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SIRP alpha mutant or fusion protein thereof, and application thereof

A fusion protein and variant technology, applied in the fields of tumor therapy and molecular immunology, can solve problems such as off-target toxicity, effectiveness and production cost limitations

Active Publication Date: 2019-03-26
江苏东抗生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This also raises the issue of CD47 binding to healthy tissue requiring higher therapeutic doses to achieve adequate receptor occupancy on cancer cells and direct inhibition of "don't eat me" signaling introducing potential off-target toxicity
At present, many companies at home and abroad are developing drugs based on CD47, and found that SIRPαFc has the characteristics of good penetration, tissue distribution, and high safety. However, the current SIRPα molecular design still has limitations in terms of effectiveness and production costs. sex

Method used

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  • SIRP alpha mutant or fusion protein thereof, and application thereof
  • SIRP alpha mutant or fusion protein thereof, and application thereof
  • SIRP alpha mutant or fusion protein thereof, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1 Construction of expression vector of SIRPα variant-Fc fusion protein

[0095] The amino acid sequences of the wild-type SIRPα and its variants prepared in the present invention are respectively shown in SEQ ID NO.1-3, wherein the amino acid sequence of the wild-type SIRPα, that is, the SIRPα parent, is SEQ ID NO.1; the amino acid sequence of the variant B2F6 It is SEQ ID NO.2; the amino acid sequence of the variant D1H4 is SEQ ID NO.3.

[0096] SIRPα and its variants are connected to IgG1Fc (amino acid sequence: SEQ ID NO.6), IgG4Fc (amino acid sequence: SEQ ID NO.7) or 6×His ( Amino acid sequence: SEQ ID NO.8) etc. are connected to form a fusion protein, and the schematic diagram of its protein structure is as follows figure 1 shown.

[0097] In order to secrete and express SIRPα and its variants in mammalian cells, it is necessary to add a secreted and expressed signal peptide (amino acid sequence: SEQ ID NO.5) to the N segment of the expression vector whe...

Embodiment 2

[0101] Example 2 Expression and purification of SIRPα variant-Fc fusion protein

[0102] Recombinant immunoglobulin variants were expressed by transient transfection of 293E cells.

[0103] 293E cells in the logarithmic growth phase were treated with 4×10 5 / ml density inoculated in a shaker flask at 37°C, 5% CO 2 Shaking, 125r / min cultured for 24 hours before transfection.

[0104] For every 100ml of 293E cells, take 5mL of OPTI-MEM and add 200μg of plasmid, shake and mix and incubate at room temperature for 5min to get the plasmid solution; take another 5mL of OPTI-MEM and add 600μg of PEI, shake and mix and incubate at room temperature for 5min to get the PEI solution ; Mix the plasmid and PEI solution, vortex and incubate at room temperature for 20 minutes, add the reaction mixture dropwise to the cells, and place at 37°C, 5% CO 2 The shaker, cultured at 125r / min, feed fed on the 4th and 6th day, harvested the supernatant on the 8th day.

[0105] Cell culture supernatant...

Embodiment 3

[0106] Example 3 Analysis of binding activity between SIRPα variant-Fc fusion protein and CD47

[0107] Dilute the CD47 antigen to 1 μg / mL in the coating solution, add 100 μL per well to the enzyme-linked plate, and place it in a humid box at 4 °C overnight. Wash the enzyme-linked plate 3 times with a plate washer, block with 1.5% casein, 200 μL per well, and block for 1 hour at 37°C in a humid box. Dilute the fusion protein of SIRPα and its variants with 1×PBS to 5 μg / mL, and after 4-fold gradient dilution, add 100 μL per well to the enzyme-linked plate, and react in a wet box at 37°C for 1 h. Wash the enzyme-linked plate 3 times, add goat anti-human Fc-HRP secondary antibody and react at room temperature for 45 minutes. Wash the enzyme-linked plate 5 times and add 100 μL TMB substrate for color development, react for 3 minutes and use 100 μL 2N H 2 SO 4 The reaction was terminated, and the enzyme-linked immunosorbent assay was read at 450nm.

[0108] The antibody-antigen...

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Abstract

The invention discloses an SIRP alpha mutant or fusion protein thereof, and application thereof. The SIRP alpha mutant has an amino acid sequence shown as SEQ ID NO. 2-3; the fusion protein comprisesthe SIRP alpha or the mutant thereof, and second protein; the second protein is IgG Fc or His6; the mutant and the fusion protein provided by the invention have high affinity to CD47 and can stop thebinding between the SIRP alpha and the CD47 so to achieve tumor treatment.

Description

technical field [0001] The invention belongs to the fields of tumor treatment and molecular immunology, and relates to SIRPα variants or fusion proteins and applications thereof. Background technique [0002] Macrophages are the main members of the body's inherent immune system. Macrophages recognize and remove foreign, damaged or aging cells through phagocytosis to maintain a stable internal environment. The high expression of CD47 on the surface of tumor cells, Inhibits the phagocytosis of tumor cells by macrophages, which in turn leads to the inability of macrophages to remove and kill tumor cells in tumor patients. Antagonists targeting CD47-SIRPα interaction (anti-CD47 antibody, anti-SIRPα-antibody, SIRPα-Fc fusion protein, etc.), can activate macrophage phagocytosis by blocking the binding of CD47 and SIRPα, and make the innate immune response system play a role function to enhance the anti-tumor immune response. [0003] The anti-CD47 antibody or SIRPα-Fc fusion pro...

Claims

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Application Information

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IPC IPC(8): C07K14/705C07K19/00C12N15/62C12N15/12C12N15/85C12N5/10A61K38/16A61P35/00
CPCA61P35/00C12N15/85C07K14/70596A61K38/00
Inventor 魏化伟黄亚杰陈春巧
Owner 江苏东抗生物医药科技有限公司
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