PTPN2-blocked immune cell and application and preparation thereof

A technology of immune cells and NKT cells, applied in the field of PTPN2-blocking immune cells and their applications and preparations, can solve the problems of solid tumors without good treatment effect, low clinical response rate, off-target, etc., to enhance the killing function and reduce exhaustion. , to avoid toxic effects

Inactive Publication Date: 2020-12-22
GUANGDONG XIANKANGDA BIOTECH CO LTD
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AI-Extracted Technical Summary

Problems solved by technology

Although CAR-T cell therapy shines in the treatment of hematological tumors, it does not have a good therapeutic effect on solid tumors. T cells are not easy to enter the...
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Method used

Term CAR: The design of chimeric antigen receptor CARs has gone through the following process: the first generation CAR has only one intracellular signaling component CD3ζ or FcγRI molecule, and because there is only one activation domain in the cell, it can only cause transient T cell proliferation and less cytokine secretion, but can not provide long-term T cell proliferation signal and sustained anti-tumor effect in vivo, so it has not achieved good clinical efficacy. The second-generation CARs introduce a co-stimulatory molecule based on the original structure, such as CD28, 4-1BB, OX40, and ICOS. Compared with the first-generation CARs, the function is greatly improved, and the persistence of CAR-T cells and the ability to treat tumor cells are further enhanced. lethality. On the basis of the second-generation CARs, some new immune co-stimulatory molecules such as CD27 and CD134 are connected in series to develop into the third-generation and fourth-generation CARs, and there are also double-ta...
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Abstract

The invention relates to the field of biological cells, and particularly relates to a PTPN2-blocked immune cell and application and a preparation thereof. The PTPN2-blocked immune cell expresses a chimeric antigen receptor, the PTPN2 function in the immune cell is blocked, and the immune cell can secrete an anti-PD-1 antibody. The invention provides a method for killing tumors and reversing exhaustion. The PTPN2-blocked function is innovatively combined with CAR and anti-PD-1 immunoregulation, so that the tumor killing activity of the immune cell is improved, and the effect of curing tumors isachieved.

Application Domain

Antibody mimetics/scaffoldsNucleic acid vector +14

Technology Topic

Biology cellCAR - Chimeric antigen receptor +8

Image

  • PTPN2-blocked immune cell and application and preparation thereof
  • PTPN2-blocked immune cell and application and preparation thereof
  • PTPN2-blocked immune cell and application and preparation thereof

Examples

  • Experimental program(1)
  • Effect test(1)

Example Embodiment

[0079]Example 1
[0080]The preparation method and function verification of immune cells in this embodiment include the following steps:
[0081]Step 1: Isolation of peripheral blood PBMC and expansion of T cells
[0082]Separate mononuclear cells from donor peripheral blood, use ficol to perform density gradient centrifugation, and use T cell sorting kit to enrich T cells (CD3 MicroBeads, human-lyophilized, 130-097-043), using coupled anti-CD3 /anti-CD28 magnetic beads activate the culture and expansion of T cells; the medium uses TexMACS GMP Medium (MiltenyiBiotec, 170-076-309), containing 10% FBS, 2mM L-glutamine, 100IU/ml rhIL2, all cells are placed Cultivate in a constant temperature incubator at 37°C and 5% CO2.
[0083]Step 2: Cell line culture
[0084]GPC3 expressing cell line: Huh-7 (human liver cancer cell), purchased from ATCC.
[0085]Cell line that does not express GPC3: A549 (human non-small cell lung cancer cells), purchased from ATCC.
[0086]Packaging cells: 293T (human embryonic kidney cell line), purchased from ATCC.
[0087]Culture medium: Huh-7, A549, and 293T are cultured in DMEM medium. All media were supplemented with 10% (v/v) fetal bovine serum.
[0088]Step 3: CAR structure design and lentivirus packaging
[0089]GPC3-CAR structure, that is, the CAR structure targeting GPC3 (glypican 3):
[0090]In an embodiment of the method of the present invention, an antibody targeting PTPN2, a second-generation CAR and a secreted PD-1 antibody are constructed on an expression frame, connected by P2A, and named PPGPC3 CAR. The core structure of CAR includes secretion signal peptide sequence; scFv from anti-GPC3 antibody (patent number: US 2007/0190599A1); CD8 transmembrane region; intracellular segment stimulation signal 4-1BB-CD3ζ.
[0091]The second CAR constructed in the present invention, that is, the second generation CAR and the secreted PD-1 antibody are placed in an expression frame, connected by P2A, and named PGPC3 CAR.
[0092]In the present invention, the expression cassette with only CAR is used as a control and named as GPC3 CAR, such asfigure 1 It is the structure diagram of 3 kinds of CAR.
[0093]The expression cassette was cloned into the PHBLV lentiviral vector backbone and placed under the EF1α(EF-1α) promoter to form PHBLV-EF1α-GPC3-CAR, PHBLV-EF1α-PGPC3-CAR and PHBLV-EF1α-PPGPC3-CAR , The PHBLV-EF1α-GPC3-CAR or PHBLV-EF1α-PGPC3-CAR or PHBLV-EF1α-PPGPC3-CAR, the lentiviral envelope plasmid pMD2.G (Addgene, Plasmid#12259) and the lentiviral packaging plasmid psPAX2 (Addgene Plasmid) #12260) The three plasmids were transferred into 293T using Lipofectamine 3000 to prepare a complete lentiviral expression vector; the virus supernatant was collected at 48h and 72h, and concentrated by ultracentration (Merck Millipore); the concentrated virus can be used to infect T cells.
[0094]Step 4: CAR-T cell preparation
[0095]4.1 Lentivirus infection
[0096]After the isolated and purified primary T cells are activated for 1 day, use the 3 lentiviruses packaged in step 3 to carry out lentiviral vector infection at MOI (1-10), transfer to a cell culture flask, and place it at 37°C, 5% CO2 constant temperature Cultivate in an incubator.
[0097]4.2 Cell proliferation and CAR positive rate detection
[0098]Using three types of lentiviral vectors, three types of CAR-T cells were successfully constructed, named PPGPC3 CAR T, PGPC3 CAR T, and GPC3 CAR T. T cells not infected with lentivirus were used as controls (NT)
[0099]On the 6, 9, 11, and 13 days, samples were taken every day to detect the number of cells. On the 6th day, the CAR positive rate of T cells was detected, and the expression of PTPN2 was detected on the 13th day. Add culture medium every 1-2 days.
[0100]Such asfigure 2 Shown: the proliferation rate of 3 kinds of CAR-T cells and NT, the sequence of the inserted antibody has no significant effect on the proliferation rate of the cells.
[0101]Such asimage 3 Shown: It is the expression of the four T cell CARs on day 6. It can be seen that the antibody has no significant effect on the CAR positive rate.

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