Anti-MUC1 CAR-T source exosome preparation method, product and application

Abstract

The invention belongs to the field of tumor immunotherapy, and particularly relates to a preparation method of an anti-MUC1 CAR-T source exosome, a product and application. The preparation method of the exosome comprises the following steps: centrifuging an anti-MUC1 CAR-T cell culture solution, collecting supernate, and filtering to obtain filtrate; adding a PEG-6000 solution into the filtrate, incubating, collecting the precipitate, and resuspending to obtain an exosome resuspension; the preparation method comprises the following steps: taking the exosome as a raw material, spreading the exosome on the top layer of an iodixanol gradient tube, carrying out gradient centrifugation, collecting components with the density of 1.13-1.19 g / mL, collecting precipitates, resuspending to obtain a purified exosome, carrying out size exclusion chromatography to obtain a size exclusion chromatography solution, and concentrating to obtain the anti-MUC1 CAR-T exosome. The prepared exosome is small in particle size, high in exosome marker protein expression and high in purity, lung cancer cells can be effectively inhibited, and the tumor size is reduced.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy, specifically relating to a method for preparing exosomes derived from anti-MUC1 CAR-T, as well as the product and its application. Background Technology

[0002] Lung cancer is one of the malignant tumors with the highest incidence and mortality rates worldwide. Mucin 1 (MUC1), a transmembrane glycoprotein, is expressed at low levels in normal epithelial cells, but is highly expressed and abnormally glycosylated in various malignant tumors such as lung cancer, breast cancer, and pancreatic cancer, forming tumor-specific antigenic epitopes and becoming an ideal target for tumor targeted therapy.

[0003] In recent years, chimeric antigen receptor T-cell (CAR-T) therapy has made breakthrough progress in hematologic malignancies, but it faces multiple bottlenecks in the treatment of solid tumors (such as lung cancer): (1) Tumor microenvironment (TME) inhibition: Immunosuppressive molecules such as PD-L1 and TGF-β present in the TME of lung cancer can lead to the depletion of CAR-T cell function and reduce killing activity; (2) Imbalance between targeting and safety: Some CAR-T cells attack normal tissues due to off-target effects, causing serious toxic side effects; (3) Insufficient penetration ability of solid tumors: As living cells, CAR-T cells have difficulty penetrating the dense stromal barrier of solid tumors in lung cancer and cannot effectively contact deep tumor cells.

[0004] Exosomes, membrane-bound vesicles with a diameter of 30-150 nm, possess advantages such as low immunogenicity, good biocompatibility, and the ability to penetrate biological barriers. They can also carry functional molecules from parental cells, making them a next-generation carrier for targeted cancer therapy. The advantages of exosomes include: no need for live cell infusion, allowing for mass production and reduced costs; no proliferative capacity, avoiding toxic side effects; and small size, enabling them to penetrate the solid tumor matrix and deliver therapeutic molecules to a targeted site.

[0005] However, the clinical translation of CAR-T cell-derived exosomes (CAR-T-Exo) is still limited by the lack of efficient preparation and purification technologies. Existing exosome preparation methods suffer from problems such as low purity, low recovery rate, poor specificity, and loss of activity.

[0006] To address the treatment needs of MUC1-positive lung cancer and the technical bottlenecks in CAR-T-Exo preparation, developing a high-purity, high-recovery, and high-activity anti-MUC1 CAR-T-Exo preparation method is crucial for promoting the clinical translation of exosome therapy for solid tumors. Summary of the Invention

[0007] To address the above shortcomings, the present invention provides a composition, a method for preparing the same, and its application in protecting brain health.

[0008] The technical solution of the present invention includes: On one hand, the present invention provides a method for preparing exosomes derived from anti-MUC1 CAR-T, characterized in that the exosome preparation method includes the following steps: S1 and anti-MUC1 CAR-T cell culture medium were centrifuged, the supernatant was collected, filtered, and the filtrate was obtained. S2. Add PEG-6000 solution to the filtrate to make the final concentration of PEG-6000 3%-4% w / v. After incubation, collect the precipitate, resuspend it, and obtain the exocrine resuspension. S3. Iodixanol solutions with final concentrations of 18% v / v, 16% v / v, 14% v / v, 12% v / v, 10% v / v, and 8% v / v are sequentially added from bottom to top into centrifuge tubes to obtain iodixanol gradient tubes. S4. The exosome resuspension was spread on the top layer of an iodixanol gradient tube and subjected to gradient centrifugation. Components with a density of 1.13-1.19 g / mL were collected, the precipitate was collected, and the resuspension was performed to obtain purified exosomes. S5. Purify the exosomes and perform size exclusion chromatography to obtain size exclusion chromatography solution, concentrate it, and obtain anti-MUC1CAR-T exosomes.

[0009] Specifically, the centrifugation in step S1 includes: centrifuging at 200-400×g at room temperature for 5-15 min, collecting the supernatant, centrifuging at 1500-3000×g at 4℃ for 15-30 min, and collecting the supernatant.

[0010] Preferably, the centrifugation in step S1 includes: centrifuging at 300×g at room temperature for 10 min, collecting the supernatant, centrifuging at 2000×g at 4℃ for 20 min, and collecting the supernatant.

[0011] Specifically, the filtration described in step S1 is filtration using a sterile filter membrane.

[0012] Preferably, the filtration in step S1 is performed using a 0.22 μm sterile filter membrane.

[0013] Specifically, the final concentration of PEG-6000 mentioned in step S2 is 3.0%-3.1%w / v, 3.1%-3.2%w / v, 3.2%-3.3%w / v, 3.3%-3.4%w / v, 3.4%-3.5%w / v, 3.5%-3.6%w / v, 3.6%-3.7%w / v, 3.7%-3.8%w / v, 3.8%-3.9%w / v, or 3.9%-4.0%w / v.

[0014] Preferably, the final concentration of PEG-6000 in step S2 is 3.0%-3.1%w / v, 3.1%-3.2%w / v, 3.2%-3.3%w / v, 3.3%-3.4%w / v, or 3.4%-3.5%w / v.

[0015] More preferably, the final concentration of PEG-6000 in step S2 is 3.3%-3.4% w / v.

[0016] Specifically, the PEG-6000 solution mentioned in step S2 is a solution with a concentration of 15%-25% w / v PEG-6000.

[0017] Preferably, the PEG-6000 solution mentioned in step S2 is a solution with a concentration of 15%-16% w / v, 16%-17% w / v, 17%-18% w / v, 18%-19% w / v, 19%-20% w / v, 20%-21% w / v, 21%-22% w / v, 22%-23% w / v, 23%-24% w / v, or 24%-25% w / v PEG-6000.

[0018] Specifically, the volume ratio of the filtrate to the PEG-6000 solution in step S2 is 4-5:1.

[0019] Preferably, the volume ratio of the filtrate to the PEG-6000 solution in step S2 is 4.0-4.1:1, 4.1-4.2:1, 4.2-4.3:1, 4.3-4.4:1, 4.4-4.5:1, 4.5-4.6:1, 4.6-4.7:1, 4.7-4.8:1, 4.8-4.9:1, or 4.9-5.0:1. More preferably, the volume ratio of the filtrate to the PEG-6000 solution in step S2 is 5:1.

[0020] Specifically, the incubation in step S2 includes incubation at 0-5°C in the dark for 2-4 hours.

[0021] Preferably, the incubation in step S2 includes incubation at 4°C in the dark for 3 hours.

[0022] Specifically, the collection of precipitate in step S2 is: centrifugation to collect the precipitate.

[0023] Preferably, the centrifugation is performed at 4000-6000×g, at 4℃ for 10-20 min, the supernatant is discarded, and the precipitate is collected.

[0024] More preferably, the centrifugation is performed at 5000×g, 4℃ for 15 min, the supernatant is discarded, and the precipitate is collected.

[0025] Specifically, the resuspension mentioned in step S2 involves resuspending the precipitate 2-3 times using sterile PBS.

[0026] Preferably, the resuspension in step S2 involves resuspending the precipitate twice using sterile PBS.

[0027] Specifically, the amount of iodixanol solution added in step S3 is 1-2 mL for each concentration.

[0028] Preferably, the amount of iodixanol solution added in step S3 is 1.5 mL for each concentration.

[0029] Specifically, after adding the iodixanol solution in step S3, allow it to stand at 0-5℃ for 20-40 minutes to stabilize the gradient. Preferably, after adding the iodixanol solution in step S3, the mixture is allowed to stand at 4°C for 30 minutes to stabilize the gradient. Specifically, the amount of exocrine body suspension added in step S4 is 0.5-1.5 mL.

[0030] Preferably, the amount of exocrine resuspension added in step S4 is 1 mL.

[0031] Specifically, the gradient centrifugation described in step S4 includes: using an SW41Ti rotor, 80,000-120,000×g, 0-5℃, for 15-20h (slow acceleration, acceleration ≤5, deceleration ≤3).

[0032] Specifically, the gradient centrifugation described in step S4 includes: using an SW41Ti rotor, 100,000×g, 4℃, for 16h (slow acceleration, acceleration ≤5, deceleration ≤3).

[0033] Preferably, the size exclusion chromatography solvent in step S5 is an eluent with A280 > 100 mA U.

[0034] Specifically, the concentration described in step S5 is ultrafiltration concentration.

[0035] Preferably, the volume of the ultrafiltration concentration is 5-10 times the volume.

[0036] More preferably, the volume of the ultrafiltration concentration is 5 times the volume.

[0037] On the other hand, the present invention provides anti-MUC1 CAR-T exosomes prepared by any of the above-described exosome preparation methods.

[0038] In another aspect, the present invention provides the use of the above-mentioned anti-MUC1 CAR-T exosomes in the preparation of drugs for the prevention, treatment or adjuvant treatment of lung cancer.

[0039] Specifically, the drug achieves its therapeutic effect by inhibiting the proliferation and invasion of lung cancer cells, inducing tumor cell apoptosis, and reducing the release of pro-inflammatory factors.

[0040] In another aspect, the present invention provides a drug comprising the above-mentioned anti-MUC1 CAR-T exosomes.

[0041] Preferably, the dosage form of the drug includes any one or more of the following: tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays, and injections.

[0042] Preferably, the drug is used for the prevention, treatment, or adjunctive treatment of lung cancer.

[0043] Specifically, the drug also includes pharmaceutically acceptable carriers.

[0044] Preferably, the pharmaceutically acceptable carrier includes any one or more of the following: diluent, binder, disintegrant, lubricant, solvent, cosolvent, stabilizer, preservative, pH adjuster, surfactant, and lipid carrier.

[0045] In another aspect, the present invention provides a method for preventing, treating or adjuvant treatment of lung cancer, the method comprising using the aforementioned anti-MUC1 CAR-T exosomes or drugs.

[0046] Specifically, the method includes administering an effective amount of anti-MUC1 CAR-T exosomes or drugs to the subject.

[0047] Preferably, the subject is a mammal.

[0048] More preferably, the mammal is a human.

[0049] The beneficial effects of this invention are: The exosomes prepared by the method of this invention have a particle size concentrated in the range of 80-200 nm, exhibit a typical disc-shaped structure, clear membrane boundaries, and no obvious aggregation. They highly express CD8, CD63, and TSG101 exosome marker proteins, with a purity of 19.92 × 10⁻⁶. 9 particles / μg.

[0050] Experiments showed that anti-MUC1 CAR-T exosomes inhibited MUC1-A549 lung cancer cells by 57.97% (cell survival rate was only 42.03% at a concentration of 500 μg / mL); and anti-MUC1 CAR-T exosomes exhibited good anti-tumor activity in vivo. The exosome preparation method of this invention enables efficient purification of exosomes, and the product can be used to prepare lung cancer therapeutic drugs, providing a new strategy for immunotherapy of solid tumors. Attached Figure Description

[0051] Figure 1 This is a particle size distribution of anti-MUC1 CAR-T exosomes.

[0052] Figure 2 TEM image of anti-MUC1 CAR-T exosomes.

[0053] Figure 3 This is a WB assay of anti-MUC1 CAR-T exosomes.

[0054] Figure 4 This image shows the in vivo tumor-inhibiting effect of anti-MUC1 CAR-T exosomes.

[0055] Figure 5 This image shows the effect of anti-MUC1 CAR-T exosomes on inhibiting tumor volume and weight in vivo.

[0056] Figure 6 The in vivo tumor inhibition rate of anti-MUC1 CAR-T exosomes. Detailed Implementation

[0057] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0058] Basic Example 1: Construction of anti-MUC1 CAR-T cells Using CRISPR / Cas9 technology, PD-1 gene-specific sgRNA was designed and co-transfected with Cas9 protein into human peripheral blood T cells. Simultaneously, a CAR expression vector containing the MUC1 scFv-4-1BB-CD3ζ structure was introduced. PD-1 was screened by flow cytometry. - / - Cell lines with CAR expression rates ≥70% were used to obtain anti-MUC1 CAR-T cells. These anti-MUC1 CAR-T cells are the PD-1 gene knockout CAR-T cells prepared in Example 3 of patent CN112940137A, and the construction method is detailed in the CN112940137A specification.

[0059] Anti-MUC1 CAR-T cells were cultured in serum-free KBM581 medium to the logarithmic growth phase. The culture medium of anti-MUC1 CAR-T cells was collected for the preparation of exosomes. Example 1: Preparation of anti-MUC1 CAR-T exosomes 1. Pretreatment of anti-MUC1 CAR-T cell supernatant Anti-MUC1 CAR-T cell culture medium was centrifuged at 300×g at room temperature for 10 min, the bottom cell pellet was discarded, and the supernatant was retained. The supernatant was centrifuged at 2000×g at 4℃ for 20 min to remove cell debris and apoptotic bodies, and the supernatant was retained. The supernatant was filtered through a 0.22μm sterile filter membrane and the filtrate was collected.

[0060] 2. PEG-6000 precipitation Weigh PEG-6000 powder and dissolve it in sterile PBS to obtain a 20% w / v PEG-6000 solution. Add the 20% w / v PEG-6000 solution to the filtrate at a volume ratio of 5:1 (5:1). Mix thoroughly and incubate at 4°C in the dark for 3 hours. Centrifuge at 5000×g at 4°C for 15 minutes, discard the supernatant, and collect the precipitate.

[0061] Resuspend the precipitate in 1 mL of sterile PBS. Gently scrape the bottom of the centrifuge tube with the pipette tip to avoid adhering the exosomes to the tube wall. Centrifuge at 5000×g, 4℃ for 15 min. Collect the precipitate and resuspend it in 1 mL of sterile PBS. Gently pipette 10-15 times and let stand for 5 min. Aspirate the supernatant to obtain the exosome resuspension.

[0062] 3. Iodixanol gradient centrifugation (1) Preparation of density gradient: In a 13 mL ultrafiltration tube, 1.5 mL of iodixanol solution of various concentrations prepared by sterile PBS (final concentration: 18% v / v, 16% v / v, 14% v / v, 12% v / v, 10% v / v, 8% v / v) was added from bottom to top, and the gradient was allowed to stand at 4℃ for 30 min to stabilize.

[0063] (2) Sample loading and centrifugation: Slowly spread 1 mL of exocrine body suspension on the top layer of the gradient, use SW41Ti rotor, 100,000×g, 4℃, 16 h (slow acceleration, acceleration ≤5, deceleration ≤3). (3) Collection of exosome layer: After centrifugation, remove the floating matter at the top of the supernatant tube, and collect 1 tube every 0.2 mL from the bottom to the top using a syringe. Measure the density of each tube with a densitometer. Collect tubes with a density of 1.13-1.19 g / mL, combine them, and add PBS to make up to 15 mL. Centrifuge at 100000×g at 4℃ for 60 min, discard the supernatant, and resuspend the precipitate in 1 mL of sterile PBS. This is the purified exosome.

[0064] 4. Size exclusion chromatography purification Pre-load a SEC column (GE Healthcare Sepharose CL-4B, 10 mL column volume, 1 cm inner diameter), fix the column vertically, connect the UV detector, and wash with sterile PBS at a flow rate of 0.3 mL / min for 10 column volumes until the UV absorbance (280 nm) of the eluent drops to the baseline.

[0065] Take 1 mL of purified exosomes and slowly inject them into the sample loading port of the chromatographic column using a syringe. After loading, rinse the syringe with 1 mL of sterile PBS to push all residual sample into the column, ensuring that the sample completely enters the stationary phase. Elute with sterile PBS at a constant flow rate of 0.3 mL / min, while simultaneously monitoring the absorbance at 280 nm using a UV detector. Once A280 > 100 mAU, begin collecting the exosome-containing fraction until A280 < 100 mAU, at which point collection stops. This is the SEC-purified exosome solution. Transfer the combined exosome solution to a 30 kDa ultrafiltration centrifuge tube and centrifuge at 3000 × g for 15-20 min at 4 °C to concentrate the volume by 5 times, obtaining the anti-MUC1 CAR-T exosome sample.

[0066] NTA analysis showed that the anti-MUC1 CAR-T exosomes were mainly concentrated in the 80-200 nm range, exhibiting a unimodal distribution with an average particle size of 173.6 nm. Representative spectra are shown below. Figure 1 As shown, the results indicate that the particle size distribution curve of anti-MUC1 CAR-T exosomes follows a normal distribution.

[0067] Morphological characteristics: TEM image ( Figure 2 The results showed that the exosomes had a typical disc-shaped structure with clear membrane boundaries and no obvious aggregation.

[0068] Western blot: Figure 3 The results showed that CD8, CD63, and TSG101 all exhibited clear and uniform positive signals with high gray values, indicating good enrichment of exosome marker proteins. Calnexin showed no obvious band, which is consistent with the "three positive and one negative" protein marker characterization of exosomes, indicating that the exosome preparation was successful.

[0069] Comparative Example 1: Preparation of anti-MUC1 CAR-T exosomes The only difference between Comparative Example 1 and Example 1 is the step "2, PEG-6000 precipitation". The specific process is as follows: 2. PEG-6000 precipitation Weigh PEG-6000 powder and dissolve it in sterile PBS to obtain a 20% w / v PEG-6000 solution. Add the 20% w / v PEG-6000 solution to the filtrate at a volume ratio of 3:1 (filtrate to 20% w / v PEG-6000 solution), mix thoroughly, incubate at 4°C in the dark for 3 hours, centrifuge at 5000×g at 4°C for 15 minutes, discard the supernatant, and collect the precipitate.

[0070] Resuspend the precipitate in 1 mL of sterile PBS. Gently scrape the bottom of the centrifuge tube with the pipette tip to avoid adhering the exosomes to the tube wall. Centrifuge at 5000×g, 4℃ for 15 min. Collect the precipitate and resuspend it in 1 mL of sterile PBS. Gently pipette 10-15 times and let stand for 5 min. Aspirate the supernatant to obtain the exosome resuspension.

[0071] Comparative Example 2: Preparation of anti-MUC1 CAR-T exosomes The only difference between Comparative Example 2 and Example 1 is the step "2, PEG-6000 precipitation". The specific process is as follows: Weigh PEG-8000 powder and dissolve it in sterile PBS to obtain a 20% w / v PEG-8000 solution. Add the 20% w / v PEG-8000 solution to the filtrate at a volume ratio of 5:1 (filtrate to PEG-8000 solution). Mix thoroughly and incubate at 4°C in the dark for 3 hours. Centrifuge at 5000×g at 4°C for 15 minutes, discard the supernatant, and collect the precipitate.

[0072] Resuspend the precipitate in 1 mL of sterile PBS. Gently scrape the bottom of the centrifuge tube with the pipette tip to avoid adhering the exosomes to the tube wall. Centrifuge at 5000×g, 4℃ for 15 min. Collect the precipitate and resuspend it in 1 mL of sterile PBS. Gently pipette 10-15 times and let stand for 5 min. Aspirate the supernatant to obtain the exosome resuspension.

[0073] Comparative Example 3: Preparation of anti-MUC1 CAR-T exosomes The only difference between Comparative Example 3 and Example 1 is the step "3. Iodixanol gradient centrifugation". The specific process is as follows: 3. Iodixanol gradient centrifugation (1) Preparation of density gradient: In a 13 mL ultrafiltration tube, 1.5 mL of each concentration of iodixanol solution prepared by sterile PBS (final concentration: 30%, 25%, 20%, 15%, 10%, 5%) was added from bottom to top, and the gradient was allowed to stabilize at 4℃ for 30 min.

[0074] (2) Sample loading and centrifugation: Slowly spread 1 mL of exocrine body suspension on the top layer of the gradient, use SW41Ti rotor, 100,000×g, 4℃, 16 h (slow acceleration, acceleration ≤5, deceleration ≤3). (3) Collection of exosome layer: After centrifugation, remove the floating matter at the top of the supernatant tube, and collect 1 tube every 0.2 mL from the bottom to the top using a syringe. Measure the density of each tube with a densitometer. Collect tubes with a density of 1.13-1.19 g / mL, combine them, and add PBS to make up to 15 mL. Centrifuge at 100000×g at 4℃ for 60 min, discard the supernatant, and resuspend the precipitate in 1 mL of sterile PBS. This is the purified exosome.

[0075] Comparative Example 4: Preparation of anti-MUC1 CAR-T exosomes The only difference between Comparative Example 4 and Example 1 is the absence of step "2, PEG-6000 precipitation". The specific process is as follows: 1. Pretreatment of anti-MUC1 CAR-T cell supernatant Refer to Example 1.

[0076] 2. Iodixanol gradient centrifugation (1) Preparation of density gradient: In a 13 mL ultrafiltration tube, 1.5 mL of each concentration of iodixanol solution prepared by sterile PBS (final concentration: 18%, 16%, 14%, 12%, 10%, 8%) was added from bottom to top, and the gradient was allowed to stabilize at 4℃ for 30 min.

[0077] (2) Sample loading and centrifugation: Slowly spread 1 mL of filtrate on the top layer of the gradient, use an SW41Ti rotor, 100,000×g, 4℃, 16 h (slow acceleration, acceleration ≤5, deceleration ≤3). (3) Collection of exosome layer: After centrifugation, remove the floating matter at the top of the supernatant tube, and collect 1 tube every 0.2 mL from the bottom to the top using a syringe. Measure the density of each tube with a densitometer. Collect tubes with a density of 1.13-1.19 g / mL, combine them, and add PBS to make up to 15 mL. Centrifuge at 100000×g at 4℃ for 60 min, discard the supernatant, and resuspend the precipitate in 1 mL of sterile PBS. This is the purified exosome.

[0078] 3. Size exclusion chromatography purification Refer to Example 1.

[0079] Preparation of anti-MUC1 CAR-T exosomes (Comparative Example 5) The only difference between Comparative Example 5 and Example 1 is the absence of step "3. Iodixanol gradient centrifugation". The specific process is as follows: 1. Pretreatment of anti-MUC1 CAR-T cell supernatant Refer to Example 1.

[0080] 2. PEG-6000 precipitation Refer to Example 1.

[0081] 3. Size exclusion chromatography purification Pre-load a SEC column (GE Healthcare Sepharose CL-4B, 10 mL column volume, 1 cm inner diameter), fix the column vertically, connect the UV detector, and wash with sterile PBS at a flow rate of 0.3 mL / min for 10 column volumes until the UV absorbance (280 nm) of the eluent drops to the baseline.

[0082] Take 1 mL of exosome resuspension and slowly inject it into the sample loading port of the chromatographic column using a syringe. After loading, rinse the syringe with 1 mL of sterile PBS to push all residual sample into the column, ensuring that the sample completely enters the stationary phase. Elute with sterile PBS at a constant flow rate of 0.3 mL / min, while simultaneously monitoring the absorbance at 280 nm using a UV detector. Once A280 > 100 mAU, begin collecting the exosome-containing fraction until A280 < 100 mAU, at which point collection stops. This is the SEC-purified exosome solution. Transfer the combined exosome solution to a 30 kDa ultrafiltration centrifuge tube and centrifuge at 3000 × g for 15-20 min at 4 °C to concentrate the volume by 5 times, obtaining the anti-MUC1 CAR-T exosome sample.

[0083] Preparation of anti-MUC1 CAR-T exosomes (Comparative Example 6) The only difference between Comparative Example 6 and Example 1 is the absence of steps "2. PEG-6000 precipitation" and "3. Iodixanol gradient centrifugation". The specific process is as follows: 1. Pretreatment of anti-MUC1 CAR-T cell supernatant Refer to Example 1.

[0084] 2. Size exclusion chromatography purification Pre-load a SEC column (GE Healthcare Sepharose CL-4B, 10 mL column volume, 1 cm inner diameter), fix the column vertically, connect the UV detector, and wash with sterile PBS at a flow rate of 0.3 mL / min for 10 column volumes until the UV absorbance (280 nm) of the eluent drops to the baseline.

[0085] Take 1 mL of filtrate and slowly inject it into the sample loading port of the chromatographic column using a syringe. After loading, rinse the syringe with 1 mL of sterile PBS to push all residual sample into the column, ensuring that the sample completely enters the stationary phase. Elute with sterile PBS at a constant flow rate of 0.3 mL / min, while simultaneously monitoring the absorbance at 280 nm using a UV detector. Once A280 > 100 mAU, begin collecting the exosome-containing fraction until A280 < 100 mAU, at which point collection stops. This is the SEC-purified exosome solution. Transfer the combined exosome solution to a 30 kDa ultrafiltration centrifuge tube and centrifuge at 3000 × g for 15-20 min at 4 °C to concentrate the volume by 5 times, obtaining the anti-MUC1CAR-T exosome sample.

[0086] Experimental Example 1: Determination of anti-MUC1 CAR-T exosome purity Exosome content / concentration was obtained using nanoflow cytometry, and protein content was determined using a BCA kit. Exosome purity can be calculated from exosome content and protein concentration: Exosome purity (particales / μg) = Exosome concentration (particales / μL) / Protein content (μg / μL).

[0087] The exosome purity of the anti-MUC1 CAR-T exosome samples prepared in Example 1 and Comparative Examples 1-6 was determined using the above method. The results are shown in Table 1. Table 1

[0088] Comparative Example 1: The final concentration of PEG-6000 was increased to 5% w / v (3:1 volume ratio). This excessively high concentration resulted in the co-precipitation of a large amount of impurities, increasing the protein content to 0.52 μg / μL and reducing the exosome purity to 12.37 × 10⁻⁶. 9 particles / μg; it is suggested that the final concentration of PEG-6000 should be controlled at 3%-4% w / v (5:1 volume ratio) in order to balance enrichment efficiency and removal of impurities.

[0089] Comparative Example 2: Replacing PEG-6000 with PEG-8000 increased exosome purity from 19.92 × 10⁻⁶. 9 The number of particles / μg decreased to 17.04 × 10⁻⁶. 9 The particle count / μg indicates that PEG-6000 is a key component.

[0090] Comparative Example 3: In a wide gradient (5%-30%), the high-concentration layer easily adsorbed cell debris, while the low-concentration layer could not effectively separate HDL and exosomes, resulting in an increase in residual protein levels to 0.68 μg / μL and a decrease in purity to 10.39 × 10⁻⁶. 9particles / μg. This demonstrates that a narrow gradient (8%-18%) is better suited to the density characteristics of anti-MUC1 CAR-T exosomes, thus improving separation specificity.

[0091] Comparative Example 4: Lacking the PEG-6000 precipitation step, the sample was directly loaded with filtrate, resulting in a low initial exosome concentration of only 3.97 × 10⁻⁶. 9 The protein concentration was as high as 0.75 μg / μL, with a large amount of other proteins entering the gradient via exosomes, resulting in a final protein content of only 5.28 × 10⁻⁶. 9 particles / μg.

[0092] Comparative Example 5: Lacking the iodixanol gradient centrifugation step, it was unable to remove microvesicles and large molecular weight proteins with particle sizes similar to exosomes. The protein content increased to 0.89 μg / μL, and the purity decreased to 5.88 × 10⁻⁶. 9 particles / μg.

[0093] Comparative Example 6 only used pretreatment + SEC purification; exosomes were not enriched, and the initial concentration was extremely low (1.2 × 10⁻⁶). 9 The protein concentration was 1.32 μg / μL, and the purity was only 0.98 × 10⁻⁶. Furthermore, a large amount of contaminating proteins directly entered the SEC column, resulting in a protein content of only 0.98 × 10⁻⁶. 9 particles / μg.

[0094] Comparative Example 6 lacked the PEG-6000 precipitation step and the iodixanol gradient centrifugation step, resulting in a purity of only 0.98 × 10⁻⁶. 9 Using particles / μg as a baseline, and comparing with Comparative Example 6, the synergistic effect of "PEG-6000 precipitation" and "iodixanol gradient centrifugation" was calculated: Compared to Comparative Example 6, Comparative Example 4 added an iodixanol gradient centrifugation step; Comparative Example 5 added a PEG-6000 precipitation step; and Example 1 added both a PEG-6000 precipitation step and an iodixanol gradient centrifugation step. The exosome purity of Comparative Example 4 increased by 439.20% compared to Comparative Example 6, and the purity of Comparative Example 5 increased by 499.89%, for a combined increase of 939.10%. In contrast, the purity of exosomes in Example 1 increased by 1932.53% compared to Comparative Example 6, which is greater than the combined increase of Comparative Examples 4 and 5 compared to Comparative Example 6. This demonstrates that the combination of the PEG-6000 precipitation step and the iodixanol gradient centrifugation step in this invention has a synergistic effect in the purification of anti-MUC1 CAR-T exosomes.

[0095] Experimental Example 2: Detection of in vitro antitumor activity of anti-MUC1 CAR-T exosomes Human lung adenocarcinoma cell line MUC1 + A549 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were used for experiments. MUC1 + A549 cells at 5 × 10 3 Cells were seeded at a density of 100 μL / well in 96-well cell culture plates and cultured for 24 h to allow cell adhesion. The anti-MUC1 CAR-T exosomes prepared in Example 1 and Comparative Examples 1-6 were diluted to 500 μg / mL with DMEM medium; a blank control group (medium only, without MUC1) was also set up. + A549 cells), cell control group (MUC1) + (A549 cells + culture medium), 3 parallel wells per group. Remove the original culture medium from the 96-well plate, add 100 μL of culture medium containing the corresponding anti-MUC1 CAR-T exosomes to each well, incubate at 37℃, 5% CO2 for 24 h, collect the supernatant, and use the CCK-8 kit to measure the absorbance (OD) of each well at 450 nm.

[0096] Cell viability (%) = (OD value of exosome group - mean OD value of blank control group) / (mean OD value of cell control group - mean OD value of blank control group) × 100%.

[0097] The measurement results are shown in Table 2: Table 2

[0098] The results showed that the anti-MUC1 CAR-T exosomes prepared in Example 1 of this invention had the best in vitro antitumor activity.

[0099] Experiment 3: Validation of the efficacy of an animal model of anti-MUC1 CAR-T exosomes 1. Laboratory animals SPF-grade NSG mice (4 weeks old, half male and half female) were used in this experiment. The temperature was 22-25℃, the humidity was 50%-60%, and the light and dark were alternated for 12 hours. They were allowed free access to sterile feed and autoclaved water and were acclimatized for 1 week before the experiment.

[0100] 2. Experimental Methods 2.1 Constructing an animal model of lung adenocarcinoma (1) Cell preparation: Logarithmic growth phase MUC1-A549 cells were used, and the concentration was adjusted to 5×10⁻⁶ after trypsin digestion. 7MUC1-A549 cell suspension was obtained by increasing cell count / mL and used for model construction.

[0101] (2) Inoculation procedure: 100 μL of MUC1-A549 cell suspension was injected subcutaneously into the right thigh of NSG mice.

[0102] (3) Model validation: Observe the injection site daily; tumor volume ≥100mm 3 MUC1-A549 lung adenocarcinoma mouse model was obtained, and the experimental animals were randomly assigned to each group.

[0103] 2.2 Grouping and Dosing MUC1-A549 lung adenocarcinoma model mice were divided into a model control group, a CAR-T treatment group, a low-dose exosome group, a medium-dose exosome group, and a high-dose exosome group, with 6 mice in each group.

[0104] Blank control group: The same volume of normal saline was injected into the tail vein once every 3 days for a total of 6 times.

[0105] CAR-T therapy group: 1×10⁻⁶ mmol / L via tail vein injection 7 One anti-MUC1 CAR-T cell per dose, 7 days apart, for a total of 2 doses.

[0106] Low-dose exosome group: 50 μg of the anti-MUC1 CAR-T exosome solution from Example 1 was injected via the tail vein once every 3 days for a total of 6 times.

[0107] Medium-dose exosome group: 100 μg of the anti-MUC1 CAR-T exosome solution from Example 1 was injected via tail vein once every 3 days for a total of 6 times.

[0108] High-dose exosome group: 200 μg of the anti-MUC1 CAR-T exosome solution from Example 1 was injected via the tail vein once every 3 days for a total of 6 times.

[0109] 3. Experimental Results The experiment was terminated after 28 days of treatment. Anti-MUC1 CAR-T exosomes showed significant in vivo tumor-suppressing activity. Figures 4-6 Throughout the treatment process, tumor growth in mice was monitored. The high-dose exosome group significantly inhibited tumor growth compared to the control group (P<0.01), and its tumor-suppressing effect was comparable to that of the CAR-T therapy group (P>0.05). The tumor volume in the high-dose exosome group was 342.80±143.19 mm. 3 Tumor volume in the CAR-T treatment group (254.67±77.2 mm) vs. CAR-T treatment group 3 The low-dose and medium-dose exosome groups also showed some tumor-suppressive effects (P<0.1).

[0110] Relative tumor weight analysis also showed that anti-MUC1 CAR-T exosomes significantly reduced tumor weight. Meanwhile, the high-dose exosome group also showed a higher tumor inhibition rate (66.2%), which was not statistically different from the tumor inhibition rate of the CAR-T treatment group (70.4%) (P>0.05).

[0111] These results all indicate that the anti-MUC1 CAR-T exosomes of the present invention have good anti-tumor activity in vivo.

[0112] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing exosomes derived from anti-MUC1 CAR-T, characterized in that, The method for preparing exosomes includes the following steps: S1 and anti-MUC1 CAR-T cell culture medium were centrifuged, the supernatant was collected, filtered, and the filtrate was obtained. S2. Add PEG-6000 solution to the filtrate to make the final concentration of PEG-6000 3%-4% w / v. After incubation, collect the precipitate, resuspend it, and obtain the exocrine resuspension. S3. Iodixanol solutions with final concentrations of 18% v / v, 16% v / v, 14% v / v, 12% v / v, 10% v / v, and 8% v / v are sequentially added from bottom to top into centrifuge tubes to obtain iodixanol gradient tubes. S4. The exosome resuspension was spread on the top layer of an iodixanol gradient tube and subjected to gradient centrifugation. Components with a density of 1.13-1.19 g / mL were collected, the precipitate was collected, and the resuspension was performed to obtain purified exosomes. S5. Purify the exosomes and perform size exclusion chromatography to obtain size exclusion chromatography solution, concentrate it, and obtain anti-MUC1 CAR-T exosomes.

2. The method for preparing exosomes according to claim 1, characterized in that, The centrifugation described in step S1 includes: centrifuging at 200-400×g at room temperature for 5-15 min, collecting the supernatant, then centrifuging at 1500-3000×g at 4℃ for 15-30 min, and collecting the supernatant.

3. The method for preparing exosomes according to claim 1, characterized in that, The PEG-6000 solution mentioned in step S2 is a solution with a concentration of 15%-25% w / v PEG-6000; the volume ratio of the filtrate to the PEG-6000 solution is 4-5:

1.

4. The method for preparing exosomes according to claim 1, characterized in that, The incubation described in step S2 includes incubation at 0-5°C in the dark for 2-4 hours.

5. The method for preparing exosomes according to claim 1, characterized in that, The amount of iodixanol solution added in step S3 is 1-2 mL for each concentration.

6. The method for preparing exosomes according to claim 1, characterized in that, The size exclusion chromatography solvent mentioned in step S5 is an eluent with A280 > 100 mAU; the concentration is ultrafiltration concentration; the volume of ultrafiltration concentration is 5-10 times the volume.

7. Anti-MUC1 CAR-T exosomes prepared by the exosome preparation method according to any one of claims 1-6.

8. The use of the anti-MUC1 CAR-T exosomes according to claim 7 in the preparation of medicaments for the prevention, treatment or adjuvant treatment of lung cancer.

9. A drug comprising the anti-MUC1 CAR-T exosome as described in claim 7.

10. The medicament according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable carriers.

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