Method for jointly preparing CAR-VGamma9VDelta2T cells and CAR-NKT cells

A NKT cell and joint preparation technology, which is applied in the field of joint preparation of CAR-Vγ9Vδ2T cells and CAR-NKT cells, can solve cytokine storm and other problems

Active Publication Date: 2017-02-15
BEIJING DOINGTIMES INST OF TRANSLATIONAL MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In order to overcome the defects in the prior art that CAR-αβT cells need to be customized, pre-purified to obtain αβT cell subsets in the preparation process, and severe cytokine storm occurs after CAR-αβT is reinfused into the body, the present invention provides a new The method for jointly preparing CAR-Vγ9Vδ2T cells and CAR-NKT cells, which uses universal immune cells γδT as effector cells to be transduced, and can also be “standardized” and “batched” using γδT from healthy donors "Preparation; in addition, this method uses in vitro specific activation technology to maximize the advantage of Vγ9Vδ2T cells and NKT cells in peripheral blood PBMCs to be transduced, and does not require pre-purification to obtain specific T cell subsets, saving time and effort The cost is reduced; and the time of CAR-Vγ9Vδ2T cells and CAR-NKT cells jointly prepared by this method can be controlled by drugs, and will not last for a long time like other CAR-Ts, so the mild effect will not cause severe lethal cytokine storm

Method used

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  • Method for jointly preparing CAR-VGamma9VDelta2T cells and CAR-NKT cells
  • Method for jointly preparing CAR-VGamma9VDelta2T cells and CAR-NKT cells
  • Method for jointly preparing CAR-VGamma9VDelta2T cells and CAR-NKT cells

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Embodiment 1

[0045] In the ultra-clean workbench of the GMP laboratory, PBMCs were isolated from 80ml of peripheral blood of healthy donors with human lymphocyte separation medium Ficoll paque plus (GE) and counted. A small amount of samples were taken for immunophenotypic analysis before PBMC culture, see figure 1After suspending the separated PBMCs, they were added to the serum-free cell culture medium AIM-V (GIBCO, USA) containing 10% plasma (plasma and peripheral blood were from the same healthy donor). Serum cell culture medium adjusted to 2 x 10 6 Each / ml, then add zoledronic acid for injection (Novartis, Switzerland) to make its concentration in the serum-free cell culture medium 2.5 μ M, and pre-activate and cultivate Vγ9Vδ2T / NKT in an incubator with 5% carbon dioxide at 37°C; After 24 hours of preactivation, add rhIL-2 to make the concentration in the serum-free cell culture medium 1000U / ml, and continue to selectively activate and expand Vγ9Vδ2T / NKT cells in vitro in an incubator...

Embodiment 2

[0050] In the ultra-clean workbench of the GMP laboratory, human lymphocyte separation medium Ficoll paque plus (GE) was used to separate and count PBMCs from 80ml of peripheral blood of healthy donors. A small amount of samples were taken for immunophenotypic analysis before PBMC culture, see figure 2 After the isolated PBMCs were suspended, they were added to the serum-free cell culture medium AIM-V (GIBCO, USA) containing 10% plasma (plasma and peripheral blood came from the same healthy donor), and the cell concentration was adjusted with the above-mentioned plasma-free medium. Serum cell culture medium adjusted to 2 x 10 6 pc / ml, add Pamidronate Disodium for Injection (Shenzhen Xinlitai Pharmaceutical Co., Ltd.) to make its concentration in serum-free cell culture medium 10 μM, and pre-activate culture in an incubator at 37°C and 5% carbon dioxide Vγ9Vδ2T / NKT cells; add rhIL-2 after 24 hours of preactivation so that the concentration in the serum-free cell culture medium...

Embodiment 3

[0054] In the ultra-clean workbench of the GMP laboratory, PBMCs were isolated and counted from 80ml of infant umbilical cord blood using Ficoll paque premium (GE), a human lymphocyte separation medium, and a small amount of samples were taken for immunophenotypic analysis before PBMCs culture, see image 3 After suspending the isolated PBMCs, add them to the serum-free cell culture medium AIM-V (GIBCO, USA) containing 10% plasma (plasma and peripheral blood come from the same infant umbilical cord blood). Serum cell culture medium adjusted to 2 x 10 6 cells / ml, and then transferred to a culture flask coated with 1 μg / ml anti-γδTCR antibody (Immunotech, USA), pre-activated and cultured Vγ9Vδ2T / NKT in an incubator with 5% carbon dioxide at 37°C; rhIL- 2 Make the concentration of 1000U / ml in the serum-free cell culture medium, selectively activate and expand Vγ9Vδ2T / NKT cells in vitro in a 5% carbon dioxide incubator at 37°C, and supplement the above-mentioned fresh culture once...

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Abstract

The invention discloses a method for jointly preparing CAR-VGamma9VDelta2T cells and CAR-NKT cells, comprising the steps of 1) adding peripheral blood mononuclear cells to a cell medium, pre-activating VGamma9VDelta2T cells and NKT cells therein, and amplifying the VGamma9VDelta2T cells and NKT cells by selective in-vitro activation; 2) transducing the mixed cells of step 1) by using a lentiviral vector carrying chimeric antigen receptor's gene sequence. It is possible to carry out standard and batch preparation herein by using GammaDeltaT of a healthy donor; the purities of the VGamma9VDelta2T cells and NKT cells in the application can reach 60% and 30% and above respectively without purification, and therefore pre-purification can be omitted; in-vivo activation proliferation level and existence time of CAR-VGamma9VDelta2T cells and CAR-NKT cells jointly prepared by using the method can be controlled through clinical medication.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for jointly preparing CAR-Vγ9Vδ2T cells and CAR-NKT cells. Background technique [0002] Leukemia is the most important hematological malignancy that seriously endangers human health. Chemotherapy is currently the main method for treating leukemia. However, chemotherapy lacks specificity and has serious side effects. Most patients eventually relapse and die. Hematopoietic stem cell transplantation is also an important treatment for leukemia. However, tumor recurrence after transplantation and graft-versus-host disease are still two difficult problems to overcome. [0003] CAR-T therapy has shown good targeting, lethality and durability in clinical trials of acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (B-CLL) and B-cell lymphoma. Immune cell therapy for B-cell leukemia and lymphoma provides new solutions and shows great development potential and applic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N15/867C12N5/10
CPCC12N5/0636C12N5/0646C12N15/86C12N2500/42C12N2501/2302C12N2501/2315C12N2501/515C12N2502/00C12N2740/15041
Inventor 盖丽云李香群李刚毅
Owner BEIJING DOINGTIMES INST OF TRANSLATIONAL MEDICINE
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