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Preparation method and application of third generation LMP2A CAR-T cells

A cell and extracellular region technology, applied in the field of preparation of third-generation LMP2ACAR-T cells, can solve problems such as LMP2ACAR-T cells without third-generation

Inactive Publication Date: 2019-02-26
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 LMP2A CAR-T cells

Method used

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  • Preparation method and application of third generation LMP2A CAR-T cells
  • Preparation method and application of third generation LMP2A CAR-T cells
  • Preparation method and application of third generation LMP2A CAR-T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1 Construction of lentiviral expression vector for anti-LMP2A CAR

[0100] 1.1 Design and synthesis of LMP2A scFv primers targeting the extracellular region of the chimeric antigen receptor LMP2A CAR targeting LMP2A

[0101] The fully human anti-LMP2A antibody Fab constructed in the laboratory was analyzed with the biological software DNAstar 8.0, and the light chain variable region (Vκ) and heavy chain variable region (Vκ) were designed to be amplified. H ) primers were optimized, and the primers were finally synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd. LMP1scFv heavy and light chain variable region primer sequences are as follows:

[0102]

[0103] The direction of the above primers are all 5'-3'

[0104] 1.2 Synthesis and amplification of the heavy and light chain variable regions of the LMP2A scFv segment of the extracellular domain of the chimeric antigen receptor LMP2A CAR targeting LMP2A

[0105] 1) Reaction system (take 50ul system a...

Embodiment 2

[0189] Example 2 LMP2A CAR virus packaging

[0190] 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 , after resuspension, incubate at room temperature for 5min; 1.5ml centrifuge tube B: 3.3ug psPAX2+3.3ug pMD2.G+3.3ug LMP2A CAR, add Opti-MEM to make the volume to 250ul; 3) Drop the reagents in tube A into tube B , 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 (164...

Embodiment 3

[0191] Example 3 Concentration of LMP2A CAR virus

[0192] method one:

[0193] 1) After the supernatant collected in Example 2 was filtered through a 0.45um membrane filter, it was transferred to a 100kD ultrafiltration tube and concentrated to 1 / 3 volume

[0194] 2) The concentrated solution is filtered through a 0.22um filter membrane, and the filtrate is collected and stored at -80°C

[0195] Method Two:

[0196] 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

Through a genetic engineering technology, a LMP2A-targeted chimeric antigen receptor extracellular region LMP2A scFv segment is constructed by using a high affinity full human anti-LMP2A Fab as a template. A third generation LMP2A-targeted chimeric antigen receptor is constructed by recombination of the LMP2A-targeted chimeric antigen receptor(LMP2A CAR) extracellular region LMP2A scFv segment andsignal peptide CD8alpha, transmembrane region CD8TM and intracellular regions CD28, CD137 and CD3 zeta. The prepared LMP2A-targeted chimeric antigen receptor is recombined into a linearized lentiviral vector by overlay PCR to construct an anti-LMP2A CAR lentiviral expression vector, a LMP2A CAR virus solution is prepared, and T cells are infected to obtain LMP2A CAR-T cells. In vitro and in vivotests validate the LMP2A CAR-T cells have specific targeted killing effects on LMP2A positive nasopharyngeal carcinoma cells, and provide a new therapeutic idea and means 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 and application of the third-generation LMP2A CAR-T cells. 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 when they see a doctor, and their five-year survival rate is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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