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Fusion protein prodrug taking interleukin 15 as active ingredient

A fusion protein and drug technology, applied in the field of medicine and biology

Active Publication Date: 2019-11-12
IMMUNE TARGETING INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] (3) May activate autoreactive T cells and participate in the process of autoimmune diseases;
[0018] (4) May cause coronary heart disease;

Method used

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  • Fusion protein prodrug taking interleukin 15 as active ingredient
  • Fusion protein prodrug taking interleukin 15 as active ingredient
  • Fusion protein prodrug taking interleukin 15 as active ingredient

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1, design and construction of fusion protein

[0079] 1. Design and construct the following four fusion proteins:

[0080] (1) Control protein of IL15-Fc:

[0081] The structure of mouse IL15 and hIgG Fc was constructed as a fusion protein (hereinafter named IL15-Fc) see figure 1 A; wherein, the amino acid sequence of murine IL15 is shown in Seq ID No.1, and the amino acid sequence of hIgG Fc is shown in Seq ID No.2;

[0082] (2) Two super IL15 fusion proteins with different connection sequences:

[0083] For the structures of IL15-IL15Rαsushi-Fc (hereinafter named IL15-RA-Fc) and IL15Rαsushi-IL15-Fc (hereinafter named RA-IL15-Fc), see figure 1 B; wherein, IL15Rα sushi is the sushi domain of the IL15 receptor α subunit, its amino acid sequence is shown in Seq ID No.3, and the amino acid sequence of the linker (Linker) between IL15Rα sushi and IL15 is shown in Seq ID No.5 Show;

[0084] (3) RB-IL15-RA-Fc (prodrug) of the extracellular domain fragment of I...

Embodiment 2

[0110] Example 2, research on the biological function of super IL15 fusion protein

[0111] 1. The function of promoting lymphocyte production,

[0112] The steps of lymphocyte proliferation-promoting test (CCK8 test) are as follows:

[0113] (1) CTLL2 cells were cultured with 1640 complete medium containing 100 U / ml commercialized recombinant IL2 cytokine;

[0114] (2) On the day of the experiment, the cells were cultured and washed twice with 1640 complete medium without IL2 cytokines, and counted to adjust the cell concentration to 20000 / ml;

[0115] (3) The diluted samples IL15-Fc, IL15-RA-Fc, RA-IL15-Fc and RB-IL15-RA-Fc were cultured with 1640 complete medium without IL2 cytokines. The initial concentration is 10 μg / ml, 10-fold dilution, and 8 dilution gradients are made;

[0116] (4) Add 100 μl of cell suspension and 100 μl of sample to the 96-well cell culture plate, and make control wells with only cells and blank wells with only medium;

[0117] (5) After culturi...

Embodiment 3

[0135] Example 3. Comparing the tumor therapeutic effects and side effects of super IL15 and RB-IL15-RA-Fc

[0136] 1. Detect the therapeutic effect and side effects of RB-IL15-RA-Fc on tumor in mouse tumor model

[0137] Method 1: 3×10 6 A20 cells were inoculated subcutaneously into the lower left flank of Balb / c mice. When the tumor grows to 60-80mm 3 , intratumoral or ocular vein administration of 25 μg super IL15 or RB-IL15-RA-Fc, administration 2 times, 2 days apart, the control group was given PBS in the same way. Blood was collected 20 hours after the second treatment, and the level of serum inflammatory factors was detected by CBA, and the tumor inhibitory effect and the survival curve of the mice were recorded separately.

[0138] The steps of the CBA experiment are as follows:

[0139] (1) Blood was collected from the eye vein of mice, and the serum was collected and stored at -80°C;

[0140] (2) The levels of cytokines IL12p70, IL-6, IFN-γ, TNFα, MCP1 and IL-10...

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Abstract

The invention relates to a fusion protein prodrug taking interleukin 15 as an active ingredient. The prodrug is a fusion protein and includes the following structural units: (1) a first structural unit is the fragment of one or a subunit of an interleukin 15 (IL15) acceptor; (2) a second structural unit is IL15 having activity; (3) a third structural unit located on the C end of the fusion proteinis an antibody Fc fragment; (4) a connection fragment 1 is used for connecting the first, second and third structural units; (5) a fourth structural unit located on the N end of the fusion protein isthe extracellular fragment of the beta subunit of the IL15 acceptor; and (6) a connection fragment 2 is used for connecting the fourth structural unit and residual structural units.

Description

technical field [0001] The invention belongs to the field of medicine and biology, and specifically relates to a fusion protein-type drug prodrug with interleukin-15 as an active ingredient. Background technique [0002] Interleukin 15: In 1994, Grabstein and other researchers found a soluble cytokine that can maintain the proliferation of CTLL-2 cells in the supernatant of monkey kidney epidermal cell line cv-1 / EBVA, named IL15, that is, interleukin 15 (interleukin-15, IL15) [1] . IL-15, similar to IL-2, belongs to a family of 4 helical cytokines. The human IL15 gene is located on chromosome 5 q25-35. IL15 is a glycoprotein with a molecular weight of 14-15ka. The mature protein consists of 112 amino acids and contains 3 possible N-linked glycosylation sites. The expression of IL15 is tightly regulated. The expression of IL15 mRNA can be detected in many tissues and cells, such as fibrous tissue, muscle cells, kidney cells, lymphocytes, mast cells, tumor cells, etc., bu...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K38/20A61P35/00
CPCC07K14/5443C07K14/7155C12N15/85A61P35/00C07K2319/00C07K2319/30A61K38/00C12N15/62C07K2319/32C07K16/00A61K38/2086A01K2267/0331A01K2227/105A01K2207/12C07K1/14C07K2319/50
Inventor 傅阳心彭华郭静雅
Owner IMMUNE TARGETING INC
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