Abstract:
A treatment for tumorous canines and a method of preparing, cultivating, and cryogenically preserving the pharmaceutical formulation used in the treatment. Canine lymphocytes are activated and expanded in the presence of a combination of anti-canine CD3 antibodies and interleukin-2 (IL-2) (preferably anti-canine CD3 antibodies and human interleukin-2 (human IL-2)) and administered to tumorous dogs. Following treatment, the canine tumors were observed to be in regression while no serious side effects were noted. Quality-of-life improvements were observed in the form of the animals&#39; ability to walk increased distances and improved coat appearance.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     The present invention relates to a method for treating canine tumor, a pharmaceutical formulation which uses canine lymphocytes to treat a tumorous canine, and a method of culturing and cryogenically preserving cells used for the treatment.  
         [0002]     J. H. Jardine et al have reported that canine lymphocytes may be expanded using human interleukin 2 (hIL-2) (Veterinary Immunology, Volume 21, 1989, pp. 153-160). Moreover, D. W. Mitchell et al have reported that propagation may be conducted using PHA (Phytohemaglutinin) and hIL-2 (American Journal of Veterinary Research, Volume 52, 1991, pp. 1,132-1,136).  
         [0003]     Furthermore, H. Itoh et al have reported that canine lymphocytes may be propagated with solid phase anti-canine CD3 antibodies and hIL-2 (Journal of Veterinary Medical Science, Volume 65, 2003, 329-333). In addition, Mizuno et al have reported that canine lymphocytes may be cultivated with PHA and IL-2 at a temperature of 38° C. (Mizuno et al, Experimental Animal, Volume 45, 1996, 292).  
         [0004]     Reports by J. H. Jardine et al and D. W. Mitchell et al, however, disclose that canine lymphocytes may be propagated with hIL-2 alone to produce LAK (Lymphokine Activated Killer) cells derived from large granular lymphocytes (LGL). A report by D. W. Mitchell discloses that canine peripheral lymphocytes have been cultivated with PHA and hIL-2, and that PHA activates T-cells via CD2 molecules on the surface of the T-cell while the anti-CD3 antibodies activate T-cells via CD3. Therefore, cells cultivated in this manner have a different activation mechanism and therefore exhibit a different T-cell population and function.  
         [0005]     Moreover, H. Itoh et al confirmed that no adverse effects were observed by the infusion of activated lymphocytes obtained by cultivating canine lymphocytes with solid-phase anti-canine CD3 antibodies and hIL-2, but they did not confirm that there was anti-tumor activity in vivo. Furthermore, Mizuno et al propagated canine lymphocytes with PHA and IL-2 at a temperature of 38° C., but did not confirm any anti-tumor effect in vivo. The Mizuno and Itoh&#39;s methods for activating lymphocytes are different from the CD3 activation method specified by the present invention in that these methods do not provide effective treatment for a canine tumor.  
         [0006]     In regard to therapies applied to tumorous canines, as of the present there have been no anti-cancer drugs developed for canines. As a result, anti-cancer drugs developed for human beings have been administered to tumorous canines at dosages calculated according to body surface area. The safety of the treatment and its anti-tumor effects, however, have not been sufficiently confirmed. Furthermore, although many clinical trials have made use of activated lymphocytes for human cancer therapies, there have been no confirmed reports regarding the safety of these therapies, improvements in the patients&#39; quality of life, nor the anti-tumor effects of activated lymphocytes on tumorous canines.  
       SUMMARY OF THE INVENTION  
       [0007]     The invention was construed through a process in which inventors activated and propagated canine peripheral blood lymphocytes in the presence of a combination of solid-phase anti-canine CD3 antibodies and interleukin-2 (IL-2) with a preferred combination being anti-canine CD3 antibodies and recombinant human interleukin-2 (hIL-2). These were administered to tumorous canines after which beneficial results were observed in the form improvements in a quality-of-life index and anti-tumor effect.  
         [0008]     The Claim  1  invention relates to a method of treating canine tumor wherein the peripheral blood lymphocytes of a canine are propagated and activated using a combination of solid-phase anti-canine CD3 antibodies and interleukin-2 (IL-2) after which the resulting lymphocytes are administered to a tumorous canine.  
         [0009]     Moreover, the Claim  2  invention relates to a method for treating canine tumor according to Claim  1  wherein the interleukin-2 (IL-2) is a human interleukin-2 (hIL-2).  
         [0010]     The Claim  3  invention relates to the method for treating canine tumor according to Claim  1  wherein the temperature under which canine peripheral blood lymphocytes are propagated and activated lies within a range of from 37° C. to 41° C.  
         [0011]     Moreover, the Claim  4  invention relates to a method for treating canine tumor according to Claim  1  wherein the temperature under which canine peripheral blood lymphocytes are propagated and activated lies within a range of from 38° C. to 40° C.  
         [0012]     The Claim  5  invention relates to a pharmaceutical formulation for treating a canine tumor wherein the formulation is obtained from the propagation and activation of canine peripheral blood lymphocytes in the presence of a combination of solid-phase anti-canine CD3 antibodies and interleukin-2 (IL-2).  
         [0013]     Moreover, the Claim  6  invention relates to a pharmaceutical formulation for treating a canine tumor according to Claim  5  wherein the interleukin-2 (IL-2) is a human interleukin-2 (hIL-2).  
         [0014]     The Claim  7  invention relates to a pharmaceutical formulation for treating a canine tumor according to Claim  5  wherein the temperature under which the canine peripheral blood lymphocytes are propagated and activated lies within a range of from 37° C. to 41° C.  
         [0015]     Furthermore, the Claim  8  invention relates to a formulation for treating a canine tumor according to Claim  5  wherein the propagation and activation temperature of the canine peripheral blood lymphocytes lies within a range of from 38° C. to 40° C.  
         [0016]     The Claim  9  invention relates to a method of preparing cryogenically preserved cells for treating a canine tumor wherein peripheral blood lymphocytes of a canine are propagated and activated in the presence of a combination of solid-phase anti-canine CD3 antibodies and interleukin-2 (IL-2) after which the resulting lymphocytes are cryogenically preserved for subsequent thawing and preparation for use as a pharmaceutical formulation.  
         [0017]     The Claim  10  invention relates to a method of cryogenically preserving cells for treating a canine tumor according to Claim  9  wherein the interleukin-2 (IL-2) is human interleukin-2 (hIL-2).  
         [0018]     The Claim  11  invention relates to a method of cryogenically preserving cells for treating a canine tumor according to Claim  9  wherein the temperature under which the canine peripheral blood lymphocytes are propagated and activated in vitro lies within a range of from 37° C. to 41° C. Moreover, the Claim  12  invention relates to a method of cryogenically preserving cells for treating a canine tumor according to Claim  9  wherein the temperature under which the canine peripheral blood lymphocytes are propagated and cultivated in vitro lies within a range of from 38° C. to 40° C.  
         [0019]     As noted above, the canine tumor treatment cells utilized in the treatment method, pharmaceutical formulation, and cryogenic preservation method specified by the invention are activated and propagated in the presence of a combination of solid-phase anti-canine CD3 antibodies and interleukin-2 (IL-2), preferably in the presence of a combination of anti-canine CD3 antibodies and human recombinant interleukin-2 (hIL-2). These cells provide a clearly recognizable tumor-reducing effect when administered to a tumorous canine as activated lymphocytes. No serious side effects were observed after administration of the formulation, an improvement in quality-of-life was noted in the form of the ability of the treated animals to walk longer distances, and an improvement in the animals&#39; health was noted in the form of a healthier appearing coat.  
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0020]      FIG. 1  is a line drawing comparing cell propagation levels at different cultivation temperatures. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0021]     A detailed description of the invention is provided by the following preferred embodiments.  
       First Embodiment  
       [0022]     Lymphocytes collected separately from two healthy test canines were cultivated twice each according to the following procedure.  
         [0000]     Procedure 1: Preparation of Cell Cultivation Flasks  
         [0023]     Five milliliters (5 ml) of an anti-canine CD3 antibody (Serotec Ltd., mouse anti-canine CD3) solution diluted to 5 μg/ml with a physiological saline solution (Hikari Pharmaceuticals, Ltd.) were placed in a 75 cm 2  culture flask (Sumitomo Bakelite Co., Ltd.) to a level which covered the bottom of the flask.  
         [0024]     The flasks were maintained at 4° C. (±2° C.) overnight after which the anti-canine CD3 antibody solution in the flask was discarded. Twenty milliliters (20 ml) of the physiological saline solution was poured into the flasks after which it was sealed with a cap and thoroughly agitated. The caps were then removed and the solution discarded. This operation was repeated after which the liquid remaining in the flask and cap was removed and the flasks placed in a refrigerator until further use.  
         [0000]     Procedure 2: Blood Collection  
         [0025]     Five milliliters (5 ml) of peripheral blood were separately collected and heparinized from healthy canine test subjects 1 and 2 weighing 2 kg and 22 kg respectively.  
         [0000]     Procedure 3: Separation of Peripheral Blood Mononuclear Cells  
         [0026]     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; No. S-1100PRV) and separated for 10 minutes in a centrifugal separator (Kokusan Co., Ltd. No. H-700FR) at 1,800 rpm at 24° C.  
         [0027]     The centrifugation process separated and precipitated the erythrocytes, leukocytes, and lymphocytes from the supernatant plasma. The plasma was then cryogenically preserved and put aside for future use. In order to separate the lymphocytes from the remaining blood cell layers, the layers were diluted with approximately twice their volume of washing solution [500 ml of RPMI-1640 cultivating medium (Nikken Bio Medical Laboratories Inc.), 3 ml of 1 N hydrochloric acid (Wako Pure Chemical Industries, Ltd.), and 10 ml of 1 M HEPES solution (Sigma-Aldrich Co., No. H0887)] and were added in a 50 ml centrifuge tube (Greiner Co., Ltd., No. 227216).  
         [0028]     Three milliliters (3 ml) of Lymphosepar 1 (Immuno-Biological Laboratories, Ltd., No. 23010) was subsequently added to a 15 ml centrifuge tube using a 5 ml pipette (Corning Japan K.K., No. 4487). Ten milliliters (10 ml) of the blood cells suspended in a washing solution were then gently layered onto the Lymphosepar 1 without disturbing the interface. The solution was then separated in a centrifuge at 1,600 rpm at 24° C. for 30 minutes with the brake off.  
         [0029]     After centrifugation, a fraction of the lymphocytes was taken from the interfacial layer and placed in a 50 ml centrifuge tube (Greiner Bio-one Co., Ltd: No. 227216) with a 5 ml pipette, the washing solution was added to attain a volume ranging from 20 to 30 ml, and the solution was mixed well. Ten microliters (10 μl) of the solution was taken for counting the peripheral blood lymphocytes, and 500 μl each of the solution was placed into each of three round bottom tubes (Beckton-Dickinson Japan Co., Ltd. No. 352008) for analyzing cell surface antigens.  
         [0000]     Procedure 4: Counting the Collected Cells  
         [0030]     The 10 μl sample collected for the purpose of counting the number of peripheral blood mononuclear cells in Procedure 3 was mixed with 40 μl of Turks solution (Muto Pure Chemicals Co., Ltd.) after which 10 μl was placed-in an improved Neubeier Hematocytometer (Erma Inc.) for counting the cell number under a microscope. A total of 2.1×10 7  and 1.1×10 7  peripheral blood mononuclear cells were counted for the healthy canine subject No. 1, and 6.4×10 6  and 6.9×10 6  were counted for the healthy canine subject No. 2.  
         [0000]     Procedure 5: Analysis of Cell Surface Antigens  
         [0031]     The samples collected for cell surface antigen analysis in Procedure 3 were separated in a centrifuge at 24° C. at 1800 rpm for 5 minutes after which the supernatants were discarded. Into each of three tubes were respectively added (1) 5 μl of control y1/y1 antibody (Becton, Dickinson and Company: 349526), (2) 5 μl of a 1:1 mixture of FITC mouse anti-canine CD3 antibody (Serotec Co., Ltd.; No. MCA1774F) and PE mouse anti-canine B cell antibody (Serotec Co., Ltd. No. MCA178PE), and (3) 5 μl of FITC rat anti-canine CD4/PE rat anti-canine CD8 antibody. These solutions where then reacted at 4° C. for at least 10 minutes.  
         [0032]     After reaction, 2 ml of IsoFlow sheath solution (Becton, Dickinson and Co., No. C412006) containing 2% human albumin were added to each test tube and the contents of each mixed with a vortex mixer (Scientific Industry Co., No. 4781487). The contents of the test tubes were then separated in a centrifuge at 24° C. and 1,800 rpm for 5 minutes after which as much of the supernatant as possible was discarded. Analysis was conducted after adding 500 μl of 2% human albumin IsoFlow sheath solution to the test tubes on the day, while 500 μl of Cell Fix (Becton, Dickinson and Company: No. 340181) was added to the tubes for the analysis on the next day. Analysis of cell surface antigens was performed with a FACS Calibur 4A (Becton, Dickinson and Company). The results are shown in Table 1.  
                                                                                     TABLE 1                           Analysis of Cell Surface Antigens Before Cultivation                Healthy Canine 1       Healthy Canine 2                    1   2   1   2                            CD3 positive T cell   67.1%   70.5%   35.7%   40.3%           B cell positive cell    9.3%   14.5%   13.3%   19.5%           CD4 positive cell   36.3%   38.1%   26.0%   30.5%           CD8 positive cell   26.3%   25.0%   21.2%   22.5%                      
 
 Procedure 6: Peripheral Blood Mononuclear Cell Cultivation 
 
         [0033]     After sampling, the remaining peripheral blood mononuclear cells were separated in a centrifuge at 24° C. at 1600 rpm for 10 minutes after which the supernatant was discarded by decanting and the cell pellets dispersed with a vortex mixer. The cells were suspended in 20 ml of the cultivating medium [400 ml of RPMI-1640+7s; (Nikken Bio Medical Laboratory Inc.), 4.5 ml of 35,000 IU/ml rIL-2 (Proleukin: Chiron Corp.), 45 ml of FBS (JRH Biosciences, Inc.)] and transferred to a flask prepared as described in Procedure 1. The flask was then put into a large air jacket CO 2  incubator and incubated at a temperature of 37° C. (±0.2°) in an environment having a CO 2  concentration of 5% (±0.2%), and 95% (±5%) humidity.  
         [0000]     Procedure 7: Propagating and Expanding Cell Culture  
         [0034]     The cells cultivated according to Procedure  6  were observed under a microscope, and 10 to 30 ml of the same cultivating medium was added three to seven days after cultivation was initiated. The cultivating medium was added until the volume in the flask reached 50 ml after which cultivation continued. The cells were transferred to a new flask, together with the cultivating medium, four to eight days after cultivation commenced. Fifty milliliters (50 ml) of a fresh cultivating medium were added after which cultivation was allowed to continue. Six to ten days from the start of cultivation, 150 ml of the cultivating medium, which contained hIL-2 175 IU/ml and 10% FBS, was added and cultivation allowed to continue for eight to sixteen days.  
         [0000]     Procedure 8: Inactivating the Autologous Serum  
         [0035]     The blood serum collected in Procedure  3  was thermally inactivated in a water bath at 57° C. for 50 minutes after which it was preserved at 4° C.  
         [0000]     Procedure 9: Tests to Verify Cell Quality  
         [0036]     In order to verify the quality of the cells, an endotoxin test, sterility test, and analysis of cell surface antigens were carried out the day before the cells were to be prepared for final administration after the eight to sixteen-day cultivation period. For verifying, approximately 3 ml of the cultivation fluid was collected, as a test sample, with a 5 ml syringe having a 38 mm by 18-gauge needle (Terumo Corporation). For the endotoxin test, 1 ml of the sample was dispensed in a 5 ml round bottom test tube (Becton, Dickinson and Company, No. 352054) and the test tube capped. Five-hundred microliters (500 μl) of the sample were spread on a chocolate agar plate (Nikken Bio Medical Laboratory Inc.) for the sterility test. Five-hundred microliters (500 μl) of the sample were dispensed to each of three 5 ml round bottom test tubes for the cell surface antigen analysis.  
         [0000]     Procedure 10: Endotoxin Test  
         [0037]     The test sample collected in Procedure 9 was centrifuged at 24° C. at 1800 rpm for 5 minutes. Before the test, 80 μl of pure water and 20 μl of the cultivated supernatant were mixed into a main reagent solution (Toxi Color LS-50S set, Seikagaku Corporation, No. 020130) prepared in a round bottom 5 ml test tube after which the solution was incubated in a water bath at 37° C. for 35 minutes. Concurrently, 100 μl of pure water and an endotoxin standard solution (CSE-L set, Seikagaku Corp., No. 020055), were mixed with the main reagent solution respectively and incubated in the water bath together with the sample for use as a blank and positive control.  
         [0038]     The reaction was terminated by the addition of 400 μl of 0.8 mol/l acetic acid solution after which 380 μl of the resulting solution was transferred to a 96-well microplate. Absorbency reference wavelengths were measured at 405 nm and 655 nm using a microplate reader. As a result of the standard solution being prepared to approximately 0.1 EU/ml, a multiple of five of the test sample measurement value was within a result of the standard, and the test sample was thus determined to meet the standard. All results were determined to have met this standard.  
         [0000]     Procedure 11: Sterility Test  
         [0039]     The sample prepared in Procedure 9 was seeded on a chocolate agar plate and stored in an incubator at 37° C. The sterility determination was made the next day, that is, before preparation of the final cell formulation which would be used for treatment. As there were no colonies of microorganisms existing on the plate, a negative determination was made, and thus rendered all results negative.  
         [0000]     Procedure 12: Cell Surface Antigen Analysis Prior to Formulating Cells for Administration  
         [0040]     Cell surface antigen analysis was conducted using the same method described in Procedure 5. The results are shown in Table 2.  
                                                                             TABLE 2                           Cell Surface Antigen Analysis After Cultivation                Healthy Canine 1   Healthy Canine 2                    1   2   1   2                        CD3 positive T cell   96.0%   92.0%   79.6%   96.6%       B cell positive cell   2.5%   2.3%   1.4%   0.9%       CD4 positive cell   4.6%   10.2%   11.4%   12.5%       CD8 positive cell   94.7%   90.0%   92.0%   91.8%                  
 
 Procedure 13: Preparing the Cell Formulation 
 
         [0041]     The formulation was prepared for administration on the day after it was determined that the cells met the endotoxin, sterility, and cell surface antigen analysis standards within a period eight to twelve days from initiation of cultivation. Preparation was as follows. The entire 250 ml quantity of the incubation broth in the flask was transferred, during cultivation, to a centrifuge tube and centrifugally separated at 24° C. at 1,000 rpm for 8 minutes. The resulting supernatant was discarded by decanting and the cells dispersed using a vortex mixer. Fifty milliliters (50 ml) of the physiological saline solution containing 0.5% of autologous serum (as prepared in Procedure 8) was then added and mixed into the solution.  
         [0042]     The resulting solution was separated in a centrifuge again at 24° C. and 1,700 rpm for 8 minutes, the supernatant discarded, and the cells dispersed using the vortex mixer. In order to assure adequate cell suspension, 10 ml of the physiological saline solution containing 2% autologous serum were added to the cell dispersion solution for treatment of canines weighing less than 10 kg, and 30 ml was added for canines weighing more than 10 kg. The cell suspensions were filtered through a cell strainer (Beckton, Dickinson &amp; Co., No. 352360) after which 10 μl of the cells were suspended in 1 ml of physiological saline solution with 2% albumin in preparation for measuring the number of cells.  
         [0000]     Procedure 14: Counting the Number of Cells after Formulation  
         [0043]     Ten microliters (10 μl) of the final formulation prepared in Procedure 13 were sampled and the number of cells measured using the same method described in Procedure 4. The 10 μl of the sample was mixed with 20 μl trypan blue staining solution (Sigma Corp., No. T8154), 10 μl of the resulting solution was placed in an improved Neubeier Hematocytometer, and then the stained cells were counted under a microscope to determine the survival rate. It was determined that the cell volume was 3.3×10 9  and 2.2×10 9  for the healthy canine subject 1, and 4.1×10 9  and 3.0×10 9  for the healthy canine subject  2 . All cell survival rates were greater than 95% and were thus considered satisfactory.  
         [0000]     Procedure 15: Administering the Activated Lymphocytes  
         [0044]     The autologous cell suspension prepared in Procedure 13 was administered to the healthy canine subjects 1 and 2. No adverse effects were observed other than a slight rise in body temperature.  
       Second Embodiment  
       [0045]     One to 12 treatments of the activated lymphocytes, which were formulated in the same manner as described in the first embodiment, were administered to the nine tumorous canines. Quality-of-life improvements were noted in the form of increased appetite, improvement in appearance of coat, and the ability of the animals to walk approximately twice as far as before treatment. The results of the administrations are shown in Table 3.  
                                                                                           TABLE 3                           Results of treating tumorous canines with lymphocytes       cultivated at 37° C.                                    Number of                                       administered   No.of   Quality of life       Subject   Canine Type   Weight   Sex   Age   Cancer Type   cells   treatments   improvement                    1   Labrador Retriever     30 Kg   F   6 years   chronic   1.9 × 10 8 ˜5.0 × 10 8     12   vigor restore,                       11 months   granulomatuous           improvement in                           hepatitis           fur appearance       2   Bernese     39 Kg   F   9 years   adenocarcinoma   0.7 × 10 8 ˜4.5 × 10 8     9   vigor restore,           Mountain Dog                           recovery of                                       appetite       3   Shetland Sheepdog   10.7 Kg   M   8 years   rectal gland cancer   1.1 × 10 8 ˜3.0 × 10 8     5   vigor restore,                       3 months               recovery of                                       appetite       4   Italian Greyhound     8 Kg   M   6 years   cervical fiber cancer   1.2 × 10 8 ˜7.6 × 10 8     4   increased energy                       11 months       5   Shetland Sheepdog    9.1 Kg   F   14 years   soft tissue sarcoma   1.0 × 10 8 ˜3.1 × 10 8     5   increased vigor,                       2 months               improved health,                                       disappearance of                                       tumor       6   Flat Coated   29.5 Kg   F   7 years   blood vessel   1.7 × 10 8 ˜3.9 × 10 8     5   excellent recovery           Retriever           11 months   sarcoma           from surgery,                                       normal vigor       7   Gold Retriever   35.6 Kg   F   12 years   blood vessel   0.7 × 10 8 ˜3.9 × 10 8     5   Maintained                       10 months   sarcoma           healthy fur and                                       vigor       8   Golden Retriever   32.7 Kg   M   10 years   rectal gland cancer   0.7 × 10 8 ˜4.7 × 10 8     4   no change                       5 months       9   Cairn Terrier    7.5 Kg   F   9 years   mammary gland   4.7 × 10 8     1   improvement in                       11 months   malignant           appearance of fur,                           mixed tumor           vigor restore                  
 
       Third Embodiment  
       [0046]     Peripheral blood lymphocytes taken from the healthy canine subjects 3 and 4 were separated by the procedure described in the first embodiment. The canine lymphocytes were cultivated through a method essentially similar to that described in the first embodiment, but at cultivation temperatures of 37° C. (±0.2° C.), 38° C. (±0.2° C.) and 39° C. (±0.2° C.). The results for lymphocyte volumes which were grown fifteen to twenty-fold are shown in Table 4 (for the healthy canine subject 3), in Table 5 (for the healthy canine subject 4), in Table 6, and in the  FIG. 1  graph which shows a comparison of cell growth at different cultivation temperatures. Among the three cultivation temperatures of 37° C., 38° C., and 39° C., optimum cell grown was achieved for the cells cultivated at 39° C.  
                                                                                   TABLE 4                           Cell quantity and surface antigens at different cultivation       temperatures (healthy canine subject 3)                Days   Cell   Survival                           cultivated   Quantity   Rate   CD3   B Cell   CD4   CD8                        cultivation temp.   0   5.00E+06   —   73.51%   24.67%   41.72%   26.14%       37° C.   9   7.50E+07   84.5%    78.5%   5.99%   46.08%   69.68%       38° C.   7   1.05E+08   93.7%   94.26%   6.74%   23.76%   83.32%       39° C.   6   8.00E+08   89.0%   99.32%   6.43%   31.38%   78.17%                  
 
         [0047]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                   
               
               
                 Cell quantities and surface antigens at different culture 
               
               
                 temperatures (healthy canine subject 4) 
               
             
          
           
               
                   
                 Days 
                 Cell 
                 Survival 
                   
                 B Cell 
                   
                   
               
               
                   
                 Cultivated 
                 Quantity 
                 Rate 
                 CD3 (%) 
                 (%) 
                 CD4 (%) 
                 CD8 (%) 
               
               
                   
                   
               
             
          
           
               
                 cultivation 
                 0 
                 5.00E+06 
                 — 
                 81.28 
                 27.17 
                 44.68 
                 24.53 
               
               
                 temp. 
               
               
                 37° C. 
                 7 
                 9.00E+07 
                 92.2% 
                 94.24 
                 5.57 
                 27.54 
                 78.58 
               
               
                 38° C. 
                 7 
                 1.05E+08 
                 92.8% 
                 95.83 
                 4.96 
                 23.62 
                 82.26 
               
               
                 39° C. 
                 6 
                 1.00E+08 
                 89.0% 
                 96.96 
                 7.04 
                 27.08 
                 80.09 
               
               
                   
               
             
          
         
       
     
         [0048]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                   
               
               
                 Days required to increase cell quantity by a factor of ten 
               
             
          
           
               
                   
                 Cultivation Temperature 
                 Sample 
                 Days 
                 Average (days) 
               
               
                   
                   
               
               
                   
                 37° C. 
                 Sample 3 
                 8.4 
                 7.3 
               
               
                   
                   
                 Sample 4 
                 6.2 
               
               
                   
                 38° C. 
                 Sample 3 
                 6.2 
                 6.2 
               
               
                   
                   
                 Sample 4 
                 6.2 
               
               
                   
                 39° C. 
                 Sample 3 
                 5.4 
                 5.3 
               
               
                   
                   
                 Sample 4 
                 5.2 
               
               
                   
                   
               
             
          
         
       
     
         [0049]     As shown in Table 6, the cultivation period required to grow the number of cells ten-fold averaged 7.3 days at 37° C., 6.2 days at 38° C., and 5.3 days at 39° C. It was determined that the time required for cultivation could be shortened by a few days.  
       Fourth Embodiment  
       [0050]     In the fourth embodiment, cultivation was carried out under the same conditions as those described in the first embodiment with the exception that the temperature was increased between 38° C. and 39° C. in an environment having a CO 2  concentration of 5.0 (±0.2%) and 95.0% (±5.0%) humidity in a large air jacket-type CO 2  incubator. The obtained activated lymphocytes were administered one and two times to five tumorous canines. Quality-of-life improvements were observed in the form of increased appetite, improvements in coat quality, and the ability of the canines to walk approximately twice as far as before treatment. The results are shown in Table 7.  
                                                                                           TABLE 7                           Results obtained from administering of lymphocytes, cultivated at a temperature over 38° C., to tumorous       canines.            Canine   Canine                   Number of   Number of           No.   Type   Weight   Sex   Age   Cancer Type   administered cells   treatments   Improvement of QOL                    1   Labrador   30 kg   F   6 years 11   chronic granulomatuous   3.7 × 10 8     12   recovery of vigor,           Retriever           months   hepatitis           improvement in quality of coat       5   Shetland   9.1 kg    F   14 years 2   soft tissue sarcoma   1.9 × 10 8     5   increased vigor,           Sheepdog           months               condition maintained                                       (disappearance of tumor)       7   Golden   35.6 kg     F   12 years   blood vessel sarcoma   1.9 × 10 8     5   Maintained coat quality and vigor           Retriever           10 months       9   Cairn   7.5 kg    F   9 years   mammary gland malignant   1.1 × 10 8     1   improvement in coat quality,           Terrier           11 months   mixed tumor           recovery of vigor       10   Labrador   31 kg   M   1 year   rhabdomyosarcoma of   2.8 × 10 8 ˜4.7 × 10 8     2   vigor maintained           Retriever           5 months   bladder                  
 
         [0051]     While the no.5 tumorous canine in Tables 3 and 7 died after six administrations of the activated lymphocytes, the death was unrelated to its tumorous condition. It was confirmed by autopsy, however, that a tumor, which could not be surgically removed, had disappeared.  
       Fifth Embodiment  
       [0052]     In the fifth embodiment, the lymphocytes were cultivated under conditions essentially the same as those described in the third embodiment that the temperature was raised between 38° C. and 39° C. in the large air jacket-type CO 2  incubator in which the environment had CO 2  concentration of 5% (±0.2%) and 95% (±5.0%) humidity. A portion of the cells were then cryogenically preserved, thawed, incubated, and then administered to the test subjects. Methods essentially similar to those described in Procedures 1 through 6 were applied before the commencement of cultivation.  
         [0000]     Procedure 16: Cryogenic Preservation of a Portion of the Cells  
         [0053]     Three to five days following the cultivation period during which the cells were activated and propagated, 10 to 30 ml of the cultivating medium was added to the same flask until the volume in the flask reached 50 ml. The number of cells was then counted during the period four to seven days after cultivation commenced. The cells were cryogenically preserved when their number exceeded 1.0×10 6 /ml.  
         [0054]     With the cells still under cultivation, 30 to 50 ml of the cultivating solution was transferred to each of 50 ml test tubes and then separated in a centrifuge at 24° C. and 1,200 rpm for 5 minutes. Their supernatants were discarded by decanting after which 1.8 ml of a cell cryopreservation medium (Bambanker™, Lymphotec Inc.) was added to each of the tubes then the solutions were thoroughly mixed with a vortex mixer. One point eight milliliters (1.8 ml) of the cell suspension was added to each of 2 ml cryotubes (Corning Incorporated, No. 430289), the tubes placed in a Bicell unit (a cryopreservation container manufactured by Nihon Freezer Co., Ltd.), and stored in a deep freezer (Sanyo Electric Co., Ltd. No. MDF-192) at a temperature of −85° C. One day later they were moved to a liquid nitrogen tank (My Sciences Co., Ltd.) for preservation of biological testing samples.  
         [0000]     Procedure 17: Thawing and Cultivating the Cryogenically Preserved Cells  
         [0055]     The cryogenically preserved cells prepared in Procedure 16 were thawed and cultivated as follows. The frozen tubes were taken out of the liquid nitrogen tank and thawed in a heating block (Taitec Co., Ltd., Dryothermic Unit No. DTU-28) for 4 minutes. The entire contents was placed in 10 ml of washing solution using a Pasteur pipette, after which the resulting cell suspension was separated in a centrifuge at 24° C. at a 1,200 rpm for 3 minutes.  
         [0056]     After the supernatant was discarded, the dispersed cell pellets were suspended in 15 to 20 ml of the cultivating medium (400 ml of RPMI1640+7 S, 4.5 ml of 35,000 IU/ml human rIL-2, and 45 ml of FBS) then transferred to a 50 ml test tube from which 10 μl was sampled into a trypan blue staining solution for cell counting. One to two milliliters (1 to 2 ml) of the remaining cell suspension were placed into each well of an untreated 24-well plate (Sumitomo Bakelite Co., Ltd., No. MS-80240) after which incubation was initiated in a large jacket-type CO 2  incubator at 37° C. (±0.5° C.) within an environment of 5% (±0.2%) CO 2  and 95% (±5.0%) humidity.  
         [0057]     Cells which began cultivation in less than 20 ml of the cultivating medium were examined with a microscope one to three days following their activation and propagation after which 1 to 2 ml of the cultivating medium was placed in each well. The cell counting solution with trypan blue staining solution was mixed sufficiently after which 10 μl of the solution was placed in an improved Neubeier Hemocytometer where the number of unstained living cells and stained dead cells were ascertained under a microscope.  
         [0000]     Procedure 18: Activating the Cryogenically Preserved Cells  
         [0058]     Cells cultivated according to the method described in Procedure 17 were observed with a microscope. Two to four days following the incubation period during which the cells were activated and propagated, 20 to 30 ml of the still cultivating cell solution was transferred from the wells to a culture flask prepared in the same manner as described in Procedure 1 using a pipette, then was added 20 to 30 ml of cultivating medium (400 ml of RPMI1640+7 S, 4.5 ml of 35,000 IU/ml human rIL-2, and 45 ml of FBS) from which cultivation continued.  
         [0000]     Procedure 19: Propagating and Expanding Culture of the Cryogenically Preserved Cells  
         [0059]     The cells cultivated in Procedure 18 were observed through a microscope. The cells adhering to the bottom of the flask were removed by tapping the flask two to four days after cultivation during which the cells were activated and propagated. All of the cells were then transferred to a 225 cm 2  culture flask (Corning Costar Company: No. 3000), an additional 50 ml of the cultivating medium (400 ml of RPMI1640+7 S, 4.5 ml of 35,000 IU/ml human rIL-2, and 45 ml of FBS) was added, and cultivation was allowed to continue.  
         [0060]     In order to prepare the formulation for administration to the canine test subjects, the cells were observed through a microscope six to eight days from the start of cultivation during which the cells were activated and propagated, after which 150 ml of the cultivating medium (400 ml of RPMI1640+7 S, 2.25 ml of 35,000 IU/ml human rIL-2, and 45 ml of FBS) were added.  
         [0000]     Procedure 20: Testing the Cryogenically Preserved Cells The cultivated and cryogenically preserved cells were tested in the same manner as described in Procedures 9 through to 12.  
         [0000]     Procedure 21: Preparation and Counting the Cryogenically Preserved Cells for Therapeutic Administration  
         [0061]     The cryogenically preserved cells, which were cultivated as described in Procedures 13 and 14, were counted and prepared for final administration.  
         [0000]     Procedure 22: Administering the Cryogenically Preserved Cells  
         [0062]     When the autologous cell suspension prepared in Procedure 13 was administered only once to the canine patient, a clear improvement in quality-of-life was noted in the form of increased appetite, improved coat appearance, and the ability of the animal to walk a distance approximately twice as far as previously. The results are shown in Table 8.  
                                                     TABLE 8                           Results of treatment in which cryogenically frozen lymphocytes       were thawed, reactivated, and administered to a canine patient.            Patient   Canine               Cancer   Quantity of   No.of   Improvement       No.   type   Weight   Sex   Age   Type   administered cells   treatments   quality-of-life               9   Cairn   7.5 Kg   F   9 years   mixed   4.7 × 10 8     1   improvement in           Terrier           11 months   malignant           coat quality,                           mammary           recovery of vigor                           gland                           tumor                  
 
  FIG. 1 : Comparison of Cell Propagation by Temperature 
        Comparison of differences in canine lymphocyte propagation according to cultivation temperature 
            healthy canine subject 3     healthy canine subject 4    
            Total Cell Count     Number of Days Under Cultivation