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Tumor-treatment adenovirus vaccine taking hTERT (human telomerase reverse transcriptase) as target point

A technology for tumor treatment and recombinant adenovirus, which is applied in anti-tumor drugs, gene therapy, virus/bacteriophage, etc., and can solve the problem of low infection efficiency of blood-derived cells

Inactive Publication Date: 2014-07-30
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most commercially available adenoviral vectors are derived from adenovirus type 5 or type 2 (Ad5 and Ad2), which have inefficient infection of blood-derived cells, including DCs

Method used

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  • Tumor-treatment adenovirus vaccine taking hTERT (human telomerase reverse transcriptase) as target point
  • Tumor-treatment adenovirus vaccine taking hTERT (human telomerase reverse transcriptase) as target point
  • Tumor-treatment adenovirus vaccine taking hTERT (human telomerase reverse transcriptase) as target point

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079]Example 1. Obtaining recombinant adenovirus co-expressing eTERTFc fusion antigen, GM / CSF and B7.1

[0080] 1. Construction of tumor therapeutic adenoviral vector Ad5F11p-eTERTFcGB

[0081] 1. Construction of recombinant shuttle vector pShuttle-CMV-eTERTFcGB

[0082] In the recombinant shuttle vector, the hTERT complex gene eTERTFcGB is composed of eTERTFc fusion antigen gene, GM / CSF gene (GenBank number: NM-000758) and B7.1 gene (GenBank number: NM-005191);

[0083] The eTERTFc fusion antigen gene consists of a human hTERT gene 1609-2628 base gene fragment (GenBank No.: NM_198253), a human IgG Fc segment gene (GenBank No.: Z17370) and a glycosylphosphatidylinositol (GPI) anchor peptide The composition of the gene (GenBank number: XM676434), and the nucleotide sequence of the eTERTFc fusion antigen gene is shown in sequence 1 in the sequence listing.

[0084] The eTERTFc fusion antigen gene is connected with the GM / CSF gene and the B7.1 gene through the IRES sequence, a...

Embodiment 2

[0205] Example 2. Amplification, purification and titer determination of tumor therapeutic adenovirus vaccine targeting hTERT

[0206] In recent years, recombinant adenovirus vectors have been increasingly used in the field of gene therapy. In order to meet the needs of preclinical animal experiments and clinical human trials, a large number of recombinant adenoviruses must be produced and purified through an effective and flexible process. product. The traditional method is cyanogen chloride density gradient ultracentrifugation, which can produce a small amount of clinical-grade adenovirus products, but with the advent of the era of gene therapy, a large amount of adenovirus products are required. The "Q Sepharose XL virus licensed" developed by GE based on the ion exchange medium can provide a fast and scalable method for purification of adenovirus. The purpose of this example is to conduct experimental research on the amplification and purification process of the recombina...

Embodiment 3

[0278] Example 3, Prokaryotic expression, purification and antigenic activity detection of telomerase reverse transcriptase hTERT antigenic fragment

[0279] At present, preclinical studies of broad-spectrum tumor immunotherapeutic vaccines targeting hTERT are mostly evaluated using mice or transgenic mouse tumor-bearing models. In order to further explore the effect of hTERT in immunotherapy of malignant tumors and its related mechanisms, The hTERT protein (1609-2628 bases) was purified and its immunogenicity was tested, which laid the foundation for the next step of the research on the immunological mechanism of Ad5F11p-eTERTFcGB in the tumor therapeutic adenovirus vaccine. .

[0280] First, the PCR method was used to amplify the hTERT1609-2628 base sequence, which was constructed into the prokaryotic expression vector pET-42a and induced to express in Escherichia coli E.coli.Rossata (DE3), and the expression conditions of hTERT protein were optimized Finally, determine the...

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Abstract

The invention discloses a tumor-treatment adenovirus vaccine taking hTERT (human telomerase reverse transcriptase) as a target point. Firstly, a hTERT composite gene eTERTFcGB is inserted to the downstream of a CMV (cytomegalovirus) promoter in a pShuttle-CMV shuttle vector to obtain a recombinant shuttle vector with the hTERT composite gene eTERTFcGB; then, the recombined shuttle vector and a virus framework vector AdEasy-1 / F11p co-transform bacteria to obtain a recombinant adenovirus vector. The recombinant adenovirus vector can inhibit growth of transplantation tumors, and can be applied to immunopotentiation of malignant tumor biological immune therapy.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an adenovirus vaccine, in particular to a tumor therapeutic adenovirus vaccine targeting hTERT. Background technique [0002] Metastasis and recurrence are the main factors of clinical death in most malignant tumor patients. Studies have shown that more than 90% of malignant tumor patients eventually die of tumor metastasis or recurrence. However, traditional treatment options, whether surgical treatment, radiotherapy or chemotherapy, are difficult to guarantee the complete eradication of advanced or metastatic malignant tumor cells. Tumor immunotherapy is currently a hot research field in the world. With the in-depth research on the anti-tumor immune mechanism and the biological characteristics of malignant tumors, especially the discovery of a variety of tumor-associated antigens, animal experiments and clinical trials for the biological treatment of malignant tumors Research has...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N7/01A61K48/00A61P35/00
Inventor 于继云阎瑾琦肖毅徐元基姜云波张巍王宇刘宁杜芝燕
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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