 days, and the cells were removed. Then, the same experiment as above was performed on single cells (Non-BBHS, Non-incorporated BBHS) and then regulated by immunofluorescent staining CD4 / Foxp3, a marker of lymphocytes, was stained. The results are shown in Fig.

In addition, the human blood-derived blood cell mass (BBHS) was cultured for 3 days and 5 days, and the supernatant was obtained and subjected to enzyme immunoassay (ELISA). The results are shown in FIG.

As a result, as shown in Fig. 9, when cultured for 3 days and 5 days, there was almost no TNF-a or IL-12p70, which is a type 1 lymphocyte-related cytokine, inflammatory lymphocytes, whereas anti- In the case of IL-5, IL-10, and IL-13, the type 2 lymphocyte-related cytokines were also present when cultured for 3 days and secreted more when cultured for 5 days.

From the above, the method of the present invention can effectively produce an anti-inflammatory mononuclear cell, a second type helper lymphocyte, or a regulated lymphocyte without using a specific cytokine, and thus can contribute to the development of a cell therapy agent such as autoimmune disease and chronic inflammatory disease It is expected to be.

The foregoing 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. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

Claims (5)

Isolating monocytes from human blood and then comprising human blood-derived cell masses formed through three-dimensional aggregation culture, pharmaceutical composition for the treatment of immune-related diseases. The method of claim 1,
The pharmaceutical composition is characterized in that it further comprises a single cell that does not form a cell mass in the three-dimensional aggregation culture. 3. The method according to claim 1 or 2,
The cell mass or single cell is characterized in that it comprises one or more cells selected from the group consisting of monocyte cells, anti-inflammatory type 2 helper lymphocytes, and regulatory lymphocytes. The method of claim 1,
The immune-related disease is characterized in that it comprises autoimmune diseases and chronic inflammatory diseases, pharmaceutical compositions. A method of treating an immune related disease by administering to a subject a pharmaceutically effective amount of the pharmaceutical composition of any one of claims 1 to 3.

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