KR20070022619A - Canine tumor treatment method, pharmaceutical formulation applied thereto, and method of cryogenically preserving cells used therewith - Google Patents

Abstract

The present invention provides a method of preparing, culturing and cryopreserving a dog having a tumor and the agent used for the treatment. Peripheral blood lymphocytes in dogs are proliferated and activated in the presence of a combination of a solid anti-canine CD3 antibody and interleukin-2 (IL-2) (preferably, anti-canine CD3 antibody and human interleukin-2 (human IL-2)) The lymphocytes are then administered to the dog with the tumor. After this treatment, tumors in dogs were reduced without serious side effects. Improvements in quality of life have been identified in terms of animal ability to increase walking distance and improve hair appearance.

Description

Tumor therapy for dogs, formulations used therein, and methods for cryopreservation of cells used with it {CANINE TUMOR TREATMENT METHOD,

1 is a diagram showing a comparison of the degree of cell proliferation at different culture temperatures.

The present invention relates to a canine tumor therapy, an agent for treating canine tumors using canine lymphocytes, and a method of culturing and cryopreserving the cells used in the treatment.

It has been reported by J. H. Jardine et al. (Veterinary Immunology, Vol. 21, pp. 153-160, 1989) that lymphocytes of dogs can be propagated using human interleukin 2 (hIL-2). It has also been reported by DW Mitchell et al. That proliferation can be performed using PHA (phytohemagglutinin) and hIL-2 (American Journal of Veterinal Research, Vol. 52, pp. 1132-1136, 1991). .

It has also been reported by H. Itoh et al (Journal of Veterinary Medical Science, Vol. 65, pp. 329-333, 2003) that lymphocytes of dogs can be proliferated by a solid anti-canine CD3 antibody and hIL-2. It has also been reported by Mizuno et al. That dog lymphocytes can be cultured at 38 ° C. using PHA and IL-2 (Mizuno et al., Experimental Animal, Vol. 45, p. 292, 1996).

However, J.H. Jardine et al. D.W. Mitchell et al. Report that hIL-2 alone can propagate lymphocytes in dogs to produce Lymphokine Activated Killer (LAK) cells derived from large granular lymphocytes (LGL). D.W. Mitchell reports that peripheral lymphocytes in dogs are cultured with PHA and hIL-2, and PHA activates T cells via CD2 molecules on the surface of T cells, whereas anti-CD3 antibodies activate T cells via CD3. Started. Therefore, cells cultured in this manner have different activation mechanisms, and thus the population and function of T cells are different.

In addition, H. Itoh et al. Confirmed that there was no side effect by injecting activated lymphocytes obtained by culturing dog lymphocytes using solid anti-dog CD3 antibody and hIL-2, but did not confirm antitumor activity in vivo. In addition, Mizuno et al proliferated the lymphocytes of dogs using PHA and IL-2 at a temperature of 38 ° C, but did not confirm the antitumor effect in vivo. The method of activating lymphocytes of Mizuno and Itoh is different from the CD3 activation method described above by the present invention, i.e., these methods do not provide an effective treatment for dog tumors.

In the treatment used for tumors of dogs, no anticancer agent has been developed for dogs so far. As a result, anticancer agents developed for humans have been administered to dogs with tumors at a dose calculated by body surface area. However, the stability of the treatment and its antitumor effects have not been fully confirmed. In addition, although a number of clinical trials have been conducted using activated lymphocytes for the treatment of human cancer, there have been no reports confirming the stability of these treatments, improving the quality of life of patients or antitumor effects of activated lymphocytes in dogs with tumors.

The present inventors have found that peripheral blood in dogs in the presence of a combination of a solid anti-dog CD3 antibody and interleukin-2 (IL-2), preferably a combination of an anti-dog CD3 antibody and recombinant human interleukin-2 (hIL-2). The present invention has been accomplished through a method of activating and propagating lymphocytes. After administering them to dogs with tumors, beneficial results in morphological improvement and antitumor effects of quality of life indicators were identified.

Claim 1 is a tumor of a dog which uses a combination of a solid anti-canine CD3 antibody and interleukin-2 (IL-2) to proliferate and activate peripheral blood lymphocytes of a dog, and then administers the obtained lymphocytes to a tumor-bearing dog. It is about treatment.

Furthermore, the invention of claim 2 relates to the tumor treatment of dogs according to claim 1, wherein the interleukin-2 (IL-2) is human interleukin-2 (hIL-2).

The invention according to claim 3 relates to a dog tumor treatment method according to claim 1, wherein the temperature at the time of proliferating and activating the peripheral blood lymphocytes of the dog is in the range of 37 to 41 ° C.

Furthermore, the invention of claim 4 relates to a dog tumor treatment method according to claim 1, wherein the temperature at the time of proliferating and activating the peripheral blood lymphocytes of the dog is in the range of 38 to 40 ° C.

The invention of claim 5 relates to an agent for the treatment of dog tumors obtained by proliferating and activating peripheral blood lymphocytes of a dog in the presence of a combination of a solid anti-dog CD3 antibody and interleukin-2 (IL-2).

Furthermore, the invention of claim 6 relates to the agent for treating tumors of dogs according to claim 5, wherein the interleukin-2 (IL-2) is human interleukin-2 (hIL-2).

The invention according to claim 7 relates to the agent for treating dog tumors according to claim 5, wherein the temperature at the time of proliferating and activating the peripheral blood lymphocytes of the dog is within the range of 37 to 41 ° C.

Furthermore, the invention of claim 8 relates to an agent for treating dog tumors according to claim 5, wherein the proliferation and activation temperature of the peripheral blood lymphocytes of the dog is within the range of 38 to 40 ° C.

The invention according to claim 9, wherein the lymphocytes obtained after the proliferation and activation of the peripheral blood lymphocytes of the dog in vitro in the presence of a combination of a solid anti-canine CD3 antibody and interleukin-2 (IL-2), The present invention relates to a cryopreservation method of cells for treating tumors in dogs which are thawed and prepared as a preparation.

The invention according to claim 10 relates to a cryopreservation method of a cell for treating tumors of a dog according to claim 9, wherein the interleukin-2 (IL-2) is human interleukin-2 (hIL-2).

The invention according to claim 11 relates to a cryopreservation method of a cell for treating dog tumors according to claim 9, wherein the temperature at the time of proliferating and activating the peripheral blood lymphocytes of the dog in vitro is in the range of 37 to 41 ° C.

The invention according to claim 12 also relates to a cryopreservation method of a cell for treating dog tumors according to claim 9, wherein the temperature at the time of proliferating and culturing the peripheral blood lymphocytes of the dog in vitro is in the range of 38 to 40 ° C.

As described above, the dog's tumor therapeutic cells used in the therapies, preparations and cryopreservation methods embodied by the invention are preferably in the presence of a combination of solid anti-dog CD3 antibodies and interleukin-2 (IL-2), preferably anti- Activated and proliferated in the presence of a combination of canine CD3 antibodies and human recombinant interleukin-2 (hIL-2). These cells provide a clear tumor shrinking effect when administered as activated lymphocytes to dogs with tumors. No serious side effects have been identified after administration of the formulation, and improved quality of life (QOL) has been confirmed in the form of the ability of the treated animal to walk long distances, and it has been confirmed that the health of the animal improves in the form of a healthy hair appearance. It became.

The present invention is described in detail by the following preferred examples.

Example 1

Lymphocytes collected individually from two healthy dogs were incubated twice each by the following procedure.

Procedure 1: Preparation of Cell Culture Flask

5 mL of anti-opened CD3 antibody (Serotec Ltd., mouse anti-opened CD3) solution diluted to 5 µg / mL in saline (Hikari Pharmaceuticals, Ltd.) was placed in a 75 cm 2 culture flask (Sumitomo Bakelite Co., Ltd.) The bottom surface of the flask was uniformly covered.

The flask was kept at 4 ° C. (± 2 ° C.) overnight before the anti-open CD3 antibody solution in the flask was removed. 20 mL of physiological saline was poured into the flask, and the lid was closed and shaken vigorously. The lid was then opened and the solution removed. After repeating this operation, the liquid remaining in the flask and in the lid was removed and the flask was stored in the refrigerator until the next use.

Course 2: Blood Collection

5 mL of peripheral blood were separately collected from healthy dogs 1 and 2 weighing 2 kg and 22 kg, respectively, and then heparinized.

Process 3: Isolation of Peripheral Blood Monocytes

The peripheral blood was aseptically placed in a 15 mL centrifuge tube (Greiner Bio-One Co., Ltd .; No.188271) in a clean bench (Showa science Co., Ltd .; S-1100PRV) and centrifuge (Kokusan Co. , Ltd. No. H-700FR) was separated for 10 minutes at 1800 rpm at 24 ℃.

In the centrifugation process, red blood cells, white blood cells, and lymphocytes were separated and precipitated from the upper plasma. The plasma was then cryopreserved for next use. In order to separate lymphocytes from the remaining blood cell layer, the layer was washed with about twice the amount of washing solution [500 mL of RPMI-1640 culture medium (Nikkon Bio Medical Laboratories Inc.), 1 N hydrochloric acid (Wako Pure Chemical Industries, Ltd.), and 10 mL of 1 M HEPES solution (Sigma-Aldrich Co., No. H0887)] and added to a 50 mL centrifuge tube.

3 mL of Lymphosepar 1 (Immuno-Biological Laboratories, Ltd., No. 23010) was then added to a 15 mL centrifuge tube using a 5 mL pipette (Corning Japan K.K., No. 4487). Next, 10 mL of blood cells suspended in the washing solution was slowly layered on the Lymphosepar 1 without breaking the interface. This solution was then separated by centrifuge at 30 ° C., 1,600 rpm for 30 minutes with a break off.

After centrifugation, a portion of lymphocytes were taken from the intermediate layer and placed in a 50 mL centrifuge tube (Greiner Bio-one Co., Ltd .; No.227216) using a 5 mL pipette, and added with a washing solution to an amount of 20 to 30 mL, and the solution Mixed well. 10 μl of the solution was taken to determine the number of peripheral blood lymphocytes, and 500 μl of the solution was respectively placed in three round bottom tubes (Bechton-Dickinson Japan Co., Ltd. No. 352008) to analyze cell surface antigens. Put in.

Step 4: Determination of Recovered Cell Number

In step 3, 10 µl sample collected to measure the number of peripheral blood monocytes was mixed with 40 µl of Turk solution (Turk solution, Muto Pure Chemicals Co., Ltd.), and then 10 µl was improved New Buyer blood cell. It was placed in a counter (Neubeier Hematocytometer, Erma Inc.) and the cell number was measured under a microscope. The number of peripheral blood monocytes was 2.1 × 10 ⁷ and 1.1 × 10 ⁷ for healthy dog 1 and 6.4 × 10 ⁶ and 6.9 × 10 ^{6 for} healthy dog 2.

Course 5: Analysis of Cell Surface Antigens

In step 3, the sample collected for cell surface antigen analysis was separated at a centrifuge at 24 ° C. and 1800 rpm for 5 minutes, and then suspended matters were removed. In three separate tubes (1) 5 μl of control γ1 / γ1 antibody (Becton, Dickinson and Company; 349526), (2) FITC mouse anti-dog CD3 antibody (Serotec Co., Ltd .: MCA1774F) and PE mouse anti-dog 5 μl of a 1: 1 mix of B cell antibody (Serotec Co., Ltd.:MCA178PE) and 5 μl of (3) FITC rat anti-dog CD4 / PE rat anti-dog CD8 antibody were added, respectively. These solutions were made to react at 4 degreeC for 10 minutes or more.

After the reaction, 2 mL of IsoFlow Sheath solution (Becton, Dickinson and Company; No. C412006) containing 2% human albumin was added to each test tube, and the contents were added to a vortex mixer (Scientific Industry Co., No.4781487). And mixed. The contents of the test tube were separated at a centrifuge at 24 ° C. and 1800 rpm for 5 minutes, and then the suspension was removed as much as possible. On the day, 500 μl of 2% human albumin IsoFlow Sheath solution was added to the test tube, while the next day analysis was performed after adding 500 μl of Cell Fix (Becton, Dickinson and Company: No.340181) to the test tube. . Cell surface antigens were analyzed using FACS Calibur 4A (Becton, Dickinson and Company). The results are shown in Table 1.

Analysis of Cell Surface Antigens Before Culture Healthy dog 1 Healthy dog 2 One 2 One 2 CD3-positive T cells 67.1% 70.5% 35.7% 40.3% B cell positive cell 9.3% 14.5% 13.3% 19.5% CD4-positive cells 36.3% 38.1% 26.0% 30.5% CD8 positive cells 26.3% 25.0% 21.2% 22.5%

Course 6: Cultivation of Peripheral Blood Monocytes

After sampling, the remaining peripheral blood monocytes were separated by centrifuge at 24 ° C. and 1600 rpm for 10 minutes, then the suspended matter was removed with a decanter, and the cell pellet was dispersed using a vortex mixer. 20 mL of the culture medium [RPMI-1640 + 7s (Nikken Bio Medical Laboratory Inc.) 400 mL, 35,000 IU / mL rIL-2 (PROLEUKIN: Chiron Corp.) 4.5 mL, FBS (JRH Biosciences, Inc.) 45 mL] Was suspended in the flask, and transferred to a flask prepared as described in Step 1. This flask was then placed in a large air jacketed CO ₂ incubator and incubated in an environment of temperature 37 ° C. (± 0.2 ° C.), CO ₂ concentration 5% (± 0.2%) and humidity 95% (± 5%).

Course 7: Cell Proliferation and Culture

The cells cultured by the above procedure 6 were observed under a microscope, and 10-30 mL of the same culture medium was added 3 to 7 days after the start of the culture. Culture medium was added until the amount in the flask became 50 mL, and the culture was continued. Four to eight days after the start of the culture, the cells were transferred to a new flask with culture medium. After adding 50 mL of fresh culture medium, the culture was continued. After 6 to 10 days from the start of the culture, 150 mL of culture medium containing hIL-2 175 IU / mL and 10% FBS was added, and the culture was continued for 8 to 16 days.

Course 8: Inactivate Serum

Serum collected in step 3 was thermally inactivated in a 57 ° C. water bath for 50 minutes and then stored at 4 ° C.

Course 9: Cell Characterization Test

In order to confirm the cell characteristics, endotoxin test, sterility test and cell surface antigen analysis were carried out after 8-16 days of culture and one day before the cells were prepared for final administration. For identification, about 3 mL of the culture was taken as a test sample with a 5 mL syringe (Terumo Corporation) with a needle of 18 gauge x 38 mm. For endotoxin testing, 1 mL of sample was placed in a 5 mL round bottom test tube (Becton, Dickinson and Company: No. 352054), and the test tube was capped. 500 μl samples were spread on a chocolate agar medium (Nikken Bio Medical Laboratory Inc.) for sterility testing. For cell surface antigen analysis, 500 μl of samples were placed in three 5 mL round bottom test tubes, respectively.

Course 10: Endotoxin Test

The test sample collected in step 8 was centrifuged at 24 ° C. and 1800 rpm for 5 minutes. Before the test, 80 μl pure water and 20 μl of culture suspension were mixed with a main reagent solution (Toxi color LS-50S set, Seikagaku Corporation, No.020130) prepared in a 5 mL round bottom test tube, and the solution was mixed in a 37 ° C. water bath. Incubate for 35 minutes. At the same time, 100 μl of pure and endotoxin standard solution (CSE-L set: Seikagaku Corp., No. 020055) were respectively mixed with the main reagent solution and incubated in a water bath with the samples used as blanks and positive controls.

After completion of the reaction by adding 400 µl of 0.8 mol / L acetic acid solution, 380 µl of the obtained solution was transferred to a 96-hole microplate. Absorbance reference wavelengths at 405 nm and 655 nm were measured using a microplate reader. Since the standard solution was prepared to be about 0.1 EU / mL, the test sample was said to meet the standard when five batches of the measured value of the test sample were within the measured value of the standard solution. The results all met this criterion.

Course 11. Sterility Test

The sample prepared in step 9 was sprinkled on chocolate agar medium and stored in an incubator at 37 ° C. Aseptic determinations were made the next day, ie, before preparation of the final cell preparation used for treatment. If no microbial colony was present on the plate, a negative judgment was made, so the results were all negative.

Course 12: Analysis of Cell Surface Antigens Before Cell Preparation for Administration

Cell surface antigen analysis was performed using the same method as described in Step 5. The results are shown in Table 2.

Analysis of Cell Surface Antigens after Culture Healthy dog 1 Healthy dog 2 One 2 One 2 CD3-positive T cells 96.0% 92.0% 79.6% 96.6% B cell positive cell 2.5% 2.3% 1.4% 0.9% CD4-positive cells 4.6% 10.2% 11.4% 12.5% CD8 positive cells 94.7% 90.0% 92.0% 91.8%

Procedure 13: Preparation of Cellular Formulations

During the 8 to 12 days after the start of the culture, the cells were determined to meet the endotoxin test, the sterility test, and the assay of the cell surface antigens. Preparation was carried out as follows. During the incubation, 250 mL of the total amount of the culture solution in the flask was transferred to a centrifuge tube and centrifuged at 1000 rpm for 24 minutes at 24 ° C. The resulting suspension was decanted and removed, and cells were dispersed using a vortex mixer. Then, 50 mL of saline containing 0.5% of autoserum (prepared in Step 8) was added and mixed.

The obtained solution was again centrifuged for 8 minutes at 1700 rpm at 24 ° C. in a centrifuge, suspended matter was removed, and cells were dispersed using a vortex mixer. In order to suspend the cells sufficiently, physiological saline containing 2% autoserum in the cell dispersion was added to 10 mL for treatment of dogs weighing less than 10 kg and 30 mL for treatment of dogs weighing 10 kg or more. The cell suspension was filtered through a cell filter (Becton, Dickinson and Company: No. 352360), and then 10 µl of the cells was suspended in 1 mL of saline containing 2% albumin to prepare a cell number.

Course 14: Cell number measurement after preparation

10 µl of the final formulation prepared in Step 13 was sampled, and the cell number was measured using the same method as described in Step 4. 10 μl of the sample was mixed with 20 μl of trypan blue staining solution (SIGMA Corp.:No.T8154), and 10 μl of the obtained solution was placed on an improved New Buyer hemocytometer, and the number of stained cells was measured under a microscope. The survival rate was determined. The number of cells was 3.3 × 10 ⁹ in healthy dog 1, 2.2 × 10 ⁹ cells, 4.1 × 10 ⁹ in healthy dog 2 and 3.0 × 10 ⁹ cells. Cell viability is considered to be a satisfactory result of 95% or more.

Course 15: Administration of Activated Lymphocytes

The magnetic cell suspension prepared in step 13 was administered to healthy dog 1 and healthy dog 2. No side effects were observed except for a slight increase in body temperature.

Example 2

Activated lymphocytes formulated in the same manner as described in Example 1 were administered 1 to 12 times to 9 dogs with tumors. Improvements in quality of life (QOL) have been identified in the form of increased appetite, improved hair appearance, and the ability of animals to increase their walking distance approximately two times than before treatment. The administration results are shown in Table 3.

Treatment Results of Dogs with Tumors from Lymphocytes incubated at 37 ° C dog Conversion weight gender age carcinoma Administration cell number Dosing frequency Improvement of QOL One Labrador Retriever 30Kg cancer 6 years 11 months Chronic Granulomatous Hepatitis 1.9 × 10 ⁸ to 5.0 × 10 ⁸ 12 Vitality recovery, improvement of hair appearance 2 Burnies Mountain Dock 39 Kg cancer 9 years Adenocarcinoma 0.7 × 10 ⁸ to 4.5 × 10 ⁸ 9 Revitalization, Restoration of Appetite 3 Shuttleland Sheepdog 10.7Kg Number 8 years 3 months Rectal adenocarcinoma 1.1 × 10 ⁸ to 3.0 × 10 ⁸ 5 Revitalization, Restoration of Appetite 4 Italian Greyhound 8Kg Number 6 years 11 months Cervical Fibrosarcoma 1.2 × 10 ⁸ to 7.6 × 10 ⁸ 4 Energy increase 5 Shuttleland Sheepdog 9.1Kg cancer 14 years 2 months Soft tissue sarcoma 1.0 × 10 ⁸ to 3.1 × 10 ⁸ 5 Vitality, Improved Health, Tumor Loss 6 Flat Coated Retriever 29.5Kg cancer 7 years 11 months Angiosarcoma 1.7 × 10 ⁸ to 3.9 × 10 ⁸ 5 Recovery after surgery, normal vitality 7 Golden Retriever 356 Kg cancer 12 years 10 months Angiosarcoma 0.7 × 10 ⁸ to 3.9 × 10 ⁸ 5 Maintain healthy hair and vitality 8 Golden Retriever 32.7Kg Number 10 years 5 months Rectal adenocarcinoma 0.7 × 10 ⁸ to 4.7 × 10 ⁸ 4 No change 9 Cairn Terrier 7.5Kg cancer 9 years 11 months Malignant mixed tumor of the mammary gland 4.7 × 10 ⁸ One Improvement of hair appearance and vitality recovery

Example 3

Peripheral blood lymphocytes taken from healthy dogs 3 and 4 were isolated by the procedure described in Example 1. Lymphocytes were cultured in essentially the same manner as described in Example 1 except that the incubation temperature was at 37 ° C (± 0.2 ° C), 38 ° C (± 0.2 ° C) and 39 ° C (± 0.2 ° C). The results for the amount of lymphocytes when grown at 15 to 20 times are shown in Table 4 (Healthy Dog 3), Table 5 (Healthy Dog 4) and Table 6. Is shown in the graph. Among three culture temperatures 37 ° C., 38 ° C., and 39 ° C., cells cultured at 39 ° C. achieved the best microproliferation.

Cell counts and cell surface antigens at different culture temperatures (healthy dogs 3) Incubation days Cell count Survival rate CD3 (%) B cell (%) CD4 (%) CD8 (%) Incubation temperature 0 5.00E + 06 - 73.51 24.67 41.72 26.14 37 ℃ 9 7.50E + 07 84.5% 78.5 5.99 46.08 69.68 38 ℃ 7 1.05E + 08 93.7% 94.26 6.74 23.76 83.32 39 ℃ 6 8.00E + 08 89.0% 99.32 6.43 31.38 78.17

Cell counts and cell surface antigens at different culture temperatures (healthy dogs 4) Incubation days Cell count Survival rate CD3 (%) B cell (%) CD4 (%) CD8 (%) Incubation temperature 0 5.00E + 06 - 81.28 27.17 44.68 24.53 37 ℃ 7 9.00E + 07 92.2% 94.24 5.57 27.54 78.58 38 ℃ 7 1.05E + 08 92.8% 95.83 4.96 23.62 82.26 39 ℃ 6 1.00E + 08 89.0% 96.96 7.04 27.08 80.09

Number of days needed to increase cell number 10 times Incubation temperature Sample Days Average days 37 ℃ Sample 3 8.4 7.3 Sample 4 6.2 38 ℃ Sample 3 6.2 6.2 Sample 4 6.2 39 ℃ Sample 3 5.4 5.3 Sample 4 5.2

As shown in Table 6, the incubation period required to multiply by 10 times the cell number is 7.3 days at 37 ℃, 6.2 days at 38 ℃, 5.3 days at 39 ℃. It was confirmed that the time required for incubation can be shortened to 1-2 days.

Example 4

In large air jacket-type CO ₂ incubator other than increasing the temperature in the 38 ℃ and 39 ℃ under the environment of CO ₂ concentration of 5.0 (± 0.2%), humidity of 95% (± 5.0%) is the same as that described in Example 1 The culture was carried out under the conditions. The resulting activated lymphocytes were administered once and twice to five tumor-bearing dogs, respectively. Improvements in quality of life (QOL) have been identified in the form of increased appetite, improved hair quality and the ability of dogs to double the walking distance before treatment. The results are shown in Table 7.

Results obtained by administration of lymphocytes cultured at a temperature of 38 ° C. or higher for tumor dogs Dog No Conversion weight gender age carcinoma Administration cell number Dosing frequency Improvement of QOL One Labrador Retriever 30Kg cancer 6 years 11 months Chronic Granulomatous Hepatitis 3.7 × 10 ⁸ 12 Vitality recovery, improvement of hair quality 5 Shuttleland Sheepdog 9.1Kg cancer 14 years 2 months Soft tissue sarcoma 1.9 × 10 ⁸ 5 Vitality, maintenance of body (tumor loss) 7 Golden Retriever 35.6Kg cancer 12 years 10 months Angiosarcoma 1.9 × 10 ⁸ 5 Maintain vitality and hair quality 9 Cairn Terrier 7.5Kg cancer 9 years 11 months Malignant mixed tumor of the mammary gland 1.1 × 10 ⁸ One Improvement of hair quality Vitality recovery 10 Labrador Retriever 31Kg Number 1 year 5 months Rhabdomyosarcoma of the bladder 2.8 × 10 ⁸ to 4.7 × 10 ⁸ 2 Vitality

In Table 3 and Table 7, the dog with tumor died after 6 administrations of activated lymphocytes, but this death was not related to the tumor. However, it was confirmed that tumors that could not be removed by surgery by autopsy dissection were lost.

Example 5

Exemplary embodiment in which Example 5 In, increase in the temperature of 38 ℃ and 39 ℃ under the environment of CO ₂ concentration of 5% (± 0.2%), humidity of 95% (± 5.0%) in a large air jacket-type CO ₂ incubator in Example 3 Lymphocytes were cultured under essentially the same conditions as described in. Then, some of the cells were cryopreserved, thawed, cultured and administered to the subjects. Prior to the initiation of the culture, a method essentially identical to that described in Processes 1 to 6 was performed.

Course 16: Partial Cryopreservation of Cells

Three to five days after the culture for activating and propagating the cells, 10-30 mL of the culture medium was added to the same flask until the amount in the flask became 50 mL. Then, the number of cells was measured 4-7 days after the start of the culture. Cells were cryopreserved when the cell number exceeded 1.0 × 10 ⁶ / mL.

While continuing culturing the cells, 30-50 mL of the culture solution was transferred to 50 mL test tubes, and then centrifuged at 24 ° C. and 1200 rpm for 5 minutes. After the suspension was removed by decant, 1.8 mL of lysate preservatives (Bambanker ^™ , Lymphotec Inc.) was added to each test tube, and the solution was mixed well with a vortex mixer. 1.8 mL of cell suspension was added to each 2 mL cryotube (cryotube, Corning Incorporated: No.430289), and the tube was placed in a bicell unit (Cryopreservation vessel from Nihon Freezer Co., Ltd.) and temperature -85 ° C. Ultra low temperature freezer (Sanyo Electric Co., Ltd. No.MDF-192). After the next day, it was transferred to a liquid nitrogen tank (My Sciences Co., Ltd.) for biological preservation.

Course 17: Thawing and Cultivating Cryopreserved Cells

Cryopreserved cells prepared in step 16 were thawed and cultured as follows. The freezing tube was removed from the liquid nitrogen tank and thawed in a heating block (Taitec Co., Ltd .: Dryothermic Unit No. DTU-28) for 4 minutes. After the whole contents were transferred to 10 mL of washing | cleaning liquid using the Pasteur pipette, the obtained cell suspension was centrifuged at 24 degreeC and 1200 rpm for 3 minutes, and isolate | separated.

After removing the suspended matter, the dispersed cell pellet was suspended in 15-20 mL of culture medium (RPMI1640 + 7S 400 mL, 35,000 IU / mL human rIL-2 4.5 mL, FBS 45 mL), and then transferred to a 50 mL test tube. 10 μl was sampled with trypan blue stain for measurement. 1 to 2 mL of the remaining cell suspension was placed in each hole of an untreated 24-hole plate (Sumitomo Bakelite Co., Ltd., No. MS-80240), followed by 37 ° C. (± 0.5 ° C.) in a large jacketed CO ₂ incubator, Incubation was started in an environment of 5% CO ₂ (± 2%) and 95% humidity (± 5.0%).

The cells which started culturing in culture medium of less than 20 mL were observed under a microscope for 1 to 3 days from its activation and proliferation, and then 1 to 2 mL of culture medium was placed in each hole. After sufficient mixing of the trypan blue staining solution and the cell number measurement solution, 10 μl of the solution was placed in an improved Neuberier Hemocytometer to measure the number of cells stained and dyed under living microscope and dying cells.

Course 18: Activation of Cryopreserved Cells

Cells cultured by the method described in Procedure 17 were observed under a microscope. From 2 to 4 days from the incubation period of activating and proliferating the cells, 20-30 mL of the cell solution still being cultured is transferred from the hole into a culture flask prepared in the same manner as described in Procedure 1 using a pipette, followed by Culture culture was continued by adding 20-30 mL of medium (400 mL of RPMI 1640 + 7S, 4.5 mL of 35,000 IU / mL human rIL-2, 45 mL FBS).

Course 19: Proliferation and Cultivation of Cryopreserved Cells

Cells cultured in step 18 were observed through a microscope. Two to four days after incubation to activate and propagate the cells, the cells attached to the bottom of the flask were removed by tapping. The whole cell was then transferred to a 225 cm 2 culture flask (Corning Coster Company: No. 3000), and 50 mL of culture medium (RPMI 1640 + 7S 400 mL, 35,000 IU / mL human rIL-2 4.5 mL, FBS 45 mL) was added. Further addition was continued to incubate.

In order to prepare a dosing formulation, the cells were observed through a microscope 6-8 days after the start of cell culture to activate and proliferate the cells, and then culture medium (RPMI 1640 + 7S 400 mL, 35,000 IU / mL human rIL- 2 2.25 mL, FBS 45 mL) 150 mL was added.

Course 20: Examination of cryopreserved cells

The cultured and cryopreserved cells were examined in the same manner as described in procedures 9-12.

Course 21: Preparation and Cell Number Measurement of Cryopreserved Cells for Therapeutic Administration

Cryopreserved cells cultured as described in procedures 13 to 14 were measured and prepared for final administration.

Course 22: Administration of Cryopreserved Cells

Administration of the magnetic cell suspension prepared in step 13 to the patient dog improved QOL in the form of animal capacity with a strong appetite, improved hair appearance, and a two-fold increase in walking distance. This was confirmed. The results are shown in Table 8.

Treatment results after thawing and reactivating frozen lymphocytes in the dog Patient Conversion weight gender age carcinoma Administration cell number Dosing frequency Improvement of QOL 9 Cairn Terrier 7.5Kg cancer 9 years 11 months Malignant mixed tumor of the mammary gland 4.7 × 10 ⁸ One Improvement of hair quality Vitality recovery

According to the present invention regarding the treatment of dog tumors, preparations for treating dog tumors using dog lymphocytes, and methods of culturing and cryopreserving the cells used in the treatment, tumors of dogs with tumors can be reduced without serious side effects. Can improve the quality of life.

Claims (12)

A method of treating dog tumors, comprising proliferating and activating peripheral blood lymphocytes of a dog in the presence of a combination of a solid anti-dog CD3 antibody and interleukin-2 (IL-2). The method of claim 1, wherein the interleukin-2 (IL-2) is human interleukin-2 (human IL-2). The method of treating dog tumors according to claim 1, wherein the temperature at the time of proliferating and activating the peripheral blood lymphocytes of the dog is in the range of 37 to 41 ° C. The method of treating dog tumors according to claim 1, wherein the temperature at the time of proliferating and activating the peripheral blood lymphocytes of the dog is in the range of 38 to 40 ° C. A preparation for treating tumors in dogs comprising peripheral blood lymphocytes proliferated and activated in the presence of a combination of a solid anti-dog CD3 antibody and interleukin-2 (IL-2). The method of claim 5, wherein the interleukin-2 (IL-2) is human interleukin-2 (human IL-2). The agent for the treatment of dog tumors according to claim 5, wherein the temperature at the time of proliferating and activating the peripheral blood lymphocytes of the dog is within a range of 37 to 41 ° C. The agent for treating dog tumors according to claim 5, wherein the temperature at the time of proliferating and activating the peripheral blood lymphocytes of the dog is in the range of 38 to 40 ° C. After proliferating and activating peripheral blood lymphocytes in dogs in the presence of a combination of solid phase anti-dog CD3 antibody and interleukin-2 (IL-2), the lymphocytes are cryopreserved, thawed and restored in use, and reactivated. Cryopreservation method of cells for the treatment of dogs, characterized in that for preparing. 10. The method of claim 9, wherein interleukin-2 (IL-2) is human interleukin-2 (human IL-2). 10. The cryopreservation method of cells for treating tumors of dogs according to claim 9, wherein the temperature when proliferating and activating peripheral blood lymphocytes in vitro is in the range of 37 to 41 ° C. The cryopreservation method of cells for treating tumors of dogs according to claim 9, wherein the temperature of proliferating and activating the peripheral blood lymphocytes of the dog in vitro is in the range of 38 to 40 ° C.

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