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Genetic engineering cell line for producing unfucosylated protein and establishment method thereof

A glycosylated protein and fucosylation technology, applied in the field of genetic engineering cell lines and their establishment, can solve the problems of low fucose removal efficiency, complex types, increased heterogeneity of glycoforms and product quality control Difficulty and other issues

Active Publication Date: 2017-09-29
江苏东抗生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are certain defects in this glycosylation technology: by introducing bisecting galactoside, the technology makes more than 30 kinds of glycoforms on the antibody, and the types are more and more complicated, which increases the glycoforms. Heterogeneity and difficulty in product quality control; in addition, the fucose removal efficiency of this method is low, and about 30% of the glycoforms still contain core fucose

Method used

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  • Genetic engineering cell line for producing unfucosylated protein and establishment method thereof
  • Genetic engineering cell line for producing unfucosylated protein and establishment method thereof
  • Genetic engineering cell line for producing unfucosylated protein and establishment method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Construction of Slc35c1 gene knockout vector

[0075] Targeting the coding region of the mRNA sequence of the Chinese hamster Slc35c1 gene (NM_001246808.1, as shown in SEQ ID NO.3), 4 gRNAs (Table 1) were designed, synthesized by Nanjing GenScript Co., Ltd., and two paired After gradient annealing of the single-strand (F and R) of BbsI restriction endonuclease, pX330-U6-Chimeric_BB-CBh-hSpCas9 (purchased from Addgene, Cat. No. Plasmid42230; Multiplex Genome Engineering Using CRISPR / CasSystems.Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Science. 2013 Jan 3.10.1126 / science. 1231143 PubMed23287718) vector ligation. Schematic diagram of vector structure and insertion site figure 1 shown.

[0076] After sequencing verification, it was confirmed that the correct clone was obtained, transfected Top-10 competent cells, picked a single clone and cultured in a shaker flask, and obtained transfection with an endotox...

Embodiment 2

[0080] Embodiment 2: Construction of FUT8 gene knockout vector

[0081] Targeting the coding region of the mRNA sequence (XM_003501735.2, shown in SEQ ID NO.14) of the Chinese hamster FUT8 gene, five gRNAs were designed (Table 2), synthesized by Nanjing GenScript Biotechnology Co., Ltd., and two paired After gradient annealing of the single-strand (F and R) of BbsI restriction endonuclease, pX330-U6-Chimeric_BB-CBh-hSpCas9 (purchased from Addgene, Cat. No. Plasmid42230; Multiplex Genome Engineering Using CRISPR / CasSystems.Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Science. 2013 Jan 3.10.1126 / science. 1231143 PubMed23287718) vector ligation.

[0082] Table 2 gRNA design targeting FUT8 gene

[0083]

[0084]

[0085] After sequencing verification, it was confirmed that the correct clone was obtained, transfected Top-10 competent cells, picked a single clone and cultured in a shaker flask, and obtained transfection wit...

Embodiment 3

[0086] Example 3 Construction of CHO Cells with Slc35c1 Gene Knockout

[0087] Inoculate 30ml density 5×10 in 125ml cell culture shake flask 5 For CHO-K1 (ATCC) cells, the cell viability is above 98%. After the cells are centrifuged, they are diluted to 1×10 with electroporation buffer. 7 Cells / ml density, add 40μg of the constructed pX330 plasmid targeting the Slc35c1 gene into the electric shock cup, then add 0.7ml of cell suspension, and finally add the electroporation buffer to 0.8ml, mix gently, and set the temperature at 300V for 20ms Electric shock once under certain conditions, place the electric shock cup in an ice box for 5 minutes, add medium after transfection, and place at 37°C, 5% CO 21. Cultivate on a cell culture shaker with a rotation speed of 130rpm. After 2 days, add lentil agglutinin LCA (Lensculinaris agglutinin) to 200 μg / ml to screen stable resistant clones; 5 days after adding the drug, collect the cells by centrifugation, and use the specific affinit...

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Abstract

The invention discloses a genetic engineering cell line for producing an unfucosylated protein and an establishment method thereof. The invention utilizes the CRISPR / Cas9 technique to knock out the Slc35cl gene and / or the Fut8 gene in host cells, and thereby the Slc35cl gene-silencing and / or Fut8 gene-silencing stable genetic engineering cell line is obtained. By utilizing the genetic engineering cell line disclosed by the invention, the protein from which fucosylation is completely removed can be produced, moreover, the protein is still stable after 30 generations of passage, and thereby the two major problems of incomplete fucosylation removal and unstable passage in the prior art are solved. An unfucosylated antibody produced by the method disclosed by the invention shows enhanced ADCC (antibody dependent cell-mediated cytotoxicity) activity, and thereby the clinical therapeutic effect of the antibody is enhanced.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a genetically engineered cell line capable of producing afucosylated protein and a method for establishing the same. technical background [0002] Since the first antibody drug was approved in 1986, antibody drugs have developed vigorously for more than 30 years. Due to the obvious advantages of antibody drugs such as high targeting, specificity, and specificity, the therapeutic field has also changed from traditional cancer, Autoimmune diseases have gradually expanded to the diagnosis and treatment of anti-infection, metabolic diseases, and cardiovascular diseases, and have achieved remarkable curative effects. While improving the quality of life of patients, they have also created huge economic benefits. In addition to blocking target-mediated signal transduction and inducing apoptosis, antibodies against tumors exert therapeutic effects through the following effector func...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12P21/00C12R1/91
CPCC07K14/47C12N9/1051C12N15/113C12N15/907C12N2310/10C12P21/00C12Y204/01068
Inventor 赵健魏化伟黄亚杰
Owner 江苏东抗生物医药科技有限公司
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