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Trimeric IL-1Ra

A technology of trimerization and fusion protein, which can be used in DNA/RNA fragments, allergic diseases, metabolic diseases, etc., and can solve problems such as the inability to block the action of IL-1α

Inactive Publication Date: 2010-11-17
ANAPHORE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in contrast to IL-1ra, these only block IL-1β, but cannot block the action of IL-1α

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1: Formation, production and purification of trimeric IL-IRa

[0136] It has been previously shown that IL-IRa can be produced as a recombinant protein in E. coli (Steinkasserer et al. 1992. FEBS 310:63-65). The protein is very stable and refolds efficiently. Isoforms of IL-IRa with additional amino acids at the N-terminus have also been described (Haskill et al. 1991, PNAS 88:3681-3685; Muzio et al. 1995, JEM 182, 623-628). These molecules bind IL-1R as well as the mature secreted form, suggesting that it may be possible to fuse an excess peptide to the N-terminus of the antagonist without affecting binding to the receptor. Structural analysis of crystals of IL-IRa that interacts with IL-IR also supports that N-terminal changes do not affect interaction with ILIR (Schreuder et al. 1997, Nature 386: 190-194). IL-IRa was cloned from human cDNA libraries derived from bone marrow and / or human placenta.

[0137] Trimeric IL-IRa was engineered to be C-terminally...

Embodiment 2

[0156] Example 2: Trimeric IL-1Ra compound inhibits IL-1 induction of IL-8 in U937 cells ability to guide

[0157] Further analysis of GG-TripV-IL-1ra (trip V-IL1Ra), GG-TripK-IL-1ra (tripK-IL1Ra), GG-TripT-IL-1ra (trip T-IL1Ra) and CII-H6-GrB-GG - Ability of TripT-IL-1ra (trip Q-IL1Ra) to inhibit IL-1 induction of IL-8 in U937 cells. The results are shown in Figure 4 middle.

[0158] Compounds were essentially equally potent in the blocking response and all exhibited (comparing w / w) works just as well. IL-8 production increased rather than continued to decrease at the highest protein concentration used (100 g / mL) due to the buffering effect in the experiment. According to several in vitro effect detection experiments and Biacore detection, according to the blocking and binding efficiency and yield, it is determined that TripT IL1Ra is the best compound.

Embodiment 3

[0159] Example 3: Polyethylene Glycol Trimeric IL-1Ra Compounds

[0160] Because the in vivo half-life is KINERET Key parameters in efficiency (KINERET The half-life in humans is only 4-6 hours, and thus can be used once a day), so the ability to pegylate TripT IL1Ra by N-terminal pegylation was tested. Trimeric IL1-Ra was PEGylated at the N-terminus. The trimeric IL1-Ra antagonist protein after the last step of the purification procedure described above was used as the starting point for pegylation. The buffer was exchanged with PBS buffer pH 6.0 for pegylation reaction. The protein concentration in the reactions was 0.5-3.5 mg / ml and a 5-10 molar excess of mPeg5K-acetaldehyde or mPeg20K-acetaldehyde (Nektar) supplemented with 20 mM sodium borocyanide was used. The reaction was carried out at 20°C for 16 hours. After the reaction, the mixture was applied to a Source 15S column (GE Healthcare) to purify the mono-PEGylated form. Such as Figure 5 As shown, the antagon...

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Abstract

Interleuekin-1 receptor antagonists (IL-1Ra) including fusion proteins having a trimerizing domain and an IL-1Ra polypeptide sequence. The fusion proteins are part of trimeric complexes that are used in pharmaceutical compositions for treating diseases mediated by IL-1. Effective treatment of inflammatory diseases, such as rheumatoid arthritis and diabetes, are described.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Patent Application No. 60 / 978,254, filed October 8, 2008, which is hereby incorporated by reference in its entirety. technical field [0003] The present invention relates to the treatment of diseases mediated by interleukin 1. More specifically, the present invention relates to interleukin 1 receptor antagonists (IL-IRa) useful in the treatment of these diseases. Background technique [0004] The IL-1 family is an important part of the innate immune system, which are regulators of the adaptive immune system. The balance between IL-1 and IL-1Ra in local tissues affects the possible development of inflammatory diseases and leads to structural damage. Inflammatory and autoimmune diseases can develop in the joints, lungs, gastrointestinal tract, central nervous system (CNS) or blood vessels in the presence of excess IL-1. Human disease has been treated with IL-IRa b...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K14/545
CPCC07K2319/31C07K14/4726C07K14/545C07K14/4713C12N15/62A61P1/04A61P1/18A61P3/00A61P3/10A61P9/10A61P11/00A61P11/06A61P13/12A61P17/00A61P17/06A61P19/00A61P19/02A61P19/06A61P19/10A61P21/00A61P25/00A61P25/04A61P25/16A61P25/28A61P29/00A61P31/04A61P35/00A61P35/02A61P35/04A61P37/06A61P43/00
Inventor 索尔·L·霍尔蒂特米凯尔·H·安德森约瑟夫斯·D·尼兰
Owner ANAPHORE INC
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