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Novel IL-15 fusion protein for resisting tumors

A fusion protein and a new type of technology, applied in the field of molecular biology, can solve the problems of systemic immune side effects, achieve the effects of improving anti-tumor efficacy, simplifying the production process, and increasing binding affinity

Pending Publication Date: 2022-06-03
LETO LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The role of IL-15 is systemic rather than tumor-specific, which can easily cause systemic immune side effects

Method used

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  • Novel IL-15 fusion protein for resisting tumors
  • Novel IL-15 fusion protein for resisting tumors
  • Novel IL-15 fusion protein for resisting tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] In the IL-15 M1 embodiment, the natural amino terminus and carboxyl terminus of IL-15 are linked with a first linker such as the sequence shown in SEQ ID NO. Cleavage occurs at the peptide bond, resulting in new amino and carboxy termini.

[0074] The IL-15Rα polypeptide is fused to the new amino terminus of the IL-15 polypeptide variant through a second linker, and then the new fusion protein is directly fused to the amino terminus of the hIgG1 Fc domain, such as figure 2 a shown. Those skilled in the art can design other IL-15 M1 variants satisfying the present invention, such as Fc domain-IL-15Rα polypeptide-second linker-IL-15 polypeptide variant, based on the teachings given in the present invention and the prior art. body.

Embodiment 2

[0076] In the IL-15 M2 embodiment, the natural amino terminus and carboxyl terminus of IL-15 are linked with a first linker such as the sequence shown in SEQ ID NO. Cleavage occurs at the peptide bond, resulting in new amino and carboxy termini.

[0077] The IL-15Rα polypeptide is directly fused to the new amino terminus of the IL-15 polypeptide variant, and then the new fusion protein is fused to the carboxyl group of the hIgG1 Fc domain through a second linker such as the sequence shown in SEQ ID NO.4 The sequence shown in SEQ ID NO.3 of the third linker is fused to the amino terminus of the Fc domain, as shown in figure 2 b shown.

Embodiment 3

[0079] IL-15 M1 and IL-15 M2 were synthesized and expressed separately, and purified and prepared by means of molecular Fc domain tags. The breaking point is the peptide bond following the E82 residue. The expression host was mammalian cells (HEK293 or CHO).

[0080] i. Expression plasmid synthesis

[0081] Suzhou Jinweizhi Biotechnology Co., Ltd. was entrusted to synthesize the genes encoding IL-15 M1 and IL-15 M2, and then according to the operation method mentioned in the "Molecular Cloning Experiment Guide", DH10B was transformed, sequenced, and bacteria were preserved to obtain the corresponding plasmids.

[0082] ii. Plasmid extraction and HEK293 cell preparation

[0083] ii-1 plasmid extraction

[0084] According to the operation method mentioned in the "Molecular Cloning Experiment Guide", DH10B was transformed, sequenced, preserved and cultured. According to the operation methods mentioned in "Qiagen Mini-prep Kit" and "Qiagen Endofree Maxi-prep Kit", IL-15 M1 and...

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Abstract

The invention discloses a novel IL-15 fusion protein, which at least comprises (1) an IL-15R alpha polypeptide, (2) an IL-15 polypeptide variant, (3) an Fc structural domain, an Fc structural domain derivative or a functional fragment thereof, and (4) a connexon or a derivative thereof, the IL-15 polypeptide variant at least comprises a first connexon or a derivative thereof, and the first connexon or the derivative thereof is connected with a natural amino terminal and a carboxyl terminal of the IL-15 polypeptide. According to the novel IL-15 fusion protein for tumor treatment, the anti-tumor effect of IL-15 can be improved. In addition, the tumor treatment recombinant protein can be efficiently expressed and purified, a foundation is laid for industrialization of IL-15 therapeutic protein, and the tumor treatment recombinant protein has the great potential of becoming an effective immune anti-tumor therapy medicine.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a novel IL-15 fusion protein for anti-tumor. Background technique [0002] Human interleukin-15 (IL-15) gene is located on chromosome 4q31, spans about 34kb, has 7 introns and 9 exons, and its mRNA is 1.5-1.8kb in length. IL-15 is a glycosylated cytokine with a relative molecular weight of about 12-14kD. The precursor molecule of IL-15 consists of 162 amino acids, with a longer leader sequence of 48 amino acid residues. Its mature protein contains 114 amino acids, including 4 cysteine ​​residues, Cys35 and Cys85, Cys42 and Cys88 are connected, and the two pairs of intramolecular disulfide bonds formed are important for maintaining the spatial conformation and biological activity of IL-15. effect. [0003] Cytokines, including IL-15, have broad immunomodulatory activities, and IL-15 has been extensively studied as a promising antitumor target. Although IL-15 and IL-2 h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/22C07K1/16C12N15/62C12N15/85A61K38/20A61P35/00
CPCC07K14/5443C07K14/7155C12N15/85A61P35/00C07K2319/30C07K2319/35A61K38/00
Inventor 张维张建军孟万利徐艳玲赵耀朴锦华冉冬王冀姝
Owner LETO LAB CO LTD
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