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Preparation method of novel cytokine fusion protein IP10 single-chain antibody

A single-chain antibody and cytokine technology, applied in the medical field, can solve the problem of not being able to enrich IP10, and achieve the effect of enhancing killing activity, reducing toxic and side effects, and maintaining natural conformation and folding.

Inactive Publication Date: 2015-07-01
卢小玲 +1
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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a novel specific cytokine fusion protein for glioma——IP10 single-chain antibody (IP10- scFv), which can not only specifically target and bind brain glioma cells, but also activate lymphocytes in the body to recruit around glioma cells, participate in the annihilation of tumor cells, and clinically treat glioma and prevent its recurrence play a fundamental role

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  • Preparation method of novel cytokine fusion protein IP10 single-chain antibody
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  • Preparation method of novel cytokine fusion protein IP10 single-chain antibody

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[0046] In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

[0047] After an in-depth understanding of glioma antigens, it was discovered that epidermal growth factor receptor mutant III (EGFRvIII) is a membrane antigen highly expressed on the cell membranes of most malignant gliomas, and its high expression can promote tumor cell proliferation, Invasion and infiltration, and no cross-reaction with neurons, glial cells, and vascular endothelial cells, etc., with high specificity (Sampson JH, Archer GE, Mitchell DA, Heimberger AB, Bigner DD.Tumor-specific immunotherapy targeting the EGFRvIII mutation in patients with malignant glioma. Semin Immunol. 2008, 20:267-75).

[0048] A single-chain antibody (scFv) is a small molecule peptide that uses a connecting peptide to connect the variable region of the heavy c...

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Abstract

The invention discloses a preparation method of a novel cytokine fusion protein-chemokine interferon inducible protein 10 single-chain antibody (IP10-scFv). The method comprises the steps of: S1. synthesizing a human IP10 extracellular terminal gene; S2. utilizing a recombinant phage antibody system to construct the single-chain antibody of an anti-epidermal growth factor receptor mutant III (EGFRvIII); S3. conducting optimization synthesis on a (Gly4Ser)3 flexible joint; S4. cloning IP10 and the anti-EGFRvIII single-chain antibody into an eukaryotic expression vector, and conducting connection through the optimized (Gly4Ser)3 flexible joint, thus obtaining an IP10-scFv gene; and S5. subjecting the gene to high expression in Escherichia coli, thus obtaining IP10-scFv with biological activity. The IP10-scFv provided in the invention has a higher antigen-antibody binding rate, can reduce the dosage of IP10 clinically and reduce the toxic and side effects, and further can reduce the adoptive input amount of glioma specific cytotoxic T lymphocyte (CTL) and increase the activity of CTL.

Description

technical field [0001] The invention belongs to the field of medicine, and in particular relates to a preparation method of a novel cytokine fusion protein-chemokine interferon-inducible protein 10 (IP10) single-chain antibody, and an in vivo activity detection method thereof. Background technique [0002] Despite comprehensive treatment methods such as surgery, radiotherapy, and chemotherapy, the prognosis of malignant glioma, especially glioblastoma multiforme, has not improved significantly in the past 20 years. Immunotherapy stimulates and supplements the body's anti-tumor immunity It has the characteristics of strong specificity, mild side effects and long-term memory. It is an ideal adjuvant therapy strategy for targeted removal of infiltrating residual tumor cells. In the past, it was believed that the central nervous system was an immune-exempt zone, and immunotherapy was difficult to be effective. However, in recent years, more and more evidences have suggested that...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C07K19/00A61K49/00C12R1/19
Inventor 卢小玲赵永祥王旋姜晓兵
Owner 卢小玲
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