Use of NK cells in treating vitiligo and psoriasis

Abstract

Provided is use of NK cells in treating vitiligo and psoriasis. Specifically, provided are use of NK cells and a cancer-targeted drug in the preparation of a drug for treating patients with primary liver cancer or hepatocellular carcinoma accompanied by psoriasis, and use of NK cells and rituximab in the preparation of a drug for treating patients with hepatic cyst accompanied by psoriasis.

Description

Application of NK cells in the treatment of vitiligo and psoriasis

[0001] Cross-referencing

[0002] This application claims priority and benefit to Chinese Patent Application No. 202411602234.3, filed on November 11, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the use of NK cells in the treatment of vitiligo and psoriasis. Background Technology

[0004] Natural killer (NK) cells originate from bone marrow lymphoid stem cells and are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs, and lymph nodes. NK cells are a type of lymphocyte that is MHC-free, requires no prior sensitization, and does not require antibody involvement; they can directly and non-specifically kill tumor cells and virus-infected cells. NK cells typically contain large amounts of perforin and granzyme B. When activated NK cells encounter target cells, the perforin and granzyme B released by the NK cells attack the target cells. NK cells can also directly kill target cells through antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells can also secrete cytokines such as IFN-γ, TNF-α, GM-CSF, and IL-3. These cytokines can act directly on target cells or activate other types of immune cells to attack target cells.

[0005] Cell therapy is a hot topic in the medical field, and NK cells, due to their immunological advantages, are a key focus in cell therapy. Developing NK cell-based therapies for various diseases is urgently needed in this field. Summary of the Invention

[0006] In one aspect, this application provides the use of NK cells in the preparation of a medicament for the treatment of vitiligo.

[0007] Secondly, this application provides the use of NK cells in the preparation of medicaments for treating psoriasis in individuals.

[0008] In some implementations, the individual also has primary liver cancer, liver cysts, or hepatocellular carcinoma.

[0009] Thirdly, this application provides the use of NK cells and cancer-targeting drugs in the preparation of a medicament for treating patients with primary liver cancer or hepatocellular carcinoma complicated with psoriasis.

[0010] In some implementations, the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

[0011] Fourthly, this application provides the use of NK cells in the preparation of a medicament for the treatment of patients with primary liver cancer or hepatocellular carcinoma complicated with psoriasis in combination with cancer-targeted drugs.

[0012] In some implementations, the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

[0013] Fifthly, this application provides the use of NK cells and rituximab in the preparation of a medicament for the treatment of patients with hepatic cysts complicated with psoriasis.

[0014] Sixthly, this application provides the use of NK cells in the preparation of a medicament for the treatment of patients with hepatic cysts and psoriasis in combination with rituximab.

[0015] In a seventh aspect, this application provides an agent for treating vitiligo in an individual, wherein the agent comprises NK cells.

[0016] Eighthly, this application provides an formulation for treating psoriasis in an individual, wherein the formulation comprises NK cells.

[0017] In some implementations, the individual also has primary liver cancer, liver cysts, or hepatocellular carcinoma.

[0018] In a ninth aspect, this application provides a composition comprising individual NK cells and a cancer-targeting drug, said composition for treating primary liver cancer or hepatocellular carcinoma with psoriasis.

[0019] In some implementations, the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

[0020] In a tenth aspect, this application provides a formulation comprising NK cells, said formulation for use in combination with cancer-targeting drugs to treat primary liver cancer or hepatocellular carcinoma with psoriasis.

[0021] In some implementations, the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

[0022] In one aspect, this application provides a composition comprising NK cells alone and rituximab, said composition for treating liver cysts with psoriasis.

[0023] In a twelfth aspect, this application provides a formulation comprising NK cells, said formulation for use in combination with rituximab for the treatment of hepatic cysts with psoriasis.

[0024] In a thirteenth aspect, this application provides a method for treating vitiligo in an individual, comprising administering a therapeutically effective amount of NK cells to the individual suffering from vitiligo.

[0025] In a fourteenth aspect, this application provides a method for treating psoriasis in an individual, comprising administering a therapeutically effective amount of NK cells to the individual suffering from psoriasis.

[0026] In some implementations, the individual also has primary liver cancer, liver cysts, or hepatocellular carcinoma.

[0027] In a fifteenth aspect, this application provides a method for treating primary liver cancer or hepatocellular carcinoma with psoriasis, comprising administering a therapeutically effective amount of NK cells and a cancer-targeting drug to a patient suffering from primary liver cancer or hepatocellular carcinoma with psoriasis.

[0028] In some implementations, the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

[0029] In a sixteenth aspect, this application provides a method for treating liver cysts with psoriasis, comprising administering a therapeutically effective amount of NK cells and rituximab to a patient suffering from liver cysts with psoriasis.

[0030] In some embodiments of any one of the first to sixteenth aspects, the NK cells are formulated as an injection solution.

[0031] In some embodiments of any one of the first to sixteenth aspects, said NK cells are live cells. In some embodiments of any one of the first to sixteenth aspects, the NK cells are expanded in vitro.

[0032] In some embodiments of any one of the first to sixteenth aspects, the in vitro expansion of NK cells is carried out in a serum-free culture system using a rocking bioreactor, and the culture process parameters for in vitro expansion include: total aeration of 20-100 mL / min, culture temperature of 36.5-37.5 °C, rocking speed of 8-10 rpm, rocking angle of 4-6°, dissolved oxygen (DO) ≥20%, and pH of 6.8-7.2.

[0033] In some embodiments of any one of the first to sixteenth aspects, the NK cells are obtained from NK cells derived from peripheral blood mononuclear cells (PBMCs).

[0034] In some implementations of any one of the first to sixteenth aspects, more than 3 billion NK cells are infused per cycle, for at least 5 cycles.

[0035] In some embodiments of any one of the first to sixteenth aspects, CD3 in the NK cells - CD56 + NK cells accounted for ≥95.0%.

[0036] In some embodiments of any one of the first to sixteenth aspects, CD3 in the NK cells - CD56 + CD16 + NK cells account for ≥90.0%.

[0037] In some embodiments of any one of the first to sixteenth aspects, CD3 in the NK cells - CD56 + NKG2D + NK cells accounted for ≥95.0%.

[0038] In some embodiments of any one of the first to sixteenth aspects, CD19 in the NK cells + B cells account for ≤2.0%.

[0039] In some embodiments of any one of the first to sixteenth aspects, CD3 in the NK cells + CD56 - The percentage of T cells is ≤3.0%.

[0040] In some embodiments of any one of aspects one through sixteen, NK cells are stored in bags, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the viable cell density in said NK cell suspension is 0.8-1.2 × 10⁻⁶. 8 per mL.

[0041] In some embodiments of any one of aspects one through sixteen, NK cells are preserved in bags, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the total number of viable cells in said NK cell suspension is 2.0-3.0 × 10⁻⁶. 9 NK cells. Attached Figure Description

[0042] Figure 1 illustrates an example of NK cells and cancer-targeted drugs used to treat a patient with primary liver cancer and psoriasis.

[0043] Figure 2 shows an example of NK cells and rituximab used to treat a patient with liver cysts and psoriasis.

[0044] Figure 3 illustrates an example of NK cell therapy and cancer-targeted drugs used to treat a patient with hepatocellular carcinoma and psoriasis.

[0045] Figure 4 shows an example of NK cell therapy for vitiligo. Detailed Implementation

[0046] In one aspect, this application provides the use of NK cells in the preparation of a medicament for the treatment of vitiligo.

[0047] Secondly, this application provides the use of NK cells in the preparation of medicaments for treating psoriasis in individuals.

[0048] In some implementations, the individual also has primary liver cancer, liver cysts, or hepatocellular carcinoma.

[0049] Thirdly, this application provides the use of NK cells and cancer-targeting drugs in the preparation of a medicament for treating patients with primary liver cancer or hepatocellular carcinoma complicated with psoriasis.

[0050] In some implementations, the cancer-targeting drug is an antibody drug.

[0051] In some implementations, the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

[0052] Fourthly, this application provides the use of NK cells in the preparation of a medicament for the treatment of patients with primary liver cancer or hepatocellular carcinoma complicated with psoriasis in combination with cancer-targeted drugs.

[0053] In some implementations, the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

[0054] Fifthly, this application provides the use of NK cells and rituximab in the preparation of a medicament for the treatment of patients with hepatic cysts complicated with psoriasis.

[0055] Sixthly, this application provides the use of NK cells in the preparation of a medicament for the treatment of patients with hepatic cysts and psoriasis in combination with rituximab.

[0056] In a seventh aspect, this application provides an agent for treating vitiligo in an individual, wherein the agent comprises NK cells.

[0057] Eighthly, this application provides an formulation for treating psoriasis in an individual, wherein the formulation comprises NK cells.

[0058] In some implementations, the individual also has primary liver cancer, liver cysts, or hepatocellular carcinoma.

[0059] In a ninth aspect, this application provides a composition comprising individual NK cells and a cancer-targeting drug, said composition for treating primary liver cancer or hepatocellular carcinoma with psoriasis.

[0060] In some implementations, the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

[0061] In a tenth aspect, this application provides a formulation comprising NK cells, said formulation for use in combination with cancer-targeting drugs to treat primary liver cancer or hepatocellular carcinoma with psoriasis.

[0062] In some implementations, the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

[0063] In one aspect, this application provides a composition comprising NK cells alone and rituximab, said composition for treating liver cysts with psoriasis.

[0064] In a twelfth aspect, this application provides a formulation comprising NK cells, said formulation for use in combination with rituximab for the treatment of hepatic cysts with psoriasis.

[0065] In a thirteenth aspect, this application provides a method for treating vitiligo in an individual, comprising administering a therapeutically effective amount of NK cells to the individual suffering from vitiligo.

[0066] In a fourteenth aspect, this application provides a method for treating psoriasis in an individual, comprising administering a therapeutically effective amount of NK cells to the individual suffering from psoriasis.

[0067] In some implementations, the individual also has primary liver cancer, liver cysts, or hepatocellular carcinoma.

[0068] In a fifteenth aspect, this application provides a method for treating primary liver cancer or hepatocellular carcinoma with psoriasis, comprising administering a therapeutically effective amount of NK cells and a cancer-targeting drug to a patient suffering from primary liver cancer or hepatocellular carcinoma with psoriasis.

[0069] In some implementations, the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

[0070] In a sixteenth aspect, this application provides a method for treating liver cysts with psoriasis, comprising administering a therapeutically effective amount of NK cells and rituximab to a patient suffering from liver cysts with psoriasis.

[0071] In some embodiments of any one of the first to sixteenth aspects, the NK cells are formulated as an injection solution.

[0072] In some embodiments of any one of aspects 1 through 16, the NK cells are living cells. The term “therapeutic effective amount” refers to the amount of NK cells, a drug, or a composition comprising the NK cells and / or drugs of this application that effectively produces some desired therapeutic effect, such as treating (or improving) the symptoms of a patient (or individual), to a reasonable benefit / risk ratio suitable for any medical treatment.

[0073] In some implementations, the therapeutically effective amount is sufficient to alleviate or eliminate at least one symptom. Those skilled in the art will recognize that even if symptoms are not completely eradicated but only partially improved, the amount can still be considered therapeutically effective.

[0074] The term "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal reduction in one or more measurable markers of the disease or condition being treated (e.g., psoriasis or vitiligo). Treatment may optionally include alleviating or improving the symptoms of the disease or condition, or slowing the progression of the disease or condition. "Treatment" does not necessarily mean the complete eradication or cure of the disease or condition or its associated symptoms.

[0075] In some embodiments of any one of the first to sixteenth aspects, NK cells are expanded in vitro.

[0076] In some embodiments of any one of the first to sixteenth aspects, the in vitro expansion of NK cells is carried out in a serum-free culture system using a rocking bioreactor, and the culture process parameters for in vitro expansion include: total aeration of 20-100 mL / min, culture temperature of 36.5-37.5 °C, rocking speed of 8-10 rpm, rocking angle of 4-6°, dissolved oxygen (DO) ≥20%, and pH of 6.8-7.2.

[0077] In some embodiments of any one of the first to sixteenth aspects, the total ventilation rate is 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mL / min, or a range between any of the above values.

[0078] In some embodiments of any of the first to sixteenth aspects, the culture temperature is 36.5, 36.6, 36.7, 36.8, 36.9, 37, 37.1, 37.2, 37.3, 37.4 or 37.5 °C, or a range between any of the above values.

[0079] In some embodiments of any of the first to sixteenth aspects, the oscillation speed is 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10 rpm, or a range between any of the above values.

[0080] In some embodiments of any of the first to sixteenth aspects, the swing angle is 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6°, or a range between any of the above values.

[0081] In some embodiments of any one of the first to sixteenth aspects, dissolved oxygen (DO) is ≥20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, or a range between any of the above values.

[0082] In some embodiments of any of the first to sixteenth aspects, the pH is 6.8, 6.9, 7, 7.1 or 7.2, or a range between any of the above values.

[0083] In some embodiments of any one of the first to sixteenth aspects, the NK cells are obtained from NK cells derived from peripheral blood mononuclear cells (PBMCs).

[0084] In some implementations of any one of the first to sixteenth aspects, more than 3 billion NK cells are infused per cycle, for at least 5 cycles.

[0085] In some embodiments of any of the first to sixteenth aspects, the NK cell infusion per cycle is greater than 3 billion, 5 billion, 6 billion, 7 billion, 7.5 billion, 8 billion, 8.5 billion, 9 billion, 9.5 billion, 10 billion, 12 billion, 13 billion, 14 billion, or 15 billion, or any range between the foregoing values.

[0086] In some embodiments of any of the first to sixteenth aspects, NK cells are infused per cycle in amounts of 3 billion, 5 billion, 6 billion, 7 billion, 7.5 billion, 8 billion, 8.5 billion, 9 billion, 9.5 billion, 10 billion, 12 billion, 13 billion, 14 billion, or 15 billion, or any range between the foregoing values.

[0087] In some embodiments of any one of the first to sixteenth aspects, NK cells are infused for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 cycles (or a range between any of the above values).

[0088] In some embodiments of any one of the first to sixteenth aspects, NK cells are infused for at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 cycles (or a range between any of the above values).

[0089] In some embodiments of any one of the first to sixteenth aspects, CD3 in the NK cells - CD56 + NK cells accounted for ≥95.0%.

[0090] In some embodiments of any one of the first to sixteenth aspects, CD3 in the NK cells - CD56 + The percentage of NK cells is ≥95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, or 99.5% (or any range between the above values).

[0091] In some embodiments of any one of the first to sixteenth aspects, CD3 in the NK cells - CD56 + CD16 + NK cells account for ≥90.0%.

[0092] In some embodiments of any one of the first to sixteenth aspects, CD3 in the NK cells - CD56 + CD16 +The percentage of NK cells is ≥90.0%, 90.5%, 91.0%, 91.5%, 92.0%, 92.5%, 93.0%, 93.5%, 94.0%, 94.5%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, or 99.5% (or any range between the above values).

[0093] In some embodiments of any one of the first to sixteenth aspects, CD3 in the NK cells - CD56 + NKG2D + NK cells accounted for ≥95.0%.

[0094] In some embodiments of any one of the first to sixteenth aspects, CD3 in the NK cells - CD56 + NKG2D + The percentage of NK cells is ≥95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, or 99.5% (or any range between the above values).

[0095] In some embodiments of any one of the first to sixteenth aspects, CD19 in the NK cells + B cells account for ≤2.0%.

[0096] In some embodiments of any one of the first to sixteenth aspects, CD19 in the NK cells + The percentage of B cells is ≤2.0%, 1.5%, 1.0%, or 0.5% (or any value between these values).

[0097] In some embodiments of any one of the first to sixteenth aspects, CD3 in the NK cells + CD56 - The percentage of T cells is ≤3.0%.

[0098] In some embodiments of any one of the first to sixteenth aspects, CD3 in the NK cells + CD56 - The percentage of T cells is ≤3.0%, 2.5%, 2.0%, 1.5%, 1.0%, or 0.5% (or any range between these values).

[0099] In some embodiments of any one of aspects one through sixteen, NK cells are stored in bags, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the viable cell density in said NK cell suspension is 0.8-1.2 × 10⁻⁶. 8 per mL.

[0100] In some embodiments of any one of the first to sixteenth aspects, NK cells are preserved in bags containing 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, or 50 mL (or any range of the above values) of NK cell suspension.

[0101] In some embodiments of any of the first to sixteenth aspects, the NK cells are stored in bags, each bag containing a suspension of NK cells with a viable cell density of 0.8, 0.9, 1.0, 1.1, or 1.2 × 10⁻⁶ cells / day. 8 per mL (or any value between the above values).

[0102] In some embodiments of any one of the first to sixteenth aspects, the NK cells are stored in bags, each bag containing a suspension of NK cells with a total live cell count of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 × 10⁻⁶ cells. 9 NK cells (or any number between the above values).

[0103] Example

[0104] This application will be described in more detail by way of specific examples. The following embodiments are provided for illustrative purposes only and are not intended to limit this application in any way. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same results.

[0105] Example 1: Preparation of NK cell injection solution

[0106] The preparation of NK cell injection solution is divided into three parts: preparation of peripheral blood mononuclear cells (PBMCs), preparation of NK cell injection solution stock solution, and preparation of formulation.

[0107] Preparation of peripheral blood mononuclear cells (PBMCs)

[0108] PBMC preparation includes hPB Leukopak pretreatment and CTS. TM Rotea TM The process involves five main steps: processing, PBMC dispensing, labeling, and PBMC freezing.

[0109] hPB Leukopak preprocessing

[0110] Dosage: 0.7-5.0 mL / 1×10 9 Cell addition amount: PPC3 dilution was added to hPB Leukopak (i.e., peripheral blood mononuclear cells) (Source: RosetteSep) TM Human CD3 Depletion Cocktail; dosage: 1.5-5.0 mL / per 1×10 9 Cells) or PPC3S dilution buffer (conjugated IgG4 antibody to erythrocytes and T cells, added at a rate of 1.5-5.0 mL / 1×10⁻⁶ cells). 9 Cells). Incubate at room temperature for 10-40 minutes. Add erythrocyte sedimentation diluent (3% m / v hydroxyethyl starch) to hPB Leukopak at a final concentration of 1.2%-2.0%. Hang vertically and incubate at room temperature for (120±45) min. After the erythrocytes have settled and separated, use blood plasma separators to press the upper cell suspension into the sample bag.

[0111] CTS TM Rotea™ processing

[0112] The cell suspension was diluted with an equal volume of sodium chloride injection. The cell suspension was circulated within the sample bag to form a stable fluidized bed. The cell suspension was then continuously loaded into the sample bag to concentrate the PBMC cells within the fluidized bed. After washing with sodium chloride injection and replacement with cryopreservation solution, the PBMC cells were collected into a harvest bag. The PBMC cells in the harvest bag were counted. Based on the count results, the viable cell density of the PBMC cell suspension was adjusted to (2.7-6.0) × 10⁻⁶. 7 Cells / mL. Cell viability should be ≥80.0%.

[0113] PBMC separation

[0114] According to 6.8×10 8 Calculate the volume of each bag to be dispensed using live cells / bag. Aseptically connect the harvest bag and the cryopreservation bag. Mix the PBMC cells to be dispensed on a shaker and then dispense them into the cryopreservation bags using a peristaltic pump. After dispensing, samples are retained for testing (such as sterility testing, mycoplasma testing, etc.).

[0115] Label

[0116] Affix PBMC labels to the bodies of the pre-packaged cryopreservation bags.

[0117] PBMC cryopreservation

[0118] Place the blood bag box horizontally into the cryopreservation chamber of the programmed cooling system and cool to -70 to -90°C. After cooling, transfer the frozen PBMC cells to a gas phase liquid nitrogen tank and store at ≤-150°C.

[0119] Preparation of NK cell injection stock solution

[0120] The preparation of NK cell injection stock solution includes three major steps: initial NK cell culture, NK cell expansion culture, NK cell concentration and washing, and stock solution preparation.

[0121] Initial culture of NK cells

[0122] PBMC recovery

[0123] Three T225cm blankets 2 Add 1 mL of coating factor mixture (CD33-CLL1 (0-50 μg / mL), CD52 (0-50 μg / mL), CD38 (0-50 μg / mL), CD3 antibody (0-50 μg / mL)) to each culture flask, then add 29 mL of sodium chloride injection solution to each flask and mix well. Place the culture flask at T225 cm⁻¹. 2 The culture flasks are placed in a biosafety cabinet and incubated at room temperature for 1-4 hours or in a carbon dioxide incubator at 37.0±0.5℃ for 1-4 hours.

[0124] Take one bag of frozen PBMC cells from the liquid nitrogen tank and place it in an anhydrous resuscitator (resuscitation temperature 37±0.5℃) for rapid thawing. Thawing time is 2-5 minutes. Transfer the thawed PBMC cells to a 50mL centrifuge tube containing 15mL of compound electrolyte injection or sodium chloride injection. Add more compound electrolyte injection or sodium chloride injection to a final volume of 45mL and mix the cells thoroughly.

[0125] Centrifuge at 600g at room temperature for 8±2 min. Discard the supernatant, add 45 mL of NK cell activation medium (1640 medium, heparin sodium (0-10 IU / mL), platelet lysis buffer (0-10% v / v), human serum albumin (1.0%-5.0% v / v), IL-2 (0-2000 IU / mL), IL-15 (0-50 ng / mL), etc.) to resuspend, mix well and count. Based on the cell count results, if the total number of viable cells is ≥3.6 × 10⁻⁶, the cell count is considered complete. 8 If the total number of viable cells is ≥80.0%, then inoculation and culture should proceed. If the total number of viable cells is <3.6×10⁶, then inoculation and culture should be performed. 8 If the number of cells or the cell viability is less than 80.0%, the PBMC cell resuscitation will be terminated.

[0126] Remove T225cm 2 Based on the cell count results, the PBMC cell suspension was evenly transferred to three coated T225cm culture flasks. 2 In each culture flask (coating agent composition: CD33-CLL1 (0-50 μg / mL), CD52 (0-50 μg / mL), CD38 (0-50 μg / mL), CD3 antibody (0-50 μg / mL), each T225cm2 The total inoculation volume of the culture flask is 120 mL, and the inoculation density of viable cells is ≥1.00 × 10⁻⁶. 6 The volume is 100 ml / mL, and the three bottles are labeled A, B, and C respectively. After labeling the bottles, place three T225cm... 2 The culture flasks were placed in a static incubator at 37.0±0.5℃ and 5.0±0.5% CO2 for 3 days.

[0127] T225cm 2 Culture flask replenishment culture

[0128] On the third day of cultivation, the above three T225cm 2 Add 60 mL of NK cell activation medium to each culture flask and continue static culture.

[0129] T225cm 2 Culture flask transfer bag culture

[0130] On days 5-7 of culture, three T225cm 2 After thoroughly mixing the cell suspension in the culture flasks and counting the cells, if the average viable cell density of the three flasks is ≥6.7 × 10⁻⁶... 5 If the number of cells / mL is 1, then transfer to 825 cm⁻¹ 2 Cells were cultured in culture bags, and NK cell activation medium was added as replenishment fluid. The replenishment volume was adjusted according to the average viable cell density of the three flasks. After replenishment, the viable cell density was ≥4.00×10⁻⁶. 5 The volume of fluid replenishment should be 900-1800 mL after adding the fluid. If the average viable cell density is below 6.70 × 10⁶ cells / mL... 5 If the cell density is [number] cells / mL, continue culturing until the second day, then take samples for counting to determine whether to transfer the cells to a bag. If transfer is not performed on the second day, take samples again on the third day for counting to determine whether to transfer the cells to a bag. If the average viable cell density on the third day is less than 6.70 × 10⁻⁶, the cell density will be determined. 5 If the count reaches 1 / mL, then terminate the culture. After replenishing the solution, continue the culture in a carbon dioxide incubator at 37.0±0.5℃ and a CO2 concentration of 5.0±0.5% for two days.

[0131] 825cm 2 Cell culture bag replenishment culture 1

[0132] Days 7-10 of cultivation: 825cm 2Mix the cell suspension in the cell culture bag thoroughly and take samples for counting. The replenishing fluid consists of NK cell expansion medium (1640 medium, heparin sodium (0-10 IU / mL), platelet lysis buffer (0-10% v / v), human serum albumin (1.0%-5.0% v / v), IL-2 (0-2000 IU / mL), IL-15 (0-50 ng / mL), etc.). Adjust the replenishing fluid volume according to the viable cell density; after replenishment, the viable cell density should be ≥4.00 × 10⁶ cells / mL. 5 Platelet count / mL, add 5-10 mL of serum substitute (II) (platelet lysis buffer and human serum albumin, volume ratio 1:1) before replenishment. The volume after replenishment is 1800 mL-2700 mL, and continue static incubation. If the culture still does not meet the 825 cm⁻¹ standard on day 10... 2 If the cell culture bag is replenished with the standard solution after one culture, the culture should be terminated.

[0133] 825cm 2 Cell culture bag replenishment culture 2

[0134] On days 9-12, the height will be 825cm. 2 Mix the cell suspension in the cell culture bag thoroughly, take a sample, and count the cells. The total number of viable cells should be ≥2.0 × 10⁻⁶. 9 If the cell viability is ≥80.0%, the sample should be sent for flow cytometry testing. If the above requirements are not met, continue culturing. If the culture volume is less than 2700 mL, add NK cell expansion medium. After adding medium, the viable cell density should be ≥6.00 × 10⁶ cells / mL. 5 The flow cytometry volume should not exceed 2700 mL after replenishment. If the flow cytometry results do not meet the standards for submission, the culture should be terminated.

[0135] The total number of viable cells, cell viability, NK cell purity, and total T cell and B cell impurities before fluid replenishment on days 9-12 were used as intermediate control indicators. If the total number of viable cells and cell viability met the intermediate control standards, samples were sent for flow cytometry analysis. If the total number of viable cells and cell viability did not meet the intermediate control standards, culture continued until day 12 until the intermediate control standards were met, at which point samples were sent for flow cytometry analysis. If the standards were still not met, culture was terminated. If the flow cytometry results met the intermediate control standards, reactor inoculation and culture were performed; if the flow cytometry results did not meet the intermediate control standards, culture was terminated. The specific intermediate control standards are shown in Table 1 below.

[0136] Table 1: Reactor Control Standards (Days 9-12)

[0137] Note 1: *The total number of live cells is calculated based on the live cell density. Total number of live cells = live cell density × volume.

[0138] Note 2: The above procedure is for a 50L reactor (working volume 25L). For the PBMC resuscitation operation in a 100L reactor, the total number of cells inoculated (and the number of culture flasks) should be twice that of the 50L reactor. Other culture and replenishment parameters should be the same. When transferring cells from D5 to D7, transfer one bag for every three culture flasks and transfer two bags of cells to the reactor for culture.

[0139] NK cell expansion culture

[0140] Reactor inoculation and culture

[0141] On days 9-12, the central control results confirmed that the total number of viable cells per bag was ≥2.0×10⁻⁶. 9 Cell viability ≥80.0%, NK cell purity (CD3+) - CD56 + ≥70.0%, total T cell impurities (CD3) + ≤9.0%, B cell impurities (CD19) + If the concentration is ≤5.0%, it can be transferred to a reactor for further amplification and culture.

[0142] Transfer all the cell suspension to the reactor cell culture bag. Adjust the replenishment volume according to the viable cell density. The replenishment volume is the same as NK cell expansion medium, with 25-100 mL of serum substitute (II) (platelet lysis buffer and human serum albumin, volume ratio 1:1). After replenishment, the viable cell density should be ≥4.00 × 10⁶ cells / mL. 5 Cells / mL, glucose concentration ≥1.00g / L. If the glucose concentration is lower than 1.00g / L after rehydration, glucose solution is added to make the glucose concentration of the cell suspension 1.00-2.00g / L.

[0143] The reactor process parameters are controlled as follows:

[0144] a. Culture parameters: total aeration rate of 20-100 mL / min, culture temperature of 37.0±0.5℃, rotation speed of 8-10 rpm, angle of 4-6°, pH: 7.0±0.2, DO: ≥20%.

[0145] b. pH control is related to carbon dioxide and 1 mol / L sodium bicarbonate, while DO control is related to oxygen and nitrogen.

[0146] reactor amplification culture

[0147] The reactor process parameters are controlled as follows:

[0148] Culture parameters: total aeration rate of 20-40 mL / min, culture temperature of 37.0±0.5℃, rotation speed of 8-10 rpm, angle of 4-6°, pH of 7.0±0.2, and DO of ≥20%.

[0149] pH / DO control: pH control is related to carbon dioxide and 1 mol / L sodium bicarbonate, and DO control is related to oxygen and nitrogen.

[0150] Sampling: After adjusting the rotation speed and angle to mix the cell suspension, samples were taken to detect pH value, viable cell density, cell viability, glucose, lactate, ammonia and glutamine. If the absolute value of the detected pH value deviates from the online pH value by ≥0.10, pH calibration was performed, and flow cytometry was performed on days 15-18.

[0151] Fluid resuscitation: Adjust the fluid volume according to the viable cell density. After fluid resuscitation, the viable cell density should be ≥5.00×10⁻⁶. 5 Cells / mL. If no fluid is added for three consecutive days, terminate this batch of culture. After the second fluid addition, add 25-50 mL of serum substitute (II). If no fluid is added and the glucose concentration in the cell suspension is below 1.00 g / L, add glucose solution to bring the glucose concentration in the cell suspension to 1.00-2.00 g / L, until the total volume is 25 L.

[0152] Perfusion: When the culture system reaches the maximum volume of the culture bag and the lactate concentration is ≥1.00 g / L, begin intermittent perfusion culture to replace the fresh NK cell expansion medium (using hollow fiber columns). After the first perfusion, add 25-100 mL of serum substitute (II). After each subsequent perfusion (e.g., the third, fifth, and seventh perfusions), add 25-50 mL of serum substitute (II). Post-perfusion requirements: Theoretical glucose concentration ≥1.00 g / L. If the glucose concentration after perfusion is <1.00 g / L, add glucose solution to bring the theoretical glucose concentration of the cell suspension to 1.00-2.00 g / L.

[0153] Cell viability, NK purity, total T cell impurities, and double-negative cell impurities before fluid replenishment on days 15-18 were used as intermediate control indicators. If the intermediate control standards were met, the next step of expansion culture could be carried out; otherwise, the culture was terminated. The specific intermediate control standards are shown in Table 2 below.

[0154] Table 2: Control Standards for Reactor Amplification Culture (Days 15-18)

[0155] Note: If cell viability and NK purity (CD3+) are measured between days 15 and 18... - CD56 + ), total T cell impurities (CD3) + ) and double-negative cell impurities (CD3) - CD56 - If the control standards in Table 2 are met, then the NK cell concentration washing and stock solution preparation process can be carried out on D18-D23.

[0156] A sample is taken for sterility testing 48 hours before the preparation of the stock solution. The sterility test is used as a mid-control indicator. If the sterility test meets the mid-control standard, the next step of Pre-stock solution preparation can be carried out.

[0157] NK cell concentration, washing, and stock solution preparation

[0158] NK cell suspension centrifugation and washing:

[0159] Concentration: The NK cell suspension obtained above was processed using a fully enclosed automated cell processing system. The temperature was set to 4℃ (temperature control range of 2-8℃). The sample was concentrated by continuous flow at an injection flow rate of 250 mL / min and a centrifugation speed of 2200 rpm.

[0160] Washing: After continuous flow concentration, proceed to the Washing Solution-1 washing step, with 6-8 washing cycles, a single washing volume of 140 mL, a flow rate of 150 mL / min, and a centrifugation speed of 1500 rpm. After the Washing Solution-1 washing step, proceed to the Freeze-Preservation Solution-2 washing step, with 1-2 washing cycles, a single washing volume of 140 mL, a flow rate of 150 mL / min, and a centrifugation speed of 1500 rpm.

[0161] Pre-concentrate preparation:

[0162] Based on the NK cell count results, the resuspension volume of cryopreservation solution-2 was calculated. The resuspension volume of the pre-stock solution was ≤600mL, the flow rate was 150mL / min, the number of cycles was 1, the mixing speed was 800rpm, the mixing acceleration was 200rpm, and the mixing time was 180-300s. After resuspension, the resuspension was transferred to the stock solution bag at a flow rate of 200mL / min to obtain the pre-stock solution.

[0163] Formula for calculating resuspension volume:

[0164] If the calculated resuspension volume is >600mL, resuspend at a volume of 600mL.

[0165] Take approximately 1 mL for cell counting. Use cell viability and viable cell density as intermediate control indicators. If the intermediate control standards are met, proceed to the next step. See Table 3 below for the specific intermediate control standards for the Pre-stock solution.

[0166] Table 3: Pre-concentrate control standards (days 18-23)

[0167] Original solution dilution:

[0168] Based on the pre-stock solution count results and the viable cell density of the stock solution ((0.8-1.2)×10⁻⁶), 8Calculate the original volume and the required volume of cryopreservation solution (volume / mL) to determine the volume of the stock solution and the volume of cryopreservation solution to be added.

[0169] Calculation formula: Volume of stock solution = (Pre - density of viable cells in stock solution × Pre - volume of stock solution) / set density of viable cells in stock solution;

[0170] The volume of cryopreservation solution - 2 = the volume of the original solution - the volume of Pre-original solution.

[0171] Based on the volume calculation of cryopreservation solution-2, the added cryopreservation solution-2 is directly introduced into the centrifuge cup. The injection flow rate of cryopreservation solution-2 is 250 mL / min. After rinsing the centrifuge cup, the cryopreservation solution-2 will be transferred into the original solution bag.

[0172] After mixing the stock solution bag, take a sample and count it. If the count result is (0.8-1.2)×10 8 If the count is [number of cells / mL], then the stock solution is obtained. If the count result exceeds the viable cell density range of the stock solution, cryopreservation solution-2 needs to be added to adjust the viable cell density to (0.8-1.2)×10⁻⁶. 8 The NK cell stock solution is obtained by obtaining cells / mL.

[0173] Formulation preparation

[0174] Filling: Use a closed dispensing system (temperature controlled 2-8℃) to dispense the stock solution. Use a peristaltic pump with a dispensing flow rate of 50-200 mL / min. Dispense 20 mL of the stock solution (viable cell density (0.8-1.2) × 10⁻⁶ cells / min). 8 25 mL (viable cell density (0.8-1.2) × 10⁻⁶ cells / mL) or 25 mL (viable cell density (0.8-1.2) × 10⁻⁶ cells / mL). 8 (cells / mL) or 30 mL (viable cell density (0.8-1.2) × 10⁻⁶) 8 (cells / mL) or 50 mL (viable cell density (0.8-1.2) × 10⁻⁶) 8 (cells / mL), filled into disposable cell cryopreservation bags.

[0175] Sealing: Use a handheld heat sealer to heat seal the opening of the long tubing in the disposable cell cryopreservation bag. After heat sealing, check that the formulation packaging is intact and there is no leakage.

[0176] Labeling: Attach labels and transfer the labeled products to cryopreservation boxes (both cryopreservation boxes and cryopreservation bags need to be labeled).

[0177] Program cooling:

[0178] a. Turn on the programmed cooling device and quickly transfer the labeled sample into the chamber of the programmed cooling device for programmed cooling (temperature drops to -80℃).

[0179] b. See Table 4 for relevant parameters of the program control system.

[0180] Table 4: Relevant Parameters of the Programmable Control Program

[0181] Cryopreservation: After the temperature-controlled process is completed, the NK cell injection solution is transferred to a gas phase liquid nitrogen tank for cryopreservation (storage conditions ≤-150℃).

[0182] The simplified preparation steps of NK cell injection solution are as follows:

[0183] NK cells are derived from peripheral blood and prepared in vitro as PBMCs, which are then stored in liquid nitrogen. The resuscitated PBMCs undergo initial and expansion cultures in vitro to obtain high-quality NK cells. After concentration and washing, the NK cell suspension is adjusted to a viable cell density of (0.8-1.2) × 10⁻⁶ cells / mL. 8 The concentration of NK cells / mL is the NK cell stock solution. Using a closed dispensing system, the NK cell stock solution is filled into disposable cell cryopreservation bags. The bags are then heat-sealed and labeled, placed in a cryopreservation box, and transferred to a programmed cooling system for programmed cooling to -80°C. After programmed cooling, the cells are transferred to a liquid nitrogen tank for storage (≤-150°C), thus obtaining the NK cell injection solution. The final cell purity quality control of the NK cell injection solution is shown in Table 5.

[0184] Table 5: Final Cell Purity Quality Control of NK Cell Injection Solution

[0185] Example 2: Application of NK cells in the treatment of psoriasis ①

[0186] Patient's past medical history

[0187] The patient was a 58-year-old male with psoriasis, primary liver cancer, and hepatitis B-related cirrhosis. He was diagnosed with primary liver cancer in 2022, accompanied by psoriasis.

[0188] Treatment status

[0189] Treatment of psoriasis with NK cell injection prepared in Example 1: Starting in August 2022, NK cell injection was administered (cycle and dosage: 21 days per cycle, administered on days 1 and 3 of each cycle, 4 billion cells / time, total infusion of 8 billion cells per cycle), in combination with bevacizumab injection (trade name: The treatment consisted of 21-day cycles, with administration on day 1 of each cycle at a dose of 15 mg / kg body weight. A total of 14 cycles of NK cell infusion were performed by July 2023.

[0190] Treatment results

[0191] The efficacy of the NK cell injection prepared in Example 1 in treating psoriasis is shown in Figure 1.

[0192] As shown in Figure 1, before NK cell therapy was administered on August 4, 2022, there were obvious psoriatic plaques. After 4 cycles of NK cell therapy, the psoriatic plaques significantly decreased in size. After 7 cycles of NK cell therapy, there were no obvious psoriatic plaques. After 10 cycles of NK cell therapy and during subsequent follow-ups, there was no recurrence of psoriasis.

[0193] Example 3: Application of NK cells in the treatment of psoriasis ②

[0194] Patient's past medical history

[0195] The patient was a 59-year-old male who had suffered from psoriasis for over 30 years. His past medical history included coronary atherosclerosis, pulmonary nodules (considered benign), arrhythmia—complete right bundle branch block, post-thyroidectomy (taking levothyroxine), enlarged and hard prostate, and liver cysts.

[0196] Treatment status

[0197] Treatment of psoriasis with NK cell injection prepared in Example 1: Starting in January 2024, NK cell injection (cycle and dosage: 30 days per cycle, 7.5 billion doses per dose on day 1 of each cycle) was administered. From the third cycle onwards, rituximab injection (trade name: Rituximab, 30 days per cycle, 100 mg dose per dose) was used in combination. A total of 7 cycles of NK cell infusion were performed by July 2024.

[0198] Treatment results

[0199] The efficacy of the NK cell injection prepared in Example 1 in treating psoriasis is shown in Figure 2.

[0200] As shown in Figure 2, before NK cell therapy was administered on December 29, 2023, there were obvious psoriatic plaques. After three cycles of NK cell therapy, the psoriatic plaques significantly decreased in size, and after seven cycles of NK cell therapy, there were no obvious psoriatic plaques.

[0201] Example 4: Application of NK cells in the treatment of psoriasis ③

[0202] Patient's past medical history

[0203] The patient, a 53-year-old male, presented with psoriasis, hepatocellular carcinoma (post-surgery, bilateral lung metastases), and hypertension. He was diagnosed with primary liver cancer in September 2014. He subsequently underwent various treatments, including hepatocellular carcinoma resection and cholecystectomy, transarterial chemoembolization (TACE), raltilimab + sorafenib (1 year), sintilimab + apatinib (10 months), and local radiotherapy, but his condition remained uncontrolled.

[0204] Treatment status

[0205] Treatment of psoriasis with NK cell injection prepared in Example 1: Starting in November 2022, NK cell injection was administered (cycle and dosage: 21 days per cycle, administered on days 1 and 3 of each cycle, 5 billion cells / time, total infusion of 10 billion cells per cycle), in combination with lenvatinib (trade name: Treatment began on day 5 of cycle 1, with a dose of 12 mg once daily. A total of 6 cycles of NK cell infusion were performed by April 2023.

[0206] Treatment results

[0207] The efficacy of the NK cell injection prepared in Example 1 in treating psoriasis is shown in Figure 3.

[0208] As shown in Figure 3, before receiving NK cell therapy on November 23, 2022, the patient had obvious psoriatic plaques. During NK cell therapy, the patient's liver cancer was effectively controlled, and the psoriatic lesions improved. After six cycles of NK cell therapy, there were no longer any obvious psoriatic plaques.

[0209] Example 5: Application of NK cells in the treatment of vitiligo

[0210] Patient's past medical history

[0211] A 17-year-old male patient presented with vitiligo lesions, approximately the size of a bean, on his eyelids in 2015. Subsequent antibiotic treatment controlled the progression of the condition, with minimal spread. In August 2021, sun exposure led to extensive sunburn on his face, resulting in ulceration after scratching. Following scab removal, the skin lost pigmentation and began to spread irregularly. From August 2021 to 2022, he received oral and topical medications (compound carrageenan tincture, dexamethasone sodium phosphate injection, folic acid tablets) as well as ultraviolet phototherapy, but his condition showed no significant improvement.

[0212] Treatment status

[0213] Vitiligo was treated with the NK cell injection prepared in Example 1: NK cell injection was administered in July 2023 (cycle and dosage: 5 billion per cycle, 28 days per cycle, once per cycle). Six cycles of treatment were completed in December 2023.

[0214] No new drugs were added during cell therapy; the existing drug treatment continued: compound calamine tincture, dexamethasone sodium phosphate injection, folic acid tablets, and occasional ultraviolet phototherapy.

[0215] Treatment results

[0216] The efficacy of the NK cell injection prepared in Example 1 in treating vitiligo is shown in Figure 4.

[0217] As shown in Figure 4, the vitiligo on the patient's face showed a fading trend during treatment, with the area of ​​the vitiligo significantly reduced and faded compared to before treatment. During follow-up, the vitiligo on the face continued to fade, and the area of ​​the vitiligo further decreased.

Claims

1. The use of NK cells in the preparation of drugs for the treatment of vitiligo.

2. Use of NK cells in the preparation of drugs for the treatment of psoriasis in individuals.

3. The use as described in claim 2, wherein the individual also suffers from primary liver cancer, liver cysts, or hepatocellular carcinoma.

4. Use of NK cells and cancer-targeting drugs in the preparation of medicines for the treatment of patients with primary liver cancer or hepatocellular carcinoma complicated with psoriasis.

5. The use as described in claim 4, wherein the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

6. Use of NK cells in the preparation of medicaments for the treatment of patients with primary liver cancer or hepatocellular carcinoma complicated with psoriasis in combination with cancer-targeted drugs.

7. The use as described in claim 6, wherein the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

8. Use of NK cells and rituximab in the preparation of a medicament for the treatment of patients with hepatic cysts and psoriasis.

9. Use of NK cells in the preparation of a drug for the treatment of patients with hepatic cysts and psoriasis in combination with rituximab.

10. A formulation for treating vitiligo in an individual, wherein the formulation comprises NK cells.

11. An formulation for treating psoriasis in an individual, wherein the formulation comprises NK cells.

12. The formulation for the use of claim 11, wherein the individual also suffers from primary liver cancer, liver cysts, or hepatocellular carcinoma.

13. A composition comprising NK cells and a cancer-targeting drug, said composition for treating primary liver cancer or hepatocellular carcinoma with psoriasis.

14. The composition for the use of claim 13, wherein the cancer-targeting drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

15. A formulation comprising NK cells, said formulation being used in combination with a cancer-targeting drug for the treatment of primary liver cancer or hepatocellular carcinoma with psoriasis.

16. The formulation for the use of claim 15, wherein the cancer-targeting drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

17. A composition comprising NK cells alone and rituximab, said composition for the treatment of hepatic cysts with psoriasis.

18. A formulation comprising NK cells, said formulation being used in combination with rituximab for the treatment of hepatic cysts with psoriasis.

19. A method for treating vitiligo in an individual, comprising administering a therapeutically effective amount of NK cells to the individual suffering from vitiligo.

20. A method for treating psoriasis in an individual, comprising administering a therapeutically effective amount of NK cells to an individual suffering from psoriasis.

21. The method of claim 20, wherein the individual also suffers from primary liver cancer, liver cysts, or hepatocellular carcinoma.

22. A method for treating primary liver cancer or hepatocellular carcinoma with psoriasis, comprising administering therapeutically effective amounts of NK cells and cancer-targeting drugs to a patient with primary liver cancer or hepatocellular carcinoma with psoriasis.

23. The method of claim 22, wherein the cancer-targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib, or sorafenib.

24. Treatment of hepatic cysts with psoriasis, which includes administering therapeutically effective doses of NK cells and rituximab to patients with hepatic cysts with psoriasis.

25. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein the NK cells are formulated as an injection.

26. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein the NK cells are living cells.

27. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein the NK cells are expanded in vitro.

28. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein, The NK cells were obtained from the in vitro induction and culture of NK cells derived from peripheral blood mononuclear cells (PBMCs).

29. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein the NK cells are infused at a rate of more than 3 billion per cycle for at least 5 cycles.

30. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein CD3 in the NK cells - CD56 + NK cells accounted for ≥95.0%.

31. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein CD3 in the NK cells - CD56 + CD16 + NK cells account for ≥90.0%.

32. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein CD3 in the NK cells - CD56 + NKG2D + NK cells accounted for ≥95.0%.

33. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein CD19 in NK cells + B cells account for ≤2.0%.

34. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein CD3 in the NK cells + CD56 - The percentage of T cells is ≤3.0%.

35. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein the in vitro expansion of NK cells is carried out in a serum-free culture system using a swing bioreactor, and the culture process parameters for in vitro expansion include: The total aeration rate is 20-100 mL / min, the culture temperature is 36.5-37.5℃, the shaking speed is 8-10 rpm, the shaking angle is 4-6°, the dissolved oxygen (DO) is ≥20%, and the pH is 6.8-7.

2.

36. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein the NK cells are stored in bags, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the viable cell density in the NK cell suspension is 0.8-1.2 × 10⁻⁶. 8 per mL.

37. The use as claimed in any one of claims 1 to 9, the formulation for the use as claimed in any one of claims 10 to 12, 15 to 16 and 18, the composition for the use as claimed in any one of claims 13 to 14 and 17, or the method as claimed in any one of claims 19 to 24, wherein the NK cells are stored in bags, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the total number of viable cells in the NK cell suspension is 2.0-3.0 × 10⁻⁶. 9 NK cells.

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