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CAR-T in treatment of hepatocellular carcinama by targeting HBV and application thereof

A targeting and chimeric antigen receptor technology, applied in the field of immunotherapy, can solve the problem of unavoidable off-target effects in multiple organs

Inactive Publication Date: 2019-09-17
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although GPC3 is highly expressed in more than 75% of HCC, it is not a tumor-specific antigen and is also expressed in normal tissues such as the lung, kidney, and female reproductive system, and potential multi-organ off-target effects are inevitable

Method used

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  • CAR-T in treatment of hepatocellular carcinama by targeting HBV and application thereof
  • CAR-T in treatment of hepatocellular carcinama by targeting HBV and application thereof
  • CAR-T in treatment of hepatocellular carcinama by targeting HBV and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of vector expressing CAR lentivirus and preparation of CAR-T cells

[0039] 1. Experimental method

[0040] (1) Gene synthesis is a DNA fragment composed of encoding CD8α signal peptide (SP), myc tag, CD8 hinge and CD8 transmembrane region (TM), 4-1BB and CD3ζ intracellular signal domain in series, and the upstream and downstream Add BamHI and SalI restriction sites, respectively, and insert the synthetic gene into the pRRL vector after double digestion to construct the plasmid pRRL-Control-CAR-M.

[0041] (2) Gene synthesis is composed of DNA fragments in series encoding CD8α signal peptide, myc tag, anti-HBVs scFv, CD8 hinge and CD8 transmembrane region, 4-1BB and CD3ζ intracellular signal domains, and the upstream and downstream are respectively added BamHI and SalI restriction sites, the synthetic gene was inserted into the pRRL vector after double digestion to construct the plasmid pRRL-HBVs-CAR-M.

[0042] (3) Gene synthesis is a DNA fragm...

Embodiment 2

[0051] Example 2 Phenotype detection of CAR-T cells

[0052] 1. Experimental method

[0053] (1) Each CAR-T cell after lentivirus infection was cultured for 10 days, and 1×10 6 Total cell protein was extracted from each cell. After the protein was separated by SDS-PAGE gel, it was transferred to the membrane, and the expression of each CAR was detected by CD3ζ chain antibody and tag Flag or myc antibody.

[0054] (2) After the CAR-T cells infected with lentivirus continued to be cultured for 10 days, 1×10 5 After being fixed with 4% paraformaldehyde, the cells were incubated with myc antibody and labeled with FITC-labeled secondary antibody. After centrifugation, the cells were transferred to glass slides, and then the cell localization of each CAR was observed by confocal laser microscopy.

[0055] (3) After each CAR-T cell was cultured for 10 days after lentivirus infection, 1×10 5 Cells were incubated with Flag or myc antibody and labeled with secondary antibody to dete...

Embodiment 3

[0058] Example 3 Detection of the in vitro killing ability of each CAR-T cell

[0059] 1. Experimental method

[0060] Subsequent experiments (including in vivo and in vitro) were carried out using the HBVs+GPC3+ cell line HepG2.2.15, the HBVs-GPC3+ cell line HepG2 and the HBVs-GPC3- cell line SK-HEP-1 to comprehensively evaluate each CAR-T cell for its target cell Assess the off-target effects on non-target cells, as follows:

[0061] (1) The proliferation activity of various CAR-T cells was detected by flow cytometry.

[0062] For each CAR-T cell labeled with CFSE, take 1×10 5 labeled cells with 1×10 5 The above three liver cancer cells were co-cultured, and the cells were collected on the 2nd, 4th, 6th, 8th, and 10th day, and the proliferation of each CAR-T cell was analyzed by flow cytometry.

[0063] (2) ELISA detection of cytokine expression of CAR-T cells

[0064] Take 1×10 4 CAR-T cells were mixed with 1×10 4 The above three liver cancer cells were co-cultured, ...

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Abstract

The invention relates to chimeric antigen receptor T cell (CAR-T) in the treatment of hepatocellular carcinama (HCC) by targeting hepatitis B virus (HBV) and an application thereof. In order to overcome a potential multiple organ off-target effect in the existing CAR-T treatment of HCC, the inventor, after repeated screening, prepares chimeric antigen receptor T cells targeting HBV surface antigen (HBVsAg). The results show that CAR-T prepared in the invention has a good killing effect on HBV positive HCC cells, and has a significantly higher killing effect than the CAR-T cells targeting GPC3 in current clinical trials and researches, showing a better application prospect.

Description

technical field [0001] The present invention relates to the field of immunotherapy, in particular to CAR-T targeting HBV for treating liver cancer and its application, more specifically to the preparation of CAR-T targeting HBV surface antigen (HBVsAg) and its application in treating liver cancer. Background technique [0002] Chimeric antigen receptor T cell (CAR-T) treatment of tumors is a milestone breakthrough in the field of tumor treatment in recent years. Among them, the CAR-T products CTL019 (Novartis) and KTE-C19 (Kite) for the treatment of leukemia and lymphoma have successively obtained the US breakthrough therapy certification, and the complete remission rate of patients in clinical trials has reached 70%-94%. The good therapeutic effect has prompted the US FDA to accelerate the drug approval process, and in 2017, the CD19CAR-T drugs of the above two companies were respectively approved for marketing. [0003] Hepatocellular carcinoma (HCC) is one of the most co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K39/00A61K38/21A61K31/513A61K31/675A61K31/7072A61K31/522A61P35/00A61P1/16A61P31/20
CPCA61K31/513A61K31/522A61K31/675A61K31/7072A61K38/21A61K39/0011A61K2039/5156A61K2039/5158A61K2039/844A61P1/16A61P31/20A61P35/00C07K14/7051C07K16/082C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043A61K2300/00
Inventor 刘泳陈全刚郑骏年张青许德勤
Owner XUZHOU MEDICAL UNIV
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