Tumor-targeted CIML NK cell as well as preparation method and application thereof

Abstract

The invention specifically discloses a tumor-targeted CIML NK cell as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The invention provides a preparation method of tumor-targeted CIML NK cells, the NK cells are endowed with memorability by utilizing cell factor combinations IL-12, IL-15 and IL-18, the CIML NK cells with sufficient quantity and high purity can be obtained by utilizing K562 transfected with CD48 / CD137L / mIL-21 for dominant amplification, and a tumor tissue capable of targeting high expression of an iRGD receptor is prepared by utilizing a high-penetrability tumor-targeted lipid insert. The CIMLNK is endowed with tumor targeting and penetrability, and after the lipid insert is transfused back into a body, more CIMLNK can be targeted and infiltrated into tumor tissues, so that a stronger anti-tumor effect is achieved. The invention provides a theoretical basis and an experimental basis for developing a new treatment strategy, and has good clinical transformation and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to tumor-targeted CIML NK cells and a preparation method and application thereof. Background Art

[0002] Natural killer (NK) cells are important effector cells in the innate immune system and play a key role in anti-tumor immune response. In recent years, with the rapid development of tumor immunotherapy, NK cell therapy, as an important supplement to T cell therapy, has shown great clinical application prospects.

[0003] NK cells have many unique advantages: First, NK cells can directly recognize and kill tumor cells, without relying on antigen pre-stimulation like T cells. Second, NK cells can exert anti-tumor effects through multiple mechanisms, including direct cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), and secretion of cytokines. In addition, NK cell therapy has better safety than CAR-T cell therapy, with less adverse reactions such as cytokine release syndrome (CRS) and neurotoxicity. This makes it an ideal choice for "off-the-shelf" cell therapy products.

[0004] Nevertheless, there are still some key issues to be solved in the field of NK cell treatment of solid tumors, such as how to improve the persistence of NK cells in the body, how to enhance their targeting of tumor tissues in the body, and how to overcome the immunosuppression of the tumor microenvironment. In addition, the selection of NK cell sources, in vitro expansion, and optimization of genetic engineering are also important research directions.

[0005] As a member of the innate immune system, NK cells are generally considered to have a short life cycle, produce nonspecific immune responses, and have no immune memory for previous stimulation. In 2009, Cooper et al. observed that initial stimulation with cytokines IL-12 / 15 / 18 can enhance the production of IFN-γ by most mouse NK cells, and after 1-3 weeks, these cells show memory-like NK cell characteristics. A 2012 study found that human NK cells pre-activated by cytokines still show functionally altered recall responses after long-term quiescence. Through identification, IL-12 / 15 / 18-induced memory NK cells, namely cytokine-induced memory-like (CIML) NK cells, have strong proliferation ability, long survival time in vivo, and more effective anti-tumor effects. In addition, CIML NK cells are ideal candidate cells for the development of chimeric antigen receptor CAR-NK cells. CIML NK cells can be engineered with CAR to make them antigen-specific, which can further enhance their anti-tumor activity. Compared with the control group CD19-CAR-NK cells, CD19-CAR-CIML NK cells showed strong anti-tumor activity against lymphoma cell lines resistant to NK cell therapy in vitro, mainly reflected in the production, degranulation and killing ability of IFN-γ. These CAR-CIML NK cells showed persistent antigen specificity and significantly reduced tumor burden in the xenograft mouse lymphoma model. CAR-CIML-NK can solve the problem of tumor tissue targeting, but NK gene editing also has certain difficulties, and the technical difficulty and high cost of transgenic technology make it difficult to carry out on a large scale.

[0006] Tumor penetrating peptide (internalizing RGD, iRGD) (CRGDK / RGPD / EC) can achieve specific and efficient drug delivery in tumors. After systemic administration, iRGD selectively targets integrins αvβ3 and αvβ5, which are selectively expressed on tumor blood vessels through the RGD motif. After targeting the tumor, iRGD is proteolytically processed into the RXXK / R sequence (C-terminal sequence), which conforms to the C-terminal rule (CendR) motif that binds to the neuropilin-1 (NRP-1) receptor on the cell surface. The CendR–NRP-1 interaction triggers vascular endocytosis and enhances vascular permeability, allowing iRGD-loaded or co-injected drugs to penetrate into tumor tissues. RGD-loaded drugs can penetrate into tumors even in the presence of abnormally proliferating connective tissue.

[0007] In the past two decades, bispecific and multispecific antibodies have made significant progress in the field of tumor treatment. These antibodies are able to bind to two or more antigens simultaneously. Bispecific immune cell engagers are particularly eye-catching, as they can direct immune cells to tumors, overcome immunosuppression in the tumor microenvironment, and restore anti-tumor activity. With the advancement of protein engineering technology, bispecific antibodies have been continuously optimized and applied in clinical practice, and a variety of drugs have been approved to date. At the same time, a large number of bispecific antibodies are in the preclinical or clinical development stage, showing broad application prospects. Future research will focus on optimizing target selection, exploring directed manipulation of the tumor microenvironment, and optimizing the spatial location of anti-tumor immune cells to improve efficacy.

[0008] The present invention proposes a method for preparing cytokine-activated memory NK cells with tumor targeting. The method uses multiple cytokines to confer NK memory, further uses K565 transfected with CD48 / CD137L / mIL-21 as trophoblast cells, and advantageously amplifies CIML NK of sufficient quantity and purity from single nuclei derived from peripheral blood or cord blood. Furthermore, CIML NK is given tumor tissue targeting and penetration in vivo with a high-penetration tumor-targeting lipid plug-in. The method proposed by the present invention is expected to overcome the main challenges faced by existing NK cell therapy, including key issues such as low in vitro amplification efficiency, short survival time after reinfusion into the body, and insufficient tumor tissue targeting. Summary of the invention

[0009] 1. Technical issues to be resolved

[0010] In view of this, one of the main purposes of the present invention is to provide a method for preparing tumor-targeted CIML NK cells. The method combines cytokine induction, uses K565 transfected with CD 48 / CD137L / mIL-21 as trophoblasts, and amplifies mononuclei from peripheral blood or cord blood, and combines with high-penetration tumor-targeted lipid plug-ins to obtain NK cells with both memory-like properties and tumor targeting. The method proposed by the present invention is expected to overcome the main challenges faced by existing NK cell therapy, including insufficient tumor targeting and poor in vivo persistence.

[0011] (II) Technical solution

[0012] In order to solve the above problems, the present invention provides a method for preparing tumor-targeted CIML NK cells, the preparation method comprising:

[0013] S1: contacting a cell population containing NK cells with a culture system containing IL-12, IL-15 and IL-18 and culturing the cell population in vitro to obtain a cell population containing memory NK cells;

[0014] S2: contacting the cell population comprising memory NK cells with a culture system comprising feeder cells and culturing the cells ex vivo to expand memory NK cells.

[0015] In one embodiment, the preparation method further comprises obtaining a cell population comprising NK cells from a subject.

[0016] In one embodiment, the subject is healthy.

[0017] In one embodiment, the subject is non-healthy.

[0018] In one embodiment, the cell population comprising NK cells is a mammalian cell population; preferably, the mammalian cells are human cells.

[0019] In one embodiment, the human cell is an engineered cell.

[0020] In one embodiment, the human cell is a non-engineered cell.

[0021] In one embodiment, the cell population comprising NK cells is a peripheral blood mononuclear cell (PBMC) population and / or an umbilical cord blood mononuclear cell (UCBMC) population.

[0022] In one embodiment, the PBMCs and / or UCBMCs are freshly obtained PBMCs and / or UCBMCs.

[0023] In one embodiment, the PBMCs and / or UCBMCs are frozen PBMCs and / or UCBMCs.

[0024] In one embodiment, the cell population comprising NK cells is derived from human tissue.

[0025] In one embodiment, the human tissue is fresh.

[0026] In one embodiment, the human tissue is frozen.

[0027] In one embodiment, peripheral blood or umbilical cord blood is collected using a purple anticoagulant tube, separated using a lymphocyte separation solution, and the white coat cells are aspirated and collected by centrifugation to obtain mononuclear cells, which are mainly T cells, NK cells, B cells and monocytes through flow cytometry detection, among which NK cells account for between 5% and 20%.

[0028] In one embodiment, the S1 is cultured for at least 3 h, or at least 5 h, or at least 7 h, or at least 9 h, or at least 11 h, or at least 13 h, or at least 15 h, or at least 17 h, or at least 19 h, or at least 21 h; preferably, the S1 is cultured for 3-25 h; preferably, the S1 is cultured for 15-17 h; preferably, the S1 is cultured for 16 h.

[0029] In one embodiment, the concentration of IL-12 in S1 is 1-100 ng / ml; preferably, the concentration is 1-50 ng / ml; preferably, the concentration is 10 ng / ml;

[0030] In one embodiment, the concentration of IL-15 is 50-150 ng / ml; preferably, the concentration is 100 ng / ml.

[0031] In one embodiment, the concentration of IL-18 is 50-150 ng / ml; preferably, the concentration is 100 ng / ml.

[0032] In one embodiment, the culture method further comprises washing the cell population comprising memory NK cells described in S1 and contacting it with the culture system comprising trophoblast cells described in S2 and culturing it in vitro to expand the memory NK cells.

[0033] In one embodiment, the trophoblast cells in S2 are K562 cells transfected with CD48, CD137L and mIL-21; preferably, the K562 cells are irradiated cells; preferably, the radiation dose is 100 Gy.

[0034] In one embodiment, the ratio of the trophoblast cells to the cells in the cell population comprising memory NK cells is 1:1-1:10; preferably, the ratio is 1:6.

[0035] In one embodiment, the S2 is cultured for at least 3 days, or at least 5 days, or at least 7 days, or at least 9 days, or at least 11 days, or at least 13 days, or at least 15 days, or at least 17 days, or at least 19 days, or at least 21 days; preferably, cultured for 3-25 days; preferably, cultured for 21 days.

[0036] In one embodiment, the feeder cells in S2 are supplemented on the second day of culture.

[0037] In one embodiment, the feeder cells in S2 are supplemented on the 9th day of culture.

[0038] In one embodiment, the culture system in S2 further comprises a serum-free culture medium.

[0039] In one embodiment, the culture system in S2 further comprises IL-2.

[0040] In one embodiment, the concentration of IL-2 in S2 is 1-200 U / ml; preferably, the concentration is 1-150 U / ml; preferably, the concentration is 100 U / ml.

[0041] In one embodiment, the method in S2 increases the total number of memory NK cells in the cell population comprising memory NK cells before contact by at least 10 times, or at least 50 times, or at least 100 times, or at least 200 times, or at least 500 times, or at least 1000 times, or at least 1100 times, or at least 11200 times, or at least 1300 times, or at least 1400 times, or at least 1500 times compared to the total number of memory NK cells in the cell population before contact; preferably, the method increases the total number of memory NK cells in the cell population by 1100-1200 times; preferably, the method increases the total number of memory NK cells in the cell population by 1122 times.

[0042] In one embodiment, the preparation method further comprises: culturing the memory NK cells in vitro in the presence of a tumor-targeting penetrating peptide to obtain memory NK cells with tumor targeting; preferably, the tumor-targeting penetrating peptide is DSPE-PEG-iRGD; preferably, the amount of DSPE-PEG-iRGD is 10-100ug / 10 6 Memory NK cells; preferably, the dosage of DSPE-PEG-iRGD is 20ug / 10 6 memory NK cells.

[0043] In one embodiment, the above preparation method further comprises enriching ex vivo expanded memory NK cells from a cell population comprising memory NK cells.

[0044] On the other hand, the present invention also provides a method for preparing tumor-targeted CIML NK cells, the preparation method specifically comprising:

[0045] S1: contacting a cell population containing NK cells with a culture system containing IL-12, IL-15 and IL-18 and culturing the cell population in vitro for 16 hours to obtain a cell population containing memory NK cells;

[0046] S2: contacting the cell population containing memory NK cells with a culture system containing K562 cells transfected with CD48, CD137L and mIL-21 and irradiated and IL-2, and culturing the cells in vitro for 21 days to expand memory NK cells;

[0047] Among them, (1) the concentration of IL-12 in S1 is 10 ng / ml; (2) the concentration of IL-15 in S1 is 100 ng / ml; (3) the concentration of IL-18 in S1 is 100 ng / ml; (4) the ratio of K562 cells to cells in the cell population containing memory NK cells in S2 is 1:6; (5) the concentration of IL-2 in S2 is 100 U / ml.

[0048] On the other hand, the present invention also provides a method for preparing tumor-targeted CIML NK cells, the preparation method specifically comprising:

[0049] S1: contacting a cell population containing NK cells with a culture system containing IL-12, IL-15 and IL-18 and culturing the cell population in vitro for 16 hours to obtain a cell population containing memory NK cells;

[0050] S2: contacting the cell population containing memory NK cells with a culture system containing K562 cells transfected with CD48, CD137L and mIL-21 and irradiated and IL-2, and culturing the cells in vitro for 21 days to expand memory NK cells;

[0051] S3: culturing the memory NK cells in vitro in the presence of DSPE-PEG-iRGD to obtain memory NK cells with tumor targeting;

[0052] Among them, (1) the concentration of IL-12 in S1 is 10 ng / ml; (2) the concentration of IL-15 in S1 is 100 ng / ml; (3) the concentration of IL-18 in S1 is 100 ng / ml; (4) the ratio of K562 cells to cells in the cell population containing memory NK cells in S2 is 1:6; (5) the concentration of IL-2 in S2 is 100 U / ml; (6) the amount of DSPE-PEG-iRGD in S3 is 20 ug / 10 6 memory NK cells.

[0053] On the other hand, the present invention also provides an isolated tumor-targeted memory NK cell population produced by the above preparation method.

[0054] In one embodiment, the percentage of memory NK cells in the isolated tumor-targeted memory NK cell population (1) is greater than 10%, or greater than 15%, or greater than 20%, or greater than 25%, or greater than 30%, or greater than 35%, or greater than 40%, or greater than 45%, or greater than 50%, or greater than 55%, or greater than 60%;

[0055] or (2) 10% to 99%, or 20% to 95%, or 30% to 95%, or 35% to 95%, or 40% to 95%, or 45% to 95%, or 50% to 95%, or 55% to 95%, or 60% to 95%, or 65% to 95%.

[0056] In another aspect, the present invention also provides a pharmaceutical composition, comprising:

[0057] (1) a therapeutically effective amount of the above-mentioned tumor-targeted memory NK cell population;

[0058] (2) A pharmaceutically or immunologically acceptable carrier or excipient.

[0059] In another aspect, the present invention also provides a pharmaceutical preparation, which comprises the above-mentioned pharmaceutical composition.

[0060] On the other hand, the present invention also provides a use of the above-mentioned tumor-targeted memory NK cell population, pharmaceutical composition, and pharmaceutical preparation in the preparation of drugs for preventing and / or treating tumors and / or tumor-related diseases and / or symptoms.

[0061] In one embodiment, the tumor includes one or a combination of breast cancer, gastric cancer, liver cancer, lymphoma, intestinal cancer, lung cancer, and cervical cancer.

[0062] (III) Beneficial effects

[0063] The present invention provides a method for preparing tumor-targeted CIML NK cells, which has the following beneficial effects compared with the prior art:

[0064] 1. The present invention uses a combination of cytokines to confer memory to NK cells, and uses K562 transfected with CD48 / CD137L / mIL-21 for advantageous amplification, thereby obtaining sufficient number of CIML NK cells with high purity.

[0065] 2. The present invention utilizes a highly penetrable tumor-targeting lipid plug-in to prepare a tumor tissue that can target iRG D receptor-highly expressed tumor tissues, giving CIMLNK tumor targeting and penetration. After being reinfused into the body, the lipid plug-in can enable more CIML NK to target and infiltrate into tumor tissues, exerting a stronger anti-tumor effect.

[0066] (IV) Terms and Definitions

[0067] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0068] As used herein, the term "separation" refers to the process of increasing the percentage of a substance in a composition. For example, separating one type of cell from a cell population refers to the process of producing a cell population in which the percentage of cells of this type is increased compared to the percentage of cells of this type in the original cell population. Therefore, when used in the context of one type of cell, the term "separated" does not mean that the isolated cell population contains 100% of this type of cell, but rather means that the percentage of this type of cell in the cell population is increased after the separation process.

[0069] As used herein, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and / or animals without excessive adverse reactions (such as toxicity, irritation, and allergic response), ie, at a reasonable benefit / risk ratio.

[0070] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is a carrier for the administration of therapeutic agents, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not necessary active ingredients themselves and are not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art, and a full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0071] Pharmaceutically acceptable carriers in the compositions include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0072] As used herein, the term "excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), carrier or vehicle. Pharmaceutical excipients can be sterile liquids, such as water and oils, including those derived from oils of petroleum, animals, plants or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous dextrose solutions and glycerol solutions can also be used as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition may also contain a small amount of wetting agent or emulsifier or pH buffer. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, etc. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Such compositions will contain a preventive or therapeutically effective amount of an active ingredient provided herein, such as a purified form, and an appropriate amount of an excipient to provide a form suitable for administration to a patient. The formulation should be compatible with the mode of administration.

[0073] In some embodiments, the choice of excipient is determined in part by the particular cell and / or method of administration.Thus, there are a variety of suitable formulations.

[0074] Generally, acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants (including ascorbic acid, methionine, vitamin E, sodium metabisulfite); preservatives, isotonic agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents such as EDTA and / or nonionic surfactants.

[0075] Buffers can be used to control pH within a range that optimizes therapeutic effectiveness, especially if stability is pH dependent. Suitable buffers for use with the present disclosure include organic and inorganic acids and their salts. For example, citrates, phosphates, succinates, tartrates, fumarates, gluconates, oxalates, lactates, acetates. Additionally, the buffer may include histidine and trimethylamine salts such as Tris.

[0076] Preservatives may be added to retard microbial growth. Suitable preservatives for use with the present disclosure include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol and m-cresol.

[0077] Tonicity agents (sometimes referred to as "stabilizers") may be present to adjust or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby reducing the possibility of intermolecular and intramolecular interactions.

[0078] Exemplary tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0079] Additional exemplary excipients include: (1) fillers, (2) solubilizers, (3) stabilizers, and (4) agents to prevent denaturation or adhesion to the container walls. Such excipients include: polyhydroxy sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbitol, xylose, ribose, ribitol, myoinisitose, inositol, galactose, dulcitol, glycerol, cyclitols (e.g., , cyclohexanehexol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0080] Nonionic surfactants or detergents (also referred to as "wetting agents") may be present to help dissolve the therapeutic agent and protect the therapeutic protein from aggregation caused by agitation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of active therapeutic proteins or antibodies. Suitable nonionic surfactants include, for example, polysorbates (20, 40, 60, 65, 80, etc.), poloxamer (184, 188, etc.), polyols, polyoxyethylene sorbitol monoethers (etc.), lauroyl alcohol 400, polyoxy stearate 40, polyoxyethylene hydrogenated castor oil 10, 50 and 60, monostearate, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0081] In order to make the pharmaceutical compositions for in vivo administration, they are preferably sterile. The pharmaceutical compositions can be sterilized by filtering through a sterile filtration membrane. The pharmaceutical compositions herein can generally be placed in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle.

[0082] The route of administration is according to known and accepted methods, such as by single or multiple boluses or infusion over a long period of time in a suitable manner, for example, injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical application, inhalation or by sustained release or long-term release means.

[0083] In another embodiment, the pharmaceutical composition can be provided as a controlled release or sustained release system. In one embodiment, a pump can be used to achieve controlled release or sustained release (see, e.g., Sefton, Crit. Ref. Biomed. Eng. 14: 201-40 (1987); Buchwald et al., Surgery 88: 507-16 (1980); and Saudek et al., N. Engl. J. Med. 321: 569-74 (1989)). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (see, e.g., Medical Applications of Controlled Release (Langer and Wise, eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball, eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126 (1983); Levy et al., Science 228:190-92 (1985); During et al., Ann. Neurol. 25:351-56 (1989); Howard et al., J. Neurosurg. 71:105-12 (1989); U.S. Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; PCT Publication Nos. WO 99 / 15154 and WO 99 / 20253). Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained-release formulation is inert, free of leachable impurities, storage-stable, sterile, and biodegradable. In another embodiment, a controlled or sustained-release system can be placed near a specific target tissue (e.g., the nasal passages or lungs), thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, Vol. 2, 115-38 (1984)).Controlled release systems are discussed, for example, by Langer, Science 249: 1527-33 (1990). Any technique known to those skilled in the art can be used to produce sustained release formulations comprising one or more agents as described herein (see, e.g., U.S. Pat. No. 4,526,938, PCT Publication Nos. WO 91 / 05548 and WO 96 / 20698, Ning et al., Radiotherapy & Oncology 39: 179-89 (1996); Song et al., PDA J. of Pharma. Sci. & Tech. 50: 372-97 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24: 853-54 (1997); and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24: 759-60 (1997)).

[0084] The pharmaceutical compositions described herein may also contain more than one active compound or agent, which is necessary for the specific indication being treated. Alternatively or in addition, the composition may include a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, or a growth inhibitory agent. Such molecules are suitable for being present in combination in an amount effective for the intended purpose.

[0085] The active ingredient can also be embedded in microcapsules, prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

[0086] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level includes the type and severity of the subject, age, sex, drug activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant medications, and other factors well known in the medical field.

[0087] As used herein, the term "treating" a condition or patient means taking steps to obtain a beneficial or desired result, including a clinical result. Beneficial or desired clinical results include, but are not limited to, or eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, condition, or disorder, substantially improving or alleviating the clinical or aesthetic symptoms of a condition, substantially preventing the clinical or aesthetic symptoms of a disease, condition, or disorder, and avoiding harmful or annoying symptoms. Treatment also refers to accomplishing one or more of the following: (a) lessening the severity of the condition; (b) limiting the development of symptoms characteristic of the condition being treated; (c) limiting the worsening of symptoms characteristic of the condition being treated; (d) limiting the recurrence of the condition in patients who previously had the condition; and / or (e) limiting the recurrence of symptoms in patients who were previously free of symptoms of the condition.

[0088] As used herein, the term "prevent" or "preventing" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms (eg, diabetes or cancer).

[0089] As used herein, the term "cell population comprising NK cells" can be obtained from a variety of sources. In some embodiments, the cell population comprising NK cells is obtained from a cultured NK cell line. In some embodiments, the cell population comprising NK cells is collected, separated, purified or induced from body fluids, tissues or organs (including but not limited to peripheral blood, umbilical cord blood, bone marrow, lymph nodes, spleen or other tissues or fluids of the subject).

[0090] In certain embodiments, the cell population comprising NK cells is peripheral blood mononuclear cells (PBMC) and / or umbilical cord blood mononuclear cells (UCBMC). In certain embodiments, the PB MC and / or UCBMC are freshly obtained PBMC and / or UCBMC. In certain embodiments, the PBMC and / or UCBMC are frozen PBMC and / or UCBMC. Various methods of collecting and preparing PBMC and / or UCBMC are known in the art.

[0091] In certain embodiments, the cell population comprising NK cells is derived from human tissue. In specific embodiments, the human tissue is fresh. In certain embodiments, the human tissue is frozen. Various methods for collecting and preparing human tissue are known in the art.

[0092] In some embodiments, the cell population comprising T cells is a mammalian cell. In certain embodiments, the mammalian cell is a human cell. In certain embodiments, the human cell is an engineered cell. In certain embodiments, the human cell is a non-engineered cell. In certain embodiments, the mammalian cell is a non-human cell. In specific embodiments, the non-human cell is an engineered cell or a non-engineered cell.

[0093] In some embodiments, the cell population comprising NK cells is obtained from a subject. In certain embodiments, the cell population comprising NK cells is obtained from a healthy subject. In certain embodiments, the cell population comprising NK cells is obtained from an unhealthy subject. In certain embodiments, the unhealthy subject suffers from a solid tumor cancer. In certain embodiments, the unhealthy subject suffers from a blood cancer. In certain embodiments, the unhealthy subject suffers from both a solid tumor cancer and a blood cancer.

[0094] The method provided by the present invention comprises the step of inducing memory NK cells in vitro in a culture system.

[0095] In some embodiments, the inducing conditions include cytokines. Non-limiting examples of cytokines that can be used with the present disclosure include lectins, liver growth factors, prostaglandins, fibroblast growth factors, prolactin, placental lactogen, OB protein, tumor necrosis factor-α, tumor necrosis factor-β, Mullerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factor (NGF), platelet growth factor, TGF-α, TGF-β, insulin-like growth factor-I, insulin-like growth factor-II, erythropoietin (EPO), osteoinductive factors, interferon-α, interferon-β, interferon IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor, and LT (lymphotoxin).

[0096] In some embodiments, the inducing conditions include IL-12, IL-15, and IL-18.

[0097] In some embodiments, the concentration of IL-12 is 10 ng / ml, the concentration of IL-15 is 100 ng / ml, and the concentration of IL-18 is 100 ng / ml.

[0098] The method provided by the present invention comprises the step of amplifying memory NK cells in vitro in a culture system.

[0099] In some embodiments, the expansion conditions include cytokines. Non-limiting examples of cytokines that can be used with the present disclosure include lectins, liver growth factors, prostaglandins, fibroblast growth factors, prolactin, placental lactogen, OB protein, tumor necrosis factor-α, tumor necrosis factor-β, Mullerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factor (NGF), platelet growth factor, TGF-α, TGF-β, insulin-like growth factor-I, insulin-like growth factor-II, erythropoietin (EPO), osteoinductive factors, interferon-α, interferon-β, interferon IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor, and LT (lymphotoxin).

[0100] In some embodiments, the live expansion conditions include substances other than cytokines. In some embodiments, the expansion conditions include trophoblasts. In some embodiments, the expansion conditions include plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0102] Figure 1 A shows the proportion of CD3-CD56+ in the mononuclear cell population derived from cord blood after CIMLNK was pre-activated by cytokines IL-12 / 15 / 18 and cultured with feeder cells in vitro for 21 days. Figure 1 B shows the proliferation of CD3-CD56+ cell numbers during culture;

[0103] Figure 2The figure shows that after 21 days of expansion, CIML NK cells induced by IL-12 / 15 / 18 activation expressed higher levels of CD107a when encountering tumor cells MKN45 compared with the control group (not pre-activated by IL-12 / 15 / 18).

[0104] Figure 3 The results show that the three-dimensional spheres were constructed in vitro using HGC27 cells with high expression of iRGD ligands, and the tumor-targeted lipid plug-in DSPE-PEG-iRGD promoted the infiltration of CIML NK in the three-dimensional spheres. The results showed that the fluorescence intensity inside the three-dimensional spheres of the CIML NK group modified by DSPE-PEG-iRGD was the strongest, indicating that DSPE-PEG-iRGD can promote more CIML NK to infiltrate into the three-dimensional spheres.

[0105] Figure 4 The results show that DSPE-PEG-iRGD modified CIML NK can target more iRGD receptor-positive tumor tissues after infusion, showing good tumor targeting

[0106] Figure 5 The figure is a flow chart of the preparation of CIML NK with tumor targeting. DETAILED DESCRIPTION

[0107] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0108] As used herein, “containing,” “having,” or “including” include “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of;” “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0109] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0110] Example 1

[0111] Cytokine-induced activation and expansion of memory NK cells:

[0112] 1. Isolation of mononuclear cells: Mononuclear cells from human peripheral blood or cord blood were isolated by Ficoll density gradient centrifugation, washed with physiological saline and resuspended in serum-free culture medium containing 10% autologous plasma.

[0113] 2. Cytokine-induced memory NK cells: Add the cytokine combination IL-12 / 15 / 18 to the above serum-free medium for induction. The concentrations of the cytokines used are: IL-12 concentration is 10 ng / ml, IL-15 concentration is 100 ng / ml, IL-18 concentration is 100 ng / ml, and the induction time is 16 hours. After 16 hours, wash and centrifuge three times and then resuspend in serum-free medium.

[0114] 3. Establish the expansion conditions of memory NK cells: thaw the frozen K562 expressing CD48 / CD137L / mIL-21 and irradiated, add it to the above complete medium, the ratio of 1:6 with the mononuclear cells induced by cytokines, and add 100U / ml IL-2 for expansion.

[0115] 4. Expansion of memory NK cells: Replace half of the medium every two days, count the cells, and use flow cytometry to detect the proportion of CD3-CD56+ cells in the cell population.

[0116] The results are as follows Figure 1 As shown, on day 21, the proportion of CD3-CD56+ NK cells in the expansion system reached 90%, and the number of NK cells expanded 1122 times in 21 days.

[0117] Example 2

[0118] Verify the memory property of the prepared CIML NK:

[0119] CIML NK cells were harvested on the 21st day of expansion culture, centrifuged and washed, and co-cultured with MKN45 cells at a ratio of 5:1. The control group cells were NK cells (c-NK cells) that were not induced by the cytokine combination IL-12 / 15 / 18. After 24 hours of culture, the expression of CD107a was detected.

[0120] The results are as follows Figure 2 As shown, CD107a is one of the surface markers of NK cell activation. CIML NK cells expanded and cultured after cytokine induction, when encountering tumor cells again, expressed higher levels of CD107a than NK cells in the control group and had stronger cytotoxicity.

[0121] Example 3

[0122] Verify the binding ability of fusion protein (DSPE-PEG-iRGD) to NK cells and tumor cells:

[0123] 1. Construct HGC27 three-dimensional spheres (HGC27 multicellular tumor spheres), and conduct experiments when the HGC27 multicellular tumor spheres proliferate to about 200-300 μm: select HGC27-MCTS (HGC27 multicellular tumor spheres) with uniform diameter, and divide HGC27-MCTS into four groups, namely c-NK cell group, c-NK-iRGD (c-NK modified by DSPE-PEG-iRGD) cell group, CIML NK cell group and CIML NK-iRGD (CIML NK modified by DSPE-PEG-iRGD) cell group.

[0124] 2. Add CFSE green fluorescence labeled c-NK cells, c-NK-iRGD cells, CIML NK cells and CIML NK-iRGD cells to the culture wells of HGC27-MCTS at an effector-target ratio of 5:1 and incubate at 37°C and 5% CO 2 After incubation in the incubator for 6 hours, HGC27-MCTS in each culture well was carefully aspirated and placed in a 1.5 ml EP tube, and washed twice with 1× PBS buffer to remove free NK cells.

[0125] 3. Place the washed HGC27-MCTS in a confocal dish and observe under a fluorescence confocal microscope. Use the "Z-stack" function to perform multi-level scanning and reconstruction of HGC27-MCTS, and use ImageJ software to analyze the NK cell infiltration in HGC27-MCTS.

[0126] The results are as follows Figure 3 As shown, the fluorescence intensity inside the three-dimensional sphere of the CIML NK group modified with DSPE-PEG-iRGD is the strongest, indicating that DSPE-PEG-iRGD can promote more CIML NK to infiltrate into the interior of the three-dimensional sphere.

[0127] Example 4

[0128] The iRGD lipid insert confers in vivo targeting to CIML NKs:

[0129] CIML NK was labeled with near-infrared DIR dye and then treated with 200 μg DSPE-PEG-i RGD and 1x10 7 After 2 hours of co-incubation, CIMLNK was intravenously injected into BALB / c nude mice bearing MKN45 subcutaneous tumors. After 24 hours, the distribution of CIMLNK in the body was observed by near-infrared.

[0130] The results are as follows Figure 4As shown, DSPE-PEG-iRGD enhanced the tumor targeting of CIML NK.

[0131] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0132] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing tumor-targeted CIML NK cells, characterized in that: The preparation method comprises: S1: contacting a cell population containing NK cells with a culture system containing IL-12, IL-15 and IL-18 and culturing the cell population in vitro to obtain a cell population containing memory NK cells; S2: contacting the cell population comprising memory NK cells with a culture system comprising feeder cells and culturing the cells ex vivo to expand memory NK cells.

2. The preparation method according to claim 1, characterized in that: The cell population containing NK cells in S1 includes a peripheral blood mononuclear cell population and / or an umbilical cord blood mononuclear cell population.

3. The preparation method according to claim 1, characterized in that: The concentration of IL-12 in S1 is 1-100 ng / ml; and / or the concentration of IL-15 is 50-150 ng / ml; And / or the concentration of IL-18 is 50-150 ng / ml.

4. The preparation method according to claim 1, characterized in that: The trophoblast cells in S2 are K562 cells transfected with CD48, CD137L and mIL-21.

5. The preparation method according to claim 4, characterized in that: The ratio of the trophoblast cells to the cells in the cell population containing memory NK cells is 1:1-1:

10.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The preparation method further comprises: culturing the memory NK cells in vitro in the presence of tumor-targeting penetrating peptides to obtain memory NK cells with tumor targeting.

7. The preparation method according to any one of claim 6, characterized in that: The tumor penetrating peptide is DSPE-PEG-iRGD.

8. The preparation method according to claim 7, characterized in that: The dosage of the tumor penetrating peptide is 10-100ug / 10 6 memory NK cells.

9. A tumor-targeted memory NK cell population, characterized in that: The memory NK cell population is prepared according to the preparation method according to any one of claims 1-8.

10. Use of the memory NK cell population according to claim 9 in the preparation of a medicament for preventing and / or treating tumors and / or tumor-related diseases and / or symptoms.

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