Preparation of mouse dry CAR-T cell and hydrogel cell bank thereof

Abstract

The invention provides a preparation method of a mouse dry CAR-T cell and a hydrogel cell bank thereof, so that the encapsulated CAR-Tscm can be prevented from being interfered by adverse factors in a tumor microenvironment to the greatest extent, a dry state is reserved, and a depletion process is slowed down, thereby realizing long-term anti-tumor activity. On one hand, in the CAR-T preparation process, the optimal IL-7 and IL-15 induction proportion of a dry CAR-T cell subset is screened out, and the CAR-T cell depletion is relieved in combination with a metabolism regulation medicine. And on the other hand, the dry CAR-T cells and other components are encapsulated by adopting the hydrogel with high biocompatibility, and the hydrogel has injectability, can form a stem cell bank in vivo, is simple to operate and has relatively high clinical application potential.

Description

Technical Field

[0001] The present invention relates to the field of cell therapy, and in particular to the preparation of mouse stem CAR-T cells and a hydrogel cell bank thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Chimeric antigen receptor T cells (CAR-T) have achieved great success in hematological tumors, marking a new era of cancer immunotherapy. CAR-T cell therapy faces huge challenges in the application of solid tumors. Under continuous chronic antigen stimulation in vivo, T cell exhaustion is an inevitable fate. In addition, tumor cells in the tumor microenvironment limit the aerobic glycolysis required for T cell glucose availability and effector function, and the hypoxic and low pH environment aggravates T cell metabolic disorders, ultimately accelerating the rate of CAR-T cell exhaustion.

[0004] Existing studies have shown that alleviating CAR-T cell exhaustion is a method to improve the persistence of the effect, and increasing the proportion of stem cells in the infused product is an effective measure to prolong the effect time. Intervening in the early stage of the CAR-T cell preparation process can make it stay more in the front stage of the differentiation trajectory. Common methods include adjusting the concentration of IL-2, selecting cytokines such as IL-7, IL-15, IL-21, inhibiting the PI3K-AKT signaling pathway or activating the WNT signaling pathway, intervening in T cell metabolism, knocking out or overexpressing certain key genes, etc. However, these methods can only increase a certain proportion of stem CAR-T cells before in vitro infusion. Once the CAR-T cells are separated from the in vitro culture conditions and face long-term antigen stimulation after entering the body, they will still go to terminal T cell exhaustion. Secondly, due to the heterogeneity of tumors, it is difficult to achieve precise regulation of the tumor suppressive microenvironment. Therefore, effective strategies that can enhance the stemness of CAR-T cells are urgently needed.

[0005] The present invention provides a method for preparing a hydrogel that can improve and maintain the dryness of CAR-T cells. First, the optimal preparation conditions of CAR-Tscm are screened, and IL-7 and IL-15 are selected for combined induction in the entire preparation process to obtain a CAR-T infusion product with a CAR-Tscm% (chimeric antigen receptor T memory stem cells) of more than 40%. Secondly, glutamine inhibitors and nicotinamide riboside (NR) are added to the culture conditions for metabolic reprogramming to alleviate CAR-T cell exhaustion. The former stimulates T cell oxidative phosphorylation while inhibiting glycolytic activity, and the latter can alleviate mitochondrial dysfunction and reduce the production of reactive oxygen species. Finally, these components are co-loaded with stem CAR-T cells in an injectable sodium alginate / hyaluronic acid hydrogel, which has biocompatibility and in vivo degradability, and provides a favorable microenvironment for maintaining the dryness of CAR-T cells. Summary of the invention

[0006] The purpose of the present invention is to provide a simple method for preparing a hydrogel that improves and maintains the dryness of CAR-T cells, so that the encapsulated CAR-Tscm can be protected from the interference of adverse factors in the tumor microenvironment to the greatest extent, retain the dry state, slow down the exhaustion process, and thus achieve long-term anti-tumor activity. On the one hand, in the CAR-T preparation process, the optimal IL-7 and IL-15 induction ratios of the stem CAR-T cell subpopulations are screened out, and metabolic regulation drugs are combined to reduce CAR-T cell exhaustion. On the other hand, a highly biocompatible hydrogel is used to encapsulate the stem CAR-T cells and other components. The hydrogel is injectable, can form a stem cell bank in the body, is simple to operate, and has a high potential for clinical application.

[0007] In one aspect, the present invention provides a hydrogel comprising CAR-T cells, a blank hydrogel and a CAR-T cell reprogramming drug, wherein the blank hydrogel is a sodium alginate and calcium cross-linked hydrogel, and the CAR-T cell reprogramming drug is a glutamine inhibitor and nicotinamide riboside.

[0008] Furthermore, the concentration ratio of the CAR-T cell reprogramming drug glutamine inhibitor and nicotinamide riboside in the hydrogel is 2 μM:5 μM, 5 μM:5 μM, or 10 μM:5 μM.

[0009] Furthermore, the loading ratio of CAR-T cells in the hydrogel is 5×10 6 CAR-T cells.

[0010] Furthermore, a method for preparing CAR-T cells is provided, and the steps are as follows: extracting mouse primary CD3+T cells is inoculated in a complete culture medium containing IL-7 and IL-15, adding activation magnetic beads Anti-CD3 / CD28 Dynabeads to activate T cells, transferring the cells to a cell culture plate coated with a recombinant human fibronectin fragment (RetroNectin) one day after T cell activation, adding an appropriate amount of mouse stem cell virus MSCV retroviral liquid, culturing for 8-12 hours after high-speed centrifugation, replacing fresh culture medium and supplementing cytokines and reprogramming drugs, removing the magnetic beads using a magnetic rack 3 days after activation, and amplifying CAR-T cells for 2-3 days to obtain a mouse CAR-T cell preparation with a CAR-Tscm% expression of more than 40%, using IL-7 and IL-15 for combined induction in the entire preparation process, and adding glutamine inhibitor (DON) and nicotinamide riboside (NR) to the culture conditions for metabolic reprogramming.

[0011] Furthermore, the concentration ratio of IL-7 and IL-15 is 1:2, 1:1, 2:1 or 4:1, and the total concentration is 100-400 ng / mL.

[0012] Furthermore, the concentration ratio of the cell reprogramming drug glutamine inhibitor and nicotinamide riboside is 2 μM:5 μM, 5 μM:5 μM, or 10 μM:5 μM.

[0013] Another aspect of the present invention provides a method for preparing a hydrogel, and the preparation method is as follows: prepare a CAR-T cell suspension and a calcium chloride phosphate buffer solution, wherein the cell suspension contains sodium alginate, hyaluronic acid and a cell reprogramming drug, respectively, and load them into two 1 mL syringes, push the syringe plunger at the same speed to inject 100 μL subcutaneously into the abdomen of a mouse, dissect the mouse subcutaneously after 5 minutes, and observe the formation of a hydrogel cell bank of CAR-T cell stemness.

[0014] Another aspect of the present invention provides a use of a hydrogel in the treatment of solid tumors.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a method for preparing a hydrogel cell bank suitable for storing and maintaining dryness of stem CAR-T cells.

[0016] (2) The present invention screened the optimal ratio of IL-7 and IL-15 for retaining Tscm, and prepared CAR-T cells with this ratio in which the CAR-Tscm accounted for more than 40%, that is, the cells had the characteristics of high stemness and low exhaustion.

[0017] (3) Based on the induction of IL-7 and IL-15, the present invention adds glutamine inhibitors and nicotinamide riboside to improve T cell metabolism. The final results show that the two drugs can reduce the expression of exhaustion indicators.

[0018] (4) The present invention uses hydrogel to encapsulate stem CAR-T cells and various components to directly shield adverse factors in the tumor microenvironment, and provides a method for injecting the hydrogel subcutaneously using a double-headed syringe to facilitate the in vivo formation of the hydrogel. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 :Tscm induction effect of different IL-7 and IL-15 ratios; Figure 2 : The proportion of each subpopulation of CAR-T under different culture conditions; Figure 3 :The effect of metabolic drugs on the expression of CAR-T cell exhaustion markers; Figure 4 : Scanning image of CAR-Tscm cell hydrogel cell bank; Figure 5 : Cell viability of the cell bank in the hydrogel; Figure 6 : Schematic diagram of the double-head syringe (left) and the hydrogel morphology after subcutaneous injection in mice (right). DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0022] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0023] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0024] Example 1: Screening for the optimal ratio of IL-7 and IL-15 to retain Tscm CD3+T cells were isolated from the spleen of 6-8 week old female C57BL / 6 mice using the MojoSort Mouse CD3+ T Cell Isolation kit, and the purity was greater than 95% as determined by flow cytometry. CD3+T cells were inoculated in a 24-well cell culture plate at a cell concentration of 1 million / mL, and different proportions of IL-7 and IL-15 were added and cultured for 3 days. The cells were collected for flow cytometry, and CD44-, CD62L+, and Sca1+ were defined as Tscm cell subsets. The optimal induction ratio of IL-7 and IL-15 was screened to be 2:1, and the Tscm ratio was about 40%. The results are as follows Figure 1 As shown. The culture medium is 1640 culture medium, containing 10% FBS, 1% penicillin-streptomycin solution, and 55 μM β-mercaptoethanol for cell culture. The IL-7 and IL-15 concentration ratio is any one of 1:2, 1:1, 2:1, and 4:1, and 2:1 is finally selected as the optimal IL-7 and IL-15 ratio.

[0025] Example 2. Preparation of stem CAR-T cells The extracted mouse primary T cells were inoculated in the culture medium. IL-7 and IL-15 were added to the culture medium at a ratio of 2:1, and IL-2 used in the traditional CAR-T preparation process was used as a control. Anti-CD3 / CD28 Dynabeads (Dynabeads Mouse T-Activator CD3 / CD28) was activated to activate T cells.

[0026] After 1 day of T cell activation, the cells were transferred to a cell culture plate coated with human fibronectin fragment (RetroNectin) for standby use. In a 24-well plate, 1 mL of cell suspension was added to each well, and the cell concentration was controlled at 500,000-1,000,000 / mL. An appropriate amount of MSCV retrovirus solution was added to each well and centrifuged at 1500 g for 60 min at 32°C. After centrifugation, the cells were cultured at 37°C and 5% CO2 for 8-12 hours. After the virus infection of the cells was completed, fresh culture medium was replaced and cytokines were supplemented. The magnetic beads were removed using a magnetic stand 3 days after activation, and the CAR-T cells were amplified for 2-3 days.

[0027] Cells were collected for flow cytometry to determine the proportion of each CAR-T cell subset, including CAR-Tscm (FITC+, CD44-, CD62L+, Sca1+), CAR-Tn (FITC+, CD44-, CD62L+), CAR-Tcm (FITC+, CD44+, CD62L+) and CAR-Tem or CAR-Teff (FITC+, CD44+, CD62L-). The results are shown in Figure 2 As shown, it indicates that IL-7 and IL-15 induction at a concentration ratio of 2:1 can retain more than 40% of the CAR-Tscm% subpopulation.

[0028] The culture medium is 1640 culture medium containing 10% FBS, 1% penicillin-streptomycin solution, and 55 μM β-mercaptoethanol for cell culture. The final concentration of IL-2 is 30 U / mL. The 24-well plate is a cell culture plate without TC treatment. The MSCV retroviral solution can be any mouse CAR target, and EGFP-EphA2-mCARMSCV virus solution is selected in the example.

[0029] Example 3: Optimizing metabolic regulation drugs to reduce the expression of exhaustion markers in stem CAR-T cells The stem CAR-T cells induced by IL-7 and IL-15 were seeded at 500,000 / mL in a 48-well cell culture plate, and glutamine antagonists and / or nicotinamide riboside were added and cultured for 2 days. The cells were collected, labeled for depletion indicators PD-1 and Tim-3, and analyzed by flow cytometry. Figure 3 As shown, both glutamine antagonists and nicotinamide riboside have the effect of slowing down the exhaustion of stem CAR-T cells. The glutamine antagonist includes 6-diazo-5-oxo-L-desleucine (DON) with a final concentration of 5 μM. The final concentration of nicotinamide riboside (NR) is 5 μM.

[0030] Example 4. Preparation and characterization of hydrogels that improve and maintain the stemness of CAR-T cells 1. In vitro preparation of hydrogels to improve and maintain the stemness of CAR-T cells Sodium alginate (ALG) and calcium ion cross-linking were used to prepare the hydrogel, and a certain proportion of hyaluronic acid (HA) was added to the sodium alginate to improve cell compatibility. A mixed solution of ALG and HA in different mass proportions with a total concentration of 12 mg / mL was prepared using the stem CAR-T cell suspension induced under IL-7 and IL-15 conditions as liquid A. An anhydrous CaCl2 solution with a concentration of 10 mg / mL was prepared as liquid B. Liquid A and liquid B were evenly mixed in the same volume to form a hydrogel that improves and maintains the dryness of CAR-T cells. By scanning the 3D image with a confocal live cell imaging microscope, it can be seen that the CAR-T cells are distributed in the hydrogel ( Figure 4 ).

[0031] MTT cytotoxicity test was performed to test the cell viability of CAR-T cells in hydrogel. In a 48-well cell culture plate, 100 μL of solution A was first added, and 100 μL of solution B was added and mixed quickly. After the hydrogel cross-linking was completed, 200 μL of culture medium was added to the upper layer and cultured for 3 days. Then the cell viability was determined according to the MTT experimental method and calculated. The results showed that the cell viability of prescriptions AH2 and AH3 was higher ( Figure 5 ). The different mass ratios of ALG:HA are 12:0, 8:1, 4:1, 2:1, corresponding to prescriptions AH1, AH2, AH3 and AH4, respectively.

[0032] 2. In vivo preparation of hydrogels to improve and maintain the stemness of CAR-T cells Since sodium alginate cross-links with calcium ions quickly, affecting needle permeability, a double-ended injection is used to complete the in vivo cell bank preparation process, such as Figure 6 As shown. Liquid A and liquid B were loaded into two 1 mL syringes respectively, and 100 μL was injected subcutaneously into the abdomen of the mouse by pushing the syringe plunger at the same speed. After 5 minutes, the subcutaneous tissue of the mouse was dissected, and the formation of a hydrogel that improved and maintained the stemness of CAR-T cells was observed.

[0033] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A hydrogel, characterized in that: It includes CAR-T cells, blank hydrogel and CAR-T cell reprogramming drugs, wherein the blank hydrogel is a sodium alginate and calcium cross-linked hydrogel, and the CAR-T cell reprogramming drugs are glutamine inhibitors and nicotinamide riboside.

2. The hydrogel according to claim 1, characterized in that: The concentration ratio of the CAR-T cell reprogramming drug glutamine inhibitor and nicotinamide riboside is 2 μM:5 μM, 5 μM:5 μM, or 10 μM:5 μM.

3. The hydrogel according to claim 1, wherein: The loading ratio of CAR-T cells in the hydrogel is 5×10 6 CAR-T cells.

4. The hydrogel according to claim 1, characterized in that: The preparation method is as follows: prepare a CAR-T cell suspension and a phosphate buffer solution of calcium chloride, wherein the cell suspension contains sodium alginate, hyaluronic acid and reprogramming drugs, respectively, and load them into two 1 mL syringes, push the syringe plunger at the same speed to inject subcutaneously into the abdomen of the mouse, and after 5 minutes, a CAR-T cell dry hydrogel cell bank is formed subcutaneously.

5. Use of the hydrogel as claimed in claim 1 in the treatment of solid tumors.

6. A method for preparing CAR-T cells, characterized in that: The extracted mouse primary CD3+T cells were inoculated in a complete culture medium containing IL-7 and IL-15, and activation magnetic beads Anti-CD3 / CD28 Dynabeads were added to activate T cells. One day after T cell activation, the cells were transferred to a cell culture plate coated with a recombinant human fibronectin fragment, and an appropriate amount of mouse stem cell virus MSCV retroviral solution was added. After high-speed centrifugation, the cells were cultured for 8-12 hours, and fresh culture medium was replaced and supplemented with cytokines and reprogramming drugs. The magnetic beads were removed by a magnetic rack 3 days after activation, and CAR-T cells were amplified for 2-3 days to obtain a mouse CAR-T cell preparation with chimeric antigen receptor T memory stem cells expressing more than 40%. IL-7 and IL-15 were used for combined induction in the entire preparation process, and glutamine inhibitors and nicotinamide riboside were added to the culture conditions for metabolic reprogramming.

7. The method for preparing CAR-T cells according to claim 6, characterized in that: The concentration ratio of IL-7 and IL-15 is 1:2, 1:1, 2:1 or 4:1, and the total concentration is 100-400 ng / mL.

8. The method for preparing CAR-T cells according to claim 6, wherein the concentration ratio of the reprogramming drug glutamine inhibitor and nicotinamide riboside is 2 μM:5 μM, 5 μM:5 μM, or 10 μM:5 μM.

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