Interleukin-2 derivative as well as preparation method and application thereof

A technology of derivatives and fatty acid derivatives, applied in the field of interleukin-2 derivatives, can solve the problems of unstable product quality, poor patient compliance, prolonging IL-2 molecules, etc., achieve good drug prospects and reduce binding capacity , the effect of prolonging the half-life

Pending Publication Date: 2022-04-15
HISUN BIORAY PHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although there are many kinds of IL-2 preparations on the market, they all have certain problems. For example, high-frequency administration leads to poor patient compliance and heavy economic burden. Clinical treatment requires a high dosage, and high-dose administration is likely to cause serious side effects, etc.
IL-2 molecules modified with PEG can reduce the toxicity problem caused by IL-2, and can effectively prolong the half-life of IL-2 molecules, but the complex production process leads to the problem of unstable product quality and properties
In addition, since some improved drugs require PEG molecules to be gradually shed in the human body before they can be converted into active forms, the shed PEG molecules may accumulate in the body and pose potential health risks

Method used

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  • Interleukin-2 derivative as well as preparation method and application thereof
  • Interleukin-2 derivative as well as preparation method and application thereof
  • Interleukin-2 derivative as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: Preparation of IL-2K35C protein

[0050] According to the IL-2 wild-type sequence (SEQ ID NO: 1), the 35th lysine was mutated into cysteine, and the mutated protein was named IL-2K35C (SEQ ID NO: 2); in addition, the The 43rd lysine and the 64th lysine were mutated into cysteine ​​respectively, and named as IL-2K43C and IL-2K64C for comparison. In order to facilitate subsequent purification, the Fc region of human IgG1 was introduced at the N-terminal of the protein, and the thrombin cleavage site LVPRGS was introduced between Fc and the target protein. The full-length sequence of IL-2K35 with Fc tag (Fc-IL-2K35C) is shown in SEQ ID NO:3.

[0051] The above clone design was entrusted to Taizhou Baiying Biotechnology Co., Ltd. to construct and perform CHO cell expression and purification. Using a 125ml disposable sterile shaker flask, thaw the frozen CHO cells in a 37-degree water bath, and dilute the cells to 0.3×10 6 pcs / ml, volume 30ml. 125rpm (19mm ...

Embodiment 2

[0053] Embodiment 2: the synthesis of fatty acid derivative

[0054] Such as image 3 Shown, with 22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetracarbonyl-3,6,12,15,42-pentaoxo-9,18,23 Triazine-tetradecanoic acid (1) is a raw material (hereinafter referred to as semaglutide side chain intermediate, synthesized with reference to Example 1 of patent document WO2011117415A) and N-(2-aminoethyl) maleimide in The condensing agent 2-bromo-1-ethylpyridine tetrafluoroborate (Bide Pharmaceuticals, BKZ779) reacted to obtain tert-butyl 25-(tert-butoxycarbonyl)-1-(2,5-carbonyl-2, 5-dihydro-1H-pyrrol-1-yl)-4,13,22,27-tetracarbonyl-6,9,15,18-tetraoxo-3,12,21,26-tetraazotetratradecanoic acid Ester (2), followed by removal of Boc protection with trifluoroacetic acid (TEDIA, TS4295-013) to give the fatty acid derivative 25-carboxy-1-(2,5-dicarbonyl-2,5-dihydro-1H-pyrrole -1-yl)-4,13,22,27-tetracarbonyl-6,9,15,18-tetraoxo-3,12,21,26-tetraazatetradecanoic acid (3). The specific oper...

Embodiment 3

[0055] Example 3: Conjugation of fatty acid derivatives and IL-2K35C

[0056] TCEP (Suzhou Haofan Biological Co., Ltd., 20190501, prepared as an aqueous solution with reducing buffer) and IL-2K35C protein were mixed at a molar ratio of 6:1, and reduced at 18°C ​​for two hours to make the mutated surface half The sulfhydryl group of cystine is in a free state. The reducing buffer was 35mM sodium citrate (Sangon, A610035), 2mM EDTA (Sangon, A610185-0500), 154mM NaCl (Sangon, A501218-0001), pH=5. After the reduction was completed, the product was ultracentrifuged 10 times at a speed of 7000 rpm / min, and 10 times the buffer volume was replaced to remove TCEP. The fatty acid derivative (FA) obtained in Example 2 was dissolved in a reducing buffer containing 5% N,N-dimethylformamide (DMA) (TEDIA, DS1441-001), and the reduced IL-2K35C The mixture was mixed at a molar ratio of 2:1, and the coupling reaction was carried out at 18° C., and the reaction time was 2 hours.

[0057] Afte...

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Abstract

The invention belongs to the field of medicines, and provides an IL-2 derivative comprising an IL-2 mutant and a fatty acid derivative, the IL-2 mutant comprises one or more cysteine mutations relative to an IL-2 wild type as shown in SEQ ID NO: 1, and the mutations at least comprise that lysine at the 35th site as shown in SEQ ID NO: 1 is mutated into cysteine; the fatty acid derivative comprises a fatty acid and a water-soluble linker. The invention also provides preparation and application of the IL-2 derivative. Compared with natural IL-2, the IL-2 derivative disclosed by the invention has a remarkably prolonged half-life period and better safety, and has a better patent medicine prospect.

Description

technical field [0001] The invention belongs to the field of medicine and relates to interleukin-2 derivatives. Background technique [0002] Interleukin-2 (IL-2) is an interleukin, a cytokine signaling molecule in the immune system. The molecular weight is about 15.5KD. It is mainly secreted by activated CD4+T cells, activated CD8+T cells, NK cells, dendritic cells and macrophages, and is responsible for regulating the activity of lymphocytes. [0003] IL-2 plays multiple regulatory roles on the immune system by binding to different receptor subunits. The trimeric receptor formed by IL-2Rα (CD25), IL-2Rβ (CD122), IL-2Rγ (CD132) is the highest affinity (KD about 10pM) receptor form of IL-2, mainly expressed in activated lymphoid Cells and CD4 positive (CD4+), CD25 positive (CD25+), FoxP3 positive (Foxp3+) suppressor regulatory T cells (Treg); dimer receptors composed of IL-2Rβγ subunits are intermediate affinity receptors (KD About 1nM), mainly expressed on cytotoxic T ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/55C07K1/107C07K1/10C07K1/06A61K38/20A61P35/00
Inventor 王晓泽聂磊吴振华王新增潘晨晓陈旭晨陈刚王海彬
Owner HISUN BIORAY PHARMA CO LTD
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