A cell drug cryopreservation preparation, a preparation method thereof and application thereof

Abstract

The application provides a cell drug cryopreservation preparation and a preparation method and application thereof, relates to the technical field of gene modified NK cell drug cryopreservation, and is characterized in that a basic cell cryopreservation solution is uniformly mixed with one or more of human blood albumin solution, polysaccharide solution, a surfactant, an apoptosis inhibitor and a compound electrolyte injection to obtain a cell drug cryopreservation preparation. The cryopreservation preparation and the cryopreservation method of the application have specific protective effects on gene modified NK cell drugs, and the cryopreservation preparation does not contain serum components, so that the possibility of animal pathogen pollution is reduced. The gene modified NK cell drugs are cryopreserved by using the cryopreservation preparation and the cryopreservation method of the application, so that the cell viability, the chimeric antigen receptor (CAR) positive rate, the long-term stability and the killing function on tumor cells can be maintained.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the cryopreservation of gene-modified immune cell drugs and the cryopreservation of immune cell drugs. Specifically, it relates to a CAR NK cell drug cryopreservation preparation and cryopreservation method and its application. Background Technology

[0002] Natural killer (NK) cells are important immune cells and a crucial component of innate immunity. Under physiological conditions, NK cell inhibitory receptors recognize major histocompatibility complex-I (MHC-I) molecules widely expressed on the surface of normal tissue cells, thus inhibiting NK cell function and preventing them from killing their own normal tissue cells. In tumor tissues, the expression of MHC-I molecules on the surface of tumor cells is usually downregulated, while the expression of ligands of activating receptors, such as NKp30, NKp44, and NKp46, is upregulated, leading to NK cell activation and ultimately the killing of tumor cells. NK cells kill target cells mainly through three pathways: 1) directly killing target cells by releasing cytoplasmic granules containing perforin and granzymes; 2) releasing cytokines, such as IFN-γ and TNF-α, which induce tumor cell apoptosis through interaction with corresponding receptors on the surface of tumor cells; and 3) the binding of the Fc receptor CD16 to the Fc fragment of antibodies, which can trigger antibody-dependent cell-mediated cytotoxicity (ADCC) to kill cells. Chimeric Antigen Receptor NK (CAR NK) cells are genetically modified NK cells that express CAR molecules on their surface. These CAR molecules recognize target antigens and activate the NK cells to kill tumor cells. The CAR structure of CAR NK cells typically consists of three parts: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular activation domain.

[0003] Cryoprotectants are generally classified into two types: permeable and non-permeable. Permeable cryoprotectants can penetrate into cells, reducing the electrolyte concentration in the intracellular and extracellular solutions, preventing intracellular water leakage, avoiding excessive cell dehydration and shrinkage, and preventing the formation of ice crystals in the intracellular and extracellular environments. Permeable cryoprotectants are generally small molecules, mainly including dimethyl sulfoxide (DMSO), glycerol (GLY), methanol (METH), ethylene glycol (GE), and propylene glycol (PG). Permeable cryoprotectants, once inside the cell, can enhance membrane fluidity and partially dehydrate the cell, leading to a lower freezing point, reducing the number and size of intracellular ice crystals, and protecting cells from damage during freezing and thawing. However, permeable cryoprotectants themselves are toxic to cells. Non-permeable cryoprotectants are generally large molecules, requiring high concentrations for good cell viability and condition after cryopreservation and thawing. However, high concentrations increase the risks of reintroduction into the human body and also increase usage costs.

[0004] In summary, there are currently no clinical-grade cryopreserved formulations of CAR NK cell therapy drugs to demonstrate their medicinal value. Therefore, all reported early "clinical" studies utilize fresh formulations for clinical exploration. Maintaining the viability, CAR positivity rate, cytotoxicity, and long-term cell stability of cryopreserved CAR NK cell therapy products remains a significant hurdle. Therefore, developing a cryopreserved formulation of CAR NK cell therapy is of great importance to the field of immunotherapy. Summary of the Invention

[0005] The thawing effect after cryopreservation of cell drugs is crucial for cryopreservation. For cell drugs, biosafety is a critical consideration. While existing cell cryopreservation formulations can preserve cells, their thawing effects on cells, especially CAR NK cell drugs, are inconsistent, and biosafety issues exist. This invention discloses a novel cell cryopreservation formulation that can maintain the viability, CAR positivity rate, cytotoxicity, and long-term cell stability of CAR NK cell drug products, which is of great significance for the field of immunocellular therapy.

[0006] The present invention adopts the following technical solution:

[0007] A cell drug cryopreservation preparation includes a basic cell cryopreservation solution and additives; the additives include one or more of human serum albumin solution, polysaccharide solution, surfactant, apoptosis inhibitor, and compound electrolyte injection.

[0008] This invention discloses a method for preparing the above-mentioned cell drug cryopreservation formulation, wherein a basic cell cryopreservation solution is mixed evenly with one or more of human serum albumin solution, polysaccharide solution, surfactant, cell apoptosis inhibitor, and compound electrolyte injection to obtain the cell drug cryopreservation formulation.

[0009] In the cell drug cryopreservation formulation of this invention, the basic cell cryopreservation solution is a conventional product with a volume percentage of 50-80%, the human serum albumin solution has a volume percentage of 0-30%, the polysaccharide solution has a volume percentage of 0-30%, the surfactant has a volume percentage of 0-10%, the compound electrolyte injection has a volume percentage of 0-30%, and the apoptosis inhibitor has a concentration of 10-80 μM. Preferably, in the cell drug cryopreservation formulation, the basic cell cryopreservation solution has a volume percentage of 50-80%, the human serum albumin solution has a volume percentage of 5-25%, the polysaccharide solution has a volume percentage of 10-25%, the surfactant has a volume percentage of 0-5%, the compound electrolyte injection has a volume percentage of 0-25%, and the apoptosis inhibitor has a concentration of 10-80 μM. In a further preferred embodiment, the volume percentage of the basal cell cryopreservation solution in the cell drug cryopreservation formulation is 50-60%, the volume percentage of the human serum albumin solution is 20-25%, the volume percentage of the polysaccharide solution is 20-25%, and the concentration of the apoptosis inhibitor is 10-80 μM; wherein the concentration of the apoptosis inhibitor can be 20-80 μM or 40-80 μM.

[0010] In this invention, the polysaccharide includes one or more of fructose, trehalose, and dextran. Preferably, the dextran includes any one or more of dextran 20, dextran 40, or dextran 70. The apoptosis inhibitor includes one or more of Baxinhibitor-5, Y-27632, and Pifithrin-μ. The compound electrolyte injection has a certain effect on maintaining cell osmotic pressure balance and acid-base balance, and its main components include one or more of sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride.

[0011] This invention discloses a method for cryopreserving cell drugs using the above-mentioned cell drug cryopreservation formulation, comprising the following steps: mixing the cell drug with the cell drug cryopreservation formulation, and then cryopreserving using a programmed freezing device to complete the cell drug cryopreservation. Preferably, the cryopreservation procedure is as follows: standing at 10-30°C, then cooling to -5--20°C, then cooling to -40--60°C, then heating to -20--40°C, then cooling to -40--60°C, further cooling to -80--100°C, then placing at -80°C, and finally placing in liquid nitrogen; thus completing the cryopreservation. A further preferred cryopreservation procedure is as follows: stand at 20°C for 10–30 min; cool to -15°C at 0.5–2°C / min; cool to -50°C at 10–30°C / min; heat to -30°C at 5–10°C / min; cool to -50°C at 0.5–2°C / min; cool to -90°C at 5–10°C / min; then transfer to a -80°C freezer for 20 hours, and finally place in a liquid nitrogen tank for long-term cryopreservation.

[0012] Preferably, when using the above-mentioned cell drug cryopreservation formulation to cryopreserve cells, the cell density is 1–10 × 10⁻⁶ cells / year. 7 per ml.

[0013] In this invention, the cell drug includes one or more of immune cells and gene-modified immune cells, and the gene-modified immune cells include CAR-modified and / or TCR-modified immune cells; preferably, the cells include one or more of NK cells and gene-modified NK cells; the gene-modified NK cells include CAR NK and / or TCR NK.

[0014] This invention discloses the application of the above-mentioned cell cryopreservation preparation in the preparation of cell drugs, especially immunotherapy cell drugs, particularly NK cell drugs.

[0015] This invention discloses the application of the above-mentioned cell drug cryopreservation formulation in the cryopreservation of genetically modified immune cells or immune cells. Preferably, this invention discloses the application of the above-mentioned cell drug cryopreservation formulation in the cryopreservation of CAR NK cell drugs.

[0016] Commonly used cryopreservation formulations for cell therapies contain a certain concentration of fetal bovine serum (FBS) or serum substitutes. FBS and its substitutes increase the introduction of animal-derived components, raising the risk of viral infection. For example, existing cryopreservation solutions consist of 50% GT-T551 H3 medium, 10% dimethyl sulfoxide (MSO), and 40% FBS; or a cryopreservation solution made from fetal bovine serum and DMSO in a 9:1 ratio. Therefore, developing cryopreservation formulations for CAR NK cell therapies that are free of animal-derived components and have clearly defined compositions will significantly improve their compliance as raw materials for cell therapy products. This invention provides a CAR NK cell drug cryopreservation preparation and cryopreservation method, and its application in the preparation of immunotherapy cell drugs. The preparation utilizes one or more of the following: a basic cell cryopreservation solution, human serum albumin, polysaccharides, surfactants, apoptosis inhibitors, and compound electrolyte injections. Preferably, the cell drug preparation includes a basic cell cryopreservation solution, human serum albumin, polysaccharides, and apoptosis inhibitors. This cryopreservation preparation ensures long-term cryopreservation of the CAR NK cell drug and maintains cell viability, CAR positivity, cytotoxicity, and long-term cell stability. Attached Figure Description

[0017] Figure 1 Cell viability and recovery rate for three cryopreservation densities.

[0018] Figure 2 To assess cell viability in different cryopreservation formulations.

[0019] Figure 3 To assess cell viability in different cryopreservation formulations, cells were stored at room temperature.

[0020] Figure 4 Cell viability of cryopreserved formulations with different proportions of HSA.

[0021] Figure 5 Recovery rates of HSA cryopreserved formulations with different proportions.

[0022] Figure 6 The CAR positivity rate was calculated for the control cryopreservation procedure group and the optimized cryopreservation procedure group.

[0023] Figure 7 To compare the viability of the cryopreservation procedure group and the optimized cryopreservation procedure group.

[0024] Figure 8 Recovery rates of the control cryopreservation procedure group and the optimized cryopreservation procedure group.

[0025] Figure 9 The cell viability of cryopreservation preparations 4 and 5 within 2 months of cryopreservation.

[0026] Figure 10 The cell recovery rate within 2 months of cryopreservation of formulations 4 and 5.

[0027] Figure 11 The CAR positivity rate of cells within 2 months of cryopreservation of formulations 4 and 5.

[0028] Figure 12 The cell-killing efficacy of cryopreservation formulations 4 and 5 within 2 months of cryopreservation.

[0029] Figure 13 Cell viability is the value of the cryopreservation formulation containing Pifithrin-μ.

[0030] Figure 14 The CAR positivity rate of cells containing Pifithrin-μ cryopreservation formulation.

[0031] Figure 15 The results are for cell killing assays using a cryopreservation formulation containing Pifithrin-μ. Detailed Implementation

[0032] This invention discloses the application of the above-mentioned cell drug cryopreservation formulation in the cryopreservation of genetically modified immune cells or immune cells. Preferably, this invention discloses the application of the above-mentioned cell cryopreservation formulation in the cryopreservation of CAR NK cell drugs.

[0033] Specifically, this invention provides a CAR NK cell drug cryopreservation formulation and cryopreservation method, and its application in the preparation of immunotherapy cell drugs. The cryopreservation formulation can ensure long-term cryopreservation of CAR NK cell drugs and maintain cell viability, CAR positivity, cytotoxicity, and long-term cell stability.

[0034] The CAR NK cell drug cryopreservation formulation provided by the present invention includes one or more components of the basic cell cryopreservation solution Cryptor CS10, human serum albumin solution, polysaccharide (fructose, trehalose, dextran 40) solution, polyvinylpyrrolidone (PVP) and apoptosis inhibitors (Bax inhibitor-5, Y-27632, Pifithrin-μ), and compound electrolyte injection.

[0035] Preferably, the volume percentage of the Cryptotor CS10 is 50-80%.

[0036] Preferably, the volume percentage of the human serum albumin solution is 0-30%; wherein the mass concentration of the human serum albumin solution is 0-30%, preferably 15-25%.

[0037] Preferably, the volume percentage of the polysaccharide solution is 0-25%; wherein the mass concentration of the polysaccharide solution is 5-20%, preferably 10-15%.

[0038] Preferably, the volume percentage of polyvinylpyrrolidone (PVP) is 0-10%.

[0039] Preferably, the concentration of the apoptosis inhibitor (preferably 2-phenylethynyl sulfonamide Pifithrin-μ) is 10–80 μM, more preferably 20–40 μM.

[0040] Preferably, the dextran includes any one or a combination of at least two of dextran 20, dextran 40, or dextran 70.

[0041] This invention provides a method for cryopreserving CAR NK cell drugs, the cryopreservation procedure being as follows:

[0042] (1) Let stand at 20℃ for 10-30 minutes;

[0043] (2) Cool down to -15℃ at a rate of 0.5–2℃ / min.

[0044] (3) Cool down to -50℃ at a rate of 10-30℃ / min;

[0045] (4) Increase the temperature to -30℃ at a rate of 5-10℃ / min;

[0046] (5) Cool down to -50℃ at a rate of 0.5–2℃ / min;

[0047] (6) Cool down to -90℃ at a rate of 5-10℃ / min;

[0048] (7) After being placed in a -80℃ freezer for 20 hours, it is then placed in a liquid nitrogen tank for long-term cryopreservation.

[0049] This invention provides a CAR NK cell drug cryopreservation formulation and cryopreservation method, wherein the CAR NK cell density is 5 × 10⁻⁶. 7 cells / ml.

[0050] This invention provides a CAR NK cell drug cryopreservation formulation and cryopreservation method, which is suitable for use in the preparation of immunotherapy cell drugs.

[0051] The following experiments demonstrate the innovation of this invention. The raw materials were commercially available products, and the preparation procedures and performance tests were performed using conventional techniques. Unless otherwise specified, all proportions and percentages in the formulation are by volume. The data underwent routine statistical analysis, and *, P < 0.05, indicating significant differences.

[0052]

[0053] The frozen anti-CD19 CAR NK cell therapy drug was removed from liquid nitrogen and thawed in a 37°C water bath. The thawed cells were then transferred to a sterile centrifuge tube containing 10 mL of PBS, centrifuged at 500 g for 5 min, and the supernatant was discarded. The cells were resuspended in 2 mL of NK cell culture medium (EcoSy NK medium + 10% FBS). 10 μL of the cell suspension was mixed with AO / PI, and cell viability was assessed using the AO / PI method. The recovery rate was calculated as: (Total number of viable cells after cryopreservation / Total number of viable cells before cryopreservation) × 100%. Unless otherwise specified, thawing time refers to thawing the cells at 37°C (approximately 2 minutes) after removal from liquid nitrogen, followed by testing.

[0054] The cryopreservation procedure is as follows: 1. Stand at 20℃ for 20 min; 2. Cool down to -15℃ at 1℃ / min; 3. Cool down to -50℃ at 20℃ / min; 4. Heat up to -25℃ at 10℃ / min; 5. Cool down to -60℃ at 1℃ / min; 6. Cool down to -90℃ at 10℃ / min; 7. After placing in a -80℃ freezer for 20 hours, place in a liquid nitrogen tank for cryopreservation.

[0055] The optimized cryopreservation procedure is as follows: 1. Stand at 20℃ for 20 min; 2. Cool down to -15℃ at 1℃ / min; 3. Cool down to -50℃ at 15℃ / min; 4. Heat up to -30℃ at 10℃ / min; 5. Cool down to -50℃ at 1℃ / min; 6. Cool down to -90℃ at 10℃ / min; 7. After placing in a -80℃ freezer for 20 hours, place in a liquid nitrogen tank for cryopreservation.

[0056] Example 1: Preparation of cryopreservation formulation of anti-CD19 CAR NK cell drug

[0057] This embodiment provides a method for preparing a cryopreservation formulation of an anti-CD19 CAR NK cell drug as follows: Under room temperature conditions, in a biosafety cabinet, according to the following volume percentages: CS10 ratio of 50-75 V / V%, HSA solution ratio of 0-25 V / V%, dextran 40 solution ratio of 0-10 V / V%, compound electrolyte injection ratio of 0-25 V / V%, and 2-phenylacetylenesulfonamide concentration of 0-80 μM, each component is added sequentially according to the required cryopreservation formulation concentration in Table 1 to prepare the cryopreservation formulation; after adding all components, gently shake to mix, and store at 4°C for later use.

[0058] Table 1 Different cryopreservation preparations

[0059]

[0060] Example 2: Preparation of anti-CD19 CAR NK cell drug using conventional methods

[0061] The experimental steps are briefly described below:

[0062] 1. Construction of CD19 CAR retroviral vector

[0063] The nucleotide sequence (DNA fragment) encoding a chimeric antigen receptor (CAR) was artificially synthesized at Beijing Qingke Biotechnology Co., Ltd. The nucleotide sequence is SEQ ID NO: 1, and the amino acid sequence is SEQ ID NO: 2. The CD19 CAR DNA fragment and the pMSCV DNA fragment were homologously recombinated using recombinase to construct a pMSCV retroviral vector plasmid containing the CD19 CAR.

[0064] 2. Preparation of retroviruses

[0065] The CD19 CAR retroviral vector plasmid and the retroviral helper packaging plasmid were co-transfected into 293T cells to prepare retroviruses. The experimental steps are briefly described below:

[0066] (1) Resuscitate 293T cells, add 5E+06 cells to a T75 cell flask, use DMEM medium containing 10% FBS, and place in a CO2 cell culture incubator for culture;

[0067] (2) When the cell confluence reaches about 80%, change to fresh cell culture medium (DMEM medium containing 10% FBS).

[0068] (3) Take 500 μL of opti-DMEM medium, add 10 μg of pMSCV CD19 CAR plasmid, 10 μg of pUMVC plasmid and 5 μg of pRD114 plasmid and mix well;

[0069] (4) Take another 500 μL of opti-DMEM medium, add 25 μg of polyetherimide (PEI) solution and mix well;

[0070] (5) Add the PEI solution to the plasmid solution in step (3) and mix well. After standing at room temperature for 20 min, add it to 293T cells. After incubation for 8 hours, remove the culture medium and add 15 ml of fresh cell culture medium.

[0071] (6) Collect the culture medium supernatant 48 hours after transfection, centrifuge at 400g for 5 minutes, and aliquot the supernatant virus solution and store it in a -80℃ freezer for NK cell transduction.

[0072] 3. Preparation of Anti-CD19 CAR NK Cell Drugs by Retroviral Transduction of NK Cells

[0073] NK cells were sorted from human peripheral blood mononuclear cells (PBMCs) according to the Miltenyi Biotec NK Cell Isolation Kit (130-092-657) instructions. After sorting, K562 feeder cells were used for activation. The experimental steps are briefly described below:

[0074] (1) Melt 25M human peripheral blood mononuclear cells (PBMCs) in a 37℃ water bath. Take 10μL of PBMCs in a biosafety cabinet and count them using a cell counter.

[0075] (2) Transfer the CBMCs to a 15ml centrifuge tube, add 6ml of pre-cooled 1X DPBS, and centrifuge at 400g for 5 minutes. After centrifugation, discard the supernatant and resuspend the CBMCs in MACS buffer (40μL / 1E+07 cells).

[0076] (3) Then add NK cell Biotin-Antibody Cocktail (10 μL / 1E+07 cells), mix well and incubate at 4 degrees Celsius for 5 minutes.

[0077] (4) Add MACS buffer (30 μL / 1E+07 cells), then add NK Cell Microbeads Cocktail (20 μL / 1E+07 cells), mix well and incubate at 4°C for 10 minutes.

[0078] (5) Rinse the LS column (Miltenyi Biotec, 130-042-401) with 3ml MACS buffer, then add the cell suspension to the LS column, and immediately add 3ml MACS buffer. After all the cell suspension has flowed into the 15ml centrifuge tube, add another 3ml MACS buffer and collect all the cells into the 15ml centrifuge tube.

[0079] (6) Centrifuge the cell suspension at 400g for 10 minutes. Resuspend the cells in Ecosai NK medium containing 10% FBS, take 10μL of cells for counting, and add K562 feeder to the NK cells at a ratio of NK:K562 feeder = 1:2 to activate cell culture.

[0080] 4. CD19 retrovirus transduction of NK cells

[0081] On day 5 of NK cell activation culture, CD19 retrovirus was added to prepare anti-CD19 CAR NK cell drug. The experimental steps are briefly described below: Activated NK cells were transferred to 6-well cell culture plates, and CD19 retrovirus was added at a multiplicity of infection (MOI) of 1. Then, 10 µg / mL of Vectofusin-1 (Miltenyi Biotec, 130-111-163) transduction aid was added. The cell culture medium was Ecosai NK cell medium (Ecosai, NE000-N012) containing 5% FBS and 200 IU IL-2, with a cell density of 5E+05 cells / ml. The cells were centrifuged at 1000g for 30 minutes and then cultured in a CO2 cell incubator.

[0082] Example 3: Effect of cryopreservation formulations on different cell cryopreservation densities

[0083] Inside the biosafety cabinet, anti-CD19 CAR NK cells were pipetted into a 50ml centrifuge tube. 20μL of the cells were counted, and the remaining cells were centrifuged at 600 rpm for 10 minutes. The supernatant was discarded, and the cells were resuspended in a cryopreservation formulation consisting of 75% CS10 and 25% HSA solution. The cell density was adjusted as shown in Table 2, and the cells were added to 1ml cryopreservation tubes. The cells were cryopreserved using the control cryopreservation procedure.

[0084] Table 2 Different cell cryopreservation densities

[0085]

[0086] After 3 days of cryopreservation in liquid nitrogen, cell viability and recovery rate were as follows: Figure 1As shown, the three cryopreservation densities did not affect the viability and recovery rate of the anti-CD19 CAR NK cell therapy. The experimental results indicate that the cryopreservation formulation is suitable for different cryopreservation densities of the anti-CD19 CAR NK cell therapy. Example 4

[0087] Cells were cryopreserved using a control cryopreservation procedure. The cryopreservation preparation and cryopreservation density are shown in Table 1. Cells were cryopreserved for 10 days. The anti-CD19 CAR NK cell drug was removed from liquid nitrogen, thawed at 37°C, and then placed at room temperature for 0, 0.5, 1, 1.5, and 2 hours, respectively, and cell viability was measured.

[0088] At 0 hours, the cell resuscitation viability of Formula 5 was significantly better than that of Formula 1, showing a statistically significant difference. (See [link to relevant documentation]). Figure 2 Furthermore, the results of formulations 2 and 3, which contain compound electrolytes, are similar to those of formulation 1, indicating that compound electrolytes cannot improve cell resuscitation survival rate in this invention.

[0089] Within 0 to 2 hours, cell viability remained highest in cryopreservation preparation group 5, and the viability stability after thawing was better than other cryopreservation preparation groups, as shown in the results. Figure 3 As shown. Example 5

[0090] Cells were cryopreserved using a control cryopreservation procedure. Cryopreservation density and freezing conditions are shown in Table 1. Cells were cryopreserved for 13 days. The anti-CD19 CAR NK cell line was retrieved from liquid nitrogen, and the cell viability and recovery rate are shown in Table 1. Figure 4 and Figure 5 As shown, different proportions of HSA cryopreservation formulations affect the viability and recovery rate of anti-CD19 CAR NK cell drugs. Formulation 5 shows relatively higher viability and recovery rate of anti-CD19 CAR NK cell drugs, which are significantly different from those with HSA proportions of 0%, 5%, 10%, 15%, and 20%. In particular, after thawing, the cell viability of anti-CD19 CAR NK cells cryopreserved using cryopreservation formulation 5 remained basically unchanged compared with that before cryopreservation.

[0091] Example 6: Cryopreservation of anti-CD19 CAR NK cell drugs using different cryopreservation procedures

[0092] 1. The experimental steps for cryopreservation of anti-CD19 CAR NK cell drugs are briefly described below:

[0093] Inside the biosafety cabinet, pipette the anti-CD19 CAR NK cell drug into a 50ml centrifuge tube, take 20μL for counting, and centrifuge the remaining cells at 600 rpm for 10 minutes.

[0094] Discard the supernatant and resuspend the cells in cryopreservation formulation (i.e., cryopreservation formulation 4) containing 75% CS10 and 25% HSA to achieve a cell density of 5E+07 cells / ml. Add the cells to 1ml cryopreservation tubes, for a total of 6 cell lines (1ml / tube).

[0095] Place the cryovials into a programmed freezing instrument (Thermo Fisher, CryoMed). Incubate the cell suspension at 4°C for 10 minutes, and then freeze 3 cell sections (1 ml / section) using both the control and optimized cryopreservation programs.

[0096] The cryopreservation procedure is as follows: 1. Stand at 20℃ for 20 min; 2. Cool down to -15℃ at 1℃ / min; 3. Cool down to -50℃ at 20℃ / min; 4. Heat up to -25℃ at 10℃ / min; 5. Cool down to -60℃ at 1℃ / min; 6. Cool down to -90℃ at 10℃ / min; 7. After placing in a -80℃ freezer for 20 hours, place in a liquid nitrogen tank for cryopreservation.

[0097] The optimized cryopreservation procedure is as follows: 1. Stand at 20℃ for 20 min; 2. Cool down to -15℃ at 1℃ / min; 3. Cool down to -50℃ at 15℃ / min; 4. Heat up to -30℃ at 10℃ / min; 5. Cool down to -50℃ at 1℃ / min; 6. Cool down to -90℃ at 10℃ / min; 7. After placing in a -80℃ freezer for 20 hours, place in a liquid nitrogen tank for cryopreservation.

[0098] 2. Detection of CAR positivity rate of anti-CD19 CAR NK cell drugs

[0099] CAR NK cells frozen for 10 days were revived at 37°C, and the CAR positivity rate was detected. The experimental procedure is briefly described as follows: 5E+05 cells were transferred to 1.5 mL EP tubes after revival and centrifuged at 400g for 5 minutes; the supernatant was discarded, and 200 μL of 1X DPBS was added to each well for washing twice (centrifuged at 400g for 5 minutes); then 100 μL of anti-CD19FMC63 antibody diluted 1:100 with DPBS was added to each well. The cells were incubated at 4°C for 30 minutes; after incubation, the cells were centrifuged at 400g for 5 minutes. The supernatant was discarded, and 200 μL of 1X DPBS was added to each well for washing once (centrifuged at 400g for 5 minutes); the supernatant was discarded, and 100 μL of 1X DPBS was added to each well to resuspend the cells. The CAR positivity rate was analyzed by flow cytometry. The CAR positivity rates of the control cryopreservation group and the optimized cryopreservation group were 57.6% and 55.6%, respectively. The results are as follows: Figure 6 As shown, optimizing the cryopreservation procedure does not affect the CAR positivity rate.

[0100] 3. Viability and recovery rate of anti-CD19 CAR NK cell drugs

[0101] After cell thawing, cells were incubated at room temperature for 0, 0.5, 1, 1.5, and 2 hours, respectively. The thawed cells were then transferred to sterile centrifuge tubes containing 10 mL of PBS, centrifuged at 500 g for 5 min, the supernatant was discarded, and the cells were resuspended in 2 mL of NK cell culture medium (EcoSy NK medium + 10% FBS). 10 μL of the cell suspension was mixed with AO / PI, and cell viability was detected and recovery rate analyzed using the AO / PI method. Cell viability remained stable within 0-1 h. Within 1-2 h, the cell viability of the optimized cryopreservation program decreased from 89.59% to 81.66%, while the viability of the control cryopreservation program decreased from 88.39% to 77.43%. The results are as follows: Figure 7 As shown in the figure. The cell recovery rate of the optimized cryopreservation program group was higher than that of the control cryopreservation program group, as shown in the figure. Figure 8 As shown.

[0102] Example 7: Effects of different cryopreservation formulations on the cryopreservation stability of anti-CD19 CAR NK cell drugs

[0103] Inside the biosafety cabinet, pipette the anti-CD19 CAR NK cell drug into a 50ml centrifuge tube, take 20μL for counting, and centrifuge the remaining cells at 600 rpm for 10 minutes. Discard the supernatant, resuspend the cells using cryopreservation formulations 4 and 5 in Table 1, adjust the cell density to 5.00E+07 (cells / ml), add the cells to 1ml cryopreservation tubes, and cryopreserve 6 cell lines (1ml / line). After preparing the cells, cryopreserve them using the optimized cryopreservation program and then place them in a liquid nitrogen tank for cryopreservation.

[0104] Anti-CD19 CARNK cell therapy drugs were frozen for 0 months (10 days), 1 month, and 2 months, respectively. The cells were then removed from liquid nitrogen and placed in a 37°C water bath to thaw them. After cell thawing, the cells were placed at room temperature for 0, 0.5, 1, 1.5, and 2 hours, respectively. The cells were then transferred to sterile centrifuge tubes containing 10 mL of PBS, centrifuged at 500 g for 5 min, and the supernatant was discarded. The cells were resuspended in 2 mL of NK cell culture medium (EcoSy NK medium + 10% FBS). 10 μL of the cell suspension was mixed with AO / PI, and cell viability was detected using the AO / PI method.

[0105] Cells thawed at 0 months (10 days), 1 month, and 2 months and then placed at room temperature for 0 hours were collected. 5E+0 cells were added to each well and centrifuged at 500g for 5 minutes. The supernatant was discarded, and each well was washed twice with 200μL of 1X DPBS (centrifuged at 500g for 5 minutes). Then, 100μL of anti-CD19 FMC63 antibody diluted 1:100 in DPBS was added to each well. The wells were incubated at 4°C for 30 minutes. After incubation, the cells were centrifuged at 400g for 5 minutes. The supernatant was discarded, and each well was washed once with 200μL of 1X DPBS (centrifuged at 400g for 5 minutes). The supernatant was discarded, and the cells were resuspended in 100μL of 1X DPBS per well. The CAR positivity rate of the cells was analyzed using flow cytometry.

[0106] Cells thawed at 0 months (10 days), 1 month, and 2 months after cryopreservation and then placed at room temperature for 0 hours were collected. 1E+07 cells from each tube were added to a 1.5 mL EP tube and centrifuged at 400g for 5 minutes. The supernatant was discarded, and each tube was washed twice with 200 μL of 1X DPBS (centrifuged at 400g for 5 minutes). Raji-ffluc cells were then revived and cultured. 20,000 cells / well were added to 100 μL of 96-well white plates. The culture medium was 1640 containing 10% FBS. Cells were added at a CAR positivity rate-to-target ratio (E:T ratio) of 9:1, using 100 μL / well of 1640 containing 10% FBS. The cells were incubated in a CO2 cell culture incubator for 4 hours. After incubation, add 50 μL of firefly luciferase substrate ONE-Glo (Promega, E6110) reagent to each well, gently shake to mix, let stand for 5 minutes, and then detect the fluorescence value using a microplate reader (TECAN, SAPRK).

[0107] Experimental Results: Cells were revived after 0, 1, and 2 months of cryopreservation. Cell viability was measured within 0-2 hours of placement, and viability and recovery rate were analyzed. Experimental data indicate that cryopreservation formulation 5 is superior to cryopreservation formulation 4, and its viability and recovery rate remain stable within 2 hours (e.g., ...). Figure 9 and Figure 10 (As shown). CAR positivity rate and kill rate test results indicate that the CAR positivity rate and kill rate of cryopreserved formulation 5 are superior to those of cryopreserved formulation 4 (as shown). Figure 11 and Figure 12 (As shown). The cryopreserved formulation 5 exhibits excellent long-term stability of the anti-CD19 CAR NK cell drug, meeting the quality standards for cell drug production.

[0108] Example 8: Viability of anti-CD19 CAR NK cell drugs before and after cryopreservation, and CAR positivity rate and cytotoxic activity after thawing.

[0109] Based on cryopreservation formulation 5, different concentrations of Pifithrin-μ were added, and the cryopreservation formulations are shown in Table 3.

[0110] Table 3. Different concentrations of Pifithrin-μ cryopreservation formulations

[0111]

[0112] Inside the biosafety cabinet, pipette the anti-CD19 CAR NK cell drug into a 50ml centrifuge tube, take 20μL for counting, and centrifuge the remaining cells at 500g for 10 minutes. Discard the supernatant, resuspend the cells using the preparations shown in Table 3 above, adjust the cell density to 5.00E+07 (cells / ml), and add the cells to 1ml cryovials, 6 cells per 1ml cryovial. After preparing the cells, cryopreserve them using the optimized cryopreservation program and then place them in a liquid nitrogen tank for 10 days.

[0113] Take the frozen anti-CD19 CAR NK cell drug from liquid nitrogen and place it in a 37°C water bath to thaw and revive the cells. Transfer the thawed cells to a sterile centrifuge tube containing 10 mL of PBS, centrifuge at 500 g for 5 min, and discard the supernatant. Add 2 mL of NK cell culture medium (EcoSy NK medium + 10% FBS) to resuspend the cells. Take 10 μL of the cell suspension and mix it with AO / PI, then count the cells using a cell counter.

[0114] The cell density was adjusted to 1E+06 cells / mL and seeded into 12-well plates. The plates were then placed in a CO2 incubator for further culture. The culture medium was: Ecosai NK medium + 10% FBS + 500 IU IL2. Cell viability was detected using the AO / PI method on a cell counter at 0h and 48h after resuscitation.

[0115] Two aliquots of 5E+05 cells were transferred to 1.5 ml centrifuge tubes: the Blank group and the sample group. Centrifuge at 500 g for 5 minutes, discard the supernatant, and add 1 ml of 1X DPBS to each well for washing twice (centrifuge at 500 g for 5 minutes). Then, add 100 μL / well of anti-CD19 FMC63 antibody (ACROBiosystems, FM3-HPY53) diluted 1:100 with DPBS to the sample group; incubate at room temperature in the dark for 15 minutes. After incubation, centrifuge at 500 g for 5 minutes; discard the supernatant, add 1 ml of 1X DPBS to each well for washing once (centrifuge at 500 g for 5 minutes); discard the supernatant, and resuspend the cells in 100 μL of 1X DPBS in each well. The CAR positivity rate of the Blank group and the sample group was analyzed using flow cytometry (Backman, Cytoflex).

[0116] Raji-ffluc cells were revived and cultured. Cells were counted using a cell counter, and 20,000 cells / well were added to 100 μL of each well in a 96-well plate. The culture medium was 1640 containing 10% FBS.

[0117] Anti-CD19 CAR NK cell drugs were counted 48 hours after resuscitation and culture. Anti-CD19 CAR NK cell drugs were added to Raji-ffluc cells according to CAR positive rate effect-to-target ratio (E:T ratio) of 9:1, 3:1, and 1:1. The culture medium was 1640 containing 10% FBS, 100 μL / well.

[0118] Incubate in a CO2 cell culture incubator for a total of 4 hours. After incubation, add 50 μL of firefly luciferase substrate ONE-Glo (Promega, E6110) reagent to each well, gently shake to mix, let stand for 5 minutes, and then detect the fluorescence value using a microplate reader (TECAN, SAPRK).

[0119] Experimental results: Cell viability was measured 48 hours after resuscitation and culture. Figure 13 As shown, compared with before cryopreservation, the cell viability of anti-CD19 CAR NK cells cryopreserved using cryopreservation formulations 5, 11, 12, 13, and 14 after 48 hours of resuscitation and culture was not significantly different (P>0.05). The CAR positivity rate of cells in the Blank group and the cryopreservation formulations 5, 11, 12, 13, and 14 groups after 48 hours of resuscitation and culture was analyzed by flow cytometry. Figure 14 As shown, the values ​​were 53.5%, 52.6%, 50.8%, 51.1%, and 47.7%, respectively, indicating that the addition of Pifithrin-μ to the frozen preparation did not affect CAR expression. The cytotoxicity assay results of the anti-CD19 CAR NK cell drug are shown below. Figure 15 As shown, under effector-to-target ratio (E:T) conditions of 9:1, 3:1, and 1:1, anti-CD19 CAR NK cell drugs cryopreserved using cryopreservation formulations 11, 12, 13, and 14 exhibited 10%–20% higher killing power against Raji-ffluc target cells after 48 hours of thawing and culture compared to cryopreservation formulation 5, which was significantly different (P<0.05). This indicates that the addition of Pifithrin-μ can enhance the killing activity of anti-CD19 CAR NK cell drugs.

[0120] The sequences involved above are as follows:

[0121] SEQ ID NO: 1

[0122]

[0123] SEQ ID NO:2

[0124] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMYRMQLLSCIALSLALVTNSGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR*。

Claims

1. A cell drug cryopreservation formulation, characterized in that, It consists of a basic cell cryopreservation solution, a human serum albumin solution, and a polysaccharide solution. In the cell drug cryopreservation preparation, the volume percentage of the basic cell cryopreservation solution is 50-60%, the volume percentage of the human serum albumin solution is 20-25%, and the volume percentage of the polysaccharide solution is 20-25%. The basic cell cryopreservation solution is CryptotorCS10, and the polysaccharide is dextran.

2. A cell drug cryopreservation formulation, characterized in that, It consists of a basic cell cryopreservation solution, a human serum albumin solution, a polysaccharide solution, and an apoptosis inhibitor. In the cell drug cryopreservation preparation, the volume percentage of the basic cell cryopreservation solution is 50-60%, the volume percentage of the human serum albumin solution is 20-25%, the volume percentage of the polysaccharide solution is 20-25%, and the concentration of the apoptosis inhibitor is 10-80 μM. The basic cell cryopreservation solution is Cryptotor CS10, and the polysaccharide is dextran.

3. The cell drug cryopreservation formulation according to claim 2, characterized in that, Apoptosis inhibitors include one or more of Baxinhibitor-5, Y-27632, and Pifithrin-μ.

4. A method for cryopreserving cell drugs using the cell drug cryopreservation formulation according to claim 1 or 2, characterized in that, The process includes the following steps: mixing the cell drug with the cell drug cryopreservation formulation, and then freezing it using a programmed freezing device to complete the cell drug cryopreservation.

5. The method for cryopreserving cell drugs according to claim 4, characterized in that, The cryopreservation procedure is as follows: stand at 10-30°C, then cool to -5--20°C, then cool to -40--60°C, then heat to -20--40°C, then cool to -40--60°C, then cool further to -80--100°C, then place at -80°C, and finally place in liquid nitrogen to complete the cryopreservation.

6. The method for cryopreserving cell drugs according to claim 5, characterized in that, Cell-based drugs include one or more types of immune cell drugs and gene-modified immune cell drugs.

7. The application of the cell drug cryopreservation formulation of claim 1 in the cryopreservation of immune cell drugs and gene-modified NK cell drugs.

8. The method for preparing the cell drug cryopreservation formulation according to claim 1, characterized in that, The basic cell cryopreservation solution, human serum albumin solution, and polysaccharide solution are mixed to obtain a cell drug cryopreservation formulation.

9. The method for preparing the cell drug cryopreservation formulation according to claim 2, characterized in that, The basic cell cryopreservation solution, human serum albumin solution, polysaccharide solution, and apoptosis inhibitor were mixed to obtain a cell drug cryopreservation formulation.

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