KR102097665B1 - Medium composition and method for massive culture of autologous activated canine cytotoxic cell - Google Patents

Abstract

The present invention relates to a composition for preventing or treating a dog's auto-derived activated cytotoxic cell medium composition, a culture method, a dog's auto-derived lymphocytes and a dog's skin disease. In particular, a dog's auto-derived dog comprising an anti-CD3 antibody, a dog's auto plasma, a human interleukin-2 (IL-2), and a beta-glucan medium composition and a culture method, and cytotoxic cells cultured therefrom It relates to a composition for preventing or treating skin diseases of dogs using lymphocytes and the active ingredient thereof.

Description

Medium composition and culture method for self-derived activation of canine cytotoxic cells in dogs {MEDIUM COMPOSITION AND METHOD FOR MASSIVE CULTURE OF AUTOLOGOUS ACTIVATED CANINE CYTOTOXIC CELL}

The present invention relates to a medium composition for proliferation of autologous activated cytotoxic cells in dogs, a culture method, a composition for preventing or treating dog autologous cytotoxic cells and skin diseases of dogs. In particular, a medium composition and a culture method comprising a dog anti-CD3 antibody, a dog auto plasma, human interleukin-2 (IL-2), and beta-glucan, and T cells and natural killer cells cultured therefrom It relates to a composition for the prevention or treatment of canine skin diseases and autologous cytotoxic cells of dogs and dog skin diseases using the same as an active ingredient.

Beyond nuclear family, the number of single-person households is increasing rapidly. However, if your dog has a strange odor, red skin on your dog's skin, a lot of dandruff, eczema, or your dog's itch is likely to have a skin disease.

Skin diseases that appear in dogs include skin infections or allergic skin diseases. The dog's skin infections are mainly caused by pyoderma, dermatophytosis, improvement, folliculosis, etc. Dermatitis is the main cause.

Skin infection disease is a skin disease caused by various causes such as malnutrition, microbial infections such as bacterial fungi, hormones, and allergies. Symptoms such as rash, erythema, hair loss, and pruritus appear. There are various microorganisms in the dog's skin and usually do not cause the disease, but if the immunity is weakened by various factors, the skin inflammatory disease is caused by the overgrowth of the microorganisms. Most of the treatments are one-dimensional treatments such as antibiotics, antifungal agents, or anti-inflammatory agents for the microorganisms that cause them. There is a reaction at the beginning of treatment, but the more antibiotics are used, the more resistant the bacteria are, and the more frequent the recurrence, the worsening of the skin condition. Since the weakening of immunity causes the development of infectious diseases, it is necessary to fundamentally treat the disease by increasing the immunity.

The incidence of atopic dermatitis in dogs is about 3 to 15%, which is one of the most common diseases in dogs. However, most drugs currently treating canine atopic dermatitis are steroids and antihistamines. These drugs have a strong anti-inflammatory effect, which has the advantage of rapid improvement of symptoms, but have the disadvantage that the effects are temporary and severe side effects such as Cushing's syndrome, vasodilation, diabetes, osteoporosis, and reduced immunity may occur upon repeated administration. Unlike humans, although these serious side effects may occur, drug development aimed at atopic dermatitis diseases in dogs is rarely performed, and thus, the above drugs are still used.

The cause of atopic dermatitis has not yet been determined, but according to scientific evidence to date, overexpression of Th2 type cytokines such as IL-4 mRNA overexpression in skin lesion sites and IL-4 mRNA overexpression in PBMCs It is known to be caused by an imbalance of the Th1 / Th2 cytokines. Therefore, it is believed that by enhancing the Th1 cytokine that can suppress the environment caused by the Th2 cytokine, the imbalance of the Th1 / Th2 cytokine can be resolved and the atopic dermatitis disease can be treated.

Interferon-gamma is a representative Th1 type cytokine, and is mainly produced by cytotoxic cytotoxic cells, type I helper cytotoxic cells, and natural killer cells (NK cells) in humans. Is known. Interferon-gamma mainly increases secretion upon viral infection, thereby activating cytotoxic cells and NK cells to kill the virus-infected cells. In particular, interferon-gamma promotes differentiation of undifferentiated cytotoxic cells into Th1 type cytotoxic cells and, conversely, inhibits differentiation into Th2 type cytotoxic cells. In addition, it plays an important role in increasing the Th1 response and inhibiting the Th2 response, such as inhibiting the production of IgE in B cells.

In a previous study, a study paper reported that atopic symptoms were improved by administering dog-derived interferon-gamma recombinant protein (KT-100TM) to patients with canine atopic dermatitis disease [Veterinary dermatology 21.1 (2010): 42-49. ]. This was intended to balance the Th1 / Th2 cytokines through recombinant interferon-gamma in atopic disease dogs with a predominant Th2 response. However, in the case of dog recombination interferon-gamma, although the effect is excellent, it has been reported that the side effects are frequent after administration to many dogs, and thus it is no longer used in real dogs.

Other targets are drugs targeting IL-31, a cytokine that plays an important role in inducing pruritus, a typical symptom of atopic disease, or JAK1 (Janus kinase 1) molecule, which plays an important role in signaling by Th2 cytokines. These drugs are being developed, but these are only temporary effects due to drug administration and do not affect overall immunity improvement, and thus there is an urgent need for the development of therapeutic agents having fundamental efficacy.

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

The present invention has been devised to solve the above problems, and to maximize the production of interferon-gamma, dog anti-CD3 antibodies, dog auto plasma, human interleukin-2 (IL-2), and beta-glucan are used. It is an object of the present invention to provide a self-derived activated cytotoxic cell medium composition comprising a dog.

In addition, another object of the present invention is to provide a culture method of culturing a dog's auto-derived activated cytotoxic cells using the medium composition for culturing a self-derived activated cytotoxic cell of the dog.

In addition, another object of the present invention is to provide a dog-derived lymphocyte of a dog including a dog-derived activated cytotoxic cell cultured with the culture method of the dog or cultured with the culture method of the dog-derived activated cytotoxic cell of the dog. .

In addition, another object of the present invention is to provide a composition for preventing or treating skin diseases of a dog comprising the dog's self-derived lymphocytes as an active ingredient.

In order to achieve the object described above, the medium composition for autologous activated cytotoxic cell culture of dogs of the present invention includes a cell culture medium and an additive added to the cell culture medium, and the autologous activated cytotoxic cell of dogs In the medium composition for culturing, the additive is characterized in that it comprises a dog anti-CD3 antibody, dog auto plasma, human interleukin-2 (IL-2), and beta-glucan.

In addition, the cytotoxic cells cultured with the medium composition for autologous activated cytotoxic cell culture of the dog have a ratio of CD3 ⁺ CD4 ^- CD8 ⁺ cytotoxic cells having an immune phenotype of 55 to 90%, preferably 60 to 80%, More preferably, it may be 65 to 75%.

On the other hand, the dog's self-derived lymphocytes of the present invention are characterized by including the dog's auto-derived activated cytotoxic cells cultured with the medium composition for culturing the self-derived activated cytotoxic cells of the dog.

On the other hand, the method of culturing autologous activated cytotoxic cells of dogs of the present invention

(A) separating and obtaining monocytes and autologous plasma from the peripheral blood of the dog, respectively;

(B) coating the cell culture vessel with a coating solution containing dog anti-CD3 antibody;

(C) The monocytes were inoculated into a cell culture container coated with the dog's anti-CD3 antibody, and the cell culture medium to which the dog's autologous plasma, human interleukin-2 (IL-2), and beta-glucan was added was added. A first culture step of culturing by adding; And

(D) characterized in that it comprises a second culture step of culturing a material selected from the group consisting of human interleukin-2, beta-glucan and mixtures thereof by adding a culture medium for cell culture of the first culture step.

In addition, in the step of obtaining the monocytes in step (A), a blood vessel containing peripheral dog blood is centrifuged to form a mononuclear layer, and then the mononuclear layer is transferred to a test tube, each buffer is filled with a buffer solution, and then centrifuged again. And washing.

In addition, the composition ratio of the dog anti-CD3 antibody to 100 parts by weight of the coating solution in step (B) is 1 × 10 ^-5 to 2 × 10 ^-4 parts by weight, preferably 2 × 10 ^-5 to 18 × 10 ^-5 It may be 5 parts by weight, more preferably 5 × 10 ^-5 to 15 × 10 ^-5 parts by weight.

In addition, the composition ratio of the dog's autologous plasma to 100 parts by weight of the dog's auto-derived activated cytotoxic cell culture medium composition may be 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 medium composition for autologous activated cytotoxic cell culture of the dog 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 beta-glucan to 100 parts by weight of the medium composition for autologous activated cytotoxic cell culture of the dog is 1 × 10 ^-4 to 2 × 10 ^-3 parts by weight, preferably 2 × 10 ^-4 to 18 × 10 ^-4 parts by weight, more preferably 5 × 10 ^-4 to 15 × 10 ^-4 parts by weight.

On the other hand, the dog's self-derived lymphocytes of the present invention are characterized by including the dog's self-derived activated cytotoxic cells cultured by the method for culturing the self-derived activated cytotoxic cells of the dog.

In addition, the cytotoxic cells may be cytotoxic T cells.

In addition, the cytotoxic cells may be natural killer cells.

On the other hand, the composition for the prevention or treatment of skin diseases of dogs of the present invention is characterized by including the self-derived lymphocytes of the dog as an active ingredient.

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

In addition, the skin infection of the dog may be selected from the group consisting of pyoderma, dermatophytosis, amelioration, folliculosis and combinations thereof.

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

Lymphocytes containing activated cytotoxic cells derived from dogs cultured by the medium composition or culture method of the present invention have a high proportion of cells producing interferon-gamma, thereby improving symptoms of skin diseases including atopic dermatitis or infectious dermatitis in dogs, and Could be eased. In addition, by controlling the overall imbalance of the immune system focused on the Th2 response to the Th1 response, there is an advantage in that the healing effect is maintained without recurring for a longer time.

1 is a schematic diagram showing the activation lymphocyte administration and blood collection time.
FIG. 2 is a photograph showing skin conditions before and after 5 weeks of 5th administration of activated lymphocytes in atopic dermatitis disease dogs.
3 is a photograph showing the skin condition before and after the 5th administration of the activated lymphocytes in dogs with skin infection disease.

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific matters such as specific components are described, which are provided to help a more comprehensive understanding of the present invention, and it is common in the art that the present invention can be practiced without these specific details. It will be obvious to those who have the knowledge of. And, in the description of the present invention, if it is determined that a detailed description of related known functions or configurations may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.

The present invention is to cultivate activated cell toxic cells for the purpose of treating skin diseases such as atopic dermatitis and infectious dermatitis caused by dogs by culturing monocyte cells derived from dogs, and anti-CD3 antibodies of dogs in a cell culture medium , Human interleukin-2 (IL-2), beta-glucan.

Monocytes isolated from the initial blood herein are peripheral blood-derived mononuclear cells (PBMC), wherein the PBMC are immune cells such as B cells, T cells, and natural killer cells. And granulocytes such as basophils, eosinophils, and neutrophils.

T cells are one of the lymphocytes that are responsible for the antigen-specific adaptive immune response, and are immune cells that express CD3 molecules on the surface. Helper T cells that significantly regulate the differentiation and activation of other immune cells through cytokines, cytotoxic T cells and cytosines that secrete cytotoxic substances and remove infected cells. It can be divided into regulatory T cells that secrete kinase and suppress over-activated immune cells.

Cytotoxic T cells are T cells that express CD3 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 that produce interferon-gamma. In addition, it is a cell capable of producing granulins that can remove microorganisms and cells infected with microorganisms, such as bacteria and fungi, along with granzyme used to remove virus-infected cells and cancer cells.

Regulatory T cells express CD4 and CD25 on the cell surface and are characterized by expressing the transcription factor Foxp3 together. Regulatory T cells produce the inhibitory, cytokine interleukin-10 and TGF beta and have the ability to suppress over-activated immune cells. In this regard, regulatory T cells can be used as one method of atopic treatment.

Natural killer cells (NK cells) are cells that can directly remove virus-infected cells or cancer cells, and when activated, are one of the main cells that produce interferon-gamma. The natural killer cells of humans have a clear phenotype, but the natural killer cells of dogs are not yet known.

'Cytotoxic cell' in the present application specification refers to a cell that removes a virus, a cell infected with a virus, or other cell that has lost its function, and was used as a term encompassing the cytotoxic T cell and natural killer cells.

Dog anti-CD3 antibodies induce activation of T cells by binding to the T cell co-receptor CD3 molecule complexed with the T cell receptor (TCR) present in dog T cells. Activation by TCR plays an important role in the activation of T cells.

IL-2 is a glycoprotein having a molecular weight of 14 to 17 kDa produced by antigen-activated T cells, and the T cell itself that produced IL-2 reacts to IL-2 again (autocrine) or surroundings It is a method of delivering IL-2 to T cells (paracrine) that induces the growth of itself and other 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.

Beta-glucan (β-glucan) is a substance present on the cell wall of microorganisms such as bacteria, yeast, and fungi. D-glucose is a polysaccharide composed of β-1,3 bonds and β-1,6 bonds. Beta-glucan is known to activate immune cells through TLR2 (toll-like receptor 2), Dectin-1 and CR3 (complement receptor type 3) molecules, and macrophages activated by beta-glucan secrete cytokines. Through this, T cells and natural killer cells can be activated. These functions are currently used as adjuvant therapy and adjuvants in chemotherapy.

The CADESI (canine atopic dermatitis extent and severity index) -03 score used in the present invention is a score of four diseases in total in 62 sites of erythema, lichenification, excoriation, and alopecia. It is an index of the severity of atopy evaluated by the total [Veterinary dermatology 18.2 (2007): 78-86.]. The score of each item is rated from 0 to 5 depending on the severity of symptoms, and the total is 1,240.

The pruritus evaluation criteria used in the present invention were evaluated by the pruritus evaluation criteria as an indicator according to the judgment of the guardian, and evaluated by dividing them into 0 to 5 points according to the severity of symptoms.

In addition, the cytotoxic cells cultured with the medium composition for autologous activated cytotoxic cell culture of the dog have a ratio of CD3 ⁺ CD4 ^- CD8 ⁺ cytotoxic cells having an immune phenotype of 55 to 90%, preferably 60 to 80%, More preferably, it is characterized in that it occupies a large proportion of other lymphocytes as 65 to 75%. When the ratio of the cytotoxic cells having the CD3 ⁺ CD4 ^- CD8 ⁺ immune phenotype is within the above range, it is possible to generate a sufficient amount of interferon-gamma at a level that does not cause excessive immune response in the body after administration.

On the other hand, the method for culturing autologous activated cells of dogs derived from the present invention starts from the step of separately obtaining mononuclear cells and autologous plasma from the peripheral blood of the dog. In this, the monocyte obtaining step is performed by centrifuging the blood collection tube containing the dog's peripheral blood to form a mononuclear layer, then transferring the mononuclear layer to a test tube, filling each test tube with a buffer solution, and centrifuging again for washing. The cell yield can be further increased.

Then, the cell culture container is coated with dog anti-CD3 antibody. The dog's anti-CD3 antibody may be diluted with a phosphate buffer solution, and it is also possible to increase the efficiency of coating using gelatin, collagen, or fibronectin.

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

In addition, the first culturing step of suspending and culturing the mononuclear cells in the cell culture medium to which the obtained dog's autologous plasma, human interleukin-2, and beta-glucan was added and inoculating them in the coated cell culture vessel was performed. do. This, the first culture step is preferably performed for 30 minutes to 24 hours.

Herein, the composition ratio of the dog's auto plasma to 100 parts by weight of the dog's auto-derived activated cytotoxic cell culture medium composition may be 2 to 20 parts by weight, preferably 3 to 18 parts by weight, and more preferably 5 to 15 parts by weight. . When the content of the dog's autogenous plasma is within the above range, sufficient proliferation of cytotoxic cells is achieved at a level where unnecessary cell proliferation does not occur.

In addition, the composition ratio of human interleukin-2 to 1 ml of the medium composition for autologous activated cytotoxic cell culture of the dog 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 where activation-induced killing by excessive stimulation does not occur.

In addition, the composition ratio of beta-glucan to 100 parts by weight of the medium composition for autologous activated cytotoxic cell culture of the dog is 1 × 10 ^-4 to 2 × 10 ^-3 parts by weight, preferably 2 × 10 ^-4 to 18 × 10 ^-4 parts by weight, more preferably 5 × 10 ^-4 to 15 × 10 ^-4 parts by weight. When the content of beta-glucan is within the above range, it is possible to generate a sufficient amount of interferon-gamma at a level that does not cause activation-induced killing by excessive stimulation.

Then, as a second culture step, a substance selected from the group consisting of human interleukin-2, beta-glucan and a mixture thereof is added to the culture medium for cell culture in the first culture step and cultured.

On the other hand, the dog's self-derived lymphocytes of the present invention include those of the dog's auto-derived activated cytotoxic cells cultured with the medium composition for self-derived activated cytotoxic cell culture or cultured by the dog's self-derived activated cytotoxic cell culture method. It is characterized by.

In addition, the cytotoxic cells may be cytotoxic T cells or natural killer cells. These cells have a particularly high interferon-gamma production capacity.

On the other hand, the composition for the prevention or treatment of skin diseases of dogs of the present invention is characterized by including the self-derived lymphocytes of the dog as an active ingredient.

In addition, the skin disease of the dog may be a skin infection or an allergic skin disease, and the skin infection of the dog may be selected from the group consisting of pyoderma, dermatophytosis, amelioration, folliculosis and combinations thereof, and the allergic skin disease of the dog is atopic It may be selected from the group consisting of sex dermatitis, flea allergic dermatitis, and combinations thereof.

Hereinafter, examples of the present invention will be described.

Example

Example Step 1: Blood sampling of a dog

For the immune cell culture, 5-10 ml of peripheral blood was collected from 10 naturally occurring atopic dermatitis dogs in a heparin tube (BD vacutainer, BD bioscience). Blood collection took place at the licensed animal hospital with the consent of the guardian. The collected heparin tube was wrapped with foil to block light, and then transported at room temperature, and upon arrival at the culture room, monocyte separation was performed.

Example Step 2: Monocyte 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 layers. The upper plasma was recovered for later incubation. In order to separate the lymphocytes, the blood cell layer was diluted with RPMI1640 (Hyclone) medium in a 1: 1 ratio, and the diluted blood cell layer was carefully placed on 4 ml of Ficoll-paque PLUS (specific gravity 1.077, GE healthcare) solution, followed by 30 minutes. The liver was centrifuged at 400 xg. The monocyte layer separated between the medium layer and the 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, and left at 37 ° C for 10 minutes, followed by 5 minutes. Centrifugation at 2000 rpm. After suspending monocytes with 10 ml of 1 x PBS solution (phosphate-buffered saline, welgene), the cells were centrifuged at 2000 rpm for 5 minutes and the supernatant was removed. After the washing process was performed once more, monocytes were suspended in 1 ml of RPMI1640 medium and used for further culture and analysis.

Example Step 3: Monocyte cell culture

One day before incubation, the dog's anti-CD3 antibody (AbD serotec) was diluted in 1 x PBS solution at 1 µg / ml, and the flask surface was covered with 5 ml of the dog's anti-CD3 antibody diluted in T-25 flask (Nunc). This was added and stored at 4 ° C. The flask was decanted with anti-CD3 antibody solution before use and washed twice with 5 ml of 1 x PBS solution.

After diluting a portion of the monocytes obtained above in a Trypan blue (Sigma Aldrich) solution, live cells were counted. 1.0 x 10 ⁷ cells were inoculated into the washed T-25 flask, and the culture solution was added to a final volume of 5 ml. At this time, 10% inactivated autologous plasma, 2 mM L-glutamic acid (Gibco), 1 x Antibiotic-Antimycotic (Gibco) was added to RPMI1640 medium with the culture medium used, and 1000 IU / mL human IL-2 (cytocaine) proleukin) and 10 μg / ml beta-glucan (Santa Cruz Biotechnology, Dallas, TX) were used. Cells were cultured in a 37 ° C., 5% by volume CO ₂ incubator.

On days 2 to 3 of culture, 1000 IU / ml human IL-2 and 10 μg / ml beta-glucan were added to further activate the cells. After the 4th day of cultivation, the same amount is given at intervals of 1 to 2 days depending on cell proliferation, except that 10% FBS (Fetal bovine serum, Gibco) is used instead of 10% inactivated autologous plasma in the culture medium. The culture solution and the three cytokines were added to a final volume of 20 ml, and at the final volume of 20 ml or more, only the same amount of the culture solution and cytokine was added to 1000 IU / ml human IL-2.

Example Step 4: After culture, cells are administered to atopic dermatitis dogs

Example Monocytes of atopic dermatitis dogs were cultured for 13 days in the same manner as described in step 3. After incubation, the cell culture solution was transferred to a 250 ml conical tube, and the supernatant was removed after centrifugation at 2000 rpm for 5 minutes. Then, the cells were suspended with 250 ml of 1 x PBS solution, centrifuged at 2000 rpm for 5 minutes, and the supernatant was removed. After the washing process was performed once more, the washed cells were suspended in 10 ml of a normal saline solution (Daehan Pharm. Co., Ltd.) and filtered through nylon mesh (40um, BD Bioscience) to remove impurities. Then, some cells were removed and stained with trypan blue solution to count the living cells. Based on living cells, up to 5.0 x 10 ⁸ cells were placed in a 20 ml to 50 ml syringe (Korean vaccine) equipped with an 18G needle (Korea vaccine), and put into a final saline solution to make a final volume of 20 ml. The productized cells were stored at 4 ° C., and stored at room temperature 30 minutes prior to administration, followed by administration after adjusting the product to the body temperature. The administration proceeded at a rate of 10 ml / 30 minutes and cells were administered intravenously.

Test Example 1: Confirmation of symptom change and pruritus of atopic dermatitis dog after administration

As shown in Figure 1, after the cultured cells were administered a total of 6 times at intervals of 2 weeks, the symptoms of atopic dermatitis and changes in pruritus were confirmed.

FIG. 2 is a photograph showing the skin condition before and 5 weeks after the 5th administration to atopic dermatitis dogs, and it was visually confirmed that erythema and wounds disappear and hairs grow evenly after administration of activated lymphocytes. From the above results, it was confirmed that atopy symptoms and pruritus were improved when the activated lymphocytes were administered, and it was confirmed that the atopy symptoms and pruritus did not worsen even during the 8-week no sagging period.

Test Example 2: Confirmation of symptoms of dog with skin infection disease after administration

As shown in Figure 1, after the cultured cells were administered a total of 6 times at 2 week intervals, changes in symptoms and pruritus of dogs with infectious skin diseases were confirmed.

3 is a photograph showing the skin condition before and 5 weeks after the 5th administration to a skin-infectious disease dog, and it can be visually confirmed that the wound heals and the hair grows evenly after administration of activated lymphocytes.

The preferred embodiments of the present invention have been described above, but the present invention is not limited to the specific embodiments described above, and those skilled in the art can perform various modifications without departing from the gist of the present invention. Of course it is possible. Therefore, the scope of the present invention should not be construed as being limited to the above embodiments, but should be defined by the claims and equivalents as well as the claims described below.

Claims (8)

A medium composition for proliferating autologous activated cytotoxic cells of a dog, comprising a medium for proliferation of monocytes and an additive added to the medium for proliferation of monocytes,
The additive is an anti-CD3 antibody, autologous plasma of dogs, human interleukin-2 (IL-2), and beta-glucan. The method according to claim 1,
Cytotoxic cells cultured with the medium composition for proliferation of autologous activated cytotoxic cells of the dog, characterized in that the proportion of cytotoxic cells having the CD3 ⁺ CD4 ^- CD8 ⁺ immune phenotype is 55 to 90%, the autologous activated cells of dog Medium composition for toxic cell proliferation. The method according to claim 1,
The cytotoxic cells are cytotoxic T cells, natural killer cells, and a cell composition characterized in that the cells selected from the group consisting of combinations, self-derived activated cytotoxic cell medium composition for dogs. Self-derived lymphocytes of dogs with increased ability to generate interferon gamma comprising self-derived activated cytotoxic cells of dogs cultured with a medium composition for self-derived activated cytotoxic cell proliferation of claim 1. (A) separating and obtaining monocytes and autologous plasma from the peripheral blood of the dog, respectively;
(B) coating the cell culture vessel with a coating solution containing dog anti-CD3 antibody;
(C) Inoculating the monocytes into a cell culture container coated with the dog's anti-CD3 antibody, and culturing by adding a medium for proliferation of the dog's autologous plasma, human interleukin-2, and beta-glucan to the monocyte proliferation medium. Cultivation step;
(D) a second culture step of culturing by further adding human interleukin-2 and beta-glucan to the culture that has undergone the first culture step;
(E) a third culture step of culturing by adding a medium for proliferation of monocytes to which FBS (Fetal bovine serum), human interleukin-2, and beta-glucan are added to the culture that has undergone the second culture step; And
(F) a self-derived activated cytotoxic cell of a dog comprising a fourth culturing step of culturing by adding a medium for proliferation of monocytes to which autologous plasma and human IL-2 has been added to the culture that has undergone the third culturing step. Proliferation method. The method according to claim 5,
The cytotoxic cell is characterized in that the cell selected from the group consisting of cytotoxic T cells, natural killer cells, and combinations thereof, a method for proliferation of autologous activated cytotoxic cells of dogs. Self-derived lymphocytes of dogs with increased ability to produce interferon gamma, including self-derived activated cytotoxic cells of dogs cultured by the method of proliferating the self-derived activated cytotoxic cells of claim 5. A composition for preventing or treating atopic dermatitis or infectious skin diseases of dogs comprising the self-derived lymphocytes of claim 4 or 7 as an active ingredient.

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