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Anti-H7N9 all-human-derived monoclonal antibody 2J17, and preparation method and application thereof

A monoclonal antibody and fully human technology, applied in the field of immunology, can solve the problems of rimantadine drug resistance and no effective treatment methods, and achieve the effect of reducing cumbersome operations and costs, low production costs, and simple and fast operation

Active Publication Date: 2017-11-10
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

H7N9 virus is a kind of influenza virus, which is resistant to the traditional antiviral drugs amantadine and rimantadine, and there is no effective treatment at present

Method used

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  • Anti-H7N9 all-human-derived monoclonal antibody 2J17, and preparation method and application thereof
  • Anti-H7N9 all-human-derived monoclonal antibody 2J17, and preparation method and application thereof
  • Anti-H7N9 all-human-derived monoclonal antibody 2J17, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] (1) Construction of NTH-3T3 cell line stably expressing CD40L

[0038]3T3-CD40L feeder cells were established using lentivirus. The lentiviral expression vector pLVX-CD40L was constructed, transfected into 293T cells, and the virus supernatant was collected on the fourth day of transfection. NIH-3T3 cells were activated, cultured for 3 generations, infected with lentivirus, continued to be cultured and passed 3 times. Use a flow cytometer to sort the cells whose FITC fluorescence intensity is near the MFI, and add them back to the culture flask at 37°C, 5% CO 2 Cultivate and detect in the incubator, and the test results are as follows: figure 1 As shown, 3T3 cells expressing CD40L and 3T3 cells transfected with empty vector pLVX (with ZxGreen) were stained with anti-CD40L with APC, and then analyzed by flow cytometry. It was found that all 3T3-CD40L feeder cells expressed CD40L. When the cells grow to 80%-90%, digest and collect the cells at a concentration of 1×10 ...

Embodiment 2

[0058] Example 2 Cloning, recombination and expression of humanized monoclonal antibody 2J17 gene

[0059] The B cells obtained in Example 1 capable of secreting antibodies binding to the H7N9 virus were lysed, and the lysate was taken for reverse transcription of RNA to obtain the PCR template cDNA of the human antibody gene. Design and synthesize primers for cloning antibody genes, clone heavy and light chain genes of antibodies using cDNA as a template, and express and purify in eukaryotic cells 293F or HEK293 recombinantly. specifically:

[0060] (1) Transfer the B cell liquid to a 96-well plate (Eppendorf, 030133366).

[0061] (2) Reverse transcription system: 150ng random primer (invitrogen, 48190-011), 0.5ul 10mMdNTP (Invitrogen, 18427-088), 1μl 0.1M DTT (Invitrogen, 18080-044), 0.5% v / v IgepalCA-630 (Sigma, I3021-50ML), 4U RNAsin (Promega), 6U Prime RNAse Inhibitor (Eppendorf) and 50U III reverse transcriptase (Invitrogen, 18080-044), add DEPC water to 14ul / well. ...

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Abstract

The invention relates to an anti-H7N9 all-human-derived monoclonal antibody 2J17, and a preparation method and application thereof. The heavy chain variable region amino acid sequence of the antibody is disclosed as SEQ ID NO:2, or an amino acid sequence with the same function, which is formed by performing substitution, deletion or addition of one or more amino acids on the sequence; and / or the light chain variable region amino acid sequence of the antibody is disclosed as SEQ ID NO:4, or an amino acid sequence with the same function, which is formed by performing substitution, deletion or addition of one or more amino acids on the sequence. The antibody 2J17 can be bound with hemagglutinin HA of the H7N9 virus in a targeted way. Compared with the rat-derived antibody, genes of the all-human-derived antibody are completely derived from human genes without components of other species, so that the all-human-derived antibody does not have rat antiantibody resistance and other toxic or side effects in the human body, has better biocompatibility, and has higher adaptability and potential to become the macromolecular drug for treating influenza viruses.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to anti-H7N9 fully human monoclonal antibody 2J17 and its preparation method and application. Background technique [0002] Among the top ten best-selling drugs in the world in 2015, 6 are fully human or humanized monoclonal antibody drugs. Ranked first is AbbVie's anti-TNFa monoclonal antibody Humira for the treatment of arthritis. This is a fully human monoclonal antibody and has been the king of drugs with sales of more than 10 billion for three consecutive years. Since the first monoclonal antibody drug was launched in 1986, monoclonal antibody drugs have experienced mouse monoclonal antibody drugs (such as Orthoclone OKT3), chimeric monoclonal antibody drugs (Rituximab), humanized monoclonal antibody drugs (Herceptin) and fully human monoclonal antibody drugs. Source monoclonal antibody drug (Humira) and other stages. Due to the anti-mouse antibody reaction (HAMA) in the...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10G01N33/577G01N33/569A61K39/42A61P31/16
CPCC07K16/1018C07K2317/24C07K2317/56C07K2317/92
Inventor 万晓春李俊鑫刘绿艳
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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