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Anti-dr5 antibody and its preparation method and application

An antibody and carrier technology, applied in the field of anti-DR5 antibody and its preparation, can solve the problems of cell apoptosis and insufficient antagonistic activity, and achieve the effects of high biological activity, excellent sensitivity and high affinity

Active Publication Date: 2020-11-03
SHENZHEN ZHONGKE AMSHENN MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art, provide an anti-DR5 antibody and its preparation method and application, aiming to solve the insufficient antagonistic activity of the existing TRAIL receptor antagonist anti-tumor drugs, and the need to add cross-linking The technical problem of the ability of the agent to induce apoptosis

Method used

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  • Anti-dr5 antibody and its preparation method and application
  • Anti-dr5 antibody and its preparation method and application
  • Anti-dr5 antibody and its preparation method and application

Examples

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Embodiment 1

[0098] Preparation of anti-DR5 monoclonal antibody. In this example, the hybridoma method was used to produce anti-DR5 monoclonal antibody using mouse ascites.

[0099] Female BALB / c mice (6 weeks old) were immunized with sDR5-Fc protein antigen (provided by Shenzhen Zhongke Aishen Pharmaceutical Co., Ltd., the preparation method refers to patent 201610067931.2), and the antigen was emulsified with Freund's complete adjuvant for the first immunization. At the beginning of the second immunization, the antigen was emulsified with Freund's incomplete adjuvant and injected subcutaneously at 5 to 6 points. The amount of antigen injected per mouse was about 100ug. Ten days after the third immunization, a small amount of blood was collected from the tail of the mice for serum titer ELISA detection, and mice with high antibody titers (>1:100000) were selected for boosting the fourth immunization, and the antigenic protein was injected intraperitoneally , each mouse was injected about ...

Embodiment 2

[0124] Isotype identification of anti-DR5 monoclonal antibodies.

[0125] Using the commercial mouse monoclonal antibody Isotyping kit (sino biological, SEK003) to identify the subtype of the monoclonal antibody, the results are as follows image 3 As shown, it can be seen from the figure that the 6C93D6 antibody is an IgG2b antibody.

Embodiment 3

[0127] Affinity determination of monoclonal antibodies against DR5.

[0128] 10ug / ml sDR5-Fc protein (provided by Shenzhen Zhongke Aishen Pharmaceutical Co., Ltd.) was used to solidify and bind to the sensor, and SD buffer (PBS+0.02%Tween 20+0.1%BSA) was used to prepare different concentrations of 6C93D6 antibody (76nM, 38nM, 19nM, 9.5nM, 4.75nM) as the mobile phase, using an intermolecular interaction instrument (OCTET K2, PALLlife science) for affinity detection, the program is set as: Baseline 240s, Loading 360s, Baseline2180s, Association 480s, Dissociation 480s, AHC (Anti-hIgG Fc Capture) sensor was used. The result is as Figure 4 As shown, the results show that the affinity of the 6C93D6 antibody to DR5 is 6.77E-09 (M), that is, the 6C93D6 antibody has a high affinity to DR5.

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Abstract

The invention particularly relates to an anti-DR5 antibody, a preparation method and application thereof. The anti-DR5 antibody comprises a constant region and a variable region. The variable region comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises the following three CDRs: CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3; the light chain variable region comprises the following three CDRs: CDR1' shown in SEQ ID NO: 4, CDR2' shown in SEQ ID NO: 5, and CDR3' shown in SEQ ID NO: 6. The antibody has an unique antibody variable region sequence, specifically binds to an antigen DR5, does not cross-react with DR4, has high affinity and biological activity, and can induce apoptosis withoutadding a cross-linking agent, and can be combined with TRAIL to synergically induce apoptosis.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an anti-DR5 antibody and its preparation method and application. Background technique [0002] According to the research of the World Health Organization, from 1990 to 2013, the cancer mortality rate has increased from 12% to 15%, and it has become a high-mortality disease second only to cardiovascular diseases. It is predicted that the number of new cancer cases in the world will reach 20 million people, 12 million people died of cancer; therefore, human beings urgently need to develop new cancer treatment drugs. [0003] Inducing cancer cell apoptosis is one of many cancer treatment methods. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) can bind to death receptor (Death receptor, DR) to induce cancer cell apoptosis. Therefore, TRAIL-DR has become a popular target for the development of anti-tumor antibody drugs. TRAIL, also known as Apo2L, wa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
Inventor 万晓春陈倩夏蒙李俊鑫刘绿艳张青梅
Owner SHENZHEN ZHONGKE AMSHENN MEDICINE CO LTD