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WO2016209021A1 - Méthode pour faire proliférer des cellules tueuses naturelles et composition pour faire proliférer des cellules tueuses naturelles - Google Patents

Méthode pour faire proliférer des cellules tueuses naturelles et composition pour faire proliférer des cellules tueuses naturelles Download PDF

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WO2016209021A1
WO2016209021A1 PCT/KR2016/006754 KR2016006754W WO2016209021A1 WO 2016209021 A1 WO2016209021 A1 WO 2016209021A1 KR 2016006754 W KR2016006754 W KR 2016006754W WO 2016209021 A1 WO2016209021 A1 WO 2016209021A1
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Prior art keywords
cells
culture
natural killer
interleukin
cell
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Korean (ko)
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백영석
최미경
강유라
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Cha Biotech Co Ltd
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Cha Biotech Co Ltd
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Priority claimed from KR1020160078622A external-priority patent/KR101909879B1/ko
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Priority to EA201890121A priority Critical patent/EA038848B1/ru
Publication of WO2016209021A1 publication Critical patent/WO2016209021A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • 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

Definitions

  • the present invention relates to a method for proliferating and killing natural killer cells, and to increasing the anticancer effect of proliferated natural killer cells.
  • NK cells Natural killer cells used in immune cell therapy are morphologically large cells having large granules in the cytoplasm, and account for about 5-15% of lymphocytes in the blood.
  • the main functions of NK cells that have been identified so far include the ability to kill tumor cells, cytotoxicity against virus infected cells, and the ability to kill bacteria and fungi.
  • NK cells are expected to play an important role in antitumor, immunity and protective immunity against microorganisms.
  • NK cells do not have cell surface receptors, such as T cell receptors, CD4 or immunoglobulins, and thus are classified as unique immune cells different from conventional T cells and B cells.
  • NK cells are known to be cells that are capable of killing cancer nonspecifically.
  • the killing ability of these NK cells can be used in the treatment of solid cancers with lymphokine activated killer cells (LAK) and tumor infiltration lymphocytes (TIL), or by donor lymphocyte infusion.
  • LAK lymphokine activated killer cells
  • TIL tumor infiltration lymphocytes
  • donor lymphocyte infusion donor lymphocyte infusion.
  • NK cells defects in the differentiation and activity of NK cells have been described in breast cancer (Konjevic G, et al., Breast Cancer Res. Treat., 66: 255-263, 2001), melanoma cancer (Ryuke Y, et al., Melanoma Res. , 13: 349-356, 2003), lung cancer (Villegas FR, et al., Lung Cancer, 35: 23-28, 2002), and has been reported to be associated with NK cells in the field of treatment of these diseases. Treatment is emerging.
  • NK cells are only 5-15% of lymphocytes in the blood, even in normal humans. Especially in cancer patients, the number, differentiation and function of NK cells are often reduced, so that the number of cells is sufficient for treating diseases. It is difficult to secure.
  • NK cells obtained according to the proliferation method of the conventionally developed NK cells have various deviations in proliferation rate and cytotoxic activity. Therefore, there is an urgent need for a method of proliferation or differentiation of new NK cells to secure a large amount of NK cells that maintain sufficient cancer cell killing ability.
  • One object of the present invention is to provide a method for propagating natural killer cells with improved anticancer effect.
  • Another object of the present invention is to provide a method for producing natural killer cells.
  • Still another object of the present invention is to provide a pharmaceutical composition for treating or preventing cancer, including natural killer cells.
  • Still another object of the present invention is to provide a composition for propagation of natural killer cells.
  • One aspect provides a NK cell proliferation method comprising culturing NK cells in a culture medium comprising a first interleukin, a second interleukin, and an anti-NKp46 antibody.
  • the first interleukin may be IL-2.
  • the second interleukin may be at least one interleukin selected from the group consisting of interleukin-12 (IL-12), interleukin-15 (IL-15), and interleukin-18 (IL-18). Also preferably, the second interleukin may be IL-12, IL-18, IL-12 / IL-18, or IL-12 / IL-15 / IL-18, and more preferably, IL-18. have.
  • IL-12 interleukin-12
  • IL-15 interleukin-15
  • IL-18 interleukin-18
  • the NK cells may be cultured in a culture medium containing the first interleukin, the second interleukin, and the anti-NKp46 antibody in the presence of gamma globulin (IgG) and fibronectin.
  • IgG gamma globulin
  • fibronectin a culture medium containing the first interleukin, the second interleukin, and the anti-NKp46 antibody in the presence of gamma globulin (IgG) and fibronectin.
  • the term “cell proliferation” means that the cells undergo a series of cell division steps to increase the number of cells in the culture or to differentiate the cells.
  • the term “natural killer cell proliferation” means an increase in the number of NK cells in a culture through a series of cell division steps or by differentiation of immature blood cells into NK cells. Therefore, the "natural killer cell proliferation” may include an increase in the number of NK cells, or a state in which undifferentiated lymphocyte cells acquire NK cells to differentiate into NK cells or NK cells become mature NK cells in immature states. have.
  • the NK cells which are the raw materials for the proliferation, can be purchased commercially or collected from humans or animals, and preferably supplied from humans in need of treatment with NK cells.
  • the NK cells can also be supplied from any tissue source in vivo.
  • the NK cells may be included in blood collected from a living body.
  • the blood can be used as an NK cell source for application to the NK cell proliferation method of the present invention, as long as the blood comprises NK cells, and may be, for example, whole blood, umbilical cord blood, bone marrow, or peripheral blood.
  • the natural killer cell proliferation may include, for example, culturing peripheral blood mononuclear cells.
  • peripheral blood mononuclear cells PBMCs
  • PBMCs peripheral blood mononuclear cells
  • the PBMC can be prepared by a conventional manufacturing method from peripheral blood collected in vivo.
  • the PBMC can be separated from peripheral blood using specific gravity centrifugation using Ficoll.
  • the peripheral blood mononuclear cells may also be those obtained from an individual in need of treatment, that is, autologous peripheral blood mononuclear cells.
  • the peripheral blood mononuclear cells are autologous peripheral blood mononuclear cells, even if some T cells exist in the proliferated NK cell population, since all cells are derived from the patient, there is an advantage of not having to remove the T cells.
  • peripheral blood mononuclear cells used in the proliferation method of NK cells of the present invention may be frozen and preserved.
  • the peripheral blood mononuclear cells can be isolated from blood, frozen and thawed again to use for NK cell proliferation.
  • the freezing may use a conventionally known method, preferably the first freezing step in the range of 4 to -42 °C, the second freezing step in the range of -42 to -15 °C, and -15 to -120 °C It can be frozen by a freezing method consisting of a third freezing step.
  • each freezing step can raise or lower the temperature to the upper limit or the lower limit of the temperature range of each freezing step by maintaining the sample sequentially for a predetermined time at several discontinuous temperatures within the temperature range.
  • the first freezing step is frozen under conditions of 10-15 minutes at 0 ° C., 5-10 minutes at ⁇ 12 ° C., and 0.5-1 minute at ⁇ 42 ° C.
  • the second freezing step comprises: After the freezing step 1 to freeze at -25 °C for 1 to 3 minutes, and -15 °C for 1 to 3 minutes, the third freezing step after the second freezing step at -42 °C 20 to 40 minutes and -120 It may be made of a process of freezing at 20 °C to 50 minutes conditions.
  • the first freezing step is frozen at 0.5-5 ° C./m in the range of 4-40 ° C.
  • the second freezing step is 1-10 in the range of ⁇ 40-90 ° C. after the first freezing step.
  • the third freezing step may be made of a process of freezing to the conditions of 1 ⁇ 10 °C / m in the range of -90 ⁇ -120 °C after the second freezing step.
  • Each freezing step of the freezing method may be performed by a controlled rate freezer (CRF).
  • CRF controlled rate freezer
  • the cryopreserved peripheral blood mononuclear cells can be provided for culturing and proliferating NK cells according to the present invention after thawing, and can be used after culturing and proliferating at a desired time after collecting blood from the patient by undergoing a freezing process.
  • a freezing method and thawing method of peripheral blood mononuclear cells that can be effectively applied to the culture method of activated NK cells. Accordingly, a large amount of blood can be secured, separated and frozen and stored when the patient's condition is good, thereby enabling regular production and administration of NK cells regardless of the patient's condition.
  • the NK cell proliferation method may culture NK cells in the presence of gamma globulin (IgG) and fibronectin.
  • the culture in the presence of gamma globulin and fibronectin is, for example, in a culture container containing gamma globulin and fibronectin in the culture solution or coated with gamma globulin and fibronectin on the culture surface (the surface of the culture container that can come into contact with the cells). It may be to culture the cells.
  • the NK cells may be cultured in a culture vessel coated with gamma globulin and fibronectin.
  • the culture vessel coated with gamma globulin and fibronectin may be prepared by adding a solution containing gamma globulin and fibronectin.
  • a known adhesive protein may be used in place of the fibronectin, for example, collagen or the like may be used.
  • a solution containing gamma globulin and fibronectin is added to a culture vessel (e.g., a T75 flask), followed by incubation at low temperature (e.g., 2-4 ° C) to coat the culture vessel with gamma globulin and fibronectin. Can be done.
  • the concentration of gamma globulin may be 0.1-1 ng / ml, 1-10 ng / ml, 10-100 ng / ml or 1-100 ng / ml.
  • Gamma globulin can activate NK cells by stimulating Fc ⁇ RIII of NK cells.
  • the concentration of fibronectin may be 0.1-50 ⁇ g / ml, 1-50 ⁇ g / ml, 5-50 ⁇ g / ml, 10-50 ⁇ g / ml. Fibronectin may promote the migration of NK cells and facilitate the interaction between cells.
  • the NK cell proliferation method includes NK cells in a first interleukin (interleukin-2 (IL-2)), a second interleukin (interleukin-12 (IL-12), interleukin-15 (IL-15), and interleukin.
  • IL-2 interleukin-2
  • IL-12 interleukin-12
  • IL-15 interleukin-15
  • interleukin interleukin.
  • the culture solution may include an anti-NKp46 antibody, a second interleukin, and IL-2 in a conventional immune cell culture medium.
  • the medium for immune cell culture include ALyS505NK-EX (Cell Science & Technology Inst.
  • Culture medium such as KBM (Kohjin bio, Japan) can be used.
  • the above-mentioned anti-NKp46 antibody, second interleukin, and IL-2 of natural killer cells can be added to the above-mentioned immune cell culture medium to prepare a culture solution for use in one embodiment of the present invention.
  • the anti-NKp46 antibody and the second interleukin can also be prepared by adding a commercial medium for immune cell culture containing IL-2.
  • the culture medium may be used without limitation as long as the medium for the immune cell culture containing the second interleukin, anti-NKp46 antibody and IL-2.
  • the concentration of IL-2 in the culture medium is 500-5,000 IU / mL, 600-4,000 IU / mL, 700-3,000 IU / mL, 800-2,000 IU / mL, 900-1,500 IU / mL, 900-1,200 IU. / Ml or 1,000-1,200 IU / ml.
  • the IL-2 may promote growth of NK cells.
  • the concentration of the anti-NKp46 antibody included in the culture solution may be 0.1 to 10 ⁇ g / ml, 0.5 to 10 ⁇ g / ml, 1 to 10 ⁇ g / ml, or 5 to 10 ⁇ g / ml.
  • NKp46 is an activating receptor for NK cells, whereby the anti-NKp46 antibodies of the present invention can stimulate NKp46 to activate NK cells.
  • the concentration of the second interleukin contained in the culture solution is 0.1-100 ng / ml, 1-100 ng / ml, 10-100 ng / ml, 20-100 ng / ml, 30-100-100 ng / ml, 50-100 ng / ml, or 70-100 ng / ml.
  • the second interleukin of the present invention is a component that can not only increase the proliferation efficiency of NK cells, but also greatly increase the activation of NK cells.
  • the present invention uses a second interleukin in addition to the IL-2 and anti-NKp46 antibodies, thereby enhancing and synergistic the proliferation and activation of NK cells possessed by the IL-2 and anti-NKp46 antibodies, thereby increasing existing NK cell proliferation. Compared to the method can have a better proliferation effect of NK cells.
  • the combination of IL-2, the second interleukin, and the anti-NKp46 antibody which are components included in the composition for NK cell proliferation of the present invention, can maximize the proliferation and culture efficiency of NK cells. It will also be very cost effective.
  • the culture may further comprise plasma or serum.
  • plasma or serum contained in the culture solution may be obtained from peripheral blood obtained by separating PMBC used for NK cell culture.
  • the concentration of plasma may be 1-20 v / v%, 1-15 v / v%, 2-15 v / v%, 5-15 v / v%, or 5-10 v / v% days for the whole culture. Can be.
  • PBMCs and plasma are respectively isolated from peripheral blood of a patient to which NK cells are administered, and the obtained PBMCs are isolated from the IL-2, second interleukin, anti-NKp46 antibody, and the peripheral blood. It may be cultured in a medium containing plasma (or serum). In addition, the culture is preferably cultured in a culture vessel coated with gamma globulin and fibronectin.
  • Cultivation of the peripheral blood mononuclear cells can be carried out under normal cell culture conditions, that is, about 37 °C, CO 2 incubator, the culture medium can be continuously added while adding every two or three days.
  • concentration of PBMC added to the medium may range from 4 X 10 5 to 5 X 10 7 cells / mL, but is not limited thereto.
  • the incubation period may be performed, for example, for 7 to 20 days, 8 to 18 days, 10 to 16 days, 10 to 15 days, or 10 to 14 days, but is not limited thereto.
  • the culture days may be appropriately set within or outside the range.
  • Culture vessels may include commercially available dishes, flasks, plates, multi-well plates, culture bags.
  • the culture can be cultured in each step in order to maximize the growth of NK cells.
  • each culture step may be the same or different culture medium components, culture vessels and culture periods, the final growth of NK cells can be achieved by finding the optimum culture period for each step.
  • the present invention is characterized in that the culture composition (or culture medium) is added to the flask at intervals of 2-3 days in the flask, thereby culturing the NK cells effectively.
  • the NK cell proliferation method of the present invention comprises a culture medium containing peripheral blood mononuclear cells, a first interleukin, a second interleukin, an anti-NKp46 antibody, and a plasma in a culture vessel coated with gamma globulin and fibronectin. 1 culture step; And a second culture step of culturing by further adding a culture solution including a first interleukin, a second interleukin, an anti-NKp46 antibody, and a plasma to the culture obtained from the first culture step.
  • the first interleukin may be interleukin-2 (IL-2).
  • the second interleukin may be at least one interleukin selected from the group consisting of interleukin-12 (IL-12), interleukin-15 (IL-15), and interleukin-18 (IL-18).
  • IL-12 interleukin-12
  • IL-15 interleukin-15
  • IL-18 interleukin-18
  • the first interleukin and the second interleukin are as described above.
  • the plasma may be obtained from peripheral blood derived peripheral blood mononuclear cells. Serum may be used instead of the plasma.
  • the concentration of IL-2 contained in the culture medium in the first culture step is 500-5000 IU / mL, 600-4000 IU / mL, 700-3000 IU / mL, 800-2000 IU / mL, 900-1500 IU / mL , 900-1,200 IU / mL or 1,000-1,200 IU / mL.
  • Said first culture step comprises a first interleukin (IL-2), a second interleukin (at least one interleukin selected from the group consisting of IL-12, IL-15, and IL-18), anti-NKp46 antibodies and plasma
  • IL-2 interleukin
  • second interleukin at least one interleukin selected from the group consisting of IL-12, IL-15, and IL-18
  • anti-NKp46 antibodies and plasma
  • Peripheral blood mononuclear cells may be cultured for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days in a culture medium containing, for example, 3 days, 4 days May be incubated for 5 days, 6 days, preferably 5 days.
  • the NK cell proliferation method according to the present invention may further include adding a culture solution including IL-2 and plasma to the culture during the first culture step.
  • the culture solution further comprising IL-2 and plasma added above may be added on the third day of culture and / or on the fourth day of culture.
  • the concentration of IL-2 included in the additionally added culture solution may be the same as the concentration of IL-2 in the culture solution of the first culture step, but is not limited thereto.
  • the volume of the culture added may be the same as the volume of the culture.
  • NK cell cell proliferation method of the present invention is a group consisting of a first interleukin (IL-2), a second interleukin (IL-12, IL-15, and IL-18) in a culture obtained from the first culture step in one embodiment And a second culture step of culturing by further adding a culture solution comprising at least one interleukin selected from C), an anti-NKp46 antibody and plasma.
  • IL-2 interleukin
  • IL-12, IL-15, and IL-18 interleukin
  • the second culture step may be performed by transferring the culture of the first culture step to a new container.
  • the new container may not contain gamma globulin and fibronectin.
  • the concentration of IL-2 in the culture medium of the second culture step may be the same as the concentration of IL-2 in the culture solution of the first culture step, but is not limited thereto.
  • the concentration of IL-2 contained in the culture medium in the second culture step is 500-5,000 IU / mL, 600-4,000 IU / mL, 700-3,000 IU / mL, 800-2,000 IU / mL, 900-1,500 IU / Ml, 900-1,200 IU / ml or 1,000-1,200 IU / ml.
  • the culture comprising the antibody and plasma may be the same volume as the culture.
  • the cells may be cultured in the culture solution for 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days, preferably 1 day, 2 days, or 3 days. have.
  • the NK cell proliferation method of the present invention may further include a third culture step of culturing after lowering the concentration of IL-2 in the culture to 4/5 or less than 1/10 of the concentration before adding the culture.
  • the IL-2 concentration is for example 1/100 to 3/5, 1/100 to 1/4, 1/100 to 2/5, 1/100 to 1/3, or 1 / 100 to 1/5.
  • Lowering the concentration of IL-2 in the culture solution may be performed by adding a culture medium not containing IL-2 to the culture.
  • the culture solution added in the third culture step may include plasma.
  • IL-2 concentration of the culture in the third culture step is 1500-1300 IU / ml, 1300-1000 IU / ml, 1000-600 IU / ml, 600-500 IU / ml, 500-400 IU / ml, 400 -300 IU / mL, 300-200 IU / mL, 200-100 IU / mL.
  • the culture solution of each culture step may contain appropriate proteins, cytokines, antibodies, compounds and other components provided that they do not impair the effect of amplifying NK cells.
  • the proliferated cells may be to express any one or more selected from the group consisting of CD56, CD16, NKG2D, Perforin and Granzyme B.
  • the expression may be to express more of the factor compared to the peripheral vascular blood cells or cell population before proliferation, or the natural killer cells or cell population before proliferation.
  • the peripheral vascular blood cells or cell populations comprising the proliferated natural killer cells, or the proliferated natural killer cells or cell population may comprise at least 70% or more of the above factors in the whole cell or in the entire cell population. It may be at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or 100%.
  • the "% expression” or “expression rate” may refer to the percentage of NK cells expressing one or more of the factors relative to the total number of cells in the sample.
  • the expression or expression rate of the factor in the cell or cell population can be determined by the value detected using a flow cytometer.
  • the present invention may further provide a method of producing the method comprising the step of suspending NK cells proliferated by the NK cell proliferation method in a solution containing dextran and albumin. .
  • NK cells proliferated by the NK cell proliferation method of the present invention expressed a high level of anticancer substances compared to PBMC, and showed excellent anticancer activity (see FIGS. 7 to 9).
  • Another aspect provides a pharmaceutical composition for treating or preventing cancer comprising the proliferated NK cells.
  • the NK cells and the proliferation method of the NK cells included in the pharmaceutical composition are as described above.
  • the subject may be a mammal and may comprise a human.
  • the cancer may be leukemia, breast cancer, ovarian cancer, brain cancer, melanoma cancer, gastric cancer, liver cancer, colon cancer or lung cancer, but is not limited thereto.
  • the pharmaceutical composition may include a cell population including NK cells cultured according to the NK cell proliferation method.
  • the composition may include a culture solution or culture used for culturing in addition to the cell population cultured according to the above method.
  • the composition may comprise a new culture or physiological saline that does not contain additional components such as cytokines and cell populations isolated from the culture.
  • the new culture medium may be a medium having the same composition as the medium used for culturing NK cells or a medium having a different composition.
  • the dosage of NK cells varies depending on the condition and weight of the cancer patient, the extent of the disease, the dosage form, the route of administration, and the duration, for example, 1 ⁇ 10 6 to 1 ⁇ 10 per day.
  • the dose may be administered at a dose of 1 ⁇ 10 7 to 1 ⁇ 10 9 cells / kg.
  • the administration may be administered once or several times a day.
  • liquid unit preparations such as liquids, suspensions, emulsions, and the like, it may also be administered to the patient at the cell concentration.
  • the pharmaceutical composition according to the present invention includes NK cells proliferated by the method of the present invention as described above, and may further include a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be formulated in parenteral formulations, such as liquids, suspensions, emulsions, lyophilizers, etc. according to conventional methods.
  • the pharmaceutically acceptable carrier may include an aqueous diluent or a solvent such as phosphate buffered saline, purified water or sterile water. And other conventional preservatives.
  • the pharmaceutical composition may include various anti-tumor agents or other therapeutic agents in addition to the NK cells or a cell population including NK cells.
  • Another aspect provides a method of treating or preventing cancer comprising administering such proliferated NK cells to a subject.
  • the subject and cancer are as described above.
  • the route of administration may be administered orally or parenterally (eg, by injection), and the injection may be intravenous, subcutaneous, intramuscular or intraperitoneal. It may be, but is not particularly limited thereto.
  • Another aspect provides the use of said propagated NK cells for the manufacture of a medicament for the treatment or prevention of cancer.
  • Another embodiment is one kind selected from the group consisting of a first interleukin (IL-2), a second interleukin (interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18) It provides a composition for NK cell proliferation comprising the above interleukin) and anti-NKp46 antibody.
  • IL-2 interleukin
  • IL-12 interleukin-12
  • IL-15 interleukin-15
  • IL-18 interleukin-18
  • Another embodiment is a first interleukin (IL-2), a second interleukin (interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-) for the preparation of a composition for NK cell proliferation 18) and at least one interleukin selected from the group consisting of 18) and anti-NKp46 antibodies.
  • IL-2 interleukin
  • IL-12 interleukin-12
  • IL-15 interleukin-15
  • IL-18 interleukin-18
  • the composition for proliferating NK cells may further include gamma globulin and fibronectin.
  • the first interleukin and the second interleukin are as described above, respectively.
  • the concentration of IL-2 in the proliferation composition is 500-5000 IU / mL, 600-4000 IU / mL, 700-3000 IU / mL, 800-2000 IU / mL, 900-1500 IU / mL, 900-1,200 IU. / Ml or 1,000-1,200 IU / ml.
  • the concentration of the second interleukin in the growth composition is 0.1-100 ng / ml, 1-100 ng / ml, 10-100 ng / ml, 20-100 ng / ml, 30-100-100 ng / ml, 50-100 ng / ml, or 70-100 ng / ml.
  • the concentration of the anti-NKp46 antibody in the proliferation composition may be 0.1 to 10 ⁇ g / ml, 0.5 to 10 ⁇ g / ml, 1 to 10 ⁇ g / ml, or 5 to 10 ⁇ g / ml.
  • composition for proliferating NK cells may be for proliferating or differentiating NK cells in peripheral blood mononuclear cells.
  • the composition for proliferating NK cells may further include plasma or serum.
  • the plasma or serum added to the composition may be obtained from blood from which NK cells are separated.
  • the plasma contained in the composition for proliferation may be obtained from peripheral blood mononuclear cells isolated peripheral blood.
  • the proliferating composition comprises gamma globulin, fibronectin, first interleukin (IL-2), second interleukin (at least one interleukin selected from the group consisting of IL-12, IL-15, IL-18) and anti-NKp46 antibodies
  • IL-2 first interleukin
  • second interleukin at least one interleukin selected from the group consisting of IL-12, IL-15, IL-18
  • anti-NKp46 antibodies In addition to the components, it may include essential components or other carriers or adjuvants for PBMC culture and NK cell proliferation.
  • Natural killer cell proliferation method or a composition for natural killer cell proliferation can promote the proliferation and differentiation of natural killer cells, and can stably provide natural killer cells with increased cytotoxic activity.
  • the natural killer cells thus prepared may be usefully used for the treatment of diseases such as anticancer treatment.
  • 1 is a result of the proliferation of the total cell number (A) and NK cell number (B) by the proliferation method according to an embodiment of the present invention.
  • 3 is a result showing the effect of NK cell proliferation according to IL-2 concentration.
  • FIG. 5 is a flow cytometry result of NK cells by the proliferation method according to an embodiment of the present invention.
  • Figure 6 shows the results showing the activation receptor of NK cells obtained according to one embodiment using a flow cytometer.
  • Figure 7 is a graph showing the flow cytometry results for confirming the expression of anticancer substances in NK cells obtained according to one embodiment.
  • FIG. 8 shows the results of evaluating cancer cell killing% of K562 cells, which are leukemia cell lines of NK cells obtained according to various embodiments, by a flow cytometry method.
  • Figure 9 shows the results of evaluating the cancer cell lysis (% cell lysis) of the NK cells obtained according to one embodiment by a flow cytometry method. Cancer cell killing for K562 (chronic leukemia), OVCAR3 (ovarian cancer), Hep3B (liver cancer), HepG2 (liver cancer), A704 (renal cancer), DU145 (prostate cancer).
  • PBMC 10 shows the pre-culture PBMC characteristics of Donor 1.
  • A. Proportion of NK cells (Q9, CD3-CD56 +), B. CD16 expressing NK cells (Q2, CD16 + CD56 +), C. B cells (Q5, CD3-CD19 +), D. K562 cells, cytotoxicity (7AAD + , E: T 10: 1)
  • Blood was prepared by collecting blood from normal veins. At this time, the blood collection vessel used a blood collection tube containing heparin. Blood from the patient was carefully transferred to 30 ml each of two tubes (# 352070, BD or equivalent) containing Ficoll (# 17-1440-02, GE Healthcare or equivalent). The tube containing blood was centrifuged at 2,500 rpm for 10 minutes in a break off state, and then the upper plasma portion was transferred to a new tube.
  • the transferred plasma was inactivated for 30 minutes in a heat block, and then centrifuged for 5 minutes at 4,000 rpm.
  • the supernatant from the centrifuged tube was transferred to a new tube, labeled as plasma, and stored at 2 to 8 ° C.
  • the cell suspension was centrifuged at 1,500 rpm for 5 minutes to remove the supernatant, and Ca / Mg free DPBS was added to the tube from which the supernatant was removed, followed by further centrifugation at 1,500 rpm for 5 minutes. The supernatant was removed and the remaining precipitated cells were suspended in 1 ml of Alys505NK-EX (# 01410P10, CSTI) medium.
  • Example 1.1 All the cell suspensions obtained in Example 1.1 were centrifuged at 1,500 rpm for 5 minutes and the supernatant was removed. The cells were suspended in a Cryostor CS10 or ALyS505NK-EX + Albumin + DMSO mixed solution stored at 2 to 8 ° C. to give a cell number of 1-100 ⁇ 10 6 cells / ml. Dispense the suspended cells in 1 ml into 2 ml cryogenic vials and then use controlled rate freezers (CRF) for 10-15 minutes at 0 ° C, 5-10 minutes at -12 ° C, and -42 ° C. Frozen in a first step under conditions of 0.5 to 1 min at 1 to 3 minutes at ⁇ 25 ° C.
  • CRF controlled rate freezers
  • the second step was frozen at a condition of 5 ° C./m in the range of ⁇ 90 ° C., and was frozen at 5 ° C./m in the range of ⁇ 90 ° to 120 ° C. after the second freezing step.
  • Frozen cells were transferred to LN 2 tanks and stored (below -130 ° C).
  • the heat block was set to 37 ° C., and then a culture solution with 10% plasma was added to the T flask. Depending on the cell concentration, the culture volume can be variously adjusted to, for example, 4ml, 6ml, 8ml, or 10ml.
  • the frozen PBMC which was frozen in Example 2.2, was placed in a heat block to dissolve frozen PBMCs. When the frozen PBMC was half dissolved, it was transferred to a T flask containing the culture. Subsequently, it was placed in a 37 ° C., 5% CO 2 incubator and incubated for one day.
  • the cultured PBMCs were collected in a tube, Ca / Mg free DPBS was added, centrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed. Cells isolated by centrifugation were suspended in a small amount of culture and cell number was measured. The viability after thawing of cryopreserved cells is shown in Table 1. As can be seen in Table 1, thawed PMBC after cryopreservation according to the present invention was found to survive 93% or more, it was confirmed that high survival rate is maintained.
  • fibronectin # FC-010, Millipore
  • 0.121 mL gamma globulin # 020A1004, Green Cross
  • the prepared coating solution was put into a T75 flask (# 156499, Nunc) using a pipette and reacted at 2 to 8 ° C. for at least 16 hours. The remaining coating solution was washed with Ca / Mg free DPBS before cell culture and then removed.
  • Example 2.1.1 10 ⁇ l of the amount of fibronectin in Example 2.1.1, A culture flask coated with fibronectin and gamma globulin was prepared in the same manner as in Example 2.1.1 except that only 1.21 ml of gamma globulin was changed.
  • fibronectin # FC-010, Millipore
  • Example 1 The cell suspension prepared in Example 1 was taken and placed in the coating flask prepared in Example 2.1, and 1.5 ml of autologous plasma and 0.03 ml of anti-NKp46 (# MAB1850, R & D) solution, IL-18 (# B003-2, R & D) 0.075 mL and 13.4625 mL of Alys505NK-EX (# 01410P10, CSTI) were added and incubated in a CO 2 incubator for 2-3 days. After that, 1.5 ml of autologous plasma and 13.5 ml of Alys505NK-EX were added to the flask and incubated in a CO 2 incubator for 1-2 days.
  • Example 2.2.1 All procedures were the same as in Example 2.2.1, except that 3 ⁇ l of anti-NKp46 (homologous) solution, 37.5 ⁇ l of IL-18 (homologous), and 2 times of Alys505NK-EX were used and a total of 35.95 mL was used. .
  • Example 2.2.1 All procedures are the same as in Example 2.2.1 except that 15 ⁇ l of anti-NKp46 (homologous) solution, 7.5 ⁇ l of IL-12 (# 554613, BD) and 2 times of Alys505NK-EX were used and a total of 26.98 ml was used. It was carried out by.
  • Example 2.2.1 All procedures are the same as in Example 2.2.1, but 0.03 ml of anti-NKp46 (homologous) solution, 7.5 ml of total IL-12 (homologous), and 12.5 ⁇ l of IL-15 (# 247-IL-0025, R & D) A total of 37.5 ⁇ l of IL-18 and 2 times of Alys505NK-EX were added and 26.913 mL of total was used.
  • Example 2.2.1 After the primary culture of Example 2.2.1, the T75 flask in which the cells were cultured was taken out of the incubator, and the cells were collected and transferred to a T175 flask (# 159910, Nunc). 3 ml of autologous plasma and 27 ml of Alys505NK-EX (# 01410P10, CSTI) were added to a T175 flask and incubated for 1-2 days in a CO 2 incubator.
  • Example 2.3.1 All procedures were the same as in Example 2.3.1 except that 0.06 ml of anti-NKp46 (# MAB1850, R & D) solution, 0.06 ml of IL-18 (# B003-2, R & D), and 53.88 ml of Alys505NK-EX were used. Secondary culture of the culture of Example 2.2.2 was performed.
  • Example 2.3.1 All procedures are the same as in Example 2.3.1, except for using 0.12 ml of anti-NKp46 (# MAB1850, R & D) solution, 0.03 ml of IL-12 (# 554613, BD), and 53.85 ml of Alys505NK-EX. Second culture of .3 culture was performed.
  • Example 2.3.1 All procedures were the same as in Example 2.3.1, using 0.06 mL of anti-NKp46 (# MAB1850, R & D) solution, 0.03 mL of IL-12 (homologous), 0.02 mL of IL-18 (homologous), 44.89 mL of Alys505NK-EX Secondary culture of the culture of Example 2.2.4 was carried out except for the following.
  • Example 2.3.1 All procedures are the same as in Example 2.3.1, except for 0.06 mL of anti-NKp46 (# MAB1850, R & D) solution, 0.03 mL of IL-12 (homologous), 0.06 mL of IL-15 (homologous), 0.02 mL of IL-18 (homologous) 0.02
  • the second culture of the culture of Example 2.2.5 was performed except that 62.83 ml of Alys505NK-EX was used.
  • the cells of the T175 flask and the autologous plasma cultured in Example 2.3 were placed in a culture solution containing 300 IU / ml IL-2, and cultured in a CO 2 incubator. After 2-3 days a new equal volume of culture (culture containing 300 IU / ml IL-2) was mixed with the cell suspension in which the cells were incubated and then cultured in a CO 2 incubator.
  • IL-2 is added to the immune cell culture medium without IL-2 in a predetermined amount instead of the culture medium to which IL-2 is added, respectively. It may be.
  • Example 2.4 After all the cells cultured in Example 2.4 were collected, the supernatant was removed by centrifugation at 1500 rpm for 5 minutes and then suspended in phosphate buffered saline. 10 ⁇ l of the cell suspension was diluted with phosphate buffered saline, and then 10 ⁇ l of the diluted solution was mixed with 10 ⁇ l of trypan blue and placed on a hemocytometer to measure cell number and viability. The cell number was determined by the formula (live cell number + dead cell number) ⁇ 1/4 ⁇ 2 ⁇ dilution factor ⁇ total volume ⁇ 10 4 , and cell viability was determined as viable cell number ⁇ (live cell number + dead cell number) ⁇ 100 Obtained by the formula
  • IL-12, IL-15, or a mixture of IL-12 and IL-15, or IL-18 has been shown to be effective in raising the NK ratio.
  • Example 1 The cell suspension prepared in Example 1 was taken and placed in the coating flask prepared in Example 2.1, and 1.5 ml of autologous plasma and 0.03 ml of anti-NKp46 (# MAB1850, R & D) solution, IL-18 (# B003-2, R & D) 0.075 mL, 13.4625 mL of Alys505NK-EX (# 01400P10, CSTI) without IL-2 was added, and IL-2 was added at a concentration of 0 to 10,000 IU / mL (see FIG. 3) in a CO 2 incubator for a total of 14 Incubated daily.
  • anti-NKp46 # MAB1850, R & D
  • IL-18 # B003-2, R & D
  • Alys505NK-EX # 01400P10, CSTI
  • NK cells cultured for 14 days were centrifuged at 1500 rpm for 5 minutes to remove supernatant and then suspended in phosphate buffered saline. Ten microliters of the suspended cells were taken, diluted with phosphate buffered saline, and 10 microliters of the diluted solution was mixed with 10 microliters of trypan blue and placed on a hemocytometer to measure the cell number. The cell number measurement method is the same as in Example 3.1. The number of NK cells was calculated from the percentage of NK cells obtained through flow cytometry. The IL-2 concentration of 100 IU / ml was set to 1, and the fold proliferation of NK cells following the remaining IL-2 concentration treatment was calculated.
  • Example 1 The cell suspension prepared in Example 1 was added to the prepared 96 well plate, and 10 ⁇ l of autologous plasma, 0.2 ⁇ l of anti-NKp46 (# MAB1850, R & D) solution, and 1000 IU / ml of IL-2 were added to Alys505NK-EX. 89.8 ⁇ l (# 01410P10, CSTI) was added, IL-12 was added at a concentration of 0-10 ng / ml (see FIG. 4A), and IL-18 was added at a concentration of 0-300 ng / ml (see FIG. 4B). Incubated for 48 hours in a CO 2 incubator.
  • NK cells were transferred to a new tube and centrifuged at 1,500 rpm for 5 minutes to transfer only the supernatant to a new tube.
  • the activity of NK cells was confirmed by IFN-gamma secretion using IFN-gamma ELISA kit (# DIF50, R & D).
  • IL-12 secreted high IFN-gamma at a concentration range of 0.1 to 10 ng / ml and IL-18 secreted high IFN-gamma at a concentration of 1 to 100 ng / ml. was measured.
  • NK cells cultured and harvested in Example 2 were analyzed using flow cytometry. Specifically harvested cells were suspended in Ca / Mg free DPBS, centrifuged at 1500 rpm for 5 minutes, then the supernatant was removed to obtain cell pellets. Antibody (CD3-FITC, CD56-APC, CD16-PE) containing fluorescent material was added to the obtained cell pellet, and then incubated at 4 ° C. for 20 minutes. FACS buffer was added and centrifuged once for 1 minute at 8,000 rpm, and analyzed by flow cytometry (FACSCalibur, BD), and the results are shown in FIG. 5.
  • cells grown according to one embodiment express more CD56 and CD16 and less CD3 than PBMCs before proliferation.
  • NK cells were stained with fluorescent materials.
  • Antibodies KIRDL1-PE, NKG2A-PE, NKG2D-PE, NKp30-PE, NKp44-PE, NKp46-PE
  • FACS buffer was added and centrifuged once for 1 minute at 8000 rpm, and analyzed by flow cytometry (Guava 8HT, merk), and the results are shown in FIG. 6.
  • the cells proliferated according to one embodiment it can be seen that express more NKG2D, NKp30, NKp44, or NKp46 than PBMCs before proliferation.
  • the proliferated cells express more perforin, granzyme B, or TRAIL than PBMCs before proliferation.
  • Hep3B, OVCAR3, HepG2, A704, DU145 various solid cancer cell lines
  • leukemia cell lines Hep3B, OVCAR3, HepG2, A704, DU145
  • Cells were prepared by adding the culture solution to the washed cell pellet at a concentration of 5 ⁇ 10 5 cells / ml. Prepared cells were treated with CFSE (# c34554, Life technologies) and incubated for 10 minutes in a CO 2 incubator.
  • the cells were washed twice with Ca / Mg free DPBS and then treated with the prepared NK cells and incubated for 4 hours in a CO 2 incubator. 10 minutes before the end of incubation, 7AAD was treated and after the end of incubation, cells were harvested in vitro and analyzed for cancer cell killing ability by flow cytometry (FACSCalibur, BD). Cancer cell killing value was calculated by the following formula.
  • Cytotixicity (%) (sample-group-natural glass) / (100-natural glass) ⁇ 100
  • K562 lysis results are shown in FIG. 9 and Table 2, and lysis results for various solid cancer cell lines are shown in FIG. 9.
  • the use of anti-NKp46 combination in a flask coated with fibronectin and gamma globulin showed high K562 elimination efficacy, OVCAR3 (ovarian cancer), Hep3B (liver cancer), HepG2 (liver cancer), A704 (renal cancer), DU145
  • OVCAR3 ovarian cancer
  • Hep3B liver cancer
  • HepG2 liver cancer
  • A704 renal cancer
  • DU145 In various solid cancers such as (prostate cancer), the removal efficiency increased according to the E: T ratio (the number of effect cells: the number of target cells).
  • the effector cell refers to NK cells
  • the target cell refers to cancer cells.

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Abstract

La présente invention concerne une méthode pour faire proliférer des cellules tueuses naturelles et une composition pour faire proliférer des cellules tueuses naturelles, capable d'améliorer l'activité de prolifération cellulaire et l'activité anticancéreuse, la méthode comprenant une étape de culture de cellules tueuses naturelles dans un milieu de culture liquide contenant une première interleukine (l'IL-2), une seconde interleukine (au moins une interleukine sélectionnée au sein du groupe constitué par l'IL-12, l'IL-15 et IL-18), et un anticorps anti-NKp46.
PCT/KR2016/006754 2015-06-24 2016-06-24 Méthode pour faire proliférer des cellules tueuses naturelles et composition pour faire proliférer des cellules tueuses naturelles Ceased WO2016209021A1 (fr)

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WO2020146795A1 (fr) * 2019-01-11 2020-07-16 Omeros Corporation Procédés et compositions pour le traitement du cancer
CN111849894A (zh) * 2020-07-31 2020-10-30 南京元荟生物科技有限公司 一种肿瘤浸润淋巴细胞的分离方法及其应用
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CN113528438A (zh) * 2021-08-09 2021-10-22 山东第一医科大学附属肿瘤医院(山东省肿瘤防治研究院、山东省肿瘤医院) 基于表面活化受体和抑制受体表达的nk细胞数量扩增方法
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CN117551608A (zh) * 2023-11-10 2024-02-13 深圳泽医细胞治疗集团有限公司 活化培养基、高分泌IFN-γ的NK细胞的培养方法及应用
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CN118389428A (zh) * 2024-06-25 2024-07-26 深圳市中佳生物医疗科技有限公司 一种自然杀伤细胞培养基及其应用
CN118956745A (zh) * 2024-09-30 2024-11-15 广东壹加再生医学研究院有限公司 一种提高nk细胞杀伤活性的方法

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