Anticancer genetic engineering bivalent antibody, preparation method thereof, and anticancer genetic engineering drug

A genetic engineering and anti-tumor technology, applied in the field of biopharmaceuticals, can solve the problems of limited specific binding between anti-tumor antibodies and tumor cells, and ineffective tumor prevention and treatment, and achieve prevention and treatment of tumor cell infection and specific binding Enhanced, crafted simple effects

Inactive Publication Date: 2014-05-21
SHENZHEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide an anti-tumor antibody that can simultaneously interact with tumor cells and T lymphocytes in view of the defects in the prior art that the specific binding effect between the anti-tumor antibody and tumor cells is limited, and the effect on the prevention and treatment of tumors is not obvious. Anti-tumor genetic engineering bivalent antibodies and anti-tumor genetic engineering drugs that specifically bind, effectively prevent and treat tumor infection

Method used

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  • Anticancer genetic engineering bivalent antibody, preparation method thereof, and anticancer genetic engineering drug
  • Anticancer genetic engineering bivalent antibody, preparation method thereof, and anticancer genetic engineering drug
  • Anticancer genetic engineering bivalent antibody, preparation method thereof, and anticancer genetic engineering drug

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preparation example Construction

[0024] The preparation method of the anti-tumor genetic engineering bivalent antibody of the present invention comprises the following steps: A: obtaining the constant region gene, the SIRPα gene and the B7 gene respectively; B: when the constant region gene is the constant region gene of a human antibody, using The eukaryotic expression vector constructs an expression plasmid comprising the constant region gene of the human antibody, the SIRPα gene and the B7 gene; or, when the constant region gene is a flexible linker sequence, a prokaryotic expression vector or the eukaryotic expression vector is used to construct an expression plasmid comprising a flexible linker sequence. The expression plasmids of the connecting sequence, SIRPα gene and B7 gene; C. Transfect the expression plasmid formed in step B into the expression cell line for culturing, and stably express the bivalent antibody; D. Collect the supernatant by centrifugation or purify the cells Obtain bivalent antibodie...

Embodiment 1

[0051] The PCR product of the constant region gene of the IgG1 antibody was digested by Xho I and Xba I, and then loaded into the eukaryotic expression vector pCDNA3.1 / His C, which was digested with the same restriction enzymes, to construct a recombinant plasmid pCDNA3 containing the constant region gene of the IgG1 antibody .1 / His C-IgG; clone the SIRPα gene and B7 gene amplified by PCR into the recombinant plasmid pCDNA3.1 / His C-IgG containing the constant region gene of IgG1 antibody, and construct the constant region gene containing IgG1 antibody Eukaryotic expression plasmids pCDNA3.1 / His C-SIRPα-IgG and pCDNA3.1 / His C-B7-IgG flexibly linked to SIRPα gene and B7 gene. Specifically, the above-mentioned flexible connection is realized through a flexible connection sequence (GAA TGT TCC).

[0052] Using 293 cells as the expression cell line, high-quality and high-concentration eukaryotic expression plasmids pCDNA3.1 / His C-SIRPα-IgG and pCDNA3.1 / His C-B7-IgG were extracted, ...

Embodiment 2

[0054] Design primers, primer sequence 9 is: 5'-CTT AAG CTT ACC ATG GGG GGT TCT-3', from the eukaryotic expression plasmid pCDNA3.1 / His C-SIRPα-IgG and pCDNA3.1 / His C- The SIRPα-IgG and B7-IgG genes of the eukaryotic expression vector pCDNA3.1 / His C with selection tags (6×His tag and Xpress Epitop tag) were amplified from B7-IgG, and these two genes were loaded into pCDNA5 / In the eukaryotic vector of FRT / TO TOPO TA, the eukaryotic expression plasmids pCDNA5 / FRT / TO TOPO TA-SIRPα-IgG and pCDNA5 / FRT / TO TOPO TA-B7-IgG were obtained.

[0055] The human kidney epithelial cell line Flp-In T-Rex 293 was used as the expression cell line to establish a stable and high-expression cell line, and extract high-quality and high-concentration eukaryotic expression plasmids pCDNA5 / FRT / TO TOPO TA-SIRPα-IgG and pCDNA5 / FRT / TO TOPO TA-B7-IgG was transfected into Flp-In T-Rex 293 cells using liposome transfection method. After 24 hours, the antibiotic Blasticidin B was used to screen for resistan...

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Abstract

The invention relates to an anticancer genetic engineering bivalent antibody, a preparation method thereof, and an anticancer genetic engineering drug. The anticancer genetic engineering bivalent antibody comprises a constant region, and SIRPa and B7 connected with the constant region. The preparation method of the anticancer genetic engineering bivalent antibody comprises following steps: acquirement of constant region gene, SIRPa gene, and B7 gene; construction of an expression plasmid containing the three genes; transfection of the expression plasmid into expression cell strain for culturing; and obtaining of the bivalent antibody via separation and purification. The bivalent antibody comprises both SIRPa and B7 which are capable of realizing specific binding with CD47 locus of tumor cells and CD28 locus of lymphocyte; specific binding performance is improved greatly; double inhibition on body tumor cells is realized; the preparation method is simple and practicable; tumor cells can be influenced by the anticancer genetic engineering drug containing the anticancer genetic engineering bivalent antibody effectively; and the anticancer genetic engineering drug is capable of preventing and treating tumor diseases effectively.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to an anti-tumor genetic engineering antibody, and more specifically, relates to an anti-tumor genetic engineering bivalent antibody, a preparation method thereof, and an anti-tumor genetic engineering drug. Background technique [0002] CD47 is also called integrin associated protein (integrinassociated protein, IAP), and its ligand is signal regulatory protein alpha chain (signal regulatory protein alpha, SIRPα). CD47 can produce inhibitory signals by combining with SIRPα to reduce the phagocytic activity of phagocytes (monocytes and macrophages), thereby inhibiting the immune system. In the study of malignant diseases such as leukemia, non-Hodgkin's lymphoma, bladder cancer, and breast cancer, it was found that tumor cells had elevated CD47, and high expression of CD47 indicated a poor clinical prognosis. CD47 also existed in some types of tumor stem cells. Overexpressi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/85C12N15/13A61K39/395A61P35/00
Inventor 周兆平易俊波王玉树雷明军苏庆宁
Owner SHENZHEN UNIV
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