KR20180077777A - Medium composition and method for massive culture of autologous activated canine cytotoxic cell - Google Patents

Abstract

The present invention relates to a culture medium composition for proliferating activated autologous cytotoxic cells of a canine; a culture method; canine autologous lymphocytes; and a composition for preventing or treating a canine skin disease. In particular, the present invention relates to a culture medium composition comprising a canine anti-CD3 antibody, a canine autologous plasma, a human interleukin-2 (IL-2), a canine interleukin-12 (IL-12), and a human interleukin-18 (IL-18); a culture method; canine autologous lymphocytes comprising cytotoxic cells cultured thereby; and a composition for preventing or treating a canine skin disease, comprising the same as an effective component.

Description

Technical Field [0001] The present invention relates to a method of culturing a cell line,

The present invention relates to a culture composition, culture method, cell-derived cytotoxic cell for dog, and composition for preventing or treating skin disease in dogs. Particularly, a culture medium composition comprising an anti-CD3 antibody, a dog plasma, human interleukin-2 (IL-2), interleukin-12 (IL-12), and human interleukin-18 (IL- A method for culturing the same, a method for culturing the same, a method for culturing the same, a method for culturing the same, a method for culturing the same, and a method for preventing or treating canine skin diseases using the same.

We are often seeing dogs around these days when the ratio of one - person households surges beyond the nuclear family. However, there is a high possibility that the dog has a skin disease if it smells strange from a dog, if it has a red flag on his skin, if it has dandruff, if it shows eczema, or if his dog is itchy.

The skin diseases that appear in dogs include skin infections or allergic skin diseases. The dog skin infections are the major causes such as peptic ulcer, dermatophyte, dermatophyte, improvement, and xanthomatosis. Dog allergic skin diseases are atopic dermatitis, flea allergy Dermatitis are the main causes.

Skin infectious diseases are skin diseases caused by various causes such as malnutrition, microbial infections such as bacterial mold parasites, hormones and allergies, and symptoms such as rash, erythema, hair loss and pruritus appear. There are various microorganisms in the skin of dogs and usually do not cause disease. However, when the immunity is weakened by various factors, skin inflammation disease is caused by overgrowth of microorganisms. Treatment is mostly one-dimensional treatments such as antibiotics, antifungal agents or anti-inflammatory drugs against causative microorganisms. There is a reaction at the beginning of the treatment, but the more the antibiotics are used, the more resistant the bacteria are, and the more often the recurrence is, the worse the skin condition becomes. Since the weakening of immunity is the cause of the infectious disease, it is necessary to treat the disease fundamentally by increasing immunity.

The incidence of atopic dermatitis is about 3 to 15%, which is one of the common diseases in dogs. However, the drugs currently treating atopic dermatitis diseases are mostly steroid drugs and antihistamines. Although these drugs have a strong anti-inflammatory effect, they have the advantage of improving the symptoms rapidly. However, they have a disadvantage that the effects are temporary and repeated administration can cause severe side effects such as Cushing's syndrome, vasodilation, diabetes, osteoporosis and immunity. Although these serious side effects may occur, unlike humans, the development of drugs for the treatment of atopic dermatitis of dogs is rarely practiced, and thus the above drugs are still used.

Although the precise mechanism of atopic dermatitis has not been elucidated yet, according to the scientific evidence to date, over-expression of Th2 type cytokines such as overexpression of IL-4 mRNA and overexpression of IL-4 mRNA in PBMC And Th1 / Th2 cytokines due to the imbalance of the Th1 / Th2 cytokine. Therefore, it may be possible to treat the atopic dermatitis disease by eliminating the imbalance of Th1 / Th2 cytokine by reinforcing Th1 cytokine which can suppress the environment by Th2 cytokine.

Interferon-gamma is a typical Th1-type cytokine produced mainly by cytotoxic cytotoxic cells, type I helper cytotoxic cells and natural killer cells (NK cells) in humans It is known. Interferon-gamma is mainly secreted during viral infection, which activates T cells and natural killer cells, killing the virus-infected cells. In particular, interferon-gamma promotes the differentiation of undifferentiated cytotoxic cells into Th1-type cytotoxic cells and, conversely, inhibits the differentiation into Th2-type cytotoxic cells. In addition, it plays an important role in increasing the Th1 response and inhibiting the Th2 response by inhibiting the production of IgE in B cells.

Previous studies have reported that atopic symptoms were improved by administering the dog-derived interferon-gamma recombinant protein (KT-100TM) to dogs with atopic dermatitis [Veterinary dermatology 21.1 (2010): 42-49. ]. This is intended to balance Th1 / Th2 cytokines through recombinant interferon-gamma in atopic disease dogs with predominant Th2 responses. However, in the case of the recombinant interferon-gamma, the effect is excellent, but many dogs have reported that the side effects are frequent after administration, so they are not used in actual dogs.

In addition, IL-31, which is a cytokine that plays an important role in the development of atopic dermatitis, is aimed at JAK1 (Janus kinase 1) molecule which plays an important role in signal transduction by Th2 cytokine. However, these drugs are temporary effects by the administration of drugs, and they do not affect overall immunity improvement. Therefore, there is a desperate need for the development of therapeutic agents having fundamental efficacy.

  Yasukawa, K., etc. (2010). Low-dose recombinant canine interferon-γ for treatment of canine atopic dermatitis: An open randomized comparative trial of two doses. Veterinary dermatology, 21 (1), 42-49.

In order to maximize the production of interferon-gamma, the present invention provides an anti-CD3 antibody, an anti-CD3 antibody, a human plasma, a human interleukin-2 (IL-2) IL-12), and human interleukin-18 (IL-18).

It is another object of the present invention to provide a culture method for culturing a cell-derived, activated cytotoxic cell of a dog using the culture medium for the cell-derived activated cytotoxic cell culture.

It is another object of the present invention to provide a cancer-derived lymphocyte comprising the above-mentioned self-derived activated cytotoxic cell culture medium or a self-induced activated cytotoxic cell cultured by the above culture method .

It is another object of the present invention to provide a composition for preventing or treating canine skin diseases, which comprises the above-mentioned self-derived lymphocyte as an active ingredient.

In order to achieve the above object, the present invention provides a culture medium for cell-free activated cytotoxic cell culture comprising a culture medium for cell culture and an additive added to the culture medium for cell culture, (IL-2), interleukin-12 (IL-12), and human interleukin-18 (IL-12) IL-18).

In addition, the additive may further include beta-glucan.

In addition, the cytotoxic cells cultured in the culture medium for activating the dog-derived activated cytotoxic cell culture may have a ratio of cytotoxic cells having a CD3 ⁺ CD4 ^- CD8 ⁺ immunophenotype of 55 to 90%, preferably 60 to 80% And more preferably 65 to 75%.

Meanwhile, the self-derived lymphocyte of the present invention is characterized in that the self-derived activated cytotoxic cell cultured with the culture medium for the cell-derived activated cytotoxic cell culture is included.

Meanwhile, the self-induced activated cytotoxic cell culture method of the present invention

(A) isolating and isolating mononuclear and autologous plasma from peripheral blood, respectively;

Coating the cell culture container with a coating solution containing (B) anti-CD3 antibodies;

(IL-2), interleukin-12 (IL-12), and human IL-12 in a cell culture container coated with said anti-CD3 antibody, Of IL-18 (IL-18) is added to a culture medium for culture; And

(D) an interleukin selected from the group consisting of human interleukin-2 and interleukin-12 (IL-12), human interleukin-18 (IL-18) And a second culturing step of culturing the cells.

In the step (A) of obtaining the mononuclear cell, the mononuclear cell layer is formed by centrifuging the blood vessel while receiving the peripheral blood, the mononuclear cell layer is transferred to the test tube, the test tube is filled with the buffer solution, And washing.

The composition ratio of the anti-CD3 antibody to 100 parts by weight of the coating liquid of the step (B) is 1 × 10 ^-5 to 2 × 10 ^-4 parts by weight, preferably 2 × 10 ^-5 to 18 × 10 ^-5 parts by weight, may Buil more preferably 5 × 10 ^-5 to 15 × 10 ^-5 wt.

In addition, the composition ratio of the dog plasma to 100 parts by weight of the culture medium for activating the dog-derived activated cytotoxic cell culture is 2 to 20 parts by weight, preferably 3 to 18 parts by weight, more preferably 5 to 15 parts by weight have.

In addition, the composition ratio of human interleukin-2 to 1 ml of the culture medium for self-induced activated cytotoxic cell culture may be 100 to 2,000 IU, preferably 200 to 1,800 IU, and more preferably 500 to 1,500 IU .

In addition, the composition ratio of interleukin-12 to 100 parts by weight of the culture medium for cell-free activated cytotoxic cell culture is 1 × 10 ^-7 to 2 × 10 ^-6 parts by weight, preferably 2 × 10 ^-7 to 18 × 10 ^-7 parts by weight, and more preferably 5 × 10 ^-7 to 15 × 10 ^-7 parts by weight.

The composition ratio of human interleukin-18 to 100 parts by weight of the culture medium for cell-free activated cytotoxic cell culture is 2 × 10 ^-7 to 7 × 10 ^-6 parts by weight, preferably 5 × 10 ^-7 to 5 × 10 ^-6 parts by weight, 55 x 10 ^-7 parts by weight, and more preferably 12 x 10 ^-7 to 45 x 10 ^-7 parts by weight.

Meanwhile, the self-derived lymphocyte of the present invention is characterized in that the cell-derived activated cytotoxic cell cultured by the above-mentioned self-induced activated cytotoxic cell culture method is included.

In addition, the cytotoxic cell may be a cytotoxic T cell.

In addition, the cytotoxic cell may be a natural killer cell.

On the other hand, the composition for preventing or treating skin diseases of dogs of the present invention is characterized by containing the above-mentioned self-derived lymphocyte as an active ingredient.

In addition, the dog skin diseases may be skin infections or allergic skin diseases.

In addition, the dog skin infections may be selected from the group consisting of arachnidosis, dermatophyte, dermatophyte, eosinophilia, and combinations thereof.

In addition, the dog's allergic skin diseases may be selected from the group consisting of atopic dermatitis, flea allergic dermatitis, and combinations thereof.

The lymphocytes containing the activated cytotoxic cells derived from the dogs cultured by the medium composition or the culture method of the present invention have a high proportion of interferon-gamma producing cells, and thus can improve symptoms of skin diseases including atopic dermatitis and infectious dermatitis, It could alleviate. In addition, the overall immune system imbalance based on Th2 response is controlled by the Th1 response, so that the healing effect is maintained without recurrence for a longer period of time.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the growth rate and survival rate of cells obtained by the culture method of the present invention. Fig.
2 is a graph showing the cell phenotype and the proportion of interferon gamma-producing cells obtained before and after the culture method of the present invention.
FIG. 3 is a graph comparing the interferon-gamma production amount of cells obtained by interleukin-2 alone culturing conditions, interleukin-2, interleukin-12 and interleukin-18 culture conditions and the ratio of interferon-gamma producing cells.
4 is a schematic diagram showing the administration of activated lymphocytes and the timing of blood collection.
FIG. 5 is a graph showing CADESI-03 score and symptom improvement rate, which are indicators of atopic symptoms after administration of activated lymphocytes to atopic dermatitis disease dogs.
FIG. 6 is a graph showing the score of leaning sensation and symptom improvement rate after the administration of activated lymphocytes to atopic dermatitis disease dogs.
FIG. 7 is a photograph showing the skin condition before the administration of the activated lymphocyte in the atopic dermatitis disease dog and after 2 weeks from the fifth injection.
FIG. 8 is a graph showing changes in the ratio of interferon-gamma-producing cells in cytotoxic cells and ancillary T cells of peripheral blood lymphocytes according to the administration of the activated lymphocytes of the present invention.
FIG. 9 is a photograph showing the skin condition before the administration of activated lymphocytes and two weeks after the fifth injection in a skin infectious disease dog.

Hereinafter, preferred embodiments of the present invention will be described in detail. In the following description, numerous specific details, such as specific elements, are set forth in order to provide a thorough understanding of the present invention, and it is to be understood that the present invention may be practiced without these specific details, It will be obvious to those who have knowledge of. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.

The present invention relates to a method of culturing an activated cytotoxic cell for the treatment of a skin disease such as atopic dermatitis or infectious dermatitis caused by a dog by culturing a mononuclear cell derived from a dog, , Human IL-2, IL-12, IL-18, and human IL-18.

Herein, the term "mononuclear cells" as used herein refers to peripheral blood-derived mononuclear cells (PBMCs), and the PBMCs are cells that are responsible for immunity such as B cells, T cells and natural killer cells And granulocytes such as basophil, eosinophil, and neutrophil.

T cells are immunocytes that express CD3 molecules on their surface, one of the lymphocytes that mediate the antigen-specific adaptive immune response. T helper T cells that regulate the differentiation and activation of other immune cells through cytokines, cytotoxic T cells that remove infectious donor cells by secretion of cytotoxic substances, And regulatory T cells that secrete cynes and inhibit hyperimmunized immune cells.

Cytotoxic T cells are T cells expressing CD3 molecules and CD8 molecules on the surface, and have the function of removing cells that have lost viral infection or function. Activated cytotoxic T cells are one of the main cells producing interferon-gamma. In addition, it is a cell that can produce granulins which can remove cells infected with microorganisms and microorganisms such as bacteria and fungi together with granzyme used for removing virus-infected cells and cancer cells.

Regulatory T cells express CD4 and CD25 on the cell surface and are characterized by the expression of the transcription factor Foxp3. Regulatory T cells have the ability to inhibit hyperactivated immune cells by producing the inhibitory, cytokines, interleukin-10 and TGF-beta. In this regard, regulatory T cells can be used as a method of atopic treatment.

Natural killer cells (NK cells) are cells capable of directly removing virus-infected cells and cancer cells, and when activated, are one of the main cells producing interferon-gamma. Although the phenotype of human natural killer cells has been clarified, the phenotypes of natural killer cells are still unknown.

In the present specification, 'cytotoxic cells' refers to cells that remove viruses, virus-infected cells, or other cells that have lost their function, and are used to encompass the cytotoxic T cells and natural killer cells.

Anti-CD3 antibody binds to CD3 molecule, a T cell receptor that complexes with T cell receptor (TCR) present in dog T cells, and induces T cell activation. Activation by TCR plays an important role in the activation of T cells.

IL-12 is a glycoprotein with a molecular weight of 14 to 17 kDa produced by antigen-activated T cells. The IL-12 is a glycoprotein produced by an autocrine or a peripheral (IL-2) -mediated pathway (paracrine) to the T cells of the T cells. In addition, IL-2 can induce the growth of B cells, natural killer cells and regulatory T cells in addition to T cells.

IL-12 is a cytokine produced by dendritic cells, macrophages and B cells, and induces the differentiation of undifferentiated T cells into type 1 T-helper cells in T cells. In particular, IL-12 is a cytokine that plays an important role in the expression of interferon-gamma in T cells and natural killer cells by phosphorylating STAT4. It is also known that IL-12 inhibits the expression of IL-4 and increases cytotoxicity of natural killer cells and cytotoxic T cells.

IL-18 is a pro-inflammatory cytokine that is mainly produced by macrophages. IL-18 induces a greater increase in interferon-gamma expression by IL-18-activated transcription factors, in addition to STAT4, a transcription factor essential for IL-12-activated interferon-gamma expression.

Β-glucan is a polysaccharide consisting of β-1,3 bonds and β-1,6 bonds in D-glucose, which is present in cell walls of microorganisms such as bacteria, yeast and mold. Beta-glucan is known to activate immune cells through toll-like receptor 2 (TLR2), Dectin-1 and CR3 (complement receptor type 3) molecules, and beta-glucan-activated macrophages secrete cytokines Lt; RTI ID = 0.0 > T cells < / RTI > and natural killer cells. These functions are currently used as adjuvant therapy and immunity enhancer in chemotherapy.

The canine atopic dermatitis extent and severity index (CADESI) -03 score used in the present invention is a score of 4 diseases including erythema, lichenification, excoriation and alopecia in 62 dogs Is an indicator of the severity of atopy [Veterinary dermatology 18.2 (2007): 78-86.]. The score of each item is evaluated from 0 to 5 according to the degree of symptom, and the sum is 1,240 points.

The pruritus evaluation criteria was used as an indicator according to the judgment of the caregiver, and the pruritus symptom score used in the present invention was divided into 0 to 5 points according to the degree of symptoms.

In addition, the cytotoxic cells cultured in the culture medium for activating the dog-derived activated cytotoxic cell culture may have a ratio of cytotoxic cells having a CD3 ⁺ CD4 ^- CD8 ⁺ immunophenotype of 55 to 90%, preferably 60 to 80% , More preferably 65 to 75%, and is characterized by occupying a larger proportion than other lymphocytes. When the ratio of the cytotoxic cells having the CD3 ⁺ CD4 ^- CD8 ⁺ immunophenotype is within the above range, it is possible to generate a sufficient amount of interferon-gamma at such a level that no excessive immune response occurs in the body after administration.

On the other hand, the self-induced activated cytotoxic cell culture method of the present invention starts from the step of separately isolating mononuclear cells and autologous plasma from dog peripheral blood. In the step of obtaining monocytes, the mononuclear cell layer is formed by centrifuging the blood vessels while receiving the peripheral blood, the mononuclear cell layer is transferred to a test tube, each test tube is filled with a buffer solution, The cell yield can be further increased.

The cell culture vessel is then coated with four anti-CD3 antibodies. The anti-CD3 antibody can be diluted with a phosphate buffer, and gelatin, collagen or fibronectin can be used to increase the efficiency of the coating.

In this case, the composition ratio of the anti-CD3 antibody to 100 parts by weight of the coating liquid is preferably 1 × 10 ^-5 to 2 × 10 ^-4 parts by weight, more preferably 2 × 10 ^-5 to 18 × 10 ^-5 parts by weight, it may be an 5 × 10 ^-5 to 15 × 10 ^-5 wt. When the content of the anti-CD3 antibody is within the above range, sufficient proliferation of cytotoxic cells occurs at a level at which activation-induced cell death (AICD) due to excessive stimulation does not occur.

Then, the mononuclear cells were suspended in the cell culture medium to which the obtained autologous plasma, human interleukin-2, interleukin-12 (IL-12), and human interleukin-18 (IL-12) The first culture step in which the coated cell culture container is inoculated and cultured is performed. Such a first incubation step is preferably carried out for 30 minutes to 24 hours.

In addition, the composition ratio of the dog plasma to 100 parts by weight of the culture medium for activating the dog-derived activated cytotoxic cell culture is 2 to 20 parts by weight, preferably 3 to 18 parts by weight, more preferably 5 to 15 parts by weight have. When the amount of autologous plasma is within the above range, sufficient proliferation of cytotoxic cells occurs at a level at which unnecessary cells do not proliferate.

In addition, the composition ratio of human interleukin-2 to 1 ml of the culture medium for self-induced activated cytotoxic cell culture may be 100 to 2,000 IU, preferably 200 to 1,800 IU, and more preferably 500 to 1,500 IU . When the content of human interleukin-2 is within the above range, activation and proliferation of cytotoxic cells are sufficiently achieved at a level at which activation induction killing by excessive stimulation does not occur.

Human interleukin-2 or human interleukin-2 and interleukin-12 as a second culture step are added to the cell culture medium for the first culturing step and cultured.

In addition, the composition ratio of interleukin-12 to 100 parts by weight of the culture medium for cell-free activated cytotoxic cell culture is 1 × 10 ^-7 to 2 × 10 ^-6 parts by weight, preferably 2 × 10 ^-7 to 18 × 10 ^-7 parts by weight, and more preferably 5 × 10 ^-7 to 15 × 10 ^-7 parts by weight. When the content of interleukin-12 is within the above range, it is possible to generate a sufficient amount of interferon-gamma at a level at which activation induction death by excessive stimulation does not occur.

The composition ratio of human interleukin-18 to 100 parts by weight of the culture medium for cell-free activated cytotoxic cell culture is 2 × 10 ^-7 to 7 × 10 ^-6 parts by weight, preferably 5 × 10 ^-7 to 5 × 10 ^-6 parts by weight, 55 x 10 ^-7 parts by weight, and more preferably 12 x 10 ^-7 to 45 x 10 ^-7 parts by weight. When the content of human interleukin-18 is within the above range, a sufficient amount of interferon-gamma can be produced at a level at which activation induction death by excessive stimulation does not occur.

In addition, the additive may further include beta-glucan.

Interleukins selected from the group consisting of human interleukin-2 and interleukin-12 (IL-12), human interleukin-18 (IL-18) Is added to a cell culture medium and cultured.

Meanwhile, the self-induced lymphocyte of the present invention includes a cell-derived activated cytotoxic cell culture medium composition or a dog-derived activated cytotoxic cell cultured by the above-mentioned self-induced activated cytotoxic cell culture method .

The cytotoxic cells may be cytotoxic T cells or natural killer cells. These cells have particularly high interferon-gamma production capacity.

On the other hand, the composition for preventing or treating skin diseases of dogs of the present invention is characterized by containing the above-mentioned self-derived lymphocyte as an active ingredient.

The dog skin disease may be a skin infection or an allergic skin disease. The dog skin infection may be selected from the group consisting of peptic ulcer, dermatophyte, dermatophyte, Dermatitis, dermatitis, flea allergic dermatitis, and combinations thereof.

Hereinafter, embodiments of the present invention will be described.

Example

Example  Step 1: Blood collection of dogs

Five to 10 ml of peripheral blood were collected from ten dogs with naturally occurring atopic dermatitis for immunocyte cultivation in a heparin tube (BD vacutainer, BD bioscience). Blood collection was conducted with the consent of the guardian at an approved veterinary hospital. Blood collected heparin tubes were wrapped in foil, blocked with light, transported at room temperature, and mononuclear cell separation was performed upon arrival at the culture room.

Example 2: Mononuclear cell separation

The collected peripheral blood was transferred to a 15 ml conical tube and centrifuged at 2000 rpm for 5 minutes to separate into plasma and blood cell layer. Plasma of the upper layer was collected for further use in culture. To separate the lymphocytes, the blood cell layer was diluted 1: 1 with RPMI1640 (Hyclone) medium. The diluted blood cell layer was carefully raised on 4 ml of Ficoll-paque PLUS (specific gravity 1.077, GE healthcare) And centrifuged at 400 xg of liver. Separate mononuclear layer between the media layer and red blood cell layer was carefully transferred to a new 15 ml conical tube. 10 ml of 0.83% ammonium chloride solution was added to remove the remaining red blood cells, incubated at 37 ° C for 10 minutes, And centrifuged at 2000 rpm. Monocytes were suspended in 10 ml of 1x PBS solution (phosphate-buffered saline, welgene), centrifuged at 2000 rpm for 5 minutes, and the supernatant was removed. After the washing step was performed once more, mononuclear cells were suspended in 1 ml of RPMI1640 medium and used for further culturing and analysis.

Example 3: Monocytic cell culture

One day prior to incubation, the anti-CD3 antibody (AbD serotec) was diluted to 1 μg / ml in 1 × PBS solution and 5 ml of anti-CD3 antibody diluted in T-25 flask (Nunc) And stored at 4 ° C. The flasks were washed twice with 5 ml of 1 x PBS solution before removing the anti-CD3 antibody solution prior to use.

A portion of the monocytes previously obtained was diluted in Trypan blue (Sigma Aldrich) solution and the living cells were counted. 1.0 x 10 < ⁷ > cells were inoculated into the washed T-25 flask, and the culture was added to a final volume of 5 ml. 2 mM L-glutamic acid (Gibco) and 1 x Antibiotic-Antimycotic (Gibco) were added to the RPMI1640 medium and the cells were incubated with 1000 IU / ml human IL-2 proleukin) and 10 ng / ml IL-12 (R & D systems) and 30 ng / ml human IL-18 (MBL) were used. The cells were cultured in a 5 volume% CO ₂ incubator at 37 ° C.

On days 2 to 3 of incubation, 1000 IU / ml human IL-2 and 10 ng / ml IL-12 and 30 ng / ml human IL-18 were added to further activate the cells. On the fourth day after culture, the same amount of cells were collected at intervals of 1 day to 2 days according to cell proliferation, except that 10% FBS (Fetal bovine serum, Gibco) was used instead of 10% The culture medium and the three cytokines were added to a final volume of 20 ml, and only 1000 IU / ml human IL-2 was added to the same volume of the culture medium and cytokine at a final volume of 20 ml or more.

Test Example 1: Determination of cell proliferation rate and survival rate

After incubation for 13 days using the blood of the naturally occurring atopic dermatitis dogs as in Example 3 above, the cells cultured in 1 x PBS solution were suspended, centrifuged at 2000 rpm for 5 minutes, and the supernatant was removed Respectively. After the washing step was further performed once, the cultured cells were suspended in RPMI 1640 medium, and some cells were stained with trypan blue solution to count the number of living cells and the total number of cells.

As a result, as shown in the left side of FIG. 1, in 60 cultures, the cell proliferation rate was increased by about 52.57 times on average, and the survival rate was as high as about 93.37% as shown in the right side of FIG.

Test Example 2: Cell phenotype analysis before and after culture

The phenotype of the monocyte cells and the cells cultured by the above Example 3 was analyzed.

Mononuclear cells and the cells cultured for 13 days according to the above Example 3 were diluted in a FACS staining buffer (ebioscience) at a concentration of 1 x 10 ⁶ / ml, and then 100 μl each was dispensed into a 5 ml sample tube. The fluorescently labeled antibody for surface antigen analysis was added to the cell-seeded tube under the same conditions as in Table 1, and the isotype antibody conjugated with only fluorescently labeled antibody was added as a control. The test tube containing the fluorescently labeled antibody was allowed to stand in the dark for 30 minutes, washed twice with 1 × PBS, and fixed in 300 μl of 4% paraformaldehyde solution. Fixed cells were analyzed for cell distribution using a Gallios (Beckman Coulter) flow cytometer and data were analyzed using KALUZA software (Beckman Coulter).

FITC PE PerCP-eF710 PE-Cy7 APC No. 1 tube IgG1-FITC IgG1-PE IgG2b APC No. 2 tube CD3-FITC CD21-PE CD45-APC Tube 3 IgG1-FITC IgG2a-PE IgG1-PerCP-eF710 IgG2a-PC7 IgG2b APC No. 4 tube CD3-FITC CD5-PE CD8-PerCP-eF710 CD4-PC7 CD45-APC

As a result, when the cell phenotypes of 60 mononuclear cells cultured according to Example 3 and the cultured cells were compared as shown in FIG. 2, the ratio of cytotoxic cells (CD3 ⁺ CD21 ^- ) after culturing was increased to 97% On the other hand, the ratio of B cells (CD3 ^- CD21 ⁺ ) is almost 0.19%, indicating that cultured cells are mostly cytotoxic cells. In addition, based on the total T cells (CD3 ^+), cytotoxic cytotoxic cells ^{(CD4-CD8 +)} ratio was significantly increased the average 68.96% and then incubated in culture The mean 27.02%, secondary cytotoxic cells (CD4 ⁺ CD8 the ^- ) Was significantly decreased from 55.33% before culturing to 3.37% after culturing. Therefore, it can be confirmed that cytotoxic cytotoxic cells are the cells cultured according to the step 3 of the Example. However, as described above, studies on the phenotype of dog have not yet been completed, so that natural killer cells may be detected as the above-mentioned phenotype. Therefore, the phenotype should be interpreted to include a part of the cytotoxic T cell, even though the natural killer cell has a small specific gravity.

Test Example 3: Analysis of interferon-gamma producing cell ratio before and after culture

Since interferon-gamma is an important cytokine to inhibit the Th2 response, the ratio of interferon-gamma producing cells to pre-incubated mononuclear cells and to cells cultured according to Example 3 was determined.

Peripheral blood was used for pre-culture of mononuclear cells. 0.5 ml of peripheral blood was diluted in 0.5 ml of RPMI 1640 medium and then 40 ng / ml of PMA (phorbol 12-myristate 13-acetate, Sigma Aldrich), 1 μl / ml of Ionomycin (Sigma Aldrich), 5 μl of Brefeldin A / Ml were added in this order and reacted at 37 DEG C for 4 hours. For the cultured cells, the cells were washed twice with 1 x PBS solution, and 5.0 x 10 ⁶ cells were suspended in 1 ml of RPMI 1640 medium, and PMA, Ionomycin, and Brefeldin A were sequentially added at the above concentrations, And reacted for 4 hours. After 4 hours of reaction, 9 ml of 0.83% ammonium chloride solution was added to remove erythrocytes, and the mixture was incubated at 37 ° C for 10 minutes and centrifuged at 2000 rpm for 5 minutes. Then, the cells were washed twice with 10 ml of 1 x PBS, and the cells were suspended in 100 占 of FACS staining buffer (ebioscience).

CD3 FITC monoclonal antibody (eBioscience), anti-CD4 PE-Cy7 monoclonal antibody (eBioscience), and anti-CD8 PerCP-eF710 monoclonal antibody (eBioscience) were each added to the diluted cells and stained in the cow for 30 minutes . The stained cells were washed once with 10 ml of 1 x PBS, and the cells were suspended in 100 μl of IC Fixation buffer (eBioscience) and fixed in a dark place for 60 minutes.

After cell fixation, 2 ml of 1 x Permeabilization buffer (eBioscience) was added and the supernatant was removed after centrifugation at 2000 rpm for 5 minutes. The cells were suspended in 2 ml of 1 x Permeabilization buffer, and then centrifuged at 2000 rpm for 5 minutes to wash the cells once. The washed cells were suspended in 100 μl of 1 × Permeabilization buffer, and then the anti-bovine interferon-gamma PE monoclonal antibody (AbD serotec) was added. Anti-bovine interferon-gamma PE monoclonal antibodies are known to bind to interferon-gamma molecules. A monoclonal antibody with fluorescent label alone was used as a control. After staining for 30 min in a cow, 0.5 ml of 1 × Permeabilization buffer was added and the supernatant was removed by centrifugation at 2000 rpm for 5 min. The cells were suspended in 0.5 ml of 1 × Permeabilization buffer, and then the cells were washed once by centrifugation at 2000 rpm for 5 minutes. The washed cells were diluted in 300 μl of FACS staining buffer (ebioscience). The percentage of interferon-gamma producing cells was measured by Gallios flow cytometry and the data were analyzed using KALUZA software.

As a result, it can be seen that the ratio of interferon-gamma producing cells (IFN-gamma producing cells) significantly increased from 13.46% before culturing to 63.44% after culturing as shown in FIG. In other words, the cells cultured by the step 3 of Example 3 are found to be interferon-gamma producing cells.

Test Example 4: Analysis of regulatory T cell ratio before and after culture

Regulatory cytotoxic cells are known to be cells that inhibit the activation of immune cells. The ratio of regulated cytotoxic cells to monocyte cells before culturing and to cells cultured according to Example 3 was measured.

Monocytes and 3.0 x 10 ⁶ cells cultured for 13 days according to the above Example 3 were washed twice with 1 x PBS, suspended in 100 μl of FACS staining buffer, and dispensed into 96-well round bottom plates. The anti-CD25 FITC monoclonal antibody (eBioscience) and the anti-CD4 PE-Cy7 monoclonal antibody (eBioscience) were stained in the wells of the cells and stained in the dark for 30 minutes. The 96-well plate was centrifuged at 2000 rpm for 5 minutes, the supernatant was removed, and the cells were suspended in 200 μl of 1 x PBS solution. After washing once more, the washed cells were suspended in 200 μl of a 1: 3 mixture of Fixation / Permeabilization Concentrate (eBioscience) and Fixation / Permeabilization Diluent (eBioscience) and fixed in a dark place for 60 minutes .

100 μl of 1 × Permeabilization buffer was added to the fixed cells, centrifuged at 2000 rpm for 5 minutes, and then the supernatant was removed. The cells were suspended in 200 μl of 1 × permeabilization buffer and centrifuged at 2000 rpm for 5 minutes. The washed cells were suspended in 100 μl of 1 × Permeabilization buffer, transferred to a 5 ml sample tube, and anti-mouse / rat Foxp3 APC monoclonal antibody (eBoscience) was added. The mouse / rat Foxp3 APC monoclonal antibody is known to bind to Foxp3 molecules. A monoclonal antibody with fluorescent label alone was used as a control. After dyeing, 500 μl of 1 × Permeabilization buffer was added to the tube containing the cells, centrifuged at 2000 rpm for 5 minutes, and then the supernatant was removed. The cells were suspended in 500 μl of 1 × Permeabilization buffer and centrifuged at 2000 rpm for 5 minutes. The washed cells were diluted in 300 μl of FACS staining buffer (ebioscience). The percentage of regulated cytotoxic cells was determined by Gallios flow cytometry and the data were analyzed using KALUZA software.

As a result, as shown in FIG. 2, regulatory T cells were significantly decreased from 1.43% before culturing to 0.12% after culturing. That is, it can be seen that cells cultured by Example 3 have almost no regulated cytotoxic cells.

Test Example 5: Confirmation of interferon-gamma production by interleukin-12 and interleukin-18

The amount of interferon-gamma and the ratio of interferon-gamma producing cells in the culture medium were measured under conditions of interleukin-2 alone culturing and interleukin-2, interleukin-12, and interleukin-18 culture.

Interferon-gamma in the culture medium produced during the culture period was measured using a Canine IFN-γ DuoSet ELISA kit (R & D Systems, Minneapolis, MN). After culturing for 13 days according to the examples, the obtained culture was centrifuged at 2,000 rpm for 5 minutes, and the supernatant was collected in a 15 ml conical tube.

First, add 56 μl of capture antibody to 10 μl of 1 × PBS solution, mix well and dispense 100 μl into a 96-well plate and incubate at room temperature for 8 hours or more. The 96-well plate was washed three times with 300 μl of washing buffer (1 x PBS solution containing 0.05% Tween 20), 300 μl of Reagent Diluent (R & D Systems) was added to the 96 well plate And incubated for 1 hour. The 96-well plate was washed three times with 300 μl of washing buffer, and the supernatant and standard solution (5.6 μl of recombinant interferon-gamma antibody solution, 1 μl of Reagent Diluent solution, 2-fold serial dilution ) Was added to 96 well plate and incubated at room temperature for 2 hours. The 96 well plate was washed three times with 300 μl of washing buffer, and 100 μl of the detection solution (10 μl of Reagent Diluent solution, 56 μl of interferon-gamma detection antibody solution) was added to a 96-well plate, And incubated for 1 hour. 100 μl of Streptavidin-HRP solution (containing 50 μl of Streptavidin-HRP in 10 μl of Reagent Diluent solution) was added to each well of a 96-well plate at room temperature And incubated for 20 minutes. The 96-well plate was washed three times with 300 μl of washing buffer, and the washing buffer was completely removed. A 1: 1 mixture of Color Reagent A (R & D Systems) and Color Reagent B And the reaction was carried out in a cow for 15 minutes. 50 μl of stop solution (R & D Systems) was added and the absorbance was measured at 450 nm using a microplate reader (Molecular device, USA).

As a result, as shown in FIG. 3, interleukin-2 production, interleukin-12 and interleukin-18 culture conditions showed that the interleukin-gamma production amount was 18,292.6 ng and the interleukin-2 alone culture condition was about 10 times higher than 1,910.7 ng. Interferon-2 and interleukin-18 were found to be 67.10% and 40.30% higher than interleukin-2 alone, respectively. That is, the above results show that interleukin-12 and interleukin-18 increase interferon-gamma production of activated lymphocytes.

Example 4: After culturing, cells were administered to atopic dermatitis dogs

Example Monocytes of atopic dermatitis were cultured for 13 days in the same manner as described in step 3. After incubation, the cell culture was transferred to a 250 ml conical tube, centrifuged at 2000 rpm for 5 min and the supernatant was removed. The cells were then suspended in 250 ml of 1 x PBS solution, centrifuged at 2000 rpm for 5 minutes, and the supernatant was removed. After washing once more, the washed cells were suspended in 10 ml of Normal Saline solution (Korea Pharmaceutical Industry Co., Ltd.), and the cells were filtered by a nylon mesh (40um, BD Bioscience) to remove impurities. Then some cells were removed and stained with trypan blue solution, and then living cells were counted. Up to 5.0 x 10 ⁸ cells were put into 20 ~ 50 ml syringes (Korean vaccine) equipped with 18G needle (Korean vaccine) based on living cells and put into a final volume of 20 ml with normal saline solution. The commercialized cells were stored at 4 ° C and stored at room temperature for 30 minutes before administration. The administration was carried out at a rate of 10 ml / 30 min and the cells were administered intravenously.

Test Example 6: After administration, symptoms of atopic dermatitis dogs and changes in their senses

As shown in FIG. 4, the cultured cells were administered at intervals of two weeks for a total of six times, and symptom changes and pruritus of atopic dermatitis were observed. And the average number of treated cells are shown in Table 2. < tb > < TABLE >

No. Bell gender age Route of administration Number of doses Average number of cells administered P-1 Maltese M / Neutralization 9y I.V 6 4.2 x 10 ⁸ P-2 Yorkshire Terrier south 7y I.V 6 2.4 x 10 ⁸ P-3 Dachshund F / neutralization 11y I.V 6 1.0 x 10 ⁸ P-4 French Bulldog F / neutralization 1y I.V 6 4.4 x 10 ⁸ P-5 French Bulldog M / Neutralization 1y I.V 6 4.6 x 10 ⁸ P-6 Shih Tzu M / Neutralization 9y I.V 6 1.6 x 10 ⁸ P-7 Shih Tzu F / neutralization 11y I.V 6 2.1 x 10 ⁸ P-8 Bull Terrier F / neutralization 2y I.V 6 4.3 x 10 ⁸ P-9 Spitz M / Neutralization 2y I.V 6 4.4 x 10 ⁸ P-10 Shi Tzu M / Neutralization 13y I.V 6 3.8 x 10 ⁸

First, the score of CADESI-03, which is an indicator of atopy severity, showed a tendency to decrease as the activated lymphocyte was administered as shown in the graph of FIG. Specifically, the mean was 101.50 at the pre-dose and 56.90 at 2 weeks after the 5th dose. The symptom improvement rate improved to 45.92% after 2 weeks of the 5th dose. The CADESI-03 score was 39.10 and the symptom improvement rate was 65.74% after 8 weeks of the 6th administration, and the symptoms improved even though the cells were not administered any more.

As shown in FIG. 6, the score of cannulation was decreased with the administration of the activated lymphocyte, and the mean score was decreased from 3.50 before the administration to 1.70 after 2 weeks from the 5th administration. The symptom improvement rate was improved to 54.83% on average after 2 weeks of the 5th administration. After 8 weeks of the 6th administration, the mean score of the cannabis was 1.70 and the symptom improvement rate was 54.33%, indicating that the leaning did not deteriorate even though the cells were not administered any more.

FIG. 7 is a photograph showing the skin condition before and 5 weeks after administration of atopic dermatitis dogs. It was visually confirmed that erythema and scarring disappeared after the administration of the activated lymphocyte and the hair was uniformly grown.

These results suggest that the active lymphocyte treatment improves atopic symptoms and pruritus, and that atopic symptoms and pruritus do not deteriorate even during the 8 - week absence period.

Test Example 7: Confirmation of the ratio of interferon-gamma-producing cells to the number of administration

Changes in the ratio of interferon-gamma producing cells in blood-assisted T cells and cytotoxic cells were measured at each sampling time.

The ratio of interferon-gamma producing cells was measured in the same manner as in Test Example 3. As shown in FIG. 8, the ratio of cells producing interferon-gamma in cytotoxic cells showed an increasing tendency according to the administration of activated lymphocytes. In the cytotoxic cells, the interferon-gamma producing cells Increased by 39% on average. On the other hand, the proportion of cells producing interferon-gamma in the sub-T cells did not change despite the activation of lymphocytes.

These results suggest that the improvement of atopic dermatitis by the administration of activated lymphocytes is induced by an increase in the ratio of cytotoxic cells producing interferon-gamma.

Test Example 8: After the administration, the symptoms of skin infectious disease were observed

As shown in FIG. 4, after the cultured cells were administered at a total of 6 times at intervals of 2 weeks, changes in the symptoms of the dogs and changes in the pruritus of the infected skin diseases were confirmed. And the average number of treated cells are shown in Table 3. < tb > < TABLE >

No. Bell gender age Disease name Route of administration Number of doses Average dose
Cell number P-11 Shih Tzu M / Neutralization 13y Agranulocytosis dermatitis I.V 6 3.8 x 10 ⁸ P-12 Rottweiler south 2y Fungus salt I.V 6 1.0 x 10 ⁸ P-13 Scottish Terrier female 6y Fungus
dermatitis I.V 6 3.0 x 10 ⁸

FIG. 9 is a photograph showing the skin condition before the administration of the skin infectious disease dog and after the 5th administration 2 weeks after the administration of the activated lymphocyte, and it is possible to visually confirm that the wound heals and the hair grows evenly after the administration of the activated lymphocyte.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Of course it is possible. Accordingly, the scope of the present invention should not be construed as being limited to the above-described embodiments, but should be determined by equivalents to the appended claims, as well as the following claims.

Claims (8)

1. A culture medium for culturing a cell-culturing animal cell and an additive added to the cell culture medium,
(IL-2), interleukin-12 (IL-12), and human interleukin-18 (IL-18) A culture medium for autologous-derived activated cytotoxic cell culture. The method according to claim 1,
The cytotoxic cells cultured in the culture medium for the cell-derived activated cytotoxic cell culture are characterized in that the ratio of the cytotoxic cells having the CD3 ⁺ CD4 ^- CD8 ⁺ immunophenotype is 55 to 90% A medium composition for cell culture. The method according to claim 1,
Wherein the cytotoxic cell is a cell selected from the group consisting of cytotoxic T cells, natural killer cells, and combinations thereof. A self-induced lymphocyte from a dog comprising the self-induced activated cytotoxic cells cultured in the culture medium for the self-induced activated cytotoxic cell culture of claim 1. (A) isolating and isolating mononuclear and autologous plasma from peripheral blood, respectively;
Coating the cell culture container with a coating solution containing (B) anti-CD3 antibodies;
(IL-2), interleukin-12 (IL-12), and human IL-12 in a cell culture container coated with said anti-CD3 antibody, Of IL-18 (IL-18) is added to a culture medium for culture; And
(D) an interleukin selected from the group consisting of human interleukin-2 and interleukin-12 (IL-12), human interleukin-18 (IL-18) And a second culturing step of culturing the host cell by adding the culture medium. The method of claim 5,
Wherein said cytotoxic cells are cells selected from the group consisting of cytotoxic T cells, natural killer cells, and combinations thereof. A dog-derived lymphocyte comprising the cancer-induced activated cell cytotoxic cells cultured by the self-induced activated cytotoxic cell culture method of claim 5. A composition for preventing or treating canine skin disease comprising the self-derived lymphocyte according to claim 4 or 7 as an active ingredient.

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