Method for preparing iTNK cell, iTNK cell, and pharmaceutical composition and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to a method for preparing iTNK cells, prepared iTNK, a pharmaceutical composition of the iTNK and application of the iTNK. Specifically, the invention relates to a method for preparing iTNK cells, which comprises the following steps: (1) adding a proper amount of anti-CD3 / CD28 / CD2T cell Activator into a culture medium for culturing T cells; culturing for 1-4 days; preferably culturing for 2 days; (2) replacing the culture medium with a culture medium which does not contain anti-CD3 / CD28 / CD2T cell Activator, and continuously culturing for 20 to 30 days; preferably, the culture lasts for 26 days. The iTNK cell prepared by the method disclosed by the invention has dual attributes of cytotoxic T cells and NK cells and a tumor killing function, and has a good anti-tumor application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a method for preparing iTNK cells, the prepared iTNK cells, pharmaceutical compositions thereof, and uses thereof. Specifically, the present invention relates to a method for preparing iTNK cells using human pluripotent stem cells. The prepared iTNK cells possess dual properties of cytotoxic T cells and NK cells and tumor-killing function. Background Art

[0002] iTNK cells (induced T-to-NK cells) are cells that possess some of the characteristics of both cytotoxic T cells and NK cells. iTNK cells are dual-attribute immune cells that combine the advantages of both cytotoxic T cells and NK cells, and hold great potential for the development of new immunotherapies.

[0003] It has been reported that iTNK cells have been used in clinical studies to treat nasopharyngeal carcinoma, colorectal cancer, melanoma, rhabdomyosarcoma, gastric cancer, appendiceal mucinous cystadenocarcinoma and non-small cell lung cancer. Among them, 6 of 9 patients showed tumor stabilization and one patient achieved partial remission (Jiang ZW, Qin L, Tang YO, Liao R, Shi JX, He BJ, et al. Humaninduced-T-to-natural killer cells have potent anti-tumour activities. BiomarkRes 2022; 10).

[0004] iTNK cells have characteristics of both T cells and NK cells, as well as TCR-independent anti-tumor cytotoxicity. When used in combination with CAR or tumor-specific monoclonal antibodies, iTNK cells show stronger anti-tumor effects (Jiang ZW, Qin L, Tang YO, Liao R, Shi JX, He BJ, et al. Human induced-T-to-natural killer cells have potent anti-tumour activities. Biomark Res 2022; 10; Forkel H, Grabarczyk P, Depke M, Troschke-Meurer S, Simm S, Hammer E, et al. BCL11B depletion induces the development of highly cytotoxic innate T cells out of IL-15 stimulated peripheral blood alphabeta CD8+ T cells. Oncoimmunology 2022; 11: 2148850).

[0005] Human iTNK cells can be obtained by knocking out the BCL11B gene in T cells derived from umbilical cord blood or peripheral blood. Knocking out the Bcl11b gene can reprogram T cells into iTNK cells, which exhibit NK cell characteristics and reduce the expression of some T cell characteristic genes (Li, P., Burke, S., Wang, J., Chen, X., Ortiz, M., Lee, SC, Lu, D., Campos, L., Goulding, D., Ng, BL, et al. (2010). Reprogramming of T cells to natural killer-like cells upon Bcl11b deletion. Science 329, 85-89.10.1126 / science.1188063.).

[0006] However, current iTNK cells are produced by gene editing and amplification of peripheral blood T cells. On the one hand, they rely on primary cell sources, which makes the source limited. On the other hand, the efficiency and stability of gene editing will limit their clinical application.

[0007] Therefore, new technical means for preparing iTNK cells still need to be developed. Summary of the Invention

[0008] After in-depth research and creative work, the inventors have developed a method for producing iTNK cells. The inventors discovered that this method eliminates the need for primary cell sources; the resulting iTNK cells possess the dual properties of cytotoxic T cells and NK cells, as well as tumor-killing functions, and have promising anti-tumor applications. This provides the following invention:

[0009] One aspect of the present invention relates to a method for preparing iTNK cells, comprising the following steps:

[0010] (1) adding an appropriate amount of anti-CD3 / CD28 / CD2 T cell activator to the culture medium for T cells; culturing for 1-4 days, preferably 2 days;

[0011] (2) replacing the culture medium with one that does not contain anti-CD3 / CD28 / CD2 T cell activator and continuing the culture for 20-30 days, preferably 26 days;

[0012] Preferably, the concentration of anti-CD3 / CD28 / CD2 T cell Activator is 20 μl / mL-30 μl / mL; more preferably 25 μl / mL.

[0013] In some embodiments of the present invention, the preparation method according to claim 1, wherein,

[0014] The culture medium is XF T expansion medium;

[0015] Preferably, the culture medium contains appropriate amounts of IL-2, IL-7, IL-15, IL-21 and Z-VAD-FMK;

[0016] Preferably, the culture medium contains 40-60 ng / mL IL-2, 8-12 ng / mL IL-7, 4-6 ng / mL IL-15, 8-12 ng / mL IL-21 and 8-12 μM Z-VAD-FMK;

[0017] Preferably, the culture medium contains 50 ng / mL IL-2, 10 ng / mL IL-7, 5 ng / mL IL-15, 10 ng / mL IL-21, and 10 μM Z-VAD-FMK;

[0018] Preferably, the culture medium further comprises appropriate amounts of FBS, Glutamax, ascorbic acid, ITS, minocycline hydrochloride and NAC;

[0019] Preferably, the culture medium contains 8%-12% FBS, 0.8%-1.2% Glutamax, 80-120 μg / mL ascorbic acid, 0.8%-1.2% ITS, 1.8-2.2 μM minocycline hydrochloride, and 20-28 μM NAC;

[0020] Preferably, the culture medium contains 10% FBS, 1% Glutamax, 100 μg / mL ascorbic acid, 1% ITS, 2 μM minocycline hydrochloride, and 24 μM NAC.

[0021] In some embodiments of the present invention, the preparation method, wherein,

[0022] The method further includes the step of isolating iTNK cells from step (2).

[0023] In some embodiments of the present invention, the preparation method, wherein,

[0024] The T cells are αβCD8 + T cells.

[0025] In some embodiments of the present invention, the preparation method, wherein,

[0026] The T cells are CD34 + Hematopoietic progenitor cells were obtained;

[0027] Preferably, the T cells (e.g., αβCD8 + T cells) by CD34 + Hematopoietic progenitor cells and MS5-DLL4 supporting cells were differentiated in the ATO system.

[0028] In some embodiments of the present invention, the preparation method, wherein,

[0029] CD34 + Hematopoietic progenitor cells are made from human pluripotent stem cells;

[0030] Preferably, the CD34 + Hematopoietic progenitor cells are derived from human pluripotent stem cells by differentiation in a 3D differentiation system or a 2D differentiation system;

[0031] Preferably, the human pluripotent stem cells are selected from human embryonic stem cells, human induced pluripotent stem cells and human chemically induced pluripotent stem cells.

[0032] The present invention establishes a step-by-step directed induction differentiation strategy that can generate iTNK cells from human pluripotent stem cells through the stages of hematopoietic progenitor cell induction, T cell induction, and iTNK cell induction. Flow cytometry was used to detect the expression of surface markers in iTNK cells; single-cell RNA sequencing and TCR sequencing were used to detect the transcriptional signatures and TCR expression profiles of iTNK cells; in vitro tumor killing experiments were used to test the killing effect of iTNK cells on various tumor cell lines, examining TCR- and NKR-mediated tumor recognition and killing functions; cytokine release experiments were used to detect the release of cytotoxic factors when iTNK cells kill tumor cells; and a mouse tumor xenograft model was used to test the in vivo tumor killing function of iTNK cells.

[0033] Another aspect of the present invention relates to an iTNK cell, which is prepared by any one of the preparation methods of the present invention;

[0034] Preferably, the iTNK cells also express a chimeric antigen receptor (CAR).

[0035] Another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of the iTNK cells of the present invention, and optionally, further comprising one or more pharmaceutically acceptable excipients;

[0036] Preferably, the pharmaceutical composition further comprises one or more anti-tumor drugs;

[0037] Preferably, the anti-tumor drug is selected from chimeric antigen receptor T cells, antibody drugs and chemotherapy drugs.

[0038] Typically, the pharmaceutical compositions of the present invention contain 0.1-90% by weight of iTNK cells. Pharmaceutical compositions can be prepared according to methods known in the art. For this purpose, if desired, iTNK cells can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to prepare an appropriate administration or dosage form for human use.

[0039] The pharmaceutical composition of the present invention can be administered in a unit dosage form, and the route of administration can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosal, cutaneous, peritoneal, or rectal. Examples of dosage forms include tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, and lyophilized powder injections. These can be conventional preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems. Various carriers known in the art can be widely used to prepare unit dosage forms into tablets. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. To prepare the dosing unit as a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, gelucine, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dry starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare the dosing unit as a suppository, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. To prepare the dosing unit as a capsule, iTNK cells are mixed with the aforementioned carriers, and the resulting mixture is placed in a hard or soft capsule. iTNK cells can also be prepared as microcapsules and suspended in an aqueous medium to form a suspension, which can then be encapsulated in a hard capsule or formulated as an injection. In order to prepare the dosing unit into an injectable preparation, such as a solution, emulsion, lyophilized powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc.In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional solubilizers, buffers, pH adjusters, etc. may also be added.

[0040] In addition, if necessary, coloring agents, preservatives, perfumes, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations.

[0041] The dosage of the pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the route of administration, and the number of doses, etc. The above dosage can be administered in a single dose or divided into several doses, such as two, three, or four doses.

[0042] The actual dosage level of iTNK cells in the pharmaceutical compositions of the present invention can be varied so that the resulting pharmaceutical composition is effective for achieving the desired therapeutic response for a specific patient and route of administration. The dosage level should be selected based on the specific route of administration, the severity of the condition being treated, and the condition and medical history of the patient being treated. However, it is common practice in the art to start the iTNK cell dosage at a level below that required for the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0043] Yet another aspect of the present invention relates to a combination pharmaceutical product comprising a first product and a second product in separate packages, wherein:

[0044] The first product comprises the iTNK cells of the present invention;

[0045] The second product comprises one or more anti-tumor drugs; preferably, the anti-tumor drugs are selected from chimeric antigen receptor T cells, antibody drugs and chemotherapy drugs;

[0046] Preferably, the first product and the second product further independently contain one or more pharmaceutically acceptable excipients;

[0047] Preferably, the combination product further comprises a package insert.

[0048] Another aspect of the present invention relates to the use of the iTNK cells of the present invention or the pharmaceutical composition of the present invention in the preparation of a drug for treating or preventing tumors;

[0049] Preferably, the tumor is selected from one or more of colorectal cancer, melanoma, breast cancer, liver cancer, lung cancer, nasopharyngeal carcinoma, rhabdomyosarcoma, gastric cancer and appendiceal mucinous cystadenocarcinoma;

[0050] Preferably, the lung cancer is non-small cell lung cancer.

[0051] Another aspect of the present invention relates to a kit comprising a first reagent combination, wherein the first reagent combination comprises:

[0052] Culture medium: XF T expansion medium, appropriate amounts of IL-2, IL-7, IL-15, IL-21, and Z-VAD-FMK, as well as appropriate amounts of FBS, Glutamax, ascorbic acid, ITS, minocycline hydrochloride, and NAC;

[0053] Preferably, it comprises 40-60 ng / mL IL-2, 8-12 ng / mL IL-7, 4-6 ng / mL IL-15, 8-12 ng / mL IL-21 and 8-12 μM Z-VAD-FMK;

[0054] Preferably, it includes 50 ng / mL IL-2, 10 ng / mL IL-7, 5 ng / mL IL-15, 10 ng / mL IL-21 and 10 μM Z-VAD-FMK;

[0055] Preferably, it comprises 8%-12% FBS, 0.8%-1.2% Glutamax, 80-120 μg / mL ascorbic acid, 0.8%-1.2% ITS, 1.8-2.2 μM minocycline hydrochloride and 20-28 μM NAC;

[0056] Preferably, it includes 10% FBS, 1% Glutamax, 100 μg / mL ascorbic acid, 1% ITS, 2 μM minocycline hydrochloride, and 24 μM NAC;

[0057] Preferably, the kit is used to prepare iTNK cells.

[0058] In some embodiments of the present invention, the kit further comprises a second reagent combination, wherein the second reagent combination comprises:

[0059] Preparation of αβCD8 from hematopoietic progenitor cells + T cell culture media and induction reagents.

[0060] In some embodiments of the present invention, the kit further comprises a third reagent combination, wherein the third reagent combination comprises:

[0061] Culture media and induction reagents for preparing hematopoietic progenitor cells from human pluripotent stem cells.

[0062] Some of the terms involved in the present invention are as follows:

[0063] Human pluripotent stem cells: A type of cell of human origin that has the ability to self-renew and differentiate into various cell types of the three germ layers.

[0064] Hematopoietic progenitor cells: A cell type that has the ability to self-renew and differentiate into various types of blood cells.

[0065] CD34 + Hematopoietic progenitor cells: CD34 is a cell surface marker of hematopoietic progenitor cells. + Hematopoietic progenitor cells are obtained by sorting hematopoietic progenitor cells using CD34 during the preparation of hematopoietic progenitor cells.

[0066] 3D differentiation system: The 3D differentiation system mentioned in the present invention refers to an embryoid body (EB) differentiation system, in which cells aggregate to form EBs, grow and differentiate in suspension in a culture medium, and present a three-dimensional pattern.

[0067] 2D differentiation system: The 2D differentiation system mentioned in the present invention refers to an adherent differentiation method, in which cells grow and differentiate on a culture plate, presenting a two-dimensional pattern in morphology.

[0068] MS5-hDLL4 and ATO system: Montel-Hagen's team established a 3D differentiation system for artificial thymic organoids (ATO), where hematopoietic progenitor cells from umbilical cord blood or embryonic mesoderm progenitor cells from human embryonic stem cell lines or human induced pluripotent stem cell lines were co-aggregated with MS5-DLL4 (or DLL1) supporting cells to form a 3D structure for T cell induction. + T cells showed diverse TCR expression and were shown to have effector functions through activation and killing experiments.

[0069] The term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0070] In the present invention, unless otherwise specified, the "first" (e.g., the first product or the first reagent combination), "second" (e.g., the second product or the second reagent combination), and "third" (e.g., the third reagent combination) are for the purpose of distinguishing between references or clarifying the expression, and do not have a typical order meaning.

[0071] Advantageous Effects of the Invention

[0072] The present invention achieves one or more of the following technical effects:

[0073] ① In the present invention, both human embryonic stem cell lines and human chemically induced pluripotent stem cell lines can be induced to differentiate into iTNK cells in a step-by-step manner, thus eliminating the dependence on primary cell sources.

[0074] ② The iTNK cells produced by the present invention express CD8 + T cell surface markers include CD8αβ, CD3, TCRαβ, etc., and NK cell surface markers include CD56, NKG2D, NKp44, CD94, etc.

[0075] ③ At the transcriptome level, the iTNK cells prepared by the present invention simultaneously transcribe CD8 + T cell characteristic genes CD8B, CD3D, CD27, CD2, etc., and NK cell characteristic genes NCAM1, NCR2, KLRK1, KLRC1, PRF1, GZMA, etc.

[0076] ④ The iTNK cells prepared by the present invention have a diverse TCR expression spectrum.

[0077] ⑤ The iTNK cells prepared by the present invention can exert their killing function through the CD3-TCR-mediated signaling pathway, and can also recognize and kill tumors through NK-related mechanisms such as NK-activating receptors, and have broad-spectrum anti-tumor properties.

[0078] ⑥ The iTNK cells prepared by the present invention release a variety of cytotoxic factors at high levels during the process of killing tumors.

[0079] ⑦ The iTNK cells prepared by the present invention significantly inhibited tumor growth in mice and have good anti-tumor application prospects.

[0080] ⑧The iTNK cells produced by the present invention have the dual properties of cytotoxic T cells and NK cells and tumor killing function. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 : Flow cytometry results of T cell-related surface marker expression in iTNK cell sample 2.

[0082] Figure 2: Flow cytometry results of NK cell-related surface marker expression in iTNK cell sample 2.

[0083] Figure 3 : Flow cytometry results of T cell-related surface marker expression in iTNK cell sample 4.

[0084] Figure 4 : Flow cytometry results of NK cell-related surface marker expression in iTNK cell sample 4.

[0085] Figure 5 : Single-cell sequencing results of the transcription of T cell and NK cell-related characteristic genes in iTNK cell sample 1.

[0086] Figure 6 : TCR expression profile results in iTNK cell sample 1.

[0087] Figure 7 : Single-cell sequencing results of the transcription of T cell and NK cell-related characteristic genes in iTNK cell sample 2.

[0088] Figure 8 : TCR expression profile results in iTNK cell sample 2.

[0089] Figure 9 Figure 2: Cytotoxicity of iTNK cell samples 2 and 4 against various solid tumor cell lines. iTNK cell samples 2 and 4 were used independently. Cytotoxicity experiments against HepG2 and A375 cell lines were performed independently using iTNK cell sample 2, while cytotoxicity experiments against MDA-MB-231 cell lines were performed independently using iTNK cell sample 4.

[0090] Figure 10 : The killing results of iTNK cell sample 1 on tumor cells through TCR-CD3-mediated signals.

[0091] Figure 11 Figure 4 shows the killing results of iTNK cell sample 4 and NK cells against tumor cells via NKG2D-mediated signaling. "NK isotype ctrl" and "iTNK isotype ctrl" represent the NK cell group and the iTNK cell group, respectively, in which an isotype control antibody was added, as controls, corresponding to the experimental group in which an anti-NKG2D antibody was added.

[0092] Figure 12Figure 2: Detection results of cytotoxic factors released by iTNK cells (iTNK cell samples 2 / 4), T cells, and NK cells during tumor cell killing. iTNK cell sample 2 and iTNK cell sample 4 were used independently. The HCT116 killing assay was performed independently using iTNK cell sample 4, while the Nalm-6-LUC-OKT3 cell line killing assay was performed independently using iTNK cell sample 2.

[0093] Figure 13 : Detection results of iTNK cell sample 1 in vivo tumor killing. DETAILED DESCRIPTION

[0094] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0095] Some of the reagents used in the following experiments are shown in Table A below.

[0096] Table A

[0097]

[0098]

[0099]

[0100]

[0101] ①Human embryonic stem cell line H1, purchased from iCell Bioscience Inc.;

[0102] ② Human chemically induced pluripotent stem cells (CiPS cells) were generated by Deng Hongkui's laboratory using a small molecule reprogramming method (Guan J, Wang G, Wang J, Zhang Z, Fu Y, Cheng L, et al. Chemical reprogramming of humansomatic cells to pluripotent stem cells. Nature 2022; 605:325-31);

[0103] ③ The human induced pluripotent stem cell line iPS-7 was prepared using a four-transcription factor reprogramming method and purchased from Cauliscell Biotechnology.

[0104] In the following experiments, unless otherwise specified, cells were cultured at 37°C and 5% CO2.

[0105] Preparation Example 1: Preparation of iTNK cell sample 1

[0106] 1. Induction of differentiation of hematopoietic progenitor cells:

[0107] In the 3D differentiation system, human embryonic stem cell line H1 was resuspended in mTesR plus medium containing 50 nM Chroman1 and 5 μM Emricasan, and the culture medium was adjusted to 2 × 10 5 Cells were seeded at a density of 1 / well in low-attachment six-well plates (Corning, 3471) and cultured for one day. On day 0 of differentiation, the medium was changed to RPMI1640 medium supplemented with 20 ng / mL BMP4, 5 μM CHIR-99021, 20 ng / mL bFGF, 1% NEAA (Gibco), 1% GlutaMAX (Gibco), 1% PS, 2% B27 (without vitamin A) (Gibco), 1-thioglycerol, and 50 μg / mL ascorbic acid. On days 2 and 4 of differentiation, the medium was changed to RPMI1640 medium supplemented with 5 ng / mL BMP4, 50 ng / mL VEGF, 50 ng / mL bFGF, 10 μM SB-431542, 1% NEAA, 1% GlutaMAX, 1% PS, 2% B27 (without vitamin A), 1-thioglycerol, and 50 μg / mL ascorbic acid. On days 6 and 8 of differentiation, the medium was changed to IMDM medium (Gibco) supplemented with 10 ng / mL VEGF, 20 ng / mL SCF, 20 ng / mL FLT3L, 20 ng / mL TPO, 5 ng / mL IL-7, 10 μM SB-431542, 1% NEAA, 1% GlutaMAX, 1% PS, 2% B27 (without vitamin A), 1-thioglycerol, 50 μg / mL ascorbic acid, 30 μM NAC, and 2 μM minocycline hydrochloride. Cultures were continued for 10 days.

[0108] On day 10 of differentiation, cells were collected, digested with Accutase, and CD34 cells containing hematopoietic progenitor cells were sorted using the MACS sorting system.+ The cells were detected by flow cytometry analysis to express blood cell markers such as CD45 and CD43.

[0109] 2. T cell differentiation induction:

[0110] T cell induction was performed in the ATO system. The preparation of the ATO system can be found in the following references: Seet CS, He C, Bethune MT, Li S, Chick B, Gschweng EH, et al. Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids. Nat Methods 2017;14:521-30; and Montel-Hagen A, Seet CS, Li S, Chick B, Zhu Y, Chang P, et al. Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells. Cell Stem Cell 2019;24:376-89e8.

[0111] The CD34 containing hematopoietic progenitor cells prepared in step 1 + The cells were co-aggregated with MS5-hDLL4 support cells and cultured at the air-liquid interface of a transwell chamber (LABSELECT). Hematopoietic progenitor cells derived from the 3D differentiation system were cultured at a rate of 1×10 4 HPCs and 1×10 5 MS5-hDLL4 cells / well. IMDM culture medium containing 5 ng / mL FLT3L, 5 ng / mL IL-7, 1% NEAA, 1% GlutaMAX, 1% PS, 2% B27 (without vitamin A), 1-thioglycerol, 50 μg / mL ascorbic acid, 30 μM NAC, and 2 μM minocycline hydrochloride was added to the lower layer. Fresh culture medium was replaced weekly. This was after 8 weeks of T cell induction.

[0112] The cells in the ATO system were collected and analyzed by flow cytometry. αβCD8, CD8α, CD8β, CD3, TCRαβ and other αβCD8 + The expression of T cell markers.+ T cells.

[0113] 3. iTNK cell differentiation induction:

[0114] The αβCD8 + T cells were activated and cultured for two days using XF T expansion medium (STEMCELL Technologies) supplemented with 10% FBS, 1% Glutamax, 100 μg / mL ascorbic acid, 1% ITS, 2 μM minocycline hydrochloride, 24 μM NAC, 50 ng / mL IL-2, 10 ng / mL IL-7, 5 ng / mL IL-15, 10 ng / mL IL-21, 10 μM Z-VAD, and anti-CD3 / CD28 / CD2 T cell activator (25 μL / mL). The T cell activator was then withdrawn and cultured for a further 26 days to obtain iTNK cells.

[0115] Among them, 50ng / mL IL-2, 10ng / mL IL-7, 5ng / mL IL-15, 10ng / mL IL-21, 10μM Z-VAD and anti-CD3 / CD28 / CD2 T cell Activator (25μL / mL) are induction components; 10% FBS, 1% Glutamax, 100μg / mL ascorbic acid, 1% ITS, 2μM minocycline hydrochloride, 24μM NAC are basic components.

[0116] iTNK cell sample 1 was prepared.

[0117] Preparation Example 2: Preparation of iTNK cell sample 2

[0118] The experimental steps were the same as those in Preparation Example 1, except that the human embryonic stem cell line H1 in step 1 was replaced by human chemically induced pluripotent stem cells CiPS-3.

[0119] iTNK cell sample 2 was prepared.

[0120] Preparation Example 3: Preparation of iTNK cell sample 3

[0121] The experimental steps were the same as those in Preparation Example 1, except that the human embryonic stem cell line H1 in step 1 was replaced by the human induced pluripotent stem cell line iPS-7.

[0122] iTNK cell sample 3 was prepared.

[0123] Preparation Example 4: Preparation of iTNK cell sample 4

[0124] Step 1 is basically the same as step 1 of Preparation Example 1, except that a 2D differentiation system is used in step 1. The differentiation process of the 2D differentiation system is basically the same as that of the 3D system, wherein: when seeding cells, human pluripotent stem cells are resuspended in mTesR plus medium supplemented with 5 μM Y-27632 and the cells are plated at 1×10 5 The cells were seeded at a density of 1 / well in a six-well plate coated with Vitronectin and the differentiation was extended to day 12 for sorting CD34 + Hematopoietic progenitor cells.

[0125] Step 2 is basically the same as that of Preparation Example 1, except that the hematopoietic progenitor cells derived from the 2D differentiation system are cultured at a rate of 2.5×10 4 HPCs and 1.25×10 5 MS5-hDLL4 / well was plated.

[0126] Step 3 is substantially the same as Step 3 of Preparation Example 1.

[0127] iTNK cell sample 4 was prepared.

[0128] Preparation Example 5: Preparation of iTNK cell sample 5

[0129] The experimental steps were the same as those in Preparation Example 4, except that the human embryonic stem cell line H1 in step 1 was replaced by human chemically induced pluripotent stem cells CiPS-3.

[0130] iTNK cell sample 5 was prepared.

[0131] Preparation Example 6: Preparation of iTNK cell sample 6

[0132] The experimental steps were the same as those in Preparation Example 4, except that the human embryonic stem cell line H1 in step 1 was replaced by the human induced pluripotent stem cell line iPS-7.

[0133] iTNK cell sample 6 was prepared.

[0134] Example 1: Flow cytometric analysis to detect the expression of surface markers of iTNK cells

[0135] At the end of differentiation, cultured cells (i.e., iTNK cell sample 2 and iTNK cell sample 4) were harvested and resuspended in PBS containing 0.5% BSA and 2 mM EDTA to form a single-cell suspension. The designated antibodies were added and incubated with the cells for 15 minutes at room temperature in the dark. 0.2 μL of each antibody was added to each sample. The antibodies are as follows:

[0136] PerCP 7-AAD (BioLegend, Cat: 420404), FITC TCRαβ (BioLegend, Cat: 306706), BV421 CD3 (BioLegend, Cat: 300434), APC CD4 (BioLegend, Cat: 300514), PE-Cy7 CD8β (Invitrogen, Cat: 25-5273-42), PE CD45RA (BioLegend, Cat: 304108), PE CD8α (BioLegend, Cat: 300908), APC-Cy7 CD27 (BioLegend, Cat: 302815), BV605 CD28(BioLegend,Cat:302968), FITC CD3(BioLegend,Cat:317306), BV650 CD56 (BioLegend, Cat: 318344), APC NKp44 (BioLegend, Cat: 325110), PE NKp46 (BioLegend, Cat: 331908), APC-Cy7 NKG2D (BioLegend, Cat: 320824), PE-Cy7 CD159a (BioLegend, Cat: 375114), APCCD94 (BioLegend, Cat: 305508).

[0137] The flow cytometer CytoFlex was used for data acquisition of flow cytometry analysis, and FlowJo software was used for data analysis and image drawing.

[0138] The results are as follows Figures 1 to 4 shown.

[0139] The results showed that the prepared cell product included a majority of iTNK cells (accounting for about 80%). iTNK cells expressed T cell-related surface markers CD3, TCRαβ, CD8α, CD8β, CD45RA, and CD27, and some expressed CD28. iTNK cells expressed NK cell-related surface markers CD56, NKp44, NKp46, NKG2D, and CD94, and some expressed CD159a.

[0140] Example 2: Single-cell RNA and TCR sequencing analysis to detect the transcriptional signature and TCR expression profile of iTNK cells

[0141] iTNK cells (iTNK cell sample 1 and iTNK cell sample 2) were collected and filtered through a 40 μm cell strainer. The single-cell suspension was used for single-cell RNA and TCR sequencing. The 10× Single-Cell Transcriptome Service and 10× Single-Cell Immune Repertoire Sequencing V2 (5'+TCR) technology service were provided by Beijing Bio-Gene Biotech Co., Ltd.

[0142] Raw sequencing data in fastq format from 10× Genomics VDJ were aligned to the hg38 genome, and the counts of UMIs per cell barcode were quantified using Cellranger version 6.0.2. Downstream analyses were performed on the output matrix using R version 4.2.2 and the Seurat software package, version 4.3.0. For each sample, low-quality cells (those expressing too few or too many genes and UMIs, or containing a high mitochondrial component) were excluded. Expression data were normalized using default parameters to eliminate the effect of variable sequencing depth between cells and then log-transformed. The top 2000 highly variable genes (HVGs) were selected using the default "vst" method in the Seurat software package, and principal component analysis (PCA) was performed on the expression of these HVGs. One to 20 PCs were used as input for nonlinear dimensionality reduction using Unified Mapping (UMAP) and visualized in two-dimensional UMAP coordinates.

[0143] FindNeighbors in the Seurat package was used to construct a KNN graph based on Euclidean distance in PCA space. Unsupervised clustering of cells was performed using the Louvain algorithm based on the KNN graph. Cells were annotated based on characteristic genes. Differentially expressed genes (DEGs) were identified using the Wilcoxon Rank Sum test in normalized data, and P values ​​were corrected using the Bonferroni correction. DEGs with adjusted P values ​​< 0.05 and log fold-change > 0.3 were retained.

[0144] The results are as follows Figures 5 to 8 shown.

[0145] The results showed that based on the single-cell transcriptional profiling analysis, T cell-related characteristic genes were analyzed, and TCR + The cell populations all co-expressed CD8A, CD8B, CD3D and TRAC, but basically did not express CD4. Most cells expressed CD27. Analysis of NK cell-related characteristic genes revealed that TCR in iTNK cells + Most of the cells expressed NCAM1, NCR2, NCR3, KLRC1 and KLRK1, and some cells expressed FCGR3A.

[0146] According to single-cell TCR sequencing analysis, TCR + The distribution of Vα and Vβ in iTNK cells showed rich diversity.

[0147] Example 3: In vitro killing assay to detect the killing effect of iTNK cells on various tumor cell lines

[0148] Human colorectal cancer cell line HCT116 (Procell), human breast cancer cell line MDA-MB-231 (Procell), human melanoma cell line A375 (National Infrastructure of Cell Line Resource), and human hepatocellular carcinoma cell line HepG2 (gifted by Xiang Kuanhui, Peking University Health Science Center. HCT116, MDA-MB-231, A375, and HepG2 cell lines were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (GeminiBio) and 1% double-antibody (Gibco). The above four tumor cell lines were infected with GFP-LUC lentivirus (pHAGE PGK-GFP-IRES-LUC-W plasmid purchased from Core Bio and then packaged for lentivirus) to construct the corresponding tumor cell lines expressing luciferase.

[0149] The human B-cell acute lymphoblastic leukemia cell line, Nalm-6-LUC (Meisen CTCC), was infected with the OKT3 lentivirus (CD5L-OKT3scFv-CD14-pCDH-CMV-MCS-EF1-Puro plasmid synthesized and packaged by Beijing Ruibo Xingke Biotechnology Co., Ltd.) to construct the Nalm-6-LUC-OKT3 cell line. Both Nalm-6-LUC and Nalm-6-LUC-OKT3 cell lines were cultured in RPMI1640 medium (Gibco) supplemented with 10% fetal bovine serum (GeminiBio) and 1% bispecific antibody (Gibco).

[0150] Resuspend the tumor cells in the corresponding tumor cell culture medium to a volume of 100 μL, 1 × 10 4 The cells / well were seeded in 96-well plates. iTNK cells ( Figure 9 iTNK cell sample 2, iTNK cell sample 4; Figure 10 iTNK cell sample 1; Figure 11 iTNK cell samples in 4), primary CB CD8 + T cells or primary CB CD56 + NK cells) were resuspended in the corresponding tumor cell culture medium and seeded at the specified effector-target ratio (E:T) with a volume of 50 μL per well.

[0151] In the neutralization experiment, an isotype control antibody (Isotype Control) (BioLegend, Cat: 400102) was added to the control group at a final concentration of 5 μg / mL, and an anti-NKG2D antibody (Biolegend, Cat: 320813) was added to the experimental group at a final concentration of 5 μg / mL. Neutralization experiments here refer to the addition of an NKG2D-specific antibody to bind to NKG2D, thereby blocking its interaction with the corresponding ligand in the system and its mediated functions. The isotype control antibody is an immunoglobulin of the same species and subtype as the anti-NKG2D antibody and is used to eliminate the potential effects of nonspecific binding of the antibody to the cell surface. After 12 hours of incubation, apoptosis of tumor target cells was quantified using a standard luciferase-based bioluminescence assay using a microplate reader (PerkinElmer). The percentage of lysed cells was calculated as follows: % lysis = 100 × (Natural Death RLU - Experimental RLU) / (Natural Death RLU).

[0152] The results are as follows Figure 9 、 Figure 10 and Figure 11 shown.

[0153] The results showed that iTNK cells were able to mediate potent cytotoxicity against various tumor cell lines, including HepG2, A375, and MDA-MB-231. iTNK cells had little cytotoxicity against NALM-6 cells, but showed potent cytotoxicity against NALM-6-OKT3 cells. Adding neutralizing antibodies against NKG2D to the cytotoxicity system reduced the cytotoxicity of HCT116 cells by iTNK cells and NK cells to a certain extent, indicating that both cells can partially mediate the recognition and cytotoxicity of HCT116 cells through the NKG2D receptor.

[0154] Example 4: Cytotoxic factor release assay to detect cytotoxic factors when iTNK cells kill tumor cells Release

[0155] Human colorectal cancer cell line HCT116 (Procell) was infected with GFP-LUC lentivirus (pHAGEPGK-GFP-IRES-LUC-W plasmid purchased from Core Bio and then packaged into lentivirus) to construct the corresponding tumor cell line expressing luciferase;

[0156] The human B-cell acute lymphoblastic leukemia cell line Nalm-6-LUC (Meisen CTCC) was infected with the OKT3 lentivirus (CD5L-OKT3scFv-CD14-pCDH-CMV-MCS-EF1-Puro plasmid synthesized by Beijing Ruibo Xingke Biotechnology Co., Ltd. and then packaged by lentivirus) to construct the Nalm-6-LUC-OKT3 cell line.

[0157] Resuspend the tumor cells in the corresponding tumor cell culture medium to a volume of 100 μL, 1 × 10 4 Cells were seeded in 96-well plates at a density of 100 cells / well. iTNK cells (iTNK cell sample 2 and iTNK cell sample 4) as effector cells, primary CB CD8 + T cells or primary CB CD56 + NK cells were resuspended in the corresponding tumor cell culture medium and seeded at an effector-to-target ratio of 4:1 in a volume of 50 μL per well. After 12 hours of incubation, the culture supernatant from each group was collected and the concentrations of various cytotoxic factors were quantified by multiplex flow cytometry using a kit (LEGENDplex™ Human CD8 / NK Panel (13-plex) with Filter Plate, biolgend, Cat: 740267). Standard curves were generated and experimental data were calculated according to the kit instructions.

[0158] The results are as follows Figure 12 shown.

[0159] The results showed that the release of IFN-γ, granzyme A, granzyme B, perforin, and IL-17A was detected in both iTNK cell and NK cell co-incubation systems, with higher levels in the iTNK cell co-incubation system. Granylysin, IL-2, and IL-10 were also detected in the iTNK cell co-incubation system. In both iTNK cell and T cell co-incubation systems with NALM-6-OKT3 cells, the release of IFN-γ, granzyme A, granzyme B, perforin, and IL-17A was detected. Except for IFN-γ, the other cytotoxic factors were higher in the iTNK cell co-incubation system. Granylysin, IL-10, and sFasL were also detected in the iTNK cell co-incubation system.

[0160] Example 5: In vivo anti-tumor experiment

[0161] There were 5 mice in the control group and 6 mice in the iTNK group. All mice used were 6 to 8 weeks old NOD.Cg-Prkdc scid Il2rg tm1Vst / Vst(NPG) immunodeficient male mice were purchased from Beijing Weitongda Company.

[0162] HCT116-GFP-LUC tumor cells were cultured at a density of 1 × 10 6 Two days later, 1×10 cells / mouse were injected into the tail vein of each mouse. 6iTNK cells (iTNK cell sample 1) were resuspended in 200 μL PBS. A control group received an equal volume of PBS alone for comparison with the experimental group. Tumor burden in each group of mice was monitored at designated times using in vivo bioluminescence imaging using a Xenogen IVIS (Caliper Life Sciences). Mice were intraperitoneally injected with 15 mg / mL D-luciferin potassium salt (Energy Chemical) and imaged 8 minutes later. Quantification and plotting were performed using Living Image software.

[0163] The results are as follows Figure 13 shown.

[0164] The results showed that the injection of iTNK cells significantly inhibited tumor growth in mice within three weeks.

[0165] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for preparing iTNK cells, comprising the following steps: (1) adding an appropriate amount of anti-CD3 / CD28 / CD2 T cell activator to the culture medium for T cells; culturing for 1-4 days, preferably 2 days; (2) replacing the culture medium with one that does not contain anti-CD3 / CD28 / CD2 T cell activator and continuing the culture for 20-30 days, preferably 26 days; Preferably, the concentration of anti-CD3 / CD28 / CD2 T cell Activator is 20 μl / mL-30 μl / mL; more preferably 25 μl / mL.

2. The preparation method according to claim 1, wherein The culture medium is XF T expansion medium; Preferably, the culture medium contains appropriate amounts of IL-2, IL-7, IL-15, IL-21 and Z-VAD-FMK; Preferably, the culture medium contains 40-60 ng / mL IL-2, 8-12 ng / mL IL-7, 4-6 ng / mL IL-15, 8-12 ng / mL IL-21 and 8-12 μM Z-VAD-FMK; Preferably, the culture medium contains 50 ng / mL IL-2, 10 ng / mL IL-7, 5 ng / mL IL-15, 10 ng / mL IL-21, and 10 μM Z-VAD-FMK; Preferably, the culture medium further comprises appropriate amounts of FBS, Glutamax, ascorbic acid, ITS, minocycline hydrochloride and NAC; Preferably, the culture medium contains 8%-12% FBS, 0.8%-1.2% Glutamax, 80-120 μg / mL ascorbic acid, 0.8%-1.2% ITS, 1.8-2.2 μM minocycline hydrochloride and 20-28 μM NAC; Preferably, the culture medium contains 10% FBS, 1% Glutamax, 100 μg / mL ascorbic acid, 1% ITS, 2 μM minocycline hydrochloride and 24 μM NAC.

3. The preparation method according to any one of claims 1 to 2, wherein: The method further includes the step of isolating iTNK cells from step (2).

4. The preparation method according to any one of claims 1 to 3, wherein: The T cells are αβCD8 + T cells.

5. The preparation method according to any one of claims 1 to 4, wherein: The T cells are CD34 + Hematopoietic progenitor cells were obtained; Preferably, the T cells (e.g., αβCD8 + T cells) by CD34 + Hematopoietic progenitor cells were differentiated from MS5-DLL4 supporting cells in the ATO system.

6. The preparation method according to claim 5, wherein CD34 + Hematopoietic progenitor cells are made from human pluripotent stem cells; Preferably, the CD34 + Hematopoietic progenitor cells are derived from human pluripotent stem cells by differentiation in a 3D differentiation system or a 2D differentiation system; Preferably, the human pluripotent stem cells are selected from human embryonic stem cells, human induced pluripotent stem cells and human chemically induced pluripotent stem cells.

7. An iTNK cell, produced by the preparation method according to any one of claims 1 to 6; Preferably, the iTNK cells also express a chimeric antigen receptor.

8. A pharmaceutical composition comprising an effective amount of the iTNK cells according to claim 7, and optionally, further comprising one or more pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition further comprises one or more anti-tumor drugs; Preferably, the anti-tumor drug is selected from chimeric antigen receptor T cells, antibody drugs and chemotherapy drugs.

9. A combination pharmaceutical product comprising a first product and a second product in separate packages, wherein: The first product comprises the iTNK cells according to claim 7; The second product comprises one or more anti-tumor drugs; preferably, the anti-tumor drugs are selected from chimeric antigen receptor T cells, antibody drugs and chemotherapy drugs; Preferably, the first product and the second product further independently contain one or more pharmaceutically acceptable excipients; Preferably, the combination product further comprises a drug package insert.

10. Use of the iTNK cell according to claim 7 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating or preventing tumors; Preferably, the tumor is selected from one or more of colorectal cancer, melanoma, breast cancer, liver cancer, lung cancer, nasopharyngeal carcinoma, rhabdomyosarcoma, gastric cancer and appendiceal mucinous cystadenocarcinoma; Preferably, the lung cancer is non-small cell lung cancer.

11. A kit comprising a first reagent combination, wherein the first reagent combination comprises: Culture medium: XF T expansion medium, appropriate amounts of IL-2, IL-7, IL-15, IL-21, and Z-VAD-FMK, as well as appropriate amounts of FBS, Glutamax, ascorbic acid, ITS, minocycline hydrochloride, and NAC; Preferably, it includes 40-60 ng / mL IL-2, 8-12 ng / mL IL-7, 4-6 ng / mL IL-15, 8-12 ng / mL IL-21 and 8-12 μM Z-VAD-FMK; Preferably, it includes 50 ng / mL IL-2, 10 ng / mL IL-7, 5 ng / mL IL-15, 10 ng / mL IL-21 and 10 μM Z-VAD-FMK; Preferably, it includes 8%-12% FBS, 0.8%-1.2% Glutamax, 80-120 μg / mL ascorbic acid, 0.8%-1.2% ITS, 1.8-2.2 μM minocycline hydrochloride and 20-28 μM NAC; Preferably, it includes 10% FBS, 1% Glutamax, 100 μg / mL ascorbic acid, 1% ITS, 2 μM minocycline hydrochloride, and 24 μM NAC; Preferably, the kit is used for preparing iTNK cells.

12. The kit according to claim 11, further comprising a second reagent combination, wherein the second reagent combination comprises: Preparation of αβCD8 from hematopoietic progenitor cells + T cell culture media and induction reagents.

13. The kit according to any one of claims 11 to 12, further comprising a third reagent combination, the third reagent combination comprising: Culture media and induction reagents for preparing hematopoietic progenitor cells from human pluripotent stem cells.

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