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Long-acting interleukin-2 capable of realizing targeted regulation of T cell, and application of long-acting interleukin-2 for treating autoimmunity disease

An interleukin and human interleukin technology, which is applied in the field of biopharmaceuticals, can solve problems such as unfavorable industrial preparation and clinical practical use, prediction and dose climbing verification, and reduce patient compliance, and achieve long-term immunogenicity. Efficacy and safety of treatment, effect of reducing dose and number of injections

Active Publication Date: 2020-01-03
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The treatment plan of low-dose IL-2 is very different for different diseases and different individuals, and it is difficult to predict and verify dose escalation in the human body; on the other hand, for chronic diseases such as autoimmune diseases, IL-2 needs long-term Injection administration also seriously reduces patient compliance; from the aspect of antibody application, the chemical dosage ratio and complex stability of IL-2 / antibody complexes make it unfavorable for industrial preparation and clinical practical use, while the application of such The immunogenicity brought by antibodies or other IL-2 mutants also limits the wide application of this type of method in the clinic

Method used

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  • Long-acting interleukin-2 capable of realizing targeted regulation of T cell, and application of long-acting interleukin-2 for treating autoimmunity disease
  • Long-acting interleukin-2 capable of realizing targeted regulation of T cell, and application of long-acting interleukin-2 for treating autoimmunity disease
  • Long-acting interleukin-2 capable of realizing targeted regulation of T cell, and application of long-acting interleukin-2 for treating autoimmunity disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Selection of modifiable sites and preparation of hIL-2 containing unnatural amino acids with azide groups at the sites

[0047] According to the crystal structure of IL-2, the binding site of IL-2 and its receptor, the inventor of the present invention has selected several suitable sites to modify, which is mainly based on the following factors: 1. amino acid is exposed on the protein surface to facilitate coupling 2. Shield the immunogenic region; 3. Shield the proteolysis region. Through more detailed literature review [Eleonora Trotta et al, NatMed.2018Jul; 24(7):1005-1014; Spangler JB.et al, Immunity.2015May 19; 42(5):815-25], the inventor selected The binding sites of IL-2 and IL-2Rα, β, and γ subunits were affected to varying degrees, including H16, D20, Y31, T51, A73, K76, and H79 ( figure 1 ). For each site, the inventors designed primers capable of mutating the codon encoding the amino acid into an amber codon, and then utilized the site-directed mu...

Embodiment 2

[0048] Embodiment 2 IL-2 preparation of site-directed modification of PEG

[0049] The above-mentioned IL-2 mutant containing an unnatural amino acid containing an azide group is coupled to a PEG modifier containing a cyclooctyne, and the azide group on the unnatural amino acid and the cyclooctyne are used to perform a coupling reaction of ring tension , to obtain IL-2 derivatives with specific site-modified PEG. After the reaction, the complex was desalted, and then purified by ion exchange (Source 15S, 20mM sodium acetate pH=4.5, 0-250mM NaCl gradient) and size exclusion chromatography to obtain specific site-modified PEG with >95% purity IL-2 derivatives, such as figure 2 shown.

Embodiment 3

[0050] Example 3 Improving the modification site of IL-2 targeting and activating Treg and the acquisition of the corresponding best modifier

[0051] The affinity of the modified IL-2 derivatives to different IL-2R receptor subunits will determine their targeting to specific immune cells. By detecting different sites, IL-2 derivatives modified with different PEG sizes and IL -2Rα and IL-2Rβ affinity changes to determine the best modification sites and corresponding PEG modifiers targeting Treg.

[0052] (1) Biofilm Interference Experiment (BLI):

[0053] IL-2 plays a biological role by binding to the IL-2R receptor complex on the cell surface. IL-2R is composed of α, β, and γ three molecular subunits, of which the α chain (CD25) does not have the function of signal transmission and can only Together with the β and γ chains, it forms a high-affinity binding (Kd=10pM) with IL-2 as a trimolecular complex; while both the β chain (CD122) and the γ (CD132) chain belong to the type...

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Abstract

The invention relates to modification sites capable of enabling human interleukin-2 to carry out targeted activation on Treg, human interleukin-2 subjected to site-directed mutation on the modification sites, and human interleukin-2 subjected to site-directed modification on the modification sites. The modified long-acting interleukin-2 can carry out targeted activation regulation on T cells in awide therapeutic window, slightly and evenly does not activate other effector cells, and performs a long-acting systematic immunorepressive effect. The invention further relates to application of thecategory of site-directed mutation or modification interleukin-2, and purposes of the site-directed mutation or modification interleukin-2 for treating various autoimmunity diseases as a stable and long-acting immunosuppressor.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and specifically relates to interleukin-2 with site-specific modification, for example, derivatives of interleukin-2 subjected to site-specific pegylation, so that it has the function of targeting and regulating T cells. The present invention further relates to the use of the above-mentioned site-modified interleukin-2 in the treatment of autoimmune diseases, such as safe and effective rheumatoid arthritis, systemic lupus erythematosus, autoimmune diabetes and the like. Background technique [0002] Interleukin-2 (interleukin-2) is the first type of cytokine, as one of the earliest discovered and medicinal cytokines, IL-2 has a vital biological role in immune regulation. The human IL-2 with the signal sequence cut out has a molecular weight of 15418.02 Daltons and consists of 133 amino acid residues. [0003] IL-2 plays a biological role by binding to the IL-2 receptor (IL-2R) on the cell membr...

Claims

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Application Information

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IPC IPC(8): C07K14/55A61K38/20A61P37/02
CPCC07K14/55A61P37/02A61K38/00A61K47/60A61K38/2013A61P37/06A61P19/02A61P17/06A61P1/16A61P1/00A61P1/04A61P3/10A61P17/00A61P13/12A61P7/06A61P17/14
Inventor 张博周德敏张烜王妍孙家琦
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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