Abstract:
A new immunotherapy to cancer patients is provided. In the therapy, lymphocytes collected from a patient by leukopheresis are treated with interferon-α in vitro for a short period of term and then returned to the patient intravenously. According to the therapy, reduction in sensitivity of tumor cells to natural killer-cell lysis due to administration of interferon-α is eliminated, the activities of NK cell and 2-5AS and interferon-producing capacity can be enhanced, a large amount of lymphocytes can be activated directly in vitro with the dose of optimally adjusted to the patient, none of side effects are found, and any other potentiators and comodulaators, which may not be allowed to use in vivo, can be added to the lymphocytes and removed therefrom before returning to the patient.

Description:
TECHNICAL FIELD 
     The invention pertains to immunotherapy to cancer patients employing lymphocytes activated with interferon-α. 
     BACKGROUND OF THE INVENTION 
     Interferon (IFN) is known to have antitumor activity through two major mechanisms, a direct cytotoxic effect by IFN on the tumor cells and an indirect cytotoxic effect through the activation of natural killer (NK) cells, macrophages or other immune cells of the host system. Activation of NK cells and other immune cells with IFN administration has been reported to be effective in the treatment of a wide variety of tumors. However, this very treatment decreases sensitivity of tumor cells to NK cell lysis due to an increase in expression of HLA antigen and sugar chains on the surface of tumor cells (Trincheri G., et. al., J. Exp. Med., 1314-1333, 1978). Moreover, high-dose IFN-α administration to patients with myclocytic leukemia or renal cancer has been reported to cause severe side effects such as fever, malaise, depilation and depression in some cases, which significantly affect the patients (Quesada JR, et. al., J. Clin Onco 4: 234-243, 1986). 
     In recent years, lymphokine-activated killer cell (LAK) therapy has been conducted in large scale clinical studies wherein lymphocytes taken outside of the body were activated using interleukin-2 (IL-2) and subsequently injected back into the body. Results from these studies have been reported to be promising but there yet remain many problems to overcome, especially in terms of cost/effectiveness and occurrence of side effects (Rosenberg, S. A., et. al.; N. Engl. J. Med. 313: 1485, 1985). 
    
    
     BRIEF DESCRIPTION OF THE INVENTION 
     The present inventors were interested in the potential of a new approach of immunotherapy using IFN-activated lymphocytes in improving the effectiveness of treatment to patients with cancer. In order to eliminate sensitivity reduction of tumor cells to NK cell lysis due to in vivo IFN administration, the inventors have aimed at the efficient activation of immune cells, mainly HK cells through in vitro IFN treatment, and designated this treatment as interferon-activated-NK-lymphocyte (IFNANK) therapy. 
     Briefly, lymphocytes are collected from a patient by leukopheresis, treated with IFN-α in vitro for a short term, and thereafter returned to the patient. 
     NK cell activity, 2-5AS ((2&#39;-5&#39;)oligoadenylate synthetase) activity and IFN-α producing capacity before and after the therapy were monitored to evaluate the effectiveness of the therapy. Among the cancer patients who received the therapy, the augmentation of this ability and capacity were proved in about half of the patients and none of the patients experienced any side effects. 
     The present invention is directed to a method for treating a patient with cancer, which comprises collecting lymphocytes from the patient by leukopheresis, contacting the lymphocytes with interferon-α in vitro for a short term which is effective to potentiate the immunological activity of the lymphocytes, and then returning the lymphocytes to the patient intravenously. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present method is applicable to a patient suffering from any kind of cancer such as gastric cancer, ovarian cancer, cervical cancer, adenocarcinoma, small cell carcinoma, melanoma, leukemia, etc. 
     Leukopheresis can be carried out using a commercially available intermittent or continuous flow cell separators for separating blood components. In the rotary chamber of the separator, peripheral blood of a patient with an anti-coagulant such as citrate is centrifuged for a short term, thereby lymphocyte-rich fraction containing leukocytes is collected into a collection bag. 
     After or during the collection, IFN-α is added to the bag in an amount to make a final concentration of not less than 100 IU/ml, preferably about 300-500 IU/ml to contact with lymphocytes. By gentle stirring the mixture for 0.5 to 1.5 hours, preferably about 30 min. at room temperature, the lymphocytes are effectively potentiated and can be returned to the patient intravenously. 
     The results obtained by the present method in Example 2 and Table 2 are summarized in the following; 
     None of the subjects treated by present method experienced any side effects. On the other hand, high-dose administration which is commonly used in patients with hepatitis C or cancer has been reported to cause severe side effects such as fever in more than 50% of cases, and malaise, depilation, leukopenia or thrombocytopenia in 10-30% of cases. No side effects, however, appeared in the present method. 
     In terms of immunological potentiation, NK cell activity was enhanced in 3 out of 4 healthy controls, and about half of the cases of cancer patients. Especially focused on the initial trial, 6 out of 8 cancer patients revealed augmented NK cell activity, although in some of those cases, increased rate of HK activity was lowered in the subsequent treatments. This fluctuation in the activity seems to be attributed to the individual difference in sensitivity to IFN-α, which may be related to the progress of cancer. 
     Increased 2-5AS activity was observed in most of the trials, although not always paralleled with the results of increased NK cell activity. 2-5AS activity may be considered as a more useful parameter to evaluate the sensitivity to IFN-α in an individual patient than OK cell activity (Shindo, M. et. al.; 
     Hepatology 8, 366-370, 1988). 
     Enhancement of IFN-α producing capacity, in the initial trial, was observed in 6 out of 8 cancer patients and in 3 out of 4 healthy controls. IFN-producing capacity has been reported to decrease with the progression of disease in patients with lung or bladder cancer (Kou J, Y, et. al.; Urol Res. 19:51-56, 1991; Onodera, H. et. al.; J. IFN Res., Suppl. 1: S132, 1993). An increase of IFN-α producing capacity may be a promising sign for the patients. 
     According to present invention, (1) a large amount of lymphocytes (up to 5×10 9 ) can be activated directly in the collection bag with the dose of IFN-α optimally adjusted to the patient, (2) no side effects are found, and (3) any other potentiators and comodulators, which may not be allowed for use in vivo, can be added in the bag. By removing them from the activated lymphocytes before re-injection, it is possible to make the lymphocytes harmless to the patient. 
     EXAMPLE 1 
     (Preliminary in vitro experiment to determine the optimal dose of IFN-α to augment the NK cell ability) 
     To find out an optimal concentration of IFN, a leukocyte fraction containing lyphocytes separated by leukopheresis were resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum and adjusted to a concentration of 5×10 6  cells/ml, and cultured with 0-1000 IU/ml human leukocyte IFN-α cells for 18 hours in a humidified incubator at 37° C. with 5% CO 2 . Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation. For the measurement of NK cell activity, K562 cells derived from erythroleukemia cell line, were used as target cells and were labeled with europium, whereas PBMCs were used as effector cells. NK cell activity was measured by the ability of PBMCs to induce release of the europium from lysed K562 cells and the results are expressed in the following Table as percentage cytotoxicity. 
     
         ______________________________________NK cell activity treated withvarious doses of IFN-α in vitroIFN (IUml)    NK activity (%)______________________________________  0           16.4 10           26 100          45.11000          45.6______________________________________ 
    
     As seen from the above Table, lymphocytes are activated in a dose dependent manner by treating in vitro with IFN-α in the range of concentration from 1 to 1000 units/ml of IFN-α. Lymphocytes activation with 1000 IU/ml of IFN-α revealed a similar result to that of 100 IU/ml of IFN-α, indicating the activation reaches the plateau at 100 IU/ml of IFN-α. Therefore, for contacting lymphocytes with IFN-α, it is preferred to add IFN-α in an amount sufficient to make a final concentration of not less than 100 IU/ml, more preferably 300 to 500 IU/ml to the lymphocytes. 
     EXAMPLE 2 
     (Lymphocyte collection and its activation with IFN) 
     Subjects: Four healthy controls and 8 cancer patients who all received IFNANK therapy for a total twenty-six times. Patients&#39; profiles are shown in Table 1. 
     To obtain a large number of lymphocytes, leukopheresis was carried out using SPECTRA® (a blood cell collection device produced by COBE Inc., U.S.A.) in the following manner. 2000-4000 ml of peripheral blood was centrifuged in the system at a flow rate of 40-60 ml/min. for about 1-1.5 hour, and 200-300 ml of lymphocyte-rich fraction containing ACD acid citrate dextrose) as a anti-coagulant was collected in the bag. Approximately 2-7×10 9  leukocytes containing 50-80% lymphocytes were recovered in the bag. After or during the collection, 1×10 5  IU of IFN-α was added into the bag, which corresponds to a final concentration of 400-500 IU/ml. After gentle stirring for 30 min. at room temperature, &#34;activated lymphocytes&#34; were returned to the subject intravenously. 
     Samples of peripheral blood were extracted from the subjects intravenously before IFNANK therapy and up to 96 hours after the therapy, in order to monitor NK cell activity, IFN-α producing capacity and 2&#39;5&#39;A oligoadenylate (2-5A) synthetase activity in the whole blood. 
     IFN-producing capacity was measured using whole blood method (Kou J. Y. et. al. Urol Res. 19: 51-56, 1991). 
     For the measurement of 2-5A synthetase activity, samples of heparinized peripheral blood were stored at 80° C. until measurement. After thawing, 2-5A synthetase activity was measured using &#34;25A&#34; kit (Eiken Co. Ltd., Japan). 
     For measuring IFN-α producing capacity, samples of heparinized peripheral blood were incubated with Sendai virus at 37° C. for 20 hours, thereafter IFN-α activity was measured by bioassay (Kohase M, et. al. Japan Medical Foundation Publication No. 15: 299-309, 1982) 
     
                                           TABLE 1__________________________________________________________________________(Patients&#39; profile)Sex    Age     Diagnosis              Medical background__________________________________________________________________________H T   male  68 gastric cancer              gastrectomy, 1992 (stage IV)              recurrence and reoperation, 1994 (stage IV)Y T   male  44 adenocarcinoma,              operation, 1993 (stage III)     lung     recurrence, 1994 (stage IV)M T   female  53 cervical cancer              operation, 1994 (stage II)N K   male  39 adenocarcinoma,              operation, 1994 (stage III)     lung     recurrence, 1995 (stage IV); radiation,              chemotherapyT A   female  47 ATL      mycosis fungoides was diagnosed in 1992Y M   female  65 melanoma first diagnosis, 1992 (stage IV);              chemotherapyY M   female  58 small cell              radiation and chemotherapy (stage III),     carcinoma, lung              complete remissionH M   female  41 ovarian cancer              operation, 1994 (stage III);              recurrence (stage IV)__________________________________________________________________________ 
    
     
                                           TABLE 2-a__________________________________________________________________________Results of IFNANK therapy   No. of   WBC(× 10.sup.9)Days   treatedfrom   with IFN  Days IFN-α                      2-5AS                           NKIFNANKfirst   (Lympho-  after                  produc-                      activity                           activity                               WBC                                  Hb Pit  Sidetimestrial   cytes × 10.sup.9)         TBVL*             IFNANK                  tion                      (pmol/dl)                           (%) (/μl)                                  (g/dl)                                     (× 10/μl)                                          effect**__________________________________________________________________________HT, Gastric cancer, male, 681st  0  3.9   2000 ml             0 d  4086                      4440 5   6400                                  10.5    ND   (2.4)     1 d  3798                      7860 26             4 d  7097                      5790 382nd  76 2.9   2448 ml             0 d  1519                      4020 31  7200                                  13.5                                     34.7 ND   (2.1)     2 d  4447                      3450 59  6400                                  11.4                                     35.43rd  130   3.9   3000 ml             0 d  1946                      4950 31  6300                                  10.8                                     37.2 ND   (2.8)     1 d  1667                      6610 19  6800                                  10.9                                     41.6             2 d  1075                      6840 19  6700                                  10.5                                     40.2died 236YT, lunge cancer, male, 441st  0  2.7   3312 ml             0 d  7148                      1650 52  5000                                  14.6                                     20.9 ND   (1.5)     4 hr 10520                      3120 66             1 d  9761     57  5100                                  14.22nd  26 2.4   2268 ml             0 d  5409                      1350 52  4800                                  14.3                                     22.9 ND             1 d  2653                      1380 37             2 d  8946     503rd  699   3.9   4503 ml             o d  4007                      3480 48  5700                                  14.9                                     31   ND   (2.0)     1 d  2619                      1950 21  4200                                  13.2                                     28.1died 197MT, Uterine cancer, female, 531st  0  2.9   3650 ml             0 d  1358     12  4200                                  12.1                                     26.7 ND   (2.2)     1 d  1790     0   3600                                  12.5                                     27.4             2 d  2425     14  4300                                  11.9                                     26.62nd  210   3.2   3593 ml             0 d  1389     27  4800                                  11.7                                     25.2 ND   (2.8)     2 d  11224    12  4100                                  11.1                                     213rd  230   3.0   3335 ml             0 d  2689     11  5700                                  11.8                                     24.4 ND   (2.3)     1 d  1466     8   4400                                  11.4                                     22.3             2 d  1316     19  4600                                  11.6                                     23NK, Lung cancer, male, 391st  0  2.2   3934 ml             0 d  5145                      3750 17  3900                                  11.5                                     22.9 ND             4 hr 22322                      6330 8   4900                                  10.9                                     23.2             1 d  7879                      19500                           23  4200                                  12.1                                     23.5             4 d           27             5 d  9317     22  4300                                  11.9                                     22.32nd  62 4.38  4002 ml             0 d  10388    35  5000                                  11.6                                     25   ND   (2.3)     1 d  14225                      4110 13  4000                                  10.8                                     23.5             2 d  9921                      4440 31  4400                                  11.2                                     25.43rd  92 3.8   4001 ml             0 d  11485                      2580 49  5300                                  11.7                                     20.4 ND   (2.9)     3 d  7075                      5640 39  5400                                  11.9                                     19.74th  116   3.5   4002 ml             0 d  6352     28  4700                                  12.7                                     18.9 ND   (3.0)     1 d  9043     20  4800                                  11.8                                     19.2             4 d  10120    22  5000                                  12 18.25th  148   3.9   3820 ml             0 d  13147    47  5900                                  12 18   ND   (2.8)     3 d  10089    22  6400                                  11.9                                     19.1             6 d           356th  175   2.7   4241 ml             0 d  6881     37  4900                                  12.7                                     16.4 ND   (1.4)     1 d  9018     27             4 d  12572    14  5000                                  11.9                                     147th  195   2.8   3943 ml             0 d  7493     12  6900                                  11.2                                     14.4 ND   (1.1)     1 d  6228     25  6200                                  11.3                                     13.4             2 d  6423     16  7200                                  11.5                                     12.2TA, ATL, female, 471st  0  3.1   3134 ml             0 d  6770                      1950 8   5500                                  12.3                                     19   ND             1 d  6883                      5910 7   5300                                  13.5                                     21.4             4 d  7369                      6191 10  7500                                  13.2                                     22.5YM, Melanoma, female, 651st  0        --  0 d  4664                      10350                           14  4600                                  9.5                                     29.7 ND             1 d  2805                      7290 5   3300                                  10.4                                     30.9             2 d  798 7620 4   4900                                  11.1                                     32.32nd  12           0 d  452 5700 16  5000                                  9.9                                     34   ND             2 d  454 3690 19  3800                                  10 32.9             4 d  704 8310 11  7400                                  10.5                                     26.8YM, Lung cancer, female, 581st  0  4.6   3533 ml             0 d  5386                      3210 37  4200                                  13.1                                     16.6 ND   (2.0)     1 d  3124                      7350 36  4300                                  12 16.8             4 d  2348                      1950 43  4900                                  13 18.2HM, Ovarian cancer, female, 411st  0  2.6   2505 ml             0 d  1904     60  5900                                  10.1                                     41.1 ND   (1.8)     1 d  3688     30  5800                                  9.1                                     39.6             4 d  7560     49  6600                                  9.4                                     39.42nd  7  1.5   2009 ml             0 d  4278     15  6900                                  8.9                                     44.5 ND   (1.3)     1 d  7344     22  7700                                  9  45.5died 73Healthy PersonSH, male, 571st  0  7.6   5200 ml             0 d  8525                      6150 53  6700                                  15.4                                     ND   (5.5)     2 h  14097                      6180 50  5200                                  12.6             17 h 1864                      6930 57             1 d  9770                      7500 61             2 d  3736                      7110 65             4 d  15809                      5520 72YR, male, 431st  0  6.0   4750 ml             0 d  8757                      1110 32  6000                                  15.2                                     ND   (4.0)     1 d  13748                      4620 39             2 d  9014                      4080 47YS, male, 591st  0  6     4600 ml             0 d  13452                      2190 50        ND             1 d  9948                      4050 45             2 d  8561                      5280 40YH, female, 321st  0  2.9   3000 ml             0 d  6872                      1710 14             1 d  7444                      3900 10             2 d  12914                      5010 34             3 d  6561                      3600 8             4 d  4890                      3180 12__________________________________________________________________________  *Total blood volume circulated through leukopheretic apparatus. **Fever(&gt;37° C.), Malaise, Leukopenia(&gt;3000/μl), Thrombocytopenia(&lt;10.sup.4 /μl)!-