WO2009151182A1

WO2009151182A1 - A method for cultivating self activated lymphocyte

Info

Publication number: WO2009151182A1
Application number: PCT/KR2008/005657
Authority: WO
Inventors: Dong Ouk Lee; Sun Min Hong; Byoung In Choi
Original assignee: Nkbio Co., Ltd
Priority date: 2008-06-10
Filing date: 2008-09-24
Publication date: 2009-12-17
Also published as: CN101603029A; KR20090127973A

Abstract

Provided is a method of culturing self-activated lymphocytes applicable to the treatment of malignant tumors. The method raises the percentage's of natural killer (NK) cells of the lymphocytes and evenly activate the NK cells, T cells and natural killer T (NKT) cells, and thus can be used to effectively eliminate various kinds of cancer cells. The method of culturing self- activated lymphocytes involves: extracting lymphocytes from human peripheral blood; performing a first culturing step of culturing the extracted lymphocytes in a culture fluid to which IL-2, L-glutamine and autochthonous plasma are added, in the presence of anti-CD3, anti- CD 16, and anti-CD56 antibodies; and performing a second culturing step of culturing the mixed culture fluid resulting from the first culturing step in the presence of anti-CD3, anti-CD16, and anti-CD56 antibodies after being admixed with a culture fluid to which IL-2, L-glutamine and autochthonous plasma are added.

Description

A METHOD FOR CULTIVATING SELF ACTIVATED

LYMPHOCYTE

Technical Field

[1] The inventive concept relates to a method of culturing self- activated lymphocytes, which are applicable to the treatment of malignant tumors, and more particularly, to a method of culturing self-activated lymphocytes, in which lymphocytes are separated from human peripheral blood and then cultured in the presence of interleukin2 (IL-2), and anti-CD3, anti-CD16 and anti-CD56 antibodies to raise a percentage of natural killer (NK) cells of the lymphocytes and evenly activate the NK cells, T cells and natural killer T (NKT) cells, and thus can be used to effectively eliminate various kinds of cancer cells.

[2]

Background Art

[3] In general, malignant tumors occurring in the human body are treated by surgery, radiation therapy, or chemotherapy. However, some malignant tumors may exist in specific anatomical sites which are difficult to treat, and in this case it is essential to apply an adjuvant therapy potent to improve the prognosis of a patient.

[4] Adaptive immunotherapy using self- activated immunocytes, which is one of the adjuvant therapies, is a method involving extracting natural killer (NK) cells, dendritic (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 fewer 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] A method of activating immunocytes which is applicable to conventional adaptive immunotherapy 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.

[6] However, the method of activating CD4 T cells selectively activates, only T cells among other immunocytes, which are involved in acquired immunity. Thus, when the method 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 method is limited when treating malignant tumors, in that various kinds of cancer cells which have never been memorized cannot be attacked.

[7]

Disclosure of Invention Technical Problem

[8] The inventive concept provides a method of culturing self-activated lymphocytes, in which lymphocytes are separated from human peripheral blood and then cultured in the presence of interleukin2 (IL-2), and anti-CD3, anti-CD 16 and anti-CD56 antibodies to raise a proportion of natural killer (NK) cells of the lymphocytes and evenly activate the NK cells, T cells and natural killer T (NKT) cells, and thus various kinds of cancer cells can be effectively eliminated.

[9]

Technical Solution

[10] A method of culturing self-activated lymphocytes, the method comprising: extracting lymphocytes from human peripheral blood; performing a first culturing step of culturing the extracted lymphocytes in a culture fluid to which interleukin2 (IL-2), L- glutamine and autochthonous plasma are added, in the presence of anti-CD3, anti- CD 16, and anti-CD56 antibodies, thereby obtaining a mixed culture fluid; and performing a second culturing step of culturing the mixed culture fluid resulting from the first culturing step in the presence of anti-CD3, anti-CD16, and anti-CD56 antibodies after being admixed with a culture fluid to which IL-2, L-glutamine and autochthonous plasma are added.

[H]

Advantageous Effects

[12] In the method of 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), are activated on a mass scale. The method 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.

[13]

Brief Description of Drawings

[14] FIG. 1 is graphs of phenotypic variations in self-activated lymphocytes acquired using a method of culturing self- activated lymphocytes according to the present invention. [15] FIG. 2 is a graph of immunocyte count with respect to time during culturing processes in the method of culturing self-activated lymphocytes according to the present invention.

[16] FIG. 3 is a graph of cytotoxicity of the self- activated lymphocytes cultured using the method of culturing self-activated lymphocytes according to the present invention.

[17] FIG. 4 is photographic images showing an anti-cancer effect in a mouse model of the self-activated lymphocytes cultured using the method of culturing self- activated lymphocytes according to the present invention.

[18]

Best Mode for Carrying out the Invention

[19] The inventive concept provides a method of culturing self-activated lymphocytes, which are highly effective in the treatment of malignant tumors, and in which in culturing lymphocytes extracted from human peripheral blood, interleukin2 (IL-2), and anti-CD3, anti-CD16 and anti-CD56 antibodies are used as additives to evenly activate various immunocytes including natural killer (NK) cells, natural killer T (NKT) cells and T cells. 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.

[20] NK cells are large granular lymphocytes (LGL), a typical kind of lymphocytes, and have the ability to kill infected viruses and tumor cells, but not to kill most normal cells. NK cells react with cytokines such as IL-2, IL- 12, Interferon, and the like, and thus have improved cytolytic, secretory and proliferative functions. In addition, an anti-tumor function of the NK cells occurs via the mechanism of either necrosis or apoptosis, or both of them. 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.

[21] 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 are non- major histocompatibility complex (MHC) -restricted killer cells and 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 ty- phimurium (Salmonella typhi.).

[22] In general, the mechanism of NK cells acting on cancer cells is antibody-dependent cell-mediated cytotoxicity (ADCC), which will be described in detail below. NK cells express CD16, known as an Fc receptor for immunoglobulin (IgG), and can perform other types of non-MHC-restricted killing due to the receptor CD16. 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.

[23] 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 due to the insufficient count of individual NK cells.

[24] 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.

[25] 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) that 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.

[26] T cells refer to cells having a T cell antigen receptor (TCR) on the cell surface. TCR forms a heterodimer with CD3 antigen, which is a 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.

[27] 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.

[28] 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 fa- cilitates growth of B cells by reacting on the B cells.

[29] 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.

[30] Thus, according to the inventive concept, IL-2 and anti-CD3, anti-CD 16 and anti-

CD56 monoclonal antibodies are used 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 im- munocytes.

[31] Hereinafter, a method of culturing self- activated lymphocytes according to an embodiment of the inventive concept will be described in detail.

[32] The method of culturing self-activated lymphocytes according to an embodiment of the inventive concept includes a lymphocytes extraction step, a first culturing step, a second culturing step and a third culturing step.

[33] Lymphocytes extraction step

[34] Blood of a target patient to be treated 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. Erythrocytes and a granulocyte layer, which have a specific gravity greater than 1.077, are separated downward, and a monocyte layer and platelets, which have a specific gravity less than or equal to 1.077, are separated upward based on a difference in specific gravity. The monocyte layer including lymphocytes is recovered, and only the lymphocytes are extracted from the monocyte layer.

[35] First culturing step

[36] The lymphocytes harvested through the lymphocytes extraction step are cultured in a culture fluid to which IL-2, L-glutamine, and autochthonous plasma are added, in the presence of anti-CD3, anti-CD16 and anti-CD56 antibodies for 3-4 days. Herein, the lymphocytes may be first cultured in the culture fluid containing IL-2, L-glutamine, and autochthonous plasma in the presence of anti-CD3, and then cultured sequentially in the presence of anti-CD 16 and then anti-CD56. This process will be described in detail below.

[37] Initially, the harvested lymphocytes are suspended in 3 m# of the culture fluid and then admixed into a 75 cm² culture bath containing a solidified anti-CD3 antigen and a mixture of 50-100 μJl of a dilute solution containing 17x106-19x106 IU (International Unit)/m# of IL-2, 100-250 μJl of L-glutamine, 2-4 m# of autochthonous plasma and 25-33 mH of the culture fluid, and then cultured in a 37⁰C CO2 incubator for 12-18 hours. As above, since the lymphocytes are initially suspended in a small amount of the culture fluid and then admixed into the culture fluid to which IL-2, L-glutamine, and autochthonous plasma are added, a loss of the lymphocytes and losses of IL-2, L- glutamine, and autochthonous plasma added in small amounts can be prevented.

[38] Then, the mixed culture fluid in the culture bath containing the solidified anti-CD3 is moved into a 75 cm² culture bath containing a solidified anti-CD56 and cultured in the 37⁰C, CO2incubator for 12-18 hours. Then, the mixed culture fluid in the culture bath containing the solidified anti-CD56 is moved into a 75 cm² culture bath containing a solidified anti-CD 16 and cultured in the 37⁰C CO2incubator for 48-60 hours.

[39] Second culturing step

[40] The mixed culture fluid after the first culturing step is completed is admixed into a culture fluid to which IL-2, L-glutamine, and autochthonous plasma are added, and further cultured in the presence of anti-CD3, anti-CD16 and anti-CD56 antibodies for 2-3 days.

[41] As in the first culturing step, in the second culturing step the mixed culture fluid resulting from the first culturing step is first cultured in the culture fluid containing IL- 2, L-glutamine, and autochthonous plasma in the presence of anti-CD3, and then cultured sequentially in the presence of anti-CD 16 and then anti-CD56. This process will be described in detail below.

[42] Initially, 30-40 m# of the mixed culture fluid resulting from the first culturing step is admixed into a 225 cm² culture bath containing a solidified anti-CD3 and a mixture of 50-200 id of a dilute solution containing 17x106-19x106 IUM of IL-2, 1-1.5 mH of L-glutamine, 5-10 mϋ, of autochthonous plasma and 50-67 mϋ, of the culture fluid, and then cultured in the 37⁰C, CO5 incubator for 8-12 hours.

[43] Then, the mixed culture fluid in the culture bath containing the solidified anti-CD3 is moved into a 225 cm² culture bath containing a solidified anti-CD56 and cultured in the 37⁰C, CO2incubator for 8-12 hours. Then, the mixed culture fluid in the culture bath containing the solidified anti-CD56 is moved into a 225cm² culture bath containing a solidified anti-CD16 and cultured in the 37⁰C, CO2incubator for 24-56 hours.

[44] The culture fluids used in the first and second culturing steps may be any culture fluid containing nutrients essential for the growth and survival of cells, for example, amino acids, vitamins, organic and inorganic compounds, proteins, and the like. In addition, the culture fluids may contain 800-1200 IU/m-6 of IL-2. In addition, in the first and second culturing steps, the monoclonal antibodies, anti-CD3, anti-CD16 and anti-CD56, solidified in the culture baths are used. Since the culturing process is sequentially performed in the presence of each of the monoclonal antibodies, it can be controlled to evenly distribute NK cells (CD56 and CD16), NKT cells (CD56 and CD 16) and T cells(CD3) in the final culture product.

[45] Third culturing step

[46] 5-10 mϋ, of autochthonous plasma is added to 86-119 mϋ, of the mixed culture fluid resulting from the second culturing step. The mixed culture fluid to which the autochthonous plasma is newly added is injected into a gas permeable culture bag containing 1 1 of a culture fluid and then further cultured for 7-9 days to proliferate the cell to a large count.

[47] The culture fluid used in the third culturing step may be any culture fluid containing nutrients essential for the growth and survival of cells, for example, amino acids, vitamins, organic and inorganic compounds, proteins, and the like. In addition, the culture fluids may contain 100-200 IUM of IL-2.

[48] The numbers and sizes of the NK cells, T cells and NKT cells increase through the first, second and third culturing steps as described above. As a result of a practical culturing experiment, the total cell count, which was 2.0x106-4.0x107 in lymphocytes separated from 60cc of blood, finally increased to 2.0x109-1.0x1010.

[49]

Mode for the Invention

[50] 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.

[51] Example 1: Preparation of self- activated lymphocytes

[52] Lymphocytes harvested from 60 cc of peripheral blood of a patient were suspended in 3 mϋ, of a culture fluid, admixed into 33 mϋ, of a culture fluid to which 70 id of IL-2, 200 id of L-glutamine and 3 m# of autochthonous plasma were added, and then cultured in the presence of anti-CD3, anti-CD16, and anti-CD56 for 4 days (first culturing step). The mixed culture fluid from the first culturing step was admixed into 60 mϋ, of a culture fluid to which 100 id of IL-2, 1 mϋ, of L-glutamine and 7 mϋ, of autochthonous plasma were added, and then further cultured for 3 days (second culturing step). 7 mϋ, of autochthonous plasma was added to the mixed culture fluid resulting from the second culturing step, injected into a gas permeable culture bag containing 1 1 of a culture fluid, and further cultured for 7 days (third culturing step).

[53] Example 2: Observation on variations in phenotvpe and cell count before and after culturing

[54] FIG. 1 is graphs of phenotypic variations in self-activated lymphocytes before and after culturing, in which Hl, H4, H2 and H3 regions plot distribution of NK cells, T cells, NKT cells and other immunocytes, respectively. FIG. 2 is a graph of im- munocyte count with respect to time during the culturing processes in the method of Example 1.

[55] A surface antigen analysis was performed on the self-activated lymphocytes cultured using the method of Example 1 by using floweytometry. The results will be analyzed with reference to FIG. 1. Referring to (a) of FIG. 1, 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, indicating that the proportion of the NK cells remarkably increased.

[56] In addition, variations in cell count were observed on each type of cells every day while culturing the self- activated lymphocytes using the method of Example 1. As a result, as shown in FIG. 2, the percentage of the NK cells, which was less than 20% before culturing, reached nearly 50% after 12 days of culturing, exceeding the percentage of the T cells. After 14 days of culturing, the percentage of each type of im- munocytes was 60+10% for NK/NKT cells, 40+10% for T cells, and less than 1.5% for B cells.

[57] Example 3: Analysis of cytotoxicity to various cancer cells

[58] FIG. 3 is a graph of cytotoxicity of the self-activated lymphocytes cultured using the method of Example 1.

[59] The self- activated lymphocytes cultured using the method of Example 1 and a blood cancer cell line (K562) were respectively used as an effector cell and a target cell to measure the cytotoxicity of the self-activated lymphocytes, i.e., the ability to kill the blood cancer cell line. The proportion of the self- activated lymphocytes to the cancer cell line was set to 10:1. As shown in FIG. 3, the cytotoxicity of the self-activated lymphocytes was 6.8-16.4 times greater than that of the lymphocytes extracted from normal blood.

[60] Example 4: Anti-cancer effect test on self-activated lymphocytes in mouse model

[61] FIG. 4 is photographic images showing an anti-cancer effect in a mouse model of the self- activated lymphocytes cultured using the method of Example 1.

[62] A tumor model was constructed by transplanting a Raji cell line into BALB/c nude mice at 3 weeks of age and letting 2 weeks pass. The self-activated lymphocytes cultured using the method of Example 1 and the lymphocytes just extracted from normal blood were respectively administered to different groups of the nude mice constructed as the tumor model to evaluate a mass reduction effect and a survival effect.

[63] After 10 days from the administration of the lymphocytes, the size of the mass considerably increased in the nude mice group administered with the lymphocytes just extracted from normal blood, as shown in (a) of FIG. 4. On the other hand, it could be seen that the size of the mass markedly reduced in the nude mice group administered with the self-activated lymphocytes cultured using the method of the present invention, as shown in (b) of FIG. 4. It was confirmed from these results that the self-activated lymphocytes administered to the nude mice led to apoptosis of Raji cells and exhibited an effective anti-cancer function.

[64] 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.

[65]

Industrial Applicability

[66] In the method of 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), are activated on a mass scale. The method can be applied to adaptive immunotherapy to effectively eliminate cancer cells.

[67]

Claims

[1] A method of culturing self-activated lymphocytes, the method comprising: extracting lymphocytes from human peripheral blood; performing a first culturing step of culturing the extracted lymphocytes in a culture fluid to which interleukin2 (IL-2), L-glutamine and autochthonous plasma are added, in the presence of anti-CD3, anti-CD16, and anti-CD56 antibodies, thereby obtaining a mixed culture fluid; and performing a second culturing step of culturing the mixed culture fluid resulting from the first culturing step in the presence of anti-CD3, anti-CD16, and anti- CD56 antibodies after being admixed with a culture fluid to which IL-2, L- glutamine and autochthonous plasma are added.

[2] The method of claim 1, wherein, in the first culturing step, the extracted lymphocytes are first cultured in the culture fluid to which IL-2, L-glutamine, and autochthonous plasma are added, in the presence of anti-CD3, and then cultured sequentially in the presence of anti-CD 16 and then anti-CD56.

[3] The method of claim 2, wherein, in the first culturing step, the extracted lymphocytes are cultured for 12-18 hours in a culture bath containing a solidified anti-CD3 and the culture fluid to which IL-2, L-glutamine, and autochthonous plasma are added, the mixed culture fluid in the culture bath containing the solidified anti-CD3 is moved into a culture bath containing a solidified anti-CD56 and then cultured for 12-18 hours, and the mixed culture fluid in the culture bath containing the solidified anti-CD56 is moved into a culture bath containing a solidified anti-CD16 and then cultured for 48-60 hours.

[4] The method of claim 1, wherein, in the second culturing step, the mixed culture fluid resulting from the first culturing step is first cultured in the culture fluid to which IL-2, L-glutamine, and autochthonous plasma are added, in the presence of anti-CD3, and then cultured sequentially in the presence of anti-CD 16 and then anti-CD56.

[5] The method of claim 4, wherein, in the second culturing step, the mixed culture fluid resulting from the first culturing step is added into a culture bath containing a solidified anti-CD3 and the culture fluid to which IL-2, L-glutamine, and autochthonous plasma are added, and then cultured for 8-12 hours, the mixed culture fluid in the culture bath containing the solidified anti-CD3 is moved into a culture bath containing a solidified anti-CD56 and then cultured for 8-12 hours, and the mixed culture fluid in the culture bath containing the solidified anti-CD56 is moved into a culture bath containing a solidified anti-CD 16 and then cultured for 24-56 hours.

[6] The method of claim 1, wherein the culture fluids used in the first and second culturing steps contain 800 to 1200 International Unit (IU)M of IL-2.

[7] The method of claim 1, further comprising a third culturing step of adding autochthonous plasma into the mixed culture fluid resulting from the second culturing step, injecting the mixed culture fluid containing the autochthonous plasma into a gas permeable culture bag, and culturing the mixed culture fluid.

[8] The method of claim 7, wherein the culture fluid used in the third culturing step contains 100 to 200 IUM of IL-2.