WO2009151183A1

WO2009151183A1 - A medium composition for cultivating self activated lymphocyte

Info

Publication number: WO2009151183A1
Application number: PCT/KR2008/005658
Authority: WO
Inventors: Dong Ouk Lee; Sun Min Hong; Byoung In Choi
Original assignee: NKBIO CO Ltd
Current assignee: NKBIO CO Ltd
Priority date: 2008-06-10
Filing date: 2008-09-24
Publication date: 2009-12-17
Anticipated expiration: 2010-12-10
Also published as: KR20090127974A; CN101603028A

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

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

[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.