CAR-T (chimeric antigen receptor-T) carrier targeted at EGFRvIII to interfere PD-1 and reduce tumor immune escape and construction method and application

A PD-1, immune escape technology, applied in the field of medical biology, to achieve the effect of reducing immune escape, improving curative effect, and enhancing killing

Inactive Publication Date: 2018-06-26
上海生博生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] At present, there are no reports about CAR-T vectors that can

Method used

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  • CAR-T (chimeric antigen receptor-T) carrier targeted at EGFRvIII to interfere PD-1 and reduce tumor immune escape and construction method and application
  • CAR-T (chimeric antigen receptor-T) carrier targeted at EGFRvIII to interfere PD-1 and reduce tumor immune escape and construction method and application
  • CAR-T (chimeric antigen receptor-T) carrier targeted at EGFRvIII to interfere PD-1 and reduce tumor immune escape and construction method and application

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Experimental program
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Embodiment 1

[0045] Below in conjunction with specific embodiment further elaborates this invention. It should be understood that the specific embodiments described herein are presented by way of example and not as limitations of the invention. The principal characteristics of this invention can be employed in various embodiments without departing from the scope of the invention. Example 1 Construction of CAR recombinant lentiviral vector

[0046] 1. Materials

[0047] 1. The lentiviral backbone plasmid pLenti-3G ​​basic (for the lentiviral vector structure, see figure 2 ), lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein plasmid pEnv-G, HEK293T / 17 cells, and homologous recombination enzymes were provided by Shiao (Shanghai) Biomedical Technology Co., Ltd.;

[0048]2. Human EF1α promoter (SEQ ID NO.1), CD8 leader chimeric receptor signal peptide (SEQ ID NO.2), EGFRvIII single-chain antibody light chain VL (SEQ ID NO.3), single-chain antibody hinge Linker (SEQ ID NO.4...

Embodiment 2

[0088] Example 2 Packaging of recombinant lentivirus lvCAR-EGFRvIII, lvCAR-EGFRvIII-NC-shRNA, lvCAR-EGFRvIII-PD1-shRNA.

[0089] (1) Complete medium: take out the preheated fresh medium, add 10% FBS + 5ml Pen-Srep, mix up and down;

[0090] (2) 1XPBS solution: Weigh NaCl 8g, KCl 0.2, NaCl 2 HPO 4 .12H 2 O 3.58g, KH 2Put 0.24g of PO4 in a 1000ml beaker, add 900ml Milli-Q grade ultrapure water to dissolve, after the dissolution is completed, use a 1000ml graduated cylinder to set the volume to 1000ml, and sterilize at 121℃ for 20min;

[0091] (3) 0.25% Trypsin solution: Weigh 2.5g of Trypsin, put 0.19729g of EDTA in a 1000ml beaker, add 900ml of 1XPBS to dissolve, after the dissolution is completed, use a 1000ml graduated cylinder to set the volume to 1000ml, 0.22μM filter sterilization, long-term use can be stored To -20 ℃ refrigerator;

[0092] (4) 0.5M CaCl2 solution: weigh 36.75g CaCl 2 Dissolve in 400ml Milli-Q grade ultrapure water; use Milli-Q grade ultrapure water ...

Embodiment 3

[0109] Example 3 Concentration and detection of recombinant lentiviral vector

[0110] 1. Purification of recombinant lentivirus by ion exchange chromatography

[0111] (1) Use a Thermo vacuum pump to filter the collected supernatant through a 0.22 μm-0.8 μm PES filter to remove impurities;

[0112] (2) Add 1.5M NaCl 250mM Tris-HCl (pH 6-8) to the supernatant at a ratio of 1:1 to 1:10;

[0113] (3) Place two ion exchange columns in series, and pass through the columns sequentially with 4ml 1M NaOH, 4ml 1M NaCl, 5ml 0.15M NaCl25mM Tris-HCl (pH6-8);

[0114] (4) The solution obtained in step 2 is loaded on the ion exchange column with a speed of 1-10ml / min by a peristaltic pump;

[0115] (5) After passing all the supernatant through the column, wash it once with 10ml 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution;

[0116] (6) Use 1-5ml 1.5M NaCl 25mM Tris-HCl (pH 6-8) for elution according to the loading amount, and collect the eluate;

[0117] (7) Divide the eluate into 25 to ...

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Abstract

The invention discloses a CAR-T carrier targeted at EGFRvIII to interfere PD-1 and reduce tumor immune escape, the CAR-T carrier comprises a human DF1 alpha promoter, PD-1shRNA in 3'UTR, a gene transfer carrier and a second generation CAR or third generation CAR, wherein the human DF1 alpha promoter is used for transcribing CAR genes and shown as SEQ ID NO. 1; and the sequence of the PD-1shRNA isshown as SEQ ID NO. 25; the second generation CAR or third generation CAR is used for integrating recognition, transmitting and starting and comprises an EGFRvIII single-chain antibody light chain VLand an EGFRvIII single-chain antibody heavy chain VH. Besides, the invention discloses a construction method of the carrier and application of the carrier in preparation of drugs for interfering the PD-1 to reduce tumor immune escape. According to the CAR-T carrier targeted at the EGFRvIII to interfere the PD-1 and reduce tumor immune escape and the construction method and the application, the CAR-T technology aiming at the SGFRvIII is used, tumour cell immune escape is inhibited by interfering the expression of the PD-1 in the T lymphocyte, killing to the tumour cells by the T lymphocyte is enhanced, and accordingly, the glioma resistance effect of the CAR-T immunotherapy is improved.

Description

technical field [0001] The invention belongs to the field of medical biology, and in particular relates to a carrier, in particular to a CAR-T carrier targeting EGFRvIII to interfere with PD-1 and reduce tumor immune escape. In addition, the present invention also relates to the construction method and application of the carrier. Background technique [0002] With the development of tumor immunology theory and technology, the role of immune cell therapy in tumor treatment has been paid more and more attention. Studies have found that T lymphocytes are the natural enemies of tumor cells, play a major role in tumor immune response, and have a strong killing effect on tumor cells. [0003] Chimeric Antigen Receptor T-Cell Immunotherapy (Chimeric Antigen Receptor T-Cell Immunotherapy, CAR-T immunotherapy) is a new type of cellular immunotherapy technology that has developed rapidly in recent years [Eleanor J.Cheadle, et al.CAR T cells : driving the road from the laboratory to ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/867A61K48/00A61K39/395A61K35/17A61P35/00
CPCA61K35/17A61K39/3955A61K48/005A61K2039/53C12N15/85C12N15/867C12N2740/15043C12N2800/107
Inventor 吴小江王聪顾莉萍
Owner 上海生博生物医药科技有限公司
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