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Methods for treating viral infection using IL-28 and IL-29 cysteine mutants

a technology of cysteine mutants and cysteine, which is applied in the direction of antibody medical ingredients, peptide/protein ingredients, drug compositions, etc., can solve the problems of significant toxicities, limited clinical use of methods, and inability to develop vaccines for many viral infections

Inactive Publication Date: 2005-11-03
ZYMOGENETICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although oftentimes efficacious, these methods have limitations in clinical use.
For instance, many viral infections are not amenable to vaccine development, nor are they treatable with antibodies alone.
In addition, IFN's are not extremely effective and they can cause significant toxicities; thus, there is a need for improved therapies.
Infected individuals are at high risk for developing liver cirrhosis, and eventually, hepatic cancer.
There are few effective treatments for hepatitis.
For example, treatment of autoimmune chronic hepatitis is generally limited to immunosuppressive treatment with corticosteroids.
However, IFN-α is associated with a number of dose-dependent adverse effects, including thrombocytopenia, leukopenia, bacterial infections, and influenza-like symptoms.
During seasonal epidemics most infants, children, and adults are at risk for infection or reinfection.

Method used

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  • Methods for treating viral infection using IL-28 and IL-29 cysteine mutants

Examples

Experimental program
Comparison scheme
Effect test

example 1

Induction of IL-28A, IL-29 and IL-28B by Poly I:C and Viral Infection

[0158] Freshly isolated human peripheral blood mononuclear cells were grown in the presence of polyinosinic acid-polycytidylic acid (poly I:C; 100 μg / ml) (SIGMA; St. Louis, Mo.), encephalomyocarditis virus (EMCV) with an MOI of 0.1, or in medium alone. After a 15 h incubation, total RNA was isolated from cells and treated with RNase-free DNase. 100 ng total RNA was used as template for one-step RT-PCR using the Superscript One-Step RT-PCR with Platinum Taq kit and gene-specific primers as suggested by the manufacturer (Invitrogen).

[0159] Low to undetectable amounts of human IL-28A, IL-28B, and IL-29, IFN-α and IFN-β RNA were seen in untreated cells. In contrast, the amount of IL-28A, IL-29, IL-28B RNA was increased by both poly I:C treatment and viral infection, as was also seen for the type I interferons. These experiments indicate that IL-28A, IL-29, IL-28B, like type I interferons, can be induced by double-str...

example 2

IL-28 and IL-29 Signaling Activity Compared to IFNα in HepG2 Cells

A. Cell Transfections

[0160] HepG2 cells were transfected as follows: 700,000 HepG2 cells / well (6 well plates) were plated approximately 18 h prior to transfection in 2 milliliters DMEM+10% fetal bovine serum. Per well, 1 microgram pISRE-Luciferase DNA (Stratagene) and 1 microgram pIRES2-EGFP DNA (Clontech,) were added to 6 microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100 microliters DMEM. This transfection mix was added 30 minutes later to the pre-plated HepG2 cells. Twenty-four hours later the transfected cells were removed from the plate using trypsin-EDTA and replated at approximately 25,000 cells / well in 96 well microtiter plates. Approximately 18 h prior to ligand stimulation, media was changed to DMEM+0.5% FBS.

B. Signal Transduction Reporter Assays

[0161] The signal transduction reporter assays were done as follows: Following an 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cell...

example 3

IL-29 Antiviral Activity Compared to IFNα in HepG2 Cells

[0162] An antiviral assay was adapted for EMCV (American Type Culture Collection # VR-129B, Manassas, Va.) with human cells (Familletti, P., et al., Methods Enzym. 78: 387-394, 1981). Cells were plated with cytokines and incubated 24 hours prior to challenge by EMCV at a multiplicity of infection of 0.1 to 1. The cells were analyzed for viability with a dye-uptake bioassay 24 hours after infection (Berg, K., et al., Apmis 98: 156-162, 1990). Target cells were given MTT and incubated at 37° C. for 2 hours. A solubiliser solution was added, incubated overnight at 37° C. and the optical density at 570 nm was determined. OD570 is directly proportional to antiviral activity.

[0163] The results show the antiviral activity when IL-29 and IFN on were tested with HepG2 cells: IL-29, IFN-β and IFNα-2a were added at varying concentration to HepG2 cells prior to EMCV infection and dye-uptake assay. The mean and standard deviation of the O...

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Abstract

IL-28A, IL-28B, IL-29, and certain mutants thereof have been shown to have antiviral activity on a spectrum of viral species. Of particular interest is the antiviral activity demonstrated on viruses that infect liver, such as hepatitis B virus and hepatitis C virus. In addition, IL-28A, IL-28B, IL-29, and mutants thereof do not exhibit some of the antiproliferative activity on hematopoietic cells that is observed with interferon treatment. Without the immunosuppressive effects accompanying interferon treatment, IL-28A, IL-28B, and IL-29 will be useful in treating immunocompromised patients for viral infections.

Description

REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the benefit of U.S. patent application Ser. Nos. 60 / 559,081, filed Apr. 2, 2004, 60 / 609,238, filed Sep. 13, 2004, and 60 / 634,144, filed Dec. 8, 2004, all of which are herein incorporated by reference in their entirety. BACKGROUND OF THE INVENTION [0002] Strategies for treating infectious disease often focus on ways to enhance immunity. For instance, the most common method for treating viral infection involves prophylactic vaccines that induce immune-based memory responses. Another method for treating viral infection includes passive immunization via immunoglobulin therapy (Meissner, J. Pediatr. 124:S17-21, 1994). Administration of Interferon alpha (IFN-α) is another method for treating viral infections such as genital warts (Reichman et al., Ann. Intern. Med. 108:675-9, 1988) and chronic viral infections like hepatitis C virus (HCV) (Davis et al., New Engl. J. Med. 339:1493-9, 1998) and hepatitis B virus (HBV). ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/20A61K38/21A61K38/57A61K39/12A61K39/21A61K39/29C07K14/54
CPCA61K38/20A61K38/21A61K38/212A61K38/57A61K47/48215C07K14/54A61K2300/00A61K47/60A61P1/16A61P29/00A61P31/12A61P31/14A61P31/16A61P31/18A61P31/20A61P31/22
Inventor KLUCHER, KEVINKINDSVOGEL, WAYNESIVAKUMAR, PALLAVURHENDERSON, KATHERINE
Owner ZYMOGENETICS INC
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