WO2017188790A1 - Method for proliferating and culturing immune cells by using hypoxic conditions - Google Patents

Method for proliferating and culturing immune cells by using hypoxic conditions Download PDF

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WO2017188790A1
WO2017188790A1 PCT/KR2017/004595 KR2017004595W WO2017188790A1 WO 2017188790 A1 WO2017188790 A1 WO 2017188790A1 KR 2017004595 W KR2017004595 W KR 2017004595W WO 2017188790 A1 WO2017188790 A1 WO 2017188790A1
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cells
conditions
peripheral blood
cancer
expression
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Korean (ko)
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이경미
박현성
문윤원
임선아
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고려대학교 산학협력단
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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
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
    • C12N2506/115—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells from monocytes, from macrophages

Definitions

  • the present invention relates to a method for proliferating and culturing immune cells using hypoxic conditions that allow peripheral blood mononuclear cell-derived NK cells to be cultured under normal oxygen conditions and further cultured under hypoxic conditions to improve cancer cell killing ability and to survive in the body for a long time. .
  • NK cells are one of the innate immune cells, which are capable of killing various types of cancer cells and recognize cancer cells regardless of the presence or absence of antigens.
  • the immune cells injected due to the immune evasion mechanism that occurs in the large tumor tissues cause deactivation, and the cancer cell attack ability is lost.
  • the major cause of this immune evasion mechanism is that the interior of the tumor is a hypoxic environment and this environment is a problem in the treatment of solid cancer patients by providing a condition that NK cells can not effectively kill cancer cells.
  • hypoxic oxygen partial pressures of 3% (peripheral tissue) to 14% (lung) depending on tissue, and lower oxygen levels are referred to as hypoxic, often 0.5% to 3%. It has been reported that tumor cells maintain and promote growth under hypoxic conditions, whereas normal cells and immune cells do not grow well under hypoxia. In addition, in the case of immune cells, hypoxic conditions are known to inhibit the function of immune cells. In NK cells, NKp30, NKp44, NKp46, and NKG2D, which are the major activation receptors, are downregulated when cultured at 1% O 2 , and cancer cell killing ability is lowered compared to NK cells grown in normal cell conditions.
  • the present inventors have previously exposed NK cells to various concentrations of hypoxic conditions in vitro expansion to adapt them in a hypoxic environment, and when the NK cells are injected into the patient's body, they can maintain anticancer activity even in hypoxic conditions in cancer tissues.
  • the present invention was completed by establishing culture conditions.
  • Another object of the present invention is to provide a prophylactic or therapeutic use of cancer of the NK cells with improved cancer cell killing ability.
  • the present invention is to culture the peripheral blood monocytes and feeder cells under cytokine for 3 to 13 days under normal oxygen conditions with an oxygen partial pressure of 20% to 22%, and then the oxygen partial pressure is increased. It provides a method for inducing and proliferating NK cells comprising the step of inducing and proliferating natural killer cells (NK cells) by further culture for 5 to 30 days in low oxygen conditions of 1% to 5%.
  • NK cells natural killer cells
  • the present invention also includes NK cells with improved cancer cell killing ability compared to peripheral blood mononuclear cells-derived NK cells cultured under normal oxygen conditions with an oxygen partial pressure of 20% to 22%, wherein the NK cells with improved cancer cell killing ability are in normal oxygen conditions.
  • the invention also provides the use of said NK cells for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer.
  • the present invention also provides a method for treating cancer comprising administering to a cancer patient a pharmaceutically effective amount of the pharmaceutical composition for preventing or treating the cancer.
  • NK cells induced and expanded under normal oxygen conditions in the case of NK cells induced and expanded by incubating for a certain period of time under normal oxygen conditions and further cultured under hypoxic conditions.
  • cell death is reduced, aging is inhibited, cancer cell killing ability can be promoted.
  • the NK cells have a long survival time and excellent cancer cell killing ability in vivo, and thus can be used as an adoptive immune cell therapy for the prevention or treatment of cancer.
  • NK cells 1 is a result of confirming the effect of oxygen conditions in the induction and proliferation of NK cells from peripheral blood monocytes, peripheral blood mononuclear cells cultured for 3 weeks in normal oxygen conditions (20%) or hypoxic conditions (0.5%, 1.5%) or After culturing for 9 days in normal oxygen conditions, and further cultured in low oxygen conditions (1.5%) shows the measured cell number.
  • Figure 2a is a graph comparing the proliferation of NK cells induced in normal oxygen conditions, and NK cells according to the present invention induced by further culture in hypoxic conditions after culture in normal oxygen conditions
  • Figure 2b is induced in normal oxygen conditions
  • Figure 2c is a graph comparing the cancer cell killing ability of NK cells induced in normal oxygen conditions and NK cells according to the present invention.
  • FIG. 3 is a graph comparing the killing ability of NK cells induced in normal oxygen conditions and A375 cells, a melanoma cell line under various oxygen conditions of the NK cells according to the present invention.
  • Figure 4 is a result confirming the change in the NK cells induced in normal oxygen conditions and the activating receptors and inhibitory receptors of NK cells according to the present invention.
  • 5 is a result confirming the expression of p16, an aging marker in NK cells induced in normal oxygen conditions and NK cells according to the present invention.
  • Figure 6 is a result confirming the cancer cell killing ability of NK cells induced in normal oxygen conditions and NK cells according to the present invention in xenograft hematological cancer model.
  • the present invention is cultured peripheral blood monocytes and feeder cells under cytokines for 3 days to 13 days under normal oxygen conditions with an oxygen partial pressure of 20% to 22%, hypoxia with an oxygen partial pressure of 1% to 5%
  • the present invention relates to a method of inducing and proliferating NK cells, which further comprises inducing and proliferating natural killer cells (NK cells) by further culturing for 5 to 30 days under conditions.
  • NK cells natural killer cells
  • the normal oxygen condition means a cell culture condition having an oxygen partial pressure of 20% to 22%
  • a low oxygen condition means a cell culture condition having an oxygen partial pressure of 1% to 5%, or 1.5% to 3%.
  • Induction and proliferation culture method of NK cells of the present invention is characterized in that the peripheral blood monocytes are cultured for a certain period of time under normal oxygen conditions, and further cultured under low oxygen conditions to induce and proliferate NK cells.
  • peripheral blood monocytes under cytokines were cultured for 3 days to 13 days under vegetative cells and normal oxygen conditions with an oxygen partial pressure of 20% to 22%, and 5 days under hypoxic conditions with an oxygen partial pressure of 1% to 5%. It may be further incubated for 30 days.
  • peripheral blood monocytes may be derived from peripheral blood of normal or cancer patients.
  • the feeder cells may be irradiated Jurkat cells and irradiated EBV-LCL cells. More specifically, the feeder cells may be a mixture of irradiated Jurkat cells and irradiated EBV-LCL cells in a ratio of about 1: 1 cell number, but is not particularly limited thereto.
  • peripheral blood monocytes and feeder cells may be co-cultured by mixing at a ratio of about 1: 1 cells, but are not particularly limited thereto.
  • the cytokine may comprise interleukin-2 (IL-2).
  • IL-2 interleukin-2
  • the NK cells derived above may be cells having a CD3 - CD56 + phenotype.
  • the medium used for the induction and proliferation culture of the NK cells can be used without limitation, known cell culture medium added with serum.
  • the serum can be used without limitation the kind of known serum used for cell culture.
  • NK cells according to the induction and proliferation culture method of the NK cells of the present invention has a larger number of cells than NK cells derived from peripheral blood monocytes cultured under normal oxygen conditions.
  • NK cells when transferred to hypoxic conditions (1.5% or 0.5%) when less than 3 days of culture
  • the growth rate of NK cells was significantly higher than that of the control group when the cells were transferred to hypoxic conditions after 9 days of culture under normal oxygen conditions.
  • the proliferation rate was increased by about 60%
  • the cell death confirmation experiment was reduced by about 50%
  • the cancer cell killing ability was increased by 20% to 40% by the investigation of cancer cell killing ability.
  • the cancer cell killing ability of the NK cells in the hypoxic condition was tested, compared with the NK cells cultured continuously in the normal oxygen condition, the cancer cell killing ability for solid cancer was superior, the NK cells have a high expression of CD107a (killing capacity measure) In addition, the secretion of IFN- ⁇ is increased.
  • NKp44 an activation receptor and molecules related to cancer cell killing ability, perforin and granzyme B are increased.
  • the expression of the aging marker p16 is reduced, it can be seen that less aging compared to NK cells continuously cultured under normal oxygen conditions.
  • NK cells induced by culturing under normal oxygen conditions and further cultured under hypoxic conditions showed significantly better cancer cell killing ability than NK cells induced under normal conditions.
  • NK cells of the present invention can be used as an active ingredient in the composition for the prevention or treatment of cancer, the present invention compared to NK cells derived from peripheral blood monocytes cultured in normal oxygen conditions with an oxygen partial pressure of 20% to 22% NK cells with improved cancer cell killing ability, wherein the NK cells with improved cancer cell killing ability compared to peripheral blood monocyte-derived NK cells cultured under normal oxygen conditions, CD107a, NKp44, perforin, and granzyme B It provides a pharmaceutical composition for the prevention or treatment of cancer, wherein the expression of is increased, the secretion of IFN-gamma is increased, and the expression of p16 is reduced.
  • the present invention provides the use of said NK cells for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer.
  • the present invention provides a method for treating cancer, comprising administering to a cancer patient a pharmaceutically effective amount of the pharmaceutical composition for preventing or treating the cancer.
  • the pharmaceutical composition may comprise an active ingredient or an active or inactive pharmaceutically acceptable carrier which constitutes a composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the cancer includes solid cancer, blood cancer, and the like, but is not particularly limited thereto.
  • treatment means any action that inhibits, alleviates or beneficially alters the clinical situation associated with a disease. Treatment can also mean increased survival compared to the expected survival if untreated. Treatment includes simultaneously prophylactic measures in addition to therapeutic means.
  • an "individual” may be a vertebrate, preferably a mammal, such as a dog, cat, mouse, human, or the like.
  • composition of the present invention may be formulated further comprising a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not significantly irritate an organism and does not inhibit the biological activity and properties of the administered component.
  • the pharmaceutically acceptable carrier in the present invention may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, If desired, other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added to formulate them in the form of injectables suitable for infusion into tissues or organs. It may also be formulated as an isotonic sterile solution, or as a dry preparation (particularly a lyophilized preparation), which may optionally be an injectable solution with the addition of sterile water or physiological saline.
  • composition of the present invention may further include a filler, an excipient, a disintegrant, a binder and a lubricant.
  • the compositions of the present invention may also be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
  • the term "administration" refers to introducing a composition of the present invention to a patient in any suitable manner, wherein the route of administration of the composition of the present invention is via oral or parenteral various routes as long as the target tissue can be reached. May be administered. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto.
  • an effective amount means an amount necessary to delay or entirely stop the onset or progression of the particular disease to be treated.
  • the composition may be administered in a pharmaceutically effective amount. It will be apparent to those skilled in the art that a suitable total daily dose may be determined by the practitioner within the correct medical judgment.
  • the specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and extent of the reaction to be achieved and whether other agents are used in some cases, the age, weight, general state of health, sex of the patient. And various factors and similar factors well known in the medicinal art, including diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used with or concurrent with the specific composition.
  • peripheral blood monocytes were isolated from the buff coat. Then, 10% FBS and 1% penicillin / streptomycin were added to RPMI1640 medium at a ratio of 1: 0.5: 0.5 in the presence of IL-2 500 U / ml using 100 Gy irradiated Jurkat cell line and irradiated EBV-LCL cell line The culture was co-cultured in hRPMI medium and exchanged with hRPMI medium added with 500 U / ml of IL-2 once every 2-3 days.
  • Ficoll Ficoll-paqueTM PLUS, GE healthcare
  • the culture was carried out for 9 days at normal oxygen conditions and then moved to 1.5% O 2 conditions for about 3 weeks. At this time, it was incubated while exchanging IL-2 with hRPMI medium added at 500 U / ml.
  • peripheral blood monocytes were cultured for 3 weeks in normal oxygen conditions (20%) and hypoxic conditions (0.5% and 1.5%), or normal oxygen as described in Example 1. After culturing for 9 days under conditions (20%), the cells were moved to hypoxic conditions (1.5%), and cultured for 3 weeks, and the cell number was measured by a hematocytometer. At this time, it was incubated while exchanging IL-2 with hRPMI medium added at 500 U / ml.
  • the number of cells increased under normal oxygen conditions, but the cells did not proliferate or exhibited insufficient growth effects under hypoxic conditions.
  • NK cells normal oxygen conditions (20% O 2 ) and NK cells cultured after 5 days of culture under normal oxygen conditions (1.5% O 2 ) (Hypoxia) Cell proliferation was confirmed. To this end, 3 ⁇ 10 5 cells were cultured from each of the cultured cells, and then stained with Percp-labeled CD3 mAb and APC-labeled CD56mAb to confirm cell proliferation of cells that are CD3 - CD56 + .
  • the cell death was confirmed by staining Annexin V and 7AAD in the experimental and control groups, respectively.
  • the cells were labeled with Chromium for 1 hour, co-cultured with NK cells at a ratio of 1: 1, and after 4 hours, the supernatant was taken to a gamma counter. Isotope values were identified.
  • the growth rate of NK cells was increased by about 60% when cultured in hypoxic conditions, and cell death was reduced by about 50% when cultured in hypoxic conditions.
  • cancer cell killing ability was increased by 20-40% compared to cells cultured under normal oxygen conditions when cultured in hypoxic conditions.
  • NK cells and target cells were mixed at a 1: 1 ratio, incubated at 37 ° C. for 1 hour, 2.5 ⁇ l of FITC labeled anti CD107a mAb, Golgi stop (BD pharmingen), and 37 for 5 hours. Incubated at °C. After incubation, Percp-labeled CD3 mAb and APC-labeled CD56mAb were added and reacted at 4 ° C. for 20 minutes.
  • NK cells cultured in a hypoxic condition had high expression of CD107a (death killing scale) and increased secretion of IFN- ⁇ .
  • NKp44 As shown in FIG. 4, when cultured in hypoxic conditions, NKp44, an NK cell activation receptor, and perforin and granzyme B, molecules related to cancer cell killing ability, were increased.
  • NK cells cultured by moving to hypoxic conditions are less aging than NK cells cultured under normal oxygen conditions.
  • PKH-labeled human T lymphoblastoid cell line CEM in 8-week-old NSG mice was injected 5 ⁇ 10 6 into the mouse abdominal cavity and 1 ⁇ 10 7 NK cells were injected in 48 hours after the cancer cells remained in the abdominal cavity. Collected and analyzed by flow cytometry.
  • NK cells cultured by moving to hypoxic conditions were superior to cancer cell killing ability against blood cancer compared to NK cells cultured under normal oxygen conditions.
  • the present invention can be used in the field of adoptive immune cell therapy.

Abstract

The present invention relates to a method for proliferating and culturing immune cells by using hypoxic conditions. When NK cells, which are induced by the co-culturing of peripheral blood mononuclear cells and feeder cells in the presence of cytokines under normoxic conditions, are transferred to hypoxic conditions so as to be proliferated and cultured, cell proliferation increases, apoptosis is mitigated, cancer cell killing ability increases, and senescence is reduced in comparison to NK cells continuously cultured under normoxic conditions only, and thus the NK cells proliferated and cultured under hypoxic conditions are usable as an excellent adoptive immune cell therapeutic agent for the prevention or treatment of cancer.

Description

저산소 조건을 이용한 면역세포의 증식 배양 방법Proliferation and cultivation method of immune cells using hypoxic conditions
본 발명은 말초혈액 단핵구 유래 NK 세포를 정상산소 조건에서 배양 후 저산소 조건에서 추가 배양하여 암세포 살상능을 향상시키고, 체내에서 오래 생존할 수 있도록 하는 저산소 조건을 이용한 면역세포의 증식 배양 방법에 관한 것이다.The present invention relates to a method for proliferating and culturing immune cells using hypoxic conditions that allow peripheral blood mononuclear cell-derived NK cells to be cultured under normal oxygen conditions and further cultured under hypoxic conditions to improve cancer cell killing ability and to survive in the body for a long time. .
입양 면역 세포치료제는 세포를 체외에서 활성화시켜 환자에게 주입하는 방법으로 수지상 세포, T 세포, NK 세포가 임상적용을 위하여 개발되고 있다. 그 중에서도 NK 세포는 선천 면역세포 중 하나로 다양한 종류의 암세포를 살상가능하고 항원 유무에 관계없이 암세포를 인식하기 때문에 항암치료제로서 각광을 받고 있으나, 실제 임상 시험에서 뚜렷한 완치 효과를 보이지는 않고 있다. 특히 말기암 환자의 경우 커다란 종양 조직에서 발생하는 면역회피 기전으로 인해 주입해준 면역세포가 활성저하를 일으키며, 암세포 공격능을 잃어버리게 된다. 이러한 면역회피 기전에 대한 커다란 원인은 종양의 내부가 저산소 환경이고 이러한 환경이 NK 세포가 암세포를 효과적으로 살상할 수 없는 여건을 제공함으로 고형암 환자의 치료에 있어서 문제가 되고 있다.Adoptive immune cell therapies have been developed for clinical application of dendritic cells, T cells, and NK cells by activating cells in vitro and injecting them into a patient. Among them, NK cells are one of the innate immune cells, which are capable of killing various types of cancer cells and recognize cancer cells regardless of the presence or absence of antigens. In particular, in the terminal cancer patients, the immune cells injected due to the immune evasion mechanism that occurs in the large tumor tissues cause deactivation, and the cancer cell attack ability is lost. The major cause of this immune evasion mechanism is that the interior of the tumor is a hypoxic environment and this environment is a problem in the treatment of solid cancer patients by providing a condition that NK cells can not effectively kill cancer cells.
일반적으로 우리의 체내는 조직에 따라 3% (말초조직)에서 14% (폐)의 산소 분압을 나타내며 이보다 낮은 산소 농도 상태를 저산소 상태로 칭하며 흔히 0.5%에서 3%를 가리킨다. 저산소 조건에서 종양세포는 성장 유지 및 촉진이 된다고 보고되고 있으며 이와 반대로 정상세포와 면역세포는 저산소 상태에서 잘 성장하지 못하는 것으로 알려져 있다. 뿐만 아니라 면역세포의 경우에 주로 저산소 조건이 면역세포의 기능을 억제하는 것으로 알려져 있다. NK 세포에서는 1% O2에서 배양 시 주요 활성화 수용체인 NKp30, NKp44, NKp46, NKG2D가 하향조절(downregulation) 되며 정상세포 조건에서 키운 NK 세포에 비해 암세포 살상능이 저하된다는 보고가 있다. In general, our systems show oxygen partial pressures of 3% (peripheral tissue) to 14% (lung) depending on tissue, and lower oxygen levels are referred to as hypoxic, often 0.5% to 3%. It has been reported that tumor cells maintain and promote growth under hypoxic conditions, whereas normal cells and immune cells do not grow well under hypoxia. In addition, in the case of immune cells, hypoxic conditions are known to inhibit the function of immune cells. In NK cells, NKp30, NKp44, NKp46, and NKG2D, which are the major activation receptors, are downregulated when cultured at 1% O 2 , and cancer cell killing ability is lowered compared to NK cells grown in normal cell conditions.
따라서, 본 발명자들은 NK 세포를 체외 확장 시 여러 농도의 저산소 조건에 미리 노출시켜 저산소 환경에서 적응시킨 후, 상기 NK 세포를 환자의 몸 속에 주입하였을 때 암 조직 내의 저산소 조건에서도 항암 활성을 유지할 수 있는 배양 조건을 확립함으로써 본 발명을 완성하였다.Therefore, the present inventors have previously exposed NK cells to various concentrations of hypoxic conditions in vitro expansion to adapt them in a hypoxic environment, and when the NK cells are injected into the patient's body, they can maintain anticancer activity even in hypoxic conditions in cancer tissues. The present invention was completed by establishing culture conditions.
본 발명의 목적은 정상산소 조건에서 배양 후 저산소 조건에서 추가 배양을 통해 유도되고, 암세포 살상능이 개선된 말초혈액 단핵구 유래의 NK 세포의 유도 및 증식 배양 방법을 제공하는 것이다.It is an object of the present invention to provide a method of inducing and proliferating culture of NK cells derived from peripheral blood monocytes induced by further culture in hypoxic conditions after the culture in normal oxygen conditions and improved cancer cell killing ability.
본 발명의 다른 목적은 상기 암세포 살상능이 개선된 NK 세포의 암의 예방 또는 치료적 용도를 제공하는 것이다.Another object of the present invention is to provide a prophylactic or therapeutic use of cancer of the NK cells with improved cancer cell killing ability.
상기 목적을 달성하기 위하여, 본 발명은 사이토카인 하에서 말초혈액 단핵구 및 영양세포(feeder cell)를 산소 분압이 20% 내지 22%인 정상산소 조건에서 3일 내지13일 동안 배양한 다음, 산소 분압이 1% 내지 5%인 저산소 조건에서 5일 내지 30일 동안 추가 배양하여 자연살해세포(NK 세포)를 유도 및 증식하는 단계를 포함하는 NK 세포의 유도 및 증식 배양 방법을 제공한다.In order to achieve the above object, the present invention is to culture the peripheral blood monocytes and feeder cells under cytokine for 3 to 13 days under normal oxygen conditions with an oxygen partial pressure of 20% to 22%, and then the oxygen partial pressure is increased. It provides a method for inducing and proliferating NK cells comprising the step of inducing and proliferating natural killer cells (NK cells) by further culture for 5 to 30 days in low oxygen conditions of 1% to 5%.
본 발명은 또한 산소 분압이 20% 내지 22%인 정상산소 조건에서 배양된 말초혈액 단핵구 유래의 NK 세포에 비해 암세포 살상능이 향상된 NK 세포를 포함하고, 상기 암세포 살상능이 향상된 NK 세포는 정상산소 조건에서 배양된 말초혈액 단핵구 유래의 NK 세포에 비해 CD107a, NKp44, 퍼포린(perforin), 그랜자임 B(Granzyme B)의 발현이 증가되고, IFN-gamma의 분비가 증가되며, p16의 발현이 감소된 것인, 암의 예방 또는 치료용 약제학적 조성물을 제공한다. The present invention also includes NK cells with improved cancer cell killing ability compared to peripheral blood mononuclear cells-derived NK cells cultured under normal oxygen conditions with an oxygen partial pressure of 20% to 22%, wherein the NK cells with improved cancer cell killing ability are in normal oxygen conditions. Increased expression of CD107a, NKp44, perforin, Granzyme B, increased secretion of IFN-gamma, and decreased expression of p16 compared to cultured peripheral blood monocyte-derived NK cells It provides a pharmaceutical composition for the prevention or treatment of phosphorus, cancer.
본 발명은 또한, 암의 예방 또는 치료를 위한 약제학적 조성물의 제조를 위한 상기 NK 세포의 용도를 제공한다.The invention also provides the use of said NK cells for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer.
본 발명은 또한 상기 암의 예방 또는 치료용 약제학적 조성물의 약제학적 유효량을 암 환자에 투여하는 단계를 포함하는 암의 치료방법을 제공한다.The present invention also provides a method for treating cancer comprising administering to a cancer patient a pharmaceutically effective amount of the pharmaceutical composition for preventing or treating the cancer.
본 발명은 말초혈액 단핵구에서 NK 세포를 유도 및 증식 배양함에 있어 먼저 정상산소 조건에서 일정 시간 동안 배양 후 저산소 조건에서 추가 배양하여 유도 및 증식된 NK 세포의 경우 정상산소 조건에서 유도 및 증식된 NK 세포에 비해 세포 증식이 촉진되고, 세포사가 감소되며, 노화가 억제되고, 암세포 살상능이 촉진될 수 있다.In the present invention, induction and proliferation of NK cells in peripheral blood monocytes, NK cells induced and expanded under normal oxygen conditions in the case of NK cells induced and expanded by incubating for a certain period of time under normal oxygen conditions and further cultured under hypoxic conditions. Compared to the cell proliferation, cell death is reduced, aging is inhibited, cancer cell killing ability can be promoted.
상기 NK 세포는 생체 내에서 생존기간이 길고 암세포 살상능이 우수하여 암의 예방 또는 치료에 효과적인 입양 면역 세포치료제로 사용할 수 있다.The NK cells have a long survival time and excellent cancer cell killing ability in vivo, and thus can be used as an adoptive immune cell therapy for the prevention or treatment of cancer.
도 1은 말초혈액 단핵구로부터 NK 세포를 유도 및 증식함에 있어 산소 조건의 효과를 확인한 결과로, 말초혈액 단핵구를 정상산소 조건(20%) 또는 저산소 조건(0.5%, 1.5%)에서 3주간 배양하거나, 정상산소 조건에서 9일간 배양한 후 저산소 조건(1.5%)에서 추가 배양하면서 측정된 세포수를 나타낸 것이다. 1 is a result of confirming the effect of oxygen conditions in the induction and proliferation of NK cells from peripheral blood monocytes, peripheral blood mononuclear cells cultured for 3 weeks in normal oxygen conditions (20%) or hypoxic conditions (0.5%, 1.5%) or After culturing for 9 days in normal oxygen conditions, and further cultured in low oxygen conditions (1.5%) shows the measured cell number.
도 2a는 정상산소 조건에서 유도된 NK 세포와, 정상산소 조건에서 배양 후 저산소 조건에서 추가 배양하여 유도된 본 발명에 따른 NK 세포의 증식을 비교한 그래프이고, 도 2b는 정상산소 조건에서 유도된 NK 세포와 본 발명에 따른 NK 세포의 세포사를 비교한 그래프이며, 도 2c는 정상산소 조건에서 유도된 NK 세포와 본 발명에 따른 NK 세포의 암세포 살상능을 비교한 그래프이다.Figure 2a is a graph comparing the proliferation of NK cells induced in normal oxygen conditions, and NK cells according to the present invention induced by further culture in hypoxic conditions after culture in normal oxygen conditions, Figure 2b is induced in normal oxygen conditions It is a graph comparing the cell death of NK cells and NK cells according to the present invention, Figure 2c is a graph comparing the cancer cell killing ability of NK cells induced in normal oxygen conditions and NK cells according to the present invention.
도 3은 정상산소 조건에서 유도된 NK 세포와 본 발명에 따른 NK 세포의 다양한 산소 조건에서의 흑색종 세포주인 A375 세포에 대한 살상능을 비교한 그래프이다.3 is a graph comparing the killing ability of NK cells induced in normal oxygen conditions and A375 cells, a melanoma cell line under various oxygen conditions of the NK cells according to the present invention.
도 4는 정상산소 조건에서 유도된 NK 세포와 본 발명에 따른 NK 세포의 활성화수용체와 억제 수용체의 변화를 확인한 결과이다.Figure 4 is a result confirming the change in the NK cells induced in normal oxygen conditions and the activating receptors and inhibitory receptors of NK cells according to the present invention.
도 5는 정상산소 조건에서 유도된 NK 세포와 본 발명에 따른 NK 세포에서 노화 마커인 p16의 발현을 확인한 결과이다.5 is a result confirming the expression of p16, an aging marker in NK cells induced in normal oxygen conditions and NK cells according to the present invention.
도 6은 이종이식 혈액암 모델에서 정상산소 조건에서 유도된 NK 세포와 본 발명에 따른 NK 세포의 암세포 살상능을 확인한 결과이다.Figure 6 is a result confirming the cancer cell killing ability of NK cells induced in normal oxygen conditions and NK cells according to the present invention in xenograft hematological cancer model.
이하, 본 발명의 구성을 구체적으로 설명한다.EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.
본 발명은 사이토카인 하에서 말초혈액 단핵구 및 영양세포(feeder cell)를 산소 분압이 20% 내지 22%인 정상산소 조건에서 3일 내지 13일 동안 배양한 다음, 산소 분압이 1% 내지 5%인 저산소 조건에서 5일 내지 30일 동안 추가 배양하여 자연살해세포(NK 세포)를 유도 및 증식하는 단계를 포함하는 NK 세포의 유도 및 증식 배양 방법에 관한 것이다.The present invention is cultured peripheral blood monocytes and feeder cells under cytokines for 3 days to 13 days under normal oxygen conditions with an oxygen partial pressure of 20% to 22%, hypoxia with an oxygen partial pressure of 1% to 5% The present invention relates to a method of inducing and proliferating NK cells, which further comprises inducing and proliferating natural killer cells (NK cells) by further culturing for 5 to 30 days under conditions.
상기 정상산소 조건은 산소 분압이 20% 내지 22%인 세포 배양 조건을 의미하고, 저산소 조건은 산소 분압이 1% 내지 5%, 또는 1.5% 내지 3%인 세포 배양 조건을 의미한다.The normal oxygen condition means a cell culture condition having an oxygen partial pressure of 20% to 22%, and a low oxygen condition means a cell culture condition having an oxygen partial pressure of 1% to 5%, or 1.5% to 3%.
본 발명의 NK 세포의 유도 및 증식 배양 방법은 정상산소 조건에서 말초혈액 단핵구를 일정 기간 배양하고, 저산소 조건에서 추가 배양하여 NK 세포를 유도 및 증식하는 것을 특징으로 한다. Induction and proliferation culture method of NK cells of the present invention is characterized in that the peripheral blood monocytes are cultured for a certain period of time under normal oxygen conditions, and further cultured under low oxygen conditions to induce and proliferate NK cells.
보다 구체적으로, 사이토카인 하에서 말초혈액 단핵구를 영양세포와 산소 분압이 20% 내지 22%인 정상산소 조건에서 3일 내지 13일 동안 배양하고, 산소 분압이 1% 내지 5%인 저산소 조건에서 5일 내지 30일 동안 추가 배양할 수 있다.More specifically, the peripheral blood monocytes under cytokines were cultured for 3 days to 13 days under vegetative cells and normal oxygen conditions with an oxygen partial pressure of 20% to 22%, and 5 days under hypoxic conditions with an oxygen partial pressure of 1% to 5%. It may be further incubated for 30 days.
상기 말초혈액 단핵구는 정상 또는 암환자의 말초혈액에서 유래된 것일 수 있다.The peripheral blood monocytes may be derived from peripheral blood of normal or cancer patients.
상기 영양세포는 방사선 조사된 Jurkat 세포 및 방사선 조사된 EBV-LCL 세포일 수 있다. 더 구체적으로, 상기 영양세포는 방사선 조사된 Jurkat 세포 및 방사선 조사된 EBV-LCL 세포를 약 1:1의 세포수의 비율로 혼합한 것일 수 있으나, 이에 특별히 제한하는 것은 아니다. The feeder cells may be irradiated Jurkat cells and irradiated EBV-LCL cells. More specifically, the feeder cells may be a mixture of irradiated Jurkat cells and irradiated EBV-LCL cells in a ratio of about 1: 1 cell number, but is not particularly limited thereto.
상기 말초혈액 단핵구와 영양세포는 약 1:1의 세포수의 비율로 혼합하여 공배양할 수 있으나, 이에 특별히 제한하는 것은 아니다.The peripheral blood monocytes and feeder cells may be co-cultured by mixing at a ratio of about 1: 1 cells, but are not particularly limited thereto.
상기 사이토카인은 인터루킨-2(IL-2)를 포함할 수 있다.The cytokine may comprise interleukin-2 (IL-2).
상기에서 유도된 NK 세포는 CD3-CD56+ 표현형을 갖는 세포일 수 있다.The NK cells derived above may be cells having a CD3 - CD56 + phenotype.
상기 NK 세포의 유도 및 증식 배양에 사용하는 배지는 혈청이 첨가된 공지의 세포배양배지를 제한 없이 사용할 수 있다. The medium used for the induction and proliferation culture of the NK cells can be used without limitation, known cell culture medium added with serum.
상기 혈청은 세포배양에 사용되는 공지의 혈청의 종류를 제한 없이 사용할 수 있다.The serum can be used without limitation the kind of known serum used for cell culture.
본 발명의 NK 세포의 유도 및 증식 배양 방법에 따른 NK 세포는 정상산소 조건에서 배양한 말초혈액 단핵구 유래의 NK 세포 대비 세포수가 많다. NK cells according to the induction and proliferation culture method of the NK cells of the present invention has a larger number of cells than NK cells derived from peripheral blood monocytes cultured under normal oxygen conditions.
본 발명의 일 구체예에 따르면, 정상산소 조건에서 동안 배양한 다음 저산소 조건에서 추가 배양한 경우의 세포수를 확인한 결과, 배양 3일 미만일 때 저산소 조건(1.5% 또는 0.5%)로 옮긴 경우 NK 세포는 거의 증식하지 않았으나, 정상산소 조건에서 9일 배양 후 저산소 조건으로 옮겨 준 경우 NK 세포의 증식이 대조군과 비교하여 세포수득율이 현저히 높았다. 세포 증식을 비교한 결과, 증식율이 약 60% 정도 증가되어 있으며, 세포사 확인 실험에서는 약 50% 정도 감소되어 있으며, 암세포 살상능 조사에서는 20% 내지 40% 정도 암세포 살상능이 증가하였다. According to one embodiment of the present invention, as a result of confirming the number of cells when cultured under normal oxygen conditions and further cultured under hypoxic conditions, NK cells when transferred to hypoxic conditions (1.5% or 0.5%) when less than 3 days of culture The growth rate of NK cells was significantly higher than that of the control group when the cells were transferred to hypoxic conditions after 9 days of culture under normal oxygen conditions. As a result of comparing the cell proliferation, the proliferation rate was increased by about 60%, the cell death confirmation experiment was reduced by about 50%, and the cancer cell killing ability was increased by 20% to 40% by the investigation of cancer cell killing ability.
또한, 상기 NK 세포를 저산소 조건에서 암세포 살상능을 실험한 결과, 정상산소 조건에서 연속 배양한 NK 세포 대비 고형암에 대한 암세포 살상능이 뛰어났고, 이 NK 세포는 CD107a(살상능 척도)의 발현이 높을 뿐만 아니라 IFN-γ의 분비가 증가되어 있다. In addition, the cancer cell killing ability of the NK cells in the hypoxic condition was tested, compared with the NK cells cultured continuously in the normal oxygen condition, the cancer cell killing ability for solid cancer was superior, the NK cells have a high expression of CD107a (killing capacity measure) In addition, the secretion of IFN-γ is increased.
아울러, 상기 NK 세포의 활성화수용체 및 억제수용체의 변화를 확인한 결과, 활성화수용체인 NKp44와 암세포 살상능에 관계된 분자인 퍼포린(perforin), 그랜자임 B(Granzyme B)의 발현이 증가되어 있다. In addition, as a result of confirming changes in the NK cell activation receptor and inhibitory receptor, the expression of NKp44, an activation receptor and molecules related to cancer cell killing ability, perforin and granzyme B are increased.
또한, 노화 마커인 p16의 발현이 감소되어 있어 정상산소 조건에서 연속 배양된 NK 세포 대비 노화가 덜 되었음을 알 수 있다.In addition, the expression of the aging marker p16 is reduced, it can be seen that less aging compared to NK cells continuously cultured under normal oxygen conditions.
또한, 이종이식 혈액암 모델에서 정상산소 조건에서 배양 후 저산소 조건에서 추가 배양하여 유도된 NK 세포는 정상조건에서 유도된 NK 세포에 비해 현저히 우수한 암세포 살상능을 나타냈다.In addition, in the xenograft hematologic cancer model, NK cells induced by culturing under normal oxygen conditions and further cultured under hypoxic conditions showed significantly better cancer cell killing ability than NK cells induced under normal conditions.
따라서, 본 발명의 NK 세포는 암의 예방 또는 치료용 조성물의 유효성분으로 사용할 수 있어, 본 발명은 산소 분압이 20% 내지 22%인 정상산소 조건에서 배양된 말초혈액 단핵구 유래의 NK 세포에 비해 암세포 살상능이 향상된 NK 세포를 포함하고, 상기 암세포 살상능이 향상된 NK 세포는 정상산소 조건에서 배양된 말초혈액 단핵구 유래의 NK 세포에 비해 CD107a, NKp44, 퍼포린(perforin), 그랜자임 B(Granzyme B)의 발현이 증가되고, IFN-gamma의 분비가 증가되며, p16의 발현이 감소된 것인, 암의 예방 또는 치료용 약제학적 조성물을 제공한다.Therefore, NK cells of the present invention can be used as an active ingredient in the composition for the prevention or treatment of cancer, the present invention compared to NK cells derived from peripheral blood monocytes cultured in normal oxygen conditions with an oxygen partial pressure of 20% to 22% NK cells with improved cancer cell killing ability, wherein the NK cells with improved cancer cell killing ability compared to peripheral blood monocyte-derived NK cells cultured under normal oxygen conditions, CD107a, NKp44, perforin, and granzyme B It provides a pharmaceutical composition for the prevention or treatment of cancer, wherein the expression of is increased, the secretion of IFN-gamma is increased, and the expression of p16 is reduced.
본 발명은 하나의 양태로서, 암의 예방 또는 치료를 위한 약제학적 조성물의 제조를 위한 상기 NK 세포의 용도를 제공한다.In one aspect, the present invention provides the use of said NK cells for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer.
또 하나의 양태로서, 본 발명은 상기 암의 예방 또는 치료용 약제학적 조성물의 약제학적 유효량을 암 환자에 투여하는 단계를 포함하는 암의 치료방법을 제공한다.In another aspect, the present invention provides a method for treating cancer, comprising administering to a cancer patient a pharmaceutically effective amount of the pharmaceutical composition for preventing or treating the cancer.
상기 약제학적 조성물은 인 비트로, 인 비보 또는 엑스 비보에서 진단적 또는 치료적 용도에 적합한 조성물을 이루는 활성성분 및 활성 또는 무활성 약제학적으로 허용가능한 담체를 포함할 수 있다.The pharmaceutical composition may comprise an active ingredient or an active or inactive pharmaceutically acceptable carrier which constitutes a composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
상기 암은 고형암, 혈액암 등을 포함하며, 이에 특별히 제한하지는 않는다.The cancer includes solid cancer, blood cancer, and the like, but is not particularly limited thereto.
본 발명에서 "치료" 란 질환과 관련된 임상적 상황을 억제하거나 완화하거나 이롭게 변경하는 모든 행위를 의미한다. 또한 치료는 치료를 받지 않은 경우 예상되는 생존율과 비교하여 증가된 생존을 의미할 수 있다. 치료는 치료적 수단 이외에 예방적 수단을 동시에 포함한다.As used herein, "treatment" means any action that inhibits, alleviates or beneficially alters the clinical situation associated with a disease. Treatment can also mean increased survival compared to the expected survival if untreated. Treatment includes simultaneously prophylactic measures in addition to therapeutic means.
본 명세서에 있어서, "개체"는 척추동물, 바람직하게는 포유동물, 예를 들어, 개, 고양이, 생쥐, 인간 등일 수 있다. As used herein, an "individual" may be a vertebrate, preferably a mammal, such as a dog, cat, mouse, human, or the like.
본 발명의 조성물은 약학적으로 허용가능한 담체를 추가로 포함하여 제제화될 수 있다. 본 발명에서 용어, "약학적으로 허용가능한 담체"란 생물체를 상당히 자극하지 않고 투여 성분의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 말한다. 본 발명에서의 약학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 또는 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액 및 정균제 등 다른 통상의 첨가제를 첨가하여, 조직 또는 장기에 주입하기에 적합한 주사제의 형태로 제형화할 수 있다. 또한, 등장성 멸균 용액, 또는 경우에 따라 멸균수나 생리식염수를 첨가하여 주사 가능한 용액이 될 수 있는 건조 제제(특히 동결 건조제제)로 제형화할 수도 있다.The composition of the present invention may be formulated further comprising a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not significantly irritate an organism and does not inhibit the biological activity and properties of the administered component. The pharmaceutically acceptable carrier in the present invention may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, If desired, other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added to formulate them in the form of injectables suitable for infusion into tissues or organs. It may also be formulated as an isotonic sterile solution, or as a dry preparation (particularly a lyophilized preparation), which may optionally be an injectable solution with the addition of sterile water or physiological saline.
또한, 바람직하게 본 발명의 조성물은 충진제, 부형제, 붕해제, 결합제 및 활택제 등을 추가로 포함할 수 있다. 또한 본 발명의 조성물은 포유동물에 투여된 후 활성성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형화 될 수 있다. In addition, preferably, the composition of the present invention may further include a filler, an excipient, a disintegrant, a binder and a lubricant. The compositions of the present invention may also be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
본 발명에서 용어, "투여"는 어떠한 적절한 방법으로 환자에게 본 발명의 조성물을 도입하는 것을 의미하며, 본 발명의 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다. 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경구 투여, 국소 투여, 비내 투여, 폐내 투여, 직장내 투여 될 수 있으나, 이에 제한되지는 않는다.As used herein, the term "administration" refers to introducing a composition of the present invention to a patient in any suitable manner, wherein the route of administration of the composition of the present invention is via oral or parenteral various routes as long as the target tissue can be reached. May be administered. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto.
본 명세서에서, 유효량은 목적하는 치료되어야 할 특정 질환의 발병 또는 진행을 지연하거나 전적으로 중지시키는 데 필요한 양을 의미한다. 본 발명에서 조성물은 약학적 유효량으로 투여될 수 있다. 적합한 총 1일 사용량은 올바른 의학적 판단범위 내에서 처치의에 의해 결정될 수 있다는 것은 당업자에게 자명한 일이다. 본 발명의 목적상, 특정 환자에 대한 구체적인 치료적 유효량은 달성하고자 하는 반응의 종류와 정도, 경우에 따라 다른 제제가 사용되는지의 여부를 비롯한 구체적 조성물, 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 구체적 조성물과 함께 사용되거나 동시 사용되는 약물을 비롯한 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 바람직하다.As used herein, an effective amount means an amount necessary to delay or entirely stop the onset or progression of the particular disease to be treated. In the present invention, the composition may be administered in a pharmaceutically effective amount. It will be apparent to those skilled in the art that a suitable total daily dose may be determined by the practitioner within the correct medical judgment. For the purposes of the present invention, the specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and extent of the reaction to be achieved and whether other agents are used in some cases, the age, weight, general state of health, sex of the patient. And various factors and similar factors well known in the medicinal art, including diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used with or concurrent with the specific composition.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
<실시예 1> NK 세포의 배양Example 1 Culture of NK Cells
사람의 혈액을 채혈한 후, Ficoll(Ficoll-paqueTM PLUS, GE healthcare)을 이용하여 2,500rpm에서 30분간 원심분리한 후 연막층(buffy coat)에서 말초혈액 단핵구를 분리하였다. 그 후 100 Gy로 방사선 조사한 Jurkat 세포주와 방사선 조사한 EBV-LCL 세포주를 사용하여 IL-2 500 U/ml 존재 하에 1:0.5:0.5의 비율로 RPMI1640 배지에 10% FBS와 1% penicillin/streptomycin을 넣은 hRPMI 배지에 공배양하여 2-3일 마다 한 번씩 IL-2가 500 U/ml로 첨가된 hRPMI 배지로 교환을 해 주면서 배양을 하였다.After collecting blood from humans, centrifuged at 2,500 rpm for 30 minutes using Ficoll (Ficoll-paqueTM PLUS, GE healthcare), peripheral blood monocytes were isolated from the buff coat. Then, 10% FBS and 1% penicillin / streptomycin were added to RPMI1640 medium at a ratio of 1: 0.5: 0.5 in the presence of IL-2 500 U / ml using 100 Gy irradiated Jurkat cell line and irradiated EBV-LCL cell line The culture was co-cultured in hRPMI medium and exchanged with hRPMI medium added with 500 U / ml of IL-2 once every 2-3 days.
상기 배양은 9일간 정상산소 조건에서 수행한 후 1.5% O2 조건으로 이동하여 약 3주간 더 수행하였다. 이때 한번씩 IL-2가 500 U/ml로 첨가된 hRPMI 배지로 교환해 주면서 배양하였다.The culture was carried out for 9 days at normal oxygen conditions and then moved to 1.5% O 2 conditions for about 3 weeks. At this time, it was incubated while exchanging IL-2 with hRPMI medium added at 500 U / ml.
<실험예 1> 세포수 측정Experimental Example 1 Measurement of Cell Number
저산소 조건이 NK 세포의 증식에 미치는 영향을 보기 위하여, 실시예 1에 기재된 방식대로 정상산소 조건(20%) 및 저산소 조건(0.5% 및 1.5%)에서 말초혈액 단핵구를 3주간 배양하거나, 정상산소 조건(20%)에서 9일간 배양한 후 저산소 조건(1.5%)으로 이동하여 3주간 배양하면서 헤마토사이토미터로 세포수를 측정하였다. 이때 한번씩 IL-2가 500 U/ml로 첨가된 hRPMI 배지로 교환해 주면서 배양하였다.To see the effect of hypoxic conditions on the proliferation of NK cells, peripheral blood monocytes were cultured for 3 weeks in normal oxygen conditions (20%) and hypoxic conditions (0.5% and 1.5%), or normal oxygen as described in Example 1. After culturing for 9 days under conditions (20%), the cells were moved to hypoxic conditions (1.5%), and cultured for 3 weeks, and the cell number was measured by a hematocytometer. At this time, it was incubated while exchanging IL-2 with hRPMI medium added at 500 U / ml.
도 1에 나타난 바와 같이, 정상산소 조건에서는 세포수가 증가하나, 저산소 조건에서는 세포가 증식하지 않거나 미비한 증식 효과를 나타내었다.As shown in FIG. 1, the number of cells increased under normal oxygen conditions, but the cells did not proliferate or exhibited insufficient growth effects under hypoxic conditions.
또한, 정상산소 조건에서 9일간 배양한 후 저산소 조건으로 옮긴 경우(20%→1.5%)에는 정상산소 조건보다 월등히 높은 정도로 지속적인 세포 증식이 관찰되었다. In addition, in the case of culturing for 9 days in normal oxygen condition and then transferred to hypoxic condition (20% → 1.5%), continuous cell proliferation was observed to a much higher level than normal oxygen condition.
<실험예 2> NK 세포의 증식, 세포사 및 암세포 살상능 측정Experimental Example 2 Measurement of NK Cell Proliferation, Cell Death and Cancer Cell Killing Capacity
정상산소 조건(20% O2)에서 연속 배양된 NK 세포(Normoxia)와, 정상산소 조건에서 배양 5일 후 저산소 조건(1.5% O2)(Hypoxia)으로 옮겨 배양한 NK 세포의 Ki67 발현을 비교하여 세포 증식을 확인하였다. 이를 위해, 배양한 세포를 각각 3×105 세포를 취한 후 Percp-labeled CD3 mAb, APC-labeled CD56mAb로 염색한 후 CD3-CD56+인 세포의 세포 증식을 확인하였다.Comparison of Ki67 expression of NK cells (Normoxia) continuously cultured under normal oxygen conditions (20% O 2 ) and NK cells cultured after 5 days of culture under normal oxygen conditions (1.5% O 2 ) (Hypoxia) Cell proliferation was confirmed. To this end, 3 × 10 5 cells were cultured from each of the cultured cells, and then stained with Percp-labeled CD3 mAb and APC-labeled CD56mAb to confirm cell proliferation of cells that are CD3 - CD56 + .
실험군과 대조군을 각각 Annexin V, 7AAD를 염색하여 세포사를 확인하였다.The cell death was confirmed by staining Annexin V and 7AAD in the experimental and control groups, respectively.
또한, 타겟 세포로 K562, A375 세포를 준비한 후 Chromium으로 1시간 동안 표지(labeling)한 후 NK 세포와 1:1의 비율로 공동 배양한 다음 4시간 후 상층액을 취해서 감마 카운터(gamma counter)로 동위원소 값을 확인하였다.In addition, after preparing K562 and A375 cells as target cells, the cells were labeled with Chromium for 1 hour, co-cultured with NK cells at a ratio of 1: 1, and after 4 hours, the supernatant was taken to a gamma counter. Isotope values were identified.
도 2a 및 도 2b에 나타난 바와 같이, 저산소 조건에서 배양한 경우 NK 세포의 증식율이 약 60% 정도 증가되었고, 저산소 조건에서 배양한 경우 세포사가 약 50% 정도 감소되었다.As shown in FIG. 2A and FIG. 2B, the growth rate of NK cells was increased by about 60% when cultured in hypoxic conditions, and cell death was reduced by about 50% when cultured in hypoxic conditions.
도 2c에 나타난 바와 같이, 저산소 조건에서 배양한 경우 암세포 살상능이 정상산소 조건에서 배양한 세포보다 20-40% 증가되었다.As shown in FIG. 2c, cancer cell killing ability was increased by 20-40% compared to cells cultured under normal oxygen conditions when cultured in hypoxic conditions.
<실험예 3> 산소 농도별로 NK 세포의 암세포 살상능 측정 실험Experimental Example 3 Experiment for Measuring Cancer Cell Killing Capacity of NK Cells by Oxygen Concentration
산소 농도별, 0.5%, 1.5% 및 20% NK 세포의 암세포 살상능을 측정하였다.Cancer cell killing ability of 0.5%, 1.5% and 20% NK cells by oxygen concentration was measured.
타겟 세포로 A375 세포를 준비한 후 NK 세포와 타겟 세포를 1:1 비율로 섞어 1시간 동안 37℃에서 배양하고 FITC labeled anti CD107a mAb를 2.5㎕ 넣고 Golgi stop(BD pharmingen)을 넣은 후 5시간 동안 37℃에서 배양하였다. 배양이 끝난 후 Percp-labeled CD3 mAb, APC-labeled CD56mAb를 넣고 4℃에서 20분간 반응시켰다. Intracellular FACS를 위해 세포를 FACS 버퍼로 세척하고, 고정시킨 후 BD cytoperm/cytofix kit(BD Pharmingen, San Diego, CA)로 투과시켜 PE-labeled IFN-g mAb로 염색한 후 유세포분석기를 이용하여 분석하였다. After preparing A375 cells as target cells, NK cells and target cells were mixed at a 1: 1 ratio, incubated at 37 ° C. for 1 hour, 2.5 μl of FITC labeled anti CD107a mAb, Golgi stop (BD pharmingen), and 37 for 5 hours. Incubated at ℃. After incubation, Percp-labeled CD3 mAb and APC-labeled CD56mAb were added and reacted at 4 ° C. for 20 minutes. Cells were washed with FACS buffer for intracellular FACS, fixed, permeabilized with BD cytoperm / cytofix kit (BD Pharmingen, San Diego, Calif.), Stained with PE-labeled IFN-g mAb, and analyzed using flow cytometry. .
도 3은 산소 농도별로 NK 세포의 암세포 살상능을 확인한 결과로, 저산소 조건에서 배양한 경우 정상산소 조건에서 배양한 NK 세포 대비 고형암에 대한 암세포 살상능이 더 뛰어났다. 3 is a result of confirming the cancer cell killing ability of NK cells by oxygen concentration, when cultured in a hypoxic condition was superior to the cancer cell killing capacity for solid cancer compared to NK cells cultured in normal oxygen conditions.
또한, 저산소 조건에서 배양한 NK 세포가 CD107a(살상능 척도)의 발현이 높고, IFN-γ의 분비가 증가되어 있었다. In addition, NK cells cultured in a hypoxic condition had high expression of CD107a (death killing scale) and increased secretion of IFN-γ.
<실험예 4> NK 세포의 활성화수용체 및 억제수용체 변화 측정 실험Experimental Example 4 Measurement of Changes in Activated and Inhibitors of NK Cells
정상산소 조건(20% O2)에서 배양된 NK 세포와 배양 5일째에 저산소 조건(1.5% O2)으로 옮겨 배양된 NK 세포의 활성화수용체 및 억제수용체의 발현 변화를 측정하였다.Changes in the expression of activated and inhibitory receptors of NK cells cultured in normal oxygen conditions (20% O 2 ) and cultured NK cells were transferred to hypoxic conditions (1.5% O 2 ) on day 5 of culture.
도 4에 나타난 바와 같이, 저산소 조건에서 배양한 경우 NK 세포의 활성화수용체인 NKp44와 암세포 살상능에 관계된 분자인 퍼포린(perforin), 그랜자임 B(Granzyme B)의 발현이 증가되었다.As shown in FIG. 4, when cultured in hypoxic conditions, NKp44, an NK cell activation receptor, and perforin and granzyme B, molecules related to cancer cell killing ability, were increased.
<실험예 5> NK 세포의 노화 마커 발현 측정 실험Experimental Example 5 Experiment for Measuring Aging Marker Expression of NK Cells
정상산소 조건(20% O2)에서 연속 배양된 NK 세포와 배양 5일째에 저산소 조건(1.5% O2)으로 옮겨 배양된 NK 세포의 노화 마커 발현 변화를 측정하였다.Changes in aging marker expression of NK cells cultured in normal oxygen conditions (20% O 2 ) and cultured NK cells were transferred to hypoxic conditions (1.5% O 2 ) on day 5 of culture.
도 5에 나타난 바와 같이, 정상산소 조건에서 배양한 경우 23일째에 p16의 발현이 현저히 증가하나, 저산소 조건에서 배양한 경우 상대적으로 발현이 감소되었다. 따라서, 저산소 조건으로 옮겨 배양한 NK 세포가 정상산소 조건에서 배양한 NK 세포에 비해 노화가 덜 되었음을 알 수 있다.As shown in FIG. 5, the expression of p16 was significantly increased at 23 days when cultured under normal oxygen conditions, but the expression was relatively decreased when cultured under hypoxic conditions. Therefore, it can be seen that NK cells cultured by moving to hypoxic conditions are less aging than NK cells cultured under normal oxygen conditions.
<실험예 6> 이종이식 혈액암 동물 모델에서 NK 세포의 암세포 살상능 측정 실험Experimental Example 6 Measurement of Cancer Cell Killing Capacity of NK Cells in Xenograft Blood Cancer Animal Model
이종이식 혈액암 동물 모델에서 정상산소 조건과 배양된 NK 세포와 정상산소 조건에서 배양 9일째에 저산소 조건(1.5% O2)으로 옮겨 배양된 NK 세포의 체내 암세포 살상능을 확인하기 위해, 7~8주령 NSG 마우스에서 PKH로 표지한 인간 T 세포 림프 아세포(T lymphoblastoid cell line) CEM을 마우스 복강내에 5×106 주입하고 NK 세포를 1×107 씩 주입하여 48시간 후에 복강내에 남아 있는 암세포를 수거하여 유세포 분석기로 분석하였다.In the xenograft hematological cancer animal model, in order to determine the cancer cell killing ability of the cultured NK cells in the normal oxygen condition and cultured NK cells and the normal oxygen condition to the low oxygen condition (1.5% O 2 ) on the 9th day of culture. PKH-labeled human T lymphoblastoid cell line CEM in 8-week-old NSG mice was injected 5 × 10 6 into the mouse abdominal cavity and 1 × 10 7 NK cells were injected in 48 hours after the cancer cells remained in the abdominal cavity. Collected and analyzed by flow cytometry.
도 6에 나타난 바와 같이, 저산소 조건으로 옮겨 배양한 NK 세포가 정상산소 조건에서 배양한 NK 세포 대비 혈액암에 대한 암세포 살상능이 더 뛰어났다. As shown in FIG. 6, NK cells cultured by moving to hypoxic conditions were superior to cancer cell killing ability against blood cancer compared to NK cells cultured under normal oxygen conditions.
본 발명은 입양 면역 세포치료제 분야에서 이용할 수 있다.The present invention can be used in the field of adoptive immune cell therapy.

Claims (6)

  1. 사이토카인 하에서 말초혈액 단핵구 및 영양세포(feeder cell)를 산소 분압이 20% 내지 22%인 정상산소 조건에서 3일 내지 13일 동안 배양한 다음, 산소 분압이 1% 내지 5%인 저산소 조건에서 5일 내지 30일 동안 추가 배양하여 자연살해세포(NK 세포)를 유도 및 증식하는 단계를 포함하는 NK 세포의 유도 및 증식 배양 방법.Under cytokines, peripheral blood monocytes and feeder cells were incubated for 3 to 13 days under normal oxygen conditions with an oxygen partial pressure of 20% to 22%, and then under 5% hypoxic conditions with an oxygen partial pressure of 1% to 5%. Induction and proliferation culture method of NK cells comprising the step of inducing and proliferating natural killer cells (NK cells) by further culture for one to 30 days.
  2. 제1항에 있어서,The method of claim 1,
    말초혈액 단핵구는 정상 또는 암환자의 말초혈액에서 유래된 것인, NK 세포의 유도 및 증식 배양 방법.Peripheral blood monocytes are derived from the peripheral blood of normal or cancer patients, induction and proliferation culture method of NK cells.
  3. 제1항에 있어서,The method of claim 1,
    영양세포는 방사선 조사된 Jurkat 세포 및 방사선 조사된 EBV-LCL 세포를 포함하는, NK 세포의 유도 및 증식 배양 방법.The feeder cell comprises irradiated Jurkat cells and irradiated EBV-LCL cells.
  4. 제1항에 있어서,The method of claim 1,
    사이토카인은 인터루킨-2(IL-2)를 포함하는, NK 세포의 유도 및 증식 배양 방법.The cytokine comprises interleukin-2 (IL-2), method of inducing and proliferating culture of NK cells.
  5. 산소 분압이 20% 내지 22%인 정상산소 조건에서 배양된 말초혈액 단핵구 유래의 NK 세포에 비해 암세포 살상능이 향상된 NK 세포를 포함하고, It includes NK cells with improved cancer cell killing ability compared to peripheral blood monocyte-derived NK cells cultured under normal oxygen conditions with an oxygen partial pressure of 20% to 22%,
    상기 암세포 살상능이 향상된 NK 세포는 정상산소 조건에서 배양된 말초혈액 단핵구 유래의 NK 세포에 비해 CD107a, NKp44, 퍼포린(perforin), 그랜자임 B(Granzyme B)의 발현이 증가되고, IFN-gamma의 분비가 증가되며, p16의 발현이 감소된 것인, 암의 예방 또는 치료용 약제학적 조성물.The NK cells with improved cancer cell killing ability increased expression of CD107a, NKp44, perforin, and granzyme B compared to peripheral blood monocyte-derived NK cells cultured under normal oxygen conditions, and the expression of IFN-gamma. A pharmaceutical composition for preventing or treating cancer, wherein the secretion is increased and the expression of p16 is reduced.
  6. 산소 분압이 20% 내지 22%인 정상산소 조건에서 배양된 말초혈액 단핵구 유래의 NK 세포에 비해 암세포 살상능이 향상된 NK 세포를 포함하는 암의 예방 또는 치료용 약제학적 조성물의 약제학적 유효량을 암 환자에 투여하는 단계를 포함하고,A pharmaceutically effective amount of a pharmaceutical composition for the prevention or treatment of cancer comprising NK cells with enhanced cancer cell killing ability compared to peripheral blood mononuclear cells-derived NK cells cultured under normal oxygen conditions with an oxygen partial pressure of 20% to 22% was provided to cancer patients. Administering,
    상기 암세포 살상능이 향상된 NK 세포는 정상산소 조건에서 배양된 말초혈액 단핵구 유래의 NK 세포에 비해 CD107a, NKp44, 퍼포린(perforin), 그랜자임 B(Granzyme B)의 발현이 증가되고, IFN-gamma의 분비가 증가되며, p16의 발현이 감소된 것인, 암의 치료방법.The NK cells with improved cancer cell killing ability increased expression of CD107a, NKp44, perforin, and granzyme B compared to peripheral blood monocyte-derived NK cells cultured under normal oxygen conditions, and the expression of IFN-gamma. The secretion is increased, the expression of p16 is reduced.
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