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Specific chimeric antigen receptor against human HER 2 antigen, coding gene, expression vector and application

A chimeric antigen receptor and expression vector technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of increasing CAR, reducing the continuous killing of target tumors, affecting the tumor killing effect, etc., achieving strong killing specificity, Improve safety and enhance the effect of specific killing effect

Active Publication Date: 2019-05-31
卢英
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Rosenberg research group used the scFv of trastuzumab as the basis to synthesize the third-generation CAR T lymphocytes targeting HER2 / neu, and fatal complications occurred in patients
[0012] The affinity of scFv for antigen determines whether CAR T cells can activate and kill tumor cells. Among the existing targets for treating solid tumors, it is difficult to penetrate into the tumor body due to the barrier of solid tumors, and the specificity of ScFv on the surface of CAR-T is not strong, which will also affect tumor killing effect
However, increasing the affinity of CAR may lead to the risk of severe off-target effects caused by on-target / off-tumor effects and reduce the ability to continuously kill target tumors. The stimulation threshold must be considered to obtain the best specificity for CAR-T cell activation. Enhanced tumor killing specificity and treatment safety

Method used

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  • Specific chimeric antigen receptor against human HER 2 antigen, coding gene, expression vector and application
  • Specific chimeric antigen receptor against human HER 2 antigen, coding gene, expression vector and application
  • Specific chimeric antigen receptor against human HER 2 antigen, coding gene, expression vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of CAR virus against human HER2

[0043] 1. Gene sequence of anti-human HER2 chimeric antigen receptor

[0044] Synthesize sequentially containing signal peptide (CD8α leader), anti-human HER2 antigen single-chain antibody scFv (HER2 scFv), human CD8α hinge region (CD8αhinge), human CD28 transmembrane region (CD28TM), co-stimulatory factor 4-1BB and intracellular signal domain CD3ζ, chimeric antigen receptor receptor structure as figure 1 shown.

[0045] Wherein the signal peptide is a leader peptide, and its nucleotide sequence is shown in SEQ NO.8

[0046] The HER2 single-chain antibody structure (HER2scFv) is a specific antigen-binding domain designed for the related antigen HER2 on the surface of HER2-positive tumor cells, and its nucleotide sequence is shown in SEQ NO.9

[0047] The nucleotide sequence of the hinge region of human CD8α is shown in SEQ ID NO.10. The nucleotide sequence of human CD28 transmembrane is shown in SEQ ID NO.11....

Embodiment 2

[0053] Example 2. Preparation of chimeric antigen receptor modified T cells against human HER2 antigen

[0054] 1. Packaging of lentivirus

[0055] The pCDH-CAR plasmid constructed in Example 1 and the packaging plasmids pSPAX2 and pMD2.G were transfected into 293T cells (from ATCC) with polyethyleneimine transfection reagent (PEI, from Sigma) in a ratio of 4:2:1, specifically For the method, see the manual of PEI transfection reagent.

[0056] 2. Purification of lentivirus

[0057] After 72 hours, the virus supernatant was collected, centrifuged at 4°C, 3000rpm for 10 minutes, filtered through a 0.45 μm filter, mixed with PEG8000 / NaCl at a volume of 4:1, left standing at 4°C for 2-3 hours, and then centrifuged at high speed for 30 minutes. Discard the supernatant, resuspend and dissolve the pellet in pre-cooled PBS to obtain a concentrated virus solution, and store it at -80°C for future use.

[0058] 3. Determination of lentivirus titer

[0059] The virus infected 293T c...

Embodiment 3

[0070] Example 3. In vitro tumoricidal activity detection of T lymphocytes modified by anti-human HER2 chimeric antigen receptor

[0071] Test cell lines: human breast cancer cell lines SKBR3, MCF-7 and ovarian cancer cell lines SKOV3, A2780, as well as HER2-negative tumor cell line MDA-MB-468 and human HER2-negative mouse lung cancer cell TC-1, Both were purchased from ATCC. 293T cells were purchased from ATCC. Among them, SKBR3 and SKOV3 are tumor cells with high expression of HER2; MCF-7 and A2780 are cell lines with moderate and low expression of HER2.

[0072] After the CAR-T cells prepared in Example 2 were co-cultured with the above cell lines at an effect-to-target ratio of 20:1 for 4 hours, 5×10 5 cell suspension of cells. The cultured group with only T cells was used as a negative control group.

[0073] 1. ELISA to detect the levels of IFN-γ, TNF-α, and IL-2 secreted by T lymphocytes expressing chimeric antigen receptors targeting HER2

[0074] Take out the ELI...

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Abstract

The invention relates to a specific chimeric antigen receptor against a human HER 2 antigen, a coding gene, an expression vector and application. The chimeric antigen receptor against the human HER 2antigen comprises a human CD8 leader chimeric receptor signal peptide, a human HER2 single chain antibody, a human CD8 alpha hinge region, a human CD28 intracellular transmembrane region, a human 41BBintracellular region inducible co-stimulator and a human CD3 zeta intracellular signaling structure domain which are sequentially connected. The invention also provides the coding sequence, the recombinant expression vector and a construction method and application of the chimeric antigen receptor against the human HER2. The chimeric antigen receptor can be stably express on T lymphocytes, can specifically recognize and kill HER 2 positive tumor cells, improve the safety of treatment, and can be used for targeted therapy of tumors.

Description

technical field [0001] The invention belongs to the technical field of biology and medicine, and relates to a specific chimeric antigen receptor for anti-human HER2 antigen, coding gene, expression vector and application. Background technique [0002] Cancer is a major disease that endangers human health today. In February 2018, statistics from the National Cancer Center showed that an average of more than 10,000 people were diagnosed with cancer every day in my country, and 7 people were diagnosed with cancer every minute. Cancer has become the most urgent problem to be overcome. [0003] The methods for treating tumors mainly include traditional and new treatment methods. The traditional ones include surgery, radiation therapy, and chemotherapy. The new treatment methods—precision therapy include: monoclonal antibody drugs, CAR-T cell immunotherapy, gene editing, etc. Among them, cellular immunotherapy is a new type of therapy that has emerged after surgery, chemotherapy...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
Inventor 卢英
Owner 卢英
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