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Improved FIX fusion protein, conjugate and application of fusion protein and conjugate

A technology of fusion protein and conjugate, which is applied in the direction of fusion of polypeptides, peptide/protein components, and prolonging the fusion of plasma life, which can solve the problems of poor physical and chemical stability, limiting patient medication compliance, and short half-life

Active Publication Date: 2021-01-05
江苏晟斯生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For hemophilia B, infusion of factor IX preparations (currently usually recombinantly expressed factor IX protein in vitro) to replenish blood coagulation factor IX levels in patients is currently the only effective treatment, but due to the ubiquity of polypeptide drugs, the half-life in vivo is relatively short , poor physical and chemical stability, easy to be degraded by various proteases in the body, etc., making these drugs usually need to be injected multiple times a day, resulting in frequent medication
Bring a greater burden to patients physically, psychologically and economically, and limit patients' medication compliance

Method used

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  • Improved FIX fusion protein, conjugate and application of fusion protein and conjugate
  • Improved FIX fusion protein, conjugate and application of fusion protein and conjugate
  • Improved FIX fusion protein, conjugate and application of fusion protein and conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1 Preparation of fusion protein FIX-L1-Fc-L2-P

[0127] 1. Construction of expression vector

[0128] The FIX fusion protein constructed in this example has a common molecular structure: FIX-L1-Fc-L2-P. Wherein, FIX represents nine natural blood coagulation factors from human, and its sequence is shown in SEQ ID NO:1.

[0129] L1 represents the first linker between FIX and Fc, and is a connecting peptide composed of a flexible peptide or a flexible peptide and a rigid structure based on multiple prolines (Pro, P). Among them, L1 uses a total of 6 sequences, respectively Represented by C1, C2, C3, C4, C5 and GS, the specific sequence is as follows:

[0130] C1: GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPA (SEQ ID NO: 8)

[0131] C2: GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPA (SEQ ID NO: 9)

[0132] C3: GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAVAPPPALPAVAPPPALPAPVRLPGPA (SEQ ID NO: 10)

[0133] C4: GGGGSGGGGSGGGGSGGGGSGGGGSVAPPPALPAPVRLPGPAVAPPPALPAVAPPPALPA (SEQ ID ...

Embodiment 2

[0212] Example 2 In vitro activity detection and comparison of FIX-L1-Fc-L2-P

[0213] This example detects FIX-C1-Fc-L2-P, FIX-C2-Fc-L2-P, FIX-C3-Fc-L2-P, FIX-C4-Fc-L2-P and The in vitro activity of FIX-C5-Fc-L2-P was compared with the in vitro activity of FIX-GS-Fc-L2-P without the rigid unit in the first linker L1.

[0214] The details are as follows: the activity of human coagulation factor IX (hFIX) was determined by a one-phase method based on activated partial thromboplastin time (APTT), with ellagic acid as the activator, and the 8thInternational Standard 2009 FIX Concentrate (WHO FIX Activity standard substance) is carried out APTT determination, takes the logarithm respectively with the activity of known standard substance and measured APTT time, then carries out linear fitting and draws standard curve. Dilute the sample to be tested to the range of the standard curve, mix it with FIX-poor plasma to measure the APPT value, and calculate the biological activity of the ...

Embodiment 3

[0218] Example 3 Preparation of fusion protein FIX-C1-Fc-L2-PEG conjugated with PEG

[0219] The C-terminus of FIX-C1-Fc-L2-P protein contains LPETG, which can be specifically recognized by Sortase A enzyme. Under the action of Sortase A enzyme, the amide bond between T and G is cut off, and T reacts with the sulfhydryl group at position 184 of Sortase enzyme to form a thioester bond intermediate, which is then attacked by GGGAA-PEG with poly-Gly at one end, and finally Able to link PEG with specific molecular weight to the C-terminus of protein.

[0220] PEGlation reaction system: Dissolve 40KD-GGGAA-PEG in buffer and adjust the pH to 7.5, then add protein and 40KD-GGGAA-PEG to the 50mM Tris 150mM NaCl pH 7.5 buffer system at a ratio of 1:20, and then add Sortase A enzyme and 10 mM CaCl 2 After 30 min, EDTA was added to terminate the reaction.

[0221] The conjugation product (FIX-C1-Fc-L2-PEG) was detected by SEC-HPLC for conjugation rate. SEC detection uses TOSOHTSKgel ...

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Abstract

The present invention relates to a blood coagulation factor IX fusion protein having an extended circulating half-life, a conjugate containing the fusion protein, a pharmaceutical composition containing the fusion protein and the conjugate and an application of the fusion protein, the conjugate and the composition in the treatment of hemorrhagic diseases (such as hemophilia B).

Description

technical field [0001] The present invention relates to bioactive polypeptide fusion protein and its conjugate with polymer that prolong half-life in vivo, particularly relate to the human coagulation factor IX that prolongs half-life in vivo, the pharmaceutical composition containing them and their in the treatment bleeding disease (such as hemophilia) Application in disease B). Background technique [0002] Hemophilia is a group of hereditary bleeding disorders, which is a severe coagulation disorder caused by the lack of certain coagulation factors in the blood. These include hemophilia A (factor VIII, AHG deficiency) and hemophilia B (factor IX, PTC deficiency). [0003] Hemophilia B (hemophilia B) is a severe coagulation disorder caused by the deficiency of human coagulation factor IX (hFIX). The gene encoding hFIX (hFIX) is located at the far end of the long arm of the human X chromosome, located in the Xq27.1 region, with a total length of 34kb, including 8 exons an...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/107C12N9/64C12N15/62A61K38/48A61K47/68A61K47/60A61P7/04
CPCC12N9/644A61K47/60A61P7/04C12Y304/21022C07K2319/30A61K38/00A61K47/68C07K2319/31C12N9/6424A61K38/36A61K38/48
Inventor 苏鸿声陈宪莫炜川朱鹿燕阎海霞姜明任子甲王亚里
Owner 江苏晟斯生物制药有限公司
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