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Screening and reforming method for super-stable immunoglobulin variable domain, and applications thereof

A domain antibody, selected technology, applied in the direction of immunoglobulin, anti-animal/human immunoglobulin, compound screening, etc.

Active Publication Date: 2017-07-28
MOGAM INST FOR BIOMEDICAL RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0024] However, the above methods for improving the folding properties of proteins have never been applied to the selection of domain antibodies, in particular, VH or VL domain antibodies, etc.

Method used

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  • Screening and reforming method for super-stable immunoglobulin variable domain, and applications thereof
  • Screening and reforming method for super-stable immunoglobulin variable domain, and applications thereof
  • Screening and reforming method for super-stable immunoglobulin variable domain, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0319] Example 1: Preparation of pET-TAPE for constructing TAPE system

[0320] In order to construct the twin arginine transporter (Tat)-associated protein engineering (TAPE) system, the pET9a vector was used to transform the pathway signal sequence of a Tat substrate protein TorA (Escherichia coli trimethylamine-N-oxide reductase) into , ssTorA, was linked to the N-terminus of the target protein and TEM-1 β-lactamase was linked to its C-terminus to make pET-TAPE.

[0321] However, the signal sequence that directs the protein to the Tat pathway is not limited to ssTorA, as it will be apparent to one of ordinary skill that the signal sequence of all Tat pathway proteins can be used, as described above. In addition, it is obvious to those of ordinary skill that, as the vector used, any vector satisfying the purpose of the present invention can be used, such as pET9a (New England Biolab), ΔpMA using an arabinose-inducible promoter (Korean Patent Application Laid-Open No. 1996-00...

Embodiment 2

[0329] Example 2: Preparation of Immunoglobulin Heavy Chain Variable Domain (VH) Libraries Derived from Human Germline

[0330] cDNA libraries were secured by reverse transcription of mRNA obtained from human liver, peripheral blood mononuclear cells (PBMC), spleen and thyroid.

[0331] To thereby ensure the DNA sequence of the human immunoglobulin heavy chain variable region domain, a mixed primer shown in SEQ ID NO: 5 to 13 was designed to ensure that all human heavy chain variable domain genes are available in human germ cells Tie. Each ensured human heavy chain variable domain gene library was inserted between the NdeI and BamHI sites of pET-TAPE to complete with about 10 8 size library.

[0332] Specifically, cDNA was prepared from RNA extracted from human spleen, peripheral blood mononuclear cells, liver and thymus (Clontech, Madison, WI, US) by reverse transcription reaction. AMV reverse transcriptase and RNase inhibitors were purchased from Promega (Madison, WI, USA...

Embodiment 3

[0342] Example 3: Screening of VH libraries derived from human germlines by TAPE (Tat-associated protein engineering)

[0343] (1) Construction of VH library derived from human germline

[0344] Transformation of Escherichia coli T7Express LysY / I with pET-TAPE library by electroporation q . Then, they were cultured at 37° C. in SOC medium for 1 hour, and then inoculated and cultured in LB medium containing 50 μg / ml of carbenicillin (1X). When the OD value of Escherichia coli was 0.6, the Escherichia coli was collected using centrifugation, and then the plasmid was isolated using a plasmid DNA purification kit (QIAGEN, Valencia, CA, USA), and then cut with restriction enzymes NcoI and BamHI. Cleavage genes include VH library and β-lactamase gene, which is used to rule out false positives that may appear in the subsequent liquid panning process. The pET-TAPE plasmid was also cut with restriction enzymes NcoI and BamHI. After cleavage, each DNA was purified using a gel extrac...

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PUM

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Abstract

The present invention relates to a method known as Tat-related protein transformation engineering (TAPE), wherein a target protein and an antibiotic resistance protein are linked to a Tat signal sequence, the obtained substance is expressed in Escherichia coli, the target protein with advantages of high solubility and excellent heat stability is screened, and particularly human or modified VH and VL domain antibody and a human or modified VH and VL domain antibody scaffold derived from the immunoglobulin variable domain (VH or VL) of human germ cells, screened by the TAPE method and having advantages of solubility and excellent thermal stability are screened. The invention further provides a library and a preparation method thereof, wherein the library has a human or modified VH and VL domain antibody or antibody scaffold screened by the TAPE method and containing a random CDR sequence. The invention further provides a VH or VL domain antibody screened by the library and having target protein binding ability, and a pharmaceutical composition containing the domain antibody.

Description

[0001] This application is a divisional application of an invention patent application submitted on August 22, 2012 with the application number 201280076586.9 and the title of the invention is "Methods for Screening and Transforming Ultra-stable Immunoglobulin Variable Domains and Its Application". technical field [0002] The following disclosure relates to a method called Tat-associated protein engineering (TAPE), by fusing target proteins and antibiotic resistance proteins to the Tat signal sequence and expressing it in E. Target proteins for stability, especially immunoglobulin variable domains (VH or VL) derived from human germline. The present invention also relates to a human heavy chain variable domain antibody (hereinafter referred to as "VH domain antibody") and a light chain variable domain antibody (hereinafter referred to as "VH domain antibody") having excellent solubility and thermal stability screened by the TAPE method. referred to as "VL domain antibodies") a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K16/18C07K16/32C07K19/00
CPCC07K16/00C07K16/18C07K16/32C07K2317/21C07K2317/94C07K2319/00C07K2319/10G01N2500/00C07K2317/569C07K19/00C12N15/62C12N15/63G01N33/6857G01N33/53C40B40/10C07K2317/56C07K2317/565
Inventor 李滢权金圣根朴荣燮南效廷金董植朴在燦尹桦
Owner MOGAM INST FOR BIOMEDICAL RES
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