WO2023096352A1 - Feeder cell line genetically engineered to express hla-e and use thereof - Google Patents

Abstract

The present invention relates to genetically-engineered cells for NK cell activation. Genetically-engineered cells for NK cell activation according to one aspect can synergistically induce the proliferation and activation of NK cells from a sample, and thus a method for proliferating NK cells or NK cells proliferated by the method can be usefully used for antibody therapeutics and the like.

Description

Feeder cell line genetically engineered to express HLA-E, and use thereof

This application claims priority to Korean Patent Application No. 10-2021-0164859 filed on November 25, 2021 and Korean Patent Application No. 10-2022-0118160 filed on September 19, 2022, the entire specification References in the application.

It relates to a feeder cell line genetically engineered to express HLA-E, and its use, specifically, a method for proliferating NK cells using the same, or a method for measuring NK cell activity using the same.

NK cells (natural killer cells) are a type of cytotoxic lymphocytes that are important for innate immunity. NK cells respond to virus-infected cells or cancer cells, and these responses are signals generated by various activating receptors and inhibitory receptors of NK cells reacting with each ligand of target cells. ) depends on Normally, NK cells can attack target cells if the activation signal is stronger than the inhibitory signal, but do not attack if the inhibitory signal is stronger. Therefore, normal cells are not attacked by NK cells because they generate inhibitory signals due to the presence of NK cell inhibitory receptor ligands (MHC, major histocompatibility complex).

Some of the cancer cells may decrease due to the occurrence of MHC abnormalities on the cell surface. In this case, there is no inhibitory signal from NK cells, so NK cells attack target cells. It has cytotoxicity by secreting perforin to pierce the cell membrane of infected cells or cancer cells and releasing granzyme to kill these cells. In the case of B-cell lymphoma and many cancer patients, defects in the number or anticancer activity of NK cells have been found, and it is known that NK cell dysfunction is closely related to the occurrence of these cancers. Therefore, adoptive cell therapy (ACT), in which a large amount of high-performance NK cells derived from the peripheral blood of healthy people are cultured in vitro and then administered to cancer patients by maximizing anticancer activity, is emerging. The treatment is Development of an amplification method is essential to secure a large amount of high-performance NK cells.

Most of the previously reported NK cell proliferation methods were methods for NK cell (conventional NK cell) amplification. However, the clinical significance of adaptive NK cells, which are one of the subtypes of NK cells involved in the adaptive immune system, has recently been emphasized as specialized natural killer cells capable of forming immunological memory. Except for one study that showed amplification (Cancer Immunol. Res. 2017, 5, 654-665.), there is no condition.

Therefore, there is a need to develop innovative methods for selectively amplifying adaptive NK cells.

One aspect is to provide feeder cells for culturing NK cells genetically engineered to express human leukocyte antigen (HLA).

Another aspect is to provide a composition for NK cell culture comprising the culture feeder cells.

Another aspect includes obtaining a blood sample containing a population of NK cells; and contacting at least a portion of the mixed population of NK cells with cells genetically engineered to activate NK cells, wherein the genetically engineered cells are strained to express Human leukocyte antigen (HLA). It is to provide a method of proliferating NK cells, comprising steps that are wholly engineered.

One aspect provides a cell line genetically engineered to express human leukocyte antigen (HLA).

The cell line may be a culture feeder cell that selectively amplifies only NK cells.

As used herein, the term "feeder cells (feeder cells)" does not have the ability to divide and proliferate by irradiation, but because it has metabolic activity, it produces various metabolites to help the proliferation of target NK cells. can do. The feeder cell that can be used in the present specification is an animal cell line into which a gene has been introduced, and may be a human chronic myelogenous leukemia cell line (eg, K562 cell), RPMI8866, EBV_LCL, HFWT, and the like. In general, since the K562 cell line, which is a representative nutritional helper cell used for NK cell proliferation, is only used for NK cell proliferation, the K562 cell line itself should not proliferate. Therefore, before culturing with NK cells, the K562 cell line is pretreated by irradiation with strong radiation (eg, 50 to 100 Gy), so that the K562 cell line itself does not proliferate at all, but to help only the proliferation of NK cells do. Meanwhile, for efficient proliferation of NK cells, most cytokines such as IL-2 and IL-15 must be continuously exposed to NK cells. Therefore, when cancer cell line-based feeder cells are genetically engineered to express cytokines such as IL-2 and/or IL-15, the cancer cell line pretreated with radiation or the like cannot survive for a long time in the culture process. There is a problem that the selective proliferation efficiency of NK cells is low. However, NK cells can be obtained selectively and more efficiently by genetically engineering the cell line according to one aspect to express human leukocyte antigen.

The NK cells may be adaptive NK cells. As used herein, the term "adaptive NK cells" are specialized natural killer cells capable of forming an immunological memory and are involved in the adaptive immune system. In addition, the adaptive NK cells do not have antigen specificity, survive longer in vivo among NK cells, and have a stronger cell subtype (Ab-dependent cell-mediated cytotoxicity, ADCC), etc. subgroup). In one embodiment, the NK cells may be NKG2C+NK cells.

As used herein, the term "Human leukocyte antigen (HLA)" is a cell membrane glycoprotein molecule expressed on the surface of human nucleated cells, which presents an antigen to T lymphocytes to induce an adaptive immune response against the invading antigen. and protects normal cells from apoptosis by NK cells. In one embodiment, the HLA may be, for example, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and the like. In another embodiment, the HLA-E is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more of the nucleic acid sequence of SEQ ID NO: 1 or its amino acid sequence. or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more sequence homology.

As used herein, the term "genetic engineering" or "genetically engineered" refers to the act of introducing one or more genetic modifications to a cell or a cell produced thereby. .

Specifically, the genetically engineered cell may contain an exogenous gene encoding the aforementioned gene. The term "exogenous" means that the referenced molecule or referenced activity has been introduced into a host cell. A molecule may be introduced as non-chromosomal genetic material such as a plasmid or introduction of an encoding nucleic acid into host genetic material, such as, for example, by insertion into a host chromosome. With regard to the expression of a coding nucleic acid, the term "exogenous" refers to introduction of the coding nucleic acid into a subject in a form capable of being expressed. With respect to biosynthetic activity, the term “exogenous” refers to an activity introduced into the host parent cell. The source may be, for example, a homologous or heterologous encoding nucleic acid that expresses the activity mentioned after being introduced into the parental cell of the host. Therefore, the term "endogenous" refers to the mentioned molecule or activity present in the host cell. Similarly, with respect to expression of an encoding nucleic acid, the term "endogenous" refers to the expression of an encoding nucleic acid contained within a subject. The term "heterologous" refers to a molecule or activity from a source other than the species mentioned and the term "homologous" refers to a molecule or activity from the host parental cell. Thus, exogenous expression of an encoding nucleic acid may utilize either or both heterologous or homologous encoding nucleic acids.

Thus, the cell may contain a nucleic acid encoding HLA. More specifically, the cells may be transformed with a vector containing a nucleic acid encoding HLA.

As used herein, the term "vector" refers to a vector capable of expressing a protein of interest in a suitable host cell, and refers to a genetic construct comprising regulatory elements operably linked to express a gene insert. A vector according to one embodiment may include expression control elements such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, and/or an enhancer, and the promoter of the vector may be constitutive or inducible. In addition, the vector may be an expression vector capable of stably expressing the fusion protein in a host cell. As the expression vector, conventional ones used in the art to express foreign proteins in plants, animals, or microorganisms may be used. The recombinant vector may be constructed through various methods known in the art. For example, the vector may include a selectable marker for selecting a host cell containing the vector and, in the case of a replicable vector, an origin of replication. In addition, the vector can replicate autonomously or be introduced into host DNA, wherein the vector is selected from the group consisting of plasmids, lentiviruses, adenoviruses, adeno-associated viruses, retroviruses, herpes simplex viruses, and vaccinia viruses. it could be

In addition, in the vector, the polynucleotide sequence encoding the aforementioned fusion protein may be operably linked to a promoter. As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby The regulatory sequence will control the transcription and/or translation of the other nucleic acid sequence.

Another aspect provides a composition for culturing NK cells comprising cells genetically engineered to express human leukocyte antigen (HLA).

Details of the genetically engineered cells are as described above.

The composition for NK cell culture comprising cells genetically engineered to express HLA according to one embodiment can induce the amplification and/or activation of NK cells (eg, natural killer cells), thereby culturing NK cells, It can be usefully used for isolation or proliferation.

Another aspect includes obtaining a blood sample containing a population of mixed immune cells; and contacting at least a portion of the mixed population of NK cells with cells genetically engineered to activate NK cells, wherein the genetically engineered cells are strained to express Human leukocyte antigen (HLA). A method of proliferating NK cells is provided, comprising the step of being wholly engineered.

In one embodiment, the contacting step is co-cultivating the population of NK cells mixed with the genetically engineered cells to stimulate, activate, or expand the subpopulation of the NK cells. It may contain.

As used herein, the term "stimulation of NK cells" refers to increasing the activity of natural killer cells, for example, cytotoxic activity, in vitro or in vivo, or generating, increasing, amplifying, or proliferating activated natural killer cells. that can mean

In one embodiment, non-limiting examples of the NK cells include macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, and neutrophils. Thus, in certain embodiments, the NK cells are selected from the group consisting of macrophages, B lymphocytes, T lymphocytes (CD8+ CTL), mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, or neutrophils. It could be any one. In certain embodiments, NK cells may be natural killer cells or T lymphocytes. The mixed NK cells may include one or more selected from the group consisting of macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, and neutrophils.

As used herein, the term "Natural Killer cells" or "NK cells" are cytotoxic lymphocytes constituting a major component of the innate immune system, and are defined as large granular lymphocytes (LGL) and lymphoid progenitor cells ( Common lymphoid progenitor (CLP) constitutes a third cell differentiated from B and T lymphocytes. The "natural killer cells" or "NK cells" include natural killer cells without additional modification derived from any tissue source, and may include mature natural killer cells as well as natural killer progenitor cells. The natural killer cells are activated in response to interferon or macrophage-derived cytokines, and natural killer cells are labeled with "activating receptors" and "inhibitory receptors", which control the cytotoxic activity of cells. Including surface receptors. Natural killer cells can be generated from hematopoietic cells, eg, hematopoietic stems or precursors, from any source, eg, placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, and the like.

In one embodiment, the natural killer cells may be activated natural killer cells. The activated natural killer cells may refer to cells in which cytotoxicity or natural killer cells' innate immunomodulatory ability is activated compared to parental cells, eg, hematopoietic cells or natural killer progenitor cells.

In one embodiment, activated natural killer cells or populations enriched in activated natural killer cells are characterized by one or more functionally relevant markers, such as CD16, CD57, CD69, CD94, CD161, CD158a, CD158b, NKp30. , NKp44, NKp46, DNAM-1, 2B4, NKp46, CD94, KIR (eg KIR2DL1, KIR2DL2/3, KIR3DL1), and the NKG2 family of activating receptors (eg NKG2A, NKG2C, NKG2D) can be evaluated.

In one embodiment, the co-culture may be performed in the presence of cytokines.

As used herein, the term "cytokine" may refer to a protein (~5-20 kDa) that plays a role in cell signaling. Cytokines are released by cells and affect the behavior of cells that release cytokines and/or other cells. Non-limiting examples of cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, monokines, and colony stimulating factors. Cytokines can be produced by a wide variety of cells including, but not limited to, immune cells such as macrophages, B lymphocytes, T lymphocytes, mast cells and monocytes, endothelial cells, fibroblasts and stromal cells. can Cytokines can be produced by more than one type of cell. Cytokines act through receptors and are of particular importance in the immune system, regulating the balance between humoral and cellular immune responses, and regulating the maturation, growth and responsiveness of cell populations. A cytokine herein may be a naturally occurring cytokine or may be a mutant version of a naturally occurring cytokine. As used in this application, “naturally occurring” may also be referred to as wild type and includes allelic variants. A mutated version or "mutation" of a naturally occurring cytokine refers to certain mutations made to a naturally occurring sequence to alter the function, activity and/or specificity of a cytokine. In one embodiment, mutations can enhance the function, activity and/or specificity of a cytokine. In another embodiment, the mutations can reduce the function, activity and/or specificity of the cytokine. A mutation may include deletion or addition of one or more amino acid residues of a cytokine.

The cytokines include BMP (Bone morphogenetic protein) family, CCL (Cheomkine ligands) family, CMTM (CKLF-like MARVEL transmembrane domain containing member) family, CXCL (CXC motif ligand ligand) family, GDF (Growth/differentiation factor) family, Growth hormone, IFN (Interferon) family, IL (Interleukin) family, TNF (Tumor necrosis factors) family, GPI (glycophosphatidylinositol), SLUPR-1 (Secreted Ly-6/uPAR -Related Protein 1), SLUPR-2 (Secreted Ly -6/uPAR -Related Protein 2) and combinations thereof.

In one embodiment, the cytokine may be an interleukin or a mutant thereof. Many interleukins are synthesized by helper CD4 T lymphocytes, as well as monocytes, macrophages, and endothelial cells. Interleukins can promote the development and differentiation of T and B lymphocytes and hematopoietic cells. Interleukins include, for example, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21 , IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, or IL36. Thus, in certain embodiments, the cytokine is IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18 , including but not limited to wildtype and mutant forms of IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, or IL36 It may be an interleukin or a mutant thereof.

In another embodiment, the cytokines added to the co-culture may be IL-2 and IL-15. The IL-2 may be used at a concentration of 1 to 20 ng/ml, 1 to 15 ng/ml, 1 to 10 ng/ml, or 2 to 8 ng/ml. The IL-15 is 10 to 200 ng/mL, 10 to 180 ng/mL, 20 to 160 ng/mL, 20 to 120 ng/mL, 20 to 40 ng/mL, 20 to 80 ng/mL, 40 to 160 ng/ml, 40 to 120 ng/ml, 40 to 80 ng/ml, 80 to 160 ng/ml, or 80 to 120 ng/ml. In one embodiment, by exposing the cells to the IL-2 and IL-15 during co-culture, the proliferation of NK cells can be further increased synergistically.

In one embodiment, the co-culture may be performed for 2 to 100 days.

In one embodiment, the genetically engineered cells may be treated with 50 Gy to 300 Gy radiation.

In one embodiment, the sample may be a biological sample derived from an individual, for example, a mammal including a human. In addition, the biological sample may be isolated from an individual, and may be blood, whole blood, serum, plasma, lymph fluid, urine, feces, tissue, cell, organ, bone marrow, saliva, sputum, cerebrospinal fluid, or a combination thereof. In addition, the biological sample may include PBMCs, purified NK cells, or primary resting cells (ie, immediately isolated from blood).

Another aspect includes obtaining a blood sample containing a mixed population of NK cells; activating NK cells by contacting at least a portion of the mixed population of NK cells with cells genetically engineered to activate NK cells, wherein the genetically engineered cells have a human leukocyte antigen (HLA) Step that is genetically engineered to express; And it provides a method for examining the activity of NK cells comprising the step of analyzing the degree of activation of the activated NK cells.

Another aspect includes obtaining a blood sample containing a mixed population of NK cells; activating NK cells by contacting at least a portion of the mixed population of NK cells with cells genetically engineered to activate NK cells, wherein the genetically engineered cells have a human leukocyte antigen (HLA) Step that is genetically engineered to express; And it provides a method for diagnosing a NK cell-related disease comprising analyzing the degree of activation of the activated NK cells or a method for providing information on the diagnosis.

In the method for examining the activity of NK cells of the present invention, the step of comparing the activation of the NK cells compared to normal NK cells is, specifically, the above-mentioned genetically engineered cells under conditions equivalent to the experimental group using normal NK cells as a control group When the activation phenomenon in normal NK cells when stimulated is remarkably high in the experimental group, when it does not appear or when the degree is remarkably low, it is judged as abnormal. Through the above steps, pathological signs of diseases related to abnormal NK cells, viral infection, presence of cancer cells, and specific cancers can be determined, and the prognosis of these diseases can be predicted. The "normal NK cell" refers to NK cells possessed by or derived from an individual without the disease, and the individual without the disease is at least a physical, genetic, or exogenous factor known to affect NK cell activity. have no conditions

In one embodiment, the method may further include isolating required NK cells from the sample. If necessary, the step of stimulation may be performed in the presence of other blood cells or lymphocytes, and the step of stimulating may be performed after the step is performed to obtain a sample containing only NK cells as lymphocytes. The degree of purification of the isolated NK cells and the composition of the sample may vary to the extent necessary for the experiment. NK cells can be used as they are purified from the sample, if necessary, or can be used after proliferating to secure conditions or cell mass suitable for the experiment.

However, since the combination of specific factors is specific to NK cells, when the combination of factors specific to NK cells is used in the method of the present invention, the step of isolating the NK cells may not be essential.

In one embodiment, measuring the degree of activation may include at least one selected from among degranulation activity, cytotoxicity activity, and measurement of cytokines secreted by NK cell stimulation.

The degranulation activity may mean, for example, induction of lysis of target cells by secretion of perforin or granzyme, and this may be analyzed using FACS. Specifically, a method of stimulating PBMCs or pure isolated NK cells isolated from a whole blood sample and then measuring CD107a expression proportional to degranulation using a fluorochrome-conjugated antibody can be used.

The cytotoxic activity, for example, after culturing with a culture containing target cells capable of activating NK cells labeled with europium fluorescent dye, the amount of fluorescent dye released through target cell lysis It can be measured using a microplate reader.

In one embodiment, the cytokine may be one selected from IFN-γ, TNF-α, TNF-β, MIP-1α, MIP-1β, PANTES, IL-8 and IL-10. Analysis of immunoactivator expression of NK cells as described above may be performed using FACS, intracellular cytokine staining, or ELISA. Specifically, after staining the NK cell surface using a specific fluorochrome-conjugated antibody, the cells are permeabilized (permeabilization), and another specific fluorochrome-conjugated immunoactivator (eg, IFN-γ antibody) A method of measuring the expression of immunoactivating factors in NK cells by staining cytokines and the like can be used.

In one embodiment, the disease associated with the activity of the NK cell is one that shows abnormal NK cell activity, for example, hyperimmune disease, autoimmune disease, immune rejection, immunodeficiency disease, histiocytosis, cancer, 2 Type 2 diabetes, parasitic infections and viral diseases. The hypersensitive immune disease is at least one selected from asthma and empyema, the autoimmune disease is at least one selected from lupus, multiple sclerosis, type 1 diabetes and rheumatoid arthritis, or the histiocytosis is HLH, XLP1 , and may be any one or more selected from XLP2. Hemophagocytic lymphohistiocytosis (HLH) is characterized by group 2 Langerhans cell histiocytosis, erythrophagocytic lymphohistiocytosis (familial, sporadic), infection-associated hemophagocytic syndrome, virus-associated hemophagocytic syndrome, and giant lymphadenopathy. Histiocytosis, or reticular histiocytosis. The "cancer" is meant to include tumor, hematological cancer, or solid cancer, and includes those that impair the synergistic activity of NK cells in an individual or do not cause synergistic activity of NK cells as target cells under specific conditions. In one embodiment, the cancer is lung cancer, liver cancer, esophageal cancer, stomach cancer, colon cancer, small intestine cancer, pancreatic cancer, melanoma, breast cancer, oral cancer, brain tumor, thyroid cancer, parathyroid cancer, kidney cancer, cervical cancer, sarcoma, prostate cancer, urethra It may be selected from the group consisting of cancer, bladder cancer, testicular cancer, blood cancer, lymphoma, skin cancer, psoriasis, and fibroadenoma. In one embodiment, the cancer may be pancreatic cancer or B cell lymphoma. In one embodiment, the viral disease may be hepatitis B. In one embodiment, the immunodeficiency disease may be DiGeorge syndrome or Chedial-Higashi syndrome.

Information on the disease related to NK cell activity can be determined as abnormal NK cells when activation of NK cells in the experimental group is not detected compared to normal NK cells, or NK cells that have lost activity for a specific receptor are abnormal for target cells. If it causes or does not cause activity as a result, it can be determined that the target cell is related to a specific disease.

Another aspect provides NK cells prepared by the method for propagating NK cells. The NK cells may be genetically engineered to express human leukocyte antigen (HLA).

Another aspect provides a cell therapy agent comprising the immune cells or cell population thereof as an active ingredient.

Another aspect provides a pharmaceutical composition for preventing or treating cancer or infectious disease using the immune cells or cell population thereof as an active ingredient.

Another aspect provides the use of the immune cells or cell population thereof for the manufacture of a medicament.

Another aspect provides a method for treating a disease comprising administering the immune cells or cell population thereof to a subject.

As used herein, the term "disease" may mean one pathological condition, particularly cancer, infectious disease, inflammatory disease, metabolic disease, autoimmune disorder, degenerative disease, apoptosis-related disease, and graft rejection.

As used herein, the term "treatment" refers to, includes, or alleviates, inhibits the progress of, or prevents a disease, disorder or condition, or one or more symptoms thereof, and an "active ingredient" or "pharmaceutically effective amount" refers to a disease, disorder or condition. , or any amount of a composition used in the practice of the invention provided herein sufficient to alleviate, inhibit the progression of, or prevent one or more symptoms thereof.

As used herein, the terms "administering," "introducing," and "implanting" are used interchangeably and according to one embodiment, the administration of a composition into a subject by a method or route that results in at least partial localization to a desired site. It may refer to the arrangement of a composition according to one embodiment. It can be administered by any suitable route that delivers at least a portion of the cells or cellular components of a composition according to one embodiment to a desired location within a living subject. The survival period of the cells after administration to the subject may be as short as several hours, for example, 24 hours to several days, or as long as several years.

As used herein, the term "isolated cell", eg, "isolated immune cell" and the like, refers to a cell substantially separated from a tissue of origin, eg, hematopoietic cell.

In one embodiment, the method of administering the pharmaceutical composition is not particularly limited, but may be administered orally or parenterally, such as intravenous, subcutaneous, intraperitoneal, inhalation or topical application, depending on the desired method. The dosage varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. A daily dose refers to an amount of a therapeutic substance according to one aspect sufficient to treat a disease state alleviated by administration to a subject in need thereof. An effective amount of a therapeutic agent will depend on the particular compound, the disease state and its severity, and the subject in need of treatment, and can be routinely determined by one skilled in the art. As a non-limiting example, the dosage of the composition according to one aspect to the human body may vary depending on the patient's age, weight, sex, dosage form, state of health, and degree of disease. Based on an adult patient weighing 70 kg, for example, about 1,000 to 10,000 cells/time, 1,000 to 100,000 cells/time, 1,000 to 1000,000 cells/time, 1,000 to 10,000,000, 1,000 to 100,000,000 cells/time, 1,000 to 1,000,000,000 cells/time, 1,000 to 10,000,000,000 cells/circuit, once or several times a day at regular time intervals, divided administration may be administered, or multiple times at regular time intervals.

'Individual' means a subject in need of treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow. .

A pharmaceutical composition according to one embodiment may include a pharmaceutically acceptable carrier and/or additives. For example, sterile water, physiological saline, common buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, tonicity agents, or preservatives, etc. can do. For topical administration, it may also include combining organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. can When the pharmaceutical composition according to one embodiment is prepared in a formulation suitable for injection, immune cells, immune cells, or substances that increase their activity are dissolved in a pharmaceutically acceptable carrier or in a dissolved solution state. may be frozen.

The pharmaceutical composition according to one embodiment, if necessary according to the administration method or dosage form, suspending agent, solubilizing agent, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, excipient, pH adjuster, analgesic agent, Buffers, reducing agents, antioxidants and the like may be appropriately included. Pharmaceutically acceptable carriers and agents suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995. The pharmaceutical composition according to one embodiment is formulated in unit dosage form by using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. It can be prepared as, or it can be prepared by inserting into a multi-dose container. The dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of a powder, granule, tablet or capsule.

According to the genetically engineered cells according to one aspect, compared to cells that are not genetically engineered, the proliferation and activity of adaptive NK cells can be increased by at least two to several times. It can be proliferated and used as a cell therapy.

Figure 1a is a result of analyzing the expression of HLA-E in genetically engineered K562 cells using RT-qPCR.

Figure 1b is the result of analyzing the expression of HLA-E using flow cytometry (FACS).

Figure 2a is a result of measuring the activation of NK cells amplified by feeder cells according to one aspect.

Figure 2b is a graph showing Fold expansion of NK cells amplified by feeder cells according to one aspect.

Figure 2c is a graph showing the long-term culture effect of the culture helper cells according to one aspect.

Figure 3a is a diagram confirming the phenotypic characteristics of NK cells amplified by feeder cells according to one aspect, specifically confirming the levels of CD16 and CD57 (dark black: K562-HLA-E, gray: K562) .

Figure 3b is a diagram confirming the phenotypic characteristics of NK cells amplified by feeder cells according to one aspect, specifically confirming the levels of CD69 and NKp30 (dark black: K562-HLA-E, gray: K562) .

Figure 3c is a diagram confirming the phenotypic characteristics of NK cells amplified by feeder cells according to one aspect, specifically, confirming the levels of NKp46 and DNAM-1 (dark black: K562-HLA-E, gray: K562).

Figure 3d is a diagram confirming the level of NKG2D and NKG2A as a result of confirming the phenotypic characteristics of NK cells amplified by feeder cells according to one aspect (dark black: K562-HLA-E, gray: K562) .

Figure 3e is a diagram confirming the level of FcRy as a result of confirming the phenotypic characteristics of NK cells amplified by feeder cells according to one aspect (dark black: K562-HLA-E, gray: K562).

Figure 3f is a graph comparing phenotypic expression levels of NK cells amplified by feeder cells and K562 feeder cells according to one aspect (dark black: K562-HLA-E, gray: K562) .

Figure 3g is a graph comparing the phenotypic expression levels of NK cells amplified by feeder cells and K562 feeder cells according to one aspect in terms of mean fluorescence intensity (MFI) (dark black : K562-HLA-E, gray: K562).

Figure 4a is a result confirming the FcεRIγ-NK cell and NKG2C + NK cell frequency correlation of NK cells amplified by feeder cells and NK cells amplified by K562 cells according to one aspect.

Figure 4b is a result of comparing FcεRIγ-expression rates of NK cells amplified by feeder cells and NK cells amplified by K562 cells according to one aspect.

Figure 4c is a result of confirming FcεRIγ cell and NKG2C expression of NK cells amplified by feeder cells and NK cells amplified by K562 cells according to one aspect.

Figure 5a is a graph confirming the cytotoxicity (ADCC) of NK cells amplified by feeder cells according to one aspect to K562 cells and Raji cells.

Figure 5b is a graph confirming the cytotoxicity according to the HLA-C type of NK cells amplified by feeder cells according to one aspect.

Figure 6a is a graph measuring CD107a expression of NK cells amplified by feeder cells according to one aspect.

Figure 6b is a graph measuring IFN-γ expression of NK cells amplified by feeder cells according to one aspect.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Preparation of genetically engineered feeder cells

Feeder cells expressing human leukocyte antigen (HLA) were prepared. Specifically, the human-derived HLA-E gene (SEQ ID NO: 1) was cloned into a lentiviral vector, pCDH-CMV-EF1-GFP, to prepare a vector for producing a recombinant lentivirus. Thereafter, the prepared recombinant gene (pCDH-CMV-HLA-E-EF1-GFP) was transfected into 293FT cells together with a packaging vector using lipofectamin3000 (Invitrogen) for virus production. After a period of time, the cells were cultured for 48 hours by replacing the medium with a fresh medium, and then the virus-containing medium was recovered. The recovered medium was centrifuged at 500 Х g for 10 minutes, and only virus-containing pure medium was separated using a 0.45 μm filter to produce HLA-E-expressing lentivirus. Thereafter, 1 ml of HLA-E expressing lentivirus was dissolved in 9 ml of medium containing K562 cells and added together with polybrene (8 μg/ml), followed by cell culture for 48 hours. Then, infected cells were selected using RT-qPCR (Quantitative reverse transcription PCR) and flow cytometry (FACS).

Figure 1a is a result of analyzing the expression of HLA-E in genetically engineered K562 cells using RT-qPCR.

Figure 1b is the result of analyzing the expression of HLA-E using flow cytometry (FACS).

As a result, as shown in FIG. 1a, it was confirmed that the expression level of HLA-E in the genetically engineered K562 cells was significantly higher than that of conventional K562 cells. In addition, as shown in FIG. 1B , it was confirmed that the conventional K562 cells did not express HLA-E, but the genetically engineered K562 cells expressed HLA-E. Therefore, the K562 cell line expressing HLA-E was named "K562-HLA-E".

Example 2. Selective Expansion of Adaptive NK Cells from Peripheral Blood Monocytes

In order to confirm the adaptive NK cell amplification effect of the genetically engineered feeder cells according to one aspect, after culturing K562-HLA-E cells and NK cells, NKG2C ⁺ and KIR expression of NK cells were confirmed.

Specifically, after isolating human peripheral blood mononuclear cells (PBMC) from healthy adult donors using Ficoll-Hypaque (d = 1.077, Lymphoprep ^TM ; Axis-Shield, Oslo, Norway), they were washed twice with PBS. Washed. Then, the PBMCs were cultured in RPMI 1640 medium containing 10 U/mL recombinant human IL-2 (containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 4 mmol/L L-glutamine) at 24 - K562 and K562-HLA-E cells (Example 1) irradiated with 100 Gy gamma rays were co-cultured in a well plate, respectively. After 7 days, the IL-2 concentration was increased from 10 U/mL to 100 U/mL, and 5 ng/mL of aqueous IL-5 was added. On days 7 and 14, feeder cells irradiated with gamma rays (K562 and K562-HLA-E) were added for re-stimulation, and cultured up to 98 days while changing the medium every 2 to 3 days. On days 0, 14, and 28 of culture, the expression levels of NKG2C, NKG2A, KIR2DL1, and KIR2DL2/3 were confirmed using flow cytometry (FACS). In addition, the expression of NKG2C, KIR2DL1, KIR2DL2/3 and KIR3DL1 was measured according to the number of culture days. In addition, the number of NK cells according to the number of culture days was divided by the absolute number of NK cells compared to day 0 and expressed as fold expansion.

Figure 2a is a result of measuring the activation of NK cells amplified by feeder cells according to one aspect.

Figure 2b is a graph showing Fold expansion of NK cells amplified by feeder cells according to one aspect.

Figure 2c is a graph showing the long-term culture effect of the culture helper cells according to one aspect.

As a result, as shown in Figure 2a, on the 14th day of culture, the feeder cells of Example 1 were significantly higher in NKG2C+ NK cells with self-specific KIR2DL2/3 expression compared to the K562 feeder cells. .

In addition, as shown in Figure 2b, it was confirmed that the feeder cells of Example 1 and the feeder cells of K562 significantly increased the fold expansion of NK cells on the 21st day of culture. In particular, on the 42nd to 49th day of culture, it was confirmed that the fold expansion of the feeder cells of Example 1 was about twice as high as that of the K562 feeder cells. Specifically, in the case of NK cells cultured with feeder cells of Example 1 in the presence of IL-2 and IL-15, 169 ± 94 times and 10311 ± 6676 times on day 14 and day 42 of culture, respectively. amplification was confirmed.

In addition, as shown in Figure 2c, in the case of K562 feeder cells, NK cells expressing NKG2C, KIR2DL1, KIR2DL2/3 and KIR3DL1 were amplified from day 42 to day 49, but no further amplification was confirmed thereafter. there was. On the other hand, in the case of feeder cells of Example 1, it was confirmed that NK cells expressing NKG2C, KIR2DL1, KIR2DL2/3 and KIR3DL1 were amplified even after 90 days.

That is, feeder cells according to one aspect not only significantly increase the activity of adaptive NK cells, but also allow long-term culture of NK cells, so that NK cells can be massively proliferated and used as a cell therapeutic agent.

Example 3. Phenotypic Characteristics of NK Cells Expanded by K562-HLA-E Cells

3-1. Identification of surface antigens of NK cells

The phenotypic characteristics of the NK cells amplified by the feeder cells according to one aspect were confirmed.

Specifically, after washing 2 Х 10 ⁵ of NK cells amplified in the same manner as in Example 2 with FACS buffer (PBS containing 1% FBS), APC-Cyanine7-conjugated mouse anti-human CD3 and PE-Cyanine7 conjugated Treatment with anti-human CD56 membrane antibody for 15 minutes. Cells were then harvested and further stained for 30 minutes with different fluorescently-conjugated anti-human CD16, CD57, CD69, NKp30, NKp46, DNAM-1, NKG2D, NKG2A and FcRγ membrane antibodies, respectively. Then, after washing the cells with FACS buffer, data were acquired using FACS Verse (BD Biosciences) and analyzed with Kaluza. As a control, NK cells amplified by K562 cells were used.

Figure 3a is a diagram confirming the phenotypic characteristics of NK cells amplified by feeder cells according to one aspect, specifically confirming the levels of CD16 and CD57 (dark black: K562-HLA-E, gray: K562) .

Figure 3b is a diagram confirming the phenotypic characteristics of NK cells amplified by feeder cells according to one aspect, specifically confirming the levels of CD69 and NKp30 (dark black: K562-HLA-E, gray: K562) .

Figure 3c is a diagram confirming the phenotypic characteristics of NK cells amplified by feeder cells according to one aspect, specifically, confirming the levels of NKp46 and DNAM-1 (dark black: K562-HLA-E, gray: K562).

Figure 3d is a diagram confirming the level of NKG2D and NKG2A as a result of confirming the phenotypic characteristics of NK cells amplified by feeder cells according to one aspect (dark black: K562-HLA-E, gray: K562) .

Figure 3e is a diagram confirming the level of FcRy as a result of confirming the phenotypic characteristics of NK cells amplified by feeder cells according to one aspect (dark black: K562-HLA-E, gray: K562).

Figure 3f is a graph comparing phenotypic expression levels of NK cells amplified by feeder cells and K562 feeder cells according to one aspect (dark black: K562-HLA-E, gray: K562) .

Figure 3g is a graph comparing the phenotypic expression levels of NK cells amplified by feeder cells and K562 feeder cells according to one aspect in terms of mean fluorescence intensity (MFI) (dark black : K562-HLA-E, gray: K562).

As a result, as shown in Figures 3a to 3e, the NK cells amplified by the feeder cells of Example 1 had surface markers such as CD16, CD57, CD69, NKp30, NKp46, DNAM-1, NKG2D, NKG2A and FcRγ. expression could be confirmed. Specifically, in the case of NKp30, NKG2A, and NKp46, expression levels were lower than that of NK cells amplified by K562 feeder cells, and CD57, a surface marker of adaptive NK cells, and intracellular receptors In the case of FcRγ, the expression level was higher than that of NK cells amplified by K562 feeder cells. On the other hand, CD16 showed a lower expression level than that of NK cells amplified by K562 feeder cells (see Figs. 3f and 3g). This is thought to be because the adaptive NK cells preferentially produce cytokines including IFN-γ in response to FcγRIIIa triggering.

That is, NK cells amplified by feeder cells according to one aspect may have complete functional characteristics of adaptive NK cells.

3-2. Confirmation of FcεRIγ and NKG2C expression in NK cells

Adaptive NK cells have an FcεRIγ-deficient phenotype. Therefore, it was confirmed whether the NK cells amplified by the feeder cells according to one aspect also had the same phenotype.

Specifically, after washing 2 Х 10 ⁵ NK cells amplified in the same manner as in Example 2 with FACS buffer (PBS containing 1% FBS), APC-Cyanine7-conjugated mouse anti-human CD3 and PE-Cyanine7 conjugated Treatment with anti-human CD56 membrane antibody for 20 minutes. Thereafter, the cells were washed with FACS buffer, permeabilization and fixation were performed to confirm the expression of FcεRIγ, an intracellular signal receptor, and then FITC-conjugated mouse anti-human FcεRIγ antibody treated for 30 minutes. Thereafter, after washing with FACS buffer, data were acquired using FACS Verse (BD Biosciences) and analyzed with Kaluza.

Figure 4a is a result confirming the FcεRIγ-NK cell and NKG2C + NK cell frequency correlation of NK cells amplified by feeder cells and NK cells amplified by K562 cells according to one aspect.

Figure 4b is a result of comparing FcεRIγ-expression rates of NK cells amplified by feeder cells and NK cells amplified by K562 cells according to one aspect.

Figure 4c is a result of confirming FcεRIγ cell and NKG2C expression of NK cells amplified by feeder cells and NK cells amplified by K562 cells according to one aspect.

As a result, as shown in Figure 4a, NKG2C positive NK cells on day 0 were confirmed to have a correlation with FcεRIγ-, but when the culture was maintained for more than 28 days, it was confirmed that no correlation between NKG2C expression rate and FcεRIγ- was observed. there was.

In addition, as shown in FIG. 4b, when the culture of NK cells was continued for more than 28 days, no correlation was observed between NKG2C-positive cells and FcεRIγ-. However, it was confirmed that the FcεRIγ- ratio of NK cells amplified by feeder cells of Example 1 was higher than the FcεRIγ- ratio of NK cells cultured with K562 feeder cells.

In addition, as shown in Figure 4c, it was confirmed that the NK cells amplified by the K562-HLA-E feeder cells were amplified into NKG2C-positive FcεRIγ+ NK cells from day 0 to day 28 of culture.

Example 4. Confirmation of cytotoxicity of NK cells amplified by K562-HLA-E cells

4-1. Confirmation of cytotoxicity of NK cells against K562 cells and Raji cells

In order to confirm the efficacy of NK cells amplified by feeder cells according to one aspect as an antibody therapeutic, the cytotoxicity of NK cells on day 14 and day 49 against target cells was measured for 4 hours by CFSE-based assay. Specifically, target cells were stained with 0.5 μM CFSE for 10 minutes at 37° C. in FACS buffer and washed twice with complete medium. Then, for cytotoxicity analysis, K562 cells stained with CFSE were placed in a 96-well round bottom plate (96-well U-bottom plate) and NK cells amplified by K562-HLA-E and K562 feeder cells The amplified NK cells were mixed at effector-to-target (E:T) ratios of 1:1, 0.5:1, and 0.25:1, respectively, and cultured in a 37°C, 5% CO ₂ incubator for 4 hours. In addition, in order to perform ADCC analysis, CFSE-stained Raji cells were cultured with 1 μg/ml of rituximab and then cultured in the same manner as above. Thereafter, the mixed cells were transferred to a FACD tube and 1 μg of 1 mg/ml PI (Invitrogen) was added to each tube, and the cells were acquired in FACS Verse and analyzed using Kaluza software.

Figure 5a is a graph confirming direct cytotoxicity of K562 cells on days 14 and 49 of culture of NK cells amplified by feeder cells according to one aspect, and cytotoxicity (ADCC) by addition of Raji cells and Rituximab. .

As a result, as shown in Figure 4a, the NK cells amplified by the feeder cells of Example 1 and the NK cells amplified by the K562 feeder cells exhibit similar cytotoxicity to Raji cells bound to K562 and Rituximab. could confirm that

That is, NK cells amplified by feeder cells according to one aspect exhibit cytotoxicity and can be used as antibody therapeutics.

4-2. Confirmation of NK cell cytotoxicity against HLA-mismatched MCF7 cells

Cytotoxicity according to the HLA-C type of NK cells amplified by feeder cells according to one aspect was confirmed.

Specifically, HLA-C1 and HLA-C2 types were analyzed using the HLA-C SSP PCR kit as a PCR-SSP (PCR amplification with sequence-specific primers) test method. The donor's CMV serology and HLA-C genotype are shown in Table 1 below. Thereafter, cytotoxicity according to the genotype was confirmed.

donor	CMV serology	HLA-C genotype
donor 1	positive	C1C1
donor 2	positive	C2C2
donor 3	positive	C1C1
donor 4	positive	C1C1
donor 5	positive	C2C2
donor 6	positive	C2C2

Figure 5b is a graph confirming the cytotoxicity according to the HLA-C type of NK cells amplified by feeder cells according to one aspect.

As a result, as shown in FIG. 5B, the NK cells amplified by the feeder cells of Example 1 showed improved cytotoxicity against MCF7 cancer cells in C1C1 NK cells compared to NK cells with an HLA-C genotype of C2C2. could confirm that

That is, the NK cells amplified by the feeder cells according to one aspect can be usefully used as a therapeutic agent having improved killing efficacy against HLA-C mismatched target cancer cells.

Example 5. Confirmation of activation of NK cells amplified by K562-HLA-E cells

5-1. NK cell activation by CD107a degranulation

In order to measure the activation of NK cells amplified by feeder cells according to one aspect, the level of CD107a expression on the surface of NK cells proportional to CD107a degranulation of NK cells was measured.

Specifically, 2 Х 10 ⁵ of NK cells, 2 Х 10 ⁵ of target cells (K562 and MCF7), and 5 μM of PE-conjugated anti-human CD107a were amplified in the same manner as in Example 2 in a 95-well round-bottom plate. cultured. After 1 hour, Monensin and brefeldin A (BD Biosciences) were added, followed by further incubation for 4 hours. NK cells were then obtained by staining with anti-human CD3 and CD56 antibodies.

Figure 6a is a graph measuring CD107a expression of NK cells amplified by feeder cells according to one aspect.

As a result, as shown in FIG. 6a, it was confirmed that the NK cells amplified by the feeder cells of Example 1 showed CD107a expression characteristics similar to those of the NK cells amplified by K562 feeder cells or MCF7 target cells. These results indicate that NK cells with increased activity were successfully generated from peripheral blood mononuclear cells of normal donors. Therefore, feeder cells according to one aspect are effective in producing NK cells with increased activity.

5-2. NK cell activation by IFN-γ production

In order to evaluate the immunoregulatory activity of NK cells amplified by the feeder cells according to one aspect, cytotoxicity of NK cells by intracellular IFN-γ production was confirmed.

Specifically, the NK cells amplified in the same manner as in Example 2 were cultured in a 96-well round bottom plate at 37° C., 5% CO ₂ for 5 hours in the presence of brefeldin A (BD Biosciences) and Monensin (BD Biosciences) did Cells were then harvested and washed by FACS before staining with anti-human CD3 and CD56 membrane antibodies for 20 minutes. After washing, fixation and permeabilization, NK cells were further stained with PE-conjugated anti-human IFN-γ antibody for 30 min on ice. Then, after washing the cells, the cells were obtained in FACS Verse and analyzed using Kaluza software.

Figure 6b is a graph measuring IFN-γ expression of NK cells amplified by feeder cells according to one aspect.

As a result, as shown in FIG. 6B, it was confirmed that the NK cells amplified by the feeder cells of Example 1 showed a similar level of IFN-γ expression to that of the NK cells amplified by the K562 feeder cells. That is, it was confirmed that NK cells with increased activity were successfully generated from peripheral blood mononuclear cells of a normal donor.

Therefore, the NK cells amplified by the feeder cells according to one aspect are effective in producing NK cells having high immunoregulatory activity by cytokines.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (13)

1. Feeder cells for culturing NK cells genetically engineered to express human leukocyte antigen (HLA).
2. The feeder cells of claim 1, wherein the feeder cells are selected from the group consisting of K562, RPMI8866, EBV_LCL, and HFWT cells.
3. The feeder cell according to claim 1, comprising a nucleic acid encoding a human leukocyte antigen.
4. The feeder cell according to claim 1, wherein the human leukocyte antigen expresses at least one selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G. .
5. A composition for culturing NK cells comprising the feeder cells of claim 1.
6. obtaining a blood sample containing a population of NK cells; and

   contacting at least a portion of the mixed population of NK cells with cells genetically engineered to activate NK cells, wherein the genetically engineered cells are genetically engineered to express Human leukocyte antigen (HLA) A method of proliferating NK cells comprising the step of being engineered.
7. 7. The method of claim 6, wherein the contacting step comprises co-culturing the population of NK cells mixed with the genetically engineered cells to expand the subpopulation of the NK cells. method.
8. The method according to claim 7, wherein the co-culture is made in the presence of cytokines.
9. The method according to claim 8, wherein the cytokines IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, At least one selected from the group consisting of IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, and IL36.
10. The method of claim 9 , wherein the cytokines are IL-2 and IL-15.
11. The method according to claim 7, wherein the co-culture is performed for 2 to 100 days.
12. The method of claim 6 , wherein the blood sample is a whole blood sample.
13. A cell therapy product containing, as an active ingredient, NK cells genetically engineered to express human leukocyte antigen (HLA).

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