Anti-human her 2 antigen specific chimeric antigen receptor, encoding gene and 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 reducing continuous killing of target tumors, increasing CAR, affecting tumor killing effect, etc., to enhance specific killing effect. , to improve safety, the effect of strong killing specificity

Active Publication Date: 2022-07-19
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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  • Anti-human her 2 antigen specific chimeric antigen receptor, encoding gene and expression vector and application
  • Anti-human her 2 antigen specific chimeric antigen receptor, encoding gene and expression vector and application
  • Anti-human her 2 antigen specific chimeric antigen receptor, encoding gene and 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 the signal peptide (CD8αleader), anti-human HER2 antigen single chain antibody scFv (HER2scFv), human CD8α hinge region (CD8αhinge), human CD28 transmembrane region (CD28TM), costimulatory factor 4-1BB and intracellular signal domain CD3ζ, a chimeric antigen receptor receptor structure such as figure 1 shown.

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

[0046] 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. The nucleotide sequence of...

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) at a ratio of 4:2:1 with polyethyleneimine transfection reagent (PEI, from Sigma company), specifically See the instructions of PEI transfection reagent for the method.

[0056] 2. Purification of lentivirus

[0057] 72 hours later, the virus supernatant was collected, centrifuged at 4°C for 10 minutes at 3000 rpm, filtered through a 0.45 μm filter, mixed with PEG8000 / NaCl at a volume of 4:1, left at 4°C for 2-3 hours, and then centrifuged at high speed for 30 minutes. The supernatant was discarded, and the precipitate was resuspended and dissolved in pre-cooled PBS to obtain a virus concentrate, which was stored at -80°C for later use.

[0058] 3. Lentiviral titer determination

[0059] The vi...

Embodiment 3

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

[0071] Tested 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, All were purchased from ATCC. 293T cells were purchased from ATCC. Among them, SKBR3 and SKOV3 are tumor cells with high HER2 expression; MCF-7 and A2780 are cell lines with moderate and low expression of HER2.

[0072] The CAR-T cells prepared in Example 2 were co-cultured with the above cell lines at a 20:1 effect-target ratio for 4 hours. 5 cell suspension of individual cells. The culture group with only T cells served 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 receptor targeting HER 2

[0074] Take out the ELISA plate that ha...

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PUM

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Abstract

The present invention relates to specific chimeric antigen receptors against human HER 2 antigen, encoding genes and expression vectors, and applications. The anti-human HER2 chimeric antigen receptor includes a human CD8 leader chimeric receptor signal peptide, an anti-human HER2 single-chain antibody, a human CD8α hinge region, a human CD28 intracellular transmembrane region, and a human 41BB intracellular region in series. Stimulatory factors and the human CD3ζ intracellular signaling domain. The present invention also provides the coding sequence of the anti-human HER2 chimeric antigen receptor, the recombinant expression vector and the construction method and application thereof. The anti-human HER2 chimeric antigen receptor provided by the invention can be stably expressed in T lymphocytes, can specifically identify and kill HER2-positive tumor cells, improve the safety effect 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 against human HER2 antigen, an encoding gene, an expression vector and applications. Background technique [0002] Cancer is the main disease endangering human health today. In February 2018, statistics from the National Cancer Center showed that on average, 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 of treating tumors mainly include traditional and new treatment methods. The traditional methods include surgery, radiation therapy, and chemotherapy. The new treatment methods-precision treatment include: monoclonal antibodies, CAR-T cell immunotherapy, and gene editing. Among them, cellular immunotherapy is a new type of therapy that emerged after surgery, chemotherapy,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
Inventor 卢英
Owner 卢英
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