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A kind of EGFR-specific chimeric antigen receptor and its application

A chimeric antigen receptor, specific technology, applied in the fields of molecular biology and immunology, can solve the problems of limited anti-tumor effect, increased expression level, strong exhaustion, etc., and achieve strong tumor-specific antigen activation, Good applicability, enhanced lasting effect

Active Publication Date: 2021-07-09
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN104087607A discloses a CAR-T that specifically recognizes human EGFR. Under the same effect-to-target ratio, the in vitro killing rate of EGFR-overexpressing glioma cells U87 is as high as 98%. The in vitro killing rate of cell CFPAC-1 is only 40%. CN103113470A also discloses a CAR-T targeting human EGFR. The in vitro killing rate of cell A431 is still only about 50%
When differentiated into exhausted T cells, their ability to proliferate and secrete cytokines is greatly reduced, while the expression level of inhibitory receptors is increased, which ultimately limits their anti-tumor effects
The modification of CAR structure usually endows T cells with the effector function of mediating powerful tumor cell killing, but the excessive activation of T cells will drive the exhaustion of T cells, so it may also limit the persistence of T cells in turn, so the existing CAR- Although T is mostly effective, it often faces the dilemma of strong depletion and poor treatment effect
The results of clinical application of EGFR-specific CAR-T in the treatment of non-small cell lung cancer showed that 2 out of 11 patients with non-small cell lung cancer had a partial response, and 5 cases were in stable condition, but the duration was only 2-8 months. Subsequently, the disease progressed (NCT01869166), which shows that the current application of EGFR-specific CAR-T needs to further improve the persistence and reduce the exhaustion rate.

Method used

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  • A kind of EGFR-specific chimeric antigen receptor and its application
  • A kind of EGFR-specific chimeric antigen receptor and its application
  • A kind of EGFR-specific chimeric antigen receptor and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] This embodiment is the packaging and preparation method of EGFR-CAR recombinant lentiviral vector, the EGFR-CRA includes EGFR-Fc-CAR (second generation) as shown in SEQ ID NO.09, EGFR as shown in SEQ ID NO.10 -Fc-CAR (third generation), EGFR-Hinge-CAR (second generation) as shown in SEQ ID NO.11 and EGFR-Hinge-CAR (third generation) as shown in SEQ ID NO.12, the nucleotide sequences are as follows Shown in SEQ ID NO.13-16.

Embodiment 2

[0082] This example is a detection method for chimeric antigen receptor expression on 293T.

[0083] (1) Flow cytometry detection of chimeric antigen receptor expression on the surface of 293T cells

[0084] Collect the 293T cells transfected for 60 h in Example 1, centrifuge at 1000 rpm for 5 min to remove the supernatant, wash twice with PBS, and then wash with 1×10 6 cells / mL were added to PBS to suspend cells, and the corresponding antibody was added to protect from light at 4°C for 40 minutes, then washed with PBS, resuspended, and finally detected by flow cytometry. Wherein Fc-EGFR-CAR is detected by anti-human IgG1 Fc antibody; Hinge-EGFR-CAR is detected by anti-human IgG(Fab')2 antibody.

[0085] (2) Western blot verification of chimeric antigen receptor expression in 293T cells

[0086] The 293T cells transfected for 60 hours in Example 1 were collected, lysed with RIPA lysate, sonicated, centrifuged to obtain the supernatant, measured for concentration and quantifi...

Embodiment 3

[0090] This example is the isolation and culture of human primary T lymphocytes.

[0091] Lymphocytes were separated by human peripheral blood lymphocyte separation medium Ficoll (produced by Tianjin Haoyang Company), and X-VIVO (produced by LONZA Company) medium containing 10% FBS, CD3, CD28 antibodies and cytokines was used as a T cell culture medium. Adjust the cell density to 1 x 10 6 Cells / mL were cultured, and the expressions of T cell surface markers CD3 and CD8 were detected by flow cytometry after stimulation for 72 hours, and the lentivirus prepared in Example 1 was used for infection.

[0092] The result is as Figure 4 As shown, 99% of the isolated and cultured lymphocytes are CD3-positive cells, and 95% are CD8-positive effector cells, indicating that effector T cells that can be used for lentivirus infection to prepare CAR-T have been isolated.

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PUM

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Abstract

The invention discloses an EGFR-specific chimeric antigen receptor and its application, which comprises a transmembrane signal region, an extracellular recognition region, a hinge region, a transmembrane region and an intracellular signal conduction region connected in sequence. The surface of the CAR-T modified with the EGFR-specific chimeric antigen receptor of the present invention has a strong tumor-specific antigen activation effect, cytokine secretion function and tumor killing ability on triple-negative breast cancer. The killing efficiency in vitro is more than 90%. Animal experiments have also verified its significant effective inhibitory effect. In vivo experiments also have a significant tumor inhibitory effect, indicating that the CAR-T of the EGFR-specific chimeric antigen receptor of the present invention is triple-negative Breast cancer has good applicability.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and immunology, and specifically relates to an EGFR-specific chimeric antigen receptor and its application. Background technique [0002] In 2014, the National Cancer Center reported that the incidence of breast cancer among Chinese women was 41.82 per 100,000 people (Cancer incidence and mortality in China, 2014. Chinese Journal of Cancer Research, 2018, 30(1): 1- 12.), ranking first among female malignant tumors. At present, the annual incidence of breast cancer in China exceeds 160,000, and it is still increasing at a rate of 3% to 4%. Statistics show that the incidence of breast cancer in major cities in China has increased by 37% in 10 years, and the death rate has increased by 38.9%. Therefore, the prevention and treatment of breast cancer has a huge medical and health demand in our country. Among them, triple negative breast cancer (TNBC) accounts for 12-17% of all types of br...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/867C12N15/62C12N5/10A61K39/00A61P35/00
CPCA61K39/0011A61P35/00C07K14/7051C07K16/2863C07K16/32C07K16/40C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 刘文夏琳郑早早刘珺懿陈宇洁
Owner XIAMEN UNIV