Tumor cell dryness restriction-type CAR and application thereof

A tumor cell, restricted technology, applied in the field of tumor cell immunotherapy, which can solve problems such as unsatisfactory, residual tumor stem cells, recurrence, etc.

Active Publication Date: 2020-07-17
赛德特生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of this technology in solid tumors is still unsatisfactory. There are still many problems to be solved, and there are still many limitations in clinical use. One of the important problems is the recurrence caused by residual tumor stem cells after treatment.

Method used

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  • Tumor cell dryness restriction-type CAR and application thereof
  • Tumor cell dryness restriction-type CAR and application thereof
  • Tumor cell dryness restriction-type CAR and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Based on the existing CAR technology research, in order to further improve the application effect of CAR-T in the treatment of solid tumors, the inventors designed a new CAR structure, the specific structure diagram is as follows figure 1 shown.

[0065] in particular:

[0066] NKG2D-41BB-CD3z is: human CD8a molecular signal peptide (CD8a), human NKG2D extracellular region, human CD8 molecular transmembrane region and 41BB molecular intracellular region (41BB), human CD3z molecular intracellular region (CD3Zeta) connected in sequence ), namely: CD8a-NKG2D-41BB-CD3Zeta (CD3ζ);

[0067] NKG2D -41BB-CD3z-IL24 is: human CD8a molecular signal peptide (CD8a), human NKG2D extracellular region, human CD8 molecular transmembrane region and 41BB molecular intracellular region (41BB), human CD3z molecular intracellular region connected in sequence (CD3Zeta), PA2 junction sequence (P2A), IL22 CDS region sequence (IL22); namely: CD8a-NKG2D-41BB-CD3Zeta (CD3ζ)-P2A-IL24.

[0068] T...

Embodiment 2

[0090] On the basis of Example 1, the inventors further constructed a lentiviral expression vector, so that it can be further used to infect T cells and prepare CAR-T. The specific construction process of the lentiviral expression vector is introduced as follows.

[0091] (1) According to the central dogma, using existing technology to obtain the coding sequence DNA of the CAR;

[0092] (2) Using the pCDH-EF1-conGFP plasmid as the expression vector, use the restriction endonuclease EcoR I (NEB) to digest it at 37°C, and then use In-Fusion HD Cloning Kits (Bao Biology) to convert the step (1 The coding sequence DNA in ) was integrated and recombined into the pCDH-EF1-conGFP plasmid;

[0093] (3) Transform the ligation product in step (2) into STABL3 competent cells, screen, expand and culture, and further extract the plasmid to obtain recombinant lentiviral expression that can express NKG2D-41BB-CD3z or NKG2D-41BB-CD3z-IL24 Plasmids (respectively named: pCDH-EF1-NKG2D-41BB-CD3...

Embodiment 3

[0096] On the basis of Example 2, the inventors further transfected the constructed lentiviral expression plasmid into CAR-T cells, and further conducted preliminary cell experiments. The specific experimental process is briefly introduced as follows.

[0097] (1) Lentiviral packaging

[0098] 293T cells are used as the primary target cells to be transfected, specifically:

[0099] First, six-well plates were plated and incubated for 24 hours to culture 293T cells (37°C, DMEM complete medium);

[0100] Subsequently, the medium was replaced with OPTI-MEM medium, and then 1.5 g of the lentiviral expression plasmid constructed in Example 2, 1.5 g of the packaging plasmid psPAX2, and 1 g of Pmd2.G, and 8 μl of liposome transfection reagent were carried out. Mix to transfect target cell 293T;

[0101] Replace with normal DMEM medium 12 hours after transfection;

[0102] Finally, after continuing to culture for 48 hours, the virus supernatant (that is, the packaged virus-like pa...

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Abstract

The invention belongs to the technical field of tumor cell immunotherapy, and particularly relates to a restriction-type CAR capable of inhibiting the dryness of tumor cells and application of the CAR. The chimeric antigen receptor is an amino acid sequence obtained by connecting a plurality of protein fragments, and is obtained by sequentially connecting a human CD8a molecule signal peptide CD8a,a human NKG2D extracellular region, a human CD8 molecule transmembrane region and a 41BB molecule intracellular region 41BB, and a human CD3z molecule intracellular region CD3Zeta. In the preferred design, the chimeric antigen receptor is further obtained by connecting a connecting sequence to an IL24 CDS region sequence IL24. By further optimizing the structure of the CAR and simultaneously in combination with SFN application, the chimeric antigen receptor has good technical effects on effectively killing tumor cells and tumor stem cells, reducing the recurrence of diseases and improving theapplication effect of CAR-T cells, so that the has good practical value and popularization and application significance.

Description

technical field [0001] This application belongs to the technical field of tumor cell immunotherapy, and specifically relates to a restricted CAR (Chimeric Antigen Receptor, Chimeric Antigen Receptor) capable of inhibiting tumor cell stemness and application for a patent for its application. Background technique [0002] As an effective treatment for tumor immunotherapy, CAR-T (Chimeric Antigen Receptor T-Cell, Chimeric Antigen Receptor T-Cell) has received more research and attention in recent years. Clinically, with CD19 as the target, use CAR-T to clear CD19 + Tumor cells have shown good effects in the treatment of acute lymphoblastic leukemia (ALL). However, the application of this technology in solid tumors is still unsatisfactory. There are still many problems to be solved, and there are still many limitations in clinical use. One of the important problems is the recurrence caused by residual tumor stem cells after treatment. [0003] MDA-7 / IL-24 (melanoma differentia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/867A61K39/00A61P35/00A61K31/26
CPCC07K14/7056C07K14/70578C07K14/7051C07K14/70517C07K14/54C12N15/86A61K39/00114A61K31/26A61P35/00C07K2319/02C07K2319/03C07K2319/00C12N2740/15043C12N2800/107A61K2039/5156A61K2039/86A61K2300/00
Inventor 张毅张凯温纯利
Owner 赛德特生物制药有限公司
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