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TEM8 (tumor endothelia marker 8) targeting chimeric antigen receptor T cell and application thereof

A chimeric antigen receptor, targeting technology, applied in the field of genetic engineering, can solve the problems of general curative effect, limited tumor types, toxic and side effects, etc., and achieve the effect of high-efficiency tumor killing activity

Pending Publication Date: 2018-10-26
廊坊康宝汇泰生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional radiotherapy and chemotherapy target limited types of tumors, have mediocre curative effects, and are accompanied by strong toxic and side effects

Method used

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  • TEM8 (tumor endothelia marker 8) targeting chimeric antigen receptor T cell and application thereof
  • TEM8 (tumor endothelia marker 8) targeting chimeric antigen receptor T cell and application thereof
  • TEM8 (tumor endothelia marker 8) targeting chimeric antigen receptor T cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of chimeric antigen receptor T cells targeting TEM8

[0037] 1. Construction of lentiviral expression vector

[0038] Synthesize a nucleic acid fragment (BamHI-Sp-EcoRI-NheI-CD28-CD3zeta-SalI, synthesized by Zhongmei Taihe Biotechnology (Beijing) Co., Ltd.) encoding CD28-CD3ζ, as shown in SEQ ID NO.6, named pGSI -seq8. 3ug pGSI-seq8 and recombinant lentiviral vector plasmid pCDH-EF1 (Addgene) were double-digested with BamHI and SalI restriction endonucleases respectively, the digested products were recovered from the gel, ligated with T4 DNA ligase, ligated overnight at 4°C, and transformed into DH5α For competent cells, take 100 μL of the bacterial solution and spread it on an LB plate containing ampicillin resistance, and culture overnight at 37°C. Single clones were picked for colony PCR, positive clones were sent for sequencing, and clones with correct sequencing results were saved and plasmids were extracted, named pCDH-EF1-emCAR.

[0039] ...

Embodiment 2

[0051] Example 2 The killing effect of targeting TEM8 chimeric antigen receptor T cells on cancer cells expressing TEM8

[0052] The liver cancer cell line HepG2 cells stably expressing TEM8 and the cervical cancer cell line Hela low expressing TEM8 were used as target cells, and the TEM8-targeting chimeric antigen receptor T cells prepared in Example 1 and the uninfected Anti-TEM8 CAR were used respectively. Lentiviral T cells (called uninfected T cells) were used to produce effector cells, and the target cells were inoculated in a 96-well plate at a density of 5000 / ml, 100 μl per well, and the target cells were 5:1, 10:1, and 20:1. Than adding effector cells to target cells, placed in 5% CO 2 , Cultivate in a 37°C incubator for 4 hours, use CCK8 to detect cell viability, and calculate the killing efficiency. The result is as Figure 3-6 As shown, the results show that chimeric antigen receptor T cells expressing TEM8 have a strong and specific killing effect on TEM8-positi...

Embodiment 3

[0053] Example 3 The killing effect of T cells targeting TEM8 chimeric antigen receptor on tumors in vivo

[0054] 30 6-week-old female SCID mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were subcutaneously injected with 5*10 6 HepG2 (liver cancer) cells, when the tumor grows to 50-100mm 3 Size, tumor model was randomly divided into 3 groups: control group, normal T cell group and anti-TEM8 chimeric antigen receptor T cell (prepared in Example 1) group; the control group was injected with normal saline 200ul / time, twice a week; In the T cell group, T cells were injected into the tail vein 1*10 7 One / time, 2 times a week; Anti-TEM8 chimeric antigen receptor T cell group Injection of anti-TEM8 chimeric antigen receptor T cells 1*10 by tail vein 7 Each time, twice a week; the survival status of the mice within 100 days was counted, and the survival rate curve was made. Figure 7 The results showed that chimeric antigen receptor T cells ta...

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Abstract

The invention belongs to the technical field of genetic engineering and discloses a TEM8 (tumor endothelia marker 8) targeting chimeric antigen receptor T cell and application thereof. The TEM8 targeting chimeric antigen receptor T cell is capable of expressing SCFV-CD28-CD3 zeta fusion protein the a T cell. The TEM8 targeting chimeric antigen receptor T cell is obtained by T cell modification andtransformation, is capable of specifically recognizing and killing tumor cells highly-expressed by TEM8, and is suitable for prevention and treatment of corresponding tumor diseases.

Description

technical field [0001] The invention relates to chimeric antigen receptor T cells targeting TEM8 and applications thereof, belonging to the technical field of genetic engineering. Background technique [0002] In 2020, the number of new cases and deaths of malignant tumors in the world will reach 20 million and 12 million, and cancer will become the number one killer of human health. Traditional radiotherapy and chemotherapy target limited types of tumors, have mediocre curative effects, and are accompanied by strong toxic and side effects. Neovascularization around the tumor plays an important role in the process of tumor formation, growth, and metastasis, and it can provide nutrients for the rapid proliferation of tumors. Tumor-induced angiogenesis is a critical step in tumor progression and is known as the "angiogenic switch". Under the pathological conditions of tumors, the uncontrolled regulation of blood vessels is the main factor contributing to the malignant growth...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867A61K35/17A61P35/00C12Q1/02
CPCA61P35/00G01N33/5011G01N33/505A61K35/17C12N5/0636C12N15/86C07K14/7051C07K14/70521C07K16/28C07K2319/02C07K2317/622C12N2510/00C12N2740/15043
Inventor 段海峰薛冰华解晶胡显文
Owner 廊坊康宝汇泰生物技术有限公司
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