Interleukin 21 (IL21) protein mutant and application thereof

A technology of IL21 and mutants, applied in the field of medicine, can solve the problems of low drugability and short plasma half-life.

Active Publication Date: 2020-05-29
HYQUO MOLECULE BEIJING TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, clinical studies have found that the plasma half-life of IL21 is relatively short, only about 3.09 hours [12] , even if the molecular weight of the fusion protein constructed by IL21 and monoclonal antibody reaches about 180kD, the plasma half-life is still relatively short, and the plasma half-life of the fusion protein constructed by IL21 and Rituximab (anti-CD20) is only about 18.94 hours in animal experiments [10] , druggability is still not high

Method used

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  • Interleukin 21 (IL21) protein mutant and application thereof
  • Interleukin 21 (IL21) protein mutant and application thereof
  • Interleukin 21 (IL21) protein mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1. Establishment of a CHO cell line capable of stably displaying chimeric IL21 / 4

[0100] The protein conformation of IL21 has two conformations: stable and unstable. A chimeric IL21 / 4 was constructed by replacing the unstable region in the protein structure of IL21 with the homologous region of IL4. The results showed that the protein conformation of the chimeric IL21 / 4 It is single and stable, and its biological activity is improved. And considering that the construction of IL21 mutants and Herceptin (Herceptin) fusion protein is an important purpose of the experiment, after choosing to display the fusion protein of chimeric IL21 / 4 and Herceptin on the surface of CHO working cells, through the detection of the constant region of Herceptin heavy chain It is also beneficial to test the display efficiency of chimeric IL21 / 4, so we first displayed chimeric IL21 / 4 and Herceptin fusion protein on the surface of CHO working cells.

[0101] In order to obtain a CHO c...

Embodiment 2

[0120] Example 2. Study on the stability of IL21 / 4 by introducing disulfide bonds at different positions through protein design

[0121] 1. Through protein structure analysis and design, different sites on the IL21 / 4 protein were selected for mutation, and disulfide bonds were introduced to obtain the following group of mutants. The introduced disulfide bonds are as follows:

[0122] (1) Both ILE at position 16 and SER at position 70 are mutated to CYS, introducing a 16-70 disulfide bond; named 16cIL21 / 4 ;

[0123] (2) Both the 17th ILE and the 106th SER are mutated to CYS, introducing a 17-106 disulfide bond; named 17cIL21 / 4 ;

[0124] (2) The 16th ILE, the 70th SER, the 17th VAL, and the 106th LYS were all mutated into CYS, and two sets of disulfide bonds were introduced; named 4cIL21 / 4 .

[0125] 2. Obtain CHO cells that can stably display IL21 / 4 mutants

[0126] In order to obtain CHO cell lines that can stably display IL21 / 4 mutants, firstly, using FRT-IL21 / 4-Her...

Embodiment 3

[0129] Example 3, Preliminary detection of the stability of IL21 / 4 mutants

[0130] In order to test the stability of IL21 / 4 mutants, CHO working cells displaying IL21 / 4-Herceptin and CHO working cells displaying mutants 16cIL21 / 4-Herceptin and 4c IL21 / 4-Herceptin were used as the experimental group. Heated at different temperature gradients, and then labeled with the IL21 receptor IL21Rα ectodomain-linker-GFP-his binding, flow cytometric detection of the display rate of each group after binding to the receptor at different temperature gradients, due to temperature changes, thermal Proteins with poor stability will be denatured first, the normal conformation will be destroyed, and the ability to bind to the receptor will be lost. The thermal stability of each mutant can be detected.

[0131] The specific experimental method is as follows:

[0132] (1) Digest CHO cells stably displaying IL21 mutants on the cell membrane surface with 0.02% EDTA-PBS, elute with medium containing...

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Abstract

The invention relates to an interleukin 21 (IL21) protein mutant and an application thereof. The mutant is obtained by mutating 16th-site ILE and 70th-site SER of a wild-type IL21 into CYS, and forming a disulfide bond between the two mutated CYS. The amino acid sequence of the wild-type IL21 protein is shown as SEQ ID NO.1. The invention also relates to a fusion protein containing the IL21 protein mutant and an application of the IL21 protein mutant or the fusion protein in preparation of a drug, preferably, the drug is a drug for regulating or activating immunity or an anti-tumor drug. The invention also relates to an application of the IL21 protein mutant or the fusion protein in preparation of a preparation for promoting B cell differentiation and proliferation, T cell differentiationand proliferation, and NK cell differentiation and proliferation.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to an interleukin 21 protein (IL21) mutant and application thereof. Background technique [0002] For the immunosuppressive problem in tumor immunotherapy research, cytokine and small molecule inhibitor therapy can play an immunomodulatory role. Cytokine and small molecule inhibitors can target suppressive cell subsets and inhibit their number or function, or Anti-tumor effector cells are activated by immunostimulatory molecules to enhance the patient's anti-tumor immune response. IL2 (interleukin 2) is an important mediator molecule in the immune response and one of the most powerful anti-tumor cytokines known, with a wide range of biological activities. IL2 directly acts on thymus T cells and promotes the differentiation of Treg cells. Suppressing autoimmunity can also promote the differentiation of initial T cells into effector T cells and memory T cells that can be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/54C07K19/00C12N15/24C12N15/62A61K38/20A61P37/02A61P37/04A61P35/00
CPCC07K14/54C07K14/5406A61P37/02A61P37/04A61P35/00A61K38/00C07K2319/00C07K16/32C07K2319/02C07K2319/60C07K2319/21C07K14/7155C07K16/24
Inventor 张信赵云杭海英
Owner HYQUO MOLECULE BEIJING TECH CO LTD
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