Methods of making nk cells, nk cells, and uses thereof

Abstract

This invention discloses a method for preparing NK cells, the NK cells obtained thereby, and their applications. The method includes: sorting NK cells from peripheral blood of an allogeneic donor; culturing them in a serum-free medium containing CD3 monoclonal antibody, CD16 antibody, and IL-2, without the need for a feeder layer; preferably, IL-21 is added for amplification on days 3-5 of culture, and the cells are cultured for 20-40 days. This method yields non-genetically modified NK cells, among which CD56... bright The NK cells comprise >90% of the cell line and highly express a variety of activating and functional molecules. These NK cells effectively clear Aβ oligomers and senescent cells, and alleviate neuroinflammation. In treatment, intracranial injection achieves equivalent or better efficacy with only about 1 / 10th the dose administered intravenously, significantly clearing Aβ deposits in the brains of AD model mice and improving pathology.

Description

Technical Field

[0001] This invention relates to the field of cell culture, and more specifically, to a method for preparing NK cells, NK cells and their uses, and more specifically, to a pharmaceutical composition and a reagent kit. Background Technology

[0002] Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), are characterized by the progressive damage and loss of function of specific neurons. The number of patients worldwide continues to rise, while clinically, there is a lack of effective means to fundamentally block or reverse the disease progression. Existing drugs are mostly limited to symptom relief or limited delay of disease progression, and are insufficient to comprehensively repair the damaged brain microenvironment.

[0003] In recent years, immunomodulatory strategies based on natural killer cells (NK cells) have brought new ideas to the treatment of neurodegenerative diseases. NK cells have innate immune killing and regulatory functions and can penetrate the blood-brain barrier, and are considered to have the potential to directly or indirectly clear abnormal proteins and regulate neuroinflammation. Currently, most NK cell therapies in clinical research are based on autologous sources, but their preparation and application have obvious limitations: First, NK cells obtained from the peripheral blood of patients (mostly the elderly) often exhibit immunosenescence, characterized by weak proliferation capacity and decreased functional activity, resulting in a low starting point for in vitro expansion and unstable product quality, directly affecting the therapeutic effect; Second, the autologous cell preparation process is highly personalized, time-consuming, and costly, making it difficult to achieve large-scale, standardized production, thus limiting its clinical accessibility; Third, in traditional intravenous administration, most cells remain in the peripheral circulation, with only a very small number entering the brain parenchyma, resulting in low targeting efficiency. Often, extremely high doses are required to achieve the local concentration needed for treatment, thus bringing potential systemic risks and high costs.

[0004] Therefore, there is an urgent need in this field to develop a new method for preparing NK cells that can achieve stable, efficient, and scalable in vitro expansion from high-quality cell sources to obtain highly active and pure NK cells, thereby providing a reliable cellular basis for developing safer and more effective treatments for neurodegenerative diseases. Summary of the Invention

[0005] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a method for preparing NK cells.

[0006] This application is based on the following discoveries of the inventors: Firstly, the inventors compared the NK cells obtained from the peripheral blood of young healthy allogeneic donors with those from older donors and found that the former had more significant expansion potential, stronger cytotoxic activity (e.g., killing ability for K562 cells), and higher cytokine secretion level of IFN-γ. This suggests that the use of young donor cells can fundamentally avoid the functional limitations caused by immunosenescence, providing a high-quality source for the preparation of efficient and off-the-shelf cell products.

[0007] Further, the inventors established an expansion system that does not require feeder cells throughout the entire process: after sorting, the allogeneic NK cells are placed in a serum-free medium containing CD3 monoclonal antibody, CD16 antibody, and IL-2 to start the culture, and IL-21 is added on the 3rd to 5th day to achieve stable and efficient expansion of the cells. Unexpectedly, this method not only makes CD3 - CD56 + The purity of the cells is more than 99%, and the proportion of CD56 bright phenotypes in the cells is increased to more than 95% — this subpopulation has stronger tissue migration and residence ability, which is highly consistent with the needs of brain disease treatment. At the same time, the expanded cells widely express activation receptors and effector molecules such as NKp30, NKp44, NKG2D, CXCR3, CD69, Granzyme B, Perforin, etc., showing the characteristics of comprehensive functional enhancement.

[0008] At the mechanism level, in vitro experiments have confirmed that the NK cells obtained in this way can effectively uptake and clear the key pathological protein Aβ oligomers of Alzheimer's disease in a concentration- and time-dependent manner. More importantly, the inventors have for the first time clearly defined that these NK cells can efficiently recognize and clear senescent microglial cells (such as etoposide-induced HMC3 cells), revealing a new action pathway of reducing neural inflammation and tissue damage by clearing senescent cells, and expanding the therapeutic mechanism of NK cells in neurodegenerative diseases.

[0009] On this basis, the inventors further verified the efficacy differences of different administration methods in AD model mice (FAD 4T ). The results showed that a single intracranial injection (intracerebroventricularly) only required 3 x 10 6 cells per mouse (about equivalent to 1 / 10 of the intravenous dose), and after one month of administration, the effect of clearing Aβ in the cortex and reducing neural inflammation (reducing the positive area of GFAP) reached or even surpassed that of the multiple intravenous administration group with a total dose of 1.2 x 10 8 cells. This proves that intracranial injection can completely bypass the blood-brain barrier, achieve precise and efficient delivery of cells at the lesion site, and thus achieve significant therapeutic advantages at extremely low doses, fundamentally breaking through the key bottleneck of low targeting efficiency of intravenous administration.

[0010] Based on this, in a first aspect of the present application, a method for preparing NK cells is provided. According to an embodiment of the present application, the method comprises: sorting and purifying NK cells from peripheral blood mononuclear cells, wherein the peripheral blood mononuclear cells are from an allogeneic donor; culturing the sorted and purified NK cells in a serum-free culture medium containing CD3 monoclonal antibody, CD16 antibody and interleukin-2, so as to harvest NK cells; wherein the method does not use feeder cells throughout the whole process. According to the method of the present application, by using allogeneic donor-derived cells and combining with a specific feeder-free expansion system, NK cells can be efficiently and stably expanded. This fundamentally avoids the problems of low expansion efficiency and poor cell activity caused by using patient's own senescent cells, and the safety risks, process complexity and batch differences that may be caused by using feeder cells, thereby laying a process foundation for preparing off-the-shelf standardized NK cells. According to the method of the present application, NK cells with high activity can be safely, quickly and stably prepared.

[0011] In some aspects of the present application, the method further comprises: adding interleukin-21 to the culture system for expansion culture on day 3-5 after the start of the initial culture.

[0012] In some aspects of the present application, the harvested NK cells are achieved by culturing continuously for 20-40 days.

[0013] In some aspects of the present application, the allogeneic donor is a young healthy donor.

[0014] In some aspects of the present application, the peripheral blood mononuclear cells are obtained by density gradient centrifugation from the peripheral blood of the allogeneic donor.

[0015] In some aspects of the present application, the concentration of the CD3 monoclonal antibody is 0.5-5 ng / mL, the concentration of the CD16 antibody is 1-5 μg / mL, and the concentration of the interleukin-2 is 3.5-6.5 ng / mL.

[0016] In some aspects of the present application, the sorting and purification is achieved by using immunomagnetic bead sorting or flow cytometry sorting.

[0017] In some aspects of the present application, the feeder cells are not used throughout the whole process by not adding or co-culturing any heterologous feeder cells during the whole culture process.

[0018] In some aspects of the present application, the concentration of the added interleukin-21 is 20-100 ng / mL.

[0019] In a second aspect, the present application provides an NK cell. According to embodiments of the present application, the NK cell is prepared by the method of the first aspect of the present application. The NK cell according to embodiments of the present application is a cell therapy product with comprehensive enhanced functions. It not only overcomes the bottleneck of cell quality and the problem of production waiting time of autologous therapy, but also has a unique advantage phenotype and function spectrum due to the specific culture conditions. The NK cell according to embodiments of the present application has a lower recognition threshold for diseased cells, a stronger killing effect, a longer survival time in vivo, and an effect of preferentially entering diseased tissues, eliminating senescent cells, and reducing neuroinflammation.

[0020] In some aspects of the present application, the NK cell is a non-genetically modified cell, and wherein CD56 bright The proportion of NK cells with the phenotype is greater than 90%, preferably greater than 95%.

[0021] In some aspects of the present application, the NK cell highly expresses at least one receptor or functional molecule selected from the group consisting of NKp30, NKp44, CD69, NKG2D, CD25, CXCR3, Ki67, CD107a, Granzyme B, Perforin, IFN-γ, and 4-1BB.

[0022] In a third aspect, the present application provides use of the NK cell of the second aspect of the present application in the preparation of a medicament for preventing and / or treating a neurodegenerative disease. The present application first explicitly and confirms that the allogeneic, functionally enhanced NK cell prepared by the above-mentioned specific method has outstanding application value in the treatment of neurodegenerative diseases. This use is based on the unique multiple mechanisms of the cell itself, such as clearing Aβ, eliminating senescent cells, and reducing neuroinflammation, and provides a new cell immunotherapy strategy for solving the problem of lack of effective disease-modifying therapy for such diseases.

[0023] In some aspects of the present application, the neurodegenerative disease is selected from at least one of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, and aging-related diseases.

[0024] In some aspects of the present application, the neurodegenerative disease is Alzheimer's disease.

[0025] In some aspects of the present application, the medicament is formulated as a preparation for intravenous injection or intracranial injection.

[0026] In some aspects of the present application, the intracranial injection includes intracerebroventricular injection or brain parenchyma injection.

[0027] In some aspects of the present application, the single effective dose of intracranial injection is lower than 1 / 5 to 1 / 20, preferably 1 / 10, of the single effective dose of intravenous injection.

[0028] In some aspects of the present application, the function of the drug comprises at least one of the following: (1) uptake of Aβ oligomers, clearance of β-amyloid deposits; (2) clearance of senescent cells, reduction of expression of senescent cell markers p16 or p21; (3) reduction of neuroinflammation, reduction of levels of markers GFAP or IBA1; (4) improvement of cognitive dysfunction.

[0029] In a fourth aspect of the present application, a pharmaceutical composition is provided. According to embodiments of the present application, the pharmaceutical composition comprises the NK cells of the second aspect of the present application, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition according to embodiments of the present application can prepare the functional enhanced NK cells of the present application into a dosage form that can be directly used for clinical administration. By compounding with a pharmaceutically acceptable carrier or excipient (such as a cryopreservation solution, a buffer for injection), the viability and functional stability of the NK cells can be maintained, ensuring the quality of the NK cells during transportation, storage and injection, which is a necessary product form to realize its therapeutic use.

[0030] In some aspects of the present application, the pharmaceutical composition further comprises interleukin-2.

[0031] In a fifth aspect of the present application, a kit is provided. According to embodiments of the present application, the kit comprises the NK cells of the second aspect of the present application, or the pharmaceutical composition of the fourth aspect of the present application. The kit according to embodiments of the present application packages the therapeutic core component (NK cells or pharmaceutical composition thereof) in a standardized, ready-to-use or easy-to-use form, possibly with necessary instructions, diluents or auxiliary tools, which helps to ensure the standardization, repeatability and convenience of the treatment process.

[0032] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter in the description of embodiments of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0033] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description of embodiments, taken in conjunction with the accompanying drawings, in which: Figure 1 Fig. 1 shows the results of NK cell receptor expression and phenotype analysis before and after expansion in Example 1.

[0034] Figure 2 Fig. 2 shows the results of preparation and identification of Aβ oligomers (AβO) in Example 2.

[0035] Figure 3 Fig. 3 shows the results of detection of NK cell uptake of AβO in Example 2.

[0036] Figure 4 The graph shows the detection results of NK cell clearance of AβO in Example 2.

[0037] Figure 5 The image shows the β-galactosidase staining results of HMC3 cells after etoposide-induced senescence in Example 2.

[0038] Figure 6 The graph shows the results of detecting the expression levels of senescence-associated secretory phenotype (SASP) factors in HMC3 cells and their induced senescence cells in Example 2.

[0039] Figure 7 The graph shows the detection results of p16 expression level during the induced senescence process of HMC3 and PLC cells in Example 2.

[0040] Figure 8 The graph shows the detection results of NKG2D ligand expression levels during the induced senescence process of HMC3 and PLC cells in Example 2.

[0041] Figure 9 The graph shows the results of the in vitro killing effect of NK cells on HMC3 cells and their induced senescent cells in Example 2.

[0042] Figure 10 The immunohistochemical results and clearance rate of Aβ-positive cells in the brains of AD model mice after intravenous injection of NK cells are shown in Example 3.

[0043] Figure 11 The immunohistochemical results of GFAP-positive cells in the brains of AD model mice after intravenous injection of NK cells in Example 3 are shown in the figure, along with the remission rate statistics.

[0044] Figure 12 The figure shows a comparison of the positive results of Aβ in the brains of AD model mice after a single intracranial injection and multiple intravenous injections of NK cells in Example 4.

[0045] Figure 13 The figure shows a comparison of the positive results of GFAP in the brains of AD model mice after a single intracranial injection and multiple intravenous injections of NK cells in Example 4.

[0046] Figure 14 A statistical comparison graph showing the in vitro expansion efficiency of NK cells from young and old donors in Example 5 is displayed.

[0047] Figure 15 The graph shows the detection results of the killing efficiency of NK cells from young and old donors against K562 cells in Example 5.

[0048] Figure 16The graph shows the results of detecting IFN-γ expression levels in NK cells from young and old donors in Example 5. Detailed Implementation

[0049] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0050] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0051] In this document, the term "NK cell (natural killer cell)" refers to a type of lymphocyte in the innate immune system that has the function of directly killing target cells (such as tumor cells, virus-infected cells, senescent cells, etc.). In this invention, it specifically refers to CD3 cells obtained by the method described above from peripheral blood of allogeneic donors, and which have not undergone genetic engineering modification. - CD56 + Lymphocyte population.

[0052] In this document, the term "allogeneic donor" refers to a healthy individual who is genetically different from the individual receiving treatment (the recipient). In this invention, young, healthy donors are preferred, typically individuals aged 18-40 years who have been medically assessed as free from infectious diseases, immune system disorders, malignant tumors, and other health conditions, and whose NK cells have been shown to have stronger in vitro expansion potential and functional activity.

[0053] In this article, the term "CD56" bright "NK cells" refers to a subset of NK cells characterized by high expression of CD56 and low or no expression of CD16. This subset is considered to have stronger cytokine (such as IFN-γ) secretion capacity and tissue migration / residential properties. In this invention, after amplification using the method described above, the proportion of this subset is significantly increased to greater than 90%, preferably greater than 95%.

[0054] In this paper, the term "feeder cells" refers to xenogeneic cells (such as K562 cells, peripheral blood mononuclear cells, etc., treated with radiation or mitomycin C, which assist and support the growth and proliferation of target cells (such as NK cells) in a cell co-culture system by providing contact support or secreting growth factors. The phrase "no feeder cells used throughout the entire culture process" in this invention means that no feeder cells of any origin are added to or co-cultured in the culture system throughout the entire culture process from inoculation to harvest.

[0055] In this paper, the term "non-genetically modified" refers to NK cells whose genomes were not artificially and permanently altered during the preparation process using techniques such as gene transduction, gene editing (e.g., CRISPR / Cas9), or transposon systems.

[0056] In this document, the term "β-amyloid oligomer (Aβ oligomer, AβO)" refers to a relatively low molecular weight, soluble oligomer form formed by the aggregation of β-amyloid monomers. In the pathology of Alzheimer's disease, Aβ oligomers are considered one of the key forms of neurotoxicity. In this invention, the NK cells possess the ability to take up and clear such Aβ oligomers.

[0057] In this paper, the term "senescent cell" refers to a cell that has entered an irreversible cell cycle arrest state due to factors such as replication senescence and stress, but still maintains metabolic activity. Its characteristics include increased activity of senescence-associated β-galactosidase (SA-β-gal), high expression of cell cycle repressor proteins (such as p16 and p21), and secretion of multiple inflammatory factors (i.e., the SASP phenotype). In this invention, it specifically refers to cells that can be recognized and eliminated by NK cell surface receptors (such as NKG2D), such as etoposide-induced senescent microglia (HMC3-ETO).

[0058] In this paper, the term "neuroinflammatory markers" refers to "GFAP (glial fibrillary acidic protein) and IBA1 (ionized calcium-binding adapter molecule 1)," where "GFAP" is a specific marker of astrocyte activation, and its expression level is positively correlated with the degree of inflammatory response in the central nervous system; and "IBA1" is a marker of microglia activation. In this invention, the NK cell therapy effectively reduces the expression levels of GFAP and IBA1 in brain tissue, indicating that it has the function of alleviating neuroinflammatory reactions.

[0059] In this document, the term "intracranial injection" refers to a method of drug administration that delivers drugs or cells directly into the cranial cavity. Specifically, in this invention, it includes: "intraventricular injection (ICV)" which refers to injecting cells into the cerebrospinal fluid of the ventricular system; and "parenchymal injection" which refers to injecting cells directly into a specific brain tissue region. This invention particularly emphasizes that the single effective dose of intracranial injection is significantly lower than that of intravenous injection, preferably about 1 / 10 of the intravenous injection dose.

[0060] In this article, the term "off-the-shelf" product refers to cell therapy products based on healthy allogeneic donor cells that have undergone standardized preparation, quality testing, and cryopreservation in advance. Compared to "autologous" products, these products do not require individualized production and can be used immediately.

[0061] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0062] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0063] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, or pharmaceutical compositions of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0064] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in an individual who is susceptible but has not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0065] This application discloses a method for preparing NK cells, NK cells and their uses, pharmaceutical compositions and kits, which will be described in detail below.

[0066] Methods for preparing NK cells In a first aspect, this application proposes a method for preparing NK cells. According to an embodiment of this application, the method includes: sorting and purifying NK cells from peripheral blood mononuclear cells derived from an allogeneic donor; culturing the sorted and purified NK cells in a serum-free medium containing CD3 monoclonal antibody, CD16 antibody, and interleukin-2 to harvest NK cells; wherein the method does not use feeder cells throughout the process. The method according to the embodiment of this application can safely, rapidly, and stably prepare highly active NK cells. By using cells derived from an allogeneic donor and combining them with a specific feeder-free expansion system, the method according to the embodiment of this application can achieve efficient and stable expansion of NK cells. This fundamentally avoids the problems of low expansion efficiency and poor cell viability caused by using autologous senescent cells from patients, as well as the safety risks, process complexity, and batch-to-batch variability that may arise from using feeder cells, laying a technological foundation for the preparation of commercially available standardized NK cells.

[0067] According to embodiments of this application, the method further includes adding interleukin-21 to the culture system on days 3 to 5 after the start of initial culture for amplification culture. According to embodiments of this application, adding interleukin-21 at a specific time point can produce a synergistic effect with CD3 monoclonal antibody, CD16 antibody, and interleukin-21, significantly promoting the continuous proliferation and functional maturation of NK cells, thereby obtaining a higher quantity and higher quality of NK cell products.

[0068] According to embodiments of this application, the harvesting of NK cells is achieved through continuous culture for 20 to 40 days. By setting the culture period to 20-40 days, sufficient time is provided for the expansion and functional maturation of NK cells, enabling stable expansion of up to several hundred times while maintaining high cell viability, thus meeting the cell quantity requirements for clinical applications.

[0069] According to embodiments of this application, the allogeneic donor is a young, healthy donor. According to embodiments of this application, selecting a young, healthy donor as the cell source results in superior initial proliferative potential and basic functional status of their NK cells. Figures 14-16 As shown, NK cells from young donors are significantly superior to those from older donors in terms of expansion fold, cytotoxic activity (killing K562 cells), and secretion of effector factors (such as IFN-γ), which ensures that the final product has stronger therapeutic potential.

[0070] According to an embodiment of this application, the peripheral blood mononuclear cells are obtained from allogeneic donor peripheral blood using density gradient centrifugation. Density gradient centrifugation is a mature and reliable cell separation technique that can efficiently and cost-effectively obtain high-purity peripheral blood mononuclear cells, providing high-quality starting material for subsequent NK cell sorting.

[0071] According to embodiments of this application, the concentration of the CD3 monoclonal antibody is 0.5-5 ng / mL, the concentration of the CD16 antibody is 1-5 μg / mL, and the concentration of interleukin-2 is 3.5-6.5 ng / mL. The concentration ranges described in these embodiments have been optimized and validated. Within these ranges, NK cells can be activated most effectively and their proliferation process initiated, while avoiding overactivation or functional exhaustion due to excessively high concentrations, and insufficient stimulation due to excessively low concentrations, thereby achieving an optimal balance between amplification efficiency and cell function.

[0072] According to embodiments of this application, the sorting and purification is achieved using immunomagnetic bead sorting or flow cytometry sorting. According to embodiments of this application, using immunomagnetic bead sorting or flow cytometry sorting can isolate target NK cells from peripheral blood mononuclear cells with high purity and high recovery rate, removing other lymphocytes (such as T cells) that may inhibit NK cell proliferation or affect product safety, ensuring the purity of the culture system and the consistency of the amplification direction.

[0073] According to embodiments of this application, the complete absence of feeder cells is achieved by not adding or co-culturing any xenogeneic feeder cells throughout the entire culture process. According to embodiments of this application, the risk of introducing exogenous cells (such as radiation-treated K562 cells) is completely eliminated. This greatly simplifies the production process and quality control procedures, reduces the risk of NK cells being contaminated or carrying exogenous antigens, and improves the safety of NK cells.

[0074] According to embodiments of this application, the added interleukin-21 concentration is 20-100 ng / mL, for example, it can be 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, or a range between the two, 25-100 ng / mL, 30-100 ng / mL. Interleukin-21 at concentrations within the ranges specified in embodiments of this application can effectively induce and maintain NK cell proliferation signals and promote their expression towards CD56, which has stronger tissue retention and cytokine secretion capabilities.bright Phenotypic differentiation, while avoiding toxicity or adverse effects on cells.

[0075] NK cells In a second aspect of this application, an NK cell is proposed. According to an embodiment of this application, the NK cell is prepared by the method described in the first aspect of this application. The NK cell according to the embodiment of this application has the effects of a lower recognition threshold for diseased cells, stronger killing effect, longer survival in vivo, superior entry into diseased tissues, clearing senescent cells, and reducing neuroinflammation. It should be explained that the NK cell according to the embodiment of this application is a cell product with comprehensively enhanced function. It not only overcomes the bottleneck of cell quality and production waiting time in autologous therapy, but also possesses unique advantageous phenotypes and functional spectra due to specific culture conditions.

[0076] According to embodiments of this application, the NK cells are non-genetically modified cells, and wherein CD56... bright The proportion of NK cells in the phenotype is greater than 90%, preferably greater than 95%. According to embodiments of this application, the NK cells of the present invention have extremely high CD56... bright Subgroup proportion. This subgroup is considered to have stronger tissue migration, infiltration and retention capabilities, as well as better immunomodulatory functions, making it particularly suitable for the treatment of neurodegenerative diseases that require entry and long-term action on specific lesion sites such as brain tissue.

[0077] According to embodiments of this application, the NK cells highly express at least one receptor or functional molecule selected from NKp30, NKp44, CD69, NKG2D, CD25, CXCR3, Ki67, CD107a, Granzyme B, Perforin, IFN-γ, and 4-1BB. According to embodiments of this application, the NK cells of the present invention simultaneously highly express multiple key activating receptors, chemokine receptors, degranulation markers, and effector molecules. This enhanced phenotype in various aspects implies that the cells have stronger target recognition capabilities (via NKp30, NKG2D, etc.), migration to inflammatory sites (via CXCR3), and tissue residency (CD56). bright The ability of cell subsets to exhibit proliferative activity (via Ki67) and the ability to perform cytotoxic and immunomodulatory functions (via Granzyme B, Perforin, IFN-γ, etc.) provides the molecular basis for its multiple therapeutic effects in complex disease microenvironments.

[0078] use In a third aspect, this application discloses the use of the NK cells described in the second aspect in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases. This invention, for the first time, clearly demonstrates and confirms the outstanding application value of allogeneic, functionally enhanced NK cells prepared by the specific method described above in the treatment of neurodegenerative diseases. This use, based on the cells' unique multiple mechanisms of clearing Aβ, eliminating senescent cells, and reducing neuroinflammation, provides a novel cellular immunotherapy strategy to address the challenge of lacking effective disease-modifying therapies for these diseases.

[0079] According to embodiments of this application, the neurodegenerative disease is selected from at least one of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, and age-related diseases.

[0080] According to embodiments of this application, the neurodegenerative disease is Alzheimer's disease. Embodiments of this application clarify the specific disease range to which the NK cells of this invention are applicable, particularly Alzheimer's disease. Experimental data directly demonstrate the NK cells' ability to clear the key pathological protein Aβ in AD, and their significant effects on clearing Aβ in the brain and alleviating neuroinflammation in AD model mice, providing solid experimental support for this specific application.

[0081] According to embodiments of this application, the drug is formulated as a preparation for intravenous or intracranial injection. Embodiments of this application provide two feasible clinical application routes. Intravenous injection is a relatively conventional and less invasive systemic administration method; while intracranial injection is an innovative local administration strategy for brain diseases.

[0082] According to embodiments of this application, the intracranial injection includes intraventricular injection or parenchymal injection. Embodiments of this application further specify feasible methods of intracranial drug delivery. Intraventricular injection can deliver cells broadly to the cerebrospinal fluid circulation area, suitable for diffuse lesions; parenchymal injection can achieve precise, high-concentration drug delivery to the lesion site. This provides refined treatment options for neurodegenerative diseases with different pathological characteristics.

[0083] According to embodiments of this application, the single effective dose of intracranial injection is less than 1 / 5 to 1 / 20 of the single effective dose of intravenous injection, preferably 1 / 10. According to embodiments of this application, in AD model mice, a single intracranial injection of only about 1 / 10 of the intravenous dose of cells can achieve or even surpass the efficacy of multiple high-dose intravenous injections. This directly demonstrates that intracranial administration can greatly improve treatment efficiency, significantly reduce the effective dose, thereby potentially reducing systemic side effects caused by large-dose cell infusion and significantly reducing treatment costs.

[0084] According to embodiments of this application, the functions of the drug include at least one of the following: (1) taking up Aβ oligomers and clearing β-amyloid protein deposits; (2) clearing senescent cells and reducing the expression of senescent cell markers p16 or p21; (3) alleviating neuroinflammation and reducing the levels of markers GFAP or IBA1; and (4) improving cognitive dysfunction. Embodiments of this application clarify the specific mechanism of action and expected efficacy of the drug of the present invention in treating neurodegenerative diseases (especially AD).

[0085] Pharmaceutical Composition In a fourth aspect, this application provides a pharmaceutical composition. According to an embodiment of this application, the pharmaceutical composition comprises the NK cells described in the second aspect of this application, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition according to the embodiment of this application prepares the functionally enhanced NK cells of the present invention into a dosage form suitable for direct clinical administration. By incorporating pharmaceutically acceptable carriers or excipients (such as cryopreservation solutions or injection buffers), the viability and functional stability of the NK cells can be maintained, ensuring their quality during transportation, storage, and injection, making it an essential product form for achieving their therapeutic use.

[0086] According to embodiments of this application, the pharmaceutical composition further comprises interleukin-2. According to embodiments of this application, the addition of interleukin-2 to the pharmaceutical composition can continue to provide survival and activation signals to NK cells after in vivo infusion, helping to maintain the persistence and functional activity of NK cells in the patient after infusion, potentially prolonging the duration of therapeutic effect and thereby enhancing long-term efficacy.

[0087] The pharmaceutical compositions of the present invention can be administered by any acceptable method of administration. The pharmaceutical compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms, such as injections or lyophilized powders, and current methods for preparing these dosage forms are known or obvious to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the bioactive components contained therein to be bioavailable after administration to a patient.

[0088] Reagent test kit In a fifth aspect of this application, a kit is provided. According to embodiments of this application, the kit comprises NK cells as described in the second aspect of this application, or a pharmaceutical composition as described in the fourth aspect of this application. The kit according to embodiments of this application packages the core therapeutic component (NK cells or their pharmaceutical composition) in a standardized, ready-to-use, or easy-to-use form, possibly accompanied by necessary instructions for use, diluents, or auxiliary tools, which helps ensure the standardization, repeatability, and convenience of the treatment process.

[0089] As previously mentioned, the NK cells are functionally enhanced allogeneic NK cells prepared using a specific method, possessing a high proportion of CD56. bright Phenotypic and multi-disease therapeutic functions; the pharmaceutical composition comprises the NK cells and a pharmaceutically acceptable carrier or excipient. Furthermore, the kit may also include auxiliary components for administering the treatment, such as a dedicated syringe for intracranial injection, a stereotactic aid, cell diluent or maintenance solution, and interleukin-2, etc. Kits containing the above components provide a standardized, ready-to-use, complete solution for the treatment of neurodegenerative diseases, particularly precision treatment via intracranial injection. The kit can be used in medical institutions or research facilities to facilitate the reproducible administration of NK cell therapy, for example, by preparing the NK cells into a formulation suitable for intracranial injection according to the manufacturer's instructions, to intervene in individuals with neurodegenerative diseases such as Alzheimer's disease at a significantly lower dose than intravenous administration, with the aim of clearing pathological proteins, reducing neuroinflammation, and improving pathological conditions.

[0090] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0091] Example: Example 1: Method for preparing non-genetically modified NK cells expanded from allogeneic peripheral blood After screening young, healthy allogeneic donors with peak immune function, peripheral blood mononuclear cells (PBMCs) were isolated from the donors' peripheral blood. NK cells were then purified through negative selection using a CD3 MicroBeads (Miltenyi Biotec, catalog number: 130-097-043) NK cell sorting and purification kit. The purified NK cells were then expanded and cultured in a factor amplification system. The specific amplification procedure was as follows: peripheral blood was collected from healthy donors, and PBMCs were isolated. Subsequently, NK cells were purified through negative selection using CD3 MicroBeads (Miltenyi Biotec, catalog number: 130-097-043). The isolated NK cells were initially cultured in KBM581 serum-free medium (Corning, catalog number: 88-581-CM) containing CD3 monoclonal antibody (0.5–5 ng / mL, preferably 1 ng / mL), CD16 antibody (1–5 μg / mL, preferably 2 μg / mL), and IL-2 (3.5–6.5 ng / mL, preferably 5 ng / mL). After 3–5 days of culture, IL-21 (20–100 ng / mL, preferably 50 ng / mL) was added to the culture medium. During the culture process, the medium was replenished every two days until the cell density reached 1.5 × 10⁶ cells / mL, depending on the cell growth status. 6 Cells were cultured at a density of 1,000 cells / mL, supplemented with fresh cytokines (5 ng / mL IL-2 and 50 ng / mL IL-21) according to the total volume, and cultured for 20-40 days. After culture, the cell population was harvested, yielding high-purity, highly viable allogeneic NK cells with enhanced cytotoxic activity. This method eliminates the need for feeder cells, enabling efficient expansion of allogeneic NK cells and providing a stable and reliable cell source for the preparation of ready-to-use NK cell immunotherapy products.

[0092] To verify the expression of important functional proteins such as NK cell receptors in vitro expanded from allogeneic peripheral blood, flow cytometry was used to detect the expression of NCR receptor family (NKp30 (Biolegend, catalog number: 325208), NKp44 (Biolegend, catalog number: 325116)) and C lectin receptor family (CD69 (Biolegend, catalog number: 310910), NKG2D (Biolegend, catalog number: 320808), NKG2C (Biolegend, catalog number: 375023)). Activation-related receptors (CD25 (Biolegend, catalog number: 356108), DNAM-1 (Biolegend, catalog number: 338316)), chemokine-related receptors (CXCR3 (Biolegend, catalog number: 353720), LFA-1 (Biolegend, catalog number: 363404)), proliferation-related receptors (Ki67 (Biolegend, catalog number: 350504)), and killing-related receptors (CD107a (Biolegend, catalog number: 328606), Granzyme) The expression levels of representative molecules such as B (Biolegend, catalog number: 515406), Perforin (Biolegend, catalog number: 353310), IFN-γ (Biolegend, catalog number: 506518), 4-1BB (Biolegend, catalog number: 309818), TRAIL (Biolegend, catalog number: 308206), and CD16 (Biolegend, catalog number: 302040) were analyzed. Specifically, allogeneic peripheral blood-derived NK cells expanded in vitro and their corresponding pre-expansion PB-NK cells (as a control group) were collected. After washing twice with PBS, the cells were resuspended in FACS solution (PBS containing 0.1% sodium azide and 0.4% BSA), counted, and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells / mL; add corresponding antibodies and isotype control antibodies respectively, incubate at 4°C for 30 minutes; wash twice with PBS and resuspend in FACS solution, analyze cell fluorescence by flow cytometry, and analyze the results using FlowJo software. Figure 1As shown in AC, compared to the PB-NK control, NK cells expanded in vitro from allogeneic peripheral blood highly expressed NKp30, NKp44, CD69, NKG2D, CD25, CXCR3, Ki67, CD107a, Granzyme B, Perforin, IFN-γ, and 4-1BB, indicating that in vitro expansion culture comprehensively enhanced the killing, activation, proliferation, and chemotaxis abilities of NK cells. Furthermore, NK cells expanded in vitro from allogeneic peripheral blood also maintained high expression levels of molecules beneficial to NK cell function, such as NKG2C, TRAIL, CD16, and DNAM-1, which are already highly expressed in PB-NK. This demonstrates that the allogeneic peripheral blood-derived NK cells expanded in vitro related to this invention possess a unique expression profile of function-related proteins, implying a significant improvement in NK cell function. CD56 bright As a characteristic of tissue-resident NK cells, CD56 highly expresses CD56. bright NK cells are considered a tissue-resident NK cell subset with dominant tissue infiltration and residence. This invention unexpectedly discovered that the NK cell expansion method of this invention can increase the expression of CD56 in peripheral blood cells that were originally low in CD56. dim NK cells, after being activated by factors in the expansion system, transform into CD56 cells. bright NK cell subsets, CD56 in NK cells after expansion culture bright NK cells reached 97.65% (as shown in Table 1 and...). Figure 1 As shown in D), this demonstrates that the NK cells amplified by the system of the present invention have the ability to enter tissues (including tissues such as the kidneys and skin in autoimmune diseases) with the advantage of tissue-resident NK cells, and have the potential to clear abnormal cells in diseased tissues more deeply.

[0093] Table 1: Proportions of NK Cell Subpopulations

[0094] Example 2: In vitro pharmacological study of NK cells against Alzheimer's disease 1. Study on the uptake and clearance effects of Aβ oligomers (AβO) by NK cells NK cells can directly take up and clear AβO, the core pathological protein of Alzheimer's disease (AD). Therefore, the inventors first explored the uptake and clearance of AβO by NK cells through in vitro experiments. The main research process is as follows: Aβ oligomer (AβO) aggregation: Take 1 mg of β-amyloid (Aβ, MCE, catalog number: HY-P1363A), centrifuge at 12000×g, 4℃ for 5 minutes, place on ice, add 222 μL of hexafluoroisopropanol (HFIP, Sigma, catalog number: 18127-10mL), seal and vortex to mix, let stand at room temperature for 60 minutes until clear, to obtain a 1 mM Aβ-HFIP solution. Aliquot 55 μL of the above Aβ-HFIP solution into four 1.5 mL centrifuge tubes, evaporate the HFIP at room temperature to obtain a colorless and transparent peptide membrane, and store at -20℃. Before the experiment, one vial of Aβ peptide was taken, and 11 μL of DMSO (Sigma, catalog number: D2650) was added. The solution was sonicated in a water bath for 10 minutes to obtain a 5 mM Aβ-DMSO solution. Then, 539 μL of DMEM medium (HyClone, catalog number: SH30243.01) was added to obtain a 100 μM Aβ solution. To obtain the Aβ oligomer, the final Aβ solution was aged at 4°C for 24 hours to obtain a 100 μM AβO solution. 20 μL of the AβO solution was taken and 5 μL of 5× loading buffer was added. The solution was boiled in a metal bath at 100°C for 5 minutes. 20 μL of the sample was then loaded into the wells of an SDS-PAGE gel (FastPAGE). TM Protein precast gel (Qingke, catalog number: TSP024-15) was run at 120V for 1-2 hours before transfer. Ponceau S (Solebo, catalog number: P0012) staining was used to check the protein transfer status. The membrane was blocked with 5% skim milk (Solebo, catalog number: D8340-100g) at room temperature for 1 hour, followed by washing three times with TBST (Solebo, catalog number: T1086) for 5 minutes each time. The membrane was incubated overnight at 4℃ with primary antibody (Anti-β-Amyloid (1-16), Biolegend, catalog number: 803014), followed by washing three times with TBST for 5 minutes each time. Secondary antibody (HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L), Proteintech, catalog number: SA00001-1) was incubated at room temperature for 1 hour, washed three times with TBST for 5 minutes each time, and then developed and exposed (ECL chemiluminescent substrate, Tanon, catalog number: 180-501).

[0095] The results are as follows Figure 2 As shown, the prepared AβO solution was incubated at 4°C for 24 hours and 1 week. WB detection results showed that the prepared AβO solution was a mixture of oligomers, with the majority of components between 40 kDa and 180 kDa.

[0096] Detection of AβO uptake and clearance by NK cells: To investigate the ability of NK cells to uptake and clear AβO, NK cells were treated with different concentrations of AβO in vitro, and the uptake of AβO by NK cells was detected after 1 hour. Then, NK cells were co-incubated with 20 µM AβO for 1 hour, washed three times with PBS to remove residual AβO from the culture medium, and then further incubated for 48 hours. NK cells were collected at each time point to observe the clearance effect of AβO protein by the cells.

[0097] Transfer the NK cell suspension to a 15 mL centrifuge tube and centrifuge at 400 × g at room temperature for 8 minutes. After centrifugation, discard the supernatant and resuspend the cells in 5 mL of 1× PBS, then centrifuge as before. Repeat the washing process once. Resuspend the cells in 2 mL of culture medium and count 20 μL of the cell suspension. Adjust the NK cell concentration to 1 × 10⁻⁶ cells / mL. 7 per mL.

[0098] AβO uptake: (1) Seed 1 mL of NK cells into a 6-well plate, and add 1, 5, 10, and 20 μM AβO to co-incubate the NK cells. After 1 hour of incubation, collect the cells, centrifuge at 300×g to remove the supernatant, and wash 3 times with PBS. (2) Seed 1 mL of NK cells into a 6-well plate, and add 20 μM AβO to co-incubate the NK cells. Collect the cells at 1, 3, 6, 24, and 48 hours after incubation, centrifuge at 300×g to remove the supernatant, and wash 3 times with PBS.

[0099] AβO clearance: 1 mL of NK cells were seeded into 6-well plates, and 20 μM of AβO was added and co-incubated with the NK cells. After 1 hour of co-incubation, the supernatant was removed, and the cells were washed three times with PBS. Cells were then collected at 0, 1, 6, 24, and 48 hours to detect the clearance effect of intracellular AβO.

[0100] Western blot (WB) assay: Add 40 μL of lysis buffer to the collected cells and lyse on ice for 45 minutes. Protein concentration was detected using the Bradford (Vazyme, E211-01-AB) method. Add 5× loading buffer and boil in a metal bath at 100°C for 5 minutes. Load equal volumes of protein sample into SDS-PAGE gel wells and run at 120V for 1-2 hours. Add 10 mL of equilibration buffer (eBlot L1 Transfer Concentration Kit, GenScript, L00724C) to the box, place an NC membrane (GenScript, L00732C), equilibrate for 1 minute, and then transfer the membrane. Check the protein transfer status with Ponceau S staining. Block the membrane with 5% skim milk at room temperature for 1 hour, then wash the membrane three times with TBST for 5 minutes each time. Incubate with primary antibody (Anti-β-Amyloid (1-16), Biolegend, 803014) at 4°C overnight, then wash three times with TBST for 5 minutes each time. The secondary antibody (HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L), Proteintech, catalog number: SA00001-1) was incubated at room temperature for 1 hour, washed three times with TBST for 5 minutes each time, and then developed and exposed.

[0101] Results of NK cell AβO uptake as follows: Figure 3 As shown, NK cells not incubated with AβO do not express AβO. With increasing AβO dosage and incubation time, a distinct intracellular AβO expression band was detected in NK cells, indicating that NK cells have the ability to take up abnormally aggregated Aβ protein. The amount of AβO taken up increases in a concentration-dependent manner, with the highest AβO uptake observed at 20 μM. Subsequently, NK cells were treated with 20 μM, and AβO uptake was detected at different time points. The results showed that the AβO uptake by NK cells was time-dependent.

[0102] The clearance effect of NK cells on AβO is as follows Figure 4 As shown, NK cells were co-incubated with 20 µM AβO for 1 hour, washed three times with PBS to remove residual AβO from the culture medium, and then the NK cells were cultured for another 48 hours. The AβO content of NK cells at each time point was detected. The results showed that the amount of AβO in NK cells gradually decreased over time, indicating that NK cells have a certain ability to clear AβO protein.

[0103] 2. Study on the clearance effect of NK cells on senescent microglia (HMC3 cells) In the aging brain and the brain of Alzheimer's disease, in addition to the accumulation of abnormal proteins, a type of dysfunctional senescent cell appears. These cells no longer divide themselves, but secrete a series of harmful inflammatory factors, damaging the surrounding tissue environment. This phenomenon is known as the senescence-associated secretory phenotype (SASP). One of the core capabilities of NK cells is to recognize and eliminate these senescent cells. Senescent cells express specific ligands (such as MICA / B, ULBP, etc.) on their surface. These molecules act as "alarm signals" for NK cell surface activating receptors (such as NKG2D). When NK cells recognize these signals through their receptors, they are activated and target these senescent cells. The activated NK cells then initiate a killing program to eliminate the senescent cells. This leads to a series of positive chain reactions: First, it directly reduces neuroinflammation by decreasing the large number of pro-inflammatory factors produced by SASP at the source, directly improving the inflammatory microenvironment of the brain; second, it promotes tissue repair, creating space for the repair and functional recovery of surrounding healthy cells after clearing senescent cells; third, it indirectly reduces abnormal proteins, as some senescent cells (such as senescent microglia) may lose the ability to effectively clear Aβ and tau proteins. Clearing them helps restore the brain's cleansing function. Therefore, the inventors explored the clearing effect of NK cells on senescent microglia (HMC3 cells) through in vitro experiments. The main research process is as follows: Cell line: HMC3 cells (microglia, procell, catalog number: CL-0620) Inducing HMC3 Cell Senescence: Etoposide (MCE, catalog number: HY-13629) is a common chemotherapy drug for tumors, which exerts its anti-cancer effect by inhibiting DNA repair and protein synthesis in tumor cells. It has been reported that high doses of etoposide can induce tumor cell apoptosis, while low doses can induce tumor cell senescence. During cell senescence, the activity level of senescence-associated β-galactosidase (SA-β-gal) is upregulated. Using X-gal as a substrate, it hydrolyzes under the catalysis of senescence-specific β-galactosidase to generate a deep blue product, which can be visually detected under an optical microscope to indicate the senescence status of cells. HMC3 cells were co-incubated with different concentrations of etoposide for 24 hours, the supernatant was removed, and the cells were washed three times with PBS. Afterwards, the cells were cultured in fresh medium for four days, and β-gal staining (Cell Senescence β-gal Staining Kit, Beyotime, catalog number: C0602) was performed. The senescence status of the cells was observed under a regular optical microscope. HMC3 cells were digested with trypsin for 5 minutes, and the digestion was stopped by adding culture medium. The cells were collected in 15 mL centrifuge tubes and centrifuged at 400×g at room temperature for 8 minutes. After centrifugation, the supernatant was discarded, and the cells were resuspended in 5 mL of 1× PBS and centrifuged again. The cells were then resuspended in 2 mL of culture medium, and 20 μL of the cell suspension was used for cell counting. The HMC3 cell concentration was adjusted to 3×10⁻⁶ cells / mL. 5 HMC3 cells were seeded at a density of 1 mL / mL into 6-well plates. 0, 5, 10, and 25 µM etoposide were added to each well, and the cells were incubated for 24 hours. The supernatant was removed, and the cells were washed three times with PBS. Fresh medium was then added, and the cells were cultured for another four days. The cell culture medium was aspirated, and the cells were washed once with PBS. 1 mL of β-gal staining fixative was added, and the cells were fixed at room temperature for 15 minutes. The cells were washed three times with PBS for 3 minutes each time. The PBS was aspirated, and 1 mL of staining working solution was added to each well. The cells were incubated overnight at 37°C. The 6-well plates could be sealed with parafilm or plastic wrap to prevent evaporation. Note: Incubation at 37°C cannot be performed in a CO2 incubator. The cells were observed under a regular optical microscope the following day.

[0104] like Figure 5 The results showed that 5 µM etoposide induced an increase in the number of blue-stained HMC3 cells, but with increasing concentration, the number of apoptotic cells increased, while the number of senescent cells induced decreased. Therefore, a 5 µM dose of etoposide was chosen to induce HMC3 cell senescence.

[0105] Western blotting was used to detect the expression level of senescence-associated secretory phenotype (SASP) in induced HMC3 senescent cells. Western blotting was used to detect the expression of SASP-related factors IL-6, MCP-1, and PAI-1 in HMC3 and HMC3-ETO cells. Collected cells were lysed on ice for 45 minutes with 40 μL of lysis buffer, and protein concentration was determined using the Bradford method. Cells were then boiled in a metal bath at 100°C for 5 minutes with 5× loading buffer. Equal volumes of protein samples were loaded into SDS-PAGE gel wells and run at 120V for 1-2 hours before transfer to a membrane. The transfer status was checked by Ponceau S staining. The membrane was blocked with 5% skim milk at room temperature for 1 hour, followed by washing three times with TBST for 5 minutes each time. Primary antibodies (IL-6 (D3K2N) Rabbit Monoclonal Antibody, CST, catalog number: 12153T; PAI-1 (D9C4) Rabbit Monoclonal Antibody, CST, catalog number: 11907T; MCP-1 Antibody (Carboxy-terminal Antigen), CST, catalog number: 39091S) were incubated overnight at 4°C, followed by washing three times with TBST for 5 minutes each time. Secondary antibodies (HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L), Proteintech, catalog number: SA00001-2) were incubated at room temperature for 1 hour, followed by washing three times with TBST for 5 minutes each time, and then developed and exposed.

[0106] The results are as follows Figure 6 , 7 As shown in Figures 8, compared with HMC3 cells, the expression levels of IL-6, MCP-1, and PAI-1 in etoposide-induced senescent HMC3 cells were significantly increased. Furthermore, the expression levels of p16 and NKG2D ligands in HMC3 and PLC cells after senescence induction were simultaneously detected. The results showed that the senescence phenotype of HMC3 and PLC cells was consistent, with significantly upregulated p16 mRNA levels and significantly increased expression levels of NKG2D ligands MICA / B (BioLegend, catalog number: 320908), ULBP1 (R&D, catalog number: FAB1380A), and ULBP2 / 5 / 6 (R&D, catalog number: FAB1298N). These results indicate that the HMC3 senescence model was successfully established.

[0107] In vitro killing assay: The in vitro killing ability of NK cells against senescent cells was evaluated using HMC3 and HMC3-ETO cells as target cells. NK cells and HMC3-ETO cells were co-incubated at effector-to-target ratios of 1:1 and 3:1, respectively, and the target cell mortality rate was measured after 4 hours. E-plate16 plates (Agilent, catalog number: 300600890) were pre-illuminated with UV light in a clean bench for at least 20 minutes, and then 50 μL of HMC3 complete culture medium was added to each well along the sidewall. The E-plate16 plates were placed on an RTCA instrument, and the system automatically scanned them. The cell index on the ordinate was observed; wells with a value not exceeding 0.2 were considered usable. Remove E-Plate 16 and add 100 μL of well-mixed cell suspension along the sidewall to each well. For the blank control wells containing only effector cells, add 100 μL of HMC3 complete culture medium (note that after every four wells, vortex the cell suspension in the centrifuge tube to mix thoroughly, avoiding air bubbles, and ensure the pipette tip does not touch the bottom of the plate). Cap the E-Plate 16 and place it in a clean bench at room temperature for 20 minutes to allow cells to settle to the bottom. Place the E-Plate 16 on the RTCA Station in the incubator, set the total monitoring time to 24 hours, and the data collection interval to 15 minutes → click “apply” → click “continue” to perform real-time dynamic monitoring of cell proliferation. After approximately 4 hours of plating, add NK cells. Click "pause" to pause monitoring. Remove the E-Plate 16 and place it in a clean bench. Mix the NK cell suspension in the EP tube by pipetting. Open the cap and add different concentrations of NK cell suspension along the sidewall of each well. Perform three replicates for each effector-to-target ratio. For the blank control wells containing only target cells, add 100 μL of HMC3 medium containing 1000 IU / mL IL-2. Place the plate back on the RTCA instrument and continue monitoring. After the system stops monitoring, click "export" to export the raw data in Excel format. Select the values ​​corresponding to each well 4 hours after adding NK cells. The specific killing efficiency is calculated using the following formula: Specific killing efficiency = [(average cell index of control wells - average cell index of experimental wells) / average cell index of target cells in control wells] × 100%.

[0108] The results are as follows Figure 9 As shown, NK cells have a strong killing efficiency against HMC3-ETO cells in vitro, and exhibit a dose-response effect. The killing efficiency against HMC3-ETO cells gradually increases with the increase of the proportion of NK cells.

[0109] Example 3: Intravenous infusion of NK cells to treat Alzheimer's disease (AD) model mice with FAD 4T pharmacological studies The experiment used 5-week-old AD model mice (FAD) 4T NK cell therapy was administered via two routes: a single intravenous infusion and four doses. The relative levels of Aβ and GFAP in the cortical and hippocampal regions of mice were measured at one week, one month, and two months post-treatment to explore the efficacy of NK cells in Aβ clearance and neuroinflammatory regulation under different treatment regimens. The main research process is as follows: Selection of laboratory animals: FAD 4T mice Brand: Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Strain number: T053302 Full strain name: C57BL / 6JGpt-Tg(Thy-APP / Thy-PSEN1)5 / Gpt Official gene names: APP, PSEN1 Full gene name: amyloid beta precursor protein, presenilin 1 Gene aliases: AAA, ABETA, ABPP, AD1, APPI, CTFgamma, CVAP, PN-II, PN2, alpha-sAPP, preA4 NCBI: 351 5663 FAD 4T The mice were constructed by transferring the human APP gene carrying Swedish and Indiana mutations and the human PSEN1 gene carrying M146L and L286V mutations into the mouse genome. Compared to other commonly used transgenic AD mouse models, FAD... 4T Aβ deposition can be detected in the cortex and hippocampus of mice at 1.5 months of age, and the deposition gradually increases with age; astrocyte and microglia activation is observed at 2.5 months of age; and spatial learning and cognitive impairment is observed at 8 months of age. These mice can simulate the pathological process of Alzheimer's disease and can serve as a high-quality animal model for preclinical research.

[0110] Intravenous injection: Irradiate the clean bench with UV light for at least 20 minutes before use. Turn on the clean bench and wait for it to stabilize before proceeding with the procedure; thoroughly mix the NK cells with a 1mL pipette and then draw 300μL of the cell suspension using a 1mL insulin syringe; disinfect the injection site on the mouse tail with a 75% alcohol swab, and administer the injection via the tail vein with the syringe held in your right hand.

[0111] Dosing regimen: 5-week-old FAD 4T Female mice, NK cell intravenous administration: 3 × 10 7One NK cell per mouse, single dose / four-times dose (once a week). Adjunctive administration: Mice are intraperitoneally injected with 50,000 IU / rhIL-2 every 2-3 days, or 125 μL rhIL-2 per mouse.

[0112] Sampling Method: The laminar flow hood should be irradiated with UV light for at least 20 minutes before use. Turn on the laminar flow hood and allow it to stabilize before proceeding with the procedure. After euthanizing the mice, dislocate their cervical spine and sever the connection between the skull and cervical vertebrae. Insert the vertebral foramen from the spinal cord end and cut along the midline of the skull, separating the skull until the entire brain is exposed. Use ophthalmic scissors to sever the cranial nerves at the base of the skull, remove the entire brain, and place it in pre-cooled PBS. Cut the brain in half along the sagittal suture, fix the hemisphere in paraformaldehyde fixative for 24 hours, then embed and section. IHC is used to detect the relative levels of Aβ and GFAP.

[0113] Immunohistochemistry: The experimental reagents are shown in Table 2. Table 2:

[0114] The experimental apparatus is shown in Table 3. Table 3:

[0115] Experimental steps: 1) Repair: Use antigen repair solution ER2 to repair tissue for 10 minutes.

[0116] 2) Blocking: Hydrogen peroxide for 10 minutes, followed by blocking with Yuanxi blocking solution for 10 minutes.

[0117] 3) Aβ primary antibody incubation: Dilute Aβ antibody with Yuanxi antibody dilution buffer / blocking buffer at a concentration of 1 μg / mL and incubate at room temperature for 60 minutes. GFAP primary antibody incubation: Dilute GFAP antibody with Yuanxi antibody dilution buffer / blocking buffer at a concentration of 1:250 and incubate at room temperature for 60 minutes.

[0118] 4) Secondary antibody incubation: Incubate with secondary antibody (Leica goat anti-rabbit poly-HRP) from Leica DS9800 staining system at room temperature for 15 minutes.

[0119] 5) After rinsing once with DAB working solution, add DAB working solution again for 5 minutes to develop the color.

[0120] 6) Stain with hematoxylin working solution for 10 minutes.

[0121] 7) Gradient ethanol dehydration, xylene clearing, neutral resin mounting.

[0122] 8) Wash with TBST 3-5 times after each step between steps (2) and (6).

[0123] The settings for the fully automated immunohistochemistry staining machine are shown in Table 4. Table 4:

[0124] Data acquisition and storage: Large-area tissue analysis was performed using Indica Labs HALO 3.6. 1) Selection of analysis scope: The tissue is automatically selected using the magic wand function, and the hippocampus and cortical areas are selected for differentiation.

[0125] 2) Analytical parameter configuration: Under the Analysis tab, load the area analysis and bright field cell analysis modules, set the working magnification, select and define DAB staining for quantification, specify the target phenotype, and optimize the following detection parameters: nucleus, cell membrane, and cytoplasm; 3) Analysis execution and result generation: Save analysis settings, start image processing, and generate a quantitative analysis report.

[0126] The results are as follows Figure 10 As shown, a single intravenous injection of 3×10 7 Following NK cell infusion, the number of Aβ-positive cells in both the cortex and hippocampus was significantly lower than that in the PBS control group. One week after NK cell infusion, the Aβ clearance rate in the cortex was 51.01%, and in the hippocampus it was 38.34%, indicating that NK cell infusion can significantly clear Aβ deposits in both the cerebral cortex and hippocampus simultaneously within one week. Increasing the number of intravenous infusions increased the Aβ clearance rate in the hippocampus from 38.34% after a single infusion to 78.16%, demonstrating a clear dose-dependent therapeutic effect of NK cells; increasing the number of infusions can further enhance efficacy. One month after four intravenous infusions (two months after the first infusion), the efficacy was effectively maintained, showing a long-term clearance effect on Aβ in both the cortex and hippocampus. Overall, the infused NK cells showed better Aβ clearance in the hippocampus than in the cortex.

[0127] The results are as follows Figure 11As shown, glial fibrillary acidic protein (GFAP), as a specific marker of reactive astrocyte activation, directly reflects the severity of neuroinflammation. Pathological results showed that GFAP expression trended in line with Aβ. One week after a single intravenous administration, the number of GFAP-positive cells in the NK cell-administered group was significantly lower than that in the PBS group, with remission rates of 62.27% and 38.20% in the cortical and hippocampal regions, respectively, indicating that NK cells can rapidly suppress neuroinflammatory responses. One month and two months after drug administration, the GFAP-positive area in the NK cell-administered group with four intravenous administrations was significantly lower than that in the PBS group, and the remission rate for GFAP was maintained, suggesting that NK cells can maintain drug efficacy and continuously suppress neuroinflammatory responses.

[0128] Example 4: Intracranial injection of NK cells to treat Alzheimer's disease (AD) model mice with FAD 4T pharmacological studies Intracranial injection can deliver immune cells directly to the brain, overcoming the blood-brain barrier and avoiding the blockage of therapeutic cells by the blood-brain barrier as seen with systemic administration. This increases local concentration, reduces the risk of systemic immune activation (such as cytokine storm), and thus enhances the clearance of Aβ plaques. Therefore, the inventors chose intracranial injection as a treatment for 5-week-old AD model mice (FAD). 4T NK cell therapy was administered to mice, and the relative levels of Aβ and GFAP in the cortical and hippocampal regions of the brain were measured one month after drug administration. This was to explore the efficacy of intracranial injection of NK cells in Aβ clearance and neuroinflammatory regulation. The main research process is as follows: Intracranial administration: FAD 4T After a week of acclimatization, mice were anesthetized with an intraperitoneal injection of 0.9% sodium pentobarbital solution (Stoelting Co, IL, Jitai Beijing) and then fixed on a stereotaxic apparatus. The mouse scalp was prepared, disinfected, and the skin was incised and the periosteum dissected. The anterior fontanelle was exposed for localization. Using a 32-gauge Hamilton 10 μL syringe, injections were made 3.16 mm dorsally and 3.75 mm to each side of the anterior fontanelle, with the anterior fontanelle as the origin, to a depth of 3.25 mm. The syringe was then withdrawn 0.5 mm to allow space for the injected drug. 5 μL of the drug was injected into each side at a rate of 0.5 μL / min, with a 5-minute pause after injection. The periosteum was sealed, and the scalp was sutured. The mice were placed in a 37°C warming blanket for postoperative observation and returned to their cages after recovery.

[0129] Dosing regimen: 5-week-old FAD 4T Female mice, single intracranial administration of NK cells: 3 × 10 6 1 NK / mice (1.5 x 10 cm each for the left and right hemispheres) 6(Number of NK cells). Adjunctive administration: Mice were intraperitoneally injected with 50,000 IU / rhIL-2 every 2-3 days, or 125 μL rhIL-2 per mouse.

[0130] The sampling method and immunohistochemical method were exactly the same as in Example 3.

[0131] The results are as follows Figure 12 , Figure 13 As shown, compared with the intravenous administration group (single dose 3×10), 7 Compared to a single intracranial injection of the same dose (3 × 10⁸ NK cells, once a week for a total of four times), a single intracranial injection of the same dose (3 × 10⁸ NK cells) is more effective. 6 One NK cell can achieve or even surpass the effect of four intravenous injections (total dose 1.2 × 10⁻⁶). 8 The therapeutic effects were assessed. One month after administration, significant efficacy was observed in both groups. The cortical Aβ protein deposition and GFAP-positive area in the NK cell therapy group were significantly lower than those in the PBS control group. Further quantitative analysis showed that the Aβ clearance rate in the cortical region of the intracranial administration group reached 51.74%, which was higher than that of the intravenous administration group; while in the hippocampus, the Aβ clearance effects of the two groups were comparable. In addition, intracranial injection of NK cells resulted in a greater degree of relief of GFAP-positive area in both the cortex and hippocampus than intravenous administration. In summary, this embodiment used only 2.5% of the total intravenous dose of NK cells to achieve stronger or comparable Aβ clearance and neuroinflammation relief through a single intracranial administration, highlighting the significant advantage of local administration in improving treatment efficiency.

[0132] Example 5: Functional comparison of NK cells from donors of different ages NK cells, as a core component of the innate immune system, experience a significant decline in function during aging. Multiple studies have shown systematic differences in phenotype, function, and molecular characteristics between NK cells in young and elderly individuals. Regarding basal immune function, aged NK cells exhibit clear functional deficiencies. Compared to functionally healthy NK cells in young individuals, the expression of key activation receptors (such as NKG2D and NKp46) on the surface of aged NK cells is significantly downregulated, directly leading to a weakened ability to recognize and kill target cells. Furthermore, the proliferation and expansion capacity of aged NK cells is severely impaired, exhibiting low expansion efficiency under external stimuli, and the differentiated cells are in a suboptimal state of function, revealing the depletion of intrinsic function in aged NK cells. From the perspective of cell population composition, the aging process triggers a remodeling of NK cell subsets. The NK cell pool in young individuals is dominated by functionally mature CD56 cells. dim Predominantly cell subsets, while older adults exhibit dysfunctional CD56. negAberrant expansion of NK cell subsets. This imbalance in subset ratios is considered a crucial basis for the overall decline in NK cell immune surveillance function. Single-cell transcriptomics studies have shown that NK cells from Alzheimer's patients exhibit unique transcriptomic characteristics at single-cell resolution, with disordered expression of key signaling pathways and effector molecules. This transcriptional reprogramming may directly lead to a decreased ability of aged NK cells to clear pathological products such as β-amyloid, thereby participating in the progression of neurodegenerative diseases.

[0133] Based on the above background, the inventors hypothesized that NK cells derived from young donors are significantly superior in function to those from older donors, and that this advantage may have greater therapeutic potential in the pathological microenvironment of Alzheimer's disease. To verify this hypothesis, the inventors designed a comparative study.

[0134] Test protocol: NK cells were isolated from PBMCs of young, healthy donors and older donors, and their function was assessed at three levels after in vitro expansion: 1. Detect the expansion efficiency of NK cells in two groups. 2. Detection of cytotoxic activity: Comparison of the in vitro killing efficiency of two groups of NK cells against tumor cell lines (such as K562). 3. Cytokine secretion: Assessing the ability to produce key effector molecules such as IFN-γ. The results are as follows Figure 14 As shown, the NK cell proliferation rate of young donors was significantly higher than that of older donors, indicating that the NK cell proliferation capacity of older donors was impaired. Next, the two groups of NK cells were co-incubated with K562 cells (Procell, catalog number: CL-0130) at an effector-to-target ratio of 1:1 for 4 hours. Flow cytometry was used to detect the NK cell killing efficiency. The specific experimental procedure is as follows: K562 cells were collected in 15mL centrifuge tubes and centrifuged at 400×g at room temperature for 8 minutes. After centrifugation, the supernatant was discarded, and the cells were resuspended in 5mL of 1×PBS and centrifuged again. The cells were resuspended in 2mL of 1640 medium, and 20μL of the cell suspension was used for cell counting. The K562 cell concentration was adjusted to 1×10⁻⁶ cells / mL. 6 Cells / mL. Add CFSE (Biolegend, catalog number: 423801) at a 1:1000 ratio, incubate at 37°C for 15 minutes, centrifuge and discard the supernatant, resuspend in the same volume of culture medium, and seed 100 μL of the cell suspension into 96-well plates. Next, prepare the NK cell suspension. Centrifuge the NK cells, discard the supernatant, and adjust the cell concentration to 1×10⁻⁶ cells / mL using 1640 medium containing 1000 IU / mL IL-2. 6100 μL of NK cell suspension was added along the sidewall of each well containing target cells, with three replicates per group for each effector-to-target ratio. After co-incubation at 37°C for 4 hours, the original culture medium and cells were completely aspirated and transferred to 2.0 ml EP tubes. 5-10 minutes before flow cytometry, 5 μL of LDAPI (Biolegend, catalog number: 422801) solution was added to each group, and the killing effect was detected. The results showed that the NK cell killing effect of young donors was more significant compared with that of older donors. (See details...) Figure 15 .

[0135] In addition, the two groups of NK cells and K562 cells were divided into two groups according to a ratio of 1×10. 5 The cells in each well were mixed at an effector-to-target ratio of 1:1, and BFA / Monensin Mixture (250X) (Biolegend, catalog number: CS1002) was added to block the transport of proteins to the Golgi complex. The mixture was incubated in vitro for 4 hours, and the cells were collected, washed once with PBS, and 1 μL of Human TruStain FcX antibody (Biolegend, catalog number: 422302) was added to each tube for blocking. The cells were then incubated at 4°C for 15 minutes. After blocking, 1 μL of CD3 antibody (Biolegend, catalog number: 300430) and CD56 antibody (Biolegend, catalog number: 362550) were added to label cell surface molecules, and the cells were incubated at 4°C for 30 minutes. After washing once with PBS, the cells were fixed and permeabilized using the FIX & PERM kit (Link Biotech, catalog number: GAS006 / 2). Then, IFN-γ antibody (Biolegend, catalog number: 506507) was added to stain for intracellular factors, and the cells were incubated at 4°C for 45 minutes. After washing once with PBS, the expression of IFN-γ in NK cells was detected by flow cytometry. The results are as follows: Figure 16 As shown, the expression level of IFN-γ in NK cells of young donors was higher than that of older donors.

[0136] NK cells from young donors showed significant advantages in cytotoxic activity and cytokine production, while those from older donors exhibited marked functional impairment. These results not only directly confirm the functional superiority of allogeneic young NK cells but also provide solid experimental evidence for the therapeutic potential of immunotherapy using allogeneic young donor NK cells for Alzheimer's disease.

[0137] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0138] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for preparing NK cells, characterized in that, include: NK cells were sorted and purified from peripheral blood mononuclear cells, which were derived from allogeneic donors; The sorted and purified NK cells were cultured in a serum-free medium containing CD3 monoclonal antibody, CD16 antibody and interleukin-2 in order to harvest NK cells. The method described herein does not use feeder cells throughout the entire process.

2. The method according to claim 1, characterized in that, Further includes: On days 3 to 5 after the start of the initial culture, interleukin-21 was added to the culture system for amplification culture. Optionally, the harvesting of NK cells is achieved by continuous culture for 20 to 40 days.

3. The method according to claim 1 or 2, characterized in that, The allogeneic donor is a young and healthy donor; Optionally, the peripheral blood mononuclear cells are obtained from allogeneic donor peripheral blood by density gradient centrifugation; Optionally, the concentration of the CD3 monoclonal antibody is 0.5-5 ng / mL, the concentration of the CD16 antibody is 1-5 μg / mL, and the concentration of the interleukin-2 is 3.5-6.5 ng / mL; Optionally, the sorting and purification is achieved by using immunomagnetic bead sorting or flow cytometry sorting. Optionally, the complete absence of feeder cells is achieved by not adding or co-culturing any heterogeneous feeder cells throughout the entire culture process.

4. The method according to claim 2, characterized in that, The concentration of the added interleukin-21 is 20-100 ng / mL.

5. An NK cell, characterized in that, The NK cells are prepared by the method described in any one of claims 1-4.

6. The NK cell according to claim 5, characterized in that, The NK cells are non-genetically modified cells, and CD56 cells are present in them. bright The proportion of NK cells in the phenotype is greater than 90%, preferably greater than 95%.

7. The NK cell according to claim 5 or 6, characterized in that, The NK cells highly express at least one receptor or functional molecule selected from NKp30, NKp44, CD69, NKG2D, CD25, CXCR3, Ki67, CD107a, Granzyme B, Perforin, IFN-γ, and 4-1BB.

8. Use of the NK cells according to any one of claims 5 to 7 in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases.

9. The use according to claim 8, characterized in that, The neurodegenerative disease is selected from at least one of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, and age-related diseases; preferably Alzheimer's disease; Optionally, the drug is formulated as a preparation for intravenous or intracranial administration.

10. The use according to claim 9, characterized in that, The intracranial injection includes intraventricular injection or brain parenchymal injection.

11. The use according to claim 10, characterized in that, The single effective dose of the intracranial injection is less than 1 / 5 to 1 / 20 of the single effective dose of the intravenous injection, preferably 1 / 10.

12. The use according to claim 8, characterized in that, The drug has at least one of the following functions: (1) Take up Aβ oligomers and clear β-amyloid protein deposits; (2) Clear senescent cells and reduce the expression of senescent cell markers p16 or p21; (3) Reduces neuroinflammation by decreasing the levels of markers GFAP or IBA1; (4) Improve cognitive impairment.

13. A pharmaceutical composition, characterized in that, It comprises NK cells as described in any one of claims 5 to 7, and a pharmaceutically acceptable carrier or excipient; Preferably, the pharmaceutical composition further comprises interleukin-2.

14. A reagent kit, characterized in that, It comprises NK cells according to any one of claims 5 to 7, or the pharmaceutical composition according to claim 13.

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