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Recombinant interferon-beta with enhanced biological activity

A biologically active, interferon-based technology, applied in drug combinations, medical preparations containing active ingredients, allergic diseases, etc., can solve problems such as lack of formation

Inactive Publication Date: 2009-08-12
NOVARTIS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Site-directed mutagenesis techniques have been successfully used to prepare mutated bioactive protein analogs (herein "protein analogs" refer to synthetic proteins in which one or more amino acids have been genetically and / or chemically modified and / or or heat modification, and the protein retains the biological activity of the parent protein), the analog retains the desired activity of its parent protein, but lacks the ability to form intermolecular linkages or undesired intramolecular disulfide bonds

Method used

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  • Recombinant interferon-beta with enhanced biological activity
  • Recombinant interferon-beta with enhanced biological activity
  • Recombinant interferon-beta with enhanced biological activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Preparation of recombinant Asp25 IFN-β1b.

[0062] A representative composition of the PCR mix used to prepare recombinant deamidated IFN-β1b and a representative PCR protocol are shown in Table 1 and Table 2, respectively.

[0063] Table 1. Composition of the PCR mix

[0064] PCR mix

(51 μL total volume) Template DNA (10

ng / μL) µL

2 µL

5

Forward primer (SEQ

ID NO: 3)(100

ng / μL) 1.25

1.25

Reverse primer (SEQ

ID NO: 4)(100

ng / μL) 1.25

1.25

Buffer 10× 5 5 dNTP mix 1 1 Quick solution 3 3 water 36.5 33.5

Pfu Turbo DNA

polymerase 1 1

[0065] Table 2. Representative PCR Protocols

[0066] PCR protocol duration temperature step 1 1 minute 95℃ step 2 50 seconds 95℃ step 3 50 seconds 60℃ step 4 1 min / kb 68°C, return to step 2, repeat 18 cycles step 5 7 minutes 95℃ ...

Embodiment 2

[0068] Example 2: Increased Potency of Recombinant Asp25 IFN-β1b

[0069] The results of the CPE and Hs294T antiproliferative bioactivity assays are summarized in Tables 3 and 4, respectively. Table 3 shows the CPE activity of Asp 25IFN-β 1b, heat treatment and high pH-treated Asp 25IFN-β 1b, and compared with ( ) compared with the CPE activity of Asn25 IFN-β 1b without HA.

[0070] Table 3: CPE biological activities of IFN-β 1b protein analogs.

[0071] Specific activity (×10 7 IU / mg)

[0072]

[0073]

[0074] Table 4 shows the Hs294T antiproliferative activity of Asp25 IFN-β 1b, compared with HA formulated Compared with the corresponding activity of Asn25 IFN-β1b without HA.

[0075]Table 4. Hs294T antiproliferative activity of IFN-β1b protein analogs

[0076] Specific activity (×10 7 IU / mg)

[0077]

[0078] 2.1. CPE bioassay

[0079] IFN-β induces an antiviral state in mammalian cells in which the replication and cytopathic effects (CPE) of some virus ...

Embodiment 3

[0086] Example 3. Characterization of recombinant Asp25 IFN-β 1b

[0087] Recombinant Asp25 IFN-βlb has a recombinant substitution of an aspartic acid residue for an asparagine residue at position 25 (numbering according to natural interferon). It also carries a Cys17Ser mutation. The primary sequence of Asp25 IFN-β1b is shown in image 3 . Aspartic acid at position 25 can be figure 2 The degradation pathway shown converts to succinimide and isoaspartate species. The recombinant IFN-β1b protein analogs of the present invention encompass Asp25 mutant proteins as well as Imide25 and Isoasp25 interferons from Asp25 mutants. The composition of the recombinant IFN-β1b protein analogs of the present invention was investigated in the following assays:

[0088] 3.1 Mapping of reduced Lys-C peptide

[0089] Peptide mapping uses enzyme digestion followed by RP-HPLC to generate fingerprints of proteins. Each peptide fragment isolated by RP-HPLC can be isolated and further charact...

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Abstract

Human interferon-ss protein analogs in which the asparagine at position 25, numbered in accordance with native human interferon-ss, is recombinantly replaced with an aspartate residue exhibit a biological activity of human interferon-ss (e.g. IFN-ss 1b) at an increased level relative to IFN-ss 1b. These analogs are obtained by introducing a gene coding for Asp25 IFN-ss into a cell and expressing the recombinant protein. The resulting IFN-ss protein analog is suitable for large scale manufacturing for incorporation in HA-containing or HA-free therapeutics for treatment of diseases including multiple sclerosis. A reduced Lys endoproteinase-C peptide map technique that produces a fingerprint profile for proteins using an enzymatic digest followed by RP-HPLC is also useful in quality control as an ID test for the IFN-ss protein analog products.

Description

1. Technical field [0001] The present invention is in the general field of bioactive protein chemistry. More specifically, it relates to interferon-beta analogs altered by mutations, which differ from the native protein by substitution, deletion or modification of cysteine, asparagine and other residues. 2. Background technology [0002] Interferon-beta has been found to be useful in the treatment of human diseases, notably multiple sclerosis. Multiple sclerosis (MS) is a chronic, often disabling disease of the central nervous system that occurs when the protective sheath around nerve fibers breaks down. About 30% of MS patients suffer from a relapsing-remitting form of the disease, in which symptoms completely or partially disappear after a flare-up, followed by a period of stabilization that can last for months or years. The FDA has approved the administration of beta interferon (interferon-beta or IFN-beta) to treat relapsing-remitting forms of MS. Currently, three int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/565
CPCC07K14/565A61P25/00A61P37/00A61K38/21
Inventor D·约翰逊-杰克逊K·古屋I·扎罗D·拉森
Owner NOVARTIS AG
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