Bispecific neutralizing antibody for resisting novel coronavirus SARS-CoV-2 and application of bispecific neutralizing antibody

A technology of bispecific antibody and coronavirus, applied in the direction of antiviral agent, virus/bacteriophage, antiviral immunoglobulin, etc.

Active Publication Date: 2022-06-10
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are many variants of SARS-CoV-2, and the neutralizing activity of existing neutralizing antibodies against different variants has appeared to a certain extent reduce

Method used

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  • Bispecific neutralizing antibody for resisting novel coronavirus SARS-CoV-2 and application of bispecific neutralizing antibody
  • Bispecific neutralizing antibody for resisting novel coronavirus SARS-CoV-2 and application of bispecific neutralizing antibody
  • Bispecific neutralizing antibody for resisting novel coronavirus SARS-CoV-2 and application of bispecific neutralizing antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Preparation of Nanobodies Recognizing SARS-CoV-2 RBD

[0076] Step 1: Rescue the phage surface-displayed nanobody library

[0077] The fully synthetic nanobody library is stored in the host bacteria in the form of phagemids. Before the panning process begins, the library is rescued to make it a phage-displayed antibody library. The specific method is as follows:

[0078] Take 1mL (OD 600 =100) antibody library Glycerol bacteria, inoculated into 5 bottles of 200mL 2TY-CARB medium (OD 600 =0.1), 37°C, 250rpm shake to OD 600 = about 0.5; add 1.6×10 12 PFU M13KO7, let stand at 37°C for 30 minutes, shake the bacteria at 37°C at 200rpm for 30 minutes; transfer the bacterial liquid to a centrifuge bottle, centrifuge at 2200g for 15 minutes, resuspend the bacteria with 400mL 2TY-CARB-KAN, 30°C, 250rpm Shake the bacteria for 14-16 hours; divide the bacteria solution into centrifuge bottles (200mL per bottle), centrifuge at 10000g, 4°C for 30 minutes, collect the ...

Embodiment 2

[0095] Embodiment 2: SARS-CoV-2 pseudotyped virus neutralizing activity assay

[0096] 10 4 293T stably transfected ACE2 cells (from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were inoculated in a 96-well plate, and SARS-CoV-2 and its mutant strain pseudotyped viruses were mixed with different dilutions of neutralizing nanobodies. , incubated at room temperature for 30 minutes, added to the cells, and after 6 hours of cultivation, the DMEM medium with 2% FBS was replaced, and the cultivation was continued for 48 hours. The pseudotyped virus of SARS-CoV-2 and its mutants contains a luciferase reporter gene, and the intracellular luciferase activity was detected using the luciferase reporter gene detection kit (Promega, catalog number: E4550), and the curve was fitted with GraphPad Prism 8 , calculate IC 50 value.

[0097] The experimental results of Nanobody A4 and Nanobody A7 inhibiting the invasion of SARS-CoV-2 pseudo...

Embodiment 3

[0105] Example 3: Preparation of bispecific neutralizing antibodies

[0106] Step 1: Amplification and recombination of gene sequence

[0107] The recombinant plasmids of Nanobody A4 and Nanobody A7 obtained in Example 1 were used as templates for amplification respectively. Using the recombinant plasmid A4 as a template, use primers F and 4R to amplify to obtain gene fragment A4-1, and use primer 4F and primer R to amplify to obtain gene fragment A4-2; using recombinant plasmid A7 as a template, use primers F and 7R Amplify to obtain gene fragment A7-1, and use primer 7F and primer R to amplify to obtain gene fragment A7-2. The primers used are shown below.

[0108] F: 5'-caagtcaagctgctctgtg-3' (SEQ ID No. 12);

[0109] 4F:

[0110] 5'-agcggaggtgggggttcaggcggcggaggcagcgaagttcaattggttgaatc-3' (SEQ ID No. 13);

[0111] 4R: 5'-tgaacccccacctccgcttccgcctcctccagaagaaacagtaacttgag-3' (SEQ ID No. 14);

[0112] 7F:

[0113] 5'-agcggaggtgggggttcaggcggcggaggcagcgaagttcaactggttgaa...

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Abstract

The invention discloses a bispecific neutralizing antibody for resisting novel coronavirus SARS-CoV-2 and application of the bispecific neutralizing antibody, and belongs to the technical field of biological medicine. The antibody is a bispecific antibody A4-A7 or a bispecific antibody A7-A4 and comprises a nano antibody A4 and a nano antibody A7, each of the nano antibody A4 and the nano antibody A7 comprises a variable region, and each of the variable regions is provided with three complementary determining regions CDR1, CDR2 and CDR3. The invention also discloses a nucleotide molecule for coding the anti-novel coronavirus SARS-CoV-2 bispecific neutralizing antibody, a recombinant plasmid, a recombinant bacterium and a transgenic cell line containing the nucleic acid molecule, and applications of the nucleotide molecule, the recombinant plasmid, the recombinant bacterium and the transgenic cell line. The bispecific neutralizing antibody for resisting the novel coronavirus SARS-CoV-2, disclosed by the invention, has a neutralizing capability on related variants of the SARS-CoV-2 and concerned variant pseudotype viruses, IC50 reaches a sub-nanomole level, and the bispecific neutralizing antibody has good broad-spectrum neutralizing activity.

Description

technical field [0001] The invention relates to a bispecific neutralizing antibody against novel coronavirus SARS-CoV-2 and application thereof, belonging to the technical field of biomedicine. Background technique [0002] The novel coronavirus SARS-CoV-2 is the causative agent of the novel coronavirus pneumonia pandemic. With the continuous expansion of the infected population, the original strain of the new coronavirus has also begun to undergo evolutionary mutations. The World Health Organization classifies these mutant strains into Variants of concern (Variants of concern, VOC) according to the ease of transmission and the severity of related diseases. ): Alpha, Beta, Gamma, Delta, Omicron; Variants of interest VOI: Lambda, Mu, etc. [0003] Neutralizing antibodies are antibodies produced by B cells that can recognize and bind to surface antigens of pathogens to prevent pathogens from infecting cells. At present, monoclonal antibodies against SARS-CoV-2 have received ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13C12N15/85C12N5/10G01N33/569A61K39/42A61P31/14
CPCC07K16/10C12N15/85C12N5/0686G01N33/56983A61P31/14G01N2333/165C07K2317/565C07K2317/31C07K2317/569C12N2510/00C12N2800/107A61K2039/505Y02A50/30
Inventor 王健伟赵振东任丽丽黄鹤朱悦
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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