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Highly expressed recombinant gutted adenovirus containing human constant region whole antibody gene and its use

A human constant region and adenovirus technology, applied in the field of life sciences, can solve the problems that recombinant empty-shell adenoviruses have not been seen, and the therapeutic concentration of antibodies cannot be reached.

Inactive Publication Date: 2005-06-29
钱其军
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although there are many applications of empty shell adenoviruses to express various therapeutic proteins in vivo, the protein concentration in serum in vivo is 0.5-10 μg / ml, while the therapeutic concentration in serum of full antibodies usually requires 40-60 μg / ml, so people generally It is believed that the application of recombinant empty adenovirus carrying full antibody gene therapy containing human constant region cannot achieve effective antibody therapeutic concentration
Therefore, so far, there have been no reports on the application of recombinant empty adenoviruses carrying full antibody genes containing human constant regions to treat diseases.

Method used

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  • Highly expressed recombinant gutted adenovirus containing human constant region whole antibody gene and its use
  • Highly expressed recombinant gutted adenovirus containing human constant region whole antibody gene and its use
  • Highly expressed recombinant gutted adenovirus containing human constant region whole antibody gene and its use

Examples

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example 1

[0319] Example 1. Humans carrying the humanized antibody SG-HER gene of anti-human epidermal growth factor receptor 2 (Her2), the human antibody SG-EGFR gene of anti-human epidermal growth factor receptor (EGFR), and the human anti-human CD20 Construction of expression vectors of chimeric antibody SG-CD20 gene and human-mouse chimeric antibody SG-anti HBV preS2 gene against human hepatitis B virus preS2

[0320] pShuttle-CMV was purchased from Qbiogene in the United States. It contains the human cytomegalovirus promoter (CMV IE) and SV40 poly A tailing signal. PCR technology was used to clone the human cytomegalovirus promoter (CMV IE) and SV40 poly A tailing signal. Bgl II restriction sites were added to the upstream and downstream positions, and multiple cloning sites were inserted between the human cytomegalovirus promoter (CMV IE) and the SV40 poly A tailing signal, respectively EcoRI, Sal I, Hind III, Xho I and BamH I restriction sites, the method uses positional mutation...

example 2

[0527] Example 2. Humans carrying the humanized antibody SG-HER gene of anti-human epidermal growth factor receptor 2 (Her2), the human antibody SG-EGFR gene of anti-human epidermal growth factor receptor (EGFR), and the human anti-human CD20 Construction and recombination of chimeric antibody SG-CD20 gene and anti-human-mouse chimeric antibody SG-anti HBV preS2 gene empty-shell adenovirus vector

[0528] The empty shell adenovirus helper virus (FL Helper) and 293 (203-FLPe6) containing yeast recombinase FLPe were donated by Professor Lowenstein PR of Cedars-Sinai Medical Center (Las Angeles CA 90048-1860) in the United States (see literature Umana P, Gerdes CA, Stone D, Davis JRE, Ward D, CastroMG, Lowenstein PR. Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination. Nature Biotechnology 2001 19 582-585).

[0529] For the construction of the empty shell adenovirus vector pSGG, the following seq...

example 3

[0560] Example 3: In vitro and in vivo expression of humanized antibody by recombinant empty adenovirus (SGG002) carrying humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2)

[0561] The normal cell line lung fibroblast WI-38, the normal cell line lung fibroblast BJ and the human epidermal growth factor receptor 2 (Her2) positive tumor cell SK-Br-3 were purchased from ATCC Company in the United States. 5×10 5 Spread the cells / well in a 6-well plate, incubate in a 37°C incubator with 5% CO2, change 1ml of serum-free liquid the next day, then add the control adenovirus Ad5-Lac Z or anti-human epidermal growth factor receptor 2 (Her2) humanized antibody SG-HER gene recombinant empty adenovirus SGG002. Its virus load is 5×10 8 VP / well, after culturing for 90 minutes, wash twice with phosphate buffered saline (PBS) to wash away the virus, culture with 3ml of culture medium with 5% fetal bovine serum, collect the supernatant at 72 hours respectiv...

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Abstract

The invention has supplied a recombinant virus containing whole antibody gene of the human constant zone with highly effective expression and its application. By bringing in the nucleotide sequence containing whole antibody gene of the human constant zone into the recombinant gene group, the invention has expressed the whole antibody of human constant zone in cell or body with high efficiency for curing illness.

Description

technical field [0001] The invention relates to the field of life sciences, in particular to a recombinant empty-shelled adenovirus capable of efficiently expressing full antibodies containing human constant regions for treating diseases and its application. Background technique [0002] The monoclonal antibody preparation technology founded by Kohler and Milstein in 1975 provides a new method for the treatment of diseases - guided therapy. In the initial stage of oriented therapy research, people gave great enthusiasm and unrealistic expectations, but the curative effect of early clinical research was far from satisfactory. The main reasons are as follows: 1. In the early clinical trials, mouse-derived antibodies were used, and antibodies against mouse-derived antibodies (HAMA) would be produced in the human body, which would neutralize therapeutic mouse-derived antibodies and make It is cleared quickly, so the half-life of the mouse-derived antibody in the human body is v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00A61K39/235A61K48/00A61P3/02A61P35/00C12N7/01C12N15/34C12N15/861C12N15/87
CPCA61K38/00A61K2039/5256A61P3/02A61P35/00C12N15/86C12N2710/10343C12N2800/30
Inventor 钱其军杨琴
Owner 钱其军
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