NKT cell preparation, method for preparing the same, and use thereof

Abstract

The application provides an NKT cell preparation and a preparation method and application thereof, and belongs to the field of biological medicine. The NKT cell preparation provided by the application comprises NKT cells, a compound electrolyte injection, 5%-10% human blood albumin, 5%-10% raffinose and 2%-5% lysine, wherein the concentration of the NKT cells in the cell preparation is 1.0x10 6 -1.0x10 8 The prepared finished preparation has a long shelf life and can be stored stably at 4 DEG C for more than 144 hours and stored stably 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 6-1.0×10 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:

[0009] 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.

[0010] Preferably, 1000 mL of 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.

[0011] Preferably, the concentration of the NKT cells in the cell preparation is 1.0×10 6 -1.0×10 8 / mL.

[0012] 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(+)≥70%, CD56(+)≥90%, CD3(+)≥80%, CD3(+) and CD8(+)≥80%.

[0013] In another aspect, the present application provides a preparation method of the above-mentioned NKT cell preparation, comprising the following steps:

[0014] S1, preparing NKT cells and compound electrolyte injection;

[0015] 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;

[0016] S3, resuspending the NKT cells with the filtrate prepared in step S2 to obtain a NKT cell preparation.

[0017] Preferably, the step S2 comprises:

[0018] 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.

[0019] 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

[0020] In another aspect, the present application provides an application of the NKT cell preparation or the preparation method described above in the preparation of NKT cell drugs or the preservation of NKT cells.

[0021] Preferably, the application comprises the storage of NKT cells.

[0022] Preferably, the storage condition of the NKT cells is -80℃ to 4℃.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] 1. The NKT cell preparation provided by the present application has fewer types of components and a simple preparation method, and does not contain DMSO, animal-derived proteins and other substances with safety risks.

[0025] 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

[0026] Unless otherwise specified, all raw materials and reagents in the present application are purchased from commercial suppliers, without further purification, and experiments are carried out according to the operation instructions. The raw materials and reagents susceptible to moisture are stored in a fully sealed bottle and are used directly, without special treatment. The instruments, equipment and devices used in the present application are all conventional instruments, equipment and devices.

[0027] Data analysis is performed using professional data processing software, and single-factor ANOVA test is used for significance analysis, and P<0.05 represents significant difference.

[0028] Table 1 explains the sources of the key raw materials involved in the present application.

[0029] Table 1 Raw material source explanation

[0030]

[0031] 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. ​

[0032] 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%.

[0033] The NKT cell preparation is prepared by the following steps:

[0034] (1) Take the compound electrolyte injection, add the human blood albumin, the raffinose and the lysine, make up the volume with the compound electrolyte injection, and filter and sterilize through a 0.22 μm filter membrane;

[0035] (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;

[0036] (3) Resuspend the NKT cells with the filtrate prepared in step (1) to obtain the NKT cell preparation;

[0037] (4) The NKT cell preparation can be filled into a 100 mL reinfusion bag with a 50 mL syringe, sealed with a heat sealer, and stored at low temperature or ultralow temperature.

[0038] Preparation Example: Preparation of NKT cells

[0039] Harvest the NKT cells cultured by the NKT cell culture kit (purchased from Shenzhen Mersel 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.

[0040] Example 1: Preparation of NKT cell preparation

[0041] The preparation method of each 100 mL cell preparation is as follows:

[0042] (1) Take 20 mL of compound electrolyte injection, add 25 mL of 20% human blood albumin, add 5 g of raffinose, and add 2 g of lysine, make up the volume 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%);

[0043] (2) Resuspend NKT cells with the filtrate prepared in step (1) to a final concentration of 1.0 x 10 8 and obtain a cell preparation.

[0044] The cell preparation is filled into a 100 mL reinfusion bag with a 50 mL syringe and sealed with a heat sealer.

[0045] Example 2: Preparation of NKT cell preparation

[0046] The preparation method of each 100 mL cell preparation is as follows:

[0047] (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 sterilize through a 0.22 μm filter (the mass fraction of human serum albumin in the cell preparation is 10%);

[0048] (2) Resuspend NKT cells with the filtrate prepared in step (1) to a final concentration of 1.0 x 10 8 and obtain a cell preparation.

[0049] Example 3: Preparation of NKT cell preparation

[0050] The difference from Example 1 is that the final concentration of NKT cells is 1.0 x 10 7 , and the others are the same.

[0051] Example 4: Preparation of NKT cell preparation

[0052] The difference from Example 1 is that the final concentration of NKT cells is 1.0 x 10 6 , and the others are the same.

[0053] Example 5: Preparation of NKT cell preparation

[0054] The difference from Example 2 is that the final concentration of NKT cells is 1.0 x 10 7 , and the others are the same.

[0055] Example 6: Preparation of NKT cell preparation

[0056] The difference from Example 2 is that the final concentration of NKT cells is 1.0 x 10 6 , and the others are the same.

[0057] Comparative Example 1: Preparation of NKT cell preparation

[0058] The difference from Example 1 is that 7 g of raffinose is added and no lysine is added, and the others are the same.

[0059] Comparative Example 2: Preparation of NKT cell preparation

[0060] The difference from Example 1 is that raffinose is not added, and 7 g of lysine is added, and the others are the same.

[0061] Comparative Example 3: Preparation of NKT cell preparation

[0062] The difference from Example 1 is that neither raffinose nor lysine is added, and the others are the same.

[0063] Comparative Example 4: Preparation of NKT cell preparation

[0064] The difference from Example 1 is that raffinose is replaced by an equal amount of trehalose, and the others are the same.

[0065] Comparative Example 5: Preparation of NKT cell preparation

[0066] The difference from Example 1 is that lysine is replaced by an equal molar amount of serine, and the others are the same.

[0067] Test Example 1: Stability test of NKT cell preparation

[0068] I. Experimental methods and standards:

[0069] (1) Bovine serum albumin detection

[0070] According to the 2020 edition of Chinese Pharmacopoeia 4 parts 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.

[0071] (2) Sterility test

[0072] According to the 2020 edition of Chinese Pharmacopoeia 4 parts 1101 sterility test method, the cell preparation is detected, and the requirement is no bacterial growth.

[0073] (3) Endotoxin test

[0074] According to the 2020 edition of Chinese Pharmacopoeia 4 parts 1143 endotoxin gel limit method, the cell preparation is detected, and the requirement is less than 0.5 EU / mL.

[0075] (4) Mycoplasma test

[0076] According to the 2020 edition of Chinese Pharmacopoeia 4 parts 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.

[0077] (5) pH value test

[0078] According to the 2020 edition of Chinese Pharmacopoeia 4 parts 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.

[0079] (6) Osmotic pressure test

[0080] According to the "Freezing point depression method" in Chinese Pharmacopoeia 2020 edition 3 part 0632, 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.

[0081] (7) NKT cell marker identification

[0082] The surface marker molecules CD3, CD56 and CD8 of the cells are detected by flow cytometry, and the requirements are that CD3 (+) and CD56 (+) ≥ 70%, CD56 (+) ≥ 90%, CD3 (+) ≥ 80%, and CD3 (+) and CD8 (+) ≥ 80%.

[0083] (8) Number of viable cells

[0084] The trypan blue staining method commonly used in the art is used to count the viable cells with a cell counter, and the percentage (%) of viable cells in the total cell count is calculated.

[0085] (9) In vitro killing (toxicity) experiment

[0086] The quantitative determination of human NKT cytotoxic activity can be determined using target cells K562. The 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 add target cells with a density of 1×10 5 (K562) cells / mL in each well, and after adding 10 ng / mL SDF-1α, adhere at 37°C for 1 hour. Then rinse the well plate with HBSS. Add NKT cells with a density of 5×10 5 cells / mL in HBSS to each well (in triplicate), and incubate at 37°C. Specifically, using a kit (LIVE / DEAD™ Cell-mediated Cytotoxicity Assay Kit, purchased from Invitrogen), fluorescence imaging detection is performed by fluorescence imaging system imaging, and image analysis software is used for analysis to obtain the in vitro killing rate of NKT cells.

[0087] Table 2 summarizes the detection items, detection methods and standards of the cell preparation involved in the examples and comparative examples.

[0088] Table 2 NKT cell preparation detection items, detection methods and standards

[0089]

[0090] II. Storage stability at 4°C

[0091] The cell preparations prepared in the examples and comparative examples were stored in a refrigerator at -80°C, taken out after 24 h, placed in a 4°C refrigerator for 6 h of recovery, and then subjected to stability testing for 144 h. Sampling was performed at 0 h, 24 h, 48 h, 72 h, 120 h, and 144 h, respectively, and 3 parallel experiments were set for each group. The stable storage refers to that in the 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, which could be maintained at more than 90% at 72 h. The cell survival rate of 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) was significantly improved, and the cell survival rate at 144 h could be maintained at more than 90%, and the storage time was significantly increased, which reflected the synergistic effect of raffinose and lysine.

[0092] Table 3 Storage stability testing of different cell preparations in a 4°C refrigerator

[0093]

[0094] Note: The same column of data is marked with the same letter to indicate 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).

[0095] III. Long-term stability

[0096] The shelf life of the cell cryopreservation preparation needs to be determined according to the long-term stability. According to the requirements of “Guiding Principles for Stability Testing of Biological Products” in 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. Three parallel experiments were set for each group. The cell preparation was taken out of the freezer and placed in a 4°C refrigerator for 6 h of recovery, and no further washing or culturing was required 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.

[0097] Table 4 Long-term stability testing of NKT cell preparation prepared in Example 1

[0098]

[0099] Table 5 Long-term stability testing of NKT cell preparation prepared in Example 2

[0100]

[0101] Table 6 Long-term stability testing of NKT cell preparation prepared in Example 3

[0102]

[0103] Table 7 Long-term stability testing of NKT cell preparation prepared in Example 4

[0104]

[0105] Table 8 Long-term stability testing of NKT cell preparation prepared in Example 5

[0106]

[0107] Table 9 Long-term stability testing of NKT cell preparation prepared in Example 6

[0108]

[0109] Table 10 Long-term stability testing of NKT cell preparation prepared in Comparative Example 1

[0110]

[0111] Table 11 Long-term stability testing of NKT cell preparation prepared in Comparative Example 2

[0112]

[0113] Table 12 Long-term stability testing of NKT cell preparation prepared in Comparative Example 3

[0114]

[0115] Table 13 Long-term stability testing of NKT cell preparation prepared in Comparative Example 4

[0116]

[0117] Table 14 Long-term stability testing of NKT cell preparation prepared in Comparative Example 5

[0118]

[0119] Table 15 Summary of cell viability for long-term stability of different cell preparations

[0120]

[0121] 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 marked with different letters means that there is a significant difference between the data (P<0.05).

[0122] As can be seen from Tables 4-14, the different cell preparations meet the standards except for the two indicators of the proportion of viable cell number and in vitro killing experiment; in terms of the viable cell number, as can be seen from Table 15, compared with Comparative Example 3, the cell survival rate of Comparative Example 1 and Comparative Example 2 is obviously improved by adding only stachyose and lysine respectively, and the cell survival rate can be maintained above 90% in the first 4 months; and the cell survival rate is obviously improved by preparing the cell preparation according to the method of Example 1, and the cell survival rate can be maintained above 90% in 12 months, the storage time is significantly increased, which reflects the synergistic effect of stachyose and lysine.

[0123] 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. A preparation of NKT cells, characterized in that, The NKT cell preparation comprises NKT cells, compound electrolyte injection, 5-10% human blood albumin, 5-10% raffinose and 2-5% lysine; 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 per 1000 mL; wherein % refers to mass fraction.

2. The NKT cell preparation of claim 1, wherein, The concentration of the NKT cells in the cell preparation is 1.0 x 10 6 -1.0 x 10 8 individuals.

3. The NKT cell preparation of claim 1, wherein, 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(+) are 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(+) are greater than or equal to 80%.

4. A method for the preparation of the NKT cell preparation according to any one of claims 1 to 3, characterized in that, The method 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.

5. The preparation method according to claim 4, characterized in that, 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.

6. The preparation method according to claim 5, characterized in that, The step S2 comprises: taking 20 mL of compound electrolyte injection, adding 20% human blood albumin 25 mL, adding raffinose 5 g, adding lysine 2 g, using compound electrolyte injection to make up the volume to 100 mL, filtering and sterilizing to obtain a filtrate. The step S3 comprises resuspending the NKT cells with the filtrate prepared in step S2 to a final concentration of 1.0 x 10 8 cells / mL to prepare the NKT cell preparation.

7. Use of the NKT cell preparation of any one of claims 1-3 or the preparation method of any one of claims 4-6 in the preparation of NKT cell drugs or the preservation of NKT cells.

8. Use according to claim 7, characterized in that, The use comprises storing NKT cells, and the storage condition of the NKT cells is at-80℃ to 4℃.

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