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Gene-modified mesenchymal stem cell and method for producing BsAb antibody therefrom

A kind of stem cell and gene modification technology, applied in the field of BsAb antibody production, can solve the problems of low transfection efficiency, destruction of cell genome, low molecular weight of protein and functional factors, etc., to reduce purification links, reduce contact probability, and increase the difficulty of preservation Effect

Inactive Publication Date: 2017-03-15
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although viral vector transfection can improve the efficiency of stem cell gene modification, this method has certain limitations due to the safety of viruses, such as strict requirements on the operating environment, such as the possibility of random integration of viral genes into the cell genome after infection , thus destroying the cell genome, causing cancer risk, and the transfection efficiency of viral vectors in animals is not high, because the body itself will start the mechanism of silencing the virus function
In addition, the existing research methods of using stem cells for gene transfection to produce cytokines and proteins produce relatively low molecular weight proteins and functional factors, and are more common in the use of ribonucleic acid (RNA) such as mRNA as gene carriers
[0005] There is no similar report on the application of stem cells to produce bispecific single chain antibodies with larger molecular weight

Method used

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  • Gene-modified mesenchymal stem cell and method for producing BsAb antibody therefrom
  • Gene-modified mesenchymal stem cell and method for producing BsAb antibody therefrom
  • Gene-modified mesenchymal stem cell and method for producing BsAb antibody therefrom

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Carrying out chemical reagent transfection for mesenchymal stem cells derived from human umbilical cord

[0039] (1) Preparation of Lipofectamine2000 / DNA transfection complex

[0040] a. Preparation of plasmid DNA containing antiCD20×antiCD3 antibody expression gene

[0041] In this example, the antiCD20×antiCD3 gene sequence (SEQ ID No.1, which is transformed into a bispecific single-chain antibody sequence based on the humanized monoclonal antibody anti-CD20 sequence) The antiCD20 sequence is derived from: GA101 (Obinutuzumab, Roche), and the antiCD3 part is derived from the patent US008076459) cloned into the vector SZ63.pMC.ZY781.CMVmax.intron.bpA. Provided by Therapeutic Research Laboratory, its detailed spectrum is as follows figure 2 As shown, the sequence is shown in SEQ ID No.2 (artificial sequence), wherein the drug screening marker is Kanamycin), and the recombinant plasmid SZ66.pMC.ZY781.CMVmax.intron.CD20Bite.bpA (6880bp) is obtained after tra...

Embodiment 2

[0062] Example 2. Electrotransfection of human umbilical cord-derived mesenchymal stem cells

[0063] 18μl Supplement solution [P3Primary Cell 4D-Nucleofector TM X Kit, Lonza Company] with 82 μl NucleofectorTM solution [P3Primary Cell 4D-Nucleofector TM X Kit, Lonza Company] was fully mixed to form an electrotransfer solution (100 μl system / electrotransfer cup); the mesenchymal stem cells (provided by Shenzhen Nanshan Hospital) were digested with trypsin to make them fall off the culture dish wall, and centrifuged (1000rpm, 5 min), the supernatant was discarded, and the mesenchymal stem cells were resuspended with 100 μl electroporation solution to make the density reach 1×10 7 pc / ml; Add 6 μg of plasmid DNA plasmid with antiCD20×antiCD3-BsAb antibody expression gene to 100 μl of mesenchymal stem cell resuspension (see Example 1 for plasmid preparation), and mix gently with a pipette to make the cells Mix the plasmid well; add the mixture into the electric transfer cup, us...

Embodiment 3

[0064] Example 3. Nanocomposite-mediated transfection of human umbilical cord-derived mesenchymal stem cells

[0065] Add 4 μg of the plasmid DNA plasmid with antiCD20×antiCD3 antibody expression gene (see Example 1 for plasmid preparation) to an appropriate amount of opti-MEM medium, with a final volume of 100 μl, and let it stand for 5 minutes; at the same time, take an appropriate amount of ε-caprolactone The modified modified polyethyleneimine (patent application No. 201310390436.1) was dissolved in opti-MEM medium, and the final volume was 100 μl to prepare a transfection agent solution. After standing for 5 minutes, the plasmid solution and transfection reagent were mixed with a pipette. The solution was gently blown up and down to mix, and left at room temperature for 30 minutes to make a plasmid DNA nanocomposite.

[0066] Human umbilical cord-derived mesenchymal stem cells were used at 5×10 per well 5 The density of cells / ml was inoculated in 6-well plates (5×10 per ...

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Abstract

The invention provides a gene-modified mesenchymal stem cell and a method for producing a bispecific single-chain antibody (BsAb antibody) therefrom through the gene-modified mesenchymal stem cell. The gene-modified mesenchymal stem cell is obtained through a nonviral vector transfection mesenchymal stem cell carrying a bispecific single-chain antibody gene. The invention further specially provides the method for producing the bispecific single-chain antibody through the gene-modified mesenchymal stem cell.

Description

technical field [0001] The present invention relates to a genetically modified mesenchymal stem cell and its method for producing BsAb antibodies. Specifically, the present invention relates to a genetically modified mesenchymal stem cell, a preparation method thereof, and a genetically modified mesenchymal stem cell. A method of producing a bispecific single chain antibody. Background technique [0002] In the immune system of the human body, cytotoxic T lymphocytes (Cytotoxic T Lymphocyte, CTL) and killer T cells (Cytokine induced killer) play an important role. They have T cell receptors (T cell receptor, TCR), which can express The cells of the target antigen specifically recognize and bind, and secrete perforin and granzyme, thereby killing the pathogenic target cells. However, lesion areas such as cancer cells often show defects in antigen expression, lack of substances such as immune adhesion molecules and co-stimulatory molecules, which make the body's immune recogn...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12P21/02
CPCC12N5/10C12N15/85C12P21/02
Inventor 王志勇陈志英谭燕杨磊何成宜钟育健
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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