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Construction of CAR-T cell for co-expressing IL18 through non-viral carrier and application of CAR-T cell

A co-expression and cell technology, applied to genetically modified cells, cells modified by introducing foreign genetic material, receptors/cell surface antigens/cell surface determinants, etc., can solve problems such as the difficulty of preparing viral vectors

Inactive Publication Date: 2018-12-07
杭州荣泽生物科技集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although viral vectors can be integrated into the host cell genome at a high copy number, the preparation of viral vectors is difficult, especially in large-scale production; and lentiviruses also have potential risks of safety

Method used

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  • Construction of CAR-T cell for co-expressing IL18 through non-viral carrier and application of CAR-T cell
  • Construction of CAR-T cell for co-expressing IL18 through non-viral carrier and application of CAR-T cell
  • Construction of CAR-T cell for co-expressing IL18 through non-viral carrier and application of CAR-T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] This example provides a method for constructing a chimeric antigen receptor molecule CD19 CAR-IL18 that co-expresses IL18 and CD19

[0037]Construction of CD19 antibody scFv: Synthesize CD19 antibody FMC63 gene fragment (Nanjing GenScript), respectively add BamH I and Xho I restriction enzyme sites in the upstream and downstream of FMC63, and construct the fragment into pUC57 vector;

[0038] Construction of FMC63-28bbZ vector: the existing vector pCDH-CMV-CSF2RA-Trop2scFv-CD8α-CD28-4-1BB-CD3ζ-T2A-CopGFP and pUC57-FMC63 vectors were subjected to double enzyme digestion by BamH I and Xho I restriction enzymes The fragments were digested and ligated with T4 ligase to obtain pCDH-CMV-CSF2RA-FMC63scFv-CD8α-CD28-4-1BB-CD3ζ-T2A-CopGFP, and then CopGFP was combined with IL18 by In-Fusion PCR. The substitution was performed to obtain pCDH-CMV-CSF2RA-FMC63scFv-CD8α-CD28-4-1BB-CD3ζ-T2A-IL18, referred to as pCDH-CMV-CD19CAR-IL18. The CD19CAR-IL18 fragment with Bag II and Not I ...

Embodiment 2

[0040] This example is the preparation of CD19 scFv-IL18 CAR-T cells

[0041] Sleeping Beauty Transposon Recombinant Plasmid / Transposase System: Extract pT2 / SVNeo-CMV-CD 19 CAR-IL18 and Sleeping Beauty’s transposase plasmid SB11 with the QIAGEN Endotoxin-Free Plasmid Large Extraction Kit to extract the recombinant transposase The transposase plasmid SB11 was mixed with the recombinant transposon plasmid pT-CMV-CD19 CAR-IL18 at a mass ratio of 1:3 to obtain a transposon recombinant plasmid / transposase system that co-expressed IL18 and CD19 scFv. T cell transduction: T cells were isolated according to the instructions of the EasySeq kit (STEMCELL), and then the T cells were cultured at 37°C for 3 h, and T cell culture medium (SuperCulture TM L100) and IL2, the virus supernatant was diluted 2-fold, and T cells were cultured overnight in the supernatant, and then used SuperCulture TM L100 (containing IL2) was cultured for 48 hours before use.

[0042] Preparation of CD19 CAR-T...

Embodiment 3

[0044] This example is the killing effect of CD19 CAR-T cells expressing IL18 on tumor cells in vitro.

[0045] Adjust the density of Daudi, Nalm-6 tumor cells to 2 × 10 in a 24-well cell culture plate 5 cells / ml, 500 μL prepared CD19 scFv-IL18 CAR-T cells were added to the 24-well plate (the number was 1×10 5 1); similarly, the ratio of effector cells: target cells (effect: target ratio) was 5:1, 2.5:1, 1:1, 0.5:1, 0:1, 1:0 ratio of effector cells and target cells The cells were mixed evenly (see Table 1), and the complete medium was supplemented to 1ml, and the cells were mixed evenly and cultured normally for 6 hours. The co-cultured cells in each group were collected, stained with human anti-CD19 fluorescent antibody, and the percentage of CD19+ positive cells was analyzed by flow cytometry. Compared with the percentage of initial target cells in each group, the killing rate of target cells in each group was calculated. From the flow cytometry scatter plot, it can be s...

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Abstract

The invention discloses a preparation method of a CD19 specific chimeric antigen receptor T cell for co-expressing IL18. According to the preparation method, a sleeping beauty plasmid transposon system containing CD19 CAR and IL18 is transfected into the T cell, and meanwhile, a specific dose of IL2 and a specific dose of anti-CD3 / CD28 magnetic bead antibodies are added, so that the CD19 CAR-T cell for co-expressing IL18 is obtained. The obtained T cell is capable of specifically killing tumor cells, and meanwhile, the killing effect of the T cells can be remarkably improved by virtue of IL18.

Description

technical field [0001] The invention relates to the field of immune cell preparation, in particular, the invention relates to an IL-18 co-expressed in CD19-specific chimeric antigen receptor T cells and its use. technical background [0002] Chimeric antigen receptor T cell therapy (Chimeric antigen receptor T cell therapy, CAR-Ttherapy) is an emerging tumor immunotherapy, which is to express a tumor on the T cell membrane by modifying the T cells in the patient's body. Specific chimeric antigen receptors (Chimeric antigen receptors, CARs) enable them to be activated through tumor-specific, MHC-independent pathways, thereby exerting anti-tumor effects. CAR is a transmembrane receptor, mainly composed of an extracellular recognition domain, a transmembrane region and an intracellular activation domain. variable fragment, scFv), the intracellular activation domain is CD3ζ of T cell receptor (TCR), when CAR extracellular single-chain antibody recognizes and binds with tumor ce...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/63C12N15/62C07K19/00
CPCC07K14/54C07K14/7051C07K16/2803C07K2319/33C12N2510/00
Inventor 陈相波冯煜雷鸣田朋飞
Owner 杭州荣泽生物科技集团有限公司
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