KR20180077863A - Medium composition and method for massive culture of autologous activated canine cytotoxic cell - Google Patents

Abstract

The present invention relates to a medium composition for proliferating autologous activated canine cytotoxic cells, a culture method, an autologous canine lymphocyte, and a composition for preventing or treating skin diseases. In particular, the present invention relates to a medium composition containing beta-glucan, human interleukin-2 (IL-2), canine plasma, and canine anti-CD3 antibodies. The present invention further relates to a culture method, an autologous canine lymphocyte including the cultured cytotoxic cells obtained thereby, and a composition for preventing or treating skin disease in dogs by containing the same as an active ingredient.

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. In particular, the present invention relates to a medium composition and a culture method comprising the anti-CD3 antibody, the dog serum, the human interleukin-2 (IL-2), and the beta-glucan, and a T cell and a naturally- The present invention relates to a self-induced cytotoxic cell and a composition for preventing or treating canine skin disease using the cell as an effective ingredient.

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 increases secretion mainly during viral infection, which activates cytotoxic and NK cells and kills 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.

Disclosure of the Invention The present invention has been conceived to solve the above-mentioned problems. In order to maximize the production of interferon-gamma, the present invention provides an anti-CD3 antibody, dog plasma, human interleukin-2 (IL- Cell-active, cytotoxic cell culture medium containing the same.

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, Characterized in that the additive comprises at least one anti-CD3 antibody, a dog plasma, human interleukin-2 (IL-2), and 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;

(C) Inoculating the monocyte into a cell culture container coated with the anti-CD3 antibody and culturing the cell culture medium containing the animal self-plasma, human IL-2, and beta-glucan, A first culturing step of culturing by addition; And

(D) a second culturing step in which a substance selected from the group consisting of human interleukin-2, beta-glucan, and a mixture thereof is cultured by adding the cell culture medium for the first culturing step.

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 cell-free 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 .

The composition ratio of the beta-glucan to 100 parts by weight of the culture medium for cell-free activated cytotoxic cell culture is 1 x ^10-4 to 2 x ^10-3 parts by weight, preferably 2 x ^10-4 to 18 x 10 ^-4 parts by weight, and more preferably 5 10 ^-4 to 15 10 ^-4 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.

FIG. 1 is a schematic diagram showing an activated lymphocyte administration and blood sampling time.
FIG. 2 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. 3 is a photograph showing the skin condition before the administration of activated lymphocytes and after 2 weeks from the fifth administration in a skin infected 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 interleukin-2 (IL-2), and beta-glucan.

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 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-2 is a glycoprotein with a molecular mass of 14 to 17 kDa produced by antigen-activated T cells. It is known that T cells producing IL-2 themselves are autocrine or 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.

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, a first incubation step of suspending the monocyte in the cell culture medium to which the autologous plasma, the human interleukin-2, and the beta-glucan were added, and inoculating the monocyte into the coated cell culture container and culturing was carried out do. Such a first incubation step is preferably carried out for 30 minutes to 24 hours.

Herein, the composition ratio of the dog plasma to 100 parts by weight of the culture medium for the cell-derived activated cytotoxic cell culture may be 2 to 20 parts by weight, preferably 3 to 18 parts by weight, more preferably 5 to 15 parts by weight . 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 cell-free 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.

The composition ratio of the beta-glucan to 100 parts by weight of the culture medium for cell-free activated cytotoxic cell culture is 1 x ^10-4 to 2 x ^10-3 parts by weight, preferably 2 x ^10-4 to 18 x 10 ^-4 parts by weight, and more preferably 5 10 ^-4 to 15 10 ^-4 parts by weight. When the content of beta-glucan is within the above range, a sufficient amount of interferon-gamma can be produced at a level at which activation induced death by excessive stimulation does not occur.

Next, as a second culturing step, a substance selected from the group consisting of human interleukin-2, beta-glucan, and a mixture thereof is added to the cell culture medium for the first culturing step 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 [mu] g / ml beta-glucan (Santa Cruz Biotechnology, Dallas, TX). 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 [mu] g / ml beta-glucan 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.

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 1: After administration, symptoms of atopic dermatitis dogs and change in sensation

As shown in Fig. 1, the cultured cells were administered at intervals of two weeks for a total of six times, and symptomatic changes in the symptoms of the atopic dermatitis and change of the cannula were observed.

FIG. 2 is a photograph showing the skin condition before and after the fifth administration of atopic dermatitis dog. It was visually confirmed that the erythema and scars disappear and the hair grows even after the administration of the activated lymphocyte. From the above results, it was confirmed that atopic symptoms and pruning were improved when the activated lymphocyte was administered, and it was confirmed that the atopic symptoms and pruritus did not deteriorate even during the 8 - week no -

Test Example 2: After the administration, the symptom change of the skin infectious disease dog was confirmed

As shown in Fig. 1, the cultured cells were administered at intervals of two weeks for a total of six times, and the changes in the symptoms of the dogs and the changes in the pruritus were confirmed.

FIG. 3 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 is healed and the hair is uniformly grown 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,
Wherein the additive comprises at least one anti-CD3 antibody, a dog plasma, human interleukin-2 (IL-2) and beta-glucan. The method according to claim 1,
Wherein the cytotoxic cells cultured in the culture medium for the cell-derived activated cytotoxic cell culture is characterized in that the ratio of the cytotoxic cells having the CD3 ⁺ CD4 ^- CD8 ⁺ immunophenotype is 55 to 90% A culture medium for toxic 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;
(C) a step of inoculating the monocyte into a cell culture container coated with the anti-CD3 antibody and incubating the cell culture medium supplemented with the animal cell culture medium supplemented with human plasma, human interleukin-2 and beta-glucan A culture step; And
(D) a second culturing step in which a substance selected from the group consisting of human interleukin-2, beta-glucan, and a mixture thereof is cultured by adding the cell culture medium for the first culturing step Toxic cell culture method. 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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