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Construction body of nano antibody R14 and application thereof

A nanobody and pentamer technology, applied in the direction of antibodies, antiviral agents, antiviral immunoglobulin, etc., can solve the problem of low drug concentration, inability to effectively reduce the viral load of the lungs, and reduce the antiviral effect of neutralizing antibodies, etc. question

Active Publication Date: 2022-07-29
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Conventional monoclonal antibodies are generally administered through intravenous injection. However, the drug concentration of monoclonal antibodies administered through the intravenous route from the systemic circulation into the lungs is very low, which greatly reduces the antiviral effect of the neutralizing antibody itself, resulting in inability to Effectively reduces the viral load in the lungs

Method used

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  • Construction body of nano antibody R14 and application thereof
  • Construction body of nano antibody R14 and application thereof
  • Construction body of nano antibody R14 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1: Construction, expression and purification of nanobody R14-based trivalent (TR14) and IgM pentameric (MR14) antibodies

[0089] The structural schematic diagrams of the monovalent Nanobody and its trivalent form and IgM pentamer form in this example are as follows: figure 1 shown.

[0090] The basic nanobody R14 used was obtained by the laboratory by simultaneously immunizing alpacas with SARS-CoV-2 RBD protein and SARS-CoV-2 NTD protein, constructing antibody library, and screening by phage display technology; the amino acid sequence of monovalent nanobody R14 As shown in SEQ ID NO: 8, it can bind SARS-CoV-2 RBD with high affinity and specificity (the binding constant is less than 1E-10M), and in the pseudovirus neutralization experiment, it can neutralize SARS with high neutralizing activity -CoV-2 pseudovirus, all of which indicate that the R14 nanobody is a novel coronavirus (SARS-CoV-2) alpaca-derived nanobody that can bind to SARS-CoV-2 RBD with high a...

Embodiment 2

[0094] Example 2: Expression and purification of SARS-CoV-2 and its variant strain RBD

[0095] The coding sequence of 6 histidine tags (hexa-His-tag) is connected to the 3' end of the coding sequence of the RBD protein of the original strain of SARS-CoV-2 (its amino acid sequence is shown in SEQ ID NO: 18) and the translation stop codon TGA, through the restriction endonuclease sites EcoRI and XhoI, it was constructed into the pCAGGS vector (purchased from Invitrogen), and then the resulting recombinant vector was transfected into 293F cells (purchased from Invitrogen), carried out Expression of SARS-CoV-2 RBD-his protein.

[0096] The coding sequence of the RBD protein (its amino acid sequence is shown in SEQ ID NO: 19) of the SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.1 and Omicron (B.1.1.529) ) The 3' end of the coding sequence of the RBD protein of subtype BA.2 (its amino acid sequence is shown in SEQ ID NO: 20) is respectively connected with the coding seq...

Embodiment 3

[0098] Example 3: Surface plasmon resonance technology to detect the binding ability of each antibody to the RBD protein of the original SARS-CoV-2 strain and its variants

[0099] Surface plasmon resonance analysis was performed using a Biacore 8K (Biacore Inc.). Specific steps are as follows:

[0100] The monovalent Nanobody R14 and its constructs TR14 and MR14 prepared in the above examples were biotinylated, and then immobilized on the Series Sensor Chip SA chip (Cytiva Life Sciences); with PBST buffer (2.7 mM KCl, 137 mM NaCl) , 4.3mM Na 2 HPO 4 , 1.4mM KH 2 PO 4 , 0.05% Tween) The RBD protein of the original SARS-CoV-2 strain prepared in the above-mentioned example and its variant strains were diluted by multiple ratios, and the samples were loaded one by one from low concentration to high concentration on the chip. Calculations of binding kinetic constants were performed using BIAevaluation software 8K (Biacore, Inc.). Equilibrium dissociation constant (K) between...

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Abstract

The invention provides a construction body (including a multivalent nano antibody and a nano antibody fusion protein) of a nano antibody R14 based on specific binding SARS-CoV-2RBD, a related product thereof and an application of the construction body. The construction body (including multivalent nano-antibody and nano-antibody fusion protein) of the nano-antibody R14 based on the specific binding SARS-CoV-2RBD can effectively inhibit SARS-CoV-2 infection and variant strain infection thereof, can be dosed by atomization, can directly reach the lung, takes effect quickly, is long in half-life period, and can be used for preparing the nano-antibody R14 of the SARS-CoV-2RBD. And a more effective treatment strategy is provided for clinically preventing or treating infection of the new coronavirus and the variant thereof.

Description

[0001] This application is a divisional application of an invention patent application with an application date of March 21, 2022, an application number of 202210278872.9, and the invention name of "Construct of Nanobody R14 and its application". technical field [0002] The present invention relates to the field of biomedicine, in particular to a nanobody R14 construct and application thereof, and more particularly, to a multivalent nanobody based on the nanobody R14 that specifically binds to SARS-CoV-2 RBD, a nanobody fusion protein, a nanobody fusion protein, and a nanobody fusion protein encoding the same. The polynucleotide, the nucleic acid construct comprising the polynucleotide, the expression vector comprising the nucleic acid construct, the transformed cell comprising the above-mentioned polynucleotide, the nucleic acid construct or the expression vector, and the pharmaceutical combination comprising any of the above-mentioned products Compounds and their application...

Claims

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Application Information

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IPC IPC(8): C07K16/10A61K39/42A61P31/14
CPCC07K16/10A61P31/14C07K2317/569C07K2317/35C07K2317/565C07K2317/567C07K2317/52C07K2317/64C07K2317/92C07K2317/76C07K2317/94C07K2317/22A61K2039/505G01N33/56983A61K2039/53G01N2333/165G01N2469/10
Inventor 王奇慧高福刘红辉刘博仵丽丽韩鹏程
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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