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A kind of chimeric antigen receptor for the treatment of liver cancer and its application

A chimeric antigen receptor and carrier technology, which is applied in liver cancer vaccines, antibody mimics/scaffolds, and introduction of foreign genetic material using carriers, which can solve the problems of increasing CAR-T cells, toxic side effects, and insufficient sustainable viability.

Active Publication Date: 2021-05-14
CARBIOGENE THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing CAR-Ts generally show the defect of insufficient survival ability in vivo
Blindly increasing the dose of CAR-T cells can easily cause strong toxic side effects, such as inflammatory factor storm and central nervous system toxicity, which in turn reduces the safety of CAR-T therapy

Method used

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  • A kind of chimeric antigen receptor for the treatment of liver cancer and its application
  • A kind of chimeric antigen receptor for the treatment of liver cancer and its application
  • A kind of chimeric antigen receptor for the treatment of liver cancer and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Example 1. Preparation of CAR-T cells

[0155] 1. Construction of retroviral vector

[0156] 1. Optimization of the full-length cDNA sequence of wild-type human IFNα2b gene

[0157] The full-length sequence of wild-type human IFNα2b gene cDNA is called nIFNα2b. In order to make nIFNα2b more suitable for expression in human cells, under the condition that the amino acid sequence encoded by nIFNα2b remains unchanged, the codon optimization of nIFNα2b sequence was carried out on the website http: / / sg.idtdna.com / site to obtain oIFNα2b, The nucleotide sequence of oIFNα2b is shown in 2701-3264 of SEQ ID NO.1.

[0158] 2. Design and synthesis of GPC3-CAR-IFN gene sequence

[0159] The GPC3-CAR-IFN gene sequence sequentially includes the coding gene sequence of human CD8 leader peptide, the coding gene sequence of GPC3 scFv, the coding gene sequence of human CD8 hinge transmembrane region, the coding gene sequence of human 4-1BB intracellular region, the coding gene sequence...

Embodiment 2

[0192] Example 2, CFSE labeling method to detect the specific killing effect of CAR-T cells on tumor cells

[0193] CFSE (CFDA-SE) is a cell staining reagent that can fluorescently label living cells. It can easily penetrate the cell membrane, covalently bind to intracellular proteins in living cells, and release green fluorescence after hydrolysis. The principle of CFSE labeling living cells can be used to label and quantify tumor cells, so as to detect the killing efficiency of CAR-T cells on tumor target cells. The specific method is: the target cells are equally divided into two groups and adjusted to the same cell density. Stained with low concentration and high concentration of CFSE respectively, wherein high concentration stained target cells and non-stained immune cells were co-cultured according to a certain ratio. After a period of incubation, the high-concentration stained tube of target cells (along with immune cells) was mixed in equal volume with the low-concent...

Embodiment 3

[0207] Example 3. Tumor transplantation model to detect the tumor killing effect of CAR-T cells in animals

[0208] Experimental materials: B-NDG severe combined immunodeficiency mice aged 5-6 weeks and weighing 18-22 g (Biocytogen Biotechnology Co., Ltd.).

[0209] Experimental grouping: The experimental materials were randomly divided into 3 experimental groups, with 5 mice in each group. The specific treatment methods for each group are as follows:

[0210] GPC3 CAR-IFN T group: B-NDG severe combined immunodeficiency mice were subcutaneously inoculated with HepG2 tumor cell solution (solvent is PBS), the inoculation volume was 0.3ml (containing 1×10 7 HepG2 cells). Five days after tumor cell inoculation, the GPC3 CAR-IFN T cell solution (solvent is PBS) prepared in Example 1 was injected into the mouse tail vein, and the injection volume was 0.2ml (containing 5×10 6 GPC3 CAR-IFN T cells).

[0211] GPC3 CAR T group: B-NDG severe combined immunodeficiency mice were subcut...

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Abstract

The invention discloses a chimeric antigen receptor for treating liver cancer and its application. The chimeric antigen receptor comprises a leader peptide, anti-GPC3 single-chain antibody, human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3ζ intracellular region, self-cleaving peptide, signal peptide, EGFRt protein, Cleavage peptide and full-length human IFN. The present invention uses GPC3 as an antigen target to design and develop a new generation of CAR-T cell therapy products, and adds a gene-optimized human full-length interferon (IFN) fragment to the C-terminus of GPC3-CAR. Compared with CAR-T cells expressing only GPC3-CAR, CAR-T cells expressing GPC3-CAR-IFN have stronger tumor killing ability, which further improves the safety and effectiveness of CAR-T cells in treating tumors.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a chimeric antigen receptor for liver cancer treatment and its application, in particular to a chimeric antigen receptor containing GPC3-CAR and secreting cytokines and its application. Background technique [0002] Liver cancer (HCC) is one of several malignant tumors with the highest morbidity and mortality worldwide, ranking fifth in incidence and third in mortality. At present, the main treatment for liver cancer is still surgical resection. However, the postoperative recurrence rate of liver cancer is extremely high, and the 5-year survival rate is only about 10%. Targeted therapy drugs (such as sorafenib) can only prolong the average survival time of patients by 2 to 3 months. In fact, there is still no more effective treatment for liver cancer. [0003] Due to its small side effects and obvious therapeutic effect, tumor immunotherapy is gradually becoming the dev...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00A61P1/16
CPCA61K2039/5156A61P1/16A61P35/00A61K39/001102A61K39/001104A61K39/001141A61K2039/844C07K14/555C07K14/56C07K14/565C07K14/7051C07K14/70517C07K14/70578C07K14/71C07K16/303C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 朱建高杨文君
Owner CARBIOGENE THERAPEUTICS CO LTD
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