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Human neutralizing antibody or antigen binding fragment thereof and application thereof

A technology for combining fragments and antibodies, applied in the direction of antibodies, applications, carriers, etc., can solve the problem of the decline of neutralizing antibody ability, and achieve the effect of high neutralizing activity and good neutralizing activity

Active Publication Date: 2022-06-03
GUANGZHOU EIGHTH PEOPLES HOSPITAL GUANGZHOU MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the neutralizing antibody ability of antibody drugs against the recently emerged Omicron (B.1.1.529) mutant strains has decreased significantly, so it is imminent to screen a broad-spectrum neutralizing antibody that can prevent known and future mutant infections

Method used

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  • Human neutralizing antibody or antigen binding fragment thereof and application thereof
  • Human neutralizing antibody or antigen binding fragment thereof and application thereof
  • Human neutralizing antibody or antigen binding fragment thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Example 1: Obtaining antibody sequence information by high-throughput single-cell sequencing

[0126] Blood was collected from patients who recovered after infection with the Delta (B.1.617.2) new coronavirus variant strain, and peripheral blood mononuclear cells (PBMCs) were separated from the fresh blood by gradient centrifugation with Ficoll. And B cells were isolated from PBMC by magnetic bead method, and antigen-binding B cells were enriched. Then, library construction was carried out in the P2 laboratory according to the kit instructions (ChromiumSingle Cell V(D)J Reagent Kits, 10X Genomics). Sequencing was performed on the MGI sequencing platform, running the MGISEQ-2000RS Sequencing Flow cell kit (made by MGI). Data were analyzed after sequencing off the machine, and antibody assembly was performed.

Embodiment 2

[0127] Example 2 Antibody protein expression and purification

[0128] GenScript was entrusted to express and purify the antibody protein.

Embodiment 3

[0129] Example 3 Analysis of the binding ability of neutralizing antibodies to antigens of original and mutant strains of SARS-CoV-2 by ELISA

[0130] Take 50ng of the new coronavirus RBD antigen or the new coronavirus S1 antigen to coat the ELISA plate, 4 ℃, overnight. Washed 3 times with PBST (0.05%). Add 100 ul of 5% nonfat milk powder to each well and block at room temperature for 2 hours. Washed 3 times with PBST. The neutralizing antibody was serially diluted with skim milk powder, the initial concentration was 1.5μg / ml (10nM), and 5-fold serial dilution was carried out in sequence, a total of 8 concentration gradients were added to the wells, each concentration gradient was repeated 3 wells, and 50ul was added. The primary antibody was prepared with skim milk powder at 2ug / ml and incubated at room temperature for 1h. Washed 3 times with PBST. Add 50ul 8000-fold diluted Anti human-FC secondary antibody, incubate at room temperature for 45min, and wash 3 times with PB...

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Abstract

The invention relates to a human neutralizing antibody or an antigen binding fragment thereof and application thereof, the human neutralizing antibody or the antigen binding fragment comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprises amino acid sequences: (I) HCDR1, HCDR2 and HCDR3 respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; or (II) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 11, 12 and 13 respectively, or (II) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 12 and 13 respectively; or (III) HCDR1, HCDR2 and HCDR3 of which the amino acid sequences are respectively shown as SEQ ID NO: 21, 22 and 23; the light chain variable region comprises amino acid sequences: (I) an LCDR1, an LCDR2 and an LCDR3 respectively shown as SEQ ID NO: 4, 5 and 6; or (II) an LCDR1, an LCDR2 and an LCDR3 as shown in SEQ ID NO: 14, 15 and 16 respectively, or (II) an LCDR1, an LCDR2 and an LCDR3 as shown in SEQ ID NO: 16 respectively; or (III) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, 35 and 26, respectively, as shown in SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 35 and SEQ ID NO: 26. The neutralizing antibody YB9-258, the neutralizing antibody YB13-292 and the neutralizing antibody YB13-208 disclosed by the invention can be used for effectively inhibiting SARS-CoV-2 pseudovirus infection, and also has relatively good neutralizing activity on an SARS-CoV-2 true virus.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a human-derived neutralizing antibody or an antigen-binding fragment thereof and application thereof, and more particularly, to a human-derived neutralizing antibody or an antigen-binding fragment thereof that binds to coronavirus S protein, Polypeptides, polynucleotides, nucleic acid constructs, expression vectors, transformed cells, pharmaceutical compositions, and their use in the preparation of medicaments for preventing or treating or detecting or diagnosing novel coronavirus infection. Background technique [0002] Coronavirus disease 2019 (COVID-19), caused by a novel coronavirus called Severe Acute Respiratory Syndrome (SARS-CoV-2), has spread severely worldwide. At present, COVID-19 has spread to more than 100 countries or regions around the world, infecting more than 200 million people and directly causing more than 4.4 million deaths. SARS-CoV-2 is an RNA virus with...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14G01N33/569G01N33/68C12N15/85
CPCC07K16/10A61P31/14G01N33/56983G01N33/6854C07K2317/24C07K2317/565C07K2317/56C07K2317/92C07K2317/76A61K2039/505G01N2333/165G01N2469/10
Inventor 唐小平谢彩霞于海生王媚娘李锋刘小盼王亚萍罗小斐石静蓉刘龙奇冯成千项海涛唐国芳石泉郭栗良子李姣姣
Owner GUANGZHOU EIGHTH PEOPLES HOSPITAL GUANGZHOU MEDICAL UNIV
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