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Anti-H7N9 fully humanized monoclonal antibody 4E18 and preparation method therefor and application of humanized monoclonal antibody

A monoclonal antibody, fully human technology, applied in the field of immunology, can solve the problems of drug resistance and no effective treatment methods

Active Publication Date: 2020-06-23
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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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 fully humanized monoclonal antibody 4E18 and preparation method therefor and application of humanized monoclonal antibody
  • Anti-H7N9 fully humanized monoclonal antibody 4E18 and preparation method therefor and application of humanized monoclonal antibody
  • Anti-H7N9 fully humanized monoclonal antibody 4E18 and preparation method therefor and application of humanized monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] (1) Construction of NTH-3T3 cell line stably expressing CD40L (3T3-CD40L)

[0055] 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.

[0056] 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 ...

Embodiment 2

[0077] Example 2 Cloning, recombination, expression and purification of humanized monoclonal antibody 4E18 gene

[0078] The B cells obtained in Example 1 capable of secreting the 4E18 antibody 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:

[0079] (1) Transfer the lysed B cell solution to a 96-well plate (Eppendorf, 030133366).

[0080] (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)...

Embodiment 3

[0134] Example 3 Neutralization and antibody affinity experiments of the purified fully human monoclonal antibody 4E18

[0135] (1) Purpose of the experiment

[0136] Using the virus-infected cell model (canine kidney cell MDCK), the inhibitory effect and effect of 4E18 antibody on H7N9 influenza virus were evaluated by microneutralization-ELISA experiment, and the anti-influenza virus activity of the antibody was detected.

[0137] (2) Experimental steps

[0138] (2.1) Cell plating

[0139] MDCK canine kidney cells in the logarithmic growth phase were digested with trypsin, collected by centrifugation after termination, blown evenly, and prepared a single cell suspension; the cell concentration was adjusted to 5×10 with cell culture medium 4 cells / ml, seeded in 96-well cell culture plate, and the cells were placed at 37°C, 5% CO 2 Incubate overnight in the incubator.

[0140] (2.2) Pretreatment of 4E18 antibody and H7N9 virus (the virus A / Anhui / 1 / 2013 was obtained from th...

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Abstract

The application relates to an anti-H7N9 fully humanized monoclonal antibody 4E18 and a preparation method therefor and application of the humanized monoclonal antibody. The fully humanized monoclonalantibody 4E18 is quickly screened from memory B cells by utilizing a polymerase chain reaction (PCR) method, and does not contain any murine component. The antibody of the application can be bound tohemagglutinin (HA) of an H7N9 virus in a targeted manner, and has a neutralizing activity of significantly resisting H7N9 virus infection; the antibody of the application does not have an anti-mouse anti-antibody toxic and side effect and the like, has higher biocompatibility, and is more suitable and has more potential to become a macromolecular drug for treating influenza viruses.

Description

technical field [0001] The application belongs to the field of immunology, and in particular relates to an anti-H7N9 fully human monoclonal antibody 4E18 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) i...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/16G01N33/577G01N33/569
CPCC07K16/1018A61P31/16G01N33/577G01N33/56983C07K2317/76C07K2317/565C07K2317/56C07K2317/21C07K2317/92A61K2039/505C07K16/10A61K39/42G01N33/569
Inventor 万晓春李俊鑫
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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