Method for preparing chimeric antigen receptor T cells through serum-free culture

A chimeric antigen receptor and serum-free medium technology, applied in the biological field, can solve problems such as large differences in serum quality, introduction of mycoplasma, viruses, and hindering the development of cell therapy

Active Publication Date: 2019-11-26
SHANGHAI CELLULAR BIOPHARMACEUTICAL GROUP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there is a large difference in quality between batches of serum
In addition, risks such as mycoplasma and viruses may be introduced during the process of serum sampling, which seriously hinder the development of cell therapy
[0005] At present, there is no satisfactory and efficient serum-free preparation of chimeric antigen receptor T cells in the market, which seriously affects the further development, promotion and application of chimeric antigen receptor T cell immunotherapy

Method used

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  • Method for preparing chimeric antigen receptor T cells through serum-free culture
  • Method for preparing chimeric antigen receptor T cells through serum-free culture
  • Method for preparing chimeric antigen receptor T cells through serum-free culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Media screening

[0070] 1.1 Frozen or fresh PBMC

[0071] Take frozen or fresh 300×10 6 -1000×10 6 Cells of PBMC were resuspended in serum-free medium.

[0072] 1.2 Sorting

[0073] PBMC were labeled with CD19 and CD14 magnetic beads and incubated for 30 minutes. Install the pipelines required for sorting on the CliniMACS as required, and perform negative sorting operation after the cell incubation to remove CD19 and CD14-labeled impurity cells, and obtain sorted cells with CD3-positive cells as the main component

[0074] 1.3 Cell activation

[0075] The sorted cells were activated with CD3 / CD28, and then 3×10 6 -6×10 6 Cell density per ml was inoculated with serum-free medium (supplemented with IL-2) containing a final concentration of 1.0% albumin, and cultured for 2 days.

[0076] 1.4 Gene transduction

[0077] After the cells were centrifuged to discard the supernatant, add the required volume of lentivirus according to MOI 2-10, resuspend in serum-free m...

Embodiment 2

[0087] Experimental Consumables Screening

[0088] 2.1 PBMC recovery, sorting, cell activation, gene transduction

[0089] The same method as in Example 1 was used to perform cryopreserved PBMC recovery, sorting, cell activation, and gene transduction.

[0090] 2.2 Virus removal

[0091] Centrifuge the above cell suspension at 200-300 g for 6-8 minutes. Aspirate the supernatant, flick and resuspend the cells to obtain virus-depleted T cells

[0092] 2.3 Expansion culture

[0093] Transfer the virus-depleted T cells to the medium (OpTmizer SFM+1.0% albumin), add IL-2 (final concentration is 25IU / ml), and then transfer to G-Rex culture flask, culture bag and T75 culture respectively bottle, then placed at 37°C, 5% CO 2 Continue to cultivate.

[0094] After continuing to culture for 3 days, samples were taken and counted from the G-Rex culture flask, culture bag and T75 culture flask, and the cell density and cell viability were recorded. Reserve sample testing.

[0095] ...

Embodiment 3

[0101] Cell culture supplement experiment

[0102] 3.1 Method

[0103] In this embodiment, different additives IL-2, IL-7, IL-15 or their combinations were added to the cell culture medium, and 5 experimental groups were set up.

[0104] Table 1

[0105] Experimental group 1 Experimental group 2 Experimental group 3 Experimental group 4 Experimental group 5 IL-2 + - + + + IL-7 - + + + - IL-15 - + + - +

[0106] The same method as in Example 1 was used to perform cryopreserved PBMC recovery, sorting, magnetic bead labeling activation, gene transduction, magnetic bead removal, virus removal and expansion culture.

[0107] 1) The medium used is OpTmizer SFM+albumin (final concentration is 1.0%)

[0108] 2) All steps of adding IL-2 were replaced with cytokines corresponding to experimental groups 2-5 in Table 1, and cultured.

[0109] 3.2 Results

[0110] Such as Figure 4 Shown: The combination of cytokines IL-2, IL-7, a...

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Abstract

The invention provides a method for preparing chimeric antigen receptor T cells through serum-free culture. Specifically, the method comprises the steps that (a) peripheral blood mononuclear cells (PBMCs) are provided; (b) the PBMCs are subjected to negative sorting treatment to obtain sorted PBMCs; (c) the sorted PBMCs are activated to obtain activated T cells; (d) the activated T cells are subjected to gene transfection through a virus vector expressing a chimeric antigen receptor, and thus transfected T cells are obtained; (e) the transfected T cells are subjected to virus removal treatment, and thus T cells with viruses removed are obtained; and (f) the T cells with viruses removed are subjected to multiplication culture, and thus the chimeric antigen receptor T cells are obtained. Through the culture process, the culture success rate of the chimeric antigen receptor T cells is greatly increased, and the safety and effectiveness of the chimeric antigen receptor T cells are greatlyimproved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing chimeric antigen receptor T cells through serum-free culture. Background technique [0002] Chimeric antigen receptor T cell immunotherapy is a method of gene editing to enable T cells to have scFv fragments and intracellular signaling domains that can specifically recognize tumor-associated antigens, thereby enhancing the targeting, killing and persistence of T cells. New medical technology. In recent years, tumor immunotherapy has performed very well clinically, bringing hope to the clinical cure of tumors. [0003] The preparation process of chimeric antigen receptor T cells is relatively complicated, involving many operations such as cell activation, sorting, transfection, expansion and culture. Insufficiency in any link, such as process flow, equipment facilities, and reagent selection, will have an important impact on the quality of cell preparation, wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867
CPCC12N5/0636C12N15/86C07K14/7051C12N2510/00C12N2740/15043A61P37/02C12N2740/16043A61K39/0011A61P35/00A61K35/17C07K2319/03A61K48/00C12N2500/90C12N2501/2301C12N2501/2307C12N2501/2315C12N5/0031C12N2501/2302
Inventor 赵荻骏李超王飞吴军峰刘晓雨赵佳维
Owner SHANGHAI CELLULAR BIOPHARMACEUTICAL GROUP LTD
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