Application of NK cells in treatment of leucoderma and psoriasis

Abstract

The invention relates to application of NK cells in treatment of leucoderma and psoriasis.

Description

Technical Field

[0001] The present application relates to the application of NK cells in the treatment of vitiligo and psoriasis. Background Art

[0002] Natural killer cells (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 lymphocytes that are not MHC-restricted, do not require prior sensitization, and do not require the participation of antibodies, and can directly kill tumor cells and virus-infected cells in a non-specific manner. NK cells usually contain a large amount of perforin and granzyme B. When activated NK cells encounter target cells, the perforin and granzyme B released by NK cells attack the target cells. NK cells 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, which can act directly on target cells or attack target cells by activating other types of immune cells.

[0003] Cell therapy is a hot topic in the current medical field, and NK cells are also a focus of cell therapy due to their own immunological advantages. The development of NK cell-based therapies for different diseases is urgently needed in this field. Summary of the invention

[0004] In a first aspect, the present application provides the use of NK cells in the preparation of a drug for treating vitiligo.

[0005] In a second aspect, the present application provides the use of NK cells in the preparation of a medicament for treating psoriasis in an individual.

[0006] In some embodiments, the individual also has primary liver cancer, liver cysts, or hepatocellular carcinoma.

[0007] In a third aspect, the present application provides the use of NK cells and cancer-targeted drugs in the preparation of drugs for treating patients with primary liver cancer or hepatocellular carcinoma associated with psoriasis.

[0008] In some embodiments, the cancer targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib or sorafenib.

[0009] In a fourth aspect, the present application provides the use of NK cells in the preparation of a drug for treating patients with primary liver cancer or hepatocellular carcinoma accompanied by psoriasis in combination with cancer targeted drugs.

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

[0011] In a fifth aspect, the present application provides the use of NK cells and rituximab in the preparation of a medicament for treating patients with liver cysts associated with psoriasis.

[0012] In a sixth aspect, the present application provides the use of NK cells in the preparation of a drug for treating patients with liver cysts and psoriasis in combination with rituximab.

[0013] In some embodiments of any one of the first to sixth aspects, the NK cells are expanded in vitro.

[0014] In some embodiments of any one of the first to sixth aspects, the in vitro expansion of NK cells is performed in a serum-free culture system using a rocking bioreactor, and the culture process parameters for the in vitro expansion include: total ventilation volume 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.

[0015] In some embodiments of any one of the first to sixth aspects, the NK cells are obtained from peripheral blood mononuclear cell (PBMC)-derived NK cells.

[0016] In some embodiments of any one of the first to sixth aspects, more than 3 billion NK cells are infused per cycle, and the infusion is performed for at least 5 cycles.

[0017] In some embodiments of any one of the first to sixth aspects, the CD3 - CD56 + NK cells account for ≥95.0%.

[0018] In some embodiments of any one of the first to sixth aspects, the CD3 - CD56 + CD16 + NK cells account for ≥90.0%.

[0019] In some embodiments of any one of the first to sixth aspects, the CD3 - CD56 + NKG2D + NK cells account for ≥95.0%.

[0020] In some embodiments of any one of the first to sixth aspects, the CD19 in the NK cells + B cells account for ≤2.0%.

[0021] In some embodiments of any one of the first to sixth aspects, the CD3 + CD56 - T cells account for ≤3.0%.

[0022] In some embodiments of any one of the first to sixth aspects, the NK cells are stored in a bag, 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 8 Pieces / mL.

[0023] In some embodiments of any one of the first to sixth aspects, the NK cells are stored in a bag, 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 9 NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 An example of the use of NK cells and cancer-targeted drugs to treat a patient with primary liver cancer and concurrent psoriasis is shown.

[0025] Figure 2 An example of NK cells and rituximab for the treatment of a patient with liver cysts associated with psoriasis is shown.

[0026] Figure 3 An example of the use of NK cells and cancer-targeted drugs to treat a patient with hepatocellular carcinoma and concurrent psoriasis is shown.

[0027] Figure 4 An example of NK cell therapy for vitiligo is shown. DETAILED DESCRIPTION

[0028] In a first aspect, the present application provides the use of NK cells in the preparation of a drug for treating vitiligo.

[0029] In a second aspect, the present application provides the use of NK cells in the preparation of a medicament for treating psoriasis in an individual.

[0030] In some embodiments, the individual also has primary liver cancer, liver cysts, or hepatocellular carcinoma.

[0031] In a third aspect, the present application provides the use of NK cells and cancer-targeted drugs in the preparation of drugs for treating patients with primary liver cancer or hepatocellular carcinoma associated with psoriasis.

[0032] In some embodiments, the cancer targeted drug is an antibody drug.

[0033] In some embodiments, the cancer targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib or sorafenib.

[0034] In a fourth aspect, the present application provides the use of NK cells in the preparation of a drug for treating patients with primary liver cancer or hepatocellular carcinoma accompanied by psoriasis in combination with cancer targeted drugs.

[0035] In some embodiments, the cancer targeted drug is bevacizumab, atezolizumab, sintilimab, apatinib mesylate, lenvatinib or sorafenib.

[0036] In a fifth aspect, the present application provides the use of NK cells and rituximab in the preparation of a medicament for treating patients with liver cysts associated with psoriasis.

[0037] In a sixth aspect, the present application provides the use of NK cells in the preparation of a drug for treating patients with liver cysts and psoriasis in combination with rituximab.

[0038] In some embodiments of any one of the first to sixth aspects, the NK cells are expanded in vitro.

[0039] In some embodiments of any one of the first to sixth aspects, the in vitro expansion of NK cells is performed in a serum-free culture system using a rocking bioreactor, and the culture process parameters for the in vitro expansion include: total ventilation volume 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.

[0040] In some embodiments of any one of the first to sixth aspects, the total ventilation volume 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.

[0041] In some embodiments of any one of the first to sixth 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.

[0042] In some embodiments of any one of the first to sixth aspects, the rocking 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.

[0043] In some embodiments of any one of the first to sixth aspects, the rocking 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.

[0044] In some embodiments of any one of the first to sixth 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.

[0045] In some embodiments of any one of the first to sixth aspects, the pH is 6.8, 6.9, 7, 7.1 or 7.2, or a range between any of the foregoing values.

[0046] In some embodiments of any one of the first to sixth aspects, the NK cells are obtained from peripheral blood mononuclear cell (PBMC)-derived NK cells.

[0047] In some embodiments of any one of the first to sixth aspects, greater than 3 billion NK cells are infused per cycle, and the infusion is performed for at least 5 cycles.

[0048] In some embodiments of any one of aspects 1 to 6, the number of NK cells infused 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 a range between any of the foregoing values.

[0049] In some embodiments of any one of aspects 1 to 6, 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 NK cells are infused per cycle, or a range between any of the foregoing values.

[0050] In some embodiments of any one of aspects 1 to 6, 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 ranges between any of the above values).

[0051] In some embodiments of any one of aspects 1 to 6, 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 ranges between any of the above values).

[0052] In some embodiments of any one of the first to sixth aspects, the CD3 - CD56 + NK cells account for ≥95.0%.

[0053] In some embodiments of any one of the first to sixth aspects, the CD3 - CD56 + The proportion 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 the range between any of the above values).

[0054] In some embodiments of any one of the first to sixth aspects, the CD3 - CD56 + CD16 + NK cells account for ≥90.0%.

[0055] In some embodiments of any one of the first to sixth aspects, the CD3 - CD56 + CD16 +The proportion 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 the range between any of the above values).

[0056] In some embodiments of any one of the first to sixth aspects, the CD3 - CD56 + NKG2D + NK cells account for ≥95.0%.

[0057] In some embodiments of any one of the first to sixth aspects, the CD3 - CD56 + NKG2D + The proportion 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 the range between any of the above values).

[0058] In some embodiments of any one of the first to sixth aspects, the CD19 in the NK cells + B cells account for ≤2.0%.

[0059] In some embodiments of any one of the first to sixth aspects, the CD19 in the NK cells + The B cell proportion is ≤ 2.0%, 1.5%, 1.0% or 0.5% (or the range between any of the above values).

[0060] In some embodiments of any one of the first to sixth aspects, the CD3 + CD56 - T cells account for ≤3.0%.

[0061] In some embodiments of any one of the first to sixth aspects, the CD3 + CD56 - The T cell proportion is ≤3.0%, 2.5%, 2.0%, 1.5%, 1.0% or 0.5% (or a range between any of the above values).

[0062] In some embodiments of any one of the first to sixth aspects, the NK cells are stored in a bag, 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 8 Pieces / mL.

[0063] In some embodiments of any one of aspects 1 to 6, the NK cells are stored in bags, each bag containing 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL or 50 mL (or a range between any of the above values) of NK cell suspension.

[0064] In some embodiments of any one of the first to sixth aspects, the NK cells are stored in a bag, and the viable cell density of the NK cell suspension contained in each bag is 0.8, 0.9, 1.0, 1.1 or 1.2×10 8 / mL (or any range between the above values).

[0065] In some embodiments of any one of the first to sixth aspects, the NK cells are stored in a bag, and the total number of viable cells in the NK cell suspension contained in each bag is 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0×10 9 NK cells (or any range between the above values).

[0066] Example

[0067] The application will be described in more detail by way of specific examples. The following examples are provided for illustrative purposes only, and are not intended to limit the application in any way. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to produce substantially the same result.

[0068] Example 1: Preparation of NK cell injection

[0069] The preparation of NK cell injection is divided into three parts: peripheral blood mononuclear cell (PBMC) preparation, NK cell injection stock solution preparation and preparation preparation.

[0070] Peripheral blood mononuclear cell (PBMC) preparation

[0071] PBMC preparation includes hPB Leukopak pretreatment, CTS TM Rotea TM There are five major steps: processing, PBMC packaging, labeling, and PBMC freezing.

[0072] hPB Leukopak Pretreatment

[0073] 0.7-5.0mL / 1×10 9 Add PPC3 dilution to hPB Leukopak (i.e., peripheral blood mononuclear cells) (Source: RosetteSep TM Human CD3 Depletion Cocktail; the amount of addition is 1.5-5.0mL / per 1×10 9 cells) or PPC3S diluent (IgG4 antibody coupled to red blood cells and T cells, added in an amount of 1.5-5.0 mL / per 1×10 9 Cells). Incubate at room temperature for 10-40 minutes. Add erythrocyte sedimentation diluent (3% m / v hydroxyethyl starch) to hPBLeukopak at a final concentration of 1.2%-2.0%. Hang vertically and let stand at room temperature for (120±45) minutes. After the erythrocyte sedimentation is layered, use a blood separator to press the upper cell suspension into the sample bag.

[0074] CTS TM RoteaTM Treatment

[0075] Dilute the cell suspension with an equal volume of sodium chloride injection. After the cell suspension in the sample bag circulates and forms a stable fluidized bed, continue to load the cell suspension in the sample bag to concentrate the PBMC cells in the fluidized bed. After washing with sodium chloride injection and replacing with cryopreservation solution, the cryopreservation solution collects the PBMC cells into the harvest bag. Count the PBMC cells in the harvest bag. According to the counting results, adjust the live cell density of the PBMC cell suspension to (2.7-6.0)×10 7 Cell viability should be ≥80.0%.

[0076] PBMC isolation

[0077] Press 6.8×10 8 Calculate the volume of each bag based on the number of live cells / bag. Aseptically dock the harvest bag with the cryopreservation bag, mix the PBMC cells to be dispensed on a shaker, and dispense them into the cryopreservation bag using a peristaltic pump. After dispensing, keep samples for inspection (such as sterility inspection, mycoplasma inspection, etc.).

[0078] Labeling

[0079] Affix PBMC labels to the above-packed cryopreservation bags.

[0080] PBMC cryopreservation

[0081] Place the blood bag box flat in the freezing chamber of the program cooling instrument and cool it to -70--90°C. After cooling, transfer the frozen PBMC cells to a gas phase liquid nitrogen tank and store them at ≤-150°C.

[0082] Preparation of NK cell injection stock solution

[0083] The preparation of NK cell injection stock solution includes three major processes: NK cell initial culture, NK cell expansion culture, NK cell concentration and cleaning, and stock solution preparation.

[0084] Initial culture of NK cells

[0085] PBMC resuscitation

[0086] Coated with three T225cm 2 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)) was added to each culture bottle. 29 mL of sodium chloride injection was added to each bottle and mixed well. 2 The culture flask was placed in a biosafety cabinet and incubated at room temperature for 1-4 h or in a carbon dioxide incubator at 37.0±0.5℃ for 1-4 h.

[0087] Take out 1 bag of frozen PBMC cells from the liquid nitrogen tank, put them into a water-free resuscitation instrument (resurrection temperature 37±0.5℃) for rapid thawing, and the thawing time is 2-5min. Transfer the thawed PBMC cells to a 50mL centrifuge tube to which 15mL of compound electrolyte injection or sodium chloride injection has been added, then add compound electrolyte injection or sodium chloride injection to 45mL and mix the cells.

[0088] Centrifuge at 600g for 8±2min at room temperature. Discard the supernatant, add 45mL of NK cell activation medium (1640 medium, sodium heparin (0-10IU / mL), platelet lysate (volume ratio 0-10%), human serum albumin (volume ratio 1.0%-5.0%), IL2 (0-2000IU / mL), IL-15 (0-50ng / mL), etc.) and resuspend, mix and count. According to the cell counting results, if the total number of viable cells is ≥3.6×10 8 If the total number of viable cells is less than 3.6×10 8 If the number of cells per well or the cell viability is <80.0%, the PBMC cell recovery is terminated.

[0089] Remove T225cm 2 According to the counting results, transfer the above PBMC cell suspension evenly to three T225cm coated 2 Culture flask (coating agent composition: CD33-CLL1 (0-50μg / mL), CD52 (0-50μg / mL), CD38 (0-50μg / mL), CD3 antibody (0-50μg / mL) antibody), each T225cm2 The total inoculation volume of the culture flask is 120 mL, and the inoculation live cell density is ≥ 1.00 × 10 6 / mL, the three bottles are marked as bottle A, bottle B, and bottle C. After the bottle is marked, place the three T225cm 2 The culture flask was placed in a carbon dioxide incubator at 37.0±0.5°C and 5.0±0.5% CO2 for 3 days.

[0090] T225cm 2 Culture bottle rehydration culture

[0091] On the third day of culture, the three T225cm 2 Add 60 mL of NK cell activation medium to each culture flask and continue static culture.

[0092] T225cm 2 Culture flask transfer bag culture

[0093] On the 5th to 7th day of culture, three T225cm 2 The cell suspension in the culture flask was mixed and sampled and counted. If the average viable cell density of the three flasks was ≥ 6.7 × 10 5 / mL, then transferred to 825cm 2 Cells were cultured in cell culture bags, and NK cell activation medium was added for rehydration. The rehydration volume was adjusted according to the average viable cell density of three bottles. The viable cell density after rehydration was ≥4.00×10 5 / mL, the volume after rehydration is 900-1800mL. If the average viable cell density is lower than 6.70×10 5 If the average viable cell density is less than 6.70×10 / mL, continue culturing until the next day and take samples for counting to determine whether to transfer the bag. If the bag is not transferred on the second day, take samples for counting again on the third day to determine whether to transfer the bag. If the average viable cell density is less than 6.70×10 / mL, take samples for counting again on the third day to determine whether to transfer the bag. 5 After the rehydration is completed, the culture is continued in a carbon dioxide incubator at 37.0±0.5℃ and a CO2 concentration of 5.0±0.5% for two days.

[0094] 825cm 2 Cell culture bag rehydration culture 1

[0095] Day 7-10: 825cm 2The cell suspension in the cell culture bag was mixed and sampled for counting. The rehydration solution was NK cell expansion medium (1640 culture medium, sodium heparin (0-10 IU / mL), platelet lysate (volume ratio 0-10%), human serum albumin (volume ratio 1.0%-5.0%), IL2 (0-2000 IU / mL), IL-15 (0-50 ng / mL), etc.), and the rehydration volume was adjusted according to the viable cell density. The viable cell density after rehydration was ≥4.00×10 5 / mL, add 5-10mL serum substitute (II) (platelet lysate and human albumin, volume ratio 1:1) before rehydration, the volume after rehydration is 1800mL-2700mL, and continue static culture. If it still does not meet 825cm on the 10th day 2 If the cell culture bag reaches 1 refill standard, the culture is terminated.

[0096] 825cm 2 Cell culture bag rehydration culture 2

[0097] Days 9-12, 825cm 2 Mix the cell suspension in the cell culture bag and take a sample for cell counting. The total number of viable cells is ≥2.0×10 9 If the above requirements are not met, continue to culture. If the culture system is less than 2700mL, add fluid and NK cell expansion medium. After fluid replenishment, the viable cell density is ≥6.00×10 5 / mL, and the total volume after rehydration should not exceed 2700mL. If the flow cytometry results do not meet the flow cytometry standards for inspection, the culture will be terminated.

[0098] The total number of live cells, cell viability, NK purity, total T cell impurities, and B cell impurities before rehydration on days 9-12 are used as in-process control indicators. If the total number of live cells and cell viability meet the in-process control standards, samples are taken for flow cytometry testing; if the total number of live cells and cell viability do not meet the in-process control standards, culture is continued until the 12th day, until samples are taken for flow cytometry testing after meeting the in-process control standards, and culture is terminated if it still does not meet the standards. In the above situation, if the flow cytometry results meet the in-process control standards, the reactor is inoculated and cultured; if the flow cytometry results do not meet the in-process control standards, the culture is terminated. The in-process control standards are shown in Table 1 below.

[0099] Table 1: Reactor in-process control standards (Day 9-12)

[0100]

[0101] Note 1: *The total number of viable cells is calculated based on the viable cell density. Total number of viable cells = viable cell density × volume.

[0102] Note 2: The above process is for a 50L reactor (working volume 25L). The total amount of cells inoculated (and the number of culture bottles inoculated) during the PBMC recovery operation in the 100L reactor is twice that of the 50L reactor. Other culture and fluid replenishment parameters are the same. During bag transfer culture on D5-D7, one bag is transferred for every three culture bottles, and two bags of cells are cultured in the reactor.

[0103] NK cell expansion and culture

[0104] Reactor inoculation culture

[0105] On the 9th to 12th day, the in-process control results confirmed that the total number of viable cells in each bag was ≥ 2.0 × 10 9 , cell viability ≥ 80.0%, NK purity (CD3 - CD56 + )≥70.0%, total T cell impurities (CD3 + )≤9.0%, B cell impurities (CD19 + )≤5.0%, the culture can be continued in the reactor.

[0106] Transfer all the cell suspension to the reactor cell culture bag, adjust the volume of the rehydration according to the viable cell density, and add NK cell expansion medium and serum substitute (II) (platelet lysate and human albumin, volume ratio 1:1) to a volume of 25-100mL. The viable cell density after rehydration is ≥4.00×10 5 / mL, glucose concentration ≥1.00g / L. If the glucose concentration after rehydration is lower than 1.00g / L, add glucose solution to make the glucose concentration of the cell suspension 1.00-2.00g / L.

[0107] The reactor process parameters are controlled as follows:

[0108] a. Culture parameters: total ventilation volume 20-100 mL / min, culture temperature 37.0±0.5°C, rotation speed 8-10 rpm, angle 4-6°, pH: 7.0±0.2, DO: ≥20%.

[0109] b. pH control is related to carbon dioxide and 1 mol / L sodium bicarbonate, and DO control is related to oxygen and nitrogen.

[0110] Bioreactor expansion culture

[0111] The reactor process parameters are controlled as follows:

[0112] Culture parameters: total ventilation volume 20-40 mL / min, culture temperature 37.0±0.5°C, rotation speed 8-10 rpm, angle 4-6°, pH: 7.0±0.2, DO: ≥20%.

[0113] 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.

[0114] Sampling: After adjusting the rotation speed and angle to mix the cell suspension, take samples to test the pH value, live cell density, cell viability, glucose, lactate, ammonia and glutamine. If the absolute value of the deviation between the detected pH value and the online pH value is ≥0.10, pH calibration is performed, and flow cytometry detection is performed on the 15th to 18th day.

[0115] Rehydration: Adjust the rehydration volume according to the viable cell density. The viable cell density after rehydration should be ≥5.00×10 5 / mL. If no fluid is replenished for three consecutive days, terminate the current batch of culture. After the second fluid replenishment, add 25-50mL of serum substitute (Ⅱ). If no fluid is replenished and the glucose concentration of the cell suspension is lower than 1.00g / L, add glucose solution to make the glucose concentration of the cell suspension 1.00-2.00g / L until the total volume is replenished to 25L.

[0116] Perfusion: When the culture system reaches the maximum volume of the culture bag and the lactate concentration is ≥1.00g / L, the intermittent perfusion culture mode is used to replace the fresh NK cell expansion medium (using a hollow fiber column). After the first perfusion, add 25-100mL of serum replacement (II), and after the interval perfusion (for example, the third, fifth, and seventh perfusions), add 25-50mL of serum replacement (II). Requirements after perfusion: Theoretical glucose concentration ≥1.00g / L. If the glucose concentration after perfusion is <1.00g / L, add glucose solution so that the theoretical glucose concentration of the cell suspension is 1.00-2.00g / L.

[0117] The cell viability, NK purity, total T cell impurities and double-negative cell impurities before rehydration on days 15-18 were used as in-process control indicators. If they met the in-process control standards, the next step of expansion and culture operation could be carried out. Otherwise, the culture would be terminated. The specific in-process control standards are shown in Table 2 below.

[0118] Table 2: In-process control standards for reactor expansion culture (Day 15-18)

[0119]

[0120] Note: If the cell viability and NK purity (CD3 - CD56 + ), total T cell impurities (CD3 + ) and double negative cell impurities (CD3 - CD56 - ), and the in-process control standards in Table 2 have been met, then the NK cell concentration, washing and stock solution preparation processes can be carried out on D18-D23.

[0121] 48 hours before the preparation of the stock solution, a sample is taken for sterility test. The sterility test is used as the in-process control indicator. If it meets the in-process control standard, the next step of Pre-stock solution preparation can be carried out.

[0122] NK cell concentration, washing and stock solution preparation

[0123] NK cell suspension centrifugation washing:

[0124] Concentration: The NK cell suspension obtained above was processed using a fully enclosed automated cell processing system, the temperature was set to 4°C (temperature control range was 2-8°C), the sample was concentrated by continuous flow, the injection flow rate was 250 mL / min, and the centrifugal speed was 2200 rpm.

[0125] Washing: After continuous flow concentration is completed, enter the washing solution-1 washing step, the washing cycle number is 6-8 times, the single washing volume is set to 140mL, the flow rate is 150mL / min, and the centrifugal speed is 1500rpm. After the washing solution-1 washing step is completed, enter the freezing solution-2 washing step, the washing number is 1-2 times, the single washing volume is set to 140mL, the flow rate is 150mL / min, and the centrifugal speed is 1500rpm.

[0126] Pre-stock solution preparation:

[0127] According to the NK cell counting results, the resuspension volume of cryopreservation solution-2 was calculated, the resuspension volume range of 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 the resuspension was completed, the resuspension was transferred to the stock solution bag at a flow rate of 200mL / min to obtain the Pre-stock solution.

[0128] The formula for calculating the resuspension volume is:

[0129]

[0130] If the calculated resuspension volume is > 600 mL, resuspend in 600 mL volume.

[0131] Take about 1 mL for cell counting. Cell viability and live cell density are used as in-process control indicators. If they meet the in-process control standards, the next step can be carried out. The specific in-process control standards of Pre-stock solution are shown in Table 3 below.

[0132] Table 3: Pre-stock solution in-process control standards (Day 18-23)

[0133]

[0134] Original solution dilution:

[0135] According to the Pre-stock solution counting results and the stock solution live cell density ((0.8-1.2)×10 8 / mL) to calculate the volume of the original solution and the volume of the freezing solution-2 to be added,

[0136] Calculation formula: stock solution volume = (Pre-stock solution viable cell density × Pre-stock solution volume) / set stock solution viable cell density;

[0137] Cryopreservation solution-2 volume = stock solution volume - Pre-stock solution volume.

[0138] According to the calculation result of the volume 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 cleaning the centrifuge cup, the cryopreservation solution-2 will enter the original solution bag.

[0139] After mixing the stock solution bag, take samples and count. If the count result is (0.8-1.2)×10 8 If the count result exceeds the live cell density range of the stock solution, it is necessary to continue to add freezing solution -2 to adjust the live cell density to (0.8-1.2)×10 8 / mL, and the NK cell stock solution was obtained.

[0140] Preparation

[0141] Filling: Use a closed filling system (temperature control 2-8°C) to fill the stock solution, the peristaltic pump filling flow rate is 50-200mL / min, and 20mL (viable cell density (0.8-1.2) × 10 8 / mL) or 25mL (viable cell density (0.8-1.2) × 10 8 / mL) or 30mL (viable cell density (0.8-1.2) × 10 8 / mL) or 50mL (viable cell density (0.8-1.2) × 10 8 / mL), and fill into disposable cell freezing bags.

[0142] Sealing: Use a handheld heat sealer to heat the long catheter tube of the disposable cell freezing bag. After the heat seal is completed, check that the preparation packaging is complete and there is no leakage.

[0143] Labeling: Attach labels and transfer the labeled products into freezing boxes (both freezing boxes and freezing bags need to be labeled).

[0144] Program cooling:

[0145] a. Turn on the programmed cooling instrument and quickly transfer the labeled samples into the programmed cooling instrument chamber for programmed cooling (the temperature drops to -80°C).

[0146] b. For program-controlled program related parameters, see Table 4.

[0147] Table 4: Program-controlled program related parameters

[0148]

[0149]

[0150] Cryopreservation: After the programmed cooling is completed, the NK cell injection solution is transferred to a gas phase liquid nitrogen tank for cryopreservation (storage conditions ≤ -150°C).

[0151] The exemplary simplified preparation steps of NK cell injection are as follows:

[0152] NK cells are derived from peripheral blood, prepared into PBMC cells in vitro, and stored in liquid nitrogen. The revived PBMC cells are cultured in vitro for initial and amplified culture to obtain high-quality NK cells. After the NK cell suspension is concentrated and washed, the NK cells are adjusted to a live cell density of (0.8-1.2)×10 8 / mL, that is, NK cell stock solution. Use a closed filling system to fill the NK cell stock solution into a disposable cell freezing bag, heat and label the disposable cell freezing bag, put it into a freezing box, and then transfer it to the programmed cooling chamber of the program cooling instrument for programmed cooling to -80°C. After the programmed cooling is completed, it is transferred to a liquid nitrogen tank for storage (storage conditions ≤-150°C), and the NK cell injection solution is obtained. The final cell purity quality control of the NK cell injection solution is shown in Table 5:

[0153] Table 5: Final cell purity quality control for NK cell injection

[0154]

[0155]

[0156] Example 2: Application of NK cells in the treatment of psoriasis①

[0157] Patient's medical history

[0158] A 58-year-old male patient suffered from psoriasis, primary liver cancer, and cirrhosis due to hepatitis B. The patient was diagnosed with primary liver cancer in 2022 and had psoriasis.

[0159] Treatment

[0160] Treatment of psoriasis with NK cell injection prepared in Example 1: Starting from August 2022, NK cell injection (cycle and dosage: 21 days as a cycle, administration on the 1st and 3rd days of each cycle, 4 billion / time, a total of 8 billion per cycle) was administered in combination with bevacizumab injection (trade name: One cycle is 21 days, and the drug is administered on the first day of each cycle. The dosage is 15 mg / kg body weight). As of July 2023, a total of 14 cycles of NK cell infusion have been performed.

[0161] Treatment Outcomes

[0162] The efficacy of the NK cell injection prepared in Example 1 in treating psoriasis is as follows: Figure 1 shown.

[0163] like Figure 1 As shown, there were obvious psoriasis plaques before NK cell treatment on August 4, 2022. After 4 cycles of NK cell treatment, the psoriasis plaques were significantly reduced. After 7 cycles of NK cell treatment, there were no obvious psoriasis plaques. After 10 cycles of NK cell treatment and during subsequent follow-up, there was no recurrence of psoriasis.

[0164] Example 3: Application of NK cells in the treatment of psoriasis②

[0165] Patient's medical history

[0166] A 59-year-old male patient has suffered from psoriasis for more than 30 years. Past medical history: coronary atherosclerosis, pulmonary nodules (considered benign), arrhythmia-complete right bundle branch block, post-thyroid surgery (taking Euthyrox), enlarged and firm prostate, liver cysts, etc.

[0167] Treatment

[0168] Use the NK cell injection prepared in Example 1 to treat psoriasis: Starting from January 2024, NK cell injection is given (cycle and dosage: 30 days as a cycle, administered on the first day of each cycle, 7.5 billion / time), and from the third cycle onwards, rituximab injection (trade name: MabThera, 30 days as a cycle, dosage of 100 mg each time) is used in combination for treatment. As of July 2024, a total of 7 cycles of NK cell infusion are performed.

[0169] Treatment Outcomes

[0170] The efficacy of the NK cell injection prepared in Example 1 in treating psoriasis is as follows: Figure 2 shown.

[0171] like Figure 2 As shown, there were obvious psoriasis plaques before NK cell treatment on December 29, 2023. After 3 cycles of NK cell treatment, the psoriasis plaques were significantly reduced, and after 7 cycles of NK cell treatment, there were no obvious psoriasis plaques.

[0172] Example 4: Application of NK cells in the treatment of psoriasis③

[0173] Patient's medical history

[0174] A 53-year-old male patient suffered from psoriasis, hepatocellular carcinoma (bilateral lung metastasis after surgery) and hypertension. The patient was diagnosed with primary liver cancer in September 2014. He subsequently underwent multiple treatments including hepatocellular carcinoma resection + cholecystectomy, TACE, retilizumab + sorafenib (1 year), sintilimab + apatinib (10 months), and local radiotherapy, but his condition was not effectively controlled.

[0175] Treatment

[0176] Treatment of psoriasis with NK cell injection prepared in Example 1: Starting from November 2022, NK cell injection (cycle and dosage: 21 days as a cycle, administration on the 1st and 3rd days of each cycle, 5 billion / time, a total of 10 billion per cycle) was administered in combination with lenvatinib (trade name: The drug was administered starting from the 5th day of the first cycle, with a dose of 12 mg once a day), and a total of 6 cycles of NK cell infusion were performed by April 2023.

[0177] Treatment Outcomes

[0178] The efficacy of the NK cell injection prepared in Example 1 in treating psoriasis is as follows: Figure 3 shown.

[0179] like Figure 3 As shown in the figure, there were obvious psoriasis plaques before NK cell treatment on November 23, 2022. During the NK cell treatment, the patient's liver cancer was effectively controlled and the psoriasis lesions improved. After 6 cycles of NK cell treatment, there were no obvious psoriasis plaques.

[0180] Example 5: Application of NK cells in the treatment of vitiligo

[0181] Patient's medical history

[0182] A 17-year-old male patient first developed vitiligo on his eyelids in 2015. He was subsequently treated with antibiotics to control the development and it hardly spread. In August 2021, he suffered from sunburn on a large area of ​​his face. He became ulcerated after scratching, and his skin lost pigment after scabbing, and it spread irregularly. From August 2021 to 2022, he took oral medications and ointments (compound calix cumin tincture, dexamethasone sodium phosphate injection, folic acid tablets) and ultraviolet light therapy, but there was no significant change in his condition.

[0183] Treatment

[0184] 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 as one cycle, 1 administration per cycle). Six cycles of treatment were completed in December 2023.

[0185] No other new drugs were added during the cell therapy, and previous drug treatment was continued: compound calciferol tincture, dexamethasone sodium phosphate injection, folic acid tablets, and occasionally ultraviolet light therapy.

[0186] Treatment Outcomes

[0187] The efficacy of the NK cell injection prepared in Example 1 in treating vitiligo is as follows: Figure 4 shown.

[0188] like Figure 4 As shown in the figure, the white spots on the patient's head and face showed a trend of regression during the treatment, and the area of ​​the white spots was significantly reduced and lightened compared with before treatment. During the follow-up, the white spots on the head and face continued to recede, and the area of ​​the white spots further shrank.

Claims

1. Use of NK cells in the preparation of drugs for treating vitiligo.

2. Use of NK cells in the preparation of a medicament for treating psoriasis in an individual.

3. The use according to claim 2, wherein the individual also suffers from primary liver cancer, liver cyst or hepatocellular carcinoma.

4. Use of NK cells and cancer-targeted drugs in the preparation of drugs for treating patients with primary liver cancer or hepatocellular carcinoma associated with psoriasis.

5. The use according to 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 a drug for treating patients with primary liver cancer or hepatocellular carcinoma accompanied by psoriasis in combination with cancer targeted drugs.

7. The use according to 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 treating patients with liver cysts associated with psoriasis.

9. Use of NK cells in the preparation of a medicament for treating patients with liver cysts associated with psoriasis in combination with rituximab.

10. The use according to any one of claims 1 to 9, wherein the NK cells are expanded in vitro.

11. The use according to any one of claims 1 to 9, wherein The NK cells are obtained from the in vitro induction and culture of NK cells derived from peripheral blood mononuclear cells (PBMC).

12. The use according to any one of claims 1 to 9, wherein the NK cells are infused in greater than 3 billion cells per cycle, and the infusion is performed for at least 5 cycles.

13. The use according to any one of claims 1 to 9, wherein the CD3 - CD56 + NK cells account for ≥95.0%.

14. The use according to any one of claims 1 to 9, wherein the CD3 - CD56 + CD16 + NK cells account for ≥90.0%.

15. The use according to any one of claims 1 to 9, wherein the CD3 - CD56 + NKG2D + NK cells account for ≥95.0%.

16. The use according to any one of claims 1 to 9, wherein the CD19 in the NK cells + B cells account for ≤2.0%.

17. The use according to any one of claims 1 to 9, wherein the CD3 + CD56 - T cells account for ≤3.0%.

18. The use according to claim 10, wherein the NK cells are expanded in vitro using a rocking bioreactor in a serum-free culture system, and the culture process parameters of the in vitro expansion include: The total aeration volume was 20-100 mL / min, the culture temperature was 36.5-37.5°C, the rocking speed was 8-10 rpm, the rocking angle was 4-6°, the dissolved oxygen (DO) was ≥20%, and the pH was 6.8-7.

2.

19. The use according to any one of claims 1 to 9, wherein the NK cells are stored in a bag, 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 8 Pieces / mL.

20. The use according to any one of claims 1 to 9, wherein the NK cells are stored in a bag, 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 9 NK cells.

Images