CAR-TICOS cell as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses a CAR-T cell with an improved preparation process as well as a preparation method and application of the CAR-T cell. The preparation method of the CAR-T cell comprises the step of stimulating T cells by using three monoclonal antibodies CD3, CD28 and ICOS to obtain the CAR-T cell (defined as a CAR-TICOS cell) with stronger functions. The invention relates to an application of CAR-TICOS cells in preparation of antitumor drugs. The invention provides the preparation method of the CAR-TICOS cells, the method endows T cells with higher lentiviral transfection efficiency, the prepared CAR-TICOS cells have stronger in-vivo and in-vitro amplification ability and better in-vivo anti-tumor ability, the survival rate of an affected body can be greatly improved, and the survival rate of the affected body can be expected to be improved to 100% under certain conditions at present.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to CAR-T ICOS Cells and preparation methods and applications thereof. Background Art

[0002] Immune cell therapy for tumors is to improve the targeting of immune cells to tumor cells and enhance their killing of tumor cells by artificially modifying immune cells. Chimeric antigen receptor (CAR) T cell therapy is considered one of the most typical examples of the "immune enhancement" strategy. CAR-T cell therapy is a therapy that uses genetic engineering technology to artificially modify the T cells of tumor patients, generate tumor-specific CAR-T cells after large-scale culture in vitro, and then inject them back into the patient's body to attack cancer cells. CAR-T cell therapy for hematological malignancies has completely changed anti-cancer treatment. Since 2010, a large number of clinical studies have shown that CAR-T has a good therapeutic effect on acute lymphocytic leukemia, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, etc. There are currently nine CAR-T drugs on the market, such as Yescarta produced by Gilead and Kymriah produced by Novartis.

[0003] CAR-T cells effectively cure a part of patients with refractory hematological malignancies, but disease recurrence after treatment remains one of the main obstacles. The proliferation and persistence of CAR-T cells in vivo are closely related to prognosis. More and more evidence shows that the poorly differentiated state and intrinsic program of T cells are related to the proliferation and anti-tumor ability of CAR-T cells. However, the current preparation process usually uses monoclonal antibodies CD3 and CD28 to co-stimulate CAR-T cells, which leads to the phenotypic characteristics of reduced number, aging and exhaustion of mitochondria in CAR-T cells. At present, studies have shown that in the preparation process of CAR-T cells, the addition of cytokines (such as IL-15, IL-21, TGF-β1), inhibitors of calcium signaling pathways, NOTCH1 agonists and PI3Kδ / γ inhibitors can optimize the phenotype and function of CAR-T cells. By optimizing the preparation process of CAR-T cells and improving the quality and metabolic adaptability of CAR-T cells, the therapeutic potential of CAR-T cells can be enhanced. The present invention uses monoclonal antibodies CD3, CD28 and ICOS to co-stimulate to prepare CAR-T cells. Summary of the invention

[0004] The present invention provides CAR-T ICOS The cells, preparation methods and applications thereof are mainly intended to solve the problems of reduced mitochondrial mass, aging and exhaustion of CAR-T cells currently prepared by CD3 combined with CD28 co-stimulation, and to provide new CAR-T cells that can inhibit tumors.

[0005] In order to solve the above problems, the present invention adopts the following technical solutions:

[0006] A first aspect of the present invention Involving CAR-T ICOS The method for preparing cells comprises the following steps

[0007] CAR-T cells are obtained by combining monoclonal antibodies CD3, CD28 and ICOS to stimulate T cells ICOS In terms of cell naming, the CAR-T cells obtained by this method are called CAR-T ICOS cell.

[0008] CAR-T ICOS As for the cell preparation method, the specific steps are as follows

[0009] S1. Place the sorted T cells in a culture plate coated with monoclonal antibodies CD3, CD28, and ICOS. After stimulation and activation, add a lentivirus containing a chimeric antigen receptor gene vector targeting a specific antigen, and continue to stimulate and culture under the same conditions.

[0010] S2. Remove the culture medium in S1 by centrifugation, re-plant the centrifuged T cells on a culture plate without monoclonal antibodies CD3, CD28 and ICOS, and culture and expand the CAR-T cells in serum-free culture medium.

[0011] The culture plate is pre-incubated and coated with monoclonal antibodies CD3, CD28 and ICOS at 37°C. The culture plate can also be replaced by other tools. The culture plate is pre-coated by adsorption. During the coating process, monoclonal antibodies CD3, CD28 and ICOS can be diluted in phosphate buffered saline (PBS).

[0012] As for the requirements for monoclonal antibodies CD3, CD28 and ICOS, the concentration of monoclonal antibody CD3 is 2±0.2μg / ml, the concentration of CD28 is 2±0.2μg / ml, and the concentration of ICOS is 0.5±0.05μg / ml, where ± is the error value and can fluctuate within a certain range. However, when the error range is exceeded, if the effects are similar, they should be regarded as equivalent.

[0013] For the aforementioned steps, the steps not specifically described in the foregoing can be prepared according to existing processes. Of course, the following preparation conditions can also be adopted, and any condition can be selected independently:

[0014] In step S1, the reaction conditions are as follows (one or more are selected according to different situations):

[0015] 1) Activated T cells and lentivirus were mixed at MOI=1,

[0016] 2) T cells are taken from the peripheral blood of the treated subjects,

[0017] 3) The culture medium is a serum-free medium containing 200 U / mL human IL-2;

[0018] In step S2, the reaction conditions are as follows (one or more are selected according to different situations):

[0019] 1) Remove the S1 medium by centrifugation and re-seed the cells on a culture plate without monoclonal antibodies CD3, CD28 and ICOS.

[0020] 2) Resuspend the T cells in serum-free medium to culture the CAR-T cells,

[0021] 3) The culture medium was a serum-free medium containing 200 U / mL human IL-2.

[0022] In some specific application scenarios, the chimeric antigen receptor gene vector targeting a specific antigen in step S1 is a gene vector carrying a humanized anti-CD19-CAR, and the T cells are CD19-CAR-T cells.

[0023] Second aspect of the present invention CAR-T prepared by any of the above preparation methods ICOS cell.

[0024] Compared with conventional CAR-T cells stimulated by monoclonal antibodies CD3 and CD28, CAR-T cells stimulated by CD3, CD28 and ICOS ICOS The cells have more obvious advantages in various aspects, such as higher lentiviral transfection efficiency, increased mitochondrial mass in CAR-T cells, and reduced senescent phenotype of CAR-T cells. ICOS The cells have strong proliferation ability both in vivo and in vitro, and have good anti-tumor effects in vivo. It can be seen that compared with the combination of monoclonal antibody CD3 / CD28 to stimulate T cells, ICOS combined with CD3 / CD28 to stimulate T cells has a synergistic effect, stimulating the obtained CAR-T ICOS The transfection efficiency, proliferation ability, anti-tumor ability and phenotypic characteristics of the cells were significantly improved.

[0025] The third aspect of the present invention Involving CAR-T ICOS Application of cells in the preparation of anti-tumor drugs.

[0026] The anti-tumor drug is an anti-lymphoma drug. The lymphoma is at least one of common lymphoma and other acute lymphocytic leukemia, diffuse large B-lymphoma, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, etc.

[0027] In the manufacturing process of CAR-T cells, monoclonal antibodies CD3, CD28, and ICOS can give T cells high lentiviral transfection efficiency; the CAR-T cells obtained by this method ICOS The cells have stronger proliferation capabilities both in vivo and in vitro, and possess better anti-tumor capabilities in vivo. In particular, they can significantly improve the survival of tumor-bearing mice, and even achieve zero mortality under certain conditions.

[0028] The beneficial effects of the present invention are:

[0029] Provides new CAR-T ICOS Cells and preparation methods thereof, and the CAR-T ICOS The cells have a higher lentiviral transfection efficiency, stronger in vivo and in vitro proliferation capabilities, and better in vivo anti-tumor capabilities; and can greatly improve the survival rate of patients. Currently, under certain conditions, it is expected that the survival rate of patients can be increased to 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the co-stimulatory molecule ICOS obtained in the present invention combined with monoclonal antibodies CD3 and CD28 to stimulate T cells;

[0031] Figure 2 The CAR-T obtained by the present invention ICOS The transfection efficiency and phenotypic characteristics of cells. Among them: A. Schematic diagram of the process of preparing CAR-T cells with monoclonal antibodies; B. The transfection efficiency of CAR-T cells prepared using monoclonal antibodies CD3 and CD28 (conventional method) and monoclonal antibodies CD3, CD28 and ICOS. CAR-T cells obtained by conventional methods are defined as conventional CAR-T cells, and CAR-T cells prepared with monoclonal antibodies CD3, CD28 and ICOS are defined as CAR-T ICOS Cells; C, conventional CAR-T cells and CAR-T ICOS Cell differentiation and memory phenotype; D. Conventional CAR-T cells and CAR-T ICOS expression levels of cellular memory and exhaustion markers;

[0032] Figure 3 The CAR-T obtained by the present invention ICOS Metabolic characteristics of cells. A. Hippocampal metabolic analysis of conventional CAR-T cells and CAR-T ICOS Cellular oxygen consumption rate (OCR) level; B, conventional CAR-T cells and CAR-T ICOS OCR levels of cell basal, maximum respiration, and spare respiratory capacity; C, conventional CAR-T cells and CAR-T ICOSCell mitochondrial quality assessment; D. Conventional CAR-T cells and CAR-T ICOS Comparison of cell mitochondrial membrane potential;

[0033] Figure 4 The CAR-T obtained by the present invention ICOS The in vitro killing ability, in vitro CAR-T expansion ability and cytokine secretion of cells. ICOS Comparison of in vitro expansion ability of cells; B, CAR-T cells and Raji cells at different effector-target ratios. ICOS Comparison of the standard killing function of cells for 24 hours; C. When the ratio of CAR-T cells to Raji cells is 1:1, the difference between conventional CAR-T cells and CAR-T ICOS Comparison of cell killing function at 72 hours; D. Conventional CAR-T cells and CAR-T ICOS Comparison of cellular cytokine secretion;

[0034] Figure 5 The CAR-T obtained by the present invention ICOS The anti-tumor effect of cells in a mouse lymphoma model. A. Schematic diagram of the animal experiment process; B. Injection of PBS, conventional CAR-T cells and CAR-T into the tail vein ICOS In vivo imaging of NSG small animals after cell transplantation; C, injection of PBS, conventional CAR-T cells and CAR-T into the tail vein ICOS Statistical diagram of tumor burden in vivo imaging of NSG small animals after cell transplantation; D, conventional CAR-T cells and CAR-T ICOS Statistical diagram of cell proliferation in NSG mice; E, mice that received tail vein injection of PBS, conventional CAR-T cells, and CAR-T ICOS Survival curve of NSG mice after cell transplantation. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with specific research projects.

[0036] Various cells and experimental animals used in the following examples:

[0037] Humanized CD19-CAR lentivirus and human lymphoma Raji cells were provided by Wuhan Sian Medical Technology Co., Ltd., and highly immunodeficient NSG mice were purchased from Shanghai Nanmo Biotechnology Co., Ltd.

[0038] Example 1: CAR-T ICOS 1. Cell Preparation

[0039] (1) The culture plates were pre-coated by adsorption, and monoclonal antibodies CD3 (OKT3; 2 μg / ml; BioLegend), CD28 (2 μg / ml; BioLegend), and ICOS (0.5 μg / ml; R&D Systems) to the corresponding concentration in the brackets, add 200 μl of the diluent to a 48-well culture plate, and incubate in a 37°C incubator for 4 hours before use. Figure 1 shown.

[0040] (2) T cells were sorted by magnetic beads and resuspended to 5 × 10 cells / mL in serum-free medium (X-VIVO 15TM, ​​Lonza, Walkersville, MD, USA) containing 200 U / mL human IL-2 (PeproTech, Cranbury, NJ, USA). 5 / mL and placed in a pre-coated 48-well culture plate.

[0041] (3) After stimulating activated T cells for 1 day, the activated T cells and lentivirus were mixed at an MOI of 1, and then stimulated and cultured at 37°C for 72 hours.

[0042] Example 2: CAR-T ICOS Cell phenotype and metabolic level detection

[0043] (1) Collect transfected CAR-T cells and detect the transfection efficiency and phenotype of CAR-T cells by flow cytometry. ICOS Cell transfection efficiency and phenotypic characteristics such as Figure 2 The results showed that compared with the combination of CD3 and CD28 antibodies, the combined stimulation of CD3, CD28 and ICOS monoclonal antibodies significantly increased the efficiency of T cell lentiviral transfection by 1.3 times ( Figure 2 B), more CAR-T cells are obtained; compared with conventional CAR-T cells (Note: CAR-T in the embodiment ICOS The concentration and reaction conditions of CD4+CAR-T cells are the same as those of CAR-T cells. ICOS The central memory cell phenotype is higher in cells and CD8+CAR-T ICOS The proportion of immature cells in the cells is higher ( Figure 2 C); CAR-T ICOS Cells expressed higher levels of memory markers CD62L and CCR7 ( Figure 2 D), which means CAR-T ICOS Lower levels of cellular senescence.

[0044] (2) Collect transfected CAR-T cells and detect CAR-T using hippocampal metabolic analyzer ICOS Cellular metabolism level. CAR-T ICOS Cellular metabolism levels Figure 3 The results showed that compared with conventional CAR-T cells, CAR-T ICOS Higher levels of cellular oxidative phosphorylation ( Figure 3 A and 3B), CAR-T ICOS The cell mitochondrial mass (Mitotracker value reflects the number of mitochondria) increased by 1.2 times ( Figure 3 C) CAR-T ICOS The cell mitochondrial membrane potential (TMRM value reflects mitochondrial membrane potential) increased by 1.2 times ( Figure 3 D).

[0045] Example 3: In vitro expansion ability and killing function

[0046] (1) The transfected CAR-T cells were centrifuged and resuspended in serum-free culture medium (X-VIVO 15TM, ​​Lonza, Walkersville, MD, USA) to expand the CAR-T cells. ICOS The difference in the number of cell proliferation gradually increased with the increase of proliferation time; after 15 days of in vitro expansion, CAR-T cells were significantly higher than conventional CAR-T cells. ICOS The number of cells increased by 2 times ( Figure 4 A).

[0047] (2) 24-hour standard killing experiment. To evaluate the cytotoxicity of CAR-T cells, Raji cells were stained with 2 μM CFSE for 10 minutes and co-cultured for cytotoxicity experiments after washing. Serially diluted CAR-T cells were incubated with CFSE-labeled Raji cells. After 24 hours of culture, live cells were quantitatively analyzed by flow cytometry; the cytotoxicity of CAR-T cells to target cells was evaluated by analyzing the lysis rate of CFSE-labeled target cells. ICOS In vitro cell killing function Figure 4 In the 24-hour standard killing experiment, the results showed that conventional CAR-T cells and CAR-T ICOS The cells have comparable ability to kill tumor cells.

[0048] (3) 72-hour extended killing experiment. To evaluate the tumor killing ability of CAR-T cells, Raji cells were stained with 2μM CFSE for 10 minutes and co-cultured with CFSE-labeled Raji cells in a 1:1 ratio. After 72 hours of culture, live cells were quantitatively analyzed by flow cytometry; the cytotoxicity of CAR-T cells to target cells was evaluated by analyzing the lysis rate of CFSE-labeled target cells. ICOS The cells have a higher ability to kill tumor cells than conventional CAR-T cells ( Figure 4 C).

[0049] (4) Flow cytometry CBA multi-factor detection of cytokine secretion in the cell supernatant after co-culture. ICOS Cytokine secretion during cell killing in vitro Figure 4 Middle D.

[0050] Example 4: Evaluation of CAR-T ICOS Anti-tumor ability of cells in vivo

[0051] (1) NSG mice were purchased from Shanghai Model Organisms Corporation (Shanghai, China). Mice were housed in microisolator cages under specific pathogen-free conditions and had free access to autoclaved food and acidified water. 3×10 6 Raji cells were used to establish xenograft tumors, and bioluminescence imaging was performed 1 week later to determine whether the tumor model was successfully established;

[0052] (2) The NSG mice with successfully established tumor models were evenly divided into three groups (8 mice in each group), namely, PBS group, conventional CAR-T cell group, and CAR-T ICOS Cell groups. The three groups of mice were injected with 100 μl PBS, 100 μl containing 5×10 6 CAR-T CD28 Cell suspension and 100 μl containing CAR-T ICOS Suspension of cells;

[0053] (3) After CAR-T cell infusion, bioluminescence imaging was performed weekly to determine the tumor burden and obtain the efficacy curves of different groups of CAR-T cell-treated mice. CAR-T cell counts were detected by flow cytometry every week. After 14 days of treatment, the death status of NSG mice in different groups was recorded every day to obtain the median survival time of different groups of CAR-T-treated mice.

[0054] The CAR-T in this example ICOS In vivo anti-tumor ability of cells and evaluation of drug efficacy in mouse lymphoma Figure 5As shown; the results showed: CAR-T ICOS Cells have better anti-tumor properties in vivo ( Figure 5 AC) and expansion capability ( Figure 5 D) CAR-T ICOS The cells increased the survival rate of tumor-bearing mice to 100% ( Figure 5 E).

[0055] Manipulating the strength of T cell priming signals can regulate the metabolic fitness and phenotype of CAR-T cells. Enhancing the strength of T cell receptor signals and ICOS co-stimulatory signals can lead to increased T cell metabolic fitness and therapeutically effective T cells. CAR-T cells were prepared using the stimulatory molecule ICOS in combination with the co-stimulatory molecules CD3 and CD28. It was found that during the manufacturing process of CAR-T cells, CD3, CD28 and ICOS can confer high lentiviral transfection efficiency to T cells. CAR-T ICOS The cells have stronger proliferation ability in vivo and in vitro, have better anti-tumor ability in vivo, and can significantly improve the survival of tumor-bearing mice. According to Example 4, CAR-T ICOS The cells have increased the survival rate of tumor-bearing mice to 100%, allowing all mice to survive.

[0056] It will be clear to those skilled in the art that various modifications to the above embodiments may be made without departing from the overall spirit and concept of the present invention. All of these modifications fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims attached to the present invention.

Claims

1. CAR-T ICOS A method for preparing a cell, characterized in that: The following steps are included CAR-T cells are obtained by stimulating T cells with monoclonal antibodies CD3, CD28 and ICOS ICOS cell.

2. The CAR-T according to claim 1 ICOS A method for preparing a cell, characterized in that: The specific steps are as follows S1. Place the sorted T cells in a culture plate pre-coated with monoclonal antibodies CD3, CD28, and ICOS. After stimulation and activation, add a lentivirus containing a chimeric antigen receptor gene vector targeting a specific antigen and continue to stimulate and culture. S2. Remove the culture medium in S1 by centrifugation, and expand the CAR-T cells in serum-free culture medium using the centrifuged T cells.

3. The CAR-T according to claim 2 ICOS A method for preparing a cell, characterized in that: The plates were pre-coated with monoclonal antibodies CD3, CD28 and ICOS at 37°C.

4. The CAR-T according to claim 1 ICOS A method for preparing a cell, characterized in that: The concentration of monoclonal antibody CD3 was 2±0.2 μg / ml, the concentration of stimulatory molecule CD28 was 2±0.2 μg / ml, and the concentration of stimulatory molecule ICOS was 0.5±0.05 μg / ml.

5. The CAR-T according to claim 2 ICOS A method for preparing a cell, characterized in that: In step S1, the reaction conditions are as follows: 1) Activated T cells and lentivirus were mixed at MOI = 1; 2) T cells are obtained from the peripheral blood of the treated subject; 3) The culture medium was a serum-free medium containing 200 U / mL human IL-2.

6. The CAR-T according to claim 2 ICOS A method for preparing a cell, characterized in that: In step S2, the reaction conditions are as follows: 1) Remove the medium in S1 by centrifugation, 2) The centrifuged T cells are resuspended in serum-free medium to culture and expand CAR-T cells. 3) The culture medium was a serum-free medium containing 200 U / mL human IL-2.

7. The CAR-T according to any one of claims 1 to 6. ICOS A method for preparing a cell, characterized in that: The chimeric antigen receptor gene vector targeting a specific antigen is a lentivirus carrying a humanized anti-CD19-CAR gene vector, and the T cells are CD19-CAR-T cells.

8. The CAR-T prepared by the preparation method of claim 7 ICOS cell.

9. The CAR-T cell of claim 8 ICOS Application of cells in the preparation of anti-tumor drugs.

10. The use according to claim 9, wherein: The anti-tumor drug is an anti-lymphoma drug.

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