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Preparation method and application of third-generation LMP1 (latent membrane protein 1) CAR-T (Chimeric Antigen Receptor T) cells

A cell and extracellular region technology, applied in the field of genetic engineering and immune targeted therapy, can solve problems such as the lack of third-generation LMP1CAR-T cells

Inactive Publication Date: 2019-03-01
THE SECOND AFFILIATED HOSPITAL OF NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the field of CAR-T cell therapy for nasopharyngeal carcinoma, there is no report on the successful preparation of third-generation LMP1 CAR-T cells

Method used

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  • Preparation method and application of third-generation LMP1 (latent membrane protein 1) CAR-T (Chimeric Antigen Receptor T) cells
  • Preparation method and application of third-generation LMP1 (latent membrane protein 1) CAR-T (Chimeric Antigen Receptor T) cells
  • Preparation method and application of third-generation LMP1 (latent membrane protein 1) CAR-T (Chimeric Antigen Receptor T) cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1 Construction of lentiviral expression vector for anti-LMP1 CAR

[0100] 1.1 Design and synthesis of LMP1 scFv primers targeting LMP1 chimeric antigen receptor LMP1 CAR extracellular region

[0101] The fully human anti-LMP1 antibody Fab constructed in the laboratory was analyzed with the biological software DNAstar 8.0, and the variable region of the light chain (Vκ) and the variable region of the heavy chain (Vκ) were designed and amplified. H ) primers were optimized, and the primers were finally synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.

[0102] LMP1scFv heavy and light chain variable region primer sequences are as follows:

[0103]

[0104] The direction of the above primers are all 5'-3'

[0105] 1.2 Synthesis and amplification of heavy and light chain variable regions of LMP1 scFv segment targeting LMP1 chimeric antigen receptor LMP1 CAR extracellular region

[0106] 1) Reaction system (take 50ul system as an example):

[0107] ...

Embodiment 2

[0191] Example 2 LMP1 CAR virus packaging

[0192] 1) Collect X-293T cells, inoculate them on a 10cm culture dish and continue culturing. When the cell confluence reaches 70%, replace with fresh incomplete medium 30 minutes before virus packaging; 2) 1.5ml centrifuge tube A: 470ul Opti-MEM+30ul PEI , resuspend and incubate at room temperature for 5min; 1.5ml centrifuge tube B: 3.3ug psPAX2+3.3ug pMD2.G+3.3ug LMP1 CAR, add Opti-MEM to 250ul; 3) Add the reagents in tube A to tube B dropwise , mix gently, and place at room temperature for 15 minutes; 4) Add the mixture dropwise to X-293T cells, shake the culture dish gently to make the mixture evenly distributed, and incubate in a 37°C incubator; 5) After 6-8 hours, replace with fresh Complete medium (1640+10% FBS); 6) Observe the transfection efficiency under a fluorescent microscope after 24 hours; 7) Collect the supernatant after 48 hours, if the cells adhere well, replace with fresh incomplete medium (1640+10% FBS) The cultu...

Embodiment 3

[0193] Example 3 Concentration of LMP1 CAR virus

[0194] method one:

[0195] 1) After the supernatant collected in Example 2 is filtered through a 0.45um filter membrane, it is transferred to a 100kD ultrafiltration tube and concentrated to 1 / 3 volume;

[0196] 2) The concentrated solution was filtered through a 0.22um filter membrane, and the filtrate was collected and frozen at -80°C.

[0197] Method Two:

[0198] 1) Dissolve 8.76g NaCl and 50g PEG-8000 in 200ml double distilled water respectively, and autoclave to prepare 5×PEG-8000 NaCl solution; 2) The virus supernatant collected in Example 2 was mixed with 5 Mix ×PEG-8000NaCl, shake once every 20-30min, 5 times in total, overnight at 4°C; 3) Centrifuge at 6000rpm for 20min, remove the supernatant; 4) Collect the precipitate, resuspend in medium, and freeze at -80°C.

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Abstract

The invention utilizes a genetic engineering technology for constructing an extracellular region LMP1 (latent membrane protein1) scFv segment of a chimeric antigen receptor targeting LMP1 with a high-affinity full human-derived anti-LMP1 Fab as a template. The extracellular region LMP1 scFv segment of LMP1 CAR is recombined with signal peptide CD8alpha, transmembrane region CD8TM, and intracellular regions CD28, CD137, and CD3zeta to construct a third-generation chimeric antigen receptor targeting LMP1. The prepared chimeric antigen receptor targeting LMP1 is recombined into a linearized lentiviral vector by overlap PCR, an anti-LMP1 CAR lentiviral recombinant expression vector is constructed, an LMP1 CAR virus solution is prepared, and T cells are infected to obtain LMP1 CAR-T cells. Thespecific targeted killing effect of the cells on LMP1 positive nasopharyngeal carcinoma cells is verified in vitro and in vivo, and new therapeutic ideas and means are provided for the treatment of nasopharyngeal carcinoma.

Description

technical field [0001] The invention belongs to the field of genetic engineering and immune targeting therapy, and relates to a preparation method of third-generation LMP1 CAR-T cells and its application in providing a novel treatment for nasopharyngeal carcinoma. Background technique [0002] Nasopharyngeal carcinoma is a highly malignant tumor derived from the nasopharyngeal epithelium. The tumor progresses and easily invades important structures such as the skull base, and early cervical lymph node metastasis and distant metastasis occur. Nasopharyngeal carcinoma is characterized by ethnic and regional distribution. It is one of the top ten high-incidence malignant tumors in my country. Its incidence rate is 20 / 100,000, which has caused serious health problems. [0003] At present, the main treatment for nasopharyngeal carcinoma is radiotherapy and chemotherapy. However, due to the atypical first symptoms, patients are often in the middle and late stages of the disease wh...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/085C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 陈仁杰冯振卿朱进唐奇陈渊张舒张大为
Owner THE SECOND AFFILIATED HOSPITAL OF NANJING MEDICAL UNIV
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