High-pressure oxygen modified CAR-T cell and application thereof in solid tumor treatment

Abstract

The invention discloses a hyperbaric oxygen modified CAR-T cell and application thereof in solid tumor treatment, and belongs to the technical field of tumor immunotherapy. The invention provides a method for enhancing the function of CAR-T (Chimeric Antigen Receptor-T) cells. The method comprises the step of treating the CAR-T cells by using hyperbaric oxygen. It is proved that the anti-tumor activity of the CAR-T cells can be enhanced through treatment of hyperbaric oxygen (HBO), and the anti-tumor activity of the CAR-T cells can be enhanced through treatment of the HBO. Different from gene editing or medicament adding, the physical means of HBO is adopted to carry out functional modification on the CAR-T cells, and additional gene modification is not needed; the HBO-treated CAR-T cells have microenvironment adaptability, and especially can aim at the microenvironment characteristics of high fibrosis and high immunosuppression of triple negative breast cancer; through HBO treatment, the functional activity of a CAR-T body is enhanced, and the microenvironment accessibility of a tumor part can be improved; meanwhile, the method has the advantage of safety, potential risks of gene editing are avoided, and the natural biological characteristics of the T cells are reserved.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor immunotherapy, and in particular to hyperbaric oxygen-modified CAR-T cells and their application in the treatment of solid tumors. Background Art

[0002] Chimeric antigen receptor T cell (CAR-T) therapy is a revolutionary immunotherapy method that genetically modifies the patient's T cells to express chimeric antigen receptors (CARs) that can specifically recognize tumor antigens, thereby achieving targeted killing of tumor cells. Since the approval of the first CAR-T product, CAR-T therapy has achieved significant therapeutic effects in hematological malignancies (such as B-cell lymphoma and acute lymphoblastic leukemia), and some patients have even achieved long-term remission. However, despite the excellent performance of CAR-T therapy in hematological tumors, CAR-T therapy still has the following problems in clinical application: (1) CAR-T cells are prone to functional exhaustion after long-term exposure to antigen stimulation in the body, resulting in decreased efficacy; (2) CAR-T cells have a limited survival time in the body, making it difficult to achieve long-term anti-tumor effects; (3) Side effects such as cytokine release syndrome (CRS) and neurotoxicity limit the widespread application of CAR-T therapy. In addition, its application in the treatment of solid tumors faces many challenges. The complex microenvironment, tumor heterogeneity and immunosuppressive mechanisms of solid tumors further limit the infiltration, persistence and killing effect of CAR-T cells.

[0003] In order to overcome the above problems, researchers have tried various strategies to enhance the efficacy of CAR-T cells, such as the optimization of co-stimulatory molecules. Researchers introduced co-stimulatory molecules (such as CD28, 4-1BB, etc.) into the second and third generation CAR-T cells to enhance the activation and persistence of T cells. However, the effects of these co-stimulatory molecules vary greatly in different tumor types, and they have not completely solved the problems of cell exhaustion and resistance of the solid tumor microenvironment. Some researchers have also used technologies such as CRISPR / Cas9 to knock out immunosuppression-related genes (such as PD-1, CTLA-4) to enhance the function of CAR-T cells. However, the efficiency and safety of gene editing still need further verification. In addition, the combination of CAR-T therapy with immune checkpoint inhibitors, chemotherapy or radiotherapy can also improve the efficacy. However, the toxic side effects and cost issues of combined therapy limit its widespread application. Although these methods have improved the effects of CAR-T therapy to a certain extent, the following unresolved issues still exist: (1) Existing technologies are difficult to effectively overcome the immunosuppressive barrier of the solid tumor microenvironment and lack specific enhancement strategies for solid tumors; (2) The proliferation and survival time of CAR-T cells in the body are limited, making it difficult to achieve long-term anti-tumor effects; (3) Side effects such as CRS and neurotoxicity are still the main obstacles to the clinical application of CAR-T therapy.

[0004] To address the above issues, there is an urgent need to develop a new method that can significantly enhance the efficacy of CAR-T cells, especially in the application of solid tumors. Summary of the Invention

[0005] The purpose of the present invention is to provide a hyperbaric oxygen-modified CAR-T cell and its application in the treatment of solid tumors to solve the problems existing in the above-mentioned prior art. The present invention treats CAR-T cells by HBO, overcoming the three major obstacles of current CAR-T treatment of solid tumors: (1) insufficient tumor infiltration; (2) microenvironment suppression; and (3) limited persistence, providing a new treatment option for the application of CAR-T in solid tumors.

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

[0007] The present invention provides a method for enhancing the function of CAR-T cells, comprising the step of treating CAR-T cells with hyperbaric oxygen.

[0008] Optionally, the conditions of the high-pressure oxygen treatment are 100-200 kPa, 30-120 min, and continuous treatment for 1-3 days.

[0009] The present invention also provides CAR-T cells prepared according to the method.

[0010] The present invention also provides the use of the CAR-T cells in the preparation of drugs for treating solid tumors.

[0011] Optionally, the solid tumor comprises melanoma or triple-negative breast cancer.

[0012] The present invention also provides a drug for treating solid tumors, comprising the CAR-T cells.

[0013] Optionally, the solid tumor comprises melanoma or triple-negative breast cancer.

[0014] Optionally, it further comprises pharmaceutically acceptable excipients.

[0015] Optionally, the auxiliary materials include at least one of a diluent, a filler, an excipient, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant and a flavoring agent.

[0016] The present invention discloses the following technical effects:

[0017] HBO treatment has been shown to enhance the anti-tumor activity of CAR-T cells. This invention utilizes a physical modification method, distinct from gene editing or drug addition, to functionally modify CAR-T cells using HBO. This allows HBO-treated CAR-T cells to adapt to the microenvironment, specifically targeting the highly fibrotic and immunosuppressive microenvironment of triple-negative breast cancer. HBO treatment enhances the functional activity of the CAR-T cells and improves their accessibility to the tumor microenvironment. This approach also offers safety advantages, avoiding the potential risks of gene editing and preserving the natural biological characteristics of T cells.

[0018] The present invention overcomes the three major obstacles of current CAR-T therapy for solid tumors: (1) insufficient tumor infiltration; (2) microenvironmental suppression; and (3) limited persistence, providing a new treatment option for the application of CAR-T in solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 Effects of different HBO treatments on CD19CAR-T (A) and MUC1CAR-T (B);

[0021] Figure 2Verification of the characteristic performance of CAR-T cells after HBO treatment; A: killing ability; B: tumor infiltration ability; C: proliferation ability;

[0022] Figure 3 The expression levels of CAR-T cell exhaustion markers PD-1, TIM-3, and LAG3 after HBO treatment;

[0023] Figure 4 Monitoring of tumor-bearing mice in CD19-CAR-T melanoma model cell animal experiments; A: Curves showing changes in weight and tumor size in each group of mice; B: Photos of mice and tumors in each group;

[0024] Figure 5 Monitoring of tumor-bearing mice in the MUC1-CAR-T triple-negative breast cancer model cell animal experiment; A: Curves of changes in weight and tumor size of mice in each group; B: Photos of mice in each group and tumors. DETAILED DESCRIPTION

[0025] Example 1: Preparation Method for HBO Enhanced CAR-T Function

[0026] 1. CAR-T cell preparation

[0027] 1.1 T cell recovery and activation

[0028] (1) Day 1: Thaw the frozen T cells at 37°C, transfer them to a centrifuge tube containing 5 mL of complete culture medium (culture medium: X-vivo15 medium + 5% FBS + 100 U / mL IL-2), mix thoroughly, centrifuge at 1200 rpm for 3 min, and discard the supernatant.

[0029] (2) Resuspend the cell pellet with complete culture medium and inoculate into a well plate, and culture in a constant temperature incubator at 37°C and 5% CO2.

[0030] (3) Magnetic bead washing: Magnetic bead washing buffer: Pre-cool PBS + 5% FBS, prepare 1 mL into an EP tube, take the corresponding volume of Dynabeads Human T-Activator CD3 / CD28 (Cat. No.: 11161D) and add it to the wash buffer. Vortex and oscillate for 15 seconds to mix thoroughly. Place the mixed magnetic beads in a magnetic rack, let it stand for 1 minute, and then remove the washing buffer.

[0031] (4) Mixing magnetic beads with cells: According to the ratio of magnetic beads to cells = 1:1, take the corresponding magnetic beads and add them to the cells, mix them evenly, and adjust the cell density to 1×10 6 / mL and continue culturing.

[0032] 1.2 T cell infection and expansion

[0033] (1) Retronectin-C plate coating (second day)

[0034] The Retronectin solution was diluted with PBS to 20-100 μg / mL. The concentration used in this example was 25 μg / mL.

[0035] Add the Retronectin solution to the well plate and place it at room temperature for 2 hours or at 4°C overnight.

[0036] (2) Cell infection (day 3)

[0037] ① Remove the Retronectin solution and block with 2% BSA solution diluted in PBS. Leave at room temperature for 30 minutes.

[0038] ② Remove the BSA solution and add an appropriate volume of HBSS / Hepes or PBS to wash the plate. After removing the washing solution, the plate coating is completed;

[0039] ③ Add 125-250 μL / cm 2 Incubate the lentivirus in a 5% CO2, 32℃ / 37℃ incubator for 4-6h to promote the binding of Retronectin reagent and viral particles;

[0040] ④ After incubation, remove the supernatant from the plate, wash the plate with PBS containing 0.1%-2% BSA / HSA, and then infect with the virus (MOI=10);

[0041] ⑤ Collect T cells and count them. Suspend T cells at a concentration of 1×10 6 cells / mL of growth medium;

[0042] ⑥ Add 0.5×10 4 -2.5×10 4 cells / cm 2 Target cells (cells with CD19 or MUC1) were cultured in a 37°C, 5% CO2 incubator for 2-3 days.

[0043] (3) T cell medium replacement and microscopic examination (fourth day)

[0044] 24 h after virus infection, the cells were examined under a microscope, centrifuged and the medium was replaced. The cells were placed in a 96-well plate and continued to be cultured. The cell density was adjusted to 1×10 6 / mL, and the positive rate was tested 72 hours after infection.

[0045] 1.3 CAR-T cell positive rate detection (fifth day)

[0046] (1) Take ≥1e 5 Place 100 cells in a 1.5 mL centrifuge tube, place the tube in a magnet for 2 minutes, transfer the supernatant to a new tube, centrifuge at 1200 rpm for 3 minutes, and discard the supernatant.

[0047] (2) Add 1 mL of PBS to resuspend the cells, centrifuge at 1200 rpm for 3 min, and discard the supernatant.

[0048] (3) Add 100 μL of the corresponding antibody diluent to resuspend the cells (for example, the CD19CAR corresponding antibody AlexFluor647-Protein L is diluted 1:1000, the MUC1CAR corresponding antibody THE TM NWSHPQFEK Tag Antibody [FITC] 1:100 dilution) and incubate at 4°C in the dark for 45 min.

[0049] (4) Add 1 mL of PBS to resuspend the cells, centrifuge at 1200 rpm for 3 min, and discard the supernatant (repeat 3 times).

[0050] (5) Add 100 μL of PBS to resuspend the cells, sieve them with a 40 μm cell sieve, and perform flow cytometry to determine the positive rate.

[0051] CD19CAR-T and MUC1CAR-T were prepared and used for subsequent operations.

[0052] 2. HBO condition optimization

[0053] (1) Treatment timing: Treatment was performed on the fifth day of CAR-T recovery and expansion (i.e., 72 h after lentiviral transduction) when cell viability was >90%.

[0054] (2) Oxygen pressure gradient test: set two sets of pressure conditions: 100 kPa and 150 kPa.

[0055] (3) Treatment duration: three groups of stabilization time: 30 min / 60 min / 90 min.

[0056] (4) Number of treatments: Continuous treatment for 1-3 days.

[0057] (5) Determine the optimal conditions using cell viability and CAR expression stability as indicators.

[0058] The results are as follows Figure 1 As shown, 100kPa has little effect on the cell viability of CART cells. When the HBO treatment time is extended to 90 minutes and the treatment is continued for 3 days, the T cell viability is enhanced. At 150kPa, the cell viability is the strongest after 90 minutes and 3 days of continuous treatment. Therefore, the optimal treatment condition is 150kPa for 90 minutes and 3 days of continuous treatment.

[0059] 3. Feature performance verification

[0060] The killing ability, invasion ability, exhaustion characteristics and molecular mechanism were tested under the conditions of 90min and 150kPa.

[0061] The results are as follows Figure 2 and Figure 3 As shown, the level of tumor killing by HBO-treated CART cells was significantly enhanced ( Figure 2 Middle A), enhanced infiltration ability to solid tumors ( Figure 2 Middle B), the proliferation level of CART increased 3-4 times ( Figure 2 Middle C), the expression of depletion markers PD-1 and TIM-3 decreased by 20-40%, and LAG3 expression decreased by 50% ( Figure 3 ).

[0062] Example 2 Validation of melanoma model

[0063] 1. Establishment of A375-CD19 transplanted tumor model

[0064] (1) Female SPF M-NSG mice, 4-6 weeks old, weighing 16-20 g, were randomly divided into groups. The feeding and experiments were carried out in a mouse room with a constant temperature (20-26°C) and constant humidity (50%-56%).

[0065] (2) Tumor cells (A375-CD19) in the logarithmic growth phase were taken, rinsed twice with serum-free PBS, suspended in serum-free PBS, and Matrigel was added at a ratio of 1:1.

[0066] (3) Inoculate mice subcutaneously with 0.1 mL of 5×10 6 The cell suspension was inoculated subcutaneously in the lower back.

[0067] (4) Tumor monitoring: The general condition, tumor formation, and tumor growth of mice were observed daily during the experiment, and the tumor size and mouse weight were measured every 3 days;

[0068] Tumor volume = length × width × width / 2.

[0069] 2. Grouped medication

[0070] Tumor size is 150 mm 3 At the same time, mice with uniform tumor sizes were selected and divided into 4 groups: control group (Blank), hyperbaric oxygen (HBO) group, CAR-T group, and CAR-T + HBO combined treatment group (HBO + CAR-T, after the injection of CAR-T, the treated mice were treated with HBO). n = 5.

[0071] CAR-T infusion: Inject an appropriate amount of CAR-T cells (e.g. 1-5×10 6 cells / cell, in this example, 5×10 6 cells / cell).

[0072] HBO treatment: The CAR-T cell + tumor-bearing mouse group was treated with HBO for 3 consecutive days. The hyperbaric oxygen chamber program was set as follows: pressure 150 kPa (equivalent to 2.5 atmospheres), pressure rise time 15 minutes, pressure stabilization time 90 minutes, and pressure reduction time: 15 minutes.

[0073] The general condition, tumor formation and tumor growth of mice were observed daily, and the tumor size and mouse weight were detected every 3 days.

[0074] 3. Results

[0075] The results are as follows Figure 4 As shown, mice injected with CAR-T cells and then treated with HBO for three consecutive days can promote the tumor inhibition effect of CAR-T. Moreover, the present invention also found that HBO treatment of tumors alone can promote tumor development.

[0076] Example 3 Triple-negative breast cancer model validation

[0077] 1. Establishment of BT549 transplanted tumor model

[0078] (1) Female SPF M-NSG mice, 4-6 weeks old, weighing 16-20 g, were randomly divided into groups. The feeding and experiments were carried out in a mouse room with a constant temperature (20-26°C) and constant humidity (50%-56%).

[0079] (2) Tumor cells (BT549) in the logarithmic growth phase were taken, rinsed twice with serum-free PBS, suspended in serum-free PBS, and Matrigel was added at a ratio of 1:1.

[0080] (3) Inoculate mice subcutaneously with 0.1 mL of 5×10 6 The cell suspension was inoculated subcutaneously in the lower back.

[0081] (4) Tumor monitoring: The general condition, tumor formation, and tumor growth of mice were observed daily during the experiment, and the tumor size and mouse weight were measured every 3 days;

[0082] Tumor volume = length × width × width / 2.

[0083] 2. Grouped medication

[0084] (1) Tumor size is 150 mm 3At the same time, mice with uniform tumor sizes were selected and divided into the following groups: control group (Blank), hyperbaric oxygen group (HBO), CAR-T group (MUC1CAR), CAR-T group pretreated with hyperbaric oxygen (HBO-CAR-T), and tumor-bearing mice hyperbaric oxygen combined with CAR-T group (HBO+CAR-T). n = 5.

[0085] (2) HBO treatment of CAR-T cell / tumor-bearing mice (DAY 8-10)

[0086] The mice in the HBO+CAR-T group were injected with appropriate amount of CAR-T cells (e.g. 1-5×10 6 cells / cell, in this example, 5×10 6 cells / cell);

[0087] The hyperbaric oxygen chamber program was set as follows: pressure 150 kPa (equivalent to 2.5 atmospheres), pressure rise time 15 min, pressure stabilization time 90 min, and pressure reduction time: 15 min; the corresponding CAR-T cells or tumor-bearing mice were treated with HBO.

[0088] After CAR-T cells were treated with HBO, the HBO-CAR-T group was injected with the same amount of CAR-T cells as the HBO+CAR-T group through the tail vein.

[0089] The general condition, tumor formation and tumor growth of mice were observed daily, and the tumor size and mouse weight were detected every 3 days.

[0090] 3. Results

[0091] The results are as follows Figure 5 As shown, HBO treatment of mouse tumors for 3 consecutive days followed by CAR-T injection can promote the tumor inhibition effect of CAR-T. HBO treatment of CAR-T for 3 consecutive days followed by CAR-T injection can also promote the tumor inhibition effect of CAR-T without producing tumor promotion effect. The effect is better than HBO treatment of mouse tumors for 3 consecutive days followed by CAR-T injection.

[0092] The key differences between this invention and the existing technology are: physical modification method: unlike gene editing or drug addition, HBO is used as a physical means to functionally modify the prepared CAR-T cells; microenvironment adaptability: a treatment scheme is designed specifically for the high fibrosis and high immunosuppression characteristics of triple-negative breast cancer microenvironment; dual-stage synergy: it not only enhances the functional activity of the CAR-T body, but also improves the accessibility of the microenvironment at the tumor site; safety advantage: it avoids the potential risks of gene editing and retains the natural biological characteristics of T cells. It has the following advantages:

[0093] (1) Process innovation: A standardized CAR-T physical processing process has been established with controllable parameters and good repeatability.

[0094] (2) Mechanism innovation: It was first demonstrated that HBO treatment can enhance CAR-T function through epigenetic modification.

[0095] (3) Application innovation: Combination treatment regimens specifically designed for solid tumors including melanoma and triple-negative breast cancer.

[0096] (4) Product advantages: The prepared HBO-CAR-T is "ready-to-use" and does not require additional genetic modification of CART cells.

[0097] The present invention overcomes the three major obstacles of current CAR-T therapy for solid tumors: (1) insufficient tumor infiltration; (2) microenvironmental suppression; and (3) limited persistence, providing a new treatment option for the application of CAR-T in solid tumors.

[0098] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for enhancing CAR-T cell function, characterized in that: The method includes the step of treating CAR-T cells with hyperbaric oxygen.

2. The method according to claim 1, wherein The conditions of the high-pressure oxygen treatment are 100-200 kPa, 30-120 min, and continuous treatment for 1-3 days.

3. CAR-T cells prepared according to the method according to any one of claims 1-2.

4. Use of the CAR-T cells according to claim 3 in the preparation of a drug for treating solid tumors.

5. The use according to claim 4, characterized in that The solid tumor includes melanoma or triple-negative breast cancer.

6. A drug for treating solid tumors, characterized in that: Comprising the CAR-T cell according to claim 4.

7. The drug according to claim 6, characterized in that The solid tumor includes melanoma or triple-negative breast cancer.

8. The drug according to claim 6, wherein Also contains pharmaceutically acceptable excipients.

9. The drug according to claim 8, wherein The auxiliary materials include at least one of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

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