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Method for preparing CD19-targeted chimeric antigen receptor NK cells from human pluripotent stem cells and application of CD19-targeted chimeric antigen receptor NK cells

A technology of pluripotent stem cells and NK cells, applied in the field of chimeric antigen receptor NK cells, which can solve problems such as long cycle, long expansion time, and unclear medium components

Pending Publication Date: 2021-11-23
广州瑞臻再生医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are several key problems in the current NK cell therapy: 1) Insufficient source, the number of NK cells collected directly from the peripheral blood lymphocytes of healthy volunteers is small, the expansion ability is poor, and the expansion time is long, and it is generally difficult to reach the required level of treatment. Cell number; 2) Tumor patients have low self-immunity, and the activity and expansion ability of autologous NK cells obtained are worse than those of healthy volunteers. It is difficult to apply to large-scale treatment, and it is difficult to meet the needs of cancer patients
However, the reported differentiation process of human pluripotent stem cells into NK cells has problems such as complex process, long cycle, dependence on feeder cells, unclear medium composition, low purity and viability, and residual undifferentiated cells are likely to cause cancer risk

Method used

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  • Method for preparing CD19-targeted chimeric antigen receptor NK cells from human pluripotent stem cells and application of CD19-targeted chimeric antigen receptor NK cells
  • Method for preparing CD19-targeted chimeric antigen receptor NK cells from human pluripotent stem cells and application of CD19-targeted chimeric antigen receptor NK cells
  • Method for preparing CD19-targeted chimeric antigen receptor NK cells from human pluripotent stem cells and application of CD19-targeted chimeric antigen receptor NK cells

Examples

Experimental program
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Effect test

Embodiment 1CD1

[0120] Example 1 Preparation of CD19-CAR-NK cells

[0121] 1. The structure and sequence of CD19-CAR (NK-19CAR) gene

[0122] A complete CAR molecule mainly includes extracellular domain, transmembrane domain and intracellular domain. The extracellular domain is composed of a single-chain antibody (scFv) of a monoclonal antibody responsible for recognizing and binding to an antigen and a connecting hinge region (Hinge). The intracellular domain consists of a co-stimulatory domain and a signal transduction domain. The optimally designed CD19-CAR gene structure of the present invention is beneficial to promote the activation and proliferation of CD19-CAR-NK cells in the later stage, and enhance the lethality of CD19-CAR-NK cells to tumors.

[0123] The structural molecule of the CD19-CAR of the present invention (also referred to as NK-19CAR, 19CAR in this text and the accompanying drawings) consists of a signal peptide, an anti-CD19 heavy chain and a connecting region, an ant...

Embodiment 2

[0200] Example 2 In vitro killing ability of CD19-CAR-NK cells (cytotoxicity test)

[0201] The K562 cells and RAJI cells in this example are from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing.

[0202] The human leukemia cell line CD19-K562, which is more sensitive to NK cell killing, and the human leukemia cell line CD19-K562 overexpressing CD19 by lentivirus, and the lymphoma RAJI cells expressing CD19 were selected as target cells, and a luciferase-based reporter gene transfection method was used to evaluate the NK- The ability of 19CAR gene-modified NK cells to kill target cells. Transfect the target cells with the reporter gene—luciferase (luc) gene, and establish a stable transfected target cell line, so as to measure the cytotoxicity and apoptosis mediated by NK cells. By measuring the activity of the reporter enzyme (representing the number of dead target cells) released into the culture medium, the percentag...

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Abstract

The invention discloses a method for preparing CD19-targeted chimeric antigen receptor NK cells from human pluripotent stem cells and application of the CD19-targeted chimeric antigen receptor NK cells. Specifically, the invention discloses the method for preparing CD19-CAR-NK cells, which comprises the following steps: directionally knocking a CD19-CAR gene and an EF1 alpha promoter into a human pluripotent stem cell genome AAVS1 region by utilizing a CRISPR / CAS9 system of a targeted AAVS1 site to obtain recombinant human pluripotent stem cells (CD19-CAR-hPSC), then inducing and culturing the CD19-CAR-hPSC to differentiate into embryoid bodies, sorting CD34+ hematopoietic precursor cells from hematopoietic precursor cells differentiated from the embryoid bodies, then directionally differentiating to the NK cells, and finally differentiating into the CD19-CAR-NK cells. The CD19-CAR-NK cells disclosed by the invention has higher multiplication capacity, higher purity and stronger anti-tumor capacity, and can be safely and effectively used for tumor immunotherapy.

Description

technical field [0001] The invention belongs to the field of cellular immunotherapy, and specifically relates to a method and application of human pluripotent stem cells for preparing chimeric antigen receptor NK cells targeting CD19. Background technique [0002] Chimeric antigen receptor (CAR) technology uses genetic engineering technology to modify immune cells to express exogenous anti-tumor genes (CAR genes), so that immune cells such as lymphocytes have the ability to recognize tumor cell surface antigens , and specifically recognize and kill tumor cells without the restriction of histocompatibility complex (MHC). The structure of CAR is mainly composed of extracellular domains that recognize cell surface antigens and intracellular signal transduction domains. The domain is used to specifically recognize the specific protein (antigen) on the surface of the tumor, and the intracellular domain contains the costimulatory molecular domain, which is used to initiate the imm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/62C12N15/867C07K19/00A61K39/00A61P35/00A61P35/02
CPCC07K16/2803C07K14/7051C12N5/0646C12N15/86A61K39/0011A61P35/00A61P35/02C07K2319/02C07K2319/03C07K2319/33C07K2317/622C12N2510/00C12N2506/45C12N2740/15043A61K2039/804
Inventor 李扬周士新
Owner 广州瑞臻再生医学科技有限公司
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