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Recombinant interleukin-15 analogue

An analogue, il-15-xa-yb-zc technology, applied in the field of molecular biology, can solve problems such as short half-life, difficult purification, and difficult industrialization

Pending Publication Date: 2021-03-26
LETO LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although IL-15 has higher safety and activity than IL-2, as a protein drug, natural wild-type IL-15 has obvious drug development bottlenecks, including low expression in prokaryotes and eukaryotes , purification difficulties, short half-life, etc., making it difficult to carry out industrialization, so it is necessary to study its exogenous expression to obtain high production efficiency

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Expression of wild-type IL-15 and IL-15 analogues in Escherichia coli

[0077] 1.1 Construction of expression vector

[0078] The wild-type IL-15 nucleotide sequence was synthesized by Shanghai Sangong Company.

[0079] (1) Primer design

[0080] The C-terminal sequence of IL-15 was changed by introducing the base sequence of different amino acids to be added in the reverse primer. The amino acid sequence, nucleotide sequence and primer sequences used during construction of wild-type IL-15 and the constructed IL-15 analogs are shown in Table 2:

[0081] The amino acid sequence of table 2 wild-type IL-15, IL-15 analogue, nucleotide sequence and the primer sequence used when constructing

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] Analogs 34-43 are formed by introducing mutations into wild-type IL-15 to form IL-15 mutants, and then adding the sequence GSLPETGGSGGSHHHHHH to the C-terminus of the IL-15 mutants. A...

Embodiment 2

[0155] Example 2 In vitro cell activity experiment of IL-15 analogues

[0156] 2.1 Preparation of IL-15 analogs

[0157] The IL-15 analog 11, analog 18, analog 21 and analog 28 expressed by inclusion bodies were dissolved in 8M urea solution, and purified by ion exchange and reversed-phase chromatography (see: Yunier et al, Preparative Biochemistry and Biotechnology, 47:9,889-900), purified to obtain a relatively pure protein, SDS-PAGE electrophoresis as shown in Figure 4 shown.

[0158] 2.2 CTLL-2 cell experiment

[0159] The proliferation test of CTLL-2 cells is a commonly used test to measure the activity of interleukin-stimulated immune cells at the cell level. Therefore, the biological activity of IL-15 analogues was examined here by wild-type IL-15 and the proliferative effect of IL-15 analogues on CTLL-2 cells.

[0160] 1) CTLL-2 cell preparation: the cells were resuspended in culture medium containing FBS and Rat-T-Stim.

[0161] 2) Adding samples: inoculate the...

Embodiment 3I

[0167] Preparation of conjugates of embodiment 3 IL-15 analogs (enzymatic reaction method)

[0168] Utilizing the purified IL-15 analog 21 (IL-15-GS-LPETG-GSGGSHHHHHH), the fatty acid chain (816366) with GGG at the N-terminus (816366) and the IL-15-GS -LPETG-GSGGSHHHHHH is connected. The reaction was carried out according to the Sortase A:IL-15:fatty acid chain ratio of 1:6:30, and the reaction buffer was 50mM Tris-HCl, 1mM CaCl 2 , 150 mM NaCl pH 8.0 at room temperature for 3 hours before purification. Purification uses reverse phase chromatography C8 (Saifen Technology) to separate the uncoupled IL-15 analog 21, unreacted fatty acid chain and the coupled product, and the final product is tested for purity by UPLC (such as Figure 5 ) and LC-MS ( Figure 6 ) identification showed that IL-15 analogue 21 has been coupled to fatty acid chains to form IL-15 analogue 21-816366.

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Abstract

The invention discloses an recombinant interleukin-15 (IL-15) analogue. The IL-15 analogue has the amino acid sequence of the IL-15, as well as one or more amino acids added at the C terminal of the IL-15 amino acid sequence; and the one or more amino acids comprise positively charged amino acids. The IL-15 analogue disclosed by the invention is highly expressed in escherichia coli that the expression level is about 20 times that of wild IL-15; and the in-vitro cell activity of the IL-15 analogue has no obvious difference. The invention further discloses a conjugate of the IL-15 analogue. Theconjugate of the IL-15 analogue can prolong half life of the IL-15 analogue by coupling a fatty acid chain so as to improve long-acting property of the IL-15 analogue. Thus, a foundation for industrialization of IL-15 protein drugs is laid.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a recombinant interleukin-15 analogue and its expression. Background technique [0002] Interleukin-15 (IL-15) is a cytokine of about 12-14kD. The natural human interleukin-15 mature peptide contains 114 amino acids, including 4 cysteine ​​residues, Cys35 and Cys85, Cys42 and Cys88 The two pairs of intramolecular disulfide bonds formed play an important role in maintaining the spatial conformation and biological activity of IL-15. [0003] Similar to most cytokines, IL-15 can play a role in the body's normal immune response, such as promoting the development of T cells, B cells, natural killer (NK) cells, etc. IL-15 and IL-2 have a common IL -2Rβ chain and γ chain receptors, so their biological activities are very similar. However, due to the different α chain receptors, IL-2 can activate Treg cells and induce Teff and NK cell apoptosis (AICD), the clinical applicat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/54C12N15/24C12N15/70C12N1/21C12R1/19
CPCC07K14/5443C12N15/70C07K2319/35C07K2319/31C12R2001/19C12N15/63A61K47/542C07K14/54C12N1/205
Inventor 赵耀张建军朱笑婷徐艳玲彭璐佳王冀姝张维
Owner LETO LAB CO LTD
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