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Use of truncated cysteine IL28 and IL29 mutants to treat cancers and autoimmune disorders

a technology of truncated cysteine and il28, which is applied in the field of use of truncated cysteine il28 and il29 mutants to treat cancers and autoimmune disorders, can solve the problems of loss of tolerance, scarring damage to nerve fibers, and disruption of neurotransmission

Inactive Publication Date: 2007-01-25
ZYMOGENETICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a study that tested a human protein called IL-29 to see if it could help treat multiple sclerosis (MS). The study used a mouse model for MS and found that IL-29 was able to delay the onset of the disease and reduce the severity of symptoms. This suggests that using IL-29 may be beneficial in treating MS. The study was designed to evaluate the effectiveness of IL-29 in treating EAE, a mouse model for MS.

Problems solved by technology

Sometimes, the immune system loses the ability to recognize “self” as normal and the subsequent response directed against the tissue or cells, results in loss of tolerance, a state of autoimmunity.
The pathologies resulting from autoimmunity often have serious clinical consequences and are one of the major health problems in the world, especially in developed nations.
The destruction of the myelin sheath leads to disruption of neurotransmission and scarring damage to the nerve fibers.
Over the course of the disease, there is loss of strength in the extremities, leading to problems with movement and in the most severe cases, leading to paralysis of the limbs.
Novantrone® is cytotoxic to most cells and therefore as one would expect, has an array of side effects and is toxic at doses required for the maximal therapeutic effects.
IFN-β is also toxic, limiting dosage of the drug in MS patients.
Furthermore, continuous use of these drugs has been shown to desensitize patients to further use of the same drug, thereby limiting the ability to use these drugs as long term therapeutics.

Method used

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  • Use of truncated cysteine IL28 and IL29 mutants to treat cancers and autoimmune disorders
  • Use of truncated cysteine IL28 and IL29 mutants to treat cancers and autoimmune disorders
  • Use of truncated cysteine IL28 and IL29 mutants to treat cancers and autoimmune disorders

Examples

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example 1

Mammalian Expression Plasmids

[0235] An expression plasmid containing zcyto20 and zcyto21 was constructed via homologous recombination. Fragments of zcyto20 and zcyto21 cDNA were generated using PCR amplification. The primers for PCR were as follows:

[0236] zcyto20 / pZMP21: zc40923, and zc43152 SEQ ID NOS: 42 and 43, respectively; and zcyto21 / pZMP21: zc40922, and zc43153 SEQ ID NOS:72 and 73, respectively.

[0237] The PCR reaction mixture was run on a 1% agarose gel and a band corresponding to the size of the insert was gel-extracted using a QIAquick™ Gel Extraction Kit (Qiagen, Valencia, Calif.).

[0238] The plasmid pZMP21, which was cut with BglII, was used for recombination with the PCR insert fragment. Plasmid pZMP21 is a mammalian expression vector containing an expression cassette having the MPSV promoter, and multiple restriction sites for insertion of coding sequences; an E. coli origin of replication; a mammalian selectable marker expression unit comprising an SV40 promoter, e...

example 2

Expression of Mammalian Constructs in CHO Cells

[0242] 200 μg of a zcyto20 / pZMP21 and zcyto21 / pZMP21 construct were digested with 200 units of Pvu I at 37° C. for three hours and then were precipitated with IPA and spun down in a 1.5 mL microfuge tube. The supernatant was decanted off the pellet, and the pellet was washed with 1 mL of 70% ethanol and allowed to incubate for 5 minutes at room temperature. The tube was spun in a microfuge for 10 minutes at 14,000 RPM and the supernatant was aspirated off the pellet. The pellet was then resuspended in 750 μl of PF-CHO media in a sterile environment, and allowed to incubate at 60° C. for 30 minutes. CHO cells were spun down and resuspended using the DNA-media solution. The DNA / cell mixture was placed in a 0.4 cm gap cuvette and electroporated using the following parameters: 950 μF, high capacitance, and 300 V. The contents of the cuvette were then removed and diluted to 25 mLs with PF-CHO media and placed in a 125 mL shake flask. The fl...

example 3

Purification and Analysis of zcyto20-CHO Protein

[0243] Purification of Zcyto20-CHO Protein

[0244] Recombinant zcyto20 (IL-28A) protein was produced from a pool of DXB11-CHO cell lines. Cultures were harvested, and the media were sterile filtered using a 0.2 μm filter.

[0245] The purification of zcyto20-CHO protein was achieved by the sequential use of a Poros HS 50 column (Applied Biosystems, Framingham, Mass.), a Monolithic WCX column (Isco, Inc., Lincoln, Nebr.), a ToyoPearl Butyl 650S column (TosoH, Montgomeryville, Pa.), and a Superdex 75 column (Amersham Biosciences, Piscataway, N.J.). Culture media from DXB111-CHO were adjusted to pH 6.0 before loading onto a Poros 50 HS column. The column was washed with 50 mM MES (2-Morpholinoethanesulfonic acid), 100 mM NaCl, pH 6 and the bound protein was eluted with a 10 column volumes (CV) linear gradient to 60% of 50 mM MES, 2 M NaCl, pH 6. The eluting fractions were collected and the presence of zcyto20 protein was confirmed by SDS-PA...

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Abstract

Methods for treating patients with cancer and autoimmune disorders using IL-28 and IL-29 molecules. The IL-28 and IL-29 molecules include polypeptides that have homology to the human IL-28 or IL-29 polypeptide sequence and proteins fused to a polypeptide with IL-28 and IL-29 functional activity. The molecules can be used as a monotherapy or in combination with other known cancer and / or autoimmune therapeutics.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This present application claims the benefit of U.S. Patent Application Ser. Nos. 60 / 771,260, filed Feb. 8, 2006, and 60 / 700,951, filed Jul. 20, 2005, both of which are herein incorporated by reference.BACKGROUND OF THE INVENTION [0002] Cytokines generally stimulate proliferation or differentiation of cells of the hematopoietic lineage or participate in the immune and inflammatory response mechanisms of the body. Examples of cytokines which affect hematopoiesis are erythropoietin (EPO), which stimulates the development of red blood cells; thrombopoietin (TPO), which stimulates development of cells of the megakaryocyte lineage; and granulocyte-colony stimulating factor (G-CSF), which stimulates development of neutrophils. These cytokines are useful in restoring normal blood cell levels in patients suffering from anemia, thrombocytopenia, and neutropenia or receiving chemotherapy for cancer. [0003] The interleukins are a family of cytokine...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/21A61K38/17A61K39/00
CPCA61K38/20A61K38/21A61K47/48215C07K14/54A61K2300/00A61K47/60A61P1/00A61P1/04A61P17/06A61P19/02A61P25/00A61P25/28A61P29/00A61P35/00A61P35/02A61P37/02A61P37/06A61P43/00
Inventor SHEPPARD, PAUL O.
Owner ZYMOGENETICS INC
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