COMPOSITION FOR CULTURING NATURAL KILLER CELLS, AND METHOD USING SAME
20220259562 · 2022-08-18
Assignee
Inventors
- KyuBum KWACK (Seongnam-si, KR)
- Sooyeon OH (Seoul, KR)
- Hae Jong LEE (Anyang-si, KR)
- Jeong Min SIM (Daejeon, KR)
- Jae Joon LIM (Suwon-si, KR)
Cpc classification
International classification
Abstract
Provided are a composition and a kit, each for culturing natural killer cells, and a method of using the same. According to the composition for culturing natural killer cells according to aspect, and a method of culturing natural killer cells using the same, when natural killer cells are cultured in peripheral blood mononuclear cells, they are cultured in a medium including the composition for culturing natural killer cells, the composition including magnetic particles, of which at least one surface is bound with an activating receptor ligand, an inhibitory receptor ligand, a costimulatory receptor ligand, a cytokine, a cytokine receptor, an immune checkpoint ligand, a blocking antibody, or a combination thereof, thereby proliferating natural killer cells in a large quantity and promoting activation or inhibition of natural killer cells, or expansion of natural killer cells. Accordingly, the natural killer cells cultured using the same may be usefully applied to immune cell therapy products. Further, the magnetic particles may be easily separated from the medium, which is a simple and economical manner. Since the safe magnetic particles are used, they are excellent in terms of clinical safety.
Claims
1. A composition for culturing natural killer cells, the composition comprising magnetic particles of which at least one surface is bound with an activating receptor ligand, an inhibitory receptor ligand, a costimulatory receptor ligand, a cytokine, a cytokine receptor, an immune checkpoint ligand, a blocking antibody, or a combination thereof.
2. The composition of claim 1, wherein the activating receptor ligand is one or more selected from the group consisting of an NCR family ligand, an NKG2 family ligand, a KIR family ligand, BAG6, AICL, MICA, MICB, CADM1, IgG, CD48, NTB-A/SLAMF6, CD70, CD155, CD319, C8, C9, and CS1.
3. The composition of claim 1, wherein the inhibitory receptor ligand is one or more selected from the group consisting of HLA-A, HLA-B, HLA-BW4, HLA-C1, HLA-C2, HLA-E, HLA-G, CD112/Nectin-2, CD112/Nectin-3, cadherin, collagen, OCIL, and CLEC2D.
4. The composition of claim 1, wherein the costimulatory receptor ligand is one or more selected from the group consisting of a TNF family ligand, a TLR family ligand, a 4-1BB ligand, CD28 ligand, NTBA, TLRL, PVR/Nectin-2, and PVR.
5. The composition of claim 1, wherein the cytokine is one or more selected from the group consisting of IFN-α, IFN-β, IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, and IL-27.
6. The composition of claim 1, wherein the cytokine receptor is IL-2Rα, IL-15Rα, or a combination thereof.
7. The composition of claim 1, wherein the immune checkpoint ligand is one or more selected from the group consisting of B7 family, galectin family, PVR family, PD-L1, a BTLA-4 ligand, a CTLA-4 ligand (CD80), a Tim-3 ligand, and a TIGIT ligand.
8. The composition of claim 1, wherein the blocking antibody is one or more selected from the group consisting of anti-KIR2DL1 monoclonal antibody (mAb), anti-KIR2DL2 mAb, anti-KIR2DL3 mAb, anti-KIR2DL5A mAb, anti-KIR2DL5B mAb, anti-KIR3DL1 mAb, anti-KIR3DL2 mAb, anti-KIR2DL4 mAb, anti-CD94/NKG2A mAb, anti-CD94/NKG2B mAb, anti-CD96 mAb, anti-CEACAM-1 mAb, anti-ILT2/LILRB mAb, anti-KLRG1 mAb, anti-LAIR1 mAb, anti-NKRP1A mAb, anti-Siglec3 mAb, anti-Siglec7 mAb, and anti-Siglec9 mAb.
9. The composition of claim 1, wherein at least one surface of the magnetic particles is coated with protein G or protein A.
10. The composition of claim 1, wherein the activating receptor ligand, the inhibitory receptor ligand, the costimulatory receptor ligand, the cytokine, the cytokine receptor, the immune checkpoint ligand, and the blocking antibody is in a fusion form with a human immunoglobulin.
11. The composition of claim 10, wherein the human immunoglobulin is human immunoglobulin G.
12. The composition of claim 1, wherein the magnetic particles have a size of 500 nm to 10 μm.
13. The composition of claim 1, wherein the culturing is for proliferating or activating natural killer cells.
14. The composition of claim 1, wherein the natural killer cells are comprised in peripheral blood mononuclear cells (PBMCs).
15. A method of culturing natural killer cells, the method comprising culturing natural killer cells in a medium comprising a composition for culturing natural killer cells, the composition comprising magnetic particles, of which at least one surface is bound with an activating receptor ligand, an inhibitory receptor ligand, a costimulatory receptor ligand, a cytokine, a cytokine receptor, an immune checkpoint ligand, a blocking antibody, or a combination thereof.
16. The method of claim 15, further comprising obtaining peripheral blood mononuclear cells, before the culturing.
17. The method of claim 15, further comprising removing the magnetic particles from the medium, after the culturing.
18. The method of claim 15, wherein the culturing is performed for 6 days to 21 days.
Description
BRIEF DESCRIPTION OF DRAWINGS
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MODE OF DISCLOSURE
[0078] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating one or more specific embodiments, and the scope of the present disclosure is not limited to these exemplary embodiments.
Example 1: Culture of Natural Killer (NK) Cells Using Magnetic Particles for Culturing NK Cells
[0079] 1.1 Preparation of Magnetic Particles for Culturing NK Cells
[0080] As magnetic particles which may serve as feed cells during culture of NK cells, 4-1BB ligand- or IL-15Rα-bound magnetic particles were prepared. To bind a specific protein (4-1BB ligand or IL-15Rα) to magnetic particles, magnetic particles coated with protein G were used. Protein G has very high binding affinity to human immunoglobulin. Therefore, when a specific protein fused with human immunoglobulin is used, the specific protein may be bound to magnetic particles through binding between protein G and the immunoglobulin.
[0081] In detail, Dynabeads (Thermo Fisher Scientific, Novex) which are magnetic particles coated with protein G were purchased. As the specific proteins, interleukin-15 receptor alpha (IL-15Rα) and 4-1BB ligand (4-1BBL) were used, and an IL-15Rα-fused immunoglobulin-tagged protein (hereinafter, referred to as ‘IL-15Rα_IgG1Fc’, R&D systems, Minneapolis, Minn., USA) and a 4-1BBL-fused immunoglobulin-tagged protein (hereinafter, referred to as ‘4-1BBL_IgG1Fc’, ACRObiosystems, Newark, Del., USA) were purchased.
[0082] The immunoglobulin-tagged protein and protein G-coated magnetic particles were allowed to react in a cold room for about 1 hour, and the supernatant was removed from MagneSphere magnetic stand (Promega, Madison, USA) by buffer aspiration, thereby preparing ‘magnetic particles for culturing NK cells’.
[0083] 1.2 Culture of NK Cells
[0084] NK cells were obtained from peripheral blood mononuclear cells (PBMCs) by culturing. PBMCs were obtained from 5 healthy blood donors under the Institutional Review Board (IRB) approval (1044308-201701-BR-005) by College of Medicine, CHA University. Each collected whole blood was diluted with phosphate buffered saline (PBS). PBMCs were isolated from each the blood sample diluted with PBS, and put in a SepMate tube (STEMCELL Technologies, Inc., Vancouver, Canada). PBMC isolation was performed by Ficoll-Hypaque density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St. Louis, USA).
[0085] Culturing of the obtained PBMCs was performed at a density of 1×10.sup.6 PBMCs/well in 24 wells. A portion of the medium was changed or added every 2 to 4 days depending on the morphology of PBMCs, and cell counting was performed every 6 days (days 6 and 12). PBMCs were maintained in a basic RPMI-1640 (Hyclone, Logan, USA) supplemented with 10% heat-inactivated FBS (Gibco, Thermo Fisher, USA), 50 μM beta-mercaptoethanol, and 1% penicillin and streptomycin (P/S).
[0086] Hereinafter, NK cells were cultured by a method of culturing NK cells in PBMCs using whole PBMCs obtained in Example 1.2, and experimental groups used for experiments were as follows: {circle around (1)} culture in a medium including soluble IL-15Rα or 4-1BBL, {circle around (2)} culture in a medium including the magnetic particles of Example 1.2, and {circle around (3)} culture in a medium including magnetic particles to which specific molecules were not bound.
Experimental Example 1: Evaluation of NK Cell Proliferation Using Magnetic Particles for Culturing NK Cells
[0087] NK cell proliferation efficiency, when the magnetic particles for culturing NK cells prepared in Example 1.1 were used, was evaluated. To measure NK cell proliferation, the three types of experimental groups were subjected to flow cytometry every 6 days (on day 0, day 6, and day 12). PBMC proliferation was measured using a hemocytometer. After PBMC counting, a portion of the sample was used for flow cytometry. PBMC samples were treated and stained with a CD3 PE antibody (Thermo Fisher Scientific) and a CD56 FITC antibody (Thermo Fisher Scientific). Through staining with these two antibodies, populations of NK cells, NKT cells, T cells, B cells, and monocytes in the obtained PBMCs may be distinguished.
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[0089] As shown in
[0090] It is known that a phenomenon of cluster formation by NK cell aggregation during culture indicates enhancement of NK cell activation. Therefore, when PBMCs were cultured using the 4-1BBL or IL-15Rα-bound magnetic particles according to an aspect, PBMCs well aggregated, indicating NK cell activation.
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[0092] As shown in
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[0094] As shown in
Experimental Example 2: Evaluation of Proportion of NK Cells in PBMCs According to Use of Magnetic Particles for Culturing NK Cells
[0095] It was evaluated whether a proportion of NK cells in PBMCs was changed, when the magnetic particles for culturing NK cells prepared in Example 1.1 were used. After PBMCs were stained with each CD marker (CD3, CD56), cell populations were examined by flow cytometry.
[0096] In detail, a proportion of NK cells in PBMCs on days 0, 6, and 12 of culture was measured using a CD3 PE antibody (Thermo Fisher Scientific) and a CD56 FITC antibody (BD Pharmingen™), which are NK cell markers, by a flow cytometer, CytoFLEX (Beckman Coulter, Inc., Brea, Calif., USA).
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[0098] As shown in
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[0100] In addition, to examine the change in the number of NK cells after culture, a percentage of NK cells obtained by antibody staining was multiplied by the number of each PBMC obtained in
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[0102] As shown in
[0103] In addition to the percentage data of NK cells (CD3-, CD56+), data on T cells (CD3+, CD56-), NKT cells (CD3+, CD56+), B cells, and monocytes (CD3-, CD56-) were also obtained in the same manner using CD3 and CD56 antibodies. The results of measuring a fold change in the percentage of whole PBMCs including NK cells (CD3-, CD56-) are shown in Table 1 below. Table 1 shows the result of charting the fold change of the increase rate of the whole PBMCs including NK cells, T cells, and NKT cells. Statistical analysis was performed by a paired t-test.
TABLE-US-00001 TABLE 1 sIL-15 sIL-15 +Magnetic sIL-15 +Magnetic particles sIL-15 +Magnetic particles (4-1BBL, (Control) particles (4-1BBL) IL-15Rα) Whole 1 ± 0.21 1.32 ± 0.25 2.07 ± 0.88 2.27 ± 0.88 PBMCs NK cells 1 ± 0.25 1.42 ± 0.31 4.90 ± 2.81* 6.08 ± 2.20* NKT cells 1 ± 0.30 1.91 ± 1.09 1.48 ± 0.30 1.97 ± 0.69 T cells 1 ± 0.46 0.93 ± 0.69 1.06 ± 0.24 1.13 ± 0.42 (*P < 0.05). Both the percentage and the number of NK cells significantly increased, when cultured with magnetic particles to which specific molecules were bound, indicating that, when PBMCs are cultured with magnetic particles to which specific molecules were bound, the environment inside PBMCs is induced to the NK cell dominant environment.
Experimental Example 3: Evaluation of PBMC-Mediated Cell Killing Ability According to Use of Magnetic Particles for Culturing NK Cells
[0104] To evaluate improvement of immune cell function of NK cells, in addition to the increase in the number of NK cells in PBMCs, by using the magnetic particles for culturing NK cells prepared in Example 1.1, PBMC-mediated cell killing ability against K562 was evaluated.
[0105] In detail, a leukemia cell line K562 was treated with PBMCs cultured for 12 days as described above. First, CFSE staining was performed to distinguish the target cell K562 from the effector cell PBMC in flow cytometry. Only the effector cells (NK cells) were gated and cultured with K562 for 4 hours, and then, 7-AAD staining was performed to detect dead K562 cells. Here, the co-culture was performed at a ratio of E:T=10:1. By detecting values of the 7-AAD staining, it was examined how many % of the target cells were killed. The dead cells were selected based on a K562 dot plot.
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[0108] As shown in
[0109] Therefore, since the cell killing ability against K562 is improved in the PBMC group cultured with magnetic beads according to an aspect, not only the number of cells but also the cytotoxic function is improved.
Experimental Example 4: Evaluation of Interferon Secretion of PBMCs According to Use of Magnetic Beads for Culturing NK Cells
[0110] To evaluate whether interferon secretion of PBMCs is improved by using the magnetic particles for culturing NK cells prepared in Example 1.1, an experiment was performed to quantify the amount of IFN-γ which is closely related to the cytotoxic function and highly secreted from activated NK cells.
[0111] The supernatant cultured for 12 days under each experimental condition as described above was used. IFN-γ was detected by enzyme-linked immune-specific assay (ELISA) using an antibody-coated IFN-γ-capture plate.
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[0113] As shown in
Experimental Example 5: Evaluation of Change in NK Cell Receptor Expression According to Use of Magnetic Particles for Culturing NK Cells
[0114] When the magnetic particles for culturing NK cells prepared in Example 1.1 were used, the proliferation and percentage of NK cells and PBMC-mediated cytotoxicity were increased, and therefore, changes in the NK cell receptor expression were analyzed.
[0115] In detail, samples obtained from 3 donors according to each group described above were cultured for 12 days, and then 6 types of receptor molecules were identified using antibodies against the receptor molecules. The used receptor molecules were DNAM1, CD27, NKG2A, NKG2D, CD69, and CD16. Experimental groups used are as follows: (A) soluble IL-15; (B) culture using soluble IL-15 and 4-1BBL_IgG1Fc-bound magnetic particles; and (C) culture using soluble IL-15 and 4-1BBL_IgG1Fc- and IL-15Rα_IgG1Fc-bound magnetic particles.
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[0117] In addition, the data on the NK cell receptor expression were prepared in a table and statistically processed. Paired t-tests were performed using data of the NK cell receptor expression rate of each donor.
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[0119] As shown in
[0120] Therefore, it was confirmed that, when NK cells are cultured using the magnetic particles according to an aspect, inhibitory receptors were decreased and activating receptors were increased in NK cells by culturing with the magnetic particles.