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Antibody neutralizing human infected H7N9 influenza A virus and use thereof

An influenza A virus, antibody technology, applied in the direction of antibodies, antiviral agents, antiviral immunoglobulins, etc., can solve the problem of no marketed antibody drugs, etc.

Active Publication Date: 2015-09-09
SINO CELL TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many different types of anti-hemagglutinin and anti-M2 antibodies in the research and development stage, a small number of antibodies have entered clinical phase I and II studies, and there are no antibody drugs on the market yet

Method used

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  • Antibody neutralizing human infected H7N9 influenza A virus and use thereof
  • Antibody neutralizing human infected H7N9 influenza A virus and use thereof
  • Antibody neutralizing human infected H7N9 influenza A virus and use thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0057] Antibody preparation

[0058] Antibodies of the invention can be prepared by any method known in the art. For example, the sequence of the antibody is inserted into the corresponding eukaryotic expression vector and transfected into cells, such as the technology of obtaining antibody by transient expression in 293 cells, and the technology of obtaining antibody by stable expression in CHO cells. High-density cell culture techniques are usually used to obtain high yields of antibodies.

[0059] Antibody purification techniques, including techniques for preparing pharmaceutical grade antibodies, are also known in the art. Antibodies can be purified by centrifugation, filtration, affinity, charge, molecular weight, hydrophobicity and other chromatography methods.

[0060] The preparation methods of the antibody fragments of the present invention are also known in the art, including enzymatic digestion with pepsin or papain, or antibody fragments can be obtained by clonin...

Embodiment 1

[0082] Example 1. Anti-H7N9 virus neutralizing antibody activity and sequence information analysis of rabbit origin

[0083] Before the outbreak of H7N9 virus, the rabbit antibody library of H7N7 (A / Netherlands / 219 / 2003) and H7N9 (A / Anhui / 1 / 2013) hemagglutinin protein was constructed, which was obtained by 293 mammalian transient expression and insect system expression The rabbit antibody library was panned for the hemagglutinin protein of the H7N9 virus (A / Anhui / 1 / 2013), and 30 specific antibodies against the hemagglutinin protein of the human-infected H7N9 virus were screened. The antibody sequence was constructed into a eukaryotic expression vector, and a milligram-scale antibody was produced in the 293 mammalian transient expression system. The binding ability of the antibody to various H7N9 virus hemagglutinin proteins was tested (Table 4). At the same time, the hemagglutination inhibition test was used to detect the activity of the antibody, and the ability of the antibo...

Embodiment 2

[0087] Example 2. Humanized Antibody Has Efficient Virus Neutralizing Activity

[0088] According to the expression level and neutralizing activity of the antibody, the rabbit antibody H7N9-R002 was selected for humanization design to obtain the H7N9-H002 humanized antibody. The humanized transformation technology is based on the most classic CDR transplantation method and is aimed at rabbit source Antibody design. The humanized antibody gene sequence was obtained by whole gene synthesis, and constructed into a eukaryotic expression vector, and antibodies in milligram to gram protein quantities were produced in the 293 mammalian transient expression system. Humanized antibodies that retain the affinity of rabbit antibodies were screened by affinity detection with H7N9 hemagglutinin protein, and the humanized antibody H7N9-H002 with the highest affinity was obtained. The amino acid sequence of the variable region of the light chain of the antibody is SEQ ID NO: 56 , the nucleo...

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Abstract

Provided is a neutralising antibody which binds to and neutralises an H7 type influenza A virus. The nucleotide sequences of the light and heavy chain variable regions of the antibody are at least 75% identical to the nucleotide sequence of any one of SEQ ID NO: 38-55, 58 or 59, and the antibody can neutralise a human infection H7N9 influenza A virus haemagglutinin protein. Also provided is an efficient expression method for integrating an antibody into a cell such as a CHO cell, and a use for the antibody, a related haemagglutinin protein antigen binding fragment and an epitope in the diagnosis, treatment and prevention of human infection H7N9 influenza A virus infection.

Description

technical field [0001] The present invention relates to a new antibody series against the hemagglutinin HA protein of type A H7N9 influenza and its humanized antibody, which can neutralize 100TCID in a small dose 50 Half of the H7N9 virus infects MDCK cells, and protects mice at a dose of 10LD50, reaching a death protection rate of 90%. Therefore, the antibody can be used for the treatment and prevention of diseases caused by H7N9 virus. The present invention also relates to the binding epitope of the antibody-bound H7N9 hemagglutinin protein and the amino acid to which the antibody binds. The present invention also relates to a method for producing the antibody in CHO cells using gene recombination technology. Background technique [0002] Influenza virus is defined as A and B by the type of hemagglutinin (HA). Type A can be subdivided into 16 subtypes, of which group I has H1, H2, H5, H6, H8, H9, HI1, H12 , H13 and H16 subtypes exist, and group II consists of H3, H4, H7,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61K48/00A61P31/16G01N33/569
CPCA61K2039/505A61P31/16C07K16/1018C07K2317/76G01N2333/11
Inventor 谢良志孙春昀张杰宋德勇饶木顶朱萍霞李成红
Owner SINO CELL TECH INC
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