A CAR-T cell freezing solution and freezing method

Abstract

The present invention discloses a freezing solution and freezing method for CAR-T cells. The freezing method includes: mixing CAR-T cells with a freezing solution to obtain a mixed solution, subjecting the mixed solution to a program cooling, and storing it in liquid nitrogen; the cooling program includes: waiting at 2 to 4 ° C, cooling to the latent heat point of the freezing solution at 20 to 15 ° C at a rate of 1.5 to 1 ° C / min, cooling to 75 to 65 ° C at a rate of 30 to 20 ° C / min, heating to 30 to 25 ° C at a rate of 8 to 10 ° C / min, and cooling to 90 to 80 ° C at a rate of 1.5 to 1 ° C / min. The present invention fully analyzes the effect of the latent heat of the freezing solution on the cooling process, rapidly cools near the latent heat point, reduces the temperature rise of the freezing solution, realizes efficient matching of the cooling program with the freezing solution, improves the activity and viability of the cells after freezing and resuscitation, thereby maintaining the therapeutic effect of CAR-T cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology and relates to a freezing solution and a freezing method for CAR-T cells. Background Art

[0002] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) utilizes a patient's own immune T cells, genetically modified ex vivo, and then infused back into the body to directly eliminate cancer cells. CAR-T immune cell therapy shows great promise in treating hematologic malignancies, but numerous limitations prevent immediate infusion of CAR-T cells after production. Instead, prepared CAR-T cells are typically cryopreserved, transported via cold chain to a hospital, and then thawed and infused. Therefore, ensuring the quality of cryopreserved CAR-T cells and their high activity for a period of time after thawing is crucial.

[0003] Cryopreservative fluid used for cell therapy needs to be directly used for patient reinfusion, so the requirements for cryopreservative fluid excipients are relatively high, and generally require the use of excipients above GMP grade. At the same time, the cell cooling program generally has a direct impact on the freezing effect. The existing cooling program still mainly adopts the traditional cooling method for frozen cells. The cooling program is usually to cool to the required temperature (such as -80°C or -120°C) in one step at a fixed rate. For example, CN108552160A discloses a CAR-T cell cryopreservative fluid for direct intravenous reinfusion, as well as its preparation method and application. The cryopreservative fluid contains dimethyl sulfoxide, invert sugar solution, dextran solution, glucose solution, human serum albumin solution, and normal saline. The cooling method of the freezing method includes: cooling to -120°C at 1°C / min. CN113841690A discloses a protective agent for cryopreservation of CAR-T cells and a cryopreservation method, wherein the protective agent includes a culture medium and the following components added to the culture medium: paulowniain, inositol nicotinate, wax gourd seed extract, hydroxytyrosol, β-glucan, insulin, and glutathione. The cryopreservation method includes the following steps: mixing CART cells and a protective agent to obtain a cell suspension, subpackaging the suspension into sterile cryopreservation tubes, cooling the cryopreservation tubes to -80°C at 3°C / min, and then transferring the suspension to liquid nitrogen for cryopreservation. However, the existing cooling program is generally difficult to match with the cryopreservation solution, resulting in a large loss of cell viability after recovery and difficulty in maintaining high activity.

[0004] In summary, providing an effective cryopreservation method for CAR-T cells and improving the activity and viability of cells after freezing and resuscitation is of great significance to the CAR-T field. Summary of the Invention

[0005] In response to the deficiencies of the existing technology and actual needs, the present invention provides a CAR-T cell freezing solution and freezing method, which efficiently matches the cooling program with the freezing solution, maintains the activity and viability of the frozen CAR-T cells, and thus maintains the therapeutic effect of the CAR-T cells.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for freezing CAR-T cells, comprising:

[0008] The CAR-T cells are mixed with the cryopreservation solution to obtain a mixed solution, and the mixed solution is subjected to a program cooling to -80°C and stored in liquid nitrogen; the cooling program includes: waiting at 2 to 4°C, cooling to a potential hotspot of the cryopreservation solution of -20 to -15°C (for example, -19°C, -18°C, -17°C or -16°C) at a rate of -1.5 to -1°C / min, cooling to a potential hotspot of the cryopreservation solution of -20 to -15°C (for example, -19°C, -18°C, -17°C or -16°C), cooling to a potential hotspot of the cryopreservation solution of -30 to -20°C / min (for example, -29°C, -28°C, -25°C, -24°C, -23°C, -22°C or -21°C) at a rate ...1.5 to -1°C / min ) is reduced to -75 to -65°C (for example, it can be -74°C, -73°C, -72°C, -71°C, -69°C, -68°C, -67°C or -66°C), the temperature is increased at 8 to 10°C / min to -30 to -25°C (for example, it can be -29°C, -28°C, -27°C or -26°C), and the temperature is reduced to -90 to -80°C (for example, it can be -89°C, -88°C, -87°C, -86°C, -85°C, -84°C, -83°C or -82°C) at -1.5 to -1°C / min.

[0009] In the present invention, the latent hot spot is when the cell freezing solution begins to freeze, which will release heat. Although the cooling device cavity is in a cooling state, the cells resuspended in the freezing solution will increase in temperature. The cooling device needs to speed up the cooling rate to remove the excess heat. The flatter the latent hot spot on the freezing curve, the smaller the temperature change and the better the freezing effect.

[0010] In the present invention, it was found that the latent heat of the cryopreservation fluid can cause a large difference between the sample cooling curve and the set cooling curve during the cooling process, thereby destroying the cell structure and resulting in poor cell freezing effect. Based on this finding, the present application fully considers the influence of the latent heat of the cryopreservation fluid on the cooling process, analyzes the temperature point (latent hot spot) when the cryopreservation fluid releases latent heat, quickly cools down near this temperature point, minimizes the temperature rise of the cryopreservation fluid, and then slowly cools down to achieve efficient matching of the cooling program and the cryopreservation fluid, thereby improving the activity and viability of the cells after freezing and resuscitation, thereby maintaining the therapeutic effect of CAR-T cells.

[0011] In the present invention, a fully adapted cooling strategy can be formulated for different cryopreservation solutions. All cryopreservation solutions for CAR-T cells applicable in the art are applicable to the method of the present invention without special limitation.

[0012] Preferably, the freezing solution may contain compound electrolytes, glucose, sodium chloride, dextran, human serum albumin and dimethyl sulfoxide (DMSO).

[0013] Preferably, the compound electrolyte comes from a compound electrolyte injection.

[0014] Preferably, the glucose and sodium chloride are from glucose and sodium chloride injection.

[0015] Preferably, the dextran is derived from Dextran 40 Glucose Injection.

[0016] Preferably, the human albumin comes from human albumin injection.

[0017] Preferably, the freezing solution contains, by volume percentage, 30% to 70% of compound electrolyte injection (for example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%), 30% to 70% of glucose and sodium chloride injection (for example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%), 10% to 30% of dextran 40 glucose injection (for example, 11%, 12%, 15%, 16%, 17%, 18%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%. 6%, 18%, 20%, 22%, 24%, 25%, 26%, 28% or 29%), human serum albumin injection 10% to 30% (for example, 11%, 12%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 26%, 28% or 29%) and dimethyl sulfoxide 5% to 15% (for example, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% or 14%), wherein the sum of the volume percentages of each component is 100%.

[0018] Preferably, the cooling procedure further includes a pre-cooling step.

[0019] Preferably, the precooling is to 2-8°C, including but not limited to 3°C, 4°C, 5°C, 6°C or 7°C.

[0020] Preferably, the density of the CAR-T cells is 0.8×10 7 ~1.2×10 7 cells / mL, including but not limited to 0.9×10 7 , 1.0×10 7 or 1.1×10 7 .

[0021] As a preferred technical solution, the CAR-T cell freezing method includes the following steps:

[0022] (1) mixing the CAR-T cells with the freezing solution to obtain a mixed solution, and precooling the mixed solution to 2-8°C;

[0023] (2) The pre-cooled mixed solution is cooled to the latent hot spot of the freezing solution at -18°C at a rate of -1°C / min, then cooled to -75°C at a rate of -30°C / min, then heated to -28°C at a rate of 10°C / min, and finally cooled to -80°C at a rate of -1°C / min. The mixture is then stored in liquid nitrogen. The latent hot spot is when the cell freezing solution begins to freeze, and heat is released. Although the cooling chamber is in a cooling state, the cells resuspended in the freezing solution will experience an increase in temperature. The cooling instrument is required to accelerate the cooling rate to remove excess heat. The flatter the latent hot spot on the freezing curve, the smaller the temperature change and the better the freezing effect.

[0024] In a second aspect, the present invention provides a CAR-T cell cryopreservation solution, wherein the cryopreservation solution contains, by volume percentage, 30% to 70% of a compound electrolyte injection (for example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%), 30% to 70% of a glucose and sodium chloride injection (for example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%), 10% to 30% of a dextran 40 glucose injection (for example, 11% , 12%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 26%, 28% or 29%), human serum albumin injection 10% to 30% (for example, it can be 11%, 12%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 26%, 28% or 29%), and dimethyl sulfoxide 5% to 15% (for example, it can be 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% or 14%), wherein the sum of the volume percentages of each component is 100%.

[0025] The freezing solution is used in the freezing method of CAR-T cells described in the first aspect.

[0026] In a third aspect, the present invention provides a cryopreservation preparation of CAR-T cells, which contains CAR-T cells and the cryopreservation solution of the CAR-T cells described in the second aspect.

[0027] Preferably, the cryopreservation preparation of CAR-T cells is prepared by the cryopreservation method of CAR-T cells described in the first aspect.

[0028] Preferably, the density of CAR-T cells in the cryopreserved preparation of CAR-T cells is 0.8×107 ~1.2×10 7 pieces / mL.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention fully considers the influence of the latent heat of the cryopreservation solution on the cooling process, analyzes the temperature point (latent hot spot) when the cryopreservation solution releases latent heat, quickly cools the solution near this temperature point to minimize the temperature rise of the cryopreservation solution, and then slowly cools the solution to achieve efficient matching of the cooling program and the cryopreservation solution, thereby improving the activity and viability of cells after freezing and resuscitation, thereby maintaining the therapeutic effect of CAR-T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1-1 is the latent hot spot temperature curve;

[0032] Figure 1-2 Repeat the experimental temperature curve for the latent hot spot;

[0033] Figure 2-1 This is a statistical chart of cell viability within 24 days after thawing after 10 days of freezing;

[0034] Figure 2-2 This is a statistical chart of cell viability within 24 days after thawing after 20 days of cryopreservation;

[0035] Figure 2-3 This is a statistical chart of cell viability within 24 days after thawing after 30 days of cryopreservation;

[0036] Figure 3 This is a statistical chart of the killing results of target cells by cells in each group;

[0037] Figure 4-1 is a statistical graph of specific cytokines (granzyme B);

[0038] Figure 4-2 It is a statistical chart of specific cytokines (IFN-r). DETAILED DESCRIPTION

[0039] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0040] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0041] Example 1

[0042] This embodiment provides a method for freezing CAR-T cells.

[0043] The freezing solution is prepared by mixing 31.25% compound electrolyte injection (V / V), 31.25% glucose and sodium chloride injection (V / V), 10% dextran 40 glucose injection (V / V), 20% human albumin injection (V / V) and 7.5% dimethyl sulfoxide (V / V) according to volume percentage.

[0044] The cryopreservative solution was precooled to 4°C, and then placed in a programmed cooling device and cooled to -80°C at 1°C / min. The potential hot spots of the cryopreservative solution were analyzed. The results were as follows: Figure 1-1 As shown, it can be observed that when the chamber temperature reaches about -22℃, the temperature of the freezing solution rises rapidly. The results are as follows: Figure 1-2 As shown, it is consistent with the results of the first experiment, so it can be determined that the latent hot spot of the frozen re-infusion solution is -22℃.

[0045] The CAR-T cell freezing method comprises the following steps:

[0046] (1) Mix CD7CAR-T cells (patient-derived CAR-T cells) with 5 mL of self-prepared frozen infusion solution and precool to 4°C;

[0047] (2) The pre-cooled mixed solution was placed in a programmed cooling apparatus and cooled at -1°C / min to the latent hot spot of the cryopreservative solution -18°C, then cooled at -30°C / min to -75°C, then heated at 10°C / min to -28°C, then cooled at -1°C / min to -80°C, and stored in liquid nitrogen.

[0048] Example 2

[0049] This embodiment provides a method for freezing CAR-T cells. The method for freezing CAR-T cells is different from that in Example 1 only in that the cooling program in step (2) is as follows: cooling to the latent hotspot of the freezing solution at -15°C at -1°C / min, cooling to -75°C at -30°C / min, heating to -25°C at 10°C / min, and cooling to -80°C at -1°C / min.

[0050] Example 3

[0051] This embodiment provides a method for freezing CAR-T cells. The method for freezing CAR-T cells is different from that in Example 1 only in that the cooling program in step (2) is as follows: cooling to the potential hotspot of the freezing solution at -20°C at -1°C / min, cooling to -75°C at -30°C / min, heating to -30°C at 10°C / min, and cooling to -80°C at -1°C / min.

[0052] Example 4

[0053] This embodiment provides a method for freezing CAR-T cells.

[0054] The freezing solution consists of 50.9% compound electrolyte injection (V / V), 23.1% human serum albumin injection (V / V), 18.5% dextran 40 glucose (5%) injection (V / V), and 7.5% DMSO (V / V).

[0055] The above-mentioned cryopreservative solution was pre-cooled to 4°C, and then placed in a programmed cooling device and cooled to -80°C at 1°C / min. The latent hot spot of the cryopreservative solution was analyzed. It was observed that when the cavity temperature reached about -18°C, the temperature of the cryopreservative solution rose rapidly. The results were repeated again and the results were consistent, that is, the latent hot spot of the cryopreservative re-infusion solution was -18°C.

[0056] The CAR-T cell freezing method comprises the following steps:

[0057] (1) Mix CD7CAR-T cells and this freezing solution and precool to 4°C;

[0058] (2) The pre-cooled mixed solution was placed in a programmed cooling apparatus and cooled at -1°C / min to the latent hot spot of the cryopreservative solution -18°C, then cooled at -30°C / min to -75°C, then heated at 10°C / min to -28°C, then cooled at -1°C / min to -80°C, and stored in liquid nitrogen.

[0059] Comparative Example 1

[0060] This comparative example provides a CAR-T cell freezing method. The CAR-T cell freezing method is different from that of Example 1 in that the cooling program in step (2) is: from 4°C to -80°C at a rate of 1°C / min.

[0061] Comparative Example 2

[0062] This comparative example provides a CAR-T cell freezing method. The CAR-T cell freezing method is different from that of Example 1 in that the cooling program in step (2) is as follows: cooling to the latent hotspot of the freezing solution at -10°C at -1°C / min, cooling to -75°C at -15°C / min, heating to -15°C at 10°C / min, and cooling to -80°C at -1°C / min.

[0063] Comparative Example 3

[0064] This comparative example provides a method for freezing CAR-T cells. The method for freezing CAR-T cells is different from that in Example 1 only in that the cooling program in step (2) is as follows: cooling to the potential hotspot of the freezing solution at -30°C at -1°C / min, cooling to -75°C at -40°C / min, heating to -35°C at 10°C / min, and cooling to -80°C at -1°C / min.

[0065] Test Example 1

[0066] This test example analyzes the freezing effects of the freezing methods of CAR-T cells in Examples 1-4 and Comparative Examples 1-3.

[0067] The cells were recovered on the 10th day (D10), 20th day (D20) and 30th day (D30) after freezing, and the cell viability was detected by trypan blue counting. The results were as follows: Figure 2-1 、 Figure 2-2 、 Figure 2-3 As shown, the cells of Example Groups 1-4 frozen had better viability within 24 hours after thawing.

[0068] Cell counting only provides the results of cell viability and does not reflect whether the killing activity of CAR-T cells after freezing is affected. In order to further analyze the tumoricidal activity of CAR-T cells after freezing and thawing, a target cell killing test was performed. The target cells corresponding to CAR-T were used and divided into 7 groups on average. The CAR-T cells that were frozen and thawed for 30 days in Example 1-4 and Comparative Example 1-3 were added to each group of target cells, and the killing efficiency of CAR-T cells was cultured and counted (the results are shown in Figure 2). Figure 3 As shown in the table) and the release of specific cytokines (results as shown in the table) Figure 4-1 and Figure 4-2 As shown in Figure 3, the cells in Example 1-4 groups showed higher killing effects on target cells, and the release of granzyme B and IFN-r in the supernatant was also higher.

[0069] In summary, the present invention fully analyzes the effect of the latent heat of the cryopreservation fluid on the cooling process, analyzes the temperature point (latent hot spot) when the cryopreservation fluid releases latent heat, quickly cools down near this temperature point, minimizes the temperature rise of the cryopreservation fluid, and then slowly cools down to achieve efficient matching of the cooling program and the cryopreservation fluid, thereby improving the activity and viability of cells after freezing and resuscitation, thereby maintaining the therapeutic effect of CAR-T cells.

[0070] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A method for freezing CAR-T cells, characterized in that: The freezing method comprises: Mixing the CAR-T cells with the freezing solution to obtain a mixed solution, subjecting the mixed solution to programmed cooling, and storing it in liquid nitrogen; The freezing solution contains, by volume percentage, 30% to 70% of compound electrolyte injection, 30% to 70% of glucose and sodium chloride injection, 10% to 30% of dextran 40 glucose injection, 10% to 30% of human albumin injection, and 5% to 15% of dimethyl sulfoxide, wherein the sum of the volume percentages of each component is 100%; The cooling program includes: pre-cooling, cooling to the latent hot spot of the freezing solution at -20~-15°C at -1.5~-1°C / min, cooling to -75~-65°C at -30~-20°C / min, heating to -30~-25°C at 8~10°C / min, and cooling to -90~-80°C at -1.5~-1°C / min.

2. The method for freezing CAR-T cells according to claim 1, characterized in that: The precooling is to 2-8°C.

3. The method for freezing CAR-T cells according to claim 1, characterized in that: The density of the CAR-T cells was 0.8×10 7 ~1.2×10 7 pieces / mL.

4. The method for freezing CAR-T cells according to claim 1, characterized in that: The freezing method comprises the following steps: (1) Mixing CAR-T cells with freezing solution to obtain a mixed solution, and precooling the mixed solution to 2-8°C; (2) The pre-cooled mixed solution was cooled to -18°C, the potential hotspot of the cryopreservative solution, at a rate of -1°C / min, cooled to -75°C at a rate of -25°C / min, heated to -28°C at a rate of 10°C / min, and cooled to -80°C at a rate of -1°C / min.

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