NKT cell preparation as well as preparation method and application thereof

Abstract

The invention provides an NKT cell preparation as well as a preparation method and application thereof, and belongs to the field of biomedical treatment. The NKT cell preparation provided by the invention comprises NKT cells, a compound electrolyte injection, 5-10% of human serum albumin, 5-10% of raffinose and 2-5% of lysine, wherein the concentration of the NKT cells in the cell preparation is 1.0 * 10 < 6 >-1.0 * 10 < 8 > / mL; the prepared finished product preparation is long in shelf life, can be stably stored at 4 DEG C for more than 144 hours, and can be stably stored at-80 DEG C for at least 12 months.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to a cell preparation, in particular to an NKT cell preparation and a preparation method and application thereof. BACKGROUND

[0002] The clinical cell preparation preparation and application technology as an effective method for preparing, storing, transporting and applying the clinical living cell preparation according to the drug standardization has been widely applied in the biological field. In the early stage, due to the limitation of key technologies, the preparation, storage, transportation and use of cell preparations cannot be standardized and standardized management and quality control according to the general biological product drug preparation, and there are inevitable quality control defects in the process operation and application, and there are many uncontrollable risks or unpredictable potential hazards in the process operation and application process, which cause great safety hazards to clinical application.

[0003] The clinical cell preservation technology has two types which are mature in clinical application: one is frozen preservation, including liquid nitrogen-196℃ preservation and-80℃ refrigerator preservation. The frozen preservation of stem cells is safe and reliable, and can be preserved for a long time under the condition of liquid nitrogen-196℃. However, the frozen preserved stem cells cannot be directly used in clinic, and need to be thawed and resuspended in cell infusion solution for use. The cell freezing and thawing process needs very professional operation, involving the addition of cryoprotectant and sub-packaging, cooling, storage, thawing, cleaning, and reinfusion stages. Each link in the process may cause cell damage and death. In addition, the frozen preserved cells need to be washed and detected before use in a sterile clean environment, which is difficult for general clinical institutions to achieve, thereby limiting the clinical use of stem cells. The other is cold storage, that is, 4℃ non-frozen preservation. The 4℃ cold storage of stem cells does not need to be thawed before reinfusion. If the components contained in the cold storage solution meet the requirements of clinical use, the cells can be directly reinfused after rewarming, and the operation method is relatively simple and convenient for clinical use. However, the current method of cold storage of cells has a limited preservation time of less than 7 days, which greatly limits the clinical application. Although some cells can be stored at 4℃ for 3 weeks, the protective solution used contains cell culture medium, which cannot be directly used for human reinfusion or injection, that is, it cannot be used in clinic. Some use dimethyl sulfoxide or animal protein, although the amount is within the clinical safety range, but since it cannot be ruled out the possibility of adverse reactions to the human body, it also limits the clinical application.

[0004] The Chinese patent application with the publication number CN110882212A discloses a kind of clinical injection cell preparation, the cell preparation includes living cells and medicinal adjuvant, based on the total amount of medicinal adjuvant, the medicinal adjuvant contains: propylene glycol, volume fraction is 10%-90%; Trehalose 0.06-0.15 g / mL;Human blood albumin 0.1-0.2 g / mL;With plasma isotonic electrolyte solution, make up to 100%, the cell preparation lacks the shortcoming of storage time.

[0005] Wang Xiujun et al. discloses a kind of human umbilical cord mesenchymal stem cell compound electrolyte injection preparation. Among them, the compound electrolyte injection preparation is lower than 0.9% sodium chloride injection preparation in the level of cell apoptosis; 2% albumin can effectively reduce the level of cell apoptosis. Light has no serious influence on the compound electrolyte injection preparation adding 2% albumin cell preparation; Temperature fluctuation can cause rapid decline of cell survival rate; High temperature has a great influence on the state of cell preparation, leading to the appearance of a large number of flocs, and rapid decline of cell survival rate; Under low temperature conditions, the storage time of the preparation is not long (Wang Xiujun, Li Zhengnan, Li Wei, et al. Preparation and stability of human umbilical cord mesenchymal stem cell compound electrolyte injection [J]. Chinese scientific and technical data catalogue-Chinese herbal medicine, 2020).

[0006] Immune cells are a huge family. Among them, NK cells are a kind of innate immune cells, CIK is a group of heterogeneous mixed T cells, and NKT cells (natural killer T cell) are a kind of T cells between innate immunity and adaptive immunity. NKT cells were discovered in 1986, mainly playing a bridging role in innate immunity and adaptive immunity. NKT cells are a special T cell subpopulation, which has T cell receptor (TCR) and NK cell receptor on the cell surface. NKT cells can produce a large amount of cytokines and can exert similar cytotoxicity to NK cells. NKT cells express "semi-constant" TCR alpha beta peptide chains, which means that the TCR alpha chain is relatively constant, while the TCR beta chain is diverse, so it is called "NK-like T cell". Due to its strong and wide anti-tumor activity, good safety and potential for allogeneic use, NKT cells are the focus of attention in various cancer immunotherapy methods, but because the preparation of NKT cells is relatively difficult, the long-term storage problem needs to be solved urgently. SUMMARY

[0007] The present application provides a kind of NKT cell preparation and its preparation method and application to solve the problem of short storage time of NKT cell in prior art. The NKT cell preparation provided by the present application includes NKT cell, compound electrolyte injection, 5%-10% human blood albumin, 5%-10% raffinose and 2%-5% lysine, and the concentration of the NKT cell in the cell preparation is 1.0×10 6-1.0 x 10 8 The prepared finished preparation can be stored stably at 4℃ for more than 144 hours, and stored stably at -80℃ for at least 12 months.

[0008] To achieve the above object, the technical scheme adopted by the present application is as follows: In one aspect, the present application provides a NKT cell preparation, which comprises NKT cells, compound electrolyte injection, 5%-10% human blood albumin, 5%-10% raffinose and 2%-5% lysine.

[0009] Preferably, 1000 mL of the compound electrolyte injection comprises 5.26 g of sodium chloride, 5.02 g of sodium gluconate, 3.68 g of sodium acetate, 0.37 g of potassium chloride and 0.30 g of magnesium chloride.

[0010] Preferably, the concentration of the NKT cells in the cell preparation is 1.0 x 10 6 -1.0 x 10 8 / mL.

[0011] Preferably, the surface marker molecules CD3, CD56 and CD8 of the NKT cells are detected by flow cytometry, and the requirements are that CD3(+) and CD56(+) is greater than or equal to 70%, CD56(+) is greater than or equal to 90%, CD3(+) is greater than or equal to 80%, and CD3(+) and CD8(+) is greater than or equal to 80%.

[0012] In another aspect, the present application provides a preparation method of the above-mentioned NKT cell preparation, which comprises the following steps: S1, preparing NKT cells and compound electrolyte injection; S2, taking the compound electrolyte injection, adding human blood albumin, raffinose and lysine, and using the compound electrolyte injection to make up the volume, filtering and sterilizing to obtain a filtrate; S3, resuspending the NKT cells with the filtrate prepared in step S2 to obtain the NKT cell preparation.

[0013] Preferably, the step S2 comprises: Taking 20-30 mL of compound electrolyte injection, adding 20% human blood albumin 25-50 mL, adding raffinose 5-10 g, adding lysine 2-5 g, using compound electrolyte injection to make up the volume to 50-175 mL, filtering and sterilizing to obtain a filtrate.

[0014] Preferably, the step S2 comprises: taking 20 mL of compound electrolyte injection, adding 25 mL of 20% human blood albumin, adding 5 g of raffinose, adding 2 g of lysine, and adding compound electrolyte injection to 100 mL, filtering and sterilizing to obtain a filtrate; and the step S3 comprises resuspending the NKT cells with the filtrate prepared in the step S2 to obtain an NKT cell preparation with a final concentration of 1.0 x 10 8 Preferably, the step S2 comprises: taking 20 mL of compound electrolyte injection, adding 25 mL of 20% human blood albumin, adding 5 g of raffinose, adding 2 g of lysine, and adding compound electrolyte injection to 100 mL, filtering and sterilizing to obtain a filtrate; and the step S3 comprises resuspending the NKT cells with the filtrate prepared in the step S2 to obtain an NKT cell preparation with a final concentration of 1.0 x 10

[0015] In another aspect, the present application provides the use of the NKT cell preparation or the preparation method in the preparation of NKT cell drugs or the preservation of NKT cells.

[0016] Preferably, the use comprises the storage of NKT cells.

[0017] Preferably, the storage condition of the NKT cells is -80℃ to 4℃.

[0018] Compared with the prior art, the present application has the following beneficial effects: 1. The NKT cell preparation provided by the present application has fewer types of components, and the preparation method is simple, without DMSO, animal-derived proteins and other substances that pose safety risks.

[0019] 2. The NKT cell preparation provided by the present application has a long shelf life, and the finished preparation can be stored stably at 4℃ for more than 144 hours and at -80℃ for at least 12 months. DETAILED DESCRIPTION

[0020] Unless otherwise specified, all raw materials and reagents in the present application are purchased from commercial suppliers, without further purification, and experiments are performed according to the operation instructions. Raw materials and reagents susceptible to moisture are stored in fully sealed bottles and used directly, without special treatment. The instruments, equipment and devices used in the present application are all conventional instruments, equipment and devices.

[0021] Data analysis is performed using professional data processing software, and single-factor ANOVA test is used for significance analysis, with P<0.05 representing significant difference.

[0022] Table 1 explains the sources of the key raw materials involved in the present application.

[0023] Table 1 Raw material source explanation

[0024] Among them, the compound electrolyte injection contains 5.26 g of sodium chloride, 5.02 g of sodium gluconate, 3.68 g of sodium acetate, 0.37 g of potassium chloride, and 0.30 g of magnesium chloride per 1000 mL.

[0025] The NKT cell preparation provided by the application comprises NKT cells, human blood albumin, compound electrolyte injection, raffinose and lysine. The concentration of the NKT cells in the cell preparation is 1.0×10 6 -1.0×10 8 The mass fraction of the human blood albumin in the cell preparation is 5%-10%, the mass fraction of the raffinose in the cell preparation is 5%-10%, and the mass fraction of the lysine in the cell preparation is 2%-5%.

[0026] The NKT cell preparation is prepared, and the implementation process comprises the following steps: (1) Take the compound electrolyte injection, add the human blood albumin, the raffinose and the lysine, make up the volume balance with the compound electrolyte injection, and filter and sterilize through a 0.22 μm filter membrane; (2) Harvest the NKT cells, collect the NKT cell suspension after culture, remove the cell culture medium after centrifugation at 1500 rpm, resuspend with normal saline, centrifuge again at 1500 rpm for 8 minutes, and wash twice; (3) Resuspend the NKT cells with the filtrate prepared in step (1) to prepare the NKT cell preparation; (4) The NKT cell preparation can be filled into a 100 mL backset bag with a 50 mL syringe, sealed with a heat sealer, and stored at low temperature or ultralow temperature.

[0027] Preparation Example: Preparation of NKT cells Harvest the NKT cells cultured by the NKT cell culture kit (purchased from Shenzhen Morse Biotechnology, product number MCF-009). After 14 days of culture, the NKT cells have a cell expansion multiple of 1000, CD3 (+) and CD56 (+) ≥70%, CD56 (+) ≥90%, CD3 (+) ≥80%, CD3 (+) and CD8 (+) ≥80%, and cell activity >95%. The harvested NKT cells are washed twice with normal saline.

[0028] Example 1: Preparation of NKT cell preparation The preparation method of each 100 mL cell preparation is as follows: (1) Take 20 mL of compound electrolyte injection, add 25 mL of 20% human blood albumin, add 5 g of raffinose, add 2 g of lysine, make up the volume balance with the compound electrolyte injection, filter and sterilize through a 0.22 μm filter membrane (the mass fraction of the human blood albumin in the cell preparation is 5%); (2) Resuspend the NKT cells with the filtrate prepared in step (1) to make the final concentration of the NKT cells be 1.0×10 8 The cell preparation is prepared.

[0029] The cell preparation is filled into a 100 mL transfusion bag with a 50 mL syringe, and the bag is sealed with a heat sealer.

[0030] Example 2: Preparation of NKT cell preparation The preparation method of each 100 mL cell preparation is as follows: (1) Take 20 mL of compound electrolyte injection, add 50 mL of 20% human serum albumin, add 10 g of raffinose, add 5 g of lysine, and make up the volume with compound electrolyte injection. Filter sterilization through a 0.22 μm filter (the mass fraction of human serum albumin in the cell preparation is 10%); (2) Resuspend the NKT cells with the filtrate prepared in step (1) to a final concentration of 1.0 x 10 8 cells / mL to prepare the cell preparation.

[0031] Example 3: Preparation of NKT cell preparation The difference from Example 1 is that the final concentration of NKT cells is 1.0 x 10 7 cells / mL, and the others are the same.

[0032] Example 4: Preparation of NKT cell preparation The difference from Example 1 is that the final concentration of NKT cells is 1.0 x 10 6 cells / mL, and the others are the same.

[0033] Example 5: Preparation of NKT cell preparation The difference from Example 2 is that the final concentration of NKT cells is 1.0 x 10 7 cells / mL, and the others are the same.

[0034] Example 6: Preparation of NKT cell preparation The difference from Example 2 is that the final concentration of NKT cells is 1.0 x 10 6 cells / mL, and the others are the same.

[0035] Comparative Example 1: Preparation of NKT cell preparation The difference from Example 1 is that 7 g of raffinose is added, and no lysine is added, and the others are the same.

[0036] Comparative Example 2: Preparation of NKT cell preparation The difference from Example 1 is that no raffinose is added, and 7 g of lysine is added, and the others are the same.

[0037] Comparative Example 3: Preparation of NKT cell preparation The difference from Example 1 is that neither raffinose nor lysine is added, and the others are the same.

[0038] Comparative Example 4: Preparation of NKT cell preparation The difference from Example 1 is that raffinose is replaced by equal mass trehalose, and the others are the same.

[0039] Preparation of NKT cell preparation The difference from Example 1 is that lysine is replaced by equal molar amount of serine, and the others are the same.

[0040] Test Example 1: NKT cell preparation stability test I. Experimental method and standard: (1) Bovine serum albumin detection According to the 2020 edition of Chinese Pharmacopoeia 4 part 3411, the ELISA method is used to detect the residual amount of BSA in the cell preparation, and the requirement is not more than 50 ng / mL.

[0041] (2) Sterility test According to the 2020 edition of Chinese Pharmacopoeia 4 part 1101 sterility test method, the cell preparation is detected, and the requirement is no bacterial growth.

[0042] (3) Endotoxin test According to the 2020 edition of Chinese Pharmacopoeia 4 part 1143 endotoxin gel limit method, the cell preparation is detected, and the requirement is less than 0.5 EU / mL.

[0043] (4) Mycoplasma test According to the 2020 edition of Chinese Pharmacopoeia 4 part 3301 culture method and instruction cell culture method, the cell preparation is detected, and the requirement is that the mycoplasma test results are all negative.

[0044] (5) pH value test According to the 2020 edition of Chinese Pharmacopoeia 4 part 0631 pH value test method, the cell preparation is detected, and the requirement is that the pH is in the range of 6.8-7.4.

[0045] (6) Osmotic pressure test According to the 2020 edition of Chinese Pharmacopoeia 3 part 0632 "freezing point depression method", the osmotic pressure molar concentration of the cell preparation is determined, and the requirement is that the osmotic pressure is in the range of 275-325 mOsmol / k.

[0046] (7) NKT cell marker identification The cell surface markers CD3, CD56 and CD8 are detected by flow cytometry, and the requirements are CD3 (+) and CD56 (+) ≥ 70%, CD56 (+) ≥ 90%, CD3 (+) ≥ 80%, CD3 (+) and CD8 (+) ≥ 80%.

[0047] (8) Number of living cells The percentage of viable cells was determined by trypan blue staining and counting using a cell counter.

[0048] (9) In vitro killing (toxicity) experiment The quantification of human NKT cytotoxic activity can be determined using target cells K562. Target cells and NKT cells should be co-cultured on cell culture treated plates (such as Thermo Scientific Nunc 96-well microplates) coated with 5 µg / mL fibronectin in Gibco HaNKTs buffer saline solution (HBSS) at 4°C overnight. Then target cells at a density of 1 x 10 5 (K562) cells / mL were added to each well, and after adding 10 ng / mL SDF-1a, adhered at 37°C for 1 hour. Then the well plates were washed with HBSS. NKT cells at a density of 5 x 10 5 cells / mL in HBSS were added to each well (in triplicate), and incubated at 37°C. Specifically, the in vitro killing rate of NKT cells was determined by fluorescence imaging detection using a kit (LIVE / DEAD™ Cell-mediated Cytotoxicity Assay Kit, purchased from Invitrogen), imaging using a fluorescence imaging system, and analysis using image analysis software.

[0049] Table 2 summarizes the detection items, detection methods, and standards for the cell preparations involved in the examples and comparative examples.

[0050] Table 2 NKT cell preparation detection items, detection methods, and standards

[0051] II. 4°C storage stability The cell preparations prepared in the examples and comparative examples were stored in a refrigerator at -80°C, and after 24 hours, they were taken out and placed in a 4°C refrigerator for 6 hours of recovery, and then stability testing was performed for 144 hours. Samples were taken at 0h, 24h, 48h, 72h, 120h, and 144h, and three parallel experiments were set for each group. The stable storage refers to that in a cell preparation stored under certain storage conditions, the number of viable cells accounts for more than 90% of the total number of cells. The experimental results are shown in Table 3. Compared with Comparative Example 3, Comparative Example 1 and Comparative Example 2 only added raffinose and lysine, respectively, and the cell survival rate was significantly improved, and could be maintained at more than 90% at 72h. And the cell preparation prepared in the manner of Example 1 (which can be regarded as replacing part of the raffinose in Comparative Example 1 with lysine, or replacing part of the lysine in Comparative Example 2 with raffinose) has a significant improvement in cell survival rate, and the cell survival rate can be maintained at more than 90% at 144h, the storage time is significantly increased, which reflects the synergistic effect of raffinose and lysine.

[0052] Table 3 Stability test of different cell preparations at 4°C refrigerator

[0053] Note: The same letter in the same column indicates that there is no significant difference between the data (P>0.05), and different letters indicate that there is a significant difference between the data (P<0.05).

[0054] III. Long-term stability The shelf life of the cell cryopreservation preparation needs to be determined according to the long-term stability. According to the requirements of the "Guiding Principles for Stability Test of Biological Products" in the Chinese Pharmacopoeia 2020 edition 9402 and the characteristics of the product itself, long-term stability research was carried out under low temperature conditions: the cell preparations in the examples and comparative examples were filled into 100 mL backset bags with 50 mL syringes, sealed with a heat sealer, and divided into several bags for each group. The backset bags were stored in a-80°C deep freezer for 12 months, and the stability of the cell preparation in each backset bag was tested every 2 months. Each group was set up 3 parallel experiments. The cell reagent was taken out of the freezer and placed in a 4°C refrigerator for 6h, and did not need to be washed or cultured before detection or application. Tables 4-14 are the long-term stability test results of the cell preparations prepared in the examples and comparative examples, and Table 15 summarizes the cell survival rate indicators of different cell preparations.

[0055] Table 4 Long-term stability test of NKT cell preparation prepared in Example 1

[0056] Table 5 Long-term stability test of NKT cell preparation prepared in Example 2

[0057] Table 6 Long-term stability test of NKT cell preparation prepared in Example 3

[0058] Table 7 Long-term stability test of NKT cell preparation prepared in Example 4

[0059] Table 8 Long-term stability test of NKT cell preparation prepared in Example 5

[0060] Table 9 Long-term stability test of NKT cell preparation prepared in Example 6

[0061] Table 10 Long-term stability test of NKT cell preparation prepared in Comparative Example 1

[0062] Table 11 Long-term stability test of NKT cell preparation prepared by Comparative Example 2

[0063] Table 12 Long-term stability test of NKT cell preparation prepared by Comparative Example 3

[0064] Table 13 Long-term stability test of NKT cell preparation prepared by Comparative Example 4

[0065] Table 14 Long-term stability test of NKT cell preparation prepared by Comparative Example 5

[0066] Table 15 Summary of cell survival rate of long-term stability of different cell preparations

[0067] Note: The same column of data marked with the same letter means that there is no significant difference between the data (P>0.05), and different letters mean that there is a significant difference between the data (P<0.05).

[0068] From Tables 4-14, it can be seen that, except for the two indicators of viable cell number ratio and in vitro killing experiment, different cell preparations meet the standard; in terms of viable cell number, from Table 15, it can be seen that, compared with Comparative Example 3, Comparative Examples 1 and 2 only added raffinose and lysine, respectively, and the cell survival rate was significantly improved, which could be maintained at more than 90% for the first 4 months; and the cell survival rate of the cell preparation prepared according to the method of Example 1 was significantly improved, which could be maintained at more than 90% for 12 months, the storage time was significantly increased, which reflected the synergistic effect of raffinose and lysine.

[0069] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. An NKT cell preparation, characterized in that, The NKT cell preparation includes NKT cells, compound electrolyte injection, 5%-10% human serum albumin, 5%-10% raffinose, and 2%-5% lysine.

2. The NKT cell preparation according to claim 1, characterized in that, Each 1000 mL of the compound electrolyte injection solution contains 5.26 g of sodium chloride, 5.02 g of sodium gluconate, 3.68 g of sodium acetate, 0.37 g of potassium chloride, and 0.30 g of magnesium chloride.

3. The NKT cell preparation according to claim 1, characterized in that, The concentration of the NKT cells in the cell preparation was 1.0 × 10⁻⁶. 6 -1.0×10 8 per mL.

4. The NKT cell preparation according to claim 1, characterized in that, The surface marker molecules CD3, CD56, and CD8 of the NKT cells were detected by flow cytometry. The requirements were that CD3 (+) and CD56 (+) ≥70%, CD56 (+) ≥90%, CD3 (+) ≥80%, and CD3 (+) and CD8 (+) ≥80%.

5. The method for preparing the NKT cell preparation according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Preparation of NKT cells and compound electrolyte injection solution; S2. Take the compound electrolyte injection solution, add human serum albumin, raffinose and lysine, make up the volume with the compound electrolyte injection solution, filter and sterilize to obtain the filtrate. S3. Resuspend NKT cells in the filtrate obtained in step S2 to prepare NKT cell preparation.

6. The preparation method according to claim 5, characterized in that, Step S2 includes: Take 20-30 mL of compound electrolyte injection, add 25-50 mL of 20% human serum albumin, 5-10 g of raffinose, 2-5 g of lysine, and adjust the volume to 50-175 mL with compound electrolyte injection. Filter to obtain the filtrate.

7. The preparation method according to claim 6, characterized in that, Step S2 includes: taking 20mL of compound electrolyte injection, adding 25mL of 20% human serum albumin, adding 5g of raffinose, adding 2g of lysine, and adjusting the volume to 100mL with compound electrolyte injection, filtering and sterilizing to obtain filtrate. Step S3 includes resuspending NKT cells in the filtrate obtained in step S2 to a final concentration of 1.0 × 10⁻⁶. 8 NKT cell preparations were obtained by using cells per mL.

8. The use of the NKT cell preparation according to any one of claims 1-4 or the preparation method according to any one of claims 5-7 in the preparation of NKT cell drugs or NKT cell preservation.

9. The application according to claim 8, characterized in that, The applications include storing NKT cells.

10. The application according to claim 9, characterized in that, The storage conditions for NKT cells are between -80°C and 4°C.

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