Anti-H7N9 whole-human monoclonal antibody 7T33 and preparation method therefor and application of anti-H7N9 whole-human monoclonal antibody 7T33

Abstract

The invention relates to an anti-H7N9 whole-human monoclonal antibody 7T33 and a preparation method therefor and application of the anti-H7N9 whole-human monoclonal antibody 7T33. The whole-human monoclonal antibody 7T33 is quickly screened by utilizing a memory B cell polymerase chain reaction (PCR) method, and does not include any murine component. The antibody disclosed by the invention can bebound to hemagglutinin HA of an H7N9 virus in a targeted manner, and has significant neutralizing activity of anti-H7N9 virus infection; and the antibody disclosed by the invention does not have toxicand side effects of anti-mouse anti-antibodies and the like, has better biocompatibility, and is more suitable and has more potential to become a macromolecular drug for treatment of influenza viruses.

Description

Technical field

[0001] The invention belongs to the field of immunology, and specifically relates to an anti-H7N9 fully human monoclonal antibody 7T33 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 treating arthritis. This is a fully human monoclonal antibody with sales of more than 10 billion yuan for three consecutive years. From the launch of the first monoclonal antibody drug in 1986, monoclonal antibody drugs have experienced mouse-derived monoclonal antibody drugs (such as Orthoclone OKT3), chimeric monoclonal antibody drugs (Rituximab), humanized monoclonal antibody drugs (Herceptin), and whole human Source monoclonal antibody drugs (Humira) and other stages. Due to the appearance of anti-mouse antibody response (HAMA) in the human body, mouse-derived monoclonal an...

Examples

Embodiment 1

[0053] (1) Construction of NTH-3T3 cell line (3T3-CD40L) stably expressing CD40L

[0054] Use lentivirus to establish 3T3-CD40L feeder cells. The lentiviral expression vector pLVX-CD40L was constructed and transfected into 293T cells. The viral supernatant was collected on the fourth day of transfection. Activated NIH-3T3 cells, cultured for 3 generations and then infected with lentivirus, continued to culture and passage 3 times. Use flow cytometry to sort cells with FITC fluorescence intensity near MFI, and re-add them to the culture flask, 37°C, 5% CO 2 Culture and test in an incubator, 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 per ml 7 cell. Place in a ...

Embodiment 2

[0075] Example 2 Cloning, Recombination, Expression and Purification of Humanized Monoclonal Antibody 7T33 Gene

[0076] The B cells that can secrete the 7T33 antibody that binds to the H7N9 virus obtained in Example 1 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, use cDNA as template to clone antibody heavy and light chain genes, and recombine them in eukaryotic cells 293F or HEK293 for expression and purification. specifically:

[0077] (1) Transfer the lysed B cell liquid to a 96-well plate (Eppendorf, 030133366).

[0078] (2) Reverse transcription system: 150ng random primers (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), s...

Embodiment 3

[0132] Example 3 Neutralization experiment and antibody affinity experiment of purified fully human monoclonal antibody 7T33

[0133] (1) Experimental purpose

[0134] Using a virus-infected cell model (canine kidney cell MDCK), the inhibitory effect and effect of 7T33 antibody on H7N9 influenza virus were evaluated by micro-neutralization-ELISA experiment, and the anti-influenza virus activity of the antibody was detected.

[0135] (2) Experimental steps

[0136] (2.1) Cell plating

[0137] Trypsin digestion of logarithmic growth phase MDCK canine kidney cells, centrifuge and collect after termination, blow evenly to prepare a single cell suspension; adjust the cell concentration to 5×10 with cell culture medium 4 Pcs / ml, seeded on 96-well cell culture plate, and placed cells at 37℃, 5% CO 2 Cultivate overnight in an incubator.

[0138] (2.2) Pretreatment of 7T33 antibody and H7N9 virus (the virus A / Anhui / 1 / 2013 was taken from the Institute of Microbiology, Chinese Academy of Sciences) ...

Technical field

[0001] The invention belongs to the field of immunology, and specifically relates to an anti-H7N9 fully human monoclonal antibody 7T33 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 treating arthritis. This is a fully human monoclonal antibody with sales of more than 10 billion yuan for three consecutive years. From the launch of the first monoclonal antibody drug in 1986, monoclonal antibody drugs have experienced mouse-derived monoclonal antibody drugs (such as Orthoclone OKT3), chimeric monoclonal antibody drugs (Rituximab), humanized monoclonal antibody drugs (Herceptin), and whole human Source monoclonal antibody drugs (Humira) and other stages. Due to the appearance of anti-mouse antibody response (HAMA) in the human body, mouse-derived monoclonal an...

Examples

Embodiment 1

[0053] (1) Construction of NTH-3T3 cell line (3T3-CD40L) stably expressing CD40L

[0054] Use lentivirus to establish 3T3-CD40L feeder cells. The lentiviral expression vector pLVX-CD40L was constructed and transfected into 293T cells. The viral supernatant was collected on the fourth day of transfection. Activated NIH-3T3 cells, cultured for 3 generations and then infected with lentivirus, continued to culture and passage 3 times. Use flow cytometry to sort cells with FITC fluorescence intensity near MFI, and re-add them to the culture flask, 37°C, 5% CO 2 Culture and test in an incubator, 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 per ml 7 cell. Place in a ...

Embodiment 2

[0075] Example 2 Cloning, Recombination, Expression and Purification of Humanized Monoclonal Antibody 7T33 Gene

[0076] The B cells that can secrete the 7T33 antibody that binds to the H7N9 virus obtained in Example 1 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, use cDNA as template to clone antibody heavy and light chain genes, and recombine them in eukaryotic cells 293F or HEK293 for expression and purification. specifically:

[0077] (1) Transfer the lysed B cell liquid to a 96-well plate (Eppendorf, 030133366).

[0078] (2) Reverse transcription system: 150ng random primers (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), s...

Embodiment 3

[0132] Example 3 Neutralization experiment and antibody affinity experiment of purified fully human monoclonal antibody 7T33

[0133] (1) Experimental purpose

[0134] Using a virus-infected cell model (canine kidney cell MDCK), the inhibitory effect and effect of 7T33 antibody on H7N9 influenza virus were evaluated by micro-neutralization-ELISA experiment, and the anti-influenza virus activity of the antibody was detected.

[0135] (2) Experimental steps

[0136] (2.1) Cell plating

[0137] Trypsin digestion of logarithmic growth phase MDCK canine kidney cells, centrifuge and collect after termination, blow evenly to prepare a single cell suspension; adjust the cell concentration to 5×10 with cell culture medium 4 Pcs / ml, seeded on 96-well cell culture plate, and placed cells at 37℃, 5% CO 2 Cultivate overnight in an incubator.

[0138] (2.2) Pretreatment of 7T33 antibody and H7N9 virus (the virus A / Anhui / 1 / 2013 was taken from the Institute of Microbiology, Chinese Academy of Sciences) ...

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