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Application of hypoxia-adjustable promoter in CAR-T

A promoter and late promoter technology, applied in the direction of animal cells, vertebrate cells, genetically modified cells, etc., can solve the problems that affect the efficacy of CAR-T cells, the effect is not as good as that of blood system tumors, and CAR-T is difficult

Pending Publication Date: 2020-09-22
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The full name of CAR-T is chimeric antigen receptor T cell immunotherapy, which has achieved good results in hematological tumors, but the effect of CAR-T in solid tumors is not as good as that of hematological tumors. It is difficult to enter the interior of solid tumors. On the one hand, even if CAR-T cells enter the interior of solid tumors, they cannot function normally due to the tumor microenvironment. These all affect the efficacy of CAR-T cells in the treatment of solid tumors; High, solid tumor targets are often expressed in normal tissues, so there are safety issues such as off-target risks
Although there are currently constructions of fourth-generation CARs, dual CARs (constructing CAR-T with dual antigens) or iCARs with activation and inhibition functions in order to improve the effectiveness and safety of tumor CAR-T therapy, but These CAR structures still have problems of safety or difficulty in being activated

Method used

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  • Application of hypoxia-adjustable promoter in CAR-T
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  • Application of hypoxia-adjustable promoter in CAR-T

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1 plasmid construction

[0087] The plasmids were constructed according to the conditions shown in Table 2 for subsequent experiments. The plasmids were constructed using the mini promoter miniCMV, lentiviral expression vector, antigen recognition region CD19 and PSCA as examples.

[0088] The synthetic hypoxia promoter sequence 5HRE-CMVmini promoter nucleotide sequence is shown in SEQ ID NO: 1, and the promoter EcoRV-5HRE-CMVmini promoter-NheI (5H1P) matched with the lentiviral vector is further constructed, and the nucleotide sequence is shown in SEQ ID NO: 2 As shown, the targeted PSCA-CAR nucleic acid sequence is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:5; the targeted CD19-CAR nucleic acid sequence is shown in SEQ ID NO:4, and the amino acid sequence is shown in SEQ ID NO:4 Shown in ID NO:6; Targeted CEA-CAR amino acid sequence is shown in SEQ ID NO.42. The fragments were cut and recovered by double enzyme digestion, the g...

Embodiment 2

[0092] Example 2 In vitro hypoxia model verification

[0093] CoCl 2 Induced hypoxia principle: cobalt dichloride (CoCl 2 ) in the divalent cobalt ions (Co2+) can replace the PHD cofactor divalent iron ions, because Co2+ has a low affinity for oxygen, so that heme cannot combine with oxygen and cannot change into an oxidized state, thus forming a hypoxic state.

[0094] 1) Hypoxia induction scheme verification:

[0095] PBKL1-5HRE-GFP virus infected and activated PBMCs were used to construct hypoxic cell models in vitro, and the medium was changed after 12-18 hours of culture. Using CoCl 2 Induce a hypoxic environment, and cultivate until the 4th day to verify whether the hypoxic model has been constructed by green fluorescence detection, and set up the control group (without CoCl 2 ), the experimental group (plus CoCl 2 ), CoCl 2 After 24 hours of drug treatment, fluorescent microscope photographs were taken to detect the expression intensity of GFP, and the positive ra...

Embodiment 3

[0099] Example 3 Preparation of lentivirus and infection of T lymphocytes

[0100] In this example, the calcium phosphate method was used to package the lentivirus, specifically: 293T cells were cultured in DMEM medium to an optimal state, and the cells were replaced in advance. Add the packaging plasmid (RRE:REV:2G) and the expression plasmid to a 1.5 centrifuge tube in a certain ratio, and add 2.5mol / LCaCl 2 , add ddH 2 O to a total volume of 600 μL, mix well, and add 2×HBS dropwise to the mixture with a pipette, mix well, let stand at room temperature for 15 minutes, add to the treated 293T cell culture medium, and change again after 3-5 hours Liquid to 10mL DMEM medium containing 10% FBS, after 48h or 72h, the cell supernatant was collected, the virus was purified, and the titer was determined.

[0101] Lymphocytes were separated by gradient centrifugation; after centrifugation, the second white lymphocyte layer was taken, washed with saline, and cultured in RPMI 1640 co...

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Abstract

The invention belongs to the technical field of immunotherapy, and particularly relates to application of a hypoxia-adjustable promoter in CAR-T, a CAR structure of the hypoxia-adjustable promoter, anexpression vector, CAR-T cells, application of the CAR-T cells, a method for improving the IFN-gamma and / or IL-2 factor secretion capacity of the CAR-T cells in a hypoxia environment, and a method for improving the CAR-T cell killing capacity in the hypoxia environment. The CAR structure contains the hypoxia-adjustable promoter, the hypoxia-adjustable promoter is formed by connecting Hif1a adjusting elements and mini promoters, and the number of repetitions of the Hif1a adjusting elements is 3-5. The CAR structure induced and started by the hypoxia microenvironment not only can be effectivelyexpressed in T lymphocytes, but also can enhance the activation of the CAR-T cells in the hypoxia environment and enhance the effectiveness and safety of the CAR-T, and can be used for targeted therapy of tumors.

Description

technical field [0001] The invention belongs to the technical field of immunotherapy, and specifically relates to the application of a hypoxia-regulatable promoter in CAR-T, a CAR structure of a hypoxia-regulatable promoter, an expression vector, CAR-T cells and applications thereof As well as a method for improving the ability of CAR-T cells to secrete IFN-γ and / or IL-2 factors in a hypoxic environment and a method for improving the killing ability of CAR-T cells in a hypoxic environment. Background technique [0002] The full name of CAR-T is chimeric antigen receptor T cell immunotherapy, which has achieved good results in hematological tumors, but the effect of CAR-T in solid tumors is not as good as that of hematological tumors. It is difficult to enter the interior of solid tumors. On the one hand, even if CAR-T cells enter the interior of solid tumors, they cannot function normally due to the tumor microenvironment. These all affect the efficacy of CAR-T cells in the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17C07K19/00C12N5/10C12N15/63A61P35/00A61P35/02
CPCA61K35/17C07K14/7051C07K16/30C12N5/0636A61P35/00A61P35/02C07K2319/33C12N2510/00
Inventor 张巍朱秀秀赵永春赵文旭陈军黄霞
Owner CHONGQING PRECISION BIOTECH CO LTD
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