Transgenic T cell of targeted CD19 antigen as well as preparation method and application of transgenic T cell

A transgenic, primitive cell technology, applied in the direction of targeting specific cell fusion, genetically modified cells, receptors/cell surface antigens/cell surface determinants, etc., can solve the problems of lack of B lymphocytes, disease recurrence, etc.

Inactive Publication Date: 2018-03-06
YINFENG BIOLOGICAL GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] 2. 40-55% of patients have grade 3-4 neurotoxicity reactions, which require clinical control
[0013] 3. After successful treatment, the patient lacks B lymphocytes for a long time, so immunoglobulin must be injected every 2 to 3 weeks to maintain basic immune requirements
[0015] 5. There are still long-term tumor seed cells in the patient's body, which re-grow after the loss of carT cells, and the clinical manifestation is disease recurrence

Method used

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  • Transgenic T cell of targeted CD19 antigen as well as preparation method and application of transgenic T cell
  • Transgenic T cell of targeted CD19 antigen as well as preparation method and application of transgenic T cell
  • Transgenic T cell of targeted CD19 antigen as well as preparation method and application of transgenic T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of lentiviral expression vector

[0048] (1) Construction of scFv-PSA-NCAM-CD8a-PD1: The structure diagram is as follows figure 1 As shown, the car molecule is designed as figure 2 shown. The nucleotide sequence of the constructed scFv-PSA-NCAM-CD8a-PD1 is shown in SEQ ID NO:2.

[0049] (2) Construction of anti-CD19 FMC63 scFv-CD27-41BB-CD3zeta: The construction method is a conventional method, and the nucleotide sequence is shown in SEQ ID NO:8.

[0050] (3) Construction of lentiviral expression vectors I and II: 293T cells were cultured with RPMI1640+10% FBS. After the cells were at 90% density, lipo2000 was used to mix the transfection plasmids and transfect 293T cells. The transfection method is operated according to the standard of the Invitrogen manufacturer. The plasmids include: expression plasmids, pMDLg / pRRE, pRSV-Rev, pMD2.G, and the molar ratio of the mixed plasmids is 2:1:1:0.5. Harvest the lentivirus after 24-48 hours supernata...

Embodiment 2

[0051] Example 2 Transfection of T cells

[0052] 50-100ml of peripheral blood was extracted from healthy people or tumor patients, or mononuclear cells were obtained with a Cobra Spectra blood cell separator, separated by Ficoll, and sorted with CD4+ or CD8+ magnetic beads (the magnetic beads were purchased from Stem Cell Technologies).

[0053] Infection of human CD4+T or CD8+T cells with lentivirus: Refer to the instructions of Takara Retronectin for the prepared and concentrated lentivirus infection, which is briefly described as follows:

[0054] Prepare Retronectin at a concentration of 20-100 μg / ml, and use a density of 4-20 μg / cm for plating 2 After 2 hours at room temperature, suck off the supernatant for later use; add lentivirus to the above plate at 125-250 μl / cm 2 , 37C warm bath for 4 to 6 hours; T cells to be infected at a density of 0.5 to 2.5×10 4 cells / cm 2 Plank. The medium was changed 24 hours after T cell infection.

[0055] The present invention uses...

Embodiment 3

[0059] Example 3 Knockout of PD1 gene and CTLA4 gene

[0060] The experiments of knocking out the PD1 gene and CTLA4 gene were carried out respectively, and the specific methods were as follows: gRNA, CRISPR-cas9mRNA, and HDR were mixed, and the recombinant T cells (T cells transfected only with lentiviral expression vector II) were electroporated (400V, 0.5ms).

[0061] After electroporation, culture the cells for 3 days (the medium used is: Lonza X vivo15, add 10% of adult serum, IL2300~500IU / ml), extract genomic DNA, perform PCR amplification, and purify the amplified product with Agarose gel for TA cloning After purifying a single clone, 100 clones were picked and sequenced to obtain Indel mutation information. According to the ratio of mutation and non-mutation, the gene editing efficiency of PD1: 17%, CTLA4: 18.3%.

[0062] The CRISPR-cas9 mRNA nucleotide sequence is shown in SEQ ID NO: 9;

[0063] When the PD1 gene is knocked out, the nucleotide sequence of the gRNA ta...

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Abstract

The invention discloses a transgenic T cell of a targeted CD19 antigen. The transgenic T cell is a primary cell which is integrated with a gene shown as SEQ ID NO:8 and encoding the targeted CD19 antigen in chromosome, and knocks out a PD1 gene and / or CTLA4 gene, or is a primary cell which is integrated with a gene shown as SEQ ID NO:2 and encoding a targeted PSA-NCAM receptor ,and a gene shown asSEQ ID NO:8 and encoding the targeted CD19 antigen in the chromosome; the primary cell is CD4+T cell or CD8+T cell. The invention also discloses application of the transgenic T cell of the targeted CD19 antigen in preparation of a medicine for treating malignant B cell lymphoma. According to the transgenic T cell disclosed by the invention, a recognition sequence of an EGFR (Epidermal Growth Factor Receptor) is introduced in carT construction; if necessary, a carT cell can be eliminated by using EGFR monoclonal antibody Cetuximab, the PD1 gene and the CTLA4 gene are silenced or knocked out, inhibition of the gene to the carT cell is eliminated, and the function of overcoming a tumor microenvironment and inhibiting immune cells by the carT cell are enhanced.

Description

technical field [0001] The invention relates to a transgenic T cell targeting CD19 antigen, a preparation method and application thereof. Background technique [0002] The incidence rate of leukemia in China from 2003 to 2007 was 5 per 100,000 people. In recent years, due to industrialization and environmental changes, the incidence has gradually increased to 8 to 10 per 100,000 people. It is conservatively estimated that 40,000 new patients are added every year. CLL is the most common type of leukemia in Western countries, accounting for 1 / 3 of all leukemia cases. The incidence rate in Asian countries is low, and CLL accounts for less than 3% of the total number of leukemias in my country. More than 90% of the onset age of CLL is over 50 years old, and the incidence rate of men is higher than that of women, male:female=2:1. Non-Hodgkin's lymphoma (NHL) has an incidence of 3 to 6 per 100,000 people in China, and 50,000 people in the United States every year. [0003] Adu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N15/90C12N5/10A61K35/17A61P35/00
CPCC07K16/2803A61K35/17C07K14/7051C07K14/70521C07K2317/622C07K2319/00C07K2319/33C12N5/0636C12N15/86C12N15/907C12N2510/00C12N2740/15043
Inventor 黄昕华于丽丽生德伟李德柱徐峰波
Owner YINFENG BIOLOGICAL GRP
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