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Method for preparing chimeric antigen receptor T cells

A chimeric antigen receptor and cell technology, applied in the field of biomedicine, can solve the problems of low lentivirus titer, low efficiency, and hinder the wide clinical application of T cells, and achieve the effect of increasing activation ability and improving infection efficiency.

Active Publication Date: 2017-07-25
ICARTAB BIOMEDICAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current clinical experiments using lentiviruses as gene transduction vectors have proved to a certain extent the safety of lentiviruses as gene therapy vectors. Although lentiviruses belong to a genus of retroviruses, they have a wider host range. Capable of infecting aperiodic and post-mitotic cells, lentiviral infection of primary T cells has been inefficient, averaging less than 20%, hindering widespread clinical adoption of T cell-based chimeric antigen receptor immune cell therapy techniques The low transfection efficiency of T cells by using lentivirus is mainly due to the following reasons. First, the titer of the prepared lentivirus is low; The exogenous gene is integrated into the genome of T cells; the third is that the experimental protocol of lentivirus infection of primary T cells is not optimized

Method used

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  • Method for preparing chimeric antigen receptor T cells
  • Method for preparing chimeric antigen receptor T cells
  • Method for preparing chimeric antigen receptor T cells

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Prepare a 15cm dish and inoculate 5*10 6 293 cells, add complete medium (DMEM high glucose, 10% FBS), place at 37°C, 5% CO 2 incubator, overnight. Take out 100 μM polyetherimide (PEI) from the refrigerator, take out the plasmids (Lenti-GFP, pGP, pVSVG) from the refrigerator, and after thawing at room temperature, take out the PBS buffer and warm to room temperature; take 2 mL of PBS to a 6-well plate Prepare 3 wells in total, add 15 μg Lenti-EF1a-GFP, 3 μg pGP, 1.5 μg pVSVG respectively, pipette up and down to mix thoroughly, add 18 μL 100 μM polyetherimide to each well, and then add three The plasmid solution was added to the PBS well, immediately mixed up and down with a pipette, and allowed to stand at room temperature for 10 minutes to prepare the PEI / DNA complex. Add the above DNA / PEI complex dropwise into a 15cm petri dish, shake the petri dish gently, and mix well. Place the Petri dish at 37°C, 5% CO 2 After culturing for 5 hours, remove the medium containing...

Embodiment 2

[0037] Based on the variable region of the antibody sequence targeting CD19, a chimeric antigen receptor is designed, which includes signal peptide, single-chain antibody sequence, transmembrane region and intracellular co-stimulatory signal region, and is synthesized by whole gene synthesis The chimeric antigen receptor sequence, and using the EcoRI-BamHI restriction site, the chimeric antigen receptor fragment was subcloned into the Lenti-puro lentiviral expression vector to prepare the Lenti-CD19-CAR lentiviral expression vector.

[0038] Prepare a 15cm dish and inoculate 5*10 6 293 cells, add complete medium (DMEM high glucose, 10% FBS), place at 37°C, 5% CO 2 incubator, overnight. Take out 100 μM polyetherimide from the refrigerator, take out the plasmids (Lenti-CD19-CAR, pGP, pVSVG) from the refrigerator, and after thawing at room temperature, take out the HBSS buffer and warm it to room temperature; take 2 mL of PBS into the 6-well plate One well, prepare 3 wells in t...

Embodiment 3

[0047]Prepare a 15cm dish and inoculate 5*10 6 293 cells, add complete medium (DMEM high glucose, 10% FBS), place at 37°C, 5% CO 2 incubator, overnight. Take out 100 μM polyetherimide from the refrigerator, take out the plasmids (Lenti-CD20-CAR, pGP, pVSVG) from the refrigerator, and after thawing at room temperature, take out the PBS buffer and warm to room temperature; Add 15 μg Lenti-CD20-CAR, 3 μg pGP, and 1.5 μg pVSVG to one well, mix well by pipetting up and down, then add 18 μL of 100 μM polyetherimide, immediately pipette up and down to mix, Let stand for 10 minutes to prepare PEI / DNA complexes. Add the above DNA / PEI complex dropwise into a 15cm petri dish, shake the petri dish gently, and mix well. Place the Petri dish at 37°C, 5% CO 2 After culturing for 10 hours, remove the medium containing the transfection reagent and replace it with fresh conditioned medium (DMEM high sugar, 5% FBS, sodium pyruvate, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) )).

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Abstract

The invention discloses a method for preparing chimeric antigen receptor T cells. The method comprises the steps of mixing a chimeric antigen receptor slow virus expression vector, pGP plasmids, pVSVG plasmids and polyetherimide firstly, and conducting uniform blow-beating to obtain a DNA / PEI compound; inoculating a culture dish with 293 cells, and then adding a complete medium for culture; adding the DNA / PEI compound to the culture dish dropwise; removing the medium after 4-10 h of culture; adding a conditioned medium, continuing culture for 8-32 h, and then collecting the medium; conducting centrifugal treatment to obtain medium supernatant containing slow viruses; adding a sucrose solution into a centrifugal tube, and then adding the medium supernatant containing the slow viruses along the wall of the centrifugal tube; conducting centrifugal treatment, removing centrifugation supernatant, and then adding a buffer solution; and then conducting T cell transfection to obtain the chimeric antigen receptor T cells through culture. Based on a slow virus packaging system, transfection efficiency can reach 60% or more.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method of chimeric antigen receptor T cells, which can obviously improve the transfection efficiency of carriers to T cells. Background technique [0002] T lymphocytes play a major role in the tumor immune response and have a strong killing effect on tumor cells. However, there are MHC limitations when using endogenous T cells for tumor immunotherapy. The process of tumor immunoediting will reduce the expression of MHC on the surface of tumor cells, destroy the antigen processing process, reduce the immunogenicity of peptides, and form an immune escape mechanism. The T cell therapy technology that enables tumor cells to successfully evade T cell attacks and rapidly proliferate by using genetic modification technology to express tumor-specific chimeric antigen receptors has shown good targeting, killing activity and persistence in vitro and clinical ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C12N5/0636C12N15/86C12N2740/15043
Inventor 岳庆许波李凡池季平
Owner ICARTAB BIOMEDICAL
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