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Targeted PD-L1 mosaic antigen receptor modified T lymphocyte as well as preparation method and application thereof

A chimeric antigen receptor, lymphocyte technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, botanical equipment and methods, etc., to achieve the effect of eliminating immunosuppressive effects

Inactive Publication Date: 2017-11-24
SHILI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the integration of PD-L1 antibody into the CAR structure to express it in T cells and the construction of PD-L1-CAR-T cells for CAR-T cell immunotherapy have not yet been reported.

Method used

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  • Targeted PD-L1 mosaic antigen receptor modified T lymphocyte as well as preparation method and application thereof
  • Targeted PD-L1 mosaic antigen receptor modified T lymphocyte as well as preparation method and application thereof
  • Targeted PD-L1 mosaic antigen receptor modified T lymphocyte as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Determination of the PD-L1-CAR gene sequence

[0037] 1.1 The GM-CSF signal peptide gene, human CD8α hinge region gene, human CD8α transmembrane region, 4 - 1BB co-stimulatory signal domain and CD3ζ intracellular signal domain gene sequence. The anti-PD-L1 single-chain antibody (anti-PD-L1 scFv) gene sequence comes from a patent (patent publication number: CN105793288A). See the sequence listing for the sequence information of each gene.

[0038] 1.2 The above gene sequences were sequentially linked according to the human GM-CSF signal peptide gene, anti-PD-L1 scFv, human CD8α hinge region gene, human CD8α transmembrane region, 4-1BB co-stimulatory signal domain and CD3ζ intracellular signal domain gene sequence, Form the final complete PD-L1-CAR gene sequence (the nucleotide sequence of PD-L1-CAR) information.

Embodiment 2

[0039] Example 2 Construction of PD-L1-CAR expression plasmid

[0040] 2.1 Whole gene synthesis:

[0041] The complete PD-L1-CAR sequence is synthesized from the whole gene, and enzyme cleavage sites are added at both ends.

[0042] 2.2 Use primers PD-L1-F and PD-L1-R to amplify the complete PD-L1-CAR gene sequence by PCR. The PCR system is:

[0043]

[0044] PCR reaction conditions: 94°C for 4 minutes; 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 90 seconds, 34 cycles; 72°C for 5 minutes;

[0045] Run the PCR product on an agarose gel for fragment size identification, and perform gel recovery to obtain the complete PD-L1-CAR gene sequence.

[0046] 2.3 Cloning the above sequence into pCDH-CMV-MCS-EF1-copGFP-T2A-puro (pCDH for short) lentiviral expression vector to obtain the expression plasmid of anti-PD-L1-CAR.

[0047] Respectively digest PD-L1-CAR and lentiviral expression vector pCDH with XbaI / EcoRI, the enzyme digestion system is as follows, 37 ° C water bath...

Embodiment 3

[0054] Packaging, concentration and titer determination of embodiment 3 lentivirus

[0055] 3.1 Packaging of lentivirus:

[0056] 3.1.1 Cell treatment: 24 hours before transfection, collect 293T cells at passage 3-10 in logarithmic growth phase, inoculate 5×10^6 293T cells in a 10cm culture plate, and store the cells in DMEM containing 10ml 10% FBS Grow in culture medium at 37°C with 5% CO 2 Cultivate in the incubator for 24 hours, and transfection can be carried out when the density reaches 60-80%.

[0057] 3.1.2 Co-transfect the lentiviral vector plasmid (pCDH-PD-L1-CAR or its empty vector pCDH) and its packaging plasmid pLP1, pLP2, pLPVSV-G into the cells at a ratio of 5:2:2:2; The dyeing system is as follows:

[0058]

[0059] After 6-8 hours, replace the medium in the 10 cm culture dish with complete medium.

[0060] 3.1.3 24 hours after transfection, the expression of GFP fluorescence in 293T cells after transfection was observed under a fluorescent microscope. C...

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Abstract

The invention relates to the technical field of biology and particularly discloses targeted PD-L1 mosaic antigen receptor modified T lymphocyte as well as a preparation method and an application thereof. The T lymphocyte surface of the targeted PD-L1 mosaic antigen receptor modified T lymphocyte expresses a targeted PD-L1 mosaic antigen receptor PD-L1-CAR. The amino acid sequence of the targeted PD-L1 mosaic antigen receptor PD-L1-CAR is shown in SEQ ID NO.2. The targeted PD-L1 mosaic antigen receptor modified T lymphocyte can have a specific killing effect on PD-L1 positive tumor through mediation by specifically recognizing tumor cell surface PD-L1 in a body and can prevent PD-1 / PD-L1 immunosuppression signal channels to eliminate the immunosuppression function on tumor, and the purpose of specific killing of tumor is achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a modified T lymphocyte targeting PD-L1 chimeric antigen receptor and its preparation method and application. Background technique [0002] With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) (June CH, Blazar BR, Riley JL. Engineering lymphocyte subsets: tools, trials and tribulations. Nat Rev Immunol. 2009; 9: 704-16) has become one of the most promising tumor immunotherapy. So far, CAR-T cell immunotherapy has achieved good results in the treatment of blood tumors, lymphoma and glioma. For solid tumors, since it is difficult for CAR-T cells to break through the tumor immunosuppressive microenvironment of solid tumors, CAR-T cell immunotherapy needs to be further optimized. [0003] Generally, a chimeric antigen receptor CAR consists of a tumor-associated antigen-binding region, an extracellular hinge regi...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K35/17A61P35/00
CPCC12N5/0636A61K35/17C12N15/86C12N2510/00C12N2740/15043
Inventor 樊克兴高同同
Owner SHILI BIOTECH CO LTD
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