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Transgenic T cell of targeted CD30 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. Problems such as low treatment efficiency

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

[0013] 2. The effective rate of treatment is lower than the 70-90% complete cure rate (CR) of typical CD19 carT. The effective rate needs to be improved and the inhibition of the tumor microenvironment needs to be overcome
[0014] 3. Long-term CD30 carT may have persistent toxicity to patients

Method used

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  • Transgenic T cell of targeted CD30 antigen as well as preparation method and application of transgenic T cell
  • Transgenic T cell of targeted CD30 antigen as well as preparation method and application of transgenic T cell
  • Transgenic T cell of targeted CD30 antigen as well as preparation method and application of transgenic T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of lentiviral expression vector

[0044] (1) Construction of anti-CD30 AC10 scFv-CD27 Hinge-TM-CD28-41BB-CD3zeta: The construction method is a conventional method.

[0045] (2) Construction of lentiviral expression vector:

[0046] 293T cells were cultured with RPMI1640+10% FBS. After the cell density is 90%, use lipo2000 293T cells. The transfection method is in accordance with the Invitrogen manufacturer's standard operation. The plasmids include: expression plasmid, pMDLg / pRRE, pRSV-Rev, pMD2.G, and mixed plasmid according to 2:1:1:0.5 Ratio of transfected 293T cells. After 24-48 hours, the lentiviral supernatant was harvested twice. After ultracentrifugation at 100,000g for 120min, the supernatant was removed, the lentivirus pellet was obtained, and serum-free T cell culture medium was added. After resuspension, the virus infection titer (IU / ml) was measured by infecting K562 with dilution method.

[0047] Obtain the lentivirus data of a certain ex...

Embodiment 2

[0058] Example 2 Transfection of T cells

[0059] Peripheral blood of healthy people or tumor patients is drawn 50-100ml, or the mononuclear cells are obtained by the Cobra Spectra blood cell separator. After Ficoll separation, they are separated by CD4+ or CD8+ magnetic beads (magnetic beads purchased from Stem CellTechnologies).

[0060] Lentivirus infects human CD4+T or CD8+T cells: Please refer to TakaraRetronectin instruction manual for lentivirus infection after preparation and concentration. The brief description is as follows:

[0061] Prepare Retronectin with a concentration of 20-100μg / ml, and use a density of 4-20μg / cm for paving 2 After 2 hours at room temperature, aspirate the supernatant for use; add lentivirus to the above plate 125-250μl / cm 2 , 37C warm bath for 4-6 hours; T cells to be infected with a density of 0.5-2.5×10 4 cells / cm 2 Planking. The medium was changed 24 hours after T cell infection.

[0062] T cells cultured for 1 day (medium formula: Lonza X vivo15,...

Embodiment 3

[0064] Example 3 Knock out PD1 gene and CTLA4 gene

[0065] The experiments to knock out the PD1 gene and CTLA4 gene were carried out respectively, and the specific methods are as follows:

[0066] The gRNA, CRISPR-cas9 mRNA, and HDR were mixed and electrotransformed into recombinant T cells (400V, 0.5ms).

[0067] After electroporation, culture the cells for 24 hours and then change the medium (the medium used is: Lonza X vivo15, adult serum 10%, IL2500IU / ml, gentamycin 100ug / ml, IL155ng / ml, IL215ng / ml OKT3antibody 50ng / ml), Pre-coated with OKT monoclonal antibody (10ug / ml, 25C 4hr) in T75flask, continue to culture, count every 2 days, observe the cell morphology and add culture medium, maintain the cell concentration at 1.0-3.0×10 6 / ml between. After the 10th day of culture, T cells were harvested by centrifugation and washed to remove the culture solution.

[0068] The nucleotide sequence of the CRISPR-cas9 mRNA is shown in SEQ ID NO: 7;

[0069] When the PD1 gene is knocked out, ...

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Abstract

The invention discloses a transgenic T cell of a targeted CD30 antigen. The transgenic T cell is a primary cell which is integrated with a gene shown as SEQ ID NO:2 and encoding the targeted CD30 antigen, and knocks out a PD1 gene and / or CTLA4 gene, or is a primary cell containing a recombinant lentivirus expression vector (including a gene which is shown as SEQ ID NO:2 and encodes the targeted CD30 antigen and shRNA of a targeted PD1 gene or / and shRNA of a targeted CTLA4 gene); the primary cell is CD4+T cell or CD8+T cell. A preparation method comprises the following steps: firstly, carryingout lentivirus infection on the CD4+T cell or the CD8+T cell; secondly, mixing gRNA, CRISPR-cas9mRNA and HDR, and carrying out electroporation recombination on the T cell to obtain a finished product.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 knocked out or silenced, 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 CD30 antigen, and a preparation method and application thereof. Background technique [0002] Malignant lymphoma is a tumor of immune cells in the lymph nodes and extranodal lymphoid tissues, derived from the malignant transformation of lymphocytes or histiocytes. It is one of the most common malignant tumors among young people. The lesions mainly occur in the lymph nodes, with cervical lymph nodes and supraclavicular lymph nodes being the most common, followed by mediastinal, retroperitoneal, and para-aortic lymph nodes. The lesion starts from one or a group of lymph nodes, and usually spreads regularly from the primary tumor along the lymphatic tract to adjacent lymph nodes. In the late stage, blood dissemination may occur, invading blood vessels, involving the spleen, liver, bone marrow, and digestive tract. [0003] In my country, the number of new cases of malignant lymphoma has been increasing ye...

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
CPCC07K16/2878A61K35/17C07K14/7051C07K14/70521C07K2317/622C07K2319/00C07K2319/33C12N5/0636C12N9/22C12N15/86C12N15/907C12N2510/00C12N2740/15043
Inventor 黄昕华生德伟于丽丽张晋伟庞勇李德柱
Owner YINFENG BIOLOGICAL GRP
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