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Anti-cMet and CD3 specific dual-targeting antibody as well as preparation method and application thereof, microcircular deoxyribonucleic acid (DNA) containing expression cassette of dual-targeting antibody, and application of microcircular DNA

A dual-targeted antibody, specific technology, used in recombinant DNA technology, botanical equipment and methods, biochemical equipment and methods, etc.

Inactive Publication Date: 2017-06-09
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are currently 18 bispecific antibodies in the clinical research stage, and no bispecific antibody has been approved for marketing by the US FDA. These 18 bispecific antibody species do not have a BsAb against the cMet antigen.

Method used

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  • Anti-cMet and CD3 specific dual-targeting antibody as well as preparation method and application thereof, microcircular deoxyribonucleic acid (DNA) containing expression cassette of dual-targeting antibody, and application of microcircular DNA
  • Anti-cMet and CD3 specific dual-targeting antibody as well as preparation method and application thereof, microcircular deoxyribonucleic acid (DNA) containing expression cassette of dual-targeting antibody, and application of microcircular DNA
  • Anti-cMet and CD3 specific dual-targeting antibody as well as preparation method and application thereof, microcircular deoxyribonucleic acid (DNA) containing expression cassette of dual-targeting antibody, and application of microcircular DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] The construction of the minicircle parent plasmid with the nucleotide sequence encoding cMet×CD3 BsAb antibody comprises the following steps:

[0142] 1) Design BsAb antibody gene expression cassette

[0143] In addition to the nucleotide sequence encoding BsAb, the BsAb antibody gene expression cassette of Example 1 of the present invention also includes a coding sequence for a promoter, a secretion signal peptide, a protein purification tag, and a polyA tailing signal.

[0144] In a specific embodiment, the BsAb antibody gene expression cassette of Example 1 of the present invention includes sequentially connected:

[0145] Nucleotide sequence (Murine Ig kapa-chain signal peptide, SEQ ID NO: 17)-BsAb antibody gene sequence (V L cMet-linker1-V H cMet-linker2-V H CD3-linker3-V L CD3; the specific nucleotide sequence corresponds to the cMet LCVR-linker1-cMet HCVR-linker2-CD3HCVR-linker3-CD3LCVR-nucleotide sequence (SEQ ID NO: 18) of the tag encoding histidine 6-His t...

Embodiment 6

[0183] Steps of Example 6 "A method for constructing a microcircular DNA recombinant mother plasmid pMC.Bab containing a genetically engineered antibody gene expression cassette"; to obtain the cMet×CD3 BsAb antibody nucleotide sequence encoding cMet×CD3 BsAb antibody in Example 2 of the present invention pMC.BESXP minicircle master plasmid.

[0184] The only difference between step 1-2) of the present invention and the above PCT application is that step 1-2) of embodiment 2 of the present invention replaces the "gene sequence of BiTE" in embodiment 1 or 6 of the above PCT application with the embodiment of the present invention "Gene sequence of cMet×CD3 BsAb" of 2.

[0185] B)

[0186] Directly adopt the method of enzyme digestion-ligation, design two restriction sites of SmaI-ApaI at both ends of the BsAb antibody gene expression cassette and pMC.BESXP empty vector or p2ФC31 empty vector, and synthesize the BsAb antibody gene expression cassette by enzyme digestion- Inser...

Embodiment 2

[0189] Construction of Microcircle DNA with Nucleotide Sequence Encoding cMet×CD3 BsAb Antibody

[0190] Referring to the description of the PCT application (application number: PCT / CN2014 / 083741) submitted by the inventor in 2014:

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Abstract

The invention provides a specific dual-targeting antibody as well as a preparation method and application thereof. The specific dual-targeting antibody comprises a first antigen binding structural domain specifically bonded with human CD3, and a second antigen binding structural domain specifically bonded with a cMet antigen, and is capable of specifically identifying and bonding tumor cells with overexpression of the cMet antigen, wherein the tumor cells are of liver cancer, other cMet-positive tumor diseases and the like; furthermore, the specific dual-targeting antibody is also capable of bonding a T cell receptor, and mediates T cells to play a killing role, thus clearing cMet infection; therefore, the specific dual-targeting antibody has a great application value in diagnosis, treatment, prevention and detection of cMet-related diseases. The invention also provides microcircular deoxyribonucleic acid (DNA) encoding the nucleotide sequence of the specific dual-targeting antibody. The specific dual-targeting antibody and the microcircular DNA which are provided by the invention can be used for preparing antibody-based pharmaceutical compositions or cMet-positive tumor cell diagnostic reagents.

Description

technical field [0001] The invention relates to the fields of biomedicine and diagnosis and treatment, in particular to an anti-cMet and CD3 specific dual-targeting antibody, its preparation method and application, and the microcircle DNA containing the dual-targeting antibody expression cassette and its application. Background technique [0002] cMet, also known as MET or hepatocyte growth factor receptor (HGFR), is a heterodimeric protein encoded by the MET proto-oncogene, consisting of two bases, A and B. It is the specific membrane surface receptor of hepatocyte growth factor HGF, expressed in various tissue cells, such as cardiomyocytes and vascular endothelial cells, and mediates all the biological effects of HGF. After cMet is stimulated, it regulates the degree of phosphorylation in epithelial cells, leading to changes in cells, such as promoting mitosis, promoting cell movement, and the formation of tubular structures. [0003] Under normal conditions in the human ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C07K16/30C12N15/13C12N15/85C12N5/10A61K39/395A61K48/00A61P35/00
Inventor 刘运洪陈志英何成宜钟育健
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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