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Method for jointly preparing CAR-NK (chimeric antigen receptor-natural killer) cells and CAR-NKT (natural killer T) cells

A technology of NKT cells and NK cells, which is applied in the field of joint preparation of CAR-NK cells and CAR-NKT cells, can solve the problems of immune rejection, low transduction efficiency of allogeneic CAR-T, and low ratio, so as to save purification costs Effect

Active Publication Date: 2017-01-25
BEIJING DOINGTIMES INST OF TRANSLATIONAL MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the transduction efficiency of allogeneic CAR-T in the current technology is not very high, and there is still a great risk of immune rejection when using allogeneic CAR-T
At the same time, the T cells from PBMCs used to prepare CAR-T contain different T cell subsets, and the proportion of CD8+ T cells actually used to prepare CAR-T is often very low, which seriously affects the efficacy of CAR-T
In addition, there are often extremely severe fatal cytokine storms in the clinical application of CAR-T, so it is even more important to seek a safer and more reliable CAR technology that can replace CAR-T

Method used

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  • Method for jointly preparing CAR-NK (chimeric antigen receptor-natural killer) cells and CAR-NKT (natural killer T) cells
  • Method for jointly preparing CAR-NK (chimeric antigen receptor-natural killer) cells and CAR-NKT (natural killer T) cells
  • Method for jointly preparing CAR-NK (chimeric antigen receptor-natural killer) cells and CAR-NKT (natural killer T) cells

Examples

Experimental program
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Effect test

Embodiment 1

[0046] PBMCs were isolated and counted from 80ml of peripheral blood of healthy donors using human lymphocyte separation medium Ficoll paque plus (GE) in the ultra-clean workbench of the GMP laboratory, and a small amount of samples were taken for immunophenotypic analysis before PBMC culture, see figure 1 Add the PBMCs to the serum-free cell culture medium AIM-V (GIBCO, U.S.) containing 10% plasma (plasma and peripheral blood come from the same healthy donor) after being suspended, and use the above-mentioned serum-free cells containing plasma to obtain the cell concentration. Medium adjusted to 2×10 6 cells / ml, and then all transferred to culture flasks coated with 20 μg / ml anti-CD16 monoclonal antibody, preactivated NK cells and NKT cells in an incubator at 37°C and 5% carbon dioxide, and added after 24 hours of preactivation Add IL-2 to make the concentration in the serum-free cell culture medium 1000U / ml, and add IL-15 to make the concentration in the serum-free cell cult...

Embodiment 2

[0050] In the ultra-clean workbench of the GMP laboratory, human lymphocyte separation medium Ficoll paque premium (GE) was used to separate and count PBMCs from 80ml of peripheral blood of healthy donors. A small amount of samples were taken for immunophenotyping analysis before PBMC culture, see figure 2 Add the PBMCs to the serum-free cell culture medium AIM-V (GIBCO, U.S.) containing 10% plasma (plasma and peripheral blood come from the same healthy donor) after being suspended, and use the above-mentioned serum-free cells containing plasma to obtain the cell concentration. Medium adjusted to 2 × 10 6 cells / ml, and then all transferred to a culture flask co-coated with 10ng / ml anti-CD3 monoclonal antibody and 10μg / ml anti-CD52 monoclonal antibody, and pre-activated in an incubator at 37°C and 5% carbon dioxide NK cells and NKT cells, after preactivation for 24h, add IL-2 to make the concentration in the serum-free cell culture medium 1000U / ml and add IL-15 to make the con...

Embodiment 3

[0054] In the ultra-clean workbench of the GMP laboratory, human lymphocyte separation medium Ficoll paque premium (GE) was used to separate and count PBMCs from 80ml of peripheral blood of healthy donors. A small amount of samples were taken for immunophenotyping analysis before PBMC culture, see image 3 After PBMC is suspended, add in the serum-free cell culture medium AIM-V (U.S. GIBCO company) that contains 10% plasma (plasma and peripheral blood come from the same patient), the cell concentration is replaced with the above-mentioned serum-free cell culture medium that contains plasma Adjust to 2×10 6 cells / ml, and then all transferred to the culture bottle, and then add the trophoblast cell K562-4-1BB-Mil-21 (purchased from Youkang Bio) to the culture bottle to make the final concentration in the serum-free cell culture medium 1× 10 5 cells / ml, preactivate NK cells and NKT cells in an incubator at 37°C and 5% carbon dioxide, add IL-2 after 24 hours of preactivation to m...

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Abstract

The invention discloses a method for jointly preparing CAR-NK (chimeric antigen receptor-natural killer) cells and CAR-NKT (natural killer T) cells. The method includes steps of 1), adding peripheral blood mononuclear cells into cell culture media, pre-activating NK cells and NKT cells in the peripheral blood mononuclear cells and selectively activating and amplifying the NK cells and the NKT cells in an in-vitro manner; 2), transducing mixed cells obtained at the step 1) by the aid of lentiviral vectors with gene sequences of chimeric antigen receptors. The method has the advantages that the CAR-NK cells and the CAR-NKT cells can be prepared from NKs and NKTs of healthy donors in a standardized and batched manner; the purity of the NK cells and the NKT cells can respectively reach 60% and 30% at least without purification, and accordingly preliminary purification can be omitted; the in-vivo activation and amplification degrees and the in-vivo existence time of the CAR-NK cells and the CAR-NKT cells which are jointly prepared by the aid of the method can be controlled by means of clinical medication.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for jointly preparing CAR-NK cells and CAR-NKT cells. Background technique [0002] B-cell malignancies include various types of heterogeneous tumors, such as non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia (B-CLL). Treatment of B-cell malignancies includes chemotherapy, radiotherapy, bone marrow transplantation, and peripheral blood stem cell transplantation, but most patients are still incurable. [0003] The full name of CAR-T technology is Chimeric Antigen Receptor T cell immunotherapy, where CAR stands for Chimeric Antigen Receptor, that is, Chimeric Antigen Receptor. This technology uses genetically modified T cells with chimeric antigen receptors to resist tumor cells, which combines the high affinity of antigen receptors for tumor antigens with the killing mechanism of T cells, and can be carried by carrying specif...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N15/867
CPCC12N5/0646C12N15/86C12N2501/2302C12N2501/2315C12N2502/1157C12N2740/15041
Inventor 盖丽云李香群李刚毅
Owner BEIJING DOINGTIMES INST OF TRANSLATIONAL MEDICINE
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