CAR-T vector capable of targeting Her2 and blocking PD-L1 to reduce tumor immune escape and construction method and use thereof

A technology of PD-L1 and immune escape, which is applied in the construction method and application of the carrier, in the field of CAR-T carrier, 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

AI Technical Summary

Problems solved by technology

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

Method used

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  • CAR-T vector capable of targeting Her2 and blocking PD-L1 to reduce tumor immune escape and construction method and use thereof
  • CAR-T vector capable of targeting Her2 and blocking PD-L1 to reduce tumor immune escape and construction method and use thereof
  • CAR-T vector capable of targeting Her2 and blocking PD-L1 to reduce tumor immune escape and construction method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of CAR recombinant lentiviral vector

[0047] 1. Materials

[0048] 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.;

[0049] 2. Human EF1α promoter (SEQ ID NO.1), CD8leader chimeric receptor signal peptide (SEQ ID NO.2), Her2 single-chain antibody light chain VL (SEQ ID NO.3), single-chain antibody hinge Linker ( SEQ ID NO.4), Her2 heavy chain VH (SEQ ID NO.5), CD8 Hinge chimeric receptor hinge (SEQ ID NO.6), CD8 Transmembrane chimeric receptor transmembrane region (SEQ ID NO.7), CD137 Chimeric receptor costimulator (SEQ ID NO.8), TCR chimeric receptor T cell activation domain (SEQ ID NO.9), IRES ribosome binding sequence (SEQ ID NO.10), PD-L1 single chain antibody Light chain VL (SEQ...

Embodiment 2

[0080] Example 2 Packaging of recombinant lentivirus lvCAR-Her2, lvCAR-Her2-IRES-PD-L1 scFv.

[0081] (1) Complete medium: take out the preheated fresh medium, add 10% FBS + 5ml Pen-Srep, mix up and down;

[0082] (2) 1XPBS solution: Weigh NaCl 8g, KCl 0.2, NaCl 2 HPO 4 .12H 2 O 3.58g, KH 2 PO4 0.24g

[0083] Place in a 1000ml beaker, add 900ml Milli-Q grade ultrapure water to dissolve, after the dissolution is complete, use a 1000ml graduated cylinder to set the volume to 1000ml, and sterilize at 121°C for 20 minutes;

[0084] (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;

[0085] (4) 0.5M CaCl2 solution: weigh 36.75g CaCl 2 Dissolve in 400ml Milli-Q grade ultrapure water; use Milli-Q grade ultrapure water to make the ...

Embodiment 3

[0099] Example 3 Concentration and detection of recombinant lentiviral vector

[0100] 1. Purification of recombinant lentivirus by ion exchange chromatography

[0101] (1) Use a Thermo vacuum pump to filter the collected supernatant through a 0.22 μm-0.8 μm PES filter to remove impurities;

[0102] (2) Add 1.5M NaCl 250mM Tris-HCl (pH 6-8) to the supernatant at a ratio of 1:1 to 1:10;

[0103] (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 (pH 6-8);

[0104] (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;

[0105] (5) After passing all the supernatant through the column, wash it once with 10ml 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution;

[0106] (6) Use 1-5ml 1.5M NaCl 25mM Tris-HCl (pH 6-8) for elution according to the loading amount, and collect the eluate;

[0107] (7) Divide the eluate into 25 to...

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Abstract

The invention discloses a CAR-T vector capable of targeting Her2 and blocking PD-L1 to reduce tumor immune escape. The CAR-T vector comprises a human EF1 alpha promoter for eukaryotic transcription ofa chimeric antigen receptor gene, wherein the human EF1 alpha promoter is shown in the formula of SEQ ID NO. 1, a second-generation CAR or third-generation CAR chimeric antigen receptor combining recognition, delivery and starting, wherein the chimeric antigen receptor comprises a Her2 single-chain antibody light chain VL and a Her2 single-chain antibody heavy chain VH, an IRES ribosome binding sequence shown in the formula of SEQ ID NO. 10, PD-L1 scFv and a gene transfer vector. The invention discloses a construction method of the vector and a use of the vector in preparation of drugs for reducing tumor immune escape. Through a Her2 CAR-T technology and PD-L1 scFv expressed in the T lymphocytes, the PD-L1 on the surface of the tumor cells is blocked, the interaction between PD-1 and PD-L1 is inhibited, tumor cell immune escape is inhibited and the T lymphocyte effects of killing tumor cells are improved so that the CAR-T immunotherapy effects of resisting brain glioma are 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 capable of targeting Her2 and blocking PD-L1 to 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 t...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/62A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/2827C07K16/2863C07K2317/622C07K2319/02C07K2319/33C07K2319/74C12N15/86C12N2740/15043
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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