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Cell line for antibody affinity maturation screening and method of use thereof

An affinity, antibody technology, applied to cells modified by introducing foreign genetic material, using a vector to introduce foreign genetic material, recombinant DNA technology, etc. And other issues

Active Publication Date: 2018-03-20
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] (1) The growth rate of these two cells is slow (the doubling time is about 22 hours), and the tolerance to foreign transfection vectors is not very strong (for example, a considerable part of the cells die after transfection of 293T with lipo2000), At the same time, the survival rate after flow sorting is also low;
[0004] (2) The antibody affinity maturation process of the two-cell-based affinity maturation system often requires more than 8 rounds of affinity maturation to obtain high-affinity antibodies. Due to slow growth and low survival rate, each round of evolution requires It takes longer and also results in inefficient experiments

Method used

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  • Cell line for antibody affinity maturation screening and method of use thereof
  • Cell line for antibody affinity maturation screening and method of use thereof
  • Cell line for antibody affinity maturation screening and method of use thereof

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Experimental program
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Embodiment 1

[0026] Embodiment 1, the construction of plasmid

[0027] In order to establish an efficient antibody affinity maturation system based on CHO / dhFr-cells, the following plasmids were constructed (Table 1, figure 1 ):

[0028] (1) Use the CDNA of CHO-K1 cells as a template to PCR out the CHO dihydrofolate reductase (DHFR) gene, and then connect the SV40 promoter (promoter) and SV40 polyA (polyA) sequences to DHFR by overlapping PCR , form SV40promoter-DHFR-SV40polyA sequence (this sequence can transcribe and express DHFR in mammalian cells), insert SV40promoter-DHFR-SV40polyA sequence into pCDNA3.1 / hygro(+) plasmid, construct pCDNA3.1 / hygro(+) - DHFR plasmid (abbreviated as pCD).

[0029] (2) The FRT-EGFP-loxP sequence was obtained by PCR using EGFP as a template, and connected to the pCD plasmid to construct the pCDNA3.1 / hygro(+)-FRT-GFP-loxP-DHFR plasmid (pCDFGL for short).

[0030] (3) Flpo-2A-iCre sequence was amplified by overlap PCR using Flpo and iCre as templates, and...

Embodiment 2

[0038]Example 2. Using the recombinase-mediated cassette replacement (RMCE) method to establish stable, high-efficiency expression and replaceable CHO cells of the gene of interest

[0039] 1. Design ideas

[0040] In order to establish CHO cells that stably and efficiently express the target gene and can perform recombinase-mediated cassette replacement (RMCE) for antibody affinity maturation, the Flp and Cre dual histone-mediated cassette replacement (dual RMCE) method was selected. Create cell lines. A series of plasmids for RMCE were established, and the structure of its functional region is as follows figure 2 shown.

[0041] figure 2 (A) shows that in the pCDFGL plasmid, the GFP gene containing FRT and loxP recombinase binding sites is inserted downstream of the CMV promoter, and the DHFR gene containing the SV40 promoter is inserted into the plasmid, so that the pCDFGL plasmid can express both GFP and DHFR .

[0042] figure 2 (B) shows that in the pF2AC plasmid...

Embodiment 3

[0066] Example 3. Affinity maturation of anti-TNF-α single-chain antibody (3f8-scFv)

[0067] Using anti-TNF-α single-chain antibody (3f8-scFv) as an example to verify the affinity maturation method of PuroR-14 cells

[0068] The basic procedure for antibody affinity maturation is: combine approximately 2x10 6 Cells displaying the antibody were transfected with the pCEP-Neo-mAID plasmid, then placed in a medium containing G148 antibiotics and cultured for 7 days (the cells proliferated to about 200 million), trypsinized to collect about 100 million cells, and used anti-HA tagged After the antibody (anti-HA-PE 1:300 dilution, BD) and TNF-α-GFP fusion protein co-labeled the cells at 4°C, the cells with the highest affinity (accounting for about 0.02-0.05% of the total cells) were sorted by flow cytometry. %), the sorted cells were cultured in a medium without G418 antibiotics for 3 days, the cells were expanded to about 200,000, and the pCEP-Neo-mAID plasmid was re-transfected ...

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Abstract

The invention relates to a cell strain for screening affinity maturation of an antibody and a use method thereof. The cell strain is stable in expression of a target antibody by virtue of a RMCE technology. Combined with a somatic hypermutation technology of activation-induced cytidine deaminase, the cell strain for screening affinity maturation of the antibody is relatively high in efficiency and the affinity of the antibody can be improved to a great extent within a relatively short time and screening turns.

Description

technical field [0001] The invention belongs to the field of biomedicine and antibody engineering, in particular, relates to a cell line used for antibody affinity maturation screening and a method for using the same Background technique [0002] The antibody affinity evolution system is an important technology that both antibody engineering and macromolecular biopharmaceutical industries are very concerned about. In the process of antibody affinity evolution screening, the display of target antibodies is a basic step. Commonly used in vitro display systems include phage display. system, yeast display system or bacterial display system, in recent years, mammalian cell-based display systems have also been greatly developed. Compared with other display systems, mammalian display systems are more important in protein folding, post-translational modification and codon There are many advantages in many aspects, such as somatic mutation (SHM, somatichypermutation) combined with cy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85
Inventor 杭海英陈川赵云
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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