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Chimeric antigen receptor for targeting CD19 and interferon synergy and application of chimeric antigen receptor

A single-chain antibody and fusion protein technology, which is applied in interferon, targeted specific cell fusion, medical preparations containing active ingredients, etc., can solve toxic and side effects, increase the dosage of CAR-T cells, and prevent tumor recurrence And other issues

Active Publication Date: 2020-11-03
CARBIOGENE THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although CAR-T cell therapy has shown great advantages in the field of hematological tumors compared with traditional therapies, its existing problems still cannot be avoided
For example, for some patients with poor T cell activity, CAR-T cells have poor proliferation ability and low tumor cell killing efficiency
In addition, most of the existing CAR-Ts generally show the defect of insufficient survival ability in vivo, which is easy to cause tumor recurrence
Blindly increasing the dose of CAR-T cells can easily cause strong toxic side effects, such as inflammatory factor storm and central nervous system toxicity

Method used

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  • Chimeric antigen receptor for targeting CD19 and interferon synergy and application of chimeric antigen receptor
  • Chimeric antigen receptor for targeting CD19 and interferon synergy and application of chimeric antigen receptor
  • Chimeric antigen receptor for targeting CD19 and interferon synergy and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Determination of CD19-CAR-EGFRt-IFNα2b gene sequence and construction of retroviral vector

[0065] The full-length cDNA sequence information of human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3ζ intracellular region, EGFRt and human IFNα2b was searched from the NCBI website database. The full-length sequence of wild-type human IFNα2b gene cDNA is called nIFNα2b. The nIFNα2b sequence was codon-optimized on the website http: / / sg.idtdna.com / site to obtain oIFNα2b, ensuring that it is more suitable for expression in human cells when the encoded amino acid sequence remains unchanged.

[0066] According to the sequence of CD19 scFv, human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3ζ intracellular region, human P2A peptide, EGFRt, human P2A peptide, oIFNα2b, the full-length polynucleoside of CD19-CAR-EGFRt-IFNα2b was obtained acid sequence. At the same time, the full-length polynucleotide sequence of...

Embodiment 2

[0071] Embodiment 2: Retrovirus packaging and establishment of toxin-producing strains

[0072] Using the retroviral vector comprising CD19-CAR-EGFRt-IFNα2b and CD19-CAR-EGFRt prepared in Example 1, the two retroviruses were packaged according to the following method:

[0073] 1. Day 1: Phoenix Ecotropic (ECO) cells should be less than 20 passages and not overgrown. Take 0.6×10 6 / ml cell density plating, add 10ml DMEM medium to a 10cm dish, mix the cells well, and culture overnight at 37°C;

[0074] 2. Day 2: ECO cell confluence reaches about 90% for transfection (usually about 14-18 hours after plating); prepare plasmid MP71-target gene 12.5μg, 1.25M CaCl 2 250 μl, H 2 O 1ml, the total volume is 1.25ml; add 2× HBS equal to the volume of the plasmid complex in another tube, and vortex for 20 seconds while adding the plasmid complex. Gently add the mixture to the ECO dish along the side, incubate at 37°C for 4 hours, remove the medium, wash with PBS, and add pre-warmed f...

Embodiment 3

[0077] Example 3: Retrovirus infection of human T cells

[0078] 1. Throw frozen PBMCs from healthy human peripheral blood, and adjust the cell density to 1-2×10 with RPMI-1640 complete medium containing 10% FBS 6 / ml.

[0079] 2. Ficoll separation solution (Tianjin Haoyang) was used to collect PBMC, and the magnetic bead method was used to obtain relatively pure CD3 + T cells, by magnetic beads: CD3 + T cells were activated by adding clinical grade Dynabeads Human T Expander CD3 / CD28 magnetic beads (Invitrogen) at a cell ratio of 3:1.

[0080] 3. On the second day after T cell activation, coat non-tissue-treated culture plates with Retronectin (Takara) diluted in PBS to a final concentration of 15 μg / ml, 1.2 ml per well of a 6-well plate. Protected from light, overnight at 4°C for later use.

[0081] 4. After T cell activation and culture for two days, take out the coated 6-well plate, discard the coating solution, and add PBS to wash the plate once.

[0082] 5. Add the re...

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Abstract

The invention relates to the field of chimeric antigen receptors, discloses a chimeric antigen receptor for targeting CD19 and interferon synergy and an application of the chimeric antigen receptor, and particularly discloses a polynucleotide sequence which is selected from (1) a coding sequence containing anti-CD19 single-chain antibodies, a coding sequence of a human CD8 hinge transmembrane region, a coding sequence of a human 4-1BB intracellular region, a coding sequence of a human CD3 zeta intracellular region, a coding sequence of a human P2A peptide, a coding sequence of an EGFRt gene, acoding sequence of the human P2A peptide and a human IFN full-length sequence, which are connected in sequence; and (2) a complementary sequence of the polynucleotide sequence in (1). The invention also discloses a related fusion protein, a nucleic acid construct, a retrovirus and a genetically modified T cell, and applications thereof to preparation of drugs for treating CD19-mediated diseases.

Description

technical field [0001] The present invention relates to the field of chimeric antigen receptors, in particular to a chimeric antigen receptor comprising CD19-CAR and secreting interferon and its application. Background technique [0002] Chimeric antigen receptor T (CAR-T) cell therapy is one of the major breakthroughs in immunomedicine in recent years. The basic principle of CAR-T technology is to genetically engineer the tumor patient's own T cells so that the T cells express the CAR gene, transform them into CAR-T cells that can specifically attack the tumor, and then infuse them back into the patient's body to ablate the tumor. CAR-T technology was first designed and reported by Israeli scientist Eshhar Zelig in 1993, and has been continuously optimized and perfected in the following two decades. In the presence of antigens, T cells need to receive three signals in a certain order to be fully activated, and to proliferate and differentiate normally. These three signals...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/867C12N7/01C12N5/10C07K19/00A61K39/00A61P35/00
CPCC07K16/2803C07K14/7051C07K14/71C07K14/555C12N5/0636C12N15/86A61K39/001104A61K39/001141A61P35/00C07K2319/02C07K2319/03C07K2319/33C07K2317/622C12N2510/00C12N2730/00043A61K2039/804
Inventor 朱建高杨文君
Owner CARBIOGENE THERAPEUTICS CO LTD
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