CN114480276A - Method for improving anti-tumor effect of CAR-T cells - Google Patents
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Abstract
The invention relates to the technical field of immunology, in particular to a method for improving the anti-tumor effect of CAR-T cells. The method comprises treating the CAR-T cells with diazo-oxonorleucine; the concentration of the diazo-oxo-norleucine is 0.1-10 mol/L. The research of the invention finds that the diazo-oxo-norleucine has the effect of promoting the differentiation of T cells into low-differentiation T cell initial T cells and central memory T cells, and further applies the diazo-oxo-norleucine to the preparation of CAR-T cells, so that the preparation efficiency of the CAR-T cells can be improved, the anti-tumor effect of the CAR-T cells can be obviously improved, and the diazo-oxo-norleucine has important significance in the field of tumor treatment.
Description
Technical Field
The invention relates to the technical field of immunology, in particular to a method for improving the anti-tumor effect of CAR-T cells.
Background
Diazo-oxo-norleucine (DON) is a non-protein amino acid isolated from Streptomyces. In the molecular structure, DON is a diazoketone compound formed by substituting a basified group for glutamine side chain amino, and is a glutamine analogue. DON can irreversibly inhibit a series of glutamine-related enzyme activities, block glutamine metabolism, and has analgesic, antibacterial, antiviral, and antitumor effects. Glutamine is an important nitrogen source and carbon source in cell metabolism, and is involved in metabolic pathways such as glycolysis, tricarboxylic acid cycle and oxidative phosphorylation. Research shows that blocking glutamine metabolism can inhibit proliferation of tumor cell and control tumor growth. In 2019, a paper published in Science entitled "blocking glutamine to induce different metabolic programs to combat tumor immune escape" revealed differences in energy supply and metabolic pathways between T cells and tumor cells after blocking glutamine.
Chimeric antigen receptor T cell (CAR-T) therapy shows better clinical therapeutic effect in the field of treatment of hematological tumors, such as relapsed or refractory B-cell acute lymphoblastic leukemia and non-hodgkin lymphoma.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for improving the anti-tumor effect of CAR-T cells, and CAR can be effectively improved by treating the CAR-T cells with diazo-oxonorleucine+T in T cellsN(CD45RA+CCR7+) And TCM(CD45RA-CCR7+) The proportion of the subgroups, and further improve the capability of the subgroups to kill tumor cells.
The nitroxide norleucine weighed by the method is 6-diazo-5-oxo-L-norleucine (DON).
The research of the invention finds that the differentiation state of the T cells determines the persistence of the T cells to a great extent, and compared with effector T cells (TEFF) and effector memory T cells (TEM) with higher differentiation degrees, the initial T cells (TN) and central memory T Cells (TCM) in a low differentiation state have stronger viability and anti-tumor functions. The differentiation process of the T cells in immune response is accompanied with the change of metabolic pathways, so that the differentiation state of the T cells can be changed and more poorly differentiated T cells can be obtained by changing the in vitro culture environment of the cells and treating the cells by DON (Donor-N), the survival time of the CAR-T cells after being returned into the body is prolonged, the anti-tumor effect of the CAR-T cells is favorably enhanced, and good news is brought to tumor patients.
In a first aspect, the invention provides a method of increasing the anti-tumor effect of a CAR-T cell, comprising:
CAR-T cells were treated with diazooxypnorleucine.
Further, the concentration of the diazo-oxo-norleucine is 1-10 mu mol/L.
Further, the treatment of CAR-T cells with diazooxypnorleucine is:
1-10 mu mol/L diazooxypnorleucine is added into a culture medium in the process of preparing CAR-T cells.
Further, the method comprises:
infecting a T cell in a T cell culture medium with a lentivirus comprising a CAR nucleotide sequence;
the T cell culture medium contains 2.5-5 mu mol/L diazo-oxo-norleucine.
Further, the MOI of the lentivirus is (5-15): 1.
further, the T cell culture medium further comprises: OKT3 and CD28 antibodies.
Further, the T cell culture medium may employ a T cell culture medium conventional in the art for preparing CAR-T cells, each containing OKT3 and CD28 antibodies for activating T cells, for example, including: X-VIVO15 serum-free medium, IL2, IL-7, IL-15, OKT3 antibody and CD28 antibody.
Furthermore, the T cell culture medium is based on an X-VIVO15 serum-free culture medium, and further comprises IL 210-100U/mL, IL 71-20 ng/mL, IL 151-20 ng/mL, OKT3 antibody 10-100 ng/mL and CD28 antibody 0.1-10 mu g/mL.
As a preferred embodiment, the present invention provides a method of increasing the anti-tumor effect of a CAR-T cell, comprising:
(1) constructing a lentiviral vector carrying a CAR nucleotide sequence targeting a tumor cell;
(2) transfecting the lentivirus vector to 293FT cells to prepare lentiviruses carrying CAR nucleotide sequences of targeted tumor cells;
(3) and (3) infecting the T cells in a T cell culture medium for 48-96 hours by adopting the lentivirus, wherein MOI (5-15): 1.
the T cell culture medium is based on an X-VIVO15 serum-free culture medium, and further comprises IL 210-100U/mL, IL 71-20 ng/mL, IL 151-20 ng/mL, OKT3 antibody 10-100 ng/mL and CD28 antibody 0.1-10 mu g/mL.
The invention further provides application of diazo-oxo-norleucine in promoting differentiation of T cells into low-differentiation T cells.
Further, the poorly differentiated T cells include: naive T cells and/or central memory T cells.
The invention further provides application of diazo-oxo-norleucine in improving the preparation efficiency of the CAR-T cells.
The invention further provides the application of diazo-oxo-norleucine in the preparation of the medicine for improving the tumor killing efficiency of CAR-T cells.
The invention has the following beneficial effects:
in the prior art, stimulation of CD3 and CD28 antibodies is generally required in the conventional process for preparing CAR-T cells, so that T cells in the obtained CAR-T cellsEMAnd TEFFThe invention finds that the T in the obtained CAR-T cells can be improved by adding DON into a culture medium cultured in vitroNAnd TCMThe ratio of (A) to (B), thereby enhancing the in vivo persistence of the DON, and improving the ability of the DON to kill tumor cells, so that the CAR-T treated in vitro by the DON has better treatment effect.
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FIG. 1 shows T in DON-treated group and control group provided in example 1 of the present inventionN(CD45RA+CCR7+) And TCM(CD45RA-CCR7+) A graph of the statistical results of the subpopulations; wherein A is TNStatistical results of the subgroups, B is TCMStatistical results of the subpopulations.
FIG. 2 is a graph showing the statistics of the effect of DON on CAR-T cells provided in example 2 of the present invention; wherein A is the proportion of CAR-T cells 72 hours after infection, and B is T72 hours after infectionN(CD45RA+CCR7+) Subgroup proportion, C is T72 hours after infectionCM(CD45RA-CCR7+) The proportion of the subpopulations is shown schematically.
FIG. 3 is a graph showing the comparison of the expression levels of IFN- γ proteins secreted from CAR-T cells in DON-treated and non-DON-treated groups at different effective target ratios against Raji cells, provided in example 2 of the present invention.
FIG. 4 is a graph comparing the ability of CAR-T cells to specifically recognize and kill target cells in DON-treated and non-DON-treated groups at different effective target ratios against Raji cells as provided in example 2 of the present invention.
FIG. 5 is a comparison of the target cell recognition and killing functions of the mouse model of lymphoma, DON-treated group and non-DON-treated group provided in example 2 of the present invention.
Fig. 6 is a functional comparison diagram of the DON-treated group and the DON-untreated group against tumor recurrence in case of tumor recurrence provided in example 2 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.
Unless otherwise indicated, the following examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: aLaboratory Manual,2001), or conditions as recommended by the manufacturer's instructions.
Lentiviral packaging plasmids (pMDL, VSV-G, REV), vector pCDH-EF1-Luc2-T2A-tdTomato plasmids were purchased from Wuhan vast Ling Biotech, Inc. XbaI and SalI endonucleases were purchased from New England Biolabs (Beijing) LTD. PEI was purchased from Sigma. The CD19-Fc fusion protein was purchased from ACRO Biosystems, Inc. X-VIVO15 medium was purchased from Lonza. IL-2, IL-7, IL-15 cytokines were purchased from Peprotech. OKT3 was purchased from ACRO Biosystems, Inc. The CD28 antibody was purchased from Hoodia. 293FT cells, Raji cells were purchased from ATCC. Raji-Luciferase cells were purchased from eastern Ling Biotechnology, Inc., Suzhou. Flow antibodies such as CD45RA, CCR7, CFSE, PI, were purchased from BD. NOG immunodeficient mice were purchased from Beijing Wintolite laboratory animal technology, Inc.
Example 1
This example demonstrates the effect of DON on T cell differentiation, and the specific procedure is as follows:
1. PBMC culture
The peripheral venous blood of tumor patients is aseptically collected, subjected to density gradient centrifugation with Ficoll-Paque PLUS (GE, USA) lymphocyte separation medium, separated to obtain Peripheral Blood Mononuclear Cells (PBMC), and cultured with T cell culture medium.
The T cell culture medium comprises: X-VIVO15 serum-free medium (Lonza, USA) and IL2 (50U/ml; Peprotech, USA), IL-7(10 ng/ml; Peprotech, USA), IL-15(10ng/ml, Peprotech, USA), OKT3 antibody (50 ng/ml; ACRO, USA) and anti-CD28 antibody (1. mu.g/ml; T & L Biotechnology, China).
2. DON-treated PBMC differentiation events
The isolated PBMCs were divided into two groups:
the Control group (Control) was cultured in T cell medium for 7 days;
DON-treated groups were cultured for 7 days in T cell medium containing DON (2.5. mu.M) (Sigma-Aldrich, USA).
The invention dyes two groups of PBMCs through the flow antibody and carries out data analysis, and the data writing result shows the T of PBMCs of a DON treatment groupN(CD45RA+CCR7+) And TCM(CD45RA-CCR7+) The proportion of subpopulations was significantly higher (a and B in fig. 1), i.e. the proportion of poorly differentiated T cells was significantly increased.
Example 2
This example demonstrates the effect of DON on CAR-T cells, with the following specific protocol:
1. construction of CD 19-Targeted lentiviral vectors for CAR-T cells
(1) The reference patent (US 10,633,442B2) synthesizes a CAR nucleotide sequence targeting CD19, adds XbaI and NotI enzyme cutting sites at two ends of the nucleotide sequence respectively, and clones the CAR nucleotide sequence into a pUC57 vector;
(2) using XbaI and NotI to double-enzyme cut the pUC57 vector containing the target gene, cutting the gel and recovering the target gene fragment;
(3) the original vector pCDH-EF1-MCS-T2A-Puro is subjected to double enzyme digestion by XbaI and NotI, and a vector fragment of about 6.5kb is recovered by cutting gel;
(4) and connecting the recovered target gene fragment and the recovered vector fragment by using DNA ligase to obtain the recombinant lentiviral vector carrying the CD19 CAR.
2. Preparation of CD19 CAR lentivirus
Transfecting 293FT cells with the recombinant lentiviral vector obtained in the step 1 by using a transfection reagent (PEI) to generate lentivirus. The specific method comprises the following steps:
the plasmid mixture (pMDL: VSV-G: REV ═ 5:3:2, mass ratio) and CD19 CAR lentiviral vector were added to 500. mu.l of serum-free medium Opti-MEM at a mass ratio of 1:1, vortexed and mixed well. 32g PEI was added to 500. mu.l serum free medium Opti-MEM and vortexed to mix well. Then 500. mu.l of the plasmid mixture was mixed with 500. mu.l of PEI, and added to 293FT cells having a confluency of about 90%, and after 48 hours, a virus supernatant was collected, and after ultracentrifugation, the virus was concentrated 100-fold to obtain a concentrated virus.
3. Preparation of CD19 CAR-T cells
Peripheral blood T cells were isolated, cultured in T cell media, and activated T cells were infected with the virus after concentration, MOI 10:1 (antibodies to OKT3 and CD28 in the T cell media components activated T cells). At the same time of infection, the infection is divided into two groups:
DON (2.5. mu.M) was added to the T cell medium in the DON-treated group (dCAR-T);
the non-DON treated group (CAR-T) was not added with DON.
The CD19 CAR expression is detected by using FITC-conjugated CD19-Fc fusion protein, and the result shows that: 72 hours after infection, the dCAR-T group CD19 CAR infection efficiency was significantly higher than the CAR-T group (A in FIG. 2). Also, T of dCAR-T groupN(CD45RA+CCR7+) And TCM(CD45RA-CCR7+) The proportion of the subpopulations was significantly increased (B and C in fig. 2).
4. CD19 CAR-T cell in vitro and in vivo functional identification
The dCAR-T group and CAR-T group cells in step 3 were separately contacted with 5x104The supernatant IFN-. gamma.concentration was measured after 24 hours co-incubation of individual Burkitt's lymphoma cells (Raji cells) at an effective target ratio of 2: 1. The results show that DON-treated CAR-T cells secrete IFN- γ higher than the untreated group (FIG. 3).
And (3) incubating the dCAR-T group cells and the CAR-T group cells in the step 3 with CFSE pre-labeled Raji cells at an effective target ratio of 1:20,1:10,1:5,1:2,1:1,2:1 respectively, and performing flow identification on the cell death ratio (PI + target cell percentage) in the CFSE-labeled target cells. The results show that upon decreasing the effective target ratio, the DON-treated CAR-T cells specifically recognized and killed the target cell function significantly higher than CAR-T cells without DON treatment (figure 4).
Cells of dCAR-T group and CAR-T group in step 3 (1X 10)6) The lymphoma mouse model constructed by the Raji-Luciferase cell strain is transfused back, and the result shows that the DON-treated CAR-T cells have remarkably stronger capability of killing corresponding tumor cells in vivo than the DON-untreated CAR-T cells (figure 5). The tumor recurrence was simulated by injecting Raji-Luciferase cells into mice with complete tumor regression, and the results showed that the tumor recurrence resistance of CAR-T cells treated with DON was significantly stronger than that of CAR-T cells not treated with DON (fig. 6), i.e., CAR-T cells treated with DON had stronger expansion ability and better persistence in vivo.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.
Claims (10)
1. A method of increasing the anti-tumor effect of a CAR-T cell, comprising:
CAR-T cells were treated with diazooxypnorleucine.
2. The method according to claim 1, wherein the concentration of diazooxyphenylleucine is 1 to 10. mu. mol/L.
3. The method according to claim 1, wherein the treatment of the CAR-T cells with diazoxynorleucine is:
1-10 mu mol/L diazooxypnorleucine is added into a culture medium in the process of preparing CAR-T cells.
4. A method according to claim 3, characterized in that the method comprises:
infecting a T cell in a T cell culture medium with a lentivirus comprising a CAR nucleotide sequence;
the T cell culture medium contains 2.5-5 mu mol/L diazo-oxo-norleucine.
5. The method of claim 4, wherein the lentivirus has an MOI of (5-15): 1.
6. the method of claim 4 or 5, wherein the T cell culture medium further comprises OKT3 and CD28 antibodies.
7. Use of diazooxyphenylleucine to promote differentiation of T cells into poorly differentiated T cells.
8. The use according to claim 4, wherein said poorly differentiated T cells comprise: naive T cells and/or central memory T cells.
9. Application of diazo-oxo-norleucine in improving CAR-T cell preparation efficiency.
10. The use of diazooxyphenylleucine for the preparation of a medicament for increasing the tumor killing efficiency of CAR-T cells.
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| ROBERT D. LEONE ET AL: "Glutamine blockade induces divergent metabolic programs to overcome tumor immune evasion", 《SCIENCE》, pages 1013 * |
| 金玉坤;陆伟;罗雁;: "不平衡氨基酸的研究进展", 医学综述, no. 11, pages 1647 - 1649 * |
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