WO2024113294A1 - Procédé de différenciation de cellules nk, milieu de culture et son utilisation - Google Patents

Procédé de différenciation de cellules nk, milieu de culture et son utilisation Download PDF

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WO2024113294A1
WO2024113294A1 PCT/CN2022/135764 CN2022135764W WO2024113294A1 WO 2024113294 A1 WO2024113294 A1 WO 2024113294A1 CN 2022135764 W CN2022135764 W CN 2022135764W WO 2024113294 A1 WO2024113294 A1 WO 2024113294A1
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cells
medium
stem
culture medium
differentiation
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PCT/CN2022/135764
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吴晨
李超
朱浩
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血霁生物科技(上海)有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present application relates to the field of biomedicine, and specifically to a method for differentiating NK cells.
  • Natural Killer (NK) cells are large granular cells of the immune system, constituting the third major lymphocyte subset in the human body and are natural cytotoxic lymphocytes. NK cells have the ability to rapidly mediate cytotoxicity against pathogen-infected or malignantly transformed cells, and can produce a variety of chemokines and cytokines that affect other immune cell populations, participating in a variety of immune surveillance and elimination of pathogens, senescent cells and cancer processes in the body. NK cells have a limited lifespan in the body and must therefore be continuously replenished to maintain homeostasis.
  • NK human natural killer
  • the present application provides a method for differentiating NK cells.
  • the method provided in the present application adds human platelet lysate (hPL) to the NK differentiation medium, which contains rich cytokines and nutrients, and can effectively improve the efficiency of stem/progenitor cells differentiating into NK cells.
  • hPL human platelet lysate
  • NK cell killing experiments have shown that the NK cells generated by this method have good killing effects.
  • the present application provides a method for differentiating NK cells, comprising adding human platelet lysate (hPL) to NK differentiation medium.
  • hPL human platelet lysate
  • the present application provides a method for inducing stem/progenitor cells to differentiate into NK cells, comprising adding human platelet lysate to NK differentiation medium.
  • the stem/progenitor cells described in the method are hematopoietic stem/progenitor cells.
  • the stem/progenitor cells in the method are CD34 + hematopoietic stem/progenitor cells.
  • the stem/progenitor cells described in the methods are lymphoid progenitor cells (CLPs).
  • CLPs lymphoid progenitor cells
  • the stem/progenitor cells described in the method are pluripotent stem cells.
  • the stem/progenitor cells described in the method are induced pluripotent stem cells.
  • the stem/progenitor cells described in the methods are human induced pluripotent stem cells.
  • the NK cell differentiation medium in the method may further comprise a basal medium, the medium may comprise serum, or the medium may be serum-free.
  • the basal culture medium in the method can be selected from one or more of the following groups: IMDM, DMEM, F12, StemSpan TM SFEM II, APEL and mTeSR.
  • the base culture medium in the method may be DMEM.
  • the NK cell differentiation medium in the method may further comprise one or more selected from the group consisting of nutrients, antibiotics, human platelet lysate, ⁇ -mercaptoethanol, ethanolamine, and cytokines.
  • the NK cell differentiation medium in the method may include DMEM+GlutaMAX TM medium, "Ham's F-12 Nutrient Mix, GlutaMAX TM Supplement medium”, human platelet lysate (hPL), L-glutamine, ⁇ -mercaptoethanol and ethanolamine.
  • the NK cell differentiation medium in the method may comprise about 56.6% DMEM+GlutaMAX TM ; about 28.3% F12+GlutaMAX TM -I; about 10% hPL; about 1% P/S; about 2 mM L-glutamine; about 25 uM ⁇ -mercaptoethanol; and about 50 uM ethanolamine.
  • the NK cell differentiation medium in the method may contain IL-3, IL7, IL-15, SCF and Flt-3.
  • the NK cell differentiation medium in the method may contain IL-7, IL-15, SCF and Flt-3.
  • the method comprises administering SCF at a concentration of about 15-25 ng/mL.
  • the method comprises administering IL3 at a concentration of about 3-7 ng/mL.
  • the method comprises administering IL7 at a concentration of about 15-25 ng/mL.
  • the method comprises administering IL15 at a concentration of about 8-12 ng/mL.
  • the method comprises administering Flt3-L at a concentration of about 8-12 ng/mL.
  • the method wherein the NK differentiation medium comprises NK differentiation medium I.
  • the NK differentiation medium I in the method comprises about 56.6% DMEM+GlutaMAX TM ; about 28.3% F12+GlutaMAX TM -I; about 10% hPL; about 1% P/S; about 2mM L-glutamine; about 25uM ⁇ -mercaptoethanol; about 50uM ethanolamine; IL-3; IL7; IL-15; SCF and Flt-3.
  • the NK differentiation medium in the method comprises NK differentiation medium II.
  • the NK differentiation medium II in the method comprises about 56.6% DMEM+GlutaMAX TM ; about 28.3% F12+GlutaMAX TM -I; about 10% hPL; about 1% P/S; about 2mM L-glutamine; about 25uM ⁇ -mercaptoethanol; about 50uM ethanolamine; IL-7, IL-15, SCF and Flt-3.
  • the method may include one or more of the following steps: (1) inoculating hematopoietic stem/progenitor cells in NK differentiation medium I and culturing for about 6 days; (2) continuing to culture using NK differentiation medium I for about 3-5 days; (3) continuing to culture using NK differentiation medium II with a full replacement of the medium, and replacing half of the medium with the NK differentiation medium II every 5-6 days during the culture period, and culturing for 30-35 days or until NK cells are obtained.
  • the culture conditions in the method can be about 35-39°C, 3-7% CO 2 .
  • the differentiated NK cells in the method may comprise modified NK cells.
  • the modified NK cells in the methods comprise CAR-NK cells.
  • the present application also provides a NK cell differentiation medium, which contains the addition of human platelet lysate (hPL).
  • hPL human platelet lysate
  • the NK cell differentiation medium may contain serum, or the medium may be serum-free.
  • the NK cell differentiation medium may further comprise a basal medium.
  • the basal medium in the NK cell differentiation medium can be selected from one or more of the following groups: IMDM, DMEM, F12, StemSpan TM SFEM II, APEL and mTeSR.
  • the basal medium in the NK cell differentiation medium comprises DMEM.
  • the NK cell differentiation medium may further comprise one or more selected from the group consisting of nutrients, antibiotics, human platelet lysate, ⁇ -mercaptoethanol, ethanolamine, and cytokines.
  • the NK cell differentiation medium may include DMEM+GlutaMAX TM medium, "Ham's F-12 Nutrient Mix, GlutaMAX TM Supplement medium”, human platelet lysate (hPL), L-glutamine, ⁇ -mercaptoethanol and ethanolamine.
  • the NK cell differentiation medium may comprise about 56.6% DMEM+GlutaMAX TM ; about 28.3% F12+GlutaMAX TM -I; about 10% hPL; about 1% P/S; about 2 mM L-glutamine; about 25 uM ⁇ -mercaptoethanol; and about 50 uM ethanolamine.
  • the NK cell differentiation medium may contain IL-3, IL7, IL-15, SCF, and Flt-3.
  • the NK cell differentiation medium may contain IL-7, IL-15, SCF, and Flt-3.
  • the concentration of SCF in the NK cell differentiation medium is about 15-25 ng/mL.
  • the concentration of IL3 in the NK cell differentiation medium is about 3-7 ng/mL.
  • the concentration of IL7 in the NK cell differentiation medium is about 15-25 ng/mL.
  • the concentration of IL15 in the NK cell differentiation medium is about 8-12 ng/mL.
  • the concentration of Flt3-L in the NK cell differentiation medium is about 8-12 ng/mL.
  • the NK cell differentiation medium comprises NK differentiation medium I.
  • the NK cell differentiation medium I in the NK cell differentiation medium comprises about 56.6% DMEM+GlutaMAX TM ; about 28.3% F12+GlutaMAX TM -I; about 10% hPL; about 1% P/S; about 2mM L-glutamine; about 25uM ⁇ -mercaptoethanol; about 50uM ethanolamine; IL-3; IL7; IL-15; SCF and Flt-3.
  • the NK cell differentiation medium comprises NK differentiation medium II.
  • the NK cell differentiation medium II in the NK cell differentiation medium comprises about 56.6% DMEM+GlutaMAX TM ; about 28.3% F12+GlutaMAX TM -I; about 10% hPL; about 1% P/S; about 2mM L-glutamine; about 25uM ⁇ -mercaptoethanol; about 50uM ethanolamine; IL-7, IL-15, SCF and Flt-3.
  • the present application also provides a set of culture medium for differentiating NK cells from hematopoietic stem/progenitor cells.
  • the culture medium set comprises a hematopoietic stem/progenitor cell differentiation medium, which can induce stem cells to differentiate into hematopoietic stem/progenitor cells.
  • the stem cells comprise induced pluripotent stem cells.
  • the stem cells comprise embryonic stem cells.
  • the hematopoietic stem/progenitor cells are CD34+ hematopoietic stem/progenitor cells.
  • the NK cells comprise modified NK cells.
  • the modified NK cells comprise CAR-NK cells.
  • the present application also provides the use of the culture medium and the set of culture medium in generating NK cells by differentiating stem/progenitor cells.
  • the stem/progenitor cells comprise hematopoietic stem/progenitor cells.
  • the stem/progenitor cells include CD34+ hematopoietic stem/progenitor cells differentiated from induced pluripotent stem cells (iPS cells).
  • iPS cells induced pluripotent stem cells
  • the stem/progenitor cells include CD34+ hematopoietic stem/progenitor cells isolated from human blood ex vivo.
  • the stem/progenitor cells comprise pluripotent stem cells.
  • the NK cells comprise modified NK cells.
  • the modified NK cells comprise CAR-NK cells.
  • the present application also provides a culture platform for differentiating NK cells, which comprises the described method, the described culture medium, the described set of culture medium, and/or stem/progenitor cells.
  • the present application also provides a composition comprising stem/progenitor cells and the culture medium and the set of culture medium.
  • FIG1 shows the flow cytometry analysis results of NK cells cultured according to the method described in the present application.
  • FIG. 2 shows the experimental results of NK cells killing A549 and HepG2 described in the present application.
  • FIG. 3 shows the experimental results of NK cell killing K562 described in the present application.
  • the term "modified" generally refers to the changed state or structure of the cells described in the present application. This modification can be artificial or it can be caused by environmental conditions compared to the natural source. In the present application, the modification can be carried out in various ways known to those skilled in the art, for example, by physical methods, biological methods, chemical methods, etc., to modify the cells structurally and/or functionally.
  • cells can be modified by introducing nucleic acids.
  • cells can be modified by genetic engineering.
  • the NK cells may include modified NK cells.
  • the modification may include modifying the NK cells so that the NK cells express a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • human platelet lysate is generally derived from human platelets, which contain a variety of cell growth factors.
  • the human platelet lysate may include platelets from various sources, for example, the human platelet lysate may be derived from human platelets collected from blood donations.
  • the human platelet lysate may be derived from platelets separated and purified from a blood sample.
  • the human platelet lysate may be derived from platelets obtained by various cell differentiations, for example, platelets obtained by differentiation of hematopoietic stem cells, for example, platelets obtained by differentiation of induced pluripotent stem cells (iPSC), for example, platelets obtained by differentiation of megakaryocyte progenitor cells (MKP).
  • iPSC induced pluripotent stem cells
  • MKP megakaryocyte progenitor cells
  • embryonic stem cells is also called “embryonic stem cells”, which can be abbreviated as “ESC”, and generally refers to cells with the characteristics of unlimited proliferation, self-renewal and multidirectional differentiation.
  • Embryonic stem cells are stem cells obtained from the undifferentiated inner cell mass of the blastocyst (early embryonic stage). There is no restriction on their source and preparation method. Embryonic stem cells can be induced to differentiate into almost all cell types of the body, whether in vitro or in vivo. For example, the cell types can be hematopoietic stem cells, neural cells, cardiomyocytes, etc.
  • NK cell is also referred to as "natural killer cell”, which generally refers to a cell with large granules in the cytoplasm.
  • NK cells develop from bone marrow lymphoid stem cells and can differentiate and develop depending on the bone marrow or thymus microenvironment. They are mainly distributed in peripheral blood and spleen, and also exist in small amounts in lymph nodes and other tissues.
  • the term covers unmodified NK cells, as well as modified NK cells.
  • the NK cells may include artificially modified cells.
  • the cells may include CAR-NK cells.
  • differentiated generally refers to the process by which a non-specific or less specific cell acquires the characteristics of a specific cell.
  • a differentiated or differentiation-induced cell is a cell that occupies a more specific position in a cell lineage.
  • stem cell generally refers to a self-replicating and multipotent cell having one or more of the following properties: (1) long-term self-renewal, or the ability to produce at least one identical copy of the original cell, (2) differentiation into multiple, and in some cases, only one specialized cell type at the single cell level, and (3) functional regeneration of tissue in vivo.
  • Stem cells are subdivided into totipotent, pluripotent, multipotent, and oligo/unipotent according to their developmental potential.
  • progenitor cells generally also have the ability to self-renew and differentiate into more mature cells, but are committed to a certain lineage (e.g., hematopoietic progenitor cells are committed to the blood lineage; myeloid progenitor cells are committed to the bone marrow lineage; lymphoid progenitor cells are committed to the lymphoid lineage), while stem cells do not necessarily have such limitations.
  • hematopoietic progenitor cells are committed to the blood lineage
  • myeloid progenitor cells are committed to the bone marrow lineage
  • lymphoid progenitor cells are committed to the lymphoid lineage
  • stem cells do not necessarily have such limitations.
  • Hematopoietic stem cells give rise to committed hematopoietic progenitor cells (HPCs) that can form the pool of mature blood cells throughout the life of an organism.
  • HPC hematopoietic progenitor cells
  • myeloid lineages e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • lymphoid lineages e.g., T-cells, B-cells, NK-cells
  • pluripotent stem cell generally refers to a cell that has the developmental potential to differentiate into one or more germ layers (ectoderm, mesoderm, and endoderm) but not all three germ layers. Pluripotent cells may also be referred to as “partially differentiated cells”. Pluripotent cells are well known in the art, and examples of pluripotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Pluripotent" indicates that the cell can form many types of cells in a given lineage, but cannot form cells of other lineages.
  • pluripotent hematopoietic cells can form many different types of blood cells (red blood cells, white blood cells, platelets, etc.), but cannot form neurons. Therefore, the term “pluripotency” refers to a cell state with a degree of developmental potential lower than that of omnipotence and pluripotency.
  • induced pluripotent stem cell can be generally abbreviated as iPS cell or iPSC, and generally refers to a type of pluripotent stem cell prepared from non-pluripotent cells in an artificial manner.
  • the artificial manner can be the introduction of reprogramming factors.
  • the non-pluripotent cells can be adult somatic cells or terminally differentiated cells, for example, fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells, etc.
  • cytokine generally refers to a compound or component (e.g., an autoimmune factor) produced by a cell and affecting the physiological state of the cell (self) or other cells that produce the cytokine.
  • Cytokines also include any compound or component produced by recombinant or synthetic processing, and the products of these processing have similar structures and/or biological activities to naturally occurring forms.
  • the cytokine also encompasses truncated forms, functionally active fragments, homologues, analogs and variants thereof.
  • the term "marker phenotype” generally refers to identifying markers or antigens on cells to determine their phenotype (e.g., differentiation state and/or cell type).
  • immunophenotyping can be used, which uses antibodies to recognize antigens presented on cells.
  • Antibodies can be monoclonal or polyclonal, and are usually selected to have less cross-reaction with other cell markers. These markers that determine the same cell type between species can be identified based on the same markers, and there may be differences in the structure of these markers between species (e.g., amino acid sequences).
  • Cell markers can include cell differentiation markers, as well as gene expression markers. Gene expression markers can include expressed genes that can indicate cell type or differentiation state.
  • composition generally refers to a product comprising a specified amount of a specified ingredient, as well as any product produced directly or indirectly by a combination of specified amounts of a specified ingredient.
  • the composition may also include other inactive ingredients, for example, carriers, excipients, adjuvants, stabilizers, etc.
  • ex vivo generally refers to operations involving cells, tissues and/or organs that have been removed from an organism.
  • the cells, tissues and/or organs can be returned to the organism by certain methods, or enter another organism.
  • in vitro generally refers to removing or releasing a part from an organism.
  • the term "about” generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.
  • the present application provides a method for obtaining NK cells by differentiation, which comprises adding platelet lysate to NK differentiation medium.
  • the platelet lysate is human platelet lysate.
  • the initiating cell of NK cell differentiation can be any cell with NK cell differentiation potential.
  • the differentiation initiating cell can be a stem/progenitor cell.
  • the differentiation initiating cell can be selected from one or more of the following groups: pluripotent cells, pluripotent stem cells, induced pluripotent stem cells, embryonic stem cells, hematopoietic stem cells, hematopoietic stem/progenitor cells, lymphoid progenitor cells (CLP), etc.
  • the hematopoietic stem/progenitor cells may be derived from induced pluripotent stem cells.
  • the hematopoietic stem/progenitor cells are derived from ex vivo human blood.
  • the hematopoietic stem/progenitor cells are derived from umbilical cord blood.
  • the hematopoietic stem/progenitor cells are derived from bone marrow.
  • the hematopoietic stem/progenitor cells are CD34+ hematopoietic stem/progenitor cells.
  • the NK differentiation initiating cells may be of natural origin or modified.
  • the NK differentiation initiating cells may be artificially modified by physical methods, chemical methods and/or biological methods.
  • the expression of certain genes of the NK differentiation initiating cells may be adjusted.
  • the source of the stem/progenitor cells in the method is not limited, and can be of mammalian or non-mammalian origin.
  • the method includes the process of differentiating stem/progenitor cells into NK cells.
  • the method includes the process of differentiating hematopoietic stem/progenitor cells into NK cells.
  • the method includes the process of differentiating CD34+ hematopoietic stem/progenitor cells into NK cells.
  • the method includes the process of differentiating pluripotent cells into NK cells.
  • the method includes the process of differentiating pluripotent stem cells into NK cells.
  • the method includes the process of differentiating induced pluripotent stem cells into NK cells.
  • the method includes the process of differentiating human induced pluripotent stem cells into NK cells.
  • the cells include the process of differentiating megakaryocyte progenitor cells into NK cells.
  • the NK differentiation medium may be a serum-free medium.
  • the NK differentiation medium may include any NK differentiation medium known in the art, and human platelet lysate is added to the NK differentiation medium to promote the differentiation of the differentiation initiating cells into NK cells.
  • the human platelet lysate in the NK differentiation medium, can be used instead of serum to promote differentiation of differentiation initiating cells into NK cells.
  • the serum can be fetal bovine serum.
  • the serum can be AB serum.
  • the culture method may include the following steps: (1) inoculating stem/progenitor cells in NK differentiation medium I; (2) continuing to culture using NK differentiation medium II; (3) continuing to culture using NK differentiation medium II to obtain NK cells.
  • the culture method may include the following steps: (1) inoculating hematopoietic stem/progenitor cells in NK differentiation medium I and culturing for about 6 days; (2) continuing to culture using NK differentiation medium I for about 3-5 days; (3) continuing to culture using NK differentiation medium II with full medium replacement, and replacing half of the NK differentiation medium II every 5-6 days during the culture period, and culturing for 30-35 days or until NK cells are obtained.
  • the culture may be cultured at a condition of about 35-39° C.
  • a condition of about 35-39° C. For example, about 34.5° C., about 35° C., about 35.5° C., about 36.° C., about 36.5° C., about 37° C., about 37.5° C., about 38° C., about 38.5° C., about 39° C., about 39.5° C.
  • the culture may be cultured under conditions of about 3-7% CO 2.
  • the culture method can be performed under culture conditions with serum.
  • the culture method can be performed under serum-free culture conditions.
  • the culture method can be performed under feeder-free culture conditions.
  • the culture method can be carried out under culture conditions with feeding.
  • the culture medium in the culture method may be supplemented with one or more substances, including but not limited to: nutrients/extracts, growth factors, hormones, cytokines and/or culture medium additives.
  • the method may be an in vitro method.
  • the method may be an in vitro method.
  • the method may be a method for the purpose of non-disease diagnosis and treatment.
  • the present application provides a culture medium comprising an NK differentiation medium supplemented with human platelet lysate (hPL).
  • hPL human platelet lysate
  • the method may include using the culture medium described in the present application for cell differentiation to obtain NK cells.
  • the NK differentiation medium may not contain serum.
  • the content of the human platelet lysate is about 10%.
  • the content of the hPL is about 8%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9.0%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%
  • the NK differentiation medium may include a basal medium.
  • the basal medium may include any medium known in the art.
  • the basal medium may include IMDM, MEM, Ham’s F12, DMEM, RPMI1640, mTeSR1, APEL.
  • the basal medium may include DMEM.
  • the NK differentiation medium further comprises one or more selected from the following group: nutrients, antibiotics, human platelet lysate, ⁇ -mercaptoethanol, ethanolamine and cytokines.
  • the nutrients may include: L-glutamine, GlutaMAX TM .
  • the concentration of the L-glutamine is about 2 mM.
  • the concentration of the L-glutamine is about 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3.0 mM.
  • the antibiotic can be selected from: penicillin, streptomycin.
  • the content of the antibiotic is about 1%.
  • the content of the antibiotic is about 0.25%, about 0.3%, about 0.35%, about 0.40%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 1.05%, about 1.1%, about 1.15%, about 1.2%, about 1.25%, about 1.3%, about 1.35%, about 1.4%, about 1.45%, about 1.5%, about 1.55%, about 1.6%, about 1.65%, about 1.7%, about 1.75%.
  • the cytokine may be selected from the group consisting of: SCF, IL3, IL7, IL15 and Flt3-L.
  • the concentration of the cytokine is about 1-50 ng/mL.
  • the concentration of the cytokine is about 1-50 ng/mL, about 1-45 ng/mL, about 1-40 ng/mL, about 1-30 ng/mL, about 3-7 ng/mL, about 8-12 ng/mL or about 15-25 ng/mL.
  • the NK differentiation medium may include DMEM+GlutaMAX TM medium, "Ham's F-12 Nutrient Mix, GlutaMAX TM Supplement medium”, human platelet lysate (hPL), L-glutamine, ⁇ -mercaptoethanol and ethanolamine.
  • the NK differentiation medium may comprise about 56.6% DMEM+GlutaMAX TM ; about 28.3% F12+GlutaMAX TM -I; about 10% hPL; about 1% P/S; about 2 mM L-glutamine; about 25 uM ⁇ -mercaptoethanol; and about 50 uM ethanolamine.
  • the concentration of ⁇ -mercaptoethanol is about 20uM, about 21uM, about 22uM, about 23uM, about 24uM, about 25uM, about 26uM, about 27uM, about 28uM, about 29uM or about 30uM.
  • the concentration of the ethanolamine is about 50uM.
  • the concentration of the ethanolamine is about 35uM, about 36uM, about 37uM, about 38uM, about 39uM, about 40uM, about 41uM, about 42uM, about 43uM, about 44uM, about 45uM, about 46uM, about 47uM, about 48uM, about 49uM, about 50uM, about 51uM, about 52uM, about 53uM, about 54uM, about 55uM, about 56uM, about 57uM, about 58uM, about 59uM, about 60uM, about 61uM, about 62uM, about 63uM, about 64uM or about 65uM.
  • the NK differentiation medium may further contain IL-3, IL7, IL-15, SCF and/or Flt-3.
  • the concentration of the SCF is about 15-25 ng/mL.
  • the concentration of the SCF is about 15-25 ng/mL, about 15-24 ng/mL, about 15-23 ng/mL, about 15-22 ng/mL, about 15-21 ng/mL, about 15-22 ng/mL, about 16-25 ng/mL, about 17-25 ng/mL, about 18-25 ng/mL, about 19-25 ng/mL, about 20-25 ng/mL, about 21-25 ng/mL, about 22-25 ng/mL, about 23-25 ng/mL or about 24-25 ng/mL.
  • the concentration of IL3 is about 3-7 ng/mL.
  • the concentration of IL3 is about 3-6.5 ng/mL, about 3-6 ng/mL, about 3-5.5 ng/mL, about 3-5 ng/mL, about 3-4.5 ng/mL, about 3-4 ng/mL, about 3.5-7 ng/mL, about 4.5-7 ng/mL, about 5.5-7 ng/mL or about 6-7 ng/mL.
  • the concentration of the IL7 is about 15-25 ng/mL.
  • the concentration of the IL7 is about 15-25 ng/mL, about 15-24 ng/mL, about 15-23 ng/mL, about 15-22 ng/mL, about 15-21 ng/mL, about 15-22 ng/mL, about 16-25 ng/mL, about 17-25 ng/mL, about 18-25 ng/mL, about 19-25 ng/mL, about 20-25 ng/mL, about 21-25 ng/mL, about 22-25 ng/mL, about 23-25 ng/mL or about 24-25 ng/mL.
  • the concentration of IL15 is about 8-12 ng/mL.
  • the concentration of IL15 is about 8-11.5 ng/mL, about 8-11 ng/mL, about 8-10.5 ng/mL, about 8-10 ng/mL, about 8-9.5 ng/mL, about 8-9 ng/mL, about 8.5-12 ng/mL, about 9-12 ng/mL, about 9.5-12 ng/mL, about 10-12 ng/mL, about 10.5-12 ng/mL, about 11-12 ng/mL or about 11.5-12 ng/mL.
  • the concentration of Flt3-L is about 8-12 ng/mL.
  • the concentration of Flt3-L is about 8-11.5 ng/mL, about 8-11 ng/mL, about 8-10.5 ng/mL, about 8-10 ng/mL, about 8-9.5 ng/mL, about 8-9 ng/mL, about 8.5-12 ng/mL, about 9-12 ng/mL, about 9.5-12 ng/mL, about 10-12 ng/mL, about 10.5-12 ng/mL, about 11-12 ng/mL, or about 11.5-12 ng/mL.
  • the NK differentiation medium may include NK differentiation medium I, which includes about 56.6% DMEM+GlutaMAX TM , about 28.3% F12+GlutaMAX TM -I, about 10% hPL, about 1% P/S, about 2mM L-glutamine, about 25uM ⁇ -mercaptoethanol, about 50uM ethanolamine, IL-3, IL7, IL-15, SCF and Flt-3.
  • NK differentiation medium I which includes about 56.6% DMEM+GlutaMAX TM , about 28.3% F12+GlutaMAX TM -I, about 10% hPL, about 1% P/S, about 2mM L-glutamine, about 25uM ⁇ -mercaptoethanol, about 50uM ethanolamine, IL-3, IL7, IL-15, SCF and Flt-3.
  • the NK differentiation medium may include NK differentiation medium II, which includes about 56.6% DMEM+GlutaMAX TM , about 28.3% F12+GlutaMAX TM -I, about 10% hPL, about 1% P/S, about 2mM L-glutamine, about 25uM ⁇ -mercaptoethanol, about 50uM ethanolamine, IL-7, IL-15, SCF and Flt-3.
  • NK differentiation medium II which includes about 56.6% DMEM+GlutaMAX TM , about 28.3% F12+GlutaMAX TM -I, about 10% hPL, about 1% P/S, about 2mM L-glutamine, about 25uM ⁇ -mercaptoethanol, about 50uM ethanolamine, IL-7, IL-15, SCF and Flt-3.
  • the concentration of the SCF is about 15-25 ng/mL.
  • the concentration of the SCF is about 15-25 ng/mL, about 15-24 ng/mL, about 15-23 ng/mL, about 15-22 ng/mL, about 15-21 ng/mL, about 15-22 ng/mL, about 16-25 ng/mL, about 17-25 ng/mL, about 18-25 ng/mL, about 19-25 ng/mL, about 20-25 ng/mL, about 21-25 ng/mL, about 22-25 ng/mL, about 23-25 ng/mL or about 24-25 ng/mL.
  • the concentration of IL3 is about 3-7 ng/mL.
  • the concentration of IL3 is about 3-6.5 ng/mL, about 3-6 ng/mL, about 3-5.5 ng/mL, about 3-5 ng/mL, about 3-4.5 ng/mL, about 3-4 ng/mL, about 3.5-7 ng/mL, about 4.5-7 ng/mL, about 5.5-7 ng/mL or about 6-7 ng/mL.
  • the concentration of IL7 is about 15-25 ng/mL.
  • the concentration of IL7 is about 15-25 ng/mL, about 15-24 ng/mL, about 15-23 ng/mL, about 15-22 ng/mL, about 15-21 ng/mL, about 15-22 ng/mL, about 16-25 ng/mL, about 17-25 ng/mL, about 18-25 ng/mL, about 19-25 ng/mL, about 20-25 ng/mL, about 21-25 ng/mL, about 22-25 ng/mL, about 23-25 ng/mL or about 24-25 ng/mL.
  • the concentration of IL15 is about 8-12 ng/mL.
  • the concentration of IL15 is about 8-11.5 ng/mL, about 8-11 ng/mL, about 8-10.5 ng/mL, about 8-10 ng/mL, about 8-9.5 ng/mL, about 8-9 ng/mL, about 8.5-12 ng/mL, about 9-12 ng/mL, about 9.5-12 ng/mL, about 10-12 ng/mL, about 10.5-12 ng/mL, about 11-12 ng/mL or about 11.5-12 ng/mL.
  • the concentration of Flt3-L is about 8-12 ng/mL.
  • the concentration of Flt3-L is about 8-11.5 ng/mL, about 8-11 ng/mL, about 8-10.5 ng/mL, about 8-10 ng/mL, about 8-9.5 ng/mL, about 8-9 ng/mL, about 8.5-12 ng/mL, about 9-12 ng/mL, about 9.5-12 ng/mL, about 10-12 ng/mL, about 10.5-12 ng/mL, about 11-12 ng/mL or about 11.5-12 ng/mL.
  • the present application also provides a set of culture medium, which may include the NK differentiation medium described in the present application.
  • the present application may also include other differentiation mediums.
  • the other differentiation mediums may include hematopoietic stem/progenitor cell differentiation medium, and the hematopoietic stem/progenitor cell culture medium can induce stem cells to differentiate into hematopoietic stem/progenitor cells.
  • the stem cell may include an induced pluripotent stem cell.
  • the stem cell may include an embryonic stem cell.
  • the hematopoietic stem/progenitor cell is a CD34+ hematopoietic stem/progenitor cell.
  • the other differentiation culture medium can be used in combination with the culture medium described in the present application to form the set of culture medium described in the present application.
  • different differentiation culture media can be used in different differentiation steps.
  • the present application also provides a NK cell, which is prepared by the method described in the present application.
  • the present application also provides a NK cell, which is prepared by administering the NK differentiation medium described in the present application.
  • the cell and/or the state of the cell can be determined by cell markers.
  • the type of cell and/or the state of the cell can be determined by marker phenotype.
  • the cells described herein are isolated.
  • the cell can be prepared pharmaceutically according to any conventional method.
  • carrier, excipient or diluent can be used to mix or dilute the active ingredient.
  • suitable carrier, excipient or diluent is lactose, dextrose, sucrose, sorbitol, mannitol, glycine, polyethylene glycol, starch, gum arabic, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.
  • the preparation can additionally include filler, anti-agglomeration agent, lubricant, wetting agent, flavoring agent, emulsifier, preservative etc.
  • prepare the composition of the present invention by using any known method in the art to provide quick, continuous or delayed release active ingredient after giving the patient.
  • the application of the cell of the present application can be by injection (e.g., muscle, vein, peritoneal cavity, subcutaneous), or by other methods such as infusion, to ensure that it enters the bloodstream in an effective form.
  • the cell can also be applied by a route within a tumor, around a tumor, within a lesion, or around a lesion to exert local and systemic therapeutic effects. For example, it can be injected locally or intravenously.
  • the administration dosage of the cells may also be a single dose or multiple doses.
  • the actual administration amount of the cells may be determined according to a variety of relevant factors, such as the type of disease; the route of administration; the age, sex and/or weight of the patient; and the severity of the patient's symptoms.
  • composition Composition, reagent, use
  • the present application also provides a composition comprising stem/progenitor cells and the culture medium or the set of culture medium.
  • the present application also provides a reagent for NK cell differentiation, which comprises human platelet lysate.
  • the present application also provides the use of human platelet lysate in the preparation of a reagent for NK cell differentiation.
  • the present application also provides the use of human platelet lysate in the differentiation and/or expansion of hematopoietic stem/progenitor cells into NK cells.
  • the present application also provides the use of human platelet lysate in producing a culture medium for differentiating stem cells into NK cells.
  • the present application also provides a culture platform for obtaining NK cells derived from hematopoietic stem/progenitor cells, which comprises the method described in the present application, the culture medium, the set of culture medium and/or hematopoietic stem/progenitor cells.
  • NK cell differentiation medium I Resuspend the cells in NK cell differentiation medium I, take samples to analyze the cell number and viability, dilute the cells to 4 ⁇ 10 4 cells/mL, 250 ⁇ L/well, inoculate into a 96-well U-shaped cell culture plate (about 250 wells), place in a CO 2 incubator, culture at 37°C, 5% CO 2 , record as day 0, and perform half-medium change every 2 days;
  • NK killing experiment was performed after 35 days of differentiation
  • NK differentiation medium I a basic medium can be used, wherein the concentration of SCF is about 15-25 ng/mL, the concentration of IL3 is about 3-7 ng/mL, the concentration of IL7 is about 15-25 ng/mL, the concentration of IL15 is about 8-12 ng/mL, and the concentration of Flt3 is about 8-12 ng/mL;
  • NK differentiation medium II a basal medium can be used, wherein the concentration of SCF is about 15-25 ng/mL, the concentration of IL15 is about 8-12 ng/mL, the concentration of IL7 is about 15-25 ng/mL, and the concentration of Flt3 is about 8-12 ng/mL;
  • Basic culture medium about 56.6% DMEM+GlutaMAX TM , about 28.3% F12+GlutaMAX TM -I, about 10% hPL, about 1% P/S, about 2 mM L-glutamine, about 25 uM ⁇ -mercaptoethanol, and about 50 uM ethanolamine.
  • NK cell killing A549 and HepG2 are shown in Figure 2, and the statistical results are shown in Table 1.
  • the experimental results of NK cell killing K562 are shown in Figure 3, and the statistical results are shown in Table 2.
  • hPL as an additive to NK cell differentiation medium, can produce NK cells with killing function.
  • NK1 culture medium supplemented with AB serum
  • NK2 culture medium supplemented with hPL
  • NK1 culture medium supplemented with AB serum
  • NK2 culture medium supplemented with hPL

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Abstract

La présente invention concerne un procédé de différenciation de cellules NK, consistant à ajouter un lysat de plaquettes humaines (hPL) à un milieu de culture de différenciation de cellules NK. L'invention concerne en outre un milieu de culture pour différencier les cellules NK et une utilisation de celui-ci.
PCT/CN2022/135764 2022-12-01 2022-12-01 Procédé de différenciation de cellules nk, milieu de culture et son utilisation WO2024113294A1 (fr)

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