JPH10167971A - Precaution for postoperative recurrence of nonviral tumor, recurrence preventive and its preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To remarkably improve the preventive effect for postoperative recurrence of nonviral tumor or brain tumor by administrating autolymphocytes specially cultured. SOLUTION: This precaution is carried out by initially taking out peripheral blood lymphocytes from nonviral tumor patients or brain tumor patients, cultureproliferating them in the medium containing solid-phased anti-CD3 antibody and interleukin-2 and finally administrating this medium which contains self-original lymphocytes as a major conponent to patients after operation for tumor or brain tumor. This precaution is not only remarkably effective for nonviral tumor or brain tumor, especially for prevention of postoperative recurrence on glioblastoma patients, but also excellent effect is displayed by using to other brain tumor or for treatment of tumor arising from cells which can not express MHC (major histocompatibility complex) or manifest the MHC in extremely minute amount even though they could manifest it.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an epoch-making method capable of preventing recurrence of a non-viral tumor after surgery, an agent for preventing recurrence, and a method for preparing the agent for preventing recurrence. The purpose is to improve.

[0002]

2. Description of the Related Art Generally, a living body is tumor (cancer) by an immune system.
Protect yourself from. As an immune response to cancer,
It is generally considered that cellular immunity mainly composed of NK cells and T cells is important. It is considered that NK cells recognize cancer cells by NK receptors on the cell surface and destroy and eliminate cancer cells. On the other hand, T cells recognize cancer cells by T cell receptors and eliminate and destroy them. In particular, killer T cells are MHC class I-restricted and destroy and eliminate MHC class I-expressing cancer cells, but cells that do not express MHC class I or have very low expression It is believed that killer T cells do not destruct and eliminate cells, for example, cancers derived from cells of the nervous system.

[0003] This is because killer T cells recognize peptides derived from viruses and the like supplemented in MHC and regard these cells as foreign substances and eliminate them. It is already generally known that virus-infected cells are already destroyed and eliminated by a combination of MHC and killer T cells. The classification of cells is performed based on surface markers expressed on the surface of cells, and mature T cells express CD3 as a surface marker. Mature T cells not only express CD3 as a surface marker, but also killer T cells having antitumor activity are CD8, and helper T cells that are closely involved in the activation of various immune system cells are CD4. Is expressed. Mature T cells express a T cell receptor, and this receptor has high affinity for a specific antigen.

[0004] T cells also express only a single T cell receptor, and specific T cells unique to an antigen are activated via the receptor. Furthermore, T cell receptors are very diverse, and there are many different types of T cells in vivo. In addition, tumors generally include viral tumors and non-viral tumors. Viral tumors include liver cancer caused by hepatitis B virus or hepatitis C virus, Burkitt lymphoma caused by Epstein-Barr virus, and the like. Nasopharyngeal cancer, gastric cancer and the like are known. Non-viral tumors include those caused by carcinogens or genetic mutations caused by ultraviolet inoculation, or those whose cause is not clear, and the types of these spread to all tumors.

[0005] Among them, those of high malignancy and fatal include brain tumor, scirrhous stomach cancer, lung cancer, skin cancer, pancreatic cancer,
Among kidney cancer, colorectal cancer, liver cancer and the like, those having no viral antigen or viral gene can be mentioned.
In viral tumors, virus-associated antigens are expressed in the tumor, which is extraneous to the living body, and is effective by activated lymphocytes, but is not effective in tumors that do not express exogenous antigens. It is not clear whether activated lymphocytes are effective. In addition, among non-viral tumors, brain tumors have a particularly difficult problem. That is, it is considered that drugs and the like hardly reach the brain, and that the immune reaction is unique to the brain. In fact, there is a concept called the "brain-blood barrier", which is thought to keep the brain isolated from other organs.

[0006] Brain tumors include glioma, meningioma, and Schwanoma. Among them, glioma is particularly known as a malignant brain tumor. There are also several types of gliomas, of which glioblastoma and medroblastoma have a poor prognosis, and the postoperative survival rate of patients with glioblastoma is 50.1% in one year,
20.5% in 2 years, 11.4% in 3 years, 8.7% in 4 years
(CANCER TREAMENT)
AND SURVIAL; JAPAN SCIEN
TIFIC SOCIETES PRESS; 19
See page 1-201).

On the other hand, it has been first reported by Rosenberg et al. That lymphocytes, particularly T cells and NK cells, of viral cancer patients stimulated with interleukin 2 have antitumor activity [New. Eng.
J. Med. , 319, 1678 (1988)]. In addition, lymphocytes collected from the patient with viral cancer
The present inventors have also proposed a proliferation stimulating method in which stimulating proliferation is carried out with 3 immobilized antibody and interleukin 2 (see JP-A-3-80076). Suzuki et al. Have tried to administer lymphocytes to glioblastoma patients, but have not clarified the efficacy of the present lymphocyte administration. Also, August et al. Reported a method for activating lymphocytes with anti-CD3 antibody and interleukin 2 in the liquid phase (US PAT: 5443983).
In addition, Skoff immobilized infiltrating cells in tumors with anti-CD
Report on the activation method with Antibody 3 and Interleukin 2 (W
O90 / 04633).

[0008]

However, the lymphocytes expanded by the above-mentioned proliferation method have been proposed to be used exclusively for the prevention of postoperative recurrence of viral liver cancer, and have also been proposed in Rosenberg, August and Scoff. These reports differ in the origin, preparation method, activation method, administration method and the like of lymphocytes, and do not relate to the prevention of postoperative recurrence of non-viral tumors or brain tumors in the present invention. Therefore, adaptation of activated lymphocytes as a means of preventing postoperative recurrence of non-viral tumors that do not express virus-related antigens, especially surgery for brain tumors that occur in the brain with a barrier system called the "brain-blood barrier" Adapting activated lymphocytes as a means of preventing post-recurrence is quite unlikely. To prevent postoperative recurrence of non-viral tumors such as brain tumors, only chemotherapeutic agents and radiation therapy, which have strong toxicity or side effects, and have limited efficacy due to their limited use. Unknown, groundbreaking means have not yet been developed.

[0009]

Accordingly, the present invention solves the above-mentioned problems in the prior art, and provides a non-viral tumor including a brain tumor which is completely different from a general viral tumor in terms of medication effect and immune response. Relapse prevention method, relapse prevention agent with extremely high effect of suppressing postoperative recurrence of viral tumors
And has established a method for its preparation,
Specifically, lymphocytes are collected from a non-viral tumor patient and cultured and cultured in a culture solution containing an immobilized anti-CD3 antibody and interleukin-2. The present invention relates to a method and a prophylactic agent for preventing postoperative recurrence of a non-viral tumor, characterized in that a preparation to be administered is administered to a patient after surgery for tumor surgery.

[0010] The present invention also provides a method for collecting lymphocytes from a patient with a brain tumor, culturing them in a culture solution containing an immobilized anti-CD3 antibody and interleukin 2, and using the autologous lymphocytes as a main component. The present invention relates to a method and a preventive agent for preventing postoperative recurrence of a brain tumor, which comprises administering the preparation to a patient after the operation of the brain tumor. The present invention also relates to a method for preventing postoperative recurrence of a brain tumor, characterized in that at least one of serum, proteins, amino acids, saccharides and antibiotics is further added to the above-mentioned culture solution. .

[0011] The present invention further comprises the addition of various cytokines, growth factors, or a conditioned medium prepared by growing appropriate cells in the above-mentioned culture medium, or the addition of a substance that stimulates lymphocytes with a specific antigen. The present invention also relates to a method for preventing postoperative recurrence of a brain tumor. Furthermore, the present invention has autologous lymphocytes grown as a main component obtained by collecting lymphocytes of a brain tumor patient and culturing them in a culture solution containing an anti-CD3 antibody and interleukin 2 having immobilized thereon. The present invention also relates to an agent for preventing postoperative recurrence of brain tumor.

[0012] Further, the present invention provides a method for treating a brain tumor, wherein the culture medium is a culture medium containing an immobilized anti-CD3 antibody and interleukin 2, when proliferating lymphocytes collected from a brain tumor patient. The present invention also relates to a method for preparing a post-recurrence preventive agent. Further, the present invention, when proliferating lymphocytes collected from a brain tumor patient, in the culture solution,
In addition to the immobilized anti-CD3 antibody and interleukin-2, the present invention also provides a method for preparing a postoperative recurrence preventive agent for brain tumor, which further comprises at least one of serum, proteins, amino acids, saccharides, and antibiotics. Related.

[0013] After a specified amount of peripheral blood is collected from a vein of a patient with a non-viral tumor or a brain tumor by a blood collection method provided with an anticoagulant such as heparin blood collection, lymphocytes are separated by centrifugation or the like. This is anti-CD3
After culturing for a certain period of time in a solid-phased culture solution containing the antibody and interleukin 2, an agent for preventing postoperative recurrence of a non-viral tumor or a brain tumor is obtained. Further, after the non-viral tumor or the brain tumor has been operated and the patient's physical strength has stabilized after a certain period of time, the above-mentioned relapse preventing agent is injected into the peripheral blood vessels of the patient to prevent post-operative recurrence.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be described with reference to the following examples. In the present invention, lymphocytes collected from a non-viral tumor patient or a brain tumor patient are cultured in a culture solution containing an immobilized anti-CD3 antibody and interleukin 2. A method for preventing recurrence of a non-viral tumor or a brain tumor, wherein the method further comprises administering the prophylactic agent based on the expanded autologous lymphocytes to a patient after surgery for the non-viral tumor or the brain tumor. -And the gist of the agent is a recurrence preventive agent and a preparation method therefor.

[0015] Peripheral blood can be collected by heparin-added blood or citrate-added blood for the purpose of preventing coagulation, but is not limited to this method. Separation of lymphocytes from peripheral blood is performed by density centrifugation in this example. As the density centrifugation in this case, Ficoll Hypak, monopoly resolution, or the like can be used. However, the present invention is not limited to this method.

The amount of blood collected at a time is as follows:
It can be performed with 0.1 to 500 ml. More preferably, the reaction can be performed with 1 to 100 ml. More preferably, the reaction can be performed with 10 to 50 ml.
The present method can be carried out using a large amount of blood or a small amount of blood as a material, but is particularly advantageous in the case of a small amount of blood because the burden on the patient can be minimized. Therefore, the amount of blood collected at one time is preferably about 50 ml. Cells that can be used for several doses can be prepared by one blood collection, but cells can be prepared by appropriately collecting blood according to the number of administrations or the administration interval.

Next, lymphocytes collected from a non-viral tumor patient or a brain tumor patient are immobilized with anti-CD3
Stimulated growth in culture containing antibody and interleukin 2. In this case, various types of flasks, dishes, plates, and bags can be used as a support on which the anti-CD3 antibody is immobilized. The antibody can be immobilized by a method using non-specific adsorption or chemical bonding, but any method may be used as long as lymphocytes can be stimulated by the anti-CD3 antibody. In the examples, O-CD3 antibody was used as an anti-CD3 antibody.
Although KT-3 (Ortho) is used, any antibody (compound) may be used as long as it can stimulate the CD3 antigen. Further, any substance that can activate lymphocytes, particularly T cells, can be used as interleukin 2.

In vitro cell culture was performed using RPMI1
640 medium, DMEM medium, and various serum-free media can be used. If necessary, add serum, protein,
Culture of lymphocytes with the addition of amino acids, saccharides, and antibiotics is even more advantageous in terms of proliferation, activation, and culture operability. Lymphocytes grown in vitro can also be administered by suspending them in various buffers such as physiological saline and phosphate buffer. Lymphocytes are
1 × 10 (4th power) / ml to 1 × 10 (8th power) / m
1 cell concentration can be used.

More preferably, 1 × 10 (sixth power) pieces / m
A cell concentration of 1 to 1 × 10 (8th power) cells / ml can also be used. In this case, it is desirable to add a protein or the like to the cell suspension for the purpose of preventing cell aggregation and adsorption to the container. As the protein, for example, human albumin or the like can be used. Human albumin can be used at a concentration of 0.1 W / V% to 10 W / V%. Further, the administration route can be any route such as intravenous, intracerebral, intraperitoneal, intraarterial, intramuscular, subcutaneous, etc., but from the viewpoint of convenience, the above-mentioned intravenous administration is particularly recommended.

Regarding the administration timing, it can be administered before or after the operation.
Administered after surgery. For administration, immobilized CD3
Lymphocytes stimulated and proliferated with an antibody and interleukin 2 and lymphocytes that have been expanded and cultured in an appropriate culture solution after stimulation are used. In the culture solution for performing expansion culture,
Those to which interleukin 2 is added as necessary can be used. The present invention also provides an anti-CD having lymphocytes immobilized thereon.
Proliferation is performed by culturing in a culture solution containing 3 antibody and interleukin 2. Various cytokines, growth factors, or a conditioning medium prepared by growing appropriate cells are added as necessary. By stimulating with a specific antigen, the proliferation rate of lymphocytes can be further improved.

[0021]

EXAMPLES [Preparation of OKT3 Antibody-Immobilized Flask] 30 ml of a PBS (−) solution containing 5 μg / ml of OKT3 antibody was added to
A culture flask (manufactured by Sumitomo Bakelite Co., Ltd.) with a bottom area of 225 cm (square) was added, and left at room temperature for 2 hours while the bottom surface was covered with the solution to prepare an OKT3 solid-phased flask. Store under a temperature atmosphere of 4 degrees Celsius.

[Preparation of lymphocytes] Heparin-added peripheral blood was obtained from the vein of a non-viral cancer patient or brain tumor patient.
0 ml of blood was collected. An equal volume of RPMI1640 medium was added to this, and overlaid on Lymphosepearl I (manufactured by Immune Biological Laboratories) which had been previously dispensed into several 15 ml centrifuge tubes.
Centrifuged at 00 rpm for 15 minutes. After centrifugation, the lymphocyte layer was collected with a pipette, and RPMI 1640 medium (30 ml) was collected.
, And centrifuged (1800 rpm, 10
Minutes).

After removing the supernatant, the cell pellet was loosened and the culture medium (kanamycin 60 μg / ml, streptomycin 20 μg / ml, glutamine 2 mM, oxaloacetic acid 1 mM, sodium pyruvate 1 mM, HE
RP containing 10 mM PES and 0.2 U / ml insulin
50 ml of MI1640 medium supplemented with human serum 10% and interleukin 2 700 U / ml) is added to prepare a cell suspension. The OKT3 solid-phased flask prepared in the above "Preparation of OKT3 antibody-solidified flask" was washed twice with PBS (-). After washing, the above cell suspension was inoculated into an OKT3 solid-phased flask, and cultured at 37 ° C. under 5% carbon dioxide gas and saturated humidity.

Three days later, 50 ml of the culture was added thereto, and four days later, 150 ml of the culture was further added. After suspension, 125 ml of the culture in the flask was replaced with a fresh OKT3.
It was transferred into a solid-phased flask and cultured. Six days later, about 250 ml of the culture solution in the flask was added to 750 ml of the LL-7 culture solution (manufactured by Niken Institute of Biomedical Research), and a gas-permeable culture bag was used in the presence of 5% carbon dioxide to obtain 3% Celsius.
The cells were cultured at 7 degrees. Further, on the eighth day, 1 liter of the culture solution was added, and this was cultured in two gas-permeable culture bags. Similarly, on the 10th day, 4 bags and 1
On the second day, the cells were cultured in 6 bags each.
On the 14th day, the cells were collected by centrifugation, and suspended in about 250 ml of physiological saline containing 2% human albumin to prepare a lymphocyte suspension.

[Administration of Lymphocytes] The lymphocyte suspension prepared in the above “Preparation of Lymphocytes” is administered three times from a vein during the first month, and thereafter from the vein once a month. Administration was continued. The first dose was started 3 to 7 months after the operation. About 0.8 × 10 for one administration
(10th power) to 1.5 × 10 (10th power) lymphocytes were used. Administration was performed to four glioblastoma patients, and the number of survivors was observed over time. As a result, one died 11 months after the operation, and the remaining three survived at the time of the present application. Yes (28, 32, and 39 months lived as of December 1996), and surprisingly, survivors have reintegrated.

Given that the statistical one-year survival rate of glioblastoma patients is about 50%, the 2-year survival rate is about 20%, and the 3-year survival rate is about 10%, the recurrence of the method of the present invention is considered. It has been shown that activated autologous lymphocytes as a preventive method and recurrence preventive agent are extremely effective in preventing postoperative recurrence of non-viral tumors, especially glioblastoma.
Note that the present invention is not limited to only the above-described embodiments.

[0027]

According to the present invention, as described above, lymphocytes collected from non-viral tumor patients or brain tumor patients
Proliferation is carried out by culturing in a culture solution containing an immobilized anti-CD3 antibody and interleukin 2, and a prophylactic agent based on the proliferated autolymphocytes as a main component is used as a non-viral tumor or brain tumor. Since it is intended to be administered to patients after surgery, it is not only very effective in preventing post-operative recurrence of non-viral tumors or brain tumors, especially glioblastoma patients, but also other brain tumors or MHC When used for the treatment of cancer derived from cells that do not express or cells that are expressed in a very small amount, good effects can be exhibited.

Furthermore, the proliferatively prepared autolymphocytes according to the present invention not only prevent postoperative recurrence of non-viral tumors and brain tumors, but also express or express cells that do not express brain tumors or MHC. Can also be used to treat cancers derived from cells that are in trace amounts. In addition, the present invention provides a method for collecting lymphocytes from a patient and administering the prepared autolymphocyte fluid, which may be performed intravenously, so that there is little pain and burden on the patient as compared with conventional treatment methods, and the work is remarkably easy. .

Further, the therapeutic method and therapeutic agent of the present invention
A great effect can be expected as a therapeutic agent for general tumors as well as non-viral tumors and brain tumors as described above. It can be used for the treatment of other brain tumors or cancers derived from cells that do not express MHC or cells that are expressed in very small amounts. In addition to preventing postoperative recurrence, the present lymphocytes can be used as a therapeutic agent. Further, not only post-operative administration but also pre-operative administration can expect a further effect. Furthermore, the prepared autologous lymphocytes of the present invention are administered particularly for the purpose of preventing recurrence after surgery, but a great effect can be expected by use even when surgery is not performed.

With respect to collection of lymphocytes from patients with non-viral tumors or brain tumors, lymphocytes can be collected from lymph nodes, thymus, tumor-infiltrating cells, etc., in addition to peripheral blood of patients. In this case, lymphocytes derived from any tissue or organ can be used as long as they are patient-derived lymphocytes. Furthermore, the quality of life of cancer (tumor) patients can be improved by the method of the present invention or the preparation of the present invention.

Claims (12)

[Claims] 1. A peripheral blood lymphocyte is collected from a non-viral tumor patient, and cultured and cultured in a culture medium containing an immobilized anti-CD3 antibody and / or interleukin-2. A method for preventing postoperative recurrence of a non-viral tumor, comprising administering a preparation containing as a main component to a patient after surgery for tumor. 2. The culture solution to which at least one of serum, proteins, amino acids, saccharides, and antibiotics is added in addition to the immobilized anti-CD3 antibody and / or interleukin 2 The method for preventing postoperative recurrence of a non-viral tumor according to claim 1, characterized in that: 3. The culture solution contains, in addition to the immobilized anti-CD3 antibody and / or interleukin-2, various cytokines, growth factors, or a conditioned medium prepared by growing appropriate cells. The method for preventing postoperative recurrence of a non-viral tumor according to claim 1 or 2, wherein a substance that stimulates lymphocytes with a specific antigen is added. 4. An autologous cell obtained by collecting peripheral blood lymphocytes from a non-viral tumor patient and culturing the same in a culture medium containing an immobilized anti-CD3 antibody and / or interleukin-2. Prevention of postoperative recurrence of non-viral tumors mainly composed of lymphocytes. 5. The method of claim 4, wherein the non-viral tumor patient is a brain tumor. 6. The preventive agent for postoperative recurrence according to claim 5, wherein the brain tumor patient is glioblastoma. 7. A method for preventing postoperative recurrence using the agent for preventing recurrence according to claim 5. 8. The culture solution to which at least one of serum, proteins, amino acids, saccharides, and antibiotics is added in addition to the immobilized anti-CD3 antibody and / or interleukin 2 The method for preventing postoperative recurrence of a brain tumor according to claim 7, characterized in that: 9. A culture medium containing, in addition to the immobilized anti-CD3 antibody and / or interleukin 2, various cytokines, growth factors, or a condition medium prepared by growing appropriate cells, or the like, The method for preventing postoperative recurrence of brain tumor according to claim 7 or 8, wherein a substance that stimulates lymphocytes with a specific antigen is added. 10. A postoperative brain tumor patient to which a preparation containing autologous lymphocytes cultured as a main component in a culture solution containing an immobilized anti-CD3 antibody and / or interleukin 2 is administered, is a glioblastoma. Claim 7 wherein
Or the method for preventing postoperative recurrence of a brain tumor according to 8 or 9. 11. In the case of expanding peripheral blood lymphocytes collected from a brain tumor patient, the culture solution is immobilized anti-CD
3. A method for preparing an agent for preventing postoperative recurrence of brain tumor, which is a culture solution containing Antibody 3 and / or Interleukin 2. 12. In the case of expanding peripheral blood lymphocytes collected from a brain tumor patient, the culture solution is immobilized anti-CD
12. The antibody according to claim 11, wherein at least one of serum, proteins, amino acids, saccharides, and antibiotics is added in addition to the antibody 3 and / or interleukin 2.
The method for preparing a postoperative recurrence preventive agent for brain tumor according to the above.