Vector containing double-target chimeric antigen receptor gene, CAR-T cell and application of CAR-T cell

A chimeric antigen receptor and dual-target technology, which is applied in the field of tumor cell immunotherapy, can solve the problem of less gastric cancer and achieve the effects of strong proliferation, reduced economic pressure, and long-lasting vitality

Pending Publication Date: 2020-08-21
WUHAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are few studies on CAR-T cell therapy for gastric cancer,

Method used

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  • Vector containing double-target chimeric antigen receptor gene, CAR-T cell and application of CAR-T cell
  • Vector containing double-target chimeric antigen receptor gene, CAR-T cell and application of CAR-T cell
  • Vector containing double-target chimeric antigen receptor gene, CAR-T cell and application of CAR-T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Dual-target chimeric antigen receptor and its coding gene for treating gastric cancer

[0027] This example provides a dual-target chimeric antigen receptor for the treatment of gastric cancer. The specific splicing method is as follows: sequentially splicing signal peptides from the N-terminal to the C-terminal, and can respectively recognize CD44 and EpCAM single chains on the surface of gastric cancer stem cells Antibody ScFv, strepII, CD8hinge, leukocyte antigen differentiation group molecule transmembrane region CD28-TM+ICD, 4-1BB, ζ chain of CD3 (leukocyte antigen differentiation group molecule 3), F2A peptide, IL-7, F2A peptide and CCL19, Finally, a complete chimeric antigen receptor (CAR) molecule capable of treating gastric cancer was obtained. Each of the above structural fragments can perform the following functions: signal peptide can secrete CAR extracellularly; CD44 single-chain antibody ScFv specifically recognizes CD44 protein on the surface o...

Embodiment 2

[0029] Example 2. Construction of a dual-target chimeric antigen receptor recombinant expression vector for the treatment of gastric cancer

[0030] This embodiment provides a dual-target chimeric antigen receptor recombinant expression vector and its construction method. The construction method of the vector specifically includes the following steps:

[0031] S1. Construction of recombinant expression plasmid pTK-881-CD44-EpCAM

[0032] (1) Preparation of experimental materials

[0033] ①Preparation method of LB liquid culture medium: Weigh 5g liquid medium dry powder into a 500mL conical flask with an electronic balance, add 100mL ultrapure water, seal it, sterilize it in a high-pressure steam sterilizer, and cool it to 40℃~50℃ Add ampicillin at a ratio of 1000:1, mix carefully, transfer to a clean 500mL reagent bottle for later use, and store at 4°C.

[0034] The preparation method of LB solid medium: Weigh 5g solid medium dry powder into a 500mL Erlenmeyer flask with an ...

Embodiment 3

[0082] Example 3. Preparation of dual-target chimeric antigen receptor T cells (CAR-T) for the treatment of gastric cancer

[0083] S1, PBMC cell separation

[0084] Use a vacuum blood collection tube containing heparin to collect about 6mL of human peripheral fresh blood;

[0085] Dilution: add an equal volume of PBS at room temperature, and gently pipette to mix;

[0086] Adding samples: Take a 50mL centrifuge tube, draw 6mL Ficoll (lymphocyte separation solution) into the centrifuge tube (the volume ratio of Ficoll to blood before dilution is 1:1), tilt the tube at 45°, and put the diluted blood on the surface of Ficoll Slowly add to the top of Ficoll along the tube wall about 1cm above;

[0087] Centrifugation: Centrifuge at 2000rpm for 30min at 18-20°C. After centrifugation, it is divided into four layers from the bottom of the tube to the liquid surface, which are red blood cell and granulocyte layer, stratified liquid layer, mononuclear cell layer, and plasma layer; ...

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Abstract

The invention discloses a vector containing a double-target chimeric antigen receptor gene, a CAR-T cell and application of the CAR-T cell. The double-target chimeric antigen receptor is formed by sequentially splicing signal peptides, CD44 single-chain antibody ScFv, EpCAM single-chain antibody ScFv, strepII tag, CD8 molecular hinge region, CD28 molecular transmembrane region, intracellular structural domain of the CD28 molecular transmembrane region, intracellular costimulatory domain 4-1BB and CD3 molecular zeta chain from an N end to a C end. Meanwhile, a lentivirus expression recombinantplasmid pTK-881-CD44-EpCAM is constructed, lentivirus packaging is carried out, T cells are infected, and the double-target-point CAR-T cells capable of removing CD44 and EpCAM at the same time are obtained. Based on a CAR-T technology, gastric cancer stem cells are killed by only utilizing autoimmunity of a patient, so that a doctor is assisted in gastric cancer treatment.

Description

technical field [0001] The invention relates to the field of tumor cell immunotherapy, in particular to a carrier containing a dual-target chimeric antigen receptor gene, CAR-T cells and applications thereof. Background technique [0002] Gastric cancer is one of the most common malignant tumors worldwide and the third leading cause of death in patients with malignant tumors. At present, multidisciplinary combined therapy is used to improve the prognosis of patients with advanced gastric cancer. Although great progress has been made in the treatment, the prognosis of gastric cancer patients is still poor. The main reason is that the diagnosis is late, which brings difficulties to diagnosis and treatment. The theory of cancer stem cells is a hypothesis recently put forward. Cancer stem cells and normal stem cells have similar characteristics, have the ability of self-renewal, and can produce different offspring. Chemotherapy drugs used to treat malignant tumors have higher d...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K16/2884C07K16/30C07K14/7051C12N15/86A61K39/001111A61P35/00C12N2740/15043C07K2317/622C07K2319/02C07K2319/03A61K2039/5156A61K2039/5158Y02A50/30
Inventor 廖兴华戴周彤王易澳项园王君张慧敏李佳蓬李慧张同存
Owner WUHAN UNIV OF SCI & TECH
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