A composition for enhancing chimeric antigen receptor t cell function and a preparation method and application thereof

Abstract

The application discloses a kind of compositions for enhancing chimeric antigen receptor T cell function and preparation method and application thereof, belong to cell immunotherapy technical field.The composition described in the application comprises subcytotoxic concentration of taxane compound and pharmaceutically acceptable carrier, wherein the concentration of taxane compound is preferably 1-50 nM.Embodiment results show that the composition is used for pretreatment CAR-T cell, induces it to enter a specific state of metabolic active and effect function enhancement.Processed CAR-T cell shows that the proportion of memory phenotype increases, the expression of exhaustion marker decreases, the expression of cytotoxicity effector molecule is up-regulated, and is accompanied by the change of specific extracellular vesicle secretion activity.The application also provides a preparation method of function-enhanced CAR-T cell;The scheme provides a non-genetic strategy, which can improve the persistence and antitumor effect of CAR-T cell in solid tumor treatment, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of cell immunotherapy technology, specifically relating to a composition that enhances the function of chimeric antigen receptor T cells, its preparation method, and its application. Background Technology

[0002] CAR-T cell therapy has made groundbreaking progress in the treatment of hematologic malignancies. However, its application in solid tumors still faces significant challenges. Solid tumors possess a complex immunosuppressive microenvironment (TME), a dense stromal barrier, and a metabolic environment that leads to rapid T cell depletion. These factors collectively limit the infiltration, survival persistence, and cytotoxic activity of CAR-T cells in tumor tissues.

[0003] To overcome these obstacles, existing technologies largely focus on complex genetic engineering modifications, such as knocking out checkpoint genes (e.g., PD-1) or forcibly expressing specific cytokines using technologies like CRISPR / Cas9. While these methods are mechanistically attractive, they often introduce extremely high manufacturing complexity, significant regulatory burdens, and potential genotoxicity risks. Furthermore, multiplex gene editing may disrupt the natural physiological homeostasis of T cells.

[0004] Traditional chemotherapy drugs, such as taxanes (docetaxel, etc.), primarily utilize their properties of inhibiting microtubule dynamics and inducing cell cycle arrest to directly kill rapidly proliferating tumor cells. At conventional therapeutic doses, these drugs are generally considered to have suppressive and toxic effects on the patient's immune system (especially lymphocytes).

[0005] Although existing research has explored the combined application of chemotherapy and immunotherapy, how to remodel the state of CAR-T cells through non-genetic means during the in vitro preparation stage, thereby enhancing their anti-tumor potential while avoiding cytotoxicity, remains a highly challenging and unsolved issue in this field. In particular, inducing CAR-T cells to produce a novel functional state that can maintain highly efficient effector function while overcoming the physical barriers of solid tumors is of great significance for improving the clinical efficacy of CAR-T cells. Summary of the Invention

[0006] The present invention aims to provide a composition for regulating and enhancing the function of chimeric antigen receptor (CAR) T cells, a method for preparing the composition, and the application of the composition in the preparation of medicaments for treating solid tumors. The composition of the present invention can non-genetically remodel CAR-T cells under extremely low dose conditions. While maintaining cell viability, it can induce CAR-T cells into a unique state of "metabolic activity and enhanced function." CAR-T cells in this state not only exhibit significantly enhanced self-killing ability and persistence, but also show significantly enhanced secretory activity of specific extracellular vesicles (such as exosomes) and optimized functional components.

[0007] To achieve the above objectives, the present invention provides the following technical solution: First aspect: A composition for enhancing CAR-T cell function, said composition comprising a taxane compound and a pharmaceutically acceptable carrier.

[0008] Preferably, the taxane compound is at least one of docetaxel, paclitaxel, cabazitaxel, and their respective pharmaceutically acceptable salts. More preferably, it is docetaxel.

[0009] Preferably, the concentration of taxane compounds in the composition is a subcytotoxic concentration, ranging from 1 nM to 50 nM, more preferably from 5 nM to 20 nM, and most preferably 10 nM.

[0010] Preferably, the pharmaceutically acceptable carrier is a pharmaceutically acceptable carrier suitable for T cell culture.

[0011] The application of the above composition in enhancing CAR-T cell function is to treat CAR-T cells with the above composition for a treatment time of 12 to 96 hours, preferably 24 to 72 hours.

[0012] Second aspect: A method for preparing functionally enhanced CAR-T cells, the method comprising the following steps: (1) Co-culture CAR-expressing T cells with the composition described in the first aspect; (2) Collect CAR-T cells after co-culture in step (1) to obtain functionally enhanced CAR-T cells.

[0013] Preferably, the enhanced CAR-T cells exhibit an increased proportion of stem cell-like memory phenotype (TSCM) and central memory phenotype (CM), and a significant decrease in the expression of exhaustion markers (such as PD-1 and CD57). Preferably, they also exhibit upregulated expression of cytotoxic effector molecules. Preferably, the enhanced CAR-T cells are in an active state of "metabolic-cycle decoupling".

[0014] Third aspect: A method for preparing extracellular vesicles (exosomes), the method comprising the following steps: (1) Culturing functionally enhanced CAR-T cells prepared by the method described in the second aspect; (2) Collect the culture supernatant of the cells described in step (1); (3) Separate and purify extracellular vesicles from the supernatant.

[0015] The extracellular vesicles prepared by the above method exhibit antitumor functional components consistent with their parent effector cells, and have the ability to penetrate the tumor matrix and kill bystander tumor cells.

[0016] Preferably, the vesicle has an average particle size distribution between 70-150 nm, expresses classic exosome markers (CD9, CD63, CD81) on its surface, and contains / carries antitumor effector components derived from parental remodeled cells. This vesicle possesses the physical and biological properties to penetrate dense tumor matrix and induce apoptosis in bystander tumor cells.

[0017] Fourth aspect: A pharmaceutical composition comprising: a) functionally enhanced CAR-T cells prepared according to the present invention; and / or b) extracellular vesicles prepared by the present invention.

[0018] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

[0019] Fifth aspect: The present invention relates to the use of the above-mentioned substances in the preparation of drugs for treating solid tumors.

[0020] Preferably, the solid tumor is breast cancer, colorectal cancer, gastric cancer, lung cancer, or pancreatic cancer.

[0021] Preferably, the enhanced CAR-T cells and the extracellular vesicles can be used in combination, sequentially or alternately to overcome tumor microenvironment barriers through direct killing and bystander effects.

[0022] The beneficial effects of this invention are: 1. Non-genetic functional reprogramming and process simplification: CAR-T cells are pharmacologically reprogrammed using the novel immunomodulatory functions of clinically approved taxanes at subcytotoxic doses. This strategy achieves significant enhancement of cell function through non-genetic means, avoiding the genotoxic risks and complex production regulatory processes associated with gene editing, and greatly improving the feasibility of clinical translation.

[0023] 2. Co-optimization of cell state: This strategy induces CAR-T cells into a unique "metabolic-cycle decoupling" state, resulting in CAR-T cell phenotypes that are more inclined towards early differentiation (significantly increased proportion of stem cell-like memory phenotype TSCM), while significantly reducing the expression of exhaustion and aging markers such as PD-1 and CD57. This gives CAR-T cells stronger infiltration ability, persistence, and anti-tumor activity in the complex solid tumor microenvironment.

[0024] 3. High safety and broad applicability: The drug dosage used (e.g., 10 nM docetaxel) is far lower than the cytotoxic dose of routine clinical chemotherapy, and both in vivo and in vitro experiments have confirmed its good safety profile. This method can be seamlessly integrated into existing GMP manufacturing processes as a standard module and is applicable to a variety of CAR-T cell products targeting different antigens.

[0025] 4. Comprehensively overcomes microenvironment barriers, enhancing broad-spectrum killing efficacy against solid tumors and hematological malignancies: CAR-T cells remodeled with the composition of this invention exhibit comprehensively upregulated expression of cytotoxic effector molecules, possessing extremely strong matrix degradation and infiltration capabilities. In in vitro models, they can effectively disrupt and penetrate the dense 3D tumor spheroid physical structure. More importantly, these functionally enhanced CAR-T cells not only demonstrate excellent efficacy in in vivo models of solid tumors, significantly inhibiting tumor growth and greatly prolonging survival, but this remodeling strategy is also applicable to the treatment of hematological malignancies, demonstrating broad-spectrum application potential in overcoming microenvironment barriers in multiple types of tumors.

[0026] 5. Inducing functional vesicle secretion to produce a potent bystander effect: In addition to enhancing cell-wide function, the remodeling process of this invention specifically promotes the secretion of extracellular vesicles (exosomes) with significant independent anti-tumor activity by CAR-T cells. These accompanying functional vesicles can serve as a highly effective supplement to cell therapy, directly penetrating the tumor matrix barrier, killing tumor cells, and targeting and destroying cancer-associated fibroblasts (CAFs), effectively addressing the challenge of tumor antigen heterogeneity, and synergistically remodeling the tumor immune microenvironment. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Multidimensional phenotypic analysis of differentiation subsets of conventional CAR-T cells and docetaxel-remodeled CAR-T cells (Doce-CAR T cells); CD4 + The distribution and quantitative statistics of specific differentiation subsets within the T cell compartments were analyzed using high-dimensional flow cytometry. Scatter plots and corresponding violin plots on the right were used to compare and analyze the differences in community frequencies of effector memory T cells (EM), central memory T cells (CM), terminally differentiated effector cells (EMRA), and stem cell-like memory T cells (SCM). The results showed that the proportions of CM and SCM subsets were significantly increased in remodeled CAR-T cells, while the proportions of EM and EMRA subsets decreased.

[0029] Figure 2 Multidimensional phenotypic analysis of differentiation subsets of conventional CAR-T cells and docetaxel-remodeled CAR-T cells (Doce-CAR T cells); CD8 + The distribution and quantitative statistics of specific differentiation subsets within the T cell compartments were analyzed using high-dimensional flow cytometry. Scatter plots and corresponding violin plots on the right were used to compare and analyze the differences in community frequencies of effector memory T cells (EM), central memory T cells (CM), terminally differentiated effector cells (EMRA), and stem cell-like memory T cells (SCM). The results showed that the proportions of CM and SCM subsets were significantly increased in remodeled CAR-T cells, while the proportions of EM and EMRA subsets decreased.

[0030] Figure 3 This study presents a multidimensional phenotypic analysis of exhaustion markers in conventional CAR-T cells and docetaxel-remodeled CAR-T cells (Doce-CAR T cells); and an analysis of the expression characteristics of the immune exhaustion marker PD-1 in the overall population and specific differentiation subpopulations. The left side shows an expression heatmap of marker-positive cell frequencies, and the right side shows the corresponding quantitative contrast violin plots. The results confirm that, compared with conventional CAR-T cells, Doce-CAR T cells exhibited highly significant downregulation of PD-1 and CD57 expression levels not only at the overall level but also in different differentiation subpopulations.

[0031] Figure 4This study presents a multidimensional phenotypic analysis of aging biomarkers in conventional CAR-T cells and docetaxel-remodeled CAR-T cells (Doce-CAR T cells); and an analysis of the expression characteristics of the aging biomarker CD57 in the overall population and specific differentiation subpopulations. The left side shows an expression heatmap of biomarker-positive cell frequencies, and the right side shows the corresponding quantitative contrast violin plots. The results confirm that, compared with conventional CAR-T cells, Doce-CAR T cells exhibited highly significant downregulation of PD-1 and CD57 expression levels not only at the overall level but also in different differentiation subpopulations.

[0032] Figure 5 This section presents the in vitro cytotoxicity and physical structure disruption effects of Doce-CAR T cells on HCT116 3D tumor spheroids. (Left panel) Representative morphological images of 3D dense tumor spheroids formed by co-culturing different effector cell groups (RPMI 1640 control group, conventional T cells, conventional CAR T cells, docetaxel-remodeled Doce-CAR T cells, and DMSO solvent control CAR T cells) with HCT116 cells. From left to right: brightfield microscopy image, enlarged image (red arrows indicate structural disintegration and effector cell infiltration at the tumor spheroid edge), and 3D surface reconstruction of the tumor spheroid based on the image. (Right panel) Quantitative analysis of tumor spheroid physical volume and metabolic activity. The upper bar chart shows the quantitative statistics of tumor spheroid volume in each treatment group (normalized to the control group); the lower bar chart shows the quantitative results of tumor relative survival based on ATP metabolic activity. Data confirms that, compared with conventional CAR T cells, Doce-CAR T cells can significantly disrupt the physical structure of dense solid tumor spheres and greatly reduce the survival rate of tumor cells.

[0033] Figure 6Physicochemical characterization and classical biomarker identification of docetaxel-remodeled CAR-T cell-derived exosomes (Doce-CAR exos). (A) Exosome particle size distribution curves measured by dynamic light scattering (DLS) show that the main peak of the extracellular vesicles is distributed at approximately 100 nm, consistent with the particle size characteristics of classical exosomes. (B) Quantitative comparative analysis of the average effective particle size of conventional CAR-T cell exosomes (CAR exos) and remodeled exosomes (Doce-CAR exos) (normalized to the CAR exos group). The results indicate that docetaxel pretreatment did not significantly change the physical size of the secreted vesicles. (C) Flow cytometry detection of the expression of classical exosomal four-transmembrane protein biomarkers on the vesicle surface. Representative histogram comparisons show the fluorescence signals of the control group (Control) and the exosome group (Exos), confirming that the isolated and purified vesicles positively express exosomal biomarkers CD9, CD63, and CD81.

[0034] Figure 7 This study evaluates the in vivo antitumor efficacy of docetaxel-remodeled CAR-T cells (Doce-CAR T cells) in a tumor-bearing mouse model. (A) Kaplan-Meier survival curves of tumor-bearing mice in each treatment group (Control, conventional T cell group, conventional CAR-T cell group, and remodeled Doce-CAR T cell group). Statistical analysis showed that the Doce-CAR T cell treatment group significantly prolonged the overall survival of mice. (B) Tumor volume growth kinetics monitoring curve over time. Quantitative results confirmed that, compared with conventional CAR-T cells, Doce-CAR T cells could continuously and significantly inhibit the growth of solid tumors and greatly reduce tumor burden. Detailed Implementation

[0035] This invention provides a composition for regulating and enhancing the function of chimeric antigen receptor (CAR) T cells, a method for preparing the composition, and the use of the composition in the preparation of a medicament for treating solid tumors. Specifically, it includes: Product 1: A composition for enhancing the function of chimeric antigen receptor T cells (CAR-T). Composition (components and dosage): Core active ingredient: Taxanes or pharmaceutically acceptable salts thereof. The taxanes are selected from docetaxel, paclitaxel, or cabazitaxel.

[0036] Carrier components: Liquid culture medium suitable for in vitro expansion and culture of T cells (containing 10% fetal bovine serum or serum substitute, recombinant human IL-2 and other necessary nutrients and cytokines).

[0037] Numerical range (dosage): The working concentration of the taxane compound in the composition is strictly limited to a subcytotoxic concentration range. The feasible numerical range is 1 nM to 50 nM; the preferred numerical range is 5 nM to 20 nM; and the optimal numerical parameter is 10 nM.

[0038] Structure and morphology: This composition is a sterile liquid and is used as a culture medium additive or remodeling inducer for in vitro cell treatment.

[0039] Product 2: A functionally enhanced chimeric antigen receptor T cell (Doce-CAR-T cell)

[0040] Composition and structure: This product is a live cell preparation obtained by remodeling T cells expressing chimeric antigen receptor (CAR) in vitro for 24-72 hours (optimal 48 hours) in the above-mentioned "Product 1", followed by washing to remove residual drugs.

[0041] The CAR targets solid tumors or hematologic malignancies-associated antigens (including but not limited to mesothelin MSLN, GD2, HER2, or EGFRvIII).

[0042] Physical properties (biological phenotype and biomarker characteristics): Cellular state: The cells are in a state of "metabolic-cycle uncoupling", maintaining extremely high cell viability (>95%) while their functions are significantly activated.

[0043] Differentiation subset ratio (flow cytometry characteristics): Compared with untreated conventional CAR-T cells, this product contains stem cell-like memory T cells (TSCM, phenotype CD45RA). + CCR7 + The proportion of T cells and central memory T cells (CM) increased significantly, while the proportion of terminally differentiated effector cells (TEMRA) decreased significantly.

[0044] Surface marker characteristics: The expression levels of cell surface depletion marker PD-1 and aging marker CD57 were significantly downregulated.

[0045] Characteristics of effector molecules: The expression of intracellular cytotoxic effector molecules (such as perforin and granzyme B) is significantly upregulated.

[0046] Product 3: A functionally enhanced extracellular vesicle (cell-free exosome preparation)

[0047] Composition and Source: The extracellular vesicle preparation was obtained by culturing "Product 2 (functionally enhanced CAR-T cells)" in a "metabolicly active" state, followed by secretion into the supernatant, and then separating and purifying it by differential ultracentrifugation.

[0048] Structural and physical property parameters (physicochemical characterization): Particle size parameters: According to nanoparticle tracking analysis (NTA), the average particle size of the vesicle is distributed between 70 nm and 150 nm (with the main peak at around 100 nm).

[0049] Biomarker characteristics: Positive expression of classic exosomal tetraspan membrane protein biomarkers: CD9, CD63, CD81.

[0050] Functional characteristics: The vesicle carries anti-tumor functional components derived from parental CAR-T cells inside or on its membrane surface, and has biological activities that induce tumor cell apoptosis and physically penetrate the dense tumor extracellular matrix (ECM) (Note: The specific enriched target and apoptosis receptor protein spectrum will be used as a point for further in-depth research, and is protected here as a higher-level functional concept).

[0051] Method 1: Preparation method of a composition to enhance CAR-T cell function

[0052] 1. Types and quantities of raw materials: Core active ingredient: Docetaxel API.

[0053] Solvent: Dimethyl sulfoxide (DMSO).

[0054] Culture medium carrier: X-VIVO 15 serum-free medium.

[0055] Additives: Fetal bovine serum (FBS, final concentration 10%), recombinant human IL-2 (final concentration 10 ng / mL).

[0056] 2. Process Flow and Process Parameters: Step 1 (Preparation of stock solution): Weigh an appropriate amount of docetaxel raw material and dissolve it fully in DMSO to prepare a high-concentration stock solution.

[0057] Step 2 (Working solution dilution): Before use, the above stock solution is serially diluted with X-VIVO 15 serum-free medium containing 10% fetal bovine serum and 10 ng / mL IL-2.

[0058] Key parameter control: Strictly control the dilution ratio to prepare a docetaxel working solution with a final concentration of 10 nM (the effective concentration protection range of taxane compounds in this composition is set to 1-50 nM).

[0059] Method 2: Preparation of enhanced chimeric antigen receptor T cells (Doce-CAR-T)

[0060] 1. Types and sources of raw materials: Cell source: peripheral blood mononuclear cells (PBMCs) isolated from healthy donors.

[0061] Activation reagent: CD3 / CD28 antibody complex.

[0062] Vector material: Lentiviral vector encoding a second-generation CAR (scFv-41BB-CD3ζ) that targets tumor antigens (such as mesothelin MSLN).

[0063] Remodeling agent: The composition containing 10 nM docetaxel prepared by method one.

[0064] 2. Process Flow and Process Parameters: Step 1 (Isolation and Activation): Primary T cells were obtained from PBMCs through negative sorting and activated in vitro using a CD3 / CD28 antibody complex.

[0065] Step 2 (Viral transduction): The activated T cells are transduced using the lentiviral vector described above to obtain T cells that conventionally express CAR (such as MSLN-CAR-T cells).

[0066] Step 3 (Pharmacological Remodeling Culture): During the CAR-T cell expansion phase, cells were resuspended in a composite culture medium containing 10 nM docetaxel. Key process parameters: The cell seeding density was adjusted to 1 × 10⁶ / mL (protection range set at 0.5 - 5 × 10⁶ cells / mL), and the cells were co-cultured at 37°C in a 5% CO₂ incubator for 48 hours (time protection range set at 24-96 hours).

[0067] Step 4 (washing and collection): After culture, collect the cell suspension and wash twice with PBS to completely remove residual docetaxel drug, resulting in docetaxel-pretreated remodeled CAR-T cells (cell viability >95% confirmed by trypan blue staining).

[0068] Method 3: Purification and Extraction Method of Functionally Enhanced Extracellular Vesicles

[0069] 1. Types of raw materials: The cell culture supernatant produced during the Doce-CAR-T cell culture expansion in Method 2.

[0070] 2. Process Flow and Parameters (Differential-Ultra-Center Centrifugation): Step 1 (Removal of whole cells): Collect the culture supernatant, centrifuge at 300×g for 5 minutes, discard the precipitate and keep the supernatant.

[0071] Step 2 (Removal of dead cells): Centrifuge the supernatant at 2,000×g for 10 minutes, discard the precipitate and keep the supernatant.

[0072] Step 3 (Removal of large particles): Centrifuge the supernatant at 16,500×g for 45 minutes, discard the precipitate and keep the clarified supernatant.

[0073] Step 4 (Vessel Precipitation): Transfer the clarified supernatant to an ultracentrifuge tube and centrifuge at 100,000×g for 2 hours. Discard the supernatant; the resulting precipitate is the functional extracellular vesicle (exosome), which can then be resuspended in an appropriate amount of PBS for storage.

[0074] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0075] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0076] The raw materials and reagents, schemes and processes in this invention are as follows: 1. Raw materials and reagents: Drugs and solvents: Docetaxel active pharmaceutical ingredient, dimethyl sulfoxide (DMSO).

[0077] Cell culture reagents: X-VIVO 15 serum-free medium, fetal bovine serum (FBS), recombinant human IL-2 (concentration used is 10 ng / mL), phosphate buffer (PBS).

[0078] Cells and transduction materials: peripheral blood mononuclear cells (PBMCs) from healthy donors, CD3 / CD28 antibody complexes, and lentiviral vectors encoding second-generation CARs (such as scFv-41BB-CD3ζ) that target specific antigens (such as mesothelin MSLN).

[0079] 2. Scheme and Process Flow: (1) Preparation of a composition to enhance CAR-T cell function (taking 10 nM concentration as an example) Preparation of stock solution: Weigh the docetaxel active pharmaceutical ingredient and dissolve it fully in DMSO solvent to prepare a high-concentration stock solution.

[0080] Preparation of working solution: Before use, dilute the above stock solution with X-VIVO 15 serum-free medium containing 10% fetal bovine serum and 10 ng / mL IL-2 to prepare a working solution with a final docetaxel concentration of 10 nM. This working solution is the composition of the present invention. (Note: Within the scope of protection, the effective concentration of taxane compounds in the composition is 1-50 nM, with 10 nM being optimal).

[0081] (2) Preparation of functionally enhanced CAR-T cells (Doce-CAR-T)

[0082] Cell isolation and activation: PBMCs were isolated from peripheral blood of healthy donors, and purified primary T cells were obtained using negative sorting technology. The primary T cells were then activated using a CD3 / CD28 antibody complex.

[0083] Viral transduction: Activated T cells are transduced using lentiviral vectors encoding target antigens (such as MSLN) to obtain CAR-expressing T cells (such as MSLN-CAR-T cells).

[0084] Pharmacological remodeling: During the CAR-T cell expansion phase, cells were resuspended in the prepared culture medium containing 10 nM docetaxel. The cell culture density was adjusted to 1 × 10⁶ cells / mL, and the cells were co-cultured at 37°C for 48 hours. (Note: The process protection range covers culture densities of 0.5-5 × 10⁶ cells / mL.) 6 (cells / mL, culture time 24-96 hours).

[0085] Product collection and washing: After the culture period, the cell suspension was collected. The cells were washed twice with PBS buffer to completely remove residual docetaxel. The final product, obtained by centrifugation and resuspending, was the docetaxel-pretreated remodeled CAR-T cells (cell viability >95%).

[0086] (3) Extraction and preparation of extracellular vesicles (exosomes)

[0087] Supernatant collection: Collect the culture supernatant of Doce-CAR-T cells during the culture and expansion process in the above steps.

[0088] Differential overspeed centrifugation: Centrifuge at 300×g to remove whole cells from the supernatant; Collect the supernatant and centrifuge at 2,000×g to remove dead cell debris; Collect the supernatant and centrifuge at 16,500×g to further remove large granular cell debris; Finally, the clarified supernatant was transferred to an ultracentrifuge tube and ultracentrifuged at 100,000×g for 2 hours. The resulting precipitate was the purified functional extracellular vesicle (exosome).

[0089] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0090] Example 1: Formulation of a composition to enhance CAR-T cell function

[0091] Weigh the docetaxel raw material and dissolve it in DMSO to prepare a stock solution. Before use, dilute it with X-VIVO 15 serum-free medium containing 10% fetal bovine serum and 10 ng / mL IL-2 to prepare a working solution of docetaxel with a final concentration of 10 nM. This working solution is the functional enhancement composition for in vitro treatment of CAR-T cells.

[0092] Example 2: Preparation of functionally enhanced CAR-T cells

[0093] (1) Cell preparation: PBMCs were isolated from peripheral blood of healthy donors and primary T cells were obtained through negative sorting.

[0094] (2) Activation and transduction: T cells are activated using a CD3 / CD28 antibody complex, utilizing the encoding of target cells.

[0095] MSLN-CAR-T cells were obtained by transducing the second-generation CAR vector (scFv-41BB-CD3ζ) of cortisol (MSLN).

[0096] (3) Pharmacological remodeling treatment: During the CAR-T cell expansion phase, the cells were resuspended in the culture medium containing 10 nM docetaxel prepared in Example 1, and the cell density was adjusted to 1×10⁻⁶ cells / year. 6 / mL, continue culturing for 48 hours.

[0097] (4) Collection: After culture, cells were collected and washed twice with PBS to remove residual drugs, resulting in docetaxel-pretreated remodeled CAR-T cells (Doce-CAR-T). Cell viability was confirmed to be greater than 95% by trypan blue staining.

[0098] Example 3: Validation of Doce-CAR-T cell phenotype and in vitro function

[0099] (1) Phenotypic analysis (results are as follows) Figure 1-4Flow cytometry analysis showed that, compared with the untreated group, the proportion of stem cell-like memory T cells (TSCM) and central memory T cells (CM) in Doce-CAR-T cells was significantly increased, while the proportion of terminally differentiated effector cells (TEMRA) was decreased. Simultaneously, the expression levels of cell surface depletion marker PD-1 and aging marker CD57 were significantly reduced.

[0100] (2) 3D tumor sphere killing (results as follows) Figure 5 ): Doce-CAR-T cells were co-cultured with 3D tumor spheres formed from HCT116 colorectal cancer cells. After 72 hours, compared with untreated CAR-T cells, the tumor sphere volume in the Doce-CAR-T group was significantly reduced (p<0.001), and the tumor survival rate based on ATP activity was significantly decreased.

[0101] Example 4: Preparation and preliminary identification of functional extracellular vesicles (results are as follows) Figure 6 )

[0102] (1) Separation and extraction: Collect the supernatant during the culture of Doce-CAR-T cells. Purify extracellular vesicles (exosomes) by differential ultracentrifugation: remove cell debris by centrifugation at 300×g, 2,000×g and 16,500×g in sequence, and finally obtain the precipitate by ultracentrifugation at 100,000×g for 2 hours.

[0103] (2) Physicochemical characterization: Particle size analysis showed that the obtained vesicles were mainly distributed between 70-150 nm in size. Flow cytometry confirmed that they expressed the classic exosome markers CD9, CD63, and CD81.

[0104] (4) Functional components: The test revealed that the vesicle contained functional components consistent with its parent effector cells, and possessed the biological activity of inducing tumor cell apoptosis.

[0105] Example 5: In vivo antitumor effect of Doce-CAR-T cells (results are as follows) Figure 7 )

[0106] The therapeutic efficacy of Doce-CAR-T cells was evaluated in a tumor-bearing NSG mouse model. Results showed: (1) Survival period: Compared with the untreated CAR-T group and the PBS group, the Doce-CAR-T cell group significantly prolonged the survival period of mice.

[0107] (2) Tumor burden: During the treatment, tumor growth in the Doce-CAR-T group mice was significantly inhibited for a sustained period of time (p<0.001).

[0108] The results of the above embodiments demonstrate that the present invention induces CAR-T cells into a "metabolically active" remodeling state by using extremely low doses of docetaxel, which significantly enhances its application value in the treatment of solid tumors.

[0109] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composition for enhancing CAR-T cell function, characterized in that, The composition comprises a taxane compound and a pharmaceutically acceptable carrier.

2. The composition according to claim 1, characterized in that, The taxanes are at least one of docetaxel, paclitaxel, cabazitaxel, and their respective pharmaceutically acceptable salts.

3. The use of the composition of claim 1 or 2 in enhancing CAR-T cell function, wherein the use is to treat CAR-T cells with the composition of claim 1 or 2 for a period of 12 to 96 hours.

4. A method for preparing functionally enhanced CAR-T cells, characterized in that, The method includes the following steps: (1) Co-culturing CAR-expressing T cells with the composition of claim 1 or 2; (2) Collect CAR-T cells after co-culture in step (1) to obtain functionally enhanced CAR-T cells.

5. A method for preparing extracellular vesicles, characterized in that, The method includes the following steps: (1) Culturing functionally enhanced CAR-T cells prepared by the method of claim 4; (2) Collect the culture supernatant of the cells described in step (1); (3) Separate and purify extracellular vesicles from the supernatant.

6. The method according to claim 5, characterized in that, The extracellular vesicles prepared by the method exhibit antitumor functional components consistent with their parental effector cells, and have the ability to penetrate the tumor matrix and kill bystander tumor cells.

7. A pharmaceutical composition, characterized in that, It comprises: a) functionally enhanced CAR-T cells prepared by the method of claim 4; and / or b) extracellular vesicles prepared by the method of claim 5.

8. The use of one or more of the compositions of claims 1-2, the functionally enhanced CAR-T cells prepared by the method of claim 4, and the extracellular vesicles prepared by the method of claim 5 in the preparation of a drug for treating solid tumors.

9. The application according to claim 8, characterized in that, The solid tumor is breast cancer, colorectal cancer, stomach cancer, lung cancer, or pancreatic cancer.

10. The application according to claim 8, characterized in that, The enhanced CAR-T cells and the extracellular vesicles can be used in combination, sequentially, or alternately.

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