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Chimeric antigen receptor T cell targeted to WT1 and application of chimeric antigen receptor T cell

A chimeric antigen receptor and targeting technology, applied in the field of genetic engineering, can solve the problems of lack of immunogenicity, high specificity, and immune escape, and achieve the effect of high tumor killing activity and simple structure

Inactive Publication Date: 2018-10-19
段海峰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many defects in adoptive immunotherapy of pure T cells: the lack of highly immunogenic and specific antigens, the existence of immune escape, the heterogeneity of tumors, and the immunosuppression of tumor microenvironment
Gaidzik et al. detected 617 AML patients, of which 78 cases (12.6%) were identified to have WT1 mutations, and those with WT1 mutations were often younger, had higher levels of lactate dehydrogenase, higher white blood cell counts, and lower complete remission rates. More likely to relapse, poor prognosis

Method used

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  • Chimeric antigen receptor T cell targeted to WT1 and application of chimeric antigen receptor T cell
  • Chimeric antigen receptor T cell targeted to WT1 and application of chimeric antigen receptor T cell
  • Chimeric antigen receptor T cell targeted to WT1 and application of chimeric antigen receptor T cell

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of chimeric antigen receptor T cells targeting WT1

[0038] 1. Construction of lentiviral expression vector

[0039] Containing the nucleic acid fragment (BamHI-Sp-EcoRI-NheI-CD28-CD3zeta-SalI, synthesized by Zhongmei Taihe Biotechnology (Beijing) Co., Ltd.) encoding CD28-CD3ζ, as shown in SEQ ID NO.6, named as pGSI -seq8. 3ug pGSI-seq8 and recombinant lentiviral vector plasmid pCDH-EF1 (Addgene) were double-enzyme-digested with BamHI and SalI restriction endonucleases respectively, and the digested products were recovered from the gel and ligated with T4 DNA ligase, ligated overnight at 4°C, and transformed For DH5α competent cells, take 100 μL of the bacterial solution and spread it on an LB plate containing ampicillin resistance, and culture it overnight at 37°C. Single clones were picked for colony PCR, positive clones were sent for sequencing, and the clones with correct sequencing results were saved and plasmids were extracted, named pCDH-E...

Embodiment 2

[0053] Example 2 In vitro anti-tumor effect of targeting WT1 chimeric antigen receptor T cells

[0054] Using the cell line K562 cells (human chronic myelogenous leukemia cell line) and Raji cells (human Burkitt's lymphoma cells) expressing WT1 in the lymphatic system as target cells, the WT1-targeting chimeric antigen receptor T cells prepared in Example 1 were used respectively Effector cells were prepared from T cells not infected with lentivirus (Anti-WT1 CAR), and the target cells were inoculated in a 96-well plate at a density of 10,000 cells / ml, 100 μl per well, according to 1:1, 5:1, and 10:1 effects. Target ratio Add effector cells to target cells, add CCK8 (Biyuntian) reagent at the same time, place in 5% CO 2 , 37 ° C incubator continuous culture for 4 hours, during which the readings were made at 450nm wavelength every 1 hour, and the killing efficiency was calculated. The result is as Figure 2-6 As shown, the results indicated that chimeric antigen receptor T c...

Embodiment 3

[0055] Example 3 In vivo anti-tumor effect of targeting WT1 chimeric antigen receptor T cells

[0056] Get 30 female NOD SCID mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) with a body weight of 25-30g at the age of 6 weeks, and inject 5*10 6 K562 cells, with a total volume of 0.1ml, 3 days after inoculation with tumor cells, mice were randomly divided into 3 groups according to body weight: control group, normal T cells (uninfected with Anti-WT1 CAR lentivirus) group and chimeric antigen receptor targeting WT1 T cell (prepared in Example 1) group; control group tail vein injection of normal saline 200ul / time, once a week, 2 times in total; normal T cell group tail vein injection of T cells 1*10 7 One per time, once a week, 2 times in total; In the group targeting WT1 chimeric antigen receptor T cells, inject anti-WT1 chimeric antigen receptor T cells 1*10 into the tail vein 7 Each time, once a week, 2 times in total; the survival status ...

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Abstract

The invention discloses a chimeric antigen receptor T cell targeted to WT1 and application of the chimeric antigen receptor T cell, and belongs to the technical field of genetic engineering. The chimeric antigen receptor T cell comprises an antigen binding area ScFv and a signal transduction area CD28-CD zeta, connected in series in sequence; and the amino acid sequence of the chimeric antigen receptor T cell is as shown in SEQ ID NO. 1. The chimeric antigen receptor T cell targeted to WT1 has high tumor-killing activity on a cancer cell for expressing TEM8, especially a cancer for expressingWT1, such as chronic myeloid leukemia and lymphoma for expressing WT1.

Description

technical field [0001] The invention relates to chimeric antigen receptor T cells targeting WT1 and applications thereof, belonging to the technical field of genetic engineering. Background technique [0002] Traditional tumor treatment methods such as surgery, radiotherapy and chemotherapy have greatly improved the survival period and quality of life of patients with malignant tumors, but there are many problems such as large toxic and side effects, and difficulty in further improving the treatment effect. In recent years, the development in the field of cell adoptive therapy has also achieved remarkable results. In the field of ACT therapy, the biggest research hotspot is the adoptive therapy of genetically modified antigen-specific T cells. There are many defects in adoptive immunotherapy of pure T cells: the lack of highly immunogenic and specific antigens, the existence of immune escape, the heterogeneity of tumors, and the immunosuppression of tumor microenvironment. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K35/17A61P35/00C12Q1/02
CPCA61K35/17A61P35/00C07K14/7051C07K16/32C12N5/0636C12N2510/00G01N33/5005
Inventor 薛冰华解晶胡显文于婷婷
Owner 段海峰
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