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Interleukin-2 derivative

A derivative, IL-2 technology, applied in the field of molecular biology, can solve the problems of immunosuppression, toxic side effects, apoptosis, etc.

Active Publication Date: 2020-04-17
LETO LAB CO LTD
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  • 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

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1 Preparation of IL-2 (C125A), IL-2 mutant and IL-2 complex

[0106] In this example, IL-2wt (C125A), IL-2 mutants 1-4 and IL-2 complexes 1-4 were selected for expression respectively, and were purified and prepared by relying on the HPC4 tag attached to the C-terminus of the molecule.

[0107] 1.1 Expression plasmid construction

[0108] Entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize IL-2wt C125A (SEQ ID NO.74), IL-2 mutant 1-4, IL-2 complex 1-4 (IL-2Pair in IL-2 complex 1~4 and CD25-ECD Pair 1~4), and then perform overlapping PCR to obtain the target fragment according to the operation method mentioned in "Molecular Cloning". Then carry out the recombination connection of the fragment and the pTT5 universal vector, transform DH10B, sequence and bacterium preservation, so as to obtain the required IL-2wt (C125A), IL-2 mutant 1~4 and IL-2 complex 1~4 plasmid (IL Plasmids of IL-2Pair 1-4 and CD25-ECDPair 1-4 in the -2 complex); or perform PCR, D...

Embodiment 2

[0133] Example 2 Determination of the affinity of IL-2wt (C125A), IL-2 mutants 1-4 and IL-2 complexes 1-4 to IL2Rβγ and IL2Rα by biolayer interferomeory (BLI)

[0134] 1. Experimental materials

[0135] The proteins used in the experiment were all produced by Beijing Zhidao Biotechnology Co., Ltd. IL2Rα-his (purchased from Beijing Zhidao Biotechnology Co., Ltd.), IL2Rβγ-Fc (purchased from Beijing Zhidao Biotechnology Co., Ltd.) and IL2 mutants were transiently expressed by HEK293 and parent-child and obtained after purification. Buffer formulation: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.1% BSA and 0.05% Tween 20; ProA sensor (Pall Fortebio, catalog #18-5010); HISIK sensor (Pall Fortebio, catalog #18- 5120); the BLI equipment was Octet RED96 produced by Pall Fortebio; data acquisition and analysis were performed using Data acquisition 11.0 and Data analysis 11.0 software, respectively.

[0136] 2. Experimental method

[0137] 1) Preparation of IL2Rβγ-Fc

[0138] Dilute IL2R...

Embodiment 3

[0151] Embodiment 3 promotes the proliferation experiment of T cell

[0152] 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 derivatives was examined here by the proliferation experiment of CTLL-2 cells.

[0153] 1) CTLL-2 cell preparation: the cells were resuspended in culture medium containing FBS and Rat-T-Stim.

[0154] 2) Adding samples: inoculate the cells in a 96-well culture plate, 0.1 ml per well. At the same time, the proteins of the IL-2 derivatives 11, 18, 21 and 28 of the samples to be tested (i.e. the proteins prepared in Example 1) were respectively diluted in multiples, and 0.1ml was added to each well, and each dilution concentration was set in 3 multiple wells. . Separately set up culture solution control wells (100ul cells + 100ul culture solution). 37 degrees, 5% CO 2 Incubate for 72 hours.

[0155] 3) Add MTS: ...

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Abstract

The invention discloses an IL-2 derivative. At least one cysteine residue is introduced on the basis of wild type IL-2, the binding plane of the IL-2 derivative and an alpha receptor subunit is partially or completely blocked, and meanwhile, the affinity to a beta and gamma receptor subunit compound is basically reserved. The invention also discloses the compound. The compound comprises: the IL-2derivative, wherein a third cysteine residue is introduced into the IL-2 derivative on the basis of wild type IL-2; and a blocking module, wherein a fourth cysteine residue is arranged on or introduced to the blocking module. The third cysteine residue on the IL-2 derivative and the fourth cysteine residue on the blocking module form an intermolecular disulfide bond in order to form the IL-2 derivative and blocking module compound, so the binding plane of the IL-2 derivative and the alpha receptor subunit is partially blocked or completely blocked, and the affinity to the beta and gamma receptor subunit compound is substantially retained.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to an interleukin-2 derivative and a compound thereof. 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 diffe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/55C07K19/00C12N15/26C12N15/62A61K38/20A61K38/17A61P37/04
CPCC07K14/55A61K38/00A61P37/04C07K14/7155C07K2319/00A61P37/00A61K38/2013C07K2319/30
Inventor 赵耀彭璐佳郭建云朱笑婷张建军魏婷婷刘慧杰郑茜王冀姝张维
Owner LETO LAB CO LTD
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