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TACI-immunoglobulin fusion proteins

An immunoglobulin and fusion protein technology, applied in the field of improved TACI-immunoglobulin fusion protein

Inactive Publication Date: 2005-05-04
ZYMOGENETICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The original version resulted in low-level expression of heterogeneous proteins

Method used

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  • TACI-immunoglobulin fusion proteins
  • TACI-immunoglobulin fusion proteins
  • TACI-immunoglobulin fusion proteins

Examples

Experimental program
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Effect test

Embodiment 1

[0137] Example 1 describes an expression vector comprising a cytomegalovirus promoter directing transgenic expression of a recombinant protein, an immunoglobulin intron, and a tissue plasminogen activator signal sequence. A suitable immunoglobulin intron is the murine 26-10V H Intron. SEQ ID NO: 66 provides mouse 26-10V H Exemplary nucleotide sequences of introns. The expression vector may also include a 5' untranslated region (UTR) upstream of the nucleotide sequence encoding the TACI-immunoglobulin protein. Suitable 5'-UTR can be from mouse 26-10V H obtained in the gene. SEQ ID NO: 63 discloses useful natural mouse 26-10V H The 5'-UTR nucleotide sequence, while SEQ ID NO: 64 shows the mouse 26-10V H When using the 5'-UTR nucleotide sequence, it has been optimized at the 3' end.

[0138] As an example, SEQ ID NO: 67 provides the nucleotide sequence comprising the following elements: native murine 26-10V H 5'-UTR (nucleotides 1 to 51), mouse 26-10V H Signal sequence...

Embodiment 2

[0295] Production of TACI-Fc Protein by Chinese Hamster Ovary Cells

[0296] By means of electroporation, the TACI-Fc expression construct was used to transfect suspension-adapted Chinese hamster ovary (CHO) DG44 cells grown in animal protein-free medium (Urlaub et al., Som. Cell. Molec. Genet. 12:555 (1986)). CHO DG44 cells lack a functional dihydrofolate reductase gene due to deletions at two dihydrofolate reductase chromosomal locations. Growth of the cells in the presence of increasing concentrations of methotrexate results in amplification of the dihydrofolate reductase gene and the linked gene encoding the recombinant protein on the expression construct.

[0297] CHO DG44 cells were passaged in PFCHO medium (JRH Biosciences, Lenexa, KS), 4mM L-glutamine (JRH Biosciences), and 1x hypothanxine-thymidine rehydration solution (Life Technologies), and then incubated at 37°C and 5% CO 2 The cells were incubated in Corning shaker flasks on a rotary shaker platform r...

Embodiment 3

[0302] Structural analysis of TACI-Fc protein

[0303] In some cases, TACI-Fc fusion proteins were partially purified prior to analysis. The conditioned medium of Chinese hamster ovary cells was sterile filtered through a 0.22 μm filter, and the TACI-Fc protein was captured on a protein A column. Material bound to protein A was eluted and passed through an S-200 size exclusion column for final purification.

[0304] Western blot analysis was performed on both the conditioned medium of the cells and the purified protein to assess the structural stability of the TACI-Fc protein. Briefly, samples of protein or supernatant were transferred to nitrocellulose membranes and treated with peroxidase-conjugated goat anti-mouse IgG2a (Boehringer Mannheim), or peroxidase-conjugated goat anti-human IgG Fc TACI-Fc protein was detected with specific antiserum (Pierce).

[0305] Amino acid sequence analysis of the amino terminus was performed on the Model 476A and 494 protein ...

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Abstract

Molecules that interfere with the binding of a tumor necrosis factor receptor with its ligand, such as a soluble receptor, have proven usefulness in both basic research and as therapeutics. The present invention provides improved soluble transmembrane activator and calcium modulator and cyclophilin ligand-interactor (TACI) receptors.

Description

technical field [0001] The present invention generally relates to improved fusion proteins comprising a tumor necrosis factor receptor portion and an immunoglobulin portion. In particular, the invention relates to improved TACI-immunoglobulin fusion proteins. Background of the invention [0002] Cytokines are small soluble proteins that mediate growth and various biological effects in many cell types, (see, eg, Arai et al., Annu. Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin . Immunol. 3:311 (1991); Paul and Seder, Cell 76:241 (1994)). The proteins that make up the cytokine group include interleukins, interferons, colony-stimulating factors, tumor necrosis factors, and other regulatory molecules. For example: human interleukin-17 is a cytokine that stimulates the expression of interleukin-6, intracellular adhesion molecule 1, interleukin-8, granulocyte-macrophage colony-stimulating factor, and prostaglandin E2 expression, and Plays a role in the preferential maturation...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K38/00A61K39/395A61K48/00A61P35/00C07K14/705C07K14/715C07K16/00C07K19/00
CPCC07K16/241C07K2319/00C07K14/70578A61K38/00C07K2319/30A61P1/00A61P11/00A61P11/06A61P13/12A61P17/06A61P19/00A61P19/02A61P21/04A61P25/00A61P29/00A61P31/04A61P35/00A61P35/02A61P37/00A61P37/02A61P37/06A61P7/00A61P7/06A61P7/10A61P9/00A61P3/10A61K39/395A61K39/3955A61K45/06
Inventor M·W·里克森J·A·格罗斯
Owner ZYMOGENETICS INC
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