Fully-human-derived anti-HCV (hepatitis C virus) neutralizing antibody-TRN1001

An antibody and whole-human technology, applied in the fields of cellular immunology and molecular biology, to prevent repeated HCV infection and reduce adverse reactions

Active Publication Date: 2017-05-31
JINAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The hypervariable region 1 (HVR1) composed of 27 amino acid residues at the N-terminal of E1 and E2 is recognized as a neutralizing epitope, but its sequence varies greatly among different genotypes and quasispecies

Method used

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  • Fully-human-derived anti-HCV (hepatitis C virus) neutralizing antibody-TRN1001
  • Fully-human-derived anti-HCV (hepatitis C virus) neutralizing antibody-TRN1001
  • Fully-human-derived anti-HCV (hepatitis C virus) neutralizing antibody-TRN1001

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Preparation of fully human monoclonal neutralizing antibody TRN1001 against HCV

[0065] 1. PBMC Isolation and Single Memory B Cell Sorting

[0066] After cell counting, cells were sorted from PBMC by flow cytometry. First, cell debris, adherent cells and dead cells were removed, and CD3- / CD14- / CD16- / IgM- cells were obtained by fluorescent antibody staining, and CD235a-expressing cells were selected. / IgD- / CD20+ B cells, CD27ALL memory B cells were circled, and E2 double-fluorescence-labeled target cells were obtained with antigens specifically labeled with fluorescein.

[0067] 2. Single-cell RT-PCR isolation of antibody variable region genes

[0068] 1) Reverse transcription (RT) of single-cell RNA: Add 0.5 μM constant region primers and Superscript IV reverse transcriptase ( Invitrogen, Carlsbad, CA), positive and negative controls were set at the same time; reverse transcription PCR conditions: 55°C for 60min, cooled to 4°C. The product cDNA is stored a...

Embodiment 2

[0079] Example 2 TRN1001 Antibody Binding Activity Detection

[0080] 1. ELISA determination of binding activity

[0081] Steps: using different HCV envelope glycoproteins as antigens, and diluting the antigens to a concentration of 100 ng / ml with the coating solution, coating them on an ELISA 96-well plate, 100 μl per well, and overnight at 4°C. The blocking solution was blocked for 2 hours at 37°C. Add the primary antibody after blocking, the initial concentration of TRN1001 is 25 μg / ml, 3-fold serial dilution, the volume of each well is 100 μl, and incubate at 37°C for 1 hour. At the same time, HCV positive patient serum is used as a positive control, and rabies antibody is used as a negative control. HRP-labeled goat anti-human IgG (diluted 1:2000) was used as a secondary antibody and incubated at 37°C for 1 hour. Add 100 μL / well of substrate chromogenic solution (TMB), place at 37°C in the dark for 5 min, stop the reaction with 2M sulfuric acid, and perform colorimetry ...

Embodiment 3

[0086] Example 3 TRN1001 Antibody Affinity Activity Detection

[0087] CM5 chip-coupled capture molecules: Goat anti-human IgG antibodies were immobilized on the CM5 sensor chip as capture molecules, and coupled to the gold film surface of the CM5 chip according to the operation of the coupling kit. The dextran surface of the chip was activated with EDC and NHS, the coupling amount was determined by the injection time, and finally the remaining activated groups on the surface were blocked with ethanolamine. Capture molecule capture ligand on CM5 chip: The prepared fully human anti-HCV neutralizing antibody is used as the ligand, and the signal value obtained by calculation is used to determine the injection concentration and contact time of the monoclonal antibody. Affinity and kinetics analysis of the binding of monoclonal antibody TRN1001 to HCV-E2 protein (antigen): HCV-E2 protein strain was diluted with HBS-EP buffer as the analyte, and the analyte flowed through the chip ...

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Abstract

The invention discloses a fully-human-derived monoclonal antibody for neutralizing HCV (hepatitis C virus) and an application thereof and particularly discloses an antibody capable of recognizing and combining HCV envelope protein E2, a coding gene thereof, an expression vector and an application. The monoclonal antibody can stop the HCV from infecting susceptible cells and is fully-human-derived, and the antibody has greatly reduced immunogenicity, good affinity, good treatment effect and low side effects as compared with other animal-derived anti-HCV molecules.

Description

technical field [0001] The invention belongs to the field of cellular immunology and molecular biology, relates to a fully human monoclonal antibody, in particular to a fully human monoclonal neutralizing antibody against HCV. In addition, the present invention also relates to the preparation method and application of the neutralizing antibody. Background technique [0002] Hepatitis C virus (Hepatitis C virus, HCV) is the only known RNA virus that can cause chronic infection except retrovirus. The source of infection of HCV is mainly acute clinical type and asymptomatic subclinical patients, chronic patients and virus Carrier. Usually the blood of the patient is infectious 12 days before the onset of the disease, and can carry the virus for more than 12 years. It is estimated that there are currently more than 200 million HCV infected people in the world and the annual growth rate is 3.5 million, of which chronic carriers account for 60% to 80%; and HCV has pantropicity an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/63G01N33/576G01N33/577A61K39/42A61P31/14
CPCC07K16/109G01N33/5767G01N33/577
Inventor 张远旭王月明袁晓辉廖化新昝利鹏刘彤洪坡
Owner JINAN UNIVERSITY
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