CD19 and CD22 double-target chimeric antigen receptor T cell and application thereof

A technology of chimeric antigen receptor and CD22, which is applied in the field of biomedicine, can solve the problems of patient tumor recurrence and poor treatment effect of CAR-T cells, and achieve high-efficiency targeting effect, high-efficiency targeting activity and killing ability, and avoid The effect of immune escape

Active Publication Date: 2021-01-08
汤朝阳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, medical diagnosis shows that tumor cells in some hematological tumors do not express CD19 molecules, but express CD22 molecules. Only CAR-T cells targeting CD19 molecules are not effective, and some patients have tumor recurrence after a period of time Phenomenon

Method used

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  • CD19 and CD22 double-target chimeric antigen receptor T cell and application thereof
  • CD19 and CD22 double-target chimeric antigen receptor T cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of CAR Molecular Carrier

[0046] In this embodiment, the coding gene (SEQ ID NO: 4) of the anti-CD19 and CD22 dual-target chimeric antigen receptor is firstly synthesized, and the restriction endonuclease Pme1 restriction site is added to the C-terminal and N-terminal of the coding gene respectively and its protective base and restriction endonuclease Spe1 restriction site and its protective base;

[0047] The coding gene was double-digested with restriction endonucleases Pme1 and Spe1, and the digested product containing cohesive ends was recovered by agar gel electrophoresis, and then ligated into the linearized pWPXLd-eGFP plasmid (containing sticky end), the ligation reaction was carried out with the participation of T4 DNA polymerase (Invitrogent), and a lentiviral vector containing the coding gene of CAR targeting CD19 and CD22 dual targets was obtained.

[0048] In this example, CARs with antigen-binding domains of anti-CD19 scFv and CARs w...

Embodiment 2

[0049] Example 2 lentiviral packaging

[0050] In this example, the lentiviral vector constructed in Example 1 is used for lentiviral packaging, using a four-plasmid system, and the steps are as follows:

[0051] Mix the helper plasmids gag / pol, Rev and VSV-G with the recombinant vector in proportion, add it to a certain volume of serum-free DMEM, mix it and let it stand for 15 minutes; add the above mixture to the cell culture flask lined with 293T cells , mixed gently, at 37°C, 5% CO 2 Cultivate in the cell incubator for 6 hours; after 6 hours, replace the fresh medium, continue to cultivate, and add 10mM sodium butyrate solution; after 72 hours, collect the lentivirus culture supernatant for purification and detection.

[0052] Recombinant vectors include lentiviral vectors containing genes encoding CARs targeting both CD19 and CD22, lentiviral vectors containing genes encoding CARs targeting CD19 single targets, and lentiviral vectors containing genes encoding CARs target...

Embodiment 3

[0053] Example 3 Preparation of CAR-T cells

[0054] Peripheral blood mononuclear cells (PBMC) were separated from whole blood using Ficoll density gradient centrifugation kit (GE Company), and after red blood cells were removed, T cells were sorted out using MACS Pan-T magnetic beads;

[0055] The sorted T cells were diluted with medium (AIM-V medium + 5% FBS + penicillin 100 U / mL + streptomycin 0.1 mg / mL) to a cell concentration of 2.5×10 6 pcs / mL for use;

[0056] CD2 / CD3 / CD28 T cell activation expansion kit (Miltenyi Company) was used to activate T cells, that is, the coated magnetic beads were mixed with T cells at a ratio of 1:2, and the final density of T cells was 5×10 6 piece / mL / cm 2 , after mixing, place at 37°C, 5% CO 2 The incubator was stimulated for 48 hours;

[0057] After T cells were activated for 48 hours, demagnetize the beads, centrifuge at 300 g for 5 min, remove the supernatant, resuspend T cells with fresh medium, add recombinant lentivirus expressin...

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Abstract

The invention provides a CD19 and CD22 double-target chimeric antigen receptor T cell and application thereof. The chimeric antigen receptor T cell expresses a chimeric antigen receptor which is specifically combined with CD19 and CD22, wherein the chimeric antigen receptor comprises a signal peptide, an antigen binding structural domain, a transmembrane structural domain and a signal transductionstructural domain; the antigen binding structural domain comprises an anti-CD19 single-chain antibody and an anti-CD22 single-chain antibody; and the signal transduction structural domain is 4-1BB, CD3 zeta and TLR2. According to the invention, 4-1BB, CD3zeta and TLR2 which are connected in series are used as a signal transduction structural domain of the chimeric antigen receptor to construct the CD19 and CD22 targeting double-target CAR-T cell, and the CAR-T cell has efficient CD19 and CD22 targeting activity and killing ability, and is beneficial to avoiding immune escape phenomenon.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to CD19 and CD22 dual-target chimeric antigen receptor T cells and applications thereof. Background technique [0002] Chimeric antigen receptor (CAR) is a recombinant receptor targeting cell surface antigens, and its structure mainly includes three parts: extracellular antigen binding region, transmembrane region and intracellular signal region. Among them, the extracellular antigen-binding region is responsible for recognizing antigens, the transmembrane region connects the extracellular antigen-binding region and the intracellular signal region, which affects the expression ability of the imported CAR gene, and the intracellular signal region is responsible for signal transmission. [0003] Chimeric antigen receptor T cell (CAR-T) immunotherapy is a cellular immunotherapy method based on chimeric antigen receptors. The gene sequence of CAR (CAR) is transfected into T cells to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/62C12N15/867C12N7/01A61K39/00A61P35/00C12R1/93
CPCC12N5/0636C07K16/2803C07K14/7051C12N15/86C12N7/00A61K39/001112A61K39/001113A61P35/00C12N2510/00C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N2740/15043C12N2740/15021C12N2800/107A61K2039/5158
Inventor 汤朝阳秦乐吴迪冯世忠冯嘉昆王艳艳其他发明人请求不公开姓名
Owner 汤朝阳
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