Targeted chimeric antigen receptor and application thereof in treating diabetes

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a targeted chimeric antigen receptor and application thereof in treating diabetes mellitus, and the targeted chimeric antigen receptor is prepared by the following steps: (1) separating from peripheral blood to prepare a mononuclear cell suspension; (2) re-suspending the mononuclear cell suspension in a first culture medium, culturing, centrifuging, and re-suspending by using a complete culture medium to adjust the cell concentration, so as to obtain a first culture solution; the first culture medium contains CD3 / CD28 monoclonal antibody coupled magnetic beads; and (3) adding lentivirus and polyetherimide into the first culture solution, culturing, centrifuging, resuspending by using a complete culture medium to adjust the cell concentration to obtain a second culture solution, and adding cell factors into the supernate to obtain the targeted chimeric antigen receptor. The targeting chimeric antigen receptor is high in positive rate and high in cell survival rate, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a kind of targeted chimeric antigen receptor and its application in treating diabetes. BACKGROUND

[0002] Chimeric antigen receptor (CAR) technology is a major breakthrough in the field of tumor immunotherapy in recent years, which fuses single-chain antibody (scFv) recognizing tumor-associated antigen (TAA) with T cell activation signal domain through genetic engineering, so that T cells can specifically recognize and kill tumor cells expressing corresponding antigens.

[0003] Traditional CAR-T therapy has achieved remarkable efficacy in the treatment of hematological tumors, but still faces many challenges in the treatment of solid tumors, such as tumor heterogeneity, immunosuppressive microenvironment, etc. The existing chimeric antigen receptor has low positive rate and insufficient cell survival rate. SUMMARY

[0004] The present application provides a kind of targeted chimeric antigen receptor and its application in treating diabetes, the targeted chimeric antigen receptor has high positive rate, and cell survival rate is high, has extensive application prospect.

[0005] The present application solves its technical problems by adopting the following technical solutions: A preparation method of a targeted chimeric antigen receptor, comprising the following steps: (1) preparing a single nuclear cell suspension from peripheral blood; (2) resuspending the single nuclear cell suspension in a first culture medium, culturing, centrifuging, resuspending in complete culture medium to adjust the cell concentration, and obtaining a first culture solution; the first culture medium contains CD3 / CD28 monoclonal antibody conjugated magnetic beads; (3) adding lentivirus and polyetherimide to the first culture solution, culturing, centrifuging, resuspending in complete culture medium to adjust the cell concentration, obtaining a second culture solution, and adding cytokines to the supernatant to obtain a targeted chimeric antigen receptor.

[0006] The present application obtains a single nuclear cell suspension from peripheral blood, cultures it with a first culture medium containing CD3 / CD28 monoclonal antibody conjugated magnetic beads, then adds lentivirus and polyetherimide for culture, and finally adds cytokines to obtain a targeted chimeric antigen receptor with high positive rate and high cell survival rate.

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

[0008] As a preferred embodiment of the present application, the first culture solution comprises the following components: 100-300 IU / mL IL-5, 100-300 IU / mL IL-4, 2-10 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 10% fetal bovine serum, and a basic culture medium.

[0009] As a preferred embodiment of the present application, the basic culture medium is CTS AIM V SFM culture medium.

[0010] As a preferred embodiment of the present application, the lentivirus is added at an MOI of 4-10.

[0011] As a preferred embodiment of the present application, the final concentration of the polyetherimide is 50-120 μM.

[0012] As a preferred embodiment of the present application, the cytokines include IL-2 and IL-7, the final concentration of the IL-2 is 100-200 IU / mL, and the final concentration of the IL-7 is 100-200 IU / mL.

[0013] As a preferred embodiment of the present application, the culture is carried out in a 36.5-37.5 ℃ incubator with 5% CO2.

[0014] The present application also provides a targeted chimeric antigen receptor prepared by the preparation method described above.

[0015] The present application also provides a use of a targeted chimeric antigen receptor in treating diabetes.

[0016] The present application has the following beneficial effects: the present application obtains a targeted chimeric antigen receptor with high positive rate and high cell survival rate by obtaining a mononuclear cell suspension from peripheral blood, culturing the mononuclear cell suspension with a first culture solution containing CD3 / CD28 monoclonal antibody conjugated magnetic beads, then adding lentivirus and polyetherimide for culture, and finally adding cytokines. DETAILED DESCRIPTION

[0017] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0018] In the present application, the technical features described in an open form include both a closed technical solution consisting of listed features and an open technical solution containing listed features.

[0019] In the present application, when a numerical range is involved, unless otherwise specified, the numerical range is considered to be continuous, and includes the minimum value and the maximum value of the range, and every value between the minimum value and the maximum value. Further, when the range refers to an integer, every 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 characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood as including any and all sub-ranges subsumed therein.

[0020] In the present application, the specific dispersion and stirring treatment method is not particularly limited.

[0021] 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.

[0022] Example 1 A preparation method of a 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 1×10 6 cells / mL with lymphocyte culture solution GT-T551 (also known as GT-T551 culture solution) to obtain a mononuclear cell suspension; (2) Resuspend the mononuclear cell suspension in a first culture medium, and culture at 37°C in a 5% CO2 incubator for 24 h, centrifuge, resuspend the supernatant with complete culture medium to adjust the cell concentration to 5×10 6 cells / mL to obtain a first culture solution; The first culture medium comprises the following components: 200 IU / mL IL-5, 200 IU / mL IL-4, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 10% fetal bovine serum, and basal culture medium.

[0023] (3) Add lentivirus to the first culture solution at an MOI of 8 and a polyetherimide final concentration of 100 μM, and culture at 37°C in a 5% CO2 incubator for 72 h, centrifuge, resuspend with complete culture medium to adjust the cell concentration to 5×10 6 cells / mL to obtain a second culture solution, and add IL-2 and IL-7 to the supernatant to obtain a targeted chimeric antigen receptor, with a final concentration of 100 IU / mL and 200 IU / mL, respectively.

[0024] Example 2 A preparation method of a 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 1x10 6 / mL by using lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution), to obtain a mononuclear cell suspension; (2) Resuspend the mononuclear cell suspension in a first culture medium, and culture at 37℃ in a 5% CO2 incubator for 24 h, centrifuge, resuspend the supernatant in complete culture medium to adjust the cell concentration to 5x10 6 / mL, to obtain a first culture solution; The first culture medium comprises the following components: 200 IU / mL IL-5, 200 IU / mL IL-4, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 10% fetal bovine serum, and a basic culture medium.

[0025] (3) Add lentivirus to the first culture solution at an MOI of 8 and a polyetherimide final concentration of 100 μM, and culture at 37℃ in a 5% CO2 incubator for 72 h, centrifuge, resuspend in complete culture medium to adjust the cell concentration to 5x10 6 / mL, to obtain a second culture solution, and add IL-2 and IL-7 to the supernatant at a final concentration of 100 IU / mL and 200 IU / mL, respectively, to obtain a targeted chimeric antigen receptor, wherein the positive rate of the targeted chimeric antigen receptor is 49.32%, and the average cell viability is 89.56%.

[0026] Example 3 A preparation method of a 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 1x10 6 / mL by using lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution), to obtain a mononuclear cell suspension; (2) Resuspend the mononuclear cell suspension in a first culture medium, and culture at 37℃ in a 5% CO2 incubator for 24 h, centrifuge, resuspend the supernatant in complete culture medium to adjust the cell concentration to 5x10 6 / mL, to obtain a first culture solution; The first culture medium comprises the following components: 200 IU / mL IL-5, 200 IU / mL IL-4, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 10% fetal bovine serum, and a basic culture medium.

[0027] (3) adding lentivirus into the first culture solution with MOI=8 and a final concentration of polyetherimide being 100 μM, culturing at 37°C in a culture box with 5% CO2 for 72h, centrifuging, resuspending with complete culture medium to adjust the cell concentration to 5×10 6 / mL to obtain a second culture solution, and adding IL-2 and IL-7 into the supernatant with final concentrations of 100 IU / mL and 200 IU / mL respectively to obtain the targeting chimeric antigen receptor, wherein the positive rate of the targeting chimeric antigen receptor reaches 47.10%, and the average cell viability is 87.59%.

[0028] Example 4 A preparation method of a targeting chimeric antigen receptor, comprising the following steps: (1) collecting 20 mL of peripheral blood, separating mononuclear cells by Ficoll density gradient centrifugation, and adjusting 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) resuspending the mononuclear cell suspension in a first culture medium, culturing at 37°C in a culture box with 5% CO2 for 24h, centrifuging, resuspending the supernatant with complete culture medium to adjust the cell concentration to 5×10 6 / mL to obtain a first culture solution; The first culture medium comprises the following components: 300 IU / mL IL-5, 100 IU / mL IL-4, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 10% fetal bovine serum, and a basic culture medium.

[0029] (3) adding lentivirus into the first culture solution with MOI=8 and a final concentration of polyetherimide being 100 μM, culturing at 37°C in a culture box with 5% CO2 for 72h, centrifuging, resuspending with complete culture medium to adjust the cell concentration to 5×10 6 / mL to obtain a second culture solution, and adding IL-2 and IL-7 into the supernatant with final concentrations of 100 IU / mL and 200 IU / mL respectively to obtain the targeting chimeric antigen receptor, wherein the positive rate of the targeting chimeric antigen receptor reaches 47.79%, and the average cell viability is 87.93%.

[0030] Example 5 A preparation method of a targeting chimeric antigen receptor, comprising the following steps: (1) collecting 20 mL of peripheral blood, separating mononuclear cells by Ficoll density gradient centrifugation, and adjusting the density of the mononuclear cells to 1×10 6cells / mL, to obtain a single nucleated cell suspension; (2) Resuspend the single nucleated cell suspension in the first culture medium, and culture in a 37℃, 5% CO2 incubator for 24h, centrifuge, resuspend the supernatant in complete culture medium to adjust the cell concentration to 5x10 6 cells / mL, to obtain a first culture solution; The first culture medium comprises the following components: 200 IU / mL IL-5, 200 IU / mL IL-4, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 10% fetal bovine serum, and a basic culture medium.

[0031] (3) Add the lentivirus to the first culture solution at an MOI of 8 and a final polyetherimide concentration of 100 μM, and culture in a 37℃, 5% CO2 incubator for 72h, centrifuge, resuspend in complete culture medium to adjust the cell concentration to 5x10 6 cells / mL, to obtain a second culture solution, and add IL-2 and IL-7 to the supernatant at final concentrations of 200 IU / mL and 100 IU / mL, respectively, to obtain a targeted chimeric antigen receptor, wherein the positive rate of the targeted chimeric antigen receptor is 48.15%, and the average cell viability is 88.54%.

[0032] Example 6 A preparation method of a targeted chimeric antigen receptor, comprising the following steps: (1) Collect 20 mL of peripheral blood, separate mononuclear cells using Ficoll density gradient centrifugation, and adjust the density of the single nucleated cells to 1x10 6 cells / mL, to obtain a single nucleated cell suspension; (2) Resuspend the single nucleated cell suspension in the first culture medium, and culture in a 37℃, 5% CO2 incubator for 24h, centrifuge, resuspend the supernatant in complete culture medium to adjust the cell concentration to 5x10 6 cells / mL, to obtain a first culture solution; The first culture medium comprises the following components: 200 IU / mL IL-5, 200 IU / mL IL-4, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 10% fetal bovine serum, and a basic culture medium.

[0033] (3) Add the lentivirus to the first culture solution at an MOI of 8 and a final polyetherimide concentration of 100 μM, and culture in a 37℃, 5% CO2 incubator for 72h, centrifuge, resuspend in complete culture medium to adjust the cell concentration to 5x10 6 / mL, to obtain the second culture solution, and IL-2 and IL-7 are added to the supernatant respectively to obtain the targeting chimeric antigen receptor, wherein the positive rate of the targeting chimeric antigen receptor reaches 48.43%, and the average cell viability is 88.76%.

[0034] Example 7 A preparation method of a 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 by using lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution) to obtain a mononuclear cell suspension; (2) The mononuclear cell suspension is resuspended in a first culture medium, and cultured in a 37℃, 5% CO2 incubator for 24 h, centrifuged, and the supernatant is resuspended in complete culture medium to adjust the cell concentration to 5×10 6 / mL to obtain a first culture solution; The first culture medium comprises the following components: 200 IU / mL IL-5, 200 IU / mL IL-4, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 10% fetal bovine serum, and a basic culture medium.

[0035] (3) The lentivirus is added to the first culture solution at an MOI of 6 and a polyetherimide final concentration of 50 μM, and cultured in a 37℃, 5% CO2 incubator for 72 h, centrifuged, and resuspended in complete culture medium to adjust the cell concentration to 5×10 6 / mL to obtain a second culture solution, and IL-2 and IL-7 are added to the supernatant respectively to obtain the targeting chimeric antigen receptor, wherein the positive rate of the targeting chimeric antigen receptor reaches 48.49%, and the average cell viability is 88.78%.

[0036] Example 8 A preparation method of a 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 by using lymphocyte culture solution GT-T551 (also referred to as GT-T551 culture solution) to obtain a mononuclear cell suspension; (2) The mononuclear cell suspension is resuspended in a first culture medium, and cultured in a 37℃, 5% CO2 incubator for 24 h, centrifuged, and the supernatant is resuspended in complete culture medium to adjust the cell concentration to 5×106 The first culture solution is obtained by adding 1.5*106cells / mL to 10 mL of RPMI 1640 medium containing 10% fetal bovine serum, 200 IU / mL IL-5, 200 IU / mL IL-4 and 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads. The first culture medium comprises the following components: 200 IU / mL IL-5, 200 IU / mL IL-4, 8 μg / mL CD3 / CD28 monoclonal antibody conjugated magnetic beads, 10% fetal bovine serum and basal medium.

[0037] (3) The lentivirus is added to the first culture solution at an MOI of 10 and a final polyetherimide concentration of 120 μM, and is cultured at 37°C in a 5% CO2 incubator for 72 h, centrifuged, resuspended with complete culture medium and adjusted to a cell concentration of 5*106 / mL. 6 The second culture solution is obtained by adding IL-2 and IL-7 to the supernatant at final concentrations of 100 IU / mL and 200 IU / mL, respectively, to obtain a targeted chimeric antigen receptor, wherein the positive rate of the targeted chimeric antigen receptor is 48.69% and the average cell viability is 88.95%.

[0038] It should be finally pointed out that the above embodiments are used to illustrate the technical solutions of the present application but not to limit the protection scope of the present application, and although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand 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 of making a targeted chimeric antigen receptor, characterized in that, The method comprises the following steps: (1) preparing a single nuclear cell suspension from peripheral blood; (2) resuspending the single nuclear cell suspension in a first culture medium, culturing, centrifuging, resuspending in complete culture medium to adjust the cell concentration, and obtaining a first culture solution; the first culture medium contains CD3 / CD28 monoclonal antibody coupled magnetic beads; (3) adding a lentivirus and a polyetherimide into the first culture solution, culturing, centrifuging, resuspending in complete culture medium to adjust the cell concentration, obtaining a second culture solution, and adding a cytokine into the supernatant to obtain a targeted chimeric antigen receptor.

2. The method for preparing the targeted chimeric antigen receptor according to claim 1, characterized in that, The cell concentration in the single nucleated cell suspension is 0.5-2 x 10 6 / mL.

3. The method for preparing the targeted chimeric antigen receptor according to claim 1, characterized in that, The first culture solution comprises the following components: 100-300 IU / mL IL-5, 100-300 IU / mL IL-4, 2-10 μg / mL CD3 / CD28 monoclonal antibody coupled magnetic beads, 10% fetal bovine serum, and a basic culture medium.

4. The method for preparing the targeted chimeric antigen receptor according to claim 3, characterized in that, The basic culture medium is a CTS AIM V SFM culture medium.

5. The method for preparing the targeted chimeric antigen receptor according to claim 1, characterized in that, The lentivirus is added at an MOI of 4-10.

6. The method for preparing the targeted chimeric antigen receptor according to claim 1, characterized in that, The final concentration of the polyetherimide is 50-120 μM.

7. The method for preparing the targeted chimeric antigen receptor according to claim 1, characterized in that, The cytokine comprises IL-2 and IL-7, the final concentration of the IL-2 is 100-200 IU / mL, and the final concentration of the IL-7 is 100-200 IU / mL.

8. The method for preparing the targeted chimeric antigen receptor according to claim 1, characterized in that, The culturing is performed in a 36.5-37.5 ℃ incubator with 5% CO2.

9. A targeted chimeric antigen receptor, characterized in that, The targeted chimeric antigen receptor is prepared by the preparation method in any one of claims 1-8.

10. The targeted chimeric antigen receptor in claim 9 is used for treating diabetes.