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Expression and export of interferon-alpha proteins as Fc fusion proteins

A fusion protein and interferon technology, applied in the field of Fc fusion protein, can solve the problems of side effects, high dosage of interferon-α, and low efficiency

Inactive Publication Date: 2002-07-31
LEXIGEN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the high dosage, low efficiency, short half-life, difficulty of purification and side effects of interferon-α, the field needs methods that can increase the yield and improve the pharmaceutical properties of this therapeutic agent

Method used

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  • Expression and export of interferon-alpha proteins as Fc fusion proteins
  • Expression and export of interferon-alpha proteins as Fc fusion proteins
  • Expression and export of interferon-alpha proteins as Fc fusion proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1. Expression of Human Fc-Human Interferon-α (huFc-IFN-α)

[0082] mRNA was prepared from human peripheral blood mononuclear cells and then reverse transcribed with reverse transcriptase. The resulting cDNA was used as a template for the polymerase chain reaction (PCR) to clone and adapt the human interferon-α cDNA for expression as a human Fc-human interferon-α (huFc-IFN-α) fusion protein. The forward primer is 5'C CCG GGT AAA TGT GAT CTG CCT CAG AC (SEQ ID NO: 5) wherein the sequence C CCG GG (XmaI restriction site) TAAA encodes the carboxyl terminus of the immunoglobulin heavy chain, and the following sequence ( Bold) encodes the N-terminus of interferon-α. The reverse primer is 5' CTC GAG TCA ATC CTT CCT CCT TAA TC (SEQ ID NO: 6) encoding the carboxy-terminal sequence of interferon alpha and its translation stop codon (anticodon, TCA), followed by an XhoI site (CTC GAG) cloned and sequenced a PCR product comprising 517 base pairs. Sequence analysis result...

Embodiment 2

[0084] Example 2. Transfection and expression of proteins

[0085] Co-precipitation of plasmid DNA with calcium phosphate (Sambrook et al.eds. (1989) "MOLECULAR CLONING-A LABORATORYMANUAL," Cold Spring Harbor Press, NY) or with Lipofectamine Plus (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions By lipofection, the plasmid pdCs-huFc-IFN-α was introduced into human kidney 293 cells by transient transfection.

[0086] To obtain stable transfected clones, plasmid DNA was introduced into mouse myeloma NS / O cells by electroporation. Briefly, NS / O cells were grown in modified Dulbecco's Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine and penicillin / streptomycin. About 5×106 cells were washed once with phosphate buffered saline (PBS), and then suspended in 0.5 mL of PBS. 10 μg of linearized plasmid DNA was incubated with the above cells in a Gene Pulser cuvette (0.4 cm electrode separation, BioRad) for 10 minutes on ice. Elect...

Embodiment 3

[0089] Embodiment 3.ELISA operation

[0090] Concentrations of human Fc fragment-containing protein products in supernatants of anti-MTX clones and other samples were identified using an anti-huFc ELISA. The steps are described in detail as follows:

[0091] A. Coated titer plate

[0092] ELISA titer plates were coated with PBS containing 5 μg / mL affinity-purified goat anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA), and each well of a 96-well plate (Nunc-Immuno plate Maxisorp) Add 100 μL of the above coating solution. The coated plate was blocked and incubated overnight at 4°C. Then the plate was washed 4 times with PBS containing 0.05% Tween (Tween20), and then blocked with 200 μL of PBS containing 1% BSA / 1% goat serum. After incubating with blocking buffer at 37°C for 2 hours, the plate was washed 4 times with PBS containing 0.05% Tween, and then the plate was patted gently on paper towels to dry the plate.

[0093] B. Incubation of Test Samp...

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Abstract

Disclosed are nucleic acid sequences, for example, DNA or RNA sequences, which encode an immunoglobulin Fc-Interferon-alpha fusion protein. The nucleic acid sequences can be inserted into a suitable expression vector and expressed in mammalian cells. Also disclosed is a family of immunoglobulin Fc-Interferon-alpha fusion proteins that can be produced by expression of such nucleic acid sequences. Also disclosed are methods of using such nucleic acid sequences and / or fusion proteins for treating conditions, for example, hepatitis, which are alleviated by the administration of interferon-alpha.

Description

[0001] related application [0002] This application claims priority to prior US application Ser. No. 60 / 134,895, filed May 19, 1999, which is incorporated herein by reference. field of invention [0003] The invention relates to a fusion protein expression system, which can increase the production of interferon-alpha protein family members. More specifically, the present invention relates to high-level expression and secretion of Fc fusion proteins, such as immunoglobulin Fc-interferon-α, in mammalian cells, various structural forms of the proteins and their applications. Background of the invention [0004] It has been proved that the interferon-α (IFN-α) protein family has application value in the treatment of various diseases. For example, interferon-alpha 2a and 2b (commercial names Roferon and Intron A, respectively) have been used against chronic hepatitis B, hepatitis C and D (life-threatening viral liver d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K35/76A61K38/00A61K38/095A61K38/21A61K48/00A61P1/16A61P31/20A61P43/00C07K14/52C07K14/56C07K14/715C07K16/18C07K19/00C12N5/10C12N15/21C12N15/62C12N15/63C12N15/86C12N15/863C12P21/02C12R1/91
CPCC07K2319/00C07K14/56A61K48/00A61K38/00C07K2319/30A61P1/16A61P31/20A61P43/00C12N15/11
Inventor 劳健明孙亚萍S·D·吉利斯
Owner LEXIGEN PHARMA
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