Preparation method and application of CAR-T cell composition targeting tumor antigen

Abstract

The invention provides a preparation method and application of a CAR-T cell composition targeting a tumor antigen, the composition comprises CAR-T cells, an immunomodulatory factor, a cell protective agent and an effect-prolonging carrier, the mass ratio of the immunomodulatory factor to the CAR-T cells is 0.1-1: 100, the volume ratio of the cell protective agent is 5-15%, and the effect-prolonging carrier is 20-50%. The preparation method comprises the following steps: resuscitating and washing the CAR-T cells, dissolving the immunoregulatory factors, ultrasonically treating the cell protective agent, filtering, mixing according to a specific sequence, and standing. Experiments show that the composition can significantly improve the survival rate of CAR-T cells, enhance the killing activity on tumor cells, reduce the tumor volume and prolong the lifetime in an animal model, and is suitable for preparation of tumor treatment drugs.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and pharmaceutical technology, and in particular to a method for preparing and applying a CAR-T cell composition targeting tumor antigens. Background Technology

[0002] CAR-T cell therapy, as a novel immunotherapy approach, modifies T cells through genetic engineering to express chimeric antigen receptors (CARs), enabling them to specifically recognize and kill tumor cells, demonstrating significant efficacy in the treatment of hematological malignancies. However, existing CAR-T cell preparation and in vivo application still face many limitations: on the one hand, CAR-T cells have short survival times in vitro, their activity is easily affected by the environment, and they are prone to apoptosis in the tumor microenvironment due to oxidative stress, nutritional deficiencies, and other factors, leading to a decline in therapeutic efficacy; on the other hand, immunomodulatory factors (such as IL-2 and IL-15) have short half-lives when applied directly, easily triggering systemic toxicity, and the lack of effective carrier systems leads to uneven distribution of CAR-T cells in vivo and limited duration of action, making it difficult to sustain anti-tumor effects.

[0003] Furthermore, insufficient cell protection measures in traditional preparation processes often lead to impaired activity of CAR-T cells during resuscitation, washing, and mixing, further limiting their clinical application. Therefore, developing a composition and its preparation method that can synergistically enhance CAR-T cell survival, increase targeted killing activity, and prolong in vivo action time has become a key direction for optimizing CAR-T cell therapy. Summary of the Invention

[0004] Therefore, this invention proposes a method for preparing and applying a CAR-T cell composition targeting tumor antigens, thereby solving the above-mentioned problems.

[0005] The technical solution of the present invention is implemented as follows: a CAR-T cell composition targeting tumor antigens, comprising CAR-T cells, immunomodulatory factors, cell protectants and extended carriers, wherein the mass ratio of immunomodulatory factors to CAR-T cells is (0.1-1):100, the volume of the cell protectant accounts for 5-15% of the total volume of the composition, and the volume of the extended carrier accounts for 20-50% of the total volume of the composition.

[0006] Furthermore, the CAR-T cells are CAR-T cells targeting CD19, CD20, or BCMA.

[0007] Furthermore, the immunomodulatory factor is at least one of IL-2, IL-7, and IL-15.

[0008] Furthermore, the cell protectant is a liposome suspension targeting the mitochondrial peptide SS-31.

[0009] Furthermore, the liposome suspension targeting the mitochondrial peptide SS-31 is prepared by the following method: soybean lecithin, cholesterol and the mitochondrial peptide SS-31 are mixed at a mass ratio of (5-10):(1-2):1, dissolved in chloroform, and the organic solvent is removed by rotary evaporation at 35-40℃ to form a lipid film. PBS buffer with pH 7.2-7.4 is added for hydration for 1-2 hours, and the mixture is homogenized in a high-pressure homogenizer at a pressure of 800-1000 bar for 3-5 cycles to obtain a liposome suspension with a particle size of 80-150 nm and an encapsulation rate ≥85%.

[0010] Furthermore, the extended carrier is at least one of hyaluronic acid, chitosan, or polyethylene glycol.

[0011] Furthermore, the method for preparing a CAR-T cell composition targeting tumor antigens includes the following steps: (1) Resuscitate CAR-T cells at 37±0.5℃ and 3-6% (v / v) CO2. After resuscitation, wash the cells 2-3 times with serum-free medium and adjust the cell concentration to 1×10⁻⁶. 6 -5×10 6 cells / mL; (2) Dissolve the immunomodulatory factor in sterile PBS buffer to prepare a solution with a concentration of 0.1-1 μg / mL; (3) The cell protectant was ultrasonically treated at 2-8℃ for 5-10 minutes with an ultrasonic power of 100-200W. After ultrasonic treatment, it was filtered through a 0.22μm filter membrane for sterilization. (4) Mix the CAR-T cell suspension from step (1), the immunomodulatory factor solution from step (2), the cell protectant from step (3), and the extended-effect carrier in the order of proportion, and let stand at 25±2℃ for 10-15 minutes to obtain the final product.

[0012] Furthermore, in step (1), the serum-free culture medium is X-VIVO15 medium, and the centrifugation speed during washing is 300-400×g, and the centrifugation time is 5-8 minutes.

[0013] Furthermore, the mixing order in step (4) is as follows: first, mix the CAR-T cell suspension with the immunomodulatory factor solution and stir magnetically for 4-8 minutes at a stirring speed of 60-80 rpm, then add the cell protectant and the extended-effect carrier in sequence, and continue stirring for 8-12 minutes.

[0014] Furthermore, the application of CAR-T cell compositions in the preparation of drugs for treating tumors.

[0015] Compared with the prior art, the beneficial effects of the present invention are: (1) Significantly improve the survival rate of CAR-T cells: By introducing a liposome suspension targeting the mitochondrial peptide SS-31 as a cell protectant, combined with a specific ultrasonic treatment process, mitochondrial damage and oxidative stress of CAR-T cells during in vitro preparation and in vivo application can be effectively reduced, and the 72-hour survival rate can be increased to more than 85%.

[0016] (2) Enhance tumor killing activity and specificity: The specific ratio of immunomodulatory factors to CAR-T cells, combined with the sustained release effect of the extended-acting carrier, can significantly improve the killing rate of CAR-T cells against targeted tumor cells to 79%-82% and prolong the in vivo action time.

[0017] (3) Improve safety and clinical applicability: By optimizing the ratio of immunomodulatory factors and encapsulation treatment, the risk of cytokine storm caused by factor overdose is avoided; at the same time, the synergistic effect of each component has achieved significant reduction in tumor volume and prolongation of survival in animal models, providing a more efficient and safer solution for tumor treatment. Detailed Implementation

[0018] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0019] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0021] The mitochondrial peptide SS-31 was purchased from Shanghai Hongtai Biotechnology Co., Ltd.

[0022] Example 1 Preparation of CD19-targeting CAR-T cell composition 1. Raw materials and proportions CAR-T cells: targeting CD19 Immunomodulatory factors: IL-2 and IL-15 were mixed at a mass ratio of 1:1, and the mass ratio of IL-2 to CAR-T cells was 0.5:100; Cell protectant: Liposome suspension targeting mitochondrial peptide SS-31, 10% by volume; Extended-effect carrier: hyaluronic acid (molecular weight 500kDa), accounting for 35% of the volume.

[0023] Preparation method: Preparation of cell protectant: Soybean lecithin, cholesterol and mitochondrial peptide SS-31 were mixed at a mass ratio of 5:2:1, dissolved in chloroform, and the organic solvent was removed by rotary evaporation at 38℃ to form a lipid film. The film was hydrated in PBS buffer (pH 7.2-7.4) for 1 hour, and then homogenized in a high-pressure homogenizer at 1000 bar for 4 cycles to obtain a liposome suspension with a particle size of 120 nm and an encapsulation rate of ≥85%.

[0024] Preparation of CAR-T cell composition: Step (1): CAR-T cells were revived at 37℃ and 5% CO2 for 1.5 h, washed twice with X-VIVO15 medium (centrifuged at 350×g for 6 minutes), and the concentration was adjusted to 3×10⁻⁶. 6 cells / mL Step (2): Dissolve IL-2 and IL-15 in sterile PBS to prepare a mixed solution of 0.5 μg / mL. Step (3): The cell protectant was sonicated at 4°C for 8 minutes (power 150W) and then filtered through a 0.22μm filter membrane. Step (4): First, mix the CAR-T cell suspension with the immunomodulatory factor solution, stir magnetically at 60 rpm for 6 minutes, then add the cell protectant and hyaluronic acid, continue stirring for 10 minutes, and let stand at 25°C for 12 minutes to obtain the composition.

[0025] Example 2 Preparation and performance of CAR-T cell compositions targeting BCMA 1. Raw materials and proportions CAR-T cells: targeting BCMA; Immunomodulatory factor: IL-7, with a mass ratio of 0.8:100 to CAR-T cells; Cell protectant: SS-31 liposome suspension (particle size 100nm, encapsulation efficiency 86%), 12% by volume; Extended carrier: polyethylene glycol (PEG2000), 40% by volume.

[0026] Preparation method: Preparation of cell protectant: Soybean lecithin, cholesterol and mitochondrial peptide SS-31 were mixed at a mass ratio of 10:2:1, dissolved in chloroform, and the organic solvent was removed by rotary evaporation at 40℃ to form a lipid film. The film was hydrated in PBS buffer at pH 7.4 for 1-2 hours, and then homogenized in a high-pressure homogenizer at a pressure of 800-1000 bar for 3-5 cycles to obtain a liposome suspension with a particle size of 80-150 nm and an encapsulation rate of ≥85%.

[0027] Preparation of CAR-T cell composition: (1) CAR-T cells were revived at 37℃ and 5% (v / v) CO2. After revival, the cells were washed three times with X-VIVO15 medium (centrifuged at 400g for 8 minutes) and the cell concentration was adjusted to 2×10⁻⁶. 6 cells / mL; (2) Dissolve the immunomodulatory factor in sterile PBS buffer to prepare a solution with a concentration of 0.8 μg / mL; (3) The cell protectant was ultrasonically treated at 6°C for 6 minutes with an ultrasonic power of 180W. After ultrasonic treatment, it was filtered through a 0.22μm filter membrane for sterilization. (4) First, mix the CAR-T cell suspension with the immunomodulatory factor solution, stir magnetically at 70 rpm for 8 minutes, then add the cell protectant and hyaluronic acid, continue stirring for 10 minutes, and let stand at 25°C for 8 minutes to obtain the composition.

[0028] Comparative Example 1 The difference between this comparative example and Example 1 is that it does not contain SS-31 liposome suspension, while the other components, proportions, and preparation steps are the same as in Example 1.

[0029] Comparative Example 2 The difference between this comparative example and Example 1 is that the mass ratio of immunomodulatory factors (IL-2+IL-15) to CAR-T cells is 2:100, while the rest is the same as in Example 1.

[0030] Comparative Example 3 The difference between this comparative example and Example 1 is that the cell protectant was not subjected to ultrasonic treatment in step (3), while the rest is the same as in Example 1.

[0031] Experiment 1 - Detection of CAR-T cell viability in vitro 1. Take the compositions prepared in Example 1, Example 2, and Comparative Examples 1-3, and incubate them in 37℃ and 5% CO2 incubators for 0h, 24h, 48h, and 72h, respectively.

[0032] 2. Samples were taken at each time point, and the survival rate of CAR-T cells was detected by flow cytometry (Annexin V / PI double staining) (proportion of viable cells = percentage of Annexin V-PI- cells).

[0033] result:

[0034] Conclusion: The 72h survival rate of SS-31 liposomes in Examples 1-2, which contained ultrasound treatment, was significantly higher than that of the control group, proving that cell protectants and ultrasound treatment can effectively maintain the activity of CAR-T cells.

[0035] Experimental Example 2 - Detection of In Vitro Killing Activity of Tumor Cells method: 1. Target cells: Example 1 corresponds to CD19⁺Raji cells (lymphoma cells), and Example 2 corresponds to BCMA⁺MM.1S cells (myeloma cells).

[0036] 2. The effective-to-target ratio was 10:1 (CAR-T cells: target cells). Each group of compositions was co-cultured with target cells for 4 hours, and the killing rate was detected by LDH release method.

[0037] Kill rate = (Experimental group OD - Negative control OD) / (Positive control OD - Negative control OD) × 100%.

[0038] 3. Results

[0039] Conclusion: The tumor cell killing rate of the example was significantly higher than that of the control group, indicating that the synergistic effect of "immunomodulatory factor + protectant + extended-effect carrier" can enhance the targeted killing ability of CAR-T cells.

[0040] Experimental Example 3 - Detection of Antitumor Efficacy in Animals 1. Model construction: NOD / SCID mice were intraperitoneally inoculated with 1×10⁻⁶ mice. 7 1 Raji cell (to construct a lymphoma model) or MM.1S cell (to construct a myeloma model), and 6 cells were randomly assigned to different groups after 7 days (n=6).

[0041] 2. Administration: Each group was administered the corresponding composition via tail vein injection (CAR-T cell dose was 2×10⁻⁶). 6 (Each animal was injected with one saline solution), while the control group was injected with an equal volume of saline.

[0042] 3. Detection indicators: Tumor volume was measured every 3 days (lymphoma model) or serum M protein level was detected (myeloma model), and mouse survival time was recorded.

[0043] 4. Results

[0044] Conclusion: The examples significantly reduced tumor volume and prolonged survival in animal models, while Comparative Example 1 (without protectant) and Comparative Example 2 (with factor overdose) showed poor efficacy, demonstrating the synergistic advantages of the composition in vivo.

[0045] Experimental Example 4 - Stability and Targeting Detection of Cell Protectants method: 1. Stability: The particle size distribution (dynamic light scattering) and encapsulation efficiency (ultrafiltration) of SS-31 liposomes in Example 1 and Comparative Example 3 were tested and retested after storage at 4°C for 7 days.

[0046] 2. Targeting: SS-31 liposomes were fluorescently labeled and co-incubated with CAR-T cells for 2 hours. The co-localization efficiency of liposomes and mitochondria (MitoTracker staining) was observed by laser confocal microscopy.

[0047] 3. Results:

[0048] Conclusion: After ultrasonic treatment, the liposomes of Example 1 showed significantly better particle size uniformity, encapsulation efficiency, and mitochondrial targeting than those of Comparative Example 3, proving that ultrasonic treatment is key to ensuring the function of the cell protectant.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A CAR-T cell composition targeting tumor antigens, characterized in that, The composition includes CAR-T cells, immunomodulatory factors, cell protectants, and extended-effect carriers. The mass ratio of immunomodulatory factors to CAR-T cells is (0.1-1):

100. The volume of the cell protectant accounts for 5-15% of the total volume of the composition, and the volume of the extended-effect carrier accounts for 20-50% of the total volume of the composition.

2. The CAR-T cell composition targeting tumor antigens as described in claim 1, characterized in that, The CAR-T cells are CAR-T cells targeting CD19, CD20, or BCMA.

3. The CAR-T cell composition targeting tumor antigens as described in claim 1, characterized in that, The immunomodulatory factor is at least one of IL-2, IL-7, and IL-15.

4. The CAR-T cell composition targeting tumor antigens as described in claim 1, characterized in that, The cell protectant is a liposome suspension targeting the mitochondrial peptide SS-31.

5. The CAR-T cell composition targeting tumor antigens as described in claim 4, characterized in that, The liposome suspension targeting mitochondrial peptide SS-31 was prepared by the following method: soybean lecithin, cholesterol and SS-31 were mixed at a mass ratio of (5-10):(1-2):1, dissolved in chloroform, and the organic solvent was removed by rotary evaporation at 35-40℃ to form a lipid film. The mixture was then hydrated in PBS buffer (pH 7.2-7.4) for 1-2 hours and homogenized in a high-pressure homogenizer at 800-1000 bar for 3-5 cycles to obtain a liposome suspension with a particle size of 80-150 nm and an encapsulation rate of ≥85%.

6. The CAR-T cell composition targeting tumor antigens as described in claim 1, characterized in that, The extended carrier is at least one of hyaluronic acid, chitosan, or polyethylene glycol.

7. The method for preparing a CAR-T cell composition targeting tumor antigens as described in claim 1, characterized in that, Includes the following steps: (1) Resuscitate CAR-T cells at 37±0.5℃ and 3-6% (v / v) CO2. After resuscitation, wash the cells 2-3 times with serum-free medium and adjust the cell concentration to 1×10⁻⁶. 6 -5×10 6 cells / mL; (2) Dissolve the immunomodulatory factor in sterile PBS buffer to prepare a solution with a concentration of 0.1-1 μg / mL; (3) The cell protectant was ultrasonically treated at 2-8℃ for 5-10 minutes with an ultrasonic power of 100-200W. After ultrasonic treatment, it was filtered through a 0.22μm filter membrane for sterilization. (4) Mix the CAR-T cell suspension from step (1), the immunomodulatory factor solution from step (2), the cell protectant from step (3), and the extended-effect carrier in the order of proportion, and let stand at 25±2℃ for 10-15 minutes to obtain the final product.

8. The method for preparing a CAR-T cell composition targeting tumor antigens as described in claim 7, characterized in that, In step (1), the serum-free culture medium is X-VIVO15 medium. During washing, the centrifugation speed is 300-400×g and the centrifugation time is 5-8 minutes.

9. The method for preparing a CAR-T cell composition targeting tumor antigens as described in claim 7, characterized in that, The mixing order in step (4) is as follows: first mix the CAR-T cell suspension with the immunomodulatory factor solution and stir magnetically for 4-8 minutes at a speed of 60-80 rpm, then add the cell protectant and the extended-effect carrier in sequence, and continue stirring for 8-12 minutes.

10. Use of the CAR-T cell composition according to any one of claims 1-6 in the preparation of a tumor-treating drug.

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