Method for treating tumors based on adenovirus vector carrier expressed full-length cetuximab

A technology of cetuximab and adenovirus, which is applied in the fields of biotechnology and virology, can solve the problems of hybridoma cell instability, complicated process, and difficult for patients to bear

Inactive Publication Date: 2017-01-04
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has defects such as long preparation cycle, complex process, unstable hybridoma cells and easy loss of resistance, which makes the preparation cost of cetuximab relatively high. The cost of a course of treatment with cetuximab is about 24,000 RMB, 1.15 million RMB per year, the high cost makes it difficult for patients benefiting from cetuximab to afford

Method used

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  • Method for treating tumors based on adenovirus vector carrier expressed full-length cetuximab
  • Method for treating tumors based on adenovirus vector carrier expressed full-length cetuximab
  • Method for treating tumors based on adenovirus vector carrier expressed full-length cetuximab

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preparation example Construction

[0103] 4. Preparation of recombinant adenovirus plasmid

[0104]The pShuttle-CTB plasmid was digested with PI-Sce I and I-Ceu I, and the adenovirus vector pAdC68-△E1 / E3 was digested with PI-Sce I and I-Ceu I to obtain the same sticky end, linear the carrier of transformation. Use agarose gel to separate and recover the target fragment. At the same time, use low-melting point agarose gel to separate the adenovirus vector, incubate the gel containing the adenovirus vector at 65°C for 5 minutes, and use T4 DNA ligase to dissociate the above two Products were subjected to sticky-end ligation. In the competent bacteria Stbl 2, the ligation product was transformed according to the conventional method, and then the transformed product was spread on the agarose plate containing ampicillin resistance, cultivated at 30°C for 24 hours, and the clones were selected and placed in the LB medium containing ampicillin resistance for 30 Cultivate overnight at ℃, extract the plasmid, use 1% a...

Embodiment 1

[0125] Example 1, construction and identification of recombinant adenovirus plasmid

[0126] Cetuximab is a human-mouse chimeric anti-EGFR antibody, a monoclonal antibody used to treat colorectal cancer, squamous cell carcinoma of the head and neck and other cancers. The present invention constructs AdC68-△E1 / E3 and Hu5-△E1 / E3 to express the full-length Cetuximab gene, and takes an animal model of colorectal cancer as an example to evaluate the effect of recombinant adenovirus expressing Cetuximab monoclonal antibody on tumors.

[0127] Such as figure 1 As shown, the heavy and light chains carrying the signal peptide (HLA-A*0201 signal peptide) are connected with F2A, and the full-length monoclonal antibody is double digested with Avr Ⅱ and Cla Ⅰ, and then connected to the shuttle carrying the CASI promoter and SV40polyA On the vector pUC57, construct the shuttle plasmid pUC57-CASI-CTB. pUC57-CASI-CTB, pAdC68-△E1 / E3 or pHu5-△E1 / E3 were digested with PI-Sce I and I-Ceu I and ...

Embodiment 2

[0129] Example 2, the acquisition and identification of recombinant adenovirus

[0130] Recombinant adenoviral plasmids pAdC68-CTB and pHu5-CTB were linearized by Pac I, and then transferred into HEK 293 cells with LipofectatimeTM 2000. After culturing for 8-12 days, plaques appeared. After the cells became round and suspended, the cells were collected and repeated. After freezing and thawing three times, the virus supernatant was taken to infect HEK 293 cells (25 cm 2 cell culture flask). Repeat the above steps to collect an appropriate amount of virus (about 27-30 150cm 2 cell culture flask), the cesium chloride density gradient centrifugation method was used to purify the adenovirus AdC68-CTB, and the preparation method of the recombinant adenovirus Hu5-CTB was the same as above. Finally, the inventors obtained the concentration of recombinant adenovirus AdC68-CTB as 8.5×10 12 vp / ml, Hu5-CTB concentration was 4.2×10 12 vp / ml.

[0131] Extract the adenovirus genome AdC6...

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Abstract

The invention relates to a method for treating tumors based on adenovirus vector carrier expressed full-length cetuximab. By adopting the method, an adnovirus vector is first adopted to successfully and efficiently express a cetuximab monoclonal antibody. The obtained monoclonal antibody is good in biological activity and can realize an effective treatment effect for animals.

Description

technical field [0001] The invention belongs to the fields of biotechnology and virology, and more specifically, the invention relates to a method for treating tumors based on adenovirus vectors expressing full-length cetuximab. Background technique [0002] Cetuximab (cetuximab) is the first human-mouse chimeric IgG1 monoclonal antibody (Iressa: firstangiogenesis inhibitor approved for the treatment of advanced NSCLC. ExpertRev Anticancer Ther, 2003.3(3):p.257). Cetuximab can specifically bind to EGFR, competitively block the binding of epidermal growth factor and its ligand, and the binding affinity to EGFR is 10 times that of the natural ligand EGF. After binding, it can inhibit the heterodimerization of EGFR, down-regulate its downstream genes, block PI3K-AKT, RAS-RAF-MAPK and other signal transduction pathways, which are involved in the proliferation, infiltration and inhibition of apoptosis of tumor cells Play an important role. Cetuximab can also endocytose EGFR, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C07K16/30C12N15/13C12N7/01A61K35/761A61P35/00
Inventor 周东明邢嫚
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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