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KR-102961531-B1 - Method for preparing natural killer cells using poloxamer, natural killer cells produced thereby, and uses thereof

KR102961531B1KR 102961531 B1KR102961531 B1KR 102961531B1KR-102961531-B1

Abstract

The present invention relates to a method for producing natural killer cells. Furthermore, it relates to the use of natural killer cells in drugs for treating diseases. The method for producing natural killer cells according to the present invention enables the production of natural killer cells with a high cell proliferation rate through a simple process, and the natural killer cells produced according to the method of the present invention have high cytotoxicity.

Inventors

  • 나득채
  • 이미연
  • 김경운
  • 박진희
  • 김정훈

Assignees

  • 주식회사 온코인사이트

Dates

Publication Date
20260507
Application Date
20251014

Claims (13)

  1. A method for producing natural killer cells (NK cells), comprising the step of culturing hematopoietic stem cells in a culture medium containing poloxamer as an effective ingredient to increase the proliferation rate of natural killer cells, wherein the culture medium does not contain feeder cells.
  2. A method for producing natural killer cells according to claim 1, further comprising the step of inducing differentiation of the cultured hematopoietic stem cells into lymphocytic progenitor cells; and the step of differentiating the lymphocytic progenitor cells into natural killer cells.
  3. A method for producing natural killer cells according to claim 1, wherein the hematopoietic stem cells are isolated from bone marrow, umbilical cord blood, peripheral blood, or induced pluripotent stem cells.
  4. A method for producing natural killer cells according to paragraph 3, wherein the hematopoietic stem cells are isolated from umbilical cord blood.
  5. A method for producing natural killer cells according to claim 2, wherein the lymphoid progenitor cells are differentiated into natural killer cells in a culture medium containing one or more selected from the group consisting of Flt-3L, IL-15, and IL-21.
  6. A method for producing natural killer cells according to claim 5, wherein the Flt-3L concentration is about 1 to 250 ng/mL, about 10 to 150 ng/mL, or about 10 to 50 ng/mL, the IL-15 concentration is about 1 to 200 ng/mL, about 10 to 100 ng/mL, or about 10 to 40 ng/mL, and the IL-21 concentration is about 1 to 300 ng/mL, about 10 to 200 ng/mL, or about 10 to 60 ng/mL.
  7. A method for producing natural killer cells according to claim 1, wherein the culture period of the hematopoietic stem cells is 1 to 15 days.
  8. A method for producing natural killer cells according to claim 2, wherein the culture period of the step of inducing differentiation of the cultured hematopoietic stem cells into lymphocytic progenitor cells is 5 to 30 days, and the culture period of the step of differentiating the lymphocytic progenitor cells into natural killer cells is 5 to 30 days.
  9. In claim 1, the poloxamer is poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331, poloxamer 333, poloxamer 334, A method for producing natural killer cells, wherein one or more are selected from the group consisting of poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, and poloxamer 407.
  10. A method for producing natural killer cells according to claim 1, wherein the concentration of the poloxamer is about 0.1 μg/ml to 10,000 μg/ml, about 1 μg/ml to 10,000 μg/ml, about 0.1 μg/ml to 5,000 μg/ml, about 1 μg/ml to 5,000 μg/ml, or about 10 μg/ml to 5,000 μg/ml.
  11. A method for producing natural killer cells according to claim 1, wherein the natural killer cells have an increased proliferation rate compared to natural killer cells produced by culturing in a culture medium that does not contain poloxamer.
  12. A method for producing natural killer cells according to claim 1, wherein the natural killer cells have a reduced expression rate of NKG2A.
  13. delete

Description

Method for preparing natural killer cells using poloxamer, natural killer cells produced thereby, and uses thereof The present invention relates to a method for producing natural killer cells, natural killer cells produced according to the method, and uses thereof. Recently, research on T-cell-based cell therapies has been actively underway, and they have been approved and utilized as actual treatments for some blood cancers. However, autologous T cells are prone to a state of "T-cell exhaustion," where their function deteriorates due to repeated exposure to antigen stimulation within the patient's body, which can limit immune defense against infections or tumors. Furthermore, with the reporting of serious immunological side effects such as cytokine release syndrome (CRS), immune cell-associated neurotoxicity (ICANS), and graft-versus-host disease (GVHD), there is a growing need for safer alternative cell therapies. Against this backdrop, natural killer (NK) cells, a key component of the innate immune system, are garnering attention as a new therapeutic strategy. NK cells possess the characteristic of being able to autonomously recognize and eliminate abnormal cells while remaining unresponsive to normal cells. This selective cell-killing capability offers the advantage that NK cell-based therapies have a lower risk of immunological side effects compared to existing T-cell-based therapies. Furthermore, since NK cells can be cultured among allogeneic cells, they possess relatively high scalability in terms of therapeutic development and commercialization. Currently, various cell origins are being utilized to apply NK cells as therapeutic agents, including cell lines such as peripheral blood, umbilical cord blood, hematopoietic stem cells, induced pluripotent stem cells (iPSCs), and NK-92. Most NK cell expansion and activation are performed using methods that rely on feeder cells. While this approach entails issues such as the potential for feeder cell retention and increased production costs due to removal processes, it also has the disadvantage of low cell proliferation rates in the absence of feeder cells. Accordingly, there is a need for the development of culture methods that can stably mass-produce NK cells using technology that does not require feeder cells. Meanwhile, poloxamer is primarily used as a surfactant and there is prior literature stating that it is used as an adjuvant to improve the efficiency of intracellular introduction of viral vectors, but it is not known whether it affects NK cell differentiation. Figure 1 is a schematic diagram showing a method for producing natural killer cells (NK cells) derived from hematopoietic stem cells (HSC) and NK cells introduced with CAR. Figure 2 is an optical microscope image of cells during the HSC proliferation and Common Lymphoid Progenitor (CLP) expansion stages in the process of differentiating HSCs into NK cells. Figure 3 is a graph showing the proliferation rate (a), survival rate (b), and number of CD56-positive cells (c) of differentiated NK cells. Figure 4 shows raw data (a) and graph (b) obtained by flow cytometry to confirm the distribution of CD45+CD56+ cells in order to confirm the purity of differentiated NK cells. Figure 5 is a table (c) showing raw data (a), a graph (b), and numerical values obtained by flow cytometry to confirm the distribution of CD45+CD3+, CD45+CD14+, and CD45+CD19+ cells in order to confirm the purity of differentiated NK cells. Figure 6 shows raw data (a), a graph (b), and a table (c) showing the expression of NK cell receptors confirmed through flow cytometry after the completion of NK cell differentiation. Figure 7 shows FACS raw data (a) and a table and graph (b) showing the values of cytotoxicity of differentiated NK cells against K562 cells, a blood cancer cell line, according to the E:T ratio. Figure 8 shows FACS raw data (a) confirming the cytotoxicity of differentiated NK cells against HER2-positive breast cancer cell line MDA-MB-453 cells according to the E:T ratio, and a table and graph (b) showing the values. The present invention will be described in more detail below through examples. These examples are provided solely as illustrations to explain the invention more specifically, and the scope of the invention is not limited by these examples. Example 1. Preparation of Natural Killer (NK) Cells 1-1. Isolation of CD34+ Hematopoietic Stem Cells (HSCs) from Umbilical Cord Blood and Flow Cytometry To isolate CD34+ HSCs from umbilical cord blood, the Ficoll-Paque method using SepMate™ (Stemcell) Tubes was employed. CD34+ HSCs were isolated from the separated buffy coat using the MACSprep kit (Miltenyi). The cells isolated from the kit were analyzed using anti-CD34-PE (Biolegend) to confirm a CD34+ HSC isolation rate of over 90%. 1-2. Differentiation from HSCs to NK Cells (a) Days 0–7: Cells were cultured for 4 days at a concentration of 1 x 10⁵ cells/mL using StemSpan SFEM II medium and CC110, a cytokine mixt