Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Interleukin-2 mutant

A mutant and IL-2 technology, applied in the field of molecular biology, can solve problems such as toxic side effects, immunosuppression, and failure to achieve therapeutic effects, and achieve the effect of eliminating toxic side effects and increasing expression levels

Pending Publication Date: 2022-04-19
LETO LAB CO LTD
View PDF4 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, under the condition of low clinical doses of IL-2, it will preferentially bind to the high-affinity receptors on the surface of Treg cells, which will cause immunosuppression and fail to achieve the therapeutic effect
High doses of IL-2 will neutralize the immune suppression caused by Treg activation by activating a large number of effector T cells, and at the same time, there will be more toxic side effects and cell apoptosis (activation induced cell apoptosis)

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Interleukin-2 mutant
  • Interleukin-2 mutant
  • Interleukin-2 mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Preparation of IL-2 and mutants

[0056] In this example, IL-2wt (C125A) and the mutant were expressed separately, and purified and prepared by relying on the HPC4 tag attached to the C-terminus of the molecule.

[0057] 1.1 Expression plasmid construction

[0058] Entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the gene with IL-2wt (C125A, SEQ ID NO.1) and the mutant, and clone it into the pTT5 universal vector. Transform DH10B, sequence and preserve bacteria to obtain the required IL-2 wild-type and mutant plasmids.

[0059] 1.2 Plasmid extraction and HEK293 cell preparation

[0060] 1.2.1 Plasmid extraction

[0061] According to the operation methods mentioned in "Qiagen Mini-prep Kit" and "Qiagen Endofree Maxi-prep Kit", prepare IL-2wt(C125A) and IL-2 mutants.

[0062] 1.2.2 HEK293 cell preparation

[0063] The density of fresh passage is 1-1.2*10 6 / ml HEK293 cells (National Research Council, Canada) were used for transient expression...

Embodiment 2

[0079] Example 2 Determination of the affinity of hIL2 mutants to IL2Rβ and IL2Rα by biolayer interferomeory (BLI)

[0080] 1. Experimental materials

[0081] The proteins used in the experiment were all produced by our company. IL2Rα-his, IL2Rβ-Fc / Fc and IL2 mutants were obtained through HEK293 transient expression and affinity purification. Buffer formulation (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.1% BSA and 0.05% Tween 20); ProA sensor (Pall Fortebio, Cat. #18-5010); HISIK sensor (Pall Fortebio, Cat. #18 -5120); the BLI equipment is OctetRED96 produced by Pall Fortebio; the data acquisition and analysis are carried out with Data acquisition 11.0 and Data analysis 11.0 software respectively.

[0082] 2. Experimental method

[0083] 2.1 Preparation of IL2Rβ-Fc / Fc

[0084] Dilute IL2Rβ-Fc / Fc with buffer solution to a concentration of 10ug / ml, add to column 2 of a 96-well assay plate, and set Loading, 600s in the control program.

[0085] 2.2 IL2Rα-his preparation

[0086...

Embodiment 3

[0100] Embodiment 3 promotes the proliferation experiment of T cell

[0101] The CTLL-2 (T cell) proliferation assay is a commonly used assay to measure the activity of interleukin-stimulated immune cells at the cell level. Therefore, the biological activity of IL-2 mutants was examined here by proliferation experiments of CTLL-2 cells.

[0102] 1. CTLL-2 cell preparation: resuspend the cells in culture medium containing FBS and Rat-T-Stim.

[0103] 2. Adding samples: Inoculate the cells in a 96-well culture plate, 0.1ml per well. At the same time, the protein of IL-2 mutant 386 of the sample to be tested (ie, the protein prepared in Example 1) was separately diluted in multiples, and 0.1 ml was added to each well, and each dilution concentration was set in three replicate wells. Separately set up culture solution control wells (100ul cells + 100ul culture solution). 37 degrees, 5% CO 2 Incubate for 72 hours.

[0104] 3. MTS addition: add 20 μl to each well AQueous One ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an IL-2 mutant, which is characterized in that on the basis of human IL-2, amino acid sites related to IL2R alpha binding are subjected to one or more mutations of replacement, deletion and addition, and the IL-2 mutant with reduced IL2R alpha binding capacity is obtained; wherein the amino acid sites related to IL2R alpha binding are the 30th to 75th sites of the wild type IL-2. The interaction between the IL-2 mutant and IL2R alpha is reduced, and the expression quantity is improved.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to an interleukin-2 mutant. Background technique [0002] Interleukin-2 (IL-2), discovered in 1976 and then known as T-cell growth factor (TCGF), is a globular glycoprotein that plays an important role in maintaining the normal function of T lymphocytes and NK cells . Natural IL-2 is a polypeptide consisting of 133 amino acid residues, with a molecular weight of about 15kD and three cysteine ​​residues located at positions 58, 105 and 125, respectively. Post-translational modifications include Thr glycosylation at position 3, disulfide bond formation at cysteine ​​residues at position 58 and 105, and the formation of four α-helices and some connecting sequences (loop ) composed of high-order structures (Bazan et al., Science 257, 410-413 (1992)). [0003] IL-2 is mainly produced by activated T cells, it can promote the proliferation and differentiation of T cells, mai...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/55C12N15/26C12N15/85C12N5/10A61K38/20A61P37/04
CPCC07K14/55C12N15/85A61P37/04A61K38/00A61K38/20C12N5/10C12N15/63
Inventor 赵耀张维彭璐佳张建军朱笑婷郭建云董明晖
Owner LETO LAB CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com