U.S. patent application number 11/858699 was filed with the patent office on 2008-03-27 for methods for treating viral infection using il-28 and il-29 cysteine mutants.
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 | 20080075693 11/858699 |
Document ID | / |
Family ID | 34965116 |
Filed Date | 2008-03-27 |
United States Patent
Application |
20080075693 |
Kind Code |
A1 |
Klucher; Kevin M. ; et
al. |
March 27, 2008 |
METHODS FOR TREATING VIRAL INFECTION USING IL-28 AND IL-29 CYSTEINE
MUTANTS
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) ; Kindsvogel; Wayne R.; (Seattle,
WA) ; Sivakumar; Pallavur V.; (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: |
34965116 |
Appl. No.: |
11/858699 |
Filed: |
September 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11098662 |
Apr 4, 2005 |
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11858699 |
Sep 20, 2007 |
|
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60559081 |
Apr 2, 2004 |
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60609238 |
Sep 13, 2004 |
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60634144 |
Dec 8, 2004 |
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Current U.S.
Class: |
424/85.2 |
Current CPC
Class: |
A61K 38/212 20130101;
A61K 38/212 20130101; A61K 38/20 20130101; A61K 38/21 20130101;
A61P 31/20 20180101; A61P 31/16 20180101; A61K 38/57 20130101; A61P
1/16 20180101; A61K 38/21 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61P 31/12 20180101; A61K 38/20 20130101; A61K
38/57 20130101; C07K 14/54 20130101; A61P 29/00 20180101; A61K
47/60 20170801; A61K 2300/00 20130101; A61K 2300/00 20130101; A61P
31/22 20180101; A61P 31/14 20180101; A61P 31/18 20180101 |
Class at
Publication: |
424/085.2 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61P 29/00 20060101 A61P029/00 |
Claims
1. A method of treating liver inflammation in a mammal, the method
comprising: administering to the mammal a therapeutically effective
amount of an isolated polypeptide comprising amino acid residues
1-176 of SEQ ID NO:134.
2. The method of claim 1 wherein the polypeptide is a recombinant
polypeptide.
3. The method of claim 1 wherein the liver inflammation is
associated with a viral infection.
4. The method of claim 1 wherein the polypeptide is conjugated to a
polyalkyl oxide moiety.
5. The method of claim 4 wherein the polyalkyl oxide moiety is
polyethylene glycol.
6. The method of claim 5 wherein the polyethylene glycol is
monomethoxy-PEG propionaldehyde.
7. The method of claim 6 wherein the monomethoxy-PEG
propionaldehyde has a molecular weight of about 20 Kd or 30 Kd.
8. The method of claim 6 wherein the monomethoxy-PEG
propionaldehyde is linear or branched.
9. The method of claim 6 wherein the monomethoxy-PEG
propionaldehyde is conjugated N-terminally to the polypeptide.
10. A method of treating liver inflammation in a mammal, the method
comprising: administering to the mammal a therapeutically effective
amount of a composition comprising: an isolated polypeptide
comprising amino acid residues 1-176 of SEQ ID NO:134; and a
pharmaceutically acceptable vehicle; and wherein after
administration of the composition the liver inflammation is
reduced.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 11/098,662, filed Apr. 4, 2005, which claims
the benefit of U.S. Patent Application Ser. Nos. 60/559,081, filed
Apr. 2, 2004, 60/609,238, filed Sep. 13, 2004, and 60/634,144,
filed Dec. 8, 2004, all of which are herein incorporated by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Strategies for treating infectious disease often focus on
ways to enhance immunity. For instance, the most common method for
treating viral infection involves prophylactic vaccines that induce
immune-based memory responses. Another method for treating viral
infection includes passive immunization via immunoglobulin therapy
(Meissner, J. Pediatr. 124:S17-21, 1994). Administration of
Interferon alpha (IFN-.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 provide methods for treating specific virual infections, for
example, 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 should be apparent to those skilled in the art from the
teachings herein.
DESCRIPTION OF THE INVENTION
Definitions
[0009] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0010] Unless otherwise specified, "a," "an," "the," and "at least
one" are used interchangeably and mean one or more than one.
[0011] 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.).
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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).
[0016] 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.
[0017] 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).
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] "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.
[0024] 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.
[0025] 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".
[0026] 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.
[0027] 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.
[0028] 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).
[0029] 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.
[0030] 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.
[0031] 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%.
[0032] "zcyto20", "zcyto21", "zcyto22" are the previous
designations for human IL-28A, IL-29, and IL-28B, respectively and
are used interchangeably herein. IL-28A polypeptides of the present
invention are shown in SEQ ID NOs:2, 18, 24, 26, 28, 30, and 36,
which are encoded by polynucleotide sequences as shown in SEQ ID
NOs:1, 17, 23, 25, 27, 29, and 35, respectively. IL-28B
polypeptides of the present invention are shown in SEQ ID NOs:6,
22, 40, 86, 88, 90, 92, 94, 96, 98, and 100, which are encoded by
polynucleotide sequences as shown in SEQ ID NOs:5, 21, 39, 85, 87,
89, 91, 93, 95, 97, and 99, respectively. IL-29 polypeptides of the
present invention are shown in SEQ ID NOs:4, 20, 32, 34, 38, 42,
44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,
78, 80, 82, 84, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,
130, 132, 134, and 136, which are encoded by polynucleotide
sequences as shown in SEQ ID NOs:3, 19, 31, 33, 37, 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,
83, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,
133, and 135, respectively.
[0033] "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.
[0034] "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.
[0035] In one aspect, the present invention provides methods for
treating viral infections comprising administering to a mammal with
a viral infection a therapeutically effective amount of a
polypeptide comprising an amino acid sequence that has at least 95%
identity to amino acid residues of SEQ ID NO:134, wherein after
administration of the polypeptide the viral infection level is
reduced. In other embodiments, the methods comprise administering a
polypeptide comprising an amino acid sequence selected from the
group of SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34,
38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110,
112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and
136. The polypeptide may optionally comprise at least 15, at least
30, at least 45, or at least 60 sequential amino acids of an amino
acid sequence selected from the group of SEQ ID NOs:2, 4, 6, 18,
20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,
90, 92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120, 122, 124,
126, 128, 130, 132, 134, and 136. In another aspect, the viral
infection can optionally cause liver inflammation, wherein
administering a therapeutically effective amount of a polypeptide
reduces the liver inflammation. In other embodiments, the
polypeptide is conjugated to a polyalkyl oxide moiety, such as
polyethylene glycol (PEG), or F.sub.c, or human albumin. The PEG
may be N-terminally conjugated to the polypeptide and may comprise,
for instance, a 20 kD or 30 kD monomethoxy-PEG propionaldehyde. 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 viral infection and/or a hepatitis C viral infection. In another
embodiment, the polypeptide may be given prior to, concurrent with,
or subsequent to, at least one additional antiviral agent selected
from the group of Interferon alpha, Interferon beta, Interferon
gamma, Interferon omega, protease inhibitor, RNA or DNA polymerase
inhibitor, nucleoside analog, antisense inhibitor, and combinations
thereof. The polypeptide may be administered intravenously,
intraperitoneally, intrathecally, intramuscularly, subcutaneously,
orally, intranasally, or by inhalation.
[0036] In one aspect, the present invention provides methods for
treating viral infections comprising administering to a mammal with
a viral infection a therapeutically effective amount of a
composition comprising a polypeptide comprising an amino acid
sequence that has at least 95% identity to amino acid residues of
SEQ ID NO:134, and a pharmaceutically acceptable vehicle, wherein
after administration of the composition the viral infection level
is reduced. In other embodiments, the methods comprise
administering composition comprising the polypeptide comprising an
amino acid sequence as shown in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24,
26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,
62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,
96, 98, 100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,
132, 134, and/or 136. The polypeptide may optionally comprise at
least 15, at least 30, at least 45, or at least 60 sequential amino
acids of an amino acid sequence as shown in SEQ ID NOs:2, 4, 6, 18,
20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,
90, 92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120, 122, 124,
126, 128, 130, 132, 134, and/or 136. In other embodiments, the
polypeptide is conjugated to a polyalkyl oxide moiety, such as PEG,
or F.sub.c, or human albumin. The PEG may be N-terminally
conjugated to the polypeptide and may comprise, for instance, a 20
kD or 30 kD monomethoxy-PEG propionaldehyde. 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. In another embodiment, the composition may further
include or, be given prior to or, be given concurrent with, or be
given subsequent to, at least one additional antiviral agent
selected from the group of Interferon alpha, Interferon beta,
Interferon gamma, Interferon omega, protease inhibitor, RNA or DNA
polymerase inhibitor, nucleoside analog, antisense inhibitor, and
combinations thereof. The composition may be administered
intravenously, intraperitoneally, intrathecally, intramuscularly,
subcutaneously, orally, intranasally, or by inhalation.
[0037] 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 composition comprising a polypeptide
comprising an amino acid sequence that has at least 95% identity to
amino acid residues of SEQ ID NO:134, and a pharmaceutically
acceptable vehicle, wherein after administration of the composition
the viral infection level or liver inflammation is reduced. In
other embodiments, the methods comprise administering composition
comprising the polypeptide comprising an amino acid sequence as
shown in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34,
38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110,
112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or
136. The polypeptide may optionally comprise at least 15, at least
30, at least 45, or at least 60 sequential amino acids of an amino
acid sequence as shown in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26,
28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,
98, 100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,
132, 134, and/or 136. In other embodiments, the polypeptide is
conjugated to a polyalkyl oxide moiety, such as PEG, or F.sub.c, or
human albumin. The PEG may be N-terminally conjugated to the
polypeptide and may comprise, for instance, a 20 kD or 30 kD
monomethoxy-PEG propionaldehyde. 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. In another embodiment, the composition may further
include or, be given prior to or, be given concurrent with, or be
given subsequent to, at least one additional antiviral agent
selected from the group of Interferon alpha, Interferon beta,
Interferon gamma, Interferon omega, protease inhibitor, RNA or DNA
polymerase inhibitor, nucleoside analog, antisense inhibitor, and
combinations thereof. The composition may be administered
intravenously, intraperitoneally, intrathecally, intramuscularly,
subcutaneously, orally, intranasally, or by inhalation.
[0038] 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 amino acid residues of SEQ ID NO:134, 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
NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,
80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116,
118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 136. The
polypeptide may optionally comprise at least 15, at least 30, at
least 45, or at least 60 sequential amino acids of an amino acid
sequence as shown in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28,
30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,
66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,
100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,
134, and/or 136. In another embodiment, the polypeptide is
conjugated to a polyalkyl oxide moiety, such as PEG, or human
albumin, or F.sub.c. The PEG may be N-terminally conjugated to the
polypeptide and may comprise, for instance, a 20 kD or 30 kD
monomethoxy-PEG propionaldehyde. 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 viral infection or a
hepatitis B viral infection. In another embodiment, the polypeptide
may be given prior to, concurrent with, or subsequent to, at least
one additional antiviral agent selected from the group of
Interferon alpha, Interferon beta, Interferon gamma, Interferon
omega, protease inhibitor, RNA or DNA polymerase inhibitor,
nucleoside analog, antisense inhibitor, and combinations thereof.
The polypeptide may be administered intravenously,
intraperitoneally, intrathecally, intramuscularly, subcutaneously,
orally, intranasally, or by inhalation.
[0039] 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
composition comprising a polypeptide comprising an amino acid
sequence that has at least 95% identity to amino acid residues of
SEQ ID NO:134, 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 NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34,
38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110,
112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or
136. The polypeptide may optionally comprise at least 15, at least
30, at least 45, or at least 60 sequential amino acids of an amino
acid sequence as shown in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26,
28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,
98, 100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,
132, 134, and/or 136. In another embodiment, the polypeptide is
conjugated to a polyalkyl oxide moiety, such as PEG, or human
albumin, or F.sub.c. The PEG may be N-terminally conjugated to the
polypeptide and may comprise, for instance, a 20 kD or 30 kD
monomethoxy-PEG propionaldehyde. 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. In another embodiment, the composition
may further include or, be given prior to or, be given concurrent
with, or be given subsequent to, at least one additional antiviral
agent selected from the group of Interferon alpha, Interferon beta,
Interferon gamma, Interferon omega, protease inhibitor, RNA or DNA
polymerase inhibitor, nucleoside analog, antisense inhibitor, and
combinations thereof. The composition may be administered
intravenously, intraperitoneally, intrathecally, intramuscularly,
subcutaneously, orally, intranasally, or by inhalation.
[0040] 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 amino acid residues of SEQ ID
NO:134, 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 NOs:2, 4, 6,
18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120, 122,
124, 126, 128, 130, 132, 134, and/or 136. The polypeptide may
optionally comprise at least 15, at least 30, at least 45, or at
least 60 sequential amino acids of an amino acid sequence as shown
in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40,
42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,
76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 136. In
another embodiment, the polypeptide is conjugated to a polyalkyl
oxide moiety, such as PEG, or human albumin, or F.sub.c. The PEG
may be N-terminally conjugated to the polypeptide and may comprise,
for instance, a 20 kD or 30 kD monomethoxy-PEG propionaldehyde. 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. In another
embodiment, the polypeptide may be given prior to, concurrent with,
or subsequent to, at least one additional antiviral agent selected
from the group of Interferon alpha, Interferon beta, Interferon
gamma, Interferon omega, protease inhibitor, RNA or DNA polymerase
inhibitor, nucleoside analog, antisense inhibitor, and combinations
thereof. The polypeptide may be administered intravenously,
intraperitoneally, intrathecally, intramuscularly, subcutaneously,
orally, intranasally, or by inhalation.
[0041] 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 amino acid residues of SEQ ID
NO:134, 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 NOs:2, 4, 6,
18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120, 122,
124, 126, 128, 130, 132, 134, and/or 136. The polypeptide may
optionally comprise at least 15, at least 30, at least 45, or at
least 60 sequential amino acids of an amino acid sequence as shown
in SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40,
42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,
76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and/or 136. In
another embodiment, the polypeptide is conjugated to a polyalkyl
oxide moiety, such as PEG, or human albumin, or F.sub.c. The PEG
may be N-terminally conjugated to the polypeptide and may comprise,
for instance, a 20 kD or 30 kD monomethoxy-PEG propionaldehyde. 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. 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. In another
embodiment, the polypeptide may be given prior to, concurrent with,
or subsequent to, at least one additional antiviral agent selected
from the group of Interferon alpha, Interferon beta, Interferon
gamma, Interferon omega, protease inhibitor, RNA or DNA polymerase
inhibitor, nucleoside analog, antisense inhibitor, and combinations
thereof. The polypeptide may be administered intravenously,
intraperitoneally, intrathecally, intramuscularly, subcutaneously,
orally, intranasally, or by inhalation.
[0042] The discovery of a new family of interferon-like molecules
was previously described in PCT applications, PCT/US01/21087 and
PCT/US02/12887, and Sheppard et al., Nature Immunol. 4:63-68, 2003;
U.S. Patent Application Ser. Nos. 60/493,194 and 60/551,841; all
incorporated by reference herein. This new family includes
molecules designated zcyto20, zcyto21, zcyto22, zcyto24, zcyto25,
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. 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. When referring to "IL-28", the term shall mean
both IL-28A and IL-28B.
[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 an IL-28A, IL-28B, and/or
IL-29 polypeptide of the present invention 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 an
IL-28A, IL-28B, and/or IL-29 polypeptide of the present invention
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, norovirus, influenza virus,
adenovirus, parainfluenza virus, rhino virus, coxsackie virus,
vaccinia virus, west nile virus, severe acute respiratory syndrome,
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,
and/or IL-29 polypeptide of the present invention 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.
[0048] IL-28 and IL-29 are known to have an odd number of cysteines
(PCT application WO 02/086087 and Sheppard et al., supra.)
Expression of recombinant IL-28 and IL-29 can result in a
heterogeneous mixture of proteins composed of intramolecular
disulfide bonding in multiple conformations. The separation of
these forms can be difficult and laborious. It is therefore
desirable to provide IL-28 and IL-29 molecules having a single
intramolecular disulfide bonding pattern upon expression and
methods for refolding and purifying these preparations to maintain
homogeneity. Thus, the present invention provides for compositions
and methods to produce homogeneous preparations of IL-28 and
IL-29.
[0049] The present invention provides polynucleotide molecules,
including DNA and RNA molecules, that encode Cysteine mutants of
IL-28 and IL-29 that result in expression of a recombinant IL-28 or
IL-29 preparation that is a homogeneous preparation. For the
purposes of this invention, a homogeneous preparation of IL-28 and
IL-29 is a preparation in which comprises at least 98% of a single
intramolecular disulfide bonding pattern in the purified
polypeptide. In other embodiments, the single disulfide
conformation in a preparation of purified polypeptide is at 99%
homogeneous. In general, these Cysteine mutants will maintain some
biological activity of the wildtype IL-28 or IL-29, as described
herein. For example, the molecules of the present invention 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, biological activity of IL-28 or IL-29
Cysteine mutants is present when the molecules are capable of some
level of antiviral activity associated with wildtype IL-28 or
IL-29. Determination of the level of antiviral activity is
described in detail herein.
[0050] An IL-28A gene encodes a polypeptide of 200 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 31 (Ala) to 45 (Leu); helix B by amino acid residues 58
(Thr) to 65 (Gln); helix C by amino acid residues 69 (Arg) to 86
(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,
a secretory signal sequence may not be required and an N-terminal
Met may be present, resulting in expression of a polypeptide such
as, for instance, as shown in SEQ ID NO:36.
[0051] IL-28A polypeptides of the present invention also include a
mutation at the second cysteine, C2, of the mature polypeptide. For
example, C2 from the N-terminus of the polypeptide of SEQ ID NO:18
is the cysteine at amino acid position 48 (position 49, additional
N-terminal Met, if expressed in E coli, see, for example, SEQ ID
NO:36). This second cysteine (of which there are seven, like
IL-28B) or C2 of IL-28A can be mutated, for example, to a serine,
alanine, threonine, valine, or asparagine. IL-28A C2 mutant
molecules of the present invention include, for example,
polynucleotide molecules as shown in SEQ ID NOs:23 and 25,
including DNA and RNA molecules, that encode IL-28A C2 mutant
polypeptides as shown in SEQ ID NOs:24 and 26, respectively.
[0052] In addition to the IL-28A C2 mutants, the present invention
also includes IL-28A polypeptides comprising a mutation at the
third cysteine position, C3, of the mature polypeptide. For
example, C3 from the N-terminus of the polypeptide of SEQ ID NO:18,
is the cysteine at position 50, (position 51, additional N-terminal
Met, if expressed in E coli, see, for example, SEQ ID NO:36).
IL-28A C3 mutant molecules of the present invention include, for
example, polynucleotide molecules as shown in SEQ ID NOs:27 and 29,
including DNA and RNA molecules, that encode IL-28A C3 mutant
polypeptides as shown in SEQ ID NOs:28 and 30, respectively (PCT
publication WO 03/066002 (Kotenko et al.)).
[0053] The IL-28A polypeptides of the present invention include,
for example, SEQ ID NOs:2, 18, 24, 26, 28, 30, 36, and biologically
active mutants, fusions, variants and fragments thereof which are
encoded by IL-28A polynucleotide molecules as shown in SEQ ID
NOs:1, 17, 23, 25, 27, 29, and 35, respectively.
[0054] An 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, for example, SEQ ID NO:20, where amino acid
residue 188 (or amino acid residue 169 of the mature polypeptide
which begins from amino acid residue 20 (Gly)) is Asn instead of
Asp. The present invention also provides a variant IL-29 gene
wherein the mature polypeptide has a Thr at amino acid residue 10
substituted with a Pro, such as, for instance, SEQ ID NOs:54, 56,
58, 60, 62, 64, 66, and 68, which are encoded by the polynucleotide
sequences as shown in SEQ ID NOs:53, 55, 57, 59, 61, 63, 65, and
67, respectively. The present invention also provides a variant
IL-29 gene wherein the mature polypeptide has a Gly at amino acid
residue 18 substituted with an Asp, such as, for instance, SEQ ID
NOs:70, 72, 74, 76, 78, 80, 82, and 84, which are encoded by the
polynucleotide sequences as shown in SEQ ID NOs:69, 71, 73, 75, 77,
79, 81, and 83, respectively. 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) of SEQ ID NO:20. 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, a secretory
signal sequence may not be required and an N-terminal Met may be
present, resulting in expression of an IL-29 polypeptide such as,
for instance, as shown in SEQ ID NO:38.
[0055] IL-29 polypeptides of the present invention also include a
mutation at the fifth cysteine, C5, of the mature polypeptide. For
example, C5 from the N-terminus of the polypeptide of SEQ ID NO:20,
is the cysteine at position 171, or position 172 (additional
N-terminal Met) if expressed in E. coli. (see, for example, SEQ ID
NO:38). This fifth cysteine or C5 of IL-29 can be mutated, for
example, to a serine, alanine, threonine, valine, or asparagine.
These IL-29 C5 mutant polypeptides have a disulfide bond pattern of
C1(Cys15 of SEQ ID NO:20)/C3(Cys112 of SEQ ID NO:20) and C2(Cys49
of SEQ ID NO:20)/C4(Cys145 of SEQ ID NO:20). IL-29 C5 mutant
molecules of the present invention include, for example,
polynucleotide molecules as shown in SEQ ID NOs:31, 33, 49, 51,
109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,
and 135, including DNA and RNA molecules, that encode IL-29 C5
mutant polypeptides as shown in SEQ ID NOs:32, 34, 50, 52, 110,
112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and
136, respectively. Additional IL-29 C5 mutant molecules of the
present invention include polynucleotide molecules as shown in SEQ
ID NOs:53, 55, 61, and 63, including DNA and RNA molecules, that
encode IL-29 C5 mutant polypeptides as shown in SEQ ID NOs:54, 55,
62, and 64, respectively (PCT publication WO 03/066002 (Kotenko et
al.)). Additional, IL-29 C5 mutant molecules of the present
invention include polynucleotide molecules as shown in SEQ ID
NOs:69, 71, 77, and 79, including DNA and RNA molecules, that
encode IL-29 C5 mutant polypeptides as shown in SEQ ID NOs:70, 72,
78, and 80, respectively (PCT publication WO 02/092762 (Baum et
al.)).
[0056] In addition to the IL-29 C5 mutants, the present invention
also includes IL-29 polypeptides comprising a mutation at the first
cysteine position, C1, of the mature polypeptide. For example, C1
from the N-terminus of the polypeptide of SEQ ID NO:20, is the
cysteine at position 15, or position 16 (additional N-terminal Met)
if expressed in E. coli (see, for example, SEQ ID NO:38). This
first cysteine or C1 of IL-29 can be mutated, for example, to a
serine, alanine, threonine, valine, or asparagines. These IL-29 C1
mutant polypeptides will thus have a predicted disulfide bond
pattern of C2(Cys49 of SEQ ID NO:20)/C4(Cys145 of SEQ ID NO:20) and
C3(Cys112 of SEQ ID NO:20)/C5(Cys171 of SEQ ID NO:20). Additional
IL-29 C1 mutant molecules of the present invention include
polynucleotide molecules as shown in SEQ ID NOs:41, 43, 45, and 47,
including DNA and RNA molecules, that encode IL-29 C1 mutant
polypeptides as shown in SEQ ID NOs:42, 44, 46, and 48,
respectively. Additional IL-29 C1 mutant molecules of the present
invention include polynucleotide molecules as shown in SEQ ID
NOs:57, 59, 65, and 67, including DNA and RNA molecules, that
encode IL-29 C1 mutant polypeptides as shown in SEQ ID NOs:58, 60,
66, and 68, respectively (PCT publication WO 03/066002 (Kotenko et
al.)). Additional, IL-29 C1 mutant molecules of the present
invention include polynucleotide molecules as shown in SEQ ID
NOs:73, 75, 81, and 83, including DNA and RNA molecules, that
encode IL-29 C1 mutant polypeptides as shown in SEQ ID NOs:74, 76,
82, and 84, respectively (PCT publication WO 02/092762 (Baum et
al.)).
[0057] The IL-29 polypeptides of the present invention include, for
example, SEQ ID NOs:4, 20, 32, 34, 38, 42, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 110,
112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,
and biologically active mutants, fusions, variants and fragments
thereof which are encoded by IL-29 polynucleotide molecules as
shown in SEQ ID NOs:3, 19, 31, 33, 37, 41, 43, 45, 47, 49, 51, 53,
55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 109,
111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and
135, respectively, may further include a signal sequence as shown
in SEQ ID NOs:102, 104, 106, or 108. A polynucleotide molecule
encoding the signal sequence polypeptides of SEQ ID NOs:102, 104,
106, and 108 are are shown as SEQ ID NOs:101, 103, 105, and 107,
respectively.
[0058] An 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-25 (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) of SEQ ID
NO:22. IL-28B helices are predicted as follow: helix A is defined
by amino acid residues 31 (Ala) to 45 (Leu); helix B by amino acid
residues 58 (Thr) 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:22. When a polynucleotide sequence encoding the mature
polypeptide is expressed in a prokaryotic system, such as E. coli,
a secretory signal sequence may not be required and an N-terminal
Met may present, resulting in expression of a polypeptide such as
is shown in SEQ ID NO:40.
[0059] IL-28B polypeptides of the present invention also include a
mutation at the second cysteine, C2, of the mature polypeptide. For
example, C2 from the N-terminus of the polypeptide of SEQ ID NO:22
is the cysteine at amino acid position 48, or position 49
(additional N-terminal Met) if expressed in E coli (see, for
example, SEQ ID NO:40). This second cysteine (of which there are
seven, like IL-28A) or C2 of IL-28B can be mutated, for example, to
a serine, alanine, threonine, valine, or asparagine. IL-28B C2
mutant molecules of the present invention include, for example,
polynucleotide molecules as shown in SEQ ID NOs:85, and 87,
including DNA and RNA molecules, that encode IL-28B C2 mutant
polypeptides as shown in SEQ ID NOs:86 and 88, respectively.
Additional IL-28B C2 mutant molecules of the present invention
include polynucleotide molecules as shown in SEQ ID NOs:93 and 95
including DNA and RNA molecules, that encode IL-28 C2 mutant
polypeptides as shown in SEQ ID NOs:94 and 96, respectively (PCT
publication WO 03/066002 (Kotenko et al.)).
[0060] In addition to the IL-28B C2 mutants, the present invention
also includes IL-28B polypeptides comprising a mutation at the
third cysteine position, C3, of the mature polypeptide. For
example, C3 from the N-terminus of the polypeptide of SEQ ID NO:22,
is the cysteine at position 50, or position 51 (additional
N-terminal Met) if expressed in E. coli (see, for example, SEQ ID
NO:40). IL-28B C3 mutant molecules of the present invention
include, for example, polynucleotide molecules as shown in SEQ ID
NOs:89 and 91, including DNA and RNA molecules, that encode IL-28B
C3 mutant polypeptides as shown in SEQ ID NOs:90 and 92,
respectively. Additional IL-28B C3 mutant molecules of the present
invention include polynucleotide molecules as shown in SEQ ID
NOs:97 and 99 including DNA and RNA molecules, that encode IL-28B
C3 mutant polypeptides as shown in SEQ ID NOs:98 and 100,
respectively (PCT publication WO 03/066002 (Kotenko et al.)).
[0061] The IL-28B polypeptides of the present invention include,
for example, SEQ ID NOs:6, 22, 40, 86, 88, 90, 92, 94, 96, 98, 100,
and biologically active mutants, fusions, variants and fragments
thereof which are encoded by IL-28B polynucleotide molecules as
shown in SEQ ID NOs:5, 21, 39, 85, 87, 89, 91, 93, 95, 97, and 99,
respectively.
[0062] 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).
[0063] 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).
[0064] The IL-28 and IL-29 cysteine mutant polypeptides of the
present invention provided for the expression of a single-disulfide
form of the IL-28 or IL-29 molecule. When IL-28 and IL-29 are
expressed in E. coli, an N-terminal Methionine is present. SEQ ID
NOs:26, and 34, for instance, show the amino acid residue numbering
for IL-28A and IL-29 mutants, respectively, 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.48- C.sub.50- C.sub.167- C.sub.16-C.sub.48 C.sub.16-C.sub.50
C.sub.48- C.sub.50- C.sub.115- SEQ ID C.sub.115 C.sub.148 C.sub.148
C.sub.174 C.sub.115 C.sub.115 C.sub.148 NO: 18 Met IL-28A C.sub.17-
C.sub.49- C.sub.51- C.sub.168- C.sub.17-C.sub.49 C.sub.17-C.sub.51
C.sub.49- C.sub.51- C.sub.116- SEQ ID C.sub.116 C.sub.149
C.sub.1498 C.sub.175 C.sub.116 C.sub.116 C.sub.149 NO: 36 IL-29
C.sub.15- C.sub.49- C.sub.112- SEQ ID C.sub.112 C.sub.145 C.sub.171
NO: 20 Met IL-29 C.sub.16- C.sub.50- C.sub.113- SEQ ID C.sub.113
C.sub.146 C.sub.172 NO: 38 IL-28B C.sub.16- C.sub.48- C.sub.50-
C.sub.167- C.sub.16-C.sub.48 C.sub.16-C.sub.50 C.sub.48- C.sub.50-
C.sub.115- SEQ ID C.sub.115 C.sub.148 C.sub.148 C.sub.174 C.sub.115
C.sub.115 C.sub.148 NO: 22 Met IL-28B C.sub.17- C.sub.49- C.sub.51-
C.sub.168- C.sub.17-C.sub.49 C.sub.17-C.sub.51 C.sub.49- C.sub.51-
C.sub.116- SEQ ID C.sub.116 C.sub.149 C.sub.1498 C.sub.175
C.sub.116 C.sub.116 C.sub.149 NO: 40
[0065] Using methods known in the art, IL-28 or IL-29 polypeptides
of the present invention can be prepared as monomers or multimers;
glycosylated or non-glycosylated; pegylated or non-pegylated;
fusion proteins; and may or may not include an initial methionine
amino acid residue. IL-28 or IL-29 polypeptides can be conjugated
to acceptable water-soluble polymer moieties for use in therapy.
Conjugation of interferons, for example, 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)).
[0066] Suitable water-soluble polymers include polyethylene glycol
(PEG), monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG,
poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG,
monomethoxy-PEG propionaldehyde, PEG propionaldehyde,
bis-succinimidyl carbonate PEG, propylene glycol homopolymers, a
polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated
polyols (e.g., glycerol), monomethoxy-PEG butyraldehyde, PEG
butyraldehyde, monomethoxy-PEG acetaldehyde, PEG acetaldehyde,
methoxyl PEG-succinimidyl propionate, methoxyl PEG-succinimidyl
butanoate, 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, 30,000, 40,000, and 50,000, which can be
linear or branched. A IL-28 or IL-29 conjugate can also comprise a
mixture of such water-soluble polymers.
[0067] One example of an IL-28 or IL-29 conjugate comprises an
IL-28 or IL-29 moiety 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 an 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)). For example, PEGylation
can be performed by an acylation reaction or by an alkylation
reaction with a reactive polyethylene glycol molecule. In an
alternative approach, IL-28 or IL-29 conjugates are formed by
condensing activated PEG, in which a terminal hydroxy or amino
group of PEG has been replaced by an activated linker (see, for
example, Karasiewicz et al., U.S. Pat. No. 5,382,657).
[0068] PEGylation by acylation typically requires reacting an
active ester derivative of PEG with an IL-28 or IL-29 polypeptide.
An example of an activated PEG ester is PEG esterified to
N-hydroxysuccinimide. As used herein, the term "acylation" includes
the following types of linkages between IL-28 or IL-29 and a
water-soluble polymer: amide, carbamate, urethane, and the like.
Methods for preparing PEGylated IL-28 or IL-29 by acylation will
typically comprise the steps of (a) reacting an IL-28 or IL-29
polypeptide with PEG (such as a reactive ester of an aldehyde
derivative of PEG) under conditions whereby one or more PEG groups
attach to IL-28 or IL-29, and (b) obtaining the reaction
product(s). Generally, the optimal reaction conditions for
acylation reactions will be determined based upon known parameters
and desired results. For example, the larger the ratio of PEG:IL-28
or IL-29, the greater the percentage of polyPEGylated IL-28 or
IL-29 product.
[0069] PEGylation by alkylation generally involves reacting a
terminal aldehyde, e.g., propionaldehyde, butyraldehyde,
acetaldehyde, and the like, derivative of PEG with IL-28 or IL-29
in the presence of a reducing agent. PEG groups are preferably
attached to the polypeptide via a --CH.sub.2--NH.sub.2 group.
[0070] Derivatization via reductive alkylation to produce a
monoPEGylated product takes advantage of the differential
reactivity of different types of primary amino groups available for
derivatization. Typically, the reaction is performed at a pH that
allows one to take advantage of the pKa differences between the
.epsilon.-amino groups of the lysine residues and the .alpha.-amino
group of the N-terminal residue of the protein. By such selective
derivatization, attachment of a water-soluble polymer that contains
a reactive group such as an aldehyde, to a protein is controlled.
The conjugation with the polymer occurs predominantly at the
N-terminus of the protein without significant modification of other
reactive groups such as the lysine side chain amino groups.
[0071] Reductive alkylation to produce a substantially homogenous
population of monopolymer IL-28 or IL-29 conjugate molecule can
comprise the steps of: (a) reacting an IL-28 or IL-29 polypeptide
with a reactive PEG under reductive alkylation conditions at a pH
suitable to permit selective modification of the .alpha.-amino
group at the amino terminus of the IL-28 or IL-29, and (b)
obtaining the reaction product(s). The reducing agent used for
reductive alkylation should be stable in aqueous solution and
preferably be able to reduce only the Schiff base formed in the
initial process of reductive alkylation. Preferred reducing agents
include sodium borohydride, sodium cyanoborohydride, dimethylamine
borane, trimethylamine borane, and pyridine borane.
[0072] For a substantially homogenous population of monopolymer
IL-28 or IL-29 conjugates, the reductive alkylation reaction
conditions are those that permit the selective attachment of the
water-soluble polymer moiety to the N-terminus of IL-28 or IL-29.
Such reaction conditions generally provide for pKa differences
between the lysine amino groups and the .alpha.-amino group at the
N-terminus. The pH also affects the ratio of polymer to protein to
be used. In general, if the pH is lower, a larger excess of polymer
to protein will be desired because the less reactive the N-terminal
.alpha.-group, the more polymer is needed to achieve optimal
conditions. If the pH is higher, the polymer: IL-28 or IL-29 need
not be as large because more reactive groups are available.
Typically, the pH will fall within the range of 3-9, or 3-6.
Another factor to consider is the molecular weight of the
water-soluble polymer. Generally, the higher the molecular weight
of the polymer, the fewer number of polymer molecules which may be
attached to the protein. For PEGylation reactions, the typical
molecular weight is about 2 kDa to about 100 kDa, about 5 kDa to
about 50 kDa, or about 12 kDa to about 40 kDa. The molar ratio of
water-soluble polymer to IL-28 or IL-29 will generally be in the
range of 1:1 to 100:1. Typically, the molar ratio of water-soluble
polymer to IL-28 or IL-29 will be 1:1 to 20:1 for polyPEGylation,
and 1:1 to 5:1 for monoPEGylation.
[0073] General methods for producing conjugates comprising
interferon and water-soluble polymer moieties are known in the art.
See, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657,
Greenwald et al., U.S. Pat. No. 5,738,846, Nieforth et al., Clin.
Pharmacol. Ther. 59:636 (1996), Monkarsh et al., Anal. Biochem.
247:434 (1997). PEGylated species can be separated from
unconjugated IL-28 or IL-29 polypeptides using standard
purification methods, such as dialysis, ultrafiltration, ion
exchange chromatography, affinity chromatography, size exclusion
chromatography, and the like.
[0074] The IL-28 or IL-29 polypeptides of the present invention are
capable of specifically binding the IL-28 receptor and/or acting as
an antiviral agent. The binding of IL-28 or Il-29 polypeptides to
the IL-28 receptor can be assayed using established approaches.
IL-28 or IL-29 polypeptides can be iodinated using an iodobead
(Pierce, Rockford, Ill.) according to manufacturer's directions,
and the .sup.125I-IL-28 or .sup.125I-IL-29 can then be used as
described below.
[0075] In a first approach fifty nanograms of .sup.125I-IL-28 or
.sup.125I-IL-29 can be combined with 1000 ng of IL-28 receptor
human IgG fusion protein, in the presence or absence of possible
binding competitors including unlabeled cysteine mutant IL-28,
cysteine mutant IL-29, IL-28, or IL-29. The same binding reactions
would also be performed substituting other cytokine receptor human
IgG fusions as controls for specificity. Following incubation at
4.degree. C., protein-G (Zymed, San Francisco, Calif.) is added to
the reaction, to capture the receptor-IgG fusions and any proteins
bound to them, and the reactions are incubated another hour at
4.degree. C. The protein-G sepharose is then collected, washed
three times with PBS and .sup.125I-IL-28 or .sup.125I-IL-29 bound
is measure by gamma counter (Packard Instruments, Downers Grove,
Ill.).
[0076] In a second approach, the ability of molecules to inhibit
the binding of .sup.125I-IL-28 or .sup.125I-IL-29 to plate bound
receptors can be assayed. A fragment of the IL-28 receptor,
representing the extracellular, ligand binding domain, can be
adsorbed to the wells of a 96 well plate by incubating 100 .mu.l of
1 g/mL solution of receptor in the plate overnight. In a second
form, a receptor-human IgG fusion can be bound to the wells of a 96
well plate that has been coated with an antibody directed against
the human IgG portion of the fusion protein. Following coating of
the plate with receptor the plate is washed, blocked with
SUPERBLOCK (Pierce, Rockford, Ill.) and washed again. Solutions
containing a fixed concentration of .sup.125I-IL-28 or
.sup.125I-IL-29 with or without increasing concentrations of
potential binding competitors including, Cysteine mutant IL-28,
cysteine mutant IL-29, IL-28 and IL-29, and 100 .mu.l of the
solution added to appropriate wells of the plate. Following a one
hour incubation at 4.degree. C. the plate is washed and the amount
.sup.125I-IL-28 or .sup.125I-IL-29 bound determined by counting
(Topcount, Packard Instruments, Downers grove, IL). The specificity
of binding of .sup.125I-IL-28 or .sup.125I-IL-29 can be defined by
receptor molecules used in these binding assays as well as by the
molecules used as inhibitors.
[0077] In addition to pegylation, human albumin can be coupled to
an IL-28 or IL-29 polypeptide of the present invention to prolong
its half-life. Human albumin is the most prevalent naturally
occurring blood protein in the human circulatory system, persisting
in circulation in the body for over twenty days. Research has shown
that therapeutic proteins genetically fused to human albumin have
longer half-lives. An IL28 or IL29 albumin fusion protein, like
pegylation, may provide patients with long-acting treatment options
that offer a more convenient administration schedule, with similar
or improved efficacy and safety compared to currently available
treatments (U.S. Pat. No. 6,165,470; Syed et al., Blood,
89(9):3243-3253 (1997); Yeh et al., Proc. Natl. Acad. Sci. USA,
89:1904-1908 (1992); and Zeisel et al., Horm. Res., 37:5-13
(1992)).
[0078] Like the aforementioned peglyation and human albumin, an Fe
portion of the human IgG molecule can be fused to a polypeptide of
the present invention. The resultant fusion protein may have an
increased circulating half-life due to the Fe moiety (U.S. Pat. No.
5,750,375, U.S. Pat. No. 5,843,725, U.S. Pat. No. 6,291,646;
Barouch et al., Journal of Immunology, 61:1875-1882 (1998); Barouch
et al., Proc. Natl. Acad. Sci. USA, 97(8):4192-4197 (Apr. 11,
2000); and Kim et al., Transplant Proc., 30(8):4031-4036 (December
1998)).
[0079] 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).
[0080] 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.
[0081] 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.
[0082] 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).
[0083] 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).
[0084] 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 IL-28, IL-29, 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.
[0085] 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.
[0086] The various codons that encode for a given amino acid are
set forth below in Table 2. TABLE-US-00002 TABLE 2 One Amino Letter
Degenerate Acid Code Codons Codon Cys C TGC TGT TGY Ser S AGC AGT
TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCA CCC CCG CCT
CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC
AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR His H
CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met
M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN
Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W
TGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X
NNN
[0087] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding each amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by referencing the sequences disclosed herein. Variant
sequences can be readily tested for functionality as described
herein.
[0088] One of ordinary skill in the art will also appreciate that
different species can exhibit "preferential codon usage." In
general, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980;
Haas, et al. Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene
13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm,
Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol.
158:573-97, 1982. As used herein, the term "preferential codon
usage" or "preferential codons" is a term of art referring to
protein translation codons that are most frequently used in cells
of a certain species, thus favoring one or a few representatives of
the possible codons encoding each amino acid (See Table 2). For
example, the amino acid Threonine (Thr) may be encoded by ACA, ACC,
ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses
or bacteria, different Thr codons may be preferential. Preferential
codons for a particular species can be introduced into the
polynucleotides of the present invention by a variety of methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Sequences containing preferential codons can
be tested and optimized for expression in various species, and
tested for functionality as disclosed herein.
[0089] As previously noted, the isolated polynucleotides of the
present invention include DNA and RNA. Methods for preparing DNA
and RNA are well known in the art. In general, RNA is isolated from
a tissue or cell that produces large amounts of IL-28 or IL-29 RNA.
Such tissues and cells are identified by Northern blotting (Thomas,
Proc. Natl. Acad. Sci. USA 77:5201, 1980), or by screening
conditioned medium from various cell types for activity on target
cells or tissue. Once the activity or RNA producing cell or tissue
is identified, total RNA can be prepared using guanidinium
isothiocyanate extraction followed by isolation by centrifugation
in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979).
Poly (A).sup.+ RNA is prepared from total RNA using the method of
Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).
Complementary DNA (cDNA) is prepared from poly(A).sup.+ RNA using
known methods. In the alternative, genomic DNA can be isolated.
Polynucleotides encoding IL-28 or IL-29 polypeptides are then
identified and isolated by, for example, hybridization or PCR.
[0090] A full-length clones encoding IL-28 or IL-29 can be obtained
by conventional cloning procedures. Complementary DNA (cDNA) clones
are preferred, although for some applications (e.g., expression in
transgenic animals) it may be preferable to use a genomic clone, or
to modify a cDNA clone to include at least one genomic intron.
Methods for preparing cDNA and genomic clones are well known and
within the level of ordinary skill in the art, and include the use
of the sequence disclosed herein, or parts thereof, for probing or
priming a library. Expression libraries can be probed with
antibodies to IL-28 receptor fragments, or other specific binding
partners.
[0091] Those skilled in the art will recognize that the sequence
disclosed in, for example, SEQ ID NOs:17, 19 and 21, represent a
single allele of human IL-28A, IL-29, and IL28B, respectively, and
that allelic variation and alternative splicing are expected to
occur. For example, an IL-29 variant has been identified where
amino acid residue 169 as shown in SEQ ID NO:19 is an Asn residue
whereas its corresponding amino acid residue in SEQ ID NO:4 is an
Arg residue, as described in WO 02/086087. Such allelic variants
are included in the present invention. Allelic variants of IL-28
and IL-29 molecules of the present invention can be cloned by
probing cDNA or genomic libraries from different individuals
according to standard procedures. Allelic variants of the DNA
sequence shown in SEQ ID NOs:17, 19, and 21, including those
containing silent mutations and those in which mutations result in
amino acid sequence changes, in addition to the cysteine mutations,
are within the scope of the present invention, as are proteins
which are allelic variants of SEQ ID NO:18, 20, and 22. cDNAs
generated from alternatively spliced mRNAs, which retain the
properties of IL-28 or IL-29 polypeptides, are included within the
scope of the present invention, as are polypeptides encoded by such
cDNAs and mRNAs. Allelic variants and splice variants of these
sequences can be cloned by probing cDNA or genomic libraries from
different individuals or tissues according to standard procedures
known in the art, and mutations to the polynucleotides encoding
cysteines or cysteine residues can be introduced as described
herein.
[0092] Within embodiments of the invention, isolated IL-28 and
IL-29-encoding nucleic acid molecules can hybridize under stringent
conditions to nucleic acid molecules having the nucleotide sequence
selected from the group of SEQ ID NOs:1, 3, 5, 17, 19, 21, 23, 25,
27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,
63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,
97, 99, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,
133, and 135, or to its complement thereof. In general, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength and pH. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe.
[0093] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA
and DNA-RNA, can hybridize if the nucleotide sequences have some
degree of complementarity. Hybrids can tolerate mismatched base
pairs in the double helix, but the stability of the hybrid is
influenced by the degree of mismatch. The T.sub.m of the mismatched
hybrid decreases by 1.degree. C. for every 1-1.5% base pair
mismatch. Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be present in
the hybrid. The degree of stringency increases as the hybridization
temperature increases and the ionic strength of the hybridization
buffer decreases.
[0094] It is well within the abilities of one skilled in the art to
adapt these conditions for use with a particular polynucleotide
hybrid. The T.sub.m for a specific target sequence is the
temperature (under defined conditions) at which 50% of the target
sequence will hybridize to a perfectly matched probe sequence.
Those conditions which influence the T.sub.m include, the size and
base pair content of the polynucleotide probe, the ionic strength
of the hybridization solution, and the presence of destabilizing
agents in the hybridization solution. Numerous equations for
calculating T.sub.m are known in the art, and are specific for DNA,
RNA and DNA-RNA hybrids and polynucleotide probe sequences of
varying length (see, for example, Sambrook et al., Molecular
Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor
Press 1989); Ausubel et al., (eds.), Current Protocols in Molecular
Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.),
Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987);
and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)). Sequence
analysis software such as OLIGO 6.0 (LSR; Long Lake, Minn.) and
Primer Premier 4.0 (Premier Biosoft International; Palo Alto,
Calif.), as well as sites on the Internet, are available tools for
analyzing a given sequence and calculating T.sub.m based on user
defined criteria. Such programs can also analyze a given sequence
under defined conditions and identify suitable probe sequences.
Typically, hybridization of longer polynucleotide sequences, >50
base pairs, is performed at temperatures of about 20-25.degree. C.
below the calculated T.sub.m. For smaller probes, <50 base
pairs, hybridization is typically carried out at the T.sub.m or
5-10.degree. C. below the calculated T.sub.m. This allows for the
maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.
[0095] Following hybridization, the nucleic acid molecules can be
washed to remove non-hybridized nucleic acid molecules under
stringent conditions, or under highly stringent conditions. Typical
stringent washing conditions include washing in a solution of
0.5.times.-2.times.SSC with 0.1% sodium dodecyl sulfate (SDS) at
55-65.degree. C. That is, nucleic acid molecules encoding an IL-28
or IL-29 polypeptide hybridize with a nucleic acid molecule having
the nucleotide sequence selected from the group of SEQ ID NOs:1, 3,
5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 49,
51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,
85, 87, 89, 91, 93, 95, 97, 99, 109, 111, 113, 115, 117, 119, 121,
123, 125, 127, 129, 131, 133, and 135, or its complement thereof,
under stringent washing conditions, in which the wash stringency is
equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree.
C., including 0.5.times.SSC with 0.1% SDS at 55.degree. C., or
2.times.SSC with 0.1% SDS at 65.degree. C. One of skill in the art
can readily devise equivalent conditions, for example, by
substituting SSPE for SSC in the wash solution.
[0096] Typical highly stringent washing conditions include washing
in a solution of 0.1.times.-0.2.times.SSC with 0.1% sodium dodecyl
sulfate (SDS) at 50-65.degree. C. In other words, nucleic acid
molecules encoding a variant of an IL-28 or IL-29 polypeptide
hybridize with a nucleic acid molecule having the nucleotide
sequence selected from the group of SEQ ID NOs:1, 3, 5, 17, 19, 21,
23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,
59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,
93, 95, 97, 99, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,
129, 131, 133, and 135, or its complement thereof, under highly
stringent washing conditions, in which the wash stringency is
equivalent to 0.1.times.-0.2.times.SSC with 0.1% SDS at
50-65.degree. C., including 0.1.times.SSC with 0.1% SDS at
50.degree. C., or 0.2.times.SSC with 0.1% SDS at 65.degree. C.
[0097] The present invention also provides isolated IL-28 or IL-29
polypeptides that have a substantially similar sequence identity to
the polypeptides of the present invention, for example, selected
from the group of SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30,
32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,
68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,
100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,
134, and 136. The term "substantially similar sequence identity" is
used herein to denote polypeptides comprising at least 80%, at
least 90%, at least 95%, at least 96%, at least 97%, at least
97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%,
or greater than 99.5% sequence identity to the amino acid sequences
selected from the group of SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26,
28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,
98, 100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,
132, 134, and 136. The present invention also includes polypeptides
that comprise an amino acid sequence having at least 80%, at least
90%, at least 95%, at least 96%, at least 97%, at least 97.5%, at
least 98%, at least 98.5%, at least 99%, at least 99.5%, or greater
than 99.5% sequence identity to a polypeptide or fragment thereof
of the present invention. The present invention further includes
nucleic acid molecules that encode such polypeptides. The IL-28 and
IL-29 polypeptides of the present invention are preferably
recombinant polypeptides. In another aspect, the IL-28 and IL-29
polypeptides of the present invention have at least 15, at least
30, at least 45, or at least 60 sequential amino acids. For
example, an IL-29 polypeptide of the present invention relates to a
polypeptide having at least 15, at least 30, at least 45, or at
least 60 sequential amino acids to an amino acid sequence selected
from the group of SEQ ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30,
32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,
68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,
100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,
134, and 136. Methods for determining percent identity are
herein.
[0098] The present invention also contemplates variant nucleic acid
molecules that can be identified using two criteria: a
determination of the similarity between the encoded polypeptide
with the amino acid sequence selected from the group of SEQ ID
NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,
80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116,
118, 120, 122, 124, 126, 128, 130, 132, 134, and 136, and/or a
hybridization assay, as described above. Such variants include
nucleic acid molecules: (1) that hybridize with a nucleic acid
molecule having the nucleotide sequence selected from the group of
SEQ ID NOs:1, 3, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41,
43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,
77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 109, 111, 113, 115,
117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, or complement
thereof, under stringent washing conditions, in which the wash
stringency is equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at
55-65.degree. C.; or (2) that encode a polypeptide having at least
80%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 97.5%, at least 98%, at least 98.5%, at least 99%, at least
99.5%, or greater than 99.5% sequence identity to the amino acid
sequence selected from the group of SEQ ID NOs:2, 4, 6, 18, 20, 22,
24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,
60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,
94, 96, 98, 100, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,
130, 132, 134, and 136. Alternatively, variants can be
characterized as nucleic acid molecules: (1) that hybridize with a
nucleic acid molecule having the nucleotide sequence selected from
the group of SEQ ID NOs:1, 3, 5, 17, 19, 21, 23, 25, 27, 29, 31,
33, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,
69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,
109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,
and 135, or its complement thereof, under highly stringent washing
conditions, in which the wash stringency is equivalent to
0.1.times.-0.2.times.SSC with 0.1% SDS at 50-65.degree. C.; and (2)
that encode a polypeptide having at least 80%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
at least 99.5%, or greater than 99.5% sequence identity to the
amino acid sequence selected from the group of SEQ ID NOs:2, 4, 6,
18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120, 122,
124, 126, 128, 130, 132, 134, and 136.
[0099] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603
(1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 (1992). Briefly, two amino acid sequences are aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 2 (amino acids are
indicated by the standard one-letter codes). [0100] Total number of
identical matches [0101] .times.100
[0102] [length of the longer sequence plus the number of gaps
introduced into the longer sequence in order to align the two
sequences] TABLE-US-00003 TABLE 3 A R N D C Q E G H I L K M F P S T
W Y V A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3
5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8
I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K
-1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5
F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2
-2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0
-1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3
-2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1
-2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2
0 -3 -1 4
[0103] Those skilled in the art appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative variant IL-28 or IL-29. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990).
[0104] Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID
NO:2) and a test sequence that have either the highest density of
identities (if the ktup variable is 1) or pairs of identities (if
ktup=2), without considering conservative amino acid substitutions,
insertions, or deletions. The ten regions with the highest density
of identities are then rescored by comparing the similarity of all
paired amino acids using an amino acid substitution matrix, and the
ends of the regions are "trimmed" to include only those residues
that contribute to the highest score. If there are several regions
with scores greater than the "cutoff" value (calculated by a
predetermined formula based upon the length of the sequence and the
ktup value), then the trimmed initial regions are examined to
determine whether the regions can be joined to form an approximate
alignment with gaps. Finally, the highest scoring regions of the
two amino acid sequences are aligned using a modification of the
Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.
Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)),
which allows for amino acid insertions and deletions. Preferred
parameters for FASTA analysis are: ktup=1, gap opening penalty=10,
gap extension penalty=1, and substitution matrix=BLOSUM62. These
parameters can be introduced into a FASTA program by modifying the
scoring matrix file ("SMATRIX"), as explained in Appendix 2 of
Pearson, Meth. Enzymol. 183:63 (1990).
[0105] FASTA can also be used to determine the sequence identity of
nucleic acid molecules using a ratio as disclosed above. For
nucleotide sequence comparisons, the ktup value can range between
one to six, preferably from three to six, most preferably three,
with other parameters set as default.
[0106] IL-28 or IL-29 polypeptides with substantially similar
sequence identity are characterized as having one or more amino
acid substitutions, deletions or additions. These changes are
preferably of a minor nature, that is conservative amino acid
substitutions (see Table 4) and other substitutions that do not
significantly affect the folding or activity of the polypeptide;
small deletions, typically of one to about 30 amino acids; and
amino- or carboxyl-terminal extensions, such as an amino-terminal
methionine residue, a small linker peptide of up to about 20-25
residues, or an affinity tag. The present invention thus includes
polypeptides that comprise a sequence that is at least 80%, at
least 90%, at least 95%, at least 96%, at least 97%, at least
97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%,
or greater than 99.5% identical to the corresponding region of SEQ
ID NOs:2, 4, 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,
80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116,
118, 120, 122, 124, 126, 128, 130, 132, 134, and 136. Polypeptides
comprising affinity tags can further comprise a proteolytic
cleavage site between the IL-28 and IL-29 polypeptide and the
affinity tag. Preferred such sites include thrombin cleavage sites
and factor Xa cleavage sites. TABLE-US-00004 TABLE 4 Conservative
amino acid substitutions Basic: arginine lysine histidine Acidic:
glutamic acid aspartic acid Polar: glutamine asparagine
Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine
tryptophan tyrosine Small: glycine alanine serine threonine
methionine
[0107] Determination of amino acid residues that comprise regions
or domains that are critical to maintaining structural integrity
can be determined. Within these regions one can determine specific
residues that will be more or less tolerant of change and maintain
the overall tertiary structure of the molecule. Methods for
analyzing sequence structure include, but are not limited to
alignment of multiple sequences with high amino acid or nucleotide
identity, secondary structure propensities, binary patterns,
complementary packing and buried polar interactions (Barton,
Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al.,
Current Opin. Struct. Biol. 6:3-10, 1996). In general, when
designing modifications to molecules or identifying specific
fragments determination of structure will be accompanied by
evaluating activity of modified molecules.
[0108] Amino acid sequence changes are made in IL-28 or IL-29
polypeptides so as to minimize disruption of higher order structure
essential to biological activity. For example, where the IL-28 or
IL-29 polypeptide comprises one or more helices, changes in amino
acid residues will be made so as not to disrupt the helix geometry
and other components of the molecule where changes in conformation
abate some critical function, for example, binding of the molecule
to its binding partners. The effects of amino acid sequence changes
can be predicted by, for example, computer modeling as disclosed
above or determined by analysis of crystal structure (see, e.g.,
Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). Other
techniques that are well known in the art compare folding of a
variant protein to a standard molecule (e.g., the native protein).
For example, comparison of the cysteine pattern in a variant and
standard molecules can be made. Mass spectrometry and chemical
modification using reduction and alkylation provide methods for
determining cysteine residues which are associated with disulfide
bonds or are free of such associations (Bean et al., Anal. Biochem.
201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and
Patterson et al., Anal. Chem. 66:3727-3732, 1994). It is generally
believed that if a modified molecule does not have the same
cysteine pattern as the standard molecule folding would be
affected. Another well known and accepted method for measuring
folding is circular dichrosism (CD). Measuring and comparing the CD
spectra generated by a modified molecule and standard molecule is
routine (Johnson, Proteins 7:205-214, 1990). Crystallography is
another well known method for analyzing folding and structure.
Nuclear magnetic resonance (NMR), digestive peptide mapping and
epitope mapping are also known methods for analyzing folding and
structurally similarities between proteins and polypeptides
(Schaanan et al., Science 257:961-964, 1992).
[0109] A Hopp/Woods hydrophilicity profile of an IL-28 or IL-29
polypeptide sequence selected from the group of SEQ ID NOs:2, 4, 6,
18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 110, 112, 114, 116, 118, 120, 122,
124, 126, 128, 130, 132, 134, and 136, can be generated (Hopp et
al., Proc. Natl. Acad. Sci. 78:3824-3828, 1981; Hopp, J. Immun.
Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering
11:153-169, 1998). The profile is based on a sliding six-residue
window. Buried G, S, and T residues and exposed H, Y, and W
residues were ignored. Those skilled in the art will recognize that
hydrophilicity or hydrophobicity will be taken into account when
designing modifications in the amino acid sequence of an IL-28 or
IL-29 polypeptide, so as not to disrupt the overall structural and
biological profile. Of particular interest for replacement are
hydrophobic residues selected from the group consisting of Val, Leu
and Ile or the group consisting of Met, Gly, Ser, Ala, Tyr and
Trp.
[0110] The identities of essential amino acids can also be inferred
from analysis of sequence similarity between IFN-.alpha. and
members of the family of IL-28A, IL-28B, and IL-29 (as shown in
Tables 1 and 2). Using methods such as "FASTA" analysis described
previously, regions of high similarity are identified within a
family of proteins and used to analyze amino acid sequence for
conserved regions. An alternative approach to identifying a variant
polynucleotide on the basis of structure is to determine whether a
nucleic acid molecule encoding a potential variant IL-28 or IL-29
gene can hybridize to a nucleic acid molecule as discussed
above.
[0111] Other methods of identifying essential amino acids in the
polypeptides of the present invention are procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et
al., Proc. Natl. Acad. Sci. USA 88:4498 (1991), Coombs and Corey,
"Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and Design, Angeletti (ed.), pages 259-311 (Academic
Press, Inc. 1998)). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant IL-28 and IL-29 molecules are tested for biological or
biochemical activity as disclosed below to identify amino acid
residues that are critical to the activity of the molecule. See
also, Hilton et al., J. Biol. Chem. 271:4699 (1996).
[0112] The present invention also includes functional fragments of
IL-28 or IL-29 polypeptides and nucleic acid molecules encoding
such functional fragments. A "functional" IL-28 or IL-29 or
fragment thereof as defined herein is characterized by its
proliferative or differentiating activity, by its ability to induce
or inhibit specialized cell functions, or by its ability to bind
specifically to an anti-IL-28 or IL-29 antibody or IL-28 receptor
(either soluble or immobilized). The specialized activities of
IL-28 or IL-29 polypeptides and how to test for them are disclosed
herein. As previously described herein, IL-28 and IL-29
polypeptides are characterized by a six-helical-bundle. Thus, the
present invention further provides fusion proteins encompassing:
(a) polypeptide molecules comprising one or more of the helices
described above; and (b) functional fragments comprising one or
more of these helices. The other polypeptide portion of the fusion
protein may be contributed by another helical-bundle cytokine or
interferon, such as IFN-.alpha., or by a non-native and/or an
unrelated secretory signal peptide that facilitates secretion of
the fusion protein.
[0113] The IL-28 or IL-29 polypeptides of the present invention,
including full-length polypeptides, biologically active fragments,
and fusion polypeptides can be produced according to conventional
techniques using cells into which have been introduced an
expression vector encoding the polypeptide. As used herein, "cells
into which have been introduced an expression vector" include both
cells that have been directly manipulated by the introduction of
exogenous DNA molecules and progeny thereof that contain the
introduced DNA. Suitable host cells are those cell types that can
be transformed or transfected with exogenous DNA and grown in
culture, and include bacteria, fungal cells, and cultured higher
eukaryotic cells. Techniques for manipulating cloned DNA molecules
and introducing exogenous DNA into a variety of host cells are
disclosed by Sambrook et al., Molecular Cloning: A Laboratory
Manual., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocols in
Molecular Biology, John Wiley and Sons, Inc., NY, 1987.
[0114] In general, a DNA sequence encoding an IL-28 or IL-29
polypeptide is operably linked to other genetic elements required
for its expression, generally including a transcription promoter
and terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers.
[0115] To direct an IL-28 or IL-29 polypeptide into the secretory
pathway of a host cell, a secretory signal sequence (also known as
a leader sequence, prepro sequence or pre sequence) is provided in
the expression vector. The secretory signal sequence may be that of
IL-28 or IL-29, e.g., SEQ ID NO:119 or SEQ ID NO:121, or may be
derived from another secreted protein (e.g., t-PA; see, U.S. Pat.
No. 5,641,655) or synthesized de novo. The secretory signal
sequence is operably linked to an IL-28 or IL-29 DNA sequence,
i.e., the two sequences are joined in the correct reading frame and
positioned to direct the newly synthesized polypeptide into the
secretory pathway of the host cell. Secretory signal sequences are
commonly positioned 5' to the DNA sequence encoding the polypeptide
of interest, although certain signal sequences may be positioned
elsewhere in the DNA sequence of interest (see, e.g., Welch et al.,
U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.
5,143,830).
[0116] A wide variety of suitable recombinant host cells includes,
but is not limited to, gram-negative prokaryotic host organisms.
Suitable strains of E. coli include W3110 and mutants-strains
thereof (e.g, an OmpT protease deficient W3110 strain, and an OmpT
protease and fhuA deficient W3110 strain), K12-derived strains
MM294, TG-1, JM-107, BL21, and UT5600. Other suitable strains
include: BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5,
DH5I, DH51F', DH51MCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101,
JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, ER1647,
E. coli K12, E. coli K12 RV308, E. coli K12 C600, E. coliHB101, E.
coli K12 C600 R.sub.k-M.sub.k-, E. coli K12 RR1 (see, for example,
Brown (ed.), Molecular Biology Labfax (Academic Press 1991)). Other
gram-negative prokaryotic hosts can include Serratia, Pseudomonas,
Caulobacter. Prokaryotic hosts can include gram-positive organisms
such as Bacillus, for example, B. subtilis and B. thuringienesis,
and B. thuringienesis var. israelensis, as well as Streptomyces,
for example, S. lividans, S. ambofaciens, S. fradiae, and S.
griseofuscus. Suitable strains of Bacillus subtilus include BR151,
YB886, M1119, M1120, and B170 (see, for example, Hardy, "Bacillus
Cloning Methods," in DNA Cloning: A Practical Approach, Glover
(ed.) (IRL Press 1985)). Standard techniques for propagating
vectors in prokaryotic hosts are well-known to those of skill in
the art (see, for example, Ausubel et al (eds.), Short Protocols in
Molecular Biology 3.sup.rd Edition (John Wiley & Sons 1995); Wu
et al., Methods in Gene Biotechnology (CRC Press, Inc. 1997)). In
one embodiment, the methods of the present invention use Cysteine
mutant IL-28 or IL-29 expressed in the W3110 strain, which has been
deposited at the American Type Culture Collection (ATCC) as ATCC #
27325.
[0117] When large scale production of an IL-28 or IL-29 polypeptide
using the expression system of the present invention is required,
batch fermentation can be used. Generally, batch fermentation
comprises that a first stage seed flask is prepared by growing E.
coli strains expressing an IL-28 or IL-29 polypeptide in a suitable
medium in shake flask culture to allow for growth to an optical
density (OD) of between 5 and 20 at 600 nm. A suitable medium would
contain nitrogen from a source(s) such as ammonium sulfate,
ammonium phosphate, ammonium chloride, yeast extract, hydrolyzed
animal proteins, hydrolyzed plant proteins or hydrolyzed caseins.
Phosphate will be supplied from potassium phosphate, ammonium
phosphate, phosphoric acid or sodium phosphate. Other components
would be magnesium chloride or magnesium sulfate, ferrous sulfate
or ferrous chloride, and other trace elements. Growth medium can be
supplemented with carbohydrates, such as fructose, glucose,
galactose, lactose, and glycerol, to improve growth. Alternatively,
a fed batch culture is used to generate a high yield of IL-28 or
IL-29 polypeptide. The IL-28 or IL-29 polypeptide producing E. coli
strains are grown under conditions similar to those described for
the first stage vessel used to inoculate a batch fermentation.
[0118] Following fermentation the cells are harvested by
centrifugation, re-suspended in homogenization buffer and
homogenized, for example, in an APV-Gaulin homogenizer (Invensys
APV, Tonawanda, N.Y.) or other type of cell disruption equipment,
such as bead mills or sonicators. Alternatively, the cells are
taken directly from the fermentor and homogenized in an APV-Gaulin
homogenizer. The washed inclusion body prep can be solubilized
using guanidine hydrochloride (5-8 M) or urea (7-8 M) containing a
reducing agent such as beta mercaptoethanol (10-100 mM) or
dithiothreitol (5-50 mM). The solutions can be prepared in Tris,
phopshate, HEPES or other appropriate buffers. Inclusion bodies can
also be solubilized with urea (2-4 M) containing sodium lauryl
sulfate (0.1-2%). In the process for recovering purified IL-28 or
IL-29 from transformed E. coli host strains in which the IL-28 or
IL-29 is accumulates as refractile inclusion bodies, the cells are
disrupted and the inclusion bodies are recovered by centrifugation.
The inclusion bodies are then solubilized and denatured in 6 M
guanidine hydrochloride containing a reducing agent. The reduced
IL-28 or IL-29 is then oxidized in a controlled renaturation step.
Refolded IL-28 or IL-29 can be passed through a filter for
clarification and removal of insoluble protein. The solution is
then passed through a filter for clarification and removal of
insoluble protein. After the IL-28 or IL-29 protein is refolded and
concentrated, the refolded IL-28 or IL-29 protein is captured in
dilute buffer on a cation exchange column and purified using
hydrophobic interaction chromatography.
[0119] Cultured mammalian cells are suitable hosts within the
present invention. Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al.,
ibid.), and liposome-mediated transfection (Hawley-Nelson et al.,
Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral
vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and
Finer, Nature Med. 2:714-6, 1996). The production of recombinant
polypeptides in cultured mammalian cells is disclosed, for example,
by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.
Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and
Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells
include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651),
BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC
No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and
Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines.
Additional suitable cell lines are known in the art and available
from public depositories such as the American Type Culture
Collection, Manassas, Va. In general, strong transcription
promoters are preferred, such as promoters from SV-40 or
cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitable
promoters include those from metallothionein genes (U.S. Pat. Nos.
4,579,821 and 4,601,978) and the adenovirus major late
promoter.
[0120] Drug selection is generally used to select for cultured
mammalian cells into which foreign DNA has been inserted. Such
cells are commonly referred to as "transfectants". Cells that have
been cultured in the presence of the selective agent and are able
to pass the gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is a gene
encoding resistance to the antibiotic neomycin. Selection is
carried out in the presence of a neomycin-type drug, such as G-418
or the like. Selection systems can also be used to increase the
expression level of the gene of interest, a process referred to as
"amplification." Amplification is carried out by culturing
transfectants in the presence of a low level of the selective agent
and then increasing the amount of selective agent to select for
cells that produce high levels of the products of the introduced
genes. A preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate. Other drug
resistance genes (e.g. hygromycin resistance, multi-drug
resistance, puromycin acetyltransferase) can also be used.
Alternative markers that introduce an altered phenotype, such as
green fluorescent protein, or cell surface proteins such as CD4,
CD8, Class I MHC, placental alkaline phosphatase may be used to
sort transfected cells from untransfected cells by such means as
FACS sorting or magnetic bead separation technology.
[0121] Other higher eukaryotic cells can also be used as hosts,
including plant cells, insect cells and avian cells. The use of
Agrobacterium rhizogenes as a vector for expressing genes in plant
cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore)
11:47-58, 1987. Transformation of insect cells and production of
foreign polypeptides therein is disclosed by Guarino et al., U.S.
Pat. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells
can be infected with recombinant baculovirus, commonly derived from
Autographa californica nuclear polyhedrosis virus (AcNPV). See,
King, L. A. and Possee, R. D., The Baculovirus Expression System: A
Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. et
al., Baculovirus Expression Vectors: A Laboratory Manual, New York,
Oxford University Press., 1994; and, Richardson, C. D., Ed.,
Baculovirus Expression Protocols. Methods in Molecular Biology,
Totowa, N.J., Humana Press, 1995. The second method of making
recombinant baculovirus utilizes a transposon-based system
described by Luckow (Luckow, V. A, et al., J Virol 67:4566-79,
1993). This system is sold in the Bac-to-Bac kit (Life
Technologies, Rockville, Md.). This system utilizes a transfer
vector, pFastBac1.TM. (Life Technologies) containing a Tn7
transposon to move the DNA encoding the Cysteine mutant IL-28 or
IL-29 polypeptide into a baculovirus genome maintained in E. coli
as a large plasmid called a "bacmid." The pFastBac1.TM. transfer
vector utilizes the AcNPV polyhedrin promoter to drive the
expression of the gene of interest, in this case IL-28 or IL-29.
However, pFastBac1.TM. can be modified to a considerable degree.
The polyhedrin promoter can be removed and substituted with the
baculovirus basic protein promoter (also known as Pcor, p6.9 or MP
promoter) which is expressed earlier in the baculovirus infection,
and has been shown to be advantageous for expressing secreted
proteins. See, Hill-Perkins, M. S, and Possee, R. D., J. Gen.
Virol. 71:971-6, 1990; Bonning, B. C. et al., J. Gen. Virol.
75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, B., J. Biol.
Chem. 270:1543-9, 1995. In such transfer vector constructs, a short
or long version of the basic protein promoter can be used.
Moreover, transfer vectors can be constructed which replace the
native IL-28 or IL-29 secretory signal sequences with secretory
signal sequences derived from insect proteins. For example, a
secretory signal sequence from Ecdysteroid Glucosyltransferase
(EGT), honey bee Melittin (Invitrogen, Carlsbad, Calif.), or
baculovirus gp67 (PharMingen, San Diego, Calif.) can be used in
constructs to replace the native IL-28 or IL-29 secretory signal
sequence. In addition, transfer vectors can include an in-frame
fusion with DNA encoding an epitope tag at the C- or N-terminus of
the expressed Cysteine mutant IL-28 or IL-29 polypeptide, for
example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc.
Natl. Acad. Sci. 82:7952-4, 1985). Using techniques known in the
art, a transfer vector containing IL-28 or IL-29 is transformed
into E. Coli, and screened for bacmids which contain an interrupted
lacZ gene indicative of recombinant baculovirus. The bacmid DNA
containing the recombinant baculovirus genome is isolated, using
common techniques, and used to transfect Spodoptera frugiperda
cells, e.g. Sf9 cells. Recombinant virus that expresses IL-28 or
IL-29 is subsequently produced. Recombinant viral stocks are made
by methods commonly used the art.
[0122] The recombinant virus is used to infect host cells,
typically a cell line derived from the fall armyworm, Spodoptera
frugiperda. See, in general, Glick and Pasternak, Molecular
Biotechnology: Principles and Applications of Recombinant DNA, ASM
Press, Washington, D.C., 1994. Another suitable cell line is the
High FiveO.TM. cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435).
[0123] Fungal cells, including yeast cells, can also be used within
the present invention. Yeast species of particular interest in this
regard include Saccharomyces cerevisiae, Pichia pastoris, and
Pichia methanolica. Methods for transforming S. cerevisiae cells
with exogenous DNA and producing recombinant polypeptides therefrom
are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311;
Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No.
4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et
al., U.S. Pat. No. 4,845,075. Transformed cells are selected by
phenotype determined by the selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular
nutrient (e.g., leucine). A preferred vector system for use in
Saccharomyces cerevisiae is the POT1 vector system disclosed by
Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed
cells to be selected by growth in glucose-containing media.
Suitable promoters and terminators for use in yeast include those
from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No.
4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter,
U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also
U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533. The use of Pichia methanolica
as host for the production of recombinant proteins is disclosed in
U.S. Pat. Nos. 5,955,349, 5,888,768 and 6,001,597, U.S. Pat. No.
5,965,389, U.S. Pat. No. 5,736,383, and U.S. Pat. No.
5,854,039.
[0124] It is preferred to purify the polypeptides and proteins of
the present invention to >80% purity, more preferably to >90%
purity, even more preferably >95% purity, and particularly
preferred is a pharmaceutically pure state, that is greater than
99.9% pure with respect to contaminating macromolecules,
particularly other proteins and nucleic acids, and free of
infectious and pyrogenic agents. Preferably, a purified polypeptide
or protein is substantially free of other polypeptides or proteins,
particularly those of animal origin.
[0125] Expressed recombinant IL-28 or IL-29 proteins (including
chimeric polypeptides and multimeric proteins) are purified by
conventional protein purification methods, typically by a
combination of chromatographic techniques. See, in general,
Affinity Chromatography: Principles & Methods, Pharmacia LKB
Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein
Purification: Principles and Practice, Springer-Verlag, New York,
1994. Proteins comprising a polyhistidine affinity tag (typically
about 6 histidine residues) are purified by affinity chromatography
on a nickel chelate resin. See, for example, Houchuli et al.,
Bio/Technol. 6: 1321-1325, 1988. Proteins comprising a glu-glu tag
can be purified by immunoaffinity chromatography according to
conventional procedures. See, for example, Grussenmeyer et al.,
supra. Maltose binding protein fusions are purified on an amylose
column according to methods known in the art.
[0126] IL-28 or IL-29 polypeptides can also be prepared through
chemical synthesis according to methods known in the art, including
exclusive solid phase synthesis, partial solid phase methods,
fragment condensation or classical solution synthesis. See, for
example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et
al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical
Co., Rockford, Ill., 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3,
1986; and Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach, IRL Press, Oxford, 1989. In vitro synthesis is
particularly advantageous for the preparation of smaller
polypeptides.
[0127] Generally, the dosage of administered IL-28 or IL29
polypeptide of the present invention will vary depending upon such
factors as the patient's age, weight, height, sex, general medical
condition and previous medical history. Typically, it is desirable
to provide the recipient with a dosage of IL-28 or IL29 polypeptide
which is in the range of from about 1 pg/kg to 10 mg/kg (amount of
agent/body weight of patient), although a lower or higher dosage
also may be administered as circumstances dictate. One skilled in
the art can readily determine such dosages, and adjustments
thereto, using methods known in the art.
[0128] Administration of an IL-28 or IL29 polypeptide to a subject
can be topical, inhalant, intravenous, intraarterial,
intraperitoneal, intramuscular, subcutaneous, intrapleural,
intrathecal, by perfusion through a regional catheter, or by direct
intralesional injection. When administering therapeutic proteins by
injection, the administration may be by continuous infusion or by
single or multiple boluses.
[0129] Additional routes of administration include oral,
mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is
suitable for polyester microspheres, zein microspheres, proteinoid
microspheres, polycyanoacrylate microspheres, and lipid-based
systems (see, for example, DiBase and Morrel, "Oral Delivery of
Microencapsulated Proteins," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). The
feasibility of an intranasal delivery is exemplified by such a mode
of insulin administration (see, for example, Hinchcliffe and Illum,
Adv. Drug Deliv. Rev. 35:199 (1999)). Dry or liquid particles
comprising IL-28 or IL29 polypeptide can be prepared and inhaled
with the aid of dry-powder dispersers, liquid aerosol generators,
or nebulizers (e.g., Pettit and Gombotz, TIBTECH 16:343 (1998);
Patton et al., Adv. Drug Deliv. Rev. 35:235 (1999)). This approach
is illustrated by the AERX diabetes management system, which is a
hand-held electronic inhaler that delivers aerosolized insulin into
the lungs. Studies have shown that proteins as large as 48,000 kDa
have been delivered across skin at therapeutic concentrations with
the aid of low-frequency ultrasound, which illustrates the
feasibility of trascutaneous administration (Mitragotri et al.,
Science 269:850 (1995)). Transdermal delivery using electroporation
provides another means to administer a molecule having IL-28 or
IL29 polypeptide activity (Potts et al., Pharm. Biotechnol. 10:213
(1997)).
[0130] A pharmaceutical composition comprising a protein,
polypeptide, or peptide having IL-28 or IL29 polypeptide activity
can be formulated according to known methods to prepare
pharmaceutically useful compositions, whereby the therapeutic
proteins are combined in a mixture with a pharmaceutically
acceptable vehicle. A composition is said to be in a
"pharmaceutically acceptable vehicle" if its administration can be
tolerated by a recipient patient. Sterile phosphate-buffered saline
is one example of a pharmaceutically acceptable vehicle. Other
suitable vehicles are well-known to those in the art. See, for
example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th
Edition (Mack Publishing Company 1995).
[0131] For purposes of therapy, molecules having IL-28 or IL29
polypeptide activity and a pharmaceutically acceptable vehicle are
administered to a patient in a therapeutically effective amount. A
combination of a protein, polypeptide, or peptide having IL-28 or
IL29 polypeptide activity and a pharmaceutically acceptable vehicle
is said to be administered in a "therapeutically effective amount"
or "effective amount" if the amount administered is physiologically
significant. An agent is physiologically significant if its
presence results in a detectable change in the physiology of a
recipient patient. For example, an agent used to treat inflammation
is physiologically significant if its presence alleviates at least
a portion of the inflammatory response.
[0132] A pharmaceutical composition comprising IL-28 or IL29
polypeptide of the present invention can be furnished in liquid
form, in an aerosol, or in solid form. Liquid forms, are
illustrated by injectable solutions, aerosols, droplets,
topological solutions and oral suspensions. Exemplary solid forms
include capsules, tablets, and controlled-release forms. The latter
form is illustrated by miniosmotic pumps and implants (Bremer et
al., Pharm. Biotechnol. 10:239 (1997); Ranade, "Implants in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 95-123 (CRC Press 1995); Bremer et al., "Protein Delivery
with Infusion Pumps," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);
Yewey et al., "Delivery of Proteins from a Controlled Release
Injectable Implant," in Protein Delivery: Physical Systems, Sanders
and Hendren (eds.), pages 93-117 (Plenum Press 1997)). Other solid
forms include creams, pastes, other topological applications, and
the like.
[0133] Liposomes provide one means to deliver therapeutic
polypeptides to a subject intravenously, intraperitoneally,
intrathecally, intramuscularly, subcutaneously, or via oral
administration, inhalation, or intranasal administration. Liposomes
are microscopic vesicles that consist of one or more lipid bilayers
surrounding aqueous compartments (see, generally, Bakker-Woudenberg
et al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1):S61
(1993), Kim, Drugs 46:618 (1993), and Ranade, "Site-Specific Drug
Delivery Using Liposomes as Carriers," in Drug Delivery Systems,
Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)).
Liposomes are similar in composition to cellular membranes and as a
result, liposomes can be administered safely and are biodegradable.
Depending on the method of preparation, liposomes may be
unilamellar or multilamellar, and liposomes can vary in size with
diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A
variety of agents can be encapsulated in liposomes: hydrophobic
agents partition in the bilayers and hydrophilic agents partition
within the inner aqueous space(s) (see, for example, Machy et al.,
Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and
Ostro et al., American J. Hosp. Pharm. 46:1576 (1989)). Moreover,
it is possible to control the therapeutic availability of the
encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
[0134] Liposomes can adsorb to virtually any type of cell and then
slowly release the encapsulated agent. Alternatively, an absorbed
liposome may be endocytosed by cells that are phagocytic.
Endocytosis is followed by intralysosomal degradation of liposomal
lipids and release of the encapsulated agents (Scherphof et al.,
Ann. N.Y. Acad. Sci. 446:368 (1985)). After intravenous
administration, small liposomes (0.1 to 1.0 .mu.m) are typically
taken up by cells of the reticuloendothelial system, located
principally in the liver and spleen, whereas liposomes larger than
3.0 .mu.m are deposited in the lung. This preferential uptake of
smaller liposomes by the cells of the reticuloendothelial system
has been used to deliver chemotherapeutic agents to macrophages and
to tumors of the liver.
[0135] The reticuloendothelial system can be circumvented by
several methods including saturation with large doses of liposome
particles, or selective macrophage inactivation by pharmacological
means (Claassen et al., Biochim. Biophys. Acta 802:428 (1984)). In
addition, incorporation of glycolipid- or polyethelene
glycol-derivatized phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the
reticuloendothelial system (Allen et al., Biochim. Biophys. Acta
1068:133 (1991); Allen et al., Biochim. Biophys. Acta 1150:9
(1993)).
[0136] Liposomes can also be prepared to target particular cells or
organs by varying phospholipid composition or by inserting
receptors or ligands into the liposomes. For example, liposomes,
prepared with a high content of a nonionic surfactant, have been
used to target the liver (Hayakawa et al., Japanese Patent
04-244,018; Kato et al., Biol. Pharm. Bull. 16:960 (1993)). These
formulations were prepared by mixing soybean phospatidylcholine,
.alpha.-tocopherol, and ethoxylated hydrogenated castor oil
(HCO-60) in methanol, concentrating the mixture under vacuum, and
then reconstituting the mixture with water. A liposomal formulation
of dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived
sterylglucoside mixture (SG) and cholesterol (Ch) has also been
shown to target the liver (Shimizu et al., Biol. Pharm. Bull 20:881
(1997)).
[0137] Alternatively, various targeting ligands can be bound to the
surface of the liposome, such as antibodies, antibody fragments,
carbohydrates, vitamins, and transport proteins. For example,
liposomes can be modified with branched type galactosyllipid
derivatives to target asialoglycoprotein (galactose) receptors,
which are exclusively expressed on the surface of liver cells (Kato
and Sugiyama, Crit. Rev. Ther. Drug Carrier Syst. 14:287 (1997);
Murahashi et al., Biol. Pharm. Bull. 20:259 (1997)). Similarly, Wu
et al., Hepatology 27:772 (1998), have shown that labeling
liposomes with asialofetuin led to a shortened liposome plasma
half-life and greatly enhanced uptake of asialofetuin-labeled
liposome by hepatocytes. On the other hand, hepatic accumulation of
liposomes comprising branched type galactosyllipid derivatives can
be inhibited by preinjection of asialofetuin (Murahashi et al.,
Biol. Pharm. Bull. 120:259 (1997)). Polyaconitylated human serum
albumin liposomes provide another approach for targeting liposomes
to liver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681
(1997)). Moreover, Geho, et al U.S. Pat. No. 4,603,044, describe a
hepatocyte-directed liposome vesicle delivery system, which has
specificity for hepatobiliary receptors associated with the
specialized metabolic cells of the liver.
[0138] In a more general approach to tissue targeting, target cells
are prelabeled with biotinylated antibodies specific for a ligand
expressed by the target cell (Harasym et al., Adv. Drug Deliv. Rev.
32:99 (1998)). After plasma elimination of free antibody,
streptavidin-conjugated liposomes are administered. In another
approach, targeting antibodies are directly attached to liposomes
(Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).
[0139] Polypeptides having IL-28 or IL29 polypeptide activity can
be encapsulated within liposomes using standard techniques of
protein microencapsulation (see, for example, Anderson et al.,
Infect. Immun. 31:1099 (1981), Anderson et al., Cancer Res. 50:1853
(1990), and Cohen et al., Biochim. Biophys. Acta 1063:95 (1991),
Alving et al. "Preparation and Use of Liposomes in Immunological
Studies," in Liposome Technology, 2nd Edition, Vol. III,
Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al., Meth.
Enzymol 149:124 (1987)). As noted above, therapeutically useful
liposomes may contain a variety of components. For example,
liposomes may comprise lipid derivatives of poly(ethylene glycol)
(Allen et al., Biochim. Biophys. Acta 1150:9 (1993)).
[0140] Degradable polymer microspheres have been designed to
maintain high systemic levels of therapeutic proteins. Microspheres
are prepared from degradable polymers such as
poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho
esters), nonbiodegradable ethylvinyl acetate polymers, in which
proteins are entrapped in the polymer (Gombotz and Pettit,
Bioconjugate Chem. 6:332 (1995); Ranade, "Role of Polymers in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable
Controlled Release Systems Useful for Protein Delivery," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92
(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney
and Burke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin.
Chem. Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated
nanospheres can also provide vehicles for intravenous
administration of therapeutic proteins (see, for example, Gref et
al., Pharm. Biotechnol. 10:167 (1997)).
[0141] Other dosage forms can be devised by those skilled in the
art, as shown, for example, by Ansel and Popovich, Pharmaceutical
Dosage Forms and Drug Delivery Systems, 5.sup.th Edition (Lea &
Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences,
19.sup.th Edition (Mack Publishing Company 1995), and by Ranade and
Hollinger, Drug Delivery Systems (CRC Press 1996).
[0142] As an illustration, pharmaceutical compositions may be
supplied as a kit comprising a container that comprises an IL-28 or
IL29 polypeptide of the present invention. Therapeutic polypeptides
can be provided in the form of an injectable solution for single or
multiple doses, or as a sterile powder that will be reconstituted
before injection. Alternatively, such a kit can include a
dry-powder disperser, liquid aerosol generator, or nebulizer for
administration of a therapeutic polypeptide. Such a kit may further
comprise written information on indications and usage of the
pharmaceutical composition. Moreover, such information may include
a statement that the IL-28 or IL29 polypeptide composition is
contraindicated in patients with known hypersensitivity to IL-28 or
IL29 polypeptide. The kit may further comprise at least one
additional antiviral agent selected from the group of Interferon
alpha, Interferon beta, Interferon gamma, Interferon omega,
protease inhibitor, RNA or DNA polymerase inhibitor, nucleoside
analog, antisense inhibitor, and combinations thereof. The
additional antiviral agent included in the kit, for example, can be
RIBAVIRIN.TM., PEG-INTRON.RTM., PEGASYS.RTM., or a combination
thereof. It can also be advantageous for patients with a viral
infection, such as hepatitis C, to take their medicine consistently
and get the appropriate dose for their individualized therapy.
Thus, a kit may optionally also include a small needle, with a
self-priming feature and a large, easy-to-read dosing knob. This
will help patients feel confident that they are getting an accurate
dose and offers an easy-to-use alternative for people who may be
intimidated by a traditional needle and syringe system. For
example, the kit may include a disposable, one-time use precision
dosing system that allows patients to administer an IL-28 or IL-29
molecule of the present invention in three easy steps: Mix, Dial
and Deliver. (1) Mixing occurs by simply pushing down on the pen to
combine the IL-28 or IL-29 molecule powder with sterile water, both
of which are stored in the body of the pen; (2) Dialing allows
patients to accurately select their predetermined individualized
dose; and (3) Delivery allows patients to inject their
individualized dose of the medication (See, for example, Schering
Plough's PEG-INTRON REDIPEN).
[0143] IL-28 and IL-29 polypeptides of the present invention can be
used in treating, ablating, curing, preventing, inhibiting,
reducing, or delaying onset of liver specific diseases, in
particular liver disease where viral infection is in part an
etiologic agent. In particular IL-28 and IL-29 polypeptides 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
transcritptase/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.
[0144] 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 be 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.
[0145] Other examples of the types of viral infections for which an
IL-28 or IL-29 molecule of the present invention can be used in
treating, ablating, curing, preventing, inhibiting, reducing, or
delaying onset of viral symptoms 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); Coronavirus (causes
Severe Acute Respiratory Syndrome (SARS)); Rhinovirus, Respiratory
Syncytial Virus, Norovirus, West Nile Virus, Yellow Fever, Rift
Vallley 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 rom 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.
[0146] 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. U S A. 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.
[0147] Noroviruses (genus Norovirus, family Caliciviridae) are a
group of related, single-stranded RNA, nonenveloped viruses that
cause acute gastroenteritis in humans. Norovirus was recently
approved as the official genus name for the group of viruses
provisionally described as "Norwalk-like viruses" (NLV).
Noroviruses are estimated to cause 23 million cases of acute
gastroenteritis in the United States per year, and are the leading
cause of gastroenteritis in the United States.
[0148] The symptoms of norovirus illness usually include nausea,
vomiting, diarrhea, and some stomach cramping. Sometimes people
additionally have a low-grade fever, chills, headache, muscle
aches, and a general sense of tiredness. The illness often begins
suddenly, and the infected person may feel very sick. The illness
is usually brief, with symptoms lasting only about 1 or 2 days. In
general, children experience more vomiting than adults. Most people
with norovirus illness have both of these symptoms. Currently,
there is no antiviral medication that works against norovirus and
there is no vaccine to prevent infection.
[0149] Therapeutics to Noroviruses have been difficult to identify
in part because of a lack of good cell culture systems and animal
models of disease. The recent identification of a murine norovirus
now allows testing of therapeutics such as IL-28 and IL-29
polypeptides of the present invention in a cell culture system
(Wobus, Karst et al., "Replication of Norovirus in Cell Culture
Reveals a Tropism for Dendritic Cells and Macrophages," PLoS Biol,
2(12):e432, (2004)) and a mouse model of disease (Karst, Wobus et
al., "STAT1-dependent innate immunity to a Norwalk-like virus,"
Science, 299(5612):1575-8 (2003)).
[0150] Karst, S. M., C. E. Wobus, et al. (2003). "STAT1-dependent
innate immunity to a Norwalk-like virus." Science, 299(5612):
1575-8.
[0151] Norwalk-like caliciviruses (Noroviruses) cause over 90% of
nonbacterial epidemic gastroenteritis worldwide, but the
pathogenesis of norovirus infection is poorly understood because
these viruses do not grow in cultured cells and there is no small
animal model. Here, we report a previously unknown murine
norovirus. Analysis of Murine Norovirus 1 infection revealed that
signal transducer and activator of transcription 1-dependent innate
immunity, but not T and B cell-dependent adaptive immunity, is
essential for norovirus resistance. The identification of host
molecules essential for murine norovirus resistance may provide
targets for prevention or control of an important human
disease.
[0152] Wobus, C. E., S. M. Karst, et al. (2004). "Replication of
Norovirus in Cell Culture Reveals a Tropism for Dendritic Cells and
Macrophages." PLoS Biol, 2(12): e432.
[0153] Noroviruses are understudied because these important enteric
pathogens have not been cultured to date. We found that the
norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells
in vivo and replicates in cultured primary dendritic cells and
macrophages. MNV-1 growth was inhibited by the interferon-alphabeta
receptor and STAT-1, and was associated with extensive
rearrangements of intracellular membranes. An amino acid
substitution in the capsid protein of serially passaged MNV-1 was
associated with virulence attenuation in vivo. This is the first
report of replication of a norovirus in cell culture. The capacity
of MNV-1 to replicate in a STAT-1-regulated fashion and the
unexpected tropism of a norovirus for cells of the hematopoietic
lineage provide important insights into norovirus biology.
[0154] IL-28 and IL-29 polypeptides of the present invention 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.
[0155] In particular, IL-28 and IL-29 polypeptides of the present
invention may be useful in monotherapy or combination therapy with
IFN-.alpha., e.g., PEGASYS.RTM. or PEG-INTRON.RTM. (with or without
a nucleoside analog, such as RIBAVIRIN.TM., lamivudine, entecavir,
emtricitabine, telbivudine and tenofovir) or with a nucleoside
analog, such as RIBAVIRIN.TM., lamivudine, entecavir,
emtricitabine, telbivudine and tenofovir in patients who do not
respond well to IFN therapy.
[0156] 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. Jun. 30(6):995-1003, 1999; Mathai
et al., J Interferon Cytokine Res. Sep. 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 a nucleoside analog, such as
RIBAVIRIN.TM., lamivudine, entecavir, emtricitabine, and
telbivudine and tenofovir) or with a nucleoside analog, such as
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).
[0157] IL-28 or IL-29 polypeptide may be used in combination with
other immunotherapies including cytokines, immunoglobulin transfer,
and various co-stimulatory molecules. In addition to antiviral
drugs, IL-28 and IL-29 polypeptides of the present invention can be
used in combination with any other immunotherapy that is intended
to stimulate the immune system. Thus, IL-28 and IL-29 polypeptides
could be used with other cytokines such as Interferon, IL-21, or
IL-2. IL-28 and IL-29 can 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 and IL-29
polypeptides can 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
[0158] 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 CM, 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 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).
[0159] IL-28 and IL-29 molecules of the present invention 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. In addition, IL-28 and IL-29 molecules of the present
invention can be administered to a mammal in combination with other
antiviral agents such as ACYCLOVIR.TM., RIBAVIRIN.TM., Interferons
(e.g., PEGINTRON.RTM. and PEGASYS.RTM.), Serine Protease
Inhibitors, Polymerase Inhibitors, Nucleoside Analogs, Antisense
Inhibitors, and combinations thereof, to treat, ablate, cure,
prevent, inhibit, reduce, or delay the onset of a viral infection
selected from the group of hepatitis A, hepatitis B, hepatitis C,
hepatitis D, respiratory syncytial virus, herpes virus,
Epstein-Barr virus, influenza virus, adenovirus, parainfluenza
virus, Severe Acute Respiratory Syndrome, rhino virus, coxsackie
virus, vaccinia virus, west nile virus, dengue virus, venezuelan
equine encephalitis virus, pichinde virus, and polio virus. IL-28
and IL-29 polypeptides of the present invention can also be used in
combination with other immunotherapies including cytokines,
immunoglobulin transfer, and various co-stimulatory molecules. In
addition, IL-28 and IL-29 molecules of the present invention can be
used 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 of hepatitis A, hepatitis B, hepatitis C, and/or
hepatitis D. IL-28 and IL-29 molecules of the present invention can
also be used 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, Severe Acute Respiratory Syndrome, rhino
virus, coxsackie virus, vaccinia virus, west nile virus, dengue
virus, venezuelan equine encephalitis virus, pichinde virus and
polio virus.
[0160] The present invention is further illustrated by the
following non-limiting examples.
EXAMPLES
Example 1
Induction of IL-28A, IL-29 and IL-28B by Poly I:C and Viral
Infection
[0161] Freshly isolated human peripheral blood mononuclear cells
were grown in the presence of polyinosinic acid-polycytidylic acid
(poly I:C; 100 .quadrature.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).
[0162] Low to undetectable amounts of human IL-28A, IL-28B, and
IL-29, IFN-.quadrature. and IFN-.quadrature. 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 and IL-29 Signaling Activity Compared to IFN.alpha. in HepG2
Cells
A. Cell Transfections
[0163] 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
[0164] 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 5
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-00005 TABLE 5 Fold Induction of
Cytokine-dependent ISRE Signaling in HepG2 Cells Cytokine Fold
Induction 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
[0165] 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.
[0166] The results show the antiviral activity when IL-29 and IFN
on were tested with HepG2 cells: IL-29, IFN.quadrature. 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
[0167] 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 6-8. The normalized
values for IFNAR2 and CRF2-4 are also shown.
[0168] Table 6: B and T cells express significant levels of IL-28RA
mRNA. Low levels are seen in dendritic cells and most monocytes.
TABLE-US-00006 TABLE 6 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 CD 19+ resting 6.2
TBD TBD Hu CD 19+ 4 hr. PMA/Iono 10.6 TBD TBD Hu CD 19+ 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
[0169] As shown in Table 7, normal liver tissue and liver derived
cell lines display substantial levels of IL-28RA and CRF2-4 mRNA.
TABLE-US-00007 TABLE 7 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
4.5 4.9 7.7 adjacent tissue Liver, NAT - Normal 2.2 6.3 10.4
adjacent tissue 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
[0170] As shown in Table 8, primary airway epithelial cells contain
abundant levels of IL-28RA and CRF2-4. TABLE-US-00008 TABLE 8
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
[0171] As shown in Table 8, 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-00009
TABLE 8 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
[0172] As shown in Tables 9-13, 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-00010 TABLE 9 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
[0173] TABLE-US-00011 TABLE 10 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 #A112
2.0 0.7 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
[0174] TABLE-US-00012 TABLE 11 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+ + 24.1 108.5 52.1 121.8 Poly I/C 18
hrs CD4+/CD3+ + 47.8 83.7 16.5 40.8 PMA/Iono 18 hrs 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
[0175] TABLE-US-00013 TABLE 12 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
[0176] TABLE-US-00014 TABLE 13 SD SD SD IL- Hprt IFNAR2 28RA SD 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+ + 1.3 9.8 1.6 3.4 Poly I/C 18 hrs CD4+/CD3+ + 4.0
10.3 0.7 3.7 PMA/Iono 18 hrs 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
[0177] 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.quadrature.
or IFN-.quadrature.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-.quadrature. was used at a concentration
range from 125 ng/ml to 0.06 ng/ml. IFN-.quadrature.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 hours 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-.quadrature.2a, has no significant
effect on proliferation of Daudi cells.
Example 6
Mouse IL-28 Does Not Have Antiproliferative Effect on Mouse B
cells
[0178] 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.
[0179] 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.
[0180] These data demonstrate that mouse IL-28 unlike IFN.alpha.
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
[0181] Fresh human marrow mononuclear cells (Poietic Technologies,
Gaithersburg, Md.) were adhered to plastic for 2 hrs in
.quadrature.MEM, 10% FBS, 50 micromolar
.quadrature.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 .quadrature.MEM, 10% FBS, 50
micromolar .quadrature.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-.quadrature.2a, 10 ng/ml
IFN-.quadrature.2a or 1 ng/ml IFN-.quadrature.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.
[0182] IFN-.quadrature.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
zctoR19/CRF2-4
A. Signal Transduction Reporter Assay
[0183] 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
[0184] 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
[0185] 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 13
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-00015 TABLE 14 Percent Inhibition of Ligand-induced
Interferon Stimulated Response Element (ISRE) Signaling by Soluble
Receptors Huzcytor19- Fc/huCRF2- Huzcytor19- HuCRF2- Muzcytor19-
Ligand 4-Fc Fc 4-Fc 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
[0186] 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.
[0187] 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.
[0188] 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.
[0189] 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
[0190] 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.
[0191] 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.TM.) 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.
[0192] 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
[0193] 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.
[0194] 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.
[0195] 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
[0196] 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.
[0197] 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.
[0198] 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
[0199] Freshly isolated human peripheral blood mononuclear cells
were grown in the presence of IL-29 (20 ng/mL), IFN.quadrature.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 15 shows that IL-29 induces
Interferon Stimulated Gene Expression in human peripheral blood
mononuclear cells at all time-points tested. TABLE-US-00016 TABLE
15 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
[0200] 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 16 shows that IL-28 and IL-29 induce Interferon Stimulated
Gene expression in activated human T cells at all time-points
tested. TABLE-US-00017 TABLE 16 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
[0201] 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 17 shows that IL-28 and IL-29 induce Interferon
Stimulated Gene expression in primary human hepatocytes following
24-hour stimulation. TABLE-US-00018 TABLE 17 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
[0202] HepG2 and HuH7 cells (ATCC NOS. 8065, Manassas, Va.) were
stimulated with IL-28A (10 ng/ml), IL-29 (10 ng/ml),
IFN.quadrature.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 18 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-00019 TABLE 18 MxA Fold Pkr Fold OAS Fold
Induction Induction 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
[0203] TABLE-US-00020 TABLE 19 MxA Fold OAS Fold Pkr Fold Induction
Induction Induction MetIL-29-PEG 36.7 6.9 2.2 MetIL-29C172S-PEG
46.1 8.9 2.8
[0204] Data shown is for 20 ng/ml metIL-29-PEG and
metIL-29C172S-PEG versions of IL-29 after culture for 24 hours.
[0205] 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
[0206] 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.
[0207] 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:
[0208] IL28 alone at increasing concentrations (6)* up to 1.0
.mu.g/ml
[0209] IL29 alone at increasing concentrations (6)* up to 1.0
.mu.g/ml
[0210] PEGIL29 alone at increasing concentrations (6)* up to 1.0
.mu.g/ml
[0211] IFN.quadrature.2A alone at 0.3, 1.0, and 3.0 IU/ml
[0212] Ribavirin alone.
[0213] The positive control is IFN.alpha. and the negative control
is ribavirin.
[0214] The cells are stained after 72 hours with Alomar Blue to
assess viability.
[0215] *The concentrations for conditions 1-3 are:
[0216] .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.
[0217] 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
[0218] 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 (log 10
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-00021 TABLE 20 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-29 >10 .mu.g/ml
>10 .mu.g/ml 0 C172S- 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-29
>10 .mu.g/ml >10 .mu.g/ml 0 virus C172S- 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-29 >10 .mu.g/ml >10
.mu.g/ml 0 syncytial C172S- 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-29 >10 .mu.g/ml >10 .mu.g/ml 0
C172S- 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-29 >10 .mu.g/ml >10 .mu.g/ml 0 C172S- 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-29 >10 .mu.g/ml >10 .mu.g/ml 0 B4 virus C172S- 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-29 >10 .mu.g/ml >10 .mu.g/ml 0 (type A Darby
C172S- [H3N2]) Canine PEG 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-29
0.045 .mu.g/ml >10 .mu.g/ml >222 (type A C172S- [H3N2]) PEG
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-29 >10 .mu.g/ml >10 .mu.g/ml 0 virus
C172S- 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-29 0.001 .mu.g/ml
>10 .mu.g/ml >10,000 virus C172S- 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-29 0.0075 .mu.g/ml >10 .mu.g/ml >1330 C172S- 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-29 0.0065 .mu.g/ml >10 .mu.g/ml >1538 equine C172S-
encephalitis PEG 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-29 >10 .mu.g/ml >10
.mu.g/ml 0 virus C172S- 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-29 >10 .mu.g/ml >10
.mu.g/ml 0 C172S- PEG
[0219] TABLE-US-00022 TABLE 21 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-29 >10 .mu.g/ml >10
.mu.g/ml 0 C172S- 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-29 >10
.mu.g/ml >10 .mu.g/ml 0 virus C172S- 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-29 5.47 .mu.g/ml >10
.mu.g/ml >2 syncytial virus C172S- 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-29 >10 .mu.g/ml >10 .mu.g/ml 0
C172S- 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-29 2.051 .mu.g/ml >10 .mu.g/ml >5 C172S- 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-29 >10 .mu.g/ml >10 .mu.g/ml 0 virus
C172S- 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-29 >10 .mu.g/ml >10
.mu.g/ml 0 A [H3N2]) Darby C172S- Canine PEG 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-29 1.4 .mu.g/ml >10 .mu.g/ml
>7 A [H3N2]) C172S- 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-29 >10
.mu.g/ml >10 .mu.g/ml 0 C172S- 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-29 0.00037 .mu.g/ml >10 .mu.g/ml >27,000 C172S-
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-29 0.06 .mu.g/ml >10 .mu.g/ml >166
C172S- 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-29 0.02 .mu.g/ml >10 .mu.g/ml >500
equine C172S- encephalitis PEG 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-29 >10
.mu.g/ml >10 .mu.g/ml 0 C172S- 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-29 >10
.mu.g/ml >10 .mu.g/ml 0 C172S- 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
[0220] 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.quadrature.1-4 and IFN.quadrature.) 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.
[0221] 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.quadrature. (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.TM. (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.
[0222] 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-00023 TABLE 22 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
[0223] 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
[0224] ng of HPRT mRNA housekeeping gene, HPRT
[0225] As an example, the data for the 48 hour time point is shown.
TABLE-US-00024 TABLE 23 AML12's Mx1 Fold OAS Fold Pkr Fold
Induction Induction Induction MetIL-29-PEG 728 614 8
MetIL-29C172S-PEG 761 657 8
[0226] Cells were stimulated with 20 ng/ml metIL-29-PEG or
metIL-29C172S-PEG for 24 hours.
[0227] 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
[0228] 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.
[0229] 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.TM. (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.
[0230] 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-00025 TABLE 24 Mice OAS PkR Mx1 Non-Tg 4.5 4.5 3.5 IL-29
Tg 12 8 21
[0231] 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
[0232] 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.
[0233] 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.
[0234] 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-00026 TABLE 25 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
[0235] 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-00027
TABLE 26 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
[0236] TABLE-US-00028 TABLE 27 MetIL-29-PEG MetIL-29C172S-PEG Naive
3 hr 6 hr 12 hr 24 hr 3 hr 6 hr 12 hr 24 hr 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
[0237] Mice were injected with 100 .mu.g of proteins i.v. Data
shown is fold 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
[0238] 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.
[0239] 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).
[0240] 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-00029 TABLE 28 Injection OAS-1 hr OAS-6 hr OAS-24 hr
OAS-48 hr None 80 80 80 80 IL-29 80 80 180 200
[0241] OAS activity is shown at pmol/dL of plasma for a single
concentration (250 .mu.g) of human IL-29.
Example 29
IL-28 and IL-29 Inhibit Adenoviral Pathology in Mice
[0242] 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.
[0243] C57B1/6 mice were given once daily intraperitoneal
injections of 50 .mu.g mouse IL-28 (zcyto24) 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.
[0244] 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
[0245] Lymphocytic choriomeningitis virus (LCMV) infections in mice
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;
Blattmank 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.
[0246] 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.
[0247] 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
[0248] 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:
[0249] Animals: 6 weeks-old female BALB/c mice (Charles River) with
148 mice, 30 per group.
[0250] Groups:
[0251] Absolute control (not infected) to run in parallel for
antibody titre and histopathology (2 animals per group)
[0252] Vehicle (i.p.) saline
[0253] Amantadine (positive control) 10 mg/day during 5 days (per
os) starting 2 hours before infection
[0254] IL-28 or IL-29 treated (5 .mu.g, i.p. starting 2 hours after
infection)
[0255] IL-28 or IL-29 (25 .mu.g, i.p. starting 2 hours after
infection)
[0256] IL-28 or IL-29 (125 .mu.g, i.p. starting 2 hours after
infection)
[0257] Day 0--Except for the absolute controls, all animals
infected with Influenza virus
[0258] For viral load (10 at LD50)
[0259] For immunology workout (LD30)
[0260] Day 0-9--daily injections of IL-28 or IL-29 (i.p.)
[0261] Body weight and general appearance recorded (3
times/week)
[0262] Day 3--sacrifice of 8 animals per group
[0263] Viral load in right lung (TCID50)
[0264] Histopathology in left lung
[0265] Blood sample for antibody titration
[0266] 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.
[0267] Study No. 2
[0268] 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).
[0269] Group 1: Vehicle (i.p.)
[0270] Group 2: Positive control: Anti-influenza neutralizing
antibody (goat anti-influenza A/USSR (HINI) (Chemicon
International, Temecula, Calif.); 40 .mu.g/mouse at 2 h and 4 h
post infection (10 .mu.l intranasal)
[0271] Group 3: IL-28 or IL-29 (5 .mu.g, i.p.)
[0272] Group 4: IL-28 or IL-29 (25 .mu.g, i.p.)
[0273] Group 5: IL-28 or IL-29 (125 .mu.g, i.p.)
[0274] Following-life observations and immunological workouts are
prepared:
[0275] Day 0--all animals infected with Influenza virus (dose
determined in experiment 2)
[0276] Day 0-9--daily injections of IL-28 or IL-29 (i.p.)
[0277] Body weight and general appearance recorded every other
day
[0278] Day 10--sacrifice of surviving animals and perform viral
assay to determine viral load in lung.
[0279] 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).
[0280] 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 (ASNENMETM), (LMCV peptide/D.sup.b).
[0281] Quantitative immunophenotyping of the following: CD8,
tetramer, intracellular IFN.quadrature., NK1.1, CD8, tetramer,
CD62L, CD44, CD3(+ or -), NK1.1(+), intracellular
IFN.quadrature.CD4, CD8, NK1.1, DX5, CD3 (+ or -), NK1.1, DX5,
tetramer, Single colour samples for cytometer adjustment.
[0282] Survival/Re-Challenge Study
[0283] 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.
[0284] Re-Challenge Study:
[0285] Day 0: Both groups will be infected with A/PR virus
(1LD30).
[0286] Group 6 will not be treated.
[0287] Group 7 will be treated for 9 days with 125 .mu.g of IL-28
or IL-29.
[0288] Day 30: Survival study
[0289] Body weight and antibody production in individual serum
samples (Total, IgG1, IgG2a, IgG2b) are measured.
[0290] Day 60: Re-challenge study
[0291] Survivors in each group will be divided into 2 subgroups
[0292] Group 6A and 7A will be re-challenge with A/PR virus (1
LD30)
[0293] Group 6B and 7B will be re-challenge with A/PR virus (1
LD30).
[0294] 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
[0295] 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.
[0296] 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.
[0297] 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
[0298] 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.
[0299] 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.
[0300] 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)
[0301] 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 (IC.sub.50)
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)
[0302] 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.
[0303] 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.quadrature. in the assay.
[0304] The complete disclosure of all patents, patent applications,
and publications, and electronically available material (e.g.,
GenBank amino acid and nucleotide sequence submissions) cited
herein are incorporated by reference. The foregoing detailed
description and examples have been given for clarity of
understanding only. No unnecessary limitations are to be understood
therefrom. The invention is not limited to the exact details shown
and described, for variations obvious to one skilled in the art
will be included within the invention defined by the claims.
Sequence CWU 1
1
136 1 618 DNA Homo sapiens CDS (1)...(618) misc_feature (0)...(0)
IL-28A 1 atg act ggg gac tgc acg cca gtg ctg gtg ctg atg gcc gca
gtg ctg 48 Met Thr Gly Asp Cys Thr Pro Val Leu Val Leu Met Ala Ala
Val Leu 1 5 10 15 acc gtg act gga gca gtt cct gtc gcc agg ctc cac
ggg gct ctc ccg 96 Thr Val Thr Gly Ala Val Pro Val Ala Arg Leu His
Gly Ala Leu Pro 20 25 30 gat gca agg ggc tgc cac ata gcc cag ttc
aag tcc ctg tct cca cag 144 Asp Ala Arg Gly Cys His Ile Ala Gln Phe
Lys Ser Leu Ser Pro Gln 35 40 45 gag ctg cag gcc ttt aag agg gcc
aaa gat gcc tta gaa gag tcg ctt 192 Glu Leu Gln Ala Phe Lys Arg Ala
Lys Asp Ala Leu Glu Glu Ser Leu 50 55 60 ctg ctg aag gac tgc agg
tgc cac tcc cgc ctc ttc ccc agg acc tgg 240 Leu Leu Lys Asp Cys Arg
Cys His Ser Arg Leu Phe Pro Arg Thr Trp 65 70 75 80 gac ctg agg cag
ctg cag gtg agg gag cgc ccc atg gct ttg gag gct 288 Asp Leu Arg Gln
Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala 85 90 95 gag ctg
gcc ctg acg ctg aag gtt ctg gag gcc acc gct gac act gac 336 Glu Leu
Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp 100 105 110
cca gcc ctg gtg gac gtc ttg gac cag ccc ctt cac acc ctg cac cat 384
Pro Ala Leu Val Asp Val Leu Asp Gln Pro Leu His Thr Leu His His 115
120 125 atc ctc tcc cag ttc cgg gcc tgt gtg agt cgt cag ggc ctg ggc
acc 432 Ile Leu Ser Gln Phe Arg Ala Cys Val Ser Arg Gln Gly Leu Gly
Thr 130 135 140 cag atc cag cct cag ccc acg gca ggg ccc agg acc cgg
ggc cgc ctc 480 Gln Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg
Gly Arg Leu 145 150 155 160 cac cat tgg ctg tac cgg ctc cag gag gcc
cca aaa aag gag tcc cct 528 His His Trp Leu Tyr Arg Leu Gln Glu Ala
Pro Lys Lys Glu Ser Pro 165 170 175 ggc tgc ctc gag gcc tct gtc acc
ttc aac ctc ttc cgc ctc ctc acg 576 Gly Cys Leu Glu Ala Ser Val Thr
Phe Asn Leu Phe Arg Leu Leu Thr 180 185 190 cga gac ctg aat tgt gtt
gcc agt ggg gac ctg tgt gtc tga 618 Arg Asp Leu Asn Cys Val Ala Ser
Gly Asp Leu Cys Val * 195 200 205 2 205 PRT Homo sapiens 2 Met Thr
Gly Asp Cys Thr Pro Val Leu Val Leu Met Ala Ala Val Leu 1 5 10 15
Thr Val Thr Gly Ala Val Pro Val Ala Arg Leu His Gly Ala Leu Pro 20
25 30 Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro
Gln 35 40 45 Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu
Glu Ser Leu 50 55 60 Leu Leu Lys Asp Cys Arg Cys His Ser Arg Leu
Phe Pro Arg Thr Trp 65 70 75 80 Asp Leu Arg Gln Leu Gln Val Arg Glu
Arg Pro Met Ala Leu Glu Ala 85 90 95 Glu Leu Ala Leu Thr Leu Lys
Val Leu Glu Ala Thr Ala Asp Thr Asp 100 105 110 Pro Ala Leu Val Asp
Val Leu Asp Gln Pro Leu His Thr Leu His His 115 120 125 Ile Leu Ser
Gln Phe Arg Ala Cys Val Ser Arg Gln Gly Leu Gly Thr 130 135 140 Gln
Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu 145 150
155 160 His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser
Pro 165 170 175 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg
Leu Leu Thr 180 185 190 Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu
Cys Val 195 200 205 3 603 DNA Homo sapiens CDS (1)...(603)
misc_feature (0)...(0) IL-29 3 atg gct gca gct tgg acc gtg gtg ctg
gtg act ttg gtg cta ggc ttg 48 Met Ala Ala Ala Trp Thr Val Val Leu
Val Thr Leu Val Leu Gly Leu 1 5 10 15 gcc gtg gca ggc cct gtc ccc
act tcc aag ccc acc aca act ggg aag 96 Ala Val Ala Gly Pro Val Pro
Thr Ser Lys Pro Thr Thr Thr Gly Lys 20 25 30 ggc tgc cac att ggc
agg ttc aaa tct ctg tca cca cag gag cta gcg 144 Gly Cys His Ile Gly
Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala 35 40 45 agc ttc aag
aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa 192 Ser Phe Lys
Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys 50 55 60 aac
tgg agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg 240 Asn
Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg 65 70
75 80 ctt ctc cag gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg
gcc 288 Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu
Ala 85 90 95 ctg acg ctg aag gtc ctg gag gcc gct gct ggc cca gcc
ctg gag gac 336 Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala
Leu Glu Asp 100 105 110 gtc cta gac cag ccc ctt cac acc ctg cac cac
atc ctc tcc cag ctc 384 Val Leu Asp Gln Pro Leu His Thr Leu His His
Ile Leu Ser Gln Leu 115 120 125 cag gcc tgt atc cag cct cag ccc aca
gca ggg ccc agg ccc cgg ggc 432 Gln Ala Cys Ile Gln Pro Gln Pro Thr
Ala Gly Pro Arg Pro Arg Gly 130 135 140 cgc ctc cac cac tgg ctg cac
cgg ctc cag gag gcc ccc aaa aag gag 480 Arg Leu His His Trp Leu His
Arg Leu Gln Glu Ala Pro Lys Lys Glu 145 150 155 160 tcc gct ggc tgc
ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc 528 Ser Ala Gly Cys
Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 165 170 175 ctc acg
cga gac ctc aaa tat gtg gcc gat ggg gac ctg tgt ctg aga 576 Leu Thr
Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg 180 185 190
acg tca acc cac cct gag tcc acc tga 603 Thr Ser Thr His Pro Glu Ser
Thr * 195 200 4 200 PRT Homo sapiens 4 Met Ala Ala Ala Trp Thr Val
Val Leu Val Thr Leu Val Leu Gly Leu 1 5 10 15 Ala Val Ala Gly Pro
Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys 20 25 30 Gly Cys His
Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala 35 40 45 Ser
Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys 50 55
60 Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg
65 70 75 80 Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu
Leu Ala 85 90 95 Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro
Ala Leu Glu Asp 100 105 110 Val Leu Asp Gln Pro Leu His Thr Leu His
His Ile Leu Ser Gln Leu 115 120 125 Gln Ala Cys Ile Gln Pro Gln Pro
Thr Ala Gly Pro Arg Pro Arg Gly 130 135 140 Arg Leu His His Trp Leu
His Arg Leu Gln Glu Ala Pro Lys Lys Glu 145 150 155 160 Ser Ala Gly
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 165 170 175 Leu
Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg 180 185
190 Thr Ser Thr His Pro Glu Ser Thr 195 200 5 615 DNA Homo sapiens
CDS (1)...(615) misc_feature (0)...(0) IL-28B 5 atg acc ggg gac tgc
atg cca gtg ctg gtg ctg atg gcc gca gtg ctg 48 Met Thr Gly Asp Cys
Met Pro Val Leu Val Leu Met Ala Ala Val Leu 1 5 10 15 acc gtg act
gga gca gtt cct gtc gcc agg ctc cgc ggg gct ctc ccg 96 Thr Val Thr
Gly Ala Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro 20 25 30 gat
gca agg ggc tgc cac ata gcc cag ttc aag tcc ctg tct cca cag 144 Asp
Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln 35 40
45 gag ctg cag gcc ttt aag agg gcc aaa gat gcc tta gaa gag tcg ctt
192 Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu
50 55 60 ctg ctg aag gac tgc aag tgc cgc tcc cgc ctc ttc ccc agg
acc tgg 240 Leu Leu Lys Asp Cys Lys Cys Arg Ser Arg Leu Phe Pro Arg
Thr Trp 65 70 75 80 gac ctg agg cag ctg cag gtg agg gag cgc ccc gtg
gct ttg gag gct 288 Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val
Ala Leu Glu Ala 85 90 95 gag ctg gcc ctg acg ctg aag gtt ctg gag
gcc acc gct gac act gac 336 Glu Leu Ala Leu Thr Leu Lys Val Leu Glu
Ala Thr Ala Asp Thr Asp 100 105 110 cca gcc ctg ggg gat gtc ttg gac
cag ccc ctt cac acc ctg cac cat 384 Pro Ala Leu Gly Asp Val Leu Asp
Gln Pro Leu His Thr Leu His His 115 120 125 atc ctc tcc cag ctc cgg
gcc tgt gtg agt cgt cag ggc ccg ggc acc 432 Ile Leu Ser Gln Leu Arg
Ala Cys Val Ser Arg Gln Gly Pro Gly Thr 130 135 140 cag atc cag cct
cag ccc acg gca ggg ccc agg acc cgg ggc cgc ctc 480 Gln Ile Gln Pro
Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu 145 150 155 160 cac
cat tgg ctg cac cgg ctc cag gag gcc cca aaa aag gag tcc cct 528 His
His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro 165 170
175 ggc tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc ctc ctc acg
576 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr
180 185 190 cga gac ctg aat tgt gtt gcc agc ggg gac ctg tgt gtc 615
Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 195 200 205 6
205 PRT Homo sapiens 6 Met Thr Gly Asp Cys Met Pro Val Leu Val Leu
Met Ala Ala Val Leu 1 5 10 15 Thr Val Thr Gly Ala Val Pro Val Ala
Arg Leu Arg Gly Ala Leu Pro 20 25 30 Asp Ala Arg Gly Cys His Ile
Ala Gln Phe Lys Ser Leu Ser Pro Gln 35 40 45 Glu Leu Gln Ala Phe
Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu 50 55 60 Leu Leu Lys
Asp Cys Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp 65 70 75 80 Asp
Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala 85 90
95 Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp
100 105 110 Pro Ala Leu Gly Asp Val Leu Asp Gln Pro Leu His Thr Leu
His His 115 120 125 Ile Leu Ser Gln Leu Arg Ala Cys Val Ser Arg Gln
Gly Pro Gly Thr 130 135 140 Gln Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Thr Arg Gly Arg Leu 145 150 155 160 His His Trp Leu His Arg Leu
Gln Glu Ala Pro Lys Lys Glu Ser Pro 165 170 175 Gly Cys Leu Glu Ala
Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 180 185 190 Arg Asp Leu
Asn Cys Val Ala Ser Gly Asp Leu Cys Val 195 200 205 7 633 DNA Mus
musculus CDS (22)...(630) 7 tcacagaccc 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 10 ctc ctc ctg ctg ttg cct ctg ctg ctg gcc gca gtg ctg aca aga
acc 99 Leu Leu Leu Leu Leu Pro Leu Leu Leu Ala Ala Val Leu Thr Arg
Thr 15 20 25 caa gct gac cct gtc ccc agg gcc acc agg ctc cca gtg
gaa gca aag 147 Gln Ala Asp Pro Val Pro Arg Ala Thr Arg Leu Pro Val
Glu Ala Lys 30 35 40 gat tgc cac att gct cag ttc aag tct ctg tcc
cca aaa gag ctg cag 195 Asp Cys His Ile Ala Gln Phe Lys Ser Leu Ser
Pro Lys Glu Leu Gln 45 50 55 gcc ttc aaa aag gcc aag gat gcc atc
gag aag agg ctg ctt gag aag 243 Ala Phe Lys Lys Ala Lys Asp Ala Ile
Glu Lys Arg Leu Leu Glu Lys 60 65 70 gac ctg agg tgc agt tcc cac
ctc ttc ccc agg gcc tgg gac ctg aag 291 Asp Leu Arg Cys Ser Ser His
Leu Phe Pro Arg Ala Trp Asp Leu Lys 75 80 85 90 cag ctg cag gtc caa
gag cgc ccc aag gcc ttg cag gct gag gtg gcc 339 Gln Leu Gln Val Gln
Glu Arg Pro Lys Ala Leu Gln Ala Glu Val Ala 95 100 105 ctg acc ctg
aag gtc tgg gag aac atg act gac tca gcc ctg gcc acc 387 Leu Thr Leu
Lys Val Trp Glu Asn Met Thr Asp Ser Ala Leu Ala Thr 110 115 120 atc
ctg ggc cag cct ctt cat aca ctg agc cac att cac tcc cag ctg 435 Ile
Leu Gly Gln Pro Leu His Thr Leu Ser His Ile His Ser Gln Leu 125 130
135 cag acc tgt aca cag ctt cag gcc aca gca gag ccc agg tcc ccg agc
483 Gln Thr Cys Thr Gln Leu Gln Ala Thr Ala Glu Pro Arg Ser Pro Ser
140 145 150 cgc cgc ctc tcc cgc tgg ctg cac agg ctc cag gag gcc cag
agc aag 531 Arg Arg Leu Ser Arg Trp Leu His Arg Leu Gln Glu Ala Gln
Ser Lys 155 160 165 170 gag acc cct ggc tgc ctg gag gcc tct gtc acc
tcc aac ctg ttt cgc 579 Glu Thr Pro Gly Cys Leu Glu Ala Ser Val Thr
Ser Asn Leu Phe Arg 175 180 185 ctg ctc acc cgg gac ctc aag tgt gtg
gcc aat gga gac cag tgt gtc 627 Leu Leu Thr Arg Asp Leu Lys Cys Val
Ala Asn Gly Asp Gln Cys Val 190 195 200 tga cct 633 * 8 202 PRT Mus
musculus 8 Met Lys Pro Glu Thr Ala Gly Gly His Met Leu Leu Leu Leu
Leu Pro 1 5 10 15 Leu Leu Leu Ala Ala Val Leu Thr Arg Thr Gln Ala
Asp Pro Val Pro 20 25 30 Arg Ala Thr Arg Leu Pro Val Glu Ala Lys
Asp Cys His Ile Ala Gln 35 40 45 Phe Lys Ser Leu Ser Pro Lys Glu
Leu Gln Ala Phe Lys Lys Ala Lys 50 55 60 Asp Ala Ile Glu Lys Arg
Leu Leu Glu Lys Asp Leu Arg Cys Ser Ser 65 70 75 80 His Leu Phe Pro
Arg Ala Trp Asp Leu Lys Gln Leu Gln Val Gln Glu 85 90 95 Arg Pro
Lys Ala Leu Gln Ala Glu Val Ala Leu Thr Leu Lys Val Trp 100 105 110
Glu Asn Met Thr Asp Ser Ala Leu Ala Thr Ile Leu Gly Gln Pro Leu 115
120 125 His Thr Leu Ser His Ile His Ser Gln Leu Gln Thr Cys Thr Gln
Leu 130 135 140 Gln Ala Thr Ala Glu Pro Arg Ser Pro Ser Arg Arg Leu
Ser Arg Trp 145 150 155 160 Leu His Arg Leu Gln Glu Ala Gln Ser Lys
Glu Thr Pro Gly Cys Leu 165 170 175 Glu Ala Ser Val Thr Ser Asn Leu
Phe Arg Leu Leu Thr Arg Asp Leu 180 185 190 Lys Cys Val Ala Asn Gly
Asp Gln Cys Val 195 200 9 632 DNA Mus musculus CDS (22)...(630) 9
tcacagaccc 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 10 ctc ctc ctg ctg ttg
cct ctg ctg ctg gcc gca gtg ctg aca aga acc 99 Leu Leu Leu Leu Leu
Pro Leu Leu Leu Ala Ala Val Leu Thr Arg Thr 15 20 25 caa gct gac
cct gtc ccc agg gcc acc agg ctc cca gtg gaa gca aag 147 Gln Ala Asp
Pro Val Pro Arg Ala Thr Arg Leu Pro Val Glu Ala Lys 30 35 40 gat
tgc cac att gct cag ttc aag tct ctg tcc cca aaa gag ctg cag 195 Asp
Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Lys Glu Leu Gln 45 50
55 gcc ttc aaa aag gcc aag ggt gcc atc gag aag agg ctg ctt gag aag
243 Ala Phe Lys Lys Ala Lys Gly Ala Ile Glu Lys Arg Leu Leu Glu Lys
60 65 70 gac atg agg tgc agt tcc cac ctc atc tcc agg gcc tgg gac
ctg aag 291 Asp Met Arg Cys Ser Ser His Leu Ile Ser Arg Ala Trp Asp
Leu Lys 75 80 85 90 cag ctg cag gtc caa gag cgc ccc aag gcc ttg cag
gct gag gtg gcc 339 Gln Leu Gln Val Gln Glu Arg Pro Lys Ala Leu Gln
Ala Glu Val Ala 95 100 105 ctg acc ctg aag gtc tgg gag aac ata aat
gac tca gcc ctg acc acc 387 Leu Thr Leu Lys Val Trp Glu Asn Ile Asn
Asp Ser Ala Leu Thr Thr 110 115 120 atc ctg ggc cag cct ctt cat aca
ctg agc cac att cac tcc cag ctg 435 Ile Leu Gly Gln Pro Leu His Thr
Leu Ser His Ile His Ser Gln Leu 125 130 135 cag acc tgt aca cag ctt
cag gcc aca gca gag
ccc aag ccc ccg agt 483 Gln Thr Cys Thr Gln Leu Gln Ala Thr Ala Glu
Pro Lys Pro Pro Ser 140 145 150 cgc cgc ctc tcc cgc tgg ctg cac agg
ctc cag gag gcc cag agc aag 531 Arg Arg Leu Ser Arg Trp Leu His Arg
Leu Gln Glu Ala Gln Ser Lys 155 160 165 170 gag act cct ggc tgc ctg
gag gac tct gtc acc tcc aac ctg ttt caa 579 Glu Thr Pro Gly Cys Leu
Glu Asp Ser Val Thr Ser Asn Leu Phe Gln 175 180 185 ctg ctc ctc cgg
gac ctc aag tgt gtg gcc agt gga gac cag tgt gtc 627 Leu Leu Leu Arg
Asp Leu Lys Cys Val Ala Ser Gly Asp Gln Cys Val 190 195 200 tga cc
632 * 10 202 PRT Mus musculus 10 Met Lys Pro Glu Thr Ala Gly Gly
His Met Leu Leu Leu Leu Leu Pro 1 5 10 15 Leu Leu Leu Ala Ala Val
Leu Thr Arg Thr Gln Ala Asp Pro Val Pro 20 25 30 Arg Ala Thr Arg
Leu Pro Val Glu Ala Lys Asp Cys His Ile Ala Gln 35 40 45 Phe Lys
Ser Leu Ser Pro Lys Glu Leu Gln Ala Phe Lys Lys Ala Lys 50 55 60
Gly Ala Ile Glu Lys Arg Leu Leu Glu Lys Asp Met Arg Cys Ser Ser 65
70 75 80 His Leu Ile Ser Arg Ala Trp Asp Leu Lys Gln Leu Gln Val
Gln Glu 85 90 95 Arg Pro Lys Ala Leu Gln Ala Glu Val Ala Leu Thr
Leu Lys Val Trp 100 105 110 Glu Asn Ile Asn Asp Ser Ala Leu Thr Thr
Ile Leu Gly Gln Pro Leu 115 120 125 His Thr Leu Ser His Ile His Ser
Gln Leu Gln Thr Cys Thr Gln Leu 130 135 140 Gln Ala Thr Ala Glu Pro
Lys Pro Pro Ser Arg Arg Leu Ser Arg Trp 145 150 155 160 Leu His Arg
Leu Gln Glu Ala Gln Ser Lys Glu Thr Pro Gly Cys Leu 165 170 175 Glu
Asp Ser Val Thr Ser Asn Leu Phe Gln Leu Leu Leu Arg Asp Leu 180 185
190 Lys Cys Val Ala Ser Gly Asp Gln Cys Val 195 200 11 1563 DNA
Homo sapiens CDS (1)...(1563) misc_feature (0)...(0) IL-28RA 11 atg
gcg ggg ccc gag cgc tgg ggc ccc ctg ctc ctg tgc ctg ctg cag 48 Met
Ala Gly Pro Glu Arg Trp Gly Pro Leu Leu Leu Cys Leu Leu Gln 1 5 10
15 gcc gct cca ggg agg ccc cgt ctg gcc cct ccc cag aat gtg acg ctg
96 Ala Ala Pro Gly Arg Pro Arg Leu Ala Pro Pro Gln Asn Val Thr Leu
20 25 30 ctc tcc cag aac ttc agc gtg tac ctg aca tgg ctc cca ggg
ctt ggc 144 Leu Ser Gln Asn Phe Ser Val Tyr Leu Thr Trp Leu Pro Gly
Leu Gly 35 40 45 aac ccc cag gat gtg acc tat ttt gtg gcc tat cag
agc tct ccc acc 192 Asn Pro Gln Asp Val Thr Tyr Phe Val Ala Tyr Gln
Ser Ser Pro Thr 50 55 60 cgt aga cgg tgg cgc gaa gtg gaa gag tgt
gcg gga acc aag gag ctg 240 Arg Arg Arg Trp Arg Glu Val Glu Glu Cys
Ala Gly Thr Lys Glu Leu 65 70 75 80 cta tgt tct atg atg tgc ctg aag
aaa cag gac ctg tac aac aag ttc 288 Leu Cys Ser Met Met Cys Leu Lys
Lys Gln Asp Leu Tyr Asn Lys Phe 85 90 95 aag gga cgc gtg cgg acg
gtt tct ccc agc tcc aag tcc ccc tgg gtg 336 Lys Gly Arg Val Arg Thr
Val Ser Pro Ser Ser Lys Ser Pro Trp Val 100 105 110 gag tcc gaa tac
ctg gat tac ctt ttt gaa gtg gag ccg gcc cca cct 384 Glu Ser Glu Tyr
Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro 115 120 125 gtc ctg
gtg ctc acc cag acg gag gag atc ctg agt gcc aat gcc acg 432 Val Leu
Val Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn Ala Thr 130 135 140
tac cag ctg ccc ccc tgc atg ccc cca ctg gat ctg aag tat gag gtg 480
Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Asp Leu Lys Tyr Glu Val 145
150 155 160 gca ttc tgg aag gag ggg gcc gga aac aag acc cta ttt cca
gtc act 528 Ala Phe Trp Lys Glu Gly Ala Gly Asn Lys Thr Leu Phe Pro
Val Thr 165 170 175 ccc cat ggc cag cca gtc cag atc act ctc cag cca
gct gcc agc gaa 576 Pro His Gly Gln Pro Val Gln Ile Thr Leu Gln Pro
Ala Ala Ser Glu 180 185 190 cac cac tgc ctc agt gcc aga acc atc tac
acg ttc agt gtc ccg aaa 624 His His Cys Leu Ser Ala Arg Thr Ile Tyr
Thr Phe Ser Val Pro Lys 195 200 205 tac agc aag ttc tct aag ccc acc
tgc ttc ttg ctg gag gtc cca gaa 672 Tyr Ser Lys Phe Ser Lys Pro Thr
Cys Phe Leu Leu Glu Val Pro Glu 210 215 220 gcc aac tgg gct ttc ctg
gtg ctg cca tcg ctt ctg ata ctg ctg tta 720 Ala Asn Trp Ala Phe Leu
Val Leu Pro Ser Leu Leu Ile Leu Leu Leu 225 230 235 240 gta att gcc
gca ggg ggt gtg atc tgg aag acc ctc atg ggg aac ccc 768 Val Ile Ala
Ala Gly Gly Val Ile Trp Lys Thr Leu Met Gly Asn Pro 245 250 255 tgg
ttt cag cgg gca aag atg cca cgg gcc ctg gac ttt tct gga cac 816 Trp
Phe Gln Arg Ala Lys Met Pro Arg Ala Leu Asp Phe Ser Gly His 260 265
270 aca cac cct gtg gca acc ttt cag ccc agc aga cca gag tcc gtg aat
864 Thr His Pro Val Ala Thr Phe Gln Pro Ser Arg Pro Glu Ser Val Asn
275 280 285 gac ttg ttc ctc tgt ccc caa aag gaa ctg acc aga ggg gtc
agg ccg 912 Asp Leu Phe Leu Cys Pro Gln Lys Glu Leu Thr Arg Gly Val
Arg Pro 290 295 300 acg cct cga gtc agg gcc cca gcc acc caa cag aca
aga tgg aag aag 960 Thr Pro Arg Val Arg Ala Pro Ala Thr Gln Gln Thr
Arg Trp Lys Lys 305 310 315 320 gac ctt gca gag gac gaa gag gag gag
gat gag gag gac aca gaa gat 1008 Asp Leu Ala Glu Asp Glu Glu Glu
Glu Asp Glu Glu Asp Thr Glu Asp 325 330 335 ggc gtc agc ttc cag ccc
tac att gaa cca cct tct ttc ctg ggg caa 1056 Gly Val Ser Phe Gln
Pro Tyr Ile Glu Pro Pro Ser Phe Leu Gly Gln 340 345 350 gag cac cag
gct cca ggg cac tcg gag gct ggt ggg gtg gac tca ggg 1104 Glu His
Gln Ala Pro Gly His Ser Glu Ala Gly Gly Val Asp Ser Gly 355 360 365
agg ccc agg gct cct ctg gtc cca agc gaa ggc tcc tct gct tgg gat
1152 Arg Pro Arg Ala Pro Leu Val Pro Ser Glu Gly Ser Ser Ala Trp
Asp 370 375 380 tct tca gac aga agc tgg gcc agc act gtg gac tcc tcc
tgg gac agg 1200 Ser Ser Asp Arg Ser Trp Ala Ser Thr Val Asp Ser
Ser Trp Asp Arg 385 390 395 400 gct ggg tcc tct ggc tat ttg gct gag
aag ggg cca ggc caa ggg ccg 1248 Ala Gly Ser Ser Gly Tyr Leu Ala
Glu Lys Gly Pro Gly Gln Gly Pro 405 410 415 ggt ggg gat ggg cac caa
gaa tct ctc cca cca cct gaa ttc tcc aag 1296 Gly Gly Asp Gly His
Gln Glu Ser Leu Pro Pro Pro Glu Phe Ser Lys 420 425 430 gac tcg ggt
ttc ctg gaa gag ctc cca gaa gat aac ctc tcc tcc tgg 1344 Asp Ser
Gly Phe Leu Glu Glu Leu Pro Glu Asp Asn Leu Ser Ser Trp 435 440 445
gcc acc tgg ggc acc tta cca ccg gag ccg aat ctg gtc cct ggg gga
1392 Ala Thr Trp Gly Thr Leu Pro Pro Glu Pro Asn Leu Val Pro Gly
Gly 450 455 460 ccc cca gtt tct ctt cag aca ctg acc ttc tgc tgg gaa
agc agc cct 1440 Pro Pro Val Ser Leu Gln Thr Leu Thr Phe Cys Trp
Glu Ser Ser Pro 465 470 475 480 gag gag gaa gag gag gcg agg gaa tca
gaa att gag gac agc gat gcg 1488 Glu Glu Glu Glu Glu Ala Arg Glu
Ser Glu Ile Glu Asp Ser Asp Ala 485 490 495 ggc agc tgg ggg gct gag
agc acc cag agg acc gag gac agg ggc cgg 1536 Gly Ser Trp Gly Ala
Glu Ser Thr Gln Arg Thr Glu Asp Arg Gly Arg 500 505 510 aca ttg ggg
cat tac atg gcc agg tga 1563 Thr Leu Gly His Tyr Met Ala Arg * 515
520 12 520 PRT Homo sapiens 12 Met Ala Gly Pro Glu Arg Trp Gly Pro
Leu Leu Leu Cys Leu Leu Gln 1 5 10 15 Ala Ala Pro Gly Arg Pro Arg
Leu Ala Pro Pro Gln Asn Val Thr Leu 20 25 30 Leu Ser Gln Asn Phe
Ser Val Tyr Leu Thr Trp Leu Pro Gly Leu Gly 35 40 45 Asn Pro Gln
Asp Val Thr Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60 Arg
Arg Arg Trp Arg Glu Val Glu Glu Cys Ala Gly Thr Lys Glu Leu 65 70
75 80 Leu Cys Ser Met Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys
Phe 85 90 95 Lys Gly Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser
Pro Trp Val 100 105 110 Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val
Glu Pro Ala Pro Pro 115 120 125 Val Leu Val Leu Thr Gln Thr Glu Glu
Ile Leu Ser Ala Asn Ala Thr 130 135 140 Tyr Gln Leu Pro Pro Cys Met
Pro Pro Leu Asp Leu Lys Tyr Glu Val 145 150 155 160 Ala Phe Trp Lys
Glu Gly Ala Gly Asn Lys Thr Leu Phe Pro Val Thr 165 170 175 Pro His
Gly Gln Pro Val Gln Ile Thr Leu Gln Pro Ala Ala Ser Glu 180 185 190
His His Cys Leu Ser Ala Arg Thr Ile Tyr Thr Phe Ser Val Pro Lys 195
200 205 Tyr Ser Lys Phe Ser Lys Pro Thr Cys Phe Leu Leu Glu Val Pro
Glu 210 215 220 Ala Asn Trp Ala Phe Leu Val Leu Pro Ser Leu Leu Ile
Leu Leu Leu 225 230 235 240 Val Ile Ala Ala Gly Gly Val Ile Trp Lys
Thr Leu Met Gly Asn Pro 245 250 255 Trp Phe Gln Arg Ala Lys Met Pro
Arg Ala Leu Asp Phe Ser Gly His 260 265 270 Thr His Pro Val Ala Thr
Phe Gln Pro Ser Arg Pro Glu Ser Val Asn 275 280 285 Asp Leu Phe Leu
Cys Pro Gln Lys Glu Leu Thr Arg Gly Val Arg Pro 290 295 300 Thr Pro
Arg Val Arg Ala Pro Ala Thr Gln Gln Thr Arg Trp Lys Lys 305 310 315
320 Asp Leu Ala Glu Asp Glu Glu Glu Glu Asp Glu Glu Asp Thr Glu Asp
325 330 335 Gly Val Ser Phe Gln Pro Tyr Ile Glu Pro Pro Ser Phe Leu
Gly Gln 340 345 350 Glu His Gln Ala Pro Gly His Ser Glu Ala Gly Gly
Val Asp Ser Gly 355 360 365 Arg Pro Arg Ala Pro Leu Val Pro Ser Glu
Gly Ser Ser Ala Trp Asp 370 375 380 Ser Ser Asp Arg Ser Trp Ala Ser
Thr Val Asp Ser Ser Trp Asp Arg 385 390 395 400 Ala Gly Ser Ser Gly
Tyr Leu Ala Glu Lys Gly Pro Gly Gln Gly Pro 405 410 415 Gly Gly Asp
Gly His Gln Glu Ser Leu Pro Pro Pro Glu Phe Ser Lys 420 425 430 Asp
Ser Gly Phe Leu Glu Glu Leu Pro Glu Asp Asn Leu Ser Ser Trp 435 440
445 Ala Thr Trp Gly Thr Leu Pro Pro Glu Pro Asn Leu Val Pro Gly Gly
450 455 460 Pro Pro Val Ser Leu Gln Thr Leu Thr Phe Cys Trp Glu Ser
Ser Pro 465 470 475 480 Glu Glu Glu Glu Glu Ala Arg Glu Ser Glu Ile
Glu Asp Ser Asp Ala 485 490 495 Gly Ser Trp Gly Ala Glu Ser Thr Gln
Arg Thr Glu Asp Arg Gly Arg 500 505 510 Thr Leu Gly His Tyr Met Ala
Arg 515 520 13 1476 DNA Homo sapiens CDS (1)...(1476) misc_feature
(0)...(0) IL-28RA splice variant 13 atg gcg ggg ccc gag cgc tgg ggc
ccc ctg ctc ctg tgc ctg ctg cag 48 Met Ala Gly Pro Glu Arg Trp Gly
Pro Leu Leu Leu Cys Leu Leu Gln 1 5 10 15 gcc gct cca ggg agg ccc
cgt ctg gcc cct ccc cag aat gtg acg ctg 96 Ala Ala Pro Gly Arg Pro
Arg Leu Ala Pro Pro Gln Asn Val Thr Leu 20 25 30 ctc tcc cag aac
ttc agc gtg tac ctg aca tgg ctc cca ggg ctt ggc 144 Leu Ser Gln Asn
Phe Ser Val Tyr Leu Thr Trp Leu Pro Gly Leu Gly 35 40 45 aac ccc
cag gat gtg acc tat ttt gtg gcc tat cag agc tct ccc acc 192 Asn Pro
Gln Asp Val Thr Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60
cgt aga cgg tgg cgc gaa gtg gaa gag tgt gcg gga acc aag gag ctg 240
Arg Arg Arg Trp Arg Glu Val Glu Glu Cys Ala Gly Thr Lys Glu Leu 65
70 75 80 cta tgt tct atg atg tgc ctg aag aaa cag gac ctg tac aac
aag ttc 288 Leu Cys Ser Met Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn
Lys Phe 85 90 95 aag gga cgc gtg cgg acg gtt tct ccc agc tcc aag
tcc ccc tgg gtg 336 Lys Gly Arg Val Arg Thr Val Ser Pro Ser Ser Lys
Ser Pro Trp Val 100 105 110 gag tcc gaa tac ctg gat tac ctt ttt gaa
gtg gag ccg gcc cca cct 384 Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu
Val Glu Pro Ala Pro Pro 115 120 125 gtc ctg gtg ctc acc cag acg gag
gag atc ctg agt gcc aat gcc acg 432 Val Leu Val Leu Thr Gln Thr Glu
Glu Ile Leu Ser Ala Asn Ala Thr 130 135 140 tac cag ctg ccc ccc tgc
atg ccc cca ctg ttt ctg aag tat gag gtg 480 Tyr Gln Leu Pro Pro Cys
Met Pro Pro Leu Phe Leu Lys Tyr Glu Val 145 150 155 160 gca ttt tgg
ggg ggg ggg gcc gga acc aag acc cta ttt cca gtc act 528 Ala Phe Trp
Gly Gly Gly Ala Gly Thr Lys Thr Leu Phe Pro Val Thr 165 170 175 ccc
cat ggc cag cca gtc cag atc act ctc cag cca gct gcc agc gaa 576 Pro
His Gly Gln Pro Val Gln Ile Thr Leu Gln Pro Ala Ala Ser Glu 180 185
190 cac cac tgc ctc agt gcc aga acc atc tac acg ttc agt gtc ccg aaa
624 His His Cys Leu Ser Ala Arg Thr Ile Tyr Thr Phe Ser Val Pro Lys
195 200 205 tac agc aag ttc tct aag ccc acc tgc ttc ttg ctg gag gtc
cca gaa 672 Tyr Ser Lys Phe Ser Lys Pro Thr Cys Phe Leu Leu Glu Val
Pro Glu 210 215 220 gcc aac tgg gct ttc ctg gtg ctg cca tcg ctt ctg
ata ctg ctg tta 720 Ala Asn Trp Ala Phe Leu Val Leu Pro Ser Leu Leu
Ile Leu Leu Leu 225 230 235 240 gta att gcc gca ggg ggt gtg atc tgg
aag acc ctc atg ggg aac ccc 768 Val Ile Ala Ala Gly Gly Val Ile Trp
Lys Thr Leu Met Gly Asn Pro 245 250 255 tgg ttt cag cgg gca aag atg
cca cgg gcc ctg gaa ctg acc aga ggg 816 Trp Phe Gln Arg Ala Lys Met
Pro Arg Ala Leu Glu Leu Thr Arg Gly 260 265 270 gtc agg ccg acg cct
cga gtc agg gcc cca gcc acc caa cag aca aga 864 Val Arg Pro Thr Pro
Arg Val Arg Ala Pro Ala Thr Gln Gln Thr Arg 275 280 285 tgg aag aag
gac ctt gca gag gac gaa gag gag gag gat gag gag gac 912 Trp Lys Lys
Asp Leu Ala Glu Asp Glu Glu Glu Glu Asp Glu Glu Asp 290 295 300 aca
gaa gat ggc gtc agc ttc cag ccc tac att gaa cca cct tct ttc 960 Thr
Glu Asp Gly Val Ser Phe Gln Pro Tyr Ile Glu Pro Pro Ser Phe 305 310
315 320 ctg ggg caa gag cac cag gct cca ggg cac tcg gag gct ggt ggg
gtg 1008 Leu Gly Gln Glu His Gln Ala Pro Gly His Ser Glu Ala Gly
Gly Val 325 330 335 gac tca ggg agg ccc agg gct cct ctg gtc cca agc
gaa ggc tcc tct 1056 Asp Ser Gly Arg Pro Arg Ala Pro Leu Val Pro
Ser Glu Gly Ser Ser 340 345 350 gct tgg gat tct tca gac aga agc tgg
gcc agc act gtg gac tcc tcc 1104 Ala Trp Asp Ser Ser Asp Arg Ser
Trp Ala Ser Thr Val Asp Ser Ser 355 360 365 tgg gac agg gct ggg tcc
tct ggc tat ttg gct gag aag ggg cca ggc 1152 Trp Asp Arg Ala Gly
Ser Ser Gly Tyr Leu Ala Glu Lys Gly Pro Gly 370 375 380 caa ggg ccg
ggt ggg gat ggg cac caa gaa tct ctc cca cca cct gaa 1200 Gln Gly
Pro Gly Gly Asp Gly His Gln Glu Ser Leu Pro Pro Pro Glu 385 390 395
400 ttc tcc aag gac tcg ggt ttc ctg gaa gag ctc cca gaa gat aac ctc
1248 Phe Ser Lys Asp Ser Gly Phe Leu Glu Glu Leu Pro Glu Asp Asn
Leu 405 410 415 tcc tcc tgg gcc acc tgg ggc acc tta cca ccg gag ccg
aat ctg gtc 1296 Ser Ser Trp Ala Thr Trp Gly Thr Leu Pro Pro Glu
Pro Asn Leu Val 420 425 430 cct ggg gga ccc cca gtt tct ctt cag aca
ctg acc ttc tgc tgg gaa 1344 Pro Gly Gly Pro Pro Val Ser Leu Gln
Thr Leu Thr Phe Cys Trp Glu 435 440 445 agc agc cct gag gag gaa gag
gag gcg agg gaa tca gaa att gag gac 1392 Ser Ser Pro Glu Glu Glu
Glu Glu Ala Arg Glu Ser Glu Ile Glu Asp 450 455 460 agc gat
gcg ggc agc tgg ggg gct gag agc acc cag agg acc gag gac 1440 Ser
Asp Ala Gly Ser Trp Gly Ala Glu Ser Thr Gln Arg Thr Glu Asp 465 470
475 480 agg ggc cgg aca ttg ggg cat tac atg gcc agg tga 1476 Arg
Gly Arg Thr Leu Gly His Tyr Met Ala Arg * 485 490 14 491 PRT Homo
sapiens 14 Met Ala Gly Pro Glu Arg Trp Gly Pro Leu Leu Leu Cys Leu
Leu Gln 1 5 10 15 Ala Ala Pro Gly Arg Pro Arg Leu Ala Pro Pro Gln
Asn Val Thr Leu 20 25 30 Leu Ser Gln Asn Phe Ser Val Tyr Leu Thr
Trp Leu Pro Gly Leu Gly 35 40 45 Asn Pro Gln Asp Val Thr Tyr Phe
Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60 Arg Arg Arg Trp Arg Glu
Val Glu Glu Cys Ala Gly Thr Lys Glu Leu 65 70 75 80 Leu Cys Ser Met
Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 85 90 95 Lys Gly
Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val 100 105 110
Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro 115
120 125 Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn Ala
Thr 130 135 140 Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Phe Leu Lys
Tyr Glu Val 145 150 155 160 Ala Phe Trp Gly Gly Gly Ala Gly Thr Lys
Thr Leu Phe Pro Val Thr 165 170 175 Pro His Gly Gln Pro Val Gln Ile
Thr Leu Gln Pro Ala Ala Ser Glu 180 185 190 His His Cys Leu Ser Ala
Arg Thr Ile Tyr Thr Phe Ser Val Pro Lys 195 200 205 Tyr Ser Lys Phe
Ser Lys Pro Thr Cys Phe Leu Leu Glu Val Pro Glu 210 215 220 Ala Asn
Trp Ala Phe Leu Val Leu Pro Ser Leu Leu Ile Leu Leu Leu 225 230 235
240 Val Ile Ala Ala Gly Gly Val Ile Trp Lys Thr Leu Met Gly Asn Pro
245 250 255 Trp Phe Gln Arg Ala Lys Met Pro Arg Ala Leu Glu Leu Thr
Arg Gly 260 265 270 Val Arg Pro Thr Pro Arg Val Arg Ala Pro Ala Thr
Gln Gln Thr Arg 275 280 285 Trp Lys Lys Asp Leu Ala Glu Asp Glu Glu
Glu Glu Asp Glu Glu Asp 290 295 300 Thr Glu Asp Gly Val Ser Phe Gln
Pro Tyr Ile Glu Pro Pro Ser Phe 305 310 315 320 Leu Gly Gln Glu His
Gln Ala Pro Gly His Ser Glu Ala Gly Gly Val 325 330 335 Asp Ser Gly
Arg Pro Arg Ala Pro Leu Val Pro Ser Glu Gly Ser Ser 340 345 350 Ala
Trp Asp Ser Ser Asp Arg Ser Trp Ala Ser Thr Val Asp Ser Ser 355 360
365 Trp Asp Arg Ala Gly Ser Ser Gly Tyr Leu Ala Glu Lys Gly Pro Gly
370 375 380 Gln Gly Pro Gly Gly Asp Gly His Gln Glu Ser Leu Pro Pro
Pro Glu 385 390 395 400 Phe Ser Lys Asp Ser Gly Phe Leu Glu Glu Leu
Pro Glu Asp Asn Leu 405 410 415 Ser Ser Trp Ala Thr Trp Gly Thr Leu
Pro Pro Glu Pro Asn Leu Val 420 425 430 Pro Gly Gly Pro Pro Val Ser
Leu Gln Thr Leu Thr Phe Cys Trp Glu 435 440 445 Ser Ser Pro Glu Glu
Glu Glu Glu Ala Arg Glu Ser Glu Ile Glu Asp 450 455 460 Ser Asp Ala
Gly Ser Trp Gly Ala Glu Ser Thr Gln Arg Thr Glu Asp 465 470 475 480
Arg Gly Arg Thr Leu Gly His Tyr Met Ala Arg 485 490 15 674 DNA Homo
sapiens CDS (1)...(636) misc_feature (0)...(0) IL-28RA soluble
variant 15 atg gcg ggg ccc gag cgc tgg ggc ccc ctg ctc ctg tgc ctg
ctg cag 48 Met Ala Gly Pro Glu Arg Trp Gly Pro Leu Leu Leu Cys Leu
Leu Gln 1 5 10 15 gcc gct cca ggg agg ccc cgt ctg gcc cct ccc cag
aat gtg acg ctg 96 Ala Ala Pro Gly Arg Pro Arg Leu Ala Pro Pro Gln
Asn Val Thr Leu 20 25 30 ctc tcc cag aac ttc agc gtg tac ctg aca
tgg ctc cca ggg ctt ggc 144 Leu Ser Gln Asn Phe Ser Val Tyr Leu Thr
Trp Leu Pro Gly Leu Gly 35 40 45 aac ccc cag gat gtg acc tat ttt
gtg gcc tat cag agc tct ccc acc 192 Asn Pro Gln Asp Val Thr Tyr Phe
Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60 cgt aga cgg tgg cgc gaa
gtg gaa gag tgt gcg gga acc aag gag ctg 240 Arg Arg Arg Trp Arg Glu
Val Glu Glu Cys Ala Gly Thr Lys Glu Leu 65 70 75 80 cta tgt tct atg
atg tgc ctg aag aaa cag gac ctg tac aac aag ttc 288 Leu Cys Ser Met
Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 85 90 95 aag gga
cgc gtg cgg acg gtt tct ccc agc tcc aag tcc ccc tgg gtg 336 Lys Gly
Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val 100 105 110
gag tcc gaa tac ctg gat tac ctt ttt gaa gtg gag ccg gcc cca cct 384
Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala Pro Pro 115
120 125 gtc ctg gtg ctc acc cag acg gag gag atc ctg agt gcc aat gcc
acg 432 Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser Ala Asn Ala
Thr 130 135 140 tac cag ctg ccc ccc tgc atg ccc cca ctg gat ctg aag
tat gag gtg 480 Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu Asp Leu Lys
Tyr Glu Val 145 150 155 160 gca ttc tgg aag gag ggg gcc gga aac aag
gtg gga agc tcc ttt cct 528 Ala Phe Trp Lys Glu Gly Ala Gly Asn Lys
Val Gly Ser Ser Phe Pro 165 170 175 gcc ccc agg cta ggc ccg ctc ctc
cac ccc ttc tta ctc agg ttc ttc 576 Ala Pro Arg Leu Gly Pro Leu Leu
His Pro Phe Leu Leu Arg Phe Phe 180 185 190 tca ccc tcc cag cct gct
cct gca ccc ctc ctc cag gaa gtc ttc cct 624 Ser Pro Ser Gln Pro Ala
Pro Ala Pro Leu Leu Gln Glu Val Phe Pro 195 200 205 gta cac tcc tga
cttctggcag tcagccctaa taaaatctga tcaaagta 674 Val His Ser * 210 16
211 PRT Homo sapiens 16 Met Ala Gly Pro Glu Arg Trp Gly Pro Leu Leu
Leu Cys Leu Leu Gln 1 5 10 15 Ala Ala Pro Gly Arg Pro Arg Leu Ala
Pro Pro Gln Asn Val Thr Leu 20 25 30 Leu Ser Gln Asn Phe Ser Val
Tyr Leu Thr Trp Leu Pro Gly Leu Gly 35 40 45 Asn Pro Gln Asp Val
Thr Tyr Phe Val Ala Tyr Gln Ser Ser Pro Thr 50 55 60 Arg Arg Arg
Trp Arg Glu Val Glu Glu Cys Ala Gly Thr Lys Glu Leu 65 70 75 80 Leu
Cys Ser Met Met Cys Leu Lys Lys Gln Asp Leu Tyr Asn Lys Phe 85 90
95 Lys Gly Arg Val Arg Thr Val Ser Pro Ser Ser Lys Ser Pro Trp Val
100 105 110 Glu Ser Glu Tyr Leu Asp Tyr Leu Phe Glu Val Glu Pro Ala
Pro Pro 115 120 125 Val Leu Val Leu Thr Gln Thr Glu Glu Ile Leu Ser
Ala Asn Ala Thr 130 135 140 Tyr Gln Leu Pro Pro Cys Met Pro Pro Leu
Asp Leu Lys Tyr Glu Val 145 150 155 160 Ala Phe Trp Lys Glu Gly Ala
Gly Asn Lys Val Gly Ser Ser Phe Pro 165 170 175 Ala Pro Arg Leu Gly
Pro Leu Leu His Pro Phe Leu Leu Arg Phe Phe 180 185 190 Ser Pro Ser
Gln Pro Ala Pro Ala Pro Leu Leu Gln Glu Val Phe Pro 195 200 205 Val
His Ser 210 17 734 DNA Homo sapiens sig_peptide (53)...(127)
mat_peptide (128)...(655) CDS (53)...(655) 17 tgggtgacag cctcagagtg
tttcttctgc tgacaaagac cagagatcag ga atg aaa 58 Met Lys -25 cta gac
atg act ggg gac tgc acg cca gtg ctg gtg ctg atg gcc gca 106 Leu Asp
Met Thr Gly Asp Cys Thr Pro Val Leu Val Leu Met Ala Ala -20 -15 -10
gtg ctg acc gtg act gga gca gtt cct gtc gcc agg ctc cac ggg gct 154
Val Leu Thr Val Thr Gly Ala Val Pro Val Ala Arg Leu His Gly Ala -5
1 5 ctc ccg gat gca agg ggc tgc cac ata gcc cag ttc aag tcc ctg tct
202 Leu Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser
10 15 20 25 cca cag gag ctg cag gcc ttt aag agg gcc aaa gat gcc tta
gaa gag 250 Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu
Glu Glu 30 35 40 tcg ctt ctg ctg aag gac tgc agg tgc cac tcc cgc
ctc ttc ccc agg 298 Ser Leu Leu Leu Lys Asp Cys Arg Cys His Ser Arg
Leu Phe Pro Arg 45 50 55 acc tgg gac ctg agg cag ctg cag gtg agg
gag cgc ccc atg gct ttg 346 Thr Trp Asp Leu Arg Gln Leu Gln Val Arg
Glu Arg Pro Met Ala Leu 60 65 70 gag gct gag ctg gcc ctg acg ctg
aag gtt ctg gag gcc acc gct gac 394 Glu Ala Glu Leu Ala Leu Thr Leu
Lys Val Leu Glu Ala Thr Ala Asp 75 80 85 act gac cca gcc ctg gtg
gac gtc ttg gac cag ccc ctt cac acc ctg 442 Thr Asp Pro Ala Leu Val
Asp Val Leu Asp Gln Pro Leu His Thr Leu 90 95 100 105 cac cat atc
ctc tcc cag ttc cgg gcc tgt atc cag cct cag ccc acg 490 His His Ile
Leu Ser Gln Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr 110 115 120 gca
ggg ccc agg acc cgg ggc cgc ctc cac cat tgg ctg tac cgg ctc 538 Ala
Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu Tyr Arg Leu 125 130
135 cag gag gcc cca aaa aag gag tcc cct ggc tgc ctc gag gcc tct gtc
586 Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala Ser Val
140 145 150 acc ttc aac ctc ttc cgc ctc ctc acg cga gac ctg aat tgt
gtt gcc 634 Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys
Val Ala 155 160 165 agt ggg gac ctg tgt gtc tga ccctcccacc
agtcatgcaa cctgagattt 685 Ser Gly Asp Leu Cys Val * 170 175
tatttataaa ttagccactt gtcttaattt attgccaccc agtcgctat 734 18 200
PRT Homo sapiens SIGNAL (1)...(25) 18 Met Lys Leu Asp Met Thr Gly
Asp Cys Thr Pro Val Leu Val Leu Met -25 -20 -15 -10 Ala Ala Val Leu
Thr Val Thr Gly Ala Val Pro Val Ala Arg Leu His -5 1 5 Gly Ala Leu
Pro Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser 10 15 20 Leu
Ser Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu 25 30
35 Glu Glu Ser Leu Leu Leu Lys Asp Cys Arg Cys His Ser Arg Leu Phe
40 45 50 55 Pro Arg Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg
Pro Met 60 65 70 Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val
Leu Glu Ala Thr 75 80 85 Ala Asp Thr Asp Pro Ala Leu Val Asp Val
Leu Asp Gln Pro Leu His 90 95 100 Thr Leu His His Ile Leu Ser Gln
Phe Arg Ala Cys Ile Gln Pro Gln 105 110 115 Pro Thr Ala Gly Pro Arg
Thr Arg Gly Arg Leu His His Trp Leu Tyr 120 125 130 135 Arg Leu Gln
Glu Ala Pro Lys Lys Glu Ser Pro Gly Cys Leu Glu Ala 140 145 150 Ser
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys 155 160
165 Val Ala Ser Gly Asp Leu Cys Val 170 175 19 856 DNA Homo sapiens
sig_peptide (98)...(154) mat_peptide (155)...(700) CDS (98)...(700)
19 aattaccttt tcactttaca cacatcatct tggattgccc attttgcgtg
gctaaaaagc 60 agagccatgc cgctggggaa gcagttgcga tttagcc atg gct gca
gct tgg acc 115 Met Ala Ala Ala Trp Thr -15 gtg gtg ctg gtg act ttg
gtg cta ggc ttg gcc gtg gca ggc cct gtc 163 Val Val Leu Val Thr Leu
Val Leu Gly Leu Ala Val Ala Gly Pro Val -10 -5 1 ccc act tcc aag
ccc acc aca act ggg aag ggc tgc cac att ggc agg 211 Pro Thr Ser Lys
Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg 5 10 15 ttc aaa tct
ctg tca cca cag gag cta gcg agc ttc aag aag gcc agg 259 Phe Lys Ser
Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg 20 25 30 35 gac
gcc ttg gaa gag tca ctc aag ctg aaa aac tgg agt tgc agc tct 307 Asp
Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser 40 45
50 cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc cag gtg agg gag
355 Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu
55 60 65 cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg ctg aag
gtc ctg 403 Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys
Val Leu 70 75 80 gag gcc gct gct ggc cca gcc ctg gag gac gtc cta
gac cag ccc ctt 451 Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu
Asp Gln Pro Leu 85 90 95 cac acc ctg cac cac atc ctc tcc cag ctc
cag gcc tgt atc cag cct 499 His Thr Leu His His Ile Leu Ser Gln Leu
Gln Ala Cys Ile Gln Pro 100 105 110 115 cag ccc aca gca ggg ccc agg
ccc cgg ggc cgc ctc cac cac tgg ctg 547 Gln Pro Thr Ala Gly Pro Arg
Pro Arg Gly Arg Leu His His Trp Leu 120 125 130 cac cgg ctc cag gag
gcc ccc aaa aag gag tcc gct ggc tgc ctg gag 595 His Arg Leu Gln Glu
Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu 135 140 145 gca tct gtc
acc ttc aac ctc ttc cgc ctc ctc acg cga gac ctc aaa 643 Ala Ser Val
Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys 150 155 160 tat
gtg gcc gat ggg aac ctg tgt ctg aga acg tca acc cac cct gag 691 Tyr
Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr His Pro Glu 165 170
175 tcc acc tga caccccacac cttatttatg cgctgagccc tactccttcc 740 Ser
Thr * 180 ttaatttatt tcctctcacc ctttatttat gaagctgcag ccctgactga
gacatagggc 800 tgagtttatt gttttacttt tatacattat gcacaaataa
acaacaagga attgga 856 20 200 PRT Homo sapiens SIGNAL (1)...(19) 20
Met Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu -15
-10 -5 Ala Val Ala Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly
Lys 1 5 10 Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu
Leu Ala 15 20 25 Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser
Leu Lys Leu Lys 30 35 40 45 Asn Trp Ser Cys Ser Ser Pro Val Phe Pro
Gly Asn Trp Asp Leu Arg 50 55 60 Leu Leu Gln Val Arg Glu Arg Pro
Val Ala Leu Glu Ala Glu Leu Ala 65 70 75 Leu Thr Leu Lys Val Leu
Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp 80 85 90 Val Leu Asp Gln
Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu 95 100 105 Gln Ala
Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly 110 115 120
125 Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu
130 135 140 Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe
Arg Leu 145 150 155 Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn
Leu Cys Leu Arg 160 165 170 Thr Ser Thr His Pro Glu Ser Thr 175 180
21 734 DNA Homo sapiens sig_peptide (53)...(127) mat_peptide
(128)...(655) CDS (53)...(655) 21 tgggtgacag cctcagagtg tttcttctgc
tgacaaagac cagagatcag ga atg aaa 58 Met Lys -25 cta gac atg acc ggg
gac tgc atg cca gtg ctg gtg ctg atg gcc gca 106 Leu Asp Met Thr Gly
Asp Cys Met Pro Val Leu Val Leu Met Ala Ala -20 -15 -10 gtg ctg acc
gtg act gga gca gtt cct gtc gcc agg ctc cgc ggg gct 154 Val Leu Thr
Val Thr Gly Ala Val Pro Val Ala Arg Leu Arg Gly Ala -5 1 5 ctc ccg
gat gca agg ggc tgc cac ata gcc cag ttc aag tcc ctg tct 202 Leu Pro
Asp Ala Arg Gly Cys His Ile Ala Gln Phe Lys Ser Leu Ser 10 15 20 25
cca cag gag ctg cag gcc ttt aag agg gcc aaa gat gcc tta gaa gag 250
Pro Gln Glu Leu Gln Ala Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu 30
35 40 tcg ctt ctg ctg aag gac tgc aag tgc cgc tcc cgc ctc ttc ccc
agg 298 Ser Leu
Leu Leu Lys Asp Cys Lys Cys Arg Ser Arg Leu Phe Pro Arg 45 50 55
acc tgg gac ctg agg cag ctg cag gtg agg gag cgc ccc gtg gct ttg 346
Thr Trp Asp Leu Arg Gln Leu Gln Val Arg Glu Arg Pro Val Ala Leu 60
65 70 gag gct gag ctg gcc ctg acg ctg aag gtt ctg gag gcc acc gct
gac 394 Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr Ala
Asp 75 80 85 act gac cca gcc ctg ggg gat gtc ttg gac cag ccc ctt
cac acc ctg 442 Thr Asp Pro Ala Leu Gly Asp Val Leu Asp Gln Pro Leu
His Thr Leu 90 95 100 105 cac cat atc ctc tcc cag ctc cgg gcc tgt
atc cag cct cag ccc acg 490 His His Ile Leu Ser Gln Leu Arg Ala Cys
Ile Gln Pro Gln Pro Thr 110 115 120 gca ggg ccc agg acc cgg ggc cgc
ctc cac cat tgg ctg cac cgg ctc 538 Ala Gly Pro Arg Thr Arg Gly Arg
Leu His His Trp Leu His Arg Leu 125 130 135 cag gag gcc cca aaa aag
gag tcc cct ggc tgc ctc gag gcc tct gtc 586 Gln Glu Ala Pro Lys Lys
Glu Ser Pro Gly Cys Leu Glu Ala Ser Val 140 145 150 acc ttc aac ctc
ttc cgc ctc ctc acg cga gac ctg aat tgt gtt gcc 634 Thr Phe Asn Leu
Phe Arg Leu Leu Thr Arg Asp Leu Asn Cys Val Ala 155 160 165 agc ggg
gac ctg tgt gtc tga cccttccgcc agtcatgcaa cctgagattt 685 Ser Gly
Asp Leu Cys Val * 170 175 tatttataaa ttagccactt ggcttaattt
attgccaccc agtcgctat 734 22 200 PRT Homo sapiens SIGNAL (1)...(25)
22 Met Lys Leu Asp Met Thr Gly Asp Cys Met Pro Val Leu Val Leu Met
-25 -20 -15 -10 Ala Ala Val Leu Thr Val Thr Gly Ala Val Pro Val Ala
Arg Leu Arg -5 1 5 Gly Ala Leu Pro Asp Ala Arg Gly Cys His Ile Ala
Gln Phe Lys Ser 10 15 20 Leu Ser Pro Gln Glu Leu Gln Ala Phe Lys
Arg Ala Lys Asp Ala Leu 25 30 35 Glu Glu Ser Leu Leu Leu Lys Asp
Cys Lys Cys Arg Ser Arg Leu Phe 40 45 50 55 Pro Arg Thr Trp Asp Leu
Arg Gln Leu Gln Val Arg Glu Arg Pro Val 60 65 70 Ala Leu Glu Ala
Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Thr 75 80 85 Ala Asp
Thr Asp Pro Ala Leu Gly Asp Val Leu Asp Gln Pro Leu His 90 95 100
Thr Leu His His Ile Leu Ser Gln Leu Arg Ala Cys Ile Gln Pro Gln 105
110 115 Pro Thr Ala Gly Pro Arg Thr Arg Gly Arg Leu His His Trp Leu
His 120 125 130 135 Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Pro Gly
Cys Leu Glu Ala 140 145 150 Ser Val Thr Phe Asn Leu Phe Arg Leu Leu
Thr Arg Asp Leu Asn Cys 155 160 165 Val Ala Ser Gly Asp Leu Cys Val
170 175 23 528 DNA Artificial Sequence IL-28A mutant C48S CDS
(1)...(528) 23 gtt cct gtc gcc agg ctc cac ggg gct ctc ccg gat gca
agg ggc tgc 48 Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala
Arg Gly Cys 1 5 10 15 cac ata gcc cag ttc aag tcc ctg tct cca cag
gag ctg cag gcc ttt 96 His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln
Glu Leu Gln Ala Phe 20 25 30 aag agg gcc aaa gat gcc tta gaa gag
tcg ctt ctg ctg aag gac tcc 144 Lys Arg Ala Lys Asp Ala Leu Glu Glu
Ser Leu Leu Leu Lys Asp Ser 35 40 45 agg tgc cac tcc cgc ctc ttc
ccc agg acc tgg gac ctg agg cag ctg 192 Arg Cys His Ser Arg Leu Phe
Pro Arg Thr Trp Asp Leu Arg Gln Leu 50 55 60 cag gtg agg gag cgc
ccc atg gct ttg gag gct gag ctg gcc ctg acg 240 Gln Val Arg Glu Arg
Pro Met Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 ctg aag gtt
ctg gag gcc acc gct gac act gac cca gcc ctg gtg gac 288 Leu Lys Val
Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Val Asp 85 90 95 gtc
ttg gac cag ccc ctt cac acc ctg cac cat atc ctc tcc cag ttc 336 Val
Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Phe 100 105
110 cgg gcc tgt atc cag cct cag ccc acg gca ggg ccc agg acc cgg ggc
384 Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly
115 120 125 cgc ctc cac cat tgg ctg tac cgg ctc cag gag gcc cca aaa
aag gag 432 Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys
Lys Glu 130 135 140 tcc cct ggc tgc ctc gag gcc tct gtc acc ttc aac
ctc ttc cgc ctc 480 Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn
Leu Phe Arg Leu 145 150 155 160 ctc acg cga gac ctg aat tgt gtt gcc
agt ggg gac ctg tgt gtc tga 528 Leu Thr Arg Asp Leu Asn Cys Val Ala
Ser Gly Asp Leu Cys Val * 165 170 175 24 175 PRT Artificial
Sequence IL-28A mutant C48S 24 Val Pro Val Ala Arg Leu His Gly Ala
Leu Pro Asp Ala Arg Gly Cys 1 5 10 15 His Ile Ala Gln Phe Lys Ser
Leu Ser Pro Gln Glu Leu Gln Ala Phe 20 25 30 Lys Arg Ala Lys Asp
Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Ser 35 40 45 Arg Cys His
Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu 50 55 60 Gln
Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70
75 80 Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Val
Asp 85 90 95 Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu
Ser Gln Phe 100 105 110 Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly
Pro Arg Thr Arg Gly 115 120 125 Arg Leu His His Trp Leu Tyr Arg Leu
Gln Glu Ala Pro Lys Lys Glu 130 135 140 Ser Pro Gly Cys Leu Glu Ala
Ser Val Thr Phe Asn Leu Phe Arg Leu 145 150 155 160 Leu Thr Arg Asp
Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170 175 25 531 DNA
Artificial Sequence met IL-28A mutant C49S CDS (1)...(531) 25 atg
gtt cct gtc gcc agg ctc cac ggg gct ctc ccg gat gca agg ggc 48 Met
Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10
15 tgc cac ata gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc
96 Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala
20 25 30 ttt aag agg gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg
aag gac 144 Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu
Lys Asp 35 40 45 tcc agg tgc cac tcc cgc ctc ttc ccc agg acc tgg
gac ctg agg cag 192 Ser Arg Cys His Ser Arg Leu Phe Pro Arg Thr Trp
Asp Leu Arg Gln 50 55 60 ctg cag gtg agg gag cgc ccc atg gct ttg
gag gct gag ctg gcc ctg 240 Leu Gln Val Arg Glu Arg Pro Met Ala Leu
Glu Ala Glu Leu Ala Leu 65 70 75 80 acg ctg aag gtt ctg gag gcc acc
gct gac act gac cca gcc ctg gtg 288 Thr Leu Lys Val Leu Glu Ala Thr
Ala Asp Thr Asp Pro Ala Leu Val 85 90 95 gac gtc ttg gac cag ccc
ctt cac acc ctg cac cat atc ctc tcc cag 336 Asp Val Leu Asp Gln Pro
Leu His Thr Leu His His Ile Leu Ser Gln 100 105 110 ttc cgg gcc tgt
atc cag cct cag ccc acg gca ggg ccc agg acc cgg 384 Phe Arg Ala Cys
Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg 115 120 125 ggc cgc
ctc cac cat tgg ctg tac cgg ctc cag gag gcc cca aaa aag 432 Gly Arg
Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140
gag tcc cct ggc tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc 480
Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg 145
150 155 160 ctc ctc acg cga gac ctg aat tgt gtt gcc agt ggg gac ctg
tgt gtc 528 Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu
Cys Val 165 170 175 tga 531 * 26 176 PRT Artificial Sequence met
IL-28A mutant C49S 26 Met Val Pro Val Ala Arg Leu His Gly Ala Leu
Pro Asp Ala Arg Gly 1 5 10 15 Cys His Ile Ala Gln Phe Lys Ser Leu
Ser Pro Gln Glu Leu Gln Ala 20 25 30 Phe Lys Arg Ala Lys Asp Ala
Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 40 45 Ser Arg Cys His Ser
Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln 50 55 60 Leu Gln Val
Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu 65 70 75 80 Thr
Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Val 85 90
95 Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln
100 105 110 Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg
Thr Arg 115 120 125 Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu
Ala Pro Lys Lys 130 135 140 Glu Ser Pro Gly Cys Leu Glu Ala Ser Val
Thr Phe Asn Leu Phe Arg 145 150 155 160 Leu Leu Thr Arg Asp Leu Asn
Cys Val Ala Ser Gly Asp Leu Cys Val 165 170 175 27 528 DNA
Artificial Sequence IL-28A mutant C50S CDS (1)...(528) 27 gtt cct
gtc gcc agg ctc cac ggg gct ctc ccg gat gca agg ggc tgc 48 Val Pro
Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys 1 5 10 15
cac ata gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc ttt 96
His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe 20
25 30 aag agg gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg aag gac
tgc 144 Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp
Cys 35 40 45 agg tcc cac tcc cgc ctc ttc ccc agg acc tgg gac ctg
agg cag ctg 192 Arg Ser His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu
Arg Gln Leu 50 55 60 cag gtg agg gag cgc ccc atg gct ttg gag gct
gag ctg gcc ctg acg 240 Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala
Glu Leu Ala Leu Thr 65 70 75 80 ctg aag gtt ctg gag gcc acc gct gac
act gac cca gcc ctg gtg gac 288 Leu Lys Val Leu Glu Ala Thr Ala Asp
Thr Asp Pro Ala Leu Val Asp 85 90 95 gtc ttg gac cag ccc ctt cac
acc ctg cac cat atc ctc tcc cag ttc 336 Val Leu Asp Gln Pro Leu His
Thr Leu His His Ile Leu Ser Gln Phe 100 105 110 cgg gcc tgt atc cag
cct cag ccc acg gca ggg ccc agg acc cgg ggc 384 Arg Ala Cys Ile Gln
Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly 115 120 125 cgc ctc cac
cat tgg ctg tac cgg ctc cag gag gcc cca aaa aag gag 432 Arg Leu His
His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu 130 135 140 tcc
cct ggc tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc ctc 480 Ser
Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 145 150
155 160 ctc acg cga gac ctg aat tgt gtt gcc agt ggg gac ctg tgt gtc
tga 528 Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val
* 165 170 175 28 175 PRT Artificial Sequence IL-28A mutant C50S 28
Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly Cys 1 5
10 15 His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala
Phe 20 25 30 Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu
Lys Asp Cys 35 40 45 Arg Ser His Ser Arg Leu Phe Pro Arg Thr Trp
Asp Leu Arg Gln Leu 50 55 60 Gln Val Arg Glu Arg Pro Met Ala Leu
Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala Thr
Ala Asp Thr Asp Pro Ala Leu Val Asp 85 90 95 Val Leu Asp Gln Pro
Leu His Thr Leu His His Ile Leu Ser Gln Phe 100 105 110 Arg Ala Cys
Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly 115 120 125 Arg
Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu 130 135
140 Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu
145 150 155 160 Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu
Cys Val 165 170 175 29 531 DNA Artificial Sequence met IL-28A
mutant C51S CDS (1)...(531) 29 atg gtt cct gtc gcc agg ctc cac ggg
gct ctc ccg gat gca agg ggc 48 Met Val Pro Val Ala Arg Leu His Gly
Ala Leu Pro Asp Ala Arg Gly 1 5 10 15 tgc cac ata gcc cag ttc aag
tcc ctg tct cca cag gag ctg cag gcc 96 Cys His Ile Ala Gln Phe Lys
Ser Leu Ser Pro Gln Glu Leu Gln Ala 20 25 30 ttt aag agg gcc aaa
gat gcc tta gaa gag tcg ctt ctg ctg aag gac 144 Phe Lys Arg Ala Lys
Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 40 45 tgc agg tcc
cac tcc cgc ctc ttc ccc agg acc tgg gac ctg agg cag 192 Cys Arg Ser
His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln 50 55 60 ctg
cag gtg agg gag cgc ccc atg gct ttg gag gct gag ctg gcc ctg 240 Leu
Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu 65 70
75 80 acg ctg aag gtt ctg gag gcc acc gct gac act gac cca gcc ctg
gtg 288 Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu
Val 85 90 95 gac gtc ttg gac cag ccc ctt cac acc ctg cac cat atc
ctc tcc cag 336 Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile
Leu Ser Gln 100 105 110 ttc cgg gcc tgt atc cag cct cag ccc acg gca
ggg ccc agg acc cgg 384 Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala
Gly Pro Arg Thr Arg 115 120 125 ggc cgc ctc cac cat tgg ctg tac cgg
ctc cag gag gcc cca aaa aag 432 Gly Arg Leu His His Trp Leu Tyr Arg
Leu Gln Glu Ala Pro Lys Lys 130 135 140 gag tcc cct ggc tgc ctc gag
gcc tct gtc acc ttc aac ctc ttc cgc 480 Glu Ser Pro Gly Cys Leu Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg 145 150 155 160 ctc ctc acg cga
gac ctg aat tgt gtt gcc agt ggg gac ctg tgt gtc 528 Leu Leu Thr Arg
Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170 175 tga 531
* 30 176 PRT Artificial Sequence met IL-28A mutant C51S 30 Met Val
Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10 15
Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala 20
25 30 Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys
Asp 35 40 45 Cys Arg Ser His Ser Arg Leu Phe Pro Arg Thr Trp Asp
Leu Arg Gln 50 55 60 Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu
Ala Glu Leu Ala Leu 65 70 75 80 Thr Leu Lys Val Leu Glu Ala Thr Ala
Asp Thr Asp Pro Ala Leu Val 85 90 95 Asp Val Leu Asp Gln Pro Leu
His Thr Leu His His Ile Leu Ser Gln 100 105 110 Phe Arg Ala Cys Ile
Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg 115 120 125 Gly Arg Leu
His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140 Glu
Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg 145 150
155 160 Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys
Val 165 170 175 31 546 DNA Artificial Sequence IL-29 mutant C171S
CDS (1)...(546) 31 ggt ccg gtt ccg acc tct aaa cca acc acc act ggt
aaa ggt tgc cac 48 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly
Lys Gly Cys His 1 5 10 15 atc ggt cgt ttc aaa tct ctg tct ccg cag
gaa ctg gct tct ttc aaa 96 Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln
Glu Leu Ala Ser Phe Lys 20 25 30 aaa gct cgt gac gct ctg gaa gaa
tct ctg aaa ctg aaa aac tgg tct 144 Lys Ala Arg Asp Ala Leu Glu Glu
Ser Leu Lys Leu Lys Asn Trp Ser 35 40 45 tgc tct tct ccg gtt
ttc ccg ggt aac tgg gat ctg cgt ctg ctg cag 192 Cys Ser Ser Pro Val
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln 50 55 60 gtt cgt gaa
cgt ccg gtt gct ctg gaa gct gaa ctg gct ctg acc ctg 240 Val Arg Glu
Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 aaa
gtt ctg gaa gct gct gca ggt cct gct ctg gaa gat gtt ctg gat 288 Lys
Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90
95 cag ccg ctg cac act ctg cac cac atc ctg tct cag ctg cag gct tgc
336 Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys
100 105 110 att caa ccg caa ccg acc gct ggt ccg cgt ccg cgt ggt cgt
ctg cac 384 Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg
Leu His 115 120 125 cac tgg ctg cat cgt ctg cag gaa gct ccg aaa aaa
gaa tct gct ggt 432 His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys
Glu Ser Ala Gly 130 135 140 tgc ctg gaa gct tct gtt acc ttc aac ctg
ttc cgt ctg ctg acc cgt 480 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu
Phe Arg Leu Leu Thr Arg 145 150 155 160 gat ctg aaa tac gtt gct gat
ggt aac ctg tct ctg cgt acc tct acc 528 Asp Leu Lys Tyr Val Ala Asp
Gly Asn Leu Ser Leu Arg Thr Ser Thr 165 170 175 cat ccg gaa tct acc
taa 546 His Pro Glu Ser Thr * 180 32 181 PRT Artificial Sequence
IL-29 mutant C171S 32 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr
Gly Lys Gly Cys His 1 5 10 15 Ile Gly Arg Phe Lys Ser Leu Ser Pro
Gln Glu Leu Ala Ser Phe Lys 20 25 30 Lys Ala Arg Asp Ala Leu Glu
Glu Ser Leu Lys Leu Lys Asn Trp Ser 35 40 45 Cys Ser Ser Pro Val
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln 50 55 60 Val Arg Glu
Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 Lys
Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90
95 Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys
100 105 110 Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg
Leu His 115 120 125 His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys
Glu Ser Ala Gly 130 135 140 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu
Phe Arg Leu Leu Thr Arg 145 150 155 160 Asp Leu Lys Tyr Val Ala Asp
Gly Asn Leu Ser Leu Arg Thr Ser Thr 165 170 175 His Pro Glu Ser Thr
180 33 549 DNA Artificial Sequence met IL-29 mutant C172S CDS
(1)...(549) 33 atg ggt ccg gtt ccg acc tct aaa cca acc acc act ggt
aaa ggt tgc 48 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly
Lys Gly Cys 1 5 10 15 cac atc ggt cgt ttc aaa tct ctg tct ccg cag
gaa ctg gct tct ttc 96 His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln
Glu Leu Ala Ser Phe 20 25 30 aaa aaa gct cgt gac gct ctg gaa gaa
tct ctg aaa ctg aaa aac tgg 144 Lys Lys Ala Arg Asp Ala Leu Glu Glu
Ser Leu Lys Leu Lys Asn Trp 35 40 45 tct tgc tct tct ccg gtt ttc
ccg ggt aac tgg gat ctg cgt ctg ctg 192 Ser Cys Ser Ser Pro Val Phe
Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60 cag gtt cgt gaa cgt
ccg gtt gct ctg gaa gct gaa ctg gct ctg acc 240 Gln Val Arg Glu Arg
Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 ctg aaa gtt
ctg gaa gct gct gca ggt cct gct ctg gaa gat gtt ctg 288 Leu Lys Val
Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95 gat
cag ccg ctg cac act ctg cac cac atc ctg tct cag ctg cag gct 336 Asp
Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105
110 tgc att caa ccg caa ccg acc gct ggt ccg cgt ccg cgt ggt cgt ctg
384 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu
115 120 125 cac cac tgg ctg cat cgt ctg cag gaa gct ccg aaa aaa gaa
tct gct 432 His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu
Ser Ala 130 135 140 ggt tgc ctg gaa gct tct gtt acc ttc aac ctg ttc
cgt ctg ctg acc 480 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe
Arg Leu Leu Thr 145 150 155 160 cgt gat ctg aaa tac gtt gct gat ggt
aac ctg tct ctg cgt acc tct 528 Arg Asp Leu Lys Tyr Val Ala Asp Gly
Asn Leu Ser Leu Arg Thr Ser 165 170 175 acc cat ccg gaa tct acc taa
549 Thr His Pro Glu Ser Thr * 180 34 182 PRT Artificial Sequence
met IL-29 mutant C172S 34 Met Gly Pro Val Pro Thr Ser Lys Pro Thr
Thr Thr Gly Lys Gly Cys 1 5 10 15 His Ile Gly Arg Phe Lys Ser Leu
Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 Lys Lys Ala Arg Asp Ala
Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45 Ser Cys Ser Ser
Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60 Gln Val
Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80
Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85
90 95 Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln
Ala 100 105 110 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg
Gly Arg Leu 115 120 125 His His Trp Leu His Arg Leu Gln Glu Ala Pro
Lys Lys Glu Ser Ala 130 135 140 Gly Cys Leu Glu Ala Ser Val Thr Phe
Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 Arg Asp Leu Lys Tyr Val
Ala Asp Gly Asn Leu Ser Leu Arg Thr Ser 165 170 175 Thr His Pro Glu
Ser Thr 180 35 531 DNA Artificial Sequence met IL-28A CDS
(1)...(531) 35 atg gtt cct gtc gcc agg ctc cac ggg gct ctc ccg gat
gca agg ggc 48 Met Val Pro Val Ala Arg Leu His Gly Ala Leu Pro Asp
Ala Arg Gly 1 5 10 15 tgc cac ata gcc cag ttc aag tcc ctg tct cca
cag gag ctg cag gcc 96 Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro
Gln Glu Leu Gln Ala 20 25 30 ttt aag agg gcc aaa gat gcc tta gaa
gag tcg ctt ctg ctg aag gac 144 Phe Lys Arg Ala Lys Asp Ala Leu Glu
Glu Ser Leu Leu Leu Lys Asp 35 40 45 tgc agg tgc cac tcc cgc ctc
ttc ccc agg acc tgg gac ctg agg cag 192 Cys Arg Cys His Ser Arg Leu
Phe Pro Arg Thr Trp Asp Leu Arg Gln 50 55 60 ctg cag gtg agg gag
cgc ccc atg gct ttg gag gct gag ctg gcc ctg 240 Leu Gln Val Arg Glu
Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu 65 70 75 80 acg ctg aag
gtt ctg gag gcc acc gct gac act gac cca gcc ctg gtg 288 Thr Leu Lys
Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Val 85 90 95 gac
gtc ttg gac cag ccc ctt cac acc ctg cac cat atc ctc tcc cag 336 Asp
Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln 100 105
110 ttc cgg gcc tgt atc cag cct cag ccc acg gca ggg ccc agg acc cgg
384 Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg
115 120 125 ggc cgc ctc cac cat tgg ctg tac cgg ctc cag gag gcc cca
aaa aag 432 Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro
Lys Lys 130 135 140 gag tcc cct ggc tgc ctc gag gcc tct gtc acc ttc
aac ctc ttc cgc 480 Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe
Asn Leu Phe Arg 145 150 155 160 ctc ctc acg cga gac ctg aat tgt gtt
gcc agt ggg gac ctg tgt gtc 528 Leu Leu Thr Arg Asp Leu Asn Cys Val
Ala Ser Gly Asp Leu Cys Val 165 170 175 tga 531 * 36 176 PRT
Artificial Sequence met IL-28A 36 Met Val Pro Val Ala Arg Leu His
Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10 15 Cys His Ile Ala Gln Phe
Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala 20 25 30 Phe Lys Arg Ala
Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 40 45 Cys Arg
Cys His Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln 50 55 60
Leu Gln Val Arg Glu Arg Pro Met Ala Leu Glu Ala Glu Leu Ala Leu 65
70 75 80 Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala
Leu Val 85 90 95 Asp Val Leu Asp Gln Pro Leu His Thr Leu His His
Ile Leu Ser Gln 100 105 110 Phe Arg Ala Cys Ile Gln Pro Gln Pro Thr
Ala Gly Pro Arg Thr Arg 115 120 125 Gly Arg Leu His His Trp Leu Tyr
Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140 Glu Ser Pro Gly Cys Leu
Glu Ala Ser Val Thr Phe Asn Leu Phe Arg 145 150 155 160 Leu Leu Thr
Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170 175 37
621 DNA Artificial Sequence met IL-29 CDS (1)...(549) 37 atg ggc
cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc tgc 48 Met Gly
Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys 1 5 10 15
cac att ggc agg ttc aaa tct ctg tca cca cag gag cta gcg agc ttc 96
His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20
25 30 aag aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac
tgg 144 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn
Trp 35 40 45 agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg
agg ctt ctc 192 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu
Arg Leu Leu 50 55 60 cag gtg agg gag cgc cct gtg gcc ttg gag gct
gag ctg gcc ctg acg 240 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala
Glu Leu Ala Leu Thr 65 70 75 80 ctg aag gtc ctg gag gcc gct gct ggc
cca gcc ctg gag gac gtc cta 288 Leu Lys Val Leu Glu Ala Ala Ala Gly
Pro Ala Leu Glu Asp Val Leu 85 90 95 gac cag ccc ctt cac acc ctg
cac cac atc ctc tcc cag ctc cag gcc 336 Asp Gln Pro Leu His Thr Leu
His His Ile Leu Ser Gln Leu Gln Ala 100 105 110 tgt atc cag cct cag
ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc 384 Cys Ile Gln Pro Gln
Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 cac cac tgg
ctg cac cgg ctc cag gag gcc ccc aaa aag gag tcc gct 432 His His Trp
Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 ggc
tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc acg 480 Gly
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150
155 160 cga gac ctc aaa tat gtg gcc gat ggg aac ctg tgt ctg aga acg
tca 528 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr
Ser 165 170 175 acc cac cct gag tcc acc tga caccccacac cttatttatg
cgctgagccc 579 Thr His Pro Glu Ser Thr * 180 tactccttcc ttaatttatt
tcctctcacc ctttatttat ga 621 38 182 PRT Artificial Sequence met
IL-29 38 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys
Gly Cys 1 5 10 15 His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu
Leu Ala Ser Phe 20 25 30 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser
Leu Lys Leu Lys Asn Trp 35 40 45 Ser Cys Ser Ser Pro Val Phe Pro
Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60 Gln Val Arg Glu Arg Pro
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 Leu Lys Val Leu
Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95 Asp Gln
Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105 110
Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115
120 125 His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser
Ala 130 135 140 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg
Leu Leu Thr 145 150 155 160 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn
Leu Cys Leu Arg Thr Ser 165 170 175 Thr His Pro Glu Ser Thr 180 39
531 DNA Artificial Sequence met IL-28B CDS (1)...(531) 39 atg gtt
cct gtc gcc agg ctc cgc ggg gct ctc ccg gat gca agg ggc 48 Met Val
Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10 15
tgc cac ata gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc 96
Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala 20
25 30 ttt aag agg gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg aag
gac 144 Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys
Asp 35 40 45 tgc aag tgc cgc tcc cgc ctc ttc ccc agg acc tgg gac
ctg agg cag 192 Cys Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp
Leu Arg Gln 50 55 60 ctg cag gtg agg gag cgc ccc gtg gct ttg gag
gct gag ctg gcc ctg 240 Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu
Ala Glu Leu Ala Leu 65 70 75 80 acg ctg aag gtt ctg gag gcc acc gct
gac act gac cca gcc ctg ggg 288 Thr Leu Lys Val Leu Glu Ala Thr Ala
Asp Thr Asp Pro Ala Leu Gly 85 90 95 gat gtc ttg gac cag ccc ctt
cac acc ctg cac cat atc ctc tcc cag 336 Asp Val Leu Asp Gln Pro Leu
His Thr Leu His His Ile Leu Ser Gln 100 105 110 ctc cgg gcc tgt atc
cag cct cag ccc acg gca ggg ccc agg acc cgg 384 Leu Arg Ala Cys Ile
Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg 115 120 125 ggc cgc ctc
cac cat tgg ctg cac cgg ctc cag gag gcc cca aaa aag 432 Gly Arg Leu
His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140 gag
tcc cct ggc tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc 480 Glu
Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg 145 150
155 160 ctc ctc acg cga gac ctg aat tgt gtt gcc agc ggg gac ctg tgt
gtc 528 Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys
Val 165 170 175 tga 531 * 40 176 PRT Artificial Sequence met IL-28B
40 Met Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly
1 5 10 15 Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu
Gln Ala 20 25 30 Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu
Leu Leu Lys Asp 35 40 45 Cys Lys Cys Arg Ser Arg Leu Phe Pro Arg
Thr Trp Asp Leu Arg Gln 50 55 60 Leu Gln Val Arg Glu Arg Pro Val
Ala Leu Glu Ala Glu Leu Ala Leu 65 70 75 80 Thr Leu Lys Val Leu Glu
Ala Thr Ala Asp Thr Asp Pro Ala Leu Gly 85 90 95 Asp Val Leu Asp
Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln 100 105 110 Leu Arg
Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg 115 120 125
Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys 130
135 140 Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe
Arg 145 150 155 160 Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly
Asp Leu Cys Val 165 170 175 41 546 DNA Artificial Sequence IL-29
Cys15 mutant, Asn169 CDS (1)...(546) variation (44)...(45) n = A,
T, G, or C 41 ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag
ggc dnn cac 48 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys
Gly Xaa His 1 5 10 15 att ggc agg ttc aaa tct ctg tca cca cag gag
cta gcg agc ttc aag 96 Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu
Leu Ala Ser Phe Lys 20 25
30 aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg agt
144 Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser
35 40 45 tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt
ctc cag 192 Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu
Leu Gln 50 55 60 gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg
gcc ctg acg ctg 240 Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu
Ala Leu Thr Leu 65 70 75 80 aag gtc ctg gag gcc gct gct ggc cca gcc
ctg gag gac gtc cta gac 288 Lys Val Leu Glu Ala Ala Ala Gly Pro Ala
Leu Glu Asp Val Leu Asp 85 90 95 cag ccc ctt cac acc ctg cac cac
atc ctc tcc cag ctc cag gcc tgt 336 Gln Pro Leu His Thr Leu His His
Ile Leu Ser Gln Leu Gln Ala Cys 100 105 110 atc cag cct cag ccc aca
gca ggg ccc agg ccc cgg ggc cgc ctc cac 384 Ile Gln Pro Gln Pro Thr
Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 120 125 cac tgg ctg cac
cgg ctc cag gag gcc ccc aaa aag gag tcc gct ggc 432 His Trp Leu His
Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140 tgc ctg
gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc acg cga 480 Cys Leu
Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150 155
160 gac ctc aaa tat gtg gcc gat ggg aay ctg tgt ctg aga acg tca acc
528 Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr
165 170 175 cac cct gag tcc acc tga 546 His Pro Glu Ser Thr * 180
42 181 PRT Artificial Sequence IL-29 Cys15 mutant, Asn169 VARIANT
(15)...(15) Xaa = Ser, Ala, Thr, Val, or Asn 42 Gly Pro Val Pro Thr
Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His 1 5 10 15 Ile Gly Arg
Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys 20 25 30 Lys
Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser 35 40
45 Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln
50 55 60 Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu
Thr Leu 65 70 75 80 Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu
Asp Val Leu Asp 85 90 95 Gln Pro Leu His Thr Leu His His Ile Leu
Ser Gln Leu Gln Ala Cys 100 105 110 Ile Gln Pro Gln Pro Thr Ala Gly
Pro Arg Pro Arg Gly Arg Leu His 115 120 125 His Trp Leu His Arg Leu
Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140 Cys Leu Glu Ala
Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150 155 160 Asp
Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr 165 170
175 His Pro Glu Ser Thr 180 43 549 DNA Artificial Sequence Met
IL-29 Cys16 mutant, Asn170 CDS (1)...(549) variation (47)...(48) n
= A, T, G, or C 43 atg ggc cct gtc ccc act tcc aag ccc acc aca act
ggg aag ggc dnn 48 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr
Gly Lys Gly Xaa 1 5 10 15 cac att ggc agg ttc aaa tct ctg tca cca
cag gag cta gcg agc ttc 96 His Ile Gly Arg Phe Lys Ser Leu Ser Pro
Gln Glu Leu Ala Ser Phe 20 25 30 aag aag gcc agg gac gcc ttg gaa
gag tca ctc aag ctg aaa aac tgg 144 Lys Lys Ala Arg Asp Ala Leu Glu
Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45 agt tgc agc tct cct gtc
ttc ccc ggg aat tgg gac ctg agg ctt ctc 192 Ser Cys Ser Ser Pro Val
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60 cag gtg agg gag
cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg 240 Gln Val Arg Glu
Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 ctg aag
gtc ctg gag gcc gct gct ggc cca gcc ctg gag gac gtc cta 288 Leu Lys
Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95
gac cag ccc ctt cac acc ctg cac cac atc ctc tcc cag ctc cag gcc 336
Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100
105 110 tgt atc cag cct cag ccc aca gca ggg ccc agg ccc cgg ggc cgc
ctc 384 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg
Leu 115 120 125 cac cac tgg ctg cac cgg ctc cag gag gcc ccc aaa aag
gag tcc gct 432 His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys
Glu Ser Ala 130 135 140 ggc tgc ctg gag gca tct gtc acc ttc aac ctc
ttc cgc ctc ctc acg 480 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu
Phe Arg Leu Leu Thr 145 150 155 160 cga gac ctc aaa tat gtg gcc gat
ggg aay ctg tgt ctg aga acg tca 528 Arg Asp Leu Lys Tyr Val Ala Asp
Gly Asn Leu Cys Leu Arg Thr Ser 165 170 175 acc cac cct gag tcc acc
tga 549 Thr His Pro Glu Ser Thr * 180 44 182 PRT Artificial
Sequence Met IL-29 Cys16 mutant, Asn170 VARIANT (16)...(16) Xaa =
Ser, Ala, Thr, Val, or Asn 44 Met Gly Pro Val Pro Thr Ser Lys Pro
Thr Thr Thr Gly Lys Gly Xaa 1 5 10 15 His Ile Gly Arg Phe Lys Ser
Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 Lys Lys Ala Arg Asp
Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45 Ser Cys Ser
Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60 Gln
Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70
75 80 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val
Leu 85 90 95 Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln
Leu Gln Ala 100 105 110 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg
Pro Arg Gly Arg Leu 115 120 125 His His Trp Leu His Arg Leu Gln Glu
Ala Pro Lys Lys Glu Ser Ala 130 135 140 Gly Cys Leu Glu Ala Ser Val
Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 Arg Asp Leu Lys
Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser 165 170 175 Thr His
Pro Glu Ser Thr 180 45 546 DNA Artificial Sequence IL-29 Cys15
mutant, Asp169 CDS (1)...(546) variation (44)...(45) n = A, T, G,
or C 45 ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc dnn
cac 48 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa
His 1 5 10 15 att ggc agg ttc aaa tct ctg tca cca cag gag cta gcg
agc ttc aag 96 Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala
Ser Phe Lys 20 25 30 aag gcc agg gac gcc ttg gaa gag tca ctc aag
ctg aaa aac tgg agt 144 Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys
Leu Lys Asn Trp Ser 35 40 45 tgc agc tct cct gtc ttc ccc ggg aat
tgg gac ctg agg ctt ctc cag 192 Cys Ser Ser Pro Val Phe Pro Gly Asn
Trp Asp Leu Arg Leu Leu Gln 50 55 60 gtg agg gag cgc cct gtg gcc
ttg gag gct gag ctg gcc ctg acg ctg 240 Val Arg Glu Arg Pro Val Ala
Leu Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 aag gtc ctg gag gcc
gct gct ggc cca gcc ctg gag gac gtc cta gac 288 Lys Val Leu Glu Ala
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90 95 cag ccc ctt
cac acc ctg cac cac atc ctc tcc cag ctc cag gcc tgt 336 Gln Pro Leu
His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys 100 105 110 atc
cag cct cag ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc cac 384 Ile
Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 120
125 cac tgg ctg cac cgg ctc cag gag gcc ccc aaa aag gag tcc gct ggc
432 His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly
130 135 140 tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc
acg cga 480 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu
Thr Arg 145 150 155 160 gac ctc aaa tat gtg gcc gat ggg gay ctg tgt
ctg aga acg tca acc 528 Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys
Leu Arg Thr Ser Thr 165 170 175 cac cct gag tcc acc tga 546 His Pro
Glu Ser Thr * 180 46 181 PRT Artificial Sequence IL-29 Cys15
mutant, Asp169 VARIANT (15)...(15) Xaa = Ser, Ala, Thr, Val, or Asn
46 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His
1 5 10 15 Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser
Phe Lys 20 25 30 Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu
Lys Asn Trp Ser 35 40 45 Cys Ser Ser Pro Val Phe Pro Gly Asn Trp
Asp Leu Arg Leu Leu Gln 50 55 60 Val Arg Glu Arg Pro Val Ala Leu
Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 Lys Val Leu Glu Ala Ala
Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90 95 Gln Pro Leu His
Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys 100 105 110 Ile Gln
Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 120 125
His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly 130
135 140 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr
Arg 145 150 155 160 Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu
Arg Thr Ser Thr 165 170 175 His Pro Glu Ser Thr 180 47 549 DNA
Artificial Sequence Met IL-29 Cys16 mutant, Asp170 CDS (1)...(549)
variation (47)...(48) n = A, T, G, or C 47 atg ggc cct gtc ccc act
tcc aag ccc acc aca act ggg aag ggc dnn 48 Met Gly Pro Val Pro Thr
Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa 1 5 10 15 cac att ggc agg
ttc aaa tct ctg tca cca cag gag cta gcg agc ttc 96 His Ile Gly Arg
Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 aag aag
gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg 144 Lys Lys
Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45
agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc 192
Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50
55 60 cag gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg gcc ctg
acg 240 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu
Thr 65 70 75 80 ctg aag gtc ctg gag gcc gct gct ggc cca gcc ctg gag
gac gtc cta 288 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu
Asp Val Leu 85 90 95 gac cag ccc ctt cac acc ctg cac cac atc ctc
tcc cag ctc cag gcc 336 Asp Gln Pro Leu His Thr Leu His His Ile Leu
Ser Gln Leu Gln Ala 100 105 110 tgt atc cag cct cag ccc aca gca ggg
ccc agg ccc cgg ggc cgc ctc 384 Cys Ile Gln Pro Gln Pro Thr Ala Gly
Pro Arg Pro Arg Gly Arg Leu 115 120 125 cac cac tgg ctg cac cgg ctc
cag gag gcc ccc aaa aag gag tcc gct 432 His His Trp Leu His Arg Leu
Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 ggc tgc ctg gag gca
tct gtc acc ttc aac ctc ttc cgc ctc ctc acg 480 Gly Cys Leu Glu Ala
Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 cga gac
ctc aaa tat gtg gcc gat ggg gay ctg tgt ctg aga acg tca 528 Arg Asp
Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser 165 170 175
acc cac cct gag tcc acc tga 549 Thr His Pro Glu Ser Thr * 180 48
182 PRT Artificial Sequence Met IL-29 Cys16 mutant, Asp170 VARIANT
(16)...(16) Xaa = Ser, Ala, Thr, Val, or Asn 48 Met Gly Pro Val Pro
Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa 1 5 10 15 His Ile Gly
Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 Lys
Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40
45 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu
50 55 60 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala
Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu
Glu Asp Val Leu 85 90 95 Asp Gln Pro Leu His Thr Leu His His Ile
Leu Ser Gln Leu Gln Ala 100 105 110 Cys Ile Gln Pro Gln Pro Thr Ala
Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 His His Trp Leu His Arg
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 Gly Cys Leu Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 Arg
Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser 165 170
175 Thr His Pro Glu Ser Thr 180 49 546 DNA Artificial Sequence
IL-29 Asp169 Cys171 mutant CDS (1)...(546) variation (512)...(513)
n = A, T, G, or C 49 ggc cct gtc ccc act tcc aag ccc acc aca act
ggg aag ggc tgc cac 48 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr
Gly Lys Gly Cys His 1 5 10 15 att ggc agg ttc aaa tct ctg tca cca
cag gag cta gcg agc ttc aag 96 Ile Gly Arg Phe Lys Ser Leu Ser Pro
Gln Glu Leu Ala Ser Phe Lys 20 25 30 aag gcc agg gac gcc ttg gaa
gag tca ctc aag ctg aaa aac tgg agt 144 Lys Ala Arg Asp Ala Leu Glu
Glu Ser Leu Lys Leu Lys Asn Trp Ser 35 40 45 tgc agc tct cct gtc
ttc ccc ggg aat tgg gac ctg agg ctt ctc cag 192 Cys Ser Ser Pro Val
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln 50 55 60 gtg agg gag
cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg ctg 240 Val Arg Glu
Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 aag
gtc ctg gag gcc gct gct ggc cca gcc ctg gag gac gtc cta gac 288 Lys
Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90
95 cag ccc ctt cac acc ctg cac cac atc ctc tcc cag ctc cag gcc tgt
336 Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys
100 105 110 atc cag cct cag ccc aca gca ggg ccc agg ccc cgg ggc cgc
ctc cac 384 Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg
Leu His 115 120 125 cac tgg ctg cac cgg ctc cag gag gcc ccc aaa aag
gag tcc gct ggc 432 His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys
Glu Ser Ala Gly 130 135 140 tgc ctg gag gca tct gtc acc ttc aac ctc
ttc cgc ctc ctc acg cga 480 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu
Phe Arg Leu Leu Thr Arg 145 150 155 160 gac ctc aaa tat gtg gcc gat
ggg gay ctg dnn ctg aga acg tca acc 528 Asp Leu Lys Tyr Val Ala Asp
Gly Asp Leu Xaa Leu Arg Thr Ser Thr 165 170 175 cac cct gag tcc acc
tga 546 His Pro Glu Ser Thr * 180 50 181 PRT Artificial Sequence
IL-29 Asp169 Cys171 mutant VARIANT (171)...(171) Xaa = Ser, Ala,
Thr, Val, or Asn 50 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly
Lys Gly Cys His 1 5 10 15 Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln
Glu Leu Ala Ser Phe Lys 20 25 30 Lys Ala Arg Asp Ala Leu Glu Glu
Ser Leu Lys Leu Lys Asn Trp Ser 35 40 45 Cys Ser Ser Pro Val Phe
Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln 50 55 60 Val Arg Glu Arg
Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 Lys Val
Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90 95
Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys 100
105 110 Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu
His 115 120 125 His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu
Ser Ala Gly 130
135 140 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr
Arg 145 150 155 160 Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu
Arg Thr Ser Thr 165 170 175 His Pro Glu Ser Thr 180 51 549 DNA
Artificial Sequence Met IL-29 Asp170 Cys172 mutant CDS (1)...(549)
variation (515)...(516) n = A, T, G, or C 51 atg ggc cct gtc ccc
act tcc aag ccc acc aca act ggg aag ggc tgc 48 Met Gly Pro Val Pro
Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys 1 5 10 15 cac att ggc
agg ttc aaa tct ctg tca cca cag gag cta gcg agc ttc 96 His Ile Gly
Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 aag
aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg 144 Lys
Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40
45 agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc
192 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu
50 55 60 cag gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg gcc
ctg acg 240 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala
Leu Thr 65 70 75 80 ctg aag gtc ctg gag gcc gct gct ggc cca gcc ctg
gag gac gtc cta 288 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu
Glu Asp Val Leu 85 90 95 gac cag ccc ctt cac acc ctg cac cac atc
ctc tcc cag ctc cag gcc 336 Asp Gln Pro Leu His Thr Leu His His Ile
Leu Ser Gln Leu Gln Ala 100 105 110 tgt atc cag cct cag ccc aca gca
ggg ccc agg ccc cgg ggc cgc ctc 384 Cys Ile Gln Pro Gln Pro Thr Ala
Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 cac cac tgg ctg cac cgg
ctc cag gag gcc ccc aaa aag gag tcc gct 432 His His Trp Leu His Arg
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 ggc tgc ctg gag
gca tct gtc acc ttc aac ctc ttc cgc ctc ctc acg 480 Gly Cys Leu Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 cga
gac ctc aaa tat gtg gcc gat ggg gay ctg dnn ctg aga acg tca 528 Arg
Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser 165 170
175 acc cac cct gag tcc acc tga 549 Thr His Pro Glu Ser Thr * 180
52 182 PRT Artificial Sequence Met IL-29 Asp170 Cys172 mutant
VARIANT (172)...(172) Xaa = Ser, Ala, Thr, Val, or Asn 52 Met Gly
Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys 1 5 10 15
His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20
25 30 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn
Trp 35 40 45 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu
Arg Leu Leu 50 55 60 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala
Glu Leu Ala Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala Ala Ala Gly
Pro Ala Leu Glu Asp Val Leu 85 90 95 Asp Gln Pro Leu His Thr Leu
His His Ile Leu Ser Gln Leu Gln Ala 100 105 110 Cys Ile Gln Pro Gln
Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 His His Trp
Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 Gly
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150
155 160 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr
Ser 165 170 175 Thr His Pro Glu Ser Thr 180 53 546 DNA Artificial
Sequence IL-29 Pro10 Asn169 Cys171 mutant CDS (1)...(546) variation
(30)...(30) n = A, T, G, or C variation (512)...(513) n = A, T, G,
or C 53 ggc cct gtc ccc act tcc aag ccc acc ccn act ggg aag ggc tgc
cac 48 Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Cys
His 1 5 10 15 att ggc agg ttc aaa tct ctg tca cca cag gag cta gcg
agc ttc aag 96 Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala
Ser Phe Lys 20 25 30 aag gcc agg gac gcc ttg gaa gag tca ctc aag
ctg aaa aac tgg agt 144 Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys
Leu Lys Asn Trp Ser 35 40 45 tgc agc tct cct gtc ttc ccc ggg aat
tgg gac ctg agg ctt ctc cag 192 Cys Ser Ser Pro Val Phe Pro Gly Asn
Trp Asp Leu Arg Leu Leu Gln 50 55 60 gtg agg gag cgc cct gtg gcc
ttg gag gct gag ctg gcc ctg acg ctg 240 Val Arg Glu Arg Pro Val Ala
Leu Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 aag gtc ctg gag gcc
gct gct ggc cca gcc ctg gag gac gtc cta gac 288 Lys Val Leu Glu Ala
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90 95 cag ccc ctt
cac acc ctg cac cac atc ctc tcc cag ctc cag gcc tgt 336 Gln Pro Leu
His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys 100 105 110 atc
cag cct cag ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc cac 384 Ile
Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 120
125 cac tgg ctg cac cgg ctc cag gag gcc ccc aaa aag gag tcc gct ggc
432 His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly
130 135 140 tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc
acg cga 480 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu
Thr Arg 145 150 155 160 gac ctc aaa tat gtg gcc gat ggg aac ctg dnn
ctg aga acg tca acc 528 Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa
Leu Arg Thr Ser Thr 165 170 175 cac cct gag tcc acc tga 546 His Pro
Glu Ser Thr * 180 54 181 PRT Artificial Sequence IL-29 Pro10 Asn169
Cys171 mutant VARIANT (171)...(171) Xaa = Ser, Ala, Thr, Val, or
Asn 54 Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Cys
His 1 5 10 15 Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala
Ser Phe Lys 20 25 30 Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys
Leu Lys Asn Trp Ser 35 40 45 Cys Ser Ser Pro Val Phe Pro Gly Asn
Trp Asp Leu Arg Leu Leu Gln 50 55 60 Val Arg Glu Arg Pro Val Ala
Leu Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 Lys Val Leu Glu Ala
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90 95 Gln Pro Leu
His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys 100 105 110 Ile
Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 120
125 His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly
130 135 140 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu
Thr Arg 145 150 155 160 Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa
Leu Arg Thr Ser Thr 165 170 175 His Pro Glu Ser Thr 180 55 549 DNA
Artificial Sequence Met IL-29 Pro11 Asn170 Cys172 mutant CDS
(1)...(549) variation 33, 515, 516 n = A, T, G, or C 55 atg ggc cct
gtc ccc act tcc aag ccc acc ccn act ggg aag ggc tgc 48 Met Gly Pro
Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Cys 1 5 10 15 cac
att ggc agg ttc aaa tct ctg tca cca cag gag cta gcg agc ttc 96 His
Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25
30 aag aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg
144 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp
35 40 45 agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg
ctt ctc 192 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg
Leu Leu 50 55 60 cag gtg agg gag cgc cct gtg gcc ttg gag gct gag
ctg gcc ctg acg 240 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu
Leu Ala Leu Thr 65 70 75 80 ctg aag gtc ctg gag gcc gct gct ggc cca
gcc ctg gag gac gtc cta 288 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro
Ala Leu Glu Asp Val Leu 85 90 95 gac cag ccc ctt cac acc ctg cac
cac atc ctc tcc cag ctc cag gcc 336 Asp Gln Pro Leu His Thr Leu His
His Ile Leu Ser Gln Leu Gln Ala 100 105 110 tgt atc cag cct cag ccc
aca gca ggg ccc agg ccc cgg ggc cgc ctc 384 Cys Ile Gln Pro Gln Pro
Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 cac cac tgg ctg
cac cgg ctc cag gag gcc ccc aaa aag gag tcc gct 432 His His Trp Leu
His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 ggc tgc
ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc acg 480 Gly Cys
Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155
160 cga gac ctc aaa tat gtg gcc gat ggg aac ctg dnn ctg aga acg tca
528 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser
165 170 175 acc cac cct gag tcc acc tga 549 Thr His Pro Glu Ser Thr
* 180 56 182 PRT Artificial Sequence Met IL-29 Pro11 Asn170 Cys172
mutant VARIANT (172)...(172) Xaa = Ser, Ala, Thr, Val, or Asn 56
Met Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Cys 1 5
10 15 His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser
Phe 20 25 30 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu
Lys Asn Trp 35 40 45 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp
Asp Leu Arg Leu Leu 50 55 60 Gln Val Arg Glu Arg Pro Val Ala Leu
Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala Ala
Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95 Asp Gln Pro Leu His
Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105 110 Cys Ile Gln
Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 His
His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135
140 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr
145 150 155 160 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu
Arg Thr Ser 165 170 175 Thr His Pro Glu Ser Thr 180 57 546 DNA
Artificial Sequence IL-29 Pro10 Cys15 mutant Asn169 CDS (1)...(546)
variation (30)...(30) n = A, T, G, or C variation (44)...(45) n =
A, T, G, or C 57 ggc cct gtc ccc act tcc aag ccc acc ccn act ggg
aag ggc dnn cac 48 Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly
Lys Gly Xaa His 1 5 10 15 att ggc agg ttc aaa tct ctg tca cca cag
gag cta gcg agc ttc aag 96 Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln
Glu Leu Ala Ser Phe Lys 20 25 30 aag gcc agg gac gcc ttg gaa gag
tca ctc aag ctg aaa aac tgg agt 144 Lys Ala Arg Asp Ala Leu Glu Glu
Ser Leu Lys Leu Lys Asn Trp Ser 35 40 45 tgc agc tct cct gtc ttc
ccc ggg aat tgg gac ctg agg ctt ctc cag 192 Cys Ser Ser Pro Val Phe
Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln 50 55 60 gtg agg gag cgc
cct gtg gcc ttg gag gct gag ctg gcc ctg acg ctg 240 Val Arg Glu Arg
Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 aag gtc
ctg gag gcc gct gct ggc cca gcc ctg gag gac gtc cta gac 288 Lys Val
Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90 95
cag ccc ctt cac acc ctg cac cac atc ctc tcc cag ctc cag gcc tgt 336
Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys 100
105 110 atc cag cct cag ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc
cac 384 Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu
His 115 120 125 cac tgg ctg cac cgg ctc cag gag gcc ccc aaa aag gag
tcc gct ggc 432 His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu
Ser Ala Gly 130 135 140 tgc ctg gag gca tct gtc acc ttc aac ctc ttc
cgc ctc ctc acg cga 480 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe
Arg Leu Leu Thr Arg 145 150 155 160 gac ctc aaa tat gtg gcc gat ggg
aay ctg tgt ctg aga acg tca acc 528 Asp Leu Lys Tyr Val Ala Asp Gly
Asn Leu Cys Leu Arg Thr Ser Thr 165 170 175 cac cct gag tcc acc tga
546 His Pro Glu Ser Thr * 180 58 181 PRT Artificial Sequence IL-29
Pro10 Cys15 mutant Asn169 VARIANT (15)...(15) Xaa = Ser, Ala, Thr,
Val, or Asn 58 Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys
Gly Xaa His 1 5 10 15 Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu
Leu Ala Ser Phe Lys 20 25 30 Lys Ala Arg Asp Ala Leu Glu Glu Ser
Leu Lys Leu Lys Asn Trp Ser 35 40 45 Cys Ser Ser Pro Val Phe Pro
Gly Asn Trp Asp Leu Arg Leu Leu Gln 50 55 60 Val Arg Glu Arg Pro
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 Lys Val Leu
Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90 95 Gln
Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys 100 105
110 Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His
115 120 125 His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser
Ala Gly 130 135 140 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg
Leu Leu Thr Arg 145 150 155 160 Asp Leu Lys Tyr Val Ala Asp Gly Asn
Leu Cys Leu Arg Thr Ser Thr 165 170 175 His Pro Glu Ser Thr 180 59
549 DNA Artificial Sequence Met IL-29 Pro11 Cys16 mutant Asn170 CDS
(1)...(549) variation (33)...(33) n = A, T, G, or C variation
(47)...(48) n = A, T, G, or C 59 atg ggc cct gtc ccc act tcc aag
ccc acc ccn act ggg aag ggc dnn 48 Met Gly Pro Val Pro Thr Ser Lys
Pro Thr Pro Thr Gly Lys Gly Xaa 1 5 10 15 cac att ggc agg ttc aaa
tct ctg tca cca cag gag cta gcg agc ttc 96 His Ile Gly Arg Phe Lys
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 aag aag gcc agg
gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg 144 Lys Lys Ala Arg
Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45 agt tgc
agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc 192 Ser Cys
Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60
cag gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg 240
Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65
70 75 80 ctg aag gtc ctg gag gcc gct gct ggc cca gcc ctg gag gac
gtc cta 288 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp
Val Leu 85 90 95 gac cag ccc ctt cac acc ctg cac cac atc ctc tcc
cag ctc cag gcc 336 Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser
Gln Leu Gln Ala 100 105 110 tgt atc cag cct cag ccc aca gca ggg ccc
agg ccc cgg ggc cgc ctc 384 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Pro Arg Gly Arg Leu 115 120 125 cac cac tgg ctg cac cgg ctc cag
gag gcc ccc aaa aag gag tcc gct 432 His His Trp Leu His Arg Leu Gln
Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 ggc tgc ctg gag gca tct
gtc acc ttc aac ctc ttc cgc ctc ctc acg 480 Gly Cys Leu Glu Ala Ser
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 cga gac ctc
aaa tat gtg gcc gat ggg aay ctg tgt ctg aga acg tca 528 Arg Asp Leu
Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser 165 170 175 acc
cac cct gag tcc acc tga 549 Thr His Pro Glu Ser Thr * 180 60 182
PRT Artificial Sequence Met IL-29 Pro11 Cys16 mutant Asn170 VARIANT
(16)...(16) Xaa = Ser, Ala, Thr, Val, or Asn
60 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Xaa
1 5 10 15 His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala
Ser Phe 20 25 30 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys
Leu Lys Asn Trp 35 40 45 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn
Trp Asp Leu Arg Leu Leu 50 55 60 Gln Val Arg Glu Arg Pro Val Ala
Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95 Asp Gln Pro Leu
His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105 110 Cys Ile
Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125
His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130
135 140 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu
Thr 145 150 155 160 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys
Leu Arg Thr Ser 165 170 175 Thr His Pro Glu Ser Thr 180 61 546 DNA
Artificial Sequence IL-29 Pro10 Asp169 Cys171 mutant CDS
(1)...(546) variation (30)...(30) n = A, T, G, or C variation
(512)...(513) n = A, T, G, or C 61 ggc cct gtc ccc act tcc aag ccc
acc ccn act ggg aag ggc tgc cac 48 Gly Pro Val Pro Thr Ser Lys Pro
Thr Pro Thr Gly Lys Gly Cys His 1 5 10 15 att ggc agg ttc aaa tct
ctg tca cca cag gag cta gcg agc ttc aag 96 Ile Gly Arg Phe Lys Ser
Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys 20 25 30 aag gcc agg gac
gcc ttg gaa gag tca ctc aag ctg aaa aac tgg agt 144 Lys Ala Arg Asp
Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser 35 40 45 tgc agc
tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc cag 192 Cys Ser
Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln 50 55 60
gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg ctg 240
Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu 65
70 75 80 aag gtc ctg gag gcc gct gct ggc cca gcc ctg gag gac gtc
cta gac 288 Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val
Leu Asp 85 90 95 cag ccc ctt cac acc ctg cac cac atc ctc tcc cag
ctc cag gcc tgt 336 Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln
Leu Gln Ala Cys 100 105 110 atc cag cct cag ccc aca gca ggg ccc agg
ccc cgg ggc cgc ctc cac 384 Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg
Pro Arg Gly Arg Leu His 115 120 125 cac tgg ctg cac cgg ctc cag gag
gcc ccc aaa aag gag tcc gct ggc 432 His Trp Leu His Arg Leu Gln Glu
Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140 tgc ctg gag gca tct gtc
acc ttc aac ctc ttc cgc ctc ctc acg cga 480 Cys Leu Glu Ala Ser Val
Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150 155 160 gac ctc aaa
tat gtg gcc gat ggg gay ctg dnn ctg aga acg tca acc 528 Asp Leu Lys
Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser Thr 165 170 175 cac
cct gag tcc acc tga 546 His Pro Glu Ser Thr * 180 62 181 PRT
Artificial Sequence IL-29 Pro10 Asp169 Cys171 mutant VARIANT
(171)...(171) Xaa = Ser, Ala, Thr, Val, or Asn 62 Gly Pro Val Pro
Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Cys His 1 5 10 15 Ile Gly
Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys 20 25 30
Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser 35
40 45 Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu
Gln 50 55 60 Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala
Leu Thr Leu 65 70 75 80 Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu
Glu Asp Val Leu Asp 85 90 95 Gln Pro Leu His Thr Leu His His Ile
Leu Ser Gln Leu Gln Ala Cys 100 105 110 Ile Gln Pro Gln Pro Thr Ala
Gly Pro Arg Pro Arg Gly Arg Leu His 115 120 125 His Trp Leu His Arg
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140 Cys Leu Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150 155 160
Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser Thr 165
170 175 His Pro Glu Ser Thr 180 63 549 DNA Artificial Sequence Met
IL-29 Pro11 Asp170 Cys172 mutant CDS (1)...(549) variation
(33)...(33) n = A, T, G, or C variation (515)...(516) n = A, T, G,
or C 63 atg ggc cct gtc ccc act tcc aag ccc acc ccn act ggg aag ggc
tgc 48 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly
Cys 1 5 10 15 cac att ggc agg ttc aaa tct ctg tca cca cag gag cta
gcg agc ttc 96 His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu
Ala Ser Phe 20 25 30 aag aag gcc agg gac gcc ttg gaa gag tca ctc
aag ctg aaa aac tgg 144 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu
Lys Leu Lys Asn Trp 35 40 45 agt tgc agc tct cct gtc ttc ccc ggg
aat tgg gac ctg agg ctt ctc 192 Ser Cys Ser Ser Pro Val Phe Pro Gly
Asn Trp Asp Leu Arg Leu Leu 50 55 60 cag gtg agg gag cgc cct gtg
gcc ttg gag gct gag ctg gcc ctg acg 240 Gln Val Arg Glu Arg Pro Val
Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 ctg aag gtc ctg gag
gcc gct gct ggc cca gcc ctg gag gac gtc cta 288 Leu Lys Val Leu Glu
Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95 gac cag ccc
ctt cac acc ctg cac cac atc ctc tcc cag ctc cag gcc 336 Asp Gln Pro
Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105 110 tgt
atc cag cct cag ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc 384 Cys
Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120
125 cac cac tgg ctg cac cgg ctc cag gag gcc ccc aaa aag gag tcc gct
432 His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala
130 135 140 ggc tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc
ctc acg 480 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu
Leu Thr 145 150 155 160 cga gac ctc aaa tat gtg gcc gat ggg gay ctg
dnn ctg aga acg tca 528 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu
Xaa Leu Arg Thr Ser 165 170 175 acc cac cct gag tcc acc tga 549 Thr
His Pro Glu Ser Thr * 180 64 182 PRT Artificial Sequence Met IL-29
Pro11 Asp170 Cys172 mutant VARIANT (172)...(172) Xaa = Ser, Ala,
Thr, Val, or Asn 64 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Pro Thr
Gly Lys Gly Cys 1 5 10 15 His Ile Gly Arg Phe Lys Ser Leu Ser Pro
Gln Glu Leu Ala Ser Phe 20 25 30 Lys Lys Ala Arg Asp Ala Leu Glu
Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45 Ser Cys Ser Ser Pro Val
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60 Gln Val Arg Glu
Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 Leu Lys
Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95
Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100
105 110 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg
Leu 115 120 125 His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys
Glu Ser Ala 130 135 140 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu
Phe Arg Leu Leu Thr 145 150 155 160 Arg Asp Leu Lys Tyr Val Ala Asp
Gly Asp Leu Xaa Leu Arg Thr Ser 165 170 175 Thr His Pro Glu Ser Thr
180 65 546 DNA Artificial Sequence IL-29 Pro10 Cys15 mutant Asp169
CDS (1)...(546) variation 30, 44, 45 n = A, T, G, or C 65 ggc cct
gtc ccc act tcc aag ccc acc ccn act ggg aag ggc dnn cac 48 Gly Pro
Val Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Xaa His 1 5 10 15
att ggc agg ttc aaa tct ctg tca cca cag gag cta gcg agc ttc aag 96
Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys 20
25 30 aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg
agt 144 Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp
Ser 35 40 45 tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg
ctt ctc cag 192 Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg
Leu Leu Gln 50 55 60 gtg agg gag cgc cct gtg gcc ttg gag gct gag
ctg gcc ctg acg ctg 240 Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu
Leu Ala Leu Thr Leu 65 70 75 80 aag gtc ctg gag gcc gct gct ggc cca
gcc ctg gag gac gtc cta gac 288 Lys Val Leu Glu Ala Ala Ala Gly Pro
Ala Leu Glu Asp Val Leu Asp 85 90 95 cag ccc ctt cac acc ctg cac
cac atc ctc tcc cag ctc cag gcc tgt 336 Gln Pro Leu His Thr Leu His
His Ile Leu Ser Gln Leu Gln Ala Cys 100 105 110 atc cag cct cag ccc
aca gca ggg ccc agg ccc cgg ggc cgc ctc cac 384 Ile Gln Pro Gln Pro
Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 120 125 cac tgg ctg
cac cgg ctc cag gag gcc ccc aaa aag gag tcc gct ggc 432 His Trp Leu
His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140 tgc
ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc acg cga 480 Cys
Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150
155 160 gac ctc aaa tat gtg gcc gat ggg gay ctg tgt ctg aga acg tca
acc 528 Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser
Thr 165 170 175 cac cct gag tcc acc tga 546 His Pro Glu Ser Thr *
180 66 181 PRT Artificial Sequence IL-29 Pro10 Cys15 mutant Asp169
VARIANT (15)...(15) Xaa = Ser, Ala, Thr, Val, or Asn 66 Gly Pro Val
Pro Thr Ser Lys Pro Thr Pro Thr Gly Lys Gly Xaa His 1 5 10 15 Ile
Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys 20 25
30 Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser
35 40 45 Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu
Leu Gln 50 55 60 Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu
Ala Leu Thr Leu 65 70 75 80 Lys Val Leu Glu Ala Ala Ala Gly Pro Ala
Leu Glu Asp Val Leu Asp 85 90 95 Gln Pro Leu His Thr Leu His His
Ile Leu Ser Gln Leu Gln Ala Cys 100 105 110 Ile Gln Pro Gln Pro Thr
Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 120 125 His Trp Leu His
Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140 Cys Leu
Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150 155
160 Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser Thr
165 170 175 His Pro Glu Ser Thr 180 67 549 DNA Artificial Sequence
Met IL-29 Pro11 Cys16 mutant Asp170 CDS (1)...(549) variation 33,
47, 48 n = A, T, G, or C 67 atg ggc cct gtc ccc act tcc aag ccc acc
ccn act ggg aag ggc dnn 48 Met Gly Pro Val Pro Thr Ser Lys Pro Thr
Pro Thr Gly Lys Gly Xaa 1 5 10 15 cac att ggc agg ttc aaa tct ctg
tca cca cag gag cta gcg agc ttc 96 His Ile Gly Arg Phe Lys Ser Leu
Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 aag aag gcc agg gac gcc
ttg gaa gag tca ctc aag ctg aaa aac tgg 144 Lys Lys Ala Arg Asp Ala
Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45 agt tgc agc tct
cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc 192 Ser Cys Ser Ser
Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60 cag gtg
agg gag cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg 240 Gln Val
Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80
ctg aag gtc ctg gag gcc gct gct ggc cca gcc ctg gag gac gtc cta 288
Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85
90 95 gac cag ccc ctt cac acc ctg cac cac atc ctc tcc cag ctc cag
gcc 336 Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln
Ala 100 105 110 tgt atc cag cct cag ccc aca gca ggg ccc agg ccc cgg
ggc cgc ctc 384 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg
Gly Arg Leu 115 120 125 cac cac tgg ctg cac cgg ctc cag gag gcc ccc
aaa aag gag tcc gct 432 His His Trp Leu His Arg Leu Gln Glu Ala Pro
Lys Lys Glu Ser Ala 130 135 140 ggc tgc ctg gag gca tct gtc acc ttc
aac ctc ttc cgc ctc ctc acg 480 Gly Cys Leu Glu Ala Ser Val Thr Phe
Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 cga gac ctc aaa tat gtg
gcc gat ggg gay ctg tgt ctg aga acg tca 528 Arg Asp Leu Lys Tyr Val
Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser 165 170 175 acc cac cct gag
tcc acc tga 549 Thr His Pro Glu Ser Thr * 180 68 182 PRT Artificial
Sequence Met IL-29 Pro11 Cys16 mutant Asp170 VARIANT (16)...(16)
Xaa = Ser, Ala, Thr, Val, or Asn 68 Met Gly Pro Val Pro Thr Ser Lys
Pro Thr Pro Thr Gly Lys Gly Xaa 1 5 10 15 His Ile Gly Arg Phe Lys
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 Lys Lys Ala Arg
Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45 Ser Cys
Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60
Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65
70 75 80 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp
Val Leu 85 90 95 Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser
Gln Leu Gln Ala 100 105 110 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Pro Arg Gly Arg Leu 115 120 125 His His Trp Leu His Arg Leu Gln
Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 Gly Cys Leu Glu Ala Ser
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 Arg Asp Leu
Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser 165 170 175 Thr
His Pro Glu Ser Thr 180 69 546 DNA Artificial Sequence IL-29 Asp18
Asn169 Cys171 mutant CDS (1)...(546) variation (512)...(513) n = A,
T, G, or C 69 ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag
ggc tgc cac 48 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys
Gly Cys His 1 5 10 15 att gay agg ttc aaa tct ctg tca cca cag gag
cta gcg agc ttc aag 96 Ile Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu
Leu Ala Ser Phe Lys 20 25 30 aag gcc agg gac gcc ttg gaa gag tca
ctc aag ctg aaa aac tgg agt 144 Lys Ala Arg Asp Ala Leu Glu Glu Ser
Leu Lys Leu Lys Asn Trp Ser 35 40 45 tgc agc tct cct gtc ttc ccc
ggg aat tgg gac ctg agg ctt ctc cag 192 Cys Ser Ser Pro Val Phe Pro
Gly Asn Trp Asp Leu Arg Leu Leu Gln 50 55 60 gtg agg gag cgc cct
gtg gcc ttg gag gct gag ctg gcc ctg acg ctg 240 Val Arg Glu Arg Pro
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 aag gtc ctg
gag gcc gct gct ggc cca gcc ctg gag gac gtc cta gac 288 Lys Val Leu
Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90 95 cag
ccc ctt cac acc ctg cac cac atc ctc tcc cag ctc cag gcc tgt 336 Gln
Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys 100 105
110
atc cag cct cag ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc cac 384
Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115
120 125 cac tgg ctg cac cgg ctc cag gag gcc ccc aaa aag gag tcc gct
ggc 432 His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala
Gly 130 135 140 tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc
ctc acg cga 480 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu
Leu Thr Arg 145 150 155 160 gac ctc aaa tat gtg gcc gat ggg aac ctg
dnn ctg aga acg tca acc 528 Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu
Xaa Leu Arg Thr Ser Thr 165 170 175 cac cct gag tcc acc tga 546 His
Pro Glu Ser Thr * 180 70 181 PRT Artificial Sequence IL-29 Asp18
Asn169 Cys171 mutant VARIANT (171)...(171) Xaa = Ser, Ala, Thr,
Val, or Asn 70 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys
Gly Cys His 1 5 10 15 Ile Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu
Leu Ala Ser Phe Lys 20 25 30 Lys Ala Arg Asp Ala Leu Glu Glu Ser
Leu Lys Leu Lys Asn Trp Ser 35 40 45 Cys Ser Ser Pro Val Phe Pro
Gly Asn Trp Asp Leu Arg Leu Leu Gln 50 55 60 Val Arg Glu Arg Pro
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 Lys Val Leu
Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90 95 Gln
Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys 100 105
110 Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His
115 120 125 His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser
Ala Gly 130 135 140 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg
Leu Leu Thr Arg 145 150 155 160 Asp Leu Lys Tyr Val Ala Asp Gly Asn
Leu Xaa Leu Arg Thr Ser Thr 165 170 175 His Pro Glu Ser Thr 180 71
549 DNA Artificial Sequence Met IL-29 Asp19 Asn170 Cys172 mutant
CDS (1)...(549) variation (515)...(516) n = A, T, G, or C 71 atg
ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc tgc 48 Met
Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys 1 5 10
15 cac att gay agg ttc aaa tct ctg tca cca cag gag cta gcg agc ttc
96 His Ile Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe
20 25 30 aag aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa
aac tgg 144 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys
Asn Trp 35 40 45 agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac
ctg agg ctt ctc 192 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp
Leu Arg Leu Leu 50 55 60 cag gtg agg gag cgc cct gtg gcc ttg gag
gct gag ctg gcc ctg acg 240 Gln Val Arg Glu Arg Pro Val Ala Leu Glu
Ala Glu Leu Ala Leu Thr 65 70 75 80 ctg aag gtc ctg gag gcc gct gct
ggc cca gcc ctg gag gac gtc cta 288 Leu Lys Val Leu Glu Ala Ala Ala
Gly Pro Ala Leu Glu Asp Val Leu 85 90 95 gac cag ccc ctt cac acc
ctg cac cac atc ctc tcc cag ctc cag gcc 336 Asp Gln Pro Leu His Thr
Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105 110 tgt atc cag cct
cag ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc 384 Cys Ile Gln Pro
Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 cac cac
tgg ctg cac cgg ctc cag gag gcc ccc aaa aag gag tcc gct 432 His His
Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140
ggc tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc acg 480
Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145
150 155 160 cga gac ctc aaa tat gtg gcc gat ggg aac ctg dnn ctg aga
acg tca 528 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg
Thr Ser 165 170 175 acc cac cct gag tcc acc tga 549 Thr His Pro Glu
Ser Thr * 180 72 182 PRT Artificial Sequence Met IL-29 Asp19 Asn170
Cys172 mutant VARIANT (172)...(172) Xaa = Ser, Ala, Thr, Val, or
Asn 72 Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly
Cys 1 5 10 15 His Ile Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu
Ala Ser Phe 20 25 30 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu
Lys Leu Lys Asn Trp 35 40 45 Ser Cys Ser Ser Pro Val Phe Pro Gly
Asn Trp Asp Leu Arg Leu Leu 50 55 60 Gln Val Arg Glu Arg Pro Val
Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 Leu Lys Val Leu Glu
Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95 Asp Gln Pro
Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105 110 Cys
Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120
125 His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala
130 135 140 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu
Leu Thr 145 150 155 160 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu
Xaa Leu Arg Thr Ser 165 170 175 Thr His Pro Glu Ser Thr 180 73 546
DNA Artificial Sequence IL-29 Cys15 mutant Asp18 Asn169 CDS
(1)...(546) variation (44)...(45) n = A, T, G, or C 73 ggc cct gtc
ccc act tcc aag ccc acc aca act ggg aag ggc dnn cac 48 Gly Pro Val
Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His 1 5 10 15 att
gay agg ttc aaa tct ctg tca cca cag gag cta gcg agc ttc aag 96 Ile
Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys 20 25
30 aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg agt
144 Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser
35 40 45 tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt
ctc cag 192 Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu
Leu Gln 50 55 60 gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg
gcc ctg acg ctg 240 Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu
Ala Leu Thr Leu 65 70 75 80 aag gtc ctg gag gcc gct gct ggc cca gcc
ctg gag gac gtc cta gac 288 Lys Val Leu Glu Ala Ala Ala Gly Pro Ala
Leu Glu Asp Val Leu Asp 85 90 95 cag ccc ctt cac acc ctg cac cac
atc ctc tcc cag ctc cag gcc tgt 336 Gln Pro Leu His Thr Leu His His
Ile Leu Ser Gln Leu Gln Ala Cys 100 105 110 atc cag cct cag ccc aca
gca ggg ccc agg ccc cgg ggc cgc ctc cac 384 Ile Gln Pro Gln Pro Thr
Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 120 125 cac tgg ctg cac
cgg ctc cag gag gcc ccc aaa aag gag tcc gct ggc 432 His Trp Leu His
Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140 tgc ctg
gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc acg cga 480 Cys Leu
Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150 155
160 gac ctc aaa tat gtg gcc gat ggg aay ctg tgt ctg aga acg tca acc
528 Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr
165 170 175 cac cct gag tcc acc tga 546 His Pro Glu Ser Thr * 180
74 181 PRT Artificial Sequence IL-29 Cys15 mutant Asp18 Asn169
VARIANT (15)...(15) Xaa = Ser, Ala, Thr, Val, or Asn 74 Gly Pro Val
Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His 1 5 10 15 Ile
Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys 20 25
30 Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser
35 40 45 Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu
Leu Gln 50 55 60 Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu
Ala Leu Thr Leu 65 70 75 80 Lys Val Leu Glu Ala Ala Ala Gly Pro Ala
Leu Glu Asp Val Leu Asp 85 90 95 Gln Pro Leu His Thr Leu His His
Ile Leu Ser Gln Leu Gln Ala Cys 100 105 110 Ile Gln Pro Gln Pro Thr
Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 120 125 His Trp Leu His
Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140 Cys Leu
Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150 155
160 Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr
165 170 175 His Pro Glu Ser Thr 180 75 549 DNA Artificial Sequence
Met IL-29 Cys16 mutant Asp19 Asn170 CDS (1)...(549) variation
(47)...(48) n = A, T, G, or C 75 atg ggc cct gtc ccc act tcc aag
ccc acc aca act ggg aag ggc dnn 48 Met Gly Pro Val Pro Thr Ser Lys
Pro Thr Thr Thr Gly Lys Gly Xaa 1 5 10 15 cac att gay agg ttc aaa
tct ctg tca cca cag gag cta gcg agc ttc 96 His Ile Asp Arg Phe Lys
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 aag aag gcc agg
gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg 144 Lys Lys Ala Arg
Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45 agt tgc
agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc 192 Ser Cys
Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60
cag gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg 240
Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65
70 75 80 ctg aag gtc ctg gag gcc gct gct ggc cca gcc ctg gag gac
gtc cta 288 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp
Val Leu 85 90 95 gac cag ccc ctt cac acc ctg cac cac atc ctc tcc
cag ctc cag gcc 336 Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser
Gln Leu Gln Ala 100 105 110 tgt atc cag cct cag ccc aca gca ggg ccc
agg ccc cgg ggc cgc ctc 384 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Pro Arg Gly Arg Leu 115 120 125 cac cac tgg ctg cac cgg ctc cag
gag gcc ccc aaa aag gag tcc gct 432 His His Trp Leu His Arg Leu Gln
Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 ggc tgc ctg gag gca tct
gtc acc ttc aac ctc ttc cgc ctc ctc acg 480 Gly Cys Leu Glu Ala Ser
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 cga gac ctc
aaa tat gtg gcc gat ggg aay ctg tgt ctg aga acg tca 528 Arg Asp Leu
Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser 165 170 175 acc
cac cct gag tcc acc tga 549 Thr His Pro Glu Ser Thr * 180 76 182
PRT Artificial Sequence Met IL-29 Cys16 mutant Asp19 Asn170 VARIANT
(16)...(16) Xaa = Ser, Ala, Thr, Val, or Asn 76 Met Gly Pro Val Pro
Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa 1 5 10 15 His Ile Asp
Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 Lys
Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40
45 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu
50 55 60 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala
Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu
Glu Asp Val Leu 85 90 95 Asp Gln Pro Leu His Thr Leu His His Ile
Leu Ser Gln Leu Gln Ala 100 105 110 Cys Ile Gln Pro Gln Pro Thr Ala
Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 His His Trp Leu His Arg
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 Gly Cys Leu Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 Arg
Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser 165 170
175 Thr His Pro Glu Ser Thr 180 77 546 DNA Artificial Sequence
IL-29 Asp18 Asp169 Cys171 mutant CDS (1)...(546) variation
(512)...(513) n = A, T, G, or C 77 ggc cct gtc ccc act tcc aag ccc
acc aca act ggg aag ggc tgc cac 48 Gly Pro Val Pro Thr Ser Lys Pro
Thr Thr Thr Gly Lys Gly Cys His 1 5 10 15 att gay agg ttc aaa tct
ctg tca cca cag gag cta gcg agc ttc aag 96 Ile Asp Arg Phe Lys Ser
Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys 20 25 30 aag gcc agg gac
gcc ttg gaa gag tca ctc aag ctg aaa aac tgg agt 144 Lys Ala Arg Asp
Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser 35 40 45 tgc agc
tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc cag 192 Cys Ser
Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln 50 55 60
gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg ctg 240
Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu 65
70 75 80 aag gtc ctg gag gcc gct gct ggc cca gcc ctg gag gac gtc
cta gac 288 Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val
Leu Asp 85 90 95 cag ccc ctt cac acc ctg cac cac atc ctc tcc cag
ctc cag gcc tgt 336 Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln
Leu Gln Ala Cys 100 105 110 atc cag cct cag ccc aca gca ggg ccc agg
ccc cgg ggc cgc ctc cac 384 Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg
Pro Arg Gly Arg Leu His 115 120 125 cac tgg ctg cac cgg ctc cag gag
gcc ccc aaa aag gag tcc gct ggc 432 His Trp Leu His Arg Leu Gln Glu
Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140 tgc ctg gag gca tct gtc
acc ttc aac ctc ttc cgc ctc ctc acg cga 480 Cys Leu Glu Ala Ser Val
Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150 155 160 gac ctc aaa
tat gtg gcc gat ggg gay ctg dnn ctg aga acg tca acc 528 Asp Leu Lys
Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser Thr 165 170 175 cac
cct gag tcc acc tga 546 His Pro Glu Ser Thr * 180 78 181 PRT
Artificial Sequence IL-29 Asp18 Asp169 Cys171 mutant VARIANT
(171)...(171) Xaa = Ser, Ala, Thr, Val, or Asn 78 Gly Pro Val Pro
Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His 1 5 10 15 Ile Asp
Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys 20 25 30
Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser 35
40 45 Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu
Gln 50 55 60 Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala
Leu Thr Leu 65 70 75 80 Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu
Glu Asp Val Leu Asp 85 90 95 Gln Pro Leu His Thr Leu His His Ile
Leu Ser Gln Leu Gln Ala Cys 100 105 110 Ile Gln Pro Gln Pro Thr Ala
Gly Pro Arg Pro Arg Gly Arg Leu His 115 120 125 His Trp Leu His Arg
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140 Cys Leu Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150 155 160
Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser Thr 165
170 175 His Pro Glu Ser Thr 180 79 549 DNA Artificial Sequence Met
IL-29 Asp19 Asp170 Cys172 mutant CDS (1)...(549) variation
(515)...(516) n = A, T, G, or C 79 atg ggc cct gtc ccc act tcc aag
ccc acc aca act ggg aag ggc tgc 48 Met Gly Pro Val Pro Thr Ser Lys
Pro Thr Thr Thr Gly Lys Gly Cys 1 5 10 15 cac att gay agg ttc aaa
tct ctg tca cca cag gag cta gcg agc ttc 96 His Ile Asp Arg Phe Lys
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 aag aag gcc agg
gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg 144
Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35
40 45 agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt
ctc 192 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu
Leu 50 55 60 cag gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg
gcc ctg acg 240 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu
Ala Leu Thr 65 70 75 80 ctg aag gtc ctg gag gcc gct gct ggc cca gcc
ctg gag gac gtc cta 288 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala
Leu Glu Asp Val Leu 85 90 95 gac cag ccc ctt cac acc ctg cac cac
atc ctc tcc cag ctc cag gcc 336 Asp Gln Pro Leu His Thr Leu His His
Ile Leu Ser Gln Leu Gln Ala 100 105 110 tgt atc cag cct cag ccc aca
gca ggg ccc agg ccc cgg ggc cgc ctc 384 Cys Ile Gln Pro Gln Pro Thr
Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 cac cac tgg ctg cac
cgg ctc cag gag gcc ccc aaa aag gag tcc gct 432 His His Trp Leu His
Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 ggc tgc ctg
gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc acg 480 Gly Cys Leu
Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160
cga gac ctc aaa tat gtg gcc gat ggg gay ctg dnn ctg aga acg tca 528
Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu Arg Thr Ser 165
170 175 acc cac cct gag tcc acc tga 549 Thr His Pro Glu Ser Thr *
180 80 182 PRT Artificial Sequence Met IL-29 Asp19 Asp170 Cys172
mutant VARIANT (172)...(172) Xaa = Ser, Ala, Thr, Val, or Asn 80
Met Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys 1 5
10 15 His Ile Asp Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser
Phe 20 25 30 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu
Lys Asn Trp 35 40 45 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp
Asp Leu Arg Leu Leu 50 55 60 Gln Val Arg Glu Arg Pro Val Ala Leu
Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala Ala
Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95 Asp Gln Pro Leu His
Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105 110 Cys Ile Gln
Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 His
His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135
140 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr
145 150 155 160 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Xaa Leu
Arg Thr Ser 165 170 175 Thr His Pro Glu Ser Thr 180 81 546 DNA
Artificial Sequence IL-29 Cys15 mutant Asp18 Asp169 CDS (1)...(546)
variation (44)...(45) n = A, T, G, or C 81 ggc cct gtc ccc act tcc
aag ccc acc aca act ggg aag ggc dnn cac 48 Gly Pro Val Pro Thr Ser
Lys Pro Thr Thr Thr Gly Lys Gly Xaa His 1 5 10 15 att gay agg ttc
aaa tct ctg tca cca cag gag cta gcg agc ttc aag 96 Ile Asp Arg Phe
Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys 20 25 30 aag gcc
agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg agt 144 Lys Ala
Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser 35 40 45
tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc cag 192
Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln 50
55 60 gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg
ctg 240 Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr
Leu 65 70 75 80 aag gtc ctg gag gcc gct gct ggc cca gcc ctg gag gac
gtc cta gac 288 Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp
Val Leu Asp 85 90 95 cag ccc ctt cac acc ctg cac cac atc ctc tcc
cag ctc cag gcc tgt 336 Gln Pro Leu His Thr Leu His His Ile Leu Ser
Gln Leu Gln Ala Cys 100 105 110 atc cag cct cag ccc aca gca ggg ccc
agg ccc cgg ggc cgc ctc cac 384 Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Pro Arg Gly Arg Leu His 115 120 125 cac tgg ctg cac cgg ctc cag
gag gcc ccc aaa aag gag tcc gct ggc 432 His Trp Leu His Arg Leu Gln
Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140 tgc ctg gag gca tct
gtc acc ttc aac ctc ttc cgc ctc ctc acg cga 480 Cys Leu Glu Ala Ser
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150 155 160 gac ctc
aaa tat gtg gcc gat ggg gay ctg tgt ctg aga acg tca acc 528 Asp Leu
Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser Thr 165 170 175
cac cct gag tcc acc tga 546 His Pro Glu Ser Thr * 180 82 181 PRT
Artificial Sequence IL-29 Cys15 mutant Asp18 Asp169 VARIANT
(15)...(15) Xaa = Ser, Ala, Thr, Val, or Asn 82 Gly Pro Val Pro Thr
Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa His 1 5 10 15 Ile Asp Arg
Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys 20 25 30 Lys
Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser 35 40
45 Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln
50 55 60 Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu
Thr Leu 65 70 75 80 Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu
Asp Val Leu Asp 85 90 95 Gln Pro Leu His Thr Leu His His Ile Leu
Ser Gln Leu Gln Ala Cys 100 105 110 Ile Gln Pro Gln Pro Thr Ala Gly
Pro Arg Pro Arg Gly Arg Leu His 115 120 125 His Trp Leu His Arg Leu
Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135 140 Cys Leu Glu Ala
Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg 145 150 155 160 Asp
Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser Thr 165 170
175 His Pro Glu Ser Thr 180 83 549 DNA Artificial Sequence Met
IL-29 Cys16 mutant Asp19 Asp170 CDS (1)...(549) variation
(47)...(48) n = A, T, G, or C 83 atg ggc cct gtc ccc act tcc aag
ccc acc aca act ggg aag ggc dnn 48 Met Gly Pro Val Pro Thr Ser Lys
Pro Thr Thr Thr Gly Lys Gly Xaa 1 5 10 15 cac att gay agg ttc aaa
tct ctg tca cca cag gag cta gcg agc ttc 96 His Ile Asp Arg Phe Lys
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 aag aag gcc agg
gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg 144 Lys Lys Ala Arg
Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45 agt tgc
agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc 192 Ser Cys
Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60
cag gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg 240
Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65
70 75 80 ctg aag gtc ctg gag gcc gct gct ggc cca gcc ctg gag gac
gtc cta 288 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp
Val Leu 85 90 95 gac cag ccc ctt cac acc ctg cac cac atc ctc tcc
cag ctc cag gcc 336 Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser
Gln Leu Gln Ala 100 105 110 tgt atc cag cct cag ccc aca gca ggg ccc
agg ccc cgg ggc cgc ctc 384 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Pro Arg Gly Arg Leu 115 120 125 cac cac tgg ctg cac cgg ctc cag
gag gcc ccc aaa aag gag tcc gct 432 His His Trp Leu His Arg Leu Gln
Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 ggc tgc ctg gag gca tct
gtc acc ttc aac ctc ttc cgc ctc ctc acg 480 Gly Cys Leu Glu Ala Ser
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 cga gac ctc
aaa tat gtg gcc gat ggg gay ctg tgt ctg aga acg tca 528 Arg Asp Leu
Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser 165 170 175 acc
cac cct gag tcc acc tga 549 Thr His Pro Glu Ser Thr * 180 84 182
PRT Artificial Sequence Met IL-29 Cys16 mutant Asp19 Asp170 VARIANT
(16)...(16) Xaa = Ser, Ala, Thr, Val, or Asn 84 Met Gly Pro Val Pro
Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Xaa 1 5 10 15 His Ile Asp
Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 Lys
Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40
45 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu
50 55 60 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala
Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu
Glu Asp Val Leu 85 90 95 Asp Gln Pro Leu His Thr Leu His His Ile
Leu Ser Gln Leu Gln Ala 100 105 110 Cys Ile Gln Pro Gln Pro Thr Ala
Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 His His Trp Leu His Arg
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 Gly Cys Leu Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 Arg
Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg Thr Ser 165 170
175 Thr His Pro Glu Ser Thr 180 85 528 DNA Artificial Sequence
IL-28B Cys48 mutant CDS (1)...(528) variation (143)...(144) n = A,
T, G, or C 85 gtt cct gtc gcc agg ctc cgc ggg gct ctc ccg gat gca
agg ggc tgc 48 Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala
Arg Gly Cys 1 5 10 15 cac ata gcc cag ttc aag tcc ctg tct cca cag
gag ctg cag gcc ttt 96 His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln
Glu Leu Gln Ala Phe 20 25 30 aag agg gcc aaa gat gcc tta gaa gag
tcg ctt ctg ctg aag gac dnn 144 Lys Arg Ala Lys Asp Ala Leu Glu Glu
Ser Leu Leu Leu Lys Asp Xaa 35 40 45 aag tgc cgc tcc cgc ctc ttc
ccc agg acc tgg gac ctg agg cag ctg 192 Lys Cys Arg Ser Arg Leu Phe
Pro Arg Thr Trp Asp Leu Arg Gln Leu 50 55 60 cag gtg agg gag cgc
ccc gtg gct ttg gag gct gag ctg gcc ctg acg 240 Gln Val Arg Glu Arg
Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 ctg aag gtt
ctg gag gcc acc gct gac act gac cca gcc ctg ggg gat 288 Leu Lys Val
Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Gly Asp 85 90 95 gtc
ttg gac cag ccc ctt cac acc ctg cac cat atc ctc tcc cag ctc 336 Val
Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu 100 105
110 cgg gcc tgt atc cag cct cag ccc acg gca ggg ccc agg acc cgg ggc
384 Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly
115 120 125 cgc ctc cac cat tgg ctg cac cgg ctc cag gag gcc cca aaa
aag gag 432 Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys
Lys Glu 130 135 140 tcc cct ggc tgc ctc gag gcc tct gtc acc ttc aac
ctc ttc cgc ctc 480 Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn
Leu Phe Arg Leu 145 150 155 160 ctc acg cga gac ctg aat tgt gtt gcc
agc ggg gac ctg tgt gtc tga 528 Leu Thr Arg Asp Leu Asn Cys Val Ala
Ser Gly Asp Leu Cys Val * 165 170 175 86 175 PRT Artificial
Sequence IL-28B Cys48 mutant VARIANT (48)...(48) Xaa = Ser, Ala,
Thr, Val, or Asn 86 Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp
Ala Arg Gly Cys 1 5 10 15 His Ile Ala Gln Phe Lys Ser Leu Ser Pro
Gln Glu Leu Gln Ala Phe 20 25 30 Lys Arg Ala Lys Asp Ala Leu Glu
Glu Ser Leu Leu Leu Lys Asp Xaa 35 40 45 Lys Cys Arg Ser Arg Leu
Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu 50 55 60 Gln Val Arg Glu
Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 Leu Lys
Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala Leu Gly Asp 85 90 95
Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu 100
105 110 Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg
Gly 115 120 125 Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro
Lys Lys Glu 130 135 140 Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe
Asn Leu Phe Arg Leu 145 150 155 160 Leu Thr Arg Asp Leu Asn Cys Val
Ala Ser Gly Asp Leu Cys Val 165 170 175 87 531 DNA Artificial
Sequence Met IL-28B Cys49 mutant CDS (1)...(531) variation
(146)...(147) n = A, T, G, or C 87 atg gtt cct gtc gcc agg ctc cgc
ggg gct ctc ccg gat gca agg ggc 48 Met Val Pro Val Ala Arg Leu Arg
Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10 15 tgc cac ata gcc cag ttc
aag tcc ctg tct cca cag gag ctg cag gcc 96 Cys His Ile Ala Gln Phe
Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala 20 25 30 ttt aag agg gcc
aaa gat gcc tta gaa gag tcg ctt ctg ctg aag gac 144 Phe Lys Arg Ala
Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 40 45 dnn aag
tgc cgc tcc cgc ctc ttc ccc agg acc tgg gac ctg agg cag 192 Xaa Lys
Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln 50 55 60
ctg cag gtg agg gag cgc ccc gtg gct ttg gag gct gag ctg gcc ctg 240
Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu 65
70 75 80 acg ctg aag gtt ctg gag gcc acc gct gac act gac cca gcc
ctg ggg 288 Thr Leu Lys Val Leu Glu Ala Thr Ala Asp Thr Asp Pro Ala
Leu Gly 85 90 95 gat gtc ttg gac cag ccc ctt cac acc ctg cac cat
atc ctc tcc cag 336 Asp Val Leu Asp Gln Pro Leu His Thr Leu His His
Ile Leu Ser Gln 100 105 110 ctc cgg gcc tgt atc cag cct cag ccc acg
gca ggg ccc agg acc cgg 384 Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr
Ala Gly Pro Arg Thr Arg 115 120 125 ggc cgc ctc cac cat tgg ctg cac
cgg ctc cag gag gcc cca aaa aag 432 Gly Arg Leu His His Trp Leu His
Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140 gag tcc cct ggc tgc ctc
gag gcc tct gtc acc ttc aac ctc ttc cgc 480 Glu Ser Pro Gly Cys Leu
Glu Ala Ser Val Thr Phe Asn Leu Phe Arg 145 150 155 160 ctc ctc acg
cga gac ctg aat tgt gtt gcc agc ggg gac ctg tgt gtc 528 Leu Leu Thr
Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170 175 tga
531 * 88 176 PRT Artificial Sequence Met IL-28B Cys49 mutant
VARIANT (49)...(49) Xaa = Ser, Ala, Thr, Val, or Asn 88 Met Val Pro
Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10 15 Cys
His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala 20 25
30 Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp
35 40 45 Xaa Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu
Arg Gln 50 55 60 Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala
Glu Leu Ala Leu 65 70 75 80 Thr Leu Lys Val Leu Glu Ala Thr Ala Asp
Thr Asp Pro Ala Leu Gly 85 90 95 Asp Val Leu Asp Gln Pro Leu His
Thr Leu His His Ile Leu Ser Gln 100 105 110 Leu Arg Ala Cys Ile Gln
Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg 115 120 125 Gly Arg Leu His
His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140 Glu Ser
Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg 145 150 155
160 Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys
Val
165 170 175 89 528 DNA Artificial Sequence IL-28B Cys50 mutant CDS
(1)...(528) variation (149)...(150) n = A, T, G, or C 89 gtt cct
gtc gcc agg ctc cgc ggg gct ctc ccg gat gca agg ggc tgc 48 Val Pro
Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly Cys 1 5 10 15
cac ata gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc ttt 96
His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe 20
25 30 aag agg gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg aag gac
tgc 144 Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp
Cys 35 40 45 aag dnn cgc tcc cgc ctc ttc ccc agg acc tgg gac ctg
agg cag ctg 192 Lys Xaa Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu
Arg Gln Leu 50 55 60 cag gtg agg gag cgc ccc gtg gct ttg gag gct
gag ctg gcc ctg acg 240 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala
Glu Leu Ala Leu Thr 65 70 75 80 ctg aag gtt ctg gag gcc acc gct gac
act gac cca gcc ctg ggg gat 288 Leu Lys Val Leu Glu Ala Thr Ala Asp
Thr Asp Pro Ala Leu Gly Asp 85 90 95 gtc ttg gac cag ccc ctt cac
acc ctg cac cat atc ctc tcc cag ctc 336 Val Leu Asp Gln Pro Leu His
Thr Leu His His Ile Leu Ser Gln Leu 100 105 110 cgg gcc tgt atc cag
cct cag ccc acg gca ggg ccc agg acc cgg ggc 384 Arg Ala Cys Ile Gln
Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly 115 120 125 cgc ctc cac
cat tgg ctg cac cgg ctc cag gag gcc cca aaa aag gag 432 Arg Leu His
His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu 130 135 140 tcc
cct ggc tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc ctc 480 Ser
Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 145 150
155 160 ctc acg cga gac ctg aat tgt gtt gcc agc ggg gac ctg tgt gtc
tga 528 Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val
* 165 170 175 90 175 PRT Artificial Sequence IL-28B Cys50 mutant
VARIANT (50)...(50) Xaa = Ser, Ala, Thr, Val, or Asn 90 Val Pro Val
Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly Cys 1 5 10 15 His
Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe 20 25
30 Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp Cys
35 40 45 Lys Xaa Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg
Gln Leu 50 55 60 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu
Leu Ala Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala Thr Ala Asp Thr
Asp Pro Ala Leu Gly Asp 85 90 95 Val Leu Asp Gln Pro Leu His Thr
Leu His His Ile Leu Ser Gln Leu 100 105 110 Arg Ala Cys Ile Gln Pro
Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly 115 120 125 Arg Leu His His
Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu 130 135 140 Ser Pro
Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 145 150 155
160 Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165
170 175 91 531 DNA Artificial Sequence Met IL-28B Cys51 mutant CDS
(1)...(531) variation (152)...(153) n = A, T, G, or C 91 atg gtt
cct gtc gcc agg ctc cgc ggg gct ctc ccg gat gca agg ggc 48 Met Val
Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly 1 5 10 15
tgc cac ata gcc cag ttc aag tcc ctg tct cca cag gag ctg cag gcc 96
Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala 20
25 30 ttt aag agg gcc aaa gat gcc tta gaa gag tcg ctt ctg ctg aag
gac 144 Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys
Asp 35 40 45 tgc aag dnn cgc tcc cgc ctc ttc ccc agg acc tgg gac
ctg agg cag 192 Cys Lys Xaa Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp
Leu Arg Gln 50 55 60 ctg cag gtg agg gag cgc ccc gtg gct ttg gag
gct gag ctg gcc ctg 240 Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu
Ala Glu Leu Ala Leu 65 70 75 80 acg ctg aag gtt ctg gag gcc acc gct
gac act gac cca gcc ctg ggg 288 Thr Leu Lys Val Leu Glu Ala Thr Ala
Asp Thr Asp Pro Ala Leu Gly 85 90 95 gat gtc ttg gac cag ccc ctt
cac acc ctg cac cat atc ctc tcc cag 336 Asp Val Leu Asp Gln Pro Leu
His Thr Leu His His Ile Leu Ser Gln 100 105 110 ctc cgg gcc tgt atc
cag cct cag ccc acg gca ggg ccc agg acc cgg 384 Leu Arg Ala Cys Ile
Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg 115 120 125 ggc cgc ctc
cac cat tgg ctg cac cgg ctc cag gag gcc cca aaa aag 432 Gly Arg Leu
His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140 gag
tcc cct ggc tgc ctc gag gcc tct gtc acc ttc aac ctc ttc cgc 480 Glu
Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg 145 150
155 160 ctc ctc acg cga gac ctg aat tgt gtt gcc agc ggg gac ctg tgt
gtc 528 Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys
Val 165 170 175 tga 531 * 92 176 PRT Artificial Sequence Met IL-28B
Cys51 mutant VARIANT (51)...(51) Xaa = Ser, Ala, Thr, Val, or Asn
92 Met Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly
1 5 10 15 Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu
Gln Ala 20 25 30 Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu
Leu Leu Lys Asp 35 40 45 Cys Lys Xaa Arg Ser Arg Leu Phe Pro Arg
Thr Trp Asp Leu Arg Gln 50 55 60 Leu Gln Val Arg Glu Arg Pro Val
Ala Leu Glu Ala Glu Leu Ala Leu 65 70 75 80 Thr Leu Lys Val Leu Glu
Ala Thr Ala Asp Thr Asp Pro Ala Leu Gly 85 90 95 Asp Val Leu Asp
Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln 100 105 110 Leu Arg
Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg 115 120 125
Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys 130
135 140 Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe
Arg 145 150 155 160 Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly
Asp Leu Cys Val 165 170 175 93 528 DNA Artificial Sequence IL-28B
Cys48 mutant T87S H135Y CDS (1)...(528) variation 143, 144, 261 n =
A, T, G, or C 93 gtt cct gtc gcc agg ctc cgc ggg gct ctc ccg gat
gca agg ggc tgc 48 Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp
Ala Arg Gly Cys 1 5 10 15 cac ata gcc cag ttc aag tcc ctg tct cca
cag gag ctg cag gcc ttt 96 His Ile Ala Gln Phe Lys Ser Leu Ser Pro
Gln Glu Leu Gln Ala Phe 20 25 30 aag agg gcc aaa gat gcc tta gaa
gag tcg ctt ctg ctg aag gac dnn 144 Lys Arg Ala Lys Asp Ala Leu Glu
Glu Ser Leu Leu Leu Lys Asp Xaa 35 40 45 aag tgc cgc tcc cgc ctc
ttc ccc agg acc tgg gac ctg agg cag ctg 192 Lys Cys Arg Ser Arg Leu
Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu 50 55 60 cag gtg agg gag
cgc ccc gtg gct ttg gag gct gag ctg gcc ctg acg 240 Gln Val Arg Glu
Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 ctg aag
gtt ctg gag gcc wsn gct gac act gac cca gcc ctg ggg gat 288 Leu Lys
Val Leu Glu Ala Xaa Ala Asp Thr Asp Pro Ala Leu Gly Asp 85 90 95
gtc ttg gac cag ccc ctt cac acc ctg cac cat atc ctc tcc cag ctc 336
Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu 100
105 110 cgg gcc tgt atc cag cct cag ccc acg gca ggg ccc agg acc cgg
ggc 384 Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg
Gly 115 120 125 cgc ctc cac cat tgg ctg tay cgg ctc cag gag gcc cca
aaa aag gag 432 Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro
Lys Lys Glu 130 135 140 tcc cct ggc tgc ctc gag gcc tct gtc acc ttc
aac ctc ttc cgc ctc 480 Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe
Asn Leu Phe Arg Leu 145 150 155 160 ctc acg cga gac ctg aat tgt gtt
gcc agc ggg gac ctg tgt gtc tga 528 Leu Thr Arg Asp Leu Asn Cys Val
Ala Ser Gly Asp Leu Cys Val * 165 170 175 94 175 PRT Artificial
Sequence VARIANT (48)...(48) Xaa = Ser, Ala, Thr, Val, or Asn
VARIANT (87)...(87) Xaa = Ser IL-28B Cys48 mutant T87S H135Y 94 Val
Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly Cys 1 5 10
15 His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe
20 25 30 Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys
Asp Xaa 35 40 45 Lys Cys Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp
Leu Arg Gln Leu 50 55 60 Gln Val Arg Glu Arg Pro Val Ala Leu Glu
Ala Glu Leu Ala Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala Xaa Ala
Asp Thr Asp Pro Ala Leu Gly Asp 85 90 95 Val Leu Asp Gln Pro Leu
His Thr Leu His His Ile Leu Ser Gln Leu 100 105 110 Arg Ala Cys Ile
Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly 115 120 125 Arg Leu
His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys Glu 130 135 140
Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 145
150 155 160 Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly Asp Leu Cys
Val 165 170 175 95 531 DNA Artificial Sequence Met IL-28B Cys49
mutant T88S H136Y CDS (1)...(531) variation 146, 147, 264 n = A, T,
G, or C 95 atg gtt cct gtc gcc agg ctc cgc ggg gct ctc ccg gat gca
agg ggc 48 Met Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala
Arg Gly 1 5 10 15 tgc cac ata gcc cag ttc aag tcc ctg tct cca cag
gag ctg cag gcc 96 Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln
Glu Leu Gln Ala 20 25 30 ttt aag agg gcc aaa gat gcc tta gaa gag
tcg ctt ctg ctg aag gac 144 Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu
Ser Leu Leu Leu Lys Asp 35 40 45 dnn aag tgc cgc tcc cgc ctc ttc
ccc agg acc tgg gac ctg agg cag 192 Xaa Lys Cys Arg Ser Arg Leu Phe
Pro Arg Thr Trp Asp Leu Arg Gln 50 55 60 ctg cag gtg agg gag cgc
ccc gtg gct ttg gag gct gag ctg gcc ctg 240 Leu Gln Val Arg Glu Arg
Pro Val Ala Leu Glu Ala Glu Leu Ala Leu 65 70 75 80 acg ctg aag gtt
ctg gag gcc wsn gct gac act gac cca gcc ctg ggg 288 Thr Leu Lys Val
Leu Glu Ala Xaa Ala Asp Thr Asp Pro Ala Leu Gly 85 90 95 gat gtc
ttg gac cag ccc ctt cac acc ctg cac cat atc ctc tcc cag 336 Asp Val
Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln 100 105 110
ctc cgg gcc tgt atc cag cct cag ccc acg gca ggg ccc agg acc cgg 384
Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg 115
120 125 ggc cgc ctc cac cat tgg ctg tay cgg ctc cag gag gcc cca aaa
aag 432 Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys
Lys 130 135 140 gag tcc cct ggc tgc ctc gag gcc tct gtc acc ttc aac
ctc ttc cgc 480 Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn
Leu Phe Arg 145 150 155 160 ctc ctc acg cga gac ctg aat tgt gtt gcc
agc ggg gac ctg tgt gtc 528 Leu Leu Thr Arg Asp Leu Asn Cys Val Ala
Ser Gly Asp Leu Cys Val 165 170 175 tga 531 * 96 176 PRT Artificial
Sequence VARIANT (49)...(49) Xaa = Ser, Ala, Thr, Val, or Asn
VARIANT (136)...(136) Xaa = Ser Met IL-28B Cys49 mutant T88S H136Y
96 Met Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly
1 5 10 15 Cys His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu
Gln Ala 20 25 30 Phe Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu
Leu Leu Lys Asp 35 40 45 Xaa Lys Cys Arg Ser Arg Leu Phe Pro Arg
Thr Trp Asp Leu Arg Gln 50 55 60 Leu Gln Val Arg Glu Arg Pro Val
Ala Leu Glu Ala Glu Leu Ala Leu 65 70 75 80 Thr Leu Lys Val Leu Glu
Ala Xaa Ala Asp Thr Asp Pro Ala Leu Gly 85 90 95 Asp Val Leu Asp
Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln 100 105 110 Leu Arg
Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg 115 120 125
Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys 130
135 140 Glu Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe
Arg 145 150 155 160 Leu Leu Thr Arg Asp Leu Asn Cys Val Ala Ser Gly
Asp Leu Cys Val 165 170 175 97 528 DNA Artificial Sequence IL-28B
Cys50 mutant T87S H135Y CDS (1)...(528) variation 149, 150, 261 n =
A, T, G, or C 97 gtt cct gtc gcc agg ctc cgc ggg gct ctc ccg gat
gca agg ggc tgc 48 Val Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp
Ala Arg Gly Cys 1 5 10 15 cac ata gcc cag ttc aag tcc ctg tct cca
cag gag ctg cag gcc ttt 96 His Ile Ala Gln Phe Lys Ser Leu Ser Pro
Gln Glu Leu Gln Ala Phe 20 25 30 aag agg gcc aaa gat gcc tta gaa
gag tcg ctt ctg ctg aag gac tgc 144 Lys Arg Ala Lys Asp Ala Leu Glu
Glu Ser Leu Leu Leu Lys Asp Cys 35 40 45 aag dnn cgc tcc cgc ctc
ttc ccc agg acc tgg gac ctg agg cag ctg 192 Lys Xaa Arg Ser Arg Leu
Phe Pro Arg Thr Trp Asp Leu Arg Gln Leu 50 55 60 cag gtg agg gag
cgc ccc gtg gct ttg gag gct gag ctg gcc ctg acg 240 Gln Val Arg Glu
Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 ctg aag
gtt ctg gag gcc wsn gct gac act gac cca gcc ctg ggg gat 288 Leu Lys
Val Leu Glu Ala Xaa Ala Asp Thr Asp Pro Ala Leu Gly Asp 85 90 95
gtc ttg gac cag ccc ctt cac acc ctg cac cat atc ctc tcc cag ctc 336
Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu 100
105 110 cgg gcc tgt atc cag cct cag ccc acg gca ggg ccc agg acc cgg
ggc 384 Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg
Gly 115 120 125 cgc ctc cac cat tgg ctg tay cgg ctc cag gag gcc cca
aaa aag gag 432 Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro
Lys Lys Glu 130 135 140 tcc cct ggc tgc ctc gag gcc tct gtc acc ttc
aac ctc ttc cgc ctc 480 Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe
Asn Leu Phe Arg Leu 145 150 155 160 ctc acg cga gac ctg aat tgt gtt
gcc agc ggg gac ctg tgt gtc tga 528 Leu Thr Arg Asp Leu Asn Cys Val
Ala Ser Gly Asp Leu Cys Val * 165 170 175 98 175 PRT Artificial
Sequence VARIANT (50)...(50) Xaa = Ser, Ala, Thr, Val, or Asn
VARIANT (87)...(87) Xaa = Ser IL-28B Cys50 mutant T87S H135Y 98 Val
Pro Val Ala Arg Leu Arg Gly Ala Leu Pro Asp Ala Arg Gly Cys 1 5 10
15 His Ile Ala Gln Phe Lys Ser Leu Ser Pro Gln Glu Leu Gln Ala Phe
20 25 30 Lys Arg Ala Lys Asp Ala Leu Glu Glu Ser Leu Leu Leu Lys
Asp Cys 35 40 45 Lys Xaa Arg Ser Arg Leu Phe Pro Arg Thr Trp Asp
Leu Arg Gln Leu 50 55 60 Gln Val Arg Glu Arg Pro Val Ala Leu Glu
Ala Glu Leu Ala Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala Xaa Ala
Asp Thr Asp Pro Ala Leu Gly Asp 85 90 95 Val Leu Asp Gln Pro Leu
His Thr Leu His His Ile Leu Ser Gln Leu 100 105 110 Arg Ala Cys Ile
Gln Pro Gln Pro Thr Ala Gly Pro Arg Thr Arg Gly 115
120 125 Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu Ala Pro Lys Lys
Glu 130 135 140 Ser Pro Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu
Phe Arg Leu 145 150 155 160 Leu Thr Arg Asp Leu Asn Cys Val Ala Ser
Gly Asp Leu Cys Val 165 170 175 99 531 DNA Artificial Sequence Met
IL-28B Cys51 mutant T88S H136Y CDS (1)...(531) variation 152, 152,
264 n = A, T, G, or C 99 atg gtt cct gtc gcc agg ctc cgc ggg gct
ctc ccg gat gca agg ggc 48 Met Val Pro Val Ala Arg Leu Arg Gly Ala
Leu Pro Asp Ala Arg Gly 1 5 10 15 tgc cac ata gcc cag ttc aag tcc
ctg tct cca cag gag ctg cag gcc 96 Cys His Ile Ala Gln Phe Lys Ser
Leu Ser Pro Gln Glu Leu Gln Ala 20 25 30 ttt aag agg gcc aaa gat
gcc tta gaa gag tcg ctt ctg ctg aag gac 144 Phe Lys Arg Ala Lys Asp
Ala Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 40 45 tgc aag dnn cgc
tcc cgc ctc ttc ccc agg acc tgg gac ctg agg cag 192 Cys Lys Xaa Arg
Ser Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln 50 55 60 ctg cag
gtg agg gag cgc ccc gtg gct ttg gag gct gag ctg gcc ctg 240 Leu Gln
Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu 65 70 75 80
acg ctg aag gtt ctg gag gcc wsn gct gac act gac cca gcc ctg ggg 288
Thr Leu Lys Val Leu Glu Ala Xaa Ala Asp Thr Asp Pro Ala Leu Gly 85
90 95 gat gtc ttg gac cag ccc ctt cac acc ctg cac cat atc ctc tcc
cag 336 Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser
Gln 100 105 110 ctc cgg gcc tgt atc cag cct cag ccc acg gca ggg ccc
agg acc cgg 384 Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Thr Arg 115 120 125 ggc cgc ctc cac cat tgg ctg tay cgg ctc cag
gag gcc cca aaa aag 432 Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln
Glu Ala Pro Lys Lys 130 135 140 gag tcc cct ggc tgc ctc gag gcc tct
gtc acc ttc aac ctc ttc cgc 480 Glu Ser Pro Gly Cys Leu Glu Ala Ser
Val Thr Phe Asn Leu Phe Arg 145 150 155 160 ctc ctc acg cga gac ctg
aat tgt gtt gcc agc ggg gac ctg tgt gtc 528 Leu Leu Thr Arg Asp Leu
Asn Cys Val Ala Ser Gly Asp Leu Cys Val 165 170 175 tga 531 * 100
176 PRT Artificial Sequence VARIANT (51)...(51) Xaa = Ser, Ala,
Thr, Val, or Asn VARIANT (88)...(88) Xaa = Ser Met IL-28B Cys51
mutant T88S H136Y 100 Met Val Pro Val Ala Arg Leu Arg Gly Ala Leu
Pro Asp Ala Arg Gly 1 5 10 15 Cys His Ile Ala Gln Phe Lys Ser Leu
Ser Pro Gln Glu Leu Gln Ala 20 25 30 Phe Lys Arg Ala Lys Asp Ala
Leu Glu Glu Ser Leu Leu Leu Lys Asp 35 40 45 Cys Lys Xaa Arg Ser
Arg Leu Phe Pro Arg Thr Trp Asp Leu Arg Gln 50 55 60 Leu Gln Val
Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu 65 70 75 80 Thr
Leu Lys Val Leu Glu Ala Xaa Ala Asp Thr Asp Pro Ala Leu Gly 85 90
95 Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln
100 105 110 Leu Arg Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg
Thr Arg 115 120 125 Gly Arg Leu His His Trp Leu Tyr Arg Leu Gln Glu
Ala Pro Lys Lys 130 135 140 Glu Ser Pro Gly Cys Leu Glu Ala Ser Val
Thr Phe Asn Leu Phe Arg 145 150 155 160 Leu Leu Thr Arg Asp Leu Asn
Cys Val Ala Ser Gly Asp Leu Cys Val 165 170 175 101 45 DNA
Artificial Sequence signal sequence CDS (1)...(45) variation 6, 9,
12, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 n = A, T, G, or C 101
atg gcn gcn gcn tgg acn gtn gtn ytn gtn acn ytn gtn ytn ggn 45 Met
Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly 1 5 10 15
102 15 PRT Artificial Sequence signal sequence 102 Met Ala Ala Ala
Trp Thr Val Val Leu Val Thr Leu Val Leu Gly 1 5 10 15 103 57 DNA
Artificial Sequence signal sequence CDS (1)...(57) variation 6, 9,
12, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57 n = A,
T, G, or C 103 atg gcn gcn gcn tgg acn gtn gtn ytn gtn acn ytn gtn
ytn ggn ytn 48 Met Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val
Leu Gly Leu 1 5 10 15 gcn gtn gcn 57 Ala Val Ala 104 19 PRT
Artificial Sequence signal sequence 104 Met Ala Ala Ala Trp Thr Val
Val Leu Val Thr Leu Val Leu Gly Leu 1 5 10 15 Ala Val Ala 105 63
DNA Artificial Sequence signal sequence CDS (1)...(63) variation 6,
9, 12, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60,
63 n = A, T, G, or C 105 atg gcn gcn gcn tgg acn gtn gtn ytn gtn
acn ytn gtn ytn ggn ytn 48 Met Ala Ala Ala Trp Thr Val Val Leu Val
Thr Leu Val Leu Gly Leu 1 5 10 15 gcn gtn gcn ggn ccn 63 Ala Val
Ala Gly Pro 20 106 21 PRT Artificial Sequence signal sequence 106
Met Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu 1 5
10 15 Ala Val Ala Gly Pro 20 107 72 DNA Artificial Sequence signal
sequence CDS (1)...(72) variation 6, 9, 12, 18, 21, 24, 27, 30, 33,
36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72 n = A, T, G, or
C 107 atg gcn gcn gcn tgg acn gtn gtn ytn gtn acn ytn gtn ytn ggn
ytn 48 Met Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly
Leu 1 5 10 15 gcn gtn gcn ggn ccn gtn ccn acn 72 Ala Val Ala Gly
Pro Val Pro Thr 20 108 24 PRT Artificial Sequence signal sequence
108 Met Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu
1 5 10 15 Ala Val Ala Gly Pro Val Pro Thr 20 109 546 DNA Artificial
Sequence IL-29 C171X CDS (1)...(546) variation (512)...(513) n = A,
T, G, or C 109 ggt ccg gtt ccg acc tct aaa cca acc acc act ggt aaa
ggt tgc cac 48 Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys
Gly Cys His 1 5 10 15 atc ggt cgt ttc aaa tct ctg tct ccg cag gaa
ctg gct tct ttc aaa 96 Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu
Leu Ala Ser Phe Lys 20 25 30 aaa gct cgt gac gct ctg gaa gaa tct
ctg aaa ctg aaa aac tgg tct 144 Lys Ala Arg Asp Ala Leu Glu Glu Ser
Leu Lys Leu Lys Asn Trp Ser 35 40 45 tgc tct tct ccg gtt ttc ccg
ggt aac tgg gat ctg cgt ctg ctg cag 192 Cys Ser Ser Pro Val Phe Pro
Gly Asn Trp Asp Leu Arg Leu Leu Gln 50 55 60 gtt cgt gaa cgt ccg
gtt gct ctg gaa gct gaa ctg gct ctg acc ctg 240 Val Arg Glu Arg Pro
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 aaa gtt ctg
gaa gct gct gca ggt cct gct ctg gaa gat gtt ctg gat 288 Lys Val Leu
Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90 95 cag
ccg ctg cac act ctg cac cac atc ctg tct cag ctg cag gct tgc 336 Gln
Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys 100 105
110 att caa ccg caa ccg acc gct ggt ccg cgt ccg cgt ggt cgt ctg cac
384 Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His
115 120 125 cac tgg ctg cat cgt ctg cag gaa gct ccg aaa aaa gaa tct
gct ggt 432 His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser
Ala Gly 130 135 140 tgc ctg gaa gct tct gtt acc ttc aac ctg ttc cgt
ctg ctg acc cgt 480 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg
Leu Leu Thr Arg 145 150 155 160 gat ctg aaa tac gtt gct gat ggt aac
ctg dnn ctg cgt acc tct acc 528 Asp Leu Lys Tyr Val Ala Asp Gly Asn
Leu Xaa Leu Arg Thr Ser Thr 165 170 175 cat ccg gaa tct acc taa 546
His Pro Glu Ser Thr * 180 110 181 PRT Artificial Sequence IL-29
C171X VARIANT (171)...(171) Xaa = Ser, Ala, Thr, Val, or Asn 110
Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His 1 5
10 15 Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe
Lys 20 25 30 Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys
Asn Trp Ser 35 40 45 Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp
Leu Arg Leu Leu Gln 50 55 60 Val Arg Glu Arg Pro Val Ala Leu Glu
Ala Glu Leu Ala Leu Thr Leu 65 70 75 80 Lys Val Leu Glu Ala Ala Ala
Gly Pro Ala Leu Glu Asp Val Leu Asp 85 90 95 Gln Pro Leu His Thr
Leu His His Ile Leu Ser Gln Leu Gln Ala Cys 100 105 110 Ile Gln Pro
Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His 115 120 125 His
Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly 130 135
140 Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg
145 150 155 160 Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg
Thr Ser Thr 165 170 175 His Pro Glu Ser Thr 180 111 549 DNA
Artificial Sequence Met IL-29 C172X CDS (1)...(549) variation
(515)...(516) n = A, T, G, or C 111 atg ggt ccg gtt ccg acc tct aaa
cca acc acc act ggt aaa ggt tgc 48 Met Gly Pro Val Pro Thr Ser Lys
Pro Thr Thr Thr Gly Lys Gly Cys 1 5 10 15 cac atc ggt cgt ttc aaa
tct ctg tct ccg cag gaa ctg gct tct ttc 96 His Ile Gly Arg Phe Lys
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 aaa aaa gct cgt
gac gct ctg gaa gaa tct ctg aaa ctg aaa aac tgg 144 Lys Lys Ala Arg
Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45 tct tgc
tct tct ccg gtt ttc ccg ggt aac tgg gat ctg cgt ctg ctg 192 Ser Cys
Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60
cag gtt cgt gaa cgt ccg gtt gct ctg gaa gct gaa ctg gct ctg acc 240
Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65
70 75 80 ctg aaa gtt ctg gaa gct gct gca ggt cct gct ctg gaa gat
gtt ctg 288 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp
Val Leu 85 90 95 gat cag ccg ctg cac act ctg cac cac atc ctg tct
cag ctg cag gct 336 Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser
Gln Leu Gln Ala 100 105 110 tgc att caa ccg caa ccg acc gct ggt ccg
cgt ccg cgt ggt cgt ctg 384 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Pro Arg Gly Arg Leu 115 120 125 cac cac tgg ctg cat cgt ctg cag
gaa gct ccg aaa aaa gaa tct gct 432 His His Trp Leu His Arg Leu Gln
Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 ggt tgc ctg gaa gct tct
gtt acc ttc aac ctg ttc cgt ctg ctg acc 480 Gly Cys Leu Glu Ala Ser
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 cgt gat ctg
aaa tac gtt gct gat ggt aac ctg dnn ctg cgt acc tct 528 Arg Asp Leu
Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser 165 170 175 acc
cat ccg gaa tct acc taa 549 Thr His Pro Glu Ser Thr * 180 112 182
PRT Artificial Sequence Met IL-29 C172X VARIANT (172)...(172) Xaa =
Ser, Ala, Thr, Val, or Asn 112 Met Gly Pro Val Pro Thr Ser Lys Pro
Thr Thr Thr Gly Lys Gly Cys 1 5 10 15 His Ile Gly Arg Phe Lys Ser
Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30 Lys Lys Ala Arg Asp
Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35 40 45 Ser Cys Ser
Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu 50 55 60 Gln
Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70
75 80 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val
Leu 85 90 95 Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln
Leu Gln Ala 100 105 110 Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg
Pro Arg Gly Arg Leu 115 120 125 His His Trp Leu His Arg Leu Gln Glu
Ala Pro Lys Lys Glu Ser Ala 130 135 140 Gly Cys Leu Glu Ala Ser Val
Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160 Arg Asp Leu Lys
Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser 165 170 175 Thr His
Pro Glu Ser Thr 180 113 543 DNA Artificial Sequence IL-29 C170X,
truncated after N-terminal Methionine and Glycine CDS (1)...(543)
variation (509)...(510) n = A, T, G, or C 113 cct gtc ccc act tcc
aag ccc acc aca act ggg aag ggc tgc cac att 48 Pro Val Pro Thr Ser
Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile 1 5 10 15 ggc agg ttc
aaa tct ctg tca cca cag gag cta gcg agc ttc aag aag 96 Gly Arg Phe
Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys 20 25 30 gcc
agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg agt tgc 144 Ala
Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys 35 40
45 agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc cag gtg
192 Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val
50 55 60 agg gag cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg
ctg aag 240 Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr
Leu Lys 65 70 75 80 gtc ctg gag gcc gct gct ggc cca gcc ctg gag gac
gtc cta gac cag 288 Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp
Val Leu Asp Gln 85 90 95 ccc ctt cac acc ctg cac cac atc ctc tcc
cag ctc cag gcc tgt atc 336 Pro Leu His Thr Leu His His Ile Leu Ser
Gln Leu Gln Ala Cys Ile 100 105 110 cag cct cag ccc aca gca ggg ccc
agg ccc cgg ggc cgc ctc cac cac 384 Gln Pro Gln Pro Thr Ala Gly Pro
Arg Pro Arg Gly Arg Leu His His 115 120 125 tgg ctg cac cgg ctc cag
gag gcc ccc aaa aag gag tcc gct ggc tgc 432 Trp Leu His Arg Leu Gln
Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys 130 135 140 ctg gag gca tct
gtc acc ttc aac ctc ttc cgc ctc ctc acg cga gac 480 Leu Glu Ala Ser
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp 145 150 155 160 ctc
aaa tat gtg gcc gat ggg aac ctg dnn ctg aga acg tca acc cac 528 Leu
Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His 165 170
175 cct gag tcc acc tga 543 Pro Glu Ser Thr * 180 114 180 PRT
Artificial Sequence IL-29 C170X, truncated after N-terminal
Methionineand Glycine VARIANT (170)...(170) Xaa = Ser, Ala, Thr,
Val, or Asn 114 Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly
Cys His Ile 1 5 10 15 Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu
Ala Ser Phe Lys Lys 20 25 30 Ala Arg Asp Ala Leu Glu Glu Ser Leu
Lys Leu Lys Asn Trp Ser Cys 35 40 45 Ser Ser Pro Val Phe Pro Gly
Asn Trp Asp Leu Arg Leu Leu Gln Val 50 55 60 Arg Glu Arg Pro Val
Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys 65 70 75 80 Val Leu Glu
Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln 85 90 95 Pro
Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile 100 105
110 Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His
115 120 125 Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala
Gly Cys 130 135 140 Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu
Leu Thr Arg Asp 145 150 155 160 Leu Lys Tyr Val Ala Asp Gly Asn Leu
Xaa Leu Arg Thr Ser Thr His 165 170 175 Pro Glu Ser Thr 180 115 540
DNA Artificial Sequence IL-29 C169X,
truncated after N-terminal Methionine, Glycine, and Proline CDS
(1)...(540) variation (506)...(507) n = A, T, G, or C 115 gtc ccc
act tcc aag ccc acc aca act ggg aag ggc tgc cac att ggc 48 Val Pro
Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly 1 5 10 15
agg ttc aaa tct ctg tca cca cag gag cta gcg agc ttc aag aag gcc 96
Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala 20
25 30 agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac tgg agt tgc
agc 144 Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys
Ser 35 40 45 tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt ctc
cag gtg agg 192 Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu
Gln Val Arg 50 55 60 gag cgc cct gtg gcc ttg gag gct gag ctg gcc
ctg acg ctg aag gtc 240 Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala
Leu Thr Leu Lys Val 65 70 75 80 ctg gag gcc gct gct ggc cca gcc ctg
gag gac gtc cta gac cag ccc 288 Leu Glu Ala Ala Ala Gly Pro Ala Leu
Glu Asp Val Leu Asp Gln Pro 85 90 95 ctt cac acc ctg cac cac atc
ctc tcc cag ctc cag gcc tgt atc cag 336 Leu His Thr Leu His His Ile
Leu Ser Gln Leu Gln Ala Cys Ile Gln 100 105 110 cct cag ccc aca gca
ggg ccc agg ccc cgg ggc cgc ctc cac cac tgg 384 Pro Gln Pro Thr Ala
Gly Pro Arg Pro Arg Gly Arg Leu His His Trp 115 120 125 ctg cac cgg
ctc cag gag gcc ccc aaa aag gag tcc gct ggc tgc ctg 432 Leu His Arg
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu 130 135 140 gag
gca tct gtc acc ttc aac ctc ttc cgc ctc ctc acg cga gac ctc 480 Glu
Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu 145 150
155 160 aaa tat gtg gcc gat ggg aac ctg dnn ctg aga acg tca acc cac
cct 528 Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His
Pro 165 170 175 gag tcc acc tga 540 Glu Ser Thr * 116 179 PRT
Artificial Sequence IL-29 C169X, truncated after N-terminal
Methionine, Glycine, and Proline VARIANT (169)...(169) Xaa = Ser,
Ala, Thr, Val, or Asn 116 Val Pro Thr Ser Lys Pro Thr Thr Thr Gly
Lys Gly Cys His Ile Gly 1 5 10 15 Arg Phe Lys Ser Leu Ser Pro Gln
Glu Leu Ala Ser Phe Lys Lys Ala 20 25 30 Arg Asp Ala Leu Glu Glu
Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser 35 40 45 Ser Pro Val Phe
Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg 50 55 60 Glu Arg
Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val 65 70 75 80
Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro 85
90 95 Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile
Gln 100 105 110 Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu
His His Trp 115 120 125 Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu
Ser Ala Gly Cys Leu 130 135 140 Glu Ala Ser Val Thr Phe Asn Leu Phe
Arg Leu Leu Thr Arg Asp Leu 145 150 155 160 Lys Tyr Val Ala Asp Gly
Asn Leu Xaa Leu Arg Thr Ser Thr His Pro 165 170 175 Glu Ser Thr 117
537 DNA Artificial Sequence IL-29 C168X, truncated after N-terminal
Methionine, Glycine, Proline, and Valine CDS (1)...(537) variation
(503)...(504) n = A, T, G, or C 117 ccc act tcc aag ccc acc aca act
ggg aag ggc tgc cac att ggc agg 48 Pro Thr Ser Lys Pro Thr Thr Thr
Gly Lys Gly Cys His Ile Gly Arg 1 5 10 15 ttc aaa tct ctg tca cca
cag gag cta gcg agc ttc aag aag gcc agg 96 Phe Lys Ser Leu Ser Pro
Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg 20 25 30 gac gcc ttg gaa
gag tca ctc aag ctg aaa aac tgg agt tgc agc tct 144 Asp Ala Leu Glu
Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser 35 40 45 cct gtc
ttc ccc ggg aat tgg gac ctg agg ctt ctc cag gtg agg gag 192 Pro Val
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu 50 55 60
cgc cct gtg gcc ttg gag gct gag ctg gcc ctg acg ctg aag gtc ctg 240
Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu 65
70 75 80 gag gcc gct gct ggc cca gcc ctg gag gac gtc cta gac cag
ccc ctt 288 Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln
Pro Leu 85 90 95 cac acc ctg cac cac atc ctc tcc cag ctc cag gcc
tgt atc cag cct 336 His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala
Cys Ile Gln Pro 100 105 110 cag ccc aca gca ggg ccc agg ccc cgg ggc
cgc ctc cac cac tgg ctg 384 Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly
Arg Leu His His Trp Leu 115 120 125 cac cgg ctc cag gag gcc ccc aaa
aag gag tcc gct ggc tgc ctg gag 432 His Arg Leu Gln Glu Ala Pro Lys
Lys Glu Ser Ala Gly Cys Leu Glu 130 135 140 gca tct gtc acc ttc aac
ctc ttc cgc ctc ctc acg cga gac ctc aaa 480 Ala Ser Val Thr Phe Asn
Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys 145 150 155 160 tat gtg gcc
gat ggg aac ctg dnn ctg aga acg tca acc cac cct gag 528 Tyr Val Ala
Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu 165 170 175 tcc
acc tga 537 Ser Thr * 118 178 PRT Artificial Sequence IL-29 C168X,
truncated after N-terminal Methionine, Glycine, Proline, and Valine
VARIANT (168)...(168) Xaa = Ser, Ala, Thr, Val, or Asn 118 Pro Thr
Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg 1 5 10 15
Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg 20
25 30 Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser
Ser 35 40 45 Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln
Val Arg Glu 50 55 60 Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu
Thr Leu Lys Val Leu 65 70 75 80 Glu Ala Ala Ala Gly Pro Ala Leu Glu
Asp Val Leu Asp Gln Pro Leu 85 90 95 His Thr Leu His His Ile Leu
Ser Gln Leu Gln Ala Cys Ile Gln Pro 100 105 110 Gln Pro Thr Ala Gly
Pro Arg Pro Arg Gly Arg Leu His His Trp Leu 115 120 125 His Arg Leu
Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu 130 135 140 Ala
Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys 145 150
155 160 Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro
Glu 165 170 175 Ser Thr 119 534 DNA Artificial Sequence IL-29
C167X, truncated after N-terminal Methionine, Glycine, Proline,
Valine, and Proline CDS (1)...(534) variation (500)...(501) n = A,
T, G, or C 119 act tcc aag ccc acc aca act ggg aag ggc tgc cac att
ggc agg ttc 48 Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile
Gly Arg Phe 1 5 10 15 aaa tct ctg tca cca cag gag cta gcg agc ttc
aag aag gcc agg gac 96 Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe
Lys Lys Ala Arg Asp 20 25 30 gcc ttg gaa gag tca ctc aag ctg aaa
aac tgg agt tgc agc tct cct 144 Ala Leu Glu Glu Ser Leu Lys Leu Lys
Asn Trp Ser Cys Ser Ser Pro 35 40 45 gtc ttc ccc ggg aat tgg gac
ctg agg ctt ctc cag gtg agg gag cgc 192 Val Phe Pro Gly Asn Trp Asp
Leu Arg Leu Leu Gln Val Arg Glu Arg 50 55 60 cct gtg gcc ttg gag
gct gag ctg gcc ctg acg ctg aag gtc ctg gag 240 Pro Val Ala Leu Glu
Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu 65 70 75 80 gcc gct gct
ggc cca gcc ctg gag gac gtc cta gac cag ccc ctt cac 288 Ala Ala Ala
Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His 85 90 95 acc
ctg cac cac atc ctc tcc cag ctc cag gcc tgt atc cag cct cag 336 Thr
Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln 100 105
110 ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc cac cac tgg ctg cac
384 Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His
115 120 125 cgg ctc cag gag gcc ccc aaa aag gag tcc gct ggc tgc ctg
gag gca 432 Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu
Glu Ala 130 135 140 tct gtc acc ttc aac ctc ttc cgc ctc ctc acg cga
gac ctc aaa tat 480 Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg
Asp Leu Lys Tyr 145 150 155 160 gtg gcc gat ggg aac ctg dnn ctg aga
acg tca acc cac cct gag tcc 528 Val Ala Asp Gly Asn Leu Xaa Leu Arg
Thr Ser Thr His Pro Glu Ser 165 170 175 acc tga 534 Thr * 120 177
PRT Artificial Sequence IL-29 C167X, truncated after N-terminal
Methionine, Glycine, Proline, Valine, and Proline VARIANT
(167)...(167) Xaa = Ser, Ala, Thr, Val, or Asn 120 Thr Ser Lys Pro
Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe 1 5 10 15 Lys Ser
Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp 20 25 30
Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro 35
40 45 Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu
Arg 50 55 60 Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys
Val Leu Glu 65 70 75 80 Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu
Asp Gln Pro Leu His 85 90 95 Thr Leu His His Ile Leu Ser Gln Leu
Gln Ala Cys Ile Gln Pro Gln 100 105 110 Pro Thr Ala Gly Pro Arg Pro
Arg Gly Arg Leu His His Trp Leu His 115 120 125 Arg Leu Gln Glu Ala
Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala 130 135 140 Ser Val Thr
Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr 145 150 155 160
Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser 165
170 175 Thr 121 531 DNA Artificial Sequence IL-29 C166X, truncated
after N-terminal Methionine, Glycine, Proline, Valine, Proline, and
Threonine CDS (1)...(531) variation (497)...(498) n = A, T, G, or C
121 tcc aag ccc acc aca act ggg aag ggc tgc cac att ggc agg ttc aaa
48 Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys
1 5 10 15 tct ctg tca cca cag gag cta gcg agc ttc aag aag gcc agg
gac gcc 96 Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg
Asp Ala 20 25 30 ttg gaa gag tca ctc aag ctg aaa aac tgg agt tgc
agc tct cct gtc 144 Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys
Ser Ser Pro Val 35 40 45 ttc ccc ggg aat tgg gac ctg agg ctt ctc
cag gtg agg gag cgc cct 192 Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu
Gln Val Arg Glu Arg Pro 50 55 60 gtg gcc ttg gag gct gag ctg gcc
ctg acg ctg aag gtc ctg gag gcc 240 Val Ala Leu Glu Ala Glu Leu Ala
Leu Thr Leu Lys Val Leu Glu Ala 65 70 75 80 gct gct ggc cca gcc ctg
gag gac gtc cta gac cag ccc ctt cac acc 288 Ala Ala Gly Pro Ala Leu
Glu Asp Val Leu Asp Gln Pro Leu His Thr 85 90 95 ctg cac cac atc
ctc tcc cag ctc cag gcc tgt atc cag cct cag ccc 336 Leu His His Ile
Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro 100 105 110 aca gca
ggg ccc agg ccc cgg ggc cgc ctc cac cac tgg ctg cac cgg 384 Thr Ala
Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg 115 120 125
ctc cag gag gcc ccc aaa aag gag tcc gct ggc tgc ctg gag gca tct 432
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser 130
135 140 gtc acc ttc aac ctc ttc cgc ctc ctc acg cga gac ctc aaa tat
gtg 480 Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr
Val 145 150 155 160 gcc gat ggg aac ctg dnn ctg aga acg tca acc cac
cct gag tcc acc 528 Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His
Pro Glu Ser Thr 165 170 175 tga 531 * 122 176 PRT Artificial
Sequence IL-29 C166X, truncated after N-terminal Methionine,
Glycine, Proline, Valine, Proline, and Threonine VARIANT
(166)...(166) Xaa = Ser, Ala, Thr, Val, or Asn 122 Ser Lys Pro Thr
Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys 1 5 10 15 Ser Leu
Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala 20 25 30
Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val 35
40 45 Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg
Pro 50 55 60 Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val
Leu Glu Ala 65 70 75 80 Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp
Gln Pro Leu His Thr 85 90 95 Leu His His Ile Leu Ser Gln Leu Gln
Ala Cys Ile Gln Pro Gln Pro 100 105 110 Thr Ala Gly Pro Arg Pro Arg
Gly Arg Leu His His Trp Leu His Arg 115 120 125 Leu Gln Glu Ala Pro
Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser 130 135 140 Val Thr Phe
Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val 145 150 155 160
Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr 165
170 175 123 528 DNA Artificial Sequence IL-29 C165X, truncated
after N-terminal Methionine, Glycine, Proline, Valine, Proline,
Threonine, and Serine CDS (1)...(528) variation (494)...(495) n =
A, T, G, or C 123 aag ccc acc aca act ggg aag ggc tgc cac att ggc
agg ttc aaa tct 48 Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly
Arg Phe Lys Ser 1 5 10 15 ctg tca cca cag gag cta gcg agc ttc aag
aag gcc agg gac gcc ttg 96 Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys
Lys Ala Arg Asp Ala Leu 20 25 30 gaa gag tca ctc aag ctg aaa aac
tgg agt tgc agc tct cct gtc ttc 144 Glu Glu Ser Leu Lys Leu Lys Asn
Trp Ser Cys Ser Ser Pro Val Phe 35 40 45 ccc ggg aat tgg gac ctg
agg ctt ctc cag gtg agg gag cgc cct gtg 192 Pro Gly Asn Trp Asp Leu
Arg Leu Leu Gln Val Arg Glu Arg Pro Val 50 55 60 gcc ttg gag gct
gag ctg gcc ctg acg ctg aag gtc ctg gag gcc gct 240 Ala Leu Glu Ala
Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala 65 70 75 80 gct ggc
cca gcc ctg gag gac gtc cta gac cag ccc ctt cac acc ctg 288 Ala Gly
Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu 85 90 95
cac cac atc ctc tcc cag ctc cag gcc tgt atc cag cct cag ccc aca 336
His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr 100
105 110 gca ggg ccc agg ccc cgg ggc cgc ctc cac cac tgg ctg cac cgg
ctc 384 Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg
Leu 115 120 125 cag gag gcc ccc aaa aag gag tcc gct ggc tgc ctg gag
gca tct gtc 432 Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu
Ala Ser Val 130 135 140 acc ttc aac ctc ttc cgc ctc ctc acg cga gac
ctc aaa tat gtg gcc 480 Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp
Leu Lys Tyr Val Ala 145 150 155 160 gat ggg aac ctg dnn ctg aga acg
tca acc cac cct gag tcc acc tga 528 Asp Gly Asn Leu Xaa Leu Arg Thr
Ser Thr His Pro Glu Ser Thr * 165 170 175 124 175 PRT Artificial
Sequence IL-29 C165X, truncated after N-terminal Methionine,
Glycine, Proline, Valine, Proline, Threonine, and Serine VARIANT
(165)...(165) Xaa = Ser, Ala, Thr, Val, or Asn 124 Lys Pro Thr Thr
Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser 1 5 10 15 Leu Ser
Pro Gln Glu Leu Ala
Ser Phe Lys Lys Ala Arg Asp Ala Leu 20 25 30 Glu Glu Ser Leu Lys
Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe 35 40 45 Pro Gly Asn
Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val 50 55 60 Ala
Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala 65 70
75 80 Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr
Leu 85 90 95 His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro
Gln Pro Thr 100 105 110 Ala Gly Pro Arg Pro Arg Gly Arg Leu His His
Trp Leu His Arg Leu 115 120 125 Gln Glu Ala Pro Lys Lys Glu Ser Ala
Gly Cys Leu Glu Ala Ser Val 130 135 140 Thr Phe Asn Leu Phe Arg Leu
Leu Thr Arg Asp Leu Lys Tyr Val Ala 145 150 155 160 Asp Gly Asn Leu
Xaa Leu Arg Thr Ser Thr His Pro Glu Ser Thr 165 170 175 125 552 DNA
Artificial Sequence IL-29 Leu insert after N-terminal Met, C173X
CDS (1)...(552) variation (6)...(6) n = A, T, G, or C variation
(518)...(519) n = A, T, G, or C 125 atg ytn ggc cct gtc ccc act tcc
aag ccc acc aca act ggg aag ggc 48 Met Leu Gly Pro Val Pro Thr Ser
Lys Pro Thr Thr Thr Gly Lys Gly 1 5 10 15 tgc cac att ggc agg ttc
aaa tct ctg tca cca cag gag cta gcg agc 96 Cys His Ile Gly Arg Phe
Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser 20 25 30 ttc aag aag gcc
agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac 144 Phe Lys Lys Ala
Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn 35 40 45 tgg agt
tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg ctt 192 Trp Ser
Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu 50 55 60
ctc cag gtg agg gag cgc cct gtg gcc ttg gag gct gag ctg gcc ctg 240
Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu 65
70 75 80 acg ctg aag gtc ctg gag gcc gct gct ggc cca gcc ctg gag
gac gtc 288 Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu
Asp Val 85 90 95 cta gac cag ccc ctt cac acc ctg cac cac atc ctc
tcc cag ctc cag 336 Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu
Ser Gln Leu Gln 100 105 110 gcc tgt atc cag cct cag ccc aca gca ggg
ccc agg ccc cgg ggc cgc 384 Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly
Pro Arg Pro Arg Gly Arg 115 120 125 ctc cac cac tgg ctg cac cgg ctc
cag gag gcc ccc aaa aag gag tcc 432 Leu His His Trp Leu His Arg Leu
Gln Glu Ala Pro Lys Lys Glu Ser 130 135 140 gct ggc tgc ctg gag gca
tct gtc acc ttc aac ctc ttc cgc ctc ctc 480 Ala Gly Cys Leu Glu Ala
Ser Val Thr Phe Asn Leu Phe Arg Leu Leu 145 150 155 160 acg cga gac
ctc aaa tat gtg gcc gat ggg aac ctg dnn ctg aga acg 528 Thr Arg Asp
Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr 165 170 175 tca
acc cac cct gag tcc acc tga 552 Ser Thr His Pro Glu Ser Thr * 180
126 183 PRT Artificial Sequence IL-29 Leu insert after N-terminal
Met, C173X VARIANT (173)...(173) Xaa = Ser, Ala, Thr, Val, or Asn
126 Met Leu Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly
1 5 10 15 Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu
Ala Ser 20 25 30 Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu
Lys Leu Lys Asn 35 40 45 Trp Ser Cys Ser Ser Pro Val Phe Pro Gly
Asn Trp Asp Leu Arg Leu 50 55 60 Leu Gln Val Arg Glu Arg Pro Val
Ala Leu Glu Ala Glu Leu Ala Leu 65 70 75 80 Thr Leu Lys Val Leu Glu
Ala Ala Ala Gly Pro Ala Leu Glu Asp Val 85 90 95 Leu Asp Gln Pro
Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln 100 105 110 Ala Cys
Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg 115 120 125
Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser 130
135 140 Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu
Leu 145 150 155 160 Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu
Xaa Leu Arg Thr 165 170 175 Ser Thr His Pro Glu Ser Thr 180 127 549
DNA Artificial Sequence IL-29 G2L C172X CDS (1)...(549) variation
(6)...(6) n = A, T, G, or C variation (515)...(516) n = A, T, G, or
C 127 atg ytn cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc
tgc 48 Met Leu Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly
Cys 1 5 10 15 cac att ggc agg ttc aaa tct ctg tca cca cag gag cta
gcg agc ttc 96 His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu
Ala Ser Phe 20 25 30 aag aag gcc agg gac gcc ttg gaa gag tca ctc
aag ctg aaa aac tgg 144 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu
Lys Leu Lys Asn Trp 35 40 45 agt tgc agc tct cct gtc ttc ccc ggg
aat tgg gac ctg agg ctt ctc 192 Ser Cys Ser Ser Pro Val Phe Pro Gly
Asn Trp Asp Leu Arg Leu Leu 50 55 60 cag gtg agg gag cgc cct gtg
gcc ttg gag gct gag ctg gcc ctg acg 240 Gln Val Arg Glu Arg Pro Val
Ala Leu Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 ctg aag gtc ctg gag
gcc gct gct ggc cca gcc ctg gag gac gtc cta 288 Leu Lys Val Leu Glu
Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95 gac cag ccc
ctt cac acc ctg cac cac atc ctc tcc cag ctc cag gcc 336 Asp Gln Pro
Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105 110 tgt
atc cag cct cag ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc 384 Cys
Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120
125 cac cac tgg ctg cac cgg ctc cag gag gcc ccc aaa aag gag tcc gct
432 His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala
130 135 140 ggc tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc
ctc acg 480 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu
Leu Thr 145 150 155 160 cga gac ctc aaa tat gtg gcc gat ggg aac ctg
dnn ctg aga acg tca 528 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu
Xaa Leu Arg Thr Ser 165 170 175 acc cac cct gag tcc acc tga 549 Thr
His Pro Glu Ser Thr * 180 128 182 PRT Artificial Sequence IL-29 G2L
C172X VARIANT (172)...(172) Xaa = Ser, Ala, Thr, Val, or Asn 128
Met Leu Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys 1 5
10 15 His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser
Phe 20 25 30 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu
Lys Asn Trp 35 40 45 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp
Asp Leu Arg Leu Leu 50 55 60 Gln Val Arg Glu Arg Pro Val Ala Leu
Glu Ala Glu Leu Ala Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala Ala
Ala Gly Pro Ala Leu Glu Asp Val Leu 85 90 95 Asp Gln Pro Leu His
Thr Leu His His Ile Leu Ser Gln Leu Gln Ala 100 105 110 Cys Ile Gln
Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 His
His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135
140 Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr
145 150 155 160 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu
Arg Thr Ser 165 170 175 Thr His Pro Glu Ser Thr 180 129 552 DNA
Artificial Sequence IL-29 Ile insert after N-terminal Met, C173X
CDS (1)...(552) variation (518)...(519) n = A, T, G, or C 129 atg
ath ggc cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc 48 Met
Ile Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly 1 5 10
15 tgc cac att ggc agg ttc aaa tct ctg tca cca cag gag cta gcg agc
96 Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser
20 25 30 ttc aag aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg
aaa aac 144 Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu
Lys Asn 35 40 45 tgg agt tgc agc tct cct gtc ttc ccc ggg aat tgg
gac ctg agg ctt 192 Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp
Asp Leu Arg Leu 50 55 60 ctc cag gtg agg gag cgc cct gtg gcc ttg
gag gct gag ctg gcc ctg 240 Leu Gln Val Arg Glu Arg Pro Val Ala Leu
Glu Ala Glu Leu Ala Leu 65 70 75 80 acg ctg aag gtc ctg gag gcc gct
gct ggc cca gcc ctg gag gac gtc 288 Thr Leu Lys Val Leu Glu Ala Ala
Ala Gly Pro Ala Leu Glu Asp Val 85 90 95 cta gac cag ccc ctt cac
acc ctg cac cac atc ctc tcc cag ctc cag 336 Leu Asp Gln Pro Leu His
Thr Leu His His Ile Leu Ser Gln Leu Gln 100 105 110 gcc tgt atc cag
cct cag ccc aca gca ggg ccc agg ccc cgg ggc cgc 384 Ala Cys Ile Gln
Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg 115 120 125 ctc cac
cac tgg ctg cac cgg ctc cag gag gcc ccc aaa aag gag tcc 432 Leu His
His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser 130 135 140
gct ggc tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc 480
Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu 145
150 155 160 acg cga gac ctc aaa tat gtg gcc gat ggg aac ctg dnn ctg
aga acg 528 Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu
Arg Thr 165 170 175 tca acc cac cct gag tcc acc tga 552 Ser Thr His
Pro Glu Ser Thr * 180 130 183 PRT Artificial Sequence IL-29 Ile
insert after N-terminal Met, C173X VARIANT (173)...(173) Xaa = Ser,
Ala, Thr, Val, or Asn 130 Met Ile Gly Pro Val Pro Thr Ser Lys Pro
Thr Thr Thr Gly Lys Gly 1 5 10 15 Cys His Ile Gly Arg Phe Lys Ser
Leu Ser Pro Gln Glu Leu Ala Ser 20 25 30 Phe Lys Lys Ala Arg Asp
Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn 35 40 45 Trp Ser Cys Ser
Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu 50 55 60 Leu Gln
Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu 65 70 75 80
Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val 85
90 95 Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu
Gln 100 105 110 Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro
Arg Gly Arg 115 120 125 Leu His His Trp Leu His Arg Leu Gln Glu Ala
Pro Lys Lys Glu Ser 130 135 140 Ala Gly Cys Leu Glu Ala Ser Val Thr
Phe Asn Leu Phe Arg Leu Leu 145 150 155 160 Thr Arg Asp Leu Lys Tyr
Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr 165 170 175 Ser Thr His Pro
Glu Ser Thr 180 131 549 DNA Artificial Sequence IL-29 G2I C172X CDS
(1)...(549) variation (515)...(516) n = A, T, G, or C 131 atg ath
cct gtc ccc act tcc aag ccc acc aca act ggg aag ggc tgc 48 Met Ile
Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys 1 5 10 15
cac att ggc agg ttc aaa tct ctg tca cca cag gag cta gcg agc ttc 96
His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20
25 30 aag aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa aac
tgg 144 Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn
Trp 35 40 45 agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg
agg ctt ctc 192 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu
Arg Leu Leu 50 55 60 cag gtg agg gag cgc cct gtg gcc ttg gag gct
gag ctg gcc ctg acg 240 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala
Glu Leu Ala Leu Thr 65 70 75 80 ctg aag gtc ctg gag gcc gct gct ggc
cca gcc ctg gag gac gtc cta 288 Leu Lys Val Leu Glu Ala Ala Ala Gly
Pro Ala Leu Glu Asp Val Leu 85 90 95 gac cag ccc ctt cac acc ctg
cac cac atc ctc tcc cag ctc cag gcc 336 Asp Gln Pro Leu His Thr Leu
His His Ile Leu Ser Gln Leu Gln Ala 100 105 110 tgt atc cag cct cag
ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc 384 Cys Ile Gln Pro Gln
Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 cac cac tgg
ctg cac cgg ctc cag gag gcc ccc aaa aag gag tcc gct 432 His His Trp
Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 ggc
tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc ctc acg 480 Gly
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150
155 160 cga gac ctc aaa tat gtg gcc gat ggg aac ctg dnn ctg aga acg
tca 528 Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr
Ser 165 170 175 acc cac cct gag tcc acc tga 549 Thr His Pro Glu Ser
Thr * 180 132 182 PRT Artificial Sequence IL-29 G2I C172X VARIANT
(172)...(172) Xaa = Ser, Ala, Thr, Val, or Asn 132 Met Ile Pro Val
Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys 1 5 10 15 His Ile
Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe 20 25 30
Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp 35
40 45 Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu
Leu 50 55 60 Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu
Ala Leu Thr 65 70 75 80 Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala
Leu Glu Asp Val Leu 85 90 95 Asp Gln Pro Leu His Thr Leu His His
Ile Leu Ser Gln Leu Gln Ala 100 105 110 Cys Ile Gln Pro Gln Pro Thr
Ala Gly Pro Arg Pro Arg Gly Arg Leu 115 120 125 His His Trp Leu His
Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala 130 135 140 Gly Cys Leu
Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr 145 150 155 160
Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser 165
170 175 Thr His Pro Glu Ser Thr 180 133 531 DNA Artificial Sequence
IL-29 after N-terminal Met amino acid residues 2-7deleted, C166X
CDS (1)...(531) variation (497)...(498) n = A, T, G, or C 133 atg
aag ccc acc aca act ggg aag ggc tgc cac att ggc agg ttc aaa 48 Met
Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys 1 5 10
15 tct ctg tca cca cag gag cta gcg agc ttc aag aag gcc agg gac gcc
96 Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala
20 25 30 ttg gaa gag tca ctc aag ctg aaa aac tgg agt tgc agc tct
cct gtc 144 Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser
Pro Val 35 40 45 ttc ccc ggg aat tgg gac ctg agg ctt ctc cag gtg
agg gag cgc cct 192 Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val
Arg Glu Arg Pro 50 55 60 gtg gcc ttg gag gct gag ctg gcc ctg acg
ctg aag gtc ctg gag gcc 240 Val Ala Leu Glu Ala Glu Leu Ala Leu Thr
Leu Lys Val Leu Glu Ala 65 70 75 80 gct gct ggc cca gcc ctg gag gac
gtc cta gac cag ccc ctt cac acc 288 Ala Ala Gly Pro Ala Leu Glu Asp
Val Leu Asp Gln Pro Leu His Thr 85 90 95 ctg cac cac atc ctc tcc
cag ctc cag gcc tgt atc cag cct cag ccc 336 Leu His His Ile Leu Ser
Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro 100 105 110 aca gca ggg ccc
agg ccc cgg ggc cgc ctc cac cac tgg ctg cac cgg 384 Thr Ala Gly Pro
Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg 115 120 125
ctc cag gag gcc ccc aaa aag gag tcc gct ggc tgc ctg gag gca tct 432
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser 130
135 140 gtc acc ttc aac ctc ttc cgc ctc ctc acg cga gac ctc aaa tat
gtg 480 Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr
Val 145 150 155 160 gcc gat ggg aac ctg dnn ctg aga acg tca acc cac
cct gag tcc acc 528 Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His
Pro Glu Ser Thr 165 170 175 tga 531 * 134 176 PRT Artificial
Sequence IL-29 after N-terminal Met amino acid residues 2-7deleted,
C166X VARIANT (166)...(166) Xaa = Ser, Ala, Thr, Val, or Asn 134
Met Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys 1 5
10 15 Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp
Ala 20 25 30 Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser
Ser Pro Val 35 40 45 Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln
Val Arg Glu Arg Pro 50 55 60 Val Ala Leu Glu Ala Glu Leu Ala Leu
Thr Leu Lys Val Leu Glu Ala 65 70 75 80 Ala Ala Gly Pro Ala Leu Glu
Asp Val Leu Asp Gln Pro Leu His Thr 85 90 95 Leu His His Ile Leu
Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro 100 105 110 Thr Ala Gly
Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg 115 120 125 Leu
Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser 130 135
140 Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val
145 150 155 160 Ala Asp Gly Asn Leu Xaa Leu Arg Thr Ser Thr His Pro
Glu Ser Thr 165 170 175 135 558 DNA Artificial Sequence IL-29 Glu,
Ala, and Glu inserted after N-terminal Met, C175X CDS (1)...(558)
variation (524)...(525) n = A, T, G, or C 135 atg gar gcn gar ggc
cct gtc ccc act tcc aag ccc acc aca act ggg 48 Met Glu Ala Glu Gly
Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly 1 5 10 15 aag ggc tgc
cac att ggc agg ttc aaa tct ctg tca cca cag gag cta 96 Lys Gly Cys
His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu 20 25 30 gcg
agc ttc aag aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg 144 Ala
Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu 35 40
45 aaa aac tgg agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg
192 Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu
50 55 60 agg ctt ctc cag gtg agg gag cgc cct gtg gcc ttg gag gct
gag ctg 240 Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala
Glu Leu 65 70 75 80 gcc ctg acg ctg aag gtc ctg gag gcc gct gct ggc
cca gcc ctg gag 288 Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly
Pro Ala Leu Glu 85 90 95 gac gtc cta gac cag ccc ctt cac acc ctg
cac cac atc ctc tcc cag 336 Asp Val Leu Asp Gln Pro Leu His Thr Leu
His His Ile Leu Ser Gln 100 105 110 ctc cag gcc tgt atc cag cct cag
ccc aca gca ggg ccc agg ccc cgg 384 Leu Gln Ala Cys Ile Gln Pro Gln
Pro Thr Ala Gly Pro Arg Pro Arg 115 120 125 ggc cgc ctc cac cac tgg
ctg cac cgg ctc cag gag gcc ccc aaa aag 432 Gly Arg Leu His His Trp
Leu His Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140 gag tcc gct ggc
tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc 480 Glu Ser Ala Gly
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg 145 150 155 160 ctc
ctc acg cga gac ctc aaa tat gtg gcc gat ggg aac ctg dnn ctg 528 Leu
Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu 165 170
175 aga acg tca acc cac cct gag tcc acc tga 558 Arg Thr Ser Thr His
Pro Glu Ser Thr * 180 185 136 185 PRT Artificial Sequence IL-29
Glu, Ala, and Glu inserted after N-terminal Met, C175X VARIANT
(175)...(175) Xaa = Ser, Ala, Thr, Val, or Asn 136 Met Glu Ala Glu
Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly 1 5 10 15 Lys Gly
Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu 20 25 30
Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu 35
40 45 Lys Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp
Leu 50 55 60 Arg Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu
Ala Glu Leu 65 70 75 80 Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala
Gly Pro Ala Leu Glu 85 90 95 Asp Val Leu Asp Gln Pro Leu His Thr
Leu His His Ile Leu Ser Gln 100 105 110 Leu Gln Ala Cys Ile Gln Pro
Gln Pro Thr Ala Gly Pro Arg Pro Arg 115 120 125 Gly Arg Leu His His
Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys 130 135 140 Glu Ser Ala
Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg 145 150 155 160
Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Xaa Leu 165
170 175 Arg Thr Ser Thr His Pro Glu Ser Thr 180 185
* * * * *