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Monoclonal antibody for neutralizing bevacizumab and application thereof

A monoclonal antibody, bevacizumab technology, applied in chemical instruments and methods, microbial-based methods, biochemical equipment and methods, etc., can solve the problem of limited yield, large activity difference, and low yield of antigen affinity purification and other problems, to achieve the effect that the preparation method is simple and efficient, and easy to popularize and apply

Active Publication Date: 2018-10-09
DRAGONBOAT BIOPHARMACEUTICAL (SHANGHAI) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently commonly used positive antibodies include affinity-purified polyclonal antibodies (generally derived from rabbits or sheep). Although such antibodies can represent the diversity of anti-drug antibodies in the body to a certain extent, due to the low yield of antigen affinity purification , the yield of each batch is limited, and the activity varies greatly between different batches, which may affect the consistency of immunogenicity evaluation from preclinical to clinical trials
The production process of monoclonal antibodies is economical and flexible, easy to purify, high yield, and different batches have stable binding activity, easy to commercial production, it is a good positive reference for evaluating the immunogenicity of antibody drugs, but there is a lack of it in the market today. Anti-bevacizumab variable domain monoclonal antibody

Method used

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  • Monoclonal antibody for neutralizing bevacizumab and application thereof
  • Monoclonal antibody for neutralizing bevacizumab and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of monoclonal antibodies

[0052] The main steps of the preparation method of this embodiment include: animal immunity, cell fusion, hybridoma cell screening, hybridoma cell cloning, and monoclonal antibody preparation;

[0053] (1) Animal immunization: 6-week-old BALB / C female mice, the antigen is commercial bevacizumab Avastin, the immunization method is conventional immunization, and the immunization route is subcutaneous injection, with 50 μg Avastin and Freund’s complete adjuvant Mix in equal volume and subcutaneously inject at 3 to 4 points on the back of the mouse; two weeks later the same dose of immunization with Freund's incomplete adjuvant; two weeks later with three immunizations with Freund's incomplete adjuvant; Boost the immunization after two weeks, use Freund’s incomplete adjuvant, and take spleen cells for fusion after 3 days;

[0054] (2) Cell fusion: prepared on the third day after the last immunization;

[0055] Preparation of spleen ...

Embodiment 2

[0081] Example 2 Specific experiment of binding between monoclonal antibody 4H7 and bevacizumab

[0082] experiment procedure:

[0083] (1) Coating: Dilute Avastin and control antibody Erbitux to 5 μg / mL with coating buffer respectively, and add them to different 96 wells, 100 μL / well, overnight at 2-8°C;

[0084] (2) Wash the plate: Wash the plate 3 times with washing solution, 300 μL / well;

[0085] (3) Blocking: 200 μL / well 3 BSA solution at room temperature for 2.5 hours;

[0086] (4) Wash the plate: Wash the plate 3 times with washing solution, 300 μL / well;

[0087] (5) Dilute the sample: Dilute the antibody 4H7 with buffer, and then serially dilute it twice to obtain 12 concentration samples, which are: 5000 ng / mL, 2500 ng / mL, 1250 ng / mL, 625 ng / mL, 312.5 ng / mL, 156.25 ng / mL, 78.12 ng / mL, 39.06 ng / mL, 19.63 ng / mL, 9.76 ng / mL, 4.88 ng / mL, 2.44 ng / mL;

[0088] (6) Transfer the diluted sample to a 96-well plate, 100 μL / well;

[0089] (7) Incubation: incubate overnight at 2-8°C;

[0090]...

Embodiment 3

[0098] Example 3 Neutralizing activity experiment of monoclonal antibody 4H7

[0099] experiment procedure:

[0100] (1) Coating: Dilute VEGF to 50 ng / mL with coating buffer, add to 96-well, 100 μL / well, overnight at 2-8°C;

[0101] (2) Wash the plate: Wash the plate 3 times with washing solution, 300 μL / well;

[0102] (3) Blocking: 200 μL / well of 3% BSA solution at room temperature for 2.5 hours;

[0103] (4) Wash the plate: Wash the plate 3 times with washing solution, 300 μL / well;

[0104] (5) Dilute Avastin sample: Dilute the antibody Avastin with buffer to 6 μg / mL, and then serially dilute it twice to obtain 12 concentration samples, in order: 3 μg / mL, 1.5 μg / mL, 750 ng / mL, 375 ng / mL, 187.5 ng / mL, 93.8 ng / mL, 46.88 ng / mL, 23.4 ng / mL, 11.72 ng / mL, 5.86 ng / mL, 2.93 ng / mL and 1.96 ng / mL;

[0105] (6) Dilute 4H7 samples: Dilute antibody 4H7 to 20 μg / mL with buffer, and then continuously dilute the antibody 2 times to obtain 12 concentration samples, in order: 20 μg / mL, 10 μg / mL, 5 μg / m...

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Abstract

The invention provides a monoclonal antibody for neutralizing a bevacizumab and the application thereof. An amino acid sequence of a light chain CDR1 of the monoclonal antibody is shown in SEQ ID NO.3; the amino acid sequence of a light chain CDR2 is shown in SEQ ID NO.4; the amino acid sequence of a light chain CDR3 is shown in SEQ ID NO.5; the amino acid sequence of a heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO.6; the amino acid sequence of a heavy chain CDR2 is shown in SEQ ID NO.7; the amino acid sequence of a heavy chain CDR3 is shown in SEQ ID NO.8; a large number of complex experiments screen and verify that the monoclonal antibody is capable of specifically binding to the bevacizumab, can block the binding of the bevacizumab to a VEGF(vascular endothelial growth factor), can be used for studying the immunogenicity of the bevacizumab in preclinical and clinical trials, monitoring and assessing the sensitivity and the specificity of immunogenicity assays, and has broad application prospects and great market value.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a monoclonal antibody that neutralizes bevacizumab and its application. Background technique [0002] In recent years, antibody drugs have been widely used in the clinical treatment of tumors, transplant rejection, infectious diseases, autoimmune diseases and other diseases due to their safety, effectiveness, and high specificity. However, the selection of antibody drugs for treatment will have a problem that cannot be ignored, that is, the immunogenicity of antibody drugs affects the treatment. These immunogenicity not only cause acute allergic reactions in the body, but also have a competitive inhibitory effect on the drug itself. The detection of the immunogenicity of antibody drugs is an important part of the application for clinical trials and registration of biotechnology drugs. Although the immunogenicity shown in animal models does not necessarily expect an immune respons...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/42C12N5/20G01N33/577C12R1/91
CPCC07K16/42C07K2317/33C07K2317/55C07K2317/565C07K2317/76G01N33/577
Inventor 邵喆刘聪刘钰山黄应峰
Owner DRAGONBOAT BIOPHARMACEUTICAL (SHANGHAI) CO LTD