CEA targeted chimeric antigen receptor and application of CEA targeted chimeric antigen receptor in preparation of anti-cancer drugs

Abstract

The invention belongs to the technical field of biology, and particularly discloses a CEA targeted chimeric antigen receptor and application of the CEA targeted chimeric antigen receptor in preparation of anti-cancer drugs, and the CEA targeted chimeric antigen receptor is prepared by the following steps: (1) separating peripheral blood to prepare a mononuclear cell suspension; (2) adding the human CD3 / CD28T cell activated magnetic beads into the mononuclear cell suspension, uniformly mixing, incubating and sorting to obtain T cells; (3) inoculating the T cells into a differential culture medium for culturing to obtain a cell suspension; and (4) adding lentivirus and polyetherimide into the cell suspension, culturing, centrifuging, and resuspending by using a complete culture medium to adjust the cell concentration, thereby obtaining the CEA targeted chimeric antigen recipient cell. The CEA targeted chimeric recipient cell provided by the invention has high chimeric antigen receptor positive rate and cell viability, and has wide application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a CEA targeting chimeric antigen receptor and application thereof in preparation of anticancer drugs. BACKGROUND

[0002] CEA is a broad-spectrum tumor marker, which is highly expressed on the surface of various solid tumors (such as colorectal cancer, lung cancer, gastric cancer), and is lowly expressed in normal tissues, thus becoming an ideal tumor immunotherapy target. Traditional CAR-T cell therapy has achieved remarkable effects in hematological tumors, but faces challenges such as antigen heterogeneity and tumor microenvironment inhibition in solid tumors.

[0003] In the prior art, the design of CEA targeting CAR-T cells has problems of insufficient positive rate and cell viability, poor cell persistence, etc. SUMMARY

[0004] The present application provides a CEA targeting chimeric antigen receptor and application thereof in preparation of anticancer drugs. The CEA targeting chimeric receptor cell has high chimeric antigen receptor positive rate and cell viability, and has wide application prospect.

[0005] The present application solves the technical problems by adopting the following technical solutions: A preparation method of a CEA targeting chimeric antigen receptor, comprising the following steps: (1) separating peripheral blood to prepare a single nuclear cell suspension; (2) adding human CD3 / CD28 T cell activation magnetic beads to the single nuclear cell suspension, mixing, incubating, sorting, and obtaining T cells; (3) inoculating the T cells in a differentiation culture medium for culture, and obtaining a cell suspension; (4) adding a lentivirus and a polyetherimide to the cell suspension, culturing, centrifuging, resuspending with complete culture medium to adjust the cell concentration, and obtaining CEA targeting chimeric antigen receptor cells.

[0006] The present application first separates peripheral blood to prepare a single nuclear cell suspension, then activates and differentiates the single nuclear cell suspension into T cells by using human CD3 / CD28 T cell activation magnetic beads, then inoculates the T cells in a differentiation culture medium for culture, and finally co-cultures a lentivirus and a polyetherimide with the T cells to obtain CEA targeting chimeric antigen receptor cells, which have high chimeric antigen receptor positive rate and cell viability.

[0007] As an embodiment of the present application, the cell concentration in the single nuclear cell suspension is 0.5-2 x 10 6 / mL.

[0008] As an embodiment of the present application, the human CD3 / CD28 T cell activation magnetic beads are added in an amount of 5-20 μg / mL.

[0009] As an embodiment of the present application, the incubation is performed in an incubator at 36.5-37.5 °C, 5% CO2.

[0010] As an embodiment of the present application, the culture is performed in an incubator at 36.5-37.5 °C, 5% CO2.

[0011] As an embodiment of the present application, the T cells are inoculated at a concentration of 0.5-2 x 10 6 / mL.

[0012] As an embodiment of the present application, the differentiation medium comprises the following components: 9.5-10.5% fetal bovine serum, 200-300 ng / mL IL-5, 150-300 ng / mL IL-14, 100-200 ng / mL IL-21, 2-5 mM nicotinamide riboside, 1-4 mM L-carnitine, and the balance of basal medium.

[0013] As an embodiment of the present application, the basal medium is TexMACS medium.

[0014] As an embodiment of the present application, the lentivirus is added at an MOI of 4-10; The final concentration of the polyetherimide is 50-120 μM.

[0015] As an embodiment of the present application, the cells are resuspended with complete medium to adjust the cell concentration to 0.5-2 x 10 6 / mL.

[0016] The present application also provides a use of a CEA-targeted chimeric antigen receptor in the preparation of an anticancer drug.

[0017] The present application has the following beneficial effects: the peripheral blood is first separated to prepare a single nuclear cell suspension, then activated by human CD3 / CD28 T cell activation magnetic beads to differentiate into T cells, then the T cells are inoculated in a differentiation medium for culture, and finally lentivirus and polyetherimide are co-cultured with the T cells to obtain CEA-targeted chimeric antigen receptor cells, which have a high chimeric antigen receptor positive rate and cell viability. DETAILED DESCRIPTION

[0018] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are some of the embodiments of the present application but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0019] In the present application, the technical features described in an open way include both the closed technical solutions consisting of the listed features and the open technical solutions containing the listed features.

[0020] In the present application, as no special description is made, the numerical range is regarded as continuous and includes the minimum value and the maximum value of the range and each value between the minimum value and the maximum value. Further, when the range refers to an integer, each integer between the minimum value and the maximum value of the range is included. In addition, when multiple ranges are provided to describe a feature or a property, the ranges can be combined. In other words, unless otherwise specified, all the ranges disclosed herein should be understood as including any and all sub-ranges falling within the range.

[0021] In the present application, the specific dispersion and stirring treatment method is not particularly limited.

[0022] The reagents or instruments used in the present application are not specified by the manufacturer, and are all conventional products that can be obtained by purchase. The raw materials used in the comparative examples and the raw materials used in the parallel experiments of the examples are the same commercially available products unless otherwise specified.

[0023] Example 1 A preparation method of a CEA-targeted chimeric antigen receptor, comprising the following steps: (1) 20 mL of peripheral blood is collected, mononuclear cells are separated by Ficoll density gradient centrifugation, and the mononuclear cell density is adjusted to 1×10 6 cells / mL by using lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution), to obtain a mononuclear cell suspension; (2) human CD3 / CD28 T cell activation magnetic beads are added to the mononuclear cell suspension at an addition amount of 15 μg / mL, mixed, incubated at 37°C in a 5% CO2 incubator for 12 h, sorted, and T cells are obtained; (3) the T cells are inoculated in a differentiation culture medium at a concentration of 1×10 6 cells / mL, cultured for 10 days, and the differentiation culture medium is replaced by 50% of the volume every two days, to obtain a cell suspension; (4) the lentivirus (BCMA-GFP lentivirus) is added into the cell suspension at MOI = 8 and a final concentration of polyetherimide is 100 μM, and is cultured for 48 h, centrifuged, resuspended with complete culture medium, and adjusted to a cell concentration of 1×10 6 / mL to obtain CEA-targeted chimeric antigen receptor cells.

[0024] The differentiation culture medium comprises the following components: 10% fetal bovine serum, 250 ng / mL IL-5, 200 ng / mL IL-14, 180 ng / mL IL-21, 4 mM nicotinamide riboside, 2 mM L-carnitine, and the balance of TexMACS culture medium.

[0025] Example 2 A preparation method of a CEA-targeted chimeric antigen receptor comprises the following steps: (1) 20 mL of peripheral blood is collected, mononuclear cells are separated by Ficoll density gradient centrifugation, and the mononuclear cell density is adjusted to 1×10 6 / mL by using lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution) to obtain a mononuclear cell suspension; (2) human CD3 / CD28 T cell activation magnetic beads are added to the mononuclear cell suspension at an addition amount of 15 μg / mL, mixed, incubated at 37°C in a 5% CO2 incubator for 12 h, sorted, and T cells are obtained; (3) the T cells are inoculated in the differentiation culture medium at a concentration of 1×10 6 / mL, and are cultured for 10 days, and the differentiation culture medium is replaced by 50% of the volume every two days to obtain a cell suspension; (4) the lentivirus (BCMA-GFP lentivirus) is added into the cell suspension at MOI = 8 and a final concentration of polyetherimide is 100 μM, and is cultured for 48 h, centrifuged, resuspended with complete culture medium, and adjusted to a cell concentration of 1×10 6 / mL to obtain CEA-targeted chimeric antigen receptor cells.

[0026] The differentiation culture medium comprises the following components: 10% fetal bovine serum, 200 ng / mL IL-5, 300 ng / mL IL-14, 150 ng / mL IL-21, 4 mM nicotinamide riboside, 2 mM L-carnitine, and the balance of TexMACS culture medium.

[0027] Example 3 A preparation method of a CEA-targeted chimeric antigen receptor comprises the following steps: (1) Collect 20 mL of peripheral blood, separate mononuclear cells by Ficoll density gradient centrifugation, and adjust the density of mononuclear cells to 1 x 10 6 / mL with lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution) to obtain a mononuclear cell suspension; (2) Add human CD3 / CD28 T cell activation magnetic beads to the mononuclear cell suspension at an addition amount of 15 μg / mL, mix well, incubate at 37°C in a 5% CO2 incubator for 12 h, and sort to obtain T cells; (3) Seed the T cells in a differentiation medium at a concentration of 1 x 10 6 / mL and culture for 10 days, replace 50% of the volume of the differentiation medium every two days, and obtain a cell suspension; (4) Add a lentivirus (BCMA-GFP lentivirus) to the cell suspension at MOI = 8 and a polyetherimide final concentration of 100 μM, culture for 48 h, centrifuge, resuspend with complete culture medium to adjust the cell concentration to 1 x 10 6 / mL, and obtain CEA-targeted chimeric antigen receptor cells.

[0028] The differentiation medium comprises the following components: 10% fetal bovine serum, 300 ng / mL IL-5, 150 ng / mL IL-14, 200 ng / mL IL-21, 4 mM nicotinamide riboside, 2 mM L-carnitine, and the balance TexMACS medium.

[0029] Example 4 A preparation method of a CEA-targeted chimeric antigen receptor, comprising the following steps: (1) Collect 20 mL of peripheral blood, separate mononuclear cells by Ficoll density gradient centrifugation, and adjust the density of mononuclear cells to 1 x 10 6 / mL with lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution) to obtain a mononuclear cell suspension; (2) Add human CD3 / CD28 T cell activation magnetic beads to the mononuclear cell suspension at an addition amount of 15 μg / mL, mix well, incubate at 37°C in a 5% CO2 incubator for 12 h, and sort to obtain T cells; (3) Seed the T cells in a differentiation medium at a concentration of 1 x 10 6 / mL and culture for 10 days, replace 50% of the volume of the differentiation medium every two days, and obtain a cell suspension; (4) Add a lentivirus (BCMA-GFP lentivirus) to the cell suspension at MOI = 8 and a polyetherimide final concentration of 100 μM, culture for 48 h, centrifuge, resuspend with complete culture medium to adjust the cell concentration to 1 x 10 6cells / mL, to obtain CEA-targeted chimeric antigen receptor cells.

[0030] The differentiation medium comprises the following components: 10% fetal bovine serum, 250 ng / mL IL-5, 200 ng / mL IL-14, 180 ng / mL IL-21, 2 mM nicotinamide riboside, 4 mM L-carnitine, and the balance TexMACS medium.

[0031] Example 5 A preparation method of a CEA-targeted chimeric antigen receptor, comprising the following steps: (1) 20 mL of peripheral blood is collected, mononuclear cells are separated by Ficoll density gradient centrifugation, and the mononuclear cell density is adjusted to 1×10 6 cells / mL with lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution), to obtain a mononuclear cell suspension; (2) human CD3 / CD28 T cell activation magnetic beads are added to the mononuclear cell suspension at an addition amount of 15 μg / mL, mixed, incubated at 37°C in a 5% CO2 incubator for 12 h, sorted, and T cells are obtained; (3) the T cells are inoculated in the differentiation medium at a concentration of 1×10 6 cells / mL, and cultured for 10 days, the medium is replaced every two days by replacing 50% of the volume of the differentiation medium, and a cell suspension is obtained; (4) a lentivirus (BCMA-GFP lentivirus) is added to the cell suspension at MOI=8 and a polyetherimide final concentration of 100 μM, cultured for 48 h, centrifuged, resuspended with complete culture medium to adjust the cell concentration to 1×10 6 cells / mL, to obtain CEA-targeted chimeric antigen receptor cells.

[0032] The differentiation medium comprises the following components: 10% fetal bovine serum, 250 ng / mL IL-5, 200 ng / mL IL-14, 180 ng / mL IL-21, 2 mM nicotinamide riboside, 4 mM L-carnitine, and the balance TexMACS medium.

[0033] Comparative Example 1

[0034] A preparation method of a CEA-targeted chimeric antigen receptor, comprising the following steps: (1) 20 mL of peripheral blood is collected, mononuclear cells are separated by Ficoll density gradient centrifugation, and the mononuclear cell density is adjusted to 1×10 6 cells / mL with lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution), to obtain a mononuclear cell suspension; (2) Add human CD3 / CD28 T cell activation magnetic beads to the mononuclear cell suspension at an addition amount of 15 μg / mL, mix well, incubate at 37°C in a 5% CO2 incubator for 12 h, sort, and obtain T cells; (3) Seed the T cells in the differentiation medium at a concentration of 1×10 6 / mL, and culture for 10 days, replace 50% of the differentiation medium every two days, and obtain a cell suspension; (4) Add the lentivirus (BCMA-GFP lentivirus) to the cell suspension at MOI=8 and a final polyetherimide concentration of 100 μM, culture for 48 h, centrifuge, resuspend with complete medium to adjust the cell concentration to 1×10 6 / mL, and obtain CEA-targeting chimeric antigen receptor cells.

[0035] The differentiation medium comprises the following components: 10% fetal bovine serum, 50 ng / mL IL-5, 400 ng / mL IL-14, 50 ng / mL IL-21, 4 mM nicotinamide riboside, 2 mM L-carnitine, and the balance TexMACS medium.

[0036] Comparative Example 2

[0037] A preparation method of a CEA-targeting chimeric antigen receptor, comprising the following steps: (1) Collect 20 mL of peripheral blood, separate mononuclear cells by Ficoll density gradient centrifugation, and adjust the mononuclear cell density to 1×10 6 / mL with lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution) to obtain a mononuclear cell suspension; (2) Add human CD3 / CD28 T cell activation magnetic beads to the mononuclear cell suspension at an addition amount of 15 μg / mL, mix well, incubate at 37°C in a 5% CO2 incubator for 12 h, sort, and obtain T cells; (3) Seed the T cells in the differentiation medium at a concentration of 1×10 6 / mL, and culture for 10 days, replace 50% of the differentiation medium every two days, and obtain a cell suspension; (4) Add the lentivirus (BCMA-GFP lentivirus) to the cell suspension at MOI=8 and a final polyetherimide concentration of 100 μM, culture for 48 h, centrifuge, resuspend with complete medium to adjust the cell concentration to 1×10 6 / mL, and obtain CEA-targeting chimeric antigen receptor cells.

[0038] The differentiation medium comprises the following components: 10% fetal bovine serum, 400 ng / mL IL-5, 500 ng / mL IL-14, 400 ng / mL IL-21, 4 mM nicotinamide riboside, 2 mM L-carnitine, and the balance TexMACS medium.

[0039] Comparative Example 3

[0040] A preparation method of a CEA-targeting chimeric antigen receptor, comprising the following steps: (1) 20 mL of peripheral blood is collected, mononuclear cells are separated by Ficoll density gradient centrifugation, and the mononuclear cell density is adjusted to 1×10 6 / mL with lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution) to obtain a mononuclear cell suspension; (2) human CD3 / CD28 T cell activation magnetic beads are added to the mononuclear cell suspension at an addition amount of 15 μg / mL, mixed, incubated at 37°C in a 5% CO2 incubator for 12 h, sorted, and T cells are obtained; (3) the T cells are inoculated in a differentiation medium at a concentration of 1×10 6 / mL and cultured for 10 days, 50% of the differentiation medium is replaced every two days, and a cell suspension is obtained; (4) a lentivirus (BCMA-GFP lentivirus) is added to the cell suspension at MOI=8 and a polyetherimide final concentration of 100 μM, cultured for 48 h, centrifuged, resuspended with complete culture medium to adjust the cell concentration to 1×10 6 / mL, and a CEA-targeting chimeric antigen receptor cell is obtained.

[0041] The differentiation medium comprises the following components: 10% fetal bovine serum, 250 ng / mL IL-5, 200 ng / mL IL-14, 180 ng / mL IL-21, 6 mM L-carnitine, and the balance TexMACS medium.

[0042] Comparative Example 4

[0043] A preparation method of a CEA-targeting chimeric antigen receptor, comprising the following steps: (1) 20 mL of peripheral blood is collected, mononuclear cells are separated by Ficoll density gradient centrifugation, and the mononuclear cell density is adjusted to 1×10 6 / mL with lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution) to obtain a mononuclear cell suspension; (2) Add human CD3 / CD28 T cell activation magnetic beads to the mononuclear cell suspension at an addition amount of 15 μg / mL, mix well, incubate at 37°C in a 5% CO2 incubator for 12 h, sort, and obtain T cells; (3) Seed the T cells in the differentiation medium at a concentration of 1×10 6 / mL, and culture for 10 days, replace 50% of the differentiation medium every two days, and obtain a cell suspension; (4) Add the lentivirus (BCMA-GFP lentivirus) to the cell suspension at an MOI of 8 and a final polyetherimide concentration of 100 μM, culture for 48 h, centrifuge, resuspend with complete medium to adjust the cell concentration to 1×10 6 / mL, and obtain CEA-targeting chimeric antigen receptor cells.

[0047] The differentiation medium comprises the following components: 10% fetal bovine serum, 250 ng / mL IL-5, 200 ng / mL IL-14, 180 ng / mL IL-21, 6 mM nicotinamide riboside, and the balance TexMACS medium.

[0048] Comparative Example 5

[0049] A preparation method of a CEA-targeting chimeric antigen receptor, comprising the following steps: (1) Collect 20 mL of peripheral blood, separate mononuclear cells by Ficoll density gradient centrifugation, and adjust the density of the mononuclear cells to 1×10 6 / mL with lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution) to obtain a mononuclear cell suspension; (2) Add human CD3 / CD28 T cell activation magnetic beads to the mononuclear cell suspension at an addition amount of 15 μg / mL, mix well, incubate at 37°C in a 5% CO2 incubator for 12 h, sort, and obtain T cells; (3) Seed the T cells in the differentiation medium at a concentration of 1×10 6 / mL, and culture for 10 days, replace 50% of the differentiation medium every two days, and obtain a cell suspension;

[0050] (4) Add the lentivirus (BCMA-GFP lentivirus) to the cell suspension at an MOI of 8 and a final polyetherimide concentration of 100 μM, culture for 48 h, centrifuge, resuspend with complete medium to adjust the cell concentration to 1×10 6 / mL, and obtain CEA-targeting chimeric antigen receptor cells.

[0051] The differentiation medium comprises the following components: 10% fetal bovine serum, 250 ng / mL IL-2, 200 ng / mL IL-4, 4 mM nicotinamide riboside, 2 mM L-carnitine, and the balance TexMACS medium.

[0048] The cell viability and the chimeric antigen receptor positive rate of the examples and the comparative examples are shown in Table 1.

[0049] Table 1

[0050] As can be seen from Table 1, the CEA-targeted chimeric receptor cells have high chimeric antigen receptor positive rate and cell viability, and have wide application prospects.

[0051] It should be finally pointed out that the above examples are intended to illustrate the technical solutions of the present application rather than limit the protection scope of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A method for preparing a CEA-targeted chimeric antigen receptor, characterized in that, Includes the following steps: (1) Peripheral blood was separated and prepared into a suspension of mononuclear cells; (2) Add human CD3 / CD28 T cell activation magnetic beads to a mononuclear cell suspension, mix well, incubate, sort, and obtain T cells; (3) T cells were seeded in differentiation medium and cultured to obtain a cell suspension; (4) Add lentivirus and polyetherimide to the cell suspension, culture, centrifuge, resuspend in complete culture medium to adjust cell concentration, and obtain CEA-targeting chimeric antigen receptor cells.

2. The method for preparing CEA-targeted chimeric antigen receptor according to claim 1, characterized in that, The cell concentration in the mononuclear cell suspension is 0.5~2×10⁻⁶. 6 per mL.

3. The method for preparing CEA-targeted chimeric antigen receptor according to claim 1, characterized in that, The amount of human CD3 / CD28T cell activating magnetic beads added is 5~20μg / mL.

4. The method for preparing CEA-targeted chimeric antigen receptor according to claim 1, characterized in that, The incubation was carried out in an incubator at 36.5~37.5℃ and 5% CO2.

5. The method for preparing CEA-targeted chimeric antigen receptor according to claim 1, characterized in that, The culture was carried out in an incubator at 36.5~37.5℃ and 5% CO2. The T cell inoculation concentration is 0.5~2×10⁻⁶. 6 per mL.

6. The method for preparing CEA-targeted chimeric antigen receptor according to claim 1, characterized in that, The differentiation medium comprises the following components: 9.5-10.5% fetal bovine serum, 200-300 ng / mL IL-5, 150-300 ng / mL IL-14, 100-200 ng / mL IL-21, 2-5 mM nicotinamide ribose, 1-4 mM L-carnitine, and the remainder being basal medium.

7. The method for preparing CEA-targeted chimeric antigen receptor according to claim 6, characterized in that, The basal culture medium is TexMACS medium.

8. The method for preparing CEA-targeted chimeric antigen receptor according to claim 1, characterized in that, The lentivirus was added with an MOI of 4 to 10. The final concentration of the polyetherimide is 50~120μM.

9. The method for preparing CEA-targeted chimeric antigen receptor according to claim 1, characterized in that, Resuspend the cells in complete culture medium to adjust the cell concentration to 0.5–2 × 10⁻⁶. 6 per mL.

10. The use of the CEA-targeted chimeric antigen receptor as described in any one of claims 1 to 9 in the preparation of anticancer drugs.

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