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Novel method for enhancing efficiency of human T cells infected by slow-viruses carrying CAR (chimeric antigene receptor) genes

A lentivirus and cell technology, applied in the direction of retroRNA virus, virus, animal cells, etc., can solve the problems of low efficiency of CAR-T cell preparation, high cost, low efficiency of lentivirus infection of T cells, etc.

Active Publication Date: 2019-04-09
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although CAR-T technology has made breakthroughs, there are still some bottlenecks in the preparation of CAR-T cells. The efficiency of CAR-T cell preparation is low and the cost is high

Method used

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  • Novel method for enhancing efficiency of human T cells infected by slow-viruses carrying CAR (chimeric antigene receptor) genes
  • Novel method for enhancing efficiency of human T cells infected by slow-viruses carrying CAR (chimeric antigene receptor) genes
  • Novel method for enhancing efficiency of human T cells infected by slow-viruses carrying CAR (chimeric antigene receptor) genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Isolation and cryopreservation of human PBMC of embodiment 1

[0042] 1. Prepare experimental reagents: lymphocyte separation medium (room temperature ≈ 20°C); sterile saline (preheated to 37°C); cell freezing solution (precooled to 2-8°C).

[0043] 2. Transfer the clinically collected human peripheral blood sample into a 15ml sterile centrifuge tube, tighten the cap, adjust the centrifugal force of the centrifuge to 800g, set the centrifugal speed to the lowest, and centrifuge the blood sample at room temperature for 20 minutes.

[0044]3. Take out the centrifuge tube after centrifugation, avoid violent shaking or inverting the centrifuge tube, and transfer the upper light yellow serum layer to a new sterile centrifuge tube (after heat inactivation at 56°C for 1 hour, transfer the serum to -80°C Then add an equal volume of normal saline to the lower layer of red peripheral blood cells, tighten the cap of the centrifuge tube, and gently invert up and down to mix.

[00...

Embodiment 2

[0050] Example 2 Recovery, purification and activation of T cells

[0051] 1. Prepare T cell culture medium: 90% X-VIVO 15, 10% BI fetal bovine serum, and add IL-2, IL-15, IL-7, so that the final concentrations are 200U / ml, 5ng / ml, 5ng / ml.

[0052] 2. Take a frozen PBMC, melt in a 37°C water bath, add 10ml of rewarmed T cell culture medium, and centrifuge at 300g for 8min.

[0053] 3. Discard the supernatant, resuspend the cells with 3ml T cell culture medium, add DNaseI to make the working concentration 100μg / ml, votex, and let stand in the cell culture incubator for 15min.

[0054] 4. Filter with a 40 μm cell mesh, and count the filtered cells.

[0055] 5. Centrifuge the cell suspension at 300g for 8min.

[0056] 6. T cell purification: according to EasySep TM Instructions for the operation of the Human T Cell Isolation Kit, resuspend the cell pellet to 5×10^7 cells / ml and add corresponding volumes of Isolation Cocktail and RapidSpheres to purify CD3+ T cells.

[0057]...

Embodiment 3

[0059] Example 3 CCK-8 detects the effect of DMF on T cell proliferation

[0060] 1. Separation of T cells from Dynabeads: Transfer T cells activated for 72 hours to a 15ml centrifuge tube, and mix well by pipetting. Place the centrifuge tube in the EasySep TM Magnetic pole, stand at room temperature for 3 minutes, transfer the cell suspension to a new 15ml centrifuge tube, and count.

[0061] 2. Inoculate T cells in a 96-well plate at 1×10^5 cells / 100 μl / well, and treat them with different concentrations of DMF (0, 25 μM, 50 μM, 100 μM, 150 μM, 200 μM), and set 3 replicate wells for each concentration , continue to cultivate for 22 hours.

[0062] 3. Add 10 μl of CCK-8 to each well, and mix well by pipetting. The culture plate was incubated in the incubator for 1-4 hours, and the absorbance at 450 nm was measured with a microplate reader.

[0063] 4. Calculate the inhibition rate at different concentrations.

[0064] The result is as figure 1 As shown, the treatment und...

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Abstract

The invention relates to a novel method for enhancing the efficiency of infecting human T cells by slow viruses carrying CAR (chimeric antigene receptor) genes. A CAR-T technology is regarded as a technology for most possibly curing tumors; although the technology has breakthrough, bottleneck problems still existing in the aspect of preparation of CAR-T cells cannot be solved, for example, the innate immune response of T cells can enable the efficiency of infecting the T cells by the slow viruses carrying CAR genes to be low, so that the CAR-T cells are low in preparation efficiency and high in cost. For the problem of improving the efficiency of infecting the T cells by the slow viruses carrying CAR genes, the applicant conducts a series of experiments and improvement and discovers that the infection efficiency of the CAR slow viruses can be remarkably improved when the T cells are treated by DMF (dimethyl fumarate). The novel method has guiding significance and application values inthe aspects of greatly improving the preparation efficiency of the CAR-T cells, shortening the in vitro amplification time of the CAR-T cells, lowering the production cost of a CAR-T therapeutic method and the like.

Description

technical field [0001] The present invention relates to the field of immunotherapy, specifically, the present invention relates to a new method for enhancing the efficiency of human T cell infection by a lentivirus carrying a chimeric antigen receptor (CAR) gene. Background technique [0002] Tumor immunotherapy is a treatment method that kills tumor cells by improving the body's own anti-tumor immunity. It is the fourth tumor treatment technology after surgery, radiotherapy, and chemotherapy. Based on the great success of immunotherapy in tumor treatment, in 2013 Science magazine ranked tumor immunotherapy as the top ten breakthroughs of the year. Chimeric Antigen Receptor modified T cell (CAR-T) immunotherapy is followed by lymphokine-activated killer cells (LAK), cytokine-induced killer cells (CIK), tumor-infiltrating lymphocytes ( TIL), the fifth generation of adoptive cellular immunotherapy after cytotoxic T cells (CTL). It is a single-chain antibody (ScFv) sequence t...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K35/17A61P35/00A61P37/02A61P37/08A61P31/00A61P29/00
CPCA61K35/17A61P29/00A61P31/00A61P35/00A61P37/02A61P37/08C07K14/7051C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 施明孙毓刘丹汤安群郑骏年
Owner XUZHOU MEDICAL UNIV
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