WO2008138214A1 - A method for activation and expansion of lymphocytes with high efficiency and the culture sysytem thereof - Google Patents

Abstract

A method for activation and expansion of lymphocytes comprises contacting biological samples containing lymphocytes with mobilized anti-cd3 antibody, and then culturing the activated lymphocytes by using mediums containing IL-2 at different concentrations to expand activated lymphocytes. A culture system for activation and expansion of lymphocytes is also provided. The expanded, activated lymphocytes can be used in the immunotherapy of tumor, infectious disease, congenital or acquired immunodeficiency.

Description

 Method and culture system for efficiently activating and activating lymphocytes

 The present invention relates to the field of immunotherapy, and in particular to methods and culture systems for amplifying activated lymphocytes. Background technique

 In recent years, due to advances in immunology, various immunotherapeutic methods have been tried for various immunodeficiency diseases caused by a decrease in the body's ability to monitor immunity. Various immunodeficiencies caused by a decrease in immune surveillance ability include cancer (malignant tumor), congenital immunodeficiency, acquired immunodeficiency, and the like. One of the purposes of immunotherapy is to restore or/and enhance the ability of immunosurveillance to treat these diseases.

As an immunotherapy for cancer, cell adoptive immunotherapy has become an important method for controlling tumor growth and even killing tumor cells. In the 1980s, it was found that after stimulation with IL-2 in vitro, some cells in peripheral blood mononuclear cells can be specifically activated, expanded, and have the effect of killing tumor cells. These cells are lymphokine activated killer cells (LAK cells) (Sinkovics JG, Horvath JC. Human natural killer cells: a comprehensive review. Int J Oncol. Jul 2005; 27(l): 5-47.). The main effector cells of LAK cells are IL-2 activated NK cells. The earliest research found that these cells have the ability to kill leukemia cells in vitro. Since LAK cells belong to non-B non-T cells, this killing is non-HLA-restricted and represents the monitoring and killing effect of the body's natural immune system on tumor cells. Normally, NK cells have no killing effect on autologous leukemia cells without interferon (IFN), IL-2 activation, or removal of inhibitory monocytes. IL-2-activated T lymphocytes and NK cells secrete γ-interferon, resulting in leukemia cells previously resistant to sputum cells being killed by activated sputum cells. In addition to leukemia, some in vitro tests have shown that LAK cells also have a certain effect on animal and human malignant melanoma, kidney cancer, non-Hodgkin's lymphoma, lung cancer and colorectal cancer. However, due to the limitation of in vitro amplifiability and the effect of chemotherapy on lymphocytes, the anti-tumor effect of LAK cells in vivo is not satisfactory. In the early 1990s, Schmidt-Wolf IGH et al. (Schmidt-Wolf IG, Negrin RS, Kiem HP, Blume KG, Weissman IL. Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity J Exp Med. 1991 Jul 1 ;174(1): 139-49. ) A new method was developed for the culture of peripheral blood lymphocytes in vitro and then for subsequent treatment for cancer patients. Unlike LAK cells, this method uses cytokines IL-2, IL-4, IL-1 α, IFN-Υ, and anti-CD3 antibodies. After in vitro culture, the cells obtained have certain effects on leukemia and some solid tumors. The therapeutic effect. In order to distinguish from LAK cells, the cells obtained by this method are called cytokine-activated killer cells, that is, CIK cells. CIK cells differ from LAK cells in that the effector cells that exert anti-tumor effects are mainly CD3+CD56+ sputum cells (>50%). Studies have shown that CIK cells have significantly higher amplification efficiency, tumor cell killing ability, and activity in vivo than LAK cells.

 The traditional CIK treatment methods mainly have the following problems:

 • Before treatment, a large number of patients' peripheral blood (3000-5000ml) should be taken to separate lymphocytes, which is harmful to the patient's own immune system;

 • Low efficiency of cultivation: generally 5-10 times amplification;

 • The patient is poorly tolerated by radiotherapy and chemotherapy;

 • After cell culture, it contains more regulatory (inhibitory) T cells of CD4+CD25+, which may inhibit the proliferation of lymphocytes in vivo after reinfusion;

 • The cells are poorly stable and must be used within 1 to 2 hours after amplification;

 • Due to the large collection of peripheral blood mononuclear cells at one time, this method cannot be treated with high frequency;

 • The patient is required to pay an additional fee for lymphocyte separation. Summary of the invention

 In a first aspect, the invention provides a method of amplifying activated lymphocytes, the method comprising:

(a) a lymphocyte activation step, wherein the biological sample containing lymphocytes is contacted with a lymphocyte activator for a time and conditions sufficient to activate lymphocytes in the biological sample; (b) a first amplification step, wherein the activated lymphocytes obtained in the step (a) are cultured in a first medium containing a high concentration of IL-2;

 (c) a second amplification step, wherein the lymphocytes obtained in the step (b) are cultured in a second medium containing a low concentration of IL-2;

 (d) a third amplification step in which the lymphocytes obtained in the step (c) are cultured in a third medium containing a medium concentration of IL-2;

 (e) Collecting Expanded Activated Lymphocytes (EAL) obtained in the step (d).

 In one embodiment of the method of the invention, the lymphocyte activation step and the first amplification step are performed simultaneously in the same culture vessel.

 Preferably, the lymphocyte activator used in the method of the invention is an immobilized anti-CD3 antibody.

 Preferably, in the method of the present invention, the concentration of IL-2 in the first medium in step (b) may be, for example, 1000 U/ml to 6000 U/ml, and the second medium in step (c) The concentration of IL-2 may be, for example, 1 U/ml to 500 U/ml, and the concentration of IL-2 in the third medium in step (d) may be, for example, 300 U/ml to 750 U/ml.

 In the method of the invention, the medium of step (b) and / or (c) and / or (d) may also contain other cytokines, such as IL-7 and / or IL-15 o, preferably, the step ( The concentration of IL-7 in the medium of b) and / or (c) and / or (d) may be, for example, 1-100 ng / ml, and the concentration of IL-15 may be, for example, 1-200 ng / ml.

 In the method of the present invention, the culture media of steps (b), (c), and (d) may be the same or different. Preferably, the first medium is RPMI-1640 medium, the second medium is AIM-V medium, and the third medium is DMEM medium. The first, second, and third media may also be mixed media.

In the method of the present invention, the first amplification step described in the step (b) may preferably be carried out by bottle amplification, and the second expansion described in the step (c) may be carried out by bag amplification. The addition step and the third amplification step described in step (d). Preferably, the first amplification step described in step (b) is carried out for 7-14 days, and the second amplification step described in step (c) is carried out for 3 to 7 days, and in step (d) The third amplification step described is carried out for 3 to 7 days. In the method of the present invention, step (d) may be repeated one or more times before step (e) is carried out. Using the method of amplifying activated lymphocytes of the present invention, lymphocytes in the biological sample can be amplified 100 to 1000 times. Preferably, wherein the expanded cells are mainly CD8+ T cells and NK cells, for example, among the activated activated lymphocytes, CD8+ T cells can be >50%.

 In a second aspect, the invention also provides a culture system for activating activated lymphocytes, comprising:

 (a) an activation culture vessel for activating lymphocytes in a biological sample, which comprises a lymphocyte activator;

 (b) a first amplification vessel for performing a first amplification step on activated lymphocytes, comprising a first medium, said first medium comprising a high concentration of IL-2;

 (c) a second amplification vessel for performing a second amplification step on the activated lymphocytes, comprising a second medium comprising a low concentration of IL-2;

 (d) A third amplification vessel for performing a third amplification step on activated lymphocytes, comprising a third medium containing a medium concentration of IL-2.

 The present invention also provides another culture system for amplifying activated lymphocytes, comprising:

(a) a first amplification container for activating a lymphocyte in a biological sample and simultaneously performing a first amplification step, comprising a lymphocyte activator and a first medium, said first medium containing a high concentration IL-2;

 (b) a second amplification vessel for performing a second amplification step on the activated lymphocytes, comprising a second medium comprising a low concentration of IL-2;

 (c) A third amplification vessel for performing a third amplification step on activated lymphocytes, comprising a third medium containing a medium concentration of IL-2.

 Preferably, the lymphocyte activator used in the culture system of the present invention is an immobilized anti-CD3 antibody.

 Preferably, in the culture system of the present invention, the concentration of IL-2 in the first medium may be, for example, 1000 U/ml to 6000 U/ml, and the concentration of IL-2 in the second medium. It may be, for example, 1 U/ml to 500 U/ml, and the concentration of IL-2 in the third medium may be, for example, 300 U/ml to 750 U/ml.

In the culture system of the present invention, the first and/or second and/or third medium may further contain other cytokines such as IL-7 and/or IL-15. Preferably, said first and / or The concentration of IL-7 in the di- and/or third medium may be, for example, 1-100 ng/ml, and the concentration of IL-15 may be, for example, 1-200 ng/ml.

 In the culture system of the present invention, the first, second, and third media may be the same or different. Preferably, the first medium is RPMI-1640 medium, the second medium is AIM-V medium, and the third medium is DMEM medium. The first, second, and third media may also be a mixed medium.

 The present invention also provides an immunotherapy method comprising administering to an individual an expanded activated lymphocyte (EAL) obtained by the above method for amplifying activated lymphocytes of the present invention.

 In one embodiment of the immunotherapy method of the present invention, the lymphocytes obtained by the above amplification method are Expanded Activated Autologous Lymphocytes (g卩EAAL), and the immunotherapy method includes the following Steps:

 (i) obtaining a biological sample containing lymphocytes from an individual;

 (ii) amplifying the lymphocytes by the method of amplifying activated lymphocytes of the present invention described above, thereby obtaining amplified activated autologous lymphocytes;

 (iii) returning the amplified activated autologous lymphocytes to the individual.

 The immunotherapeutic methods of the invention are useful for treating individuals suffering from, for example, tumors, infectious diseases, congenital or acquired immunodeficiency. Tumors that can be treated using the immunotherapeutic methods of the invention are, for example, lung cancer, cardiac cancer, colon cancer, breast cancer, medulloblastoma, gastric cancer, renal cancer, and malignant melanoma, and individuals with tumors may be receiving Individuals who receive radiation and/or chemotherapy. Infectious diseases which can be treated using the immunotherapy method of the present invention are, for example, viral infections, particularly hepatitis B and C virus infections. DRAWINGS

 Figure 1: Analysis of cell expansion folds before and after cell culture in 30 cases of 2-week activation/amplification culture. Figure 2: Analysis of lymphocyte phenotype before and after cell culture in 25 cases of 2-week activation/amplification culture. Figure 3: After EAAL reinfusion, the proportion of peripheral blood lymphocytes secreting IFN-γ in patients is significantly increased.

Figure 4: After EAAL reinfusion, the proportion of CD8+ T cells in peripheral blood of patients was significantly higher (p=0.0036). Figure 5: After EAAL reinfusion, the proportion of CD4+ T cells and CD8+ T cells in the peripheral blood of patients was significantly reduced (p=0.0333).

 Figure 6: Recurrent breast cancer, with decreased levels of tumor markers after treatment with EAAL of the invention (5 reinfusions). detailed description

 The present invention provides a method for efficiently amplifying activated lymphocytes and a culture system for the same, which is also called an Expanded Activated Lymphocyte Kit (EAL it) o The amplification method of the present invention is improved based on the conventional CIK method and is a highly efficient CIK cell culture method. Culture/amplification method

 The method of amplifying activated lymphocytes of the present invention comprises -

(a) a lymphocyte activation step, wherein the biological sample containing lymphocytes is contacted with a lymphocyte activator for a time and conditions sufficient to activate lymphocytes in the biological sample;

 (b) a first amplification step in which the activated lymphocytes obtained in the step (a) are cultured in a first medium containing a high concentration of IL-2;

 (c) a second amplification step, wherein the lymphocytes obtained in the step (b) are cultured in a second medium containing a low concentration of IL-2;

 (d) a third amplification step in which the lymphocytes obtained in the step (c) are cultured in a third medium containing a medium concentration of IL-2;

 (e) Collecting the amplified activated lymphocytes obtained in the step (d).

 In one embodiment of the method of the invention, the lymphocyte activation step and the first amplification step are performed simultaneously in the same culture vessel. Lymphocyte

Lymphocytes that can be cultured and expanded using the methods of the invention can be derived from peripheral blood lymphocytes. The lymphocytes to be expanded may also be lymphocytes present in other biological samples, such as epithelial lymphocytes, intratumoral infiltrating lymphocytes, cancerous ascites or pleural immersion. Run lymphocytes and so on. Lymphocytes isolated from fresh peripheral blood are preferably used because of the ease of separation of lymphocytes in peripheral blood, high lymphocyte survival rate after isolation, and the like. Lymphocyte activation

 The term "lymphocyte activation" refers to the use of specific antigens or non-specific lymphocyte stimulators/molecules to stimulate lymphocytes, resulting in the synthesis of macromolecular substances (RNA, protein, DNA) and cytokines by lymphocytes. After lymphocyte activation, the cells proliferate and differentiate into progeny effector cells and memory cells of different functions.

 The term "lymphocyte activator" refers to a reagent/molecule that is capable of stimulating lymphocyte activation. For example, an anti-CD3 antibody can be used as a lymphocyte activating agent which stimulates CD3 molecules on the surface of T cells and efficiently activates T lymphocytes through T cell receptors. Lymphocyte mitogens are another class of potent lymphocyte activators. These molecules rapidly activate lymphocytes and do not pass through T lymphocyte receptors.

 An example of a suitable lymphocyte activator is an anti-CD3 antibody. In a specific embodiment of the lymphocyte expansion method of the present invention, the lymphocyte activating agent is an anti-CD3 antibody immobilized on a surface of a substrate. The use of an immobilized anti-CD3 antibody is preferred because it mainly activates CD8+ T cells having a cell killing function, which is particularly advantageous for enhancing the antitumor activity of the expanded lymphocytes. Methods of preparing culture vessels containing immobilized anti-CD3 antibodies are known and are described in detail below. Cytokine

 The cytokines used to promote lymphocyte proliferation by the conventional lymphocyte expansion method are mainly IL-2, IL-4, IL-l and IFN-γ. The compatible form of this cytokine primarily causes proliferation of CD3+CD56+ cells, CD4+ T cells, and CD8+ T cells.

 In the present invention, cytokines mainly use IL-2. IL-2 can amplify CD8+ T cells with high efficiency and specificity, and CD8+ T cells are the most important cellular components of the immune system to exert anti-tumor effects. The concentration of the cytokine IL-2 used in the conventional lymphocyte expansion method is always the same concentration from the start to the end of the culture. Unlike the conventional method, the present invention uses different concentrations of IL-2 in different culture stages.

According to the present invention, in order to obtain a large amount of lymphocytes having rapid proliferative ability in a short time The activated lymphocytes obtained in the step (a) are cultured in the first medium containing the high concentration of IL-2 in the first amplification step (b). Preferably, the concentration of IL-2 in the first medium ranges, for example, from 1000 U/ml to 6000 U/ml. The cells were grown to the 7-14th day in the first amplification step. Considering the nutrient consumption of the culture medium and the decrease in the activity of IL-2 and other cytokines, it is very important to add a proper amount of fresh culture solution every day. In the second amplification step (c), the lymphocytes obtained in the step (b) are further cultured in a second medium containing a low concentration of IL-2, so that the cells maintain the proliferation rate in the culture vessel. Not overactivated. Preferably, the concentration of IL-2 in the second medium ranges, for example, from 1 U/ml to 500 U/ml. The cells are grown for 3-7 days in the second amplification step. The 10th-14th day of the culture enters a third amplification step (d) in which the lymphocytes obtained in the step (c) are cultured in a third medium containing a medium concentration of IL-2. Preferably, the concentration of IL-2 in the third medium ranges, for example, from 300 U/ml to 750 U/ml. The cells are further cultured for 3 - 7 days in the third amplification step to extensively expand the cells to meet the clinical use requirements. With the method of the present invention, lymphocytes can be amplified by a factor of 100 or more.

 In addition to IL-2, other cytokines capable of promoting proliferation of lymphocytes may be further added to the amplification method of the present invention. For example, interleukin-7 (IL-7) and/or interleukin-15 (IL-15) can also be added to the culture medium to maintain memory cell viability and reduce death after cell activation. Achieve the best cell expansion and maintenance of cell viability. In the method of the present invention, the concentration of IL-7 in the medium of the step (b) and/or (c) and/or (d) is preferably from 1 to 100 ng/ml, and the concentration of IL-15 is preferably The ground is l-200ng/ml. Medium

The lymphocyte culture/amplification protocol used in the conventional method employs a fixed cell culture medium such as RPMI-1640 or DMEM. However, in the method of the present invention, the medium of the steps (b), (c), and (d) may be different. Preferably, the first medium is RPMI-1640 medium, the second medium is AIM-V medium, and the third medium is DMEM medium. By changing the cell culture medium used in different cell culture stages, it is possible to provide more abundant nutrients to the cells, increase the cell's proliferative capacity, and increase the amplification factor. Further, in the method of the present invention, the first, second, and third medium may also be a mixed medium. In addition, the first, second, and third media may also be added Into other components that help lymphocyte expansion, such as fetal bovine serum or human serum. The concentration of serum in the medium is 1 - 20%. The serum was thawed at 37 °C before use and inactivated by heating at 56 ° C for 30 minutes.

 The term "mixed medium" as used herein refers to a mixture of one or more of conventional media such as RPMI-1640, DMEM, IMEM, AIM-V, and the like. Training method

 In the method of the present invention, any culture vessel suitable for lymphocyte activation and amplification can be used. For example, the method of the present invention may employ a cell culture flask for cell culture, or a closed cell culture bag system for cell expansion and culture. The volume of the culture vessel should meet the need for extensive expansion of the cells. The culture bag which can be used in the present invention can, for example, accommodate the number of cells accommodated in 10-20 T225 cell culture flasks, which greatly reduces the workload of the cultured cells, while the closed system greatly reduces the possibility of contamination during the operation. For example, the first amplification step can be performed by bottle amplification, and the second amplification step and the third amplification step can be performed by bag amplification. Preferably, in the method of the invention, the lymphocyte activation step and the first amplification step are performed simultaneously in the same vial amplification vessel.

 Preferably, the first amplification step is performed for 7-14 days, the second amplification step is performed for 3 to 7 days, and the third amplification step is performed for 3 to 7 days. In the method of the present invention, the third amplification step may be repeated one or more times before performing step (e). Using the method of amplifying activated lymphocytes of the present invention, lymphocytes in the biological sample can be amplified at least 10 fold, preferably at least 100 fold, more preferably at least 1000 fold. Preferably, wherein the expanded cells are mainly CD8+ T cells and NK cells, for example, CD8+ T cells can be >50% in the expanded activated lymphocytes. Culture/amplification system

 Preparation of activated culture vessel

The activation culture vessel is used to activate lymphocytes in a biological sample containing a lymphocyte activator. In a specific embodiment, the activation culture vessel used in the culture/amplification system of the present invention contains an anti-CD3 antibody immobilized on an insoluble carrier such as the surface of the container as a lymphocyte activating agent.

 In the preparation of an activated culture vessel containing an anti-CD3. antibody immobilized on an insoluble carrier, the anti-CD3 antibody can be diluted with a sterile sodium phosphate buffer (PBS) to a concentration of 1 - 50 w g/ml. When the concentration is less than 1 μg/ml, the proliferation of T cells tends to be insufficient. When the proliferation of T cells exceeds 50 μg/ml, the proliferation of T cells is not sufficient, but the free anti-CD3 antibody which is not immobilized on the insoluble carrier tends to increase. In addition, the diluent is not particularly limited as long as it is a physiological solution such as a sterilized sodium phosphate buffer solution, and as a protective agent, white protein, agarose or the like may be added.

 The diluted anti-CD3 antibody was aseptically plated on the bottom surface of the culture flask. The reaction temperature at the time of fixation is 4 to 40 ° C, more preferably 4 to 25 V, and most preferably 4 to 10 ° C. At less than 4 °C, the immobilization efficiency of the anti-CD3 antibody tends to decrease, and when it exceeds 40 °C, the activity of the anti-CD3 antibody tends to decrease. The reaction time in the immobilization is from 30 minutes to 24 hours, preferably from 1 hour to 24 hours. At less than 30 minutes, the immobilization amount of the anti-CD3 antibody tends to decrease, and when it exceeds 24 hours, the fixation time is too long, and the production efficiency tends to decrease.

 Immediately following the immobilization of the above anti-CD3 antibody, the residual anti-CD3 antibody dilution which was not immobilized was removed. The anti-CD3 antibody which has not been immobilized is washed with an appropriate washing solution such as sodium phosphate buffer (PBS) and dried. The drying method can be carried out by natural drying in a 4-10 ° C cold storage, heating drying at 10-40 ° C, lyophilization, etc. after removing the cleaning liquid. In view of the activity and stability of the anti-CD3 antibody, lyophilization is preferred, and natural drying in a 4-10 °C cold storage is most preferred. As described above, the anti-CD3 antibody is immobilized on the surface of the insoluble carrier, and a carrier for T cell culture in which the immobilized surface of the anti-CD3 antibody is dried is obtained, and the culture vessel is obtained aseptically sealed. Preparation of the first amplification container

 The first amplification vessel is used to perform a first amplification step on the activated lymphocytes, which contains a first medium containing a high concentration of IL-2. Preferably, the concentration of IL-2 in the first medium is from 1000 U/ml to 6000 U/ml.

The IL-2 concentration was diluted 1000-6000 U/ml with basal medium RPMI-1640 medium. To increase cell viability and survival time, IL-7 and/or IL-15 can be added. The concentrations were 1-100 ng/ml and 1-200 ng/ml, respectively. Subsequently, the medium is formulated into a cell culture medium having a volume ratio of 1 - 20% fetal calf serum or human serum (the serum is thawed at 37 ° C and heated at 56 ° C for 30 minutes), but In order to promote lymphocyte proliferation, it is generally preferably 2 to 10 (V/V)%. The culture medium is preferably a sterilized RPMI-1640 medium, but is not limited thereto, and other medium suitable for lymphocytes or a mixed medium can be used. The first medium for the first amplification vessel is thus prepared.

 When the activation step is performed simultaneously with the first amplification step, the medium prepared as described above can be directly added to the above-described activated culture vessel. Preparation of a second amplification container

 The second amplification vessel is for performing a second amplification step on the activated lymphocytes, which contains a second culture medium, and the second medium contains a low concentration of IL-2. Preferably, the concentration of IL-2 in the second medium is from 1 U/ml to 500 U/ml. Preferably, the second expansion vessel may use a bag culture vessel.

 The IL-2 concentration was diluted 1 - 500 U/ml with AIM-V medium. To increase cell viability and survival time, IL-7 and / or IL-15 o concentrations of 1-100 ng / ml and 1- 200 ng / ml can be added. Subsequently, the medium was formulated into a cell culture medium having a volume ratio of 1 - 20% fetal calf serum (the serum was thawed at 37 ° C and heated at 56 ° C for 30 minutes). A second medium for the second amplification vessel is thus prepared. The second culture medium preferably uses AIM-V medium, but is not limited thereto, and other medium or mixed medium suitable for lymphocytes may also be used. Preparation of a third amplification container

 The third amplification vessel is used to perform a third amplification step on the activated lymphocytes, which contains a third medium containing a medium concentration of IL-2. Preferably, the concentration of IL-2 in the third medium is from 300 U/ml to 750 U/ml. Preferably, the third amplification container can use a bag culture container.

The IL-2 concentration was diluted from DMEM medium to 300 - 750 U/ml. In order to improve cell survival and survival time, IL-7 and / or IL-15 can be added at concentrations of 1-100 ng/ml and 1- 200 ng/ml. Subsequently, the medium was formulated into a cell culture medium in a volume ratio of 1 - 20% fetal calf serum (the serum was thawed at 37 ° C and heated at 56 ° C for 30 minutes). The third medium for the third amplification vessel was thus prepared. The third medium is best used in DMEM medium, but it is not limited thereto, and other medium suitable for lymphocytes or mixed medium may also be used. Immunotherapy

 The present invention also provides an immunotherapy method comprising administering to an individual an expanded activated lymphocyte (EAL) obtained by the method of amplifying activated lymphocytes of the present invention described above.

 In one embodiment of the immunotherapy method of the present invention, the lymphocytes obtained by the above amplification method are Expanded Activated Autologous Lymphocytes (gp EAAL), and the immunotherapy method comprises the following steps :

 (i) obtaining a biological sample containing lymphocytes from an individual;

 (ii) amplifying the lymphocytes by the method of amplifying activated lymphocytes of the present invention described above, thereby obtaining amplified activated autologous lymphocytes;

 (iii) returning the amplified activated autologous lymphocytes to the individual.

 In the immunotherapy method of the present invention, when the amplified lymphocytes used are autologous lymphocytes, we call them Expanded Activated Autologous Lymphocytes (EAAL) Therapy. The lymphocytes used in the treatment are derived from a small amount of peripheral blood of the patient. These lymphocytes can be activated and amplified 100-1000 times in a short period of about 2 to 4 weeks and then returned to the patient. The immunotherapy method of the present invention can be used for treating an individual suffering from, for example, a tumor, an infectious disease, a congenital or acquired immunodeficiency defect, and an infectious disease caused by an organ transplant and an infection-induced tumor.

Unlike conventional CIK cells, the activated cells obtained by this method are mostly CD8+ T lymphocytes (>50%). CD8+ T lymphocytes play an important role in killing virus-infected cells and cancer cells. Human T lymphocytes include many specific cells directed against different antigens. These cells, after culturing, theoretically have broad recognition and killing effects against different tumor antigens/viral antigens. The purpose of treatment is to activate and proliferate such lymphocytes in vitro to enhance their function and to be used in therapy to kill tumor cells. Tumors that can be treated using the immunotherapeutic methods of the invention are, for example, lung cancer, cardiac cancer, colon cancer, breast cancer, medulloblastoma, gastric cancer, renal cancer, and malignant melanoma, and individuals with tumors may be receiving Individuals who receive radiation and/or chemotherapy.

 For some patients with chronic viral infections, such as hepatitis B and C, this method can also effectively remove infected virus. Therefore, the immunotherapy method of the present invention can also be used to treat infectious diseases such as viral infections, particularly hepatitis B virus infection and hepatitis C virus infection. The main advantages of the invention are:

 • The culture system can be a standardized kit system;

 • Only need to collect 20-50ml donor or self-peripheral blood for lymphocyte separation; • Little damage to the donor or patient lymphatic system;

 • Saving costs;

 • Improve patient tolerance to chemoradiotherapy, combined with radiotherapy and chemotherapy;

 • High frequency treatment;

 • High amplification efficiency, up to 100-1000 times cell expansion;

 • Stable reproducibility at different times, between individuals, and between laboratories;

 • The cells are stable, maintain cell viability within 24 hours after amplification, and can be returned within 12 hours;

 • Long distance transportation. Example

 The following examples are intended to illustrate the invention and are not intended to limit the invention in any way.

 1. Cell activation:

First, a culture flask coated with an immobilized anti-CD3 antibody was prepared. Anti-CD3 antibody (Ortho) was diluted to 10 wg/ml in sterile sodium phosphate buffer (PBS). The diluted anti-CD3 antibody was aseptically plated on the bottom of a culture flask (Costa). 4 ° C overnight. Wash the anti-CD3 antibody without immobilization with sodium phosphate buffer (PBS) and dry naturally in a cold storage at 4-10 °C. After drying, the immobilized surface of the anti-CD3 antibody was dried, and the carrier for T cell culture was aseptically sealed to obtain an activated culture vessel.

Human hemorrhage whole blood 20 ml was collected with a heparin-treated syringe, 20 ml of physiological saline was added, and 2-fold dilution was performed. Each 10 ml of diluted blood was added to a 3 ml density gradient centrifuge Ficoll-Paque (Sigma-Aldrich Corp., St . Louis, MO, USA) Upper layer, centrifuged at 1600 rpm, 25 minutes, 20 °C (Beckman, Fullerton, CA, USA, S4750A) to recover lymphocytes. The isolated lymphocytes were washed 3 times with RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA), and finally a cell suspension having a cell density of about 2×10 ⁶ /ml was prepared. 5 ml of this cell suspension (1×10 ⁷ ) was added to the above activated culture vessel (culture flask coated with immobilized anti-CD3 antibody) in 50 ml of IL-2 containing 6000 U/ml as described above. The culture is started in the first amplification reagent.

 2. Cell expansion - On the second day of culture, observe the cells under the microscope (Chongqing Optical Instrument Factory, CKX-41). If the cells proliferate well, add 180ml of the first amplification reagent as described above. Growth was carried out until day 7 under conditions. Considering the nutrient consumption of the culture medium and the reduction of IL-2 (Nova Chemical Factory, Beijing Sihuan Pharmaceutical Factory, China) and other cytokine activities, it is very important to add a proper amount of fresh culture solution every day.

 The cells and the medium were transferred to the second amplification reagent as described above after the 7th day of the culture, and growth was continued for 3 days in the cell culture bag.

 On the 10th day of the culture, the cells in the second amplification reagent as described above were uniformly mixed with the third amplification reagent as described above, and then cultured in the two cell culture bags for 4 days. If a larger number of cells are required, the cells in the two cell culture bags can be further mixed with a reagent in the third amplification reagent as described above, and the culture is continued in three culture bags.

 3. Cell harvesting:

 The cells in the culture bag were transferred to a centrifuge tube, centrifuged at 1600 rpm for 10 minutes. The cells were washed twice with a solution of 1% albumin (Beijing Tiantan Biological Products Co., Ltd., China), and resuspended in 5% albumin-physiological saline for the following tests.

 4. Multiples of autologous lymphocyte expansion:

The initial number of human lymphocytes cultured using the present invention was 20 ml of peripheral venous blood. The initial number of mononuclear cells before culture is about 0.5 - 5 X 10 ⁷ . After culture, the number of cells is flat within two weeks. Both can be increased by 323 times (Fig. 1), and the cells can be amplified more than 1000 times after three weeks to four weeks of culture.

 5. The phenotype of autologous lymphocytes after expansion:

 The phenotypic detection method of lymphocytes is as follows:

The labeled cells were 10 ⁶ cells per tube, and the antibodies of the different combinations below were labeled according to the required cell phenotype. Cell markers were labeled with FITC-labeled anti-CD25, PE-CY5-labeled anti-CD4, FITC-labeled anti-CD16, PE-labeled anti-CD56, PE-CY5-labeled anti-CD8 (Becton-Dickinson) , Mountain View, CA, USA), PE-labeled anti-CD8 (ebioscience, San Diego, CA, USA) and ECD-labeled anti-CD3 (Beckman Coulter, Fullerton, CA, USA) o corresponding isotype antibody for Background correction. Phenotypic analysis was performed using a Coulter flow cytometer (COULTER EPICS XL) with the software COULTER EXP032 ADC (Beckman Coulter, Fullerton, CA, USA).

 The starting cells used in the present invention are peripheral blood mononuclear cells, wherein the T lymphocytes are about 40-70%, and the NK cells are about 1-5%. After culture, the main components of the cells are T lymphocytes and NK cells (>95%). The proportion of CD8+ T cells is about 50-95% (Fig. 2). CD8+ T lymphocytes and NK cells are the most important lymphocyte components of the body to exert anti-tumor and anti-viral effects. At the same time, the ratio of CD4+ T cells and CD8+ T cells in the cells cultured by the method of the invention is significantly decreased, which is beneficial to the anti-tumor effect of lymphocytes.

 6. Effect for patients with cancer:

The lymphocytes prepared according to the method of the present invention are returned to the patient, and the amount of the cells administered can be determined according to clinical needs, preferably 5 X 10 ⁹ per cell.

 1) Improve the level of anti-tumor cytokine Y interferon (IFN-γ) in tumor patients.

 The detection method of intracellular cytokine Y interferon (IFN-γ) level is as follows:

Lymphocytes were collected from patients receiving EAAL reinfusion. 2 X 10 ⁶ /well cells were plated in 24-well plates using PMA (10 ng/ml; Sigma-Aldrich Corp., St. Louis, MO, USA) and Ionomycin (1 μg/ml; Sigma-Aldrich Corp. , St. Louis, MO, USA), stimulated for 6 hours in the presence of monensin (10 μM; Sigma-Aldrich Corp., St. Louis, MO, USA). Cells were harvested and stained with monoclonal antibodies against surface molecules such as ECD-CD3 and PE-CY5-CD8 for 15 minutes at room temperature. After washing with PBS, treated with a permeate containing Cytoperm (Becton-Dickinson, Mountain View, CA, USA) and 0.1% saponin (Sigma-Aldrich Corp., St. Louis, MO, USA) for 20 minutes, then added to PE. -combined Anti-IFN-γ antibody (ebioscience, San Diego, CA, USA) or isotype control, after incubation at 4 ° C for 30 minutes, washed with PBS, and analyzed by flow cytometry.

 In tumor patients treated with the EAAL method, the number of lymphocytes secreting the anti-tumor cytokine DFN-Y was significantly increased after reinfusion (Fig. 3, left). At the same time, the amount of secreted IFN-γ increased significantly (Fig. 3, right).

 In addition to its role in inhibiting and killing tumors, IFN-γ also plays an important role in regulating the body's immune system. It can make the immune system's function tend to balance Thl, which is a killing function. In the tumor patients, due to the action of immunosuppressive factors secreted by tumors, the immune environment of the body is mostly an immunosuppressive environment, which hinders the killing of tumor cells by killer lymphocytes. Therefore, in addition to direct killing or inhibition of tumor growth, the mechanism of action of EAAL in anti-tumor therapy may include improving the immunosuppressive environment in patients and optimizing the immune environment for tumor killing (see (2) below).

 2) Regulate the immune system of tumor patients, and significantly increase the proportion of lymphocytes with anti-tumor effects in the immune system.

 The analysis of cell phenotype is the same as before.

 After EAAL reinfusion, the proportion of CD8+ T cells in peripheral blood lymphocytes of patients increased significantly (Fig. 4), and the ratio of CD4+ T cells to CD8+ T cells decreased significantly (Fig. 5). This indicates that the patient's immune system tends to balance lymphocytes to the immune killing function.

 3) It shows the effect of eliminating tumors in some tumor patients.

 After EAAL reinfusion treatment, some patients showed partial regression of tumors, stable tumors, and a significant improvement in quality of life (see case report and Table 1). Case report 1

 Patients with non-small cell lung cancer with brain metastases: The patient did not receive any other treatment during the first three months of treatment with EAAL. Brain CT before and after treatment showed a reduction in metastases. Case report 2

 Recurrent breast cancer, after treatment (5 times return) tumor markers CA15.3 levels decreased (Figure

6). Case report 3

 After the second epithelial medulloblastoma of the cerebellum, the brain has been replanted extensively, and the blood picture has been low. It is impossible to complete the complete course of chemotherapy. The doctor in charge used EAAL therapy in combination with chemotherapy, ie 7 days after each chemotherapy, EAAL was returned, once a week, several times in succession. Beginning 2 weeks after the last return, do the next chemotherapy. After 5 years of chemotherapeutic treatment and 6 EAAL treatments in 5 months, the NMR results showed that most of the metastases disappeared.

 The patient's conscious quality of life during the chemotherapy interval was significantly improved compared with the first relapse treatment. During the treatment period, there was no need for aseptic isolation like the first relapse chemotherapy. The patient insisted on climbing the mountain during the chemotherapy interval, and there was no abnormality due to anemia. Symptoms such as palpitation, shortness of breath, and other symptoms of chemotherapy side effects such as decreased appetite, weight loss, and poor sleep. Case report 4

 Colon cancer with double lung metastases, female, 65 years old, chemotherapy + EAAL reinfusion treatment

The patient had normal food and sleep, good mental strength, and cough disappeared. The review showed that the lung lesions had shrunk and multiple lymph node metastases had improved.

Table 1. Clinical effects of some patients after EAAL reinfusion treatment.

Patient Number Tumor Type Other Treatment Clinical Effects

 0002 Non-small cell lung cancer with pleural effusion 4 months before EAAL treatment 1. After 5 times of cell reinfusion, brain lesions shrink after 12 days, no progress in lung lesions

 Immobilization, post-brain transfer, and radiotherapy 2. Improvement in quality of life: After the cell was returned, it continued for one year of daily low heat loss, appetite and sleep improvement.

 0005 Non-small cell lung cancer with lymph nodes surgery, chemotherapy 1. No progress after 6 months of surgery

 Infiltration 2. Quality of life improvement: adverse reactions caused by chemotherapy are alleviated

 0015 Non-small cell lung cancer with bone metastasis Thymosin treatment 1. Hepatitis B surface antigen decreased after 6 times of reinfusion, pain relief, pleural effusion disappeared

 Molecular targeted therapy 2. After 4 months of treatment, the cough gradually worsened but the chest radiograph showed no obvious lesions.

 0017 Non-small cell lung cancer None 1. Significant reduction in pleural effusion after the first 2 reinfusions

 2. There is no change in pleural effusion after the third and fourth reincarnation, no progress in disease

 0010 Cardiac cancer with liver metastasis, 1 month before treatment of abdominal cavity 1. After the first 2 times of cell reinfusion, the ascites was significantly reduced, and the liver and peritoneal metastases were partially reduced. After treatment, there was no other treatment.

 0032 Colon cancer with abdominal cavity transfer Chinese medicine treatment 1. Quality of life improvement

 2. Weight gain

 0031 Breast cancer with liver and bone metastasis Just before the treatment of chemotherapy 1. Large tumors shrink, small tumors have no change

0027 Postoperative medulloblastoma, multiple disease chemotherapy 1. Two chemotherapy combined with six high-efficiency CIK treatments, the brain lesions are completely complete.

The second recurrence of the stove

0029 Lung cancer postoperative involvement of visceral chest Oral molecular targeted drugs 1. Double lung metastasis nodules improved significantly, no chemotherapy after double lung metastasis 2. Left pleural thickening nodules improved

 Method control 3. bilateral pleural effusion

 0035 Postoperative recurrence of gastric cancer, peritoneal rotation None 1. After eating, the pharyngeal sensation disappears and the appetite increases.

 Move 2. CT review no progress

 0039 Right lung cancer postoperative, liver metastasis None 1. Quality of life improved

 2. Weight gain

 0041 Right lung cancer, mediastinal lymphadenopathy None 1. Quality of life improvement

 Large, pericardial effusion 2. Reexamination showed a decline in tumor markers

 0046 Colon cancer double lung metastasis Oral chemotherapy, Chinese medicine 1. Cough disappears

 2. The lung lesions shrink, and multiple lymph node metastases have improved.

Claims

Claim 1. A method of amplifying activated lymphocytes, comprising:  (a) a lymphocyte activation step in which a biological sample containing lymphocytes is contacted with a lymphocyte activator;  (b) a first amplification step in which the activated lymphocytes obtained in the step (a) are cultured in a first medium containing IL-2 having a concentration of from 1000 U/ml to 6000 U/ml;  (c) a second amplification step, wherein the lymphocytes obtained in the step (b) are cultured in a second medium containing IL-2 at a concentration of 1 U/ml to 750 U/ml;  (d) a third amplification step, wherein the lymphocytes obtained in the step (c) are cultured in a third medium containing IL-2 at a concentration of 300 U/ml to 1000 U/ml;  (e) Collecting the amplified activated lymphocytes obtained in the step (d).  The method of claim 1, wherein said lymphocyte activation step (a) is carried out simultaneously with the first amplification step (b).  3. The method of claim 1 or 2, wherein the lymphocyte activator is an immobilized anti-CD3 antibody.  4. The method of claim 1 or 2, wherein the medium of step (b) and / or (c) and / or (d) further comprises other cytokines.  5. The method of claim 4 wherein said other cytokine is IL-7 and / or IL- 15.  6. The method of claim 5, wherein the concentration of IL-7 is from 1 to 100 ng/ml and/or the concentration of IL-15 is from 1 to 200 ng/ml.  7. The method of claim 1 or 2, wherein the first medium is RPMI-1640 medium.  8. The method of claim 1 or 2, wherein the second medium is an AIM-V medium.  9. The method of claim 1 or 2, wherein the third medium is a DMEM medium. 10. The method of claim 1 or 2, wherein the first, second, and third media are the same medium or mixed medium. 11. The method of claim 1 or 2, wherein the first amplification step described in step (b) is carried out for 7-14 days, and the second amplification step described in step (c) is carried out for 3 to 7 days. And the third amplification step described in step (d) is carried out for 3 to 7 days.  12. The method of claim 11 wherein step (d) is optionally repeated one or more times prior to performing step (e).  13. The method of any one of claims 1 to 12, wherein the lymphocytes are amplified 100 to 1000 times.  14. The method of claim 13 wherein the expanded cells are predominantly CD8+ T cells (>50%) and NK cells.  15. A culture system for amplifying activated lymphocytes, comprising:  (a) an activation culture vessel for activating lymphocytes in a biological sample, which comprises a lymphocyte activator;  (b) a first amplification vessel for performing a first amplification step on activated lymphocytes, comprising a first medium comprising IL at a concentration of from 1000 U/ml to 6000 U/ml -2;  (c) a second amplification vessel for performing a second amplification step on activated lymphocytes, comprising a second medium comprising IL at a concentration of from 1 U/ml to 750 U/ml -2; and  (d) a third amplification vessel for performing a third amplification step on activated lymphocytes, comprising a third medium comprising IL at a concentration of from 300 U/ml to 1000 U/ml - 2.  16. A culture system for activating activated lymphocytes, comprising:  (a) a first amplification container for activating a lymphocyte in a biological sample and simultaneously performing a first amplification step, comprising a lymphocyte activator and a first medium, the first medium having a concentration of IL-2 from 1000 U/ml to 6000 U/ml;  (b) a second amplification vessel for performing a second amplification step on activated lymphocytes, comprising a second medium comprising IL at a concentration of from 1 U/ml to 750 U/ml -2; and (c) a third amplification vessel for performing a third amplification step on activated lymphocytes, comprising a third medium containing IL at a concentration of from 300 U/ml to 1000 U/ml - 2.  The culture system according to claim 15 or 16, wherein said lymphocyte activating agent is an anti-CD3 antibody immobilized on the surface of said container.  18. The culture system of claim 15 or 16, wherein said first and / or second and / or third medium further comprise other cytokines.  19. The culture system of claim 18, wherein said other cytokine is IL-7 and / or IL-15 o  20. The culture system of claim 19, wherein the concentration of IL-7 is from 1 to 100 ng/ml and/or the concentration of IL-15 is from 1 to 200 ng/ml.  21. The culture system of claim 15 or 16, wherein said first medium is RPMI-1640 medium.  22. The culture system of claim 15 or 16, wherein said second medium is AIM-V medium.  23. The culture system of claim 15 or 16, wherein said third medium is DMEM medium.  24. The culture system of claim 15 or 16, wherein said first, second, and third media are the same medium or mixed medium.