WO2009151183A1 - A medium composition for cultivating self activated lymphocyte - Google Patents

A medium composition for cultivating self activated lymphocyte Download PDF

Info

Publication number
WO2009151183A1
WO2009151183A1 PCT/KR2008/005658 KR2008005658W WO2009151183A1 WO 2009151183 A1 WO2009151183 A1 WO 2009151183A1 KR 2008005658 W KR2008005658 W KR 2008005658W WO 2009151183 A1 WO2009151183 A1 WO 2009151183A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
medium composition
antibodies
lymphocytes
cell culture
Prior art date
Application number
PCT/KR2008/005658
Other languages
French (fr)
Inventor
Dong Ouk Lee
Sun Min Hong
Byoung In Choi
Original Assignee
Nkbio Co., Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nkbio Co., Ltd filed Critical Nkbio Co., Ltd
Publication of WO2009151183A1 publication Critical patent/WO2009151183A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere

Definitions

  • the inventive concept relates to a medium composition for culturing self- activated lymphocytes applicable to the treatment of malignant tumors, and more particularly, to a medium composition to which anti-CD3, anti-CD16 and anti-CD56 antibodies are added along with interleukin2 (IL-2) to evenly activate natural killer (NK) cells, T cells and natural killer T (NKT) cells, and thus the medium composition can be used to culture immunocytes that can effectively treat various kinds of malignant tumors.
  • IL-2 interleukin2
  • Adaptive immunotherapy is a method involving extracting natural killer (NK) cells, dedritic (De) cells, B cells, T cells, and the like, which are the most crucial immunocytes for the treatment of cancers, from blood of a patient, growing the extracted immunocytes to be able to withstand cancer cells by using different kinds of stimulants, and then injecting them back into the patient. Since the blood of the patient is used, the adaptive immunotherapy leads to less side effects and can be applied along with a more convenient administration method than conventional chemotherapies and the like. For these reasons, adaptive immunotherapy is currently being vigorously researched.
  • NK natural killer
  • De dedritic
  • NK cells among immunocytes activated in adaptive immunotherapy are a kind of lymphocytes that have an excellent ability to kill infected viruses and tumor cells, but not to kill most normal cells.
  • NK cells are known to play an important role in an initial stage of the body defensive mechanism and in the human tumor immunity. That is, NK cells can kill specific cancer cells, homologous cells and even heterologous cancer cells without a sensitizing process, i.e., without an acquisition of immunity to the expression of MHC.
  • NK cells can more effectively kill target cells which do not or less express Class 1 MHC.
  • NK cells can effectively kill most cancer cells that do not express MHC, cells infected with several viruses, and bacteria such as Salmonella typhimurium (Salmonella typhi.).
  • NK cells which have a superior ability to kill cancer cells, constitute only
  • NK cells cannot effectively attack cancer cells without an additional amplification process using adaptive immunotherapy.
  • the method of activating CD4 T cells involves separating CD4 T cells from a biological sample such as blood and culturing the separated CD4 T cells in vitro with a cytokine- added culture medium containing GM-CSF, IFN-gamma, TNF-alpha, lectin and IL-4 to activate the CD4 T cells. As a result, a composition for preventing or treating bacterial infectious diseases is acquired.
  • the medium composition used in the method of activating CD4 T cells only T cells among other immunocytes, which are involved in acquired immunity, are selectively activated.
  • the T cells activated on a mass scale can effectively attack and kill cancer cells which are memorized previously.
  • the medium composition is limited when treating malignant tumors, in that various kinds of cancer cells which have never been memorized cannot be attacked.
  • the inventive concept provides a medium composition for culturing self- activated lymphocytes, to which anti-CD3, anti-CD16 and anti-CD56 antibodies are added along with interleukin2 (IL-2) to evenly activate natural killer (NK) cells, T cells and natural killer T (NKT) cells, and thus the medium composition can be used to culture immunocytes that can effectively treat various kinds of malignant tumors.
  • IL-2 interleukin2
  • a medium composition for culturing self-activated lymphocytes comprising a cell culture medium and additives added to the cell culture medium, wherein the additives comprise interleukin2 (IL-2), anti-CD3 antibodies, anti-CD16 antibodies, and anti-CD56 antibodies.
  • IL-2 interleukin2
  • anti-CD3 antibodies anti-CD3 antibodies
  • anti-CD16 antibodies anti-CD56 antibodies
  • NK cells that can effectively kill most cancer cells, which do not express a major histocompatability complex (MHC), can be activated on a mass scale.
  • the medium composition can be applied to adaptive immunotherapy, which causes fewer side effects and uses a convenient administration method, to maximize the prognosis of patients suffering from various kinds of malignant tumors.
  • FIG. 1 is a graph of immunocyte count with respect to experimental group cultured in a medium composition according to the present invention.
  • FIG. 2 is graphs of phenotypic variations in self- activated lymphocytes acquired using the medium composition according to the present invention.
  • FIG. 3 is a graph of cytotoxicity of the self-activated lymphocytes cultured using the medium composition according to the present invention.
  • FIG. 4 is a comparative graph of cancer cell killing abilities of self-activated lymphocytes cultured using the medium composition according to the present invention and the self-activated lymphocytes cultured using a conventional T-cell culture medium composition.
  • the inventive concept provides a medium composition to which anti-CD3, anti-
  • CD16 and anti-CD56 antibodies are added along with interleukin2(IL-2), and which is used to culture lymphocytes extracted from human peripheral blood to produce self- activated lymphocytes in a specific composition of immunocytes that is effective in the treatment of malignant tumors.
  • IL-2 interleukin2
  • NK cells are large granular lymphocytes (LGL), a typical kind of lymphocytes, and their anti-tumor function occurs via the mechanism of either necrosis or apoptosis, or both of them.
  • LGL large granular lymphocytes
  • NK cells react with cytokines such as IL-2, IL- 12, Interferon, and the like, and thus have improved cytolytic, secretory and proliferative functions.
  • Phenotypes of NK cells in humans are CD 16 (FcrRIII) and CD56, which have no T- cell receptor complex (TRC) on the cell surface and thus are used as NK cell markers.
  • NK cells express CD 16, known as a Fc receptor for immunoglobulin (IgG), and can perform other types of non-MHC -restricted killing due to the receptor CD 16. That is, the ADCC of NK cells depends on the presence of an antibody recognizing a target cell. The Fc region of the antibody is exposed as the antibody binds to an antigen. When the exposed Fc region forms a bridge by binding to the receptor of the NK cells, cell killer substances are released from the NK cells as a result of signal transduction activated due to the binding to the receptor, to attack the target cell.
  • ADCC antibody-dependent cell- mediated cytotoxicity
  • CD 16 antigen is added to the NK cells, which induces signal transduction. Due to the stimulation by signals, transferrin receptors, such as ⁇ chain of IL-2 receptor, may be expressed in the NK cells, or tumor necrosis factor (tnf) or interferon- ⁇ (IFN- ⁇ ) may be produced.
  • transferrin receptors such as ⁇ chain of IL-2 receptor, may be expressed in the NK cells, or tumor necrosis factor (tnf) or interferon- ⁇ (IFN- ⁇ ) may be produced.
  • CD56 antigen (Leul9/NKH1) is a 180-200 kDa glycoprotein, and is expressed in all the NK cells and some T cells.
  • CD56 antigen has the same molecular formula as neural cell adhesion molecule- 1 (NCAM-I) which is widely distributed in the human nervous tissues. NCAM-I is known as molecules involved in intercellular adhesion in nervous or muscular tissues. Thus, CD56 expressed in the NK cells may be determined as functioning in intercellular adhesion.
  • NCAM-I neural cell adhesion molecule- 1
  • T cells refer to cells having a T cell antigen receptor (TCR) on the cell surface.
  • TCR forms a heterodimer with CD3 antigen, dimer of ⁇ chain and ⁇ chain.
  • Some T cells (about 5% of T cells in peripheral blood) consist of ⁇ chain and ⁇ chain dimers, not ⁇ chain and ⁇ chain dimers.
  • TCR forms a complex with CD3 antigen ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ or ⁇ ), and the CD3 antigen transfers signals into the cell when the TCR recognizes the antigen.
  • helper T cells facilitating immunological reaction to cancer cells activate killer T cells, B cells, macrophages, and the like by releasing various kinds of cytokines.
  • cytokines include IN-I, IN-2, IN-3, TNF- ⁇ , interferon- ⁇ , which are intercellular information transfer substances, and the like.
  • IL-2 is added to a culture fluid to activate immunocytes such as killer T cells, B cells, macrophages, and the like.
  • IL-2 is a 14-17 kDa glycoprotein generated as T cells are activated upon recognition of an antigen. IL-2 is secreted outside a T cell and reacts to the T cell itself from which IL-2 has secreted, to facilitate growth of the T cell. IL-2 also facilitates growth of NK cells by reacting on the NK cells, enhances the killing ability of NK cells, and facilitates growth of B cells by reacting on the B cells.
  • NKT cells are a kind of T cells and contribute to innate immunity. Functions of the
  • NKT cells have been found recently. As can be inferred from the name, NKT cells express T cell receptors and NK cell-specific surface markers. A distinctive feature of NKT cells is that NKT cells secrete various cytokines such as IL-4, IL-IO, IL- 13, IFN- ⁇ , TNF- ⁇ , and the like within a very short time after being activated. This feature suggests that the NKT cells may significantly affect adaptive immune reaction.
  • CD56 monoclonal antibodies are used as additives to the medium composition in culturing the lymphocytes extracted from human peripheral blood, to evenly activate NK cells, T cells and NKT cells.
  • IL-2 contributes to the proliferation of the T cells and the NK cells, and the monoclonal antibodies function as antigens respectively expressing CD3, CD16 and CD56 in the immunocytes.
  • lymphocytes to be cultured may be harvested from peripheral blood of a target patient to be treated, using the following method.
  • the blood of the patient is overlaid onto a Ficoll-Paque Plus solution having a specific gravity of 1.077 based on the feature of human lymphocytes or monocytes having a specific gravity less than 1.077 to sediment granules by centrifugal force.
  • a monocyte layer including lymphocytes is recovered based on a difference in specific gravity, and only the lymphocytes are extracted from the monocyte layer.
  • a medium composition (hereinafter, "first medium composition”) for culturing the self-activated lymphocytes comprises a cell culture medium and various additives added to the cell culture medium.
  • the medium composition includes, based on 39 m# of the cell culture medium, 20-500 j ⁇ of 190-21OmM L-glutamine and 2-5 m ⁇ , of autochthonous plasma as nutrients for the lymphocytes, 20 - 300 [d of 17x106-19x106 IUM of IL-2 for activating the lymphocytes, 20 - 30 j ⁇ of anti-CD3 antibodies in a concentration of 0.9-1.1 mg/m#, 20 - 30 ⁇ A of anti-CD16 antibodies in a concentration of 0.9-1.1 mg/m#, and 20 - 30 j ⁇ of anti-CD56 antibodies in a concentration of 0.9-1.1 mg/m#, wherein the anti-CD3, anti-CD 16 and anti-CD56 antibodies are for controlling a relative composition of the different lymphocytes (NK cells,
  • subculture may be used to raise culturing efficiency.
  • the lymphocytes are cultured using the medium composition as described above for 3-4 days, and the resulting medium composition is admixed to a medium composition (hereinafter, "second medium composition") described below and further cultured for 2-3 days.
  • second medium composition a medium composition
  • the second medium composition for subculturing includes, based on 67 m ⁇ , of the cell culture medium, 20-500 j ⁇ of 190-21OmM L-glutamine and 2-5 v ⁇ i of autochthonous plasma as nutrients for the lymphocytes, 20 ⁇ 300 ⁇ of 17x106-19x106 IUM of IL-2 for activating the lymphocytes, 20 ⁇ 30 ⁇ A of anti-CD3 antibodies in a concentration of 0.9-1.1 mg/m#, 20 - 30 j ⁇ of anti-CD 16 antibodies in a concentration of 0.9- 1.1 mg/m#, and 20 - 30 j ⁇ of anti-CD56 antibodies in a concentration of 0.9-1.1 mg/m#, wherein the anti-CD3, anti- CD 16 and anti-CD56 antibodies are for controlling a relative composition of the different lymphocytes (NK cells, NKT cells and T cells) in the final culture medium.
  • the cell culture media used in the first and second medium compositions may be any culture medium containing 800-1200 International Unit (IU)/m-6 of IL-2 in addition to nutrients essential for the growth and survival of cells, for example, amino acids, vitamins, organic and inorganic compounds, proteins, and the like.
  • the cell culture media may be NKB6040 (NKBIO, KOREA).
  • Example 1 Proliferative culturing of lymphocytes
  • FIG. 1 is a graph of immunocyte count with respect to experimental group cultured in a medium composition according to the present invention.
  • Lymphocytes extracted from 60 cc of peripheral blood of a healthy person (normal), a solid tumor patient and a leukemia patient were used as experimental groups.
  • the lymphocytes of each experimental group was cultured in 39 m ⁇ , of a first medium composition for 4 days.
  • 67 m# of the resulting first medium composition containing the cultured lymphocytes was admixed to a second medium composition and further cultured for 2 days.
  • 7 m ⁇ , of autochthonous was added to the second medium composition containing the cultured lymphocytes and admixed to a gas permeable bag containing 10-20 IU/m# IL- 2 in 1 I of a cell culture medium and then cultured for 8 days.
  • all the culturing steps were performed at 37 0 C in a CO2 incubator.
  • the total lymphocyte count i.e., the total number of the lymphocytes, including NK cells, T cells and NKT cells, increased from 2.0x106- 4.0x107 before culturing to 2.0x109 or greater after culturing.
  • FIG. 2 shows the graphs of phenotypic variations in self-activated lymphocytes cultured in a medium composition according to the present invention, in which Hl, H4, H2 and H3 regions plot distribution of NK cells, T cells, NKT cells and other im- munocytes, respectively.
  • Lymphocytes were harvested from 60 cc of peripheral blood of a normal person and cultured according to the culturing processes of Example 1. A surface antigen analysis was performed on the self- activated lymphocytes cultivated according to Example 1 using floweytometry. The results will be analyzed with reference to FIG. 2. Referring to (a) of FIG.
  • Example 3 Analysis of cytotoxicity to various cancer cells
  • Lymphocytes were harvested from 60 cc of peripheral blood of a lymphosarcoma patient and cultured through the culturing processes of Example 1 as described above to obtain self-activated lymphocytes.
  • the cancer cells as a target cell and the cultured self-activated lymphocytes as an effector cell were mixed in different ratios, reacted 6 hours later with lactate dehydronase (LDH) and subjected to cytotoxicity analysis by enzyme-linked immunosorbent assay (ELISA). As shown in FIG.
  • PBMC peripheral blood monocytes
  • Comparative Example 1 Comparison of cancer cell killing ability with T-cell culture medium composition
  • the T-cell culture medium composition used for the comparison was prepared by conventional CAT (anti-CD3 ANtibody- activity T cells), widely used in Japan, and anti-CD antibodies, IL-2, and the like were added thereto.
  • CAT anti-CD3 ANtibody- activity T cells
  • MCF-7 breast carcinoma cell line
  • MDA-MB-231 breast carcinoma cell line
  • SK-HEP-I hepatoma cell line
  • K562 leukemia cell line
  • the cytotoxicities of the self-activated lymphocytes cultured in the medium composition according to the inventive concept were 6-64% greater than the cytotocixities of the self- While the inventive concept has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood that various changes in form and details may be made therein without departing from the spirit and scope of the following claims.
  • the medium composition for culturing self-activated lymphocytes according to the present invention enables NK cells, that can effectively skill most cancer cells which do not express a major histocompatability complex (MHC), to be activated on a mass scale. Thus, it can be applied to adaptive immunotherapy to effectively eliminate cancer cells.
  • MHC major histocompatability complex

Abstract

Provided is a medium composition for culturing self- activated lymphocytes applicable to the treatment of malignant tumors, to which anti-CD3, anti-CD16 and anti-CD56 antibodies are added along with interleukin2 (IL-2) to evenly activate natural killer (NK) cells, T cells and natural killer T (NKT) cells, and thus the medium composition can be used to culture im- munocytes that can effectively treat various kinds of malignant tumors. The medium composition includes a cell culture medium and additives added to the cell culture medium, wherein the additives include interleukin2 (IL-2), anti-CD3 antibodies, anti-CD16 antibodies, and anti-CD56 antibodies.

Description

Description
A MEDIUM COMPOSITION FOR CULTIVATING SELF ACTIVATED LYMPHOCYTE
Technical Field
[1] The inventive concept relates to a medium composition for culturing self- activated lymphocytes applicable to the treatment of malignant tumors, and more particularly, to a medium composition to which anti-CD3, anti-CD16 and anti-CD56 antibodies are added along with interleukin2 (IL-2) to evenly activate natural killer (NK) cells, T cells and natural killer T (NKT) cells, and thus the medium composition can be used to culture immunocytes that can effectively treat various kinds of malignant tumors.
[2]
Background Art
[3] Recently, adaptive immunotherapy has drawn attention as a new, alternative cancer treatment method to surgery, radiation therapy or chemotherapy conventionally used in the treatment of cancers.
[4] Adaptive immunotherapy is a method involving extracting natural killer (NK) cells, dedritic (De) cells, B cells, T cells, and the like, which are the most crucial immunocytes for the treatment of cancers, from blood of a patient, growing the extracted immunocytes to be able to withstand cancer cells by using different kinds of stimulants, and then injecting them back into the patient. Since the blood of the patient is used, the adaptive immunotherapy leads to less side effects and can be applied along with a more convenient administration method than conventional chemotherapies and the like. For these reasons, adaptive immunotherapy is currently being vigorously researched.
[5] Specifically, NK cells among immunocytes activated in adaptive immunotherapy are a kind of lymphocytes that have an excellent ability to kill infected viruses and tumor cells, but not to kill most normal cells. Such NK cells are known to play an important role in an initial stage of the body defensive mechanism and in the human tumor immunity. That is, NK cells can kill specific cancer cells, homologous cells and even heterologous cancer cells without a sensitizing process, i.e., without an acquisition of immunity to the expression of MHC. In particular, NK cells can more effectively kill target cells which do not or less express Class 1 MHC. Thus, NK cells can effectively kill most cancer cells that do not express MHC, cells infected with several viruses, and bacteria such as Salmonella typhimurium (Salmonella typhi.).
[6] However, NK cells, which have a superior ability to kill cancer cells, constitute only
5-15% of the amount of peripheral blood lymphocytes in healthy (normal) humans, and their proportion is reduced to 1% or less in cancer patients. Thus, NK cells cannot effectively attack cancer cells without an additional amplification process using adaptive immunotherapy.
[7] Thus, to apply adaptive immunotherapy in the cancer treatment, it is essential to conduct research into a medium for culturing and activating immunocytes, including NK cells, on a mass scale according to the stages of the patient's cancer. However, conventional immunocyte culture media used in adaptive immunotherapy are focused just on amplifying mainly T cells of other immunocytes on a mass scale.
[8] A conventional immunocyte culture medium is disclosed in Korean Patent No.
0735081, entitled "A method for activating CD4 T cells". The method of activating CD4 T cells involves separating CD4 T cells from a biological sample such as blood and culturing the separated CD4 T cells in vitro with a cytokine- added culture medium containing GM-CSF, IFN-gamma, TNF-alpha, lectin and IL-4 to activate the CD4 T cells. As a result, a composition for preventing or treating bacterial infectious diseases is acquired.
[9] However, in the medium composition used in the method of activating CD4 T cells, only T cells among other immunocytes, which are involved in acquired immunity, are selectively activated. Thus, when the medium composition is applied in oncotherapy, the T cells activated on a mass scale can effectively attack and kill cancer cells which are memorized previously. However, the medium composition is limited when treating malignant tumors, in that various kinds of cancer cells which have never been memorized cannot be attacked.
[10]
Disclosure of Invention
Technical Problem
[11] The inventive concept provides a medium composition for culturing self- activated lymphocytes, to which anti-CD3, anti-CD16 and anti-CD56 antibodies are added along with interleukin2 (IL-2) to evenly activate natural killer (NK) cells, T cells and natural killer T (NKT) cells, and thus the medium composition can be used to culture immunocytes that can effectively treat various kinds of malignant tumors.
[12]
Technical Solution
[13] According to an aspect of the inventive concept, there is provided a medium composition for culturing self-activated lymphocytes, the medium composition comprising a cell culture medium and additives added to the cell culture medium, wherein the additives comprise interleukin2 (IL-2), anti-CD3 antibodies, anti-CD16 antibodies, and anti-CD56 antibodies. [14]
Advantageous Effects
[15] Using the medium composition for culturing self- activated lymphocytes according to the inventive concept, NK cells that can effectively kill most cancer cells, which do not express a major histocompatability complex (MHC), can be activated on a mass scale. The medium composition can be applied to adaptive immunotherapy, which causes fewer side effects and uses a convenient administration method, to maximize the prognosis of patients suffering from various kinds of malignant tumors.
[16]
Brief Description of Drawings
[17] FIG. 1 is a graph of immunocyte count with respect to experimental group cultured in a medium composition according to the present invention.
[18] FIG. 2 is graphs of phenotypic variations in self- activated lymphocytes acquired using the medium composition according to the present invention.
[19] FIG. 3 is a graph of cytotoxicity of the self-activated lymphocytes cultured using the medium composition according to the present invention.
[20] FIG. 4 is a comparative graph of cancer cell killing abilities of self-activated lymphocytes cultured using the medium composition according to the present invention and the self-activated lymphocytes cultured using a conventional T-cell culture medium composition.
[21]
Best Mode for Carrying out the Invention
[22] The inventive concept provides a medium composition to which anti-CD3, anti-
CD16 and anti-CD56 antibodies are added along with interleukin2(IL-2), and which is used to culture lymphocytes extracted from human peripheral blood to produce self- activated lymphocytes in a specific composition of immunocytes that is effective in the treatment of malignant tumors. Prior to a detailed description of the inventive concept, the mechanism of each additive used for the amplification and activation of the immunocytes will be described along with features of each of the immunocytes.
[23] NK cells are large granular lymphocytes (LGL), a typical kind of lymphocytes, and their anti-tumor function occurs via the mechanism of either necrosis or apoptosis, or both of them. NK cells react with cytokines such as IL-2, IL- 12, Interferon, and the like, and thus have improved cytolytic, secretory and proliferative functions. Phenotypes of NK cells in humans are CD 16 (FcrRIII) and CD56, which have no T- cell receptor complex (TRC) on the cell surface and thus are used as NK cell markers.
[24] In general, the mechanism of NK cells acting on cancer cells is antibody-dependent cell- mediated cytotoxicity (ADCC). NK cells express CD 16, known as a Fc receptor for immunoglobulin (IgG), and can perform other types of non-MHC -restricted killing due to the receptor CD 16. That is, the ADCC of NK cells depends on the presence of an antibody recognizing a target cell. The Fc region of the antibody is exposed as the antibody binds to an antigen. When the exposed Fc region forms a bridge by binding to the receptor of the NK cells, cell killer substances are released from the NK cells as a result of signal transduction activated due to the binding to the receptor, to attack the target cell.
[25] When lymphocytes are cultured in the presence of the anti-CD 16 antibody or an antigen- antibody complex, CD 16 antigen is added to the NK cells, which induces signal transduction. Due to the stimulation by signals, transferrin receptors, such as α chain of IL-2 receptor, may be expressed in the NK cells, or tumor necrosis factor (tnf) or interferon-γ (IFN-γ) may be produced.
[26] CD56 antigen (Leul9/NKH1) is a 180-200 kDa glycoprotein, and is expressed in all the NK cells and some T cells. CD56 antigen has the same molecular formular as neural cell adhesion molecule- 1 (NCAM-I) which is widely distributed in the human nervous tissues. NCAM-I is known as molecules involved in intercellular adhesion in nervous or muscular tissues. Thus, CD56 expressed in the NK cells may be determined as functioning in intercellular adhesion.
[27] T cells refer to cells having a T cell antigen receptor (TCR) on the cell surface. TCR forms a heterodimer with CD3 antigen, dimer of α chain and β chain. Some T cells (about 5% of T cells in peripheral blood) consist of γ chain and δ chain dimers, not α chain and β chain dimers. TCR forms a complex with CD3 antigen (γ, δ, ε, ζ, ζ or η), and the CD3 antigen transfers signals into the cell when the TCR recognizes the antigen.
[28] In general, helper T cells facilitating immunological reaction to cancer cells activate killer T cells, B cells, macrophages, and the like by releasing various kinds of cytokines. These kinds of cytokines include IN-I, IN-2, IN-3, TNF-α, interferon-γ, which are intercellular information transfer substances, and the like. In the inventive concept, IL-2 is added to a culture fluid to activate immunocytes such as killer T cells, B cells, macrophages, and the like.
[29] IL-2 is a 14-17 kDa glycoprotein generated as T cells are activated upon recognition of an antigen. IL-2 is secreted outside a T cell and reacts to the T cell itself from which IL-2 has secreted, to facilitate growth of the T cell. IL-2 also facilitates growth of NK cells by reacting on the NK cells, enhances the killing ability of NK cells, and facilitates growth of B cells by reacting on the B cells.
[30] NKT cells are a kind of T cells and contribute to innate immunity. Functions of the
NKT cells have been found recently. As can be inferred from the name, NKT cells express T cell receptors and NK cell-specific surface markers. A distinctive feature of NKT cells is that NKT cells secrete various cytokines such as IL-4, IL-IO, IL- 13, IFN- γ, TNF-α, and the like within a very short time after being activated. This feature suggests that the NKT cells may significantly affect adaptive immune reaction.
[31] Thus, according to the inventive concept, IL-2 and anti-CD3, anti-CD 16 and anti-
CD56 monoclonal antibodies are used as additives to the medium composition in culturing the lymphocytes extracted from human peripheral blood, to evenly activate NK cells, T cells and NKT cells. IL-2 contributes to the proliferation of the T cells and the NK cells, and the monoclonal antibodies function as antigens respectively expressing CD3, CD16 and CD56 in the immunocytes.
[32] Hereinafter, a medium composition for culturing self-activated lymphocytes according to an embodiment of the inventive concept will be described in detail.
[33] Initially, lymphocytes to be cultured may be harvested from peripheral blood of a target patient to be treated, using the following method. To do this, the blood of the patient is overlaid onto a Ficoll-Paque Plus solution having a specific gravity of 1.077 based on the feature of human lymphocytes or monocytes having a specific gravity less than 1.077 to sediment granules by centrifugal force. As a result, a monocyte layer including lymphocytes is recovered based on a difference in specific gravity, and only the lymphocytes are extracted from the monocyte layer.
[34] A medium composition (hereinafter, "first medium composition") for culturing the self-activated lymphocytes according to an embodiment of the inventive concept comprises a cell culture medium and various additives added to the cell culture medium. The medium composition includes, based on 39 m# of the cell culture medium, 20-500 jΛ of 190-21OmM L-glutamine and 2-5 mϋ, of autochthonous plasma as nutrients for the lymphocytes, 20 - 300 [d of 17x106-19x106 IUM of IL-2 for activating the lymphocytes, 20 - 30 jΛ of anti-CD3 antibodies in a concentration of 0.9-1.1 mg/m#, 20 - 30 μA of anti-CD16 antibodies in a concentration of 0.9-1.1 mg/m#, and 20 - 30 jΛ of anti-CD56 antibodies in a concentration of 0.9-1.1 mg/m#, wherein the anti-CD3, anti-CD 16 and anti-CD56 antibodies are for controlling a relative composition of the different lymphocytes (NK cells, NKT cells and T cells) in the final culture medium.
[35] In culturing the lymphocytes using the medium composition, subculture may be used to raise culturing efficiency. Initially, the lymphocytes are cultured using the medium composition as described above for 3-4 days, and the resulting medium composition is admixed to a medium composition (hereinafter, "second medium composition") described below and further cultured for 2-3 days.
[36] The second medium composition for subculturing (hereinafter, "first medium composition") includes, based on 67 mϋ, of the cell culture medium, 20-500 jΛ of 190-21OmM L-glutamine and 2-5 vλi of autochthonous plasma as nutrients for the lymphocytes, 20 ~ 300 ≠ of 17x106-19x106 IUM of IL-2 for activating the lymphocytes, 20 ~ 30 μA of anti-CD3 antibodies in a concentration of 0.9-1.1 mg/m#, 20 - 30 jΛ of anti-CD 16 antibodies in a concentration of 0.9- 1.1 mg/m#, and 20 - 30 jΛ of anti-CD56 antibodies in a concentration of 0.9-1.1 mg/m#, wherein the anti-CD3, anti- CD 16 and anti-CD56 antibodies are for controlling a relative composition of the different lymphocytes (NK cells, NKT cells and T cells) in the final culture medium.
[37] The cell culture media used in the first and second medium compositions may be any culture medium containing 800-1200 International Unit (IU)/m-6 of IL-2 in addition to nutrients essential for the growth and survival of cells, for example, amino acids, vitamins, organic and inorganic compounds, proteins, and the like. For example, the cell culture media may be NKB6040 (NKBIO, KOREA).
[38]
Mode for the Invention
[39] The inventive concept will now be described in greater detail with reference to the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the inventive concept.
[40] Example 1 : Proliferative culturing of lymphocytes
[41] FIG. 1 is a graph of immunocyte count with respect to experimental group cultured in a medium composition according to the present invention.
[42] Lymphocytes extracted from 60 cc of peripheral blood of a healthy person (normal), a solid tumor patient and a leukemia patient were used as experimental groups. The lymphocytes of each experimental group was cultured in 39 mϋ, of a first medium composition for 4 days. 67 m# of the resulting first medium composition containing the cultured lymphocytes was admixed to a second medium composition and further cultured for 2 days. In addition, to induce massive proliferation of the lymphocytes, 7 mϋ, of autochthonous was added to the second medium composition containing the cultured lymphocytes and admixed to a gas permeable bag containing 10-20 IU/m# IL- 2 in 1 I of a cell culture medium and then cultured for 8 days. Herein, all the culturing steps were performed at 370C in a CO2 incubator.
[43] As can be confirmed from FIG. 1, the total lymphocyte count, i.e., the total number of the lymphocytes, including NK cells, T cells and NKT cells, increased from 2.0x106- 4.0x107 before culturing to 2.0x109 or greater after culturing.
[44] Example 2: Observation on phenotvpe before and after culturing
[45] FIG. 2 shows the graphs of phenotypic variations in self-activated lymphocytes cultured in a medium composition according to the present invention, in which Hl, H4, H2 and H3 regions plot distribution of NK cells, T cells, NKT cells and other im- munocytes, respectively. [46] Lymphocytes were harvested from 60 cc of peripheral blood of a normal person and cultured according to the culturing processes of Example 1. A surface antigen analysis was performed on the self- activated lymphocytes cultivated according to Example 1 using floweytometry. The results will be analyzed with reference to FIG. 2. Referring to (a) of FIG. 2, surface antigens are distributed most densely in the H4 region before culturing, whereas the distribution of the surface antigens after culturing is most dense in the Hl region. A proportion of the T cells positive to CD3 and a proportion of the NK cells positive to CD 16 and CD56 were calculated. The proportions were calculated to be 53.60% and 12.74% respectively before culturing and 39.74% and 69.03% respectively after culturing.
[47] The results confirm that the proportion of NK cells markedly increased in the lymphocytes cultured using the medium composition according to the embodiment of the inventive concept. In addition, in order to evaluate an effect of variation in the composition of the final culture product on the ability to kill various cancer cells, cytotoxicity analysis was performed as follows.
[48] Example 3: Analysis of cytotoxicity to various cancer cells
[49] Lymphocytes were harvested from 60 cc of peripheral blood of a lymphosarcoma patient and cultured through the culturing processes of Example 1 as described above to obtain self-activated lymphocytes. The cancer cells as a target cell and the cultured self-activated lymphocytes as an effector cell were mixed in different ratios, reacted 6 hours later with lactate dehydronase (LDH) and subjected to cytotoxicity analysis by enzyme-linked immunosorbent assay (ELISA). As shown in FIG. 3, specifically for a leukemia cell line (K562), a human lymphoma cell line (U937) and a human cervical epidermoid cell line (ME 180), the abilities of the self-activated lymphocytes to kill cancer cells markedly increased as compared to peripheral blood monocytes (PBMC).
[50] Comparative Example 1 : Comparison of cancer cell killing ability with T-cell culture medium composition
[51] Cytotoxicities of the self- activated lymphocytes (NKM) containing NK/NKT cells and T cells in a proportion of 6:4, which were cultured through the culturing processes of Example 1 from the lymphocytes harvested from 66 cc of peripheral blood of a lymphoma patient, were analyzed on different target cells using a calcein-Am dying method and were compared with cytotoxicities of self- activated lymphocytes (CAT) cultured using a conventional T-cell culture medium composition to include 90% or greater of T cells. Herein, the T-cell culture medium composition used for the comparison was prepared by conventional CAT (anti-CD3 ANtibody- activity T cells), widely used in Japan, and anti-CD antibodies, IL-2, and the like were added thereto. In addition, a human gastric carcinoma cell line (AGS, NCI-N87), a breast carcinoma cell line (MCF-7, MDA-MB-231), a hepatoma cell line (SK-HEP-I, HepG2), and a leukemia cell line (K562) were used as the target cells.
[52] As shown in FIG. 4, for most of the cancer cell lines, except for the human gastric carcinoma cell line (AGS), the cytotoxicities of the self-activated lymphocytes cultured in the medium composition according to the inventive concept were 6-64% greater than the cytotocixities of the self- While the inventive concept has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood that various changes in form and details may be made therein without departing from the spirit and scope of the following claims.
[53]
Industrial Applicability
[54] The medium composition for culturing self-activated lymphocytes according to the present invention enables NK cells, that can effectively skill most cancer cells which do not express a major histocompatability complex (MHC), to be activated on a mass scale. Thus, it can be applied to adaptive immunotherapy to effectively eliminate cancer cells.
[55]

Claims

Claims
[1] A medium composition for culturing self-activated lymphocytes, comprising a cell culture medium and additives added to the cell culture medium, wherein the additives comprise interleukin2 (IL-2), anti-CD3 antibodies, anti-CD 16 antibodies, and anti-CD56 antibodies.
[2] The medium composition of claim 1, wherein the cell culture medium contain
800 to 1200 International Unit (IU)M of IL-2.
[3] The medium composition of claim 1, wherein the additives added to the cell culture medium include 20 ~ 300 [d of 17x106-19x106 HM of IL-2, 20 ~ 30 /d of anti-CD3 antibodies in a concentration of 0.9-1.1 mg/m#, 20 ~ 30 μA of anti-CD 16 antibodies in a concentration of 0.9-1.1 mg/m#, and 20 - 30 id of anti- CD56 antibodies in a concentration of 0.9-1.1 mg/m#, based on 39 mi of the cell culture medium.
[4] The medium composition of claim 3, wherein the additives further include
20-500 [d of 190-21OmM L-glutamine and 2-5 mk of autochthonous plasma, based on 39 m# of the cell culture medium.
[5] The medium composition of claim 1, wherein the additives added to the cell culture medium include 20 - 300 ≠ of 17x106 - 19x106 HM of IL-2, 20 - 30 id of anti-CD3 antibodies in a concentration of 0.9-1.1 mg/m#, 20 - 30 id of anti-CD 16 antibodies in a concentration of 0.9-1.1 mg/m#, and 20 - 30 id of anti- CD56 antibodies in a concentration of 0.9-1.1 mg/m#, based on 67 mϋ, of the cell culture medium.
[6] The medium composition of claim 5, wherein the additives further include
20-500 id of 190-21OmM L-glutamine and 2-5 m# of autochthonous plasma, based on 67 mϋ, of the cell culture medium.
PCT/KR2008/005658 2008-06-10 2008-09-24 A medium composition for cultivating self activated lymphocyte WO2009151183A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020080053930A KR20090127974A (en) 2008-06-10 2008-06-10 A medium composition for cultivating self activated lymphocyte
KR10-2008-0053930 2008-06-10

Publications (1)

Publication Number Publication Date
WO2009151183A1 true WO2009151183A1 (en) 2009-12-17

Family

ID=41416872

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2008/005658 WO2009151183A1 (en) 2008-06-10 2008-09-24 A medium composition for cultivating self activated lymphocyte

Country Status (3)

Country Link
KR (1) KR20090127974A (en)
CN (1) CN101603028A (en)
WO (1) WO2009151183A1 (en)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010110734A1 (en) * 2009-03-26 2010-09-30 Avaris Ab Expansion of nk cells
EP2535052A1 (en) * 2010-02-08 2012-12-19 Biotherapy Institute Of Japan Method for producing nk cell enhancement-type blood product
US20130142766A1 (en) * 2010-08-10 2013-06-06 Katsuyuki Dodo Production method for cell populations
US20130157364A1 (en) * 2010-08-30 2013-06-20 Celltech Co., Ltd. Medium composition for culturing self-activated lymphocytes and method for culturing self-activated lymphocytes using same
WO2013094988A1 (en) 2011-12-22 2013-06-27 Mogam Biotechnology Research Institute Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
CN107083363A (en) * 2017-06-29 2017-08-22 青岛麦迪赛斯医疗技术有限公司 A kind of external Efficient amplification method of peripheral blood NK cell
CN111548994A (en) * 2020-04-24 2020-08-18 广东华夏健康生命科学有限公司 Cell culture medium and method for culturing NK cells by using same
WO2021085504A1 (en) 2019-11-01 2021-05-06 京都府公立大学法人 B-cell antibody receptor and use thereof
CN113736733A (en) * 2020-05-29 2021-12-03 基亚细胞科技有限公司 Culture medium for in vitro activation of immune cells
US11766456B2 (en) 2014-11-26 2023-09-26 GC Cell Corporation Method for culturing natural killer cells using T cells

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105462923B (en) * 2014-12-17 2018-11-02 山东大学第二医院 A kind of external Efficient amplification method of people's natural killer cells
KR101683614B1 (en) * 2016-02-15 2016-12-07 신동혁 Culture Media Kits for NK cell Cultivation and NK cell Culture Method using the Same
KR101969045B1 (en) * 2016-11-22 2019-04-19 신동혁 Culture media kits for NK cell Cultivation, NK cell culture method using the same, NK Cell media solution without blood serum, and cosmetic composition comprising the serum free conditioned media
CN106754704B (en) * 2016-12-28 2020-10-16 华南生物医药研究院 Method for inducing and expanding immune cells in vitro
CN108251365B (en) * 2016-12-28 2020-10-16 华南生物医药研究院 Immune cell culture medium system
CN107488630B (en) * 2017-10-09 2018-05-29 天津长和生物技术有限公司 The amplification cultivation method of natural killer T cells culture substrate and natural killer T cells
WO2019168222A1 (en) * 2018-02-28 2019-09-06 주식회사 제넨셀 Culture medium composition for culturing of anti-cancer activated lymphocytes derived from peripheral blood mononuclear cells and method for culturing anti-cancer activated lymphocytes using same
KR102137954B1 (en) * 2019-05-16 2020-07-27 (주)녹십자셀 Activated lymphocyte including cytokine induced killer cell and preparing method thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5443983A (en) * 1986-08-08 1995-08-22 Regents Of The University Of Minnesota Method of culturing lymphocytes and method of treatment using such lymphocytes
US6316257B1 (en) * 1996-03-04 2001-11-13 Targeted Genetics Corporation Modified rapid expansion methods (“modified-REM”) for in vitro propagation of T lymphocytes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5443983A (en) * 1986-08-08 1995-08-22 Regents Of The University Of Minnesota Method of culturing lymphocytes and method of treatment using such lymphocytes
US6316257B1 (en) * 1996-03-04 2001-11-13 Targeted Genetics Corporation Modified rapid expansion methods (“modified-REM”) for in vitro propagation of T lymphocytes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MALYGIN, A.M. ET AL.: "Promotion of natural killer cell growth in vitro by bispecific (anti-CD3 x anti-CD16) antibody.", IMMUNOLOGY, vol. 81, 1994, pages 92 - 95, XP009030030 *
MORETTA, A. ET AL.: "CD69-mediated Pathway of Lymphocyte Activation: Anti-CD69 Monoclonal Antibodies Trigger the Cytolytic Activity of Different Lymphoid Effector Cells with the Exception of Cytolytic T Lymphocytes Expressing T Cell Receptor alpha/beta.", J. EXP. MED., vol. 174, 1991, pages 1393 - 1398, XP055219293, DOI: doi:10.1084/jem.174.6.1393 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8877182B2 (en) 2009-03-26 2014-11-04 Cellprotect Nordic Pharmaceuticals Ab Expansion of NK cells
WO2010110734A1 (en) * 2009-03-26 2010-09-30 Avaris Ab Expansion of nk cells
EP2535052A1 (en) * 2010-02-08 2012-12-19 Biotherapy Institute Of Japan Method for producing nk cell enhancement-type blood product
EP2535052A4 (en) * 2010-02-08 2014-01-22 Biotherapy Inst Of Japan Method for producing nk cell enhancement-type blood product
US20130142766A1 (en) * 2010-08-10 2013-06-06 Katsuyuki Dodo Production method for cell populations
US9670459B2 (en) * 2010-08-10 2017-06-06 Takara Bio Inc. Production method for cell populations
US20130157364A1 (en) * 2010-08-30 2013-06-20 Celltech Co., Ltd. Medium composition for culturing self-activated lymphocytes and method for culturing self-activated lymphocytes using same
EP2794859A4 (en) * 2011-12-22 2015-05-27 Mogam Biotechnology Inst Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
EP2794859A1 (en) * 2011-12-22 2014-10-29 Mogam Biotechnology Research Institute Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
WO2013094988A1 (en) 2011-12-22 2013-06-27 Mogam Biotechnology Research Institute Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same
US11766456B2 (en) 2014-11-26 2023-09-26 GC Cell Corporation Method for culturing natural killer cells using T cells
CN107083363A (en) * 2017-06-29 2017-08-22 青岛麦迪赛斯医疗技术有限公司 A kind of external Efficient amplification method of peripheral blood NK cell
CN107083363B (en) * 2017-06-29 2020-07-24 青岛麦迪赛斯医疗技术有限公司 Peripheral blood NK cell in-vitro efficient amplification method
WO2021085504A1 (en) 2019-11-01 2021-05-06 京都府公立大学法人 B-cell antibody receptor and use thereof
CN111548994A (en) * 2020-04-24 2020-08-18 广东华夏健康生命科学有限公司 Cell culture medium and method for culturing NK cells by using same
CN111548994B (en) * 2020-04-24 2021-05-25 广东华夏健康生命科学有限公司 Cell culture medium and method for culturing NK cells by using same
CN113736733A (en) * 2020-05-29 2021-12-03 基亚细胞科技有限公司 Culture medium for in vitro activation of immune cells
CN113736733B (en) * 2020-05-29 2023-04-25 基亚细胞科技有限公司 Culture medium for in vitro activation of immune cells

Also Published As

Publication number Publication date
CN101603028A (en) 2009-12-16
KR20090127974A (en) 2009-12-15

Similar Documents

Publication Publication Date Title
WO2009151183A1 (en) A medium composition for cultivating self activated lymphocyte
WO2009151182A1 (en) A method for cultivating self activated lymphocyte
KR101039843B1 (en) A medium composition for cultivating self activated lymphocyte and the cultivation method using the same
CN108699523B (en) NK cell culture medium addition kit and NK cell culture method using same
CN102597223B (en) Process for production of natural killer cells
Smith et al. Signals through 4-1BB inhibit T regulatory cells by blocking IL-9 production enhancing antitumor responses
EP3247791B1 (en) Universal killer t-cell
CN109385403B (en) GPC 3-targeting CAR NK cells
Sonabend et al. The role of regulatory T cells in malignant glioma
CN109721659B (en) Novel Chimeric Antigen Receptor (CAR) targeting CD19 and application thereof
JP2017524031A (en) Gamma delta T cells and uses thereof
CN114761027A (en) Cbl inhibitors and compositions for immune cell expansion
CN108276497A (en) Target Chimeric antigen receptor T cell and its application of CD19
WO2011052638A1 (en) Process for production of cytokine-induced killer cells
CN105296431A (en) Tumor binding specific gamma delta TCR gene modified alpha beta T cell and cancer suppression application thereof
CN110404061A (en) Improved T cell treatment method
JP2022519436A (en) Mucosa-related invariant T (MATI) cells expressing chimeric antigen receptors
Shirjang et al. Promising immunotherapy: Highlighting cytokine‐induced killer cells
CN107557337A (en) A kind of immunocyte of safety-type Chimeric antigen receptor modifications of anti-ROR1 and its application
CN112426526B (en) Preparation method of NK (natural killer) cells and application of NK cells in treatment of cancers
EP2606125B1 (en) Cells expressing th1 characteristics and cytolytic properties
CN111683971A (en) Pharmaceutical recombinant receptor compositions and methods
CN108949759A (en) Strike the siRNA for subtracting human IL-15, CD19 CAR expression vector, CAR-T cell and construction method and application
Zhu et al. CD137 enhances cytotoxicity of CD3+ CD56+ cells and their capacities to induce CD4+ Th1 responses
WO2021141986A1 (en) Chimeric gmcsf-il18 receptor

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08812150

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08812150

Country of ref document: EP

Kind code of ref document: A1