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Immune cell expressing hyaluronidase and application thereof

A technology of hyaluronidase and immune cells, applied in the field of immune cells and its application in anti-tumor therapy, can solve the problem that the anti-tumor function of immune cells cannot be fully exerted, cannot specifically degrade hyaluronic acid, and cannot exert infiltration To achieve good tumor infiltration ability, enhance anti-tumor activity, and improve transduction efficiency

Active Publication Date: 2020-01-21
SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Malignant solid tumors will form a dense extracellular matrix (such as hyaluronic acid, collagen, heparan sulfate polysaccharides, etc.) to block immune cells in the tumor stroma and cannot reach the tumor parenchyma, so that the anti-tumor function of immune cells cannot be fully exerted
[0010] CN102307993A discloses an extended soluble PH20 polypeptide and its use; however, this method cannot specifically degrade hyaluronic acid at the targeted site
However, there are two types of hyaluronidase, free type and membrane-bound type. Among them, the free type hyaluronidase lacks a stable sequence, so the half-life is relatively short. According to the existing literature, the half-life of hyaluronidase PH20 is 3 minutes ; while the membrane-bound hyaluronidase not only lacks a stable sequence, but the transmembrane region, hinge region and intracellular segment in the design make the recombined hyaluronidase gene larger and reduce the transduction efficiency of the gene. Hyaluronidase is connected to T cells and cannot play an infiltration role at all in practical applications

Method used

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  • Immune cell expressing hyaluronidase and application thereof
  • Immune cell expressing hyaluronidase and application thereof
  • Immune cell expressing hyaluronidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Construction of Hyaluronidase Overexpression Plasmid

[0063] Such as figure 1 As shown, the hyaluronidase overexpression gene includes a signal peptide, a hyaluronidase catalytic activity sequence and a stable sequence, and the construction steps include:

[0064] (1) Obtain the DNA of each region required by the PH20 overexpression plasmid through gene synthesis, including: signal peptide, PH20 hyaluronidase catalytic activity region and stable sequence, and the fusion gene constructed is as follows: Figure 2A-Figure 2C As shown, wherein the signal peptide includes tPA signal peptide, IL-2 signal peptide, IL-7 signal peptide, IFNγ signal peptide, PH20 original signal peptide, GMCSF signal peptide, IL-6 signal peptide or IL-15 signal peptide; stable The sequence includes a human IgG1 Fc region sequence, a human IgG2 Fc region sequence, a human IgG3 Fc region sequence or a human IgG4 Fc region, and the amino acid sequence of the constructed fusion gene is shown in SEQ...

Embodiment 2

[0082] PH20 overexpression vector lentiviral packaging

[0083] (1) Cultivate 293T cells in a 10cm culture dish, the culture medium is: DMEM high glucose medium + 10% FBS (fetal bovine serum) + 1% double antibody (100 × penicillin-streptomycin mixed solution);

[0084] (2) When the 293T cell density in the culture dish reaches 80%, replace the medium: DMEM high glucose medium + 1% FBS + 1% double antibody;

[0085] (3) After replacing the culture medium for 2 hours, start step (4);

[0086] (4) Take 500 μL of opti-DMEM into a 15 mL centrifuge tube, and add 7.2 μL of PEI (linear polyethyleneimine) with a concentration of 10 μg / μL, mix slightly, and let stand for 5 minutes;

[0087] (5) Put 500 μL of opti-DMEM into a 15 mL centrifuge tube, take 9 μg of the target plasmid, 3 μg of the pMD2.G helper plasmid, and 12 μg of psPAX, add it to the centrifuge tube, and mix well;

[0088] (6) After step (4) is completed, mix the solution of step (5) with it, mix it upside down, and let ...

Embodiment 3

[0095] PH20 Gene Overexpression Lentivirus Transduced T Cells

[0096] (1) T cell activation: After sorting out PanT from umbilical cord blood, count the cells and adjust the concentration to 1×10 6 cells / mL, then add 10 μL of Miltenyi TransAct T cell reagent to each ml of cell suspension, and replace with fresh medium after 48 hours of activation (medium: IMDM medium + 5% FBS (fetal bovine serum) + 1% Double antibody (100×penicillin-streptomycin mixed solution)+IL-2);

[0097] (2) Convert 4.8×10 7 The activated T cells were centrifuged at 300g for 5min, and resuspended with 3mL medium (medium: IMDM medium + 5% FBS (fetal bovine serum) + 1% double antibody (100×penicillin-streptomycin mixed solution)) ;

[0098] (3) Inoculate 3 mL of T cell suspension in a 6-well plate, 1 mL per well;

[0099] (4) Add the packaged PH20 gene overexpression lentiviral vector to a 6-well plate, 9 mL per well;

[0100] (5) Add 10 μL polybreen to each well;

[0101] (6) After culturing for 8 ...

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Abstract

The present invention provides an immune cell expressing hyaluronidase and an application thereof. The immune cell comprises a fusion gene and the fusion gene mainly comprises a signal peptide, a hyaluronic acid catalytic active region, and a stable sequence in series. The signal peptide is the signal peptide of secreted proteins. The provided immune cell expressing the hyaluronidase can express and secrete the hyaluronidase as a protein, the hyaluronidase is secreted outside the cell to degrade extracellular matrix, in a natural state, half-life of the hyaluronidase is relatively short, so that hyaluronidase active sequence is fused with a signal peptide sequence and the stable sequence, which can extend the half-life of the hyaluronidase in body, promotes infiltration of the immune cellto specific tissues and thus increases functional activity of the immune cell against solid tumors.

Description

technical field [0001] The present invention relates to the field of tumor cell immunotherapy, in particular to an immune cell expressing hyaluronidase and its application, in particular to an immune cell overexpressing hyaluronidase and its application in anti-tumor therapy. Background technique [0002] Tumor immune cell therapy is a new type of tumor treatment method, which transforms immune cells by expanding body cells in vitro, cytokine treatment or gene modification, so that immune cells can gain anti-tumor ability and then reinfuse them into patients in vivo to remove tumors. [0003] There are many types of immune cells that can be used to treat tumors, including (1) T lymphocytes (Tlymphocytes), (2) Chimeric Antigen Receptor T cells (CAR-T), (3) T cell receptor modified T cells (T cell receptor, TCR-T), (4) natural killer cells (Natural killer cells, NK), (5) dendritic cells (Dendritic cells, DC), (6) NK-T Cells (Natural killersT cells, NKT), (7) tumor infiltrati...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62A61K38/47A61P35/00
CPCC12N5/0636C12N9/2474C12N9/6459C12Y304/21068C12Y302/01035A61K38/47A61P35/00C12N2510/00C07K2319/02C07K2319/30
Inventor 李鹏赵若聪崔元彬林思妙姚瑶汤朝阳
Owner SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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