Chimeric antigen receptor immune cell as well as preparation method and application thereof

A chimeric antigen receptor and immune cell technology, applied in the field of chimeric antigen receptor immune cells and its preparation, can solve the problems of T cell exhaustion, difficulty in obtaining therapeutic effect, limited number of TILs, etc., and avoid immune cell apoptosis , enhanced lethality, moderate affinity effect

Pending Publication Date: 2018-09-04
SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, simply taking antibody drugs targeting the PD-1 pathway relies on T cells (Tumor infiltration T cells, TILs) infiltrating into the tumor microenvironment to kill tumor cells. The number of TILs is limited, and it is difficult to obtain a good therapeutic effect. In addition, Antibody drug therapy needs to be taken continuously. After stopping the drug, T cells will be exhausted immediately. This program cannot fundamentally reverse the fate of T cells

Method used

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  • Chimeric antigen receptor immune cell as well as preparation method and application thereof
  • Chimeric antigen receptor immune cell as well as preparation method and application thereof
  • Chimeric antigen receptor immune cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Embodiment 1 plasmid construction

[0115] (1) if figure 1 Shown are anti-PD-L1 chimeric antigen receptor (PD1.BBT2), anti-Mesothelin chimeric antigen receptor (Meso.28zT2), anti-PSCA chimeric antigen receptor (PSCA.28zT2), anti-MUC1 chimeric antigen receptor (MUC1.28zT2), anti-HER2 chimeric antigen receptor (HER2.28zT2) and anti-GPC3 chimeric antigen receptor (GPC3.28zT2) molecular structure, synthetic nucleic acid sequence of the above chimeric antigen receptor, such as SEQ ID As shown in NO.1-15, the 3' end of all synthetic sequences contains a restriction endonuclease PmeI cutting site, and the 5' end contains a restriction endonuclease SpeI cutting site;

[0116] (2) Restriction endonucleases PmeI and SpeI were used to double-digest the nucleic acid sequences of the lentiviral expression vector pwpxld-eGFP and the synthetic chimeric antigen receptor;

[0117] (3) Agarose gel electrophoresis to recover target genes containing cohesive ends: PD1.BBT2, Meso.28zT2, P...

Embodiment 2

[0119] Example 2 lentiviral packaging

[0120] Use 293T cells for virus packaging, and when the cell confluence reaches 80-90%, perform lentivirus packaging:

[0121] (1) 2 hours before virus packaging, the cell culture medium was replaced with DMEM containing 1% fetal bovine serum, and the amount added was 6mL / 100mm culture dish;

[0122] (2) Add the plasmids to 500 μL opti-MEM medium according to Table 1, wherein the pWPXLd-CAR-eGFP plasmids are pwpxld-PD1.BBT2, pwpxld-Meso.28zT2, pwpxld-PSCA.28zT2, pwpxld-MUC1.28zT2, Either of pwpxld-HER2.28zT2 or pwpxld-GPC3.28zT2;

[0123] (3) Add 36 μg PEI to another 500 μL opti-MEM medium, mix well, and let stand at room temperature for 5 minutes;

[0124] (4) After mixing the plasmid and PEI, pipette and mix well, and let stand at room temperature for 25-30min;

[0125] (5) Add the mixed solution dropwise to the 293T cells, and the 293T cells are cultured in a 100mm culture dish;

[0126] (6) After 6 hours, the culture medium was r...

Embodiment 3

[0131]Example 3 Preparation of CAR-T cells

[0132] (1) Peripheral blood mononuclear cells (PBMC) were separated from whole blood using Ficoll density gradient centrifugation kit (GE Company);

[0133] (2) Using the Pan T cell magnetic bead sorting kit (Miltenyi Company) to separate T cells from PBMC;

[0134] (3) CD2 / CD3 / CD28 T cell activation expansion kit (Miltenyi Company) was used to activate T cells for 36 hours;

[0135] (4) After T cells were activated for 36 hours, discard the magnetic beads, resuspend T cells in 1640 medium containing 10% fetal bovine serum, 1000IU / mL IL2, add lentivirus according to Table 2, and add lentivirus every 10 6 Add 1mL lentivirus and 8μg / mL polybrene to each T cell, at 37℃, 5%CO 2 Infect twice in the incubator, each interval 8-12h;

[0136] (5) After virus infection, T cells were resuspended in 1640 medium containing 10% fetal bovine serum and 1000IU / mL IL-2, and every 10 6 Add 1mL lentivirus to each T cell, add fresh medium every 48h,...

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Abstract

The invention provides a chimeric antigen receptor immune cell as well as a preparation method and application thereof. An anti-PD-L1 chimeric antigen receptor is expressed on the surface of the chimeric antigen receptor immune cell, and an extracellular antigen binding region of the anti-PD-L1 chimeric antigen receptor comprises PD-1 extracellular fragment and PD-1 signal peptides. The anti-PD-L1chimeric antigen receptor is expressed on the surface of the chimeric antigen receptor immune cell, so that immune cell depletion caused by binding of the PD-1 on the surface of the immune cell and PD-L1 on the surface of tumor cells is avoided, an inhibiting signal initiated by a PD-1 channel is transformed into an immune cell activating signal, and the killing ability of the chimeric antigen receptor immune cell on tumor cells is obviously enhanced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a chimeric antigen receptor immune cell and its preparation method and application. Background technique [0002] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) immunotherapy is a popular immunotherapy method for hematological tumors in recent years, mainly through chimeric antigen receptor T cells (CAR-T). Recognition and killing of cancer cell antigens. In recent years, CAR-T immunotherapy has achieved excellent therapeutic effects in multiple clinical trials. According to reports, after CAR-T immunotherapy, 90% of acute lymphoblastic leukemia patients were completely remission (S.L.Maude et al.Chimeric antigen receptor T cells for sustained remissions in leukemia.Engl.J.Med.,371,1507-1517( 2014)). The success of targeting CD19 antigen in the treatment of B lymphocyte leukemia and lymphoma is exciting. In 2017, the US Food and Drug Administration successively approved Nova...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/62C12N15/867A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K14/70521C07K14/70578C07K14/70596C07K16/303C07K16/3069C07K16/3092C07K16/32C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 李鹏秦乐魏新茹赵若聪程琳姚瑶
Owner SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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