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Antibody for specifically binding to lysyl-trna synthetase n-terminal domain exposed to extracellular membrane

A lysyl and synthetase technology, applied in the direction of antibodies, specific peptides, drug combinations, etc., can solve problems such as low affinity

Pending Publication Date: 2021-07-30
塞梅蒂股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the affinity of antibodies targeting the N-terminus of conventional lysyl-tRNA synthetases is lower than that of various antibodies in the form of intact IgG

Method used

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  • Antibody for specifically binding to lysyl-trna synthetase n-terminal domain exposed to extracellular membrane
  • Antibody for specifically binding to lysyl-trna synthetase n-terminal domain exposed to extracellular membrane
  • Antibody for specifically binding to lysyl-trna synthetase n-terminal domain exposed to extracellular membrane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0181] Example 1: Construction of Yeast Cell Surface Expression Library for Increased Affinity

[0182] The affinity for the N-terminus of antibody N3 antibody (Korean Patent Application No.: 10-2018-0035446) targeting the N-terminus of conventional lysyl-tRNA synthetase is about 150nM, which is lower than that of various antibodies in the form of intact IgG affinity. Therefore, in order to prepare an antibody with a better effect by increasing the affinity, the light chain variable region and the heavy chain variable region of the N3 antibody were improved.

[0183] The approximate structure of the N3 antibody was predicted using a homology (homology) model, and random mutants were introduced into the complementarity determining region predicted to play an important role in antigen binding. Specifically, in the library based on the heavy chain variable region, NNK, which is a degenerated codon that can include all 20 amino acid sequences of the complementarity determining re...

Embodiment 2

[0186] Example 2: Screening for light and heavy chain variable regions with improved affinity for GST-bound lysyl-tRNA synthetase peptides (residues 1-72)

[0187] The two N3 antibody-based affinity-improved libraries constructed in Example 1 were screened using the GST-bound lysyl-tRNA synthetase peptide (residues 1-72) as an antigen.

[0188] Specifically, for the first fluorescence-activated cell sorting screening, SG-CAA (20 g / L of galactose, 6.7 g / L of yeast nitrogen base without amino acids, 5.4 g / L of Na 2 HPO 4 , 8.6g / L NaH 2 PO 4 , 5g / L casamino acid) culture medium, under normal temperature conditions, the peptide (residue 1-72) of the lysyl-tRNA synthetase (residue 1-72) of the lysyl-tRNA synthetase that combines the purification of about 10nM with GST and induces single-chain on the cell surface The yeast expressing the light chain variable region library in Fab (scFab) form was reacted. Afterwards, at a temperature of 4°C, the GST-bound lysyl-tRNA synthetase p...

Embodiment 3

[0201] Example 3: Affinity comparison between N3 antibody and N3-1 antibody

[0202] 3-1. Cell migration inhibitory effect of antibodies

[0203] In the N3 mutant antibody of Example 2, the N3-1 antibody was converted into an IgG antibody by a conventional method. Using the transformed IgG antibody, the following experiment was carried out.

[0204] Cell migration was measured using a commonly used 24-well transfer well chamber of polycarbonate membrane (8.0 μm pore size, Costar). In the transfer well chamber, the lower well was coated with 10 μg of laminin. Afterwards, A549 cells were suspended in serum-free RPMI medium at 1×10 per well. 5 The concentration of cells is placed in the upper segment chamber. In the chamber, N3 antibody, N3-1 IgG, and human mock IgG (control group) were treated at 10 nM or 100 nM, respectively, and incubated for 24 hours. Non-migrating cells present on the upper side of the film were removed using a cotton swab. Thereafter, it was washed tw...

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Abstract

The present invention relates to an antibody for specifically binding to a Lysyl-tRNA synthetase N-terminal domain exposed to an extracellular membrane and, more specifically, to an antibody or a fragment thereof that specifically binds to a Lysyl-tRNA synthetase (KRS) N-terminal domain exposed to an extracellular membrane having a specific complementarity determining region (CDR) sequence described in the present specification, and use of a composition comprising the antibody or the fragment thereof as an active ingredient for preventing, treating, or diagnosing cancer, cancer metastasis, or immune cell migration-related diseases. The method of the present invention may be utilized to prepare an antibody with a higher affinity at a KRS N-terminus than existing antibodies.

Description

[0001] This application claims priority from Korean Patent Application No. 10-2018-0111046 filed on September 17, 2018, the entirety of which is incorporated herein by reference. technical field [0002] The present invention relates to an antibody that specifically binds to the N-terminal region of lysyl-tRNA synthetase exposed outside the cell. An antibody or fragment thereof that specifically binds to the N-terminal region of extracellular lysyl-tRNA synthetase (KRS, Lysyl-tRNA synthetase) and has high affinity and stability, and contains the antibody or fragment thereof as an effective Use of the composition of ingredients for the prevention, treatment or diagnosis of cancer, cancer metastasis or immune cell migration related diseases. Background technique [0003] Recent studies have determined that human lysyl-tRNA synthetase, normally present in the cytosol, translocates to the plasma membrane (cell membrane) and is bound to the 67-kDa laminin present on the plasma me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40G01N33/574A61P35/00
CPCA61P35/00C07K16/40G01N33/57492G01N2800/52C07K2317/92C07K2317/56C07K2317/565C07K2317/76C07K2317/94C07K2317/77C07K2317/71C07K2317/732A61K2039/505G01N33/57415G01N33/57423G01N2333/9015
Inventor 金圣勋权南勋
Owner 塞梅蒂股份有限公司
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