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Humanized anti-human von willebrand disease factor monoclonal antibody and application thereof

A technology of hemophilia factor and monoclonal antibody, applied in the direction of anti-coagulation factor immunoglobulin, application, antibody, etc., can solve the problems of unsatisfactory effect and failure of monoclonal antibody, achieve inhibition of adhesion and aggregation, inhibition of Thrombosis, low immunogenicity effect

Inactive Publication Date: 2013-05-29
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Monoclonal antibodies produced by traditional hybridoma technology are not ideal for direct use in disease treatment. The main reason is that the immunogenicity of mouse-derived antibodies causes the human body to produce neutralizing antibodies that render it ineffective.

Method used

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  • Humanized anti-human von willebrand disease factor monoclonal antibody and application thereof
  • Humanized anti-human von willebrand disease factor monoclonal antibody and application thereof
  • Humanized anti-human von willebrand disease factor monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Structural analysis of the binding of the murine antibody variable region to the vWF A1 region

[0036] The crystal structure of the vWF A1 and NMC-4Fab complex in the PDB database shows that the fourth α-helix of the A1 region is the main site for binding antibodies, and the arginines at positions 632 and 636 play an important role. The CDR2 and CDR3 of the variable region of the heavy chain of the antibody and the CDR3 of the variable region of the light chain are mainly involved in the combination with A1, so the three CDRs of the heavy chain are kept unchanged during the humanization process, while the CDRs of the light chain have A large degree of trade-offs, in order to achieve a higher degree of humanization.

Embodiment 2

[0037] Example 2: Humanized transformation of murine vWF antibody

[0038] (1) By comparing the amino acid sequences of the variable regions of NMC-4 and the human germline antibody family, it was found that the framework region of the heavy chain variable region has a high homology with the amino acid sequence encoded by the human germline antibody gene 4-59 Sex, and the length of the CDR is also the same, it is speculated that their variable regions have similar spatial structures, so the framework region of human germline antibody gene 4-59 was selected as the framework region of the humanized antibody heavy chain. Using the same method, it was found that the framework region of the light chain variable region was close to the amino acid sequence encoded by the human germline antibody gene O8, so the framework region of the human germline antibody gene O8 was selected as the framework region of the humanized antibody light chain.

[0039] (2) All the CDRs of the heavy chain...

Embodiment 3

[0048] Example 3: Expression of humanized antibody and detection of antibody activity

[0049] (1) Expression and purification of antibodies

[0050] 293F cells were inoculated into 293SFMII medium (GIBCO) in 8% CO 2 Suspension culture can be subcultured in an incubator, and transient transfection can be performed when the culture density reaches 10E6 / ml. Dilute the humanized antibody plasmid with OPTI-MEMI (GIBCO), dilute PEI (POLYSCIENCE) with the same medium, mix the plasmid and PEI at a mass ratio of 1:2 and let it stand for 5 minutes, then drop it into the suspension cells, and culture After 3-5 days, the cell supernatant was collected, concentrated and purified with a protein A affinity chromatography column.

[0051] (2) Platelet aggregation test

[0052] Freeze-dried platelet powder (Biopool) was dissolved in TBS, and distributed into 96-well plates at 60ul per well. Dilute vWF (Calbiochem) to 10ug / ml with PBS, and distribute to 96-well plates at 10ul per well. Th...

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Abstract

The invention discloses a humanized anti-human von willebrand disease factor monoclonal antibody and an application thereof. The antibody consists of a heavy chain variable region of mouse antibody NMC-4 and a light chain variable region of mouse antibody NMC-4, wherein amino acid sequences of CDR1, CDR2 and CDR3 contained in the heavy chain variable region are shown as serial numbers 1, 2 and 3 in sequence; and the light chain variable region comprises CDR1, CDR2 and CDR3 or CDR2 and CDR3 or CDR3, and the amino acid sequences of CDR1, CDR2 and CDR3 are shown as serial numbers 4, 5 and 6 in sequence. The monoclonal antibody is characterized by having vWF bonding capability, inhibiting thrombopoiesis from formation, having low immunogenicity and the like, can be used for preparing drugs for preventing and curing various thrombus diseases, and provides a possibility for preventing and curing human thrombus diseases, particularly thrombus of artery.

Description

technical field [0001] The invention relates to a humanized anti-human von Willebrand Factor (von Willebrand Factor, vWF) A1 region monoclonal antibody and application thereof. technical background [0002] The lesions caused or complicated by thromboembolism are one of the most common causes of death. With the development of society and the process of population aging, as well as the influence of factors such as diet and lifestyle changes, the incidence of thromboembolic diseases has increased year by year, which has brought huge losses and pains to society, families and individuals. Among cardiovascular and cerebrovascular diseases, thrombotic diseases rank first in morbidity, disability and mortality. Therefore, antithrombotic drugs have become the fastest growing class of drugs in the current cardiovascular and cerebrovascular drug market. [0003] Antithrombotic drugs for the treatment of thrombotic diseases are mainly divided into three categories: antiplatelet, antit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/36C12N15/13A61K39/395A61P7/02
Inventor 陈昌友吴朝霞
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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