HK1190432B - Method for amplifying nk cells - Google Patents

Description

Method for amplifying NK cells

Technical Field

The present invention relates to a method for amplifying natural killer cells (NK cells) having a high cytolytic activity (cell damage activity) with high purity and high amplification factor, and a pharmaceutical composition comprising NK cells obtained by the method.

Background

NK cells do not attack normal cells expressing MHC class I molecules, but rather attack cells with reduced expression and defects of MHC class I molecules. Therefore, when such NK cells are used for cell therapy of cancer and infectious diseases, there is an advantage that side effects of GVH (Graft-versus-host) disease can be avoided. Actually, according to the reports of Miller et al (non-patent document 1) and Rubnitz et al (non-patent document 2), when a cancer patient is used as a recipient and fresh peripheral blood mononuclear cells of a donor of a healthy person who is close to the cancer patient are transplanted after concentrating NK cells, the transplanted NK cells temporarily survive without causing side effects on the recipient, and cytolytic activity is maintained. However, no clinical treatment trials showing the effectiveness of NK cell transplantation therapy have been reported. One reason for this is that the number of cells that can be collected from a donor by apheresis is limited, and therefore, a sufficient number of NK cells required for the purpose of killing target cells such as cancer cells and pathogen-infected cells cannot be retained in the recipient until the target cells are killed.

About 1X 10 times of blood components can be recovered from 1 blood component of peripheral blood of normal adult¹⁰When the percentage of NK cells constituting peripheral blood mononuclear cells is about 7%, 7X 10 mononuclear cells can be obtained⁸Individual NK cells (non-patent document 3). On the other hand, for NK cell transplantation, 1X 10 was used⁵Per kg to 2X 10⁷One (non-patent document 1) or 5X 10⁵Per kg to 8.1X 10⁷NK cells of the order of one cell/kg (non-patent document 2). If the patient's weight is set to 60kg, then 6X 10 will be required⁶To 4.8 × 10⁹Individual NK cells. This corresponds to 0.0086-fold to 6.86-fold of NK cells obtained from 1 apheresis of peripheral blood of normal adults. However, for example, according to non-patent document 2, it is not found that the survival time of NK cells is related to the number of NK cells administered, and the median value of the survival time is only 10 days in 2 days to 189 days. Therefore, it is necessary to frequently repeat the transplantation of NK cells until the target cells are extinct in order to retain a sufficient number of NK cells required for the extinction of target cells such as cancer cells and pathogen-infected cells in the recipient, which is a significant burden on the patient.

Therefore, a technique has been developed in which NK cells obtained from a donor are first cultured and expanded in vitro to obtain NK cells sufficient for the purpose of killing target cells. Terunuma, H.et al (patent document 1) cultured peripheral blood mononuclear cells of healthy subjects for 13 days in the presence of OKT3, IL-2, and anti-CD 16 antibodies, which are agonist antibodies to human CD3, to expand NK cells to 81.2% and 130-fold purity. In addition, the NK cell cytolytic activity against K562 cells (E: T = 3: 1) was 66%. Tanaka, j. et al (patent document 2) cultured peripheral blood mononuclear cells of healthy subjects for 21 days by using a medium to which IL-2, IL-15, an anti-CD 3 antibody, 5% human AB-type serum, tacrolimus (tacrolimus) and dalteparin sodium (dalteparin) were added, thereby amplifying NK cells to 73.4% and 6268 times in purity. Furthermore, the cytolytic activity of the NK cells on K562 cells (E: T = 1: 1) was about 55%. Carlens, S. et al (non-patent document 4) reported that: in the presence of OKT3 and IL-2 which are agonist antibodies to human CD3, peripheral blood mononuclear cells of healthy subjects were cultured for 21 days to expand NK cells to 55% and 193-fold purity. In addition, the NK cell cytolytic activity against K562 cells (E: T = 1: 1) was 45%. Alici, e. et al (non-patent document 5) reported that: peripheral blood mononuclear cells of myeloma patients were cultured under the same conditions for 20 days to expand NK cells to 65% and 1625-fold purity. Furthermore, the cytolytic activity of the NK cells on K562 cells (E: T = 1: 1) was about 10%. Fujisaki, h. et al (non-patent document 6) reported that: NK cells were expanded to 96.8% purity and 277-fold by culturing peripheral blood mononuclear cells of healthy subjects for 21 days under culture conditions using leukemia cells genetically altered to express factors activating NK cells as feeder cells. Furthermore, the maximum cytolytic activity of the NK cells on K562 cells (E: T = 1: 1) was about 90%.

The cytolytic activity (E: T = 1: 1) of NK cells expanded by the methods of terenuma, h, et al (patent document 1), Tanaka, j, et al (patent document 2), carons, s, et al (non-patent document 4) and Alici, E, et al (non-patent document 5) was 66%, about 55%, 45% and about 10%, respectively. Therefore, in the prior art, the cytolytic activity of NK cells is low, and therefore the therapeutic effect is low, and the number of administered NK cells is increased, which is not preferable. In the method of Fujisaki, H.et al (non-patent document 6), the cytolytic activity of the NK cells after amplification is about 90% (maximum). However, since genetically modified tumor cells are used as feeder cells, there is a risk that the cells are mixed into the final product, which is not preferable.

Documents of the prior art

Patent document

Patent document 1: japanese laid-open patent publication No. 2007-297291

Patent document 2: japanese patent application No. 2011-

Non-patent document

Non-patent document 1: miller, j.s. et al, Blood, 105: 3051(2005)

Non-patent document 2: rubnitz, j.e. et al, j.clin.oncol., 28: 955(2010)

Non-patent document 3: cho, d. and Campana, d., Korean j.lab.med., 29: 89(2009)

Non-patent document 4: carlens, s. et al, hum.immunol., 62: 1092(2001)

Non-patent document 5: alici, e, et al, Blood, 111: 3155(2008)

Non-patent document 6: fujisaki, h. et al, Cancer res, 69: 4010(2009)

Disclosure of Invention

Problems to be solved by the invention

Therefore, there is a need for the development of a technique that can amplify NK cells having high cytolytic activity from umbilical cord blood or peripheral blood with high purity without using feeder cells.

Means for solving the problems

The present invention provides a method for amplifying NK cells. The method for amplifying NK cells of the present invention comprises: a step of preparing a cell population containing NK cells; a step of removing T cells from the cell population containing NK cells; and culturing the cells remaining after removal of the T cells in a medium containing 2500IU/mL to 2813IU/mL of IL-2.

In the NK cell expansion method of the present invention, the step of removing the T cells from the NK cell-containing cell population may be achieved by a step of removing CD 3-positive cells.

The NK cell expansion method of the present invention may include a step of removing hematopoietic precursor cells from the NK cell-containing cell population.

In the NK cell expansion method of the present invention, the step of removing the hematopoietic precursor cells from the NK cell-containing cell population may be achieved by a step of removing CD 34-positive cells.

In the NK cell expansion method of the present invention, the medium may contain autologous serum, AB-type serum, and/or serum albumin.

In the method for amplifying NK cells of the present invention, the step of preparing the cell population containing NK cells may be achieved by a step of separating monocytes from blood cells collected from the subject.

In the method for amplifying NK cells of the present invention, the blood cells are sometimes collected from peripheral blood, cord blood, bone marrow and/or lymph nodes.

In the NK cell expansion method of the present invention, the blood cells may be collected from peripheral blood by apheresis.

In the method for amplifying NK cells of the present invention, the cell population including NK cells may be prepared from at least 1 cell selected from the group consisting of stem cell-derived hematopoietic stem cells, cord blood-derived hematopoietic stem cells, peripheral blood-derived hematopoietic stem cells, bone marrow blood-derived hematopoietic stem cells, cord blood mononuclear cells, and peripheral blood mononuclear cells, wherein the stem cells are any one selected from the group consisting of embryonic stem cells, adult stem cells, and artificial pluripotent stem (iPS) cells. Sometimes the donor of the population of cells comprising NK cells is derived from: the patient who is the recipient himself, the close relative of the patient, or a person who is not related to the patient. The NK cells are sometimes derived from donors whose receptors are not the same as the major histocompatibility antigen (MHC) of the receptor and the Immunoglobulin-like receptor of the Killer cell (Killer Immunoglobulin-like receptor: KIR).

The present invention provides a pharmaceutical composition for cell therapy comprising NK cells prepared by the amplification method of the present invention. The pharmaceutical composition of the present invention may contain NK cell precursors, T cells, NKT cells, hematopoietic precursor cells, and the like, in addition to the expanded NK cells.

Sometimes the pharmaceutical compositions of the present invention are used to treat infectious diseases and/or cancer.

The pharmaceutical composition of the present invention may be administered to a patient having an HLA genotype different from that of the NK cells prepared by the amplification method of the present invention.

The present invention provides a cell therapy comprising: a step of preparing a cell population containing the NK cells; a step of removing T cells from the cell population containing NK cells; culturing the cells remaining after removal of the T cells in a medium containing 2500IU/mL to 2813IU/mL of IL-2; transplanting the NK cells expanded by the remaining cells to a patient. Sometimes the cell therapy comprises: removing hematopoietic precursor cells from the population of cells comprising NK cells. In the step of transplanting the NK cells to a patient, the expanded NK cells may be transplanted together with NK cell precursors, T cells, NKT cells, hematopoietic precursor cells, and the like. Sometimes the cell therapy of the invention is used to treat and/or prevent infectious diseases and/or cancer. Sometimes the cell therapy of the invention comprises: a step of transplanting to a patient having an HLA genotype different from that of the NK cells prepared by the amplification method of the present invention. In the cell therapy of the present invention, the step of transplanting the NK cells to a patient may be achieved by a step of administering the pharmaceutical composition of the present invention to a patient.

In the cell therapy of the present invention, the cell population containing NK cells may be prepared from at least 1 cell selected from the group consisting of stem cell-derived hematopoietic stem cells, cord blood-derived hematopoietic stem cells, peripheral blood-derived hematopoietic stem cells, bone marrow blood-derived hematopoietic stem cells, cord blood mononuclear cells, and peripheral blood mononuclear cells, wherein the stem cells are any one selected from the group consisting of embryonic stem cells, adult stem cells, and artificial pluripotent stem (iPS) cells. Sometimes the donor of the population of cells comprising NK cells is derived from: the patient who is the recipient himself, the close relative of the patient, or a person who is not related to the patient. Sometimes the NK cells are derived from donors that are not identical to the recipient's major histocompatibility antigen (MHC), and the Killer Immunoglobulin-like Receptor (Killer Immunoglobulin-like Receptor: KIR).

In the present specification, "NK cell" means a monocyte which is CD 3-negative and positive for CD56, and particularly has cytolytic activity against a cell which expresses little or no MHC class I molecule.

In the amplification method of the present invention, the cell population comprising NK cells may be prepared using various steps known to those skilled in the art. For example, density gradient centrifugation can be used for recovering monocytes from blood such as umbilical cord blood and peripheral blood. In addition, NK cells can be collected using immunomagnetic beads. The NK cells may be isolated and identified by immunofluorescent staining using an antibody specific for a cell surface marker, and by FACS (Fluorescence activated cell sorter) or flow cytometry. In addition, the NK cells can also be prepared as follows: the cells expressing the cell surface antigens CD3 and/or CD34 were separated and removed using immunomagnetic beads including, but not limited to, Dynabeads (trademark) manufactured by Dynal corporation, which is commercially available from Invitrogen corporation, and clini macs (trademark) manufactured by Miltenyi Biotec corporation. In addition, T cells and/or hematopoietic precursor cells are sometimes selectively injured or killed using specific binding partners for the T cells and/or hematopoietic precursor cells. The step of removing the T cells from the monocytes may be a step of removing other cell types, for example, hematopoietic precursor cells, B cells and/or NKT cells together with T cells. The step of removing hematopoietic precursor cells from the monocytes may be a step of removing other cell types such as T cells, B cells and/or NKT cells together with the hematopoietic precursor cells.

In the amplification method of the present invention, the monocytes separated from the umbilical cord blood and the peripheral blood may be cryopreserved, thawed according to the time of transplantation into a patient, and subjected to the amplification of NK cells. Alternatively, the monocytes may be frozen during the expansion by the NK cell expansion method of the present invention or after the expansion is completed, thawed according to the time for transplantation into a patient, and then transplanted into the patient. The blood cells may be frozen and thawed by methods known to those skilled in the art. In some cases, any commercially available cell cryopreservation solution may be used for freezing the cells.

In the cell therapy of the present invention, as a solution for suspending live NK cells, for example, normal saline, Phosphate Buffered Saline (PBS), a culture medium, serum, and the like are generally used. The solution may contain a pharmaceutically acceptable carrier as a pharmaceutical or quasi-pharmaceutical. The NK cell therapy of the present invention may be applicable to the treatment and/or prevention of various diseases to which NK cells are sensitive. The diseases include, but are not limited to, for example, oral cancer, gallbladder cancer, bile duct cancer, lung cancer, liver cancer, large intestine cancer, kidney cancer, bladder cancer, leukemia, or infectious diseases caused by viruses, bacteria, etc. Sometimes the cell therapy of the present invention can be carried out by surgical therapy, chemotherapy, radiotherapy, etc., alone or in combination. In the cell therapy of the present invention, sometimes NK cells are transplanted by, for example, administration into a vein, an artery, subcutaneously, intraperitoneally, or the like.

The cell culture medium for preparing NK cells of the present invention includes, but is not limited to, KBM501 medium (Kohjin Bio Inc.), CellGro SCGM medium (CellGenix, Kyowa Chemicals Co., Ltd.), X-VIVO15 medium (Lonza, TakaraBio Inc.), IMDM, MEM, DMEM, RPMI-1640, and the like.

Interleukin-2 (IL-2) may be added to the medium at a concentration that can achieve the object of the present invention. Sometimes the concentration of IL-2 is 2500IU/mL to 2813 IU/mL. The IL-2 preferably has a human amino acid sequence, and is preferably produced by recombinant DNA techniques from the viewpoint of safety.

In the present specification, the concentration of IL-2 is sometimes expressed in Japanese Standard Unit (JRU) and International Unit (IU). Since 1IU is about 0.622JRU, 1750JRU/mL is about 2813 IU/mL.

The culture medium may contain autologous serum of the subject, human AB serum available from BioWhittaker, etc., or blood-donated human serum albumin available from the japan red cross. The autologous serum and the human AB-type serum are preferably added at a concentration of 1 to 10%, and the blood-donated human serum albumin is preferably added at a concentration of 1 to 10%. Sometimes the subject is a healthy person, and a patient suffering from the disease.

The medium may contain appropriate components such as proteins, cytokines, antibodies, and compounds, provided that the effect of amplifying NK cells is not impaired. The cytokine is sometimes interleukin 3 (IL-3), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), Stem Cell Factor (SCF), and/or FMS-like tyrosine kinase 3 ligand (Flt 3L). The IL-3, IL-7, IL-12, IL-15, IL-21, SCF and Flt3L preferably have human amino acid sequences and are preferably produced by recombinant DNA techniques from the viewpoint of safety. The medium may be replaced at any time after the start of culture, preferably every 3 to 5 days, under the condition that the number of cells from which desired NK cells can be obtained is as small as possible.

In the amplification method of the present invention, the culture vessel includes, but is not limited to, commercially available dishes, flasks, plates, multi-well plates. The culture conditions are not particularly limited as long as the NK cell amplification effect is not impaired, and are usually 37 ℃ and 5% CO₂And culture conditions under saturated water vapor environment. Since the present invention aims to produce a large amount of NK cells, it is advantageous that more NK cells are obtained as the culture time in the medium is longer. The culture time is not particularly limited as long as it is a condition capable of expanding NK cells to a desired number of cells.

In the amplification method of the present invention, the cell population containing NK cells may contain NK cell precursors, T cells, NKT cells, hematopoietic precursor cells, and the like, in addition to NK cells. After amplification, the desired NK cells may be selected by using, for example, density gradient centrifugation, immunomagnetic beads, FACS, flow cytometry, or the like. For example, sometimes the NK cells are selectively isolated from the cell population by using anti-CD 3 antibody, anti-CD 16 antibody, anti-CD 34 antibody, anti-CD 56 antibody, anti-CD 69 antibody, anti-CD 94 antibody, anti-CD 107a antibody, anti-KIR 3DL1 antibody, anti-KIR 3DL2 antibody, anti-KIR 2DL3 antibody, anti-KIR 2DL1 antibody, anti-KIR 2DS1 antibody, anti-KIR 2DL5 antibody, anti-NKp 46 antibody, anti-NKp 30 antibody, anti-NKG 2D antibody, and the like. Sometimes the antibody is a monoclonal antibody, a polyclonal antibody, or the like. NK cells may be selected by selectively removing cells such as T cells, NKT cells, and hematopoietic precursor cells.

The method and the pharmaceutical composition of the present invention are preferably produced under conditions (good manufacturing practice, GMP) suitable for production control and quality control rules of pharmaceuticals and quasi-pharmaceuticals.

The cytolytic activity of the expanded NK cells can be assessed by methods well known to those skilled in the art. The cytolytic activity is generally quantified by: the NK cells (effector cells) and the target cells labeled with a radioactive substance, a fluorescent dye, or the like are incubated, and then the amount of radioactivity or fluorescence intensity is measured. The target cells may be K562 cells, acute myelogenous leukemia cells, and chronic myelogenous leukemia cells, but are not limited thereto. The nature of the amplified NK cells may be examined by RT-PCR, solid phase hybridization, ELISA, Western blotting, immunoprecipitation, immunoturbidimetry, FACS, flow cytometry, or the like.

In the present invention, the collection of whole umbilical cord blood and peripheral blood, the preparation of autologous serum, the preparation of monocytes derived from the whole blood, the measurement of the number of cells before and after the culture of the monocytes, the measurement of the constituent ratio of cell types such as NK cells, T cells, hematopoietic precursor cells and the like in the monocytes before and after the culture, the calculation of the amplification factor of NK cells, and the statistical analysis of measurement errors and significance can be performed by any method known to those skilled in the art.

All documents mentioned in this specification are incorporated in their entirety by reference into the present specification.

Drawings

Fig. 1A is a graph showing the results of flow cytometry measurement using double staining with antibodies to CD3 and CD56 before removing CD3 positive cells.

Fig. 1B is a graph showing the results of double staining using antibodies to CD3 and CD56 and measurement by flow cytometry after removal of CD 3-positive cells.

Fig. 2A is a graph showing the respective proliferation curves of the cell numbers of CD 3-negative cells isolated from monocytes in the peripheral blood of 5 healthy persons.

Fig. 2B is a graph showing the average cell number of CD 3-negative cells isolated from monocytes in the peripheral blood of 5 healthy persons.

Fig. 3A is a graph showing respective proliferation curves of the amplification rates of CD 3-negative cells isolated from monocytes in the peripheral blood of 5 healthy persons.

Fig. 3B is a mean proliferation curve of the expansion rate of CD3 negative cells isolated from monocytes in peripheral blood of 5 healthy persons.

Fig. 4A is a proliferation curve of each amplification factor of NK cells (CD 3 negative/CD 56 positive) isolated from monocytes in peripheral blood of 5 healthy persons.

FIG. 4B is a mean proliferation curve of the amplification rate of NK cells (CD 3 negative/CD 56 positive) isolated from monocytes in peripheral blood of 5 healthy persons.

Fig. 5A is a graph showing the results of measuring the temporal change in the composition ratio of NK cells (CD 3 negative/CD 56 positive) isolated from 5 healthy persons with respect to the total cultured cells by the flow cytometry.

Fig. 5B is a graph showing the results of measuring and averaging the temporal changes in the composition ratio of NK cells (CD 3 negative/CD 56 positive) isolated from 5 healthy persons with respect to the total cultured cells by the flow cytometry.

Fig. 6A is a graph showing the results of measuring the temporal change in the composition ratio of NK cells (CD 3 negative/CD 56 positive) isolated from 3 patients with advanced cancer (oral cancer, cystic cancer, and biliary duct cancer) to the total cultured cells by flow cytometry.

FIG. 6B is a mean proliferation curve of the amplification rate of NK cells (CD 3 negative/CD 56 positive) isolated from 3 patients with advanced cancer (oral cancer, cholecystcancer and cholangiocarcinoma).

Fig. 7 is a graph comparing the results of flow cytometry analysis of CD 69.

Fig. 8 is a graph showing measured values of Mean Fluorescence Intensity (MFI) for comparison of flow cytometry analysis results of CD 69.

Fig. 9 is a graph comparing the results of flow cytometry analysis of CD 16.

Fig. 10 is a graph showing measured values of Mean Fluorescence Intensity (MFI) for comparison of flow cytometry analysis results of CD 16.

FIG. 11 is a graph comparing the results of flow cytometry analysis of various cell surface markers.

FIG. 12 is a proliferation curve of the amplification rate of NK cells cultured in KBM medium and CellGro medium.

FIG. 13 is a graph showing the results of an experiment for examining the cytolytic activity of K562 cells of NK cells derived from peripheral blood after expansion by the method of the present invention.

Fig. 14 is a graph showing the results of measuring the change with time in the composition ratio of CD107a positive cells isolated from healthy persons to the total cultured cells by the flow cytometry method.

Fig. 15 is a bar graph showing the composition ratio of NK cells (CD 3 negative/CD 56 positive) to the total cultured cells after removal of 1 and 2 CD3 positive cells.

Fig. 16A is a histogram showing the composition ratio of CD34 positive cells among CD3 negative cells and CD3 and CD34 negative cells before expansion.

Fig. 16B is a histogram showing the composition ratio of CD3 positive cells among CD3 negative cells and CD3 and CD34 negative cells before expansion.

Fig. 17 is a histogram showing the composition ratio of NK cells (CD 3 negative/CD 56 positive) in CD 3-negative cells and CD3 and CD 34-negative cells after expansion, relative to the whole cultured cells.

Detailed Description

The following examples of the present invention are intended to be illustrative only, and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description of the claims. Changes of the present invention, for example, addition, deletion, and replacement of constituent elements of the present invention may be made without departing from the gist of the present invention.

Example 1

Amplification of NK cells (1)

1. Materials and methods

(1) Blood sampling from peripheral blood

Peripheral blood was collected from healthy people, and patients with advanced cancers (oral cancer, bladder cancer, and bile duct cancer). The experiment was carried out with approval from the ethical review committee of the regional local office clinical study in Kyushu university of medicine (approval No. 22-176, approval date: 3 months and 31 days in 23 years). Obtaining written consent from the healthy person and the patient. Blood collection, cryopreservation, and thawing are performed by methods known to those skilled in the art.

(2) Isolation of monocytes from peripheral blood

The obtained blood was diluted 2-fold with a diluent (PBS containing 1mM EDTA and 2% fetal bovine serum) stored at room temperature, and 20 to 35mL of the diluted blood was covered with 10 to 15mL of Ficoll Paque (specific gravity 1.077) in each centrifuge tube. Centrifugation was carried out at 500 Xg for 20 minutes at room temperature, and the centrifugation was stopped without braking. The supernatant (plasma fraction) was removed in a remaining number of mL, and the intermediate layer was recovered. And collecting the middle layer recovered from 1-2 centrifuge tubes into 1 new centrifuge tube, and adjusting the volume to 50mL by using the diluent. The second centrifugation is carried out at 500 Xg, room temperature, 5 minutes or 15 minutes. The supernatant was removed and the pellet suspended in 30mL of the dilution. The third centrifugation was carried out at 280 Xg for 10 minutes at room temperature. The supernatant was removed so that the cell concentration was 1X 10⁷The pellet was suspended in PBS supplemented with 2mM EDTA and 0.1% BSA (hereinafter referred to as "monocyte suspension") per mL.

(3) Removal of CD 3-positive cells

The magnetic beads (Dynabeads CD 3) on which the anti-CD 3 antibody was immobilized were washed 1 time with PBS supplemented with 0.1% BSA and then every 10 times⁷The cells were added to the monocyte suspension in 50. mu.L aliquots. The monocyte suspension containing magnetic beads was stirred at 4 ℃ for 30 minutes using a rotator. Thereafter, the magnetic beads were separated from the suspension by a magnet, and cells expressing CD3 on the cell surface (CD 3 positive cells) were removed.

(4) Culture of cell population from which CD 3-positive cells were removed

The remaining cells in the suspension (hereinafter, referred to as "CD 3 negative cells") were diluted to 5X 10 using a cell culture medium (KBM 501, 16025015, Kohjin Bio Inc.; IL-2 containing 1750 JRU/mL) (hereinafter, referred to as "KBM medium") supplemented with 5% autologous serum⁵one/mL, and inoculated in a 6-well plate (140675, nunc, ThermoFisher Scientific Co., Ltd.). At 37 deg.C, 5% CO₂And carrying out 21 days of cell culture under a saturated water vapor environment. Medium replacement was performed on days 5, 9, 13 and 17 of the culture. The cells are cultured in the absence of feeder cells.

(5) Analysis of cell number and cell surface markers

The cell number of the peripheral blood mononuclear cells was determined by counting the number of viable cells by a hemocytometer from the start of culture to day 21. The antibody was analyzed by using an anti-CD 3 antibody (317308, BioLegend Japan K.K., Japan Dickinson corporation), an anti-CD 16 antibody (556618, BD Pharmingen, Japan, Becton Dickinson, Ltd.), an anti-CD 56 antibody (304607, 318321, BioLegend Japan K.K.), an anti-CD 69 antibody (310905 BioLegend Japan K.K.), an anti-KIR 3DL1/KIR3DL2 antibody (130. flate 095. K.205, Miltenyi Biotec Co., Ltd.), an anti-KIR 2DL3 antibody (FAB 2014P, R & D SYSTEMS, COSMIO. K.K.), an anti-KIR 2DL1/KIR2DS1 antibody (339505, BioLegend Japan K.K.K., Japan), an anti-KIR 2DL5 antibody (42, Biogend. K.K.K., Biogend 46), an anti-NKp 584648 antibody (Biogend P. K.K.K.K.K., Biogend 4624, Biogend 30), and an anti-NKp 4624, Biogend 30 antibody (Biogend P., NKp., NKp.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.

2. Results

(1) Amplification of NK cells in healthy humans

Fig. 1A shows the results of an experiment in which antibodies to CD3 and CD56 were double-stained and measured by a flow cytometry method before CD 3-positive cells were removed. Fig. 1B is an experimental result of double staining using antibodies to CD3 and CD56 and measurement by flow cytometry after removal of CD3 positive cells. The "percentage of CD 3-positive cells" is the percentage of CD 3-positive cells in the total cultured cells of each experimental group measured by flow cytometry. The composition ratio (%) of CD 3-positive cells was 69.37% before removal of CD 3-positive cells and 0.68% after removal of CD 3-positive cells. From the above results, it can be seen that: CD3 positive cells were significantly removed from the monocyte suspension.

Fig. 2A is a graph showing the respective proliferation curves of the cell numbers of CD 3-negative cells isolated from monocytes in the peripheral blood of 5 healthy persons. Fig. 2B is a graph showing the average cell number of CD 3-negative cells isolated from monocytes in the peripheral blood of 5 healthy persons. The number of CD 3-negative cells per 1mL of peripheral blood collected from 5 healthy persons was measured at the start of culture, after 5 days of culture, after 9 days of culture, after 13 days of culture, after 17 days of culture, and after 21 days of culture. The standard deviation of each experimental condition was calculated from the measured values of the experimental results repeated 5 times under the same condition. CD3 negative cells continued to increase from the start of culture until day 21. The rate of increase continued to increase by day 13 and decreased after day 13. CD3 negative cells approximately 5X 10 from the start of culture⁵Increasing to about 700X 10 after 21 days of culture⁵And (4) respectively.

Fig. 3A is a graph showing respective proliferation curves of the amplification rates of CD 3-negative cells isolated from monocytes in the peripheral blood of 5 healthy persons. Fig. 3B is a mean proliferation curve of the expansion rate of CD3 negative cells isolated from monocytes in peripheral blood of 5 healthy persons. The amplification factor was calculated as a quotient of the number of cells of CD3 negative cells after 5 days, 9 days, 13 days, 17 days, and 21 days of culture divided by the number of cells of CD3 negative cells at the start of culture. The standard deviation of each experimental condition was calculated from the measured values of the experimental results repeated 5 times under the same condition. The amplification rate of CD 3-negative cells continued to increase from the start of culture to day 21. The amplification rate continued to increase significantly until day 13, and increased to about 150-fold after 21 days of culture.

Fig. 4A is a proliferation curve of each amplification factor of NK cells (CD 3 negative/CD 56 positive) isolated from monocytes in peripheral blood of 5 healthy persons. FIG. 4B is a mean proliferation curve of the amplification rate of NK cells (CD 3 negative/CD 56 positive) isolated from monocytes in peripheral blood of 5 healthy persons. In fig. 4A and 4B, CD 3-negative cells were analyzed by flow cytometry using double staining for antibodies to CD3 and CD 56. The amplification factor was calculated as a quotient obtained by dividing the number of NK cells after 7 days of culture, 14 days of culture, and 21 days of culture by the number of NK cells at the start of culture. The standard deviation of each experimental condition was calculated from the measured values of the experimental results repeated 5 times under the same condition. The amplification rate of NK cells continued to increase from the start of culture to day 21. The amplification rate continued to increase significantly up to day 14, and increased to about 400-fold after 21 days of culture.

Fig. 5A is an experimental result of measuring a change with time in the composition ratio of NK cells (CD 3 negative/CD 56 positive) isolated from 5 healthy persons with respect to the total cultured cells by a flow cytometry. Fig. 5B shows the results of experiments in which the temporal change in the average value of the composition ratio of NK cells (CD 3 negative/CD 56 positive) isolated from 5 healthy persons to the total cultured cells was measured by flow cytometry and averaged. In fig. 5A and 5B, CD 3-negative cells were analyzed by flow cytometry using double staining for antibodies to CD3 and CD 56. The "percentage of NK cell composition" is the percentage of NK cells in all cultured cells of each experimental group measured by flow cytometry. The ordinate of the graph indicates the percentage (%) of NK cells (CD 3-negative/CD 56-positive) to the total cultured cells, and the abscissa indicates the number of days in culture. The standard deviation of each experimental condition was calculated from the measured values of the experimental results repeated 5 times under the same condition. The percentage of NK cell formation increased continuously from the start of culture to day 21. The NK cell constitutive rate continued to increase significantly until day 14, and increased to about 90 after 14 days of culture. The present invention shows that NK cells can be selectively and temporally expanded.

(2) Expansion of NK cells of patients

Fig. 6A shows the results of an experiment in which the temporal change in the composition ratio of NK cells (CD 3 negative/CD 56 positive) isolated from 3 patients with advanced cancer (oral cancer, cystic cancer, and biliary duct cancer) to the total cultured cells was measured by flow cytometry. FIG. 6B is a mean proliferation curve of the amplification rate of NK cells (CD 3 negative/CD 56 positive) isolated from 3 patients with advanced cancer (oral cancer, cholecystcancer and cholangiocarcinoma). The "percentage of NK cells" is the percentage of NK cells in all cultured cells of each experimental group measured by flow cytometry. In the graph of fig. 6A, the vertical axis represents the composition ratio (%) of NK cells (CD 3 negative/CD 56 positive) to the whole cultured cells, and the horizontal axis represents the number of days in culture. The "amplification rate of NK cells" is expressed as the number of NK cells after amplification divided by the number of NK cells present in peripheral blood mononuclear cells before amplification. In the graph of FIG. 6B, the vertical axis represents the amplification rate of NK cells, and the horizontal axis represents the number of days of culture. The standard deviation of each experimental condition was calculated from the measured values of the experimental results repeated 3 times under the same condition. As shown in fig. 6A, the percentage of NK cells that constituted significantly continued to increase from the start of culture to day 14, and increased to about 85% after 14 days of culture. As shown in fig. 6B, the amplification factor of NK cells significantly continued to increase from the start of culture to day 14, and increased to about 140-fold after 14 days of culture. After 21 days of culture, the CD 3-positive cells proliferated, and thus the NK cell constitutive rate decreased. However, the proliferation of the CD 3-positive cells had little effect on the expansion of NK cells. The above results show that: NK cells isolated from patients with advanced cancer (oral, cystic and biliary tract cancer) can expand over time. Furthermore, the present invention shows: can amplify NK cells isolated from patients with cancer, infectious disease, etc. over time.

(3) Expression of differentiation markers for NK cells

Fig. 7, 9 and 11 show graphs comparing the results of flow cytometry analysis of each cell surface marker. Fig. 8 and 10 show graphs of measured values of Mean Fluorescence Intensity (MFI) obtained by comparing flow cytometry analysis results of CD69 and CD 16. The standard deviation of each experimental condition was calculated from the measured values of the experimental results repeated 3 times under the same condition. As can be seen from fig. 7 to 11: cells expanded by the method of the present invention strongly express CD69, KIR2DL3, KIR2DL1/KIR2DS1, KIR2DL5, NKp30, and NKG2D, as compared to cells before expansion. In particular, the expression of CD69 was about 100% in the expanded cells. These figures show that: the cells prepared by the method of the present invention can express differentiation markers as NK cells. Furthermore, it was shown that the NK cells possess high cytolytic activity.

The experimental results of this example show that: by removing the CD 3-positive cells, i.e., T cells, and then culturing them in the KBM medium, only NK cells can be selectively and efficiently expanded basically. Shows that: a large amount of NK cells can be produced not only from healthy persons but also from patients suffering from cancer, infectious diseases, and the like. It is also shown that: the method of the present invention can significantly expand not only peripheral blood-derived NK cells but also cells derived from other tissues and organs, particularly cord blood-derived NK cells.

Example 2

Amplification of NK cells (2)

1. Materials and methods

NK cells were prepared from healthy humans according to the method described in example 1. As a medium for cell culture, CellGro SCGM (2001, CellGenix, Kyowa Kagaku K.K.) (hereinafter, referred to as "CellGro medium") supplemented with 2500IU/mL of IL-2 (AF-200-02-2, PeproTech, Toyo Boseki Co., Ltd.) and 5% autologous serum was prepared. The NK cells were expanded in the KBM medium and the CellGro medium according to the method described in example 1.

2. Results

FIG. 12 is a proliferation curve of the amplification rate of NK cells cultured in KBM medium and CellGro medium. The amplification factor was calculated as a quotient obtained by dividing the number of NK cells after 7 days of culture, 14 days of culture, and 21 days of culture by the number of NK cells at the start of culture. The standard deviation of each experimental condition was calculated from the measured values of the experimental results repeated 2 times under the same condition. The amplification rate of NK cells continued to increase in KBM medium and CellGro medium from the start of culture to day 21. After 21 days of culture, the amplification rate was about 670-fold in KBM medium and about 140-fold in CellGro medium.

The experimental results of this example show that: NK cells were well expanded in the KBM medium, and the CellGro medium. Thus, it appears that: NK cells can be expanded in a medium containing 2500IU/mL to 2813IU/mL of IL-2, regardless of the type of medium used for cell culture.

Example 3

Cytolytic activity of expanded NK cells

1. Materials and methods

(1) Quantification of cytolytic Activity

NK cells were prepared as described in example 1 and used as effector cells. K562 cells (chronic myelogenous leukemia cells) were prepared by a method known to those skilled in the art and used as target cells. The cytolytic activity of the amplified NK cells, and non-amplified NK cells (hereinafter, referred to as "non-amplified NK cells") was quantified by methods well known to those skilled in the art. Briefly, the target cells were identified by culturing them in RPMI-1640 medium supplemented with 3,3 '-dioctadecyloxocyanine (3, 3' -dioctadecyloxycarbocyanine) (D4292, Sigma-Aldrich Japan) (final concentration: 0.01 mM) for 10 minutes. The target cells were washed 3 times after identification using PBS (-) and serum-free IMDM medium. The effector cells and the target cells were seeded in a round-bottomed 96-well culture plate and co-cultured in serum-free IMDM medium for 2 hours. In terms of the ratio of effector cells to target cells (E: T ratio), the ratio of 3: 1. 2: 1. 1: 1. 1: 5. and 1: 10, respectively. Cytolytic activity (%) was quantified by flow cytometry using an anti-MHC class I antibody (311409, BioLegend Japan) and 7-amino-actinomycin D (A9400, Sigma-Aldrich Japan).

(2) Expression of differentiation markers for NK cells

NK cells were amplified according to the method described in example 1. At the beginning of the culture, after 3 days of culture, after 7 days of culture, after 14 days of culture and after 21 days of culture, the ratio of 2: 1E: t ratio the NK cells and the K562 cells were co-cultured for 2 hours. Thereafter, the composition ratio of CD107a positive cells among the NK cells was analyzed by a flow cytometry method using an anti-CD 107a antibody (328606, BioLegendJapan).

2. Results

(1) Quantification of cytolytic Activity

FIG. 13 is a graph showing the results of an experiment for examining the cytolytic activity of K562 cells of NK cells derived from peripheral blood after expansion by the method of the present invention. The vertical axis represents cytolytic activity (unit:%). The white bars indicate cytolytic activity of non-expanded NK cells, and the black bars indicate cytolytic activity of expanded NK cells. The horizontal axis shows E of expanded NK cells or non-expanded NK cells, and K562 cells: the ratio of T. When E: the T ratio is 3: 1, the non-expanded NK cells are about 30% and the expanded NK cells are about 110% with respect to said cytolytic activity. When E: the T ratio is 2: 1, the non-expanded NK cells were about 20% and the expanded NK cells were about 107% with respect to said cytolytic activity. When E: the ratio T is 1: 1, the non-expanded NK cells are about 10% and the expanded NK cells are about 100% with respect to said cytolytic activity. When E: the ratio T is 1: 5 and 1: 10, the cytolytic activity of the expanded NK cells is about 25% and about 15%, respectively.

(2) Expression of differentiation markers for NK cells

Fig. 14 shows the results of an experiment in which the change with time in the composition ratio of CD107a positive cells isolated from healthy persons to the total cultured cells was measured by flow cytometry. The standard deviation of each experimental condition was calculated from the measured values of the experimental results repeated 5 times under the same condition. The "composition ratio of CD107a positive cells" indicates the percentage of CD107a positive cells in the total cultured cells of each experimental group measured by flow cytometry. In the graph of fig. 14, the vertical axis represents the composition ratio (%) of CD107 a-positive cells to the whole cultured cells, and the horizontal axis represents the number of days of culture. The constitutive rate of CD107a positive cells increased to about 35% from the start of culture to day 3, and was maintained at day 21.

The experimental results of this example show that: the NK cells amplified by the present invention have high cytolytic activity. Thus, for the purposes of the present invention, it shows: NK cells having high cytolytic activity can be selectively and efficiently expanded without using feeder cells, NK cells obtained by transfecting foreign molecules, or the like. Further, NK cells exhibit high cytolytic activity not only when they are amplified from cells derived from peripheral blood, but also when they are amplified from cells derived from other tissues and organs, particularly, cells derived from umbilical cord blood.

Example 4

Expansion of NK cells (3) (repeated removal of CD 3-positive cells)

After the experiments of examples 1 to 3, in further repeating the expansion experiments of NK cells, the following were found: the CD 3-positive cells were non-selectively increased, and the percentage of CD 3-positive cells in the total cultured cells was sometimes more than 30% as shown in the results of this example. The frequency of non-selective increase in the CD3 positive cells was approximately 30% in experiments in which NK cells were expanded using peripheral blood mononuclear cells collected from patients with advanced cancer by apheresis (data not shown). Therefore, in order to selectively expand NK cells, an attempt was made to repeat the step of removing CD 3-positive cells.

1. Materials and methods

NK cells were expanded and analyzed for cell number and cell surface markers according to the method described in example 1. Monocyte suspensions were prepared from patients with advanced cancers (oral, cystic and biliary tract). Removal of CD3 positive cells was performed 1 or 2 times. CD3 negative cells were cultured in the KBM medium for 14 days.

2. Results

Fig. 15 is a bar graph showing the composition ratio of NK cells (CD 3 negative/CD 56 positive) to the total cultured cells after removal of 1 and 2 CD3 positive cells. The error bars for each experimental condition represent the standard error of the measured values of the experimental results repeated 3 times under the same condition. The component ratios of NK cells, CD 3-positive cells, and other cells were expressed as percentages of NK cells, CD 3-positive cells, and other cells in the total cultured cells of each experimental group, which were measured by flow cytometry. The ordinate of the graph indicates the percentage (%) of NK cells, CD 3-positive cells, and other cells relative to the total cultured cells, and the abscissa indicates the number of removal of CD 3-positive cells. The percentage (%) of NK cells relative to the total cultured cells was about 50% in the case of 1 removal of CD 3-positive cells and about 65% in the case of 2 removals of CD 3-positive cells.

The experimental results of this example show that: repeated removal of CD 3-positive cells decreases the composition ratio of CD 3-positive cells to the whole cultured cells, and increases the composition ratio of NK cells to the whole cultured cells. However, the effect of the repeated removal of the CD 3-positive cells is not sufficient for selectively expanding NK cells. Therefore, other treatments than the repeated removal of the CD3 positive cells were tried and used.

Example 5

Amplification of NK cells (4) (removal of CD 3-Positive cells and CD 34-Positive cells)

1. Materials and methods

NK cells were expanded and analyzed for cell number and cell surface markers according to the method described in example 1. Monocyte suspensions were prepared from patients with advanced cancers (oral, cystic and biliary tract). After removal of CD3 positive cells, hematopoietic precursor cells were removed. The removal of the hematopoietic precursor cells was performed by removing cells (CD 34-positive cells) expressing CD34 on the cell surface using a biotinylated anti-CD 34 antibody (343523, BioLegend Japan ltd.) and magnetic beads (dynabeads biotin binder, 110-47, Life Technologies Japan ltd.). Briefly, the CD34 positive cells were reacted with the biotinylated anti-CD 34 antibody. Thereafter, centrifugation was performed to remove the supernatant, thereby preparing a suspension of cells to which the antibody had been bound. The magnetic beads are addedAfter washing 1 time with 0.1% BSA in PBS, every 10⁷The cells were added to the suspension in 50. mu.L. The suspension containing the magnetic beads was stirred at 4 ℃ for 30 minutes using a rotator. The magnetic beads were separated from the suspension by a magnet to remove CD34 positive cells. The remaining cells in the suspension (hereinafter, referred to as "CD 3 and CD34 negative cells") were cultured in the KBM medium for 14 days. An anti-CD 34 antibody (343505, BioLegend Japan) was additionally used for the measurement by the flow cytometry method.

2. Results

Fig. 16A is a histogram showing the composition ratio of CD34 positive cells among CD3 negative cells and CD3 and CD34 negative cells before expansion. Fig. 16B is a histogram showing the composition ratio of CD3 positive cells among CD3 negative cells and CD3 and CD34 negative cells before expansion. The error bars for each experimental condition represent the standard error of the measured values of the experimental results repeated 3 times under the same condition. The percentage of the CD 34-positive cells and the CD 3-positive cells in the total cells in each experimental group, which were measured by flow cytometry, was expressed as a percentage of CD 34-positive cells and CD 3-positive cells. The vertical axis of the graph represents the composition ratio (%) of CD 34-positive cells and CD 3-positive cells before expansion to the total cells. The horizontal axis of the graph indicates the cell types of each experimental group for amplification. Regarding the composition ratio (%) of CD 34-positive cells before amplification, it was about 0.15% in CD 3-negative cells, and about 0.02% in CD3 and CD 34-negative cells. The composition ratio (%) of CD 3-positive cells before amplification was about 0.15% in CD 3-negative cells and about 0.25% in CD 3-and CD 34-negative cells.

Fig. 17 is a histogram showing the composition ratio of NK cells (CD 3 negative/CD 56 positive) in CD 3-negative cells and CD3 and CD 34-negative cells after expansion, relative to the whole cultured cells. The error bars for each experimental condition represent the standard error of the measured values of the experimental results repeated 3 times under the same condition. The component ratios of NK cells, CD 3-positive cells, and other cells were expressed as percentages of NK cells, CD 3-positive cells, and other cells in the total cultured cells of each experimental group, which were measured by flow cytometry. The vertical axis of the graph represents the percentage (%) of NK cells, CD 3-positive cells, and other cells relative to the total cultured cells. The horizontal axis of the graph indicates the cell types of each experimental group used for amplification. Regarding the composition ratio (%) of the expanded NK cells to the whole cultured cells, it was about 60% in CD 3-negative cells and about 90% in CD3 and CD 34-negative cells.

The experimental results of this example show that: the component ratio of NK cells (CD 3 negative/CD 56 positive) to the total cultured cells was significantly increased by removing CD3 positive cells and CD34 positive cells. Furthermore, even when NK cells were expanded using peripheral blood mononuclear cells collected by apheresis, it was shown that NK cells could be expanded with high purity by removing CD 3-positive cells and CD 34-positive cells.

Conclusion

From the above experimental results, it was found that a large amount of NK cells can be produced by removing CD3 positive cells (T cells) from peripheral blood-derived monocytes. Furthermore, it is clear from the experimental results of this example that the cells amplified by the method of the present invention have very high cytolytic activity. Further, by removing CD 3-positive cells (T cells) and CD 34-positive cells (hematopoietic precursor cells) from peripheral blood-derived monocytes, NK cells can be produced with high purity.

It is known that the cytolytic activity of NK cells is low in the currently reported method for amplifying NK cells. For example, with respect to NK cells derived from peripheral blood of healthy people, the amplification results of NK cells of Terunuma, h. The purity was 81.2%, the amplification factor was 130-fold, and the cytolytic activity was 66% (E: T = 3: 1) (patent document 1). In the case of peripheral blood mononuclear cells derived from healthy persons, the amplification results of NK cells of Tanaka, j. et al were: the purity was 73.4%, the amplification rate was 6268-fold, and the cytolytic activity was about 55% (E: T = 1: 1) (patent document 2). The amplification results of NK cells from peripheral blood of myeloma patients in Carlens, s, et al were: the purity was 55%, the amplification factor was 193-fold, and the cytolytic activity was 45% (E: T = 1: 1) (non-patent document 4). The amplification of NK cells from peripheral blood of myeloma patients, Alici, e, et al, resulted in: the purity was 65%, the amplification rate was 1625-fold, and the cytolytic activity was about 10% (E: T = 1: 1) (non-patent document 5). As for NK cells derived from peripheral blood of healthy persons, amplification results of NK cells of Fujisaki, h. et al were: when tumor cells genetically modified were used as feeder cells, the purity was 96.8%, the amplification factor was 277-fold, and the maximum cytolytic activity was about 90% (E: T = 1: 1) (non-patent document 6). In contrast, the NK cells derived from peripheral blood of healthy persons were amplified as follows: purity was about 90%, amplification rate was 400-fold, and cytolytic activity was about 100% (E: T = 1: 1). In the prior art, the cytolytic activity of NK cells against K562 cells is about 90% (E: T = 1: 1) at the maximum when genetically modified tumor cells are used as feeder cells, and 66% (E: T = 3: 1) when feeder cells are not used. However, the NK cells of the present invention were not expanded using feeder cells, and the cytolytic activity against K562 cells was about 100% (E: T = 1: 1). Therefore, the present invention is remarkably superior to the prior art because the cytolytic activity of NK cells is high and there is no risk of feeder cells mixing into the final product. Therefore, the present invention is useful for producing NK cells having high cytolytic activity in large quantities with high purity from collected blood cells.

Claims (11)

1. A method for amplifying NK cells, comprising:

a step of preparing a cell population containing NK cells;

a step of removing T cells from the cell population containing NK cells; and

culturing the remaining cells from which the T cells have been removed, without using feeder cells, in a medium containing only 2500 to 2813IU/mL of IL-2 as a cytokine.

2. The method for expanding NK cells according to claim 1, wherein the step of removing the T cells from the NK cell-containing cell population is performed by a step of removing CD3 positive cells.

3. The method for expanding NK cells according to claim 1 or 2, comprising a step of removing hematopoietic precursor cells from the cell population containing NK cells between the preparing step and the culturing step.

4. The method for expanding NK cells according to claim 3, wherein the step of removing the hematopoietic precursor cells from the NK cell-containing cell population is performed by a step of removing CD 34-positive cells.

5. The method for expanding NK cells according to claim 1 or 2, wherein the medium comprises autologous serum, AB-type serum, and/or serum albumin.

6. The method for expanding NK cells according to claim 1 or 2, wherein the step of preparing the cell population containing NK cells is performed by a step of separating monocytes from blood cells collected from a subject.

7. The method for expanding NK cells according to claim 6, wherein the blood cells are collected from peripheral blood, cord blood, bone marrow and/or lymph nodes.

8. The method for expanding NK cells according to claim 7, wherein the blood cells are collected from peripheral blood by apheresis.

9. The method according to claim 1 or 2, wherein the cell population containing NK cells is prepared from at least 1 cell selected from the group consisting of a stem cell-derived hematopoietic stem cell, a cord blood-derived hematopoietic stem cell, a peripheral blood-derived hematopoietic stem cell, a bone marrow blood-derived hematopoietic stem cell, a cord blood mononuclear cell, and a peripheral blood mononuclear cell, wherein the stem cell is any one selected from the group consisting of an embryonic stem cell, an adult stem cell, and an artificial pluripotent stem (iPS) cell.

10. A pharmaceutical composition for cell therapy, comprising the NK cell population prepared by the amplification method according to claim 1 or 2.

11. The pharmaceutical composition according to claim 10, for use in the treatment of infectious diseases and/or cancer.