Humanized antibody targeted to RANKL (Receptor Activator Of Nuclear Factor Kappa B Ligand) and TNF-alpha (Tumor Necrosis Factor) and application of humanized antibody

A humanized antibody and targeting technology, applied in the fields of genetic engineering and immunology, can solve limitations and other problems, achieve the effect of inhibiting differentiation and alleviating bone loss

Inactive Publication Date: 2014-08-27
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the species specificity of murine monoclonal antibodies, its application in the clinical treatment of human rheumatoid arthritis is limited.

Method used

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  • Humanized antibody targeted to RANKL (Receptor Activator Of Nuclear Factor Kappa B Ligand) and TNF-alpha (Tumor Necrosis Factor) and application of humanized antibody
  • Humanized antibody targeted to RANKL (Receptor Activator Of Nuclear Factor Kappa B Ligand) and TNF-alpha (Tumor Necrosis Factor) and application of humanized antibody
  • Humanized antibody targeted to RANKL (Receptor Activator Of Nuclear Factor Kappa B Ligand) and TNF-alpha (Tumor Necrosis Factor) and application of humanized antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Cloning and Determination of the cDNA Sequence of the Variable Region of Animal Immunization Mouse Origin in Example 1

[0066] Extract the total mRNA (SV Total RNA, Promega) of hybridoma cells (preservation number is CGMCC NO.6853) according to the operation procedure; RT-PCR reverse transcription system (promega) obtains the cDNA of the variable region of the mouse antibody; according to the following procedures murine antibody variable region gene amplification

[0067] 1. Primers (synthesized by INVIVOGEN)

[0068] (1) Heavy chain upstream primer:

[0069] 5'-CCGCTCGAGTCATGCTCTTCTTGGTAGCAACAGCT-3'

[0070] (2) Heavy chain downstream primer:

[0071] 5'-CTAGCTAGCTGCAGAGACAGTGAGAGTGG-3'

[0072] (3) Light chain upstream primer:

[0073] 5'-CATGCCATGGAAAATGGAGACAGACACACTCCTGC-3'

[0074] (4) Light chain downstream primer: 5'-CTACGTACGCCCGTTTGATTTCCAACTT-3'

[0075] 2. PCR reaction cycle conditions

[0076] The PCR conditions for the heavy chain were: 94°C, 2min...

Embodiment 2

[0081] Example 2 Design and Synthesis of Humanized Antibody Variable Region

[0082] Design of recombinant humanized antibody: compare the heavy chain and light chain sequences of the murine antibody variable region obtained in Example 1 with the database information of the human antibody heavy chain and light chain in the BLAST program (http: / / www.ncbi.nlm.nih.gov / ). The sequences of the heavy chain (pdb:3DGG_B) and light chain (pdb:1I9R_L) of the humanized antibody with the highest homology to the variable region heavy chain and light chain of the murine antibody were respectively established.

[0083] Sequences of the heavy and light chains of the humanized antibody screened were analyzed by the VBASE2 program (http: / / www.vbase2.org / ), and the structures of their CDR and FR regions were respectively established: the CDR1 of 3DGG_B is GYTFTSYV, CDR2 is NPYNDDT, CDR3 is CAREDYYGSRWGYW. The CDR1 of 1I9R_L is QRVSSSTYSY, the CDR2 is IKAS, and the CDR3 is HSWEIPPTF. While fu...

Embodiment 3

[0084] Example 3 Construction and expression of recombinant humanized antibody expression vector

[0085] Construction of recombinant humanized antibody expression vector: The recombinant antibody heavy chain variable region sequence and pFUSEss-CHIg-hG1 (Catalog#pfusess-hchg1, purchased from InvivoGen, USA) were applied with restriction enzymes XhoI and NheI in a 37°C water bath for 1 hours for double digestion. The light chain variable region sequence of the recombinant antibody and pFUSE2ss-CLIg-hk (Catalog#pfuse2ss-hchlk, purchased from InvivoGen, USA) were subjected to double digestion with restriction enzyme NcoI in a 37°C water bath for 1 hour and BsiWI in a 55°C water bath for 1 hour. The digested products were subjected to agarose gel electrophoresis (1.2% agarose gel). Positive bands were cut out under ultraviolet light, and gel recovery was carried out according to the procedures in the kit instructions (QIAquick Gel Extraction Kit, purchased from QIAGEN, USA).

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Abstract

The invention relates to a humanized antibody targeted to RANKL (Receptor Activator Of Nuclear Factor Kappa B Ligand) and TNF-alpha (Tumor Necrosis Factor) and an application of the humanized antibody. The antibody provided by the invention is obtained by carrying out humanized modification on a region V of a murine monoclonal antibody aiming at RANKL-TNF sample region epitope through a CDR (Complementarity Determining Region) replacement technology. The invention further provides an expression vector for expressing the humanized antibody targeted to RANKL and TNF-alpha and a construction method of the expression vector. The humanized antibody provided by the invention can effectively neutralize TNF-alpha in vitro, inhibit apoptosis caused by TNF-alpha, neutralize RANKL and inhibit generation of osteoclast and meanwhile can antagonize inflammations caused by TNF-alpha and bone destructive effect caused by RANKL. The humanized antibody provides a base for research on novel biological preparations for treating rheumatoid arthritis.

Description

technical field [0001] The present invention relates to the fields of genetic engineering and immunology, in particular to humanized antibodies targeting RANKL and TNF-α and applications thereof. Background technique [0002] Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic, progressive and aggressive arthritis. It can often affect multiple tissues and organs throughout the body, especially joint involvement is the most common. It can be manifested as persistent synovitis of the affected joints, abnormal proliferation of synovial tissue, infiltration of a large number of inflammatory cells, formation of pannus, and damage to bone, cartilage, and ligaments, eventually leading to joint deformity, dysfunction, and even disability. [0003] The pathological mechanism of RA is extremely complex and has not yet been fully elucidated. However, a large number of studies have shown that in the pathological process of RA, the immune and inflammatory ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C12N15/13C12N15/85A61K39/395A61P29/00A61P19/02
CPCC07K16/464A61K2039/505C07K2317/24C12N15/85
Inventor 赵文明王君袁慧慧杜雨轩
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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