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Fully-humanized monoclonal antibody for RSV (respiratory syncytial virus) adhesion protein G surface antigen

A monoclonal antibody, fully human technology, applied in the direction of antibodies, immunoglobulins, antibody medical components, etc., to achieve the effects of improving immune function, reducing viral load, and reducing side effects

Active Publication Date: 2016-05-04
REYOUNG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The anti-RSV monoclonal antibody currently on the market is only approved for the prevention of RSV infection in premature infants. It is an antibody against the F protein, and the anti-G protein antibody is still in the development stage

Method used

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  • Fully-humanized monoclonal antibody for RSV (respiratory syncytial virus) adhesion protein G surface antigen
  • Fully-humanized monoclonal antibody for RSV (respiratory syncytial virus) adhesion protein G surface antigen
  • Fully-humanized monoclonal antibody for RSV (respiratory syncytial virus) adhesion protein G surface antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of fully human anti-RSV antibodies RSVG71 and RSVG78

[0028] 1. Artificially synthesized heavy and light chains of RSVG71 and RSVG78

[0029] According to the nucleic acid sequence of the heavy chain of the monoclonal antibody RSVG71 shown in SEQIDNO:1, the light chain (KAPPA) of the monoclonal antibody RSVG71 shown in SEQIDNO:2; the monoclonal antibody RSVG78 shown in SEQIDNO:3 Heavy chain, the nucleotide sequence of monoclonal antibody RSVG78 light chain (KAPPA) as shown in SEQIDNO:4, send GENEWIZ company artificial synthesis.

[0030] The artificially synthesized RSVG71 and RSVG78 heavy chains and light chains were used as templates, and Taq enzyme, dNTPs, and primers were added, respectively, for PCR to obtain PCR products.

[0031]2. Construction of expression vectors for recombinant antibodies

[0032] The PCR product was recovered using a rapid DNA product purification kit (purchased from Kangwei Century), and 40 μl of the PCR product was...

Embodiment 2

[0040] Expression and purification of embodiment 2 monoclonal antibodies RSVG71 and RSVG78

[0041] Using the plasmid containing the heavy chain (RH71) of RSV G71 obtained in Example 1 and the plasmid containing the light chain (RK71) of RSV G71, and the plasmid containing the heavy chain (RH78) of RSV G78 and the light chain (RK78) containing RSV G78 The plasmids were transfected into 293T cells. Dilute the plasmid and PEI with Opti-MeM (1X) buffer, then slowly add the PEI-Opti-MeM mixture into the plasmid-Opti-MeM mixture tube, let it stand at room temperature for 20 minutes, then add the PEI and plasmid mixture to the in the cell suspension. The cell concentration at the time of transfection was 0.25~0.5×10 6 cells / ml, transfection of cells per well uses 2.5 μg plasmid containing RSV G71 heavy chain + 2.5 μg plasmid containing RSV G71 light chain + 10 μg PEI, and 2.5 μg plasmid containing RSV G78 heavy chain + 2.5 μg plasmid containing RSV G78 light chain +10 μg PEI. Af...

Embodiment 3

[0044] Expression and purification of embodiment 3 RSV G antigen

[0045] (1) Construction of G protein expression vector

[0046] First, the G protein target sequence is artificially synthesized by genetic engineering, and 6 His are added to the N-terminus of the sequence, which can be combined with nickel in the nickel column, so that it can be purified by affinity chromatography, and NheI / NotI are added at both ends Two enzyme cutting sites. Both the synthesized G protein DNA fragment and the expression vector pcDNA3.1-Zeo(+) (Invitrogen Company) were digested with NheI / NotI, and the G protein target fragment and the expression vector fragment were recovered, ligated, transformed, and PCR and Positive clones were identified by restriction enzyme digestion, and the correctness of the expression vector was finally verified by sequencing. A large number of plasmids were extracted by alkaline lysis for transient transfection.

[0047] (2) Transient transfection of 293F cells...

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Abstract

The invention provides a fully-humanized monoclonal antibody RSVG71 or RSVG78 specifically combined with adhesion protein (G) of RSV (respiratory syncytial virus) and discloses encoded nucleic acid of the fully-humanized monoclonal antibody, a carrier or a host cell containing the fully-humanized monoclonal antibody and a preparation method of the fully-humanized monoclonal antibody. The invention further discloses an application of the RSV-resistant fully-humanized monoclonal antibody RSVG71 or RSVG78 in prevention and treatment of RSV related diseases and in detection of RSV.

Description

technical field [0001] The invention relates to a fully human antibody, in particular to a fully human monoclonal antibody against the G surface glycoprotein of respiratory syncytial virus (RSV). Background technique [0002] Respiratory syncytial virus (respiratory syncytial virus, RSV) is the main pathogen that causes acute lower respiratory tract infection in infants and adolescents, and is also the main viral antigen that causes children with bronchiolitis and epidemic asthmatic pneumonia. , adult individuals with chronic lung disease, and immunocompromised adults (eg, bone marrow transplant patients). The World Health Organization estimates that 160,000 to 1,000,000 deaths from respiratory diseases worldwide are caused by RSV infection. [0003] The development of RSV vaccines has a history of more than 40 years, and has gone through stages such as inactivated / recombinant vaccines, antiviral complexes, antisense drugs, RNA interference technology, and antibody drugs (s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14A61P11/00G01N33/577G01N33/569
CPCA61K39/00C07K16/1027C07K2317/54C07K2317/55C07K2317/56C07K2317/622C07K2317/624
Inventor 戴凯凡刘国华李蓬飞王全英
Owner REYOUNG PHARMA
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