Method for treating cancer patients

Abstract

A new immunotherapy to cancer patients is provided. In the therapy, lymphocytes collected from a patient by leukopheresis are treated with interferon-.alpha. 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-.alpha. 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-.alpha..

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-.alpha. 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-.alpha. in vitro for a short term, and thereafter returned to the patient.

NK cell activity, 2-5AS ((2'-5')oligoadenylate synthetase) activity and IFN-.alpha. 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-.alpha. 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-.alpha. 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-.alpha., 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-.alpha. in an individual patient than OK cell activity (Shindo, M. et. al.;

Hepatology 8, 366-370, 1988).

Enhancement of IFN-.alpha. 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-.alpha. producing capacity may be a promising sign for the patients.

According to present invention, (1) a large amount of lymphocytes (up to 5.times.10.sup.9) can be activated directly in the collection bag with the dose of IFN-.alpha. 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-.alpha. 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.times.10.sup.6 cells/ml, and cultured with 0-1000 IU/ml human leukocyte IFN-.alpha. cells for 18 hours in a humidified incubator at 37.degree. C. with 5% CO.sub.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-.alpha. 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-.alpha. in the range of concentration from 1 to 1000 units/ml of IFN-.alpha.. Lymphocytes activation with 1000 IU/ml of IFN-.alpha. revealed a similar result to that of 100 IU/ml of IFN-.alpha., indicating the activation reaches the plateau at 100 IU/ml of IFN-.alpha.. Therefore, for contacting lymphocytes with IFN-.alpha., it is preferred to add IFN-.alpha. 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' profiles are shown in Table 1.

To obtain a large number of lymphocytes, leukopheresis was carried out using SPECTRA.RTM. (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.times.10.sup.9 leukocytes containing 50-80% lymphocytes were recovered in the bag. After or during the collection, 1.times.10.sup.5 IU of IFN-.alpha. 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, "activated lymphocytes" 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-.alpha. producing capacity and 2'5'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.degree. C. until measurement. After thawing, 2-5A synthetase activity was measured using "25A" kit (Eiken Co. Ltd., Japan).

For measuring IFN-.alpha. producing capacity, samples of heparinized peripheral blood were incubated with Sendai virus at 37.degree. C. for 20 hours, thereafter IFN-.alpha. activity was measured by bioassay (Kohase M, et. al. Japan Medical Foundation Publication No. 15: 299-309, 1982)

TABLE 1__________________________________________________________________________(Patients' 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(.times. 10.sup.9) Days treated from with IFN Days IFN-.alpha. 2-5AS NKIFNANK first (Lympho- after produc- activity activity WBC Hb Pit Sidetimes trial cytes .times. 10.sup.9) TBVL* IFNANK tion (pmol/dl) (%) (/.mu.l) (g/dl) (.times. 10/.mu.l) 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(>37.degree. C.), Malaise, Leukopenia(>3000/.mu.l), Thrombocytopenia(<10.sup.4 /.mu.l)!-

Claims

1. A method for treating a patient with cancer, which comprises collecting lymphocytes from the patient by leukopheresis, contacting the lymphocytes with interferon-.alpha. in vitro for a term effective to potentiate the immunological activity of the lymphocytes, and then returning the lymphocytes to the patient intravenously.

2. A method according to claim 1, wherein the interferon-.alpha. is in a concentration of 10 to 1000 IU/ml.

3. A method according to claim 1, wherein the immunological activity is the activity of natural killer cells or (2'-5')-oligoadenylate, or the ability of producing interferon-.alpha..

4. A method according to claim 1, wherein the term for contacting lymphocytes with interferon-.alpha. is 1 to 2 hours.

5. A method according to claim 2 wherein the interferon-.alpha. is in a concentration of 300 to 500 IU/ml.

6. A method according to claim 4 wherein the term is about 1.5 hours.

7. A method for treating a patient with cancer, which comprises collecting lymphocytes from the patient by leukopheresis, contacting in vitro, the lymphocytes with interferon-alpha in an amount which is effective to potentiate the immunological activity of the lymphocytes when administered to the patient for a short term, and then returning, intravenously, the lymphocytes together with the interferon-alpha to the patient.

8. A method according to claim 7, wherein the lymphocytes are contacted with interferon-alpha in a concentration of 100 to 500 IU/ml.

9. A method according to claim 7, wherein the lymphocytes are contacted with interferon-alpha in a concentration of 300 to 500 IU/ml.

10. A method according to claim 7, wherein about 200-300 ml. of the lymphocyte-rich fraction is collected by leukopheresis.

11. A method according to claim 7, wherein about 100,000 IU of interferon-alpha is contacted with the collected lymphocytes.

12. A method according to claim 7, wherein the term for contacting lymphocytes with interferon-alpha is 1 to 2 hours.

13. A method according to claim 12 wherein the term is about 1.5 hours.

14. A method for potentiating lymphocytes which comprises collecting lymphocytes from a patient by leukopheresis, contacting the collected lymphocytes with interferon-alpha in an amount which is effective to potentiate the immunological activity of the lymphocytes when administered to the patient in vitro for a short term effective to potentiate the immunological activity of the lymphocytes, and returning the treated lymphocytes to the patient.

15. A method according to claim 14 wherein the immunological activity is the activity of natural killer cells or (2'-5')-oligoadenylase, or the ability of producing interferon-.alpha..

16. A method according to claim 14 wherein the term for contacting lymphocytes with interferon-.alpha. is 1 to 2 hours.

17. A method according to claim 16 wherein the term is about 1.5 hours.

18. A method according to claim 14 wherein the interferon-alpha is in a concentration of 100 to 500 IU/ml.

19. A method according to claim 18 wherein the interferon-.alpha. is in aconcentration of 300 to 500 IU/ml.

20. A method according claim 14 wherein about 200-300 ml of the lymphocyte-rich fraction is collected by leukopheresis.

21. A method according to claim 14, wherein about 100,000 IU of interferon-alpha is contacted with the collected lymphocytes.