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Neutralizing antibody of EB (Epstein-Barr) virus and application thereof

A technology of Epstein-Barr virus and bispecific antibody, which is applied in the fields of application, antibody, antiviral agent, etc., can solve the problems of late start of bispecific antibody, and achieve the effects of inhibiting growth and metastasis, reducing titer, and high affinity

Active Publication Date: 2022-04-29
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bispecific antibodies started late in antiviral therapy, and no research reports on EBV-related bispecific antibodies have been found so far. Therefore, the development of bispecific antibodies to inhibit and eliminate EBV and treat EBV-related tumors is an important strategy for the treatment of EBV-related tumors. important strategies for disease

Method used

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  • Neutralizing antibody of EB (Epstein-Barr) virus and application thereof
  • Neutralizing antibody of EB (Epstein-Barr) virus and application thereof
  • Neutralizing antibody of EB (Epstein-Barr) virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: Preparation of EBVgp350 monoclonal antibody of the present invention

[0064] This example describes the generation of high affinity neutralizing monoclonal antibodies against the glycoprotein gp350 of Epstein-Barr virus.

[0065] Two New Zealand white rabbits about 6 weeks old (about 2kg in weight) were immunized with recombinant gp350 protein every 14 days. After 3 times of immunization, the spleens of the rabbits were collected, the total RNA in the spleen was extracted, and reverse-transcribed into cDNA. A phage-displayed antibody library was constructed, and monoclonal R1 and R9 that specifically bind gp350 were obtained after four rounds of affinity screening.

[0066] The heavy chain variable region amino acid sequence of the R1 antibody is shown in SEQ ID NO: 1, and the light chain variable amino acid sequence is shown in

[0067] Shown in SEQ ID NO:2.

[0068] The amino acid sequence of the heavy chain variable region of the R9 antibody is shown i...

Embodiment 2

[0072] Example 2: Determination of the binding activity of the monoclonal antibody R1 of the present invention

[0073] The scFv of R1 / R9 monoclonal antibody was fused to FLAG tag sequence or hFc and expressed in mammalian cell 293F. After the expression product was purified, the binding of the purified antibody to gp350 protein and EBV virus particles was tested by sandwich ELISA method. Coat gp350-hFc or EBV granule protein on the ELISA plate, add monoclonal antibody R1 / R9 at serial dilution for incubation, and use HRP-mouse anti-FLAG or HRP-goat anti-human antibody to detect R1 / R9, gp350 and EBV binding capacity ( figure 1 ). The results show that R1, R9 and CR2 all have high affinity with recombinant protein gp350 and EBV particles, and the binding activity of R1 is better than that of R9 and CR2

Embodiment 3

[0074] Example 3: Virus neutralizing activity test of the monoclonal antibody R1 of the present invention

[0075] 1. Monoclonal antibody R1 / R9 of the present invention blocks the binding activity of gp350 and CR2

[0076] ELISA verified the neutralizing activity of the monoclonal antibody in vitro. Coat gp350-hFc on the bottom of the plate, add serially diluted R1-hFc for binding, add 2 μg / ml recombinant CR2 protein, and determine whether the antibody can effectively block the combination of the two by detecting the binding of CR2. The results show that R1 / R9 can effectively block the binding of CR2 and gp350, and the neutralizing activity of R1 is better than that of R9 ( figure 2 ).

[0077] R1 / R9 blocks the binding of gp350 and CR2 by flow cytometry. The CR2-overexpressing cell line G4 was constructed on the basis of CR2-negative epithelial cells A431. Label gp350-His with biotin, then co-incubate with G4, add different concentrations of R1 antibody to block, and fin...

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Abstract

The invention provides a neutralizing monoclonal antibody capable of recognizing EB (Epstein-Barr) virus gp350 protein with high affinity. The antibody not only has high affinity, but also can block the combination of the gp350 protein and the ligand CR2 of the gp350 protein. The neutralizing monoclonal antibody provided by the invention not only has good in-vitro EB virus infection neutralizing activity, but also can remove EB virus particles and prevent EB viruses from infecting a plurality of solid organs and tissues in vivo. The invention further provides a bispecific antibody composed of the neutralizing monoclonal antibody and a monoclonal antibody targeting CD89. According to the present invention, the bispecific antibody can significantly kill EB virus positive lymphoma cells, can inhibit the growth and the metastasis of the tumor cells, and can be used for the prevention and the treatment of post-transplantation lymphoproliferative diseases (post-transplantation lymphoproliferative diseases (post-transplantation lymphoproliferative diseases), lymphoproliferative diseases (post-transplantation lymphoproliferative diseases), monocytosis, haemophilus syndrome (HLH) and lymphoma, and can be used for the prevention and the treatment of the post-transplantation lymphoproliferative diseases (post-transplantation lymphoproliferative diseases), the post-transplantation lymphoproliferative diseases (post-transplantation lymphoproliferative diseases (post-transplantation lymphoproliferative diseases (post-transplantation lymphoproliferative diseases) and the post-transplantation lymphoma.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a high-affinity monoclonal antibody for recognizing Epstein-Barr virus gp350 protein and application thereof. Background technique [0002] Epstein-Barr virus (EBV) has an extremely high infection rate to the human body, and its latent infection and activation are closely related to the occurrence of various human tumor types, which can cause nasopharyngeal carcinoma (NPC), gastric cancer, infectious Diseases such as mononucleosis (IM). EBV has been shown to be a pathogen responsible for approximately one percent of the global human cancer burden. In particular, EBV infection has been associated with tumor formation of lymphoid and epithelial origin. Tumors of lymphoid origin include endemic Burkitt lymphoma (eBL) and Hodgkin lymphoma (HL), as well as nasopharyngeal and gastric carcinomas of epithelial origin. Furthermore, multiple sclerosis (MS), an inflammatory autoi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C07K16/46C07K19/00C12N15/13A61K39/42A61K39/395A61K47/68A61P37/02A61P31/12A61P35/00A61P35/04G01N33/577G01N33/574G01N33/68G01N33/569
CPCC07K16/085C07K16/283C07K16/2809C07K14/7051A61K47/6801A61K47/6839A61P37/02A61P31/12A61P35/00A61P35/04G01N33/577G01N33/57484G01N33/6893G01N33/56994C07K2317/56C07K2317/565C07K2317/622C07K2317/31C07K2317/76C07K2317/92C07K2319/33G01N2333/05A61K2039/505G01N2800/24
Inventor 丰明乾何慧侠
Owner HUAZHONG AGRI UNIV
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