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Anti-H7N9 full-human-derived monoclonal antibody 5J13 and preparation method and application thereof

A monoclonal antibody, 5J13 technology, applied in the field of immunology, can solve the problems of no effective treatment and drug resistance, and achieve the effect of reducing cumbersome operations and costs, low production costs, and simple and fast operations

Active Publication Date: 2017-03-22
SHENZHEN INST OF ADVANCED TECH
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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 full-human-derived monoclonal antibody 5J13 and preparation method and application thereof
  • Anti-H7N9 full-human-derived monoclonal antibody 5J13 and preparation method and application thereof
  • Anti-H7N9 full-human-derived monoclonal antibody 5J13 and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] (1) Construction of NTH-3T3 cell line stably expressing CD40L (3T3-CD40L)

[0048] 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 5 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 concentr...

Embodiment 2

[0068] Example 2 Cloning, recombination, expression and purification of humanized monoclonal antibody 5J13 gene

[0069] 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:

[0070] (1) Transfer the lysed B cell solution to a 96-well plate (Eppendorf, 030133366).

[0071] (2) Reverse transcription system: 150ng random primer (invitrogen, 48190-011), 0.5μl 10mM dNTP (Invitrogen, 18427-088), 1μl 0.1M DTT (Invitrogen, 18080-044), 0.5% v / v Igepal CA -630 (Sigma, I3021-50ML), 4U RNAsin (Promega), 6U Prime RNAse Inhibitor (Eppendorf) and 50U III reverse transcriptase (Invitrogen, 18080-044), add ...

Embodiment 3

[0124] Example 3 Neutralization and antibody affinity experiments of the purified fully human monoclonal antibody 5J13

[0125] Neutralization experiment of fully human monoclonal antibody 5J13

[0126] (1) Hemagglutination test

[0127] (a) Take a 96-well V-shaped micro-reaction plate, add 25 μL PBS to each well of 1-12 wells with a micropipette, drop 8 rows in total, and add 25 μL PBS to the first row of wells in the last 4 rows.

[0128] (b) Pipette 25 μL of standard avian influenza antigen (H7N9 virus) into the wells of the first row, pipette 3 to 5 times and mix well.

[0129] (c) Pipette 25 μL of the mixed antigen solution from the wells in the first column and add it to the wells in the second column, then pipette 25 μL after mixing and add it to the wells in the third column, and serially dilute to the wells in the 11th column, and finally Aspirate 25 μL from the wells in the 11th column and discard, and set the wells in the 12th column as the red blood cell control....

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Abstract

The invention relates to an anti-H7N9 full-human-derived monoclonal antibody 5J13 and a preparation method and application thereof. Amino acid sequences of heavy and light chain CDR1, CDR2 and CDR3 of the antibody are GFSFSNYG in the heavy chain CDR1 area, ISYDGTNK in the heavy chain CDR2 area, AKGRGPYCSSSICYHGMDV in the heavy chain CDR3 area, QSVLSGSINMNY in the light chain CDR1 area, WAS in the light chain CDR2 area and QQYYSTPLT in the light chain CDR3 area correspondingly. The antibody can be combined with hemagglutinin A of the H7N9virus in a targeted mode and has remarkable neutralization activity in resisting H7N9 virus infection. Compared with a murine antibody, genes of the full-human-derived antibody are completely from human genes and have no components of other species, toxic and side effects such as the anti-mouse anti-antibody are avoided, the biocompatibility is better, and the anti-H7N9 full-human-derived monoclonal antibody 5J13 is more suitable and has more potential in becoming a macromolecular drug for treating influenza virus.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to the anti-H7N9 fully human monoclonal antibody 5J13 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...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/16G01N33/577G01N33/569
Inventor 万晓春李俊鑫刘绿艳
Owner SHENZHEN INST OF ADVANCED TECH
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