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Efficient IL-2 (interleukin-2) mutant fusion protein and application thereof

An interleukin and fusion protein technology, applied in the field of human interleukin II mutant fusion protein, can solve the problems of application and curative effect limitations, short half-life of interleukin II, etc., to eliminate toxic and side effects, improve clinical efficacy and application scope, Enhance the effect of attributes

Inactive Publication Date: 2017-10-20
南京康志制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, due to the short half-life of injected interleukin II in vivo, its clinical application and curative effect are limited, and its clinical application also has very large limitations.

Method used

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  • Efficient IL-2 (interleukin-2) mutant fusion protein and application thereof
  • Efficient IL-2 (interleukin-2) mutant fusion protein and application thereof
  • Efficient IL-2 (interleukin-2) mutant fusion protein and application thereof

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Experimental program
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Effect test

Embodiment 1

[0019] Construction of Human Interleukin II Mutant Library

[0020] According to the amino acid sequence of human interleukin II (gene bank NM-000586) ( figure 1 ) to mutate the gene sequence. Synthesis of human interleukin II mutant fusion protein gene by overlap extension method ( figure 2 ), and further cloned into a multifunctional expression vector. These vectors were transfected into Chinese hamster ovary cells, and the cells were placed at 37°C, 5% CO 2 After culturing in an incubator, the supernatant was taken after 72 hours, and further purified into an interleukin II mutant fusion protein by immobilized metal ion affinity chromatography ( image 3 ). The purified protein was detected by electrophoresis to confirm the molecular weight, and was further used in the study of immune cell proliferation in vivo.

Embodiment 2

[0022] Mutant interleukin II to promote suppressor (CD4CD25+) T cells, NK cells and CD8+ T cell activity assay

[0023] Black mice were injected intraperitoneally with interleukin II protozoa or interleukin II mutant fusion protein. The daily dose of protozoa was 20 micrograms for a total of 4 days, and the interleukin II mutant fusion protein was 4 micrograms for a total of 4 days. The spleen was collected on the fifth day. Splenocytes were stained using CD25, CD8, NK1.1 and CD4 antibody staining. Finally, flow cytometry was used to measure and data analysis. Figure 4 and 5 showed that compared with protozoa, interleukin II mutant fusion protein enhanced the function of NK (from 14.7% to 26.8%) and CD8 (from 14.9% to 23.7%) cell expansion, while attenuating the effect on CD4+CD25+ Expansion of regulatory cells (from 4.11% to 0.97%).

Embodiment 3

[0025] Effects of Mutant Interleukin II Fusion Protein and Prototype Interleukin II Protein on the Ratio of Effector and Inhibitor Cells

[0026] as attached Figure 6 , as shown in 7, mice were injected intraperitoneally with interleukin II protobody or interleukin II mutant fusion protein, compared with the original body, the interleukin II mutant fusion protein increased the ratio of NK cells to regulatory suppressor cells (from 3.8 to 24.1)( Figure 7 ), increased the ratio of CD8 to regulatory suppressor cells (from 4.0 to 23.8) ( Figure 6 ).

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Abstract

The invention provides an efficient IL-2 (interleukin-2) mutant fusion protein and an application thereof, belongs to a novel medical product in biotechnology and particularly discloses a structure and an application of the efficient IL-2 mutant fusion protein with low toxicity. The efficient IL-2 mutant fusion protein is characterized in that an amino acid sequence constituting polypeptide has a mutation on an amino acid site of IL-2, and the fusion protein is formed by the mutated polypeptide and human IgG Fc. The efficient IL-2 mutant fusion protein has the advantages that the function for inducing suppressor regulatory T cells is reduced, and immunocompetence of the IL-2 for natural killer cells and T killer cells is improved substantially. The IL-2 mutant fusion protein can be used for stimulating the immune system, and the tumor growth suppressing function is enhanced by increasing the ratio of tumor-killing effector cells to suppressor cells, so that the clinical effect of the IL-2 mutant fusion protein is improved and the application range of the IL-2 mutant fusion protein is enlarged.

Description

technical field [0001] The invention belongs to new medical products of biotechnology, in particular to a human interleukin II mutant fusion protein with high-efficiency immune activation function and its application. Background technique [0002] Interleukin II (interleukin-2, IL-2, interleukin II) is a lymphokine secreted by T lymphocytes (human interleukin II is a protein polypeptide chain composed of 133 amino acids, with a molecular weight of 15KD), and is a key factor in maintaining the immune system. The most important activator for the normal function of T cells and NK cells. Interleukin II can promote the maturation of T cells, maintain the activity of T cells, and promote the activity of cytotoxic T lymphocytes and transform them into tumor infiltrating lymphocytes (TIL); promote the activity of natural killer (NK) cells and induce killing Growth of tumor lymphokinetic cells and killer cells (CTL). Interleukin II can enhance human immune function, activate natura...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/55C07K19/00A61K38/20A61P35/00
CPCC07K14/55A61K38/00C07K2319/30
Inventor 岳喜连刘根桃翟渊粉
Owner 南京康志制药有限公司
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