U.S. patent application number 11/929063 was filed with the patent office on 2008-05-29 for methods for treating viral infection using il-28 and il-29.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Katherine E. Henderson, Wayne R. Kindsvogel, Kevin M. Klucher, Pallavur V. Sivakumar.
Application Number | 20080124299 11/929063 |
Document ID | / |
Family ID | 32180694 |
Filed Date | 2008-05-29 |
United States Patent
Application |
20080124299 |
Kind Code |
A1 |
Klucher; Kevin M. ; et
al. |
May 29, 2008 |
METHODS FOR TREATING VIRAL INFECTION USING IL-28 AND IL-29
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.
Inventors: |
Klucher; Kevin M.;
(Bellevue, WA) ; Sivakumar; Pallavur V.; (Seattle,
WA) ; Kindsvogel; Wayne R.; (Seattle, WA) ;
Henderson; Katherine E.; (Seattle, WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
32180694 |
Appl. No.: |
11/929063 |
Filed: |
October 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11548487 |
Oct 11, 2006 |
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11929063 |
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11531646 |
Sep 13, 2006 |
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11548487 |
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10691923 |
Oct 23, 2003 |
7135170 |
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11531646 |
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60420714 |
Oct 23, 2002 |
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60463939 |
Apr 18, 2003 |
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60420713 |
Oct 23, 2002 |
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60463982 |
Apr 18, 2003 |
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Current U.S.
Class: |
424/78.27 ;
514/19.3; 514/3.7; 514/4.3; 514/7.9 |
Current CPC
Class: |
A61P 31/12 20180101;
C07K 14/54 20130101; Y02A 50/385 20180101; A61P 1/16 20180101; A61K
38/21 20130101; Y02A 50/30 20180101; A61K 47/60 20170801; A61K
38/20 20130101; Y02A 50/393 20180101; A61P 31/14 20180101; A61P
31/20 20180101; A61P 7/00 20180101 |
Class at
Publication: |
424/78.27 ;
514/12 |
International
Class: |
A61K 38/17 20060101
A61K038/17 |
Claims
1. A method of treating a viral infection in a mammal comprising
administering to the mammal a therapeutically effective amount of a
polypeptide comprising an amino acid sequence having at least 95%
sequence identity to one or more of the polypeptides selected from
the group consisting of amino acid residues 22-205 of SEQ ID NO:2,
amino acid residues 20-200 of SEQ ID NO:4, amino acid residues
22-205 of SEQ ID NO:6, amino acid residues 1-175 of SEQ ID NO:18,
amino acid residues 1-181 of SEQ ID NO:20, amino acid residues
1-175 of SEQ ID NO:22, amino acid residues 1-175 of SEQ ID NO:24,
amino acid residues 1-176 of SEQ ID NO:26, amino acid residues
1-175 of SEQ ID NO:28, amino acid residues 1-176 of SEQ ID NO:30,
amino acid residues 1-181 of SEQ ID NO:32, amino acid residues
1-182 of SEQ ID NO:34, amino acid residues 1-176 of SEQ ID NO:36,
amino acid residues 1-182 of SEQ ID NO:38, and amino acid residues
1-176 of SEQ ID NO:40, wherein the virus infecting the mammal is
selected from the group consisting of respiratory syncytial virus,
herpes virus, Epstein-Barr virus, cytomegalovirus, small pox virus,
adenovirus, influenza virus, parainfluenza virus, rhino virus,
yellow fever, rift valley virus, human immunodeficiency virus,
coxsackie virus, vaccinia virus, measles virus, west nile virus,
lassa fever virus, ebola virus, lymphocytic choriomeningitis virus,
dengue virus, venezuelan equine encephalitis virus, pichinde virus
and polio virus, and wherein after administration of the
polypeptide the viral infection is reduced.
2. The method of claim 1 wherein the polypeptide is conjugated to a
polyalkyl oxide moiety.
3. The method of claim 2 wherein the polyalkyl oxide moiety is a
polyethylene glycol.
4. A method of treating a patient having hepatocellular carcinoma
who also suffers from a chronic hepatitis C infection comprising
administering to the patient a therapeutically effective amount of
a polypeptide comprising an amino acid sequence having at least 95%
sequence identity to one or more of the polypeptides selected from
the group consisting of amino acid residues 22-205 of SEQ ID NO:2,
amino acid residues 20-200 of SEQ ID NO:4, amino acid residues
22-205 of SEQ ID NO:6, amino acid residues 1-175 of SEQ ID NO:18,
amino acid residues 1-181 of SEQ ID NO:20, amino acid residues
1-175 of SEQ ID NO:22, amino acid residues 1-175 of SEQ ID NO:24,
amino acid residues 1-176 of SEQ ID NO:26, amino acid residues
1-175 of SEQ ID NO:28, amino acid residues 1-176 of SEQ ID NO:30,
amino acid residues 1-181 of SEQ ID NO:32, amino acid residues
1-182 of SEQ ID NO:34, amino acid residues 1-176 of SEQ ID NO:36,
amino acid residues 1-182 of SEQ ID NO:38, and amino acid residues
1-176 of SEQ ID NO:40, and wherein after administration of the
polypeptide the hepatocellular carcinoma is reduced.
5. The method of claim 4 wherein the polypeptide is conjugated to a
polyalkyl oxide moiety.
6. The method of claim 5 wherein the polyalkyl oxide moiety is a
polyethylene glycol.
7. A method of treating a hepatitis B or hepatitis C infection in a
mammal comprising administering to the mammal a therapeutically
effective amount of a polypeptide comprising amino acid residues
1-175 of SEQ ID NO:2 or amino acid residues 1-176 of SEQ ID
NO:40.
8. The method of claim 7 wherein the polypeptide is conjugated to a
polyalkyl oxide moiety.
9. The method of claim 8 wherein the polyalkyl oxide moiety is a
polyethylene glycol.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 11/548,487, filed Oct. 11, 2006, which is a
divisional of U.S. patent application Ser. No. 11/531,646, filed
Sep. 13, 2006, which is a divisional of U.S. patent application
Ser. No. 10/691,923, now U.S. Pat. No. 7,135,170, filed Oct. 23,
2003, which claims the benefit of U.S. Provisional Application Ser.
No. 60/420,714, filed Oct. 23, 2002, U.S. Provisional Application
Ser. No. 60/463,939, filed Apr. 18, 2003, U.S. Provisional
Application Ser. No. 60/420,713, filed Oct. 23, 2002, and U.S.
Provisional Application Ser. No. 60/463,982, filed Apr. 18, 2003,
all of which are herein incorporated by reference.
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-.alpha.) 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). For instance, IFN-.alpha. and IFN-.beta.
are critical for inhibiting virus replication (reviewed by Vilcek
et al., (Eds.), Interferons and other cytokines. In Fields
Fundamental Virology., 3.sup.rd ed., Lippincott-Raven Publishers
Philadelphia, Pa., 1996, pages 341-365). In response to viral
infection, CD4+ T cells become activated and initiate a T-helper
type I (TH1) response and the subsequent cascade required for
cell-mediated immunity. That is, following their expansion by
specific growth factors like the cytokine IL-2, T-helper cells
stimulate antigen-specific CD8+ T-cells, macrophages, and NK cells
to kill virally infected host cells. 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.
[0003] Not all viruses and viral diseases are treated identically
because factors, such as whether an infection is acute or chronic
and the patient's underlying health, influence the type of
treatment that is recommended. Generally, treatment of acute
infections in immunocompetent patients should reduce the disease's
severity, decrease complications, and decrease the rate of
transmission. Safety, cost, and convenience are essential
considerations in recommending an acute antiviral agent. Treatments
for chronic infections should prevent viral damage to organs such
as liver, lungs, heart, central nervous system, and
gastrointestinal system, making efficacy the primary
consideration.
[0004] Chronic hepatitis is one of the most common and severe viral
infections of humans worldwide belonging to the Hepadnaviridae
family of viruses. Infected individuals are at high risk for
developing liver cirrhosis, and eventually, hepatic cancer. Chronic
hepatitis is characterized as an inflammatory liver disease
continuing for at least six months without improvement. The
majority of patients suffering from chronic hepatitis are infected
with either chronic HBV, HCV or are suffering from autoimmune
disease. The prevalence of HCV infection in the general population
exceeds 1% in the United States, Japan, China and Southeast
Asia.
[0005] Chronic HCV can progress to cirrhosis and extensive necrosis
of the liver. Although chronic HCV is often associated with
deposition of type I collagen leading to hepatic fibrosis, the
mechanisms of fibrogenesis remain unknown. Liver (hepatic) fibrosis
occurs as a part of the wound-healing response to chronic liver
injury. Fibrosis occurs as a complication of haemochromatosis,
Wilson's disease, alcoholism, schistosomiasis, viral hepatitis,
bile duct obstruction, toxin exposure, and metabolic disorders.
This formation of scar tissue is believed to represent an attempt
by the body to encapsulate the injured tissue. Liver fibrosis is
characterized by the accumulation of extracellular matrix that can
be distinguished qualitatively from that in normal liver. Left
unchecked, hepatic fibrosis progresses to cirrhosis (defined by the
presence of encapsulated nodules), liver failure, and death.
[0006] There are few effective treatments for hepatitis. For
example, treatment of autoimmune chronic hepatitis is generally
limited to immunosuppressive treatment with corticosteroids. For
the treatment of HBV and HCV, the FDA has approved administration
of recombinant IFN-.alpha.. However, IFN-.alpha. is associated with
a number of dose-dependent adverse effects, including
thrombocytopenia, leukopenia, bacterial infections, and
influenza-like symptoms. Other agents used to treat chronic HBV or
HCV include the nucleoside analog RIBAVIRIN.TM. and ursodeoxycholic
acid; however, neither has been shown to be very effective.
RIBAVIRIN.TM.+IFN combination therapy for results in 47% rate of
sustained viral clearance (Lanford, R. E. and Bigger, C. Virology
293: 1-9 (2002). (See Medicine, (D. C. Dale and D. D. Federman,
eds.) (Scientific American, Inc., New York), 4:VIII:1-8
(1995)).
[0007] Respiratory syncytial virus is the major cause of pneumonia
and bronchiolitis in infancy. RSV infects more than half of infants
during their first year of exposure, and nearly all are infected
after a second year. During seasonal epidemics most infants,
children, and adults are at risk for infection or reinfection.
Other groups at risk for serious RSV infections include premature
infants, immune compromised children and adults, and the elderly.
Symptoms of RSV infection range from a mild cold to severe
bronchiolitis and pneumonia. Respiratory syncytial virus has also
been associated with acute otitis media and RSV can be recovered
from middle ear fluid. Herpes simplex virus-1 (HSV-1) and herpes
simplex virus-2 (HSV-2) may be either lytic or latent, and are the
causative agents in cold sores (HSV-1) and genital herpes,
typically associated with lesions in the region of the eyes, mouth,
and genitals (HSV-2). These viruses are a few examples of the many
viruses that infect humans for which there are few adequate
treatments available once infection has occurred.
[0008] The demonstrated activities of the IL-28 and IL-29 cytokine
family provides methods for treating specific virual infections, in
particular, liver specific viral infections. The activity of IL-28
and IL-29 also demonstrate that these cytokines provide methods for
treating immunocompromised patients. The methods for these and
other uses that should be apparent to those skilled in the art from
the teachings herein.
SUMMARY OF THE INVENTION
[0009] In one aspect, the present invention provides methods for
treating viral infections comprising administering to a mammal with
a viral infection causing liver inflammation a therapeutically
effective amount of a polypeptide comprising an amino acid sequence
that has at least 95% identity to SEQ ID NO:2 from amino acid
residue 22 to residue 205, wherein after administration of the
polypeptide the viral infection level or liver inflammation is
reduced. In other embodiments, the methods comprise administering
polypeptide comprising an amino acid sequence as shown in SEQ ID
NO:18, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or
SEQ ID NO:36. In other embodiments, the polypeptide is conjugated
to a polyalkyl oxide moiety, such as PEG. In another embodiment, a
reduction in the viral infection level is measured as a decrease in
viral load, an increase in antiviral antibodies, a decrease in
serological levels of alanine aminotransferase or histological
improvement. In another embodiment, the mammal is a human. In
another embodiment, the present invention provides that the viral
infection is a hepatitis B virus infection or a hepatitis C virus
infection.
[0010] In another aspect, the present invention provides methods of
treating a viral infection comprising administering to a mammal
with a viral infection causing liver inflammation a therapeutically
effective amount of polypeptide comprising an amino acid sequence
that has at least 95% identity to SEQ ID NO:4 from amino acid
residue 20 to residue 200, wherein after administration of the
polypeptide the viral infection is reduced. In another embodiments,
the polypeptide comprises an amino acid sequence as shown in SEQ ID
NO:20, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:38. In another
embodiment, the polypeptide is conjugated to a polyalkyl oxide
moiety, such as PEG. In another embodiment, the reduction in the
viral infection levels is measured as a decrease in viral load, an
increase in antiviral antibodies, a decrease in serological levels
of alanine aminotransferase or histological improvement. In another
embodiment, the mammal is a human. In another embodiment, the viral
infection is a hepatitis B virus infection or a hepatitis C virus
infection.
[0011] In another aspect, the present invention provides methods
for treating liver inflammation comprising administering to a
mammal in need thereof a therapeutically effective amount of a
polypeptide comprising an amino acid sequence that has at least 95%
identity to SEQ ID NO:2 from amino acid residue 22 to residue 205,
wherein after administration of the polypeptide the liver
inflammation is reduced. In one embodiment, the invention provides
that the polypeptide comprises an amino acid sequence as shown in
SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:30; SEQ ID NO:24, SEQ ID
NO:26 or SEQ ID NO:36. In another embodiment, the polypeptide is
conjugated to a polyalkyl oxide moiety, such as PEG. In another
embodiment, the present invention provides that the reduction in
the liver inflammation is measured as a decrease in serological
level of alanine aminotransferase or histological improvement. In
another embodiment, the mammal is a human. In another embodiment,
the liver inflammation is associated with a hepatitis C virus
infection or a hepatitis B virus infection.
[0012] In another aspect, the present invention provides methods of
treating a viral infection comprising administering to an
immunocompromised mammal with an viral infection a therapeutically
effective amount of a polypeptide comprising an amino acid sequence
that has at least 95% identity to SEQ ID NO:2 from amino acid
residue 22 to amino acid residue 205, wherein after administration
of the polypeptide the viral infection is reduced. In another
embodiment, the polypeptide comprises an amino acid sequence as
shown in SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:30; SEQ ID NO:24,
SEQ ID NO:26 or SEQ ID NO:36. In another embodiment, the
polypeptide is conjugated to a polyalkyl oxide moiety, such as PEG.
In another embodiment, a reduction in the viral infection level is
measured as a decrease in viral load, an increase in antiviral
antibodies, a decrease in serological levels of alanine
aminotransferase or histological improvement. In another
embodiment, the mammal is a human. In another embodiment, the
present invention provides that the viral infection is a hepatitis
B virus infection or a hepatitis C virus infection.
[0013] In another aspect, the present invention provides methods of
treating a viral infection comprising administering to an
immunocompromised mammal with a viral infection a therapeutically
effective amount of a polypeptide comprising an amino acid sequence
that has at least 95% identity to SEQ ID NO:4 from amino acid
residue 20 to residue 200, wherein after administration of the
polypeptide the viral infection is reduced. In other embodiments,
the polypeptide comprises an amino acid sequence as shown in SEQ ID
NO:20, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:38. In another
embodiment, the polypeptide is conjugated to a polyalkyl oxide
moiety, such as PEG. In another embodiment, the reduction in the
viral infection levels is measured as a decrease in viral load, an
increase in antiviral antibodies, a decrease in serological levels
of alanine aminotransferase or histological improvement. In another
embodiment, the mammal is a human. In another embodiment, the viral
infection is a hepatitis B virus infection or a hepatitis C virus
infection.
[0014] In another aspect, the present invention provides methods of
treating liver inflammation comprising administering to an
immunocompromised mammal with liver inflammation a therapeutically
effective amount of a polypeptide comprising an amino acid sequence
that has at least 95% identity to SEQ ID NO:2 from amino acid
residue 22 to amino acid residue 205, wherein after administration
of the polypeptide the liver inflammation is reduced. In another
embodiment, the polypeptide comprises an amino acid sequence as
shown in SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:30; SEQ ID NO:24,
SEQ ID NO:26 or SEQ ID NO:36. In another embodiment, the
polypeptide is conjugated to a polyalkyl oxide moiety, such as PEG.
In another embodiment, a reduction in the liver inflammation level
is measured as a decrease in serological levels of alanine
aminotransferase or histological improvement. In another
embodiment, the mammal is a human. In another embodiment, the
present invention provides that the viral infection is a hepatitis
B virus infection or a hepatitis C virus infection.
[0015] In another aspect, the present invention provides methods of
treating liver inflammation comprising administering to an
immunocompromised mammal with liver inflammation a therapeutically
effective amount of a polypeptide comprising an amino acid sequence
that has at least 95% identity to SEQ ID NO:4 from amino acid
residue 20 to residue 200, wherein after administration of the
polypeptide the liver inflammation is reduced. In other
embodiments, the polypeptide comprises an amino acid sequence as
shown in SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:38.
In another embodiment, the polypeptide is conjugated to a polyalkyl
oxide moiety, such as PEG. In another embodiment, the reduction in
the liver inflammation is measured as a decrease in serological
levels of alanine aminotransferase or histological improvement. In
another embodiment, the mammal is a human. In another embodiment,
the viral infection is a hepatitis B virus infection or a hepatitis
C virus infection.
DESCRIPTION OF THE INVENTION
[0016] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0017] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,
Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and
Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et
al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P,
Flag.TM. peptide (Hopp et al., Biotechnology 6:1204-10, 1988),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2: 95-107, 1991. DNAs encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0018] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0019] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0020] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complement pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0021] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide). Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0022] The term "expression vector" is used to denote a DNA
molecule, linear or circular, that comprises a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription. Such additional segments include
promoter and terminator sequences, and may also include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may contain
elements of both.
[0023] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0024] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a highly
purified form, i.e. greater than 95% pure, more preferably greater
than 99% pure. When used in this context, the term "isolated" does
not exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0025] The term "level" when referring to immune cells, such as NK
cells, T cells, in particular cytotoxic T cells, B cells and the
like, an increased level is either increased number of cells or
enhanced activity of cell function.
[0026] The term "level" when referring to viral infections refers
to a change in the level of viral infection and includes, but is
not limited to, a change in the level of CTLs or NK cells (as
described above), a decrease in viral load, an increase antiviral
antibody titer, decrease in serological levels of alanine
aminotransferase, or improvement as determined by histological
examination of a target tissue or organ. Determination of whether
these changes in level are significant differences or changes is
well within the skill of one in the art.
[0027] The term "operably linked", when referring to DNA segments,
indicates that the segments are arranged so that they function in
concert for their intended purposes, e.g., transcription initiates
in the promoter and proceeds through the coding segment to the
terminator.
[0028] The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of speciation.
[0029] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, .beta.-globin, and
myoglobin are paralogs of each other.
[0030] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When the term is applied to
double-stranded molecules it is used to denote overall length and
will be understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the two strands
of a double-stranded polynucleotide may differ slightly in length
and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired.
[0031] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides".
[0032] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
[0033] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0034] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule (i.e., a ligand) and mediates the
effect of the ligand on the cell. Membrane-bound receptors are
characterized by a multi-peptide structure comprising an
extracellular ligand-binding domain and an intracellular effector
domain that is typically involved in signal transduction. Binding
of ligand to receptor results in a conformational change in the
receptor that causes an interaction between the effector domain and
other molecule(s) in the cell. This interaction in turn leads to an
alteration in the metabolism of the cell. Metabolic events that are
linked to receptor-ligand interactions include gene transcription,
phosphorylation, dephosphorylation, increases in cyclic AMP
production, mobilization of cellular calcium, mobilization of
membrane lipids, cell adhesion, hydrolysis of inositol lipids and
hydrolysis of phospholipids. In general, receptors can be membrane
bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating
hormone receptor, beta-adrenergic receptor) or multimeric (e.g.,
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF
receptor, G-CSF receptor, erythropoietin receptor and IL-6
receptor).
[0035] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0036] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a protein encoded by a splice
variant of an mRNA transcribed from a gene.
[0037] Molecular weights and lengths of polymers determined by
imprecise analytical methods (e.g., gel electrophoresis) will be
understood to be approximate values. When such a value is expressed
as "about" X or "approximately" X, the stated value of X will be
understood to be accurate to .+-.10%.
[0038] "zcyto20", "zcyto21", "zcyto22" are the previous
designations for human IL-28A, IL-29, and IL-28B. IL-28A nucleotide
and amino acid sequences are shown in SEQ ID NOS: 1, 2, 17, 18, 35,
36. IL-28B nucleotide and amino acid sequences are shown in SEQ ID
NOS: 5, 6, 21, 22, 39, 40. IL-29 nucleotide and amino acid
sequences are shown in SEQ ID NOS: 3, 4, 19, 20, 37, 38. These
sequences are described in PCT application WO 02/086087 and U.S.
Provisional Patent Application No. 60/493,194, both commonly
assigned to ZymoGenetics, Inc., incorporated herein by
reference.
[0039] "zcyto24" and "zcyto25" are the previous designations for
mouse IL-28A and IL-28B, and are shown in SEQ ID NOS: 7, 8, 9, 10,
respectively. The polynucleotide and polypeptides are fully
described in PCT application WO 02/086087 commonly assigned to
ZymoGenetics, Inc., incorporated herein by reference.
[0040] "zcytor19" is the previous designation for IL-28 receptor
.alpha.-subunit, and is shown in SEQ ID NOS: 11, 12, 13, 14, 15,
16. The polynucleotides and polypeptides are described in PCT
application WO 02/20569 on behalf of Schering, Inc., and WO
02/44209 assigned to ZymoGenetics, Inc and incorporated herein by
reference. "IL-28 receptor" denotes the IL-28 .alpha.-subunit and
CRF2-4 subunit forming a heterodimeric receptor.
[0041] All references cited herein are incorporated by reference in
their entirety.
[0042] We have previously reported the discovery of a new family of
interferon-like molecules in PCT applications, PCT/US01/21087 and
PCT/US02/12887, and Sheppard et al., Nature Immunol. 4:63-68, 2003;
herein all incorporated by reference. This new family includes
molecules designated zcyto20 (SEQ ID NOS: 1 and 2; 17 and 18),
zcyto21 (SEQ ID NOS:3 and 4; 19 and 20), zcyto22 (SEQ ID NOS:5 and
6; 21 and 22), zcyto24 (SEQ ID NOS:7 and 8), zcyto25 (SEQ ID NOS: 9
and 10), where zcyto20, 21, and 22 are human sequences, and zcyto24
and 25 are mouse sequences. HUGO designations have been assigned to
the interferon-like proteins. Zcyto20 has been designated IL-28A,
zycto22 has been designated IL-28B, zycto21 has been designated
IL-29, and will be referred to by the HUGO names hereafter. Kotenko
et al., Nature Immunol. 4:69-77, 2003, have identified IL-28A as
IFN.lamda.2, IL-28B as IFN.lamda.3, and IL-29 as IFN.lamda.1. The
receptor for these proteins, originally designated zcytor19 (SEQ ID
NOS: 11 and 12), has been designated as IL-28RA by HUGO.
[0043] The present invention provides methods for using IL-28 and
IL-29 as an antiviral agent in a broad spectrum of viral
infections. In certain embodiments, the methods include using IL-28
and IL-29 in viral infections that are specific for liver, such as
hepatitis. Furthermore, data indicate that IL-28 and IL-29 exhibit
these antiviral activities without some of the toxicities
associated with the use of IFN therapy for viral infection. One of
the toxicities related to type I interferon therapy is
myelosuppression. This is due to type I interferons suppression of
bone marrow progenitor cells. Because IL-29 does not significantly
suppress bone marrow cell expansion or B cell proliferation as is
seen with IFN-.alpha., IL-29 will have less toxicity associated
with treatment. Similar results would be expected with IL-28A and
IL-28B.
[0044] IFN-.alpha. may be contraindicated in some patients,
particularly when doses sufficient for efficacy have some toxicity
or myelosuppressive effects. Examples of patients for which IFN is
contraindicated can include (1) patients given previous
immunosuppressive medication, (2) patients with HIV or hemophilia,
(3) patients who are pregnant, (4) patients with a cytopenia, such
as leukocyte deficiency, neutropenia, thrombocytopenia, and (5)
patients exhibiting increased levels of serum liver enzymes.
Moreover, IFN therapy is associated with symptoms that are
characterized by nausea, vomiting, diarrhea and anorexia. The
result being that some populations of patients will not tolerate
IFN therapy, and IL-28A, IL-28B, and IL-29 can provide an
alternative therapy for some of those patients.
[0045] The methods of the present invention comprise administering
a therapeutically effective amount of IL-28A, IL-28B, IL-29, or
mutant of said molecules that have retained some biological
activity associated with IL-28A, IL-28B or IL-29, alone or in
combination with other biologics or pharmaceuticals. The present
invention provides methods of treating a mammal with a chronic or
acute viral infection, causing liver inflammation, thereby reducing
the viral infection or liver inflammation. In another aspect, the
present invention provides methods of treating liver specific
diseases, in particular liver disease where viral infection is in
part an etiologic agent. These methods are based on the discovery
that IL-28 and IL-29 have antiviral activity on hepatic cells.
[0046] As stated above, the methods of the present invention
provide administering a therapeutically effective amount of IL-28A,
IL-28B, IL-29, or mutant of said molecules that have retained some
biological activity associated with IL-28A, IL-28B or IL-29, alone
or in combination with other biologics or pharmaceuticals. The
present invention provides methods of treatment of a mammal with a
viral infection selected from the group consisting of hepatitis A,
hepatitis B, hepatitis C, and hepatitis D. Other aspects of the
present invention provide methods for using IL-28 or IL-29 as an
antiviral agent in viral infections selected from the group
consisting of respiratory syncytial virus, herpes virus,
Epstein-Barr virus, influenza virus, adenovirus, parainfluenza
virus, rhino virus, coxsackie virus, vaccinia virus, west nile
virus, dengue virus, Venezuelan equine encephalitis virus, pichinde
virus and polio virus. In certain embodiments, the mammal can have
either a chronic or acute viral infection.
[0047] In another aspect, the methods of the present invention also
include a method of treating a viral infection comprising
administering a therapeutically effective amount of IL-28A, IL-28B,
IL-29, or mutant of said molecules that have retained some
biological activity associated with IL-28A, IL-28B or IL-29, alone
or in combination with other biologics or pharmaceuticals, to an
immunompromised mammal with a viral infection, thereby reducing the
viral infection, such as is described above. All of the above
methods of the present invention can also comprise the
administration of zcyto24 or zcyto25 as well.
Description of IL-28A, IL-28B, and IL-29 Polynucleotides and
Polypeptides
[0048] An IL-28A gene encodes a polypeptide of 205 amino acids, as
shown in SEQ ID NO:2. The signal sequence for IL-28A comprises
amino acid residue 1 (Met) through amino acid residue 21 (Ala) of
SEQ ID NO:2. The mature peptide for IL-28A begins at amino acid
residue 22 (Val). A variant IL-28A gene encodes a polypeptide of
200 amino acids, as shown in SEQ ID NO:18. The signal sequence for
IL-28A can be predicted as comprising amino acid residue -25 (Met)
through amino acid residue -1 (Ala) of SEQ ID NO:18. The mature
peptide for IL-28A begins at amino acid residue 1 (Val). IL-28A
helices are predicted as follow: helix A is defined by amino acid
residues 24 (Leu) to 40 (Glu); helix B by amino acid residues 58
(Thr) to 65 (Gln); helix C by amino acid residues 69 (Arg) to 85
(Ala); helix D by amino acid residues 95 (Val) to 114 (Ala); helix
E by amino acid residues 126 (Thr) to 142 (Lys); and helix F by
amino acid residues 148 (Cys) to 169 (Ala); as shown in SEQ ID NO:
18. When a polynucleotide sequence encoding the mature polypeptide
is expressed in a prokaryotic system, such as E. coli, the a
secretory signal sequence may not be required and the an N-terminal
Met will be present, resulting in expression of a polypeptide such
as is shown in SEQ ID NO:36.
[0049] The IL-29 gene encodes a polypeptide of 200 amino acids, as
shown in SEQ ID NO:4. The signal sequence for IL-29 comprises amino
acid residue 1 (Met) through amino acid residue 19 (Ala) of SEQ ID
NO:4. The mature peptide for IL-29 begins at amino acid residue 20
(Gly). IL-29 has been described in published PCT application WO
02/02627. A variant IL-29 gene encodes a polypeptide of 200 amino
acids, as shown in SEQ ID NO:20, where amino acid residue 169 is
Asn instead of Asp. The signal sequence for IL-29 can be predicted
as comprising amino acid residue -19 (Met) through amino acid
residue -1 (Ala) of SEQ ID NO:20. The mature peptide for IL-29
begins at amino acid residue 1 (Gly). IL-29 has been described in
PCT application WO 02/02627. IL-29 helices are predicted as
follows: helix A is defined by amino acid residues 30 (Ser) to 44
(Leu); helix B by amino acid residues 57 (Asn) to 65 (Val); helix C
by amino acid residues 70 (Val) to 85 (Ala); helix D by amino acid
residues 92 (Glu) to 114 (Gln); helix E by amino acid residues 118
(Thr) to 139 (Lys); and helix F by amino acid residues 144 (Gly) to
170 (Leu); as shown in SEQ ID NO: 20. When a polynucleotide
sequence encoding the mature polypeptide is expressed in a
prokaryotic system, such as E. coli, the a secretory signal
sequence may not be required and the an N-terminal Met will be
present, resulting in expression of a polypeptide such as is shown
in SEQ ID NO:38.
[0050] The IL-28B gene encodes a polypeptide of 205 amino acids, as
shown in SEQ ID NO:6. The signal sequence for IL-28B comprises
amino acid residue 1 (Met) through amino acid residue 21 (Ala) of
SEQ ID NO:6. The mature peptide for IL-28B begins at amino acid
residue 22 (Val). A variant IL-28B gene encodes a polypeptide of
200 amino acids, as shown in SEQ ID NO:22. The signal sequence for
IL-28B can be predicted as comprising amino acid residue -21 (Met)
through amino acid residue -1 (Ala) of SEQ ID NO:22. The mature
peptide for IL-28B begins at amino acid residue 1 (Val). IL-28B
helices are predicted as follow: helix A is defined by amino acid
residues 8 (Leu) to 41 (Glu); helix B by amino acid residues 58
(Trp) to 65 (Gln); helix C by amino acid residues 69 (Arg) to 86
(Ala); helix D by amino acid residues 95 (Gly) to 114 (Ala); helix
E by amino acid residues 126 (Thr) to 142 (Lys); and helix F by
amino acid residues 148 (Cys) to 169 (Ala); as shown in SEQ ID NO:
2. When a polynucleotide sequence encoding the mature polypeptide
is expressed in a prokaryotic system, such as E. coli, the a
secretory signal sequence may not be required and the an N-terminal
Met will be present, resulting in expression of a polypeptide such
as is shown in SEQ ID NO:40.
[0051] Zcyto24 gene encodes a polypeptide of 202 amino acids, as
shown in SEQ ID NO:8. Zcyto24 secretory signal sequence comprises
amino acid residue 1 (Met) through amino acid residue 28 (Ala) of
SEQ ID NO:8. An alternative site for cleavage of the secretory
signal sequence can be found at amino acid residue 24 (Thr). The
mature polypeptide comprises amino acid residue 29 (Asp) to amino
acid residue 202 (Val).
[0052] Zcyto25 gene encodes a polypeptide of 202 amino acids, as
shown in SEQ ID NO:10. Zcyto25 secretory signal sequence comprises
amino acid residue 1 (Met) through amino acid residue 28 (Ala) of
SEQ ID NO:10. An alternative site for cleavage of the secretory
signal sequence can be found at amino acid residue 24 (Thr). The
mature polypeptide comprises amino acid residue 29 (Asp) to amino
acid residue 202 (Val).
[0053] The present invention provides methods comprising
administration of polypeptides having mutations in the IL-28 and
IL-29 wildtype sequence that result in expression of single forms
of the IL-28 or IL-29 molecule. Exemplary mutants are shown in SEQ
ID NOS: 24, 28, and 32. When IL-28 and IL-29 are expressed in E.
coli, an N-terminal Methionine is present. SEQ ID NOS: 26, 30 and
34 show the amino acid residue numbering for IL-28A and IL-29
mutants when the N-terminal Met is present. Table 1 shows the
possible combinations of intramolecular disulfide bonded cysteine
pairs for wildtype IL-28A, IL-28B, and IL-29.
TABLE-US-00001 TABLE 1 IL-28A C.sub.16-C.sub.115 C.sub.48-C.sub.148
C.sub.50-C.sub.148 C.sub.167-C.sub.174 C.sub.16-C.sub.48
C.sub.16-C.sub.50 C.sub.48-C.sub.115 C.sub.50-C.sub.115
C.sub.115-C.sub.148 SEQ ID NO: 18 Met IL- C.sub.17-C.sub.116
C.sub.49-C.sub.149 C.sub.51-C.sub.1498 C.sub.168-C.sub.175
C.sub.17-C.sub.49 C.sub.17-C.sub.51 C.sub.49-C.sub.116
C.sub.51-C.sub.116 C.sub.116-C.sub.149 28A SEQ ID NO: 36 IL-29
C.sub.15-C.sub.112 C.sub.49-C.sub.145 C.sub.112-C.sub.171 SEQ ID
NO: 20 Met IL-29 C.sub.16-C.sub.113 C.sub.50-C.sub.146
C.sub.113-C.sub.172 SEQ ID NO: 38 IL-28B C.sub.16-C.sub.115
C.sub.48-C.sub.148 C.sub.50-C.sub.148 C.sub.167-C.sub.174
C.sub.16-C.sub.48 C.sub.16-C.sub.50 C.sub.48-C.sub.115
C.sub.50-C.sub.115 C.sub.115-C.sub.148 SEQ ID NO: 22 Met IL-28B
C.sub.17-C.sub.116 C.sub.49-C.sub.149 C.sub.51-C.sub.1498
C.sub.168-C.sub.175 C.sub.17-C.sub.49 C.sub.17-C.sub.51
C.sub.49-C.sub.116 C.sub.51-C.sub.116 C.sub.116-C.sub.149 SEQ ID
NO: 40
[0054] Conjugation of interferons with water-soluble polymers has
been shown to enhance the circulating half-life of the interferon,
and to reduce the immunogenicity of the polypeptide (see, for
example, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996), and
Monkarsh et al., Anal. Biochem. 247:434 (1997)). Suitable
water-soluble polymers include polyethylene glycol (PEG),
monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-1-vinyl
pyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde,
bis-succinimidyl carbonate PEG, propylene glycol homopolymers, a
polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or
other carbohydrate-based polymers. Suitable PEG may have a
molecular weight from about 600 to about 60,000, including, for
example, 5,000, 12,000, 20,000 and 40,000. A Cysteine mutant IL-28
or IL-29 conjugate can also comprise a mixture of such
water-soluble polymers.
[0055] One example of a IL-28 or IL-29 conjugate comprises a IL-28
or IL-29 moiety, or mutant thereof, and a polyalkyl oxide moiety
attached to the N-terminus of the IL-28 or IL-29 moiety. PEG is one
suitable polyalkyl oxide. As an illustration, IL-28 or IL-29 can be
modified with PEG, a process known as "PEGylation." PEGylation of
IL-28 or IL-29 can be carried out by any of the PEGylation
reactions known in the art (see, for example, EP 0 154 316, Delgado
et al., Critical Reviews in Therapeutic Drug Carrier Systems 9:249
(1992), Duncan and Spreafico, Clin. Pharmacokinet. 27:290 (1994),
and Francis et al., Int J Hematol 68:1 (1998)). The methods of the
present invention include administration of IL-28, IL-29, and
mutants thereof conjugated to water-soluble polymers, such as
PEG.
[0056] IL-28A, IL-29, IL-28B, zcyto24 and zcyto25, each have been
shown to form a complex with the orphan receptor designated
zcytor19 (IL-28RA). IL-28RA is described in a commonly assigned
patent application PCT/US01/44808. IL-28B, IL-29, zcyto24, and
zcyto25 have been shown to bind or signal through IL-28RA as well,
further supporting that IL-28A, IL-29, IL-28B, zcyto24 and zcyto25
are members of the same family of cytokines. IL-28RA receptor is a
class II cytokine receptor. Class II cytokine receptors usually
bind to four-helix-bundle cytokines. For example, interleukin-10
and the interferons bind receptors in this class (e.g.,
interferon-gamma receptor, alpha and beta chains and the
interferon-alpha/beta receptor alpha and beta chains).
[0057] Class II cytokine receptors are characterized by the
presence of one or more cytokine receptor modules (CRM) in their
extracellular domains. Other class II cytokine receptors include
zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4 (Genbank
Accession No. Z17227), IL-10R (Genbank Accession No.s U00672 and
NM.sub.--001558), DIRS1, zcytor7 (commonly owned U.S. Pat. No.
5,945,511), and tissue factor. IL-28RA, like all known class II
receptors except interferon-alpha/beta receptor alpha chain, has
only a single class II CRM in its extracellular domain.
[0058] Analysis of a human cDNA clone encoding IL-28RA (SEQ ID
NO:11) revealed an open reading frame encoding 520 amino acids (SEQ
ID NO:12) comprising a secretory signal sequence (residues 1 (Met)
to 20 (Gly) of SEQ ID NO:12) and a mature IL-28RA cytokine receptor
polypeptide (residues 21 (Arg) to 520 (Arg) of SEQ ID NO:12) an
extracellular ligand-binding domain of approximately 206 amino acid
residues (residues 21 (Arg) to 226 (Asn) of SEQ ID NO:12), a
transmembrane domain of approximately 23 amino acid residues
(residues 227 (Trp) to 249 (Trp) of SEQ ID NO:12), and an
intracellular domain of approximately 271 amino acid residues
(residues 250 (Lys) to 520 (Arg) of SEQ ID NO:12). Within the
extracellular ligand-binding domain, there are two fibronectin type
III domains and a linker region. The first fibronectin type III
domain comprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:12,
the linker comprises residues 120 (Leu) to 124 (Glu) of SEQ ID
NO:12, and the second fibronectin type III domain comprises
residues 125 (Pro) to 223 (Pro) of SEQ ID NO:12.
[0059] In addition, a human cDNA clone encoding a IL-28RA variant
with a 29 amino acid deletion was identified. This IL-28RA variant
(as shown in SEQ ID NO:13) comprises an open reading frame encoding
491 amino acids (SEQ ID NO:14) comprising a secretory signal
sequence (residues 1 (Met) to 20 (Gly) of SEQ ID NO:14) and a
mature IL-28RA cytokine receptor polypeptide (residues 21 (Arg) to
491 (Arg) of SEQ ID NO:14) an extracellular ligand-binding domain
of approximately 206 amino acid residues (residues 21 (Arg) to 226
(Asn) of SEQ ID NO:14, a transmembrane domain of approximately 23
amino acid residues (residues 227 (Trp) to 249 (Trp) of SEQ ID
NO:14), and an intracellular domain of approximately 242 amino acid
residues (residues 250 (Lys) to 491 (Arg) of SEQ ID NO:14).
[0060] A truncated soluble form of the IL-28RA receptor mRNA
appears to be naturally expressed. Analysis of a human cDNA clone
encoding the truncated soluble IL-28RA (SEQ ID NO:15) revealed an
open reading frame encoding 211 amino acids (SEQ ID NO:16)
comprising a secretory signal sequence (residues 1 (Met) to 20
(Gly) of SEQ ID NO:16) and a mature truncated soluble IL-28RA
receptor polypeptide (residues 21 (Arg) to 211 (Ser) of SEQ ID
NO:16) a truncated extracellular ligand-binding domain of
approximately 143 amino acid residues (residues 21 (Arg) to 163
(Trp) of SEQ ID NO:16), no transmembrane domain, but an additional
domain of approximately 48 amino acid residues (residues 164 (Lys)
to 211 (Ser) of SEQ ID NO:16).
[0061] IL-28RA is a member of the same receptor subfamily as the
class II cytokine receptors, and receptors in this subfamily may
associate to form homodimers that transduce a signal. Several
members of the subfamily (e.g., receptors that bind interferon,
IL-10, IL-19, and IL-TIF) combine with a second subunit (termed a
.beta.-subunit) to bind ligand and transduce a signal. However, in
many cases, specific .beta.-subunits associate with a plurality of
specific cytokine receptor subunits. For example, class II cytokine
receptors, such as, zcytor11 (U.S. Pat. No. 5,965,704) and CRF2-4
receptor heterodimerize to bind the cytokine IL-TIF (See, WIPO
publication WO 00/24758; Dumontier et al., J. Immunol.
164:1814-1819, 2000; Spencer, S D et al., J. Exp. Med. 187:571-578,
1998; Gibbs, V C and Pennica Gene 186:97-101, 1997 (CRF2-4 cDNA);
Xie, M H et al., J. Biol. Chem. 275: 31335-31339, 2000).
IL-10.beta. receptor is believed to be synonymous with CRF2-4
(Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149,
2000; Liu Y et al, J Immunol. 152; 1821-1829, 1994 (IL-10R cDNA).
Therefore, one could expect that zcyto20, zcyto21, zcyto22, zcyto24
and zcyto25 would bind either monomeric, homodimeric, heterodimeric
and multimeric zcytor19 receptors. Experimental evidence has
identified CRF2-4 as the putative binding partner for IL-28RA.
[0062] Examples of biological activity for molecules used to
identify IL-28 or IL-29 molecules that are useful in the methods of
the present invention include molecules that can bind to the IL-28
receptor with some specificity. Generally, a ligand binding to its
cognate receptor is specific when the K.sub.D falls within the
range of 100 nM to 100 pM. Specific binding in the range of 100 mM
to 10 nM K.sub.D is low affinity binding. Specific binding in the
range of 2.5 pM to 100 pM K.sub.D is high affinity binding. In
another example, biologically active IL-28 or IL-29 molecules are
capable of some level of antiviral activity associated with
wildtype IL-28 or IL-29.
[0063] Use of IL-28A, IL-28B, and IL-29 for Viral Infections
[0064] IL-28 and IL-29 can be used in treating liver specific
diseases, in particular liver disease where viral infection is in
part an etiologic agent. In particular IL-28 and IL-29 will be used
to treat a mammal with a viral infection selected from the group
consisting of hepatitis A, hepatitis B, hepatitis C, and hepatitis
D. When liver disease is inflammatory and continuing for at least
six months, it is generally considered chronic hepatitis. Hepatitis
C virus (HCV) patients actively infected will be positive for
HCV-RNA in their blood, which is detectable by reverse
transcriptase/polymerase chain reaction (RT-PCR) assays. The
methods of the present invention will slow the progression of the
liver disease. Clinically, diagnostic tests for HCV include
serologic assays for antibodies and molecular tests for viral
particles. Enzyme immunoassays are available (Vrielink et al.,
Transfusion 37:845-849, 1997), but may require confirmation using
additional tests such as an immunoblot assay (Pawlotsky et al.,
Hepatology 27:1700-1702, 1998). Qualitative and quantitative assays
generally use polymerase chain reaction techniques, and are
preferred for assessing viremia and treatment response (Poynard et
al., Lancet 352:1426-1432, 1998; McHutchinson et al., N. Engl. J.
Med. 339:1485-1492, 1998). Several commercial tests are available,
such as, quantitative RT-PCR (Amplicor HCV Monitor.TM., Roche
Molecular Systems, Branchburg, N.J.) and a branched DNA
(deoxyribonucleic acid) signal amplification assay (Quantiplex.TM.
HCV RNA Assay [bDNA], Chiron Corp., Emeryville, Calif.). A
non-specific laboratory test for liver inflammation or necrosis
measures alanine aminotransferase level (ALT) and is inexpensive
and readily available (National Institutes of Health Consensus
Development Conference Panel, Hepatology 26 (Suppl. 1):2S-10S,
1997). Histologic evaluation of liver biopsy is generally
considered the most accurate means for determining hepatitis
progression (Yano et al., Hepatology 23:1334-1340, 1996.) For a
review of clinical tests for HCV, see, Lauer et al., N. Engl. J.
Med. 345:41-52, 2001.
[0065] There are several in vivo models for testing HBV and HCV
that are known to those skilled in art. For example, the effects of
IL-28 or IL-29 on mammals infected with HBV can accessed using a
woodchuck model. Briefly, woodchucks chronically infected with
woodchuck hepatitis virus (WHV) develop hepatitis and
hepatocellular carcinoma that is similar to disease in humans
chronically infected with HBV. The model has been used for the
preclinical assessment of antiviral activity. A chronically
infected WHV strain has been established and neonates are
inoculated with serum to provide animals for studying the effects
of certain compounds using this model. (For a review, see, Tannant
et al., ILAR J. 42 (2):89-102, 2001). Chimpanzees may also be used
to evaluate the effect of IL-28 and IL-29 on HBV infected mammals.
Using chimpanzees, characterization of HBV was made and these
studies demonstrated that the chimpanzee disease was remarkably
similar to the disease in humans (Barker et al., J. Infect. Dis.
132:451-458, 1975 and Tabor et al., J. Infect. Dis. 147:531-534,
1983.) The chimpanzee model has been used in evaluating vaccines
(Prince et al., In: Vaccines 97 Cold Spring Harbor Laboratory
Press, 1997.) Therapies for HIV are routinely tested using
non-human primates infected with simian immunodeficiency viruses
(for a review, see, Hirsch et al., Adv. Pharmcol. 49:437-477, 2000
and Nathanson et al., AIDS 13 (suppl. A):S113-S120, 1999.) For a
review of use of non-human primates in HIV, hepatitis, malaria,
respiratory syncytial virus, and other diseases, see, Sibal et al.,
ILAR J. 42 (2):74-84, 2001.
[0066] Other examples of the types of viral infections for which
IL-28A, IL-28B, and IL-29 can be useful include, but are not
limited to: infections caused by DNA Viruses (e.g., Herpes Viruses
such as Herpes Simplex viruses, Epstein-Barr virus,
Cytomegalovirus; Pox viruses such as Variola (small pox) virus;
Hepadnaviruses (e.g, Hepatitis B virus); Papilloma viruses;
Adenoviruses); RNA Viruses (e.g., HIV I, II; HTLV I, II;
Poliovirus; Hepatitis A; Orthomyxoviruses (e.g., Influenza
viruses); Paramyxoviruses (e.g., Measles virus); Rabies virus;
Hepatitis C); Rhinovirus, Respiratory Syncytial Virus, West Nile
Virus, Yellow Fever, Rift Valley Virus, Lassa Fever Virus, Ebola
Virus, Lymphocytic Choriomeningitis Virus, which replicates in
tissues including liver, and the like. Moreover, examples of the
types of diseases for which IL-28 and IL-29 could be used include,
but are not limited to: Acquired immunodeficiency; Hepatitis;
Gastroenteritis; Hemorrhagic diseases; Enteritis; Carditis;
Encephalitis; Paralysis; Brochiolitis; Upper and lower respiratory
disease; Respiratory Papillomatosis; Arthritis; Disseminated
disease, hepatocellular carcinoma resulting from chronic Hepatitis
C infection. In addition, viral disease in other tissues may be
treated with IL-28A, IL-28B, and IL-29, for example viral
meningitis, and HIV-related disease. For example, a transgenic
model for testing the activity of a therapeutic sample is described
in the following examples and described in Morrey, et al.,
Antiviral Ther., 3 (Suppl 3):59-68, 1998.
[0067] Animal models that are used to test for efficacy in specific
viruses are known. For example, Dengue Virus can be tested using a
model as such as described in Huang et al., J. Gen. Virol.
September; 81(Pt 9):2177-82, 2000. West Nile Virus can be tested
using the model as described in Xiao et al., Emerg. Infect. Dis.
July-August; 7(4):714-21, 2001 or Mashimo et al., Proc. Natl. Acad.
Sci. USA. August 20:99(17):11311-6, 2002. Venezuelan equine
encephalitis virus model is described in Jackson et al., Veterinary
Pathology, 28 (5): 410-418, 1991; Vogel et al., Arch. Pathol. Lab.
Med. February; 120(2):164-72, 1996; Lukaszewski and Brooks, J. of
Virology, 74(11):5006-5015, 2000. Rhinoviruses models are described
in Yin and Lomax, J. Gen. Virol. 67 (Pt 11):2335-40, 1986. Models
for respiratory syncytial virus are described in Byrd and Prince,
Clin. Infect. Dis. 25(6):1363-8, 1997. Other models are known in
the art and it is well within the skill of those ordinarily skilled
in the art to know how to use such models.
[0068] IL-28A, IL-28B, and IL-29 can be used in combination with
antiviral agents, including those described above. Some of the more
common treatments for viral infection include drugs that inhibit
viral replication such as ACYCLOVIR.TM.. In addition, the combined
use of some of these agents form the basis for highly active
antiretroviral therapy (HAART) used for the treatment of AIDS.
Examples in which the combination of immunotherapy (i.e. cytokines)
and antiviral drugs shows improved efficacy include the use of
interferon plus RIBAVIRIN.TM. for the treatment of chronic
hepatitis C infection (Maddrey, Semin. Liver. Dis. 19 Suppl
1:67-75, 1999) and the combined use of IL-2 and HAART (Ross, et al,
ibid.) Thus, as IL-28 and IL-29 can stimulate the immune system
against disease, it can similarly be used in HAART
applications.
[0069] In particular, IL-28A, IL-28B, and IL-29 may be useful in
monotherapy or combination therapy with IFN-.alpha. (with or
without RIBAVIRIN.TM.) in patients who do not respond well to IFN
therapy. These patients may not respond to IFN therapy due to
having less type I interferon receptor on the surface of their
cells (Yatsuhashi H, et al., J Hepatol. June 30(6):995-1003, 1999;
Mathai et al., J Interferon Cytokine Res. September 19(9):1011-8,
1999; Fukuda et al., J. Med. Virol. 63(3):220-7, 2001). IL-28A,
IL-28B, and IL-29 may also be useful in monotherapy or combination
therapy with IFN-.alpha. (with or without RIBAVIRIN.TM.) in
patients who have less type I interferon receptor on the surface of
their cells due to down-regulation of the type I interferon
receptor after type I interferon treatment (Dupont et al., J.
Interferon Cytokine Res. 22(4):491-501, 2002).
[0070] IL-28 or IL-29 can be used in combination with other
immunotherapies including cytokines, immunoglobulin transfer, and
various co-stimulatory molecules. In addition to antiviral drugs,
IL-28, IL-29, or mutants thereof could be used in combination with
any other immunotherapy that is intended to stimulate the immune
system. Thus, IL-28, IL-29, or mutants thereof could be used with
other cytokines such as Interferon or IL-2. IL-28, IL-29, or
mutants thereof could also be added to methods of passive
immunization that involve immunoglobulin transfer, one example
bring the use of antibodies to treat RSV infection in high risk
patients (Meissner HC, ibid.). In addition, IL-28, IL-29, or
mutants thereof could be used with additional co-stimulatory
molecules such as 4-1BB ligand that recognize various cell surface
molecules like CD137 (Tan, J T et al., J Immunol. 163:4859-68,
1999).
C. Use of IL-28A, IL-28B, and IL-29 in Immunocompromised
Patients
[0071] IL-28 and IL-29 can be used as a monotherapy for acute and
chronic viral infections and for immunocompromised patients.
Methods that enhance immunity can accelerate the recovery time in
patients with unresolved infections. Immunotherapies can have an
even greater impact on subsets of immunocompromised patients such
as the very young or elderly as well as patients that suffer
immunodeficiencies acquired through infection, or induced following
medical interventions such as chemotherapy or bone marrow ablation.
Examples of the types of indications being treated via
immune-modulation include; the use of IFN-.alpha. for chronic
hepatitis (Perry C M, and Jarvis B, Drugs 61:2263-88, 2001), the
use of IL-2 following HIV infection (Mitsuyasu R., J. Infect. Dis.
185 Suppl 2:S115-22, 2002; and Ross R W et al., Expert Opin. Biol.
Ther. 1:413-24, 2001), and the use of either IFN-.alpha. (Faro A,
Springer Semin. Immunopathol. 20:425-36, 1998) for treating Epstein
Barr Virus infections following transplantation. Experiments
performed in animal models indicate that IL-2 and GM-CSF may also
be efficacious for treating EBV related diseases (Baiocchi R A et
al., J. Clin. Invest. 108:887-94, 2001).
[0072] In summary, IL-28, IL-29, or mutants thereof can be
used:
[0073] as a monotherapy for acute and chronic viral infections and
for immunocompromised patients. Methods that enhance immunity can
accelerate the recovery time in patients with unresolved
infections.
[0074] in combination with other antiviral agents such as
ACYCLOVIR.TM., interferon plus RIBAVIRIN.TM..
[0075] in combination with other immunotherapies including
cytokines, immunoglobulin transfer, and various co-stimulatory
molecules.
[0076] to treat a mammal with a chronic or acute viral infection
that has resulted liver inflammation, thereby reducing the viral
infection and/or liver inflammation. In particular IL-28 and IL-29
will be used to treat a mammal with a viral infection selected from
the group consisting of hepatitis A, hepatitis B, hepatitis C, and
hepatitis D.
[0077] as an antiviral agent in viral infections selected from the
group consisting of respiratory syncytial virus, herpes virus,
Epstein-Barr virus, influenza virus, adenovirus, parainfluenza
virus, rhino virus, coxsackie virus, vaccinia virus, west nile
virus, dengue virus, Venezuelan equine encephalitis virus, pichinde
virus and polio virus.
EXAMPLES
Example 1
Induction of IL-28A, IL-29, IL-28B by Poly I:C and Viral
Infection
[0078] Freshly isolated human peripheral blood mononuclear cells
were grown in the presence of polyinosinic acid-polycytidylic acid
(poly I:C; 100 .mu.g/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).
[0079] Low to undetectable amounts of human IL-28A, IL-28B, and
IL-29, IFN-.alpha. and IFN-.beta. 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-stranded RNA or viral infection.
Example 2
IL-28, IL-29 Signaling Activity Compared to IFN.alpha. in HepG2
Cells
A. Cell Transfections
[0080] 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
[0081] The signal transduction reporter assays were done as
follows: Following an 18 h incubation at 37.degree. C. in DMEM+0.5%
FBS, transfected cells were stimulated with 100 ng/ml IL-28A,
IL-29, IL-28B, zcyto24, zcyto25 and huIFN-.alpha.2a ligands.
Following a 4-hour incubation at 37.degree. degrees, the cells were
lysed, and the relative light units (RLU) were measured on a
luminometer after addition of a luciferase substrate. The results
obtained are shown as the fold induction of the RLU of the
experimental samples over the medium alone control (RLU of
experimental samples/RLU of medium alone=fold induction). Table 2
shows that IL-28A, IL-29, IL-28B, zcyto24 and zcyto25 induce ISRE
signaling in human HepG2 liver cells transfected with
ISRE-luciferase.
TABLE-US-00002 TABLE 2 Fold Induction of Cytokine-dependent ISRE
Signaling in HepG2 Cells Cytokine Fold Inductn. IL-28A 5.6 IL-29 4
IL-28B 5.8 Zcyto24 4.7 Zcyto25 3 HuIFN-a2a 5.8
Example 3
IL-29 Antiviral Activity Compared to IFN.alpha. in HepG2 Cells
[0082] 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.degree. C. for 2
hours. A solubiliser solution was added, incubated overnight at
37.degree. C. and the optical density at 570 nm was determined.
OD570 is directly proportional to antiviral activity.
[0083] The results show the antiviral activity when IL-29 and IFN
on were tested with HepG2 cells: IL-29, IFN-.beta. and IFN
.alpha.-2a were added at varying concentration to HepG2 cells prior
to EMCV infection and dye-uptake assay. The mean and standard
deviation of the OD570 from triplicate wells is plotted. OD570 is
directly proportional to antiviral activity. For IL-29, the EC50
was 0.60 ng/ml; for IFN-.alpha.2a, the EC50 was 0.57 ng/ml; and for
IFN-.beta., the EC50 was 0.46 ng/ml.
Example 4
IL-28RA mRNA Expression in Liver and Lymphocyte Subsets
[0084] In order to further examine the mRNA distribution for
IL-28RA, semi-quantitative RT-PCR was performed using the SDS
7900HT system (Applied Biosystems, CA). One-step RT-PCR was
performed using 100 ng total RNA for each sample and gene-specific
primers. A standard curve was generated for each primer set using
Bjab RNA and all sample values were normalized to HPRT. The
normalized results are summarized in Tables 2-4. The normalized
values for IFNAR2 and CRF2-4 are also shown.
[0085] Table 3: B and T cells express significant levels of IL-28RA
mRNA. Low levels are seen in dendritic cells and most
monocytes.
TABLE-US-00003 TABLE 3 Cell/Tissue IL-28RA IFNAR2 CRF2-4 Dendritic
Cells unstim .04 5.9 9.8 Dendritic Cells + IFNg .07 3.6 4.3
Dendritic Cells .16 7.85 3.9 CD14+ stim'd with LPS/IFNg .13 12 27
CD14+ monocytes resting .12 11 15.4 Hu CD14+ Unact. 4.2 TBD TBD Hu
CD14+ 1 ug/ml LPS act. 2.3 TBD TBD H. Inflamed tonsil 3 12.4 9.5 H.
B-cells + PMA/Iono 4 & 24 hrs 3.6 1.3 1.4 Hu CD19+ resting 6.2
TBD TBD Hu CD19+ 4 hr. PMA/Iono 10.6 TBD TBD Hu CD19+ 24 hr Act.
PMA/Iono 3.7 TBD TBD IgD+ B-cells 6.47 13.15 6.42 IgM+ B-cells 9.06
15.4 2.18 IgD- B-cells 5.66 2.86 6.76 NKCells + PMA/Iono 0 6.7 2.9
Hu CD3+ Unactivated 2.1 TBD TBD CD4+ resting .9 8.5 29.1 CD4+
Unstim 18 hrs 1.6 8.4 13.2 CD4+ + Poly I/C 2.2 4.5 5.1 CD4+ +
PMA/Iono .3 1.8 .9 CD3 neg resting 1.6 7.3 46 CD3 neg unstim 18 hrs
2.4 13.2 16.8 CD3 neg + Poly I/C 18 hrs 5.7 7 30.2 CD3 neg + LPS 18
hrs 3.1 11.9 28.2 CD8+ unstim 18 hrs 1.8 4.9 13.1 CD8+ stim'd with
PMA/Ion 18 hrs .3 .6 1.1
[0086] As shown in Table 4, normal liver tissue and liver derived
cell lines display substantial levels of IL-28RA and CRF2-4
mRNA.
TABLE-US-00004 TABLE 4 Cell/Tissue IL-28RA IFNAR2 CRF2-4 HepG2 1.6
3.56 2.1 HepG2 UGAR 5/10/02 1.1 1.2 2.7 HepG2, CGAT HKES081501C 4.3
2.1 6 HuH7 5/10/02 1.63 16 2 HuH7 hepatoma - CGAT 4.2 7.2 3.1
Liver, normal - CGAT 11.7 3.2 8.4 #HXYZ020801K Liver, NAT--Normal
adjacent tissue 4.5 4.9 7.7 Liver, NAT--Normal adjacent tissue 2.2
6.3 10.4 Hep SMVC hep vein 0 1.4 6.5 Hep SMCA hep. Artery 0 2.1 7.5
Hep. Fibro 0 2.9 6.2 Hep. Ca. 3.8 2.9 5.8 Adenoca liver 8.3 4.2
10.5 SK-Hep-1 adenoca. Liver .1 1.3 2.5 AsPC-1 Hu. Pancreatic
adenocarc. .7 .8 1.3 Hu. Hep. Stellate cells .025 4.4 9.7
[0087] As shown in Table 5, primary airway epithelial cells contain
abundant levels of IL-28RA and CRF2-4.
TABLE-US-00005 TABLE 5 Cell/Tissue IL-28RA IFNAR2 CRF2-4 U87MG -
glioma 0 .66 .99 NHBE unstim 1.9 1.7 8.8 NHBE + TNF-alpha 2.2 5.7
4.6 NHBE + poly I/C 1.8 nd nd Small Airway Epithelial Cells 3.9 3.3
27.8 NHLF--Normal human lung fibroblasts 0 nd nd
[0088] As shown in Table 6, IL-28RA is present in normal and
diseased liver specimens, with increased expression in tissue from
Hepatitis C and Hepatitis B infected specimens.
TABLE-US-00006 TABLE 6 Cell/Tissue IL-28RA CRF2-4 IFNAR2 Liver with
Coagulation Necrosis 8.87 15.12 1.72 Liver with Autoimmune
Hepatitis 6.46 8.90 3.07 Neonatal Hepatitis 6.29 12.46 6.16
Endstage Liver disease 4.79 17.05 10.58 Fulminant Liver Failure
1.90 14.20 7.69 Fulminant Liver failure 2.52 11.25 8.84 Cirrhosis,
primary biliary 4.64 12.03 3.62 Cirrhosis Alcoholic (Laennec's)
4.17 8.30 4.14 Cirrhosis, Cryptogenic 4.84 7.13 5.06 Hepatitis C+,
with cirrhosis 3.64 7.99 6.62 Hepatitis C+ 6.32 11.29 7.43
Fulminant hepatitis secondary to Hep A 8.94 21.63 8.48 Hepatitis C+
7.69 15.88 8.05 Hepatitis B+ 1.61 12.79 6.93 Normal Liver 8.76 5.42
3.78 Normal Liver 1.46 4.13 4.83 Liver NAT 3.61 5.43 6.42 Liver NAT
1.97 10.37 6.31 Hu Fetal Liver 1.07 4.87 3.98 Hepatocellular
Carcinoma 3.58 3.80 3.22 Adenocarcinoma Liver 8.30 10.48 4.17 hep.
SMVC, hep. Vein 0.00 6.46 1.45 Hep SMCA hep. Artery 0.00 7.55 2.10
Hep. Fibroblast 0.00 6.20 2.94 HuH7 hepatoma 4.20 3.05 7.24 HepG2
Hepatocellular carcinoma 3.40 5.98 2.11 SK-Hep-1 adenocar. Liver
0.03 2.53 1.30 HepG2 Unstim 2.06 2.98 2.28 HepG2 + zcyto21 2.28
3.01 2.53 HepG2 + IFN.alpha. 2.61 3.05 3.00 Normal Female Liver -
degraded 1.38 6.45 4.57 Normal Liver - degraded 1.93 4.99 6.25
Normal Liver - degraded 2.41 2.32 2.75 Disease Liver - degraded
2.33 3.00 6.04 Primary Hepatocytes from Clonetics 9.13 7.97
13.30
[0089] As shown in Tables 7-11, IL-28RA is detectable in normal B
cells, B lymphoma cell lines, T cells, T lymphoma cell lines
(Jurkat), normal and transformed lymphocytes (B cells and T cells)
and normal human monocytes.
TABLE-US-00007 TABLE 7 HPRT IL-28RA IL-28RA IFNR2 CRF2-4 Mean Mean
norm IFNAR2 norm CRF2-4 Norm CD14+ 24 hr unstim #A38 13.1 68.9 5.2
92.3 7.0 199.8 15.2 CD14+ 24 hr stim #A38 6.9 7.6 1.1 219.5 31.8
276.6 40.1 CD14+ 24 hr unstim #A112 17.5 40.6 2.3 163.8 9.4 239.7
13.7 CD14+ 24 hr stim #A112 11.8 6.4 0.5 264.6 22.4 266.9 22.6
CD14+ rest #X 32.0 164.2 5.1 1279.7 39.9 699.9 21.8 CD14+ + LPS #X
21.4 40.8 1.9 338.2 15.8 518.0 24.2 CD14+ 24 hr unstim #A39 26.3
86.8 3.3 297.4 11.3 480.6 18.3 CD14+ 24 hr stim #A39 16.6 12.5 0.8
210.0 12.7 406.4 24.5 HL60 Resting 161.2 0.2 0.0 214.2 1.3 264.0
1.6 HL60 + PMA 23.6 2.8 0.1 372.5 15.8 397.5 16.8 U937 Resting
246.7 0.0 0.0 449.4 1.8 362.5 1.5 U937 + PMA 222.7 0.0 0.0 379.2
1.7 475.9 2.1 Jurkat Resting 241.7 103.0 0.4 327.7 1.4 36.1 0.1
Jurkat Activated 130.7 143.2 1.1 Colo205 88.8 43.5 0.5 HT-29 26.5
30.5 1.2
TABLE-US-00008 TABLE 8 HPRT SD IL-28RA SD Mono 24 hr unstim #A38
0.6 2.4 Mono 24 hr stim #A38 0.7 0.2 Mono 24 hr unstim 2.0 0.7
#A112 Mono 24 hr stim #A112 0.3 0.1 Mono rest #X 5.7 2.2 Mono + LPS
#X 0.5 1.0 Mono 24 hr unstim #A39 0.7 0.8 Mono 24 hr stim #A39 0.1
0.7 HL60 Resting 19.7 0.1 HL60 + PMA 0.7 0.4 U937 Resting 7.4 0.0
U937 + PMA 7.1 0.0 Jurkat Resting 3.7 1.1 Jurkat Activated 2.4 1.8
Colo205 1.9 0.7 HT-29 2.3 1.7
TABLE-US-00009 TABLE 9 Mean Mean Mean IL- Mean Hprt IFNAR2 28RA CRF
CD3+/CD4+ 0 10.1 85.9 9.0 294.6 CD4/CD3+ Unstim 18 hrs 12.9 108.7
20.3 170.4 CD4+/CD3+ + Poly I/C 18 hrs 24.1 108.5 52.1 121.8
CD4+/CD3+ + PMA/Iono 18 hrs 47.8 83.7 16.5 40.8 CD3 neg 0 15.4
111.7 24.8 706.1 CD3 neg unstim 18 hrs 15.7 206.6 37.5 263.0 CD3
neg + Poly I/C 18 hrs 9.6 67.0 54.7 289.5 CD3 neg + LPS 18 hrs 14.5
173.2 44.6 409.3 CD8+ Unstim. 18 hrs 6.1 29.7 11.1 79.9 CD8+ +
PMA/Iono 18 hrs 78.4 47.6 26.1 85.5 12.8.1-NHBE Unstim 47.4 81.1
76.5 415.6 12.8.2-NHBE + TNF-alpha 42.3 238.8 127.7 193.9 SAEC 15.3
49.9 63.6 426.0
TABLE-US-00010 TABLE 10 IL-28RA CRF IFNAR2 IL-28RA CRF IFNAR2 Norm
Norm Norm SD SD SD CD3+/CD4+ 0 0.9 29.1 8.5 0.1 1.6 0.4 CD4/CD3+
Unstim 18 hrs 1.6 13.2 8.4 0.2 1.6 1.4 CD4+/CD3+ + Poly I/C 18 hrs
2.2 5.1 4.5 0.1 0.3 0.5 CD4+/CD3+ + PMA/Iono 18 hrs 0.3 0.9 1.8 0.0
0.1 0.3 CD3 neg 0 1.6 46.0 7.3 0.2 4.7 1.3 CD3 neg unstim 18 hrs
2.4 16.8 13.2 0.4 2.7 2.3 CD3 neg + Poly I/C 18 hrs 5.7 30.2 7.0
0.3 1.7 0.8 CD3 neg + LPS 18 hrs 3.1 28.2 11.9 0.4 5.4 2.9 CD8+
Unstim. 18 hrs 1.8 13.1 4.9 0.1 1.1 0.3 CD8+ + PMA/Iono 18 hrs 0.3
1.1 0.6 0.0 0.1 0.0 12.8.1-NHBE Unstim 1.6 8.8 1.7 0.1 0.4 0.1
12.8.2-NHBE + TNF-alpha 3.0 4.6 5.7 0.1 0.1 0.1 SAEC 4.1 27.8 3.3
0.2 1.1 0.3
TABLE-US-00011 TABLE 11 SD SD SD IL- SD Hprt IFNAR2 28RA CRF
CD3+/CD4+ 0 0.3 3.5 0.6 12.8 CD4/CD3+ Unstim 18 hrs 1.4 13.7 1.1
8.5 CD4+/CD3+ + Poly I/C 18 hrs 1.3 9.8 1.6 3.4 CD4+/CD3+ +
PMA/Iono 18 hrs 4.0 10.3 0.7 3.7 CD3 neg 0 1.4 16.6 1.6 28.6 CD3
neg unstim 18 hrs 2.4 16.2 2.7 12.6 CD3 neg + Poly I/C 18 hrs 0.5
7.0 1.0 8.3 CD3 neg + LPS 18 hrs 1.0 39.8 5.6 73.6 CD8+ Unstim. 18
hrs 0.2 1.6 0.5 6.1 CD8+ + PMA/Iono 18 hrs 1.3 1.7 0.2 8.1
12.8.1-NHBE Unstim 2.4 5.6 2.7 2.8 12.8.2-NHBE + TNF-alpha 0.5 3.4
3.5 3.4 SAEC 0.5 4.8 1.8 9.9
Example 5
Mouse IL-28 does not Effect Daudi Cell Proliferation
[0090] Human Daudi cells were suspended in RPMI+10% FBS at 50,000
cells/milliliter and 5000 cells were plated per well in a 96 well
plate. IL-29-CEE (IL-29 conjugated with glu tag), IFN-.gamma. or
IFN-.alpha.2a was added in 2-fold serial dilutions to each well.
IL-29-CEE was used at a concentration range of from 1000 ng/ml to
0.5 ng/ml. IFN-.gamma. was used at a concentration range from 125
ng/ml to 0.06 ng/ml. IFN-.alpha.2a was used at a concentration
range of from 62 ng/ml to 0.03 ng/ml. Cells were incubated for 72 h
at 37.degree. C. After 72 h. Alamar Blue (Accumed, Chicago, Ill.)
was added at 20 microliters/well. Plates were further incubated at
37.degree. C., 5% CO, for 24 hours. Plates were read on the
Fmax.TM. plate reader (Molecular Devices, Sunnyvale, Calif.) using
the SoftMax.TM. Pro program, at wavelengths 544 (Excitation) and
590 (Emission). Alamar Blue gives a fluourometric readout based on
the metabolic activity of cells, and is thus a direct measurement
of cell proliferation in comparison to a negative control. The
results indicate that IL-29-CEE, in contrast to IFN-.alpha.2a, has
no significant effect on proliferation of Daudi cells.
Example 6
Mouse IL-28 does not have Antiproliferative Effect on Mouse B
Cells
[0091] Mouse B cells were isolated from 2 Balb/C spleens (7 months
old) by depleting CD43+ cells using MACS magnetic beads. Purified B
cells were cultured in vitro with LPS, anti-IgM or anti-CD40
monoclonal antibodies. Mouse IL-28 or mouse IFN.alpha. was added to
the cultures and .sup.3H-thymidine was added at 48 hrs. and
.sup.3H-thymidine incorporation was measured after 72 hrs.
culture.
[0092] IFN.alpha. at 10 ng/ml inhibited .sup.3H-thymidine
incorporation by mouse B cells stimulated with either LPS or
anti-IgM. However mouse IL-28 did not inhibit .sup.3H-thymidine
incorporation at any concentration tested including 1000 ng/ml. In
contrast, both mIFN.alpha. and mouse IL-28 increased .sup.3H
thymidine incorporation by mouse B cells stimulated with anti-CD40
MAb.
[0093] These data demonstrate that mouse IL-28 unlike IFNa displays
no antiproliferative activity even at high concentrations. In
addition, zcyto24 enhances proliferation in the presence of
anti-CD40 MAbs. The results illustrate that mouse IL-28 differs
from IFN.alpha. in that mouse IL-28 does not display
antiproliferative activity on mouse B cells, even at high
concentrations. In addition, mouse IL-28 enhances proliferation in
the presence of anti-CD40 monoclonal antibodies.
Example 7
Bone Marrow Expansion Assay
[0094] Fresh human marrow mononuclear cells (Poietic Technologies,
Gaithersburg, Md.) were adhered to plastic for 2 hrs in .alpha.MEM,
10% FBS, 50 micromolar .beta.-mercaptoethanol, 2 ng/ml FLT3L at
37.degree. C. Non adherent cells were then plated at 25,000 to
45,000 cells/well (96 well tissue culture plates) in .alpha.MEM,
10% FBS, 50 micromolar .beta.-mercaptoethanol, 2 ng/ml FLT3L in the
presence or absence of 1000 ng/ml IL-29-CEE, 100 ng/ml IL-29-CEE,
10 ng/ml IL-29-CEE, 100 ng/ml IFN-.alpha.2a, 10 ng/ml IFN-.alpha.2a
or 1 ng/ml IFN-.alpha.2a. These cells were incubated with a variety
of cytokines to test for expansion or differentiation of
hematopoietic cells from the marrow (20 ng/ml IL-2, 2 ng/ml IL-3,
20 ng/ml IL-4, 20 ng/ml IL-5, 20 ng/ml IL-7, 20 ng/ml IL-10, 20
ng/ml IL-12, 20 ng/ml IL-15, 10 ng/ml IL-21 or no added cytokine).
After 8 to 12 days Alamar Blue (Accumed, Chicago, Ill.) was added
at 20 microliters/well. Plates were further incubated at 37.degree.
C., 5% CO, for 24 hours. Plates were read on the Fmax.TM. plate
reader (Molecular Devices Sunnyvale, Calif.) using the SoftMax.TM.
Pro program, at wavelengths 544 (Excitation) and 590 (Emission).
Alamar Blue gives a fluourometric readout based on the metabolic
activity of cells, and is thus a direct measurement of cell
proliferation in comparison to a negative control.
[0095] IFN-.alpha.2a caused a significant inhibition of bone marrow
expansion under all conditions tested. In contrast, IL-29 had no
significant effect on expansion of bone marrow cells in the
presence of IL-3, IL-4, IL-5, IL-7, IL-10, IL-12, IL-21 or no added
cytokine. A small inhibition of bone marrow cell expansion was seen
in the presence of IL-2 or IL-15.
Example 8
Inhibition of IL-28 and IL-29 Signaling with Soluble Receptor
(zcytoR19/CRF2-4)
A. Signal Transduction Reporter Assay
[0096] A signal transduction reporter assay can be used to show the
inhibitor properties of zcytor19-Fc4 homodimeric and
zcytor19-Fc/CRF2-4-Fc heterodimeric soluble receptors on zcyto20,
zcyto21 and zcyto24 signaling. Human embryonal kidney (HEK) cells
overexpressing the zcytor19 receptor are transfected with a
reporter plasmid containing an interferon-stimulated response
element (ISRE) driving transcription of a luciferase reporter gene.
Luciferase activity following stimulation of transfected cells with
ligands (including zcyto20 (SEQ ID NO:18), zcyto21 (SEQ ID NO:20),
zcyto24 (SEQ ID NO: 8)) reflects the interaction of the ligand with
soluble receptor.
B. Cell Transfections
[0097] 293 HEK cells overexpressing zcytor19 were transfected as
follows: 700,000 293 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 293 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.
C. Signal Transduction Reporter Assays
[0098] The signal transduction reporter assays were done as
follows: Following an 18 h incubation at 37.degree. C. in DMEM+0.5%
FBS, transfected cells were stimulated with 10 ng/ml zcyto20,
zcyto21 or zcyto24 and 10 micrograms/ml of the following soluble
receptors; human zcytor19-Fc homodimer, human zcytor19-Fc/human
CRF2-4-Fc heterodimer, human CRF2-4-Fc homodimer, murine
zcytor19-Ig homodimer. Following a 4-hour incubation at 37.degree.
C., the cells were lysed, and the relative light units (RLU) were
measured on a luminometer after addition of a luciferase substrate.
The results obtained are shown as the percent inhibition of
ligand-induced signaling in the presence of soluble receptor
relative to the signaling in the presence of PBS alone. Table 11
shows that the human zcytor19-Fc/human CRF2-4 heterodimeric soluble
receptor is able to inhibit zcyto20, zcyto21 and zcyto24-induced
signaling between 16 and 45% of control. The human zcytor19-Fc
homodimeric soluble receptor is also able to inhibit
zcyto21-induced signaling by 45%. No significant effects were seen
with huCRF2-4-Fc or muzcytor19-Ig homodimeric soluble
receptors.
TABLE-US-00012 TABLE 12 Percent Inhibition of Ligand-induced
Interferon Stimulated Response Element (ISRE) Signaling by Soluble
Receptors Huzcytor19-Fc/ HuCRF2- Ligand huCRF2-4-Fc Huzcytor19-Fc
4-Fc Muzcytor19-Ig Zcyto20 16% 92% 80% 91% Zcyto21 16% 45% 79% 103%
Zcyto24 47% 90% 82% 89%
Example 9
IL-28 and IL-29 Inhibit HIV Replication in Fresh Human PBMCs
[0099] Human immunodeficiency virus (HIV) is a pathogenic
retrovirus that infects cells of the immune system. CD4 T cells and
monocytes are the primary infected cell types. To test the ability
of IL-28 and IL-29 to inhibit HIV replication in vitro, PBMCs from
normal donors were infected with the HIV virus in the presence of
IL-28, IL-29 and MetIL-29C172S-PEG.
[0100] Fresh human peripheral blood mononuclear cells (PBMCs) were
isolated from whole blood obtained from screened donors who were
seronegative for HIV and HBV. Peripheral blood cells were
pelleted/washed 2-3 times by low speed centrifugation and
resuspended in PBS to remove contaminating platelets. The washed
blood cells were diluted 1:1 with Dulbecco's phosphate buffered
saline (D-PBS) and layered over 14 mL of Lymphocyte Separation
Medium ((LSM; cellgro.TM. by Mediatech, Inc. Herndon, Va.); density
1.078+/-0.002 g/ml) in a 50 mL centrifuge tube and centrifuged for
30 minutes at 600.times.G. Banded PBMCs were gently aspirated from
the resulting interface and subsequently washed 2.times. in PBS by
low speed centrifugation. After the final wash, cells were counted
by trypan blue exclusion and resuspended at 1.times.10.sup.7
cells/mL in RPMI 1640 supplemented with 15% Fetal Bovine Serum
(FBS), 2 mM L-glutamine, 4 .mu.g/mL PHA-P. The cells were allowed
to incubate for 48-72 hours at 37.degree. C. After incubation,
PBMCs were centrifuged and resuspended in RPMI 1640 with 15% FBS, 2
mM L-glutamine, 100 U/mL penicillin, 100 .mu.g/mL streptomycin, 10
.mu.g/mL gentamycin, and 20 U/mL recombinant human IL-2. PBMCs were
maintained in the medium at a concentration of 1-2.times.10.sup.6
cells/mL with biweekly medium changes until used in the assay
protocol. Monocytes were depleted from the culture as the result of
adherence to the tissue culture flask.
[0101] For the standard PBMC assay, PHA-P stimulated cells from at
least two normal donors were pooled, diluted in fresh medium to a
final concentration of 1.times.10.sup.6 cells/mL, and plated in the
interior wells of a 96 well round bottom microplate at 50
.mu.L/well (5.times.10.sup.4 cells/well). Test dilutions were
prepared at a 2.times. concentration in microtiter tubes and 100
.mu.L of each concentration was placed in appropriate wells in a
standard format. IL-28, IL-29 and MetIL-29C172S-PEG were added at
concentrations from 0-10 .mu.g/ml, usually in 1/2 log dilutions. 50
.mu.L of a predetermined dilution of virus stock was placed in each
test well (final MOI of 0.1). Wells with only cells and virus added
were used for virus control. Separate plates were prepared
identically without virus for drug cytotoxicity studies using an
MTS assay system. The PBMC cultures were maintained for seven days
following infection, at which time cell-free supernatant samples
were collected and assayed for reverse transcriptase activity and
p24 antigen levels.
[0102] A decrease in reverse transcriptase activity or p24 antigen
levels with IL-28, IL-29 and MetIL-29C172S-PEG would be indicators
of antiviral activity. Result would demonstrate that IL-28 and
IL-29 may have therapeutic value in treating HIV and AIDS.
Example 10
IL-28 and IL-29 Inhibit GBV-B Replication in Marmoset Liver
Cells
[0103] HCV is a member of the Flaviviridae family of RNA viruses.
HCV does not replicate well in either ex-vivo or in vitro cultures
and therefore, there are no satisfactory systems to test the
anti-HCV activity of molecules in vitro. GB virus B (GBV-B) is an
attractive surrogate model for use in the development of anti-HCV
antiviral agents since it has a relatively high level of sequence
identity with HCV and is a hepatotropic virus. To date, the virus
can only be grown in the primary hepatocytes of certain non-human
primates. This is accomplished by either isolating hepatocytes in
vitro and infecting them with GBV-B, or by isolating hepatocytes
from GBV-B infected marmosets and directly using them with
antiviral compounds.
[0104] The effects of IL-28, IL-29 and MetIL-29C172S-PEG are
assayed on GBV-B extracellular RNA production by TaqMan RT-PCR and
on cytotoxicity using CellTiter96.RTM. reagent (Promega, Madison,
Wis.) at six half-log dilutions IL-28, IL-29 or MetIL-29C172S-PEG
polypeptide in triplicate. Untreated cultures serve as the cell and
virus controls. Both RIBAVIRIN.RTM. (200 .mu.g/ml at the highest
test concentration) and IFN-.alpha. (5000 IU/ml at the highest
test) are included as positive control compounds. Primary
hepatocyte cultures are isolated and plated out on collagen-coated
plates. The next day the cultures are treated with the test samples
(IL-28, IL-29, MetIL-29C172S-PEG, IFN.alpha., or RIBAVIRIN.RTM.)
for 24 hr before being exposed to GBV-B virions or treated directly
with test samples when using in vivo infected hepatocytes. Test
samples and media are added the next day, and replaced three days
later. Three to four days later (at day 6-7 post test sample
addition) the supernatant is collected and the cell numbers
quantitated with CellTiter96.RTM.. Viral RNA is extracted from the
supernatant and quantified with triplicate replicates in a
quantitative TaqMan RT-PCR assay using an in vitro transcribed RNA
containing the RT-PCR target as a standard. The average of
replicate samples is computed. Inhibition of virus production is
assessed by plotting the average RNA and cell number values of the
triplicate samples relative to the untreated virus and cell
controls. The inhibitory concentration of drug resulting in 50%
inhibition of GBV-B RNA production (IC50) and the toxic
concentration resulting in destruction of 50% of cell numbers
relative to control values (TC50) are calculated by interpolation
from graphs created with the data.
[0105] Inhibition of the GBV-B RNA production by IL-28 and 29 is an
indication of the antiviral properties of IL-28 and IL-29 on this
Hepatitis C-like virus on hepatocytes, the primary organ of
infection of Hepatitis C, and positive results suggest that IL-28
or IL-29 may be useful in treating HCV infections in humans.
Example 11
IL-28, IL-29 and MetIL-29C172S-PEG Inhibit HBV Replication in WT10
Cells
[0106] Chronic hepatitis B (HBV) is one of the most common and
severe viral infections of humans belonging to the Hepadnaviridae
family of viruses. To test the antiviral activities of IL-28 and
IL-29 against HBV, IL-28, IL-29 and MetIL-29C172S-PEG were tested
against HBV in an in vitro infection system using a variant of the
human liver line HepG2. IL-28, IL-29 and MetIL-29C172S-PEG
inhibited viral replication in this system, suggesting therapeutic
value in treating HBV in humans.
[0107] WT10 cells are a derivative of the human liver cell line
HepG2 2.2.15. WT10 cells are stably transfected with the HBV
genome, enabling stable expression of HBV transcripts in the cell
line (Fu and Cheng, Antimicrobial Agents Chemother.
44(12):3402-3407, 2000). In the WT10 assay the drug in question and
a 3TC control will be assayed at five concentrations each, diluted
in a half-log series. The endpoints are TaqMan PCR for
extracellular HBV DNA (IC50) and cell numbers using CellTiter96
reagent (TC50). The assay is similar to that described by Korba et
al. Antiviral Res. 15(3):217-228, 1991 and Korba et al., Antiviral
Res. 19(1):55-70, 1992. Briefly, WT10 cells are plated in 96-well
microtiter plates. After 16-24 hours the confluent monolayer of
HepG2-2.2.15 cells is washed and the medium is replaced with
complete medium containing varying concentrations of a test samples
in triplicate. 3TC is used as the positive control, while media
alone is added to cells as a negative control (virus control, VC).
Three days later the culture medium is replaced with fresh medium
containing the appropriately diluted test samples. Six days
following the initial addition of the test compound, the cell
culture supernatant is collected, treated with pronase and DNAse,
and used in a real-time quantitative TaqMan PCR assay. The
PCR-amplified HBV DNA is detected in real-time by monitoring
increases in fluorescence signals that result from the
exonucleolytic degradation of a quenched fluorescent probe molecule
that hybridizes to the amplified HBV DNA. For each PCR
amplification, a standard curve is simultaneously generated using
dilutions of purified HBV DNA. Antiviral activity is calculated
from the reduction in HBV DNA levels (IC.sub.50). A dye uptake
assay is then employed to measure cell viability which is used to
calculate toxicity (TC.sub.50). The therapeutic index (TI) is
calculated as TC.sub.50/IC.sub.50.
[0108] IL-28, IL-29 and MetIL-29C172S-PEG inhibited HepB viral
replication in WT10 cells with an IC50<0.032 ug/ml. This
demonstrates antiviral activity of IL-28 and IL-29 against HBV
grown in liver cell lines, providing evidence of therapeutic value
for treating HBV in human patients.
Example 12
IL-28, IL-29 and MetIL-29C172S-PEG Inhibit BVDV Replication in
Bovine Kidney Cells
[0109] HCV is a member of the Flaviviridae family of RNA viruses.
Other viruses belonging to this family are the bovine viral
diarrhea virus (BVDV) and yellow fever virus (YFV). HCV does not
replicate well in either ex vivo or in vitro cultures and therefore
there are no systems to test anti-HCV activity in vitro. The BVDV
and YFV assays are used as surrogate viruses for HCV to test the
antiviral activities against the Flavivirida family of viruses.
[0110] The antiviral effects of IL-28, IL-29 and MetIL-29C172S-PEG
were assessed in inhibition of cytopathic effect assays (CPE). The
assay measured cell death using Madin-Darby bovine kidney cells
(MDBK) after infection with cytopathic BVDV virus and the
inhibition of cell death by addition of IL-28, IL-29 and
MetIL-29C172S-PEG. The MDBK cells were propagated in Dulbecco's
modified essential medium (DMEM) containing phenol red with 10%
horse serum, 1% glutamine and 1% penicillin-streptomycin. CPE
inhibition assays were performed in DMEM without phenol red with 2%
FBS, 1% glutamine and 1% Pen-Strep. On the day preceding the
assays, cells were trypsinized (1% trypsin-EDTA), washed, counted
and plated out at 10.sup.4 cells/well in a 96-well flat-bottom
BioCoat.RTM. plates (Fisher Scientific, Pittsburgh, Pa.) in a
volume of 100 .mu.l/well. The next day, the medium was removed and
a pre-titered aliquot of virus was added to the cells. The amount
of virus was the maximum dilution that would yield complete cell
killing (>80%) at the time of maximal CPE development (day 7 for
BVDV). Cell viability was determined using a CellTiter96.RTM.
reagent (Promega) according to the manufacturer's protocol, using a
Vmax plate reader (Molecular Devices, Sunnyvale, Calif.). Test
samples were tested at six concentrations each, diluted in assay
medium in a half-log series. IFN.alpha. and RIBAVIRIN.RTM. were
used as positive controls. Test sample were added at the time of
viral infection. The average background and sample color-corrected
data for percent CPE reduction and percent cell viability at each
concentration were determined relative to controls and the
IC.sub.50 calculated relative to the TC.sub.50.
[0111] IL-28, IL-29 and MetIL-29C172S-PEG inhibited cell death
induced by BVDV in MDBK bovine kidney cells. IL-28 inhibited cell
death with an IC.sub.50 of 0.02 .mu.g/ml, IL-29 inhibited cell
death with an IC.sub.50 of 0.19 .mu.g/ml, and MetIL-29C172S-PEG
inhibited cell death with an IC.sub.50 of 0.45 .mu.g/ml. This
demonstrated that IL-28 and IL-29 have antiviral activity against
the Flavivirida family of viruses.
Example 13
Induction of Interferon Stimulated Genes by IL-28 and IL-29
A. Human Peripheral Blood Mononuclear Cells
[0112] Freshly isolated human peripheral blood mononuclear cells
were grown in the presence of IL-29 (20 ng/mL), IFN.alpha.2a (2
ng/ml) (PBL Biomedical Labs, Piscataway, N.J.), or in medium alone.
Cells were incubated for 6, 24, 48, or 72 hours, and then total RNA
was isolated and treated with RNase-free DNase. 100 ng total RNA
was used as a template for One-Step Semi-Quantitative RT-PCR.RTM.
using Taqman One-Step RT-PCR Master Mix.RTM. Reagents and gene
specific primers as suggested by the manufacturer. (Applied
Biosystems, Branchburg, N.J.) Results were normalized to HPRT and
are shown as the fold induction over the medium alone control for
each time-point. Table 13 shows that IL-29 induces Interferon
Stimulated Gene Expression in human peripheral blood mononuclear
cells at all time-points tested.
TABLE-US-00013 TABLE 13 MxA Fold Pkr Fold OAS Fold induction
Induction Induction 6 hr IL29 3.1 2.1 2.5 6 hr IFN.alpha.2a 17.2
9.6 16.2 24 hr IL29 19.2 5.0 8.8 24 hr IFN.alpha.2a 57.2 9.4 22.3
48 hr IL29 7.9 3.5 3.3 48 hr IFN.alpha.2a 18.1 5.0 17.3 72 hr IL29
9.4 3.7 9.6 72 hr IFN.alpha.2a 29.9 6.4 47.3
B. Activated Human T Cells
[0113] Human T cells were isolated by negative selection from
freshly harvested peripheral blood mononuclear cells using the Pan
T-cell Isolation.RTM. kit according to manufacturer's instructions
(Miltenyi, Auburn, Calif.). T cells were then activated and
expanded for 5 days with plate-bound anti-CD3, soluble anti-CD28
(0.5 ug/ml), (Pharmingen, San Diego, Calif.) and Interleukin 2
(IL-2; 100 U/ml) (R&D Systems, Minneapolis, Minn.), washed and
then expanded for a further 5 days with IL-2. Following activation
and expansion, cells were stimulated with IL-28A (20 ng/ml), IL-29
(20 ng/ml), or medium alone for 3, 6, or 18 hours. Total RNA was
isolated and treated with RNase-Free DNase. One-Step
Semi-Quantitative RT-PCR.RTM. was performed as described in the
example above. Results were normalized to HPRT and are shown as the
fold induction over the medium alone control for each time-point.
Table 14 shows that IL-28 and IL-29 induce Interferon Stimulated
Gene expression in activated human T cells at all time-points
tested.
TABLE-US-00014 TABLE 14 MxA Fold Pkr Fold OAS Fold Induction
Induction Induction Donor #1 3 hr IL28 5.2 2.8 4.8 Donor #1 3 hr
IL29 5.0 3.5 6.0 Donor #1 6 hr IL28 5.5 2.2 3.0 Donor #1 6 hr IL29
6.4 2.2 3.7 Donor #1 18 hr IL28 4.6 4.8 4.0 Donor #1 18 hr IL29 5.0
3.8 4.1 Donor #2 3 hr IL28 5.7 2.2 3.5 Donor #2 3 hr IL29 6.2 2.8
4.7 Donor #2 6 hr IL28 7.3 1.9 4.4 Donor #2 6 hr IL29 8.7 2.6 4.9
Donor #2 18 hr IL28 4.7 2.3 3.6 Donor #2 18 hr IL29 4.9 2.1 3.8
C. Primary Human Hepatocytes
[0114] Freshly isolated human hepatocytes from two separate donors
(Cambrex, Baltimore, Md. and CellzDirect, Tucson, Ariz.) were
stimulated with IL-28A (50 ng/ml), IL-29 (50 ng/ml), IFN.alpha.2a
(50 ng/ml), or medium alone for 24 hours. Following stimulation,
total RNA was isolated and treated with RNase-Free DNase. One-step
semi-quantitative RT-PCR was performed as described previously in
the example above. Results were normalized to HPRT and are shown as
the fold induction over the medium alone control for each
time-point. Table 15 shows that IL-28 and IL-29 induce Interferon
Stimulated Gene expression in primary human hepatocytes following
24-hour stimulation.
TABLE-US-00015 TABLE 15 MxA Fold Pkr Fold OAS Fold Induction
Induction Induction Donor #1 IL28 31.4 6.4 30.4 Donor #1 IL29 31.8
5.2 27.8 Donor #1 IFN- 63.4 8.2 66.7 .alpha.2a Donor #2 IL28 41.7
4.2 24.3 Donor #2 IL29 44.8 5.2 25.2 Donor #2 IFN- 53.2 4.8 38.3
.alpha.2a
D. HepG2 and HuH7: Human Liver Hepatoma Cell Lines
[0115] HepG2 and HuH7 cells (ATCC NOS. 8065, Manassas, Va.) were
stimulated with IL-28A (10 ng/ml), IL-29 (10 ng/ml), IFN.alpha.2a
(10 ng/ml), IFNB (1 ng/ml) (PBL Biomedical, Piscataway, N.J.), or
medium alone for 24 or 48 hours. In a separate culture, HepG2 cells
were stimulated as described above with 20 ng/ml of
MetIL-29C172S-PEG or MetIL-29-PEG. Total RNA was isolated and
treated with RNase-Free DNase. 100 ng Total RNA was used as a
template for one-step semi-quantitative RT-PCR as described
previously. Results were normalized to HPRT and are shown as the
fold induction over the medium alone control for each time-point.
Table 16 shows that IL-28 and IL-29 induce ISG expression in HepG2
and HuH7 liver hepatoma cell lines after 24 and 48 hours.
TABLE-US-00016 TABLE 16 MxA Fold Pkr Fold Induction Induction OAS
Fold Induction HepG2 24 hr IL28 12.4 0.7 3.3 HepG2 24 hr IL29 36.6
2.2 6.4 HepG2 24 hr IFN.alpha.2a 12.2 1.9 3.2 HepG2 24 hr IFN.beta.
93.6 3.9 19.0 HepG2 48 hr IL28 2.7 0.9 1.1 HepG2 48 hr IL29 27.2
2.1 5.3 HepG2 48 hr IFN.alpha.2a 2.5 0.9 1.2 HepG2 48 hr IFN.beta.
15.9 1.8 3.3 HuH7 24 hr IL28 132.5 5.4 52.6 HuH7 24 hr IL29 220.2
7.0 116.6 HuH7 24 hr IFN.alpha.2a 157.0 5.7 67.0 HuH7 24 hr
IFN.beta. 279.8 5.6 151.8 HuH7 48 hr IL28 25.6 3.4 10.3 HuH7 48 hr
IL29 143.5 7.4 60.3 HuH7 48 hr IFN.alpha.2a 91.3 5.8 32.3 HuH7 48
hr IFN.beta. 65.0 4.2 35.7
TABLE-US-00017 TABLE 17 MxA Fold Pkr Induction OAS Fold Induction
Fold Induction MetIL-29-PEG 36.7 6.9 2.2 MetIL-29C172S-PEG 46.1 8.9
2.8
[0116] Data shown is for 20 ng/ml metIL-29-PEG and
metIL-29C172S-PEG versions of IL-29 after culture for 24 hours.
[0117] Data shown is normalized to HPRT and shown as fold induction
over unstimulated cells.
Example 14
Antiviral Activity of IL-28 and IL-29 in HCV Replicon System
[0118] The ability of antiviral drugs to inhibit HCV replication
can be tested in vitro with the HCV replicon system. The replicon
system consists of the Huh7 human hepatoma cell line that has been
transfected with subgenomic RNA replicons that direct constitutive
replication of HCV genomic RNAs (Blight, K. J. et al. Science
290:1972-1974, 2000). Treatment of replicon clones with IFN.alpha.
at 10 IU/ml reduces the amount of HCV RNA by 85% compared to
untreated control cell lines. The ability of IL-28A and IL-29 to
reduce the amount of HCV RNA produced by the replicon clones in 72
hours indicates the antiviral state conferred upon Huh7 cells by
IL-28A/IL-29 treatment is effective in inhibiting HCV replicon
replication, and thereby, very likely effective in inhibiting HCV
replication.
[0119] The ability of IL-28A and IL-29 to inhibit HCV replication
as determined by Bayer Branched chain DNA kit, is be done under the
following conditions:
[0120] IL28 alone at increasing concentrations (6)* up to 1.0
.mu.g/ml
[0121] IL29 alone at increasing concentrations (6)* up to 1.0
.mu.g/ml
[0122] PEGIL29 alone at increasing concentrations (6)* up to 1.0
.mu.g/ml
[0123] IFN.alpha.2A alone at 0.3, 1.0, and 3.0 IU/ml
[0124] Ribavirin alone.
[0125] The positive control is IFN.alpha. and the negative control
is ribavirin.
[0126] The cells are stained after 72 hours with Alomar Blue to
assess viablility.
[0127] *The concentrations for conditions 1-3 are:
[0128] .mu.g/ml, 0.32 .mu.g/ml, 0.10 .mu.g/ml, 0.032 .mu.g/ml,
0.010 .mu.g/ml, 0.0032 .mu.g/ml.
[0129] The replicon clone (BB7) is treated 1.times. per day for 3
consecutive days with the doses listed above. Total HCV RNA is
measured after 72 hours.
Example 15
IL-28 and IL-29 have Antiviral Activity Against Pathogenic
Viruses
[0130] Two methods are used to assay in vitro antiviral activity of
IL-28 and IL-29 against a panel of pathogenic viruses including,
among others, adenovirus, parainfluenza virus, respiratory
syncytial virus, rhino virus, coxsackie virus, influenza virus,
vaccinia virus, west nile virus, dengue virus, Venezuelan equine
encephalitis virus, pichinde virus and polio virus. These two
methods are inhibition of virus-induced cytopathic effect (CPE)
determined by visual (microscopic) examination of the cells and
increase in neutral red (NR) dye uptake into cells. In the CPE
inhibition method, seven concentrations of test drug (log10
dilutions, such as 1000, 100, 10, 1, 0.1, 0.01, 0.001 ng/ml) are
evaluated against each virus in 96-well flat-bottomed microplates
containing host cells. The compounds are added 24 hours prior to
virus, which is used at a concentration of approximately 5 to 100
cell culture infectious doses per well, depending upon the virus,
which equates to a multiplicity of infection (MOI) of 0.01 to
0.0001 infectious particles per cell. The tests are read after
incubation at 37.degree. C. for a specified time sufficient to
allow adequate viral cytopathic effect to develop. In the NR uptake
assay, dye (0.34% concentration in medium) is added to the same set
of plates used to obtain the visual scores. After 2 h, the color
intensity of the dye absorbed by and subsequently eluted from the
cells is determined using a microplate autoreader. Antiviral
activity is expressed as the 50% effective (virus-inhibitory)
concentration (EC50) determined by plotting compound concentration
versus percent inhibition on semilogarithmic graph paper. The
EC50/IC50 data in some cases may be determined by appropriate
regression analysis software. In general, the EC50s determined by
NR assay are two- to fourfold higher than those obtained by the CPE
method.
TABLE-US-00018 TABLE 18 Visual Assay SI Visual (IC50/ Virus Cell
line Drug EC50 Visual IC50 Visual EC50) Adenovirus A549 IL-28A
>10 .mu.g/ml >10 .mu.g/ml 0 Adenovirus A549 IL-29 >10
.mu.g/ml >10 .mu.g/ml 0 Adenovirus A549 MetIL-29C172S- >10
.mu.g/ml >10 .mu.g/ml 0 PEG Parainfluenza MA-104 IL-28A >10
.mu.g/ml >10 .mu.g/ml 0 virus Parainfluenza MA-104 IL-29 >10
.mu.g/ml >10 .mu.g/ml 0 virus Parainfluenza MA-104
MetIL-29C172S- >10 .mu.g/ml >10 .mu.g/ml 0 virus PEG
Respiratory MA-104 IL-28A >10 .mu.g/ml >10 .mu.g/ml 0
syncytial virus Respiratory MA-104 IL-29 >10 .mu.g/ml >10
.mu.g/ml 0 syncytial virus Respiratory MA-104 MetIL-29C172S- >10
.mu.g/ml >10 .mu.g/ml 0 syncytial PEG virus Rhino 2 KB IL-28A
>10 .mu.g/ml >10 .mu.g/ml 0 Rhino 2 KB IL-29 >10 .mu.g/ml
>10 .mu.g/ml 0 Rhino 2 KB MetIL-29C172S- >10 .mu.g/ml >10
.mu.g/ml 0 PEG Rhino 9 HeLa IL-28A >10 .mu.g/ml >10 .mu.g/ml
0 Rhino 9 HeLa IL-29 >10 .mu.g/ml >10 .mu.g/ml 0 Rhino 9 HeLa
MetIL-29C172S- >10 .mu.g/ml >10 .mu.g/ml 0 PEG Coxsackie KB
IL-28A >10 .mu.g/ml >10 .mu.g/ml 0 B4 virus Coxsackie KB
IL-29 >10 .mu.g/ml >10 .mu.g/ml 0 B4 virus Coxsackie KB
MetIL-29C172S- >10 .mu.g/ml >10 .mu.g/ml 0 B4 virus PEG
Influenza Maden- IL-28A >10 .mu.g/ml >10 .mu.g/ml 0 (type A
Darby [H3N2]) Canine Kidney Influenza Maden- IL-29 >10 .mu.g/ml
>10 .mu.g/ml 0 (type A Darby [H3N2]) Canine Kidney Influenza
Maden- MetIL-29C172S- >10 .mu.g/ml >10 .mu.g/ml 0 (type A
Darby PEG [H3N2]) Canine Kidney Influenza Vero IL-28A 0.1 .mu.g/ml
>10 .mu.g/ml >100 (type A [H3N2]) Influenza Vero IL-29 >10
.mu.g/ml >10 .mu.g/ml 0 (type A [H3N2]) Influenza Vero
MetIL-29C172S- 0.045 .mu.g/ml >10 .mu.g/ml >222 (type A PEG
[H3N2]) Vaccinia Vero IL-28A >10 .mu.g/ml >10 .mu.g/ml 0
virus Vaccinia Vero IL-29 >10 .mu.g/ml >10 .mu.g/ml 0 virus
Vaccinia Vero MetIL-29C172S- >10 .mu.g/ml >10 .mu.g/ml 0
virus PEG West Nile Vero IL-28A 0.00001 .mu.g/ml >10 .mu.g/ml
>1,000,000 virus West Nile Vero IL-29 0.000032 .mu.g/ml >10
.mu.g/ml >300,000 Virus West Nile Vero MetIL-29C172S- 0.001
.mu.g/ml >10 .mu.g/ml >10,000 virus PEG Dengue virus Vero
IL-28A 0.01 .mu.g/ml >10 .mu.g/ml >1000 Dengue virus Vero
IL-29 0.032 .mu.g/ml >10 .mu.g/ml >312 Dengue virus Vero
MetIL-29C172S- 0.0075 .mu.g/ml >10 .mu.g/ml >1330 PEG
Venezuelan Vero IL-28A 0.01 .mu.g/ml >10 .mu.g/ml >1000
equine encephalitis virus Venezuelan Vero IL-29 0.012 .mu.g/ml
>10 .mu.g/ml >833 equine encephalitis virus Venezuelan Vero
MetIL-29C172S- 0.0065 .mu.g/ml >10 .mu.g/ml >1538 equine PEG
encephalitis virus Pichinde BSC-1 IL-28A >10 .mu.g/ml >10
.mu.g/ml 0 virus Pichinde BSC-1 IL-29 >10 .mu.g/ml >10
.mu.g/ml 0 virus Pichinde BSC-1 MetIL-29C172S- >10 .mu.g/ml
>10 .mu.g/ml 0 virus PEG Polio virus Vero IL-28A >10 .mu.g/ml
>10 .mu.g/ml 0 Polio virus Vero IL-29 >10 .mu.g/ml >10
.mu.g/ml 0 Polio virus Vero MetIL-29C172S- >10 .mu.g/ml >10
.mu.g/ml 0 PEG
TABLE-US-00019 TABLE 19 Neutral Red Assay SI NR (IC50/ Virus Cell
line Drug EC50 NR IC50 NR EC50) Adenovirus A549 IL-28A >10
.mu.g/ml >10 .mu.g/ml 0 Adenovirus A549 IL-29 >10 .mu.g/ml
>10 .mu.g/ml 0 Adenovirus A549 MetIL-29C172S- >10 .mu.g/ml
>10 .mu.g/ml 0 PEG Parainfluenza MA-104 IL-28A >10 .mu.g/ml
>10 .mu.g/ml 0 virus Parainfluenza MA-104 IL-29 >10 .mu.g/ml
>10 .mu.g/ml 0 virus Parainfluenza MA-104 MetIL-29C172S- >10
.mu.g/ml >10 .mu.g/ml 0 virus PEG Respiratory MA-104 IL-28A
>10 .mu.g/ml >10 .mu.g/ml 0 syncytial virus Respiratory
MA-104 IL-29 >10 .mu.g/ml >10 .mu.g/ml 0 syncytial virus
Respiratory MA-104 MetIL-29C172S- 5.47 .mu.g/ml >10 .mu.g/ml
>2 syncytial virus PEG Rhino 2 KB IL-28A >10 .mu.g/ml >10
.mu.g/ml 0 Rhino 2 KB IL-29 >10 .mu.g/ml >10 .mu.g/ml 0 Rhino
2 KB MetIL-29C172S- >10 .mu.g/ml >10 .mu.g/ml 0 PEG Rhino 9
HeLa IL-28A 1.726 .mu.g/ml >10 .mu.g/ml >6 Rhino 9 HeLa IL-29
0.982 .mu.g/ml >10 .mu.g/ml >10 Rhino 9 HeLa MetIL-29C172S-
2.051 .mu.g/ml >10 .mu.g/ml >5 PEG Coxsackie B4 KB IL-28A
>10 .mu.g/ml >10 .mu.g/ml 0 virus Coxsackie B4 KB IL-29
>10 .mu.g/ml >10 .mu.g/ml 0 virus Coxsackie B4 KB
MetIL-29C172S- >10 .mu.g/ml >10 .mu.g/ml 0 virus PEG
Influenza (type Maden- IL-28A >10 .mu.g/ml >10 .mu.g/ml 0 A
[H3N2]) Darby Canine Kidney Influenza (type Maden- IL-29 >10
.mu.g/ml >10 .mu.g/ml 0 A [H3N2]) Darby Canine Kidney Influenza
(type Maden- MetIL-29C172S- >10 .mu.g/ml >10 .mu.g/ml 0 A
[H3N2]) Darby PEG Canine Kidney Influenza (type Vero IL-28A 0.25
.mu.g/ml >10 .mu.g/ml >40 A [H3N2]) Influenza (type Vero
IL-29 2 .mu.g/ml >10 .mu.g/ml >5 A [H3N2]) Influenza (type
Vero MetIL-29C172S- 1.4 .mu.g/ml >10 .mu.g/ml >7 A [H3N2])
PEG Vaccinia virus Vero IL-28A >10 .mu.g/ml >10 .mu.g/ml 0
Vaccinia virus Vero IL-29 >10 .mu.g/ml >10 .mu.g/ml 0
Vaccinia virus Vero MetIL-29C172S- >10 .mu.g/ml >10 .mu.g/ml
0 PEG West Nile virus Vero IL-28A 0.0001 .mu.g/ml >10 .mu.g/ml
>100,000 West Nile virus Vero IL-29 0.00025 .mu.g/ml >10
.mu.g/ml >40,000 West Nile virus Vero MetIL-29C172S- 0.00037
.mu.g/ml >10 .mu.g/ml >27,000 PEG Dengue virus Vero IL-28A
0.1 .mu.g/ml >10 .mu.g/ml >100 Dengue virus Vero IL-29 0.05
.mu.g/ml >10 .mu.g/ml >200 Dengue virus Vero MetIL-29C172S-
0.06 .mu.g/ml >10 .mu.g/ml >166 PEG Venezuelan Vero IL-28A
0.035 .mu.g/ml >10 .mu.g/ml >286 equine encephalitis virus
Venezuelan Vero IL-29 0.05 .mu.g/ml >10 .mu.g/ml >200 equine
encephalitis virus Venezuelan Vero MetIL-29C172S- 0.02 .mu.g/ml
>10 .mu.g/ml >500 equine PEG encephalitis virus Pichinde
virus BSC-1 IL-28A >10 .mu.g/ml >10 .mu.g/ml 0 Pichinde virus
BSC-1 IL-29 >10 .mu.g/ml >10 .mu.g/ml 0 Pichinde virus BSC-1
MetIL-29C172S- >10 .mu.g/ml >10 .mu.g/ml 0 PEG Polio virus
Vero IL-28A >1.672 .mu.g/ml >10 .mu.g/ml >6 Polio virus
Vero IL-29 >10 .mu.g/ml >10 .mu.g/ml 0 Polio virus Vero
MetIL-29C172S- >10 .mu.g/ml >10 .mu.g/ml 0 PEG
Example 16
IL-28, IL-29, metIL-29-PEG and metIL-29C172S-PEG Stimulate ISG
Induction in the Mouse Liver Cell Line AML-12
[0131] Interferon stimulated genes (ISGs) are genes that are
induced by type I interferons (IFNs) and also by the IL-28 and
IL-29 family molecules, suggesting that IFN and IL-28 and IL-29
induce similar pathways leading to antiviral activity. Human type I
IFNs (IFN.alpha.1-4 and IFN.beta.) have little or no activity on
mouse cells, which is thought to be caused by lack of species
cross-reactivity. To test if human IL-28 and IL-29 have effects on
mouse cells, ISG induction by human IL-28 and IL-29 was evaluated
by real-time PCR on the mouse liver derived cell line AML-12.
[0132] AML-12 cells were plated in 6-well plates in complete DMEM
media at a concentration of 2.times.10.sup.6 cells/well.
Twenty-four hours after plating cells, human IL-28 and IL-29 were
added to the culture at a concentration of 20 ng/ml. As a control,
cells were either stimulated with mouse IFN.alpha. (positive
control) or unstimulated (negative). Cells were harvested at 8, 24,
48 and 72 hours after addition of CHO-derived human IL-28A (SEQ ID
NO:18) or IL-29 (SEQ ID NO:20). RNA was isolated from cell pellets
using RNAEasy-Kit.RTM. (Qiagen, Valencia, Calif.). RNA was treated
with DNase (Millipore, Billerica, Mass.) to clean RNA of any
contaminating DNA. cDNA was generated using Perkin-Elmer RT mix.
ISG gene induction was evaluated by real-time PCR using primers and
probes specific for mouse OAS, Pkr and Mx1. To obtain quantitative
data, HPRT real-time PCR was duplexed with ISG PCR. A standard
curve was obtained using known amounts of RNA from IFN-stimulated
mouse PBLs. All data are shown as expression relative to internal
HPRT expression.
[0133] Human IL-28A and IL-29 stimulated ISG induction in the mouse
hepatocyte cell line AML-12 and demonstrated that unlike type I
IFNs, the IL-28/29 family proteins showed cross-species
reactivity.
TABLE-US-00020 TABLE 20 Stimulation OAS PkR Mx1 None 0.001 0.001
0.001 Human IL-28 0.04 0.02 0.06 Human IL-29 0.04 0.02 0.07 Mouse
IL-28 0.04 0.02 0.08 Mouse IFN.alpha. 0.02 0.02 0.01
[0134] All data shown were expressed as fold relative to HPRT gene
expression ng of OAS mRNA=normalized value of OAS mRNA amount
relative to internal
[0135] ng of HPRT mRNA housekeeping gene, HPRT
[0136] As an example, the data for the 48 hour time point is
shown.
TABLE-US-00021 TABLE 21 AML12's Mx1 Fold Pkr Induction OAS Fold
Induction Fold Induction MetIL-29-PEG 728 614 8 MetIL-29C172S-PEG
761 657 8
[0137] Cells were stimulated with 20 ng/ml metIL-29-PEG or
metIL-29C172S-PEG for 24 hours.
[0138] Data shown is normalized to HPRT and shown as fold induction
over unstimulated cells.
Example 17
ISGs are Efficiently Induced in Spleens of Transgenic Mice
Expressing Human IL-29
[0139] Transgenic (Tg) mice were generated expressing human IL-29
under the control of the Eu-lck promoter. To study if human IL-29
has biological activity in vivo in mice, expression of ISGs was
analyzed by real-time PCR in the spleens of Eu-lck IL-29 transgenic
mice.
[0140] Transgenic mice (C3H/C57BL/6) were generated using a
construct that expressed the human IL-29 gene under the control of
the Eu-lck promoter. This promoter is active in T cells and B
cells. Transgenic mice and their non-transgenic littermates
(n=2/gp) were sacrificed at about 10 weeks of age. Spleens of mice
were isolated. RNA was isolated from cell pellets using
RNAEasy-Kit.RTM. (Qiagen). RNA was treated with DNase to clean RNA
of any contaminating DNA. cDNA was generated using Perkin-Elmer
RT.RTM. mix. ISG gene induction was evaluated by real-time PCR
using primers and probes (5' FAM, 3' NFQ) specific for mouse OAS,
Pkr and Mx1. To obtain quantitative data, HPRT real-time PCR was
duplexed with ISG PCR. Furthermore, a standard curve was obtained
using known amounts of IFN stimulated mouse PBLs. All data are
shown as expression relative to internal HPRT expression.
[0141] Spleens isolated from IL-29 Tg mice showed high induction of
ISGs OAS, Pkr and Mx1 compared to their non-Tg littermate controls
suggesting that human IL-29 is biologically active in vivo in
mice.
TABLE-US-00022 TABLE 22 Mice OAS PkR Mx1 Non-Tg 4.5 4.5 3.5 IL-29
Tg 12 8 21
[0142] All data shown are fold expression relative to HPRT gene
expression. The average expression in two mice is shown
Example 18
Human IL-28 and IL-29 Protein Induce ISG Gene Expression in Liver,
Spleen and Blood of Mice
[0143] To determine whether human IL-28 and IL-29 induce interferon
stimulated genes in vivo, CHO-derived human IL-28A and IL-29
protein were injected into mice. In addition, E. coli derived IL-29
was also tested in in vivo assays as described above using
MetIL-29C172S-PEG and MetIL-29-PEG. At various time points and at
different doses, ISG gene induction was measured in the blood,
spleen and livers of the mice.
[0144] C57BL/6 mice were injected i.p or i.v with a range of doses
(10 .mu.g-250 .mu.g) of CHO-derived human IL-28A and IL-29 or
MetIL-29C172S-PEG and MetIL-29C16-C113-PEG. Mice were sacrificed at
various time points (1 hr-48 hr). Spleens and livers were isolated
from mice, and RNA was isolated. RNA was also isolated from the
blood cells. The cells were pelleted and RNA isolated from pellets
using RNAEasy.RTM.-kit (Qiagen). RNA was treated with DNase
(Amicon) to rid RNA of any contaminating DNA. cDNA was generated
using Perkin-Elmer RT mix (Perkin-Elmer). ISG gene induction was
measured by real-time PCR using primers and probes specific for
mouse OAS, Pkr and Mx1. To obtain quantitative data, HPRT real-time
PCR was duplexed with ISG PCR. A standard curve was calculated
using known amounts of IFN-stimulated mouse PBLs. All data are
shown as expression relative to internal HPRT expression.
[0145] Human IL-29 induced ISG gene expression (OAS, Pkr, Mx1) in
the livers, spleen and blood of mice in a dose dependent manner.
Expression of ISGs peaked between 1-6 hours after injection and
showed sustained expression above control mice upto 48 hours. In
this experiment, human IL-28A did not induce ISG gene
expression.
TABLE-US-00023 TABLE 23 Injection OAS-1 hr OAS-6 hr OAS-24 hr
OAS-48 hr None - liver 1.6 1.6 1.6 1.6 IL-29 liver 2.5 4 2.5 2.8
None - spleen 1.8 1.8 1.8 1.8 IL-29 - spleen 4 6 3.2 3.2 None -
blood 5 5 5 5 IL-29 blood 12 18 11 10
[0146] Results shown are fold expression relative to HPRT gene
expression. A sample data set for IL-29 induced OAS in liver at a
single injection of 250 .mu.g i.v. is shown. The data shown is the
average expression from 5 different animals/group.
TABLE-US-00024 TABLE 24 Injection OAS (24 hr) None 1.8 IL-29 10
.mu.g 3.7 IL-29 50 .mu.g 4.2 IL-29 250 .mu.g 6
TABLE-US-00025 TABLE 25 MetIL-29-PEG MetIL-29C172S-PEG 3 hr 6 hr 12
hr 24 hr 3 hr 6 hr 12 hr 24 hr Naive 24 hr PKR 18.24 13.93 4.99
3.77 5.29 5.65 3.79 3.55 3.70 OAS 91.29 65.93 54.04 20.81 13.42
13.02 10.54 8.72 6.60 Mx1 537.51 124.99 33.58 35.82 27.89 29.34
16.61 0.00 10.98
[0147] Mice were injected with 100 .mu.g of proteins i.v. Data
shown is told expression over HPRT expression from livers of mice.
Similar data was obtained from blood and spleens of mice.
Example 19
IL-28 and IL-29 Induce ISG Protein in Mice
[0148] To analyze of the effect of human IL-28 and IL-29 on
induction of ISG protein (OAS), serum and plasma from IL-28 and
IL-29 treated mice were tested for OAS activity.
[0149] C57BL/6 mice were injected i.v with PBS or a range of
concentrations (10 .mu.g-250 .mu.g) of human IL-28 or IL-29. Serum
and plasma were isolated from mice at varying time points, and OAS
activity was measured using the OAS radioimmunoassay (RIA) kit from
Eiken Chemicals (Tokyo, Japan).
[0150] IL-28 and IL-29 induced OAS activity in the serum and plasma
of mice showing that these proteins are biologically active in
vivo.
TABLE-US-00026 TABLE 26 Injection OAS-1 hr OAS-6 hr OAS-24 hr
OAS-48 hr None 80 80 80 80 IL-29 80 80 180 200
[0151] OAS activity is shown at pmol/dL of plasma for a single
concentration (250 .mu.g) of human IL-29.
Example 20
IL-28 and IL-29 Inhibit Adenoviral Pathology in Mice
[0152] To test the antiviral activities of IL-28 and IL-29 against
viruses that infect the liver, the test samples were tested in mice
against infectious adenoviral vectors expressing an internal green
fluorescent protein (GFP) gene. When injected intravenously, these
viruses primarily target the liver for gene expression. The
adenoviruses are replication deficient, but cause liver damage due
to inflammatory cell infiltrate that can be monitored by
measurement of serum levels of liver enzymes like AST and ALT, or
by direct examination of liver pathology.
[0153] C57B1/6 mice were given once daily intraperitoneal
injections of 50 .mu.g mouse IL-28 (zcyto24 as shown in SEQ ID
NO:9) or metIL-29C172S-PEG for 3 days. Control animals were
injected with PBS. One hour following the 3.sup.rd dose, mice were
given a single bolus intravenous tail vein injection of the
adenoviral vector, AdGFP (1.times.10.sup.9 plaque-forming units
(pfu)). Following this, every other day mice were given an
additional dose of PBS, mouse IL-28 or metIL-29C172S-PEG for 4 more
doses (total of 7 doses). One hour following the final dose of PBS,
mouse IL-28 or metIL-29C172S-PEG mice were terminally bleed and
sacrificed. The serum and liver tissue were analyzed. Serum was
analyzed for AST and ALT liver enzymes. Liver was isolated and
analyzed for GFP expression and histology. For histology, liver
specimens were fixed in formalin and then embedded in paraffin
followed by H&E staining. Sections of liver that had been
blinded to treat were examined with a light microscope. Changes
were noted and scored on a scale designed to measure liver
pathology and inflammation.
[0154] Mouse IL-28 and IL-29 inhibited adenoviral infection and
gene expression as measured by liver fluorescence. PBS-treated mice
(n=8) had an average relative liver fluorescence of 52.4 (arbitrary
units). In contrast, IL-28-treated mice (n=8) had a relative liver
fluorescence of 34.5, and IL-29-treated mice (n=8) had a relative
liver fluorescence of 38.9. A reduction in adenoviral infection and
gene expression led to a reduced liver pathology as measured by
serum ALT and AST levels and histology. PBS-treated mice (n=8) had
an average serum AST of 234 U/L (units/liter) and serum ALT of 250
U/L. In contrast, IL-28-treated mice (n=8) had an average serum AST
of 193 U/L and serum ALT of 216 U/L, and IL-29-treated mice (n=8)
had an average serum AST of 162 U/L and serum ALT of 184 U/L. In
addition, the liver histology indicated that mice given either
mouse IL-28 or IL-29 had lower liver and inflammation scores than
the PBS-treated group. The livers from the IL-29 group also had
less proliferation of sinusoidal cells, fewer mitotic figures and
fewer changes in the hepatocytes (e.g. vacuolation, presence of
multiple nuclei, hepatocyte enlargement) than in the PBS treatment
group. These data demonstrate that mouse IL-28 and IL-29 have
antiviral properties against a liver-trophic virus.
Example 21
LCMV Models
[0155] Lymphocytic choriomeningitis virus (LCMV) infections in mice
are an excellent model of acture and chronic infection. These
models are used to evaluate the effect of cytokines on the
antiviral immune response and the effects IL-28 and IL-29 have
viral load and the antiviral immune response. The two models used
are: LCMV Armstrong (acute) infection and LCMV Clone 13 (chronic)
infection. (See, e.g., Wherry et al., J. Virol. 77:4911-4927, 2003;
Blattman et al., Nature Med. 9(5):540-547, 2003; Hoffman et al., J.
Immunol. 170:1339-1353, 2003.) There are three stages of CD8 T cell
development in response to virus: 1) expansion, 2) contraction, and
3) memory (acute model). IL-28 or IL-29 is injected during each
stage for both acute and chronic models. In the chronic model,
IL-28 or IL-29 is injected 60 days after infection to assess the
effect of IL-28 or IL-29 on persistent viral load. For both acute
and chronic models, IL-28 or IL-29 is injected, and the viral load
in blood, spleen and liver is examined. Other paramenter that can
be examined include: tetramer staining by flow to count the number
of LCMV-specific CD8+ T cells; the ability of tetramer+ cells to
produce cytokines when stimulated with their cognate LCMV antigen;
and the ability of LCMV-specific CD8+ T cells to proliferate in
response to their cognate LCMV antigen. LCMV-specific T cells are
phenotyped by flow cytometry to assess the cells activation and
differentiation state. Also, the ability of LCMV-specific CTL to
lyse target cells bearing their cognate LCMV antigen is examined.
The number and function of LCMV-specific CD4+ T cells is also
assessed.
[0156] A reduction in viral load after treatment with IL-28 or
IL-29 is determined. A 50% reduction in viral load in any organ,
especially liver, would be significant. For IL-28 or IL-29 treated
mice, a 20% increase in the percentage of tetramer positive T cells
that proliferate, make cytokine, or display a mature phenotype
relative to untreated mice would also be considered
significant.
[0157] IL-28 or IL-29 injection leading to a reduction in viral
load is due to more effective control of viral infection especially
in the chronic model where untreated the viral titers remain
elevated for an extended period of time. A two fold reduction in
viral titer relative to untreated mice is considered
significant.
Example 22
Influenza Model of Acute Viral Infection
A. Preliminary Experiment to test antiviral activity.
[0158] To determine the antiviral activity of IL-28 or IL-29 on
acute infection by Influenza virus, an in vivo study using
influenza infected c57B1/6 mice is performed using the following
protocol:
[0159] Animals: 6 weeks-old female BALB/c mice (Charles River) with
148 mice, 30 per group.
[0160] Groups:
[0161] Absolute control (not infected) to run in parallel for
antibody titre and histopathology (2 animals per group)
[0162] Vehicle (i.p.) saline
[0163] Amantadine (positive control) 10 mg/day during 5 days (per
os) starting 2 hours before infection
[0164] IL-28 or IL-29 treated (5 .mu.g, i.p. starting 2 hours after
infection)
[0165] IL-28 or IL-29 (25 .mu.g, i.p. starting 2 hours after
infection)
[0166] IL-28 or IL-29 (125 .mu.g, i.p. starting 2 hours after
infection)
[0167] Day 0--Except for the absolute controls, all animals
infected with Influenza virus
[0168] For viral load (10 at LD50)
[0169] For immunology workout (LD30)
[0170] Day 0-9--daily injections of IL-28 or IL-29 (i.p.)
[0171] Body weight and general appearance recorded (3
times/week)
[0172] Day 3--sacrifice of 8 animals per group
[0173] Viral load in right lung (TCID50)
[0174] Histopathology in left lung
[0175] Blood sample for antibody titration
[0176] Day 10--sacrifice of all surviving animals collecting blood
samples for antibody titration, isolating lung lymphocytes (4 pools
of 3) for direct CTL assay (in all 5 groups), and quantitative
immunophenotyping for the following markers: CD3/CD4, CD3/CD8,
CD3/CD8/CD11b, CD8/CD44/CD62L, CD3/DX5, GR-1/F480, and CD19.
[0177] Study No. 2
[0178] Efficacy study of IL-28 or IL-29 in C57B1/6 mice infected
with mouse-adapted virus is done using 8 weeks-old female C57B1/6
mice (Charles River).
[0179] Group 1: Vehicle (i.p.)
[0180] Group 2: Positive control: Anti-influenza neutralizing
antibody (goat anti-influenza A/USSR (H1N1) (Chemicon
International, Temecula, Calif.); 40 .mu.g/mouse at 2 h and 4 h
post infection (10 .mu.l intranasal)
[0181] Group 3: IL-28 or IL-29 (5 .mu.g, i.p.)
[0182] Group 4: IL-28 or IL-29 (25 .mu.g, i.p.)
[0183] Group 5: IL-28 or IL-29 (125 .mu.g, i.p.)
[0184] Following-life observations and immunological workouts are
prepared:
[0185] Day 0--all animals infected with Influenza virus (dose
determined in experiment 2)
[0186] Day 0-9--daily injections of IL-28 or IL-29 (i.p.)
[0187] Body weight and general appearance recorded every other
day
[0188] Day 10--sacrifice of surviving animals and perform viral
assay to determine viral load in lung.
[0189] Isolation of lung lymphocytes (for direct CTL assay in the
lungs using EL-4 as targets and different E:T ratio (based on best
results from experiments 1 and 2).
[0190] Tetramer staining: The number of CD8+ T cells binding MHC
Class I tetramers containing influenza A nucleoprotein (NP) epitope
are assessed using complexes of MHC class I with viral peptides:
FLU-NP.sub.366-374/D.sup.b (ASNENME.TM.), (LMCV
peptide/D.sup.b).
[0191] Quantitative immunophenotyping of the following: CD8,
tetramer, intracellular IFN.gamma., NK1.1, CD8, tetramer, CD62L,
CD44, CD3(+ or -), NK1.1(+), intracellular IFN.gamma., CD4, CD8,
NK1.1, DX5, CD3 (+ or -), NK1.1, DX5, tetramer, Single colour
samples for cytometer adjustment.
[0192] Survival/Re-Challenge Study
[0193] Day 30: Survival study with mice are treated for 9 days with
different doses of IL-28 or IL-29 or with positive anti-influenza
antibody control. Body weight and antibody production in individual
serum samples (Total, IgG1, IgG2a, IgG2b) are measured.
[0194] Re-Challenge Study:
[0195] Day 0: Both groups will be infected with A/PR virus
(1LD30).
[0196] Group 6 will not be treated.
[0197] Group 7 will be treated for 9 days with 125 .mu.g of IL-28
or IL-29.
[0198] Day 30: Survival study
[0199] Body weight and antibody production in individual serum
samples (Total, IgG1, IgG2a, IgG2b) are measured.
[0200] Day 60: Re-Challenge Study
[0201] Survivors in each group will be divided into 2 subgroups
[0202] Group 6A and 7A will be re-challenge with A/PR virus (1
LD30)
[0203] Group 6B and 7B will be re-challenge with A/PR virus (1
LD30).
[0204] Both groups will be followed up and day of sacrifice will be
determined. Body weight and antibody production in individual serum
samples (Total, IgG1, IgG2a, IgG2b) are measured.
Example 21
IL-28 and IL-29 have Antiviral Activity Against Hepatitis B Virus
(HBV) In Vivo
[0205] A transgenic mouse model (Guidotti et al., J. Virology
69:6158-6169, 1995) supports the replication of high levels of
infectious HBV and has been used as a chemotherapeutic model for
HBV infection. Transgenic mice are treated with antiviral drugs and
the levels of HBV DNA and RNA are measured in the transgenic mouse
liver and serum following treatment. HBV protein levels can also be
measured in the transgenic mouse serum following treatment. This
model has been used to evaluate the effectiveness of lamivudine and
IFN-.alpha. in reducing HBV viral titers.
[0206] HBV TG mice (male) are given intraperitoneal injections of
2.5, 25 or 250 micrograms IL-28 or IL-29 every other day for 14
days (total of 8 doses). Mice are bled for serum collection on day
of treatment (day 0) and day 7. One hour following the final dose
of IL-29 mice undergo a terminal bleed and are sacrificed. Serum
and liver are analyzed for liver HBV DNA, liver HBV RNA, serum HBV
DNA, liver HBc, serum Hbe and serum HBs.
[0207] Reduction in liver HBV DNA, liver HBV RNA, serum HBV DNA,
liver HBc, serum Hbe or serum HBs in response to IL-28 or IL-29
reflects antiviral activity of these compounds against HBV.
Example 22
IL-28 and IL-29 Inhibit Human Herpesvirus-8 (HHV-8) Replication in
BCBL-1 Cells
[0208] The antiviral activities of IL-28 and IL-29 were tested
against HHV-8 in an in vitro infection system using a B-lymphoid
cell line, BCBL-1.
[0209] In the HHV-8 assay the test compound and a ganciclovir
control were assayed at five concentrations each, diluted in a
half-log series. The endpoints were TaqMan PCR for extracellular
HHV-8 DNA (IC50) and cell numbers using CellTiter96.RTM. reagent
(TC50; Promega, Madison, Wis.). Briefly, BCBL-1 cells were plated
in 96-well microtiter plates. After 16-24 hours the cells were
washed and the medium was replaced with complete medium containing
various concentrations of the test compound in triplicate.
Ganciclovir was the positive control, while media alone was a
negative control (virus control, VC). Three days later the culture
medium was replaced with fresh medium containing the appropriately
diluted test compound. Six days following the initial
administration of the test compound, the cell culture supernatant
was collected, treated with pronase and DNAse and then used in a
real-time quantitative TaqMan PCR assay. The PCR-amplified HHV-8
DNA was detected in real-time by monitoring increases in
fluorescence signals that result from the exonucleolytic
degradation of a quenched fluorescent probe molecule that
hybridizes to the amplified HHV-8 DNA. For each PCR amplification,
a standard curve was simultaneously generated using dilutions of
purified HHV-8 DNA. Antiviral activity was calculated from the
reduction in HHV-8 DNA levels (IC.sub.50). A novel dye uptake assay
was then employed to measure cell viability which was used to
calculate toxicity (TC.sub.50). The therapeutic index (TI) is
calculated as TC.sub.50/IC.sub.50.
[0210] IL-28 and IL-29 inhibit HHV-8 viral replication in BCBL-1
cells. IL-28A had an IC.sub.50 of 1 .mu.g/ml and a TC.sub.50 of
>10 .mu.g/ml (TI>10). IL-29 had an IC.sub.50 of 6.5 .mu.g/ml
and a TC.sub.50 of >10 .mu.g/ml (TI>1.85). MetIL-29C172S-PEG
had an IC.sub.50 of 0.14 .mu.g/ml and a TC.sub.50 of >10
.mu.g/ml (TI>100).
Example 23
IL-28 and IL-29 Antiviral Activity Against Epstein Barr Virus
(EBV)
[0211] The antiviral activities of IL-28 and IL-29 are tested
against EBV in an in vitro infection system in a B-lymphoid cell
line, P3HR-1. In the EBV assay the test compound and a control are
assayed at five concentrations each, diluted in a half-log series.
The endpoints are TaqMan PCR for extracellular EBV DNA (IC50) and
cell numbers using CellTiter96.RTM. reagent (TC50; Promega).
Briefly, P3HR-1 cells are plated in 96-well microtiter plates.
After 16-24 hours the cells are washed and the medium is replaced
with complete medium containing various concentrations of the test
compound in triplicate. In addition to a positive control, media
alone is added to cells as a negative control (virus control, VC).
Three days later the culture medium is replaced with fresh medium
containing the appropriately diluted test compound. Six days
following the initial administration of the test compound, the cell
culture supernatant is collected, treated with pronase and DNAse
and then used in a real-time quantitative TaqMan PCR assay. The
PCR-amplified EBV DNA is detected in real-time by monitoring
increases in fluorescence signals that result from the
exonucleolytic degradation of a quenched fluorescent probe molecule
that hybridizes to the amplified EBV DNA. For each PCR
amplification, a standard curve was simultaneously generated using
dilutions of purified EBV DNA. Antiviral activity is calculated
from the reduction in EBV DNA levels (IC.sub.50). A novel dye
uptake assay was then employed to measure cell viability which was
used to calculate toxicity (TC.sub.50). The therapeutic index (TI)
is calculated as TC.sub.50/IC.sub.50.
Example 24
IL-28 and IL-29 Antiviral Activity Against Herpes Simplex Virus-2
(HSV-2)
[0212] The antiviral activities of IL-28 and IL-29 were tested
against HSV-2 in an in vitro infection system in Vero cells. The
antiviral effects of IL-28 and IL-29 were assessed in inhibition of
cytopathic effect assays (CPE). The assay involves the killing of
Vero cells by the cytopathic HSV-2 virus and the inhibition of cell
killing by IL-28 and IL-29. The Vero cells are propagated in
Dulbecco's modified essential medium (DMEM) containing phenol red
with 10% horse serum, 1% glutamine and 1% penicillin-streptomycin,
while the CPE inhibition assays are performed in DMEM without
phenol red with 2% FBS, 1% glutamine and 1% Pen-Strep. On the day
preceding the assays, cells were trypsinized (1% trypsin-EDTA),
washed, counted and plated out at 10.sup.4 cells/well in a 96-well
flat-bottom BioCoat.RTM. plates (Fisher Scientific, Pittsburgh,
Pa.) in a volume of 100 .mu.l/well. The next morning, the medium
was removed and a pre-titered aliquot of virus was added to the
cells. The amount of virus used is the maximum dilution that would
yield complete cell killing (>80%) at the time of maximal CPE
development. Cell viability is determined using a CellTiter 96.RTM.
reagent (Promega) according to the manufacturer's protocol, using a
Vmax plate reader (Molecular Devices, Sunnyvale, Calif.). Compounds
are tested at six concentrations each, diluted in assay medium in a
half-log series. Acyclovir was used as a positive control.
Compounds are added at the time of viral infection. The average
background and drug color-corrected data for percent CPE reduction
and percent cell viability at each concentration are determined
relative to controls and the IC.sub.50 calculated relative to the
TC.sub.50.
[0213] IL-28A, IL-29 and MetIL-29C172S-PEG did not inhibit cell
death (IC.sub.50 of >10 ug/ml) in this assay. There was also no
antiviral activity of IFN.alpha. in the is assay.
[0214] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
401618DNAHomo sapiensCDS(1)...(618)misc_feature(0)...(0)IL-28A 1atg
act ggg gac tgc acg cca gtg ctg gtg ctg atg gcc gca gtg ctg 48Met
Thr Gly Asp Cys Thr Pro Val Leu Val Leu Met Ala Ala Val Leu 1 5 10
15acc gtg act gga gca gtt cct gtc gcc agg ctc cac ggg gct ctc ccg
96Thr Val Thr Gly Ala Val Pro Val Ala Arg Leu His Gly Ala Leu Pro
20 25 30gat gca agg ggc tgc cac ata gcc cag ttc aag tcc ctg tct cca
cag 144Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro
Gln 35 40 45gag ctg cag gcc ttt aag agg gcc aaa gat gcc tta gaa gag
tcg ctt 192Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu
Ser Leu 50 55 60ctg ctg aag gac tgc agg tgc cac tcc cgc ctc ttc ccc
agg acc tgg 240Leu Leu Lys Asp Cys Arg Cys His Ser Arg Leu Phe Pro
Arg Thr Trp 65 70 75 80gac ctg agg cag ctg cag gtg agg gag cgc ccc
atg gct ttg gag gct 288Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro
Met Ala Leu Glu Ala 85 90 95gag ctg gcc ctg acg ctg aag gtt ctg gag
gcc acc gct gac act gac 336Glu Leu Ala Leu Thr Leu Lys Val Leu Glu
Ala Thr Ala Asp Thr Asp 100 105 110cca gcc ctg gtg gac gtc ttg gac
cag ccc ctt cac acc ctg cac cat 384Pro Ala Leu Val Asp Val Leu Asp
Gln Pro Leu His Thr Leu His His 115 120 125atc ctc tcc cag ttc cgg
gcc tgt gtg agt cgt cag ggc ctg ggc acc 432Ile Leu Ser Gln Phe Arg
Ala Cys Val Ser Arg Gln Gly Leu Gly Thr 130 135 140cag atc cag cct
cag ccc acg gca ggg ccc agg acc cgg ggc cgc ctc 480Gln Ile Gln Pro
Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu145 150 155 160cac
cat tgg ctg tac cgg ctc cag gag gcc cca aaa aag gag tcc cct 528His
His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro 165 170
175ggc tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc ctc ctc acg
576Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr
180 185 190cga gac ctg aat tgt gtt gcc agt ggg gac ctg tgt gtc tga
618Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val * 195 200
2052205PRTHomo sapiens 2Met Thr Gly Asp Cys Thr Pro Val Leu Val Leu
Met Ala Ala Val Leu 1 5 10 15Thr Val Thr Gly Ala Val Pro Val Ala
Arg Leu His Gly Ala Leu Pro 20 25 30Asp Ala Arg Gly Cys His Ile Ala
Gln Phe Lys Ser Leu Ser Pro Gln 35 40 45Glu Leu Gln Ala Phe Lys Arg
Ala Lys Asp Ala Leu Glu Glu Ser Leu 50 55 60Leu Leu Lys Asp Cys Arg
Cys His Ser Arg Leu Phe Pro Arg Thr Trp65 70 75 80Asp Leu Arg Gln
Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala 85 90 95Glu Leu Ala
Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp 100 105 110Pro
Ala Leu Val Asp Val Leu Asp Gln Pro Leu His Thr Leu His His 115 120
125Ile Leu Ser Gln Phe Arg Ala Cys Val Ser Arg Gln Gly Leu Gly Thr
130 135 140Gln Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly
Arg Leu145 150 155 160His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro
Lys Lys Glu Ser Pro 165 170 175Gly Cys Leu Glu Ala Ser Val Thr Phe
Asn Leu Phe Arg Leu Leu Thr 180 185 190Arg Asp Leu Asn Cys Val Ala
Ser Gly Asp Leu Cys Val 195 200 2053603DNAHomo
sapiensCDS(1)...(603)misc_feature(0)...(0)IL-29 3atg gct gca gct
tgg acc gtg gtg ctg gtg act ttg gtg cta ggc ttg 48Met Ala Ala Ala
Trp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu 1 5 10 15gcc gtg
gca ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag 96Ala Val
Ala Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys 20 25 30ggc
tgc cac att ggc agg ttc aaa tct ctg tca cca cag gag cta gcg 144Gly
Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala 35 40
45agc ttc aag aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa
192Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys
50 55 60aac tgg agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg
agg 240Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu
Arg 65 70 75 80ctt ctc cag gtg agg gag cgc cct gtg gcc ttg gag gct
gag ctg gcc 288Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala
Glu Leu Ala 85 90 95ctg acg ctg aag gtc ctg gag gcc gct gct ggc cca
gcc ctg gag gac 336Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro
Ala Leu Glu Asp 100 105 110gtc cta gac cag ccc ctt cac acc ctg cac
cac atc ctc tcc cag ctc 384Val Leu Asp Gln Pro Leu His Thr Leu His
His Ile Leu Ser Gln Leu 115 120 125cag gcc tgt atc cag cct cag ccc
aca gca ggg ccc agg ccc cgg ggc 432Gln Ala Cys Ile Gln Pro Gln Pro
Thr Ala Gly Pro Arg Pro Arg Gly 130 135 140cgc ctc cac cac tgg ctg
cac cgg ctc cag gag gcc ccc aaa aag gag 480Arg Leu His His Trp Leu
His Arg Leu Gln Glu Ala Pro Lys Lys Glu145 150 155 160tcc gct ggc
tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc 528Ser Ala Gly
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 165 170 175ctc
acg cga gac ctc aaa tat gtg gcc gat ggg gac ctg tgt ctg aga 576Leu
Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg 180 185
190acg tca acc cac cct gag tcc acc tga 603Thr Ser Thr His Pro Glu
Ser Thr * 195 2004200PRTHomo sapiens 4Met Ala Ala Ala Trp Thr Val
Val Leu Val Thr Leu Val Leu Gly Leu 1 5 10 15Ala Val Ala Gly Pro
Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys 20 25 30Gly Cys His Ile
Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala 35 40 45Ser Phe Lys
Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys 50 55 60Asn Trp
Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg65 70 75
80Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala
85 90 95Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu
Asp 100 105 110Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu
Ser Gln Leu 115 120 125Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly
Pro Arg Pro Arg Gly 130 135 140Arg Leu His His Trp Leu His Arg Leu
Gln Glu Ala Pro Lys Lys Glu145 150 155 160Ser Ala Gly Cys Leu Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 165 170 175Leu Thr Arg Asp
Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg 180 185 190Thr Ser
Thr His Pro Glu Ser Thr 195 2005615DNAHomo
sapiensCDS(1)...(615)misc_feature(0)...(0)IL-28B 5atg acc ggg gac
tgc atg cca gtg ctg gtg ctg atg gcc gca gtg ctg 48Met Thr Gly Asp
Cys Met Pro Val Leu Val Leu Met Ala Ala Val Leu 1 5 10 15acc gtg
act gga gca gtt cct gtc gcc agg ctc cgc ggg gct ctc ccg 96Thr Val
Thr Gly Ala Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro 20 25 30gat
gca agg ggc tgc cac ata gcc cag ttc aag tcc ctg tct cca cag 144Asp
Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln 35 40
45gag ctg cag gcc ttt aag agg gcc aaa gat gcc tta gaa gag tcg ctt
192Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu
50 55 60ctg ctg aag gac tgc aag tgc cgc tcc cgc ctc ttc ccc agg acc
tgg 240Leu Leu Lys Asp Cys Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr
Trp 65 70 75 80gac ctg agg cag ctg cag gtg agg gag cgc ccc gtg gct
ttg gag gct 288Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala
Leu Glu Ala 85 90 95gag ctg gcc ctg acg ctg aag gtt ctg gag gcc acc
gct gac act gac 336Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr
Ala Asp Thr Asp 100 105 110cca gcc ctg ggg gat gtc ttg gac cag ccc
ctt cac acc ctg cac cat 384Pro Ala Leu Gly Asp Val Leu Asp Gln Pro
Leu His Thr Leu His His 115 120 125atc ctc tcc cag ctc cgg gcc tgt
gtg agt cgt cag ggc ccg ggc acc 432Ile Leu Ser Gln Leu Arg Ala Cys
Val Ser Arg Gln Gly Pro Gly Thr 130 135 140cag atc cag cct cag ccc
acg gca ggg ccc agg acc cgg ggc cgc ctc 480Gln Ile Gln Pro Gln Pro
Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu145 150 155 160cac cat tgg
ctg cac cgg ctc cag gag gcc cca aaa aag gag tcc cct 528His His Trp
Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro 165 170 175ggc
tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc ctc ctc acg 576Gly
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 180 185
190cga gac ctg aat tgt gtt gcc agc ggg gac ctg tgt gtc 615Arg Asp
Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 195 200 2056205PRTHomo
sapiens 6Met Thr Gly Asp Cys Met Pro Val Leu Val Leu Met Ala Ala
Val Leu 1 5 10 15Thr Val Thr Gly Ala Val Pro Val Ala Arg Leu Arg
Gly Ala Leu Pro 20 25 30Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys
Ser Leu Ser Pro Gln 35 40 45Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp
Ala Leu Glu Glu Ser Leu 50 55 60Leu Leu Lys Asp Cys Lys Cys Arg Ser
Arg Leu Phe Pro Arg Thr Trp65 70 75 80Asp Leu Arg Gln Leu Gln Val
Arg Glu Arg Pro Val Ala Leu Glu Ala 85 90 95Glu Leu Ala Leu Thr Leu
Lys Val Leu Glu Ala Thr Ala Asp Thr Asp 100 105 110Pro Ala Leu Gly
Asp Val Leu Asp Gln Pro Leu His Thr Leu His His 115 120 125Ile Leu
Ser Gln Leu Arg Ala Cys Val Ser Arg Gln Gly Pro Gly Thr 130 135
140Gln Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg
Leu145 150 155 160His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys
Lys Glu Ser Pro 165 170 175Gly Cys Leu Glu Ala Ser Val Thr Phe Asn
Leu Phe Arg Leu Leu Thr 180 185 190Arg Asp Leu Asn Cys Val Ala Ser
Gly Asp Leu Cys Val 195 200 2057633DNAMus musculusCDS(22)...(630)
7tcacagaccc cggagagcaa c atg aag cca gaa aca gct ggg ggc cac atg 51
Met Lys Pro Glu Thr Ala Gly Gly His Met 1 5 10ctc ctc ctg ctg ttg
cct ctg ctg ctg gcc gca gtg ctg aca aga acc 99Leu Leu Leu Leu Leu
Pro Leu Leu Leu Ala Ala Val Leu Thr Arg Thr 15 20 25caa gct gac cct
gtc ccc agg gcc acc agg ctc cca gtg gaa gca aag 147Gln Ala Asp Pro
Val Pro Arg Ala Thr Arg Leu Pro Val Glu Ala Lys 30 35 40gat tgc cac
att gct cag ttc aag tct ctg tcc cca aaa gag ctg cag 195Asp Cys His
Ile Ala Gln Phe Lys Ser Leu Ser Pro Lys Glu Leu Gln 45 50 55gcc ttc
aaa aag gcc aag gat gcc atc gag aag agg ctg ctt gag aag 243Ala Phe
Lys Lys Ala Lys Asp Ala Ile Glu Lys Arg Leu Leu Glu Lys 60 65 70gac
ctg agg tgc agt tcc cac ctc ttc ccc agg gcc tgg gac ctg aag 291Asp
Leu Arg Cys Ser Ser His Leu Phe Pro Arg Ala Trp Asp Leu Lys 75 80
85 90cag ctg cag gtc caa gag cgc ccc aag gcc ttg cag gct gag gtg
gcc 339Gln Leu Gln Val Gln Glu Arg Pro Lys Ala Leu Gln Ala Glu Val
Ala 95 100 105ctg acc ctg aag gtc tgg gag aac atg act gac tca gcc
ctg gcc acc 387Leu Thr Leu Lys Val Trp Glu Asn Met Thr Asp Ser Ala
Leu Ala Thr 110 115 120atc ctg ggc cag cct ctt cat aca ctg agc cac
att cac tcc cag ctg 435Ile Leu Gly Gln Pro Leu His Thr Leu Ser His
Ile His Ser Gln Leu 125 130 135cag acc tgt aca cag ctt cag gcc aca
gca gag ccc agg tcc ccg agc 483Gln Thr Cys Thr Gln Leu Gln Ala Thr
Ala Glu Pro Arg Ser Pro Ser 140 145 150cgc cgc ctc tcc cgc tgg ctg
cac agg ctc cag gag gcc cag agc aag 531Arg Arg Leu Ser Arg Trp Leu
His Arg Leu Gln Glu Ala Gln Ser Lys155 160 165 170gag acc cct ggc
tgc ctg gag gcc tct gtc acc tcc aac ctg ttt cgc 579Glu Thr Pro Gly
Cys Leu Glu Ala Ser Val Thr Ser Asn Leu Phe Arg 175 180 185ctg ctc
acc cgg gac ctc aag tgt gtg gcc aat gga gac cag tgt gtc 627Leu Leu
Thr Arg Asp Leu Lys Cys Val Ala Asn Gly Asp Gln Cys Val 190 195
200tga cct 633*8202PRTMus musculus 8Met Lys Pro Glu Thr Ala Gly Gly
His Met Leu Leu Leu Leu Leu Pro 1 5 10 15Leu Leu Leu Ala Ala Val
Leu Thr Arg Thr Gln Ala Asp Pro Val Pro 20 25 30Arg Ala Thr Arg Leu
Pro Val Glu Ala Lys Asp Cys His Ile Ala Gln 35 40 45Phe Lys Ser Leu
Ser Pro Lys Glu Leu Gln Ala Phe Lys Lys Ala Lys 50 55 60Asp Ala Ile
Glu Lys Arg Leu Leu Glu Lys Asp Leu Arg Cys Ser Ser65 70 75 80His
Leu Phe Pro Arg Ala Trp Asp Leu Lys Gln Leu Gln Val Gln Glu 85 90
95Arg Pro Lys Ala Leu Gln Ala Glu Val Ala Leu Thr Leu Lys Val Trp
100 105 110Glu Asn Met Thr Asp Ser Ala Leu Ala Thr Ile Leu Gly Gln
Pro Leu 115 120 125His Thr Leu Ser His Ile His Ser Gln Leu Gln Thr
Cys Thr Gln Leu 130 135 140Gln Ala Thr Ala Glu Pro Arg Ser Pro Ser
Arg Arg Leu Ser Arg Trp145 150 155 160Leu His Arg Leu Gln Glu Ala
Gln Ser Lys Glu Thr Pro Gly Cys Leu 165 170 175Glu Ala Ser Val Thr
Ser Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu 180 185 190Lys Cys Val
Ala Asn Gly Asp Gln Cys Val 195 2009632DNAMus
musculusCDS(22)...(630) 9tcacagaccc cggagagcaa c atg aag cca gaa
aca gct ggg ggc cac atg 51 Met Lys Pro Glu Thr Ala Gly Gly His Met
1 5 10ctc ctc ctg ctg ttg cct ctg ctg ctg gcc gca gtg ctg aca aga
acc 99Leu Leu Leu Leu Leu Pro Leu Leu Leu Ala Ala Val Leu Thr Arg
Thr 15 20 25caa gct gac cct gtc ccc agg gcc acc agg ctc cca gtg gaa
gca aag 147Gln Ala Asp Pro Val Pro Arg Ala Thr Arg Leu Pro Val Glu
Ala Lys 30 35 40gat tgc cac att gct cag ttc aag tct ctg tcc cca aaa
gag ctg cag 195Asp Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Lys
Glu Leu Gln 45 50 55gcc ttc aaa aag gcc aag ggt gcc atc gag aag agg
ctg ctt gag aag 243Ala Phe Lys Lys Ala Lys Gly Ala Ile Glu Lys Arg
Leu Leu Glu Lys 60 65 70gac atg agg tgc agt tcc cac ctc atc tcc agg
gcc tgg gac ctg aag 291Asp Met Arg Cys Ser Ser His Leu Ile Ser Arg
Ala Trp Asp Leu Lys 75 80 85 90cag ctg cag gtc caa gag cgc ccc aag
gcc ttg cag gct gag gtg gcc 339Gln Leu Gln Val Gln Glu Arg Pro Lys
Ala Leu Gln Ala Glu Val Ala 95 100 105ctg acc ctg aag gtc tgg gag
aac ata aat gac tca gcc ctg acc acc 387Leu Thr Leu Lys Val Trp Glu
Asn Ile Asn Asp Ser Ala Leu Thr Thr 110 115 120atc ctg ggc cag cct
ctt cat aca ctg agc cac att cac tcc cag ctg 435Ile Leu Gly Gln Pro
Leu His Thr Leu Ser His Ile His Ser Gln Leu 125 130 135cag acc tgt
aca cag ctt cag gcc aca gca gag ccc aag ccc ccg agt 483Gln Thr Cys
Thr Gln Leu Gln Ala Thr Ala Glu Pro Lys Pro Pro Ser 140 145 150cgc
cgc ctc tcc cgc tgg ctg cac agg ctc cag gag gcc cag agc aag 531Arg
Arg Leu Ser Arg Trp
Leu His Arg Leu Gln Glu Ala Gln Ser Lys155 160 165 170gag act cct
ggc tgc ctg gag gac tct gtc acc tcc aac ctg ttt caa 579Glu Thr Pro
Gly Cys Leu Glu Asp Ser Val Thr Ser Asn Leu Phe Gln 175 180 185ctg
ctc ctc cgg gac ctc aag tgt gtg gcc agt gga gac cag tgt gtc 627Leu
Leu Leu Arg Asp Leu Lys Cys Val Ala Ser Gly Asp Gln Cys Val 190 195
200tga cc 632*10202PRTMus musculus 10Met Lys Pro Glu Thr Ala Gly
Gly His Met Leu Leu Leu Leu Leu Pro 1 5 10 15Leu Leu Leu Ala Ala
Val Leu Thr Arg Thr Gln Ala Asp Pro Val Pro 20 25 30Arg Ala Thr Arg
Leu Pro Val Glu Ala Lys Asp Cys His Ile Ala Gln 35 40 45Phe Lys Ser
Leu Ser Pro Lys Glu Leu Gln Ala Phe Lys Lys Ala Lys 50 55 60Gly Ala
Ile Glu Lys Arg Leu Leu Glu Lys Asp Met Arg Cys Ser Ser65 70 75
80His Leu Ile Ser Arg Ala Trp Asp Leu Lys Gln Leu Gln Val Gln Glu
85 90 95Arg Pro Lys Ala Leu Gln Ala Glu Val Ala Leu Thr Leu Lys Val
Trp 100 105 110Glu Asn Ile Asn Asp Ser Ala Leu Thr Thr Ile Leu Gly
Gln Pro Leu 115 120 125His Thr Leu Ser His Ile His Ser Gln Leu Gln
Thr Cys Thr Gln Leu 130 135 140Gln Ala Thr Ala Glu Pro Lys Pro Pro
Ser Arg Arg Leu Ser Arg Trp145 150 155 160Leu His Arg Leu Gln Glu
Ala Gln Ser Lys Glu Thr Pro Gly Cys Leu 165 170 175Glu Asp Ser Val
Thr Ser Asn Leu Phe Gln Leu Leu Leu Arg Asp Leu 180 185 190Lys Cys
Val Ala Ser Gly Asp Gln Cys Val 195 200111563DNAHomo
sapiensCDS(1)...(1563)misc_feature(0)...(0)IL-28RA 11atg gcg ggg
ccc gag cgc tgg ggc ccc ctg ctc ctg tgc ctg ctg cag 48Met Ala Gly
Pro Glu Arg Trp Gly Pro Leu Leu Leu Cys Leu Leu Gln 1 5 10 15gcc
gct cca ggg agg ccc cgt ctg gcc cct ccc cag aat gtg acg ctg 96Ala
Ala Pro Gly Arg Pro Arg Leu Ala Pro Pro Gln Asn Val Thr Leu 20 25
30ctc tcc cag aac ttc agc gtg tac ctg aca tgg ctc cca ggg ctt ggc
144Leu Ser Gln Asn Phe Ser Val Tyr Leu Thr Trp Leu Pro Gly Leu Gly
35 40 45aac ccc cag gat gtg acc tat ttt gtg gcc tat cag agc tct ccc
acc 192Asn Pro Gln Asp Val Thr Tyr Phe Val Ala Tyr Gln Ser Ser Pro
Thr 50 55 60cgt aga cgg tgg cgc gaa gtg gaa gag tgt gcg gga acc aag
gag ctg 240Arg Arg Arg Trp Arg Glu Val Glu Glu Cys Ala Gly Thr Lys
Glu Leu 65 70 75 80cta tgt tct atg atg tgc ctg aag aaa cag gac ctg
tac aac aag ttc 288Leu Cys Ser Met Met Cys Leu Lys Lys Gln Asp Leu
Tyr Asn Lys Phe 85 90 95aag gga cgc gtg cgg acg gtt tct ccc agc tcc
aag tcc ccc tgg gtg 336Lys Gly Arg Val Arg Thr Val Ser Pro Ser Ser
Lys Ser Pro Trp Val 100 105 110gag tcc gaa tac ctg gat tac ctt ttt
gaa gtg gag ccg gcc cca cct 384Glu Ser Glu Tyr Leu Asp Tyr Leu Phe
Glu Val Glu Pro Ala Pro Pro 115 120 125gtc ctg gtg ctc acc cag acg
gag gag atc ctg agt gcc aat gcc acg 432Val Leu Val Leu Thr Gln Thr
Glu Glu Ile Leu Ser Ala Asn Ala Thr 130 135 140tac cag ctg ccc ccc
tgc atg ccc cca ctg gat ctg aag tat gag gtg 480Tyr Gln Leu Pro Pro
Cys Met Pro Pro Leu Asp Leu Lys Tyr Glu Val145 150 155 160gca ttc
tgg aag gag ggg gcc gga aac aag acc cta ttt cca gtc act 528Ala Phe
Trp Lys Glu Gly Ala Gly Asn Lys Thr Leu Phe Pro Val Thr 165 170
175ccc cat ggc cag cca gtc cag atc act ctc cag cca gct gcc agc gaa
576Pro His Gly Gln Pro Val Gln Ile Thr Leu Gln Pro Ala Ala Ser Glu
180 185 190cac cac tgc ctc agt gcc aga acc atc tac acg ttc agt gtc
ccg aaa 624His His Cys Leu Ser Ala Arg Thr Ile Tyr Thr Phe Ser Val
Pro Lys 195 200 205tac agc aag ttc tct aag ccc acc tgc ttc ttg ctg
gag gtc cca gaa 672Tyr Ser Lys Phe Ser Lys Pro Thr Cys Phe Leu Leu
Glu Val Pro Glu 210 215 220gcc aac tgg gct ttc ctg gtg ctg cca tcg
ctt ctg ata ctg ctg tta 720Ala Asn Trp Ala Phe Leu Val Leu Pro Ser
Leu Leu Ile Leu Leu Leu225 230 235 240gta att gcc gca ggg ggt gtg
atc tgg aag acc ctc atg ggg aac ccc 768Val Ile Ala Ala Gly Gly Val
Ile Trp Lys Thr Leu Met Gly Asn Pro 245 250 255tgg ttt cag cgg gca
aag atg cca cgg gcc ctg gac ttt tct gga cac 816Trp Phe Gln Arg Ala
Lys Met Pro Arg Ala Leu Asp Phe Ser Gly His 260 265 270aca cac cct
gtg gca acc ttt cag ccc agc aga cca gag tcc gtg aat 864Thr His Pro
Val Ala Thr Phe Gln Pro Ser Arg Pro Glu Ser Val Asn 275 280 285gac
ttg ttc ctc tgt ccc caa aag gaa ctg acc aga ggg gtc agg ccg 912Asp
Leu Phe Leu Cys Pro Gln Lys Glu Leu Thr Arg Gly Val Arg Pro 290 295
300acg cct cga gtc agg gcc cca gcc acc caa cag aca aga tgg aag aag
960Thr Pro Arg Val Arg Ala Pro Ala Thr Gln Gln Thr Arg Trp Lys
Lys305 310 315 320gac ctt gca gag gac gaa gag gag gag gat gag gag
gac aca gaa gat 1008Asp Leu Ala Glu Asp Glu Glu Glu Glu Asp Glu Glu
Asp Thr Glu Asp 325 330 335ggc gtc agc ttc cag ccc tac att gaa cca
cct tct ttc ctg ggg caa 1056Gly Val Ser Phe Gln Pro Tyr Ile Glu Pro
Pro Ser Phe Leu Gly Gln 340 345 350gag cac cag gct cca ggg cac tcg
gag gct ggt ggg gtg gac tca ggg 1104Glu His Gln Ala Pro Gly His Ser
Glu Ala Gly Gly Val Asp Ser Gly 355 360 365agg ccc agg gct cct ctg
gtc cca agc gaa ggc tcc tct gct tgg gat 1152Arg Pro Arg Ala Pro Leu
Val Pro Ser Glu Gly Ser Ser Ala Trp Asp 370 375 380tct tca gac aga
agc tgg gcc agc act gtg gac tcc tcc tgg gac agg 1200Ser Ser Asp Arg
Ser Trp Ala Ser Thr Val Asp Ser Ser Trp Asp Arg385 390 395 400gct
ggg tcc tct ggc tat ttg gct gag aag ggg cca ggc caa ggg ccg 1248Ala
Gly Ser Ser Gly Tyr Leu Ala Glu Lys Gly Pro Gly Gln Gly Pro 405 410
415ggt ggg gat ggg cac caa gaa tct ctc cca cca cct gaa ttc tcc aag
1296Gly Gly Asp Gly His Gln Glu Ser Leu Pro Pro Pro Glu Phe Ser Lys
420 425 430gac tcg ggt ttc ctg gaa gag ctc cca gaa gat aac ctc tcc
tcc tgg 1344Asp Ser Gly Phe Leu Glu Glu Leu Pro Glu Asp Asn Leu Ser
Ser Trp 435 440 445gcc acc tgg ggc acc tta cca ccg gag ccg aat ctg
gtc cct ggg gga 1392Ala Thr Trp Gly Thr Leu Pro Pro Glu Pro Asn Leu
Val Pro Gly Gly 450 455 460ccc cca gtt tct ctt cag aca ctg acc ttc
tgc tgg gaa agc agc cct 1440Pro Pro Val Ser Leu Gln Thr Leu Thr Phe
Cys Trp Glu Ser Ser Pro465 470 475 480gag gag gaa gag gag gcg agg
gaa tca gaa att gag gac agc gat gcg 1488Glu Glu Glu Glu Glu Ala Arg
Glu Ser Glu Ile Glu Asp Ser Asp Ala 485 490 495ggc agc tgg ggg gct
gag agc acc cag agg acc gag gac agg ggc cgg 1536Gly Ser Trp Gly Ala
Glu Ser Thr Gln Arg Thr Glu Asp Arg Gly Arg 500 505 510aca ttg ggg
cat tac atg gcc agg tga 1563Thr Leu Gly His Tyr Met Ala Arg * 515
52012520PRTHomo sapiens 12Met Ala Gly Pro Glu Arg Trp Gly Pro Leu
Leu Leu Cys Leu Leu Gln 1 5 10 15Ala Ala Pro Gly Arg Pro Arg Leu
Ala Pro Pro Gln Asn Val Thr Leu 20 25 30Leu Ser Gln Asn Phe Ser Val
Tyr Leu Thr Trp Leu Pro Gly Leu Gly 35 40 45Asn Pro Gln Asp Val Thr
Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60Arg Arg Arg Trp Arg
Glu Val Glu Glu Cys Ala Gly Thr Lys Glu Leu65 70 75 80Leu Cys Ser
Met Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 85 90 95Lys Gly
Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val 100 105
110Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro
115 120 125Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn
Ala Thr 130 135 140Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Asp Leu
Lys Tyr Glu Val145 150 155 160Ala Phe Trp Lys Glu Gly Ala Gly Asn
Lys Thr Leu Phe Pro Val Thr 165 170 175Pro His Gly Gln Pro Val Gln
Ile Thr Leu Gln Pro Ala Ala Ser Glu 180 185 190His His Cys Leu Ser
Ala Arg Thr Ile Tyr Thr Phe Ser Val Pro Lys 195 200 205Tyr Ser Lys
Phe Ser Lys Pro Thr Cys Phe Leu Leu Glu Val Pro Glu 210 215 220Ala
Asn Trp Ala Phe Leu Val Leu Pro Ser Leu Leu Ile Leu Leu Leu225 230
235 240Val Ile Ala Ala Gly Gly Val Ile Trp Lys Thr Leu Met Gly Asn
Pro 245 250 255Trp Phe Gln Arg Ala Lys Met Pro Arg Ala Leu Asp Phe
Ser Gly His 260 265 270Thr His Pro Val Ala Thr Phe Gln Pro Ser Arg
Pro Glu Ser Val Asn 275 280 285Asp Leu Phe Leu Cys Pro Gln Lys Glu
Leu Thr Arg Gly Val Arg Pro 290 295 300Thr Pro Arg Val Arg Ala Pro
Ala Thr Gln Gln Thr Arg Trp Lys Lys305 310 315 320Asp Leu Ala Glu
Asp Glu Glu Glu Glu Asp Glu Glu Asp Thr Glu Asp 325 330 335Gly Val
Ser Phe Gln Pro Tyr Ile Glu Pro Pro Ser Phe Leu Gly Gln 340 345
350Glu His Gln Ala Pro Gly His Ser Glu Ala Gly Gly Val Asp Ser Gly
355 360 365Arg Pro Arg Ala Pro Leu Val Pro Ser Glu Gly Ser Ser Ala
Trp Asp 370 375 380Ser Ser Asp Arg Ser Trp Ala Ser Thr Val Asp Ser
Ser Trp Asp Arg385 390 395 400Ala Gly Ser Ser Gly Tyr Leu Ala Glu
Lys Gly Pro Gly Gln Gly Pro 405 410 415Gly Gly Asp Gly His Gln Glu
Ser Leu Pro Pro Pro Glu Phe Ser Lys 420 425 430Asp Ser Gly Phe Leu
Glu Glu Leu Pro Glu Asp Asn Leu Ser Ser Trp 435 440 445Ala Thr Trp
Gly Thr Leu Pro Pro Glu Pro Asn Leu Val Pro Gly Gly 450 455 460Pro
Pro Val Ser Leu Gln Thr Leu Thr Phe Cys Trp Glu Ser Ser Pro465 470
475 480Glu Glu Glu Glu Glu Ala Arg Glu Ser Glu Ile Glu Asp Ser Asp
Ala 485 490 495Gly Ser Trp Gly Ala Glu Ser Thr Gln Arg Thr Glu Asp
Arg Gly Arg 500 505 510Thr Leu Gly His Tyr Met Ala Arg 515
520131476DNAHomo sapiensCDS(1)...(1476)misc_feature(0)...(0)IL-28RA
splice variant 13atg gcg ggg ccc gag cgc tgg ggc ccc ctg ctc ctg
tgc ctg ctg cag 48Met Ala Gly Pro Glu Arg Trp Gly Pro Leu Leu Leu
Cys Leu Leu Gln 1 5 10 15gcc gct cca ggg agg ccc cgt ctg gcc cct
ccc cag aat gtg acg ctg 96Ala Ala Pro Gly Arg Pro Arg Leu Ala Pro
Pro Gln Asn Val Thr Leu 20 25 30ctc tcc cag aac ttc agc gtg tac ctg
aca tgg ctc cca ggg ctt ggc 144Leu Ser Gln Asn Phe Ser Val Tyr Leu
Thr Trp Leu Pro Gly Leu Gly 35 40 45aac ccc cag gat gtg acc tat ttt
gtg gcc tat cag agc tct ccc acc 192Asn Pro Gln Asp Val Thr Tyr Phe
Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60cgt aga cgg tgg cgc gaa gtg
gaa gag tgt gcg gga acc aag gag ctg 240Arg Arg Arg Trp Arg Glu Val
Glu Glu Cys Ala Gly Thr Lys Glu Leu 65 70 75 80cta tgt tct atg atg
tgc ctg aag aaa cag gac ctg tac aac aag ttc 288Leu Cys Ser Met Met
Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 85 90 95aag gga cgc gtg
cgg acg gtt tct ccc agc tcc aag tcc ccc tgg gtg 336Lys Gly Arg Val
Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val 100 105 110gag tcc
gaa tac ctg gat tac ctt ttt gaa gtg gag ccg gcc cca cct 384Glu Ser
Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro 115 120
125gtc ctg gtg ctc acc cag acg gag gag atc ctg agt gcc aat gcc acg
432Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn Ala Thr
130 135 140tac cag ctg ccc ccc tgc atg ccc cca ctg ttt ctg aag tat
gag gtg 480Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Phe Leu Lys Tyr
Glu Val145 150 155 160gca ttt tgg ggg ggg ggg gcc gga acc aag acc
cta ttt cca gtc act 528Ala Phe Trp Gly Gly Gly Ala Gly Thr Lys Thr
Leu Phe Pro Val Thr 165 170 175ccc cat ggc cag cca gtc cag atc act
ctc cag cca gct gcc agc gaa 576Pro His Gly Gln Pro Val Gln Ile Thr
Leu Gln Pro Ala Ala Ser Glu 180 185 190cac cac tgc ctc agt gcc aga
acc atc tac acg ttc agt gtc ccg aaa 624His His Cys Leu Ser Ala Arg
Thr Ile Tyr Thr Phe Ser Val Pro Lys 195 200 205tac agc aag ttc tct
aag ccc acc tgc ttc ttg ctg gag gtc cca gaa 672Tyr Ser Lys Phe Ser
Lys Pro Thr Cys Phe Leu Leu Glu Val Pro Glu 210 215 220gcc aac tgg
gct ttc ctg gtg ctg cca tcg ctt ctg ata ctg ctg tta 720Ala Asn Trp
Ala Phe Leu Val Leu Pro Ser Leu Leu Ile Leu Leu Leu225 230 235
240gta att gcc gca ggg ggt gtg atc tgg aag acc ctc atg ggg aac ccc
768Val Ile Ala Ala Gly Gly Val Ile Trp Lys Thr Leu Met Gly Asn Pro
245 250 255tgg ttt cag cgg gca aag atg cca cgg gcc ctg gaa ctg acc
aga ggg 816Trp Phe Gln Arg Ala Lys Met Pro Arg Ala Leu Glu Leu Thr
Arg Gly 260 265 270gtc agg ccg acg cct cga gtc agg gcc cca gcc acc
caa cag aca aga 864Val Arg Pro Thr Pro Arg Val Arg Ala Pro Ala Thr
Gln Gln Thr Arg 275 280 285tgg aag aag gac ctt gca gag gac gaa gag
gag gag gat gag gag gac 912Trp Lys Lys Asp Leu Ala Glu Asp Glu Glu
Glu Glu Asp Glu Glu Asp 290 295 300aca gaa gat ggc gtc agc ttc cag
ccc tac att gaa cca cct tct ttc 960Thr Glu Asp Gly Val Ser Phe Gln
Pro Tyr Ile Glu Pro Pro Ser Phe305 310 315 320ctg ggg caa gag cac
cag gct cca ggg cac tcg gag gct ggt ggg gtg 1008Leu Gly Gln Glu His
Gln Ala Pro Gly His Ser Glu Ala Gly Gly Val 325 330 335gac tca ggg
agg ccc agg gct cct ctg gtc cca agc gaa ggc tcc tct 1056Asp Ser Gly
Arg Pro Arg Ala Pro Leu Val Pro Ser Glu Gly Ser Ser 340 345 350gct
tgg gat tct tca gac aga agc tgg gcc agc act gtg gac tcc tcc 1104Ala
Trp Asp Ser Ser Asp Arg Ser Trp Ala Ser Thr Val Asp Ser Ser 355 360
365tgg gac agg gct ggg tcc tct ggc tat ttg gct gag aag ggg cca ggc
1152Trp Asp Arg Ala Gly Ser Ser Gly Tyr Leu Ala Glu Lys Gly Pro Gly
370 375 380caa ggg ccg ggt ggg gat ggg cac caa gaa tct ctc cca cca
cct gaa 1200Gln Gly Pro Gly Gly Asp Gly His Gln Glu Ser Leu Pro Pro
Pro Glu385 390 395 400ttc tcc aag gac tcg ggt ttc ctg gaa gag ctc
cca gaa gat aac ctc 1248Phe Ser Lys Asp Ser Gly Phe Leu Glu Glu Leu
Pro Glu Asp Asn Leu 405 410 415tcc tcc tgg gcc acc tgg ggc acc tta
cca ccg gag ccg aat ctg gtc 1296Ser Ser Trp Ala Thr Trp Gly Thr Leu
Pro Pro Glu Pro Asn Leu Val 420 425 430cct ggg gga ccc cca gtt tct
ctt cag aca ctg acc ttc tgc tgg gaa 1344Pro Gly Gly Pro Pro Val Ser
Leu Gln Thr Leu Thr Phe Cys Trp Glu 435 440 445agc agc cct gag gag
gaa gag gag gcg agg gaa tca gaa att gag gac 1392Ser Ser Pro Glu Glu
Glu Glu Glu Ala Arg Glu Ser Glu Ile Glu Asp 450 455 460agc gat gcg
ggc agc tgg ggg gct gag agc acc cag agg acc gag gac 1440Ser Asp Ala
Gly Ser Trp Gly Ala Glu Ser Thr Gln Arg Thr Glu Asp465 470 475
480agg ggc cgg aca ttg ggg cat tac atg gcc agg tga 1476Arg Gly Arg
Thr Leu Gly His Tyr Met Ala Arg * 485 49014491PRTHomo sapiens 14Met
Ala Gly Pro Glu Arg Trp Gly
Pro Leu Leu Leu Cys Leu Leu Gln 1 5 10 15Ala Ala Pro Gly Arg Pro
Arg Leu Ala Pro Pro Gln Asn Val Thr Leu 20 25 30Leu Ser Gln Asn Phe
Ser Val Tyr Leu Thr Trp Leu Pro Gly Leu Gly 35 40 45Asn Pro Gln Asp
Val Thr Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60Arg Arg Arg
Trp Arg Glu Val Glu Glu Cys Ala Gly Thr Lys Glu Leu65 70 75 80Leu
Cys Ser Met Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 85 90
95Lys Gly Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val
100 105 110Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala
Pro Pro 115 120 125Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser
Ala Asn Ala Thr 130 135 140Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu
Phe Leu Lys Tyr Glu Val145 150 155 160Ala Phe Trp Gly Gly Gly Ala
Gly Thr Lys Thr Leu Phe Pro Val Thr 165 170 175Pro His Gly Gln Pro
Val Gln Ile Thr Leu Gln Pro Ala Ala Ser Glu 180 185 190His His Cys
Leu Ser Ala Arg Thr Ile Tyr Thr Phe Ser Val Pro Lys 195 200 205Tyr
Ser Lys Phe Ser Lys Pro Thr Cys Phe Leu Leu Glu Val Pro Glu 210 215
220Ala Asn Trp Ala Phe Leu Val Leu Pro Ser Leu Leu Ile Leu Leu
Leu225 230 235 240Val Ile Ala Ala Gly Gly Val Ile Trp Lys Thr Leu
Met Gly Asn Pro 245 250 255Trp Phe Gln Arg Ala Lys Met Pro Arg Ala
Leu Glu Leu Thr Arg Gly 260 265 270Val Arg Pro Thr Pro Arg Val Arg
Ala Pro Ala Thr Gln Gln Thr Arg 275 280 285Trp Lys Lys Asp Leu Ala
Glu Asp Glu Glu Glu Glu Asp Glu Glu Asp 290 295 300Thr Glu Asp Gly
Val Ser Phe Gln Pro Tyr Ile Glu Pro Pro Ser Phe305 310 315 320Leu
Gly Gln Glu His Gln Ala Pro Gly His Ser Glu Ala Gly Gly Val 325 330
335Asp Ser Gly Arg Pro Arg Ala Pro Leu Val Pro Ser Glu Gly Ser Ser
340 345 350Ala Trp Asp Ser Ser Asp Arg Ser Trp Ala Ser Thr Val Asp
Ser Ser 355 360 365Trp Asp Arg Ala Gly Ser Ser Gly Tyr Leu Ala Glu
Lys Gly Pro Gly 370 375 380Gln Gly Pro Gly Gly Asp Gly His Gln Glu
Ser Leu Pro Pro Pro Glu385 390 395 400Phe Ser Lys Asp Ser Gly Phe
Leu Glu Glu Leu Pro Glu Asp Asn Leu 405 410 415Ser Ser Trp Ala Thr
Trp Gly Thr Leu Pro Pro Glu Pro Asn Leu Val 420 425 430Pro Gly Gly
Pro Pro Val Ser Leu Gln Thr Leu Thr Phe Cys Trp Glu 435 440 445Ser
Ser Pro Glu Glu Glu Glu Glu Ala Arg Glu Ser Glu Ile Glu Asp 450 455
460Ser Asp Ala Gly Ser Trp Gly Ala Glu Ser Thr Gln Arg Thr Glu
Asp465 470 475 480Arg Gly Arg Thr Leu Gly His Tyr Met Ala Arg 485
49015674DNAHomo sapiensCDS(1)...(636)misc_feature(0)...(0)IL-28RA
soluble variant 15atg gcg ggg ccc gag cgc tgg ggc ccc ctg ctc ctg
tgc ctg ctg cag 48Met Ala Gly Pro Glu Arg Trp Gly Pro Leu Leu Leu
Cys Leu Leu Gln 1 5 10 15gcc gct cca ggg agg ccc cgt ctg gcc cct
ccc cag aat gtg acg ctg 96Ala Ala Pro Gly Arg Pro Arg Leu Ala Pro
Pro Gln Asn Val Thr Leu 20 25 30ctc tcc cag aac ttc agc gtg tac ctg
aca tgg ctc cca ggg ctt ggc 144Leu Ser Gln Asn Phe Ser Val Tyr Leu
Thr Trp Leu Pro Gly Leu Gly 35 40 45aac ccc cag gat gtg acc tat ttt
gtg gcc tat cag agc tct ccc acc 192Asn Pro Gln Asp Val Thr Tyr Phe
Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60cgt aga cgg tgg cgc gaa gtg
gaa gag tgt gcg gga acc aag gag ctg 240Arg Arg Arg Trp Arg Glu Val
Glu Glu Cys Ala Gly Thr Lys Glu Leu 65 70 75 80cta tgt tct atg atg
tgc ctg aag aaa cag gac ctg tac aac aag ttc 288Leu Cys Ser Met Met
Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 85 90 95aag gga cgc gtg
cgg acg gtt tct ccc agc tcc aag tcc ccc tgg gtg 336Lys Gly Arg Val
Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val 100 105 110gag tcc
gaa tac ctg gat tac ctt ttt gaa gtg gag ccg gcc cca cct 384Glu Ser
Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro 115 120
125gtc ctg gtg ctc acc cag acg gag gag atc ctg agt gcc aat gcc acg
432Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn Ala Thr
130 135 140tac cag ctg ccc ccc tgc atg ccc cca ctg gat ctg aag tat
gag gtg 480Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Asp Leu Lys Tyr
Glu Val145 150 155 160gca ttc tgg aag gag ggg gcc gga aac aag gtg
gga agc tcc ttt cct 528Ala Phe Trp Lys Glu Gly Ala Gly Asn Lys Val
Gly Ser Ser Phe Pro 165 170 175gcc ccc agg cta ggc ccg ctc ctc cac
ccc ttc tta ctc agg ttc ttc 576Ala Pro Arg Leu Gly Pro Leu Leu His
Pro Phe Leu Leu Arg Phe Phe 180 185 190tca ccc tcc cag cct gct cct
gca ccc ctc ctc cag gaa gtc ttc cct 624Ser Pro Ser Gln Pro Ala Pro
Ala Pro Leu Leu Gln Glu Val Phe Pro 195 200 205gta cac tcc tga
cttctggcag tcagccctaa taaaatctga tcaaagta 674Val His Ser *
21016211PRTHomo sapiens 16Met Ala Gly Pro Glu Arg Trp Gly Pro Leu
Leu Leu Cys Leu Leu Gln 1 5 10 15Ala Ala Pro Gly Arg Pro Arg Leu
Ala Pro Pro Gln Asn Val Thr Leu 20 25 30Leu Ser Gln Asn Phe Ser Val
Tyr Leu Thr Trp Leu Pro Gly Leu Gly 35 40 45Asn Pro Gln Asp Val Thr
Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60Arg Arg Arg Trp Arg
Glu Val Glu Glu Cys Ala Gly Thr Lys Glu Leu65 70 75 80Leu Cys Ser
Met Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 85 90 95Lys Gly
Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val 100 105
110Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro
115 120 125Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn
Ala Thr 130 135 140Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Asp Leu
Lys Tyr Glu Val145 150 155 160Ala Phe Trp Lys Glu Gly Ala Gly Asn
Lys Val Gly Ser Ser Phe Pro 165 170 175Ala Pro Arg Leu Gly Pro Leu
Leu His Pro Phe Leu Leu Arg Phe Phe 180 185 190Ser Pro Ser Gln Pro
Ala Pro Ala Pro Leu Leu Gln Glu Val Phe Pro 195 200 205Val His Ser
21017734DNAHomo
sapienssig_peptide(53)...(127)mat_peptide(128)...(655)CDS(53)...(655)
17tgggtgacag cctcagagtg tttcttctgc tgacaaagac cagagatcag ga atg aaa
58 Met Lys -25cta gac atg act ggg gac tgc acg cca gtg ctg gtg ctg
atg gcc gca 106Leu Asp Met Thr Gly Asp Cys Thr Pro Val Leu Val Leu
Met Ala Ala -20 -15 -10gtg ctg acc gtg act gga gca gtt cct gtc gcc
agg ctc cac ggg gct 154Val Leu Thr Val Thr Gly Ala Val Pro Val Ala
Arg Leu His Gly Ala -5 1 5ctc ccg gat gca agg ggc tgc cac ata gcc
cag ttc aag tcc ctg tct 202Leu Pro Asp Ala Arg Gly Cys His Ile Ala
Gln Phe Lys Ser Leu Ser 10 15 20 25cca cag gag ctg cag gcc ttt aag
agg gcc aaa gat gcc tta gaa gag 250Pro Gln Glu Leu Gln Ala Phe Lys
Arg Ala Lys Asp Ala Leu Glu Glu 30 35 40tcg ctt ctg ctg aag gac tgc
agg tgc cac tcc cgc ctc ttc ccc agg 298Ser Leu Leu Leu Lys Asp Cys
Arg Cys His Ser Arg Leu Phe Pro Arg 45 50 55acc tgg gac ctg agg cag
ctg cag gtg agg gag cgc ccc atg gct ttg 346Thr Trp Asp Leu Arg Gln
Leu Gln Val Arg Glu Arg Pro Met Ala Leu 60 65 70gag gct gag ctg gcc
ctg acg ctg aag gtt ctg gag gcc acc gct gac 394Glu Ala Glu Leu Ala
Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp 75 80 85act gac cca gcc
ctg gtg gac gtc ttg gac cag ccc ctt cac acc ctg 442Thr Asp Pro Ala
Leu Val Asp Val Leu Asp Gln Pro Leu His Thr Leu 90 95 100 105cac
cat atc ctc tcc cag ttc cgg gcc tgt atc cag cct cag ccc acg 490His
His Ile Leu Ser Gln Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr 110 115
120gca ggg ccc agg acc cgg ggc cgc ctc cac cat tgg ctg tac cgg ctc
538Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu
125 130 135cag gag gcc cca aaa aag gag tcc cct ggc tgc ctc gag gcc
tct gtc 586Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala
Ser Val 140 145 150acc ttc aac ctc ttc cgc ctc ctc acg cga gac ctg
aat tgt gtt gcc 634Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu
Asn Cys Val Ala 155 160 165agt ggg gac ctg tgt gtc tga ccctcccacc
agtcatgcaa cctgagattt 685Ser Gly Asp Leu Cys Val *170 175tatttataaa
ttagccactt gtcttaattt attgccaccc agtcgctat 73418200PRTHomo
sapiensSIGNAL(1)...(25) 18Met Lys Leu Asp Met Thr Gly Asp Cys Thr
Pro Val Leu Val Leu Met-25 -20 -15 -10Ala Ala Val Leu Thr Val Thr
Gly Ala Val Pro Val Ala Arg Leu His -5 1 5Gly Ala Leu Pro Asp Ala
Arg Gly Cys His Ile Ala Gln Phe Lys Ser 10 15 20Leu Ser Pro Gln Glu
Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu 25 30 35Glu Glu Ser Leu
Leu Leu Lys Asp Cys Arg Cys His Ser Arg Leu Phe40 45 50 55Pro Arg
Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Met 60 65 70Ala
Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr 75 80
85Ala Asp Thr Asp Pro Ala Leu Val Asp Val Leu Asp Gln Pro Leu His
90 95 100Thr Leu His His Ile Leu Ser Gln Phe Arg Ala Cys Ile Gln
Pro Gln 105 110 115Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His
His Trp Leu Tyr120 125 130 135Arg Leu Gln Glu Ala Pro Lys Lys Glu
Ser Pro Gly Cys Leu Glu Ala 140 145 150Ser Val Thr Phe Asn Leu Phe
Arg Leu Leu Thr Arg Asp Leu Asn Cys 155 160 165Val Ala Ser Gly Asp
Leu Cys Val 170 17519856DNAHomo
sapienssig_peptide(98)...(154)mat_peptide(155)...(700)CDS(98)...(700)
19aattaccttt tcactttaca cacatcatct tggattgccc attttgcgtg gctaaaaagc
60agagccatgc cgctggggaa gcagttgcga tttagcc atg gct gca gct tgg acc
115 Met Ala Ala Ala Trp Thr -15gtg gtg ctg gtg act ttg gtg cta ggc
ttg gcc gtg gca ggc cct gtc 163Val Val Leu Val Thr Leu Val Leu Gly
Leu Ala Val Ala Gly Pro Val -10 -5 1ccc act tcc aag ccc acc aca act
ggg aag ggc tgc cac att ggc agg 211Pro Thr Ser Lys Pro Thr Thr Thr
Gly Lys Gly Cys His Ile Gly Arg 5 10 15ttc aaa tct ctg tca cca cag
gag cta gcg agc ttc aag aag gcc agg 259Phe Lys Ser Leu Ser Pro Gln
Glu Leu Ala Ser Phe Lys Lys Ala Arg 20 25 30 35gac gcc ttg gaa gag
tca ctc aag ctg aaa aac tgg agt tgc agc tct 307Asp Ala Leu Glu Glu
Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser 40 45 50cct gtc ttc ccc
ggg aat tgg gac ctg agg ctt ctc cag gtg agg gag 355Pro Val Phe Pro
Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu 55 60 65cgc cct gtg
gcc ttg gag gct gag ctg gcc ctg acg ctg aag gtc ctg 403Arg Pro Val
Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu 70 75 80gag gcc
gct gct ggc cca gcc ctg gag gac gtc cta gac cag ccc ctt 451Glu Ala
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu 85 90 95cac
acc ctg cac cac atc ctc tcc cag ctc cag gcc tgt atc cag cct 499His
Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro100 105
110 115cag ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc cac cac tgg
ctg 547Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp
Leu 120 125 130cac cgg ctc cag gag gcc ccc aaa aag gag tcc gct ggc
tgc ctg gag 595His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly
Cys Leu Glu 135 140 145gca tct gtc acc ttc aac ctc ttc cgc ctc ctc
acg cga gac ctc aaa 643Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu
Thr Arg Asp Leu Lys 150 155 160tat gtg gcc gat ggg aac ctg tgt ctg
aga acg tca acc cac cct gag 691Tyr Val Ala Asp Gly Asn Leu Cys Leu
Arg Thr Ser Thr His Pro Glu 165 170 175tcc acc tga caccccacac
cttatttatg cgctgagccc tactccttcc 740Ser Thr *180ttaatttatt
tcctctcacc ctttatttat gaagctgcag ccctgactga gacatagggc
800tgagtttatt gttttacttt tatacattat gcacaaataa acaacaagga attgga
85620200PRTHomo sapiensSIGNAL(1)...(19) 20Met Ala Ala Ala Trp Thr
Val Val Leu Val Thr Leu Val Leu Gly Leu -15 -10 -5Ala Val Ala Gly
Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys 1 5 10Gly Cys His
Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala 15 20 25Ser Phe
Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys30 35 40
45Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg
50 55 60Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu
Ala 65 70 75Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu
Glu Asp 80 85 90Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu
Ser Gln Leu 95 100 105Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly
Pro Arg Pro Arg Gly110 115 120 125Arg Leu His His Trp Leu His Arg
Leu Gln Glu Ala Pro Lys Lys Glu 130 135 140Ser Ala Gly Cys Leu Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 145 150 155Leu Thr Arg Asp
Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg 160 165 170Thr Ser
Thr His Pro Glu Ser Thr 175 18021734DNAHomo
sapienssig_peptide(53)...(127)mat_peptide(128)...(655)CDS(53)...(655)
21tgggtgacag cctcagagtg tttcttctgc tgacaaagac cagagatcag ga atg aaa
58 Met Lys -25cta gac atg acc ggg gac tgc atg cca gtg ctg gtg ctg
atg gcc gca 106Leu Asp Met Thr Gly Asp Cys Met Pro Val Leu Val Leu
Met Ala Ala -20 -15 -10gtg ctg acc gtg act gga gca gtt cct gtc gcc
agg ctc cgc ggg gct 154Val Leu Thr Val Thr Gly Ala Val Pro Val Ala
Arg Leu Arg Gly Ala -5 1 5ctc ccg gat gca agg ggc tgc cac ata gcc
cag ttc aag tcc ctg tct 202Leu Pro Asp Ala Arg Gly Cys His Ile Ala
Gln Phe Lys Ser Leu Ser 10 15 20 25cca cag gag ctg cag gcc ttt aag
agg gcc aaa gat gcc tta gaa gag 250Pro Gln Glu Leu Gln Ala Phe Lys
Arg Ala Lys Asp Ala Leu Glu Glu 30 35 40tcg ctt ctg ctg aag gac tgc
aag tgc cgc tcc cgc ctc ttc ccc agg 298Ser Leu Leu Leu Lys Asp Cys
Lys Cys Arg Ser Arg Leu Phe Pro Arg 45 50 55acc tgg gac ctg agg cag
ctg cag gtg agg gag cgc ccc gtg gct ttg 346Thr Trp Asp Leu Arg Gln
Leu Gln Val Arg Glu Arg Pro Val Ala Leu 60 65 70gag gct gag ctg gcc
ctg acg ctg aag gtt ctg gag gcc acc gct gac 394Glu Ala Glu Leu Ala
Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp 75 80 85act gac cca gcc
ctg ggg gat gtc ttg gac cag ccc ctt cac acc ctg 442Thr Asp
Pro Ala Leu Gly Asp Val Leu Asp Gln Pro Leu His Thr Leu 90 95 100
105cac cat atc ctc tcc cag ctc cgg gcc tgt atc cag cct cag ccc acg
490His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr
110 115 120gca ggg ccc agg acc cgg ggc cgc ctc cac cat tgg ctg cac
cgg ctc 538Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu His
Arg Leu 125 130 135cag gag gcc cca aaa aag gag tcc cct ggc tgc ctc
gag gcc tct gtc 586Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu
Glu Ala Ser Val 140 145 150acc ttc aac ctc ttc cgc ctc ctc acg cga
gac ctg aat tgt gtt gcc 634Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg
Asp Leu Asn Cys Val Ala 155 160 165agc ggg gac ctg tgt gtc tga
cccttccgcc agtcatgcaa cctgagattt 685Ser Gly Asp Leu Cys Val *170
175tatttataaa ttagccactt ggcttaattt attgccaccc agtcgctat
73422200PRTHomo sapiensSIGNAL(1)...(25) 22Met Lys Leu Asp Met Thr
Gly Asp Cys Met Pro Val Leu Val Leu Met-25 -20 -15 -10Ala Ala Val
Leu Thr Val Thr Gly Ala Val Pro Val Ala Arg Leu Arg -5 1 5Gly Ala
Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser 10 15 20Leu
Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu 25 30
35Glu Glu Ser Leu Leu Leu Lys Asp Cys Lys Cys Arg Ser Arg Leu Phe40
45 50 55Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro
Val 60 65 70Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu
Ala Thr 75 80 85Ala Asp Thr Asp Pro Ala Leu Gly Asp Val Leu Asp Gln
Pro Leu His 90 95 100Thr Leu His His Ile Leu Ser Gln Leu Arg Ala
Cys Ile Gln Pro Gln 105 110 115Pro Thr Ala Gly Pro Arg Thr Arg Gly
Arg Leu His His Trp Leu His120 125 130 135Arg Leu Gln Glu Ala Pro
Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala 140 145 150Ser Val Thr Phe
Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys 155 160 165Val Ala
Ser Gly Asp Leu Cys Val 170 17523528DNAArtificial SequenceIL-28A
mutant C48S 23gtt cct gtc gcc agg ctc cac ggg gct ctc ccg gat gca
agg ggc tgc 48Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala
Arg Gly Cys 1 5 10 15cac ata gcc cag ttc aag tcc ctg tct cca cag
gag ctg cag gcc ttt 96His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln
Glu Leu Gln Ala Phe 20 25 30aag agg gcc aaa gat gcc tta gaa gag tcg
ctt ctg ctg aag gac tcc 144Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser
Leu Leu Leu Lys Asp Ser 35 40 45agg tgc cac tcc cgc ctc ttc ccc agg
acc tgg gac ctg agg cag ctg 192Arg Cys His Ser Arg Leu Phe Pro Arg
Thr Trp Asp Leu Arg Gln Leu 50 55 60cag gtg agg gag cgc ccc atg gct
ttg gag gct gag ctg gcc ctg acg 240Gln Val Arg Glu Arg Pro Met Ala
Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80ctg aag gtt ctg gag gcc
acc gct gac act gac cca gcc ctg gtg gac 288Leu Lys Val Leu Glu Ala
Thr Ala Asp Thr Asp Pro Ala Leu Val Asp 85 90 95gtc ttg gac cag ccc
ctt cac acc ctg cac cat atc ctc tcc cag ttc 336Val Leu Asp Gln Pro
Leu His Thr Leu His His Ile Leu Ser Gln Phe 100 105 110cgg gcc tgt
atc cag cct cag ccc acg gca ggg ccc agg acc cgg ggc 384Arg Ala Cys
Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly 115 120 125cgc
ctc cac cat tgg ctg tac cgg ctc cag gag gcc cca aaa aag gag 432Arg
Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu 130 135
140tcc cct ggc tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc ctc
480Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg
Leu145 150 155 160ctc acg cga gac ctg aat tgt gtt gcc agt ggg gac
ctg tgt gtc tga 528Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp
Leu Cys Val * 165 170 17524175PRTArtificial SequenceIL-28A mutant
C48S 24Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly
Cys 1 5 10 15His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu
Gln Ala Phe 20 25 30Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu
Leu Lys Asp Ser 35 40 45Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp
Asp Leu Arg Gln Leu 50 55 60Gln Val Arg Glu Arg Pro Met Ala Leu Glu
Ala Glu Leu Ala Leu Thr65 70 75 80Leu Lys Val Leu Glu Ala Thr Ala
Asp Thr Asp Pro Ala Leu Val Asp 85 90 95Val Leu Asp Gln Pro Leu His
Thr Leu His His Ile Leu Ser Gln Phe 100 105 110Arg Ala Cys Ile Gln
Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly 115 120 125Arg Leu His
His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu 130 135 140Ser
Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu145 150
155 160Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val
165 170 17525531DNAArtificial Sequencemet IL-28A mutant C49S 25atg
gtt cct gtc gcc agg ctc cac ggg gct ctc ccg gat gca agg ggc 48Met
Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10
15tgc cac ata gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc
96Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala
20 25 30ttt aag agg gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg aag
gac 144Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys
Asp 35 40 45tcc agg tgc cac tcc cgc ctc ttc ccc agg acc tgg gac ctg
agg cag 192Ser Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu
Arg Gln 50 55 60ctg cag gtg agg gag cgc ccc atg gct ttg gag gct gag
ctg gcc ctg 240Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu
Leu Ala Leu 65 70 75 80acg ctg aag gtt ctg gag gcc acc gct gac act
gac cca gcc ctg gtg 288Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr
Asp Pro Ala Leu Val 85 90 95gac gtc ttg gac cag ccc ctt cac acc ctg
cac cat atc ctc tcc cag 336Asp Val Leu Asp Gln Pro Leu His Thr Leu
His His Ile Leu Ser Gln 100 105 110ttc cgg gcc tgt atc cag cct cag
ccc acg gca ggg ccc agg acc cgg 384Phe Arg Ala Cys Ile Gln Pro Gln
Pro Thr Ala Gly Pro Arg Thr Arg 115 120 125ggc cgc ctc cac cat tgg
ctg tac cgg ctc cag gag gcc cca aaa aag 432Gly Arg Leu His His Trp
Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140gag tcc cct ggc
tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc 480Glu Ser Pro Gly
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg145 150 155 160ctc
ctc acg cga gac ctg aat tgt gtt gcc agt ggg gac ctg tgt gtc 528Leu
Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170
175tga 531*26176PRTArtificial Sequencemet IL-28A mutant C49S 26Met
Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10
15Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala
20 25 30Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys
Asp 35 40 45Ser Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu
Arg Gln 50 55 60Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu
Leu Ala Leu65 70 75 80Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr
Asp Pro Ala Leu Val 85 90 95Asp Val Leu Asp Gln Pro Leu His Thr Leu
His His Ile Leu Ser Gln 100 105 110Phe Arg Ala Cys Ile Gln Pro Gln
Pro Thr Ala Gly Pro Arg Thr Arg 115 120 125Gly Arg Leu His His Trp
Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140Glu Ser Pro Gly
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg145 150 155 160Leu
Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170
17527528DNAArtificial SequenceIL-28A mutant C50S 27gtt cct gtc gcc
agg ctc cac ggg gct ctc ccg gat gca agg ggc tgc 48Val Pro Val Ala
Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys 1 5 10 15cac ata
gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc ttt 96His Ile
Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe 20 25 30aag
agg gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg aag gac tgc 144Lys
Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys 35 40
45agg tcc cac tcc cgc ctc ttc ccc agg acc tgg gac ctg agg cag ctg
192Arg Ser His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu
50 55 60cag gtg agg gag cgc ccc atg gct ttg gag gct gag ctg gcc ctg
acg 240Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu
Thr 65 70 75 80ctg aag gtt ctg gag gcc acc gct gac act gac cca gcc
ctg gtg gac 288Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala
Leu Val Asp 85 90 95gtc ttg gac cag ccc ctt cac acc ctg cac cat atc
ctc tcc cag ttc 336Val Leu Asp Gln Pro Leu His Thr Leu His His Ile
Leu Ser Gln Phe 100 105 110cgg gcc tgt atc cag cct cag ccc acg gca
ggg ccc agg acc cgg ggc 384Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala
Gly Pro Arg Thr Arg Gly 115 120 125cgc ctc cac cat tgg ctg tac cgg
ctc cag gag gcc cca aaa aag gag 432Arg Leu His His Trp Leu Tyr Arg
Leu Gln Glu Ala Pro Lys Lys Glu 130 135 140tcc cct ggc tgc ctc gag
gcc tct gtc acc ttc aac ctc ttc cgc ctc 480Ser Pro Gly Cys Leu Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg Leu145 150 155 160ctc acg cga
gac ctg aat tgt gtt gcc agt ggg gac ctg tgt gtc tga 528Leu Thr Arg
Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val * 165 170
17528175PRTArtificial SequenceIL-28A mutant C50S 28Val Pro Val Ala
Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys 1 5 10 15His Ile
Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe 20 25 30Lys
Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys 35 40
45Arg Ser His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu
50 55 60Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu
Thr65 70 75 80Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala
Leu Val Asp 85 90 95Val Leu Asp Gln Pro Leu His Thr Leu His His Ile
Leu Ser Gln Phe 100 105 110Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala
Gly Pro Arg Thr Arg Gly 115 120 125Arg Leu His His Trp Leu Tyr Arg
Leu Gln Glu Ala Pro Lys Lys Glu 130 135 140Ser Pro Gly Cys Leu Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg Leu145 150 155 160Leu Thr Arg
Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170
17529531DNAArtificial Sequencemet IL-28A mutant C51S 29atg gtt cct
gtc gcc agg ctc cac ggg gct ctc ccg gat gca agg ggc 48Met Val Pro
Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10 15tgc
cac ata gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc 96Cys
His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala 20 25
30ttt aag agg gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg aag gac
144Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp
35 40 45tgc agg tcc cac tcc cgc ctc ttc ccc agg acc tgg gac ctg agg
cag 192Cys Arg Ser His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg
Gln 50 55 60ctg cag gtg agg gag cgc ccc atg gct ttg gag gct gag ctg
gcc ctg 240Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu
Ala Leu 65 70 75 80acg ctg aag gtt ctg gag gcc acc gct gac act gac
cca gcc ctg gtg 288Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp
Pro Ala Leu Val 85 90 95gac gtc ttg gac cag ccc ctt cac acc ctg cac
cat atc ctc tcc cag 336Asp Val Leu Asp Gln Pro Leu His Thr Leu His
His Ile Leu Ser Gln 100 105 110ttc cgg gcc tgt atc cag cct cag ccc
acg gca ggg ccc agg acc cgg 384Phe Arg Ala Cys Ile Gln Pro Gln Pro
Thr Ala Gly Pro Arg Thr Arg 115 120 125ggc cgc ctc cac cat tgg ctg
tac cgg ctc cag gag gcc cca aaa aag 432Gly Arg Leu His His Trp Leu
Tyr Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140gag tcc cct ggc tgc
ctc gag gcc tct gtc acc ttc aac ctc ttc cgc 480Glu Ser Pro Gly Cys
Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg145 150 155 160ctc ctc
acg cga gac ctg aat tgt gtt gcc agt ggg gac ctg tgt gtc 528Leu Leu
Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170
175tga 531*30176PRTArtificial Sequencemet IL-28A mutant C51S 30Met
Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10
15Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala
20 25 30Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys
Asp 35 40 45Cys Arg Ser His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu
Arg Gln 50 55 60Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu
Leu Ala Leu65 70 75 80Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr
Asp Pro Ala Leu Val 85 90 95Asp Val Leu Asp Gln Pro Leu His Thr Leu
His His Ile Leu Ser Gln 100 105 110Phe Arg Ala Cys Ile Gln Pro Gln
Pro Thr Ala Gly Pro Arg Thr Arg 115 120 125Gly Arg Leu His His Trp
Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140Glu Ser Pro Gly
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg145 150 155 160Leu
Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170
17531546DNAArtificial SequenceIL-29 mutant C171S 31ggt ccg gtt ccg
acc tct aaa cca acc acc act ggt aaa ggt tgc cac 48Gly Pro Val Pro
Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His 1 5 10 15atc ggt
cgt ttc aaa tct ctg tct ccg cag gaa ctg gct tct ttc aaa 96Ile Gly
Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys 20 25 30aaa
gct cgt gac gct ctg gaa gaa tct ctg aaa ctg aaa aac tgg tct 144Lys
Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser 35 40
45tgc tct tct ccg gtt ttc ccg ggt aac tgg gat ctg cgt ctg ctg cag
192Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln
50 55 60gtt cgt gaa cgt ccg gtt gct ctg gaa gct gaa ctg gct ctg acc
ctg 240Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr
Leu 65 70 75 80aaa gtt ctg gaa gct gct gca ggt cct gct ctg gaa gat
gtt ctg gat 288Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp
Val Leu Asp 85 90 95cag ccg ctg cac act ctg cac cac atc ctg tct cag
ctg cag gct tgc 336Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln
Leu Gln Ala Cys 100 105 110att caa ccg caa ccg acc gct ggt ccg cgt
ccg cgt ggt cgt ctg cac 384Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg
Pro Arg Gly Arg Leu His 115 120
125cac tgg ctg cat cgt ctg cag gaa gct ccg aaa aaa gaa tct gct ggt
432His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly
130 135 140tgc ctg gaa gct tct gtt acc ttc aac ctg ttc cgt ctg ctg
acc cgt 480Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu
Thr Arg145 150 155 160gat ctg aaa tac gtt gct gat ggt aac ctg tct
ctg cgt acc tct acc 528Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Ser
Leu Arg Thr Ser Thr 165 170 175cat ccg gaa tct acc taa 546His Pro
Glu Ser Thr * 18032181PRTArtificial SequenceIL-29 mutant C171S
32Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His 1
5 10 15Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe
Lys 20 25 30Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn
Trp Ser 35 40 45Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg
Leu Leu Gln 50 55 60Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu
Ala Leu Thr Leu65 70 75 80Lys Val Leu Glu Ala Ala Ala Gly Pro Ala
Leu Glu Asp Val Leu Asp 85 90 95Gln Pro Leu His Thr Leu His His Ile
Leu Ser Gln Leu Gln Ala Cys 100 105 110Ile Gln Pro Gln Pro Thr Ala
Gly Pro Arg Pro Arg Gly Arg Leu His 115 120 125His Trp Leu His Arg
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140Cys Leu Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg145 150 155
160Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Ser Leu Arg Thr Ser Thr
165 170 175His Pro Glu Ser Thr 18033549DNAArtificial Sequencemet
IL-29 mutant C172S 33atg ggt ccg gtt ccg acc tct aaa cca acc acc
act ggt aaa ggt tgc 48Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr
Thr Gly Lys Gly Cys 1 5 10 15cac atc ggt cgt ttc aaa tct ctg tct
ccg cag gaa ctg gct tct ttc 96His Ile Gly Arg Phe Lys Ser Leu Ser
Pro Gln Glu Leu Ala Ser Phe 20 25 30aaa aaa gct cgt gac gct ctg gaa
gaa tct ctg aaa ctg aaa aac tgg 144Lys Lys Ala Arg Asp Ala Leu Glu
Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45tct tgc tct tct ccg gtt ttc
ccg ggt aac tgg gat ctg cgt ctg ctg 192Ser Cys Ser Ser Pro Val Phe
Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60cag gtt cgt gaa cgt ccg
gtt gct ctg gaa gct gaa ctg gct ctg acc 240Gln Val Arg Glu Arg Pro
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80ctg aaa gtt ctg
gaa gct gct gca ggt cct gct ctg gaa gat gtt ctg 288Leu Lys Val Leu
Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95gat cag ccg
ctg cac act ctg cac cac atc ctg tct cag ctg cag gct 336Asp Gln Pro
Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105 110tgc
att caa ccg caa ccg acc gct ggt ccg cgt ccg cgt ggt cgt ctg 384Cys
Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120
125cac cac tgg ctg cat cgt ctg cag gaa gct ccg aaa aaa gaa tct gct
432His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala
130 135 140ggt tgc ctg gaa gct tct gtt acc ttc aac ctg ttc cgt ctg
ctg acc 480Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu
Leu Thr145 150 155 160cgt gat ctg aaa tac gtt gct gat ggt aac ctg
tct ctg cgt acc tct 528Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu
Ser Leu Arg Thr Ser 165 170 175acc cat ccg gaa tct acc taa 549Thr
His Pro Glu Ser Thr * 18034182PRTArtificial Sequencemet IL-29
mutant C172S 34Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly
Lys Gly Cys 1 5 10 15His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln
Glu Leu Ala Ser Phe 20 25 30Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser
Leu Lys Leu Lys Asn Trp 35 40 45Ser Cys Ser Ser Pro Val Phe Pro Gly
Asn Trp Asp Leu Arg Leu Leu 50 55 60Gln Val Arg Glu Arg Pro Val Ala
Leu Glu Ala Glu Leu Ala Leu Thr65 70 75 80Leu Lys Val Leu Glu Ala
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95Asp Gln Pro Leu His
Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105 110Cys Ile Gln
Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125His
His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135
140Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu
Thr145 150 155 160Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Ser
Leu Arg Thr Ser 165 170 175Thr His Pro Glu Ser Thr
18035531DNAArtificial Sequencemet IL-28A 35atg gtt cct gtc gcc agg
ctc cac ggg gct ctc ccg gat gca agg ggc 48Met Val Pro Val Ala Arg
Leu His Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10 15tgc cac ata gcc
cag ttc aag tcc ctg tct cca cag gag ctg cag gcc 96Cys His Ile Ala
Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala 20 25 30ttt aag agg
gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg aag gac 144Phe Lys Arg
Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 40 45tgc agg
tgc cac tcc cgc ctc ttc ccc agg acc tgg gac ctg agg cag 192Cys Arg
Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln 50 55 60ctg
cag gtg agg gag cgc ccc atg gct ttg gag gct gag ctg gcc ctg 240Leu
Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu 65 70
75 80acg ctg aag gtt ctg gag gcc acc gct gac act gac cca gcc ctg
gtg 288Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu
Val 85 90 95gac gtc ttg gac cag ccc ctt cac acc ctg cac cat atc ctc
tcc cag 336Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu
Ser Gln 100 105 110ttc cgg gcc tgt atc cag cct cag ccc acg gca ggg
ccc agg acc cgg 384Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly
Pro Arg Thr Arg 115 120 125ggc cgc ctc cac cat tgg ctg tac cgg ctc
cag gag gcc cca aaa aag 432Gly Arg Leu His His Trp Leu Tyr Arg Leu
Gln Glu Ala Pro Lys Lys 130 135 140gag tcc cct ggc tgc ctc gag gcc
tct gtc acc ttc aac ctc ttc cgc 480Glu Ser Pro Gly Cys Leu Glu Ala
Ser Val Thr Phe Asn Leu Phe Arg145 150 155 160ctc ctc acg cga gac
ctg aat tgt gtt gcc agt ggg gac ctg tgt gtc 528Leu Leu Thr Arg Asp
Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170 175tga
531*36176PRTArtificial Sequencemet IL-28A 36Met Val Pro Val Ala Arg
Leu His Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10 15Cys His Ile Ala
Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala 20 25 30Phe Lys Arg
Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 40 45Cys Arg
Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln 50 55 60Leu
Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu65 70 75
80Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Val
85 90 95Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser
Gln 100 105 110Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Thr Arg 115 120 125Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln
Glu Ala Pro Lys Lys 130 135 140Glu Ser Pro Gly Cys Leu Glu Ala Ser
Val Thr Phe Asn Leu Phe Arg145 150 155 160Leu Leu Thr Arg Asp Leu
Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170
17537621DNAArtificial Sequencemet IL-29 37atg ggc cct gtc ccc act
tcc aag ccc acc aca act ggg aag ggc tgc 48Met Gly Pro Val Pro Thr
Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys 1 5 10 15cac att ggc agg
ttc aaa tct ctg tca cca cag gag cta gcg agc ttc 96His Ile Gly Arg
Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30aag aag gcc
agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg 144Lys Lys Ala
Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45agt tgc
agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc 192Ser Cys
Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60cag
gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg 240Gln
Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70
75 80ctg aag gtc ctg gag gcc gct gct ggc cca gcc ctg gag gac gtc
cta 288Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val
Leu 85 90 95gac cag ccc ctt cac acc ctg cac cac atc ctc tcc cag ctc
cag gcc 336Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu
Gln Ala 100 105 110tgt atc cag cct cag ccc aca gca ggg ccc agg ccc
cgg ggc cgc ctc 384Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro
Arg Gly Arg Leu 115 120 125cac cac tgg ctg cac cgg ctc cag gag gcc
ccc aaa aag gag tcc gct 432His His Trp Leu His Arg Leu Gln Glu Ala
Pro Lys Lys Glu Ser Ala 130 135 140ggc tgc ctg gag gca tct gtc acc
ttc aac ctc ttc cgc ctc ctc acg 480Gly Cys Leu Glu Ala Ser Val Thr
Phe Asn Leu Phe Arg Leu Leu Thr145 150 155 160cga gac ctc aaa tat
gtg gcc gat ggg aac ctg tgt ctg aga acg tca 528Arg Asp Leu Lys Tyr
Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser 165 170 175acc cac cct
gag tcc acc tga caccccacac cttatttatg cgctgagccc 579Thr His Pro Glu
Ser Thr * 180tactccttcc ttaatttatt tcctctcacc ctttatttat ga
62138182PRTArtificial Sequencemet IL-29 38Met Gly Pro Val Pro Thr
Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys 1 5 10 15His Ile Gly Arg
Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30Lys Lys Ala
Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45Ser Cys
Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60Gln
Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr65 70 75
80Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu
85 90 95Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln
Ala 100 105 110Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg
Gly Arg Leu 115 120 125His His Trp Leu His Arg Leu Gln Glu Ala Pro
Lys Lys Glu Ser Ala 130 135 140Gly Cys Leu Glu Ala Ser Val Thr Phe
Asn Leu Phe Arg Leu Leu Thr145 150 155 160Arg Asp Leu Lys Tyr Val
Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser 165 170 175Thr His Pro Glu
Ser Thr 18039531DNAArtificial Sequencemet IL-28B 39atg gtt cct gtc
gcc agg ctc cgc ggg gct ctc ccg gat gca agg ggc 48Met Val Pro Val
Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10 15tgc cac
ata gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc 96Cys His
Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala 20 25 30ttt
aag agg gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg aag gac 144Phe
Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 40
45tgc aag tgc cgc tcc cgc ctc ttc ccc agg acc tgg gac ctg agg cag
192Cys Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln
50 55 60ctg cag gtg agg gag cgc ccc gtg gct ttg gag gct gag ctg gcc
ctg 240Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala
Leu 65 70 75 80acg ctg aag gtt ctg gag gcc acc gct gac act gac cca
gcc ctg ggg 288Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro
Ala Leu Gly 85 90 95gat gtc ttg gac cag ccc ctt cac acc ctg cac cat
atc ctc tcc cag 336Asp Val Leu Asp Gln Pro Leu His Thr Leu His His
Ile Leu Ser Gln 100 105 110ctc cgg gcc tgt atc cag cct cag ccc acg
gca ggg ccc agg acc cgg 384Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr
Ala Gly Pro Arg Thr Arg 115 120 125ggc cgc ctc cac cat tgg ctg cac
cgg ctc cag gag gcc cca aaa aag 432Gly Arg Leu His His Trp Leu His
Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140gag tcc cct ggc tgc ctc
gag gcc tct gtc acc ttc aac ctc ttc cgc 480Glu Ser Pro Gly Cys Leu
Glu Ala Ser Val Thr Phe Asn Leu Phe Arg145 150 155 160ctc ctc acg
cga gac ctg aat tgt gtt gcc agc ggg gac ctg tgt gtc 528Leu Leu Thr
Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170 175tga
531*40176PRTArtificial Sequencemet IL-28B 40Met Val Pro Val Ala Arg
Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10 15Cys His Ile Ala
Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala 20 25 30Phe Lys Arg
Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 40 45Cys Lys
Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln 50 55 60Leu
Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu65 70 75
80Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Gly
85 90 95Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser
Gln 100 105 110Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Thr Arg 115 120 125Gly Arg Leu His His Trp Leu His Arg Leu Gln
Glu Ala Pro Lys Lys 130 135 140Glu Ser Pro Gly Cys Leu Glu Ala Ser
Val Thr Phe Asn Leu Phe Arg145 150 155 160Leu Leu Thr Arg Asp Leu
Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170 175
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