U.S. patent application number 11/945975 was filed with the patent office on 2008-09-25 for anti-il-20 antibodies and binding partners and methods of using in inflammation.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Yasmin A. Chandrasekher, Stacey R. Dillon, Wayne Kindsvogel, Joyce M. Lehner, Margaret D. Moore, Anthony W. Siadak, Pallavur V. Sivakumar, Wenfeng Xu.
Application Number | 20080233115 11/945975 |
Document ID | / |
Family ID | 33101289 |
Filed Date | 2008-09-25 |
United States Patent
Application |
20080233115 |
Kind Code |
A1 |
Xu; Wenfeng ; et
al. |
September 25, 2008 |
ANTI-IL-20 ANTIBODIES AND BINDING PARTNERS AND METHODS OF USING IN
INFLAMMATION
Abstract
The present invention relates to blocking, inhibiting, reducing,
antagonizing or neutralizing the activity of IL-20 polypeptide
molecules. IL-20 and IL-22 are cytokines that are involved in
inflammatory processes and human disease. The present invention
includes anti-IL-20 and anti-IL-22RA antibodies and binding
partners, as well as methods for antagonizing IL-20 using such
antibodies and binding partners.
Inventors: |
Xu; Wenfeng; (Seattle,
WA) ; Kindsvogel; Wayne; (Seattle, WA) ;
Chandrasekher; Yasmin A.; (Saratoge, CA) ; Dillon;
Stacey R.; (Seattle, WA) ; Lehner; Joyce M.;
(Seattle, WA) ; Siadak; Anthony W.; (Seattle,
WA) ; Sivakumar; Pallavur V.; (Seattle, WA) ;
Moore; Margaret D.; (Seattle, WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
33101289 |
Appl. No.: |
11/945975 |
Filed: |
November 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10807997 |
Mar 24, 2004 |
|
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11945975 |
|
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|
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60457481 |
Mar 24, 2003 |
|
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60523295 |
Nov 17, 2003 |
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Current U.S.
Class: |
424/133.1 ;
424/139.1; 424/178.1 |
Current CPC
Class: |
A61P 11/06 20180101;
A61P 19/02 20180101; A61P 37/04 20180101; C07K 14/54 20130101; C07K
2317/73 20130101; A61P 7/00 20180101; A61P 43/00 20180101; A61P
11/00 20180101; A61P 17/00 20180101; A61P 31/04 20180101; A61P 1/00
20180101; A61P 37/08 20180101; A61P 31/00 20180101; A61P 37/00
20180101; A61P 37/02 20180101; C07K 14/7155 20130101; C07K 2319/30
20130101; A61P 1/04 20180101; A61P 29/00 20180101; A61P 17/06
20180101; A61K 2039/505 20130101; C07K 16/244 20130101; C07K
2317/34 20130101; C07K 2319/00 20130101; C07K 16/2866 20130101 |
Class at
Publication: |
424/133.1 ;
424/139.1; 424/178.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/44 20060101 A61K039/44; A61P 29/00 20060101
A61P029/00 |
Claims
1. A method for treating endotoxemia, wherein the method comprises
administering to a mammal an effective amount of an antibody or
antibody fragment that specifically binds to a polypeptide
comprising the amino acid sequence of SEQ ID NO:8.
2. The method according to claim 1, wherein said administration of
the antibody or antibody fragment reduces the pro-inflammatory
activity of SEQ ID NO:8 in endotoxemia.
3. The method according to claim 1, wherein the antibody or
antibody fragment is selected from the group consisting of: (a) a
polyclonal antibody, (b) a murine monoclonal antibody, (c) a
humanized antibody derived from (b), (d) an antibody fragment, or
(e) a human monoclonal antibody.
4. The method according to claim 1, wherein the antibody or
antibody fragment further comprises a conjugate.
5. The method according to claim 4, wherein the conjugate comprises
PEG.
6. The method according to claim 4, wherein the conjugate is
selected from the group consisting of: a radionuclide, an enzyme, a
substrate, a cofactor, a fluorescent marker, a chemiluminescent
marker, a peptide tag, a magnetic particle, a drug, and a
toxin.
7. A method for treating septicemia, wherein the method comprises
administering to a mammal an effective amount of an antibody or
antibody fragment that specifically binds to a polypeptide
comprising the amino acid sequence of SEQ ID NO:8.
8. The method according to claim 1, wherein said administration of
the antibody or antibody fragment reduces the pro-inflammatory
activity of SEQ ID NO:8 in septicemia.
9. The method according to claim 1, wherein the antibody or
antibody fragment is selected from the group consisting of: (a) a
polyclonal antibody, (b) a murine monoclonal antibody, (c) a
humanized antibody derived from (b), (d) an antibody fragment, or
(e) a human monoclonal antibody.
10. The method according to claim 1, wherein the antibody or
antibody fragment further comprises a conjugate.
11. The method according to claim 10, wherein the conjugate
comprises PEG.
12. The method according to claim 10, wherein the conjugate is
selected from the group consisting of: a radionuclide, an enzyme, a
substrate, a cofactor, a fluorescent marker, a chemiluminescent
marker, a peptide tag, a magnetic particle, a drug, and a
toxin.
13. A method for treating toxic shock syndrome, wherein the method
comprises administering to a mammal an effective amount of an
antibody or antibody fragment that specifically binds to a
polypeptide comprising the amino acid sequence of SEQ ID NO:8.
14. The method according to claim 1, wherein said administration of
the antibody or antibody fragment reduces the pro-inflammatory
activity of SEQ ID NO:8 in toxic shock syndrome.
15. The method according to claim 1, wherein the antibody or
antibody fragment is selected from the group consisting of: (a) a
polyclonal antibody, (b) a murine monoclonal antibody, (c) a
humanized antibody derived from (b), (d) an antibody fragment, or
(e) a human monoclonal antibody.
16. The method according to claim 1, wherein the antibody or
antibody fragment further comprises a conjugate.
17. The method according to claim 16, wherein the conjugate
comprises PEG.
18. The method according to claim 16, wherein the conjugate is
selected from the group consisting of: a radionuclide, an enzyme, a
substrate, a cofactor, a fluorescent marker, a chemiluminescent
marker, a peptide tag, a magnetic particle, a drug, and a toxin.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/807,997, filed Mar. 24, 2004, which claims the benefit of
U.S. Provisional Application Ser. No. 60/457,481, filed Mar. 24,
2003, and U.S. Provisional Application Ser. No. 60/523,295, filed
Nov. 17, 2003, all of which are herein incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] Cytokines are soluble, small proteins that mediate a variety
of biological effects, including the regulation of the growth and
differentiation of many cell types (see, for example, Arai et al.,
Annu. Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol
3:311 (1991); Paul and Seder, Cell 76:241 (1994)). Proteins that
constitute the cytokine group include interleukins, interferons,
colony stimulating factors, tumor necrosis factors, and other
regulatory molecules. For example, human interleukin-17 is a
cytokine which stimulates the expression of interleukin-6,
intracellular adhesion molecule 1, interleukin-8, granulocyte
macrophage colony-stimulating factor, and prostaglandin E2
expression, and plays a role in the preferential maturation of
CD34+ hematopoietic precursors into neutrophils (Yao et al., J.
Immunol. 155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593
(1996)).
[0003] Receptors that bind cytokines are typically composed of one
or more integral membrane proteins that bind the cytokine with high
affinity and transduce this binding event to the cell through the
cytoplasmic portions of the certain receptor subunits. Cytokine
receptors have been grouped into several classes on the basis of
similarities in their extracellular ligand binding domains. For
example, the receptor chains responsible for binding and/or
transducing the effect of interferons are members of the class II
cytokine receptor family, based upon a characteristic 200 residue
extracellular domain.
[0004] The demonstrated in vivo activities of cytokines and their
receptors illustrate the clinical potential of, and need for, other
cytokines, cytokine receptors, cytokine agonists, and cytokine
antagonists. For example, demonstrated in vivo activities of the
pro-inflammatory cytokine family illustrates the enormous clinical
potential of, and need for antagonists of pro-inflammatory
molecules. The present invention addresses these needs by providing
antagonists to pro-inflammatory cytokines IL-20 and IL-22. Such
antagonists of the present invention, which may block, inhibit,
reduce, antagonize or neutralize the activity of IL-22, IL-20, or
both IL-20 and IL-22, include soluble IL-22RA receptors and
neutralizing anti-IL-22RA antibodies. The invention further
provides uses therefore in inflammatory disease, as well as related
compositions and methods.
DETAILED DESCRIPTION OF THE INVENTION
1. Overview
[0005] Amongst other inventions, the present invention provides
novel uses for a soluble receptor, designated "Zcytor11" or
"IL-22RA" and neutralizing antibodies to IL-22RA cytokine
receptors. The present invention also provides soluble IL-22RA
polypeptide fragments and fusion proteins, for use in human
inflammatory and autoimmune diseases. The anti-IL-22RA antibodies,
and soluble IL-22RA receptors of the present invention, including
the neutralizing anti-IL-22RA antibodies of the present invention,
can be used to block, inhibit, reduce, antagonize or neutralize the
activity of either IL-22 or IL-20, or both IL-20 and IL-22 in the
treatment of specific human diseases such as psoriasis, psoriatic
arthritis, arthritis, endotoxemia, inflammatory bowel disease
(IBD), colitis, and other inflammatory conditions disclosed
herein.
[0006] An illustrative nucleotide sequence that encodes human
Zcytor11 (IL-22RA) is provided by SEQ ID NO:1; the encoded
polypeptide is shown in SEQ ID NO:2. IL-22RA is a receptor subunit
for both IL-20 and IL-22. Zcytor11 (IL-22RA) is disclosed in
commonly owned U.S. Pat. No. 5,965,704, commonly owned WIPO
publication WO 02/12345, and commonly owned WIPO publication WO
02/072607. Analysis of a human cDNA clone encoding IL-22RA (SEQ ID
NO:1) revealed an open reading frame encoding 574 amino acids (SEQ
ID NO:2) comprising an extracellular ligand-binding domain of
approximately 211 amino acid residues (residues 18-228 of SEQ ID
NO:2; SEQ ID NO:3), a transmembrane domain of approximately 23
amino acid residues (residues 229-251 of SEQ ID NO:2), and an
intracellular domain of approximately 313 amino acid residues
(residues 252 to 574 of SEQ ID NO:2). Thus molecules of the present
invention include polypeptides that include a cytokine binding
domain comprising amino acids residues 18-228 of SEQ ID NO:2; SEQ
ID NO:3. In one embodiment of the soluble receptor of the present
invention, the soluble IL-22R is fused to the constant region of
the heavy chain (representative shown in SEQ ID NO:4). Those
skilled in the art will recognize that these domain boundaries are
approximate. Deletion of residues from the ends of the domains is
possible.
[0007] As described below, the present invention provides isolated
polypeptides comprising an amino acid sequence that is at least
70%, at least 80%, or at least 90%, or greater than 95%, such as
96%, 97%, 98%, or greater than 99% or more identical to a reference
amino acid sequence of 18-228 of SEQ ID NO:2, which is also shown
as SEQ ID NO:3, wherein the isolated polypeptide specifically binds
with an antibody that specifically binds with a polypeptide
comprising the amino acid sequence of SEQ ID NO:3. Illustrative
polypeptides include polypeptides comprising either amino acid
residues SEQ ID NO:3 or amino acid residues SEQ ID NO:3. Moreover,
the present invention also provides isolated polypeptides as
disclosed above that bind IL-22 (e.g., human IL-22 polypeptide
sequence as shown in SEQ ID NO:6). The human IL-22 polynucleotide
sequence is shown in SEQ ID NO:5. The mouse IL-22 polynucleotide
sequence is shown in SEQ ID NO:10, and corresponding polypeptide is
shown in SEQ ID NO:1. The present invention also provides isolated
polypeptides as disclosed above that bind IL-20 (e.g., human IL-20
polypeptide sequence as shown in SEQ ID NO:8; WIPO Publication No.
WO 99/27103). The human IL-20 polynucleotide sequence is shown in
SEQ ID NO:7.
[0008] The present invention also provides isolated polypeptides
and epitopes comprising at least 15 contiguous amino acid residues
of an amino acid sequence of SEQ ID NO:3. Illustrative polypeptides
include polypeptides that either comprise, or consist of SEQ ID
NO:3, an antigenic epitope thereof, or a functional IL-20 or IL-22
binding fragment thereof. Moreover, the present invention also
provides isolated polypeptides as disclosed above that bind to,
block, inhibit, reduce, antagonize or neutralize the activity of
IL-22 or IL-20.
[0009] The present invention also includes variant IL-22RA
polypeptides, wherein the amino acid sequence of the variant
polypeptide shares an identity with the amino acid residues of SEQ
ID NO:3 selected from the group consisting of at least 70%
identity, at least 80% identity, at least 90% identity, at least
95% identity, or greater than 95% identity, such as 96%, 97%, 98%,
or greater than 99% or more identity, and wherein any difference
between the amino acid sequence of the variant polypeptide and the
corresponding amino acid sequence of SEQ ID NO:3 is due to one or
more conservative amino acid substitutions. Such conservative amino
acid substitutions are described herein. Moreover, the present
invention also provides isolated polypeptides as disclosed above
that bind to, block, inhibit, reduce, antagonize or neutralize the
activity of IL-22 or IL-20.
[0010] The present invention further provides antibodies and
antibody fragments that specifically bind with such polypeptides.
Exemplary antibodies include neutralizing antibodies, polyclonal
antibodies, murine monoclonal antibodies, humanized antibodies
derived from murine monoclonal antibodies, and human monoclonal
antibodies. Illustrative antibody fragments include F(ab').sub.2,
F(ab).sub.2, Fab', Fab, Fv, scFv, and minimal recognition units.
Neutralizing antibodies preferably bind IL-22RA such that the
interaction of IL-20 and IL-22 with IL-22RA is blocked, inhibited,
reduced, antagonized or neutralized; anti-IL-22RA neutralizing
antibodies such that the binding of either IL-20 or IL-22 to
IL-22RA is blocked, inhibited, reduced, antagonized or neutralized
are also encompassed by the present invention. That is, the
neutralizing anti-IL-22RA antibodies of the present invention can
either bind, block, inhibit, reduce, antagonize or neutralize each
of IL-20 or IL-22 singly, or bind, block, inhibit, reduce,
antagonize or neutralize IL-20 and IL-22 together. The present
invention further includes compositions comprising a carrier and a
peptide, polypeptide, or antibody described herein.
[0011] In addition, the present invention provides pharmaceutical
compositions comprising a pharmaceutically acceptable carrier and
at least one of such an expression vector or recombinant virus
comprising such expression vectors. The present invention further
includes pharmaceutical compositions, comprising a pharmaceutically
acceptable carrier and a polypeptide or antibody described
herein.
[0012] The present invention also contemplates anti-idiotype
antibodies, or anti-idiotype antibody fragments, that specifically
bind an antibody or antibody fragment that specifically binds a
polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a
fragment thereof. An exemplary anti-idiotype antibody binds with an
antibody that specifically binds a polypeptide consisting of SEQ ID
NO:3.
[0013] The present invention also provides fusion proteins,
comprising a IL-22RA polypeptide and an immunoglobulin moiety. In
such fusion proteins, the immunoglobulin moiety may be an
immunoglobulin heavy chain constant region, such as a human F.sub.c
fragment. The present invention further includes isolated nucleic
acid molecules that encode such fusion proteins.
[0014] The present invention also provides polyclonal and
monoclonal antibodies that bind to polypeptides comprising an
IL-22RA extracellular domain such as monomeric, homodimeric,
heterodimeric and multimeric receptors, including soluble
receptors. Moreover, such antibodies can be used antagonize the
binding of IL-22RA ligands, IL-22 (SEQ ID NO:6), and IL-20 (SEQ ID
NO:8), individually or together to the IL-22RA receptor.
[0015] Moreover, over expression or upregulation of IL-22 and IL-20
was shown in human psoriatic lesions and human atopic dermatitis
skin samples, suggesting that IL-22, like IL-20 is also involved in
human psoriasis, atopic dermatitis or other inflammatory diseases
of the skin and epithelial tissues. Moreover, as described herein,
over expression of IL-20 or IL-22 in transgenic mice showed
epidermal thickening and immune cell involvement indicative of a
psoriatic phenotype; and in addition injection of IL-22 into normal
mice showed epidermal thickening and immune cell involvement
indicative of a psoriatic phenotype which was ablated by the
soluble receptor antagonist IL-22RA2 (zcytor16; WIPO Publication
No. WO 01/40467). Such in vivo data further suggests that the
pro-inflammatory IL-22 is involved in psoriasis, atopic dermatitis
or other inflammatory diseases of the skin and epithelial tissues.
As such, antagonists to IL-22 and IL-20 activity, such as IL-22RA
soluble receptors and antibodies thereto including the
anti-human-IL-22RA monoclonal and neutralizing antibodies of the
present invention, are useful in therapeutic treatment of
inflammatory diseases, particularly as antagonists to both IL-22
and IL-20 singly or together in the treatment of psoriasis.
Moreover, antagonists to IL-22 activity, such as IL-22RA soluble
receptors and antibodies thereto including the anti-human-IL-22RA
monoclonal and neutralizing antibodies of the present invention,
are useful in therapeutic treatment of other inflammatory diseases
for example as bind, block, inhibit, reduce, antagonize or
neutralize IL-22 and IL-20 (either individually or together) in the
treatment of atopic dermatitis, IBD, colitis, Endotoxemia,
arthritis, rheumatoid arthritis, and psoriatic arthritis adult
respiratory disease (ARD), septic shock, multiple organ failure,
inflammatory lung injury such as asthma or bronchitis, bacterial
pneumonia, psoriasis, eczema, atopic and contact dermatitis, and
inflammatory bowel disease such as ulcerative colitis and Crohn's
disease.
[0016] These and other aspects of the invention will become evident
upon reference to the following detailed description. In addition,
various references are identified below and are incorporated by
reference in their entirety.
2. Definitions
[0017] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0018] As used herein, "nucleic acid" or "nucleic acid molecule"
refers to polynucleotides, such as deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA), oligonucleotides, fragments generated by
the polymerase chain reaction (PCR), and fragments generated by any
of ligation, scission, endonuclease action, and exonuclease action.
Nucleic acid molecules can be composed of monomers that are
naturally-occurring nucleotides (such as DNA and RNA), or analogs
of naturally-occurring nucleotides (e.g., .alpha.-enantiomeric
forms of naturally-occurring nucleotides), or a combination of
both. Modified nucleotides can have alterations in sugar moieties
and/or in pyrimidine or purine base moieties. Sugar modifications
include, for example, replacement of one or more hydroxyl groups
with halogens, alkyl groups, amines, and azido groups, or sugars
can be functionalized as ethers or esters. Moreover, the entire
sugar moiety can be replaced with sterically and electronically
similar structures, such as aza-sugars and carbocyclic sugar
analogs. Examples of modifications in a base moiety include
alkylated purines and pyrimidines, acylated purines or pyrimidines,
or other well-known heterocyclic substitutes. Nucleic acid monomers
can be linked by phosphodiester bonds or analogs of such linkages.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like. The term "nucleic acid molecule" also includes so-called
"peptide nucleic acids," which comprise naturally-occurring or
modified nucleic acid bases attached to a polyamide backbone.
Nucleic acids can be either single stranded or double stranded.
[0019] The term "complement of a nucleic acid molecule" refers to a
nucleic acid molecule having a complementary nucleotide sequence
and reverse orientation as compared to a reference nucleotide
sequence. For example, the sequence 5' ATGCACGGG 3' is
complementary to 5' CCCGTGCAT 3'.
[0020] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons as
compared to a reference nucleic acid 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).
[0021] The term "structural gene" refers to a nucleic acid molecule
that is transcribed into messenger RNA (mRNA), which is then
translated into a sequence of amino acids characteristic of a
specific polypeptide.
[0022] An "isolated nucleic acid molecule" is a nucleic acid
molecule that is not integrated in the genomic DNA of an organism.
For example, a DNA molecule that encodes a growth factor that has
been separated from the genomic DNA of a cell is an isolated DNA
molecule. Another example of an isolated nucleic acid molecule is a
chemically-synthesized nucleic acid molecule that is not integrated
in the genome of an organism. A nucleic acid molecule that has been
isolated from a particular species is smaller than the complete DNA
molecule of a chromosome from that species.
[0023] A "nucleic acid molecule construct" is a nucleic acid
molecule, either single- or double-stranded, that has been modified
through human intervention to contain segments of nucleic acid
combined and juxtaposed in an arrangement not existing in
nature.
[0024] "Linear DNA" denotes non-circular DNA molecules having free
5' and 3' ends. Linear DNA can be prepared from closed circular DNA
molecules, such as plasmids, by enzymatic digestion or physical
disruption.
[0025] "Complementary DNA (cDNA)" is a single-stranded DNA molecule
that is formed from an mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand. The term "cDNA" also
refers to a clone of a cDNA molecule synthesized from an RNA
template.
[0026] A "promoter" is a nucleotide sequence that directs the
transcription of a structural gene. Typically, a promoter is
located in the 5' non-coding region of a gene, proximal to the
transcriptional start site of a structural gene. Sequence elements
within promoters that function in the initiation of transcription
are often characterized by consensus nucleotide sequences. These
promoter elements include RNA polymerase binding sites, TATA
sequences, CAAT sequences, differentiation-specific elements (DSEs;
McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclic AMP response
elements (CREs), serum response elements (SREs; Treisman, Seminars
in Cancer Biol. 1.47 (1990)), glucocorticoid response elements
(GREs), and binding sites for other transcription factors, such as
CRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye
et al., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response
element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and
octamer factors (see, in general, Watson et al., eds., Molecular
Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing
Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1
(1994)). If a promoter is an inducible promoter, then the rate of
transcription increases in response to an inducing agent. In
contrast, the rate of transcription is not regulated by an inducing
agent if the promoter is a constitutive promoter. Repressible
promoters are also known.
[0027] A "core promoter" contains essential nucleotide sequences
for promoter function, including the TATA box and start of
transcription. By this definition, a core promoter may or may not
have detectable activity in the absence of specific sequences that
may enhance the activity or confer tissue specific activity.
[0028] A "regulatory element" is a nucleotide sequence that
modulates the activity of a core promoter. For example, a
regulatory element may contain a nucleotide sequence that binds
with cellular factors enabling transcription exclusively or
preferentially in particular cells, tissues, or organelles. These
types of regulatory elements are normally associated with genes
that are expressed in a "cell-specific," "tissue-specific," or
"organelle-specific" manner.
[0029] An "enhancer" is a type of regulatory element that can
increase the efficiency of transcription, regardless of the
distance or orientation of the enhancer relative to the start site
of transcription.
[0030] "Heterologous DNA" refers to a DNA molecule, or a population
of DNA molecules, that does not exist naturally within a given host
cell. DNA molecules heterologous to a particular host cell may
contain DNA derived from the host cell species (i.e., endogenous
DNA) so long as that host DNA is combined with non-host DNA (i.e.,
exogenous DNA). For example, a DNA molecule containing a non-host
DNA segment encoding a polypeptide operably linked to a host DNA
segment comprising a transcription promoter is considered to be a
heterologous DNA molecule. Conversely, a heterologous DNA molecule
can comprise an endogenous gene operably linked with an exogenous
promoter. As another illustration, a DNA molecule comprising a gene
derived from a wild-type cell is considered to be heterologous DNA
if that DNA molecule is introduced into a mutant cell that lacks
the wild-type gene.
[0031] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides."
[0032] 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.
[0033] A peptide or polypeptide encoded by a non-host DNA molecule
is a "heterologous" peptide or polypeptide.
[0034] A "cloning vector" is a nucleic acid molecule, such as a
plasmid, cosmid, or bacteriophage, that has the capability of
replicating autonomously in a host cell. Cloning vectors typically
contain one or a small number of restriction endonuclease
recognition sites that allow insertion of a nucleic acid molecule
in a determinable fashion without loss of an essential biological
function of the vector, as well as nucleotide sequences encoding a
marker gene that is suitable for use in the identification and
selection of cells transformed with the cloning vector. Marker
genes typically include genes that provide tetracycline resistance
or ampicillin resistance.
[0035] An "expression vector" is a nucleic acid molecule encoding a
gene that is expressed in a host cell. Typically, an expression
vector comprises a transcription promoter, a gene, and a
transcription terminator. Gene expression is usually placed under
the control of a promoter, and such a gene is said to be "operably
linked to" the promoter. Similarly, a regulatory element and a core
promoter are operably linked if the regulatory element modulates
the activity of the core promoter.
[0036] A "recombinant host" is a cell that contains a heterologous
nucleic acid molecule, such as a cloning vector or expression
vector. In the present context, an example of a recombinant host is
a cell that produces IL-22RA from an expression vector. In
contrast, IL-22RA can be produced by a cell that is a "natural
source" of IL-22RA, and that lacks an expression vector.
[0037] "Integrative transformants" are recombinant host cells, in
which heterologous DNA has become integrated into the genomic DNA
of the cells.
[0038] A "fusion protein" is a hybrid protein expressed by a
nucleic acid molecule comprising nucleotide sequences of at least
two genes. For example, a fusion protein can comprise at least part
of a IL-22RA polypeptide fused with a polypeptide that binds an
affinity matrix. Such a fusion protein provides a means to isolate
large quantities of IL-22RA using affinity chromatography.
[0039] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule termed a "ligand." This interaction
mediates the effect of the ligand on the cell. 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). Membrane-bound receptors are characterized by a
multi-domain structure comprising an extracellular ligand-binding
domain and an intracellular effector domain that is typically
involved in signal transduction. In certain membrane-bound
receptors, the extracellular ligand-binding domain and the
intracellular effector domain are located in separate polypeptides
that comprise the complete functional receptor.
[0040] In general, the 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,
which in turn leads to an alteration in the metabolism of the cell.
Metabolic events that are often 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.
[0041] A "soluble receptor" is a receptor polypeptide that is not
bound to a cell membrane. Soluble receptors are most commonly
ligand-binding receptor polypeptides that lack transmembrane and
cytoplasmic domains, and other linkage to the cell membrane such as
via glycophosphoinositol (gpi). Soluble receptors can comprise
additional amino acid residues, such as affinity tags that provide
for purification of the polypeptide or provide sites for attachment
of the polypeptide to a substrate, or immunoglobulin constant
region sequences. Many cell-surface receptors have naturally
occurring, soluble counterparts that are produced by proteolysis or
translated from alternatively spliced mRNAs. Soluble receptors can
be monomeric, homodimeric, heterodimeric, or multimeric, with
multimeric receptors generally not comprising more than 9 subunits,
preferably not comprising more than 6 subunits, and most preferably
not comprising more than 3 subunits. Receptor polypeptides are said
to be substantially free of transmembrane and intracellular
polypeptide segments when they lack sufficient portions of these
segments to provide membrane anchoring or signal transduction,
respectively. Soluble receptors of class I and class II cytokine
receptors generally comprise the extracellular cytokine binding
domain free of a transmembrane domain and intracellular domain. For
example, representative soluble receptors include soluble receptors
for CRF2-4 (a.k.a., IL-10RB) (Genbank Accession No. Z17227) as
shown in SEQ ID NO:44 and SEQ ID NO:45; a soluble receptor for
IL-10RA (Genbank Accession No.s U00672 and NM.sub.--001558) as
shown in SEQ ID NO:46; a soluble receptor for pDIRS1 (a.k.a.,
IL-20RB) (Genbank Accession No. AY358305) as shown in SEQ ID NO:47;
and a soluble receptor for IL-22RA (U.S. Pat. No. 5,965,704) as
shown in SEQ ID NO:3. It is well within the level of one of skill
in the art to delineate what sequences of a known class I or class
II cytokine sequence comprise the extracellular cytokine binding
domain free of a transmembrane domain and intracellular domain.
Moreover, one of skill in the art using the genetic code can
readily determine polynucleotides that encode such soluble receptor
polypeptides.
[0042] The term "secretory signal sequence" denotes a DNA sequence
that encodes a peptide (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.
[0043] An "isolated polypeptide" is a polypeptide that is
essentially free from contaminating cellular components, such as
carbohydrate, lipid, or other proteinaceous impurities associated
with the polypeptide in nature. Typically, a preparation of
isolated polypeptide contains the polypeptide in a highly purified
form, i.e., at least about 80% pure, at least about 90% pure, at
least about 95% pure, greater than 95% pure, such as 96%, 97%, or
98% or more pure, or greater than 99% pure. One way to show that a
particular protein preparation contains an isolated polypeptide is
by the appearance of a single band following sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis of the protein preparation
and Coomassie Brilliant Blue staining of the gel. However, 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.
[0044] 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.
[0045] The term "expression" refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0046] 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 polypeptide encoded by a
splice variant of an mRNA transcribed from a gene.
[0047] As used herein, the term "immunomodulator" includes
cytokines, stem cell growth factors, lymphotoxins, co-stimulatory
molecules, hematopoietic factors, and the like, and synthetic
analogs of these molecules.
[0048] 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 less than 10.sup.9 M.sup.-1.
[0049] An "anti-idiotype antibody" is an antibody that binds with
the variable region domain of an immunoglobulin. In the present
context, an anti-idiotype antibody binds with the variable region
of an anti-IL-22RA antibody, and thus, an anti-idiotype antibody
mimics an epitope of IL-22RA.
[0050] An "antibody fragment" is a portion of an antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the intact antibody. For example, an anti-IL-22RA
monoclonal antibody fragment binds with an epitope of IL-22RA.
[0051] The term "antibody fragment" also includes a synthetic or a
genetically engineered polypeptide that binds to a specific
antigen, such as polypeptides consisting of the light chain
variable region, "Fv" fragments consisting of the variable regions
of the heavy and light chains, recombinant single chain polypeptide
molecules in which light and heavy variable regions are connected
by a peptide linker ("scFv proteins"), and minimal recognition
units consisting of the amino acid residues that mimic the
hypervariable region.
[0052] A "chimeric antibody" is a recombinant protein that contains
the variable domains and complementary determining regions derived
from a rodent antibody, while the remainder of the antibody
molecule is derived from a human antibody.
[0053] "Humanized antibodies" are recombinant proteins in which
murine complementarity determining regions of a monoclonal antibody
have been transferred from heavy and light variable chains of the
murine immunoglobulin into a human variable domain. Construction of
humanized antibodies for therapeutic use in humans that are derived
from murine antibodies, such as those that bind to or neutralize a
human protein, is within the skill of one in the art.
[0054] As used herein, a "therapeutic agent" is a molecule or atom
which is conjugated to an antibody moiety to produce a conjugate
which is useful for therapy. Examples of therapeutic agents include
drugs, toxins, immunomodulators, chelators, boron compounds,
photoactive agents or dyes, and radioisotopes.
[0055] A "detectable label" is a molecule or atom which can be
conjugated to an antibody moiety to produce a molecule useful for
diagnosis. Examples of detectable labels include chelators,
photoactive agents, radioisotopes, fluorescent agents, paramagnetic
ions, or other marker moieties.
[0056] 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 (1985)), substance P, FLAG
peptide (Hopp et al., Biotechnology 6:1204 (1988)), streptavidin
binding peptide, or other antigenic epitope or binding domain. See,
in general, Ford et al., Protein Expression and Purification 2:95
(1991). DNA molecules encoding affinity tags are available from
commercial suppliers (e.g., Pharmacia Biotech, Piscataway,
N.J.).
[0057] A "naked antibody" is an entire antibody, as opposed to an
antibody fragment, which is not conjugated with a therapeutic
agent. Naked antibodies include both polyclonal and monoclonal
antibodies, as well as certain recombinant antibodies, such as
chimeric and humanized antibodies.
[0058] As used herein, the term "antibody component" includes both
an entire antibody and an antibody fragment.
[0059] An "immunoconjugate" is a conjugate of an antibody component
with a therapeutic agent or a detectable label.
[0060] As used herein, the term "antibody fusion protein" refers to
a recombinant molecule that comprises an antibody component and a
IL-22RA polypeptide component. Examples of an antibody fusion
protein include a protein that comprises a IL-22RA extracellular
domain, and either an Fc domain or an antigen-binding region.
[0061] A "target polypeptide" or a "target peptide" is an amino
acid sequence that comprises at least one epitope, and that is
expressed on a target cell, such as a tumor cell, or a cell that
carries an infectious agent antigen. T cells recognize peptide
epitopes presented by a major histocompatibility complex molecule
to a target polypeptide or target peptide and typically lyse the
target cell or recruit other immune cells to the site of the target
cell, thereby killing the target cell.
[0062] An "antigenic peptide" is a peptide which will bind a major
histocompatibility complex molecule to form an MHC-peptide complex
which is recognized by a T cell, thereby inducing a cytotoxic
lymphocyte response upon presentation to the T cell. Thus,
antigenic peptides are capable of binding to an appropriate major
histocompatibility complex molecule and inducing a cytotoxic T
cells response, such as cell lysis or specific cytokine release
against the target cell which binds or expresses the antigen. The
antigenic peptide can be bound in the context of a class I or class
II major histocompatibility complex molecule, on an antigen
presenting cell or on a target cell.
[0063] In eukaryotes, RNA polymerase II catalyzes the transcription
of a structural gene to produce mRNA. A nucleic acid molecule can
be designed to contain an RNA polymerase II template in which the
RNA transcript has a sequence that is complementary to that of a
specific mRNA. The RNA transcript is termed an "anti-sense RNA" and
a nucleic acid molecule that encodes the anti-sense RNA is termed
an "anti-sense gene." Anti-sense RNA molecules are capable of
binding to mRNA molecules, resulting in an inhibition of mRNA
translation.
[0064] An "anti-sense oligonucleotide specific for IL-22RA" or a
"IL-22RA anti-sense oligonucleotide" is an oligonucleotide having a
sequence (a) capable of forming a stable triplex with a portion of
the IL-22RA gene, or (b) capable of forming a stable duplex with a
portion of an mRNA transcript of the IL-22RA gene.
[0065] A "ribozyme" is a nucleic acid molecule that contains a
catalytic center. The term includes RNA enzymes, self-splicing
RNAs, self-cleaving RNAs, and nucleic acid molecules that perform
these catalytic functions. A nucleic acid molecule that encodes a
ribozyme is termed a "ribozyme gene."
[0066] An "external guide sequence" is a nucleic acid molecule that
directs the endogenous ribozyme, RNase P, to a particular species
of intracellular mRNA, resulting in the cleavage of the mRNA by
RNase P. A nucleic acid molecule that encodes an external guide
sequence is termed an "external guide sequence gene."
[0067] The term "variant IL-22RA gene" refers to nucleic acid
molecules that encode a polypeptide having an amino acid sequence
that is a modification of SEQ ID NO:3. Such variants include
naturally-occurring polymorphisms of IL-22RA genes, as well as
synthetic genes that contain conservative amino acid substitutions
of the amino acid sequence of SEQ ID NO:3. Additional variant forms
of IL-22RA genes are nucleic acid molecules that contain insertions
or deletions of the nucleotide sequences described herein. A
variant IL-22RA gene can be identified, for example, by determining
whether the gene hybridizes with a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:1, or its complement, under
stringent conditions.
[0068] Alternatively, variant IL-22RA genes can be identified by
sequence comparison. Two amino acid sequences have "100% amino acid
sequence identity" if the amino acid residues of the two amino acid
sequences are the same when aligned for maximal correspondence.
Similarly, two nucleotide sequences have "100% nucleotide sequence
identity" if the nucleotide residues of the two nucleotide
sequences are the same when aligned for maximal correspondence.
Sequence comparisons can be performed using standard software
programs such as those included in the LASERGENE bioinformatics
computing suite, which is produced by DNASTAR (Madison, Wis.).
Other methods for comparing two nucleotide or amino acid sequences
by determining optimal alignment are well-known to those of skill
in the art (see, for example, Peruski and Peruski, The Internet and
the New Biology: Tools for Genomic and Molecular Research (ASM
Press, Inc. 1997), Wu et al. (eds.), "Information Superhighway and
Computer Databases of Nucleic Acids and Proteins," in Methods in
Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997), and
Bishop (ed.), Guide to Human Genome Computing, 2nd Edition
(Academic Press, Inc. 1998)). Particular methods for determining
sequence identity are described below.
[0069] Regardless of the particular method used to identify a
variant IL-22RA gene or variant IL-22RA polypeptide, a variant gene
or polypeptide encoded by a variant gene may be functionally
characterized the ability to bind specifically to an anti-IL-22RA
antibody. A variant IL-22RA gene or variant IL-22RA polypeptide may
also be functionally characterized the ability to bind to its
ligand, IL-22, using a biological or biochemical assay described
herein.
[0070] 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.
[0071] 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.
[0072] "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.
[0073] The present invention includes functional fragments of
IL-22RA genes. Within the context of this invention, a "functional
fragment" of a IL-22RA gene refers to a nucleic acid molecule that
encodes a portion of a IL-22RA polypeptide which is a domain
described herein or at least specifically binds with an
anti-IL-22RA antibody.
[0074] Due to the imprecision of standard analytical methods,
molecular weights and lengths of polymers are 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%.
3. Production of IL-22RA Polynucleotides or Genes
[0075] Nucleic acid molecules encoding a human IL-22RA gene can be
obtained by screening a human cDNA or genomic library using
polynucleotide probes based upon SEQ ID NO:1. These techniques are
standard and well-established, and may be accomplished using
cloning kits available by commercial suppliers. 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; Aviv and Leder, Proc.
Nat'l Acad. Sci. USA 69:1408 (1972); Huynh et al., "Constructing
and Screening cDNA Libraries in .lamda.gt10 and .lamda.gt11," in
DNA Cloning: A Practical Approach Vol I, Glover (ed.), page 49 (IRL
Press, 1985); Wu (1997) at pages 47-52.
[0076] Nucleic acid molecules that encode a human IL-22RA gene can
also be obtained using the polymerase chain reaction (PCR) with
oligonucleotide primers having nucleotide sequences that are based
upon the nucleotide sequences of the IL-22RA gene or cDNA. General
methods for screening libraries with PCR are provided by, for
example, Yu et al., "Use of the Polymerase Chain Reaction to Screen
Phage Libraries," in Methods in Molecular Biology, Vol. 15. PCR
Protocols: Current Methods and Applications, White (ed.), Humana
Press, Inc., 1993. Moreover, techniques for using PCR to isolate
related genes are described by, for example, Preston, "Use of
Degenerate Oligonucleotide Primers and the Polymerase Chain
Reaction to Clone Gene Family Members," in Methods in Molecular
Biology, Vol. 15: PCR Protocols: Current Methods and Applications,
White (ed.), Humana Press, Inc. 1993. As an alternative, a IL-22RA
gene can be obtained by synthesizing nucleic acid molecules using
mutually priming long oligonucleotides and the nucleotide sequences
described herein (see, for example, Ausubel (1995)). Established
techniques using the polymerase chain reaction provide the ability
to synthesize DNA molecules at least two kilobases in length (Adang
et al., Plant Molec. Biol. 21:1131 (1993), Bambot et al., PCR
Methods and Applications 2:266 (1993), Dillon et al., "Use of the
Polymerase Chain Reaction for the Rapid Construction of Synthetic
Genes," in Methods in Molecular Biology, Vol. 15. PCR Protocols:
Current Methods and Applications, White (ed.), pages 263-268,
(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.
4:299 (1995)). For reviews on polynucleotide synthesis, see, for
example, Glick and Pasternak, Molecular Biotechnology, Principles
and Applications of Recombinant DNA (ASM Press 1994), Itakura et
al., Annu. Rev. Biochem. 53:323 (1984), and Climie et al., Proc.
Nat'l Acad. Sci. USA 87:633 (1990).
4. Production of IL-22RA Gene Variants
[0077] The present invention provides a variety of nucleic acid
molecules, including DNA and RNA molecules, that encode the IL-22RA
polypeptides disclosed herein. Those skilled in the art will
readily recognize that, in view of the degeneracy of the genetic
code, considerable sequence variation is possible among these
polynucleotide molecules. Moreover, the present invention also
provides isolated soluble monomeric, homodimeric, heterodimeric and
multimeric receptor polypeptides that comprise at least one IL-22RA
receptor subunit that is substantially homologous to the receptor
polypeptide of SEQ ID NO:3. Thus, the present invention
contemplates IL-22RA polypeptide-encoding nucleic acid molecules
comprising degenerate nucleotides of SEQ ID NO:1, and their RNA
equivalents.
[0078] Table 1 sets forth the one-letter codes to denote degenerate
nucleotide positions. "Resolutions" are the nucleotides denoted by
a code letter. "Complement" indicates the code for the
complementary nucleotide(s). For example, the code Y denotes either
C or T, and its complement R denotes A or G, A being complementary
to T, and G being complementary to C.
TABLE-US-00001 TABLE 1 Nucleotide Resolution Complement Resolution
A A T T C C G G G G C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T
K G|T M A|C S C|G S C|G W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G
V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T
[0079] The degenerate codons, encompassing all possible codons for
a given amino acid, are set forth 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 .cndot. TAA TAG
TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN
[0080] 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 an 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 reference to the amino acid sequences of SEQ ID NO:3.
Variant sequences can be readily tested for functionality as
described herein.
[0081] Different species can exhibit "preferential codon usage." In
general, see, Grantham et al., Nucl. Acids Res. 8:1893 (1980), Haas
et al. Curr. Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355
(1981), Grosjean and Fiers, Gene 18:199 (1982), Holm, Nuc. Acids
Res. 14:3075 (1986), Ikemura, J. Mol. Biol. 158:573 (1982), Sharp
and Matassi, Curr. Opin. Genet. Dev. 4:851 (1994), Kane, Curr.
Opin. Biotechnol 6:494 (1995), and Makrides, Microbiol Rev. 60:512
(1996). 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. Therefore, the degenerate codon sequences
disclosed herein serve as a template for optimizing expression of
polynucleotides in various cell types and species commonly used in
the art and disclosed herein. Sequences containing preferential
codons can be tested and optimized for expression in various
species, and tested for functionality as disclosed herein.
[0082] A IL-22RA-encoding cDNA can be isolated by a variety of
methods, such as by probing with a complete or partial human cDNA
or with one or more sets of degenerate probes based on the
disclosed sequences. A cDNA can also be cloned using the polymerase
chain reaction with primers designed from the representative human
IL-22RA sequences disclosed herein. In addition, a cDNA library can
be used to transform or transfect host cells, and expression of the
cDNA of interest can be detected with an antibody to IL-22RA
polypeptide.
[0083] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 represents a single allele of human
IL-22RA, and that allelic variation and alternative splicing are
expected to occur. Allelic variants of this sequence can be cloned
by probing cDNA or genomic libraries from different individuals
according to standard procedures. Allelic variants of the
nucleotide sequences disclosed herein, including those containing
silent mutations and those in which mutations result in amino acid
sequence changes, are within the scope of the present invention, as
are proteins which are allelic variants of the amino acid sequences
disclosed herein. cDNA molecules generated from alternatively
spliced mRNAs, which retain the properties of the IL-22RA
polypeptide 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.
[0084] Using the methods discussed above, one of ordinary skill in
the art can prepare a variety of polypeptides that comprise a
soluble IL-22RA receptor subunit that is substantially homologous
to SEQ ID NO:1, or that encodes amino acids of SEQ ID NO:3, or
allelic variants thereof and retain the ligand-binding properties
of the wild-type IL-22RA receptor. Such polypeptides may also
include additional polypeptide segments as generally disclosed
herein.
[0085] Within certain embodiments of the invention, the isolated
nucleic acid molecules can hybridize under stringent conditions to
nucleic acid molecules comprising nucleotide sequences disclosed
herein. For example, such nucleic acid molecules can hybridize
under stringent conditions to nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NO:1, or to nucleic acid molecules
comprising a nucleotide sequence complementary to SEQ ID NO:1, or
fragments thereof.
[0086] 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.
Following hybridization, the nucleic acid molecules can be washed
to remove non-hybridized nucleic acid molecules under stringent
conditions, or under highly stringent conditions. 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. It
is well within the abilities of one skilled in the art to
adapthybridization and wash conditions for use with a particular
polynucleotide hybrid.
[0087] The present invention also provides isolated IL-22RA
polypeptides that have a substantially similar sequence identity to
the polypeptides of SEQ ID NO:3, or their orthologs. The term
"substantially similar sequence identity" is used herein to denote
polypeptides having at least 70%, at least 80%, at least 90%, at
least 95%, such as 96%, 97%, 98%, or greater than 95% sequence
identity to the sequences shown in SEQ ID NO:3, or their orthologs.
For example, variant and orthologous IL-22RA receptors can be used
to generate an immune response and raise cross-reactive antibodies
to human IL-22RA. Such antibodies can be humanized, and modified as
described herein, and used therapeutically to treat psoriasis,
psoriatic arthritis, IBD, colitis, endotoxemia as well as in other
therapeutic applications described herein.
[0088] The present invention also contemplates IL-22RA 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 of SEQ ID NO:3, and a hybridization
assay. Such IL-22RA variants include nucleic acid molecules (1)
that remain hybridized with a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:1 (or its complement) 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., and (2) that encode a polypeptide having at least 70%, at least
80%, at least 90%, at least 95%, or greater than 95% such as 96%,
97%, 98%, or 99%, sequence identity to the amino acid sequence of
SEQ ID NO:3. Alternatively, IL-22RA variants can be characterized
as nucleic acid molecules (1) that remain hybridized with a nucleic
acid molecule having the nucleotide sequence of SEQ ID NO:1 (or its
complement) 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 70%, at least 80%, at least 90%, at least 95% or greater
than 95%, such as 96%, 97%, 98%, or 99% or greater, sequence
identity to the amino acid sequence of SEQ ID NO:3.
[0089] 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 3 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: ([Total number of identical matches]/[length
of the longer sequence plus the number of gaps introduced into the
longer sequence in order to align the two sequences])(100).
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
[0090] 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 IL-22RA variant. 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). Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID NO:2
or SEQ ID NO:3) 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. Illustrative 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. Enzymo. 183:63
(1990).
[0091] 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 described above.
[0092] The present invention includes nucleic acid molecules that
encode a polypeptide having a conservative amino acid change,
compared with an amino acid sequence disclosed herein. For example,
variants can be obtained that contain one or more amino acid
substitutions of SEQ ID NO:3, in which an alkyl amino acid is
substituted for an alkyl amino acid in a IL-22RA amino acid
sequence, an aromatic amino acid is substituted for an aromatic
amino acid in a IL-22RA amino acid sequence, a sulfur-containing
amino acid is substituted for a sulfur-containing amino acid in a
IL-22RA amino acid sequence, a hydroxy-containing amino acid is
substituted for a hydroxy-containing amino acid in a IL-22RA amino
acid sequence, an acidic amino acid is substituted for an acidic
amino acid in a IL-22RA amino acid sequence, a basic amino acid is
substituted for a basic amino acid in a IL-22RA amino acid
sequence, or a dibasic monocarboxylic amino acid is substituted for
a dibasic monocarboxylic amino acid in a IL-22RA amino acid
sequence. Among the common amino acids, for example, a
"conservative amino acid substitution" is illustrated by a
substitution among amino acids within each of the following groups:
(1) glycine, alanine, valine, leucine, and isoleucine, (2)
phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,
(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)
lysine, arginine and histidine. The BLOSUM62 table is an amino acid
substitution matrix derived from about 2,000 local multiple
alignments of protein sequence segments, representing highly
conserved regions of more than 500 groups of related proteins
(Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915
(1992)). Accordingly, the BLOSUM62 substitution frequencies can be
used to define conservative amino acid substitutions that may be
introduced into the amino acid sequences of the present invention.
Although it is possible to design amino acid substitutions based
solely upon chemical properties (as discussed above), the language
"conservative amino acid substitution" preferably refers to a
substitution represented by a BLOSUM62 value of greater than -1.
For example, an amino acid substitution is conservative if the
substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
According to this system, preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 1
(e.g., 1, 2 or 3), while more preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 2
(e.g., 2 or 3). Particular variants of IL-22RA are characterized by
having at least 70%, at least 80%, at least 90%, at least 95% or
greater than 95% such as 96%, 97%, 98%, or 99% or greater sequence
identity to the corresponding amino acid sequence (e.g., SEQ ID
NO:3), wherein the variation in amino acid sequence is due to one
or more conservative amino acid substitutions.
[0093] Conservative amino acid changes in a IL-22RA gene can be
introduced, for example, by substituting nucleotides for the
nucleotides recited in SEQ ID NO:1. Such "conservative amino acid"
variants can be obtained by oligonucleotide-directed mutagenesis,
linker-scanning mutagenesis, mutagenesis using the polymerase chain
reaction, and the like (see Ausubel (1995); and McPherson (ed.),
Directed Mutagenesis: A Practical Approach (IRL Press 1991)). A
variant IL-22RA polypeptide can be identified by the ability to
specifically bind anti-IL-22RA antibodies.
[0094] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is typically carried out in a
cell-free system comprising an E. coli S30 extract and commercially
available enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722 (1991), Ellman et al., Methods Enzymol 202:301
(1991), Chung et al., Science 259:806 (1993), and Chung et al.,
Proc. Nat'l Acad. Sci. USA 90:10145 (1993).
[0095] In a second method, translation is carried out in Xenopus
oocytes by microinjection of mutated mRNA and chemically
aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.
271:19991 (1996)). Within a third method, E. coli cells are
cultured in the absence of a natural amino acid that is to be
replaced (e.g., phenylalanine) and in the presence of the desired
non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the
protein in place of its natural counterpart. See, Koide et al.,
Biochem. 33:7470 (1994). Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in vitro
chemical modification. Chemical modification can be combined with
site-directed mutagenesis to further expand the range of
substitutions (Wynn and Richards, Protein Sci. 2:395 (1993)).
[0096] A limited number of non-conservative amino acids, amino
acids that are not encoded by the genetic code, non-naturally
occurring amino acids, and unnatural amino acids may be substituted
for IL-22RA amino acid residues.
[0097] Essential amino acids in the polypeptides of the present
invention can be identified according to 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. Nat'l 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 mutant molecules are tested for biological activity 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).
[0098] Although sequence analysis can be used to further define the
IL-22RA ligand binding region, amino acids that play a role in
IL-22RA binding activity (such as binding of IL-22RA to ligand
IL-22, or to an anti-IL-22RA antibody) can also be determined by
physical analysis of structure, as determined by such techniques as
nuclear magnetic resonance, crystallography, electron diffraction
or photoaffinity labeling, in conjunction with mutation of putative
contact site amino acids. See, for example, de Vos et al., Science
255:306 (1992), Smith et al., J. Mol. Biol. 224:899 (1992), and
Wlodaver et al., FEBS Lett. 309:59 (1992).
[0099] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53 (1988)) or
Bowie and Sauer (Proc. Nat'l Acad. Sci. USA 86:2152 (1989)).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner et
al., U.S. Pat. No. 5,223,409, Huse, international publication No.
WO 92/06204, and region-directed mutagenesis (Derbyshire et al.,
Gene 46:145 (1986), and Ner et al., DNA 7:127, (1988)). Moreover,
IL-22RA labeled with biotin or FITC can be used for expression
cloning of IL-22RA ligands.
[0100] Variants of the disclosed IL-22RA nucleotide and polypeptide
sequences can also be generated through DNA shuffling as disclosed
by Stemmer, Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci.
USA 91:10747 (1994), and international publication No. WO 97/20078.
Briefly, variant DNA molecules are generated by in vitro homologous
recombination by random fragmentation of a parent DNA followed by
reassembly using PCR, resulting in randomly introduced point
mutations. This technique can be modified by using a family of
parent DNA molecules, such as allelic variants or DNA molecules
from different species, to introduce additional variability into
the process. Selection or screening for the desired activity,
followed by additional iterations of mutagenesis and assay provides
for rapid "evolution" of sequences by selecting for desirable
mutations while simultaneously selecting against detrimental
changes.
[0101] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode biologically active polypeptides, or
polypeptides that bind with anti-IL-22RA antibodies, can be
recovered from the host cells and rapidly sequenced using modern
equipment. These methods allow the rapid determination of the
importance of individual amino acid residues in a polypeptide of
interest, and can be applied to polypeptides of unknown
structure.
[0102] The present invention also includes "functional fragments"
of IL-22RA polypeptides and nucleic acid molecules encoding such
functional fragments. Routine deletion analyses of nucleic acid
molecules can be performed to obtain functional fragments of a
nucleic acid molecule that encodes a IL-22RA polypeptide. As an
illustration, DNA molecules having the nucleotide sequence of SEQ
ID NO:1 can be digested with Bal31 nuclease to obtain a series of
nested deletions. The fragments are then inserted into expression
vectors in proper reading frame, and the expressed polypeptides are
isolated and tested for the ability to bind anti-IL-22RA
antibodies. One alternative to exonuclease digestion is to use
oligonucleotide-directed mutagenesis to introduce deletions or stop
codons to specify production of a desired fragment. Alternatively,
particular fragments of a IL-22RA gene can be synthesized using the
polymerase chain reaction.
[0103] This general approach is exemplified by studies on the
truncation at either or both termini of interferons have been
summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507
(1995). Moreover, standard techniques for functional analysis of
proteins are described by, for example, Treuter et al., Molec. Gen.
Genet. 240:113 (1993), Content et al., "Expression and preliminary
deletion analysis of the 42 kDa 2-5 A synthetase induced by human
interferon," in Biological Interferon Systems, Proceedings of
ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72
(Nijhoff 1987), Herschman, "The EGF Receptor," in Control of Animal
Cell Proliferation, Vol. 1, Boynton et al., (eds.) pages 169-199
(Academic Press 1985), Coumailleau et al., J. Biol. Chem. 270:29270
(1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi
et al., Biochem. Pharmacol 50:1295 (1995), and Meisel et al., Plant
Molec. Biol. 30:1 (1996).
[0104] Analysis of the particular sequences disclosed herein
provide a set of illustrative functional fragments presented in
Table 4. The nucleotides encoding additional human IL-22RA
functional variant domains described herein, not show in Table 4,
can be determined with reference to SEQ ID NO:1. Such functional
fragments include for example, the following nucleotide sequences
of SEQ ID NO:1: nucleotides 85-381, 206-717, and 85-717 of SEQ ID
NO:1 and corresponding amino acid sequences encoded thereby as
shown in SEQ ID NO:2 and SEQ ID NO:3 respectively.
TABLE-US-00004 TABLE 4 Amino acid residues Nucleotides IL-22RA
Feature (SEQ ID NO: 2) (SEQ ID NO: 1) First Ig Domain 18-116 85-381
Second Ig Domain 125-228 206-717 Both Ig Domains 18-228 85-717
[0105] The present invention also contemplates functional fragments
of a IL-22RA gene that have amino acid changes, compared with an
amino acid sequence disclosed herein. A variant IL-22RA gene can be
identified on the basis of structure by determining the level of
identity with disclosed nucleotide and amino acid sequences, as
discussed above. An alternative approach to identifying a variant
gene on the basis of structure is to determine whether a nucleic
acid molecule encoding a potential variant IL-22RA gene can
hybridize to a nucleic acid molecule comprising a nucleotide
sequence, such as SEQ ID NO:1.
[0106] The present invention also includes using functional
fragments of IL-22RA polypeptides, antigenic epitopes,
epitope-bearing portions of IL-22RA polypeptides, and nucleic acid
molecules that encode such functional fragments, antigenic
epitopes, epitope-bearing portions of IL-22RA polypeptides. Such
fragments are used to generate polypeptides for use in generating
antibodies and binding partners that bind, block, inhibit, reduce,
antagonize or neutralize activity of IL-22 or both IL-20 and IL-22.
A "functional" IL-22RA polypeptide or fragment thereof as defined
herein is characterized by its ability to block, inhibit, reduce,
antagonize or neutralize IL-20 or IL-22 inflammatory, 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-22RA antibody, cell, IL-20 or IL-22. As previously
described herein, IL-22RA is characterized by a class II cytokine
receptor structure and domains as described herein. Thus, the
present invention further contemplates using fusion proteins
encompassing: (a) polypeptide molecules comprising one or more of
the domains described above; and (b) functional fragments
comprising one or more of these domains. The other polypeptide
portion of the fusion protein may be contributed by another class
II cytokine receptor, such as IL-10R, IL-113R, IL-20RA, IL-20RB,
IL-10RB (CRF2-4), IL-22RA2, or by a non-native and/or an unrelated
secretory signal peptide that facilitates secretion of the fusion
protein.
[0107] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a IL-22RA
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0108] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides, antigenic
peptides, epitopes, and polypeptides of the present invention are
useful to raise antibodies that bind with the polypeptides
described herein, as well as to identify and screen anti-IL-22RA
monoclonal antibodies that are neutralizing, and that may bind,
block, inhibit, reduce, antagonize or neutralize the activity of
IL-22 and IL-20 (individually or together). Such neutralizing
monoclonal antibodies of the present invention can bind to an
IL-22RA antigenic epitope. Hopp/Woods hydrophilicity profiles can
be used to determine regions that have the most antigenic potential
within SEQ ID NO:3 (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. In IL-22RA
these regions can be determined by one of skill in the art.
Moreover, IL-22RA antigenic epitopes within SEQ ID NO:3 as
predicted by a Jameson-Wolf plot, e.g., using DNASTAR Protean
program (DNASTAR, Inc., Madison, Wis.) serve as preferred antigenic
epitopes, and can be determined by one of skill in the art. Such
antigenic epitopes include (1) amino acid residues 1 (Pro) to 6
(Asp) of SEQ ID NO:3; (2) amino acid residues 26 (Ser) to 32 (Pro)
of SEQ ID NO:3; (3) amino acid residues 41 (Lys) to 47 (Asp) of SEQ
ID NO:3; (4) amino acid residues 49 (Val) to 62 (Cys) of SEQ ID
NO:3; (5) amino acid residues 41 (Lys) to 62 (Cys) of SEQ ID NO:3;
(6) amino acid residues 84 (Ala) to 97 (Ser) of SEQ ID NO:3; (7)
amino acid residues 103 (Thr) to 108 (Asp) of SEQ ID NO:3; (8)
amino acid residues 130 (Arg) to 135 (His) of SEQ ID NO:3; (9)
amino acid residues 164 (Gly) to 166 (Lys) of SEQ ID NO:3; (10)
amino acid residues 175 (Tyr) to 179 (Glu) of SEQ ID NO:3; (11)
amino acid residues 193 (Lys) to 196 (Ala) of SEQ ID NO:3; (12)
amino acid residues 203 (Lys) to 209 (Thr) of SEQ ID NO:3.
Additional epitopes include the following peptides are potentially
generated from non-reduced full-length human IL-22RA cleaved with
CnBr: peptide 6 (SEQ ID NO:56), peptide 7 (SEQ ID NO:57); peptide 8
(SEQ ID NO:58); peptide 9 (SEQ ID NO:59); peptide 10 (SEQ ID
NO:60); and peptide 11 (SEQ ID NO:61). Cysteines are
disulfide-bonded, which results in a possible link between peptides
7 (SEQ ID NO:57) and 10 (SEQ ID NO:60. Specifically, SEQ ID NO:56
corresponds to amino acid residues 1 (Pro) to 92 (Met) of SEQ ID
NO:3; SEQ ID NO:57 corresponds to amino acid residues 93 (Thr) to
120 (Met) of SEQ ID NO:3, SEQ ID NO:58 corresponds to amino acid
residues 121 (Ile) to 160 (Met) of SEQ ID NO:3, SEQ ID NO:59
corresponds to amino acid residues 161 (His) to 185 (Met) of SEQ ID
NO:3, SEQ ID NO:60 corresponds to amino acid residues 186 (Ile) to
199 (Met) of SEQ ID NO:3 and SEQ ID NO:61 corresponds to amino acid
residues 200 (Cys) to 211 (Thr) of SEQ ID NO:3. In addition,
residues of SEQ ID NO:2 (and corresponding residues of SEQ ID NO:3)
that are important to ligand-receptor binding comprise Tyr-60, and
Phe-164, Tyr-93, Arg-112, Lys-210, and Glu-211 of SEQ ID NO:2 and
(and corresponding residues of SEQ ID NO:3). Moreover, primary
residues of SEQ ID NO:2 (and corresponding residues of SEQ ID NO:3)
that are important to direct ligand-receptor binding comprise
Tyr-60, and Phe-164 of SEQ ID NO:2 (and corresponding residues of
SEQ ID NO:3), and secondary residues comprise residues Tyr-93,
Arg-112, Lys-210, and Glu-211 of SEQ ID NO:2 and (and corresponding
residues of SEQ ID NO:3). In preferred embodiments, antigenic
epitopes to which neutralizing antibodies of the present invention
bind would contain residues of SEQ ID NO:2 (and corresponding
residues of SEQ ID NO:3) that are important to ligand-receptor
binding, for example, with IL-22RA and IL-20 or IL-22 (individually
or together).
[0109] Antigenic epitope-bearing peptides and polypeptides can
contain at least four to ten amino acids, at least ten to fifteen
amino acids, or about 15 to about 30 amino acids of an amino acid
sequence disclosed herein. Such epitope-bearing peptides and
polypeptides can be produced by fragmenting a IL-22RA polypeptide,
or by chemical peptide synthesis, as described herein. Moreover,
epitopes can be selected by phage display of random peptide
libraries (see, for example, Lane and Stephen, Curr. Opin. Immuno.
5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol 7:616
(1996)). Standard methods for identifying epitopes and producing
antibodies from small peptides that comprise an epitope are
described, for example, by Mole, "Epitope Mapping," in Methods in
Molecular Biology, Vol 10, Manson (ed.), pages 105-116 (The Humana
Press, Inc. 1992), Price, "Production and Characterization of
Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies:
Production, Engineering, and Clinical Application, Ritter and
Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), and
Coligan et al. (eds.), Current Protocols in Immunology, pages
9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons
1997).
[0110] For any IL-22RA polypeptide, including variants and fusion
proteins, one of ordinary skill in the art can readily generate a
fully degenerate polynucleotide sequence encoding that variant
using the information set forth in Tables 1 and 2 above. Moreover,
those of skill in the art can use standard software to devise
IL-22RA variants based upon the nucleotide and amino acid sequences
described herein.
5. Production of IL-22RA Polypeptides
[0111] The polypeptides of the present invention, including
full-length polypeptides; soluble monomeric, homodimeric,
heterodimeric and multimeric receptors; full-length receptors;
receptor fragments (e.g. ligand-binding fragments and antigenic
epitopes), functional fragments, and fusion proteins, can be
produced in recombinant host cells following conventional
techniques. To express a IL-22RA gene, a nucleic acid molecule
encoding the polypeptide must be operably linked to regulatory
sequences that control transcriptional expression in an expression
vector and then, introduced into a host cell. In addition to
transcriptional regulatory sequences, such as promoters and
enhancers, expression vectors can include translational regulatory
sequences and a marker gene which is suitable for selection of
cells that carry the expression vector.
[0112] Expression vectors that are suitable for production of a
foreign protein in eukaryotic cells typically contain (1)
prokaryotic DNA elements coding for a bacterial replication origin
and an antibiotic resistance marker to provide for the growth and
selection of the expression vector in a bacterial host; (2)
eukaryotic DNA elements that control initiation of transcription,
such as a promoter; and (3) DNA elements that control the
processing of transcripts, such as a transcription
termination/polyadenylation sequence. As discussed above,
expression vectors can also include nucleotide sequences encoding a
secretory sequence that directs the heterologous polypeptide into
the secretory pathway of a host cell. For example, an IL-22RA
expression vector may comprise a IL-22RA gene and a secretory
sequence derived from any secreted gene.
[0113] IL-22RA proteins of the present invention may be expressed
in mammalian cells. Examples of suitable mammalian host cells
include African green monkey kidney cells (Vero; ATCC CRL 1587),
human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster
kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314),
canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary
cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin et al., Som. Cell.
Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1; ATCC
CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;
ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC
CRL 1650) and murine embryonic cells (1H-3T3; ATCC CRL 1658).
[0114] For a mammalian host, the transcriptional and translational
regulatory signals may be derived from mammalian viral sources, for
example, adenovirus, bovine papilloma virus, simian virus, or the
like, in which the regulatory signals are associated with a
particular gene which has a high level of expression. Suitable
transcriptional and translational regulatory sequences also can be
obtained from mammalian genes, for example, actin, collagen,
myosin, and metallothionein genes.
[0115] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene (Hamer et al., J. Molec. Appl. Genet. 1:273
(1982)), the TK promoter of Herpes virus (McKnight, Cell 31:355
(1982)), the SV40 early promoter (Benoist et al., Nature 290:304
(1981)), the Rous sarcoma virus promoter (Gorman et al., Proc.
Nat'l Acad. Sci. USA 79:6777 (1982)), the cytomegalovirus promoter
(Foecking et al., Gene 45:101 (1980)), and the mouse mammary tumor
virus promoter (see, generally, Etcheverry, "Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein
Engineering Principles and Practice, Cleland et al. (eds.), pages
163-181 (John Wiley & Sons, Inc. 1996)).
[0116] Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA polymerase promoter, can be used to control
IL-22RA gene expression in mammalian cells if the prokaryotic
promoter is regulated by a eukaryotic promoter (Zhou et al., Mol.
Cell. Biol. 10:4529 (1990), and Kaufman et al., Nucl Acids Res.
19:4485 (1991)).
[0117] In certain embodiments, a DNA sequence encoding a IL-22RA
soluble receptor polypeptide, or a fragment of IL-22RA 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. Multiple
components of a soluble receptor complex can be co-transfected on
individual expression vectors or be contained in a single
expression vector. Such techniques of expressing multiple
components of protein complexes are well known in the art.
[0118] An expression vector can be introduced into host cells using
a variety of standard techniques including calcium phosphate
transfection, liposome-mediated transfection,
microprojectile-mediated delivery, electroporation, and the like.
The transfected cells can be selected and propagated to provide
recombinant host cells that comprise the expression vector stably
integrated in the host cell genome. Techniques for introducing
vectors into eukaryotic cells and techniques for selecting such
stable transformants using a dominant selectable marker are
described, for example, by Ausubel (1995) and by Murray (ed.), Gene
Transfer and Expression Protocols (Humana Press 1991).
[0119] For example, one suitable selectable marker is a gene that
provides resistance to the antibiotic neomycin. In this case,
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 suitable amplifiable selectable marker is
dihydrofolate reductase (DHFR), which confers resistance to
methotrexate. Other drug resistance genes (e.g., hygromycin
resistance, multi-drug resistance, puromycin acetyltransferase) can
also be used. Alternatively, 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.
[0120] IL-22RA polypeptides can also be produced by cultured
mammalian cells using a viral delivery system. Exemplary viruses
for this purpose include adenovirus, retroviruses, herpesvirus,
vaccinia virus and adeno-associated virus (AAV). Adenovirus, a
double-stranded DNA virus, is currently the best studied gene
transfer vector for delivery of heterologous nucleic acid (for a
review, see Becker et al., Meth. Cell Biol. 43:161 (1994), and
Douglas and Curiel, Science & Medicine 4.44 (1997)). Advantages
of the adenovirus system include the accommodation of relatively
large DNA inserts, the ability to grow to high-titer, the ability
to infect a broad range of mammalian cell types, and flexibility
that allows use with a large number of available vectors containing
different promoters.
[0121] By deleting portions of the adenovirus genome, larger
inserts (up to 7 kb) of heterologous DNA can be accommodated. These
inserts can be incorporated into the viral DNA by direct ligation
or by homologous recombination with a co-transfected plasmid. An
option is to delete the essential E1 gene from the viral vector,
which results in the inability to replicate unless the E1 gene is
provided by the host cell. Adenovirus vector-infected human 293
cells (ATCC Nos. CRL-1573, 45504, 45505), for example, can be grown
as adherent cells or in suspension culture at relatively high cell
density to produce significant amounts of protein (see Garnier et
al., Cytotechnol. 15:145 (1994)).
[0122] IL-22RA can also be expressed in other higher eukaryotic
cells, such as avian, fungal, insect, yeast, or plant cells. The
baculovirus system provides an efficient means to introduce cloned
IL-22RA genes into insect cells. Suitable expression vectors are
based upon the Autographa californica multiple nuclear polyhedrosis
virus (AcMNPV), and contain well-known promoters such as Drosophila
heat shock protein (hsp) 70 promoter, Autographa californica
nuclear polyhedrosis virus immediate-early gene promoter (ie-1) and
the delayed early 39K promoter, baculovirus p10 promoter, and the
Drosophila metallothionein promoter. A second method of making
recombinant baculovirus utilizes a transposon-based system
described by Luckow (Luckow, et al., J. Virol. 67:4566 (1993)).
This system, which utilizes transfer vectors, is sold in the
BAC-to-BAC kit (Life Technologies, Rockville, Md.). This system
utilizes a transfer vector, PFASTBAC (Life Technologies) containing
a Tn7 transposon to move the DNA encoding the IL-22RA polypeptide
into a baculovirus genome maintained in E. coli as a large plasmid
called a "bacmid." See, Hill-Perkins and Possee, J. Gen. Virol
71:971 (1990), Bonning, et al., J. Gen. Virol 75:1551 (1994), and
Chazenbalk, and Rapoport, J. Biol. Chem. 270:1543 (1995). 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
IL-22RA polypeptide, for example, a Glu-Glu epitope tag
(Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952 (1985)). Using
a technique known in the art, a transfer vector containing a
IL-22RA gene 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 then isolated using common techniques.
[0123] The illustrative PFASTBAC vector can be modified to a
considerable degree. For example, 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, for example,
Hill-Perkins and Possee, J. Gen. Virol 71:971 (1990), Bonning, et
al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk and Rapoport, J.
Biol. Chem. 270:1543 (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-22RA 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 Corporation; Carlsbad, Calif.), or
baculovirus gp67 (PharMingen: San Diego, Calif.) can be used in
constructs to replace the native IL-22RA secretory signal
sequence.
[0124] The recombinant virus or bacmid is used to transfect host
cells. Suitable insect host cells include cell lines derived from
IPLB-Sf-21, a Spodoptera frugiperda pupal ovarian cell line, such
as Sf9 (ATCC CRL 1711), Sf21AE, and Sf21 (Invitrogen Corporation;
San Diego, Calif.), as well as Drosophila Schneider-2 cells, and
the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
can be used to grow and to maintain the cells. Suitable media are
Sf900 II.TM. (Life Technologies) or ESF 921.TM. (Expression
Systems) for the Sf9 cells; and Ex-cellO405.TM. (JRH Biosciences,
Lenexa, Kans.) or Express FiveO.TM. (Life Technologies) for the T.
ni cells. When recombinant virus is used, the cells are typically
grown up from an inoculation density of approximately
2-5.times.10.sup.5 cells to a density of 1-2.times.10.sup.6 cells
at which time a recombinant viral stock is added at a multiplicity
of infection (MOI) of 0.1 to 10, more typically near 3.
[0125] Established techniques for producing recombinant proteins in
baculovirus systems are provided by Bailey et al., "Manipulation of
Baculovirus Vectors," in Methods in Molecular Biology, Volume 7:
Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168
(The Humana Press, Inc. 1991), by Patel et al., "The baculovirus
expression system," in DNA Cloning 2: Expression Systems, 2nd
Edition, Glover et al. (eds.), pages 205-244 (Oxford University
Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, by
Richardson (ed.), Baculovirus Expression Protocols (The Humana
Press, Inc. 1995), and by Lucknow, "Insect Cell Expression
Technology," in Protein Engineering: Principles and Practice,
Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc.
1996).
[0126] Fungal cells, including yeast cells, can also be used to
express the genes described herein. Yeast species of particular
interest in this regard include Saccharomyces cerevisiae, Pichia
pastoris, and Pichia methanolica. Suitable promoters for expression
in yeast include promoters from GAL1 (galactose), PGK
(phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1
(alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.
Many yeast cloning vectors have been designed and are readily
available. These vectors include YIp-based vectors, such as YIp5,
YRp vectors, such as YRp17, YEp vectors such as YEp 13 and YCp
vectors, such as YCp 19. 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 suitable 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. Additional 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.
[0127] 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 (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
chysogenum 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.
[0128] For example, the use of Pichia methanolica as host for the
production of recombinant proteins is disclosed by Raymond, U.S.
Pat. No. 5,716,808, Raymond, U.S. Pat. No. 5,736,383, Raymond et
al., Yeast 14:11-23 (1998), and in international publication Nos.
WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA
molecules for use in transforming P. methanolica will commonly be
prepared as double-stranded, circular plasmids, which are
preferably linearized prior to transformation. For polypeptide
production in P. methanolica, the promoter and terminator in the
plasmid can be that of a P. methanolica gene, such as a P.
methanolica alcohol utilization gene (AUG1 or AUG2). Other useful
promoters include those of the dihydroxyacetone synthase (DHAS),
formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. A suitable selectable
marker for use in Pichia methanolica is a P. methanolica ADE2 gene,
which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC
4.1.1.21), and which allows ade2 host cells to grow in the absence
of adenine. For large-scale, industrial processes where it is
desirable to minimize the use of methanol, host cells can be used
in which both methanol utilization genes (AUG1 and AUG2) are
deleted. For production of secreted proteins, host cells can be
deficient in vacuolar protease genes (PEP4 and PRB1).
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. P. methanolica cells can be transformed by
electroporation using an exponentially decaying, pulsed electric
field having a field strength of from 2.5 to 4.5 kV/cm, preferably
about 3.75 kV/cm, and a time constant (t) of from 1 to 40
milliseconds, most preferably about 20 milliseconds.
[0129] Expression vectors can also be introduced into plant
protoplasts, intact plant tissues, or isolated plant cells. Methods
for introducing expression vectors into plant tissue include the
direct infection or co-cultivation of plant tissue with
Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA
injection, electroporation, and the like. See, for example, Horsch
et al., Science 227:1229 (1985), Klein et al., Biotechnology 10:268
(1992), and Miki et al., "Procedures for Introducing Foreign DNA
into Plants," in Methods in Plant Molecular Biology and
Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,
1993).
[0130] Alternatively, IL-22RA genes can be expressed in prokaryotic
host cells. Suitable promoters that can be used to express IL-22RA
polypeptides in a prokaryotic host are well-known to those of skill
in the art and include promoters capable of recognizing the T4, T3,
Sp6 and T7 polymerases, the P.sub.R and P.sub.L promoters of
bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac,
lpp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B.
subtilis, the promoters of the bacterio-phages of Bacillus,
Streptomyces promoters, the int promoter of bacteriophage lambda,
the bla promoter of pBR322, and the CAT promoter of the
chloramphenicol acetyl transferase gene. Prokaryotic promoters have
been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson et
al., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins
1987), and by Ausubel et al. (1995).
[0131] Suitable prokaryotic hosts include E. coli and Bacillus
subtilus. Suitable strains of E. coli include BL21(DE3),
BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF',
DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109,
JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for
example, Brown (ed.), Molecular Biology Labfax (Academic Press
1991)). Suitable strains of Bacillus subtilus include BR151, YB886,
MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning
Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL
Press 1985)).
[0132] When expressing a IL-22RA polypeptide in bacteria such as E.
coli, the polypeptide may be retained in the cytoplasm, typically
as insoluble granules, or may be directed to the periplasmic space
by a bacterial secretion sequence. In the former case, the cells
are lysed, and the granules are recovered and denatured using, for
example, guanidine isothiocyanate or urea. The denatured
polypeptide can then be refolded and dimerized by diluting the
denaturant, such as by dialysis against a solution of urea and a
combination of reduced and oxidized glutathione, followed by
dialysis against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic space in a
soluble and functional form by disrupting the cells (by, for
example, sonication or osmotic shock) to release the contents of
the periplasmic space and recovering the protein, thereby obviating
the need for denaturation and refolding.
[0133] Methods for expressing proteins in prokaryotic hosts are
well-known to those of skill in the art (see, for example, Williams
et al., "Expression of foreign proteins in E. coli using plasmid
vectors and purification of specific polyclonal antibodies," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
page 15 (Oxford University Press 1995), Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc.
1995), and Georgiou, "Expression of Proteins in Bacteria," in
Protein Engineering: Principles and Practice, Cleland et al.
(eds.), page 101 (John Wiley & Sons, Inc. 1996)).
[0134] Standard methods for introducing expression vectors into
bacterial, yeast, insect, and plant cells are provided, for
example, by Ausubel (1995).
[0135] General methods for expressing and recovering foreign
protein produced by a mammalian cell system are provided by, for
example, Etcheverry, "Expression of Engineered Proteins in
Mammalian Cell Culture," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996).
Standard techniques for recovering protein produced by a bacterial
system is provided by, for example, Grisshammer et al.,
"Purification of over-produced proteins from E. coli cells," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
pages 59-92 (Oxford University Press 1995). Established methods for
isolating recombinant proteins from a baculovirus system are
described by Richardson (ed.), Baculovirus Expression Protocols
(The Humana Press, Inc. 1995).
[0136] As an alternative, polypeptides of the present invention can
be synthesized by exclusive solid phase synthesis, partial solid
phase methods, fragment condensation or classical solution
synthesis. These synthesis methods are well-known to those of skill
in the art (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149
(1963), Stewart et al., "Solid Phase Peptide Synthesis" (2nd
Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept.
Prot. 3:3 (1986), Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach (IRL Press 1989), Fields and Colowick,
"Solid-Phase Peptide Synthesis," Methods in Enzymology Volume 289
(Academic Press 1997), and Lloyd-Williams et al., Chemical
Approaches to the Synthesis of Peptides and Proteins (CRC Press,
Inc. 1997)). Variations in total chemical synthesis strategies,
such as "native chemical ligation" and "expressed protein ligation"
are also standard (see, for example, Dawson et al., Science 266:776
(1994), Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997),
Dawson, Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l
Acad. Sci. USA 95:6705 (1998), and Severinov and Muir, J. Biol.
Chem. 273:16205 (1998)).
[0137] Peptides and polypeptides of the present invention comprise
at least six, at least nine, or at least 15 contiguous amino acid
residues of SEQ ID NO:3. As an illustration, polypeptides can
comprise at least six, at least nine, or at least 15 contiguous
amino acid residues of SEQ ID NO:3. Within certain embodiments of
the invention, the polypeptides comprise 20, 30, 40, 50, 100, or
more contiguous residues of these amino acid sequences. Nucleic
acid molecules encoding such peptides and polypeptides are useful
as polymerase chain reaction primers and probes.
[0138] Moreover, IL-22RA polypeptides and fragments thereof can be
expressed as monomers, homodimers, heterodimers, or multimers
within higher eukaryotic cells. Such cells can be used to produce
IL-22RA monomeric, homodimeric, heterodimeric and multimeric
receptor polypeptides that comprise at least one IL-22RA
polypeptide ("IL-22RA-comprising receptors" or "IL-22RA-comprising
receptor polypeptides"), or can be used as assay cells in screening
systems. Within one aspect of the present invention, a polypeptide
of the present invention comprising the IL-22RA extracellular
domain is produced by a cultured cell, and the cell is used to
screen for ligands for the receptor, including the natural ligand,
IL-22, as well as agonists and antagonists of the natural ligand.
To summarize this approach, a cDNA or gene encoding the receptor is
combined with other genetic elements required for its expression
(e.g., a transcription promoter), and the resulting expression
vector is inserted into a host cell. Cells that express the DNA and
produce functional receptor are selected and used within a variety
of screening systems. Each component of the monomeric, homodimeric,
heterodimeric and multimeric receptor complex can be expressed in
the same cell. Moreover, the components of the monomeric,
homodimeric, heterodimeric and multimeric receptor complex can also
be fused to a transmembrane domain or other membrane fusion moiety
to allow complex assembly and screening of transfectants as
described above.
[0139] To assay the IL-20 and IL-22 antagonist polypeptides and
antibodies of the present invention, mammalian cells suitable for
use in expressing IL-22RA-comprising receptors or other receptors
known to bind IL-20 or IL-22 (e.g., cells expressing
IL-22RA/CRF2-4; and IL-20RA, IL-20RB, IL-22RA/IL-20RB, or
IL-20RA/IL-20RB) and transducing a receptor-mediated signal include
cells that express other receptor subunits that may form a
functional complex with IL-22RA (or IL-20RA). These subunits may
include those of the interferon receptor family or of other class
II or class I cytokine receptors, e.g., CRF2-4 (Genbank Accession
No. Z17227), IL-10R (Genbank Accession No.s U00672 and
NM.sub.--001558), IL-22RA (commonly owned U.S. Pat. No. 5,965,704),
zcytor7 (IL-20RA) (commonly owned U.S. Pat. No. 5,945,511),
IL-20RA/IL-20RB (WIPO Publication No. WO 01/46232), and IL-9R. It
is also preferred to use a cell from the same species as the
receptor to be expressed. Within a preferred embodiment, the cell
is dependent upon an exogenously supplied hematopoietic growth
factor for its proliferation. Preferred cell lines of this type are
the human TF-1 cell line (ATCC number CRL-2003) and the AML-193
cell line (ATCC number CRL-9589), which are GM-CSF-dependent human
leukemic cell lines and BaF3 (Palacios and Steinmetz, Cell 41:
727-734, (1985)) which is an IL-3 dependent murine pre-B cell line.
Other cell lines include BHK, COS-1 and CHO cells. Suitable host
cells can be engineered to produce the necessary receptor subunits
or other cellular component needed for the desired cellular
response. This approach is advantageous because cell lines can be
engineered to express receptor subunits from any species, thereby
overcoming potential limitations arising from species specificity.
Species orthologs of the human receptor cDNA can be cloned and used
within cell lines from the same species, such as a mouse cDNA in
the BaF3 cell line. Cell lines that are dependent upon one
hematopoietic growth factor, such as GM-CSF or IL-3, can thus be
engineered to become dependent upon another cytokine that acts
through the IL-22RA receptor, such as IL-22.
[0140] Cells expressing functional receptor are used within
screening assays. A variety of suitable assays are known in the
art. These assays are based on the detection of a biological
response in a target cell. One such assay is a cell proliferation
assay. Cells are cultured in the presence or absence of a test
compound, and cell proliferation is detected by, for example,
measuring incorporation of tritiated thymidine or by colorimetric
assay based on the metabolic breakdown of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
(Mosman, J. Immunol. Meth. 65: 55-63, (1983)). An alternative assay
format uses cells that are further engineered to express a reporter
gene. The reporter gene is linked to a promoter element that is
responsive to the receptor-linked pathway, and the assay detects
activation of transcription of the reporter gene. A preferred
promoter element in this regard is a serum response element, or
SRE. See, e.g., Shaw et al., Cell 56:563-572, (1989). A preferred
such reporter gene is a luciferase gene (de Wet et al., Mol Cell.
Biol. 7:725, (1987)). Expression of the luciferase gene is detected
by luminescence using methods known in the art (e.g., Baumgartner
et al., J. Biol. Chem. 269:29094-29101, (1994); Schenborn and
Goiffin, Promega.sub.--Notes 41:11, 1993). Luciferase activity
assay kits are commercially available from, for example, Promega
Corp., Madison, Wis. Target cell lines of this type can be used to
screen libraries of chemicals, cell-conditioned culture media,
fungal broths, soil samples, water samples, and the like. For
example, a bank of cell-conditioned media samples can be assayed on
a target cell to identify cells that produce ligand. Positive cells
are then used to produce a cDNA library in a mammalian expression
vector, which is divided into pools, transfected into host cells,
and expressed. Media samples from the transfected cells are then
assayed, with subsequent division of pools, re-transfection,
subculturing, and re-assay of positive cells to isolate a cloned
cDNA encoding the ligand.
[0141] Several IL-20 responsive cell lines are known in the art or
can be constructed, for example, the Baf3/DIRS1/cytoR11 cell line
(WIPO Publication No. WO 02/072607). Moreover several IL-22
responsive cell lines are known (Dumontier et al., J. Immunol.
164:1814-1819, 2000; Dumoutier, L. et al., Proc. Nat'l. Acad. Sci.
97:10144-10149, 2000; Xie M H et al., J. Biol. Chem. 275:
31335-31339, 2000; Kotenko S V et al., J. Biol. Chem.
276:2725-2732, 2001), as well as those that express the IL-22
receptor subunit IL-22RA. For example, the following cells are
responsive to IL-22: TK-10 (Xie M H et al., supra.) (human renal
carcinoma); SW480 (ATCC No. CCL-228) (human colon adenocarcinoma);
HepG2 (ATCC No. HB-8065) (human hepatoma); PC12 (ATCC No. CRL-1721)
(murine neuronal cell model; rat pheochromocytoma); and MES13 (ATCC
No. CRL-1927) (murine kidney mesangial cell line). In addition,
some cell lines express IL-22RA (IL-22 receptor) are also
candidates for responsive cell lines to IL-22: A549 (ATCC No.
CCL-185) (human lung carcinoma); G-361 (ATCC No. CRL-1424) (human
melanoma); and Caki-1 (ATCC No. HTB-46) (human renal carcinoma). In
addition, IL-22-responsive cell lines can be constructed, for
example, the Baf3/cytoR11/CRF2-4 cell line described herein (WIPO
Publication No. WO 02/12345). These cells can be used in assays to
assess the functionality of IL-22RA as an IL-20 or IL-22 antagonist
or anti-inflammatory factor.
[0142] An additional screening approach provided by the present
invention includes the use of hybrid receptor polypeptides. These
hybrid polypeptides fall into two general classes. Within the first
class, the intracellular domain of IL-22RA, is joined to the
ligand-binding domain of a second receptor. A second class of
hybrid receptor polypeptides comprise the extracellular
(ligand-binding) domain of IL-22RA (SEQ ID NO:3) with an
intracellular domain of a second receptor, preferably a
hematopoietic cytokine receptor, and a transmembrane domain. Hybrid
IL-22RA monomers, homodimers, heterodimers and multimers of the
present invention receptors of this second class are expressed in
cells known to be capable of responding to signals transduced by
the second receptor. Together, these two classes of hybrid
receptors enable the identification of a responsive cell type for
the development of an assay for detecting IL-22 or IL-20. Moreover,
such cells can be used in the presence of IL-22 or IL-20 to assay
the soluble receptor antagonists of the present invention in a
competition-type assay. In such assay, a decrease in the
proliferation or signal transduction activity of IL-22 or IL-20 in
the presence of a soluble receptor of the present invention
demonstrates antagonistic activity. Moreover IL-22RA-soluble
receptor binding assays, an cell-based assays, can also be used to
assess whether a soluble receptor binds, blocks, inhibits, reduces,
antagonizes or neutralizes IL-22 or IL-20 activity.
6. Production of IL-22RA Fusion Proteins and Conjugates
[0143] One general class of IL-22RA analogs are variants having an
amino acid sequence that is a mutation of the amino acid sequence
disclosed herein. Another general class of IL-22RA analogs is
provided by anti-idiotype antibodies, and fragments thereof, as
described below. Moreover, recombinant antibodies comprising
anti-idiotype variable domains can be used as analogs (see, for
example, Monfardini et al., Proc. Assoc. Am. Physicians 108:420
(1996)). Since the variable domains of anti-idiotype IL-22RA
antibodies mimic IL-22RA, these domains can provide IL-22RA binding
activity. Methods of producing anti-idiotypic catalytic antibodies
are known to those of skill in the art (see, for example, Joron et
al., Ann. NY Acad. Sci. 672:216 (1992), Friboulet et al., Appl.
Biochem. Biotechnol 47:229 (1994), and Avalle et al., Ann. NY Acad.
Sci. 864:118 (1998)).
[0144] Another approach to identifying IL-22RA analogs is provided
by the use of combinatorial libraries. Methods for constructing and
screening phage display and other combinatorial libraries are
provided, for example, by Kay et al., Phage Display of Peptides and
Proteins (Academic Press 1996), Verdine, U.S. Pat. No. 5,783,384,
Kay, et. al, U.S. Pat. No. 5,747,334, and Kauffman et al., U.S.
Pat. No. 5,723,323.
[0145] IL-22RA polypeptides have both in vivo and in vitro uses. As
an illustration, a soluble form of IL-22RA can be added to cell
culture medium to inhibit the effects of the IL-22RA ligand
produced by the cultured cells.
[0146] Fusion proteins of IL-22RA can be used to express IL-22RA in
a recombinant host, and to isolate the produced IL-22RA. As
described below, particular IL-22RA fusion proteins also have uses
in diagnosis and therapy. One type of fusion protein comprises a
peptide that guides a IL-22RA polypeptide from a recombinant host
cell. To direct a IL-22RA polypeptide into the secretory pathway of
a eukaryotic host cell, a secretory signal sequence (also known as
a signal peptide, a leader sequence, prepro sequence or pre
sequence) is provided in the IL-22RA expression vector. While the
secretory signal sequence may be derived from IL-22RA, a suitable
signal sequence may also be derived from another secreted protein
or synthesized de novo. The secretory signal sequence is operably
linked to a IL-22RA-encoding sequence such that 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 nucleotide sequence encoding the polypeptide of interest,
although certain secretory signal sequences may be positioned
elsewhere in the nucleotide 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).
[0147] Although the secretory signal sequence of IL-22RA or another
protein produced by mammalian cells (e.g., tissue-type plasminogen
activator signal sequence, as described, for example, in U.S. Pat.
No. 5,641,655) is useful for expression of IL-22RA in recombinant
mammalian hosts, a yeast signal sequence is preferred for
expression in yeast cells. Examples of suitable yeast signal
sequences are those derived from yeast mating phermone
.alpha.-factor (encoded by the MF.alpha.1 gene), invertase (encoded
by the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene).
See, for example, Romanos et al., "Expression of Cloned Genes in
Yeast," in DNA Cloning 2: A Practical Approach, 2.sup.nd Edition,
Glover and Hames (eds.), pages 123-167 (Oxford University Press
1995).
[0148] IL-22RA soluble receptor polypeptides can be prepared by
expressing a truncated DNA encoding the extracellular domain, for
example, a polypeptide which contains SEQ ID NO:3, or the
corresponding region of a non-human receptor. It is preferred that
the extracellular domain polypeptides be prepared in a form
substantially free of transmembrane and intracellular polypeptide
segments. To direct the export of the receptor domain from the host
cell, the receptor DNA is linked to a second DNA segment encoding a
secretory peptide, such as a t-PA secretory peptide. To facilitate
purification of the secreted receptor domain, a C-terminal
extension, such as a poly-histidine tag, substance P, Flag.TM.
peptide (Hopp et al., Biotechnology 6:1204-1210, (1988); available
from Eastman Kodak Co., New Haven, Conn.) or another polypeptide or
protein for which an antibody or other specific binding agent is
available, can be fused to the receptor polypeptide. Moreover,
IL-22RA antigenic epitopes from the extracellular cytokine binding
domains are also prepared as described above.
[0149] In an alternative approach, a receptor extracellular domain
of IL-22RA or other class I or II cytokine receptor component can
be expressed as a fusion with immunoglobulin heavy chain constant
regions, typically an F.sub.c fragment, which contains two constant
region domains and a hinge region but lacks the variable region
(See, Sledziewski, A Z et al., U.S. Pat. Nos. 6,018,026 and
5,750,375). The soluble IL-22RA polypeptides of the present
invention include such fusions. One such fusion is shown in SEQ ID
NO:4. Such fusions are typically secreted as multimeric molecules
wherein the Fc portions are disulfide bonded to each other and two
receptor polypeptides are arrayed in closed proximity to each
other. Fusions of this type can be used to affinity purify the
cognate ligand from solution, as an in vitro assay tool, to block,
inhibit or reduce signals in vitro by specifically titrating out
ligand, and as antagonists in vivo by administering them
parenterally to bind circulating ligand and clear it from the
circulation. To purify ligand, a IL-22RA-Ig chimera is added to a
sample containing the ligand (e.g., cell-conditioned culture media
or tissue extracts) under conditions that facilitate
receptor-ligand binding (typically near-physiological temperature,
pH, and ionic strength). The chimera-ligand complex is then
separated by the mixture using protein A, which is immobilized on a
solid support (e.g., insoluble resin beads). The ligand is then
eluted using conventional chemical techniques, such as with a salt
or pH gradient. In the alternative, the chimera itself can be bound
to a solid support, with binding and elution carried out as above.
The chimeras may be used in vivo to regulate inflammatory responses
including acute phase responses such as serum amyloid A (SAA),
C-reactive protein (CRP), and the like. Chimeras with high binding
affinity are administered parenterally (e.g., by intramuscular,
subcutaneous or intravenous injection). Circulating molecules bind
ligand and are cleared from circulation by normal physiological
processes. For use in assays, the chimeras are bound to a support
via the F.sub.c region and used in an ELISA format.
[0150] To assist in isolating anti-IL-22RA and binding partners of
the present invention, an assay system that uses a ligand-binding
receptor (or an antibody, one member of a
complement/anti-complement pair) or a binding fragment thereof, and
a commercially available biosensor instrument (BIAcore, Pharmacia
Biosensor, Piscataway, N.J.) may be advantageously employed. Such
receptor, antibody, member of a complement/anti-complement pair or
fragment is immobilized onto the surface of a receptor chip. Use of
this instrument is disclosed by Karlsson, J. Immunol. Methods
145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol.
234:554-63, 1993. A receptor, antibody, member or fragment is
covalently attached, using amine or sulfhydryl chemistry, to
dextran fibers that are attached to gold film within the flow cell.
A test sample is passed through the cell. If a ligand, epitope, or
opposite member of the complement/anti-complement pair is present
in the sample, it will bind to the immobilized receptor, antibody
or member, respectively, causing a change in the refractive index
of the medium, which is detected as a change in surface plasmon
resonance of the gold film. This system allows the determination of
on- and off-rates, from which binding affinity can be calculated,
and assessment of stoichiometry of binding. Alternatively,
ligand/receptor binding can be analyzed using SELDI.TM. technology
(Ciphergen, Inc., Palo Alto, Calif.). Moreover, BIACORE technology,
described above, can be used to be used in competition experiments
to determine if different monoclonal antibodies bind the same or
different epitopes on the IL-22RA polypeptide, and as such, be used
to aid in epitope mapping of neutralizing antibodies of the present
invention that bind, block, inhibit, reduce, antagonize or
neutralize IL-22 or both IL-20 and IL-22.
[0151] Ligand-binding receptor polypeptides can also be used within
other assay systems known in the art. Such systems include
Scatchard analysis for determination of binding affinity (see
Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetric
assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et
al., Science 245:821-25, 1991).
[0152] The present invention further provides a variety of other
polypeptide fusions and related multimeric proteins comprising one
or more polypeptide fusions. For example, a soluble IL-22RA
receptor can be prepared as a fusion to a dimerizing protein as
disclosed in U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred
dimerizing proteins in this regard include immunoglobulin constant
region domains, e.g., IgG.gamma.1, and the human .kappa. light
chain. Immunoglobulin-soluble IL-22RA fusions can be expressed in
genetically engineered cells to produce a variety of multimeric
IL-22RA receptor analogs. Auxiliary domains can be fused to soluble
IL-22RA receptor to target them to specific cells, tissues, or
macromolecules (e.g., collagen, or cells expressing the IL-22RA
ligands, IL-22 or IL-20). A IL-22RA polypeptide can be fused to two
or more moieties, such as an affinity tag for purification and a
targeting domain. Polypeptide fusions can also comprise one or more
cleavage sites, particularly between domains. See, Tuan et al.,
Connective Tissue Research 34:1-9, 1996.
[0153] In bacterial cells, it is often desirable to express a
heterologous protein as a fusion protein to decrease toxicity,
increase stability, and to enhance recovery of the expressed
protein. For example, IL-22RA can be expressed as a fusion protein
comprising a glutathione S-transferase polypeptide. Glutathione
S-transferease fusion proteins are typically soluble, and easily
purifiable from E. coli lysates on immobilized glutathione columns.
In similar approaches, a IL-22RA fusion protein comprising a
maltose binding protein polypeptide can be isolated with an amylose
resin column, while a fusion protein comprising the C-terminal end
of a truncated Protein A gene can be purified using IgG-Sepharose.
Established techniques for expressing a heterologous polypeptide as
a fusion protein in a bacterial cell are described, for example, by
Williams et al., "Expression of Foreign Proteins in E. coli Using
Plasmid Vectors and Purification of Specific Polyclonal
Antibodies," in DNA Cloning 2: A Practical Approach, 2.sup.nd
Edition, Glover and Hames (Eds.), pages 15-58 (Oxford University
Press 1995). In addition, commercially available expression systems
are available. For example, the PINPOINT Xa protein purification
system (Promega Corporation; Madison, Wis.) provides a method for
isolating a fusion protein comprising a polypeptide that becomes
biotinylated during expression with a resin that comprises
avidin.
[0154] Peptide tags that are useful for isolating heterologous
polypeptides expressed by either prokaryotic or eukaryotic cells
include polyHistidine tags (which have an affinity for
nickel-chelating resin), c-myc tags, calmodulin binding protein
(isolated with calmodulin affinity chromatography), substance P,
the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu
tag, and the FLAG tag (which binds with anti-FLAG antibodies). See,
for example, Luo et al., Arch. Biochem. Biophys. 329:215 (1996),
Morganti et al., Biotechnol. Appl. Biochem. 23:67 (1996), and Zheng
et al., Gene 186:55 (1997). Nucleic acid molecules encoding such
peptide tags are available, for example, from Sigma-Aldrich
Corporation (St. Louis, Mo.).
[0155] Another form of fusion protein comprises a IL-22RA
polypeptide and an immunoglobulin heavy chain constant region,
typically an F.sub.c fragment, which contains two or three constant
region domains and a hinge region but lacks the variable region. As
an illustration, Chang et al., U.S. Pat. No. 5,723,125, describe a
fusion protein comprising a human interferon and a human
immunoglobulin Fc fragment. The C-terminal of the interferon is
linked to the N-terminal of the Fc fragment by a peptide linker
moiety. An example of a peptide linker is a peptide comprising
primarily a T cell inert sequence, which is immunologically inert.
An exemplary peptide linker has the amino acid sequence: GGSGG
SGGGG SGGGG S (SEQ ID NO:9). In this fusion protein, an
illustrative Fc moiety is a human .gamma.4 chain, which is stable
in solution and has little or no complement activating activity.
Accordingly, the present invention contemplates a IL-22RA fusion
protein that comprises a IL-22RA moiety and a human Fc fragment,
wherein the C-terminus of the IL-22RA moiety is attached to the
N-terminus of the Fc fragment via a peptide linker, such as a
peptide comprising the amino acid sequence of SEQ ID NO:4. The
IL-22RA moiety can be a IL-22RA molecule or a fragment thereof. For
example, a fusion protein can comprise the amino acid of SEQ ID
NO:3 and an Fc fragment (e.g., a human Fc fragment) (SEQ ID
NO:4).
[0156] In another variation, a IL-22RA fusion protein comprises an
IgG sequence, a IL-22RA moiety covalently joined to the
aminoterminal end of the IgG sequence, and a signal peptide that is
covalently joined to the aminoterminal of the IL-22RA moiety,
wherein the IgG sequence consists of the following elements in the
following order: a hinge region, a CH.sub.2 domain, and a CH.sub.3
domain. Accordingly, the IgG sequence lacks a CH.sub.1 domain. The
IL-22RA moiety displays a IL-22RA activity, as described herein,
such as the ability to bind with a IL-22RA ligand. This general
approach to producing fusion proteins that comprise both antibody
and nonantibody portions has been described by LaRochelle et al.,
EP 742830 (WO 95/21258).
[0157] Fusion proteins comprising a IL-22RA moiety and an Fc moiety
can be used, for example, as an in vitro assay tool. For example,
the presence of a IL-22RA ligand in a biological sample can be
detected using a IL-22RA-immunoglobulin fusion protein, in which
the IL-22RA moiety is used to bind the ligand, and a macromolecule,
such as Protein A or anti-Fc antibody, is used to bind the fusion
protein to a solid support. Such systems can be used to identify
agonists and antagonists that interfere with the binding of a
IL-22RA ligands, e.g., IL-22 or both IL-20 and IL-22, to their
receptor.
[0158] Other examples of antibody fusion proteins include
polypeptides that comprise an antigen-binding domain and a IL-22RA
fragment that contains a IL-22RA extracellular domain. Such
molecules can be used to target particular tissues for the benefit
of IL-22RA binding activity.
[0159] The present invention further provides a variety of other
polypeptide fusions. For example, part or all of a domain(s)
conferring a biological function can be swapped between IL-22RA of
the present invention with the functionally equivalent domain(s)
from another member of the cytokine receptor family. Polypeptide
fusions can be expressed in recombinant host cells to produce a
variety of IL-22RA fusion analogs. A IL-22RA polypeptide can be
fused to two or more moieties or domains, such as an affinity tag
for purification and a targeting domain. Polypeptide fusions can
also comprise one or more cleavage sites, particularly between
domains. See, for example, Tuan et al., Connective Tissue Research
34:1 (1996).
[0160] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. General methods for
enzymatic and chemical cleavage of fusion proteins are described,
for example, by Ausubel (1995) at pages 16-19 to 16-25.
[0161] IL-22RA binding domains can be further characterized by
physical analysis of structure, as determined by such techniques as
nuclear magnetic resonance, crystallography, electron diffraction
or photoaffinity labeling, in conjunction with mutation of putative
contact site amino acids of IL-22RA ligand agonists. See, for
example, de Vos et al., Science 255:306 (1992), Smith et al., J.
Mol. Biol. 224:899 (1992), and Wlodaver et al., FEBS Lett. 309:59
(1992).
[0162] The present invention also contemplates chemically modified
IL-22RA compositions, in which a IL-22RA polypeptide is linked with
a polymer. Illustrative IL-22RA polypeptides are soluble
polypeptides that lack a functional transmembrane domain, such as a
polypeptide consisting of amino acid residues SEQ ID NO:3.
Typically, the polymer is water soluble so that the IL-22RA
conjugate does not precipitate in an aqueous environment, such as a
physiological environment. An example of a suitable polymer is one
that has been modified to have a single reactive group, such as an
active ester for acylation, or an aldehyde for alkylation. In this
way, the degree of polymerization can be controlled. An example of
a reactive aldehyde is polyethylene glycol propionaldehyde, or
mono-(C1-C10) alkoxy, or aryloxy derivatives thereof (see, for
example, Harris, et al., U.S. Pat. No. 5,252,714). The polymer may
be branched or unbranched. Moreover, a mixture of polymers can be
used to produce IL-22RA conjugates.
[0163] IL-22RA conjugates used for therapy can comprise
pharmaceutically acceptable water-soluble polymer moieties.
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, PEG
propionaldehyde, bis-succinimidyl carbonate PEG, propylene glycol
homopolymers, a polypropylene oxide/ethylene oxide co-polymer,
polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol,
dextran, cellulose, or other carbohydrate-based polymers. Suitable
PEG may have a molecular weight from about 600 to about 60,000,
including, for example, 5,000, 12,000, 20,000 and 25,000. A IL-22RA
conjugate can also comprise a mixture of such water-soluble
polymers.
[0164] One example of a IL-22RA conjugate comprises a IL-22RA
moiety and a polyalkyl oxide moiety attached to the N-terminus of
the IL-22RA moiety. PEG is one suitable polyalkyl oxide. As an
illustration, IL-22RA can be modified with PEG, a process known as
"PEGylation." PEGylation of IL-22RA 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-22RA
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).
[0165] PEGylation by acylation typically requires reacting an
active ester derivative of PEG with a IL-22RA 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-22RA and a water soluble
polymer: amide, carbamate, urethane, and the like. Methods for
preparing PEGylated IL-22RA by acylation will typically comprise
the steps of (a) reacting a IL-22RA 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-22RA, 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-22RA, the greater the percentage of polyPEGylated IL-22RA
product.
[0166] The product of PEGylation by acylation is typically a
polyPEGylated IL-22RA product, wherein the lysine .epsilon.-amino
groups are PEGylated via an acyl linking group. An example of a
connecting linkage is an amide. Typically, the resulting IL-22RA
will be at least 95% mono-, di-, or tri-pegylated, although some
species with higher degrees of PEGylation may be formed depending
upon the reaction conditions. PEGylated species can be separated
from unconjugated IL-22RA polypeptides using standard purification
methods, such as dialysis, ultrafiltration, ion exchange
chromatography, affinity chromatography, and the like.
[0167] PEGylation by alkylation generally involves reacting a
terminal aldehyde derivative of PEG with IL-22RA in the presence of
a reducing agent. PEG groups can be attached to the polypeptide via
a --CH.sub.2--NH group.
[0168] Moreover, anti-IL-22RA antibodies or antibody fragments of
the present invention can be PEGylated using methods in the art and
described herein.
[0169] 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. The
present invention provides a substantially homogenous preparation
of IL-22RA monopolymer conjugates.
[0170] Reductive alkylation to produce a substantially homogenous
population of monopolymer IL-22RA conjugate molecule can comprise
the steps of: (a) reacting a IL-22RA 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-22RA, and (b) obtaining the reaction
product(s). The reducing agent used for reductive alkylation should
be stable in aqueous solution and able to reduce only the Schiff
base formed in the initial process of reductive alkylation.
Illustrative reducing agents include sodium borohydride, sodium
cyanoborohydride, dimethylamine borane, trimethylamine borane, and
pyridine borane.
[0171] For a substantially homogenous population of monopolymer
IL-22RA 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-22RA. 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-22RA need not be as
large because more reactive groups are available. Typically, the pH
will fall within the range of 3 to 9, or 3 to 6. This method can be
employed for making IL-22RA-comprising homodimeric, heterodimeric
or multimeric soluble receptor conjugates.
[0172] 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 25 kDa. The molar ratio of
water-soluble polymer to IL-22RA will generally be in the range of
1:1 to 100:1. Typically, the molar ratio of water-soluble polymer
to IL-22RA will be 1:1 to 20:1 for polyPEGylation, and 1:1 to 5:1
for monoPEGylation.
[0173] General methods for producing conjugates comprising a
polypeptide 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)). This method can be employed for making
IL-22RA-comprising homodimeric, heterodimeric or multimeric soluble
receptor conjugates.
[0174] The present invention contemplates compositions comprising a
peptide or polypeptide, such as a soluble receptor or antibody
described herein. Such compositions can further comprise a carrier.
The carrier can be a conventional organic or inorganic carrier.
Examples of carriers include water, buffer solution, alcohol,
propylene glycol, macrogol, sesame oil, corn oil, and the like.
7. Isolation of IL-22RA Polypeptides
[0175] The polypeptides of the present invention can be purified to
at least about 80% purity, to at least about 90% purity, to at
least about 95% purity, or greater than 95%, such as 96%, 97%, 98%,
or greater than 99% purity with respect to contaminating
macromolecules, particularly other proteins and nucleic acids, and
free of infectious and pyrogenic agents. The polypeptides of the
present invention may also be purified to a pharmaceutically pure
state, which is greater than 99.9% pure. In certain preparations,
purified polypeptide is substantially free of other polypeptides,
particularly other polypeptides of animal origin.
[0176] Fractionation and/or conventional purification methods can
be used to obtain preparations of IL-22RA purified from natural
sources (e.g., human tissue sources), synthetic IL-22RA
polypeptides, and recombinant IL-22RA polypeptides and fusion
IL-22RA polypeptides purified from recombinant host cells. In
general, ammonium sulfate precipitation and acid or chaotrope
extraction may be used for fractionation of samples. Exemplary
purification steps may include hydroxyapatite, size exclusion, FPLC
and reverse-phase high performance liquid chromatography. Suitable
chromatographic media include derivatized dextrans, agarose,
cellulose, polyacrylamide, specialty silicas, and the like. PEI,
DEAE, QAE and Q derivatives are suitable. Exemplary chromatographic
media include those media derivatized with phenyl, butyl, or octyl
groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl
650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia)
and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso
Haas) and the like. Suitable solid supports include glass beads,
silica-based resins, cellulosic resins, agarose beads, cross-linked
agarose beads, polystyrene beads, cross-linked polyacrylamide
resins and the like that are insoluble under the conditions in
which they are to be used. These supports may be modified with
reactive groups that allow attachment of proteins by amino groups,
carboxyl groups, sulfhydryl groups, hydroxyl groups and/or
carbohydrate moieties.
[0177] Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl and amino
derivatives for carbodiimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers. Selection of a particular
method for polypeptide isolation and purification is a matter of
routine design and is determined in part by the properties of the
chosen support. See, for example, Affinity Chromatography:
Principles & Methods (Pharmacia LKB Biotechnology 1988), and
Doonan, Protein Purification Protocols (The Humana Press 1996).
[0178] Additional variations in IL-22RA isolation and purification
can be devised by those of skill in the art. For example,
anti-IL-22RA antibodies, obtained as described below, can be used
to isolate large quantities of protein by immunoaffinity
purification.
[0179] The polypeptides of the present invention can also be
isolated by exploitation of particular properties. For example,
immobilized metal ion adsorption (IMAC) chromatography can be used
to purify histidine-rich proteins, including those comprising
polyhistidine tags. Briefly, a gel is first charged with divalent
metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1
(1985)). Histidine-rich proteins will be adsorbed to this matrix
with differing affinities, depending upon the metal ion used, and
will be eluted by competitive elution, lowering the pH, or use of
strong chelating agents. Other methods of purification include
purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (M. Deutscher,
(ed.), Meth. Enzymo. 182:529 (1990)). Within additional embodiments
of the invention, a fusion of the polypeptide of interest and an
affinity tag (e.g., maltose-binding protein, an immunoglobulin
domain) may be constructed to facilitate purification. Moreover,
the ligand-binding properties of IL-22RA extracellular domain can
be exploited for purification, for example, of IL-22RA-comprising
soluble receptors; for example, by using affinity chromatography
wherein IL-22 ligand is bound to a column and the
IL-22RA-comprising receptor is bound and subsequently eluted using
standard chromatography methods.
[0180] IL-22RA polypeptides or fragments thereof may also be
prepared through chemical synthesis, as described above. IL-22RA
polypeptides may be monomers or multimers; glycosylated or
non-glycosylated; PEGylated or non-PEGylated; and may or may not
include an initial methionine amino acid residue.
8. Production of Antibodies to IL-22RA Proteins
[0181] Antibodies to IL-22RA can be obtained, for example, using
the product of a IL-22RA expression vector or IL-22RA isolated from
a natural source as an antigen. Particularly useful anti-IL-22RA
antibodies "bind specifically" with IL-22RA. Antibodies are
considered to be specifically binding if the antibodies exhibit at
least one of the following two properties: (1) antibodies bind to
IL-22RA with a threshold level of binding activity, and (2)
antibodies do not significantly cross-react with polypeptides
related to IL-22RA.
[0182] With regard to the first characteristic, antibodies
specifically bind if they bind to a IL-22RA polypeptide, peptide or
epitope with a binding affinity (K.sub.a) of 10.sup.6 M.sup.-1 or
greater, preferably 10.sup.7 M.sup.-1 or greater, more preferably
10.sup.8 M.sup.-1 or greater, and most preferably 10.sup.9 M.sup.-1
or greater. The binding affinity of an antibody can be readily
determined by one of ordinary skill in the art, for example, by
Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660 (1949)).
With regard to the second characteristic, antibodies do not
significantly cross-react with related polypeptide molecules, for
example, if they detect IL-22RA, but not presently known
polypeptides using a standard Western blot analysis. Examples of
known related polypeptides include known cytokine receptors.
[0183] Anti-IL-22RA antibodies can be produced using antigenic
IL-22RA epitope-bearing peptides and polypeptides. Antigenic
epitope-bearing peptides and polypeptides of the present invention
contain a sequence of at least nine, or between 15 to about 30
amino acids contained within SEQ ID NO:3 or another amino acid
sequence disclosed herein. However, peptides or polypeptides
comprising a larger portion of an amino acid sequence of the
invention, containing from 30 to 50 amino acids, or any length up
to and including the entire amino acid sequence of a polypeptide of
the invention, also are useful for inducing antibodies that bind
with IL-22RA. It is desirable that the amino acid sequence of the
epitope-bearing peptide is selected to provide substantial
solubility in aqueous solvents (i.e., the sequence includes
relatively hydrophilic residues, while hydrophobic residues are
typically avoided). Moreover, amino acid sequences containing
proline residues may be also be desirable for antibody
production.
[0184] As an illustration, potential antigenic sites in IL-22RA
were identified using the Jameson-Wolf method, Jameson and Wolf,
CABIOS 4:181, (1988), as implemented by the PROTEAN program
(version 3.14) of LASERGENE (DNASTAR; Madison, Wis.). Default
parameters were used in this analysis.
[0185] The Jameson-Wolf method predicts potential antigenic
determinants by combining six major subroutines for protein
structural prediction. Briefly, the Hopp-Woods method, Hopp et al.,
Proc. Nat'l Acad. Sci. USA 78:3824 (1981), was first used to
identify amino acid sequences representing areas of greatest local
hydrophilicity (parameter: seven residues averaged). In the second
step, Emini's method, Emini et al., J. Virology 55:836 (1985), was
used to calculate surface probabilities (parameter: surface
decision threshold (0.6)=1). Third, the Karplus-Schultz method,
Karplus and Schultz, Naturwissenschaften 72:212 (1985), was used to
predict backbone chain flexibility (parameter: flexibility
threshold (0.2)=1). In the fourth and fifth steps of the analysis,
secondary structure predictions were applied to the data using the
methods of Chou-Fasman, Chou, "Prediction of Protein Structural
Classes from Amino Acid Composition," in Prediction of Protein
Structure and the Principles of Protein Conformation, Fasman (ed.),
pages 549-586 (Plenum Press 1990), and Garnier-Robson, Garnier et
al., J. Mol. Biol. 120:97 (1978) (Chou-Fasman parameters:
conformation table=64 proteins; .alpha. region threshold=103;
.beta. region threshold=105; Garnier-Robson parameters: .alpha. and
.beta. decision constants=0). In the sixth subroutine, flexibility
parameters and hydropathy/solvent accessibility factors were
combined to determine a surface contour value, designated as the
"antigenic index." Finally, a peak broadening function was applied
to the antigenic index, which broadens major surface peaks by
adding 20, 40, 60, or 80% of the respective peak value to account
for additional free energy derived from the mobility of surface
regions relative to interior regions. This calculation was not
applied, however, to any major peak that resides in a helical
region, since helical regions tend to be less flexible.
[0186] The results of this analysis indicated that the following
amino acid sequences of SEQ ID NO:3 would provide suitable
antigenic peptides: Hopp/Woods hydrophilicity profiles can be used
to determine regions that have the most antigenic potential within
SEQ ID NO:3 (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. Moreover, IL-22RA antigenic
epitopes within SEQ ID NO:3 as predicted by a Jameson-Wolf plot,
e.g., using DNASTAR Protean program (DNASTAR, Inc., Madison, Wis.)
serve as preferred antigenic epitopes, and can be determined by one
of skill in the art. Such antigenic epitopes include (1) amino acid
residues 1 (Pro) to 6 (Asp) of SEQ ID NO:3; (2) amino acid residues
26 (Ser) to 32 (Pro) of SEQ ID NO:3; (3) amino acid residues 41
(Lys) to 47 (Asp) of SEQ ID NO:3; (4) amino acid residues 49 (Val)
to 62 (Cys) of SEQ ID NO:3; (5) amino acid residues 41 (Lys) to 62
(Cys) of SEQ ID NO:3; (6) amino acid residues 84 (Ala) to 97 (Ser)
of SEQ ID NO:3; (7) amino acid residues 103 (Thr) to 108 (Asp) of
SEQ ID NO:3; (8) amino acid residues 130 (Arg) to 135 (His) of SEQ
ID NO:3; (9) amino acid residues 164 (Gly) to 166 (Lys) of SEQ ID
NO:3; (10) amino acid residues 175 (Tyr) to 179 (Glu) of SEQ ID
NO:3; (11) amino acid residues 193 (Lys) to 196 (Ala) of SEQ ID
NO:3; (12) amino acid residues 203 (Lys) to 209 (Thr) of SEQ ID
NO:3. The present invention contemplates the use of any one of
antigenic peptides 1 to 12 to generate antibodies to IL-22RA or as
a tool to screen or identify neutralizing monoclonal antibodies of
the present invention. The present invention also contemplates
polypeptides comprising at least one of antigenic peptides 1 to 10.
The present invention contemplates the use of any antigenic
peptides or epitopes described herein to generate antibodies to
IL-22RA, as well as to identify and screen anti-IL-22RA monoclonal
antibodies that are neutralizing, and that may bind, block,
inhibit, reduce, antagonize or neutralize the activity of IL-22 and
IL-20 (individually or together).
[0187] Moreover, suitable antigens also include the IL-22RA
polypeptides comprising a IL-22RA cytokine binding, or
extracellular domain disclosed above in combination with another
class I or II cytokine extracellular domain, such as those that
form soluble IL-22RA heterodimeric or multimeric polypeptides, such
as soluble IL-22RA/CRF2-4, IL-22RA/zcytor11, IL-22RA/zcytor7, and
the like.
[0188] Polyclonal antibodies to recombinant IL-22RA protein or to
IL-22RA isolated from natural sources can be prepared using methods
well-known to those of skill in the art. See, for example, Green et
al., "Production of Polyclonal Antisera," in Immunochemical
Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and
Williams et al., "Expression of foreign proteins in E. coli using
plasmid vectors and purification of specific polyclonal
antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition,
Glover et al. (eds.), page 15 (Oxford University Press 1995). The
immunogenicity of a IL-22RA polypeptide can be increased through
the use of an adjuvant, such as alum (aluminum hydroxide) or
Freund's complete or incomplete adjuvant. Polypeptides useful for
immunization also include fusion polypeptides, such as fusions of
IL-22RA or a portion thereof with an immunoglobulin polypeptide or
with maltose binding protein. The polypeptide immunogen may be a
full-length molecule or a portion thereof. If the polypeptide
portion is "hapten-like," such portion may be advantageously joined
or linked to a macromolecular carrier (such as keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for
immunization.
[0189] Although polyclonal antibodies are typically raised in
animals such as horses, cows, dogs, chicken, rats, mice, rabbits,
guinea pigs, goats, or sheep, an anti-IL-22RA antibody of the
present invention may also be derived from a subhuman primate
antibody. General techniques for raising diagnostically and
therapeutically useful antibodies in baboons may be found, for
example, in Goldenberg et al., international patent publication No.
WO 91/11465, and in Losman et al., Int. J. Cancer 46:310
(1990).
[0190] Alternatively, monoclonal anti-IL-22RA antibodies can be
generated. Rodent mono-clonal antibodies to specific antigens may
be obtained by methods known to those skilled in the art (see, for
example, Kohler et al., Nature 256:495 (1975), Coligan et al.
(eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991) ["Coligan"], Picksley et al.,
"Production of monoclonal antibodies against proteins expressed in
E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover
et al. (eds.), page 93 (Oxford University Press 1995)).
[0191] Briefly, monoclonal antibodies can be obtained by injecting
mice with a composition comprising a IL-22RA gene product,
verifying the presence of antibody production by removing a serum
sample, removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0192] In addition, an anti-IL-22RA antibody of the present
invention may be derived from a human monoclonal antibody. Human
monoclonal antibodies are obtained from transgenic mice that have
been engineered to produce specific human antibodies in response to
antigenic challenge. In this technique, elements of the human heavy
and light chain locus are introduced into strains of mice derived
from embryonic stem cell lines that contain targeted disruptions of
the endogenous heavy chain and light chain loci. The transgenic
mice can synthesize human antibodies specific for human antigens,
and the mice can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described, for example, by Green et al., Nature Genet.
7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et
al, Int. Immun. 6:579 (1994).
[0193] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography (see, for example, Coligan at pages
2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10,
pages 79-104 (The Humana Press, Inc. 1992)).
[0194] For particular uses, it may be desirable to prepare
fragments of anti-IL-22RA antibodies. Such antibody fragments can
be obtained, for example, by proteolytic hydrolysis of the
antibody. Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. As an
illustration, antibody fragments can be produced by enzymatic
cleavage of antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent to produce 3.5S Fab' monovalent fragments.
Optionally, the cleavage reaction can be performed using a blocking
group for the sulfhydryl groups that result from cleavage of
disulfide linkages. As an alternative, an enzymatic cleavage using
pepsin produces two monovalent Fab fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys.
89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman et al.,
in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967),
and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
[0195] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0196] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association can be noncovalent, as
described by Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech.
12:437 (1992)).
[0197] The Fv fragments may comprise V.sub.H and V.sub.L chains
which are connected by a peptide linker. These single-chain antigen
binding proteins (scFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains which are connected by an oligonucleotide. The structural
gene is inserted into an expression vector which is subsequently
introduced into a host cell, such as E. coli. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing scFvs are
described, for example, by Whitlow et al., Methods: A Companion to
Methods in Enzymology 2:97 (1991) (also see, Bird et al., Science
242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack et
al., Bio/Technology 11:1271 (1993), and Sandhu, supra).
[0198] As an illustration, a scFV can be obtained by exposing
lymphocytes to IL-22RA polypeptide in vitro, and selecting antibody
display libraries in phage or similar vectors (for instance,
through use of immobilized or labeled IL-22RA protein or peptide).
Genes encoding polypeptides having potential IL-22RA polypeptide
binding domains can be obtained by screening random peptide
libraries displayed on phage (phage display) or on bacteria, such
as E. coli. Nucleotide sequences encoding the polypeptides can be
obtained in a number of ways, such as through random mutagenesis
and random polynucleotide synthesis. These random peptide display
libraries can be used to screen for peptides which interact with a
known target which can be a protein or polypeptide, such as a
ligand or receptor, a biological or synthetic macromolecule, or
organic or inorganic substances. Techniques for creating and
screening such random peptide display libraries are known in the
art (Ladner et al., U.S. Pat. No. 5,223,409, Ladner et al., U.S.
Pat. No. 4,946,778, Ladner et al., U.S. Pat. No. 5,403,484, Ladner
et al., U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of
Peptides and Proteins (Academic Press, Inc. 1996)) and random
peptide display libraries and kits for screening such libraries are
available commercially, for instance from CLONTECH Laboratories,
Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New
England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB
Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the IL-22RA sequences disclosed
herein to identify proteins which bind to IL-22RA.
[0199] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Methods
in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation
of Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995), and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)).
[0200] Alternatively, an anti-IL-22RA antibody may be derived from
a "humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse complementary determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain. Typical residues of
human antibodies are then substituted in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA 86:3833 (1989). Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA
89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer
et al., J. Immun. 150:2844 (1993), Sudhir (ed.), Antibody
Engineering Protocols (Humana Press, Inc. 1995), Kelley,
"Engineering Therapeutic Antibodies," in Protein Engineering:
Principles and Practice, Cleland et al. (eds.), pages 399-434 (John
Wiley & Sons, Inc. 1996), and by Queen et al., U.S. Pat. No.
5,693,762 (1997).
[0201] Moreover, anti-IL-22RA antibodies or antibody fragments of
the present invention can be PEGylated using methods in the art and
described herein.
[0202] Polyclonal anti-idiotype antibodies can be prepared by
immunizing animals with anti-IL-22RA antibodies or antibody
fragments, using standard techniques. See, for example, Green et
al., "Production of Polyclonal Antisera," in Methods In Molecular
Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana
Press 1992). Also, see Coligan at pages 2.4.1-2.4.7. Alternatively,
monoclonal anti-idiotype antibodies can be prepared using
anti-IL-22RA antibodies or antibody fragments as immunogens with
the techniques, described above. As another alternative, humanized
anti-idiotype antibodies or subhuman primate anti-idiotype
antibodies can be prepared using the above-described techniques.
Methods for producing anti-idiotype antibodies are described, for
example, by Irie, U.S. Pat. No. 5,208,146, Greene, et. al, U.S.
Pat. No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol
77:1875 (1996).
[0203] An anti-IL-22RA antibody can be conjugated with a detectable
label to form an anti-IL-22RA immunoconjugate. Suitable detectable
labels include, for example, a radioisotope, a fluorescent label, a
chemiluminescent label, an enzyme label, a bioluminescent label or
colloidal gold. Methods of making and detecting such
detectably-labeled immunoconjugates are well-known to those of
ordinary skill in the art, and are described in more detail
below.
[0204] The detectable label can be a radioisotope that is detected
by autoradiography. Isotopes that are particularly useful for the
purpose of the present invention are .sup.3H, .sup.125I, .sup.131I,
.sup.35S and .sup.14C.
[0205] Anti-IL-22RA immunoconjugates can also be labeled with a
fluorescent compound. The presence of a fluorescently-labeled
antibody is determined by exposing the immunoconjugate to light of
the proper wavelength and detecting the resultant fluorescence.
Fluorescent labeling compounds include fluorescein isothiocyanate,
rhodamine, phycoerytherin, phycocyanin, allophycocyanin,
o-phthaldehyde and fluorescamine.
[0206] Alternatively, anti-IL-22RA immunoconjugates can be
detectably labeled by coupling an antibody component to a
chemiluminescent compound. The presence of the
chemiluminescent-tagged immunoconjugate is determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of chemiluminescent labeling compounds
include luminol, isoluminol, an aromatic acridinium ester, an
imidazole, an acridinium salt and an oxalate ester.
[0207] Similarly, a bioluminescent compound can be used to label
anti-IL-22RA immunoconjugates of the present invention.
Bioluminescence is a type of chemiluminescence found in biological
systems in which a catalytic protein increases the efficiency of
the chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of luminescence.
Bioluminescent compounds that are useful for labeling include
luciferin, luciferase and aequorin.
[0208] Alternatively, anti-IL-22RA immunoconjugates can be
detectably labeled by linking an anti-IL-22RA antibody component to
an enzyme. When the anti-IL-22RA-enzyme conjugate is incubated in
the presence of the appropriate substrate, the enzyme moiety reacts
with the substrate to produce a chemical moiety which can be
detected, for example, by spectrophotometric, fluorometric or
visual means. Examples of enzymes that can be used to detectably
label polyspecific immunoconjugates include .beta.-galactosidase,
glucose oxidase, peroxidase and alkaline phosphatase.
[0209] Those of skill in the art will know of other suitable labels
which can be employed in accordance with the present invention. The
binding of marker moieties to anti-IL-22RA antibodies can be
accomplished using standard techniques known to the art. Typical
methodology in this regard is described by Kennedy et al., Clin.
Chim. Acta 70:1 (1976), Schurs et al., Clin. Chim. Acta 81:1
(1977), Shih et al., Int'l J. Cancer 46:1101 (1990), Stein et al.,
Cancer Res. 50:1330 (1990), and Coligan, supra.
[0210] Moreover, the convenience and versatility of immunochemical
detection can be enhanced by using anti-IL-22RA antibodies that
have been conjugated with avidin, streptavidin, and biotin (see,
for example, Wilchek et al. (eds.), "Avidin-Biotin Technology,"
Methods In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et
al., "Immunochemical Applications of Avidin-Biotin Technology," in
Methods In Molecular Biology, Vol. 10, Manson (ed.), pages 149-162
(The Humana Press, Inc. 1992).
[0211] Methods for performing immunoassays are well-established.
See, for example, Cook and Self, "Monoclonal Antibodies in
Diagnostic Immunoassays," in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman (eds.),
pages 180-208, (Cambridge University Press, 1995), Perry, "The Role
of Monoclonal Antibodies in the Advancement of Immunoassay
Technology," in Monoclonal Antibodies: Principles and Applications,
Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc. 1995), and
Diamandis, Immunoassay (Academic Press, Inc. 1996).
[0212] The present invention also contemplates kits for performing
an immunological diagnostic assay for IL-22RA gene expression. Such
kits comprise at least one container comprising an anti-IL-22RA
antibody, or antibody fragment. A kit may also comprise a second
container comprising one or more reagents capable of indicating the
presence of IL-22RA antibody or antibody fragments. Examples of
such indicator reagents include detectable labels such as a
radioactive label, a fluorescent label, a chemiluminescent label,
an enzyme label, a bioluminescent label, colloidal gold, and the
like. A kit may also comprise a means for conveying to the user
that IL-22RA antibodies or antibody fragments are used to detect
IL-22RA protein. For example, written instructions may state that
the enclosed antibody or antibody fragment can be used to detect
IL-22RA. The written material can be applied directly to a
container, or the written material can be provided in the form of a
packaging insert.
9. Use of Anti-IL-22RA Antibodies to Antagonize IL-22RA Binding to
IL-22 or Both IL-20 and IL-22
[0213] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to soluble IL-22RA receptor polypeptides or fragments thereof, such
as antigenic epitopes, and selection of antibody display libraries
in phage or similar vectors (for instance, through use of
immobilized or labeled soluble IL-22RA receptor polypeptides or
fragments thereof, such as antigenic epitopes). Genes encoding
polypeptides having potential binding domains such as soluble
soluble IL-22RA receptor polypeptides or fragments thereof, such as
antigenic epitopes can be obtained by screening random peptide
libraries displayed on phage (phage display) or on bacteria, such
as E. coli. Nucleotide sequences encoding the polypeptides can be
obtained in a number of ways, such as through random mutagenesis
and random polynucleotide synthesis. These random peptide display
libraries can be used to screen for peptides that interact with a
known target that can be a protein or polypeptide, such as a ligand
or receptor, a biological or synthetic macromolecule, or organic or
inorganic substances. Techniques for creating and screening such
random peptide display libraries are known in the art (Ladner et
al., U.S. Pat. No. 5,223,409; Ladner et al., U.S. Pat. No.
4,946,778; Ladner et al., U.S. Pat. No. 5,403,484 and Ladner et
al., U.S. Pat. No. 5,571,698) and random peptide display libraries
and kits for screening such libraries are available commercially,
for instance from Clontech (Palo Alto, Calif.), Invitrogen Inc.
(San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.) and
Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide
display libraries can be screened using the soluble IL-22RA
receptor polypeptides or fragments thereof, such as antigenic
epitope polypeptide sequences disclosed herein to identify proteins
which bind to IL-22RA-comprising receptor polypeptides. These
"binding polypeptides," which interact with soluble
IL-22RA-comprising receptor polypeptides, can be used for tagging
cells; for isolating homolog polypeptides by affinity purification;
they can be directly or indirectly conjugated to drugs, toxins,
radionuclides and the like. These binding polypeptides can also be
used in analytical methods such as for screening expression
libraries and neutralizing activity, e.g., for binding, blocking,
inhibiting, reducing, antagonizing or neutralizing interaction
between IL-22 ligand and receptor, or viral binding to a receptor.
The binding polypeptides can also be used for diagnostic assays for
determining circulating levels of soluble IL-22RA-comprising
receptor polypeptides; for detecting or quantitating soluble or
non-soluble IL-22RA-comprising receptors as marker of underlying
pathology or disease. These binding polypeptides can also act as
"antagonists" to block or inhibit soluble or membrane-bound IL-22RA
monomeric receptor or IL-22RA homodimeric, heterodimeric or
multimeric polypeptide binding (e.g. to ligand) and signal
transduction in vitro and in vivo. Again, these binding
polypeptides serve as anti-IL-22RA monomeric receptor or
anti-IL-22RA homodimeric, heterodimeric or multimeric polypeptides
and are useful for inhibiting IL-22 or both IL-20 and IL-22
activity, as well as receptor activity or protein-binding.
Antibodies raised to the natural receptor complexes of the present
invention, and IL-22RA-epitope-binding antibodies, and anti-IL-22RA
neutralizing monoclonal antibodies may be preferred embodiments, as
they may act more specifically against the IL-22RA and can inhibit
IL-22 or both IL-20 and IL-22. Moreover, the antagonistic and
binding activity of the antibodies of the present invention can be
assayed in an IL-20 or IL-22 proliferation, signal trap, luciferase
or binding assays in the presence of IL-20 or IL-22 respectively,
and IL-22RA-comprising soluble receptors, and other biological or
biochemical assays described herein.
[0214] Antibodies to soluble IL-22RA receptor polypeptides (e.g.,
antibodies to SEQ ID NO:3) or fragments thereof, such as antigenic
epitopes may be used for inhibiting the inflammatory effects of
IL-20, IL-22, or both IL-20 and IL-22 in vivo, for theraputic use
against psoriasis, atopic dermatitis, inflammatory skin conditions,
endotoxemia, arthritis, asthma, IBD, colitis, psoriatic arthritis,
rheumatoid arthritis or other IL-20 and IL-22-induced inflammatory
conditions; tagging cells that express IL-22RA receptors; for
isolating soluble IL-22RA-comprising receptor polypeptides by
affinity purification; for diagnostic assays for determining
circulating levels of soluble IL-22RA-comprising receptor
polypeptides; for detecting or quantitating soluble
IL-22RA-comprising receptors as marker of underlying pathology or
disease; in analytical methods employing FACS; for screening
expression libraries; for generating anti-idiotypic antibodies that
can act as IL-22 or IL-20 agonists; and as neutralizing antibodies
or as antagonists to bind, block, inhibit, reduce, or antagonize
IL-22RA receptor function, or to bind, block, inhibit, reduce,
antagonize or neutralize IL-22 and/or IL-20 activity (either
individually or together) in vitro and in vivo. Suitable direct
tags or labels include radionuclides, enzymes, substrates,
cofactors, biotin, inhibitors, fluorescent markers,
chemiluminescent markers, magnetic particles and the like; indirect
tags or labels may feature use of biotin-avidin or other
complement/anti-complement pairs as intermediates. Antibodies
herein may also be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like, and these conjugates used for
in vivo diagnostic or therapeutic applications. Moreover,
antibodies to soluble IL-22RA-comprising receptor polypeptides, or
fragments thereof may be used in vitro to detect denatured or
non-denatured IL-22RA-comprising receptor polypeptides or fragments
thereof in assays, for example, Western Blots or other assays known
in the art.
[0215] Antibodies to soluble IL-22RA receptor or soluble IL-22RA
homodimeric, heterodimeric or multimeric receptor polypeptides are
useful for tagging cells that express the corresponding receptors
and assaying their expression levels, for affinity purification,
within diagnostic assays for determining circulating levels of
receptor polypeptides, analytical methods employing
fluorescence-activated cell sorting. Moreover, divalent antibodies,
and anti-idiotypic antibodies may be used as agonists to mimic the
effect of the IL-22RA ligand, IL-22 or IL-20.
[0216] Antibodies herein can also be directly or indirectly
conjugated to drugs, toxins, radionuclides and the like, and these
conjugates used for in vivo diagnostic or therapeutic applications.
For instance, antibodies or binding polypeptides which recognize
soluble IL-22RA receptor or soluble IL-22RA homodimeric,
heterodimeric or multimeric receptor polypeptides can be used to
identify or treat tissues or organs that express a corresponding
anti-complementary molecule (i.e., a IL-22RA-comprising soluble or
membrane-bound receptor). More specifically, antibodies to soluble
IL-22RA-comprising receptor polypeptides, or bioactive fragments or
portions thereof, can be coupled to detectable or cytotoxic
molecules and delivered to a mammal having cells, tissues or organs
that express the IL-22RA-comprising receptor such as
IL-22RA-expressing cancers.
[0217] Suitable detectable molecules may be directly or indirectly
attached to polypeptides that bind IL-22RA-comprising receptor
polypeptides, such as "binding polypeptides," (including binding
peptides disclosed above), antibodies, or bioactive fragments or
portions thereof. Suitable detectable molecules include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent markers, chemiluminescent markers, magnetic particles
and the like. Suitable cytotoxic molecules may be directly or
indirectly attached to the polypeptide or antibody, and include
bacterial or plant toxins (for instance, diphtheria toxin,
Pseudomonas exotoxin, ricin, abrin and the like), as well as
therapeutic radionuclides, such as iodine-131, rhenium-188 or
yttrium-90 (either directly attached to the polypeptide or
antibody, or indirectly attached through means of a chelating
moiety, for instance). Binding polypeptides or antibodies may also
be conjugated to cytotoxic drugs, such as adriamycin. For indirect
attachment of a detectable or cytotoxic molecule, the detectable or
cytotoxic molecule can be conjugated with a member of a
complementary/anticomplementary pair, where the other member is
bound to the binding polypeptide or antibody portion. For these
purposes, biotin/streptavidin is an exemplary
complementary/anticomplementary pair.
[0218] In another embodiment, binding polypeptide-toxin fusion
proteins or antibody-toxin fusion proteins can be used for targeted
cell or tissue inhibition or ablation (for instance, to treat
cancer cells or tissues). Alternatively, if the binding polypeptide
has multiple functional domains (i.e., an activation domain or a
ligand binding domain, plus a targeting domain), a fusion protein
including only the targeting domain may be suitable for directing a
detectable molecule, a cytotoxic molecule or a complementary
molecule to a cell or tissue type of interest. In instances where
the fusion protein including only a single domain includes a
complementary molecule, the anti-complementary molecule can be
conjugated to a detectable or cytotoxic molecule. Such
domain-complementary molecule fusion proteins thus represent a
generic targeting vehicle for cell/tissue-specific delivery of
generic anti-complementary-detectable/cytotoxic molecule
conjugates.
[0219] In another embodiment, IL-22RA binding polypeptide-cytokine
or antibody-cytokine fusion proteins can be used for enhancing in
vivo killing of target tissues (for example, spleen, pancreatic,
blood, lymphoid, colon, and bone marrow cancers), if the binding
polypeptide-cytokine or anti-IL-22RA receptor antibody targets the
hyperproliferative cell (See, generally, Hornick et al., Blood
89:4437-47, 1997). The described fusion proteins enable targeting
of a cytokine to a desired site of action, thereby providing an
elevated local concentration of cytokine. Suitable anti-IL-22RA
monomer, homodimer, heterodimer or multimer antibodies target an
undesirable cell or tissue (i.e., a tumor or a leukemia), and the
fused cytokine mediates improved target cell lysis by effector
cells. Suitable cytokines for this purpose include interleukin 2
and granulocyte-macrophage colony-stimulating factor (GM-CSF), for
instance.
[0220] Alternatively, IL-22RA receptor binding polypeptides or
antibody fusion proteins described herein can be used for enhancing
in vivo killing of target tissues by directly stimulating a IL-22RA
receptor-modulated apoptotic pathway, resulting in cell death of
hyperproliferative cells expressing IL-22RA-comprising
receptors.
10. Therapeutic Uses of Polypeptides Having IL-22RA Activity or
Antibodies to IL-22RA
[0221] Amino acid sequences having soluble IL-22RA activity can be
used to modulate the immune system by binding IL-22RA ligands IL-20
and IL-22 (either singly or together), and thus, preventing the
binding of IL-22RA ligand with endogenous IL-22RA receptor. IL-22RA
antagonists, such as anti-IL-22RA antibodies, can also be used to
modulate the immune system by inhibiting the binding of IL-22RA
ligand with the endogenous IL-22RA receptor. Accordingly, the
present invention includes the use of proteins, polypeptides, and
peptides having IL-22RA activity (such as soluble IL-22RA
polypeptides, IL-22RA polypeptide fragments, IL-22RA analogs (e.g.,
anti-IL-22RA anti-idiotype antibodies), and IL-22RA fusion
proteins) to a subject which lacks an adequate amount of this
polypeptide, or which produces an excess of IL-22RA ligand. IL-22RA
antagonists (e.g., anti-IL-22RA antibodies) can be also used to
treat a subject which produces an excess of either IL-22RA ligand
or IL-22RA. Suitable subjects include mammals, such as humans. For
example, such IL-22RA polypeptides and anti-IL-22RA antibodies are
useful in binding, blocking, inhibiting, reducing, antagonizing or
neutralizing IL-20 and IL-22 (either singly or together), in the
treatment of psoriasis, atopic dermatitis, inflammatory skin
conditions, psoriatic arthritis, arthritis, endotoxemia, asthma,
inflammatory bowel disease (IBD), colitis, and other inflammatory
conditions disclosed herein.
[0222] Moreover, we have shown that the IL-22RA receptor binds a
ligand called T-cell inducible Factor (IL-22) (SEQ ID NO:6;
Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000;
mouse IL-22 sequence is shown in Dumontier et al., J. Immunol.
164:1814-1819, 2000). Moreover, commonly owned zcytor11 (IL-22RA)
(U.S. Pat. No. 5,965,704) and CRF2-4 receptor also bind IL-22 as a
heterodimer (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; and Kotenko, S V et al., J. Biol. Chem. 276:2725-2732, 2001).
Moreover, IL-10.beta. receptor may be involved as a receptor for
IL-22, and it 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). Moreover, we
have shown that IL-22RA receptor binds a ligand called IL-20 (SEQ
ID NO:8; WIPO Publication No. WO 99/27103). Within preferred
embodiments, the soluble receptor form of IL-22RA, SEQ ID NO:3) is
a monomer, homodimer, heterodimer, or multimer that binds to,
blocks, inhibits, reduces, antagonizes or neutralizes IL-22 and
IL-20 in vivo. Antibodies and binding polypeptides to such IL-22RA
monomer, homodimer, heterodimer, or multimers also serve as
antagonists of IL-22RA activity, and as IL-20 and IL-22 antagonists
(singly or together), as described herein.
[0223] In addition, we have described herein, and have demonstrated
that both polyclonal and monoclonal neutralizing anti-IL-22
antibodies bind to, block, inhibit, reduce, antagonize or
neutralize IL-22 and IL-20 activity in cell based neutralization
assays.
[0224] IL-22 has been shown to be induced in the presence of IL-9,
and is suspected to be involved in promoting Th1-type immune
responses, and inflammation. IL-9 stimulates proliferation,
activation, differentiation and/or induction of immune function in
a variety of ways and is implicated in asthma, lung mastocytosis,
and other diseases, as well as activates STAT pathways. Antagonists
of IL-22 or IL-9 function can have beneficial use against such
human diseases. The present invention provides such novel
antagonists of IL-22.
[0225] IL-22 has been show to be involved in up-regulate the
production of acute phase reactants, such as serum amyloid A (SAA),
.alpha.1-antichymotrypsin, and haptoglobin, and that IL-22
expression is increased upon injection of lipopolysaccharide (LPS)
in vivo suggesting that IL-22 is involved in inflammatory response
(Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149,
2000). Production of acute phase proteins, such as SAA, is
considered s short-term survival mechanism where inflammation is
beneficial; however, maintenance of acute phase proteins for longer
periods contributes to chronic inflammation and can be harmful to
human health. For review, see Uhlar, C M and Whitehead, A S, Eur.
J. Biochem. 265:501-523, 1999, and Baumann H. and Gauldie, J.
Immunology Today 15:74-80, 1994. Moreover, the acute phase protein
SAA is implicated in the pathogenesis of several chronic
inflammatory diseases, is implicated in atherosclerosis and
rheumatoid arthritis, and is the precursor to the amyloid A protein
deposited in amyloidosis (Uhlar, C M and Whitehead, supra.). Thus,
as IL-22 acts as a pro-inflammatory molecule and induces production
of SAA, antagonists would be useful in treating inflammatory
disease and other diseases associated with acute phase response
proteins induced by IL-22. Such antagonists are provided by the
present invention. For example, method of reducing IL-22-induced or
IL-9 induced inflammation comprises administering to a mammal with
inflammation an amount of a composition of soluble
IL-22RA-comprising receptor sufficient to reduce inflammation.
Moreover, a method of suppressing an inflammatory response in a
mammal with inflammation can comprise: (1) determining a level of
serum amyloid A protein; (2) administering a composition comprising
a soluble IL-22RA cytokine receptor polypeptide as described herein
in an acceptable pharmaceutical vehicle; (3) determining a post
administration level of serum amyloid A protein; (4) comparing the
level of serum amyloid A protein in step (1) to the level of serum
amyloid A protein in step (3), wherein a lack of increase or a
decrease in serum amyloid A protein level is indicative of
suppressing an inflammatory response. Experimental evidence
described herein shows that IL-22 antagonists, such as soluble
receptors and antibodies, indeed reduce SAA levels in vivo models
for inflammatory diseases, showing that binding, blocking,
inhibiting, reducing, antagonizing or neutralizing IL-22 has
anti-inflammatory effects.
[0226] Evidence indicates that a role IL-20 and its receptors are
involved in psoriasis. This multigenic skin disease is
characterized by increased keratinocyte proliferation, altered
keratinocyte differentiation, and infiltration of immune cells into
the skin. The first line of evidence for a role of IL-20 in
psoriasis is that the observed hyperkeratosis and thickened
epidermis in the transgenic mice that resemble human psoriatic
abnormalities. Decreased numbers of tonofilaments, thought to be
related to defective keratinization, are a striking feature of
human psoriasis. Intramitochondrial inclusions have been found in
both chemically induced and naturally occurring hyperplastic skin
conditions in mice. The cause of the inclusions and their effects
on mitochondrial function, if any, are unknown. IL-20 transgenic
mice exhibit many of the characteristics observed in human
psoriasis.
[0227] Moreover, IL-20 receptor mRNA (both IL-20RA and IL-20RB
mRNA) are markedly upregulated in human psoriatic skin compared to
normal skin further suggesting a role for IL-20 in psoriasis. Both
IL-20 receptor subunits are expressed in keratinocytes throughout
the epidermis and are also expressed in a subset of immune and
endothelial cells. We propose that increased expression of an
activated IL-20 receptor may alter the interactions between
endothelial cells, immune cells and keratinocytes, leading to
dysregulation of keratinocyte proliferation and differentiation. In
addition, mouse knockout data described herein, wherein the IL-22RA
receptor is knocked out, show that IL-22RA was necessary for the
IL-20-induced inflammatory effects in skin in transgenic animals.
These results provided evidence that effectively blocking IL-22RA
activity, for example via an IL-22RA gene knockout, or similarly
via a neutralizing monoclonal antibody to IL-22RA of the present
invention, would similarly reduce IL-20-induced skin effects, as
well as IL-22-induced skin effects, for example in psoriasis, IBD,
colitis, or other inflammatory diseases induced by IL-20, and or
IL-22 including IBD, arthritis, asthma, psoriatic arthritis,
colitis, inflammatory skin conditions, and atopic dermatitis.
[0228] Moreover, IL-20 stimulates signal transduction in the human
keratinocyte HaCaT cell line, supporting a direct action of this
novel ligand in skin. In addition, IL-1.beta., EGF and TNF-.alpha.,
proteins known to be active in keratinocytes and to be involved
with proliferative and pro-inflammatory signals in skin, enhance
the response to IL-20. In both HaCaT and BHK cells expressing the
IL-20 receptor, IL-20 signals through STAT3.
[0229] As indicated in the discussion above and the examples below,
IL-20 is involved in the pathology of psoriasis. The present
invention is in particular a method for treating psoriasis by
administering agents that bind, block, inhibit, reduce, antagonize
or neutralize IL-20. The antagonists to IL-20 can either be a
soluble receptor that binds to IL-20, such a soluble IL-22RA, or
antibodies, single chain antibodies or fragments of antibodies that
bind to either IL-20 or the IL-20 receptor, e.g., anti-IL-22RA
antibodies. The antagonists will thus prevent activation of the
IL-20 receptor. Moreover, because IL-20 and IL-22 share IL-22RA as
a common receptor, antagonists such as soluble IL-22RA, or
antibodies, single chain antibodies or fragments of antibodies that
bind to IL-22RA receptor can be used to concurrently bind to,
block, inhibit, reduce, antagonize or neutralize IL-22 or both
IL-20 and IL-22 activity.
[0230] Psoriasis is one of the most common dermatologic diseases,
affecting up to 1 to 2 percent of the world's population. It is a
chronic inflammatory skin disorder characterized by erythematous,
sharply demarcated papules and rounded plaques, covered by silvery
micaceous scale. The skin lesions of psoriasis are variably
pruritic. Traumatized areas often develop lesions of psoriasis.
Additionally, other external factors may exacerbate psoriasis
including infections, stress, and medications, e.g. lithium, beta
blockers, and anti-malarials.
[0231] The most common variety of psoriasis is called plaque type.
Patients with plaque-type psoriasis will have stable, slowly
growing plaques, which remain basically unchanged for long periods
of time. The most common areas for plaque psoriasis to occur are
the elbows knees, gluteal cleft, and the scalp. Involvement tends
to be symmetrical. Inverse psoriasis affects the intertriginous
regions including the axilla, groin, submammary region, and navel,
and it also tends to affect the scalp, palms, and soles. The
individual lesions are sharply demarcated plaques but may be moist
due to their location. Plaque-type psoriasis generally develops
slowly and runs an indolent course. It rarely spontaneously
remits.
[0232] Eruptive psoriasis (guttate psoriasis) is most common in
children and young adults. It develops acutely in individuals
without psoriasis or in those with chronic plaque psoriasis.
Patients present with many small erythematous, scaling papules,
frequently after upper respiratory tract infection with
beta-hemolytic streptococci. Patients with psoriasis may also
develop pustular lesions. These may be localized to the palms and
soles or may be generalized and associated with fever, malaise,
diarrhea, and arthralgias.
[0233] About half of all patients with psoriasis have fingernail
involvement, appearing as punctate pitting, nail thickening or
subungual hyperkeratosis. About 5 to 10 percent of patients with
psoriasis have associated joint complaints, and these are most
often found in patients with fingernail involvement. Although some
have the coincident occurrence of classic Although some have the
coincident occurrence of classic rheumatoid arthritis, many have
joint disease that falls into one of five type associated with
psoriasis: (1) disease limited to a single or a few small joints
(70 percent of cases); (2) a seronegative rheumatoid arthritis-like
disease; (3) involvement of the distal interphalangeal joints; (4)
severe destructive arthritis with the development of "arthritis
mutilans"; and (5) disease limited to the spine.
[0234] Psoriasis can be treated by administering agents that bind
to, block, inhibit, reduce, antagonize or neutralize to IL-22,
IL-20, or both IL-20 and IL-22. The preferred antagonists are
either a soluble receptor to IL-20 and IL-22, such as IL-22RA (SEQ
ID NO:3) or antibodies, antibody fragments or single chain
antibodies that bind to the IL-22RA receptor, such as the
neutralizing antibodies of the present invention. Such antagonists
can be administered alone or in combination with other established
therapies such as lubricants, keratolytics, topical
corticosteroids, topical vitamin D derivatives, anthralin, systemic
antimetabolites such as methotrexate, psoralen-ultraviolet-light
therapy (PUVA), etretinate, isotretinoin, cyclosporine, and the
topical vitamin D3 derivative calcipotriol. Moreover, such
antagonists can be administered to individual subcutaneously,
intravenously, or transdermally using a cream or transdermal patch
that contains the antagonist. If administered subcutaneously, the
antagonist can be injected into one or more psoriatic plaques. If
administered transdermally, the antagonists can be administered
directly on the plaques using a cream, ointment, salve, or solution
containing the antagonist.
[0235] Antagonists to IL-20 or IL-22 can be administered to a
person who has asthma, bronchitis or cystic fibrosis or other
inflammatory lung disease to treat the disease. The antagonists can
be administered by any suitable method including intravenous,
subcutaneous, bronchial lavage, and the use of inhalant containing
the antagonist.
[0236] Analysis of the tissue distribution of the mRNA
corresponding IL-22RA cDNA showed that mRNA level was highest in
placenta and spleen, and the ligand to which IL-22RA binds (IL-22)
is implicated in inducing inflammatory response including induction
of the acute-phase response (Dumoutier, L. et al., Proc. Nat'l.
Acad. Sci. 97:10144-10149, 2000). Thus, particular embodiments of
the present invention are directed toward use of soluble IL-22RA
and anti-IL-22RA antibodies as antagonists in inflammatory and
immune diseases or conditions such as psoriasis, psoriatic
arthritis, atopic dermatitis, inflammatory skin conditions,
rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's
Disease, diverticulosis, asthma, pancreatitis, type I diabetes
(IDDM), pancreatic cancer, pancreatitis, Graves Disease, colon and
intestinal cancer, autoimmune disease, sepsis, organ or bone marrow
transplant; inflammation due to endotoxemia, trauma, surgery or
infection; amyloidosis; splenomegaly; graft versus host disease;
and where inhibition of inflammation, immune suppression, reduction
of proliferation of hematopoietic, immune, inflammatory or lymphoid
cells, macrophages, T-cells (including Th1 and Th2 cells),
suppression of immune response to a pathogen or antigen, or other
instances where inhibition of IL-22 or IL-20 cytokines is
desired.
[0237] Moreover, antibodies or binding polypeptides that bind
IL-22RA polypeptides described herein, and IL-22RA polypeptides
themselves are useful to:
[0238] 1) Block, inhibit, reduce, antagonize or neutralize
signaling via IL-20 or IL-22 receptors in the treatment of acute
inflammation, inflammation as a result of trauma, tissue injury,
surgery, sepsis or infection, and chronic inflammatory diseases
such as asthma, inflammatory bowel disease (IBD), chronic colitis,
splenomegaly, rheumatoid arthritis, recurrent acute inflammatory
episodes (e.g., tuberculosis), and treatment of amyloidosis, and
atherosclerosis, Castleman's Disease, asthma, and other diseases
associated with the induction of acute-phase response.
[0239] 2) Block, inhibit, reduce, antagonize or neutralize
signaling via IL-20 or IL-22 receptors in the treatment of
autoimmune diseases such as IDDM, multiple sclerosis (MS), systemic
Lupus erythematosus (SLE), myasthenia gravis, rheumatoid arthritis,
and IBD to prevent or inhibit signaling in immune cells (e.g.
lymphocytes, monocytes, leukocytes) via IL-22RA (Hughes C et al.,
J. Immunol 153: 3319-3325, 1994). Alternatively antibodies, such as
monoclonal antibodies (MAb) to IL-22RA-comprising receptors, can
also be used as an antagonist to deplete unwanted immune cells to
treat autoimmune disease. Asthma, allergy and other atopic disease
may be treated with an MAb against, for example, soluble IL-22RA
soluble receptors to inhibit the immune response or to deplete
offending cells. Blocking, inhibiting, reducing, or antagonizing
signaling via IL-22RA, using the polypeptides and antibodies of the
present invention, may also benefit diseases of the pancreas,
kidney, pituitary and neuronal cells. IDDM, NIDDM, pancreatitis,
and pancreatic carcinoma may benefit. IL-22RA may serve as a target
for MAb therapy of cancer where an antagonizing MAb inhibits cancer
growth and targets immune-mediated killing. (Holliger P, and
Hoogenboom, H: Nature Biotech. 16: 1015-1016, 1998). Mabs to
soluble IL-22RA may also be useful to treat nephropathies such as
glomerulosclerosis, membranous neuropathy, amyloidosis (which also
affects the kidney among other tissues), renal arteriosclerosis,
glomerulonephritis of various origins, fibroproliferative diseases
of the kidney, as well as kidney dysfunction associated with SLE,
IDDM, type II diabetes (NIDDM), renal tumors and other
diseases.
[0240] 3) Agonize, enhance, increase or initiate signaling via
IL-20 or IL-22 receptors in the treatment of autoimmune diseases
such as IDDM, MS, SLE, myasthenia gravis, rheumatoid arthritis, and
IBD. Anti-IL-22RA neutralizing and monoclonal antibodies may signal
lymphocytes or other immune cells to differentiate, alter
proliferation, or change production of cytokines or cell surface
proteins that ameliorate autoimmunity. Specifically, modulation of
a T-helper cell response to an alternate pattern of cytokine
secretion may deviate an autoimmune response to ameliorate disease
(Smith J A et al., J. Immunol. 160:4841-4849, 1998). Similarly,
agonistic Anti-soluble IL-22RA, anti-soluble IL-22RA/CRF2-4
heterodimers and multimer monoclonal antibodies may be used to
signal, deplete and deviate immune cells involved in asthma,
allergy and atopic disease. Signaling via IL-22RA may also benefit
diseases of the pancreas, kidney, pituitary and neuronal cells.
IDDM, NIDDM, pancreatitis, and pancreatic carcinoma may benefit.
IL-22RA may serve as a target for MAb therapy of pancreatic cancer
where a signaling MAb inhibits cancer growth and targets
immune-mediated killing (Tutt, A L et al., J Immunol. 161:
3175-3185, 1998). Similarly renal cell carcinoma may be treated
with monoclonal antibodies to IL-22RA-comprising soluble receptors
of the present invention.
[0241] Soluble IL-22RA polypeptides described herein can be used to
bind, block, inhibit, reduce, antagonize or neutralize IL-22 or
IL-20 activity, either singly or together, in the treatment of
autoimmune disease, atopic disease, NIDDM, pancreatitis and kidney
dysfunction as described above. A soluble form of IL-22RA may be
used to promote an antibody response mediated by Th cells and/or to
promote the production of IL-4 or other cytokines by lymphocytes or
other immune cells.
[0242] The soluble IL-22RA-comprising receptors of the present
invention are useful as antagonists of IL-20 or IL-22 cytokine.
Such antagonistic effects can be achieved by direct neutralization
or binding of IL-20 or IL-22. In addition to antagonistic uses, the
soluble receptors of the present invention can bind IL-22 and act
as carrier proteins for IL-20 or IL-22 cytokine, in order to
transport the Ligand to different tissues, organs, and cells within
the body. As such, the soluble receptors of the present invention
can be fused or coupled to molecules, polypeptides or chemical
moieties that direct the soluble-receptor-Ligand complex to a
specific site, such as a tissue, specific immune cell, or tumor.
For example, in acute infection or some cancers, benefit may result
from induction of inflammation and local acute phase response
proteins by the action of IL-22. Thus, the soluble receptors of the
present invention can be used to specifically direct the action of
IL-20 or IL-22. See, Cosman, D. Cytokine 5: 95-106, 1993; and
Fernandez-Botran, R. Exp. Opin. Invest. Drugs 9:497-513, 2000.
[0243] Moreover, the soluble receptors of the present invention can
be used to stabilize the IL-22 or IL-20, to increase the
bioavailability, therapeutic longevity, and/or efficacy of the
Ligand by stabilizing the Ligand from degradation or clearance, or
by targeting the ligand to a site of action within the body. For
example the naturally occurring IL-6/soluble IL-6R complex
stabilizes IL-6 and can signal through the gp130 receptor. See,
Cosman, D. supra., and Fernandez-Botran, R. supra. Moreover,
IL-22RA may be combined with a cognate ligand such as IL-22 to
comprise a ligand/soluble receptor complex. Such complexes may be
used to stimulate responses from cells presenting a companion
receptor subunit such as, for example, pDIRS1 (IL-20RB) or CRF2-4
(IL-10RB). The cell specificity of IL-22RA/ligand complexes may
differ from that seen for the ligand administered alone.
Furthermore the complexes may have distinct pharmacokinetic
properties such as affecting half-life, dose/response and organ or
tissue specificity. IL-22RA/IL-22 or IL-22RA/IL-20 complexes thus
may have agonist activity to enhance an immune response or
stimulate mesangial cells or to stimulate hepatic cells.
Alternatively only tissues expressing a signaling subunit the
heterodimerizes with the complex may be affected analogous to the
response to IL6/IL6R complexes (Hirota H. et al., Proc. Nat'l.
Acad. Sci. 92:4862-4866, 1995; Hirano, T. in Thomason, A. (Ed.)
"The Cytokine Handbook", 3.sup.rd Ed., p. 208-209). Soluble
receptor/cytokine complexes for IL12 and CNTF display similar
activities.
[0244] Moreover Inflammation is a protective response by an
organism to fend off an invading agent. Inflammation is a cascading
event that involves many cellular and humoral mediators. On one
hand, suppression of inflammatory responses can leave a host
immunocompromised; however, if left unchecked, inflammation can
lead to serious complications including chronic inflammatory
diseases (e.g., psoriasis, arthritis, rheumatoid arthritis,
multiple sclerosis, inflammatory bowel disease and the like),
septic shock and multiple organ failure. Importantly, these diverse
disease states share common inflammatory mediators. The collective
diseases that are characterized by inflammation have a large impact
on human morbidity and mortality. Therefore it is clear that
anti-inflammatory proteins, such as IL-22RA, and anti-IL-22RA
antibodies, could have crucial therapeutic potential for a vast
number of human and animal diseases, from asthma and allergy to
autoimmunity and septic shock.
[0245] 1. Arthritis
[0246] Arthritis, including osteoarthritis, rheumatoid arthritis,
arthritic joints as a result of injury, and the like, are common
inflammatory conditions which would benefit from the therapeutic
use of anti-inflammatory proteins, such as IL-22RA polypeptides of
the present invention. For example, rheumatoid arthritis (RA) is a
systemic disease that affects the entire body and is one of the
most common forms of arthritis. It is characterized by the
inflammation of the membrane lining the joint, which causes pain,
stiffness, warmth, redness and swelling. Inflammatory cells release
enzymes that may digest bone and cartilage. As a result of
rheumatoid arthritis, the inflamed joint lining, the synovium, can
invade and damage bone and cartilage leading to joint deterioration
and severe pain amongst other physiologic effects. The involved
joint can lose its shape and alignment, resulting in pain and loss
of movement.
[0247] Rheumatoid arthritis (RA) is an immune-mediated disease
particularly characterized by inflammation and subsequent tissue
damage leading to severe disability and increased mortality. A
variety of cytokines are produced locally in the rheumatoid joints.
Numerous studies have demonstrated that IL-1 and TNF-alpha, two
prototypic pro-inflammatory cytokines, play an important role in
the mechanisms involved in synovial inflammation and in progressive
joint destruction. Indeed, the administration of TNF-alpha and IL-1
inhibitors in patients with RA has led to a dramatic improvement of
clinical and biological signs of inflammation and a reduction of
radiological signs of bone erosion and cartilage destruction.
However, despite these encouraging results, a significant
percentage of patients do not respond to these agents, suggesting
that other mediators are also involved in the pathophysiology of
arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149, 2002).
One of those mediators could be IL-20 or IL-22, and as such a
molecule that binds or inhibits IL-22 or IL-20 activity, such as
IL-22RA polypeptides, or anti IL-22RA antibodies or binding
partners, could serve as a valuable therapeutic to reduce
inflammation in rheumatoid arthritis, and other arthritic
diseases.
[0248] There are several animal models for rheumatoid arthritis
known in the art. For example, in the collagen-induced arthritis
(CIA) model, mice develop chronic inflammatory arthritis that
closely resembles human rheumatoid arthritis. Since CIA shares
similar immunological and pathological features with RA, this makes
it an ideal model for screening potential human anti-inflammatory
compounds. The CIA model is a well-known model in mice that depends
on both an immune response, and an inflammatory response, in order
to occur. The immune response comprises the interaction of B-cells
and CD4+ T-cells in response to collagen, which is given as
antigen, and leads to the production of anti-collagen antibodies.
The inflammatory phase is the result of tissue responses from
mediators of inflammation, as a consequence of some of these
antibodies cross-reacting to the mouse's native collagen and
activating the complement cascade. An advantage in using the CIA
model is that the basic mechanisms of pathogenesis are known. The
relevant T-cell and B-cell epitopes on type II collagen have been
identified, and various immunological (e.g., delayed-type
hypersensitivity and anti-collagen antibody) and inflammatory
(e.g., cytokines, chemokines, and matrix-degrading enzymes)
parameters relating to immune-mediated arthritis have been
determined, and can thus be used to assess test compound efficacy
in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20, 1999;
Williams et al., Immunol. 89:9784-788, 1992; Myers et al., Life
Sci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959,
1995).
[0249] The administration of soluble IL-22RA2 comprising
polypeptides (zcytor16), such as zcytor16-Fc4 or other IL-22RA2
soluble and fusion proteins to these CIA model mice was used to
evaluate the use of IL-22RA2 as an antagonist to IL-22 used to
ameliorate symptoms and alter the course of disease. Moreover, the
results showing inhibition of IL-22 by IL-22RA2 provide proof of
concept that other IL-22 antagonists, such as IL-22RA or antibodies
thereto, can also be used to ameliorate symptoms and alter the
course of disease. Since the ligand of IL-22RA2, IL-22, induces
production of SAA, which is implicated in the pathogenesis of
rheumatoid arthritis, and IL-22RA2 was demonstrated to be able to
inhibit IL-22 and SAA activity in vitro and in vivo, the systemic
or local administration of IL-22RA2 comprising polypeptides, such
as zcytor16-Fc4 or other IL-22 soluble receptors (e.g., IL-22RA;
SEQ ID NO:3) and anti-IL-22RA antibodies, and fusion proteins can
potentially suppress the inflammatory response in RA. The injection
of 10 ug zcytor16-Fc (three times a week for 4 weeks) significantly
reduced the disease score (paw score, incident of inflammation or
disease). Other potential therapeutics include IL-22RA
polypeptides, anti-IL-22RA antibodies, or anti IL-22 antibodies or
binding partners, and the like.
[0250] One group has shown that an anti-mouse IL-22 antibody may
reduce symptoms in a mouse CIA-model relative to control mice, thus
showing conceptually that soluble IL-22RA polypeptides and
neutralizing antibodies to IL-22RA may be beneficial in treating
human disease. The administration of a single mouse-IL-22-specific
rat monoclonal antibody (P3/1) reduced the symptoms of arthritis in
the animals when introduced prophylactically or after CIA-induced
arthritis was induced in the model (WIPO Publication 02/068476;
published Sep. 9, 2002). Therefore, the soluble IL-22RA
polypeptides and anti-IL-22RA antibodies of the present invention,
including the neutralizing anti-human IL-22RA antibodies of the
present invention, can be used to neutralize IL-22 and IL-20 in the
treatment of specific human diseases such as psoriasis, psoriatic
arthritis, arthritis, endotoxemia, inflammatory bowel disease
(IBD), colitis, and other inflammatory conditions disclosed
herein.
[0251] 2. Endotoxemia
[0252] Endotoxemia is a severe condition commonly resulting from
infectious agents such as bacteria and other infectious disease
agents, sepsis, toxic shock syndrome, or in immunocompromised
patients subjected to opportunistic infections, and the like.
Therapeutically useful of anti-inflammatory proteins, such as
IL-22RA polypeptides and antibodies of the present invention, could
aid in preventing and treating endotoxemia in humans and animals.
IL-22RA polypeptides, anti-IL22RA antibodies, or anti IL-22
antibodies or binding partners, could serve as a valuable
therapeutic to reduce inflammation and pathological effects in
endotoxemia.
[0253] Lipopolysaccharide (LPS) induced endotoxemia engages many of
the proinflammatory mediators that produce pathological effects in
the infectious diseases and LPS induced endotoxemia in rodents is a
widely used and acceptable model for studying the pharmacological
effects of potential pro-inflammatory or immunomodulating agents.
LPS, produced in gram-negative bacteria, is a major causative agent
in the pathogenesis of septic shock (Glausner et al., Lancet
338:732, 1991). A shock-like state can indeed be induced
experimentally by a single injection of LPS into animals. Molecules
produced by cells responding to LPS can target pathogens directly
or indirectly. Although these biological responses protect the host
against invading pathogens, they may also cause harm. Thus, massive
stimulation of innate immunity, occurring as a result of severe
Gram-negative bacterial infection, leads to excess production of
cytokines and other molecules, and the development of a fatal
syndrome, septic shock syndrome, which is characterized by fever,
hypotension, disseminated intravascular coagulation, and multiple
organ failure (Dumitru et al. Cell 103:1071-1083, 2000).
[0254] These toxic effects of LPS are mostly related to macrophage
activation leading to the release of multiple inflammatory
mediators. Among these mediators, TNF appears to play a crucial
role, as indicated by the prevention of LPS toxicity by the
administration of neutralizing anti-TNF antibodies (Beutler et al.,
Science 229:869, 1985). It is well established that lug injection
of E. coli LPS into a C57B1/6 mouse will result in significant
increases in circulating IL-6, TNF-alpha, IL-1, and acute phase
proteins (for example, SAA) approximately 2 hours post injection.
The toxicity of LPS appears to be mediated by these cytokines as
passive immunization against these mediators can result in
decreased mortality (Beutler et al., Science 229:869, 1985). The
potential immunointervention strategies for the prevention and/or
treatment of septic shock include anti-TNF mAb, IL-1 receptor
antagonist, LIF, IL-10, and G-CSF.
[0255] The administration of soluble IL-22RA2 comprising
polypeptides, such as Zcytor16-Fc4 or other IL-22RA soluble and
fusion proteins to these LPS-induced model was used to evaluate the
use of IL-22RA2 to ameliorate symptoms and alter the course of
LPS-induced disease. Moreover, the results showing inhibition of
IL-22 by IL-22RA2 provide proof of concept that other IL-22
antagonists, such as IL-22RA or antibodies thereto, can also be
used to ameliorate symptoms in the LPS-induced model and alter the
course of disease. The model showed induction of IL-22 by LPS
injection and the potential treatment of disease by IL-22RA2
polypeptides. Since LPS induces the production of pro-inflammatory
IL-22, SAA or other pro-inflammatory factors possibly contributing
to the pathology of endotoxemia, the neutralization of IL-22
activity, SAA or other pro-inflammatory factors by an antagonist
IL-22RA2 polypeptide can be used to reduce the symptoms of
endotoxemia, such as seen in endotoxic shock. Other potential
therapeutics include IL-22RA polypeptides, anti-IL-22RA antibodies,
or anti IL-22 antibodies or binding partners, and the like.
[0256] 3. Inflammatory Bowel Disease. IBD
[0257] In the United States approximately 500,000 people suffer
from Inflammatory Bowel Disease (IBD) which can affect either colon
and rectum (Ulcerative colitis) or both, small and large intestine
(Crohn's Disease). The pathogenesis of these diseases is unclear,
but they involve chronic inflammation of the affected tissues.
IL-22RA polypeptides, anti-IL-22RA antibodies, or anti IL-22
antibodies or binding partners, could serve as a valuable
therapeutic to reduce inflammation and pathological effects in IBD
and related diseases.
[0258] Ulcerative colitis (UC) is an inflammatory disease of the
large intestine, commonly called the colon, characterized by
inflammation and ulceration of the mucosa or innermost lining of
the colon. This inflammation causes the colon to empty frequently,
resulting in diarrhea. Symptoms include loosening of the stool and
associated abdominal cramping, fever and weight loss. Although the
exact cause of UC is unknown, recent research suggests that the
body's natural defenses are operating against proteins in the body
which the body thinks are foreign (an "autoimmune reaction").
Perhaps because they resemble bacterial proteins in the gut, these
proteins may either instigate or stimulate the inflammatory process
that begins to destroy the lining of the colon. As the lining of
the colon is destroyed, ulcers form releasing mucus, pus and blood.
The disease usually begins in the rectal area and may eventually
extend through the entire large bowel. Repeated episodes of
inflammation lead to thickening of the wall of the intestine and
rectum with scar tissue. Death of colon tissue or sepsis may occur
with severe disease. The symptoms of ulcerative colitis vary in
severity and their onset may be gradual or sudden. Attacks may be
provoked by many factors, including respiratory infections or
stress.
[0259] Although there is currently no cure for UC available,
treatments are focused on suppressing the abnormal inflammatory
process in the colon lining. Treatments including corticosteroids
immunosuppressives (eg. azathioprine, mercaptopurine, and
methotrexate) and aminosalicytates are available to treat the
disease. However, the long-term use of immunosuppressives such as
corticosteroids and azathioprine can result in serious side effects
including thinning of bones, cataracts, infection, and liver and
bone marrow effects. In the patients in whom current therapies are
not successful, surgery is an option. The surgery involves the
removal of the entire colon and the rectum.
[0260] There are several animal models that can partially mimic
chronic ulcerative colitis. The most widely used model is the
2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis
model, which induces chronic inflammation and ulceration in the
colon. When TNBS is introduced into the colon of susceptible mice
via intra-rectal instillation, it induces T-cell mediated immune
response in the colonic mucosa, in this case leading to a massive
mucosal inflammation characterized by the dense infiltration of
T-cells and macrophages throughout the entire wall of the large
bowel. Moreover, this histopathologic picture is accompanies by the
clinical picture of progressive weight loss (wasting), bloody
diarrhea, rectal prolapse, and large bowel wall thickening (Neurath
et al. Intern. Rev. Immunol. 19:51-62, 2000).
[0261] Another colitis model uses dextran sulfate sodium (DSS),
which induces an acute colitis manifested by bloody diarrhea,
weight loss, shortening of the colon and mucosal ulceration with
neutrophil infiltration. DSS-induced colitis is characterized
histologically by infiltration of inflammatory cells into the
lamina propria, with lymphoid hyperplasia, focal crypt damage, and
epithelial ulceration. These changes are thought to develop due to
a toxic effect of DSS on the epithelium and by phagocytosis of
lamina propria cells and production of TNF-alpha and IFN-gamma.
Despite its common use, several issues regarding the mechanisms of
DSS about the relevance to the human disease remain unresolved. DSS
is regarded as a T cell-independent model because it is observed in
T cell-deficient animals such as SCID mice.
[0262] The administration of soluble IL-22RA2 comprising
polypeptides, such as zcytor16-Fc4 or other IL-22RA soluble and
fusion proteins to these TNBS or DSS models can be used to evaluate
the use of IL-22RA to ameliorate symptoms and alter the course of
gastrointestinal disease. Moreover, the results showing inhibition
of IL-22 by IL-22RA2 provide proof of concept that other IL-22
antagonists, such as IL-22RA or antibodies thereto, can also be
used to ameliorate symptoms in the colitis/IBD models and alter the
course of disease. We observed the increased expression of IL-22 in
colon tissues of DSS-mice by RT-PCR, and the synergistic activity
of IL-22 with IL-1beta on intestinal cell lines. It indicates IL-22
may play a role in the inflammatory response in colitis, and the
neutralization of IL-22 activity by administrating IL-22RA2
polypeptides is a potential therapeutic approach for IBD. Other
potential therapeutics include IL-22RA polypeptides, anti-IL-22RA
antibodies, or anti IL-22 antibodies or binding partners, and the
like.
[0263] 4. Psoriasis
[0264] Psoriasis is a chronic skin condition that affects more than
seven million Americans. Psoriasis occurs when new skin cells grow
abnormally, resulting in inflamed, swollen, and scaly patches of
skin where the old skin has not shed quickly enough. Plaque
psoriasis, the most common form, is characterized by inflamed
patches of skin ("lesions") topped with silvery white scales.
Psoriasis may be limited to a few plaques or involve moderate to
extensive areas of skin, appearing most commonly on the scalp,
knees, elbows and trunk. Although it is highly visible, psoriasis
is not a contagious disease. The pathogenesis of the diseases
involves chronic inflammation of the affected tissues. IL-22RA
polypeptides, anti-IL-22RA antibodies, or anti IL-22 and anti IL-20
antibodies or binding partners, could serve as a valuable
therapeutic to reduce inflammation and pathological effects in
psoriasis, other inflammatory skin diseases, skin and mucosal
allergies, and related diseases.
[0265] Psoriasis is a T-cell mediated inflammatory disorder of the
skin that can cause considerable discomfort. It is a disease for
which there is no cure and affects people of all ages. Psoriasis
affects approximately two percent of the populations of European
and North America. Although individuals with mild psoriasis can
often control their disease with topical agents, more than one
million patients worldwide require ultraviolet or systemic
immunosuppressive therapy. Unfortunately, the inconvenience and
risks of ultraviolet radiation and the toxicities of many therapies
limit their long-term use. Moreover, patients usually have
recurrence of psoriasis, and in some cases rebound, shortly after
stopping immunosuppressive therapy.
[0266] IL-20 is a novel IL-10 homologue that was shown to cause
neonatal lethality with skin abnormalities including aberrant
epidermal differentiation in IL-20 transgenic mice (Blumberg H et
al., Cell 104:9-19, 2001) IL-20 receptor is dramatically
upregulated in psoriatic skin. Since IL-22 shares a receptor
subunit (zcytor11) with IL-20 receptor, and IL-22 transgenic mice
display a similar phenotype, it is possible that IL-22 is also
involved in the inflammatory skin diseases such as psoriasis. The
administration of IL-22RA polypeptide or an anti-IL-22RA antibody
antagonist, either subcutaneous or topically, may potential reduce
the inflammation and symptom. Other potential therapeutics include
IL-22RA polypeptides, soluble zcytor11/CRF2-4 receptor
polypeptides, or anti IL-22 antibodies or binding partners, and the
like.
[0267] Moreover, over expression of IL-22 and IL-20 was shown in
human psoriatic lesions, suggesting that IL-22, like IL-20 is also
involved in human psoriasis. Moreover, as described herein, over
expression of IL-20 or IL-22 in transgenic mice showed epidermal
thickening and immune cell involvement indicative of a psoriatic
phenotype; and in addition injection of IL-22 into normal mice
showed epidermal thickening and immune cell involvement indicative
of a psoriatic phenotype which was ablated by the soluble receptor
antagonist IL-22RA2 (zcytor16; WIPO Publication No. WO 01/40467).
Such in vivo data further suggests that the pro-inflammatory IL-22
is involved in psoriasis. As such, antagonists to IL-22 and IL-20
activity, such as IL-22RA soluble receptors and antibodies thereto
including the anti-human-IL-22RA monoclonal and neutralizing
antibodies of the present invention, are useful in therapeutic
treatment of inflammatory diseases, particularly as antagonists to
both IL-22 and IL-20 singly or together in the treatment of
psoriasis. Moreover, antagonists to IL-22 activity, such as IL-22RA
soluble receptors and antibodies thereto including the
anti-human-IL-22RA monoclonal and neutralizing antibodies of the
present invention, are useful in therapeutic treatment of other
inflammatory diseases for example as agents that bind to, block,
inhibit, reduce, antagonize or neutralize IL-22 and IL-20 in the
treatment of atopic dermatitis, IBD, colitis, Endotoxemia,
arthritis, rheumatoid arthritis, and psoriatic arthritis adult
respiratory disease (ARD), septic shock, multiple organ failure,
inflammatory lung injury such as asthma or bronchitis, bacterial
pneumonia, psoriasis, eczema, atopic and contact dermatitis, and
inflammatory bowel disease such as ulcerative colitis and Crohn's
disease.
[0268] Moreover, anti-IL-22RA antibodies and IL-22RA soluble
receptors of the present invention can be used in the prevention
and therapy against weight loss associated with a number of
inflammatory diseases described herein, as well as for cancer
(e.g., chemotherapy and cachexia), and infectious diseases. For
example, severe weight loss is a key marker associated with models
for septicemia, MS, RA, and tumor models. In addition, weight loss
is a key parameter for many human diseases including cancer,
infectious disease and inflammatory disease. Weight loss was shown
in mice injected with IL-22Adenovirus described herein. Anti-IL-22
antibodies and IL-22 antagonists such as the soluble IL-22RA
receptors and antibodies thereto of the present invention, as well
as zcytor16 (IL-22RA2) receptors, can be tested for their ability
to prevent and treat weight loss in mice injected with IL-22
adenovirus described herein. Methods of determining a prophylactic
or therapeutic regimen for such IL-22 antagonists is known in the
art and can be determined using the methods described herein.
[0269] IL-22RA soluble receptor polypeptides and antibodies thereto
may also be used within diagnostic systems for the detection of
circulating levels of IL-22 or IL-20 ligand, and in the detection
of IL-22 associated with acute phase inflammatory response. Within
a related embodiment, antibodies or other agents that specifically
bind to IL-22RA soluble receptors of the present invention can be
used to detect circulating receptor polypeptides; conversely,
IL-22RA soluble receptors themselves can be used to detect
circulating or locally-acting IL-22 or IL-20 polypeptides. Elevated
or depressed levels of ligand or receptor polypeptides may be
indicative of pathological conditions, including inflammation or
cancer. IL-22 is known to induce associated acute phase
inflammatory response. Moreover, detection of acute phase proteins
or molecules such as IL-20 or IL-22 can be indicative of a chronic
inflammatory condition in certain disease states (e.g., psoriasis,
rheumatoid arthritis, colitis, IBD). Detection of such conditions
serves to aid in disease diagnosis as well as help a physician in
choosing proper therapy.
[0270] In utero administration of neutralizing anti-IL-22 or IL-20
antibodies can be used to show efficacy in vivo in disease models
by reducing or eliminating the skin phenotype found IL-22
transgenic pups which over express IL-22, or IL-20 transgenic pups
which over express IL-20. There are precedents in the art for in
utero treatment with neutralizing monoclonal antibodies (mAbs). In
one case, the development of the B-1 subset of B cells was
dramatically affected by treating pregnant female mice with a mAb
specific for the B cell-specific molecule, CD19 (e.g., Krop I. Et
al., Eur. J. Immunol. 26(1):238-42, 1996). Krop et al. injected
timed pregnant mice intraperitoneally with 500 ug of rat anti-mouse
CD19 mAb (or a rat isotype-matched control Ab) in PBS beginning on
day 9 of gestation, with subsequent injections every other day
until birth. Pups were also injected once with 500 ug of these
antibodies at 10 days of age. In another case, Tanaka et al., found
that in utero treatment with monoclonal antibody to IL-2 receptor
beta-chain completely abrogates development of Thy-1+ dendritic
epidermal cells. The two distinct subunits of the IL-2 receptor,
i.e. the alpha-chain (IL-2R alpha) and the beta-chain (IL-2R beta),
are expressed in an almost mutually exclusive fashion throughout
fetal thymus ontogeny. Blocking IL-2R beta, a signal transducing
component of IL-2R, by administering a neutralizing mAb to IL-2R
beta, resulted in the complete and selective disappearance of
Thy-1+ skin dendritic epidermal cells. Development of any other T
cell subsets was uncompromised. This indicated that IL-2 plays a
crucial role in the development of fetal V gamma 5+ cells and their
descendants (see, Tanaka, T. et al., Int Immunol. 4(4):487-9,
1992). In addition, Schattemann G C et al., showed that PDGF-A is
required for normal murine cardiovascular development using an in
utero system. Several lines of evidence suggest that
platelet-derived growth factor A chain (PDGF-A) is required for
normal embryonic cardiovascular development. Introduction of
anti-PDGF-A neutralizing antibodies into mouse deciduas in utero
resulted in the selective disruption of PDGF-A ligand-receptor
interactions in vivo for a period of 18-24 hr and allowed
assessment of whether PDGF-A is required for cardiovascular
development and when it is required (see, Schattemann G C et al.,
Dev. Biol. 176(1):133-42, 1996). These results, as well as others
described in the art, provide evidence that neutralizing mAbs can
elicit strong effects in utero. Similarly, data showing the
efficacy of neutralizing IL-20 or IL-22 with monoclonal antibodies
in vivo in disease models to reduce or eliminate the skin phenotype
found in IL-20 and IL-22 transgenic pups which over express IL-20
and IL-22 respectively can be shown. These transgenic mice are born
with a "shiny" skin appearance, due at least in part to a
thickening of the epidermis as described herein. The IL-20 TG pups
expressing fairly low levels of the transgenic cytokine can recover
and do survive to breed, but the IL-22 TG mice die shortly after
birth, generally before 5 days of age.
[0271] For example, neutralizing antibodies to IL-20 include
antibodies, such as neutralizing monoclonal antibodies that can
bind IL-20 antigenic epitopes and neutralize IL-20 activity.
Accordingly, antigenic epitope-bearing peptides and polypeptides of
IL-20 are useful to raise antibodies that bind with the IL-20
polypeptides described herein, as well as to identify and screen
anti-IL-20 monoclonal antibodies that are neutralizing, and that
may bind, block, inhibit, reduce, antagonize or neutralize the
activity of IL-20. Such neutralizing monoclonal antibodies of the
present invention can bind to an IL-20 antigenic epitope. Such
epitopes within SEQ ID NO:8 as predicted by a Jameson-Wolf plot,
e.g., using DNASTAR Protean program (DNASTAR, Inc., Madison, Wis.)
serve as preferred antigenic epitopes, and can be determined by one
of skill in the art. Such antigenic epitopes include: amino acid
residues 42 (Ile) to 102 (Asp) of SEQ ID NO:8; amino acid residues
42 (Ile) to 60 (Ile) of SEQ ID NO:8; amino acid residues 42 (Ile)
to 69 (Glu) of SEQ ID NO:8; amino acid residues 42 (Ile) to 81
(Cys) of SEQ ID NO:8; amino acid residues 42 (Ile) to 96 (Lys) of
SEQ ID NO:8; amino acid residues 42 (Ile) to 102 (Asp) of SEQ ID
NO:8; amino acid residues 60 (Ile) to 69 (Glu) of SEQ ID NO:8;
amino acid residues 60 (Ile) to 81 (Cys) of SEQ ID NO:8; amino acid
residues 60 (Ile) to 96 (Lys) of SEQ ID NO:8; amino acid residues
60 (Ile) to 102 (Asp) of SEQ ID NO:8; amino acid residues 69 (Glu)
to 81 (Cys) of SEQ ID NO:8; amino acid residues 69 (Glu) to 96
(Lys) of SEQ ID NO:8; amino acid residues 69 (Glu) to 102 (Asp) of
SEQ ID NO:8; amino acid residues 81 (Cys) to 96 (Lys) of SEQ ID
NO:8; amino acid residues 81 (Cys) to 102 (Asp) of SEQ ID NO:8; and
amino acid residues 96 (Lys) to 102 (Asp) of SEQ ID NO:8.
[0272] In addition to other disease models described herein, the
activity of anti-IL-22RA antibodies on inflammatory tissue derived
from human psoriatic lesions can be measured in vivo using a severe
combined immune deficient (SCID) mouse model. Several mouse models
have been developed in which human cells are implanted into
immunodeficient mice (collectively referred to as xenograft
models); see, for example, Cattan A R, Douglas E, Leuk. Res.
18:513-22, 1994 and Flavell, D J, Hematological Oncology 14:67-82,
1996. As an in vivo xenograft model for psoriasis, human psoriatic
skin tissue is implanted into the SCID mouse model, and challenged
with an appropriate antagonist. Moreover, other psoriasis animal
models in the art may be used to evaluate IL-20 and IL-22
antagonists, such as human psoriatic skin grafts implanted into
AGR129 mouse model, and challenged with an appropriate antagonist
(e.g., see, Boyman, O. et al., J. Exp. Med. Online publication
#20031482, 2004, incorporated herein by reference). Anti-IL-22RA
antibodies that bind, block, inhibit, reduce, antagonize or
neutralize the activity of IL-22 or both IL-20 and IL-22 are
preferred antagonists, however, anti-IL-20 and anti-IL-22
antibodies (alone or in combination), soluble IL-22RA, as well as
other IL-20 and IL-22 antagonists can be used in this model.
Similarly, tissues or cells derived from human colitis, IBD,
arthritis, or other inflammatory lesions can be used in the SCID
model to assess the anti-inflammatory properties of the IL-20 and
IL-22 antagonists described herein.
[0273] Therapies designed to abolish, retard, or reduce
inflammation using anti-IL-22RA antibodies or its derivatives,
agonists, conjugates or variants can be tested by administration of
anti-IL-22RA antibodies or soluble IL-22RA compounds to SCID mice
bearing human inflammatory tissue (e.g., psoriatic lesions and the
like), or other models described herein. Efficacy of treatment is
measured and statistically evaluated as increased anti-inflammatory
effect within the treated population over time using methods well
known in the art. Some exemplary methods include, but are not
limited to measuring for example, in a psoriasis model, epidermal
thickness, the number of inflammatory cells in the upper dermis,
and the grades of parakeratosis. Such methods are known in the art
and described herein. For example, see Zeigler, M. et al. Lab
Invest 81:1253, 2001; Zollner, T. M. et al. J. Clin. Invest.
109:671, 2002; Yamanaka, N. et al. Microbiol. Immunol. 45:507,
2001; Raychaudhuri, S. P. et al. Br. J. Dermatol. 144:931, 2001;
Boehncke, W. H et al. Arch. Dermatol. Res. 291:104, 1999; Boehncke,
W. H et al. J. Invest. Dermatol. 116:596, 2001; Nickoloff, B. J. et
al. Am. J. Pathol. 146:580, 1995; Boehncke, W. H et al. J. Cutan.
Pathol. 24:1, 1997; Sugai, J., M. et al. J. Dermatol. Sci. 17:85,
1998; and Villadsen L. S. et al. J. Clin. Invest. 112:1571, 2003.
Inflammation may also be monitored over time using well-known
methods such as flow cytometry (or PCR) to quantitate the number of
inflammatory or lesional cells present in a sample, score (weight
loss, diarrhea, rectal bleeding, colon length) for IBD, paw disease
score and inflammation score for CIA RA model. For example,
therapeutic strategies appropriate for testing in such a model
include direct treatment using anti-IL-22RA antibodies, other IL-20
and IL-22 antagonists (singly or together), or related conjugates
or antagonists based on the disrupting interaction of anti-IL-22RA
antibodies with its ligands IL-20 and IL-22, or for cell-based
therapies utilizing anti-IL-22RA antibodies or its derivatives,
agonists, conjugates or variants.
[0274] Moreover, Psoriasis is a chronic inflammatory skin disease
that is associated with hyperplastic epidermal keratinocytes and
infiltrating mononuclear cells, including CD4+ memory T cells,
neutrophils and macrophages (Christophers, Int. Arch. Allergy
Immunol., 110: 199, 1996). It is currently believed that
environmental antigens play a significant role in initiating and
contributing to the pathology of the disease. However, it is the
loss of tolerance to self-antigens that is thought to mediate the
pathology of psoriasis. Dendritic cells and CD4.sup.+ T cells are
thought to play an important role in antigen presentation and
recognition that mediate the immune response leading to the
pathology. We have recently developed a model of psoriasis based on
the CD4+CD45RB transfer model (Davenport et al., Internat.
Immunopharmacol., 2:653-672). Anti-IL20, anti-IL22 or antibodies to
IL20R, and/or IL22R, such as anti-IL-22RA antibodies of the present
invention, or soluble IL-22RA, are administered to the mice.
Inhibition of disease scores (skin lesions, inflammatory cytokines)
indicates the effectiveness of IL-20 and IL-22 antagonists in
psoriasis, e.g., anti-IL-22RA antibodies or IL-22RA soluble
receptors, or other antagonists such as antibodies against IL20
and/or IL-22 or their receptors.
[0275] 5. Atopic Dermatitis.
[0276] Both IL-20 and IL-22 are upregulated in human atopic
dermatitis (AD) patient samples. AD is a common chronic
inflammatory disease that is characterized by hyperactivated
cytokines of the helper T cell subset 2 (Th2). Although the exact
etiology of AD is unknown, multiple factors have been implicated,
including hyperactive Th2 immune responses, autoimmunity,
infection, allergens, and genetic predisposition. Key features of
the disease include xerosis (dryness of the skin), pruritus
(itchiness of the skin), conjunctivitis, inflammatory skin lesions,
Staphylococcus aureus infection, elevated blood eosinophilia,
elevation of serum IgE and IgG1, and chronic dermatitis with T
cell, mast cell, macrophage and eosinophil infiltration.
Colonization or infection with S. aureus has been recognized to
exacerbate AD and perpetuate chronicity of this skin disease.
[0277] AD is often found in patients with asthma and allergic
rhinitis, and is frequently the initial manifestation of allergic
disease. About 20% of the population in Western countries suffer
from these allergic diseases, and the incidence of AD in developed
countries is rising for unknown reasons. AD typically begins in
childhood and can often persist through adolescence into adulthood.
Current treatments for AD include topical corticosteroids, oral
cyclosporin A, non-corticosteroid immunosuppressants such as
tacrolimus (FK506 in ointment form), and interferon-gamma. Despite
the variety of treatments for AD, many patients' symptoms do not
improve, or they have adverse reactions to medications, requiring
the search for other, more effective therapeutic agents. The
soluble IL-22RA polypeptides and anti-IL-22RA antibodies of the
present invention, including the neutralizing anti-human IL-22RA
antibodies of the present invention, can be used to neutralize
IL-22 and IL-20 in the treatment of specific human diseases such as
atoptic dermatitis, inflammatory skin conditions, and other
inflammatory conditions disclosed herein.
[0278] For pharmaceutical use, the soluble IL-22RA or anti-IL-22RA
antibodies of the present invention are formulated for parenteral,
particularly intravenous or subcutaneous, delivery according to
conventional methods. Intravenous administration will be by bolus
injection, controlled release, e.g, using mini-pumps or other
appropriate technology, or by infusion over a typical period of one
to several hours. In general, pharmaceutical formulations will
include a hematopoietic protein in combination with a
pharmaceutically acceptable vehicle, such as saline, buffered
saline, 5% dextrose in water or the like. Formulations may further
include one or more excipients, preservatives, solubilizers,
buffering agents, albumin to provent protein loss on vial surfaces,
etc. When utilizing such a combination therapy, the cytokines may
be combined in a single formulation or may be administered in
separate formulations. Methods of formulation are well known in the
art and are disclosed, for example, in Remington's Pharmaceutical
Sciences, Gennaro, ed., Mack Publishing Co., Easton Pa., 1990,
which is incorporated herein by reference. Therapeutic doses will
generally be in the range of 0.1 to 100 mg/kg of patient weight per
day, preferably 0.5-20 mg/kg per day, with the exact dose
determined by the clinician according to accepted standards, taking
into account the nature and severity of the condition to be
treated, patient traits, etc. Determination of dose is within the
level of ordinary skill in the art. The proteins will commonly be
administered over a period of up to 28 days following chemotherapy
or bone-marrow transplant or until a platelet count of
>20,000/mm.sup.3, preferably >50,000/mm.sup.3, is achieved.
More commonly, the proteins will be administered over one week or
less, often over a period of one to three days. In general, a
therapeutically effective amount of soluble IL-22RA or anti-IL-22RA
antibodies of the present invention is an amount sufficient to
produce a clinically significant increase in the proliferation
and/or differentiation of lymphoid or myeloid progenitor cells,
which will be manifested as an increase in circulating levels of
mature cells (e.g. platelets or neutrophils). Treatment of platelet
disorders will thus be continued until a platelet count of at least
20,000/mm.sup.3, preferably 50,000/mm.sup.3, is reached. The
soluble IL-22RA or anti-IL-22RA antibodies of the present invention
can also be administered in combination with other cytokines such
as IL-3, -6 and -11; stem cell factor; erythropoietin; G-CSF and
GM-CSF. Within regimens of combination therapy, daily doses of
other cytokines will in general be: EPO, 150 U/kg; GM-CSF, 5-15
lg/kg; IL-3, 1-5 lg/kg; and G-CSF, 1-25 lg/kg. Combination therapy
with EPO, for example, is indicated in anemic patients with low EPO
levels.
[0279] Generally, the dosage of administered soluble IL-22RA (or
IL-22RA analog or fusion protein) or anti-IL-22RA antibodies 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 soluble IL-22RA or anti-IL-22RA antibodies 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.
[0280] Administration of soluble IL-22RA or anti-IL-22RA antibodies
to a subject can be 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.
[0281] 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-22RA 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-22RA binding
activity (Potts et al., Pharm. Biotechnol. 10:213 (1997)).
[0282] A pharmaceutical composition comprising a soluble IL-22RA or
anti-IL-22RA antibody 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 carrier. A composition is said to be a
"pharmaceutically acceptable carrier" if its administration can be
tolerated by a recipient patient. Sterile phosphate-buffered saline
is one example of a pharmaceutically acceptable carrier. Other
suitable carriers are well-known to those in the art. See, for
example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th
Edition (Mack Publishing Company 1995).
[0283] For purposes of therapy, soluble IL-22RA or anti-IL-22RA
antibody molecules and a pharmaceutically acceptable carrier are
administered to a patient in a therapeutically effective amount. A
combination of a therapeutic molecule of the present invention and
a pharmaceutically acceptable carrier is said to be administered in
a "therapeutically 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 the inflammatory response.
[0284] A pharmaceutical composition comprising IL-22RA (or IL-22RA
analog or fusion protein) or neutralizing anti-IL-22RA antibody can
be furnished in liquid form, in an aerosol, or in solid form.
Liquid forms, are illustrated by injectable 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)).
[0285] 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.
[0286] 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.
[0287] 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)).
[0288] 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)).
[0289] 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. 20:259 (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.
[0290] 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)).
[0291] Polypeptides and antibodies 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)).
[0292] 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 carriers for intravenous
administration of therapeutic proteins (see, for example, Gref et
al, Pharm. Biotechnol 10:167 (1997)).
[0293] The present invention also contemplates chemically modified
polypeptides having binding IL-22RA activity such as IL-22RA
monomeric, homodimeric, heterodimeric or multimeric soluble
receptors, and IL-22RA antagonists, for example anti-IL-22RA
antibodies or binding polypeptides, or neutralizing anti-IL-22RA
antibodies, which a polypeptide is linked with a polymer, as
discussed above.
[0294] 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).
[0295] As an illustration, pharmaceutical compositions may be
supplied as a kit comprising a container that comprises a
polypeptide with a IL-22RA extracellular domain, e.g., IL-22RA
monomeric, homodimeric, heterodimeric or multimeric soluble
receptors, or a IL-22RA antagonist (e.g., an antibody or antibody
fragment that binds a IL-22RA polypeptide, or neutralizing
anti-IL-22RA antibody). 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-22RA composition is contraindicated in patients with
known hypersensitivity to IL-22RA.
[0296] A pharmaceutical composition comprising Anti-IL-22RA
antibodies or binding partners (or Anti-IL-22RA antibody fragments,
antibody fusions, humanized antibodies and the like), or IL-22RA
soluble receptor, 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.
[0297] 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.
[0298] 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.
[0299] 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)).
[0300] 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)).
[0301] 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. 20:259 (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.
[0302] 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)).
[0303] Anti-IL-22RA neutralizing antibodies and binding partners
with IL-220R IL-20 binding activity, or IL-22RA soluble receptor,
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)).
[0304] 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 carriers for intravenous
administration of therapeutic proteins (see, for example, Gref et
al., Pharm. Biotechnol. 10:167 (1997)).
[0305] The present invention also contemplates chemically modified
Anti-IL-22RA antibody or binding partner, for example
anti-Anti-IL-22RA antibodies or IL-22RA soluble receptor, linked
with a polymer, as discussed above.
[0306] 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).
[0307] The present invention contemplates compositions of
anti-IL-22 antibodies, and methods and therapeutic uses comprising
an antibody, peptide or polypeptide described herein. Such
compositions can further comprise a carrier. The carrier can be a
conventional organic or inorganic carrier. Examples of carriers
include water, buffer solution, alcohol, propylene glycol,
macrogol, sesame oil, corn oil, and the like.
11. Production of Transgenic Mice
[0308] Over expression of both IL-20 and IL-22 was shown in human
psoriatic lesions, suggesting that both IL-20 and IL-22 are
involved in human psoriasis. Moreover, as described herein, over
expression of IL-20 and IL-22 in transgenic mice showed epidermal
thickening and immune cell involvement indicative of a psoriatic
phenotype; and in addition injection of IL-22 into normal mice
showed epidermal thickening and immune cell involvement indicative
of a psoriatic phenotype which was ablated by the soluble receptor
antagonist zcytor16 (IL-22RA2). Such in vivo data further suggests
that the pro-inflammatory IL-22 is involved in psoriasis. As such,
antagonists to IL-22 activity, such as the anti-human-IL-22RA
neutralizing and monoclonal antibodies of the present invention, as
well as soluble IL-22RA receptors, are useful in therapeutic
treatment of inflammatory diseases, particularly as antagonists to
IL-22 and IL-20 in the treatment of psoriasis. Moreover, agents
that bind to, block, inhibit, reduce, antagonize or neutralize
IL-22 or both IL-20 and IL-22 activity, such as the
anti-human-IL-22RA neutralizing and monoclonal antibodies of the
present invention, as well as soluble IL-22RA receptors, are useful
in therapeutic treatment of other inflammatory diseases for example
as antagonists to IL-22 or both IL-20 and IL-22 in the treatment of
atopic dermatitis, IBD, colitis, Endotoxemia, arthritis, rheumatoid
arthritis, and psoriatic arthritis adult respiratory disease (ARD),
septic shock, multiple organ failure, inflammatory lung injury such
as asthma or bronchitis, bacterial pneumonia, psoriasis, eczema,
atopic and contact dermatitis, and inflammatory bowel disease such
as ulcerative colitis and Crohn's disease, and the like.
[0309] Within one aspect, the present invention provides a method
of producing an antibody to a polypeptide comprising: inoculating
an animal with a polypeptide selected from the group consisting of:
a polypeptide consisting of amino acid residues 42 (Ile) to 102
(Asp) of SEQ ID NO:8; a polypeptide consisting of amino acid
residues 42 (Ile) to 60 (Ile) of SEQ ID NO:8; a polypeptide
consisting of amino acid residues 42 (Ile) to 69 (Glu) of SEQ ID
NO:8; a polypeptide consisting of amino acid residues 42 (Ile) to
81 (Cys) of SEQ ID NO:8; a polypeptide consisting of amino acid
residues 42 (Ile) to 96 (Lys) of SEQ ID NO:8; a polypeptide
consisting of amino acid residues 42 (Ile) to 102 (Asp) of SEQ ID
NO:8; a polypeptide consisting of amino acid residues 60 (Ile) to
69 (Glu) of SEQ ID NO:8; a polypeptide consisting of amino acid
residues 60 (Ile) to 81 (Cys) of SEQ ID NO:8; a polypeptide
consisting of amino acid residues 60 (Ile) to 96 (Lys) of SEQ ID
NO:8; a polypeptide consisting of amino acid residues 60 (Ile) to
102 (Asp) of SEQ ID NO:8; a polypeptide consisting of amino acid
residues 69 (Glu) to 81 (Cys) of SEQ ID NO:8; a polypeptide
consisting of amino acid residues 69 (Glu) to 96 (Lys) of SEQ ID
NO:8; a polypeptide consisting of amino acid residues 69 (Glu) to
102 (Asp) of SEQ ID NO:8; a polypeptide consisting of amino acid
residues 81 (Cys) to 96 (Lys) of SEQ ID NO:8; a polypeptide
consisting of amino acid residues 81 (Cys) to 102 (Asp) of SEQ ID
NO:8; and a polypeptide consisting of amino acid residues 96 (Lys)
to 102 (Asp) of SEQ ID NO:8, wherein the polypeptide elicits an
immune response in the animal to produce the antibody; and
isolating the antibody from the animal; and wherein the antibody
specifically binds to an IL-20 polypeptide (SEQ ID NO:8).
[0310] Within one embodiment is provided the method as described
above, wherein the antibody produced by the method inhibits the
pro-inflammatory activity of IL-20 (SEQ ID NO:8). Within another
embodiment is provided the method as described above, wherein the
antibody reduces the pro-inflammatory activity of IL-20 (SEQ ID
NO:8). Within yet another embodiment is provided the method as
described above, wherein the antibody neutralizes the interaction
of IL-20 (SEQ ID NO:8) with IL-22RA (SEQ ID NO:2). Specifically,
the neutralization by the antibody is measured by showing
neutralization of IL-20 (SEQ ID NO:8) in an in vitro a cell-based
neutralization assay.
[0311] Within a second aspect, the present invention provides an
antibody produced by the method as disclosed above, which binds to
a polypeptide of SEQ ID NO:8. Within one embodiment is provided the
antibody as described above, wherein the antibody is selected from
the group consisting of: (a) a polyclonal antibody, (b) a murine
monoclonal antibody, (c) a humanized antibody derived from (b), (d)
an antibody fragment, and (e) a human monoclonal antibody. Within
one embodiment, the antibody further comprises a radionuclide,
enzyme, substrate, cofactor, fluorescent marker, chemiluminescent
marker, peptide tag, magnetic particle, or toxin. Within another
embodiment, the antibody further comprises PEGylation.
[0312] Within a third aspect, the present invention provides an
antibody or antibody fragment that binds to a polypeptide
comprising a sequence of amino acid residues as shown in SEQ ID
NO:8; and reduces the pro-inflammatory activity of IL-20 (SEQ ID
NO:8). Within one embodiment is provided an antibody or antibody
fragment that reduces the pro-inflammatory activity of either IL-20
(SEQ ID NO:8) or IL-22 (SEQ ID NO:6). Within one embodiment is
provided the antibody as described above, wherein the antibody is
selected from the group consisting of: (a) a polyclonal antibody,
(b) a murine monoclonal antibody, (c) a humanized antibody derived
from (b), (d) an antibody fragment, and (e) a human monoclonal
antibody. Within one embodiment, the antibody further comprises a
radionuclide, enzyme, substrate, cofactor, fluorescent marker,
chemiluminescent marker, peptide tag, magnetic particle, or toxin.
Within another embodiment, the antibody further comprises
PEGylation.
[0313] Within a fourth aspect, the present invention provides a
method for reducing or inhibiting IL-20-induced proliferation or
differentiation of hematopoietic cells and hematopoietic cell
progenitors comprising culturing bone marrow or peripheral blood
cells with a composition comprising an amount of an antibody as
described above, sufficient to reduce proliferation or
differentiation of the hematopoietic cells in the bone marrow or
peripheral blood cells as compared to bone marrow or peripheral
blood cells cultured in the absence of the antibody. Within one
embodiment, the hematopoietic cells and hematopoietic progenitor
cells are lymphoid cells. Within yet another embodiment, the
lymphoid cells are macrophages or T cells.
[0314] Within a fifth aspect, the present invention provides a
method of reducing IL-20-induced inflammation comprising
administering to a mammal with inflammation an amount of a
composition of an antibody as described above, sufficient to reduce
inflammation.
[0315] Within a sixth aspect, the present invention provides a
method of suppressing an inflammatory response in a mammal with
inflammation comprising: (1) determining a level of serum amyloid A
protein; (2) administering a composition comprising an antibody
according to claim 3 in an acceptable pharmaceutical vehicle; (3)
determining a post administration level of serum amyloid A protein;
(4) comparing the level of serum amyloid A protein in step (1) to
the level of serum amyloid A protein in step (3), wherein a lack of
increase or a decrease in serum amyloid A protein level is
indicative of suppressing an inflammatory response.
[0316] Within a seventh aspect, the present invention provides a
method of suppressing an inflammatory response in a mammal with
inflammation comprising: (1) determining a level of serum amyloid A
protein; (2) administering a composition comprising an antibody
according to claim 5 in an acceptable pharmaceutical vehicle; (3)
determining a post administration level of serum amyloid A protein;
(4) comparing the level of serum amyloid A protein in step (1) to
the level of serum amyloid A protein in step (3), wherein a lack of
increase or a decrease in serum amyloid A protein level is
indicative of suppressing an inflammatory response.
[0317] Within an eight aspect, the present invention provides a
method of suppressing an inflammatory response in a mammal with
inflammation comprising: (1) determining a level of serum amyloid A
protein; (2) administering a composition comprising an antibody
according to claim 16 in an acceptable pharmaceutical vehicle; (3)
determining a post administration level of serum amyloid A protein;
(4) comparing the level of serum amyloid A protein in step (1) to
the level of serum amyloid A protein in step (3), wherein a lack of
increase or a decrease in serum amyloid A protein level is
indicative of suppressing an inflammatory response.
[0318] Within a ninth aspect, the present invention provides a
method of treating a mammal afflicted with an inflammatory disease
in which IL-20 plays a role, comprising administering an antagonist
IL-20 to the mammal such that the inflammation is reduced, wherein
the antagonist comprising and antibody, antibody fragment, or
binding polypeptide that specifically binds a polypeptide or
polypeptide fragment of IL-22RA (SEQ ID NO:3) or is a polypeptide
or polypeptide fragment of IL-22RA (SEQ ID NO:3); and wherein the
inflammatory activity of IL-20 (SEQ ID NO:8) is reduced. Within one
embodiment, the disease is a chronic inflammatory disease. Within
another embodiment, the chronic inflammatory disease is selected
from inflammatory bowel disease, ulcerative colitis, Crohn's
disease, arthritis, atopic dermatitis, or psoriasis. Within yet
another embodiment, the disease is an acute inflammatory disease.
Within another embodiment the acute inflammatory disease is
selected from endotoxemia, septicemia, toxic shock syndrome or
infectious disease. Within still another embodiment, the method
provided the method as described above, wherein the antibody
further comprises a radionuclide, enzyme, substrate, cofactor,
fluorescent marker, chemiluminescent marker, peptide tag, magnetic
particle, drug, or toxin.
[0319] Within a tenth aspect, the present invention provides an
antibody comprising a monoclonal antibody that that binds to an
epitope of human IL-20 (SEQ ID NO:8) selected from the group
consisting of: a polypeptide consisting of amino acid residues 42
(Ile) to 102 (Asp) of SEQ ID NO:8; a polypeptide consisting of
amino acid residues 42 (Ile) to 60 (Ile) of SEQ ID NO:8; a
polypeptide consisting of amino acid residues 42 (Ile) to 69 (Glu)
of SEQ ID NO:8; a polypeptide consisting of amino acid residues 42
(Ile) to 81 (Cys) of SEQ ID NO:8; a polypeptide consisting of amino
acid residues 42 (Ile) to 96 (Lys) of SEQ ID NO:8; a polypeptide
consisting of amino acid residues 42 (Ile) to 102 (Asp) of SEQ ID
NO:8; a polypeptide consisting of amino acid residues 60 (Ile) to
69 (Glu) of SEQ ID NO:8; a polypeptide consisting of amino acid
residues 60 (Ile) to 81 (Cys) of SEQ ID NO:8; a polypeptide
consisting of amino acid residues 60 (Ile) to 96 (Lys) of SEQ ID
NO:8; a polypeptide consisting of amino acid residues 60 (Ile) to
102 (Asp) of SEQ ID NO:8; a polypeptide consisting of amino acid
residues 69 (Glu) to 81 (Cys) of SEQ ID NO:8; a polypeptide
consisting of amino acid residues 69 (Glu) to 96 (Lys) of SEQ ID
NO:8; a polypeptide consisting of amino acid residues 69 (Glu) to
102 (Asp) of SEQ ID NO:8; a polypeptide consisting of amino acid
residues 81 (Cys) to 96 (Lys) of SEQ ID NO:8; a polypeptide
consisting of amino acid residues 81 (Cys) to 102 (Asp) of SEQ ID
NO:8; and a polypeptide consisting of amino acid residues 96 (Lys)
to 102 (Asp) of SEQ ID NO:8. Within another embodiment is provided
the antibody as described above, wherein the antibody reduces or
neutralizes the activity of human IL-20 (SEQ ID NO:8). Within yet
another embodiment, the antibody is selected from the group
consisting of: (a) a murine monoclonal antibody, (b) a humanized
antibody derived from (a), (c) an antibody fragment, and (d) a
human monoclonal antibody. Within another embodiment, the antibody
further comprises PEGylation.
[0320] Within an eleventh aspect, a method is provided for treating
a pathological condition in a subject associated with IL-20
activity comprising administering an effective amount of the
antibody as described above, thereby treating said pathological
condition. Within one embodiment, the pathological condition is a
chronic inflammatory condition. Within another embodiment, the
chronic inflammatory condition is selected from inflammatory bowel
disease, ulcerative colitis, Crohn's disease, arthritis, atopic
dermatitis, or psoriasis. Within another embodiment, the
pathological condition is an acute inflammatory condition. Within
still another embodiment, the acute inflammatory condition is
selected from endotoxemia, septicemia, toxic shock syndrome, or
infectious disease.
[0321] The invention is further illustrated by the following
non-limiting examples.
EXAMPLE 1
Purification of IL-22RA2-Fc4 Polypeptide from Transfected BHK 570
Cells
[0322] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying
IL-22RA2 polypeptide (mature soluble receptor polypeptide from
residues 23 to 231 of SEQ ID NO:13; polynucleotides as shown in SEQ
ID NO:12) containing C-terminal fusion to human Fc4 (SEQ ID NO:14),
designated IL-22RA2-Fc4. About 16,500 ml of conditioned media from
BHK 570 cells transfected with IL-22RA2-Fc4 was filtered through a
0.2 um sterilizing filter and then supplemented with a solution of
protease inhibitors, to final concentrations of, 0.001 mM leupeptin
(Boerhinger-Mannheim, Indianapolis, Ind.), 0.001 mM pepstatin
(Boerhinger-Mannheim) and 0.4 mM Pefabloc (Boerhinger-Mannheim). A
Poros protein A50 column (20 ml bed volume, Applied Biosystems) was
packed and washed with 400 ml PBS (Gibco/BRL) The supplemented
conditioned media was passed over the column with a flow rate of 15
ml/minute, followed by washing with 800 ml PBS (BRL/Gibco).
IL-22RA2-Fc4 was eluted from the column with 0.1 M Glycine pH 3.0
and 5 ml fractions were collected directly into 0.5 ml 2M Tris pH
7.8, to adjust the final pH to 7.4 in the fractions.
[0323] Column performance was characterized through western
blotting of reducing SDS-PAGE gels of the starting media and column
pass through. Western blotting used anti-human IgG HRP (Amersham)
antibody, which showed an immunoreactive protein at 60,000 Da in
the starting media, with nothing in the pass through, suggesting
complete capture. The protein A50 eluted fractions were
characterized by reducing SDS PAGE gel. This gel showed an
intensely Coomassie stained band at 60,000 Da in fractions 3 to 11.
Fractions 3 to 11 were pooled.
[0324] Protein A 50 elution pool was concentrated from 44 ml to 4
ml using a 30,000 Da Ultrafree Biomax centrifugal concentrator (15
ml volume, Millipore). A Sephacryl S-300 gel filtration column (175
ml bed volume; Pharmacia) was washed with 350 ml PBS (BRL/Gibco).
The concentrated pool was injected over the column with a flow rate
of 1.5 ml/min, followed by washing with 225 ml PBS (BRL/Gibco).
Eluted peaks were collected into 2 ml fractions.
[0325] Eluted fractions were characterized by reducing and
non-reducing silver stained (Geno Technology) SDS PAGE gels.
Reducing silver stained SDS PAGE gels showed an intensely stained
band at 60,000 Da in fractions 14-31, while non-reducing silver
stained SDS PAGE gels showed an intensely stained band at 160,000
Da in fractions 14-31. Fractions 1-13 showed many bands of various
sizes. Fractions 14-31 were pooled, concentrated to 22 ml using
30,000 Da Ultrafree Biomax centrifugal concentrator (15 ml volume,
Millipore). This concentrate was filtered through a 0.2 .mu.m
Acrodisc sterilizing filter (Pall Corporation).
[0326] The protein concentration of the concentrated pooled
fractions was performed by BCA analysis (Pierce, Rockford, Ill.)
and the material was aliquoted, and stored at -80.degree. C.
according to our standard procedures. The concentration of the
pooled fractions was 1.50 mg/ml.
EXAMPLE 2
Construction of BaF3 Cells Expressing the CRF2-4 Receptor
(BaF3/CRF2-4 Cells) and BaF3 Cells Expressing the CRF2-4 Receptor
with the IL-22RA Receptor (BaF3/CRF2-4/IL-22RA cells)
[0327] BaF3 cells expressing the full-length CFR2-4 receptor were
constructed, using 30 .mu.g of a CFR2-4 expression vector,
described below. The BaF3 cells expressing the CFR2-4 receptor were
designated as BaF3/CFR2-4. These cells were used as a control, and
were further transfected with full-length IL-22RA receptor (U.S.
Pat. No. 5,965,704) and used to construct a screen for IL-22
activity as described below.
A. Construction of BaF3 Cells Expressing the CRF2-4 Receptor
[0328] The full-length cDNA sequence of CRF2-4 (Genbank Accession
No. Z17227) was isolated from a Daudi cell line cDNA library, and
then cloned into an expression vector pZP7P.
[0329] BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell
line derived from murine bone marrow (Palacios and Steinmetz, Cell
41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6:
4133-4135, 1986), was maintained in complete media (RPMI medium
(JRH Bioscience Inc., Lenexa, Kans.) supplemented with 10%
heat-inactivated fetal calf serum, 2 ng/ml murine IL-3 (mIL-3) (R
& D, Minneapolis, Minn.), 2 mM L-glutaMax-1.TM. (Gibco BRL), 1
mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics (GIBCO BRL)).
Prior to electroporation, CRF2-4/pZP7P was prepared and purified
using a Qiagen Maxi Prep kit (Qiagen) as per manufacturer's
instructions. For electroporation, BaF3 cells were washed once in
serum-free RPMI media and then resuspended in serum-free RPMI media
at a cell density of 10.sup.7 cells/ml. One ml of resuspended BaF3
cells was mixed with 30 .mu.g of the CRF2-4/pZP7P plasmid DNA and
transferred to separate disposable electroporation chambers (GIBCO
BRL). Following a 15-minute incubation at room temperature the
cells were given two serial shocks (800 lFad/300 V.; 1180 lFad/300
V.) delivered by an electroporation apparatus (CELL-PORATOR.TM.;
GIBCO BRL). After a 5-minute recovery time, the electroporated
cells were transferred to 50 ml of complete media and placed in an
incubator for 15-24 hours (37.degree. C., 5% CO.sub.2). The cells
were then spun down and resuspended in 50 ml of complete media
containing 2 .mu.g/ml puromycin in a T-162 flask to isolate the
puromycin-resistant pool. Pools of the transfected BaF3 cells,
hereinafter called BaF3/CRF2-4 cells, were assayed for signaling
capability as described below. Moreover these cells were further
transfected with IL-22RA receptor as described below.
B. Construction of BaF3 Cells Expressing CRF2-4 and IL-22RA
Receptors
[0330] BaF3/CRF2-4 cells expressing the full-length IL-22RA
receptor were constructed as per above, using 30 .mu.g of a IL-22RA
expression vector. Following recovery, transfectants were selected
using 200 .mu.g/ml zeocin and 2 .mu.g/ml puromycin. The BaF3/CRF2-4
cells expressing the IL-22RA receptor were designated as
BaF3/CRF2-4/IL-22RA cells. These cells were used to screen for
IL-22 activity as well as IL-22RA2 antagonist activity described
herein.
EXAMPLE 3
Screening for IL-22 Antagonist Activity Using BaF3/CRF2-4/IL-22RA
Cells Using an Alamar Blue Proliferation Assay
A. Screening for IL-22 Activity Using BaF3/CRF2-4/IL-22RA Cells
Using an Alamar Blue Proliferation Assay
[0331] Purified IL-22-CEE (Example 4) was used to test for the
presence of proliferation activity as described below. Purified
IL-22RA2-Fc4 (Example 1) was used to antagonize the proliferative
response of the IL-22 in this assay as described below.
[0332] BaF3/CRF2-4/IL-22RA cells were spun down and washed in the
complete media, (RPMI medium (JRH Bioscience Inc., Lenexa, Kans.)
supplemented with 10% heat-inactivated fetal calf serum, 2 ng/ml
murine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2 mM
L-glutaMax-1.TM. (Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL), and
PSN antibiotics (GIBCO BRL)), but without mIL-3 (hereinafter
referred to as "mIL-3 free media"). The cells were spun and washed
3 times to ensure the removal of the mIL-3. Cells were then counted
in a hemacytometer. Cells were plated in a 96-well format at 5000
cells per well in a volume of 100 .mu.l per well using the mIL-3
free media.
[0333] Proliferation of the BaF3/CRF2-4/IL-22RA cells was assessed
using IL-22-CEE protein diluted with mIL-3 free media to 50, 10, 2,
1, 0.5, 0.25, 0.13, 0.06 ng/ml concentrations. 100 .mu.l of the
diluted protein was added to the BaF3/CRF2-4/IL-22RA cells. The
total assay volume is 200 .mu.l. The assay plates were incubated at
37.degree. C., 5% CO.sub.2 for 3 days at which time Alamar Blue
(Accumed, Chicago, Ill.) was added at 20 .mu.l/well. Plates were
again incubated at 37.degree. C., 5% CO.sub.2 for 24 hours. Alamar
Blue gives a fluourometric readout based on number of live cells,
and is thus a direct measurement of cell proliferation in
comparison to a negative control. Plates were again incubated at
37.degree. C., 5% CO.sub.2 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 (Emmission). Results confirmed the dose-dependent proliferative
response of the BaF3/CRF2-4/IL-22RA cells to IL-22-CEE. The
response, as measured, was approximately 15-fold over background at
the high end of 50 ng/ml down to a 2-fold induction at the low end
of 0.06 ng/ml. The BaF3 wild type cells, and BaF3/CRF2-4 cells did
not proliferate in response to IL-22-CEE, showing that IL-22 is
specific for the CRF2-4/IL-22RA heterodimeric receptor.
[0334] In order to determine if IL-22RA2 is capable of antagonizing
IL-22 activity, the assay described above was repeated using
purified soluble IL-22RA2/Fc4. When IL-22 was combined with
IL-22RA2 at 10 .mu.g/ml, the response to IL-22 at all
concentrations was brought down to background. That the presence of
soluble IL-22RA2 ablated the proliferative effects of IL-22
demonstrates that it is a potent antagonist of the IL-22 ligand.
This assay can be used to test other antagonists of IL-22 activity
described herein, such as anti-IL-22RA antibodies.
EXAMPLE 4
Purification of IL-22-CEE from BHK 570 Cells
[0335] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying IL-22
polypeptide containing C-terminal GluGlu (EE) tag (SEQ ID NO:15; or
SEQ ID NO:16). Conditioned media from BHK cells expressing
IL-22-CEE was concentrated with an Amicon S10Y3 spiral cartridge on
a ProFlux A30. A Protease inhibitor solution was added to the
concentrated conditioned media to final concentrations of 2.5 mM
ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St.
Louis, Mo.), 0.003 mM leupeptin (Boehringer-Mannheim, Indianapolis,
Ind.), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc
(Boehringer-Mannheim). Samples were removed for analysis and the
bulk volume was frozen at -80.degree. C. until the purification was
started. Total target protein concentrations of the concentrated
conditioned media were determined via SDS-PAGE and Western blot
analysis with the anti-EE HRP conjugated antibody.
[0336] About 100 ml column of anti-EE G-Sepharose (prepared as
described below) was poured in a Waters AP-5, 5 cm.times.10 cm
glass column. The column was flow packed and equilibrated on a
BioCad Sprint (PerSeptive BioSystems, Framingham, Mass.) with
phosphate buffered saline (PBS) pH 7.4. The concentrated
conditioned media was thawed, 0.2 micron sterile filtered, pH
adjusted to 7.4, then loaded on the column overnight with about 1
ml/minute flow rate. The column was washed with 10 column volumes
(CVs) of phosphate buffered saline (PBS, pH 7.4), then plug eluted
with 200 ml of PBS (pH 6.0) containing 0.5 mg/ml EE peptide
(Anaspec, San Jose, Calif.) at 5 ml/minute. The EE peptide used has
the sequence EYMPME (SEQ ID NO:15). The column was washed for 10
CVs with PBS, then eluted with 5 CVs of 0.2M glycine, pH 3.0. The
pH of the glycine-eluted column was adjusted to 7.0 with 2 CVs of
5.times.PBS, then equilibrated in PBS (pH 7.4). Five ml fractions
were collected over the entire elution chromatography and
absorbance at 280 and 215 nM were monitored; the pass through and
wash pools were also saved and analyzed. The EE-polypeptide elution
peak fractions were analyzed for the target protein via SDS-PAGE
Silver staining and Western Blotting with the anti-EE HRP
conjugated antibody. The polypeptide elution fractions of interest
were pooled and concentrated from 60 ml to 5.0 ml using a 10,000
Dalton molecular weight cutoff membrane spin concentrator
(Millipore, Bedford, Mass.) according to the manufacturer's
instructions.
[0337] To separate IL-22-CEE from other co-purifying proteins, the
concentrated polypeptide elution pooled fractions were subjected to
a POROS HQ-50 (strong anion exchange resin from PerSeptive
BioSystems, Framingham, Mass.) at pH 8.0. A 1.0.times.6.0 cm column
was poured and flow packed on a BioCad Sprint. The column was
counter ion charged then equibrated in 20 mM TRIS pH 8.0 (Tris
(Hydroxymethyl Aminomethane)). The sample was diluted 1:13 (to
reduce the ionic strength of PBS) then loaded on the Poros HQ
column at 5 ml/minute. The column was washed for 10 CVs with 20 mM
Tris pH 8.0 then eluted with a 40 CV gradient of 20 mM Tris/1 M
sodium chloride (NaCl) at 10 ml/minute. 1.5 ml fractions were
collected over the entire chromatography and absorbance at 280 and
215 nM were monitored. The elution peak fractions were analyzed via
SDS-PAGE Silver staining. Fractions of interest were pooled and
concentrated to 1.5-2 ml using a 10,000 Dalton molecular weight
cutoff membrane spin concentrator (Millipore, Bedford, Mass.)
according to the manufacturer's instructions.
[0338] To separate IL-22-CEE polypeptide from free EE peptide and
any contaminating co-purifying proteins, the pooled concentrated
fractions were subjected to size exclusion chromatography on a
1.5.times.90 cm Sephadex S200 (Pharmacia, Piscataway, N.J.) column
equilibrated and loaded in PBS at a flow rate of 1.0 ml/min using a
BioCad Sprint. 1.5 ml fractions were collected across the entire
chromatography and the absorbance at 280 and 215 nM were monitored.
The peak fractions were characterized via SDS-PAGE Silver staining,
and only the most pure fractions were pooled. This material
represented purified IL-22-CEE polypeptide.
[0339] This purified material was finally subjected to a 4 ml
ActiClean Etox (Sterogene) column to remove any remaining
endotoxins. The sample was passed over the PBS equilibrated gravity
column four times then the column was washed with a single 3 ml
volume of PBS, which was pooled with the "cleaned" sample. The
material was then 0.2 micron sterile filtered and stored at
-80.degree. C. until it was aliquoted.
[0340] On Western blotted, Coomassie Blue and Silver stained
SDS-PAGE gels, the IL-22-CEE polypeptide was one major band. The
protein concentration of the purified material was performed by BCA
analysis (Pierce, Rockford, Ill.) and the protein was aliquoted,
and stored at -80.degree. C. according to standard procedures.
[0341] To prepare anti-EE Sepharose, a 100 ml bed volume of protein
G-Sepharose (Pharmacia, Piscataway, N.J.) was washed 3 times with
100 ml of PBS containing 0.02% sodium azide using a 500 ml Nalgene
0.45 micron filter unit. The gel was washed with 6.0 volumes of 200
mM triethanolamine, pH 8.2 (TEA, Sigma, St. Louis, Mo.), and an
equal volume of EE antibody solution containing 900 mg of antibody
was added. After an overnight incubation at 4.degree. C., unbound
antibody was removed by washing the resin with 5 volumes of 200 mM
TEA as described above. The resin was resuspended in 2 volumes of
TEA, transferred to a suitable container, and
dimethylpimilimidate-2HCl (Pierce, Rockford, Ill.) dissolved in
TEA, was added to a final concentration of 36 mg/ml of protein
G-Sepharose gel. The gel was rocked at room temperature for 45 min
and the liquid was removed using the filter unit as described
above. Nonspecific sites on the gel were then blocked by incubating
for 10 min. at room temperature with 5 volumes of 20 mM
ethanolamine in 200 mM TEA. The gel was then washed with 5 volumes
of PBS containing 0.02% sodium azide and stored in this solution at
4.degree. C.
EXAMPLE 5
In Vivo Affects of IL-22 Polypeptide
[0342] Mice (female, C57BL/6N, 8 weeks old; Charles River Labs,
Kingston, N.Y.) were divided into three groups. An adenovirus
expressing an IL-22 polypeptide (SEQ ID NO:6) was previously made
using standard methods. On day 0, parental or IL-22 adenovirus was
administered to the first (n=8) and second (n=8) groups,
respectively, via the tail vein, with each mouse receiving a dose
of .about.1.times.10.sup.11 particles in 0.1 ml volume. The third
group (n=8) received no treatment. On days 12, mice were weighed
and blood was drawn from the mice. Samples were analyzed for
complete blood count (CBC) and serum chemistry. Statistically
significant elevations in neutrophil and platelet counts were
detected in the blood samples from the IL-22 adenovirus
administered group relative to the parental adenovirus treated
group. Also, lymphocyte and red blood cell counts were
significantly reduced from the IL-22 adenovirus administered group
relative to the parental adenovirus treated group. In addition, the
IL-22 adenovirus treated mice decreased in body weight, while
parental adenovirus treated mice gained weight. Also the serum
IL-22 level was increased and the glucose level decreased at day 3.
In summary, IL-22 adeno-mice displayed acute phase response that
can also be initiated by other pro-inflammatory cytokines such as
TNF-alpha, IL-1beta, and gp130 cytokines. The acute phase response
is the set of immediate inflammatory responses initiated by pattern
recognition molecules. The acute phase proteins provide enhanced
protection against microorganisms and modify inflammatory responses
by effects on cell trafficking and mediator release. For example,
SAA has potent leukocyte activating function including induction of
chemotaxis, enhancement of leukocyte adhesion to endothelial cells,
and increased phagocytosis. Understanding the factors that initiate
and alter the magnitude and duration of the acute phase response
represents an important step in the development of new therapies
for infectious and inflammatory diseases.
[0343] The results suggested that IL-22 affects hematopoiesis,
i.e., blood cell formation in vivo. As such, IL-22 could have
biological activities effecting different blood stem cells, thus
resulting increase or decrease of certain differentiated blood
cells in a specific lineage. For instance, IL-22 appears to reduce
lymphocytes, which is likely due to inhibition of the committed
progenitor cells that give rise to lymphoid cells. IL-22 also
decreases red blood cells, supporting the notion that IL-22 could
play a role in anemia, infection, inflammation, and/or immune
diseases by influencing blood cells involved in these process.
Antagonists against IL-22, such as antibodies or its soluble
receptor IL-22RA2, could be used as therapeutic reagents in these
diseases.
[0344] Moreover, these experiments using IL-22 adenovirus in mice
suggest that IL-22 over-expression increases the level of
neutrophils and platelets in vivo. It is conceivable that there are
other factors (such as cytokines and modifier genes) involved in
the responses to IL-22 in the whole animal system. Nevertheless,
these data strongly support the involvement of IL-22 in
hematopoiesis. Thus, IL-22 and its receptors are suitable
reagents/targets for the diagnosis and treatment in variety of
disorders, such as inflammation, immune disorders, infection,
anemia, hematopoietic and other cancers, and the like.
EXAMPLE 6
IL-22-Expressing Transgenic Mice
A. Generation of Transgenic Mice Expressing Mouse IL-22
[0345] DNA fragments from a transgenic vector containing 5' and 3'
flanking sequences of the lymphoid specific E.mu.LCK promoter,
mouse IL-22 (SEQ ID NO:10; polypeptide shown in SEQ ID NO:11), the
rat insulin II intron, IL-22 cDNA and the human growth hormone poly
A sequence were prepared using standard methods, and used for
microinjection into fertilized B6C3f1 (Taconic, Germantown, N.Y.)
murine oocytes, using a standard microinjection protocol. See,
Hogan, B. et al., Manipulating the Mouse Embryo, A Laboratory
Manual, Cold Spring Harbor Laboratory Press, 1994.
[0346] Twenty-five mice transgenic for mouse IL-22 with the
lymphoid-specific E.mu.LCK promoter were identified among 154 pups.
Eleven of the transgenic pups died within hours of birth, 9
transgenic pups with a shiny appearance were necropsied the day of
birth, and 2 grew to adulthood. Expression levels were low in one
adult animal. Tissues from the necropsied pups were prepared and
histologically examined as described below.
[0347] The shiny appearance of the neonate pups appeared to be
associated with a stiffening of the skin, as if they were drying
out, resulting in a reduction of proper nursing. Their movements
became stiffened in general.
B. Genotypic and Expression Analysis from Transgenic Mice
[0348] From the mouse IL-22 transgenic line driven by the E.mu.Lck
promoter, described above, newborn pups were observed for
abnormalities on day one (day of birth) and sacrificed for tissue
collection. All pups were given a unique ear tag number, and those
designated as having a shiny skin phenotype at the time of
sacrifice were noted. Of the twelve pups, six were observed to have
the shiny skin phenotype, with two designated as "severe"
phenotypes. Severe phenotypes were defined as small pups with
little mobility whose skin was especially shiny and very dry. Skin
was collected from the left lateral side of each pup, and frozen in
Tissue-Tek embedding medium.
[0349] Genotyping confirmed that shiny skin was a good indicator of
transgenic status, although no expression data was collected.
Frozen skin blocks were sectioned to 7 microns on a cryostat and
stained to look for the presence of CD3, CD4, CD8, mouse
macrophages, B-cells, CD80, and MHC class II. The staining protocol
involved binding of commercially available antibodies to the
tissue, detection with a peroxidase labeled secondary antibody, and
DAB chromogen reaction to visualize staining.
[0350] Transgenic animals were found to be higher in MHC class II
and CD80, which stain for antigen-presenting cells and dendritic
cells respectively. The macrophage marker also detected more cells
in the severe and non-severe transgenics than in the wild type
animals, although the distribution of these cells was very
localized in the high dermis. Animals classified as severe
phenotypes had the most robust staining with all three of these
markers, showing a dramatic increase in cell intensity and number
when compared to the wild type. This variability may be due to a
difference in expression level of IL-22 in these transgenic founder
pups. The MHC class II positive cells were located in the lower
dermis arranged in loose open clusters, while the CD80 positive
cells were predominantly below the dermis either in or just above
the muscle/fat layer. These two cell populations do not appear to
overlap. All other markers were of equivalent staining in all
animals. Toluidine blue staining for mast cells revealed slight to
no difference between wild type and transgenic animals.
C. Microscopic Evaluation of Tissues from Transgenic Mice: IL-22 TG
with EuLck Promoter has a Neonatal Lethal-Histology
[0351] On the day of birth, pups from litters containing IL-22
transgenics were humanely euthanized and the whole body immersion
fixed in 10% buffered formalin. Six transgenic and two
non-transgenic pups were submitted for further workup. Four of the
six transgenics were noted to have shiny skin at the time of
euthanasia. The fixed tissues were trimmed into 5 sections
(longitudinal section of the head and cross sections of the upper
and lower thorax and upper and lower abdomen). The tissues were
embedded in paraffin, routinely processed, sectioned at 5 um (Jung
2065 Supercut microtome, Leica Microsystems, Wetzlar, Germany) and
stained with H&E. The stained tissues were evaluated under a
light microscope (Nikon Eclipse E600, Nikon Inc., Melville, N.Y.)
by a board (ACVP) certified veterinary pathologist.
[0352] On microscopic examination, the epidermis of two of the
transgenic pups was observed to be thicker than the epidermis of
the other six mice including the controls. No other abnormalities
were noted in the skin and other tissues of any of the mice.
Representative areas of skin from corresponding regions of the
thorax and abdomen were imaged with the 40.times. objective lens
and with a CoolSnap digital camera (Roper Scientific, Inc., San
Diego, Calif.) that was attached to the microscope. The thickness
of the epidermis was then determined using histomorphometry
software (Scion Image for Windows (NIH Image), Scion Corp.,
Frederick, Md., v. B4.0.2). The results shown in Table 5 were as
follows:
TABLE-US-00005 TABLE 5 Average thoracic skin Average abdominal skin
Genotype/phenotype thickness (.mu.m) thickness (.mu.m)
Non-transgenic/normal 5.2 5.4 Transgenic/non-shiny 5.0 6.7
Transgenic/shiny 8.2 7.4 Transgenic/all 7.1 7.1
[0353] There were insufficient numbers of mice to determine
statistical significance; however, the transgenics, especially
those with shiny skin, tended to have a thicker epidermis than the
non-shiny transgenics and non-transgenic controls. The shiny
transgenics may have a higher expression level of IL-22 than the
non-shiny transgenics.; however, expression levels were not
determined for these mice. These suggested a role for IL-22 in
psoriasis, psoriatic arthritis, or other inflammatory skin
conditions or other inflammatory diseases.
EXAMPLE 7
In Vivo Affects of IL-22 Polypeptide
[0354] A. Mice Infected with IL-22 Adenovirus Show Induction of
SAA
[0355] Mice (female, C57BL/6N, 8 weeks old; Charles River Labs,
Kingston, N.Y.) were divided into three groups. An adenovirus
expressing an IL-22 polypeptide (SEQ ID NO:6) was previously made
using standard methods. On day 0, parental or IL-22 adenovirus was
administered to the first (n=8) and second (n=8) groups,
respectively, via the tail vein, with each mouse receiving a dose
of .about.1.times.10.sup.11 particles in .about.0.1 ml volume. The
third group (n=8) received no treatment. On day 12, mice were
weighed and blood was drawn from the mice. On day 20 of the study,
mice were sacrificed, body weight was recorded, and blood and
tissues were collected for analysis.
[0356] All blood samples were analyzed for complete blood count
(CBC) and serum chemistry. At both day 12 and 20, statistically
significant elevations in neutrophil and platelet counts were
detected in the blood samples from the IL-22 adenovirus
administered group relative to the parental adenovirus treated
group. Also, lymphocyte counts were significantly reduced from the
IL-22 adenovirus administered group relative to the parental
adenovirus treated group at day 12, but at day 20 the opposite
effect was observed. In addition, the IL-22 adenovirus treated mice
decreased in body weight, while parental adenovirus treated mice
gained weight. Glucose was significantly reduced at both time
points in the serum samples from the IL-22 adenovirus administered
group relative to the parental adenovirus treated group. Serum
albumin was also significantly reduced at both time points. Blood
urea nitrogen levels were significantly reduced at day 20. Serum
globulin levels were significantly increased the IL-22 adenovirus
administered group relative to the parental adenovirus treated
group at both time points. Microscopically, one observed
histomorphological change attributed to IL-22 was tubular
regeneration in the kidney. While not uncommon in mice, there was
an increased incidence and severity in this group of animals.
Nephropathy is characterized as multifocal areas of basophilia of
cortical tubular epithelial cells.
[0357] An additional experiment, identical in design to the one
described above, was carried out in order to verify results and
collect additional samples. In this study, body weight was recorded
every three days, blood was collected from the mice 3 days
following adenovirus injection, and mice were sacrificed for blood
and tissue collection on day 10 (n=4 per group) and day 20 (n=4 per
group). Elevated neutrophil and platelet counts were again detected
in blood samples from the IL-22 adenovirus administered group
relative to the parental adenovirus treated group. This effect was
evident for neutrophils by day 3, but platelet count was not
significantly different until day 10. Also, lymphocyte counts were
significantly reduced from the IL-22 adenovirus administered group
relative to the parental adenovirus treated group at 3 and 10, but
they were not elevated on day 20 as in the previous study. Again,
mice given IL-22 adenovirus lost weight over the course of the
study, while control virus treated and untreated mice gained
weight. Serum chemistry parameters were consistent with the
previous study. Histological findings of tubular regeneration in
the kidney associated with IL-22 adenovirus treatment were also
confirmed in this study. This was consistent with the additional
finding of moderate proteinurea in mice given IL-22 adenovirus (day
20).
[0358] The results suggested that IL-22 affects hematopoiesis,
i.e., blood cell formation in vivo. As such, IL-22 could have
biological activities effecting different blood stem cells, thus
resulting in an increase or decrease of certain differentiated
blood cells in a specific lineage. For instance, IL-22 appears to
reduce lymphocytes, which is likely due to inhibition of the
committed progenitor cells that give rise to lymphoid cells,
supporting the notion that IL-22 could play a role in anemia,
infection, inflammation, and/or immune diseases by influencing
blood cells involved in these processes. Antagonists against IL-22,
such as antibodies or its soluble receptor IL-22RA2, could be used
as therapeutic reagents in these diseases.
[0359] Moreover, these experiments using IL-22 adenovirus in mice
suggest that IL-22 over-expression increases the level of
neutrophils and platelets in vivo. It is conceivable that there are
other factors (such as cytokines and modifier genes) involved in
the responses to IL-22 in the whole animal system. Nevertheless,
these data strongly support the involvement of IL-22 in
hematopoiesis. Thus, IL-22, anti-IL-22 antibodies, IL-22RA soluble
receptors (e.g., SEQ ID NO:3), and anti-IL-22RA antibodies are
suitable reagents/targets for the diagnosis and treatment in
variety of disorders, such as inflammation, immune disorders,
infection, anemia, hematopoietic and other cancers, and the
like.
[0360] Association of IL-22 expression with weight loss, appearance
of acute phase protein SAA, and metabolic perturbations evidenced
by decreased serum glucose, albumin and urea nitrogen suggest that
IL-22 is a cytokine which acts early in certain inflammatory
responses. Mice given IL-22 adenovirus may represent a state of
chronic inflammation, such as that observed in IBD, ulcerative
colitis, arthritis, psoriasis, psoriatic arthritis, asthma, and the
like. Certain detrimental inflammatory processes might be inhibited
by use of an antagonist to IL-22, such as anti-IL-22 antibodies,
and its receptors, such as IL-22RA soluble receptors (e.g., SEQ ID
NO:3), and anti-IL-22RA antibodies and the like.
B. IL-22 is a Pro-Inflammatory Cytokine: Serum Level of SAA in
Adeno-IL-22 Mice:
[0361] An ELISA was performed to determine the level of SAA in
IL-22-Adeno mice, using a Mouse SAA Immunoassay Kit and protocol
(Biosource International, California, USA). Diluted standards and
unknowns were plated along with HRP-anti-mouse SAA into assay
plates pre-coated with anti-mouse SAA antibody. Plates were
incubated for one hour at 37 degrees C. and then washed according
to kit instructions. Plates were developed for 15 minutes at room
temperature using TMB and stopped with 2M H.sub.2S0.sub.4, The
absorbance at 450 nm was read using a Spectromax 190 (Molecular
Devices, California, USA). The resulting data was analyzed using
Softmax Pro (Molecular Devices, California, USA) and Excel
(Microsoft Corp., Washington, USA).
[0362] Mice infected with IL-22-Adenovirus had highly elevated
levels of mSAA, over 10-fold, relative to the Parental-Adenovirus
control.
C. Flow Cytometry Analysis of IL-22-Adenovirus Infected Mice
[0363] To analyze the effects of IL-22 expression in vivo by
adenovirus, we isolated peripheral blood, spleen, and bone marrow
from IL-22-adenovirus infected C57BL/6N mice, at day 10 and day 20
after infection. Approximately 100 .mu.l of blood was collected in
heparinized tubes, then depleted of red blood cells by hypotonic
lysis (cells were lysed in 4.5 ml dH.sub.2O for .about.5 seconds
before adding 1.5 ml 3.6% NaCl). Spleens were crushed between two
frosted glass slides, and the cells released were passed over a
Nytex membrane (cell strainer) and pelleted. Bone marrow was
obtained by crushing one femur in a mortar and pestle and passing
the cells over a cell strainer (Falcon). Cells were resuspended in
FACS wash buffer (WB=HBSS/1% BSA/10 mM hepes), counted in trypan
blue, and 1.times.10.sup.6 viable cells of each type were aliquoted
into 5 ml polystyrene tubes. Cells were washed and pelleted, then
incubated for 20 min on ice with cocktails of fluorescently-labeled
(FITC, PE, and CyChrome) monoclonal antibodies (PharMingen, San
Diego, Calif.) recognizing various cell surface markers used to
identify particular immune cell subsets. These markers include the
following (listed in the groups of 3 we tested). For blood
staining: CD3, Gr1, and B220; for spleen staining: CD62L, CD44, and
CD3; CD21, CD23, and B220; IgD, IgM, and B220; CD11b, Gr1, and CD8;
for bone marrow staining: CD11b, Gr1, CD3; IgD, IgM, and B220.
Cells were washed with 1.5 ml WB and pelleted, then resuspended in
0.4 ml of WB and analyzed on a FACScan using CellQuest software
(Becton Dickinson, Mountain View, Calif.).
[0364] We found that the fraction of neutrophils in the blood of
IL-22-adeno-treated mice was elevated 4-13 fold at Day 10 and
2-3-fold at Day 20. At Day 10, this difference resulted in a
concomitant decrease in the fraction of lymphocytes and monocytes
in the blood. In the bone marrow, we found that the total number of
B cells decreased .about.1.5-fold while the percentage of mature
recirculating B cells increased and the total number of immature B
cells dropped slightly at Day 10. At Day 20, many of these
differences were not apparent, though we did find a slight increase
in the fraction of mature recirculating B cells. In the spleen, the
total number of B cells decreased slightly (1.5-2-fold) on both
days tested, while on Day 20, the fraction of marginal zone B cells
(CD21+CD23-B220+) increased by 2-fold and the number of follicular
B cells (CD21+CD23+B220+) dropped 2-fold. Marginal zone B cells are
considered to be the first line of defense against pathogens, as
they are more sensitive to B cell mitogens (e.g. LPS) than the more
common follicular B cells, and when they encounter their cognate
antigen they differentiate very quickly into antibody-secreting
cells. It is possible that IL-22 either enhances the conversion of
follicular to marginal zone B cells, or that it selectively
depletes the less mature follicular cells. The changes in B cell
numbers found in the bone marrow may reflect an enhanced
differentiation of pre/pro and/or immature B cells, or an increased
influx of recirculating B cells from the blood/spleen, and perhaps
a coincident increase in export of immature B cells to the
periphery. The actual number of mature BM B cells does not
increase, so IL-22 may not enhance their proliferation.
Alternatively, IL-22 may block, reduce or inhibit differentiation
of immature B cells and thereby increase the relative
representation of mature B cells.
D. IL-22RA2-Fc4 Neutralizes IL-22 Activity In Vivo: SAA ELISA
Showing SAA Expression Induced by IL-22 is Inhibited by
IL-22RA2-Fc4 Injection:
[0365] To assess whether IL-22RA2 could inhibit the SAA induction
by IL-22 mice (female, C3H/HEJ, 8 weeks old; Jackson Labs, Bar
Harbor, Me.) were divided into five groups of three animals each
and treated by IP injection of proteins as shown in Table 6
below:
TABLE-US-00006 TABLE 6 Group # IL-22 IL-22RA2 Group 1: -- -- Group
2: -- 100 .mu.g Group 3: 3 .mu.g -- Group 4: 3 .mu.g 20 .mu.g Group
5: 3 .mu.g 100 .mu.g
[0366] The IL-22RA2 injections preceded the IL-22 injection by 15
minutes. Both protein injections were given by the intraperitoneal
route. A blood sample was taken from each mouse prior to treatment,
then at 2 and 6 hours after treatment. Serum was prepared from each
of the samples for measurement of SAA and IL-22.
[0367] An ELISA was performed as described previously to determine
the level of SAA in mice treated with IL-22 and a soluble receptor
for IL-22, IL-22RA2-Fc4 described herein. Mice treated with 3 .mu.g
IL-22 in conjunction with IL-22RA2-Fc4 at concentrations between
20-100 ug showed a reduction in the level of SAA induced by IL-22
alone to background levels, demonstrating that IL-22RA2 inhibited
the SAA induction activity of IL-22 in vivo.
EXAMPLE 8
Expression of IL-22 in Inflammatory Bowel Disease Mouse Model
[0368] Inflammatory Bowel disease (IBD) is a multifactorial
disease, divided into two types, ulcerative colitis (UC) and
Crohn's Disease (CD). The etiology of these diseases is currently
not known and clinical manifestations differ. UC is restricted to
the colon, and symptoms include bloody diarrhea, weight loss and
abdominal pain. Macroscopic features of UC include punctuated
ulcers and a shortened colon. In contrast, Crohn's Disease can also
affect other parts of the bowel. Symptoms include diarrhea (which
is less often bloody than seen in UC), a low-grade fever and pain.
Macroscopic features include fibrotic and stenotic bowel with
strictures, deep ulcers, fissures and fistulas.
[0369] Several animal models are available that mimic these human
diseases. Three commonly used models of colitis for new drug
screening are the 2,4,6-trinitrobenzene sulphonic acid (TNBS)
induced rat model, the mouse T-cell transfer model, and the dextran
sodium sulfate, or DSS-induced mouse model. The DSS model was
derived from a model by Dr. S. Murthy, using a disease activity
index scoring system (S. N. S. Murthy, Treatment of Dextran Sulfate
Sodium-Induced Murine Colitis by Intracolonic Cyclosporin,
Digestive Diseases and Sciences, Vol. 38, No. 9 (September 1993),
pp. 1722-1734).
[0370] In the present study, an acute colitis resulted when mice
were fed DSS in their drinking water for 6 days. The animals
exhibited weight loss and bloody diarrhea, mimicking the condition
of UC patients. The mechanism of the DSS injury is not well
characterized, but it is thought that it induces a nonspecific
inflammatory immune response and mimics environmental effects on
the bowel. It is possible that H.sub.2S is produced, which could be
toxic to cells. In addition, changes in luminal bacterial flora
occur. Activated monocytes, macrophages and mast cells have been
demonstrated in the colon. Mediators for all three animal models
include inflammatory prostaglandins, leukotriene metabolites and
cytokines.
A. Method
[0371] Colitis was induced by DSS ingestion in Swiss Webster female
mice from Charles River Laboratories. The mice were 10 and 11 weeks
old at the start of the study. Mice were given 4% DSS in the
drinking water for a period of 6 days (treated mice), or were given
only normal drinking water (control mice). A Disease Activity Index
clinical score (DAI) was used, which comprises a combination of
measurements including stool quality, occult blood and weight loss.
DAI was obtained daily for each mouse beginning one day after DSS
treatment. After 6 days, DSS was removed from the drinking water of
the treated mice. All mice were monitored by DAI clinical score
until sacrifice at either 2, 7 or 10 days from the start of the
study. On each of days 2 and 7, four DSS-treated mice and one
control mouse were sacrificed. On day 10, four DSS-treated mice and
two control mice were sacrificed. For all animals after sacrifice,
the colon length was measured. Colon sections were fixed in 10%
neutral buffered formalin for histologic analysis or frozen for
mRNA extraction.
B. Histologic Scoring and Disease Activity Index (DAI) Scoring
[0372] Histologic index scores were obtained following the method
in reference 1. Generally, the colon sections were scored blinded
by a pathologist for crypt scores, hyperplastic epithelium, crypt
distortion and inflammation.
[0373] Daily, each mouse was graded as to a clinical score based on
weight loss, stool consistence and intestinal bleeding. Higher
scores were assigned for increasing amounts of weight loss,
diarrhea and bleeding. The daily score for each mouse was the mean
grade obtained from the three results/observations.
C. Results
[0374] The colon lengths for DSS-treated mice were somewhat shorter
on days 7 and 10 than non-treated controls, but the results may not
have been significant (not checked by a statistical application).
The clinical DAI scores reflected a rise in disease symptoms in the
DSS-treated mice similar to that seen in past studies using this
model. Occult blood was greatest on approximately days 4 and 5,
while loose stools were more prevalent on days 6 and 7.
Histopathology results show that disease scores were different from
the controls on all sacrifice days, especially days 7 (peak) and
10. The histopathology screening scores were: controls=0.5, day 2
DSS-treated mice=8.8, day 7 DSS-treated mice=21, day 10 DSS-treated
mice=18. Clinical and histopathology scores show that the
DSS-treated mice had significant colon disease relative to the
non-treated controls. The frozen tissue samples were used later for
mRNA determinations as described below.
D. Tissue Expression of IL-22 RNA in Murine IBD Colon Samples Using
RT-PCR:
[0375] To determine the relative expression of mouse IL-22 RNA (SEQ
ID NO:10; SEQ ID NO:11) in an inflammatory bowel disease model, the
distal colons of DSS-treated mice were collected and snap frozen in
liquid nitrogen. In this experiment mice were treated with DSS and
samples were taken on days 2, 7 and 10 post-treatment. Samples from
normal untreated mice were collected as well. RNA was then isolated
from the samples using the standard RNeasy Midiprep.TM. Kit
(Qiagen, Valencia, Calif.) as per manufacturer's instructions.
[0376] The RT-PCR reactions used the `Superscript One-Step RT-PCR
System with Platinum Taq.` (Life Technologies, Gaithersburg, Md.)
Each 25 .mu.l reaction consisted of the following: 12.5 .mu.l of
2.times. Reaction Buffer, 0.5 ul (20 pmol/.mu.l) ZC39,289 (SEQ ID
NO:17), 0.5 .mu.l (20 pmol/.mu.l) ZC39,290 (SEQ ID NO:18), 0.4
.mu.l RT/Taq polymerase mix, 10 ul RNase-free water, 1.0 .mu.l
total RNA (100 ng/.mu.l). The amplification was carried out as
follows: one cycle at 500 for 30 minutes followed by 35 cycles of
94.degree., 30 seconds; 580, 30 seconds; 720, 60 seconds; then
ended with a final extension at 720 for 7 minutes. 8 to 10 .mu.l of
the PCR reaction product was subjected to standard agarose gel
electrophoresis using a 2% agarose gel. The correct predicted cDNA
fragment size was observed as follows: There was a faint band in
both day 2 samples. Two of three day 7 samples generated a strong
band while the third day 7 sample generated a very strong band. The
three day 10 samples generated a strong band. Finally, the two
`normal` control samples did not generate any band. These results
suggest that there may be an upregulation of IL-22 in certain types
of inflammatory responses in the colon, including those associated
with IBD, UC, and CD. The data is summarized in Table 7 below where
Relative Expression was scored as follows: 0=No band, 1=faint band,
2=strong band, 3=very strong band.
TABLE-US-00007 TABLE 7 Tissue Relative Expression (0-3) Normal
Colon 0 Normal Colon 0 Day 2 Post Treatment 1 Day 2 Post Treatment
1 Day 7 Post Treatment 3 Day 7 Post Treatment 2 Day 7 Post
Treatment 2 Day 10 Post Treatment 2 Day 10 Post Treatment 2 Day 10
Post Treatment 2
EXAMPLE 9
IL-22RA2 Decreases IL-6 and SAA Levels in Mouse Collagen Induced
Arthritis (CIA) Model
A. Mouse Collagen Induced Arthritis (CIA) Model
[0377] Ten week old male DBA/1J mice (Jackson Labs) were divided
into 3 groups of 13 mice/group. On day-21, animals were given a
subcutaneous injection of 50-100 .mu.l of 1 mg/ml chick Type II
collagen formulated in Complete Freund's Adjuvant (prepared by
Chondrex, Redmond, Wash.), and three weeks later on Day 0 they were
given a 100 .mu.l (25 .mu.g) injection of LPS from E. coli 0111:B4,
prepared as 250 .mu.g/ml from a lyophilized aliquot (Sigma, St.
Louis, Mo.). IL-22RA2 was administered as an intraperitoneal
injection 3 times a week for 4 weeks, from Day 0 to Day 25. The
first two groups received either 100 or 10 .mu.g of IL-22RA2 per
animal per dose, and the third group received the vehicle control,
PBS (Life Technologies, Rockville, Md.). Animals began to show
symptoms of arthritis following the LPS injection, with most
animals developing inflammation within 2-3 weeks. The extent of
disease was evaluated in each paw by using a caliper to measure paw
thickness, and by assigning a clinical score (0-3) to each paw:
0=Normal, 0.5=Toe(s) inflamed, 1=Mild paw inflammation, 2=Moderate
paw inflammation, and 3=Severe paw inflammation as detailed
below.
Monitoring Disease:
[0378] Animals can begin to show signs of paw inflammation soon
after the second collagen injection, and some animals may even
begin to have signs of toe inflammation prior to the second
collagen injection. Most animals develop arthritis within 2-3 weeks
of the boost injection, but some may require a longer period of
time. Incidence of disease in this model is typically 95-100%, and
0-2 non-responders (determined after 6 weeks of observation) are
typically seen in a study using 40 animals. Note that as
inflammation begins, a common transient occurrence of variable
low-grade paw or toe inflammation can occur. For this reason, an
animal is not considered to have established disease until marked,
persistent paw swelling has developed.
[0379] All animals were observed daily to assess the status of the
disease in their paws, which was done by assigning a qualitative
clinical score to each of the paws. Every day, each animal has its
4 paws scored according to its state of clinical disease. To
determine the clinical score, the paw can be thought of as having 3
zones, the toes, the paw itself (manus or pes), and the wrist or
ankle joint. The extent and severity of the inflammation relative
to these zones was noted including observation all the toes for any
joint swelling, torn nails, or redness, notation of any evidence of
edema or redness in any of the paws, and notation any loss of fine
anatomic demarcation of tendons or bones, and evaluation the wrist
or ankle for any edema or redness, and notation if the inflammation
extends proximally up the leg. A paw a score of 1, 2, or 3 was
based first on the overall impression of severity, and second on
how many zones were involved. The scale used for clinical scoring
is shown below.
[0380] Clinical Score:
[0381] 0=Normal
[0382] 0.5=One or more toes involved, but only the toes are
inflamed
[0383] 1=mild inflammation involving the paw (1 zone), and may
include a toe or toes
[0384] 2=moderate inflammation in the paw & may include some of
the toes and/or the wrist/ankle (2 zones)
[0385] 3=severe inflammation in the paw, wrist/ankle, and some or
all of the toes (3 zones)
[0386] Established disease is defined as a qualitative score of paw
inflammation ranking 2 or more, that persists overnight (two days
in a row). Once established disease is present, the date is
recorded and designated as that animal's first day with
"established disease".
[0387] Blood was collected throughout the experiment to monitor
serum levels of anti-collagen antibodies. Animals were euthanized
on Day 21, and blood was collected for serum and for CBC's. From
each animal, one affected paw was collected in 10% NBF for
histology and one was frozen in liquid nitrogen and stored at
-80.degree. C. for mRNA analysis. Also, 1/2 spleen, 1/2 thymus, 1/2
mesenteric lymph node, one liver lobe and the left kidney were
collected in RNA later for RNA analysis, and 1/2 spleen, 1/2
thymus, 1/2 mesenteric lymph node, the remaining liver, and the
right kidney were collected in 10% NBF for histology. Serum was
collected and frozen at -80.degree. C. for immunoglobulin and
cytokine assays.
[0388] No statistically significant differences were found between
the groups when the paw scores and measurements data were analyzed,
although there was a suggestion that one treatment group receiving
IL-22RA2 may have had a delay in the onset and progression of paw
inflammation. There were no significant differences between the
groups for changes in body weight, CBC parameters, or anti-collagen
antibody levels. These early results indicate that IL-22RA2 does
not adversely effect body weight, red or white blood cells, or
antibody production, but may be able to reduce inflammation.
Further investigations into dosing, mechanism of action, and
efficacy are under way (e.g., Example 10).
B. Anti-Collagen ELISA Data in Mouse CIA Model
[0389] Serum samples were collected on days 0, 7, 14, 21 and 28
relative to date of LPS challenge (day 0) from the murine model of
collagen induced arthritis (Example 9A above). The serum samples
were screened by ELISA for anti-collagen antibody titers. There
were no statistically significant effects of IL-22RA2 treatment in
100 .mu.g or 10 .mu.g treatment groups on levels of anti-collagen
antibodies compared with PBS controls. Below is a description of
anti-collagen ELISA methods and materials.
[0390] Reagents used for anti-collagen ELISAs were Maxisorp 96-well
microtiter plates (NUNC, Rochester, N.Y.), chick type-II collagen
(Chondrex, Redmond, Wash.), Super Block (Pierce, Rockford, Ill.),
horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG+A+M
(H+L) (Zymed, South San Francisco, Calif.) and o-phenylenediamine
dihydrochloride substrate (Pierce, Rockford, Ill.). Buffers used in
all assays were ELISA B diluent buffer (PBS+0.1% BSA+0.05% Tween
(Sigma, St. Louis, Mo.)), ELISA C wash buffer (PBS+0.05% Tween) and
NovoD developing buffer (0.063M sodium citrate, 0.037M citric
acid), H.sub.2O.sub.2 (Sigma) and 1N H.sub.2SO.sub.4 (VWR,
Tukwilla, Wash.).
[0391] Approximately 100 .mu.L of peripheral blood was collected by
retro-orbital bleed into serum separator tubes (Becton Dickinson).
Serum was collected by centrifugation (2-3 min, 16,000.times.g,
4-6.degree. C.) and stored at -20.degree. C. until analyzed. To
determine anti-collagen Ig antibody levels, NUNC plates were coated
with 10 .mu.g/mL chick type-II collagen (Chondrex, Redmond Wash.)
and incubated overnight at 4.degree. C. Plates were washed with
ELISA C, blocked (5 minutes, room temperature) with Super Block
(Pierce, Rockford, Ill.), and washed with ELISA C. Diluted serum
samples (diluted in ELISA B 5-fold from 1:5000 to 1:625,000) were
added to ELISA plates in triplicate and the plates were incubated
overnight at 4.degree. C. After incubation, the plates were washed
with ELISA C, and peroxidase-labeled goat anti-mouse Ig Fc (Zymed,
1:2000 in ELISA B) was added. The plates were incubated (room
temperature, 90 minutes), rinsed again using ELISA C, and HRP
activity was developed using o-phenylenediamine dihydrochloride
substrate (10 mL NovoD+1 tablet OPD+10 .mu.L H.sub.2O.sub.2,
Pierce). The reaction was stopped with 1N H.sub.2SO.sub.4. Relative
optical density measurements of serum samples at 1:25,000 dilution
were taken at 490 nm using a Spectra MAX 190, and data were
analyzed using SoftMax Pro software (Molecular Devices Corporation,
Palo Alto, Calif.).
C. IL-6 and SAA Analysis in Mouse CIA Model
[0392] Day 0 serum samples were harvested from CIA mice (Example 9A
above) 4 hr post administration of 25 .mu.g LPS intraperitoneally.
Samples were screened for IL-6 and serum amyloid A (SAA)
concentrations by commercial ELISA kits purchased for Biosource
International (Camarillo, Calif.) as per manufacturer's
instructions.
[0393] The IL-6 levels were 9651+/-1563 pg/ml, 10,865+/-1478 pg/ml
and 15,006+/-2,099 pg/ml in the mice groups subjected to 100 .mu.g
IL-22RA2, 10 .mu.g IL-22RA2 and PBS control, respectively. The IL-6
concentration in the group of CIA mice exposed to the 100 .mu.g
dose of IL-22RA2 was significantly lower compared to PBS control
mice with p=0351. Statistical significance was calculated using
Fisher's PLSD with a significance level of 5% (ABACUS Concepts,
INC, Berkeley, Calif.).
[0394] In addition, SAA concentrations were 381+/-40 .mu.g/ml,
348+/-37 .mu.g/ml and 490+/-50 .mu.g/ml in the mice groups
subjected to 100 .mu.g IL-22RA2, 10 .mu.g IL-22RA2 and PBS control
groups, respectively. The SAA concentration in the group of CIA
mice exposed to the 10 .mu.g dose of IL-22RA2 was significantly
lower compared with PBS control mice with p=0.0257. Statistical
significance was calculated using Fisher's PLSD with a significance
level of 5% (ABACUS Concepts, INC, Berkeley, Calif.).
EXAMPLE 10
Anti-IL-22RA mAbs or Anti-IL-22 mAbs Inhibit Disease Severity in a
Mouse CIA Model
[0395] The collagen-induced arthritis (CIA) model is a mouse model
for rheumatoid arthritis that reflects to large extent the disease
seen in humans. (Moore, Methods Mol. Biol. 225:175-179, 2003:
Waksman, Scand. J. Immunol., 56:12-34, 2002). Mice are immunized
with 2 doses of collagen emulsified in CFA at the base of the tail.
This results in swelling of the paws that increases over a period
of time and can be both visually scored and measured using
calipers. Furthermore, serum anti-collagen antibodies correlates
well with severity of disease. Based on data showing IL-20 and
IL-22 induce inflammation, anti-IL-22RA and anti-IL-22 mAbs are
administered to groups of collagen-immunized mice, and effects on
disease scores are evaluated. A decrease in paw scores and paw
thickness after administration of anti-IL-22RA mAbs or anti-IL-22
mAbs-suggests IL-20 and IL-22 promote ongoing immune response in a
model for autoimmunity and blocking, inhibiting, reducing,
antagonizing or neutralizing their function may inhibit autoimmune
disorders. Inhibition of serum TNFa and anti-collagen antibodies
also suggests that blocking IL-22RA may be beneficial in autoimmune
disease.
[0396] Thus, to determine if anti-IL-22RA mAbs or anti-IL-22 mAbs
have an effect on autoimmunity, they are tested in a mouse model
for rheumatoid arthritis--collagen-induced arthritis (CIA).
Specifically, DBA1J mice are given collagen injections to induce
rheumatoid-like arthritis. The inoculation on Day 0 is a
subcutaneous injection of a homogenate consisting of Complete
Freund's Adjuvant (CFA) and Type II collagen (50-100 .mu.l,
prepared as 2 mg/ml of collagen). The injection is given near the
base of the tail. On Day 21, a second inoculation is administered,
the only difference being that the homogenate is prepared using
Incomplete Freund's Adjuvant (IFA), instead of the CFA. Paw scores
and thickness are measured daily. Groups of mice receive PBS,
20-200 ug control isotype matched monoclonal antibody or 20-200 ug
anti-IL-22RA mAb or anti-IL-22 mAb i.p 2.times. or 3.times./week
for 1-4 weeks starting at second collagen injection. Mice are
monitored daily till day 30. Mice are sacrificed on day 30, serum
taken for anti-collagen antibody analysis and serum cytokine
analysis (TNF.quadrature.).
[0397] Inhibition of paw scores, paw thickness, serum TNFa and
serum anti-collagen antibodies by administration of anti-IL-22RA or
anti-IL-22 mAbs suggests that blocking IL-22RA can bind, block,
inhibit, reduce, antagonize or neutralize IL-22, and inhibit an
ongoing immune response in a model for autoimmunity and may inhibit
autoimmune disorders.
EXAMPLE 11
Expression of IL-22 Receptor, IL-22RA, in the DSS Mouse Model
[0398] Quantitative RT-PCR was performed to measure expression
levels of mouse IL-22RA in the colons of mice with DSS-induced IBD
(Example 8). RNA was isolated from normal mouse colon and from the
distal colons of DSS-treated mice from treatment days 2, 7 and 10.
RT-PCR was performed using Applied Biosystems 7700 TAQMAN.RTM.
instrument and protocols. Briefly, "Primer Express" software was
used to designed primers against the mouse IL-22RA sequence
(ZC39776 (SEQ ID NO:19) and ZC39777 (SEQ ID NO:20)) and a FAM/TAMRA
labeled TAQMAN.RTM. probe (ZC38752 (SEQ ID NO:21)) according to
Applied Biosystems guidelines. 25 ng of RNA was added to each
reaction, along with PE/Applied Biosystems TAQMAN.RTM. EZ RT-PCR
Core Reagents and the above mentioned primers and probe. RT-PCR
reactions were run in duplicate under the following conditions:
50.degree. C. for 2 minutes, 60.degree. C. for 30 minutes,
95.degree. C. for 5 minutes, 40 cycles of 94.degree. C. for 20
seconds and 60.degree. C. for 1 minute. Expression values were
compared to a standard curve of known numbers of molecules of a
synthetic mouse IL-22RA RNA transcript, and expression is reported
as absolute number of molecules of mouse IL-22RA per reaction.
Preliminary data suggests that mouse IL-22RA expression may be
slightly down-regulated in the distal colons of day 7 and day 10
mice with DSS-induced IBD when compared to expression levels in
normal mouse colon.
EXAMPLE 12
IL-22 and Proinflammatory Indicators in Mild Endotoxemia Model:
LPS-Induced Endotoxemia Mouse Model
A. LPS-Induced Endotoxemia Mouse Model: Assessment Proinflammatory
Cytokines and Body Temperature in the LPS-Induced Endotoxemia Mouse
Model
[0399] An in vivo experiment was designed to examine the effect of
IL-22RA2 (IL-22RA2) in a mouse LPS model of mild endotoxemia. To
initially assess the model, we measured proinflammatory cytokines
and body temperature to collect reference data for the model.
[0400] Briefly, six month Balb/c (CRL) female mice were injected
with 25 .mu.g LPS (Sigma) in sterile PBS intraperitoneally (IP).
Serum samples were collected at 0, 1, 4, 8, 16, 24, 48 and 72 hr
from groups of 8 mice for each time point. Serum samples were
assayed for inflammatory cytokine levels. IL-1b, IL-6, TNFa, IL-10
and serum amyloid A protein (SAA) levels were measured using
commercial ELISA kits purchased from Biosource International
(Camarillo, Calif.).
[0401] TNFa levels peaked to 4000 pg/ml and IL-10 levels were 341
pg/ml at 1 hr post LPS injection. At 4 hr post LPS injection, IL-6,
IL-1b and IL-10 were 6,100 pg/ml, 299 pg/ml and 229 pg/ml,
respectively. The SAA levels in serum were 0.405 mg/ml by 4 hr post
LPS injection. SAA concentrations in serum continued to increase to
3.9 mg/ml by 24 hr post LPS, however SAA levels greater than 1 to 2
mg/ml in serum are difficult to measure accurately or reproducibly
with the existing ELISA kit due to interactions between SAA and
other serum components. These results indicated that
proinflammatory cytokines, in addition to IL-22 (Example 11B), were
indeed produced in this model. Thus the following criteria were
established as biological markers for the LPS model of mild
endotoxemia: TNFa serum levels 1 hr post LPS, IL-6 serum levels 4
hr post LPS and SAA serum levels 4 and 8 hr post LPS.
[0402] Body temperatures in a separate group of animals were
monitored by surgically implanted telemetry devices over the course
of the 72 hr experiment. Body temperatures in mice dropped
maximally by 2.degree. C. from 37.07.degree. C. to 34.98.degree. C.
30 minutes after LPS injection.
[0403] Injection of 100 ug IL-22RA2-Fc fusion protein 30 minutes
prior to the LPS injection significantly reduced about 50% of the
SAA induction at 4 hr and 8 hr time point, while 10 ug IL-22RA2-Fc
did not have significant effect. There is no significant change to
the TNF-alpha and IL-6 level. IL-22RA2-Fc injection reduced
neutrophil count in circulation at 1 hr time point. It showed the
administration of IL-22RA2-Fc can neutralize IL-22 activity in
terms of SAA induction.
B. Detection of IL-22 Activity in Mouse Serum from LPS-Induced
Endotoxemia Mouse Model Using BaF3/CRF2-4/IL-22RA Cells in an
Alamar Blue Proliferation Assay
[0404] BaF3/CRF2-4/IL-22RA cells, described herein, were spun down
and washed in PBS 2 times to ensure the removal of the mIL-3, and
then spun a third time and re-suspended in the complete media (RPMI
1640, 10% FBS, 1% GlutaMAX, 1% Sodium Pyruvate), but without mIL-3
(hereinafter referred to as "mIL-3 free media"). Cells were then
counted in a hemocytometer. Cells were plated in a 96-well format
at 5000 cells per well in a volume of 100 .mu.l per well using the
mIL-3 free media.
[0405] Serum from the LPS-induced endotoxemia mice from the
experiment described in Example 11A above, was diluted to 2% in
mIL-3 free media on the top row of the plate and then diluted
serially 1:2 down the remaining 7 rows on the 96-well plate,
leaving a volume of 100 .mu.l in each well. This was then added to
the 100 .mu.l of cells, for final serum concentrations of 1%, 0.5%,
0.25%, 0.125%, 0.063%, 0.031%, 0.016%, and 0.018% in a total assay
volume of 200 .mu.l. The assay plates were incubated at 37.degree.
C., 5% CO.sub.2 for 4 days at which time Alamar Blue (Accumed,
Chicago, Ill.) was added at 20 .mu.l/well. Plates were again
incubated at 37.degree. C., 5% CO.sub.2 for 16 hours. Alamar Blue
gives a fluourometric readout based on number of live cells, and is
thus a direct measurement of cell proliferation in comparison to a
negative control. Plates were read on the Wallac Victor 2 1420
Multilabel Counter (Wallac, Turku, Finland) at wavelengths 530
(Excitation) and 590 (Emission).
[0406] Results showed no significant proliferation above background
levels in the 0 hour, 1 hour, 8 hour, and 16 hour time points.
Serum samples from the 4 hour time point showed 4-fold to greater
than 10-fold increases in proliferation above background,
indicating the presence of IL-22 in those samples.
C. LPS-Induced Endotoxemia Mouse Model: Experiment to Assess
Effects of IL-22RA2
[0407] The ability of IL-22RA2 treatment to effect proinflammatory
indicators induced with a single 25 .mu.g LPS dose IP in mice was
tested. All samples were analyzed for SAA, IL-22 and circulating
neutrophil counts. Subsets from each group were analyzed for
particular cytokine levels (1 hour samples were screened for TNF
alpha, 4 hour samples were analyzed for IL-6). Animals were
sacrificed at indicated time points in Table 8 below and whole
blood and serum were collected and aliquoted for analysis.
[0408] 72 C57BL/6N female mice (CRL) were given a single IP dose of
IL-22RA2 as described in Table 8, below. Control mice were
C57BL/6N(CRL).
[0409] 30 minutes later, they received another IP injection of 25
.mu.g LPS (Sigma) in 100 .mu.l, to initiate an endotoxemia cascade.
Mice in each group were sacrificed at corresponding time points as
indicated in Table 8, 50 .mu.l whole blood were collected to
measure total numbers of circulating neutrophils and the rest were
spun for serum and aliquoted for various assays described
herein.
TABLE-US-00008 TABLE 8 Group No Treatment LPS Sacrifice Samples A 8
100 .mu.g IL-22RA2 IP 25 .mu.g IP 1 hour Serum aliquots Blood 30
min post tx for CBC B 8 10 .mu.g IL-22RA2 IP 25 .mu.g IP 1 hour
Serum aliquots 30 min post tx Blood for CBC C 8 200 .mu.l PBS IP 25
.mu.g IP 1 hour Serum aliquots 30 min post tx Blood for CBC D 8 100
.mu.g IL-22RA2 IP 25 ug IP 4 hours Serum aliquots 30 min post tx
Blood for CBC E 8 10 .mu.g IL-22RA2 IP 25 .mu.g IP 4 hours Serum
aliquots 30 min post tx Blood for CBC F 8 200 .mu.l PBS IP 25 .mu.g
IP 4 hours Serum aliquots 30 min post tx Blood for CBC G 8 100
.mu.g IL-22RA2 IP 25 .mu.g IP 8 hours Serum aliquots 30 min post tx
Blood for CBC H 8 10 .mu.g IL-22RA2 IP 25 .mu.g IP 8 hours Serum
aliquots 30 min post tx Blood for CBC J 8 200 .mu.l PBS IP 25 .mu.g
IP 8 hours Serum aliquots 30 min post tx Blood for CBC K 5 controls
none Pre LPS Serum aliquots Blood for CBC
D. IL-22RA2-Fc4 Neutralizes SAA Induction In Vivo: SAA ELISA
Showing SAA Expression Induced by LPS in LPS-Induced Endotoxemia
Mouse Model is Inhibited by IL-22RA2-Fc4 Injection:
[0410] To assess whether IL-22RA2 could inhibit the SAA induction
in the LPS-induced endotoxemia mouse model, mice were injected with
IL-22RA2, 30 minutes prior to LPS injection, as shown in Table 8 in
Example 11C above.
[0411] An ELISA to determine SAA levels in the 4 hour and 8 hour
samples was performed using the Mouse SAA Immunoassay Kit
(BioSource International, California) following the manufacturer's
directions. At the 4 hour time point, mice treated with 100 .mu.g
or 10 .mu.g of IL-22RA2 showed a dose-dependant, statistically
significant reduction in SAA levels relative to the PBS injected
mice. At the 8 hour time point, mice treated with 100 .mu.g,
continued to show a statistically significant reduction in SAA
levels relative to the PBS injected mice. This indicates that the
presence of IL-22RA2 is able to inhibit the induction of SAA by LPS
in vivo.
EXAMPLE 13
In Vivo Effects of IL-22 Polypeptide on Skin
A. IL-22-Induced Acanthosis
[0412] Mice (female, C3H/HEJ, 8 weeks old; Jackson Labs, Bar
Harbor, Me.) were divided into three groups of six animals and one
group of 4. Human BHK-produced IL-22 was administered by constant
infusion via mini-osmotic pumps, resulting in local and steady
state serum concentrations proportional to the concentration of the
IL-22 contained in the pump. Alzet mini-osmotic pumps (model 2002;
Alza corporation Palo Alto, Calif.) were loaded under sterile
conditions with IL-22 protein (A601F, 0.22 mL) diluted in phosphate
buffered saline (pH 7.0) to a concentration within the pump of 2
mg/mL for group 1 mice, 0.2 mg/mL for group 2 mice, 0.02 mg/mL for
group 3 mice, or 0 mg/mL (diluent only) for group 4 mice. Pumps
were implanted subcutaneously in mice through a 1 cm incision in
the dorsal skin, and the skin was closed with sterile wound
closures. These pumps are designed to deliver their contents at a
rate of 0.5 .mu.l per hour over a period of 14 days. Using this
nominal rate of infusion, dose levels were calculated to be 24
.mu.g/day, 2.4 .mu.g/day, 0.24 .mu.g/day and 0 .mu.g/day for groups
1-4, respectively.
[0413] At the end of the 14-day period, the mice were euthanized
and an approximately 1 cm square sample of skin surrounding the
pump area was collected from each mouse. The skin was fixed in 10%
neutral buffered formalin. Formalin fixed samples of skin were
embedded in paraffin, routinely processed, sectioned at 5 um and
stained with hematoxylin and eosin. The tissues were
microscopically examined in blinded fashion by an ACVP board
certified veterinary pathologist. Histological changes were noted,
and the severity of acanthosis (i.e. epidermal thickening) scored
in a subjective manner using the following scoring system:
0-normal, 1-minimal acanthosis, 2-mild acanthosis, 3-moderate
acanthosis and 4-severe acanthosis. In addition, the skin of
selected groups was imaged with a CoolSnap digital camera (Roper
Scientific, Inc., San Diego, Calif.) and epidermal thickness
measured using histomorphometry software (Scion Image for Windows,
v. 4.02, Scion Corp., Frederick, Md.).
[0414] Administration of IL-22 at 2.4, and 24 .mu.g/day resulted in
epidermal thickening as shown by the average acanthosis score (see
s) consistently greater than observed in control group skin.
Moreover, IL-22 treated animals also had mononuclear cell
infiltrates in the epidermis. These infiltrates were not observed
in the vehicle treated controls.
[0415] Acanthosis scores of epidermal thickness and measurements of
skin thickness (in generic units of pixels) by groups are shown in
Table 9 below, as follows:
TABLE-US-00009 TABLE 9 Average Measured Group # n = Pump Acanthosis
Thickness 1 6 24 .mu.g IL-22/day 3.0 ND 2 6 2.4 .mu.g IL-22/day 2.4
67.5 3 6 0.24 .mu.g IL-22/day 2.2 ND 4 4 PBS infusion 1.8 45.6
B. Effect of IL-22RA2 on IL-22-Induced Acanthosis
[0416] Mice (female, C3H/HEJ, 8 weeks old; Jackson Labs, Bar
Harbor, Me.) were divided into eight groups of eight animals each.
IL-22 was administered by constant infusion via mini-osmotic pumps,
as described in Example 12A. Alzet mini-osmotic pumps (model 2001;
Alza corporation Palo Alto, Calif.) were loaded under sterile
conditions with IL-22 protein (A#601F, 0.22 mL) diluted in
phosphate buffered saline (pH 7.0) to a concentration within the
pump of 0.22 mg/mL for group 1-2 mice, 0.45 mg/mL for group 3-4
mice, 0.9 mg/mL for group 5-6 mice, or 0 mg/mL (diluent only) for
group 7-8 mice. These pumps are designed to deliver their contents
at a rate of 0.5 .mu.l per hour over a period of 14 days. Using
this nominal rate of infusion, dose levels were calculated to be 10
.mu.g/day in groups 1-2, 5 .mu.g/day on groups 3-4, 2.5 .mu.g/day
in groups 5-6 and 10 .mu.g/day for groups 7-8. For each pair of
groups at a given dose level of IL-22, one of the groups was
injected three times (days 1, 3, and 5) with 0.1 mg of human
IL-22RA2 Fc protein (described herein) by the interperitoneal
route. The other group was injected in the same fashion with
vehicle (PBS).
[0417] On day 8 of the study, mice were euthanized and an
approximately 1 cm square sample of skin surrounding the pump area
was collected from each mouse. The skin was fixed in 10% neutral
buffered formalin. Formalin fixed samples of skin were embedded in
paraffin, routinely processed, sectioned at 5 um and stained with
hematoxylin and eosin. The tissues were microscopically examined in
blinded fashion by an ACVP board certified veterinary pathologist.
This study was scored in a different manner than the previous
example. The number of layers in the epidermis, from stratum
basalis to stratum granulosum, was determined. Based on the
results, the sections were scored as follows: O-normal (2-3
layers), 1-mild thickening (3-4 layers), 2-moderate thickening (4-6
layers) and 3-severe thickening (>6 layers).
[0418] Administration of IL-22 at 2.5, 5, 10 .mu.g/day resulted in
epidermal thickening (see Table 10). Moreover, IL-22 treated
animals also had mononuclear cell infiltrates in the epidermis.
These infiltrates were not observed in the vehicle treated
controls. Concurrent administration of 100 .mu.g IL-22RA2 (3
injections) decreased the amount of epidermal thickening in mice
treated with 5 .mu.g IL-22/day.
[0419] Acanthosis scores of epidermal thickness by groups are shown
in Table 10, below, as follows:
TABLE-US-00010 TABLE 10 Average Group # n = Pump Injection
Acanthosis 1 8 2.5 .mu.g IL-22/day 100 .mu.L vehicle 1.1 (3
injections) 2 8 2.5 .mu.g IL-22/day 100 .mu.g IL-22RA2 0.8 (3
injections) 3 8 5 .mu.g IL-22/day 100 .mu.L vehicle 2.0 (3
injections) 4 8 5 .mu.g IL-22/day 100 .mu.g IL-22RA2 0.6 (3
injections) 5 8 10 .mu.g IL-22/day 100 .mu.L vehicle 2.0 (3
injections) 6 8 10 .mu.g IL-22/day 100 .mu.g IL-22RA2 1.9 (3
injections) 7 8 Vehicle 100 .mu.L vehicle 0.0 (3 injections) 8 8
Vehicle 100 .mu.g IL-22RA2 0.0 (3 injections)
[0420] Epidermal thickening and immune infiltrates were also
observed in human psoriatic skins. The skin phenotype observed in
IL-22 subcutaneous injection further indicated the potential role
of IL-22 in the pathogenesis of psoriasis. The fact that
IL-22RA2-Fc can neutralize the IL-22 induced skin phenotype
suggests the potential use of other IL-22 antagonists such as and
anti-IL-22 neutralizing antibody or soluble receptor for the
treatment of psoriasis and other IL-22 induced inflammatory
diseases.
C. Effect of IL-22RA Soluble Receptors and Anti-IL-22RA Antibodies
on IL-22-Induced or IL-20-Induced Acanthosis
[0421] The activity of IL-22RA soluble receptors, or an antibody to
IL-22RA, to inhibit the in vivo activity of IL-22 or IL-20 is
evaluated in a similar manner, using the histological endpoint of
acanthosis caused by subcutaneous infusion of IL-22 or IL-20
protein. In an example of this model C3H/HEJ mice are implanted
with subcutaneous mini-osmotic pumps as described in examples 12(A)
and 12(B) above. During the period of exposure to IL-22 or IL-20,
the mice are treated by injection with the purified monoclonal
antibody to IL-22 or similarly injected with vehicle as control. At
the end of the IL-22 infusion period, skin would be sampled from
the pump area for histological analysis. Similar to the IL-22RA2
soluble receptor IL-22 antagonist, IL-22 or IL-20 antagonist
IL-22RA soluble receptors, or anti-IL-22RA antibodies of the
present invention are expected to show reduction in epidermal
thickening and immune cell infiltrates caused by IL-22 or IL-20,
and hence be useful as IL-22 or IL-20 antagonists as a therapeutic
for psoriasis and other IL-22 or IL-20 induced inflammatory
disease.
EXAMPLE 14
IL-22 is Upregulated in Human Psoriatic Skin Samples
A. RNA Samples
[0422] Normal skin samples as well as skin from psoriasis patients
were obtained. The latter included involved skin from stable
plaque-type psoriasis and from adjacent uninvolved skin. RNA was
isolated from human skin samples using conventional methods. The
integrity and quality of RNA samples was tested on the Agilent 2100
Bioanalyzer (Agilent Technologies, Waldbronn Germany).
B. Primers and Probes for Quantitative RT-PCR-
[0423] Real-time quantitative RT-PCR using the ABI PRISM 7700
Sequence Detection System (PE Applied Biosystems, Inc., Foster
City, Calif.) has been previously described (See, Heid, C. A. et
al., Genome Research 6:986-994, 1996; Gibson, U. E. M. et al.,
Genome Research 6:995-1001, 1996; Sundaresan, S. et al.,
Endocrinology 139:4756-4764, 1998. This method incorporates use of
a gene specific probe containing both reporter and quencher
fluorescent dyes. When the probe is intact the reporter dye
emission is negated due to the close proximity of the quencher dye.
During PCR extension using additional gene-specific forward and
reverse primers, the probe is cleaved by the 5' to 3' nucleolytic
activity of the rTth DNA Polymerase which releases the reporter dye
from the probe resulting in an increase in fluorescent
emission.
[0424] The primers and probes used for real-time quantitative
RT-PCR analyses of IL-22 expression were designed using the primer
design software Primer Express.TM. (PE Applied Biosystems, Foster
City, Calif.). Primers for human IL-22 were designed spanning an
intron-exon junction to eliminate amplification of genomic DNA. The
forward primer, ZC42459 (SEQ ID NO:22) and the reverse primer,
ZC42458 (SEQ ID NO:23) were used in a PCR reaction (below) at a 800
nM concentration to synthesize a 72 bp product. The corresponding
IL-22 probe, ZC42460 (SEQ ID NO:24) was synthesized and labeled in
house at ZymoGenetics. The IL-22 probe was labeled at the 5' end
with a reporter fluorescent dye (6-carboxy-fluorescein) (FAM) (PE
Applied Biosystems) and at the 3' end with a quencher fluorescent
dye (6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied
Biosystems).
C. Real-Time Quantitative RT-PCR-
[0425] Relative levels of IL-22 mRNA were determined by analyzing
total RNA samples using the TAQMAN.RTM. EZ RT-PCR Core Reagents Kit
(PE Applied Biosystems). Runoff IL-22 transcript was made to
generate a standard curve used for quantitation. The curve
consisted of 10-fold serial dilutions ranging from about 1e8 to 1e3
total copies of whole message for IL-22 with each standard curve
point analyzed in triplicate. The total RNA samples from skin were
also analyzed in triplicate for human IL-22 transcript levels and
for levels of hGUS as an endogenous control. In a total volume of
25 .mu.l, each RNA sample was subjected to TAQMAN.RTM. EZ RT-PCR
reaction (PE Applied Biosystems) containing: approximately 25 ng of
total RNA in DEPC treated water (Dnase/Rnase free); appropriate
primers (approximately 800 nM ZC 42459 (SEQ ID NO:22) and ZC 42458
(SEQ ID NO:23); appropriate probe (approximately 100 nM ZC 42460
(SEQ ID NO:24); 1.times. TAQMAN.RTM. EZ Buffer; 3 mM Manganese
acetate; 300 .mu.M each d-CTP, d-ATP, and d-GTP and 600 .mu.M of
d-UTP; rTth DNA Polymerase (0.1 U/.mu.l); and AMPERASE.RTM. UNG
(0.01 U/.mu.l). PCR thermal cycling conditions were as follows: an
initial UNG treatment step of one cycle at 50.degree. C. for 2
minutes; followed by a reverse transcription (RT) step of one cycle
at 60.degree. C. for 30 minutes; followed by a deactivation of UNG
step of one cycle at 95.degree. C. for 5 minutes; followed by 40
cycles of amplification at 94.degree. C. for 20 seconds and
60.degree. C. for 1 minute.
[0426] Relative IL-22 RNA levels were determined by using the
Standard Curve Method as described by the manufacturer, PE
Biosystems (User Bulletin #2: ABI Prism 7700 Sequence Detection
System, Relative Quantitation of Gene Expression, Dec. 11, 1997).
The hGUS measurements were used to normalize the IL-22 levels. Data
are shown in Table 11 below.
TABLE-US-00011 TABLE 11 Skin Sample IL-22 Normal 0 Uninvolved 0
Involved 1149
[0427] IL-22 mRNA was undetectable in skin samples from normal
patients or from uninvolved areas. In contrast, there was dramatic
upregulation for IL-22 message in involved skin from psoriasis
patients. These data support a strong disease association for IL-22
to human psoriasis.
[0428] Over expression of IL-22 was shown in human psoriatic
lesions, suggesting that IL-22 is involved in human psoriasis.
Moreover, as described herein, over expression of IL-22 in
transgenic mice showed epidermal thickening and immune cell
involvement indicative of a psoriatic phenotype, and in addition
injection of IL-22 into normal mice showed epidermal thickening and
immune cell involvement indicative of a psoriatic phenotype which
was ablated by the soluble receptor antagonist IL-22RA2. Such in
vivo data further suggests that the pro-inflammatory IL-22 is
involved in psoriasis. As such, antagonists to IL-22 activity, such
as the anti-human-IL-22 monoclonal antibodies of the present
invention, as well as soluble receptors and antibodies thereto, are
useful in therapeutic treatment of inflammatory diseases,
particularly as antagonists to IL-22 in the treatment of psoriasis.
Moreover, antagonists to IL-22 activity, such as the
anti-human-IL-22 monoclonal antibodies of the present invention, as
well as soluble receptors and antibodies thereto, are useful in
therapeutic treatment of other inflammatory diseases for example as
antagonists to IL-22 in the treatment of atopic dermatitis, IBD,
colitis, Endotoxemia, arthritis, rheumatoid arthritis, and
psoriatic arthritis, adult respiratory disease (ARD), septic shock,
multiple organ failure, inflammatory lung injury such as asthma or
bronchitis, bacterial pneumonia, psoriasis, eczema, atopic and
contact dermatitis, and inflammatory bowel disease such as
ulcerative colitis and Crohn's disease.
EXAMPLE 15
IL-22 is Upregulated in Human Atopic Dermatitis Skin Samples
A. RNA Samples
[0429] Normal skin samples (n=4) as well as skin from atopic
dermatitis patients (n=4) were obtained. RNA was isolated from
human skin samples using conventional methods. The integrity and
quality of RNA samples was tested on the Agilent 2100 Bioanalyzer
(Agilent Technologies, Waldbronn Germany).
B. Primers and Probes for Quantitative RT-PCR-
[0430] Real-time quantitative RT-PCR using the ABI PRISM 7700
Sequence Detection System (PE Applied Biosystems, Inc., Foster
City, Calif.) has been previously described (See, Heid, C. A. et
al., Genome Research 6:986-994, 1996; Gibson, U. E. M. et al.,
Genome Research 6:995-1001, 1996; Sundaresan, S. et al.,
Endocrinology 139:4756-4764, 1998. This method incorporates use of
a gene specific probe containing both reporter and quencher
fluorescent dyes. When the probe is intact the reporter dye
emission is negated due to the close proximity of the quencher dye.
During PCR extension using additional gene-specific forward and
reverse primers, the probe is cleaved by the 5' to 3' nucleolytic
activity of the rTth DNA Polymerase which releases the reporter dye
from the probe resulting in an increase in fluorescent
emission.
[0431] The primers and probes used for real-time quantitative
RT-PCR analyses of IL-22 expression were designed using the primer
design software Primer Express.TM. (PE Applied Biosystems, Foster
City, Calif.). Primers for human IL-22 were designed spanning an
intron-exon junction to eliminate amplification of genomic DNA. The
forward primer, ZC42459 (SEQ ID NO:22) and the reverse primer,
ZC42458 (SEQ ID NO:23) were used in a PCR reaction (below) at a 800
nM concentration to synthesize a 72 bp product. The corresponding
IL-22 probe, ZC42460 (SEQ ID NO:24) was synthesized and labeled in
house at ZymoGenetics. The IL-22 probe was labeled at the 5' end
with a reporter fluorescent dye (6-carboxy-fluorescein) (FAM) (PE
Applied Biosystems) and at the 3' end with a quencher fluorescent
dye (6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied
Biosystems).
C. Real-Time Quantitative RT-PCR-
[0432] Relative levels of IL-22 mRNA were determined by analyzing
total RNA samples using the TAQMAN.RTM. EZ RT-PCR Core Reagents Kit
(PE Applied Biosystems). Runoff IL-22 transcript was made to
generate a standard curve used for quantitation. The curve
consisted of 10-fold serial dilutions ranging from about 1e8 to 1e3
total copies of whole message for IL-22 with each standard curve
point analyzed in triplicate. The total RNA samples from skin were
also analyzed in triplicate for human IL-22 transcript levels and
for levels of hGUS as an endogenous control. In a total volume of
25 .mu.l, each RNA sample was subjected to TAQMAN.RTM. EZ RT-PCR
reaction (PE Applied Biosystems) containing: approximately 25 ng of
total RNA in DEPC treated water (Dnase/Rnase free); appropriate
primers (approximately 800 nM ZC 42459 (SEQ ID NO:22) and ZC 42458
(SEQ ID NO:23); appropriate probe (approximately 100 nM ZC 42460
(SEQ ID NO:24); 1.times. TAQMAN.RTM. EZ Buffer; 3 mM Manganese
acetate; 300 .mu.M each d-CTP, d-ATP, and d-GTP and 600 .mu.M of
d-UTP; rTth DNA Polymerase (0.1 U/.mu.l); and AMPERASE.RTM. UNG
(0.01 U/.mu.l). PCR thermal cycling conditions were as follows: an
initial UNG treatment step of one cycle at 50.degree. C. for 2
minutes; followed by a reverse transcription (RT) step of one cycle
at 60.degree. C. for 30 minutes; followed by a deactivation of UNG
step of one cycle at 95.degree. C. for 5 minutes; followed by 40
cycles of amplification at 94.degree. C. for 20 seconds and
60.degree. C. for 1 minute.
[0433] Relative IL-22 RNA levels were determined by using the
Standard Curve Method as described by the manufacturer, PE
Biosystems (User Bulletin #2: ABI Prism 7700 Sequence Detection
System, Relative Quantitation of Gene Expression, Dec. 11, 1997).
The hGUS measurements were used to normalize the IL-22 levels.
[0434] IL-22 mRNA was undetectable in skin samples from normal
patients. In contrast, there was dramatic upregulation for IL-22
message in 3 out of 4 skin samples from atopic dermatitis patients
(about 400-2300 copies). These data support a strong disease
association for IL-22 to human atopic dermatitis.
[0435] Over expression of IL-22 was shown in human atopic
dermatitis skins, suggesting that IL-22 is involved in human atopic
dermatitis. Moreover, as described herein, over expression of IL-22
in transgenic mice showed epidermal thickening and immune cell
involvement indicative of an atopic dermatitis phenotype, and in
addition injection of IL-22 into normal mice showed epidermal
thickening and immune cell involvement indicative of a atopic
dermatitis phenotype which was ablated by the soluble receptor
antagonist IL-22RA2. Such in vivo data further suggests that the
pro-inflammatory IL-22 is involved in atopic dermatitis. As such,
antagonists to IL-22 activity, such as the anti-human-IL-22
monoclonal antibodies of the present invention, as well as soluble
receptors and antibodies thereto, are useful in therapeutic
treatment of inflammatory diseases, particularly as antagonists to
IL-22 in the treatment of atopic dermatitis. Moreover, antagonists
to IL-22 activity, such as the anti-human-IL-22 monoclonal
antibodies of the present invention, as well as soluble receptors
and antibodies thereto, are useful in therapeutic treatment of
other inflammatory diseases for example as antagonists to IL-22 in
the treatment of atopic dermatitis, IBD, colitis, Endotoxemia,
arthritis, rheumatoid arthritis, and psoriatic arthritis, adult
respiratory disease (ARD), septic shock, multiple organ failure,
inflammatory lung injury such as asthma or bronchitis, bacterial
pneumonia, atopic dermatitis, eczema, atopic and contact
dermatitis, and inflammatory bowel disease such as ulcerative
colitis and Crohn's disease.
EXAMPLE 16
Human IL-22 Polyclonal Antibodies
[0436] Anti IL-22 Polyclonal antibodies were prepared by immunizing
2 female New Zealand white rabbits with the purified mature
recombinant human IL-22 polypeptide (amino acid residues 22 (Ala)
to 167 (Ile) of SEQ ID NO:6), produced from BHK cells (IL-22-BHK).
The rabbits were each given an initial intraperitoneal (ip)
injection of 200 .mu.g of purified protein in Complete Freund's
Adjuvant followed by booster IP injections of 100 .mu.g peptide in
Incomplete Freund's Adjuvant every three weeks. Seven to ten days
after the administration of the second booster injection (3 total
injections), the animals were bled and the serum was collected. The
animals were then boosted and bled every three weeks.
[0437] The human IL-22-specific polyclonal antibodies were affinity
purified from the immune rabbit serum using a CNBr-SEPHAROSE 4B
protein column (Pharmacia LKB) that was prepared using 10 mg of the
specific antigen purified recombinant protein human IL-22-BHK per
gram of CNBr-SEPHAROSE, followed by 20.times. dialysis in PBS
overnight. Human IL-22-specific antibodies were characterized by
ELISA using 500 ng/ml of the purified recombinant protein human
IL-22-BHK as antibody target. The lower limit of detection (LLD) of
the rabbit anti-human IL-22 affinity purified antibody is 280 pg/ml
on its specific purified recombinant antigen human IL-22-BHK.
[0438] The human IL-22-specific polyclonal antibodies were
characterized further for their ability to block the
cell-proliferative activity ("neutralization assay") of purified
recombinant human IL-22-BHK on BaF3/CRF2-4/IL-22RA cells (Example 2
and Example 3). A 50.times. molar excess of the human
IL-22-specific polyclonal antibodies was sufficient to inhibit cell
proliferation.
EXAMPLE 17
Anti-Human IL-22 Monoclonal Antibodies
[0439] Monoclonal antibodies were prepared by immunizing 4 female
Sprague-Dawley Rats (Charles River Laboratories, Wilmington,
Mass.), with the purified mature recombinant human IL-22
polypeptide (amino acid residues 22 (Ala) to 167 (Ile) of SEQ ID
NO:6), produced from BHK cells (IL-22-BHK). The rats were each
given an initial intraperitoneal (IP) injection of 100 .mu.g of the
purified human recombinant IL-22 protein in Complete Freund's
Adjuvant (Pierce, Rockford, Ill.) followed by booster IP injections
of 50 .mu.g of the purified recombinant protein in Incomplete
Freund's Adjuvant every two weeks. Seven to ten days after the
administration of the third booster injection, the animals were
bled and the serum was collected.
[0440] The human IL-22-specific rat sera samples were characterized
by ELISA using 500 ng/ml biotinylated human IL-22-BHK and 500 ng/ml
biotinylated mouse IL-22, biotinylated muIL-22-E. coli (R+D
Systems, Minneapolis, Minn.) antibody targets. Three rat serum
samples had titer to the specific antibody target biotinylated
human IL-22-BHK at a dilution of 1:1E5 and to the specific antibody
target biotinylated muIL-22-E. coli at a dilution of 1:1E4.
[0441] Splenocytes and lymphatic node cells were harvested from 2
high-titer rats and fused to SP2/0 (mouse) myeloma cells using PEG
1500 in two separate fusion procedures (4:1 fusion ratio,
splenocytes to myeloma cells, "Antibodies A Laboratory Manual, E.
Harlow and D. Lane, Cold Spring Harbor Press). Following 10 days
growth post-fusion, specific antibody-producing hybridoma pools
were identified by ELISA using the biotinylated recombinant protein
human IL-22-BHK and the biotinylated recombinant protein muIL-22-E.
coli as separate antibody targets. Hybridoma pools positive in both
ELISA protocols were analyzed further for their ability to block or
reduce the cell-proliferative activity ("neutralization assay") of
purified recombinant muIL-22-E. coli on BaF3/CRF2-4/IL-22RA cells
(Example 2 and Example 3).
[0442] Hybridoma pools yielding positive results by ELISA only or
ELISA and the "neutralization assay" were cloned at least two times
by limiting dilution.
[0443] Monoclonal antibodies purified from tissue culture media
were characterized for their utility in an ELISA for the
quantitative determination of recombinant and native human IL-22 in
mouse and human serum samples. The two antibodies selected resulted
in a quantitative assay with a lower limit of detection of
approximately 1 ng/ml recombinant huIL-22-E. coli in 100% human
serum.
[0444] Monoclonal antibodies purified from tissue culture media
were characterized for their ability to block or reduce the
cell-proliferative activity ("neutralization assay") of purified
recombinant huIL-22-E. coli or muIL-22-E. coli on
BaF3/CRF2-4/IL-22RA cells (Example 2 and Example 3). Six
"neutralizing" monoclonal antibodies were identified in this
manner. Hybridomas expressing the neutralizing monoclonal
antibodies to human IL-22 described above were deposited with the
American Type Tissue Culture Collection (ATCC; Manassas Va.) patent
depository as original deposits under the Budapest Treaty and were
given the following ATCC Accession No.s: clone 266.16.1.4.4.1 (ATCC
Patent Deposit Designation PTA-5035); clone 266.5.1.2.2.3 (ATCC
Patent Deposit Designation PTA-5033); clone 267.17.1.1.4.1 (ATCC
Patent Deposit Designation PTA-5038); clone 267.4.1.1.4.1 (ATCC
Patent Deposit Designation PTA-5037); clone 266.12.6.1.3.2.1 (ATCC
Patent Deposit Designation PTA-5034); clone 266.19.1.10.5.2 (ATCC
Patent Deposit Designation PTA-5036); and clone 267.9.1.1.4.1 (ATCC
Patent Deposit Designation PTA-5353).
EXAMPLE 18
Anti-IL-22RA Monoclonal Antibodies
[0445] Monoclonal antibodies were prepared by immunizing 4 Lewis
Rats (Rockland Immunochemicals, Gilbertsville, Pa.), with the
cleaved and purified recombinant fusion protein, muIL-22RA-Fc (SEQ
ID NO:4). The rats were each given an initial intraperitoneal (IP)
injection of 100 .mu.g of the purified recombinant fusion protein
in Complete Freund's Adjuvant (Pierce, Rockford, Ill.) followed by
booster IP injections of 50 .mu.g of the purified recombinant
protein in Incomplete Freund's Adjuvant every two weeks for four
weeks. Following the first four weeks of immunizations, booster IP
injections of 50 ug of the cleaved purified recombinant protein
coupled to the carrier protein keyhole limpet hemocyanin (KLH,
Pierce, Rockford, Ill.) in Incomplete Freund's were administered
every two weeks for four weeks. Seven to ten days after the
administration of the fourth booster injection, the animals were
bled and the serum was collected.
[0446] The muIL-22RA-specific rat serum samples were characterized
by ELISA using 500 ng/ml of the purified recombinant fusion protein
muIL-22RA-Fc as the specific antibody target and an unrelated
fusion protein as a non-specific antibody target.
[0447] Splenocytes were harvested from one high-titer rat and fused
to SP2/0 (mouse) myeloma cells in an optimized PEG-mediated fusion
protocol (Rockland Immunochemicals). Following 12 days growth
post-fusion, specific antibody-producing hybridoma pools were
identified by ELISA using 500 ng/ml each of the purified
recombinant fusion protein muIL-22RA-Fc-Bv as the specific antibody
target and an unrelated fusion protein as a non-specific antibody
target. Hybridoma pools positive to the specific antibody target
only were analyzed further for their ability to block or reduce the
cell-proliferative activity ("neutralization assay") of purified
recombinant muIL-22-E. coli on BaF3/CRF2-4/IL-22RA cells (Example 2
and Example 3) and an ability to bind via FACS analysis to
BaF3/CRF2-4/IL-22RA cells (Example 2 and Example 3) as antibody
target.
[0448] Hybridoma pools yielding a specific positive result in the
ELISA assay and positive results in either the FACS or
"neutralization assay" were cloned at least two times by limiting
dilution.
[0449] Monoclonal antibodies in tissue culture media were
characterized for their ability to block or reduce proliferation of
BaF3/CRF2-4/IL-22RA cells (Example 2 and Example 3), grown in the
presence of the purified recombinant proteins muIL-22-E. coli or
huIL-22-BHK. Fourteen "neutralizing" monoclonal antibodies have
been identified and nine monoclonal antibodies have been
cloned.
[0450] Hybridomas expressing the neutralizing monoclonal antibodies
to mouse IL-22RA described above were deposited with the American
Type Tissue Culture Collection (ATCC; Manassas Va.) patent
depository as original deposits under the Budapest Treaty and were
given the following ATCC Accession No.s: clone R2.1.1G11.1 (ATCC
Patent Deposit Deposit Designation [PTA-6035]); clone R2.1.5F4.1
(ATCC Patent Deposit Designation[PTA-6024]); clone R2.1.5H8.1 (ATCC
Patent Deposit Designation[PTA-6025]); clone R2.1.12G7.1 (ATCC
Patent Deposit Designation [PTA-6036]); clone R2.1.13C8.1 (ATCC
Patent Deposit Designation PTA-5037); clone R2.1.15E2.1 (ATCC
Patent Deposit Designation[PTA-6038]); clone R2.1.16C11.1 (ATCC
Patent Deposit Designation [PTA-6039]); clone R2.1.18C8.1 (ATCC
Patent Deposit Deposit Designation [PTA-6048]); and clone
R2.1.21G8.2 (ATCC Patent Deposit Deposit
Designation[PTA-6111]).--
EXAMPLE 19
Binding Affinity of Two Rat-Anti-Ms-IL-22RA MAb
[0451] Goat-anti-Rat IgG-Fc gamma specific Antibody (Jackson) was
immobilized onto a CM5 Biacore chip. The assay was optimized to
bind each mAb onto the anti-Rat capture surface and then a
concentration series of IL-22RA was injected across the mAb to see
association (Ka) and dissociation (Kd). After preliminary testing,
non-specific binding was observed between the fusion protein and
the capture surface on the chip. A vial of IL-22RA that had the Fc4
tag cleaved by thrombin was acquired and subsequently tested to
show no background effects. After each run, the surface was
regenerated back to the anti-Rat Antibody with 2 injections of 20
mM HCl. Data was generated for each MAb and evaluation software
(BIAevaluation software version 3.2, Pharmacia BIAcore, Uppsala,
Sweden) was used to assess the kinetics of the anti-IL-22RA
antibody binding to the IL-22RA protein, as shown in Table 12
below:
TABLE-US-00012 TABLE 12 Clone R2.1.5F4.1** Clone R2.1.15E2.1** ka
(M-1s-1) 1.49E+06 ka (M-1s-1) 1.76E+06 kd (s-1) 1.70E-04 kd (s-1)
2.55E-04 KA (M-1) 8.76E+09 KA (M-1) 6.66E+09 KD (M) 1.14E-10 KD (M)
1.504E-10 Chi2 2.08 Chi2 1.5 **Equilibrium association (Ka) and
dissociation (Kd) rate constants for each anti IL-22RA MAB are
shown and values fall in machine limits. Chi2 refers to the sum of
the square of the residuals between the binding curves and the
evaluation fitting curves. The closer the 0, the more confidence in
the data.
[0452] As shown by Table 12, both anti-IL-22RA MAb's bind strongly
to the IL-22RA protein, as evinced by the binding in pico-molar
concentration to the IL-22RA (thrombin-cleaved Fc4 tag). This data
is shown with good confidence based on the low Chi.sup.2 values and
shows mAb Clone R2.1.5F4.1 to have a slightly stronger affinity for
the IL-22RA receptor.
EXAMPLE 20
Immunohistochemical Analysis of IL-22 Protein Expression In Vivo in
Tissue Samples
A. Summary
[0453] Immunohistochemical (IHC) analysis of IL-22 protein
expression and localization was achieved using anti-human IL-22
(anti-hIL-22) monoclonal antibody (Mab 266.19.1.10.5.2) in the
following tissue samples: a Human multi-Normal Grid and Tumor Grid:
Human pancreatitis, lung and renal disease samples; Human psoriasis
skin samples; INS IL-22 TG (expressed from the rat insulin
promoter) and WT mouse pancreas; muIL-22-EuLCK TG and WT mouse skin
sample; and DSS (WT and IL-22 KO) mouse colon sample. Moreover the
staining pattern of anti-hIL-22 monoclonal antibody MAB
266.19.1.10.5.2 (Example 17) vs. polyclonal antibody (rabbit
anti-hIL-22) (Example 16) was compared.
[0454] The rat anti-Human IL-22 monoclonal antibodies MAb
266.16.1.4.4.1, and MAb 266.19.1.10.5.2 (Example 17) were tested
were shown to stain the majority of BHK/human IL-22 (>50%) but
also some BHK/mouse IL-22 cells (1-5%), and were used to
investigate the tissue distribution and expression of IL-22 in both
human patient and animal model samples and used to compare the
staining pattern with polyclonal rabbit antibody to confirm the
results.
B. Materials and Methods
[0455] Formalin-fixed and paraffin-embedded cells and tissues from
human sources and mouse animal models were sectioned at 5 .mu.m.
The cells included BHK cells expressing either human or mouse IL-22
and wild type as positive control and negative control,
respectively. The human tissues included a Multi-tissue control
slide (NormalGrid.TM.; Biomeda, Foster City, Calif.) with 50
sections of various normal human tissues (e.g., brain, pituitary
gland, adrenal gland, breast, kidney, heart, stomach, small
intestine, large intestine, fetal liver, liver, skin, pancreas,
lung, tonsil, ovary, testis, prostate, uterus, placenta, thyroid
and spleen); a Multi-tissue control slide (TumorGrid.TM.; Biomeda,
Foster City, Calif.) with 50 sections of various human tumors
(e.g., lung adeno Ca., liver adeno Ca., kidney adeno Ca., colon
adeno Ca., breast adeno Ca., thyroid adeno Ca., stomach adeno Ca.,
prostate adeno Ca., pancreas adeno Ca., ovary adeno Ca., lymphoma,
melanoma, sarcoma ewings, sarcoma epithelioid, sarcoma MFH, sarcoma
Rhabdo, carcinoid, undiff. Ca., mesothelioma, teretoma and
seminoma); lung carcinoma from CHTN (Cooperation Human Tissue
Network, Cleveland, Ohio); normal pancreas, pancreas with chronic
pancreatitis, lung with chronic perivascular inflammation, kidneys
with either multifocal glomerulosclerosis, mesangioproliferative
glomerulonephritis, or sclerotic glomeruli interstitial fibrosis
from NDRI (National Disease Research Interchange, Philadelphia,
Pa.); and psoriatic skin samples from human. The mouse tissues
included colons from inflammatory bowel disease animal model (DSS
model disclosed herein, Swiss Webster female mice) and from IL-20
WT and KO colitis animal model (DSS mice, wild type and IL-20
(IL-20) knock out female mice) treated with either vehicle or 4%
DSS in drinking water for 7 days; and skin samples from transgenic
(TG) animal models including mIL-22-EuLCK TG and mIL-22-INS control
and TG animals. One section per block/slide was stained with
hematoxylin and eosin (H&E) for histologic examination and the
subsequent section were immunohistochemically stained for IL-22
protein expression and localization.
[0456] For immunohistochemistry, the cell and tissue sections were
placed on ChemMate.TM. Capillary Gap Plus microscope slides
(BioTek, Winooski, Vt.), dried at 60.degree. C. oven for 60 minutes
and dewaxed using standard conditions of 3.times.5 minutes in
xylene, 4 minutes in 100% EtOH, 3 minutes in 100% EtOH, and 2
minutes in 95% EtOH. The tissue sections were then subjected to a
20-minute enzyme-induced epitope retrieval process at 37.degree. C.
with pepsin (NeoMarkers Fremont Calif.) followed by an
avidin/biotin-blocking step done according to the manufacturers
instructions (Zymed, South San Francisco, Calif.). TechMate 500.TM.
Automated Immunostainer and Immunoperoxidase (IP)
immunohistochemical protocol with avidin-biotin-complex detection
system (Ventana Biotek Systems, Tucson, Ariz.) were employed for
the staining. The TechMate 500.TM. Automated Immunostainer employed
the principle of capillary action and the IP protocol utilized a
type of immunostaining referred to as a "sandwich" technique. The
sections were preblocked with 5% normal goat serum (Vector,
Burlingame Calif.) in PBS for 10 minutes followed by 1.times.
buffer1 wash (Signet, Dedham Mass.) and then incubated with a
primary antibody against IL-22 (MAB 266.19.1.10.5.2) (Example 17),
PAS purified at 2.04 mg/ml) diluted at 1:800 for 30 minutes at room
temperature followed by 5.times. buffer1 wash. The primary antibody
was diluted in TechMate 500.TM. antibody dilution buffer (Ventana).
Biotinylated goat anti-rat IgG (Vector) diluted at 1:200 plus 5%
normal goat serum and 2.5% nonfat dry milk in PBS was used as the
secondary-linking antibodies for 25 minutes at room temperature
followed by 1.times. buffer1 wash and 1.times. Buffer2&3 wash
(Signet). The tissues sections were then subjected to a 3.times.7
minutes 3% hydrogen peroxide (HP) blocking (Ventana) followed by
3.times. buffer2&3 wash. Immunoperoxidase labeling was
performed with a peroxides DAB kit (Ventana), incubating with
avidin-biotin-complex (ABC) for 30 minutes followed by 5.times.
buffer2&3 wash and diaminobenzidine (DAB) for 4.times.4 minutes
followed by 2.times. buffer2&3 wash and 1.times. water wash
(Signet, Cat. No. 2340). Tissues were then counter stained with
methyl green (Dako, Cat. No. S1962) for 10 minutes followed by
2.times. buffer2&3 wash and 3.times. water wash. Control
included non-immune primary sera using rat primary antibody isotype
control (Zymed) to replace the primary antibody.
[0457] Immunostaining was observed using an Olympus BH-2 microscope
and images were captured by CoolSNAP HQ digital camera (Roper
Scientific, Tucson, Ariz.).
C. Results
[0458] Positive and negative control cell lines: MAB
266.19.1.10.5.2, an anti-hIL-22 monoclonal antibody, demonstrated
positive staining on both human IL-22 expressing BHK cells (+++)
and murine IL-22 expressing BHK cells (+), and no staining on the
wild type BHK cells (-). All the positive and negative BHK cell
lines stained with rat isotype negative control to replace the
primary antibody showed no staining (-) which indicated that the
antibody is specific to IL-22 ligand. The antibody had cross
immunoreactivity to both human and mouse IL-22.
[0459] Human tissues: Human multi-Normal Grid and Tumor Grid;
pancreas, lung and renal disease samples; and human psoriasis skin
samples were examined. These human tissues included 1). Brain,
pituitary gland, adrenal gland, breast, kidney, heart, stomach,
small intestine, large intestine, fetal liver, liver, skin,
pancreas, lung, tonsil, ovary, uterus, testis, placenta, thyroid
and spleen on the Multi-tissue control slides
(NormalGrid.TM.)/normal human tissues; 2). Lung adeno Ca., liver
adeno Ca., kidney adeno Ca., thyroid adeno Ca., stomach adeno Ca.,
prostate adeno Ca., pancreas adeno Ca., ovary adeno Ca., lymphoma,
melanoma, sarcoma ewings, sarcoma epithelioid, sarcoma MFH, sarcoma
Rhabdo, carcinoid, undiff. Ca., mesothelioma, teratoma, and
seminoma, on the Multi-tissue control slides (TumorGrid.TM.)/human
abnormal tissues/tumor; 3). Normal pancreas, pancreas with chronic
pancreatitis, lung with chronic perivascular inflammation, lung
Ca., kidney with multifocal glomerulosclerosis, kidney with
mesangioproliferative glomerulonephritis, kidney with sclerotic
glomeruli interstitial fibrosis from CHTN and/or NDRI; 4).
[0460] Mouse tissues: INS IL-22 TG and WT mouse pancreas were
examined. Scattered cells throughout the islets in the INS IL-22 TG
pancreas demonstrated strong positive staining (+++) with Mab MAB
266.19.1.10.5.2 and WT pancreas showed no staining (-).
[0461] Comparison of polyclonal and monoclonal antibodies. The
anti-IL-22 polyclonal antibody (Example 16) was shown to be
sensitive, whereas monoclonal antibody MAB 266.19.1.10.5.2 was
specific. The polyclonal antibody showed positive staining on human
IL-22 expressing BHK cells (+++), on murine IL-22 expressing BHK
cells (+), in various human and mouse tissue samples (+), and in
the islets of INS mIL-22 TG mice (+++). A greater percentage of the
islets of the transgenics (vs. wild-type) contained positive
staining. The staining in the transgenic islets was generally
distributed throughout the islet (+++) while staining in the
wild-type islets was generally limited to the periphery of the
islet (+). However, this antibody also showed non-specific staining
on the WT BHK negative control cells (+).
[0462] MAB 266.19.1.10.5.2 showed positive staining on human IL-22
expressing BHK cells on murine IL-22 expressing BHK cells (+), and
in the islets of INC mIL-22 TG mice (+++). The staining in the
transgenic islets was generally distributed throughout the islet
(+++) while the wild-type islets demonstrated negative staining
(-).
EXAMPLE 21
IL-20 is Upregulated in Human Psoriatic Skin Samples
A. RNA Samples:
[0463] Normal skin samples as well as skin from psoriasis patients
were obtained. The latter included involved skin from psoriasis and
from adjacent uninvolved skin. RNA was isolated from human skin
samples using conventional methods. The integrity and quality of
RNA samples was tested on the Agilent 2100 Bioanalyzer (Agilent
Technologies, Waldbronn Germany).
B. Primers and Probes for Quantitative RT-PCR-
[0464] Real-time quantitative RT-PCR using the ABI PRISM 7700
Sequence Detection System (PE Applied Biosystems, Inc., Foster
City, Calif.) has been previously described (See, Heid, C. A. et
al., Genome Research 6:986-994, 1996; Gibson, U. E. M. et al.,
Genome Research 6:995-1001, 1996; Sundaresan, S. et al.,
Endocrinology 139:4756-4764, 1998. This method incorporates use of
a gene specific probe containing both reporter and quencher
fluorescent dyes. When the probe is intact the reporter dye
emission is negated due to the close proximity of the quencher dye.
During PCR extension using additional gene-specific forward and
reverse primers, the probe is cleaved by the 5' to 3' nucleolytic
activity of the rTth DNA Polymerase which releases the reporter dye
from the probe resulting in an increase in fluorescent
emission.
[0465] The primers and probes used for real-time quantitative
RT-PCR analyses of IL-20 expression were designed using the primer
design software Primer Express.TM. (PE Applied Biosystems, Foster
City, Calif.). The forward primer, ZC40541 (SEQ ID NO:25) and the
reverse primer, ZC 40542 (SEQ ID NO:26) were used in a PCR reaction
(below) at a 800 nM concentration to synthesize a 71 bp product.
The corresponding IL-20 TaqMan.RTM. probe, ZC 40544 (SEQ ID NO:27)
was synthesized and labeled by PE Applied Biosystems. The IL-20
probe was labeled at the 5' end with a reporter fluorescent dye
(6-carboxy-fluorescein) (FAM) (PE Applied Biosystems) and at the 3'
end with a quencher fluorescent dye
(6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied
Biosystems).
C. Real-Time Quantitative RT-PCR
[0466] Relative levels of IL-20 mRNA were determined by analyzing
total RNA samples using the TAQMAN.RTM. EZ RT-PCR Core Reagents Kit
(PE Applied Biosystems). Runoff IL-20 transcript was made to
generate a standard curve used for quantitation. The curve
consisted of 10-fold serial dilutions ranging from about 1e8 to 1e3
total copies of whole message for IL-20 with each standard curve
point analyzed in triplicate. The total RNA samples from skin were
also analyzed in triplicate for human IL-20 transcript levels and
for levels of hGUS as an endogenous control. In a total volume of
25 .mu.l, each RNA sample was subjected to TAQMAN.RTM. EZ RT-PCR
reaction (PE Applied Biosystems) containing: approximately 25 ng of
total RNA in DEPC treated water (Dnase/Rnase free); appropriate
primers (approximately 800 nM ZC40541 (SEQ ID NO:25) and ZC40542
(SEQ ID NO:26); appropriate probe (approximately 100 nM ZC40544
(SEQ ID NO:27); 1.times. TAQMAN.RTM. EZ Buffer; 3 mM Manganese
acetate; 300 .mu.M each d-CTP, d-ATP, and d-GTP and 600 .mu.M of
d-UTP; rTth DNA Polymerase (0.1 U/.mu.l); and AMPERASE.RTM. UNG
(0.01 U/.mu.l). PCR thermal cycling conditions were as follows: an
initial UNG treatment step of one cycle at 50.degree. C. for 2
minutes; followed by a reverse transcription (RT) step of one cycle
at 60.degree. C. for 30 minutes; followed by a deactivation of UNG
step of one cycle at 95.degree. C. for 5 minutes; followed by 40
cycles of amplification at 94.degree. C. for 20 seconds and
60.degree. C. for 1 minute.
[0467] Relative IL-20 RNA levels were determined by using the
Standard Curve Method as described by the manufacturer, PE
Biosystems (User Bulletin #2: ABI Prism 7700 Sequence Detection
System, Relative Quantitation of Gene Expression, Dec. 11, 1997).
The hGUS measurements were used to normalize IL-20 levels. Data are
shown in Table 13 below.
TABLE-US-00013 TABLE 13 Skin Sample IL-20 Normal 2903 Uninvolved
7233 Involved 27,695
[0468] Although IL-20 mRNA was detectable in skin samples from
normal patients or from uninvolved areas, there was upregulation
for IL-20 message in involved skin from psoriasis patients. The
receptor subunits for IL-20, including IL-20RA, IL-22RA (IL-22RA),
and IL-20RB were expressed in human normal and diseased skin. These
data support a strong disease association for IL-20 to human
psoriasis.
[0469] Overexpression of IL-20 was shown in human psoriatic
lesions, suggesting that IL-20 is involved in human psoriasis.
Moreover, as described herein, over expression of IL-20 in
transgenic mice showed epidermal thickening and immune cell
involvement indicative of a psoriatic phenotype. Such in vivo data
further suggests that IL-20 is involved in psoriasis. As such,
antagonists to IL-20 activity, such as the anti-human-IL-22RA
monoclonal antibodies of the present invention, as well as soluble
receptors and antibodies thereto, and anti-IL-20 neutralizing and
monoclonal antibodies, are useful therapeutically as antagonists to
IL-20 in the treatment of inflammatory diseases, such as psoriasis,
as well as other indications as disclosed herein.
EXAMPLE 22
IL-20 is Upregulated in Human Atopic Dermatitis Skin Samples
A. RNA Samples
[0470] Normal skin samples as well as skin from atopic dermatitis
patients were obtained. RNA was isolated from human skin samples
using conventional methods. The integrity and quality of RNA
samples was tested on the Agilent 2100 Bioanalyzer (Agilent
Technologies, Waldbronn Germany).
B. Primers and Probes for Quantitative RT-PCR-
[0471] Real-time quantitative RT-PCR using the ABI PRISM 7700
Sequence Detection System (PE Applied Biosystems, Inc., Foster
City, Calif.) has been previously described (See, Heid, C. A. et
al., Genome Research 6:986-994, 1996; Gibson, U. E. M. et al.,
Genome Research 6:995-1001, 1996; Sundaresan, S. et al.,
Endocrinology 139:4756-4764, 1998. This method incorporates use of
a gene specific probe containing both reporter and quencher
fluorescent dyes. When the probe is intact the reporter dye
emission is negated due to the close proximity of the quencher dye.
During PCR extension using additional gene-specific forward and
reverse primers, the probe is cleaved by the 5' to 3' nucleolytic
activity of the rTth DNA Polymerase which releases the reporter dye
from the probe resulting in an increase in fluorescent
emission.
[0472] The primers and probes used for real-time quantitative
RT-PCR analyses of IL-20 expression were designed using the primer
design software Primer Express.TM. (PE Applied Biosystems, Foster
City, Calif.). The forward primer, ZC40541 (SEQ ID NO:25) and the
reverse primer, ZC 40542 (SEQ ID NO:26) were used in a PCR reaction
(below) at a 800 nM concentration to synthesize a 71 bp product.
The corresponding IL-20 TaqMan.RTM. probe, ZC 40544 (SEQ ID NO:27)
was synthesized and labeled by PE Applied Biosystems. The IL-20
probe was labeled at the 5' end with a reporter fluorescent dye
(6-carboxy-fluorescein) (FAM) (PE Applied Biosystems) and at the 3'
end with a quencher fluorescent dye
(6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied
Biosystems).
C. Real-Time Quantitative RT-PCR
[0473] Relative levels of IL-20 mRNA were determined by analyzing
total RNA samples using the TAQMAN.RTM. EZ RT-PCR Core Reagents Kit
(PE Applied Biosystems). Runoff IL-20 transcript was made to
generate a standard curve used for quantitation. The curve
consisted of 10-fold serial dilutions ranging from about 1e8 to 1e3
total copies of whole message for IL-20 with each standard curve
point analyzed in triplicate. The total RNA samples from skin were
also analyzed in triplicate for human IL-20 transcript levels and
for levels of hGUS as an endogenous control. In a total volume of
25 .mu.l, each RNA sample was subjected to TAQMAN.RTM. EZ RT-PCR
reaction (PE Applied Biosystems) containing: approximately 25 ng of
total RNA in DEPC treated water (Dnase/Rnase free); appropriate
primers (approximately 800 nM ZC40541 (SEQ ID NO:25) and ZC40542
(SEQ ID NO:26); appropriate probe (approximately 100 nM ZC40544
(SEQ ID NO:27); 1.times. TAQMAN.RTM. EZ Buffer; 3 mM Manganese
acetate; 300 .mu.M each d-CTP, d-ATP, and d-GTP and 600 .mu.M of
d-UTP; rTth DNA Polymerase (0.1 U/.mu.l); and AMPERASE.RTM. UNG
(0.01 U/.mu.l). PCR thermal cycling conditions were as follows: an
initial UNG treatment step of one cycle at 50.degree. C. for 2
minutes; followed by a reverse transcription (RT) step of one cycle
at 60.degree. C. for 30 minutes; followed by a deactivation of UNG
step of one cycle at 95.degree. C. for 5 minutes; followed by 40
cycles of amplification at 94.degree. C. for 20 seconds and
60.degree. C. for 1 minute.
[0474] Relative IL-20 RNA levels were determined by using the
Standard Curve Method as described by the manufacturer, PE
Biosystems (User Bulletin #2: ABI Prism 7700 Sequence Detection
System, Relative Quantitation of Gene Expression, Dec. 11, 1997).
The hGUS measurements were used to normalize IL-20 levels.
[0475] IL-20 mRNA was detectable at a low level (about 800 copies)
in skin samples. In contrast, there was upregulation for IL-20
message in skins from atopic dermatitis patients (about 8600
copies). The receptor subunits for IL-20, including IL-20RA),
IL-22RA, and IL-20RB are expressed in human normal and diseased
skin. These data support a strong disease association for IL-20 to
human atopic dermatitis.
[0476] Overexpression of IL-20 was shown in human atopic dermatitis
skins, suggesting that IL-20 is involved in human atopic
dermatitis. Moreover, as described herein, over expression of IL-20
in transgenic mice showed epidermal thickening and immune cell
involvement indicative of an atopic dermatitis phenotype. Such in
vivo data further suggests that IL-20 is involved in atopic
dermatitis. As such, antagonists to IL-20 activity, such as the
anti-human-IL-22RA monoclonal antibodies of the present invention,
as well as soluble receptors and antibodies thereto, and anti-IL-20
neutralizing and monoclonal antibodies, are useful therapeutically
as antagonists to IL-20 in the treatment of inflammatory diseases,
such as atopic dermatitis, as well as other indications as
disclosed herein.
EXAMPLE 23
Up-Regulation of IL-8 by IL-20
[0477] Normal Human Epidermal neonatal keratinocytes (NHEK) (from
Clonetics) at passage 2 were plated and grown to confluency in 12
well tissue culture plates. KGM (Keratinocyte growth media) was
purchased from Clonetics. When cells reached confluency, they were
washed with KGM media minus growth factors=KBM (keratinocyte basal
media). Cells were serum starved in KBM for 72 hours prior to the
addition of test compounds. Thrombin at 1 I.U./mL and trypsin at 25
nM were used as positive controls. One mL of media/well was added.
KBM only was used as the negative control.
[0478] IL-20 was made up in KBM media and added at varying
concentrations, from 2.5 .mu.g/ml down to 618 ng/mL in a first
experiment and from 2.5 .mu.g/mL down to 3 ng/mL in a second
experiment.
[0479] Cells were incubated at 37.degree. C., 5% CO.sub.2 for 48
hours. Supernatants were removed and frozen at -80.degree. C. for
several days prior to assaying for IL-8 and GM-CSF levels. Human
IL-8 Immunoassay kit #D8050 (RandD Systems, Inc.) and human GM-CSF
Immunoassay kit #HSGMO (RandD Systems, Inc.) were used to determine
cytokine production following manufacturer's instructions.
[0480] The results indicated that the expression of IL-8 and GM-CSF
were induced by IL-20.
EXAMPLE 24
Up-Regulation of Inflammatory Cytokines by IL-20
[0481] The human keratinocyte cell line, HaCaT was grown at
37.degree. C. to several days post-confluence in T-75 tissue
culture flasks. At this point, normal growth media (DMEM+10% FBS)
was removed and replaced with serum-free media. Cells were then
incubated for two days at 37.degree. C. DMEM was then removed and
four flasks of cells per treatment were treated with one of each of
the following conditions for four hours at 37.degree. C.:
recombinant human (rh) IL-1 alpha at 5 ng/mL, rh IL-1 alpha at 20
ng/mL, rh IL-1 alpha at 5 ng/mL+IL-20 at 1 .mu.g/mL, IL-20 at 1
.mu.g/mL, or rh IL-10 at 10 ng/mL.
[0482] Following cytokine treatment, media was removed and cells
were lysed using a guanidium thiocyanate solution. Total RNA was
isolated from the cell lysate by an overnight spin on a cesium
chloride gradient. The following day, the RNA pellet was
resuspended in a TE/SDS solution and ethanol precipitated. RNA was
then quantitated using a spectrophotometer, followed by a DNase
treatment as per Section V.B. of Clontech's Atlas.TM. cDNA
Expression Arrays User Manual (version PT3140-1/PR9X390, published
Nov. 5, 1999). Quality of RNA samples was verified by purity
calculations based on spec readings, and by visualization on
agarose gel. Genomic contamination of the RNA samples was ruled out
by PCR analysis of the beta-actin gene.
[0483] Clontech's protocols for polyA+ enrichment, probe synthesis
and hybridization to Atlas.TM. arrays were followed (see above,
plus Atlas.TM. Pure Total RNA Labeling System User Manual,
PT3231-1/PR96157, published Jun. 22, 1999). Briefly, polyA+ RNA was
isolated from 50 mg of total RNA using streptavidin coated magnetic
beads (by Clontech, Paolo Alto, Calif.) and a magnetic particle
separator. PolyA+ RNA was then labeled with .sup.alpha32P-dATP via
RT-PCR. Clontech CDS primers specific to the 268 genes on the
Atlas.TM. human cytokine/receptor array (Cat. #7744-1) were used in
the reaction. Labeled probe was isolated using column
chromatography and counted in scintillation fluid.
[0484] Atlas.TM. arrays were pre-hybridized with Clontech
ExpressHyb plus 100 mg/mL heat denatured salmon sperm DNA for at
least thirty minutes at 68.degree. C. with continuous agitation.
Membranes were then hybridized with 1.9.times.10.sup.6 CPM/mL (a
total of 1.14.times.10.sup.7 CPM) overnight at 68.degree. C. with
continuous agitation. The following day, membranes were washed for
thirty minutes.times.4 in 2.times.SSC, 1% SDS at 68.degree. C.,
plus for thirty minutes.times.1 in 0.1.times.SSC, 0.5% SDS at
68.degree. C., followed by one final room temperature wash for five
minutes in 2.times.SSC. Array membranes were then placed in Kodak
plastic pouches sealed and exposed to a phosphor imager screen
overnight at room temperature. The next day, phosphor screens were
scanned on a phosphor imager and analyzed using Clontech's
AtlasImage.TM. 1.0 software.
Genes Up-Regulated by IL-20:
[0485] 1. Tumor necrosis factor (TNF) was up-regulated 1.9-2.4 fold
by IL-20. [0486] 2. Placental growth factors 1 & 2 (PLGF) were
up-regulated 1.9-2.0 fold by IL-20. [0487] 3. Coagulating factor II
receptor was up-regulated 2.0-2.5 fold by IL-20. [0488] 4.
Calcitonin receptor was up-regulated 2.2-2.3 fold by IL-20. [0489]
5. TNF-inducible hyaluronate-binding protein TSG-6 was up-regulated
2.1-2.2 fold by IL-20. [0490] 6. Vascular endothelial growth factor
(VEGF) receptor-1 precursor, tyrosine-protein kinase receptor
(FLT-1) (SFLT) was up-regulated 2.1-2.7 fold by IL-20. [0491] 7.
MRP-8 (calcium binding protein in macrophages MIF-related) was
up-regulated 2.9-4.1 fold by IL-20. [0492] 8. MRP-14 (calcium
binding protein in macrophages MIF-related) was up-regulated
3.0-3.8 fold by IL-20. [0493] 9. Relaxin H2 was up-regulated 3.14
fold by IL-20. [0494] 10. Transforming growth factor beta
(TGF.beta.) receptor III 300 kDa was up-regulated 2.4-3.6 fold by
IL-20. Genes Showing Synergy with IL-20+IL-1 Treatment: [0495] 1.
Bone morphogenic protein 2a was up-regulated 1.8 fold with IL-20
treatment alone, 2.5 fold with IL-1 treatment alone, and 8.2 fold
with both IL-20 and IL-1 treatment together. [0496] 2. MRP-8 was
up-regulated 2.9 fold with IL-20 treatment alone, 10.7 fold with
IL-1 treatment alone and 18.0 fold with both IL-20 and IL-1
treatment together. [0497] 3. Erythroid differentiation protein
(EDF) was up-regulated 1.9 fold with IL-20 treatment alone, 9.7
fold with IL-1 treatment alone and 19.0 fold with both IL-20 and
IL-1 treatment together. [0498] 4. MRP-14 (calcium binding protein
in macrophages, MIF related) was up-regulated 3.0 fold with IL-20
treatment alone, 12.2 fold with IL-1 treatment alone and 20.3 fold
with both IL-20 and IL-1 treatment together. [0499] 5.
Heparin-binding EGF-like growth factor was up-regulated 2.0 fold
with IL-20 treatment alone, 14 fold with IL-1 treatment alone and
25.0 fold with both IL-20 and IL-1 treatment together. [0500] 6.
Beta-thromboglobulin-like protein was up-regulated 1.5 fold with
IL-20 treatment alone, 15 fold with IL-1 treatment alone and 27
fold with both IL-20 and IL-1 treatment together. [0501] 7.
Brain-derived neurotrophic factor (BDNF) was up-regulated 1.7 fold
with IL-20 treatment alone, 25 fold with IL-1 treatment alone and
48 fold with both IL-20 and IL-1 treatment together. [0502] 8.
Monocyte chemotactic and activating factor MCAF was up-regulated
1.3 fold with IL-20 treatment alone, 32 fold with IL-1 treatment
alone and 56 fold with both IL-20 and IL-1 treatment together.
EXAMPLE 25
IL-20 Transgenic Phenotype
[0503] Both human and mouse IL-20 were overexpressed in transgenic
mice using a variety of promoters. The liver-specific mouse albumin
promoter, directing expression of human IL-20, was used initially
in an attempt to achieve circulating levels of protein. Subsequent
studies were conducted using the keratin 14 (K14) promoter, which
primarily targets expression to the epidermis and other stratified
squamous epithelia; the mouse metallothionein-1 promoter, which
gives a broad expression pattern; and the E.mu.LCK promoter, which
drives expression in cells of the lymphoid lineage. Similar results
were obtained in all four cases, possibly because these promoters
all give rise to circulating levels of IL-20.
[0504] In all cases, transgenic pups expressing the IL-20 transgene
were smaller than non-transgenic littermates, had a shiny
appearance with tight, wrinkled skin and died within the first few
days after birth. Pups had milk in their stomachs indicating that
they were able to suckle. These mice had swollen extremities, tail,
nostril and mouth regions and had difficulty moving. In addition,
the mice were frail, lacked visible adipose tissue and had delayed
ear and toe development. Low expression levels in liver (less than
100 mRNA molecules/cell) were sufficient for both the neonatal
lethality and skin abnormalities. Transgenic mice without a visible
phenotype either did not express the transgene, did not express it
at detectable levels, or were mosaic.
[0505] Histologic analysis of the skin of the IL-20 transgenic mice
showed a thickened epidermis, hyperkeratosis and a compact stratum
corneum compared to non-transgenic littermates. Serocellular crusts
(scabs) were observed occasionally. Electron microscopic (EM)
analysis of skin from transgenic mice showed intramitochondrial
lipoid inclusions, mottled keratohyaline granules, and relatively
few tonofilaments similar to that observed in human psoriatic skin
and in mouse skin disease models. In addition, many of the
transgenic mice had apoptotic thymic lymphocytes. No other
abnormalities were detected by histopathological analysis. These
histological and EM results support and extend the observed gross
skin alterations.
EXAMPLE 26
Construction of Expression Vector for Expression of Soluble Human
IL-22RA-muFc
[0506] A human IL-22RA soluble receptor-muFc fusion (denoted as
IL-22RA-C(mG2a) containing the extracellular domain of IL-22RA
fused to the murine gamma 2a heavy chain Fc region (mG2a), was
prepared. An expression plasmid containing IL-22RA-C(mG2a) was
constructed via homologous recombination using two separate DNA
fragments and the expression vector pZMP40. Fragments of
polynucleotide sequence of IL-22RA (SEQ ID NO:1), and mG2a SEQ ID
NO:39 were generated by PCR amplification using the following
primers: (a) IL-22RA primers ZC45,593 (SEQ ID NO:28), and ZC45,592
(SEQ ID NO:29); and (b) mG2a primers ZC45,591 (SEQ ID NO:30), and
ZC45,594 (SEQ ID NO:31).
[0507] The first fragment contained the IL-22RA extracellular
domain coding region, which was made using an IL-22RA
polynucleotide (e.g., SEQ ID NO:1) as the template. The first
fragment included a 5' overlap with a partial pZMP40 vector
sequence, the IL-22RA segment, and a 3' overlap containing a linker
sequence and partial mG2a sequence. PCR conditions: 1 cycle,
94.degree. C., 5 minutes; 35 cycles, 94.degree. C., 1 minute,
followed by 55.degree. C., 2 minutes, followed by 72.degree. C., 3
minutes; 1 cycle, 72.degree. C., 10 minutes.
[0508] The second fragment included a 5' overlap with a linker
sequence and partial IL-22RA sequence, the mG2a segment, and a 3'
overlap containing a partial pZMP40 vector sequence. The murine
gamma 2a heavy chain Fc region (mG2a) (SEQ ID NO:39) was generated
from a clone of murine Ig gamma 2a heavy chain cDNA. The mG2a
contains the hinge, C.sub.H2, and C.sub.H3 domains of the murine
immunoglobulin gamma 2a heavy chain constant region. PCR
conditions: 1 cycle, 94.degree. C., 5 minutes; 35 cycles,
94.degree. C., 1 minute, followed by 55.degree. C., 2 minutes,
followed by 72.degree. C., 3 minutes; 1 cycle, 72.degree. C., 10
minutes.
[0509] The PCR reaction mixtures were run on a 1% agarose gel and a
band corresponding to the sizes of the inserts were gel-extracted
using a QIAquick.TM. Gel Extraction Kit (Qiagen).
[0510] The plasmid pZMP40, which was cut with BglII, was used in a
three-way recombination with both of the PCR insert fragments.
Plasmid pZMP40 is a mammalian expression vector containing an
expression cassette having the MPSV promoter, and multiple
restriction sites for insertion of coding sequences; an E. coli
origin of replication; a mammalian selectable marker expression
unit comprising an SV40 promoter, enhancer and origin of
replication, a DHFR gene, and the SV40 terminator; and URA3 and
CEN-ARS sequences required for selection and replication in S.
cerevisiae. Plasmid pZMP40 was constructed from pZMP21 (deposited
at the American Type Culture Collection, 10801 University
Boulevard, Manassas, Va. 20110-2209, and designated No. PTA-5266)
by addition of several restriction enzyme sites to the
polylinker.
[0511] One hundred microliters of competent yeast (S. cerevisiae)
cells were independently combined with 10 .mu.l of the insert DNA
and 100 ng of cut pZMP40 vector, and the mix was transferred to a
0.2-cm electroporation cuvette. The yeast/DNA mixture was
electropulsed using power supply (BioRad Laboratories, Hercules,
Calif.) settings of 0.75 kV (5 kV/cm), .infin. ohms, and 25 .mu.F.
Six hundred .mu.l of 1.2 M sorbitol was added to the cuvette, and
the yeast was plated in a 100-.mu.l and 300 .mu.l aliquot onto two
URA-D plates and incubated at 30.degree. C. After about 72 hours,
the Ura.sup.+ yeast transformants from a single plate were
resuspended in 1 ml H.sub.2O and spun briefly to pellet the yeast
cells. The cell pellet was resuspended in 0.5 ml of lysis buffer
(2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM
EDTA). The five hundred microliters of the lysis mixture was added
to an Eppendorf tube containing 250 .mu.l acid-washed glass beads
and 300 .mu.l phenol-chloroform, was vortexed for 3 minutes, and
spun for 5 minutes in an Eppendorf centrifuge at maximum speed.
Three hundred microliters of the aqueous phase was transferred to a
fresh tube, and the DNA was precipitated with 600 .mu.l ethanol
(EtOH) and 30 .mu.l 3M sodium acetate, followed by centrifugation
for 30 minutes at maximum speed. The tube was decanted and the
pellet was washed with 1 mL of 70% ethanol. The tube was decanted
and the DNA pellet was resuspended in 30 .mu.l TE.
[0512] Transformation of electrocompetent E. coli host cells
(DH12S) was done using 5 .mu.l of the yeast DNA prep and 50 .mu.l
of cells. The cells were electropulsed at 2.0 kV, 25 .mu.F, and 400
ohms. Following electroporation, 1 ml SOC (2% Bacto.TM. Tryptone
(Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) was
added and then the cells were plated in a 50 .mu.l and 200 .mu.l
aliquot on two LB AMP plates (LB broth (Lennox), 1.8% Bacto.TM.
Agar (Difco), 100 mg/L Ampicillin).
[0513] The inserts of three clones for the construct was subjected
to sequence analysis and one clone for each construct, containing
the correct sequence, was selected. Larger scale plasmid DNA was
isolated using a commercially available kit (QIAGEN Plasmid Mega
Kit, Qiagen, Valencia, Calif.) according to manufacturer's
instructions.
EXAMPLE 27
Expression and Purification of Human Soluble IL-22RA-muFc
Polypeptide
[0514] Three sets of 200 .mu.g of the IL-22RA-C(mG2a) construct
(Example 22) were each digested with 200 units of Pvu I at
37.degree. C. for three hours and then were precipitated with IPA
and spun down in a 1.5 mL microfuge tube. The supernatant was
decanted off the pellet, and the pellet was washed with 1 mL of 70%
ethanol and allowed to incubate for 5 minutes at room temperature.
The tube was spun in a microfuge for 10 minutes at 14,000 RPM and
the supernatant was decanted off the pellet. The pellet was then
resuspended in 750 .mu.l of PF-CHO media in a sterile environment,
and allowed to incubate at 60.degree. C. for 30 minutes. 5E6
APFDXB11 cells were spun down in each of three tubes and were
resuspended using the DNA-media solution. The DNA/cell mixtures
were placed in a 0.4 cm gap cuvette and electroporated using the
following parameters: 950 .mu.F, high capacitance, and 300 V. The
contents of the cuvettes were then removed, pooled, and diluted to
25 mLs with PF-CHO media and placed in a 125 mL shake flask. The
flask was placed in an incubator on a shaker at 37.degree. C., 6%
CO.sub.2, and shaking at 120 RPM.
[0515] The cell line was subjected to nutrient selection followed
by step amplification to 100 nM methotrexate (MTX), then to 500 nM
MTX, and finally to 1 .mu.M MTX. Step amplification was followed by
a CD8 cell sort. The CD8 cell sort was accomplished by taking a
stable 1 .mu.M MTX amplified pool and staining approximately 5E6
cells with a monoclonal FITC anti-CD8 antibody (BD PharMingen,
cat#30324.times.) using manufacturers recommended concentration.
The stained cells were processed and sorted on a FACS Vantage (BD)
flow cytometer. The top 5% of cells were collected and outgrown.
Expression was confirmed by western blot, and the cell line was
scaled-up and protein purification using standard methods
followed.
EXAMPLE 28
Neutralization of huIL-22RA by Sera from Mice Immunized with
huL22RA-mG2a
[0516] A. Cell-Based Neutralization Assay to Test for Inhibition of
IL-20 and/or IL-22.
[0517] The factor-dependent pre-B cell line BaF3 co-transfected
with IL-22RA and IL-20RB (PDIRS1) (BAF/IL-22RA/IL-20RB cells;
Example 38) was used to assess neutralization potential of
anti-IL-22RA antibodies by antagonizing IL-20 on the
IL-22RA/IL-20RB receptor. Similarly, BaF3 co-transfected with
IL-22RA and IL-10RB (CRF2-4) (BAF/IL-22RA/CRF2-4 cells; Example 2)
was used to assess neutralization potential of anti-IL-22RA
antibodies by antagonizing IL-22 on the IL-22RA/IL10RB receptor.
Proliferation in the presence of IL-20 or IL22 on its respective
receptor-expressing cell line, and inhibition of such proliferation
in the presence of the antagonist antibodies, was assessed using an
Alamar blue assay as described in Example 3. Inhibition of
proliferation on these cells is indicative of neutralizing activity
in this assay.
B. Anti-IL-22RA Serum Neutralizes Both IL-20 and IL-22 in
Cell-Based Neutralization Assay.
[0518] Using the assay described in Example 28A, serum from IL-22RA
knockout mice immunized with huIL-22RA-muG2a (Example 30(A)(1)) was
added as a serial dilution at 1%, 0.5%, 0.25%, 0.13%, 0.06%, 0.03%,
0.02%, and 0%. The assay plates were incubated at 37.quadrature.C,
5% CO.sub.2 for 4 days at which time Alamar Blue (Accumed, Chicago,
Ill.) was added at 20 .mu.l/well. Plates were again incubated at
37.quadrature.C, 5% CO.sub.2 for 16 hours. Alamar Blue gives a
fluorometric readout based on number of live cells, and is thus a
direct measurement of cell proliferation in comparison to a
negative control. Plates were read on the Wallac Victor 2 1420
Multilabel Counter (Wallac, Turku, Finland) at wavelengths 530
(Excitation) and 590 (Emission). Results showed that serum from all
seven immunized animals could neutralize signaling of both huIL-22
and huIL20 through huIL-22RA. For example, at the 1% concentration,
serum from five animals (16517, 16518, 16519, 16520, and 16527)
completely neutralized proliferation induced by huIL-22, with the
inhibition of proliferation decreasing in a dose dependent fashion
at the lower concentrations. Moreover, at the 1% concentration,
serum from the other two animals (16471 and 16701) inhibited about
90% of the proliferation induced by huIL-22, with the inhibition of
proliferation decreasing in a dose dependent fashion at the lower
concentrations. Similarly, at the 1% and 0.5% concentrations, serum
from five animals (16517, 16518, 16519, 16520, and 16527)
completely neutralized proliferation induced by huIL-20, with the
inhibition of proliferation decreasing in a dose dependent fashion
at the lower concentrations. Moreover, at the 1% concentration,
serum from animal 16701 completely neutralized proliferation
induced by huIL-20, with the inhibition of proliferation decreasing
in a dose dependent fashion at the lower concentrations. At the 1%
concentration, serum from animal 16471 neutralized about 95% of the
proliferation induced by huIL-20, with the inhibition of
proliferation decreasing in a dose dependent fashion at the lower
concentrations. Thus, sera from all seven animals were able to
neutralize the proliferation induced by either IL-20 or IL-22
through the huIL-22RA receptor. These results further demonstrated
that antibodies to IL-22RA could indeed antagonize the activity of
the pro-inflammatory ligands, IL-20 and IL-22 at low
concentrations.
[0519] These results provided additional evidence that effectively
blocking IL-22RA activity by binding, blocking, inhibiting,
reducing, antagonizing or neutralizing IL-20 or IL-22 activity
(individually or together), for example via a neutralizing
monoclonal antibody to IL-22RA of the present invention, could be
advantageous in reducing the effects of IL-20 and IL-22 (alone or
together) in vivo and may be reduce IL-20 and/or IL-22-induced
inflammation, such as that seen in IL-20-induced skin effects, as
well as IL-22-induced skin effects, for example in psoriasis, IBD,
colitis, or other inflammatory diseases induced by IL-20, and or
IL-22 including IBD, arthritis, asthma, psoriatic arthritis,
colitis, inflammatory skin conditions, and atopic dermatitis.
EXAMPLE 29
Generation of P815/hIL-22RA Cells and Immunization of Mice
[0520] A. P815/hIL-22RA Cell Generation and Injection into Mice for
Generation of Anti-hIL-22RA Antibodies
[0521] WT P815 Cells (ATCC No. TIB-64) were transfected with a
plasmid vector containing the hIL-22RA cDNA sequence (e.g., SEQ ID
NO:1) and a selectable puromycin-resistance marker, using Fugene
Reagent according to the manufacturer's protocol (Roche,
Indianapolis, Ind.). Cells were placed under Puromycin selection 48
hours following transfection. Puromycin-resistant transfectants
were cloned by limiting dilution, and screened for their level of
hIL-22RA cell surface expression by flow cytometry, using
biotinylated human IL-22 (huIL-22-biotin). Briefly, cells were
incubated with 5 .mu.g/ml huIL-22-biotin for 30 minutes on ice and
then washed. Binding of huIL-22-biotin to the cells was then
detected using PE-labeled streptavidin at 1:500. Cells were
analyzed on a Facscan flow cytometer using Cellquest software.
(Becton Dickinson, San Jose, Calif.).
[0522] The selected P815/IL-22RA cell clone was grown up and then
harvested for injection. Cells were collected, washed three times
in PBS, counted, resuspended at 1.times.10.sup.8 cells per
milliliter, and irradiated with 10,000 rads. The cell suspension
was then transferred to a 1 ml syringe, and injected by the
intra-peritoneal route into DBA/2 Mice. Mice were boosted in an
identical manner 3 weeks later and sera were screened for binding
to hIL-22RA transfectant cell line. Briefly, sera were diluted
1:100 in Facs buffer (HBSS, 2% BSA, 0.02% NaN.sub.3), and then
incubated with Fc-blocked 293 human kidney cells over-expressing
hIL-22RA. Binding of anti-IL-22RA antibodies to the cells was then
detected using fluorescein-conjugated Goat-anti-Mouse IgG diluted
to 1:200. (Southern Biotech, Birmingham, Ala.) Cells were analyzed
as described previously. Mice were boosted again a total of 3 more
times and sera were screened as described. Two mice were selected
for hybridoma fusion, using standard methods in the art for
generation of monoclonal antibodies (Example 25), based on the
level of their serum binding to the hIL-22RA transfectants.
[0523] The above method is also used for generation of P815 cells
expressing heterodimeric IL-22RA receptors, such as IL-22RA/CRF2-4
(P815/IL-22RA/CRF2-4 cells), IL-22RA/pDIRS1 (P815/IL-22RA/pDIRS1
cells), or IL-22RA/CRF2-4/pDIRS1 (P815/IL-22RA/CRF2-4/pDIRS1
cells), for example to immunize mice for the generation of
monoclonal antibodies against IL-22RA and IL-22RA-comprising
heterodimeric receptors.
EXAMPLE 30
Generation of Murine Anti-human IL-22RA (IL-22RA) mAbs
A. Immunization for Generation of Anti-IL-22RA Antibodies
(1) Using Soluble IL-22RA-muFc
[0524] Six to twelve week old IL-22RA knockout mice (Example 26)
were immunized by intraperitoneal injection with 25-50 ug of
soluble human IL-22RA-muFc protein (Example 23) mixed 1:1 (v:v)
with Ribi adjuvant (Sigma) on a biweekly schedule. Seven to ten
days following the third immunization, blood samples were taken via
retroorbital bleed, the serum harvested and evaluated for its
ability to inhibit the binding of IL-22 or both IL-20 and IL-22 to
IL-22RA in neutralization assays (e.g., described herein) and to
stain IL-22RA transfected versus untransfected 293 cells in a FACS
staining assay. Mice continued to be immunized and blood samples
taken and evaluated as described above until neutralization titers
reached a plateau. At that time, mice with the highest
neutralization titers were injected intravascularly with 25-50 ug
of soluble IL-22RA-Fc protein in PBS. Three days later, the spleen
and lymph nodes from these mice were harvested and used for
hybridoma generation, for example using mouse myeloma
(P3-X63-Ag8.653.3.12.11) cells or other appropriate cell lines in
the art, using standard methods known in the art (e.g., see
Kearney, J. F. et al., J Immunol. 123:1548-50, 1979; and Lane, R.
D. J Immunol Methods 81:223-8, 1985).
(2) Using P815 Transfectants that Express the IL-22RA Receptor.
[0525] Six to ten week old female DBA/2 mice are immunized by
intraperitoneal injection of 1.times.10.sup.5 live, transfected
P815 cells, for example P815/IL-22RA cells, P815/IL-22RA/CRF2-4,
P815/IL-22RA/pDIRS1 or P815/IL-22RA/CRF2-4/pDIRS1 cells (Example
24) (e.g., 0.5 ml at a cell density of 2.times.10.sup.5 cells/ml).
Prior to injection, the cells are maintained in the exponential
growth phase. For injection the cells are harvested, washed three
times with PBS and then resuspended in PBS to a density of
2.times.10.sup.5 cells/ml. In this model, the mice develop an
ascites tumor within 2-3 weeks and progress to death by 4-6 weeks
unless an immune response to the transfected target antigen has
been mounted. At three weeks mice with no apparent abdominal
swelling (indicative of ascites) are re-immunized as above at 2-3
week intervals. Seven to ten days following the second
immunization, blood samples are taken via retroorbital bleed, the
serum harvested and evaluated for its ability to inhibit the
binding of IL-22 or both IL-20 and IL-22 to IL-22RA in
neutralization assays (e.g., described herein) and to stain IL-22RA
transfected versus untransfected 293 cells in a FACS staining
assay. Mice continue to be immunized and blood samples taken and
evaluated as described above until neutralization titers reach a
plateau. At that time, the mice with the highest neutralization
titers are injected intraperitonealy with 1.times.10.sup.5 live,
transfected P815 cells. Four days later, the spleen and lymph nodes
from these mice are harvested and used for hybridoma generation,
for example using mouse myeloma (P3-X63-Ag8.653.3.12.11) cells or
other appropriate cell lines in the art, using standard methods
known in the art (e.g., see Kearney, J. F. et al., supra.; and
Lane, R. D. supra.).
[0526] An alternative to the above immunization scheme with live,
transfected P815 cells involves intraperitoneal injection of
1-5.times.10.sup.6 irradiated, transfected cells every 2-3 weeks.
In this approach, no animals develop and die of ascites. Instead,
animals are monitored for a neutralizing immune response to IL-22RA
in their serum as outlined above, starting with a bleed after the
second immunization. Once neutralization titers have reached a
maximal level, the mice with highest titers are given a pre-fusion,
intraperitoneal injection of 5.times.10.sup.6 irradiated cells and
four days later, the spleen and lymph nodes from these mice are
harvested and used for hybridoma generation, for example using
mouse myeloma (P3-X63-Ag8.653.3.12.11) cells or other appropriate
cell lines in the art, using standard methods known in the art
(e.g., see Kearney, J. F. et al., supra.; and Lane, R. D.
supra.).
B. Screening the Hybridoma Fusions for Antibodies that Bind IL-22RA
and Inhibit the Binding of IL-22 to IL-22RA
[0527] Two different primary screens were performed on the
hybridoma supernatants at 8-10 days post-fusion. For the first
assay, antibodies in supernatants were tested for their ability to
bind to plate bound soluble human IL-22RA-muFc protein by ELISA
using HRP-conjugated goat anti-mouse kappa and anti-lambda light
chain second step reagents to identify bound mouse antibodies. To
demonstrate specificity for the IL-22RA portion of the IL-22RA-muFc
protein, positive supernatants in the initial assay were evaluated
on an irrelevant protein fused to the same murine Fc region (mG2a).
Antibody in those supernatants that bound to IL-22RA-muFc and not
the irrelevant muFc containing fusion protein were deemed to be
specific for IL-22RA. For the second assay, antibodies in all
hybridoma supernatants were evaluated by ELISA for their ability to
inhibit the binding of biotinylated human IL-22 to plate bound
IL-22RA-muFc.
[0528] All supernatants containing antibodies that bound
specifically to IL-22RA, whether they inhibited the binding of
IL-22 to IL-22RA or not in the ELISA assay, were subsequently
tested for their ability to inhibit the binding (and concomitant
pro-proliferative effect) of IL-20 or IL-22 to IL-22RA/IL-20RB and
IL-22RA/CRF2-4 transfected Baf3 cells, respectively. All
supernatants that were neutralization positive in either the IL-22
inhibition assay or both the IL-20 and IL-22 inhibition assays were
subsequently evaluated for their ability to stain IL-22RA
transfected versus untransfected Baf3 cells by FACS analysis. This
analysis was designed to confirm that inhibition of IL-22 binding
to IL-22RA/CRF2-4, or IL-20 binding to IL-22RA/IL-20RB, was indeed
due to an antibody that specifically binds the IL-22RA receptor.
Additionally, since the FACS analysis was performed with an
anti-IgG second step reagent, specific FACS positive results
indicate that the neutralizing antibody was likely to be of the IgG
class. By these means, a master well was identified that bound
IL-22RA in the plate bound ELISA, inhibited the binding of IL-22 to
IL-22RA in the ELISA based inhibition assay, blocked the
interaction of IL-20 and IL-22 with IL-22RA/IL-20RB and
IL-22RA/CRF2-4 transfected Baf3 cells (Example 28), respectively,
and was strongly positive for the staining of both IL-22RA/IL-20RB
and IL-22RA/CRF2-4 transfected Baf3 cells with an anti-mouse IgG
second step reagent.
D. Cloning Anti-IL-22RA Specific Antibody Producing Hybridomas:
[0529] A hybridoma producing an anti-IL-22RA mAb that
cross-neutralized the binding of IL-20 and IL-22 to appropriately
transfected BaF3 cells was cloned by a standard low-density
dilution (less than 1 cell per well) approach. Approximately 5-7
days after plating, the clones were screened by ELISA on plate
bound human IL-22RA-muFc followed by a retest of positive wells by
ELISA on irrelevant muFc containing fusion protein as described
above. Selected clones, whose supernatants bound to IL-22RA-muFc
and not the irrelevant muFc containing fusion protein, were further
confirmed for specific antibody activity by repeating both
neutralization assays as well as the FACS analysis. All selected
IL-22RA antibody positive clones were cloned a minimum of two times
to help insure clonality and to assess stability of antibody
production. Further rounds of cloning were performed and screened
as described until, preferably, at least 95% of the resulting
clones were positive for neutralizing anti-IL-22RA antibody
production.
E. Biochemical Characterization of the Molecule Recognized by
Anti-IL-22RA mAbs:
[0530] Biochemical confirmation that the target molecule, IL-22RA,
recognized by the putative anti-IL-22RA mAbs is indeed IL-22RA are
performed by standard immunoprecipitation followed by SDS-PAGE
analysis or western blotting procedures, both employing soluble
membrane preparations from IL-22RA transfected versus untransfected
Baf3 cells. Moreover, soluble membrane preparations of
non-transfected cell lines that express IL-22RA are used show that
the mAbs recognize the native receptor chain as well as the
transfected one. Alternatively, the mAbs are tested for their
ability to specifically immunoprecipitate or western blot the
soluble IL-22RA-muFc protein.
EXAMPLE 31
Neutralization of huL22RA by Sera from Mice Injected with P815
Cells Transfected with huL22RA
[0531] Using the cell based neutralization assay described in
Example 28, Serum from mice injected with live huIL-22RA
transfected P815 cells (Example 30.A.2) was added as a serial
dilution at 1%, 0.5%, 0.25%, 0.13%, 0.06%, 0.03%, 0.02%, and 0%.
The assay plates were incubated at 37.quadrature.C, 5% CO.sub.2 for
4 days at which time Alamar Blue (Accumed, Chicago, Ill.) was added
at 20 .mu.l/well. Plates were again incubated at 37.quadrature.C,
5% CO.sub.2 for 16 hours. Results showed that serum from four of
the animals could neutralize signaling of both huIL-22 and huIL20
through huIL-22RA.
[0532] At the 1% concentration, serum from six animals (7125, 7127,
7128, 7118, 7124 and 7117) neutralized between 50% and 80% of the
proliferation induced by huIL-22, with the inhibition of
proliferation decreasing in a dose dependent fashion at the lower
concentrations. Moreover, at the 1% concentration, serum from four
animals (7125, 7127, 7118, and 7117) neutralized between 40% and
70% of the proliferation induced by huIL20, with the inhibition of
proliferation decreasing in a dose dependent fashion at the lower
concentrations. These results further demonstrated that antibodies
to IL-22RA could indeed antagonize the activity of the
pro-inflammatory ligands, IL-20 and IL-22 at low
concentrations.
[0533] These results provided additional evidence that effectively
blocking IL-22RA activity by binding, blocking, inhibiting,
reducing, antagonizing or neutralizing IL-20 or IL-22 activity
(individually or together), for example via a neutralizing
monoclonal antibody to IL-22RA of the present invention, could be
advantageous in reducing the effects of IL-20 and IL-22 (alone or
together) in vivo and may be reduce IL-20 and/or IL-22-induced
inflammation, such as that seen in IL-20-induced skin effects, as
well as IL-22-induced skin effects, for example in psoriasis, IBD,
colitis, or other inflammatory diseases induced by IL-20, and or
IL-22 including IBD, arthritis, asthma, psoriatic arthritis,
colitis, inflammatory skin conditions, and atopic dermatitis.
EXAMPLE 32
Phenotype of IL-22RA Knockout Mice
A. Generation of Mice Carrying Genetic Modifications
[0534] 1. Generation of Transgenic Mice Expressing Murine IL-20
with a Neonate Shine [0535] a). Construct for Expressing Murine
IL-20 from the K14 Promoter.
[0536] In order to investigate biological function of IL-20 in
vivo, a transgenic construct was made, in which murine IL-20 was
driven by human K14 promoter (also see, Example 21).
Oligonucleotides were designed to generate a PCR fragment
containing a consensus Kozak sequence and the murine IL-20 coding
region. These oligonucleotides were designed with an FseI site at
the 5' end and an AscI site at the 3' end to facilitate cloning
into pRSK14, a standard transgenic vector, containing a human
keratinocyte and epithelial cell-specific promoter.
[0537] PCR reactions were carried out with about 200 ng murine
IL-20 template (SEQ ID NO:33) and oligonucleotides designed to
amplify the full-length of the IL-20 (SEQ ID NO:34). PCR reaction
conditions were determined using methods known in the art. PCR
products were separated by agarose gel electrophoresis and purified
using a QiaQuick.TM. (Qiagen) gel extraction kit. The isolated,
correct sized DNA fragment was digested with FseI and AscI
(Boerhinger-Mannheim), ethanol precipitated and ligated into
pRSK14, previously digested with FseI and AscI. The pRSK14 plasmid,
designed for expressing a gene of interest in keratinocyte and
epithelial in transgenic mice, contains an expression cassette
flanked by about 3 Kb human keratin specific K14 promoter.
[0538] About one microliter of ligation reaction was electroporated
into DH10B ElectroMax.TM. competent cells (GIBCO BRL, Gaithersburg,
Md.) according to manufacturer's direction and plated onto LB
plates containing 100 .mu.g/ml ampicillin, and incubated overnight.
Colonies were picked and grown in LB media containing 100 .mu.g/ml
ampicillin. Miniprep DNA was prepared from the picked clones and
screened for the murine IL-20 insert by restriction digestion FseI
and AscI combined, and subsequent agarose gel electrophoresis. The
TG construct with correct cDNA inserts were confirmed by sequencing
analysis. Maxipreps of the correct pRSK14-murine IL-20 were
performed.
[0539] b) Generation and Characterization of K14 IL-20 Transgenic
Mice.
[0540] A NotI fragment of about 4 Kb in length was isolated from
the transgenic (TG) vector containing 5' and 3' flanking sequences
of the keratin specific K14 promoter, mouse IL-20 (SEQ ID NO:33;
polypeptide shown in SEQ ID NO:34), the Gormon intron, IL-20 cDNA
and the human growth hormone polyA signal sequences. It was used
for microinjection into fertilized B6C3f1 (Taconic, Germantown,
N.Y.) murine oocytes. Microinjection and production of transgenic
mice were produced as described in Hogan, B. et al. Manipulating
the Mouse Embryo, 2.sup.nd ed., Cold Spring Harbor Laboratory
Press, NY, 1994.
[0541] A TaqMan.TM. RT-PCR reaction was used to quantitate
expression of TG RNA by using PCR primers specific to the human
growth hormone polyA signal portion of the transgene.
[0542] All TG constructs expressing IL-20 exhibit a high rate of
paranatal mortality, and the TG pups that were born typically
exhibits a "shiny" phenotype. The shiny appearance of the neonate
pups appeared to be associated with a stiffening of the skin, as if
they were drying out, resulting in a reduction of proper nursing.
Their movements become stiffened in general. HistoPathologically
the shiny pups have a thickened epidermis and the keratin layer was
compacted. Most of these shiny founder pups died within the first 5
days, and the surviving and weaned pups were in general not
expressing the transgene (per transcript analysis), or they were
chimeric (per low transmittion of the transgene to the
offspring).
[0543] One line expressing murine IL-20, driven by the K14
promoter, was established. The expression level in the skin and the
thymus was low, and all the neonates were born with a shiny
phenotype. In general this line had 20% TG offspring, indicating
50-60% of the transgenic pups die in utero. (In a Hemizygous mating
50% of the offspring should be TG.)
[0544] 2. Generation of Mice with Ablated IL-22RA Expression;
IL-22RA Knockout Mice
[0545] a). Generation of Knockout (KO) Construct for Murine
IL-22RA.
[0546] To further study biological function of IL-22RA in vivo, a
mouse Knockout (KO) strain was created to ablate IL-22RA
expression. First, Mouse IL-22RA cDNA probes were used to screen a
mouse 129/SvJ genomic BAC library. Clones containing IL-22RA
genomic locus were identified and characterized. Murine IL-22RA
polynucleotide is shown in SEQ ID NO:41 and polypeptide in SEQ ID
NO:42.
[0547] To create a knockout construct for ablation of IL-22RA, a
Knockout vector was made by using ET cloning technique (Zhang et
al. 1998. A new logic for DNA engineering using recombination in E.
coli. Nat. Genet. Vol. 20:123-8). Briefly, the KO vector contains a
1.8 kb 5' arm (short arm), an IRES-LacZ/MC1neo Selectable marker,
and a 10 Kb 3' arm (long arm) of IL-22RA gene. In the KO vector,
exons 2, 3 and 4 as well as Introns 2 and 3 of IL-22RA genomic
sequence were replace by the IRES-LacZ/MC1neo Selectable marker so
that a deletion of about 4.4 Kb was generated by homologous
recombination in ES cells.
[0548] After linearization of the KO vector by restriction enzyme
PmeI, it was electroporated into 129/SvJ ES cells. Selection of
homologous recombination events, as well as identification of
recombinant ES clones were performed as described in Robertson, E.
J. et al. Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, 2.sup.nd ed., IRL Press Limited, Oxford, 1987.
[0549] b). Creation and Analysis of Mice with Ablated IL-22RA
Expression.
[0550] Positive ES clones, in which deletion of Exons 2-4 and
Introns 2-3 of IL-22RA genomic locus occurs, were expanded. They
were injected into blastocysts of C57B1/6j mice. After brief
re-expansion of the injected blastocysts, they were introduced into
pseudo-pregnant foster mothers to generate chimeras. Blastocyst
injection, chimera breeding and subsequent germline transmission of
mutated IL-22-RA were performed as described in Robertson, E. J. et
al. Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, 2.sup.nd ed., IRL Press Limited, Oxford, 1987.
[0551] The KO mutant mice were identified by PCR genotyping
strategy. Three PCR primers, ZC22901 (SEQ ID NO:35), ZC45039 ((SEQ
ID NO:36), ZC38573 (SEQ ID NO:37) were used in a multiplex PCR
reaction to detect wild-type allele and mutant allele. The
wild-type (WT) allele yields a DNA fragment of 229 bp in length,
while the mutant allele generates a DNA fragment of 371 bp in
length.
[0552] The pairing of Hemizygote mice produce a normal ratio of
Homozygote (HOM), Heterozygote (Het), and wild type (WT) offspring,
as well as a normal sex ratio. Inspecting the mice through a
PhysioScreen (Collecting body weight, tissue weight, complete blood
count (CBC), clinical chemistry, gross observation, and
HistoPathology) revealed no apparent differences between HOM, Het,
and WT animals.
B. IL-22RA was Necessary for IL-22 Induced SAA: SAA ELISA Showing
SAA Expression Induced by IL-22 was Absent in IL-22RA Knockout
Mice:
[0553] To assess whether IL-22RA was necessary for SAA induction in
mice injected with IL-22, IL-22RA KO mice were injected with 5 ug
IL-22 and bled 6 hr later.
[0554] An Elisa to determine SAA levels in the serum samples was
performed using the Mouse SAA Immunoassay Kit (BioSource
International, California) following the manufacturer's directions,
with the serum diluted 1:1000. Four out of five WT mice showed
elevated SAA levels in response to IL-22 injection, while four out
of five HOM IL-22RA KO mice showed basal levels of SAA. Both Het
IL-22RA KO mice tested have elevated SAA levels, but lower than the
SAA levels in the elevated WT mice. This indicates that IL-22RA was
necessary for the induction of SAA by IL-22.
[0555] These results provided evidence that effectively blocking
IL-22RA activity, for example via an IL-22RA gene knockout or
similarly via a neutralizing monoclonal antibody to IL-22RA of the
present invention, would similarly reduce IL-22-induced
inflammation, for example in psoriasis, IBD, colitis, endotoxemia,
or other inflammatory diseases induced by IL-22.
C. IL-22RA was Necessary for IL-22 Induced Epithelial Thickening:
Administration of IL-22 Pure Protein Via Osmotic Mini-Pump
Implanted Sub-Cutaneous does not Cause Thickening of the Epidermis
in IL-22R KO Mice.
[0556] To assess whether IL-22RA was necessary for the IL-22
induced epithelial thickening, IL-22 was administered
subcutaneously to IL-22RA HOM and WT KO mice via osmotic mini-pumps
The pumps delivered IL-22 at a rate of 18.4 .mu.L per day for 7
days. Four HOM and 6 WT IL-22RA KO mice received IL-22 protein,
while 3 HOM and 1 WT received PBS.
[0557] Serum samples from IL-22 treated mice were tested in BaF3
proliferation assay to confirm the presence of IL-22. BaF3 cells
transfected with IL-22RA and CRF2-4 require the presence of either
IL-22 or murine IL3 to proliferate. These cells were spun down and
washed in the complete media, without mIL-3 (RPMI medium (JRH
Bioscience Inc., Lenexa, Kans.) supplemented with 10%
heat-inactivated fetal calf serum, 2 mM L-glutaMax-1.TM. (Gibco
BRL), 1 mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics (GIBCO
BRL)) (hereinafter referred to as "mIL-3 free media"). The cells
were spun and washed 3 times to ensure the removal of mIL-3. Cells
were then counted in a hemacytometer and plated in a 96-well format
at 5000 cells per well in a final volume of 200 ul per well using
the mIL-3 free media. Mouse serum was present in the wells at 1%,
0.5%, 0.25% or 0.125%. The assay plates were incubated at
37.degree. C., 5% CO.sub.2 for 3 days at which time Alamar Blue
(Accumed, Chicago, Ill.) was added at 20 .mu.l &well. Plates
were again incubated at 37.degree. C., 5% CO.sub.2 for 24 hours.
Alamar Blue gives a fluorometric readout based on number of live
cells, and was thus a direct measurement of cell proliferation in
comparison to a negative control. Plates were again incubated at
37.degree. C., 5% CO.sub.2 for 24 hours. Plates were read on the
Wallac Victor 2 1420 Multilabel Counter (Wallac, Turku, Finland) at
wavelengths 530 (Excitation) and 590 (Emission). Results showed
none of the PBS injected animals had IL-22 activity, while 1 of 1
Het animals, 2 of 4 HOM animals, and 3 of 6 WT animals had
detectable IL-22 activity. Proliferation induced by this serum was
blocked by the presence of 1 .mu.g/ml IL-22BP, proving that it was
IL-22 specific.
[0558] Skin samples from IL-22 treated and untreated IL-22RA HOM
and Het knockout (KO) and WT control mice were immersion fixed in
10% buffered formalin. The tissues were trimmed and embedded in
paraffin, routinely processed, sectioned at 5 .mu.m (Jung 2065
Supercut microtome, Leica Microsystems, Wetzlar, Germany) and
stained with H&E. The stained tissues were evaluated under a
light microscope (Nikon Eclipse E600, Nikon Inc., Melville, N.Y.)
by an ACVP board certified veterinary pathologist.
[0559] Each skin sample was evaluated on a 0 (none) to 4 (severe)
scale for severity of inflammation in the tissue bordering the pump
implantation site in the hypodermis, on a 0 (none) to 3 (diffuse)
scale for extent of epidermal thickening (acanthosis), and the
number of epithelial layers were counted in the thickest part of
the epidermis. No difference was found between the HOM mice and the
WT mice that had been given PBS. The results from these two groups
were pooled into one PBS group. The mean and standard deviation was
determined for each treatment group and was shown in Table 14
below.
TABLE-US-00014 TABLE 14 Treatment PBS control HOM KO: IL-22 WT:
IL-22 Number of mice 4 4 6 Epithelial thickness 3.5 .+-. 1.0 3.2
.+-. 0.5 5.9 .+-. 2.3 Extent of acanthosis 0.5 .+-. 1.0 0.2 .+-.
0.5 1.9 .+-. 1.3 Inflammation 1.5 .+-. 1.0 1.2 .+-. 1.0 2.0 .+-.
1.0
[0560] Results showed a trend toward increased epithelial thickness
and acanthosis in WT mice treated with IL-22 and less epithelial
thickness and acanthosis in IL-22RA HOM mice when exposed to
IL-22.
[0561] These results provide evidence that effectively blocking
IL-22RA activity, for example via an IL-22RA gene knockout or
similarly via a neutralizing monoclonal antibody to IL-22RA of the
present invention, would similarly reduce IL-22-induced skin
effects, for example in psoriasis, IBD, colitis, or other
inflammatory diseases induced by IL-22.
D. IL-22RA was Necessary for IL-20 Induced Shine of Neonate Pups
Skin: Crossbreeding of Transgenic Mice Expressing Murine IL-20 with
IL-22RA KO Mice Produce Transgenic Pups that do not Shine
[0562] To assess whether IL-22RA was necessary for the IL-20
induced shine of neonate TG pups, the K14 muIL-20 transgene was
crossed into the IL-22RA KO line, and the neonates were observed
for the shiny phenotype.
[0563] Sixty-nine pups have been born with a Mendelian genotype
ratio. All the TG on a Het KO background were shiny, while none of
the non-TG, nor the TG on the HOM KO background were shiny.
[0564] An alamar blue proliferation assay using BaF3 cells
expressing IL-20RA and IL-20RB was performed to assess the presence
of IL-20 in the mouse serum. These cells will proliferate in
response to either IL-20 or murine IL3. Procedure was the same as
the one described in Section C, above. Results of the assay showed
that all the TG mice had comparable IL-20 activity, and at the same
level as IL-20 TG on the C57BL/6N background. The absence of any
shiny neonate phenotype indicate that skinny neonate phenotype was
dependent on the presence of IL-22RA. The proliferation assay
showed that all the TG mice had comparable IL-20 activity, and at
the same level as IL-20 TG on the C57BL/6N background. The absence
of any shiny neonate phenotype indicate that skinny neonate
phenotype was dependent on the presence of IL-22RA.
[0565] On day three post partum, pups from litters containing K14
muIL-20 TG on the IL-22RA KO background were humanely euthanized
and the whole body immersion fixed in 10% buffered formalin. The
fixed tissues were trimmed into cross-sections of the thorax and
abdomen, embedded in paraffin, routinely processed, sectioned at 5
um (Jung 2065 Supercut microtome, Leica Microsystems, Wetzlar,
Germany) and stained with H&E. The stained tissues were
evaluated under a light microscope (Nikon Eclipse E600, Nikon Inc.,
Melville, N.Y.) in blinded fashion by an ACVP board certified
veterinary pathologist. Tissue abnormalities were noted and the
number of epithelial layers in the epidermis of the dorsal anterior
thorax counted.
[0566] Tissues from three IL-20 TG on HOM IL-22RA KO background
(IL-20 TG/IL-22RA KO HOM) and three non-TG on IL-22RRA HOM KO
background (non-TG/IL-22RA KO HOM) mice were microscopically
examined and found to contain no abnormalities. Tissues from two
IL-20 TG on Het IL-22RA KO background (IL-20 TG/IL-22RA KO Het)
mice were also examined. The numbers of epithelial layers in the
epidermis was similar in all animals. However, the epidermis of the
two IL-20 TG/IL-22RA KO Het mice was hypereosinophilic as compared
to the other animals and exhibited loss of granularity in the
stratum granulosum. No other abnormalities were noted in the skin
or other tissues of any of the mice.
[0567] These results provide evidence that effectively blocking
IL-22RA activity, for example via an IL-22RA gene knockout or
similarly via a neutralizing monoclonal antibody to IL-22RA of the
present invention, would similarly reduce IL-20-induced skin
effects, as well as IL-22-induced skin effects, for example in
psoriasis, IBD, colitis, or other inflammatory diseases induced by
IL-20, and or IL-22 including IBD, arthritis, asthma, psoriatic
arthritis, colitis, inflammatory skin conditions, and atopic
dermatitis.
EXAMPLE 33
Histomorphometric Image Analysis of IL-22RA Knockout Mice
[0568] A line of k14 IL-20 m transgenic (TG) mice has been
established, and the TG neonates exhibit a shiny phenotype. The
transgene is expressed by the k14 promoter, which directs
expression to the keratin producing cells in the skin. A line of
IL-22RA knock out (KO) mice has also been established, and no
significant changes have been observed in the un-challenged mice.
The two lines were crossed together and neonates were collected
having the following four different genotypes: (1) TG/- HOM:
expressing the k14 IL-20 m transgene on a background not expressing
IL-22RA; (2) TG/- Het: expressing the k14 IL-20 m transgene on a
background expressing some IL-22RA from one copy of the IL-22RA
gene; (3) WT/HOM: not expressing the k14 IL-20 m transgene on a
background not expressing IL-22RA; and (4) WT/Het: not expressing
the k14 IL-20 m transgene on a background expressing some IL-22RA
from one copy of the IL-22RA gene. Thirty-four neonate pups of
these various genotypes were euthanized at day 3, approximately 48
hours post partum (Table 15):
TABLE-US-00015 TABLE 15 TG/- HOM* TG/- Het* WT/HOM* WT/Het* (Group
1) (Group 2) (Group 3) (Group 4) Total n = 10 n = 10 n = 9 n = 5 TG
= transgenic; WT = wild type; HOM = homozygous; Het = heterozygous;
and n = number of pups.
[0569] Each pup was transversely cut into three sections (cranial
thorax, caudal thorax and abdomen) through the body and the head
was discarded. The tissue specimens, 4.0-5.0 mm in thickness, were
fixed in 10% neutral buffered formalin, processed into paraffin
blocks and stained with hematoxylin and eosin (H&E) for routine
histological examination and histomorphometric image analysis.
Epidermis from the dorsal area of spinal cord in each tissue sample
was chosen for histomorphometric image analysis using an Olympus
BH-2 microscope, a video camera (Dage-MTI, Michigan City, Ind.) and
BioQuant True Color windows 98 software (R&M Biometrics, Inc.
Nashville, Tenn. 37209) with the following set up: Parameter: mag.
10.times., Z off set 0; Array: length (.quadrature.m); Measure:
manual and additive mode. The thickness (.quadrature.m) of
epidermis and stratum corneum or cornified layer from each skin
sample were individually measured 10 times, with about 0.1 mm
interval between each measurement, in each 10.times. microscopic
field and the mean value, SD and SEM were obtained by Excel
calculation. All of the sections were randomized and measured in a
blinded fashion. After the measurement, the sections were
unblinded, and the results matched to treatment groups. Final
results by treatment group were classified as follows: 1. Average
epidermal thickness (.quadrature.m) in cranial thorax, caudal
thorax and abdomen, and then sub-classified as (a) Average
epidermal thickness in cranial thorax; (b) Average epidermal
thickness in caudal thorax; and (c) Average epidermal thickness in
abdomen. 2. Average thickness of stratum corneum (.quadrature.m) in
cranial thorax, caudal thorax and abdomen, and sub-classified as
(a) Average thickness of stratum corneum in cranial thorax; (b)
Average thickness of stratum corneum in caudal thorax; and (c)
Average thickness of stratum corneum in abdomen. 3. Average
thickness of epidermis plus stratum corneum in cranial thorax,
caudal thorax and abdomen. The resulting data was analyzed using
GraphPad InStat software (GraphPad Software, Inc., San Diego,
Calif. 92121). One-way analysis of variance (ANOVA) was applied to
examine the statistical significance of differences in mean values
from group1 to group 4. Tukey-Kramer Multiple Comparisons Test was
used for the determination of statistical differences in mean
values between two groups (*P<0.05; **P<0.01; ***P<0.001;
****P<0.0001). Observations of P<0.05 were considered
significant.
[0570] (1) Histomorphometric Results
(a) Average Epidermal Thickness (.mu.m) in Cranial Thorax, Caudal
Thorax and Abdomen
[0571] Epidermal thickness increased significantly in IL-20
transgenic pups lacking one copy of the IL-22RA gene (TG/- Het)
versus the IL-20 transgenic pups with no expression of IL-22RA
(TG/- HOM, P=0.001***) and versus the control littermates (WT/HOM,
P=0.001*** and WT/Het, P=0.001***), respectively (Table 16). The
TG/- et pups showed increased thickness of non-keratinized
epidermis possibly due to keratinocyte hypertrophy. This increase
might involve all three nonkeratinized layers (basal, prickle, and
granular) but most often affected the prickle cell layer. The
epidermis of the TG/- Het pup increased about 25% in thickness and
the prickle became prominent. Whereas the epidermis of TG/- HOM
pups were slightly thicker than the controls (WT/HOM and WT/Het)
and statistics indicated no significant difference between the
groups (P>0.05). The epidermal thickness in cranial thorax,
caudal thorax and abdomen were also compared. The normally thin
epidermis of the abdomen is thicker than caudal thorax and the
caudal thorax is thicker than the cranial thorax (Table 16).
TABLE-US-00016 TABLE 16 TG/- HOM TG/- Het WT/HOM WT/Het (N = 28) (N
= 30) (N = 27) (N = 15) Mean 32.58 .+-. 1.25 41.05 .+-. 2.04 31.31
.+-. 1.08 30.83 .+-. 1.43 Results represent mean values .+-. SEM. N
= number of sections measured.
[0572] The squamous epithelium of the skin in the cranial thorax
from the TG/- Het pups showed increase in thickness accompanied by
hypertrophy of the epidermal cells (keratinocytes); however there
was no statistical difference compared with other groups, the TG/-
HOM, WT/HOM and WT/Het (P=0.1565, Table 17). This seems to result
from either histological artifact, e.g., section-to-section
variability, the nature architecture of the epidermis, or there was
not much effect in the thin skin in the cranial thorax. Note: the
histology procedure or tissue section of the cranial thorax might
disqualify for histomorphometric analysis to obtain statistical
significance.
TABLE-US-00017 TABLE 17 TG/- HOM TG/- Het WT/HOM WT/Het (N = 10) (N
= 10) (N = 9) (N = 5) Mean 29.18 .+-. 2.24 33.20 .+-. 2.24 27.28
.+-. 0.62 29.38 .+-. 1.77 Results represent mean values .+-. SEM. N
= number of sections measured
[0573] The IL-20 (TG/-) with one copy of the IL-22RA gene (Het)
showed increased mean value of the epidermal thickness compared to
the TG/- HOM (P<0.05*), WT/HOM (P<0.001***), and WT/Het
(P<0.01*), respectively (Table 18). Statistics indicated
extremely significant among the groups (P<0.0001****). The TG/-
Het epidermis increased about 29% than that of WT/Het. The
phenotype of IL-20 (TG/-) pups with absence of IL-22RA (HOM) in
part resembled to that of the pups lacking one copy of the IL-22RA
gene (Het) associated with thicker epidermis than that of the
control littermates (WT/HOM and WT/Het), however, it demonstrated
no statistical difference compared to the controls (P>0.05). The
TG/- HOM epidermis increased about 14% than that of WT/HOM. Unlike
the IL-20 TG/- pups, the IL-22RAm receptor-deficient pups (WT/HOM
and WT/Het) demonstrated relatively thinner epidermal thickness.
Noticeably, the histomorphometric result of epidermal thickness in
caudal thorax was a consistent finding correlated to the average
epidermal thickness in the cranial thorax, caudal thorax and
abdomen (Table 15), which indicated that the histological procedure
and tissue section of the caudal thorax carried out the best
quality for histomorphometric image analysis.
TABLE-US-00018 TABLE 18 TG/- HOM TG/- Het WT/HOM WT/Het (N = 10) (N
= 10) (N = 9) (N = 5) Mean 35.91 .+-. 1.37 43.79 .+-. 2.35 30.83
.+-. 1.86 30.94 .+-. 2.83 Results represent mean values .+-. SEM. N
= number of sections measured.
[0574] The results of average epidermal thickness in abdomen (Table
19) were similar to that in the caudal thorax (Table 18) except
that the TG/- HOM showed no differences compared to the control
littermates (WT/HOM and WT/Het, P>0.05). There were some
variations in the tissue sections and also two sections were
missing, i.e. without epidermis covering the dorsal area in the
TG/- HOM group.
TABLE-US-00019 TABLE 19 TG/- HOM TG/- Het WT/HOM WT/Het (N = 8) (N
= 10) (N = 9) (N = 5) Mean 32.35 .+-. 1.44 46.33 .+-. 3.10 35.81
.+-. 1.90 32.16 .+-. 2.97 Results represent mean values .+-. SEM. N
= number of sections measured.
(b) Average Thickness (.mu.m) of Stratum Corneum in Cranial Thorax,
Caudal Thorax and Abdomen.
[0575] Despite the increased epidermal thickness in the IL-20
transgenic pups (TG/-) on a background either not expressing
IL-22RA (HOM) or expressing one copy of the gene (Het), predominate
reduction of stratum corneum or cornified layer thickness was
observed in the TG/- HOM and TG/- Het skins compared to the control
littermates (WT/HOM and WT/Het) and statistics indicated extremely
significant among the groups (P<0.0001****, Table 20). The TG/-
Het pups showed about 36%, 50% and 49% decreased amounts of keratin
on the surface of the epidermis versus the TG/- HOM (P<0.01**),
WT/HOM (P<0.001***) and WT/Het (P<0.001***), respectively.
The TG/- HOM pups showed about 22% significant reduction in the
stratum corneum thickness compared with its control (WT/HOM,
P<0.05*) and only 17% reduction versus the WT/Het that revealed
no statistical significance (P>0.05). The thickness of stratum
corneum in the control pups, WT/HOM and WT/Het were about the same.
Apparently, the stratum corneum in the caudal thorax is thicker
than that in the abdomen and the abdomen is thicker than that in
the cranial thorax.
TABLE-US-00020 TABLE 20 TG/- HOM TG/- Het WT/HOM WT/Het (N = 8) (N
= 10) (N = 9) (N = 5) Mean 33.26 .+-. 2.69 21.41 .+-. 1.27 42.54
.+-. 2.01 40.31 .+-. 3.82 Results represent mean values .+-. SEM. N
= number of sections measured.
[0576] The average thickness of stratum corneum in the cranial
thorax (Table 21) resembled to that in the cranial thorax, caudal
thorax and abdomen (Table 20), however significant reduction of
stratum corneun was only found in the TG/- Het vs. TG/- HOM
(P<0.05*) and vs. WT/HOM (P<0.01**), respectively. The
standard deviation and standard error of the mean were high which
might be due to poor section, missing skin samples, nature
architecture of the epidermis, or there was not much effect in the
cranial thorax. Note: the histology procedure or tissue section of
the cranial thorax might disqualify for histomorphometric analysis
in order to obtain quality result.
TABLE-US-00021 TABLE 21 TG/- HOM TG/- Het WT/HOM WT/Het (N = 28) (N
= 30) (N = 26) (N = 14) Mean 34.96 .+-. 3.53 18.14 .+-. 3.99 40.47
.+-. 4.38 32.96 .+-. 8.11 Results represent mean values .+-. SEM. N
= number of sections measured
[0577] The result of average thickness of stratum corneum in the
caudal thorax (Table 22) was similar to that in the cranial thorax,
caudal thorax and abdomen but with three exceptions: (1) TG/- HOM
vs. TG/- Het and TG/- HOM vs. WT/HOM showed no statistical
differences (P>0.05); (2) TG/- HOM vs. WT/Het showed significant
difference (P<0.01**); (3) The stratum corneum in the WT/Het
remarkably thickened which might be the consequence of tissue
processing artifact, e.g., the keratin swelled or expanded when
placed it in hypotonic solution or left in the water bath too
long.
TABLE-US-00022 TABLE 22 TG/- HOM TG/- Het WT/HOM WT/Het (N = 10) (N
= 10) (N = 8) (N = 4) Mean 35.64 .+-. 3.4 24.22 .+-. 1.54 44.35
.+-. 3.51 53.77 .+-. 7.21 Results represent mean values .+-. SEM. N
= number of sections measured.
[0578] Only the TG/- HOM vs. WT/HOM and TG/- Het vs. WT/HOM showed
statistical significant difference, P<0.05* and P<0.001***,
respectively (Table 23). The TG/- pups displayed a reduction in the
thickness of stratum corneum in the abdomen compared to its control
littermates (WT/HOM and WT/Het).
TABLE-US-00023 TABLE 23 TG/- HOM TG/- Het WT/HOM WT/Het (N = 8) (N
= 10) (N = 9) (N = 4) Mean 28.84 .+-. 4.36 21.86 .+-. 1.30 42.45
.+-. 3.15 33.25 .+-. 3.96 Results represent mean values .+-. SEM. N
= number of sections measured
[0579] (c) Average thickness (.mu.m) of epidermis plus stratum
corneum in cranial thorax, caudal thorax and abdomen
[0580] TG/- Het pups displayed a significant increase in the
epidermal thickness and a significant decrease in the thickness of
stratum corneum compared with the control littermates (WT/HOM and
WT/Het) and the TG/- HOM pups produced a similar result but with a
minimal effect (Table 24).
TABLE-US-00024 TABLE 24 TG/- HOM TG/- Het WT/HOM WT/Het (N = 10) (N
= 10) (N = 9) (N = 4) Stratum corneum 32.58 41.05 31.31 30.83
Epidermis 33.26 21.41 42.54 40.31 Results represent mean values. N
= number of pups
(d) Signaling of IL-20 Through Both IL-20RA and IL-22RA
[0581] The epidermis is a stratified, continually renewing
epithelium dependent on a balance among cell proliferation,
differentiation, and death for homeostasis. In normal epidermis, a
mitotically active basal layer gives rise to terminally
differentiating keratinocytes that migrate outward and are
ultimately sloughed from the skin surface as enucleated squames,
the keratin or cornified layer located in the stratum corneum.
Although many proteins are known to function in maintaining
epidermal homeostasis, the molecular coordination of these events
is poorly understood. IL-20 is a novel receptor-interacting protein
and it signals through either IL20RA or IL-22RA receptors (IL-22RA)
expressed in a layer of skin associated with the proliferation of
keratinocytes. IL-20 transgenic neonates display abnormal thickened
and shiny skin phenotype. IL-22RAm (HOM) deficiency in mice showed
no response to IL-22 treatment, whereas wild type mice with the
IL-22RA gene and treated with IL-22 demonstrated significant
increase in the epidermal thickness (P<0.001***, see the results
in IL-22RAm KO/IL-22 histomorphometric image analysis, PID 59.2).
To investigate whether the absence of IL-22RA has an effect on the
shiny phenotype observed in the K14 IL-20m TG neonates, transgenic
mice ectopically expressing IL-20 were mated with IL-22RA
homozygous (HOM) or IL-22RA heterozygous (Het) deficient. A
quantitative image analysis of epidermal thickness was previously
performed on fewer pups in the caudal thorax from this study (i.e.
19 pups, 1 section per pup, for a total of 19 sections) but no
statistical significance was obtained due to the limited number of
animals studied and the variation within the groups. The aim of the
present study was to histomorphometrically quantitate more skin
samples in cranial thorax, caudal thorax and abdomen from each pup
from the same study (i.e. 34 pups, 3 section per pup, for a total
of 102 sections) to explore the biology of IL-20 and obtain
reliable quantitative results. For effective image analysis, we
made sure that the orientation of the skin in the paraffin block
was consistent and the skin samples were measured from the same
respective locations in all individual and groups of pups. Two
kinds of measurements were performed: (1) The thickness of
epidermis was measured 10 times per 10.times. microscopic field in
each skin sample, each at the dorsal side of spinal cord, to
investigate the role of IL-20 in mediating keratinocyte
proliferation and differentiation; (2) The thickness of cornified
layer or the stratum corneum was measured in the same manner to
correlate the results with the shiny skin appearance in the IL-20
TG neonates.
[0582] Histomorphometric image analysis of the epidermal thickness
revealed that the TG/- Het neonates, expressing the k14 IL-20m
transgene on a background expressing some IL-22RA from one copy of
the IL-22RA gene displayed thickened epidermis and the TG/- HOM
neonates, expressing the k14 IL-20m transgene on a background not
expressing IL-22RA had no significant change. The epidermal
thickness increased significantly in IL-20 transgenic pups lacking
one copy of the IL-22RA gene (TG/- Het) versus the IL-20 transgenic
pups lacking both copy of the IL-22RA genes (TG/- HOM, P=0.001***)
and versus the control littermates (WT/HOM, P=0.001*** and WT/Het,
P=0.001***). The TG/- Het pups showed increased thickness of the
non-keratinized epidermis mainly due to hypertrophy of the
keratinocytes in the prickle layer. The epidermis of the TG/- Het
pup increased about 25% in thickness, whereas the epidermis of TG/-
HOM pups were only slightly thicker, increased about 4-5%, than the
controls (WT/HOM and WT/Het) and statistics indicated no
significant difference between the TG/- HOM and its control WT/HOM
(P>0.05).
[0583] Histomorphometric results of the stratum corneum showed that
despite the epidermal thickening in the TG/- Het neonates,
predominate reduction of keratin or cornified layer thickness was
observed in TG/- HOM and TG/- Het skins compared to the control
littermates (WT/HOM and WT/Het) and statistics indicated extremely
significant among the groups (P<0.0001****). The TG/- Het pups
showed about 36%, 50% and 49% decreased amounts of keratin on the
surface of the epidermis versus the TG/- HOM (P<0.01**), WT/HOM
(P<0.001***) and WT/Het (P<0.001***), respectively. The TG/-
HOM pups showed about 22% significant reduction in the stratum
corneum thickness compared to its control (WT/HOM, P<0.05*) and
only 17% reduction versus the WT/Het (P>0.05). The thickness of
stratum corneum in the control pups, WT/HOM and WT/Het were about
the same. The reduction in average thickness of stratum corneum in
the TG/- HOM and TG/- Het neonates seemed to correlate the gross
finding at sac, in which at gross level the IL-20 (TG)/IL-22RA
(Het) neonates appear to have reduced shine (e.g., with less
keratin), called a sheen, while the IL-20 (TG)/IL-22RA (HOM)
neonates do not shine (e.g., with more keratin). Histologically,
the keratin in the stratum corneum in the TG/- pups appeared to be
more compact than that in the WT pups. Together, the thickened
epidermis associated with hypertrophic keratinocytes and the thin
layer of stratum corneum in the IL-20 transgenic neonates might
explain why they displayed shiny skin phenotype.
[0584] Increased hypertrophy and disturbed terminal differentiation
of keratinocytes were observed in the IL-20 transgenic neonates
with a targeted knock out of one copy of the IL-22RA gene (Het).
The skin exhibited hypertrophy in keratinocytes but fails fully
differentiate, lacking keratin or the stratum corneum. The IL-20
transgenic neonates with disruption of two copy of the IL-22RA
genes (HOM) displayed a phenotype that resembled the TG/- Het skin
but showed less or minimal effect (FIG. 12-15). It seems that the
absence of IL-22RA (HOM) has a partial effect on the shiny
phenotype observed in the K14 IL-20m TG neonates and the absence of
IL-22RA (Het) has minimal or no effect on the shiny phenotype. In
other words, the signaling of IL-20, a novel receptor-interacting
protein which signals through either IL-20RA or IL-22RA receptor
(IL-22RA) is probably not obstructed by deficient expression of one
copy of the IL-22RA gene (Het) but is partially obstructed by
deficient expression of two copies of the IL-22RA gene (HOM).
[0585] These results provide evidence that effectively blocking
IL-22RA activity, for example via an IL-22RA gene knockout or
similarly via a neutralizing monoclonal antibody to IL-22RA of the
present invention, would similarly reduce IL-20-induced skin
effects, as well as IL-22-induced skin effects, for example in
psoriasis, IBD, colitis, or other inflammatory diseases induced by
IL-20, and or IL-22.
EXAMPLE 34
Effect of IL-22 on IL-22RA Knock Out Mice
[0586] Thirty-six mice including 23 IL-22RA KO (HOM) and 13
controls (WT) were treated with either IL-22 or PBS administered
subcutaneously by implanted a minipump with tube or a minipump
alone (Table 25):
TABLE-US-00025 TABLE 25 HOM/PBS HOM/IL-22 WT/PBS WT/IL-22 (Group1)
(Group 2) (Group 3) (Group 4) Male n = 3 n = 10 n = 3 n = 8 Female
n = 0 n = 10 n = 0 n = 2 Total n = 3 n = 20 n = 3 n = 10
[0587] Skin sample, 1.5-2.5 cm in length and 4.0-5.0 mm in
thickness, from the pumping site of each animal was obtained for
routine histological examination and histomorphometric image
analysis. All tissue specimens were fixed in 10% neutral buffered
formalin and processed into paraffin blocks. Six segmental
sections, 5 um in thickness and 10 um interval between adjacent
sections with epithelium covering the entire surface, from each
skin sample per animal were stained with hematoxylin and eosin
(H&E). Histomorphometric image analysis of the skin samples was
performed using an Olympus BH-2 microscope, a video camera
(Dage-MTI, Michigan City, Ind.) and BioQuant True Color windows 98
software (R&M Biometrics, Inc. Nashville, Tenn. 37209) with the
was following set up: Parameter: mag. 10.times., Z off set 0;
Array: length (um); Measure: manual and additive mode. The
thickness (um) of epidermis was measured 5 times in each 10.times.
microscopic field from a total of 4 fields captured from the center
0.4 cm of each skin section (e.g., one 10.times. microscopic
field=0.1 cm and four 10.times. microscopic fields=0.4 cm). Total
of 6 sections from each animal were measured and the mean value, SD
and SEM were obtained by Excel calculation. All of the sections
were randomized and measured in a blinded fashion. After the
measurement, the sections were unblended, and the results matched
to treatment groups. Final results by treatment group were
classified as follows: 1. Epidermal thickness from HOM and WT male
and female mice. 2. Epidermal thickness from HOM and WT male mice.
The resulting data was analyzed using GraphPad InStat software
(GraphPad Software, Inc., San Diego, Calif. 92121). One-way
analysis of variance (ANOVA) was applied to examine the statistical
significance of differences in mean values from group1 to group 4.
Tukey-Kramer Multiple Comparisons Test and Unpaired-T test were
applied to analyze the significance in mean values between two
groups. Observations of P<0.05 were considered significant.
III. Histomorphometric Results (1) Epidermal Thickness (.mu.m) from
HOM and WT Male and Female Mice
[0588] Epidermal thickness increased significantly in the WT mouse
skins treated with IL-22 (WT/IL-22) versus the WT/PBS controls
(P=0.0001). IL-22RAm KO mouse skin treated with IL-22 (HOM/IL-22)
showed increased mean value of the epidermal thickness compared
with the HOM/PBS controls, however statistics indicated no
significant difference between the two groups (P>0.05).
Predominate reduction of epidermal thickness was observed in the
IL-22RA KO mice compared with the WT mice (e.g., HOM/IL-22 vs.
WT/IL-22: P<0.001) (Table 26).
TABLE-US-00026 TABLE 26 HOM/PBS HOM/IL-22 WT/PBS WT/IL-22 (N = 3)
(N = 19) (N = 3) (N = 10) Mean 14.15 .+-. 0.19 19.01 .+-. 1.03
23.34 .+-. 5.49 43.08 .+-. 1.85 Results represent mean values .+-.
SEM. N = animal number.
(2) Epidermal Thickness (um) from HOM and WT Male Mice
[0589] Epidermal thickness increased about 2-fold in the WT male
mouse skins treated with IL-22 (WT/IL-22) when compared with WT/PBS
male controls (P=0.0001), however, IL-22RAm KO male mouse epidermis
treated with IL-22 (HOM/IL-22) only showed slightly increase
compared with the HOM/PBS male controls (P>0.05). Noticeably,
IL-22RAm KO mice exhibited marked reduction of epidermal thickness
when compared with its control, the WT male mice (e.g., HOM/PBS VS
WT/PBS: P<0.05; HOM/IL-22 VS WT/IL-22: P<0.001) (Table
27).
TABLE-US-00027 TABLE 27 HOM/PBS HOM/IL-22 WT/PBS WT/IL-22 (N = 3)
(N = 9) (N = 3) (N = 8) Mean 14.15 .+-. 0.19 15.86 .+-. 0.75 23.34
.+-. 5.49 41.41 .+-. 1.71 Results represent mean values .+-.
SEM.
(3) Epidermal Thickness (um) from HOM and WT mice male vs.
female
[0590] Epidermis of the female mice was found thicker than that of
the male mice (e.g., HOM/IL-22/male VS HOM/IL-22/female: P<0.01;
WT/IL-22/male VS WT/IL-22/female: P<0.05) (Table 28).
TABLE-US-00028 TABLE 28 WT/IL-22 HOM/IL-22 HOM/IL-22 WT/IL-22
(Female, (Male, N = 9) (Female, N = 10) (Male, N = 8) N = 2) Mean
15.86 .+-. 0.75 21.85 .+-. 1.3 41.41 .+-. 1.71 49.75 .+-. 4.82
Results represent mean values .+-. SEM.
(4) Epidermal Thickness (.mu.m) from HOM Mice, IL-22 Pump vs. IL-22
Pump with Tube
[0591] Epidermis from IL-22RAm KO (HOM) mice with IL-22 pump &
tube were found significantly thicker than that of the IL-22RAm KO
(HOM) mice with pump only (P<0.0001, by unpaired-T test) (Table
29).
TABLE-US-00029 TABLE 29 HOM w/IL-22 pump HOM w/IL-22 pump with tube
(M = 8 & F = 2, N = 10) (M = 2 & F = 8, N = 10) Mean 15.85
+ 0.65 23.30 + 1.36 Results represent mean values .+-. SEM. M:
male; F: female; N: total number of mice.
IV. Discussion:
[0592] Taken together, the aim of this study is to characterize the
epidermal effects in the IL-22 treated skins from both IL-22RAm KO
and WT mice and relates these findings to clinical indications. A
quantitative image analysis was performed to determine the
thickness of the epidermis in H&E stained skin sections. The
skin samples from each animal were histomorphometrically measured
120 times (i.e. 20 times/each section X 6 segmental sections from
each mouse=120 measurements) and the average epidermal thickness
was obtained by Excel calculation. Histomorphometric study
demonstrated that IL-22 resulted in significant increase in the
epidermal thickness especially in the WT mice with presence of the
IL-22RA receptor (P<0.0001 by ANOVA, considered extremely
significant) and showed less or minimal effects on the IL-22RAm KO
(HOM) mice with absence of the IL-22RA receptor (P>0.05). The
epidermal thickness in the IL-22 treated WT mice was increased
about 43% than that treated with PBS (e.g., WT/PBS, P<0.001),
whereas the IL-22 treated IL-22RAm KO (HOM) mice only showed 26%
increase in epidermal thickness compared with the control (HOM/PBS,
P>0.05). IL-22RAm KO mice exhibited thinner epidermis when
compared with the WT mice (P<0.001). Overall, the biologic
effects of IL-22 on mouse skin suggest that this factor might be
involved in the regulation of epidermal growth and
proliferation.
EXAMPLE 35
Pharmacokinetics of an Anti-Human IL-20 Monoclonal Antibody (Clone
#262.7.1.3.2.4)
[0593] The test monoclonal antibody, anti-human IL-20 mAb, (clone
#262.7.1.3.2.4) was provided in 3.times.3 mL aliquots at a
concentration of 1.08 mg/mL (determined by UV Absorbance at 280 nM)
and was stored at -80.degree. C. until use. The vehicle was
1.times.PBS (50 mM NaPO4, 109 mM NaCl), pH 7.3. The mAb was thawed
at room temperature before use and aliquots 1 and 2 were used as
provided for the 100 .mu.g IV and SC dosing groups, respectively.
Half of aliquot 3 was diluted 1:2 in 1.times.PBS for the 50 .mu.g
SC dose group and the second half of aliquot 3 was diluted 1:10 in
1.times.PBS for the 10 .mu.g SC dose group. Female SCID mice
(n=96), were received from Charles River Labs. Animals were checked
for health on arrival and group-housed (3 animals per cage). The
mice were 12 weeks old with an average body weight of 22 g at the
beginning of the study.
A. Dosing Protocol
[0594] Female SCID mice (n=24/dose group) were randomly placed into
four dosing groups (see Table 30). Group 1 was administered the
anti-huIL-20 mAb via IV injection of approximately 93 .mu.L in a
tail vein and Groups 2, 3, and 4 were administered the mAb via SC
injection of approximately 93 .mu.L in the scruff of the neck.
B. Sample Collection
[0595] Prior to blood collection, mice were fully anesthetized with
halothane or isofluorane. Blood samples were collected via cardiac
stick for all timepoints except the 168 hr timepoint (collected via
eye bleed and the same animals were bled again at the 504 hr
timepoint via cardiac stick). Blood was collected into serum
separator tubes and allowed to clot for 15 minutes. Samples were
subsequently centrifuged for 3 minutes at 14,000 rpm. Following
centrifugation, aliquots of 125-150 uL were dispensed into labeled
eppendorf tubes and immediately stored at -80.degree. C. until
analysis (Table 30).
TABLE-US-00030 TABLE 30 Group # Dose (ROA) Animals PK Timepoints 1
100 .mu.g (IV) 3 mice/timepoint* 0.25, 1, 4, 8, 24, 72, 168, 336
and 504 hr 2 100 .mu.g (SC) 3 mice/timepoint* 0.25, 1, 4, 8, 24,
72, 168, 336 and 504 hr 3 50 .mu.g (SC) 3 mice/timepoint* 0.25, 1,
4, 8, 24, 72, 168, 336 and 504 hr 4 10 .mu.g (SC) 3 mice/timepoint*
0.25, 1, 4, 8, 24, 72, 168, 336 and 504 hr *The same animals were
used for the 168 and 504 hr timepoints.
C. Quantification of Serum Anti-huIL-20 mAb Concentrations by
ELISA
[0596] An Enzyme Linked Immunosorbant Assay (ELISA) was developed
and qualified to analyze mouse serum samples from animals dosed
with anti-IL-20 mAb 267.7.1.3.2.4 during pharmacokinetic studies.
This assay was designed to take advantage of a commercially
available secondary antibody and calorimetric detection using TMB.
The dilutions used for the standard curve were modified to improve
the definition of the linear portion of the standard curve. A
standard curve in the range of 100 ng/mL to 0.231 ng/mL with 2-fold
dilutions allowed for quantitation of the mouse serum samples. QC
samples were diluted to 1:100, 1:1000 and 1:10000 in 10% SCID mouse
serum and back calculated from the standard curve.
D. Pharmacokinetic Analysis
[0597] Serum concentration versus time data were downloaded into
WinNonlin Professional 4.0 software (Pharsight, Inc.; Cary, N.C.)
for pharmacokinetic analysis. Noncompartmental analysis was used to
determine pharmacokinetic parameters based on the mean data at each
time point.
E. Results
[0598] Mean serum anti-human IL-20 mAb concentrations following
administration of 100 .mu.g IV and 100, 50, and 10 .mu.g SC are
shown in Table 31:
TABLE-US-00031 TABLE 31 100 .mu.g IV Time Conc 10 .mu.g SC 50 .mu.g
SC 100 .mu.g SC (hr) (.mu.g/mL) Conc (.mu.g/mL) Conc (.mu.g/mL)
Conc (.mu.g/mL) 0.25 196 (12) LTR 0.101 (0.065) 0.481 (0.485) 1 154
(18) 0.356 (0.146) 1.61 (0.52) 3.48 (1.72) 4 118 (20) 2.42 (0.53)
10.4 (3.4) 19.7 (4.7) 8 112 (20) 3.41 (0.30) 18.9 (3.6) 40.2 (6.4)
24 103 (13) 4.95 (0.05) 26.3 (0.7) 50.1 (6.2) 72 101 (16) 4.27
(0.79) 21.0 (3.4) 43.4 (2.7) 168 45.6 (15.4) 2.92 (0.53) 19.6 (2.7)
37.6 (3.4) 336 36.4 (16.6) 3.60 (0.31) 23.5 (3.5) 34.4 (5.8) 504
28.8 (3.8) 2.74 (0.39) 20.5 (3.6) 25.7 (2.1) LTR: less than
reportable
[0599] Following IV administration, the mAb concentration versus
time profile demonstrated a biexponential decline. Following SC
administration, the mAb appeared to have a slow absorption phase,
with absorption rate-limited elimination. The serum pharmacokinetic
parameters based on the mean data at each time point are shown in
Table 32:
TABLE-US-00032 TABLE 32 10 50 Parameter Units 100 .mu.g IV .mu.g SC
.mu.g SC 100 .mu.g SC C.sub.0(IV); C.sub.max (SC) .mu.g/mL 212 4.95
26.3 50.1 T.sub.max hr N/A 24 24 24 t.sub.1/2, .lamda.z hr 509 ND
ND 612 AUC.sub.(0-t) hr .mu.g/mL 27059 1730 10845 18110
AUC.sub.(0-inf) hr .mu.g/mL 48269 ND ND 41561 AUC % 43.9 ND ND 56.4
(% extrapolated) V.sub.ss (IV); mL 1.34 ND ND 2.12 V.sub.z/F (SC)
Cl (IV); Cl/F mL/hr 0.002 ND ND 0.002 (SC) F % N/A ND ND 86.1
(bioavailability) ND: not determinable due to lack of data in the
terminal elimination phase of the concentration versus time
profile
[0600] Following IV administration, the mAb demonstrated a very low
clearance (Cl=0.002 mL/hr) and long elimination half-life
(t.sub.1/2, .lamda.z.apprxeq.21 days). The mAb demonstrated a
steady-state volume of distribution (V.sub.ss=1.3 mL) that is less
than the blood volume in a mouse (.apprxeq.1.7 mL), suggesting that
the mAb did not distribute substantially out of the vascular
compartment. The back-calculated maximum concentration (C.sub.0)
was higher than expected based on the injected dose and the blood
volume in the mouse. This, along with the small VS, suggests that
the mAb may be confined, to a large extent, in the serum fraction
of the blood.
[0601] Following SC administration, C.sub.max values increased
linearly with dose. At the 100 .mu.g SC dose, the mAb had a
t.sub.1/2, .mu.z of approximately 25 days with clearance and an
apparent volume of distribution similar to that following IV
dosing. Bioavailability was 86%. At the lower two SC doses, most
pharmacokinetic parameters could not be estimated due to the lack
of a measurable terminal elimination phase, even though samples
were taken out to 504 hours. The absorption of the mAb following SC
dosing appears to reach a steady-state with elimination throughout
the duration of the study.
EXAMPLE 36
IL-20 and IL-22 Antagonists in CD4.sup.+ CD45RB.sup.hi (CD25.sup.-)
colitis and psoriasis Model
[0602] A. Summary
[0603] Transfer of CD4+ CD45RB.sup.hi or CD4+CD25- T cells into
syngeneic SCID mice results in colitis in the mice. Co-transfer of
regulatory T cells (CD4+CD25+ or CD4+CD45RB.sup.lo) inhibits this
colitis. After transfer of CD4+CD25- T cells into mice, if mice are
additionally injected with staphylococcal enterotoxin B (SEB), mice
not only develop colitis, but also psoriasis. Antibodies against
IL-22RA, IL-20, IL-22, IL20R and/or IL-22R, or soluble IL-22RA
receptors are administered from days 0-21 after cell transfer and
symptoms for colitis and psoriasis are monitored. Inhibition of
psoriatic score or colitis (histology) indicates that IL-21 can
inhibit these autoimmune diseases.
[0604] B. Study Design
[0605] Spleens and inguinal lymph nodes are isolated from B10.D2
mice. Single cell suspensions are formed and counted. Using the
Miltenyi Bead system, CD25+ cells are sorted out by positive
selection. Cells are stained with CD25-PE (BD Pharmingen) at 1:100
dilution and incubated for 15 minutes. Excess antibody is washed
out and the cells are incubated with 10 ul anti-PE beads/10.sup.6
cells for 20 minutes. The cells are washed with PBS and passed over
an LS column (Miltenyi Biotech). Cells that pass through the column
(CD25-) are retained for further analysis. A CD4 enrichment
cocktail (Stem Cell technologies) is added (1:100) to these CD25-
cells and incubated for 15 minutes. Cells are washed with PBS. A
1:10 dilution of anti-biotin tetramer is added to the cells for 15
minutes followed by a magnetic colloid (60 .mu.l/10.sup.6 cells)
for 15 minutes (all from Stem Cell Technologies). Cells are passed
through a negative selection column (0.5'', Stem cell
Technologies). Cells that pass through are the CD4+CD25- cells.
Purity is analyzed using flow cytometry. 0.4.times.10.sup.6 cells
are injected i.v. into naive CB-17 SCID mice in a total volume of
200 .quadrature.l. Mice are injected i.p with 10 .quadrature.g SEB
the following day (d1). Symptoms for psoriasis and colitis are
followed from 2-5 weeks. Mice are scored for psoriasis disease
under the following criteria. 0--no lesions, 1--mild lesions on the
neck, 2--severe lesions on the neck and back (trunk) 3--very severe
lesions on the neck, back and the belly of mice. Ear thickening is
also measured as a measure of disease severity. Groups of mice are
injected i.p. with PBS, 100 .quadrature.g control antibody or
10-100 .quadrature.g antibodies against IL-22RA, IL-20, IL-22,
IL-20R or IL-22R, or soluble IL-22RA from days 1-30 under different
dosing regimen (3.times./week or 2.times./week).
[0606] C. Results and Conclusion
[0607] Inhibition of psoriatic and colitis symptoms in antibody
treated mice indicates that inhibition of IL-20 and/or IL-22
function can inhibit autoimmune symptoms in this model for
psoriasis and colitis.
EXAMPLE 37
IL-20, and IL-22 Antagonists in SCID-hu Transplant Psoriasis
Model
[0608] A. Summary
[0609] Human psoriasis skin grafted on SCID mouse can maintain its
clinical, light microscopic, and immunohistochemical psoriatic
features for several weeks. This model provides a system for
evaluating therapies intended to restore lesional tissue to a
normal phenotype. Once the human skin is successfully grafted,
antibodies against IL-22RA, IL-20, IL-22, IL-20R and/or IL-22R, or
soluble IL-20 or IL-22 receptors can be administered for several
weeks, and the epidermal thickness can be analyzed to evaluate the
effect of these antagonists on psoriasis.
[0610] B. Study Design
[0611] Full-thickness 6-mm punch biopsies consisting of the entire
epidermis and several mm of dermis are obtained healthy adult
volunteers and psoriatic lesional skins. Four to six biopsies are
obtained from each donor. One punch biopsy from each donor is
transplanted onto the dorsal surface of recipient SCID mouse
(CB-17, Taconic). The animals are maintained in a pathogen-free
environment. The treatment is initiated after a successful grafting
(2-3 weeks post-transplantation) as following: one biopsy for
negative control (PBS or isotype mAb), one biopsy for positive
control (Cyclosporin A), and 2-3 biopsies for treatment with
anti-human IL-22RA, anti-human IL-20, anti-human IL-22 mAb or
soluble receptors for IL-20 or IL-22 (intraperitoneal injection,
three times a week for 2-4 weeks on a M-W-F schedule).
C. Quantitative Analysis:
[0612] Clinical observations and assessments will be made regularly
throughout the experiments, and will be recorded. The severity of
the psoriatic lesions is assessed for scaliness, induration, and
erythema in a blinded fashion. The parameters can be scored using
the three-point scale: 0=complete lack of cutaneous involvement;
1=slight involvement; 2=moderate involvement; 3=severe involvement.
At the end of the dosing period each animal is euthanized and
tissues are collected for histology and IHC. (1) Part of the tissue
is fixed in 10% formalin and stained with hematoxylin and eosin.
Epidermal area is measured as a function of changes in epidermal
thickness per unit length using NIH Image software. Multiple areas
from each transplant are quantified to provide a high n value and
mean epidermal area. (2) number of inflammatory mononuclear cells
per high-power field (0.103.times.0.135 mm) in the upper dermis;
(3) the grade of parakeratosis is rated on an arbitrary scale from
0 to 3, where 0 is no parakeratosis, 1 is parakeratosis in less
than one third of the section, 2 was parakeratosis in more than one
third but less than two thirds of the section, a d 3 is
parakeratosis in more than two thirds of the section. (4) The
remaining of the tissue will be stained for Ki67 (marker of
proliferating keratinocytes), to evaluate the number of Ki67
cycling keratinocytes-per-millimeter length of the section. The
reduced severity of psoriasis as measured by epidermal thickness,
indicates the neutralization of IL-20 and IL-22 function can be
effective in this psoriasis model. To quantify the reduced severity
of psoriasis, we measure epidermal thickness, the number of
inflammatory cells in the upper dermis, the numbers of Ki67 cycling
keratinocytes, and the grades of parakeratosis. The significantly
reduced all four parameters for the treated groups compared to the
control mice, indicate the potential therapeutic use of IL-20,
IL-22 antagonists.
EXAMPLE 38
Screening for IL-20 Antagonist Activity Using BaF3/IL-22RA/IL-20RB
Cells Using an Alamar Blue Proliferation Assay
[0613] The factor-dependent pre-B cell line BaF3 was co-transfected
with IL-22RA and IL-20RB (see, method in Example 3) and treated
with IL-20 at various concentrations. Proliferation was assessed
using an alamar blue assay as described in Example 3. IL-20
stimulated proliferation in a dose-dependent manner at
concentrations expected for a cytokine, demonstrating that IL-20
binds and activates the heterodimeric IL-22RA/IL-20RB receptor at
concentrations expected for a cytokine. The negative controls
containing untransfected BaF3 did not proliferate.
[0614] In order to determine if anti-IL-22RA antibodies are capable
of antagonizing IL-20 activity, the assay described above is
performed using anti-IL-22RA antibodies as an antagonist to IL-20
activity. When IL-20 is combined with such antagonist, the response
to IL-20 is brought down to background levels. That the presence of
an antagonist that ablates or reduces the proliferative effects of
IL-20 demonstrates that it is an antagonist of the IL-20 ligand.
This assay can be used to test other antagonists of IL-20 activity
described herein, such as antagonist polypeptides comprising a
soluble IL-22RA receptor.
EXAMPLE 39
Neutralization of IL-20 and IL-22 Activity by Anti-huL22RA
Monoclonal Antibody
[0615] Using the cell-based neutralization assay described in
Example 28, a purified mouse anti-huIL-22RA monoclonal antibody
(Example 30(D)) was added as a serial dilution, for example, at 10
.mu.g/ml, 5 .mu.g/ml, 2.5 .mu.g/ml, 1.25 .mu.g/ml, 625 ng/ml, 313
ng/ml, 156 ng/ml and 78 ng/ml. The assay plates were incubated at
37.degree. C., 5% CO.sub.2 for 4 days at which time Alamar Blue
(Accumed, Chicago, Ill.) was added at 20 .mu.l/well. Plates were
again incubated at 37.quadrature.C, 5% CO.sub.2 for 16 hours.
Results showed that the purified anti-huIL-22RA monoclonal antibody
could neutralize signaling of both huIL-22 and huIL-20 through
huIL-22RA. At the 10 .mu.g/ml concentration, the antibody
completely neutralized proliferation induced by huIL-22 or huIL-20,
with the inhibition of proliferation decreasing in a dose dependent
fashion at the lower concentrations. An isotype-matched negative
control mouse mAb, tested at the concentrations described above,
provided no inhibition of proliferation of either cytokine. These
results further demonstrate that monoclonal antibodies to IL-22RA
could indeed antagonize the activity of the pro-inflammatory
ligands, IL-20 and IL-22 at low concentrations.
[0616] These results provided additional evidence that effectively
blocking IL-22RA activity, for example via a neutralizing
monoclonal antibody to IL-22RA of the present invention, could be
advantageous in blocking, inhibiting, reducing, antagonizing or
neutralizing the effects of IL-20 and IL-22 (alone or together) in
vivo and maybe reduce IL-20 and/or IL-22-induced inflammation, such
as that seen in IL-20-induced skin effects, as well as
IL-22-induced skin effects, for example in psoriasis, IBD, colitis,
or other inflammatory diseases induced by IL-20, and or IL-22
including IBD, arthritis, asthma, psoriatic arthritis, colitis,
inflammatory skin conditions, and atopic dermatitis.
EXAMPLE 40
Treatment of Pregnant IL-20 and IL-22 Transgenic Mice with
Neutralizing Anti-IL-22RA Monoclonal Antibody
[0617] To test the rat anti-mouse IL-22RA monoclonal antibody (mAb)
for neutralizing activity in vivo, pregnant IL-20 transgenic (Tg)
and IL-22 Tg mice are injected intraperitoneally with an anti-mouse
IL-22RA mAb. The newborn pups are then assessed for the presence or
absence of the "shiny" skin phenotype that normally characterizes
these strains of mice.
[0618] Specifically, male IL-20 Tg (which are generated using the
keratin-14) or IL-22 Tg (using the insulin promoter) mice are bred
to C57BL/6N females in estrus and the bred females are identified
by the presence of a vaginal plug the following day. Each pregnant
female is set aside in a separate cage and monitored daily.
Treatment groups include at least 4 pregnant females each, to allow
for a statistically significant analysis of both Tg and nonTg pups.
Based on prior experience with these Tg mice, a litter usually
ranges between approximately 6 to 8 pups per litter, of which
between 2 to 3 are Tg+.
[0619] Seven to nine days after the mice are bred (embryonic age
7-9; e7-9), the females are injected intraperitoneally with 250-500
ug of the rat anti-mouse IL-22RA mAb (rat IgG2a isotype) in a
volume of 200-250 ul of PBS. Short needles are used at a shallow
injection angle to avoid directly injecting the uterus. The
pregnant females are injected in this manner 3 days a week (Monday,
Wednesday, and Friday) for 2 weeks (until birth) in order to
successfully access the developing embryos. Control groups (of not
less than 4 pregnant female mice each) include the following:
isotype control rat IgG2a mAb, anti-human/mouse IL-22 mAb (rat IgG1
isotype), and an isotype control rat IgG1 mAb. As a control for
neutralization of murine IL-20, pregnant females are injected with
either a soluble IL-20R-Fc4 fusion protein that can bind and
neutralize both human and murine IL-20 or an Fc4 control
protein.
[0620] From days 1 to 2 after birth, the pups are closely monitored
for the appearance of the shiny skin phenotype. On day 2, the pups
are euthanized and a portion of the tail is collected for DNA
isolation to determine the genotype (Tg or nonTg) of each pup. Skin
samples are collected for histological analysis in order to assess
whether the pups exhibit the thickened epidermal cell layers that
usually characterize these Tg mice. Trunk blood is also collected
from the pups (and an eyebleed from the dams one day after birth)
to quantitate, via ELISA, the levels of anti-IL-22RA mAb in the
serum of each mouse. Because these mAbs are potent inhibitors of
IL-20 and/or IL-22 in vivo, the Tg pups have normal skin (i.e. no
epidermal thickening or "shiny" appearance).
EXAMPLE 41
IL-20 and IL-22 Antagonists in Organ Culture Psoriasis Model
[0621] Human psoriatic plaque skin can be maintained in organ
culture, and the abnormal histological features of lesional skin
are maintained in the absence of exogenous growth factors.
Antibodies against IL-22RA, IL-20, IL-22, IL20R and/or IL-22R, or
soluble IL-20 or IL-22 receptors can be administered, and the
histological features of psoriatic lesional skin can be
ameliorated.
[0622] A. Study Design
[0623] Full-thickness 2-mm punch biopsies consisting of the entire
epidermis and several mm of dermis are obtained from either healthy
adult volunteers or from psoriatic lesional skin. Immediately upon
biopsy, the tissue is immersed in culture medium consisting of
Keratinocyte Basal Medium (KBM) (Clonetics Inc, Walkersville, Md.).
The culture medium is supplemented with CaCl2 to bring the final
Ca2+ concentration to 1.4 mM (Varani et al, 1993, 1994). The
biopsies are then incubated in wells of a 96-well dish containing
200 ul of Ca2+ supplemented KBM with or without additional
treatments of antibodies against human IL-20, IL-22, IL-22RA, or
soluble receptors of IL-20 or IL-22. Cultures are incubated at
37.degree. C. in an atmosphere of 95% air and 5% CO.sub.2 for 8
days.
[0624] B. Quantitative Analysis:
[0625] At the end of incubation period, tissue is fixed in 10%
buffered formalin and examined histologically after staining with
hematoxylin and eosin. The appearance of psoriatic tissue exposed
to the antibodies or soluble receptors could be more closely
resembled that of normal tissues, including the following
observation: the initially disorganized, irregular-shaped basal
epithelial cells developed a more columnar appearance with restored
polarity; epidermal rete ridges regressed, with fewer areas of
epithelial cell expansion into the dermal space; and there was less
overall degeneration of the upper epidermal layers. The organ
culture model provides a rapid and sensitive means for determining
if a particular compound has potential as an
anti-hyperproliferative agent. The abnormal histological feature
may be ameliorated in the presence of an IL-20, IL-22 antagonist,
suggesting the effectiveness of such agent in the treatment of
psoriasis.
EXAMPLE 42
Mapping of mIL22RA (zCytoR11m) Regions Binding to Neutralizing mAbs
R2.1.5F4.1 and R2.1.15E2.1
A. Epitopes on Murine IL-22RA Wherein Neutralizing Monoclonal
Antibodies Bind.
[0626] The experiments described below were aimed at identifying a
region or regions in the amino acid sequence of murine IL-22RA
soluble receptor protein (SEQ ID NO:62) that were important for
receptor activity, or for antagonist or neutralizing antibody
binding. The murine IL-22RA-Fc protein, which was previously
cleaved with thrombin to remove the Fc, was then cleaved
C-terminally to the methionine residues in the sequence by
incubation with cyanogen bromide (CNBr). The CNBr-generated
peptides were fractionated, and fractions were tested for binding
activity as detected by ELISA and reactivity by Western analysis
using monoclonal antibodies with neutralizing properties, clones
R2.1.5F4.1 and R2.1.15E2.1.
[0627] Upon cleavage with CNBr, the following peptides were
potentially generated from non-reduced full-length mIL-22RA (Table
33). Under non-reducing conditions, cysteines are disulfide-bonded,
which may result in an internal linkage in peptide 1 and a link
between peptides 3 and 5. The residues in bold font are potentially
involved in ligand binding that correspond with human IL-22RA
residues potentially involved in ligand binding in SEQ ID NO:2 or
SEQ ID NO:3, as described in Example 42B. Specifically, SEQ ID
NO:48 corresponds to amino acid residues 16 (His) to 83 (Met) of
SEQ ID NO:42; SEQ ID NO:49 corresponds to amino acid residues 84
(Glu) to 109 (Met) of SEQ ID NO:42, SEQ ID NO:50 corresponds to
amino acid residues 110 (Thr) to 137 (Met) of SEQ ID NO:42, SEQ ID
NO:51 corresponds to amino acid residues 138 (Leu) to 177 (Met) of
SEQ ID NO:42, and SEQ ID NO:52 which corresponds to amino acid
residues 163 (His) to 208 (Pro) of SEQ ID NO:42 or 163 (His) to 212
(Arg) of SEQ ID NO:62.
TABLE-US-00033 TABLE 33 Peptide Number From To Sequence CNBr
Peptide 1 1 68 HTTVDTSGLLQHVKFQSSNFENILTWD
GGPASTSDTVYSVEYKKYGERKWLAKA GCQRITQKFCNLTM (SEQ ID NO: 48)
non-reduced: cysteines in peptide 1 are linked CNBr Peptide 2 69 94
ETRNHTEFYYAKVTAVSAGGPPVTKM (SEQ ID NO: 49) CNBr Peptide 3 95 122
TDRFSSLQHTTIKPPDVTCIPKVRSIQ M (SEQ ID NO: 50) non-reduced: peptides
3-5 are linked CNBr Peptide 4 123 162 LVHPTLTPVLSEDGHQLTLEEIFHDLF
YRLELHVNHTYQM (SEQ ID NO: 51) CNBr Peptide 5 163 212
HLEGKQREYEFLGLTPDTEFLGSITIL TPILSKESAPYVCRVKTLPLVPR (SEQ ID NO:
52)
[0628] 1. CNBr Cleavage and Isolation of Peptide Fractions
[0629] 50 .mu.g of mIL22RA was lyophilized and reconstituted in 180
.mu.L of formic acid (70%). 1 .mu.L of 5M CNBr dissolved in
acetonitrile was added. The sample was mixed and left to react for
18 hours at room temperature in the dark. 150 .mu.L of the reaction
mixture were fractionated by reversed-phase HPLC fitted with an
analytical Zorbax SB300-C8 column. Peaks were separated using a
gradient starting at 25% acetonitrile (0.085% TFA) and 75% water
(0.1% TFA) and finishing at 95% acetonitrile (0.085% TFA) and 5%
water (0.1% TFA). UV analysis showed three main and two minor
peaks, which were collected. Each fraction was divided in half; one
portion was submitted to ELISA, the other portion was lyophilized
and reconstituted in 150 .mu.L of phosphate-buffered saline
solution (PBS). UV analysis of the PBS fractions confirmed the
recovery of all peaks collected from the analytical column. The PBS
fractions were submitted for Western analysis.
[0630] 2. ELISA
[0631] HPLC fractions containing peptide sequences from IL-22RA
cleaved with CNBr were diluted to an estimated equal concentration
using HPLC buffer (90% acetonitrile, 10% H.sub.2O, 0.09%
trifluoroacetic acid). Samples were loaded to ninety-six-well
microtiter plates in 4 wells each at 100 .mu.L/well and allowed to
dry down overnight at room temperature in a fume hood. The plates
were washed with ELISA C buffer (PBS, 0.05% Tween-20), and then
blocked with ELISA B buffer (PBS, 0.1% BSA, 0.05% Tween-20) for 2
hours at 37.degree. C. Two monoclonal antibodies (mAb) to IL22RA
(Clone R2.1.5F4.1, and Clone R2.1.15E2.1) were diluted to 2
.mu.g/mL in ELISA B. Each mAb was added to each peptide sequence
sample at 100 .mu.L/well and plates were incubated for 60 minutes
at 37.degree. C. The plates were washed to remove unbound antibody,
and a secondary antibody (goat anti-rat IgG conjugated to
horseradish peroxidase (Jackson)) was diluted to 1 .mu.g/mL in
ELISA B buffer and added to all wells at 100 .mu.L/well. Plates
were incubated for 1 hour at 37.degree. C. The wells were washed
with ELISA C buffer, and then incubated with TMB 1 Component HRP
Microwell Substrate (BioFx) for 5 minutes. The reaction was stopped
by the addition of 450 nm Stop Reagent for TMB Microwell (BioFx)
and the plates read at absorbance 450 nm in a Dynatech ELISA plate
reader (Molecular Devices).
[0632] Results indicate mAb R2.1.5F4.1 reacted with HPLC fraction
#4 of the mIL22RA CNBr reaction, which also produced a band in the
Western blotting experiments.
[0633] 3. Western
[0634] HPLC fractions containing peptide sequences from IL22RA
cleaved with CNBr were lyophilized over night at room temp, and
reconstituted in PBS. Samples were then mixed with non-reducing
sample buffer (Invitrogen) and boiled for 10 min. Samples were
loaded and electrophoresed by SDS-PAGE on 4-12% Bis-Tris gels
(Invitrogen) using 1.times.MES-SDS Running Buffer (Invitrogen) and
transferred to nitrocellulose (0.2 .quadrature.m; Bio-Rad) in 20%
Methanol transfer buffer, all at room temperature. Filters were
allowed to dry over night at room temperature. The filters were
blocked with 10% non-fat dry milk in buffer A (50 mM Tris, pH 7.4,
5 mM EDTA, 0.05% Igepal CA-630, 150 mM NaCl, 0.25% gelatin) for 30
minutes at room temperature. A monoclonal antibody (mAb) to IL22RA
(Clone R2.1.5F4.1) was diluted to 2 .mu.g/mL in buffer A containing
2.5% non-fat dry milk. Blots were incubated in this primary
antibody for 1 hour at room temperature. Following incubation,
blots were washed three times in buffer A and incubated 1 hour at
room temperature with a 1:5000 dilution of secondary antibody (Goat
anti-Rat IgG-horseradish peroxidase; Jackson, Inc) in buffer A with
2.5% non-fat dry milk. The blots were then washed, developed with a
chemiluminescent substrate (Lumi-Light Western Blotting Substrate;
Roche), and exposed using a luminescent imager (Mannheim Boehringer
Lumi-Imager).
[0635] Using a 30 minute exposure, the non-reducing gel showed very
strong bands for fractions #4 and #5, along with a faint band for
fraction #3. Fraction #4 also tested positive in the ELISA.
N-Terminal Sequencing of Active Fraction #4
[0636] Of the five CNBr peptide fractions collected from the
analytical reversed-phase column, fraction #4 showed activity in
the ELISA and was also positive by Western blotting. To identify
the peptides present in the active fraction #4, the sample was
submitted to Edman degradation using well-known methods. Three
N-termini were identified from the active fraction that were
consistent with peptides 2 (SEQ ID NO:49), 3 (SEQ ID NO:50), and 5
(SEQ ID NO:52). These results indicated that the antibodies bound
to peptides 2 (SEQ ID NO:49), 3 (SEQ ID NO:50), and 5 (SEQ ID
NO:52).
TABLE-US-00034 TABLE 34 Peptide Edman Degradation N-Terminal
Sequence Identification First Sequence Obtained HLEGK QREYE FLGLT
CNBr Peptide 5 from Fraction #4 PDTEF (SEQ ID NO: 52)
CNBr-generated HLEGK QREYE FLGLT mIL22RA Sequence PDTEF LGSIT ILTPI
LSKES APYVC RVKTL PLVPR (SEQ ID NO: 53) Second Sequence Obtained
ETRNH TEFYY AKVTA CNBr Peptide 2 from Fraction #4 VSAGG (SEQ ID NO:
49) CNBr-generated ETRNH TEFYY AKVTA mIL22RA Sequence VSAGG PPVTK M
(SEQ ID NO: 54) Third Sequence Obtained TDRFS XLQHT XIXPX CNBr
Peptide 3 from Fraction #4 DXXXI (SEQ ID NO: 50) CNBr-generated
TDRFS SLQHT TIKPP mIL22RA Sequence DVTCI PKVRS IQM (SEQ ID NO:
55)
Discussion
[0637] Five fractions were isolated from a mixture of CNBr-cleaved
mIL22RA peptides. Of these, only fraction #4 was active in an ELISA
and positive by Western. Edman degradation identified three
N-termini consistent with CNBr peptides 2 (SEQ ID NO:49), 3 (SEQ ID
NO:50), and 5 (SEQ ID NO:52) in fraction #4. Within these regions,
six residues are potentially involved in ligand binding. These
residues are Y93, R112, K210, and E211 of SEQ ID NO:42, which also
correspond to residues Y78, R97, K195, and E196 of SEQ ID NO:62.
Residues Y60 and F164 of SEQ ID NO:42 are also involved in ligand
binding.
B. Epitopes on Human IL-22RA Wherein Neutralizing Monoclonal
Antibodies Bind.
[0638] The experiments described below are aimed at identifying a
region or regions in the extracellular domain for amino acid
sequence of human IL-22RA protein (SEQ ID NO:2) that are important
for receptor activity, or for antagonist or neutralizing antibody
binding. A human soluble receptor IL-22RA protein (e.g., comprising
SEQ ID NO:3, such as, IL-22RA-Fc cleaved with thrombin to remove
the Fc) is then cleaved C-terminally to the methionine residues in
the sequence by incubation with cyanogen bromide (CNBr), or other
agent known in the art that cleaves the human protein into defined
fragments. The CNBr-generated peptides are fractionated, and the
resulting fractions are tested for binding activity as detected by
ELISA and reactivity by Western analysis using monoclonal
antibodies with neutralizing properties.
[0639] Four cysteines are predicted to be disulfide-bonded with a
linkage pattern of Cys71-Cys79 and Cys204-Cys217 of SEQ ID NO:2.
Upon cleavage with CNBr, the following peptides are potentially
generated from non-reduced full-length human IL-22RA: peptide 6
(SEQ ID NO:56), peptide 7 (SEQ ID NO:57); peptide 8 (SEQ ID NO:58);
peptide 9 (SEQ ID NO:59); peptide 10 (SEQ ID NO:60); and peptide 11
(SEQ ID NO:61) (Table 35). Cysteines are disulfide-bonded, which
results in a possible link between peptides 7 (SEQ ID NO:57) and 10
(SEQ ID NO:60. Specifically, SEQ ID NO:56 corresponds to amino acid
residues 1 (Pro) to 92 (Met) of SEQ ID NO:3; SEQ ID NO:57
corresponds to amino acid residues 93 (Thr) to 120 (Met) of SEQ ID
NO:3, SEQ ID NO:58 corresponds to amino acid residues 121 (Ile) to
160 (Met) of SEQ ID NO:3, SEQ ID NO:59 corresponds to amino acid
residues 161 (His) to 185 (Met) of SEQ ID NO:3, SEQ ID NO:60
corresponds to amino acid residues 186 (Ile) to 199 (Met) of SEQ ID
NO:3 and SEQ ID NO:61 corresponds to amino acid residues 200 (Cys)
to 211 (Thr) of SEQ ID NO:3.
TABLE-US-00035 TABLE 35 Peptide Number From To Sequence CNBr
Peptide 6 1 92 Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val Lys Phe
Gln Ser Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly
Thr Pro Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg Asp
Trp Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn Leu
Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala Arg Val Thr Ala
Val Ser Ala Gly Gly Arg Ser Ala Thr Lys Met (SEQ ID NO: 56) CNBr
Peptide 7 93 120 Thr Asp Arg Phe Ser Ser Leu Gln His Thr Thr Leu
Lys Pro Pro Asp Val Thr Cys Ile Ser Lys Val Arg Ser Ile Gln Met
(SEQ ID NO: 57) CNBr Peptide 8 121 160 Ile Val His Pro Thr Pro Thr
Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu Glu Asp Ile Phe His
Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn Arg Thr Tyr Gln Met
(SEQ ID NO: 58) CNBr Peptide 9 161 185 His Leu Gly Gly Lys Gln Arg
Glu Tyr Glu Phe Phe Gly Leu Thr Pro Asp Thr Glu Phe Leu Gly Thr Ile
Met (SEQ ID NO: 59) CNBr Peptide 10 186 199 Ile Cys Val Pro Thr Trp
Ala Lys Glu Ser Ala Pro Tyr Met (SEQ ID NO: 60) CNBr Peptide 11 200
211 Cys Arg Val Lys Thr Leu Pro Asp Arg Thr Trp Thr (SEQ ID NO:
61)
[0640] 4. CNBr Cleavage and Isolation of Peptide Fractions, Western
and ELISA, and N-Terminal Sequencing
[0641] About 50 .mu.g of human IL22RA is lyophilized and is
reconstituted, fractionated, collected and analyzed using Western
analysis, and ELISA as described in EXAMPLE 42A, to identify
fractions containing anti-IL-22RA monoclonal antibodies, and those
that bind IL-22RA as shown by ELISA and Western analysis. The CNBr
peptide fractions that are collected from the analytical
reversed-phase column, are then tested for activity in the ELISA
and are confirmed as positive by Western blotting. For positive
fractions, peptides are identified via Edman degradation using
well-known methods.
Discussion
[0642] The mouse CNBr peptide #5 (SEQ ID NO:52) corresponds to
human CNBr peptides #9, and #10 (SEQ ID NO:59 and SEQ ID NO:60);
mouse CNBr peptide #2 (SEQ ID NO:49) corresponds to human CNBr #6
(SEQ ID NO:56); and mouse CNBr peptide #3 (SEQ ID NO:50)
corresponds to human CNBr #7 (SEQ ID NO:57). Of the fractions that
are isolated from a mixture the CNBr-cleaved human IL-22RA
peptides, six residues within the possible regions are potentially
involved in ligand binding: Residues of SEQ ID NO:2 (and
corresponding residues of SEQ ID NO:3) that are important to
ligand-receptor binding comprise Tyr-60, and Phe-164, Tyr-93,
Arg-112, Lys-210, and Glu-211 of SEQ ID NO:2 and (and corresponding
residues of SEQ ID NO:3). Moreover, primary residues of SEQ ID NO:2
(and corresponding residues of SEQ ID NO:3) that are important to
direct ligand-receptor binding comprise Tyr-60, and Phe-164 of SEQ
ID NO:2 (and corresponding residues of SEQ ID NO:3), and secondary
residues comprise residues Tyr-93, Arg-112, Lys-210, and Glu-211 of
SEQ ID NO:2 and (and corresponding residues of SEQ ID NO:3).
[0643] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 62 <210> SEQ ID NO 1 <211> LENGTH: 2831
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (34)...(1755) <400> SEQUENCE: 1 tagaggccaa
gggagggctc tgtgccagcc ccg atg agg acg ctg ctg acc atc 54 Met Arg
Thr Leu Leu Thr Ile 1 5 ttg act gtg gga tcc ctg gct gct cac gcc cct
gag gac ccc tcg gat 102 Leu Thr Val Gly Ser Leu Ala Ala His Ala Pro
Glu Asp Pro Ser Asp 10 15 20 ctg ctc cag cac gtg aaa ttc cag tcc
agc aac ttt gaa aac atc ctg 150 Leu Leu Gln His Val Lys Phe Gln Ser
Ser Asn Phe Glu Asn Ile Leu 25 30 35 acg tgg gac agc ggg cca gag
ggc acc cca gac acg gtc tac agc atc 198 Thr Trp Asp Ser Gly Pro Glu
Gly Thr Pro Asp Thr Val Tyr Ser Ile 40 45 50 55 gag tat aag acg tac
gga gag agg gac tgg gtg gca aag aag ggc tgt 246 Glu Tyr Lys Thr Tyr
Gly Glu Arg Asp Trp Val Ala Lys Lys Gly Cys 60 65 70 cag cgg atc
acc cgg aag tcc tgc aac ctg acg gtg gag acg ggc aac 294 Gln Arg Ile
Thr Arg Lys Ser Cys Asn Leu Thr Val Glu Thr Gly Asn 75 80 85 ctc
acg gag ctc tac tat gcc agg gtc acc gct gtc agt gcg gga ggc 342 Leu
Thr Glu Leu Tyr Tyr Ala Arg Val Thr Ala Val Ser Ala Gly Gly 90 95
100 cgg tca gcc acc aag atg act gac agg ttc agc tct ctg cag cac act
390 Arg Ser Ala Thr Lys Met Thr Asp Arg Phe Ser Ser Leu Gln His Thr
105 110 115 acc ctc aag cca cct gat gtg acc tgt atc tcc aaa gtg aga
tcg att 438 Thr Leu Lys Pro Pro Asp Val Thr Cys Ile Ser Lys Val Arg
Ser Ile 120 125 130 135 cag atg att gtt cat cct acc ccc acg cca atc
cgt gca ggc gat ggc 486 Gln Met Ile Val His Pro Thr Pro Thr Pro Ile
Arg Ala Gly Asp Gly 140 145 150 cac cgg cta acc ctg gaa gac atc ttc
cat gac ctg ttc tac cac tta 534 His Arg Leu Thr Leu Glu Asp Ile Phe
His Asp Leu Phe Tyr His Leu 155 160 165 gag ctc cag gtc aac cgc acc
tac caa atg cac ctt gga ggg aag cag 582 Glu Leu Gln Val Asn Arg Thr
Tyr Gln Met His Leu Gly Gly Lys Gln 170 175 180 aga gaa tat gag ttc
ttc ggc ctg acc cct gac aca gag ttc ctt ggc 630 Arg Glu Tyr Glu Phe
Phe Gly Leu Thr Pro Asp Thr Glu Phe Leu Gly 185 190 195 acc atc atg
att tgc gtt ccc acc tgg gcc aag gag agt gcc ccc tac 678 Thr Ile Met
Ile Cys Val Pro Thr Trp Ala Lys Glu Ser Ala Pro Tyr 200 205 210 215
atg tgc cga gtg aag aca ctg cca gac cgg aca tgg acc tac tcc ttc 726
Met Cys Arg Val Lys Thr Leu Pro Asp Arg Thr Trp Thr Tyr Ser Phe 220
225 230 tcc gga gcc ttc ctg ttc tcc atg ggc ttc ctc gtc gca gta ctc
tgc 774 Ser Gly Ala Phe Leu Phe Ser Met Gly Phe Leu Val Ala Val Leu
Cys 235 240 245 tac ctg agc tac aga tat gtc acc aag ccg cct gca cct
ccc aac tcc 822 Tyr Leu Ser Tyr Arg Tyr Val Thr Lys Pro Pro Ala Pro
Pro Asn Ser 250 255 260 ctg aac gtc cag cga gtc ctg act ttc cag ccg
ctg cgc ttc atc cag 870 Leu Asn Val Gln Arg Val Leu Thr Phe Gln Pro
Leu Arg Phe Ile Gln 265 270 275 gag cac gtc ctg atc cct gtc ttt gac
ctc agc ggc ccc agc agt ctg 918 Glu His Val Leu Ile Pro Val Phe Asp
Leu Ser Gly Pro Ser Ser Leu 280 285 290 295 gcc cag cct gtc cag tac
tcc cag atc agg gtg tct gga ccc agg gag 966 Ala Gln Pro Val Gln Tyr
Ser Gln Ile Arg Val Ser Gly Pro Arg Glu 300 305 310 ccc gca gga gct
cca cag cgg cat agc ctg tcc gag atc acc tac tta 1014 Pro Ala Gly
Ala Pro Gln Arg His Ser Leu Ser Glu Ile Thr Tyr Leu 315 320 325 ggg
cag cca gac atc tcc atc ctc cag ccc tcc aac gtg cca cct ccc 1062
Gly Gln Pro Asp Ile Ser Ile Leu Gln Pro Ser Asn Val Pro Pro Pro 330
335 340 cag atc ctc tcc cca ctg tcc tat gcc cca aac gct gcc cct gag
gtc 1110 Gln Ile Leu Ser Pro Leu Ser Tyr Ala Pro Asn Ala Ala Pro
Glu Val 345 350 355 ggg ccc cca tcc tat gca cct cag gtg acc ccc gaa
gct caa ttc cca 1158 Gly Pro Pro Ser Tyr Ala Pro Gln Val Thr Pro
Glu Ala Gln Phe Pro 360 365 370 375 ttc tac gcc cca cag gcc atc tct
aag gtc cag cct tcc tcc tat gcc 1206 Phe Tyr Ala Pro Gln Ala Ile
Ser Lys Val Gln Pro Ser Ser Tyr Ala 380 385 390 cct caa gcc act ccg
gac agc tgg cct ccc tcc tat ggg gta tgc atg 1254 Pro Gln Ala Thr
Pro Asp Ser Trp Pro Pro Ser Tyr Gly Val Cys Met 395 400 405 gaa ggt
tct ggc aaa gac tcc ccc act ggg aca ctt tct agt cct aaa 1302 Glu
Gly Ser Gly Lys Asp Ser Pro Thr Gly Thr Leu Ser Ser Pro Lys 410 415
420 cac ctt agg cct aaa ggt cag ctt cag aaa gag cca cca gct gga agc
1350 His Leu Arg Pro Lys Gly Gln Leu Gln Lys Glu Pro Pro Ala Gly
Ser 425 430 435 tgc atg tta ggt ggc ctt tct ctg cag gag gtg acc tcc
ttg gct atg 1398 Cys Met Leu Gly Gly Leu Ser Leu Gln Glu Val Thr
Ser Leu Ala Met 440 445 450 455 gag gaa tcc caa gaa gca aaa tca ttg
cac cag ccc ctg ggg att tgc 1446 Glu Glu Ser Gln Glu Ala Lys Ser
Leu His Gln Pro Leu Gly Ile Cys 460 465 470 aca gac aga aca tct gac
cca aat gtg cta cac agt ggg gag gaa ggg 1494 Thr Asp Arg Thr Ser
Asp Pro Asn Val Leu His Ser Gly Glu Glu Gly 475 480 485 aca cca cag
tac cta aag ggc cag ctc ccc ctc ctc tcc tca gtc cag 1542 Thr Pro
Gln Tyr Leu Lys Gly Gln Leu Pro Leu Leu Ser Ser Val Gln 490 495 500
atc gag ggc cac ccc atg tcc ctc cct ttg caa cct cct tcc ggt cca
1590 Ile Glu Gly His Pro Met Ser Leu Pro Leu Gln Pro Pro Ser Gly
Pro 505 510 515 tgt tcc ccc tcg gac caa ggt cca agt ccc tgg ggc ctg
ctg gag tcc 1638 Cys Ser Pro Ser Asp Gln Gly Pro Ser Pro Trp Gly
Leu Leu Glu Ser 520 525 530 535 ctt gtg tgt ccc aag gat gaa gcc aag
agc cca gcc cct gag acc tca 1686 Leu Val Cys Pro Lys Asp Glu Ala
Lys Ser Pro Ala Pro Glu Thr Ser 540 545 550 gac ctg gag cag ccc aca
gaa ctg gat tct ctt ttc aga ggc ctg gcc 1734 Asp Leu Glu Gln Pro
Thr Glu Leu Asp Ser Leu Phe Arg Gly Leu Ala 555 560 565 ctg act gtg
cag tgg gag tcc tgaggggaat gggaaaggct tggtgcttcc 1785 Leu Thr Val
Gln Trp Glu Ser 570 tccctgtccc tacccagtgt cacatccttg gctgtcaatc
ccatgcctgc ccatgccaca 1845 cactctgcga tctggcctca gacgggtgcc
cttgagagaa gcagagggag tggcatgcag 1905 ggcccctgcc atgggtgcgc
tcctcaccgg aacaaagcag catgataagg actgcagcgg 1965 gggagctctg
gggagcagct tgtgtagaca agcgcgtgct cgctgagccc tgcaaggcag 2025
aaatgacagt gcaaggagga aatgcaggga aactcccgag gtccagagcc ccacctccta
2085 acaccatgga ttcaaagtgc tcagggaatt tgcctctcct tgccccattc
ctggccagtt 2145 tcacaatcta gctcgacaga gcatgaggcc cctgcctctt
ctgtcattgt tcaaaggtgg 2205 gaagagagcc tggaaaagaa ccaggcctgg
aaaagaacca gaaggaggct gggcagaacc 2265 agaacaacct gcacttctgc
caaggccagg gccagcagga cggcaggact ctagggaggg 2325 gtgtggcctg
cagctcattc ccagccaggg caactgcctg acgttgcacg atttcagctt 2385
cattcctctg atagaacaaa gcgaaatgca ggtccaccag ggagggagac acacaagcct
2445 tttctgcagg caggagtttc agaccctatc ctgagaatgg ggtttgaaag
gaaggtgagg 2505 gctgtggccc ctggacgggt acaataacac actgtactga
tgtcacaact ttgcaagctc 2565 tgccttgggt tcagcccatc tgggctcaaa
ttccagcctc accactcaca agctgtgtga 2625 cttcaaacaa atgaaatcag
tgcccagaac ctcggtttcc tcatctgtaa tgtggggatc 2685 ataacaccta
cctcatggag ttgtggtgaa gatgaaatga agtcatgtct ttaaagtgct 2745
taatagtgcc tggtacatgg gcagtgccca ataaacggta gctatttaaa aaaaaaaaaa
2805 aaaaaaaaaa atagcggccg cctcga 2831 <210> SEQ ID NO 2
<211> LENGTH: 574 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 2 Met Arg Thr Leu Leu Thr Ile
Leu Thr Val Gly Ser Leu Ala Ala His 1 5 10 15 Ala Pro Glu Asp Pro
Ser Asp Leu Leu Gln His Val Lys Phe Gln Ser 20 25 30 Ser Asn Phe
Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr 35 40 45 Pro
Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg Asp 50 55
60 Trp Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn
65 70 75 80 Leu Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala
Arg Val 85 90 95 Thr Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys
Met Thr Asp Arg 100 105 110 Phe Ser Ser Leu Gln His Thr Thr Leu Lys
Pro Pro Asp Val Thr Cys 115 120 125 Ile Ser Lys Val Arg Ser Ile Gln
Met Ile Val His Pro Thr Pro Thr 130 135 140 Pro Ile Arg Ala Gly Asp
Gly His Arg Leu Thr Leu Glu Asp Ile Phe 145 150 155 160 His Asp Leu
Phe Tyr His Leu Glu Leu Gln Val Asn Arg Thr Tyr Gln 165 170 175 Met
His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe Phe Gly Leu Thr 180 185
190 Pro Asp Thr Glu Phe Leu Gly Thr Ile Met Ile Cys Val Pro Thr Trp
195 200 205 Ala Lys Glu Ser Ala Pro Tyr Met Cys Arg Val Lys Thr Leu
Pro Asp 210 215 220 Arg Thr Trp Thr Tyr Ser Phe Ser Gly Ala Phe Leu
Phe Ser Met Gly 225 230 235 240 Phe Leu Val Ala Val Leu Cys Tyr Leu
Ser Tyr Arg Tyr Val Thr Lys 245 250 255 Pro Pro Ala Pro Pro Asn Ser
Leu Asn Val Gln Arg Val Leu Thr Phe 260 265 270 Gln Pro Leu Arg Phe
Ile Gln Glu His Val Leu Ile Pro Val Phe Asp 275 280 285 Leu Ser Gly
Pro Ser Ser Leu Ala Gln Pro Val Gln Tyr Ser Gln Ile 290 295 300 Arg
Val Ser Gly Pro Arg Glu Pro Ala Gly Ala Pro Gln Arg His Ser 305 310
315 320 Leu Ser Glu Ile Thr Tyr Leu Gly Gln Pro Asp Ile Ser Ile Leu
Gln 325 330 335 Pro Ser Asn Val Pro Pro Pro Gln Ile Leu Ser Pro Leu
Ser Tyr Ala 340 345 350 Pro Asn Ala Ala Pro Glu Val Gly Pro Pro Ser
Tyr Ala Pro Gln Val 355 360 365 Thr Pro Glu Ala Gln Phe Pro Phe Tyr
Ala Pro Gln Ala Ile Ser Lys 370 375 380 Val Gln Pro Ser Ser Tyr Ala
Pro Gln Ala Thr Pro Asp Ser Trp Pro 385 390 395 400 Pro Ser Tyr Gly
Val Cys Met Glu Gly Ser Gly Lys Asp Ser Pro Thr 405 410 415 Gly Thr
Leu Ser Ser Pro Lys His Leu Arg Pro Lys Gly Gln Leu Gln 420 425 430
Lys Glu Pro Pro Ala Gly Ser Cys Met Leu Gly Gly Leu Ser Leu Gln 435
440 445 Glu Val Thr Ser Leu Ala Met Glu Glu Ser Gln Glu Ala Lys Ser
Leu 450 455 460 His Gln Pro Leu Gly Ile Cys Thr Asp Arg Thr Ser Asp
Pro Asn Val 465 470 475 480 Leu His Ser Gly Glu Glu Gly Thr Pro Gln
Tyr Leu Lys Gly Gln Leu 485 490 495 Pro Leu Leu Ser Ser Val Gln Ile
Glu Gly His Pro Met Ser Leu Pro 500 505 510 Leu Gln Pro Pro Ser Gly
Pro Cys Ser Pro Ser Asp Gln Gly Pro Ser 515 520 525 Pro Trp Gly Leu
Leu Glu Ser Leu Val Cys Pro Lys Asp Glu Ala Lys 530 535 540 Ser Pro
Ala Pro Glu Thr Ser Asp Leu Glu Gln Pro Thr Glu Leu Asp 545 550 555
560 Ser Leu Phe Arg Gly Leu Ala Leu Thr Val Gln Trp Glu Ser 565 570
<210> SEQ ID NO 3 <211> LENGTH: 211 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3 Pro
Glu Asp Pro Ser Asp Leu Leu Gln His Val Lys Phe Gln Ser Ser 1 5 10
15 Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr Pro
20 25 30 Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg
Asp Trp 35 40 45 Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys
Ser Cys Asn Leu 50 55 60 Thr Val Glu Thr Gly Asn Leu Thr Glu Leu
Tyr Tyr Ala Arg Val Thr 65 70 75 80 Ala Val Ser Ala Gly Gly Arg Ser
Ala Thr Lys Met Thr Asp Arg Phe 85 90 95 Ser Ser Leu Gln His Thr
Thr Leu Lys Pro Pro Asp Val Thr Cys Ile 100 105 110 Ser Lys Val Arg
Ser Ile Gln Met Ile Val His Pro Thr Pro Thr Pro 115 120 125 Ile Arg
Ala Gly Asp Gly His Arg Leu Thr Leu Glu Asp Ile Phe His 130 135 140
Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn Arg Thr Tyr Gln Met 145
150 155 160 His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe Phe Gly Leu
Thr Pro 165 170 175 Asp Thr Glu Phe Leu Gly Thr Ile Met Ile Cys Val
Pro Thr Trp Ala 180 185 190 Lys Glu Ser Ala Pro Tyr Met Cys Arg Val
Lys Thr Leu Pro Asp Arg 195 200 205 Thr Trp Thr 210 <210> SEQ
ID NO 4 <211> LENGTH: 541 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: A Soluble IL-22RA-Fc Fusion Polypeptide
<400> SEQUENCE: 4 Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val
Lys Phe Gln Ser Ser 1 5 10 15 Asn Phe Glu Asn Ile Leu Thr Trp Asp
Ser Gly Pro Glu Gly Thr Pro 20 25 30 Asp Thr Val Tyr Ser Ile Glu
Tyr Lys Thr Tyr Gly Glu Arg Asp Trp 35 40 45 Val Ala Lys Lys Gly
Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn Leu 50 55 60 Thr Val Glu
Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala Arg Val Thr 65 70 75 80 Ala
Val Ser Ala Gly Gly Arg Ser Ala Thr Lys Met Thr Asp Arg Phe 85 90
95 Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro Asp Val Thr Cys Ile
100 105 110 Ser Lys Val Arg Ser Ile Gln Met Ile Val His Pro Thr Pro
Thr Pro 115 120 125 Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu Glu
Asp Ile Phe His 130 135 140 Asp Leu Phe Tyr His Leu Glu Leu Gln Val
Asn Arg Thr Tyr Gln Met 145 150 155 160 His Leu Gly Gly Lys Gln Arg
Glu Tyr Glu Phe Phe Gly Leu Thr Pro 165 170 175 Asp Thr Glu Phe Leu
Gly Thr Ile Met Ile Cys Val Pro Thr Trp Ala 180 185 190 Lys Glu Ser
Ala Pro Tyr Met Cys Arg Val Lys Thr Leu Pro Asp Arg 195 200 205 Thr
Trp Thr Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 210 215
220 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
225 230 235 240 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala 245 250 255 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly 260 265 270 Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly 275 280 285 Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys 290 295 300 Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 305 310 315 320 Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 325 330 335
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 340
345 350 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys 355 360 365 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys 370 375 380 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu 385 390 395 400 Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys 405 410 415 Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 420 425 430 Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 435 440 445 Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 450 455 460
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 465
470 475 480 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly 485 490 495 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln 500 505 510 Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn 515 520 525 His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 530 535 540 <210> SEQ ID NO 5 <211>
LENGTH: 1116 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (21)...(557) <400> SEQUENCE: 5 tcgagttaga
attgtctgca atg gcc gcc ctg cag aaa tct gtg agc tct ttc 53 Met Ala
Ala Leu Gln Lys Ser Val Ser Ser Phe 1 5 10 ctt atg ggg acc ctg gcc
acc agc tgc ctc ctt ctc ttg gcc ctc ttg 101 Leu Met Gly Thr Leu Ala
Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu 15 20 25 gta cag gga gga
gca gct gcg ccc atc agc tcc cac tgc agg ctt gac 149 Val Gln Gly Gly
Ala Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp 30 35 40 aag tcc
aac ttc cag cag ccc tat atc acc aac cgc acc ttc atg ctg 197 Lys Ser
Asn Phe Gln Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu 45 50 55
gct aag gag gct agc ttg gct gat aac aac aca gac gtt cgt ctc att 245
Ala Lys Glu Ala Ser Leu Ala Asp Asn Asn Thr Asp Val Arg Leu Ile 60
65 70 75 ggg gag aaa ctg ttc cac gga gtc agt atg agt gag cgc tgc
tat ctg 293 Gly Glu Lys Leu Phe His Gly Val Ser Met Ser Glu Arg Cys
Tyr Leu 80 85 90 atg aag cag gtg ctg aac ttc acc ctt gaa gaa gtg
ctg ttc cct caa 341 Met Lys Gln Val Leu Asn Phe Thr Leu Glu Glu Val
Leu Phe Pro Gln 95 100 105 tct gat agg ttc cag cct tat atg cag gag
gtg gtg ccc ttc ctg gcc 389 Ser Asp Arg Phe Gln Pro Tyr Met Gln Glu
Val Val Pro Phe Leu Ala 110 115 120 agg ctc agc aac agg cta agc aca
tgt cat att gaa ggt gat gac ctg 437 Arg Leu Ser Asn Arg Leu Ser Thr
Cys His Ile Glu Gly Asp Asp Leu 125 130 135 cat atc cag agg aat gtg
caa aag ctg aag gac aca gtg aaa aag ctt 485 His Ile Gln Arg Asn Val
Gln Lys Leu Lys Asp Thr Val Lys Lys Leu 140 145 150 155 gga gag agt
gga gag atc aaa gca att gga gaa ctg gat ttg ctg ttt 533 Gly Glu Ser
Gly Glu Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe 160 165 170 atg
tct ctg aga aat gcc tgc att tgaccagagc aaagctgaaa aatgaataac 587
Met Ser Leu Arg Asn Ala Cys Ile 175 taaccccctt tccctgctag
aaataacaat tagatgcccc aaagcgattt tttttaacca 647 aaaggaagat
gggaagccaa actccatcat gatgggtgga ttccaaatga acccctgcgt 707
tagttacaaa ggaaaccaat gccacttttg tttataagac cagaaggtag actttctaag
767 catagatatt tattgataac atttcattgt aactggtgtt ctatacacag
aaaacaattt 827 attttttaaa taattgtctt tttccataaa aaagattact
ttccattcct ttaggggaaa 887 aaacccctaa atagcttcat gtttccataa
tcagtacttt atatttataa atgtatttat 947 tattattata agactgcatt
ttatttatat cattttatta atatggattt atttatagaa 1007 acatcattcg
atattgctac ttgagtgtaa ggctaatatt gatatttatg acaataatta 1067
tagagctata acatgtttat ttgacctcaa taaacacttg gatatccta 1116
<210> SEQ ID NO 6 <211> LENGTH: 179 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 6 Met
Ala Ala Leu Gln Lys Ser Val Ser Ser Phe Leu Met Gly Thr Leu 1 5 10
15 Ala Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu Val Gln Gly Gly Ala
20 25 30 Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp Lys Ser Asn
Phe Gln 35 40 45 Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu Ala
Lys Glu Ala Ser 50 55 60 Leu Ala Asp Asn Asn Thr Asp Val Arg Leu
Ile Gly Glu Lys Leu Phe 65 70 75 80 His Gly Val Ser Met Ser Glu Arg
Cys Tyr Leu Met Lys Gln Val Leu 85 90 95 Asn Phe Thr Leu Glu Glu
Val Leu Phe Pro Gln Ser Asp Arg Phe Gln 100 105 110 Pro Tyr Met Gln
Glu Val Val Pro Phe Leu Ala Arg Leu Ser Asn Arg 115 120 125 Leu Ser
Thr Cys His Ile Glu Gly Asp Asp Leu His Ile Gln Arg Asn 130 135 140
Val Gln Lys Leu Lys Asp Thr Val Lys Lys Leu Gly Glu Ser Gly Glu 145
150 155 160 Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu
Arg Asn 165 170 175 Ala Cys Ile <210> SEQ ID NO 7 <211>
LENGTH: 926 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (45)...(575) <221> NAME/KEY: variation <222>
LOCATION: (188)...(188) <223> OTHER INFORMATION: Nucleotide
may be C or G at position 188 <400> SEQUENCE: 7 ctttgaattc
ctagctcctg tggtctccag atttcaggcc taag atg aaa gcc tct 56 Met Lys
Ala Ser 1 agt ctt gcc ttc agc ctt ctc tct gct gcg ttt tat ctc cta
tgg act 104 Ser Leu Ala Phe Ser Leu Leu Ser Ala Ala Phe Tyr Leu Leu
Trp Thr 5 10 15 20 cct tcc act gga ctg aag aca ctc aat ttg gga agc
tgt gtg atc gcc 152 Pro Ser Thr Gly Leu Lys Thr Leu Asn Leu Gly Ser
Cys Val Ile Ala 25 30 35 aca aac ctt cag gaa ata cga aat gga ttt
tct gas ata cgg ggc agt 200 Thr Asn Leu Gln Glu Ile Arg Asn Gly Phe
Ser Xaa Ile Arg Gly Ser 40 45 50 gtg caa gcc aaa gat gga aac att
gac atc aga atc tta agg agg act 248 Val Gln Ala Lys Asp Gly Asn Ile
Asp Ile Arg Ile Leu Arg Arg Thr 55 60 65 gag tct ttg caa gac aca
aag cct gcg aat cga tgc tgc ctc ctg cgc 296 Glu Ser Leu Gln Asp Thr
Lys Pro Ala Asn Arg Cys Cys Leu Leu Arg 70 75 80 cat ttg cta aga
ctc tat ctg gac agg gta ttt aaa aac tac cag acc 344 His Leu Leu Arg
Leu Tyr Leu Asp Arg Val Phe Lys Asn Tyr Gln Thr 85 90 95 100 cct
gac cat tat act ctc cgg aag atc agc agc ctc gcc aat tcc ttt 392 Pro
Asp His Tyr Thr Leu Arg Lys Ile Ser Ser Leu Ala Asn Ser Phe 105 110
115 ctt acc atc aag aag gac ctc cgg ctc tgt cat gcc cac atg aca tgc
440 Leu Thr Ile Lys Lys Asp Leu Arg Leu Cys His Ala His Met Thr Cys
120 125 130 cat tgt ggg gag gaa gca atg aag aaa tac agc cag att ctg
agt cac 488 His Cys Gly Glu Glu Ala Met Lys Lys Tyr Ser Gln Ile Leu
Ser His 135 140 145 ttt gaa aag ctg gaa cct cag gca gca gtt gtg aag
gct ttg ggg gaa 536 Phe Glu Lys Leu Glu Pro Gln Ala Ala Val Val Lys
Ala Leu Gly Glu 150 155 160 cta gac att ctt ctg caa tgg atg gag gag
aca gaa tag gaggaaagtg 585 Leu Asp Ile Leu Leu Gln Trp Met Glu Glu
Thr Glu * 165 170 175 atgctgctgc taagaatatt cgaggtcaag agctccagtc
ttcaatacct gcagaggagg 645 catgacccca aaccaccatc tctttactgt
actagtcttg tgctggtcac agtgtatctt 705 atttatgcat tacttgcttc
cttgcatgat tgtctttatg catccccaat cttaattgag 765 accatacttg
tataagattt ttgtaatatc tttctgctat tggatatatt tattagttaa 825
tatatttatt tattttttgc tattaatgta tttaattttt tacttgggca tgaaacttta
885 aaaaaaattc acaagattat atttataacc tgactagagc a 926 <210>
SEQ ID NO 8 <211> LENGTH: 176 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: VARIANT <222> LOCATION: (48)...(48) <223>
OTHER INFORMATION: Amino acid at position 48 can be a D (Asp) or E
(Glu) <221> NAME/KEY: VARIANT <222> LOCATION: 48
<223> OTHER INFORMATION: Xaa = Any Amino Acid <400>
SEQUENCE: 8 Met Lys Ala Ser Ser Leu Ala Phe Ser Leu Leu Ser Ala Ala
Phe Tyr 1 5 10 15 Leu Leu Trp Thr Pro Ser Thr Gly Leu Lys Thr Leu
Asn Leu Gly Ser 20 25 30 Cys Val Ile Ala Thr Asn Leu Gln Glu Ile
Arg Asn Gly Phe Ser Xaa 35 40 45 Ile Arg Gly Ser Val Gln Ala Lys
Asp Gly Asn Ile Asp Ile Arg Ile 50 55 60 Leu Arg Arg Thr Glu Ser
Leu Gln Asp Thr Lys Pro Ala Asn Arg Cys 65 70 75 80 Cys Leu Leu Arg
His Leu Leu Arg Leu Tyr Leu Asp Arg Val Phe Lys 85 90 95 Asn Tyr
Gln Thr Pro Asp His Tyr Thr Leu Arg Lys Ile Ser Ser Leu 100 105 110
Ala Asn Ser Phe Leu Thr Ile Lys Lys Asp Leu Arg Leu Cys His Ala 115
120 125 His Met Thr Cys His Cys Gly Glu Glu Ala Met Lys Lys Tyr Ser
Gln 130 135 140 Ile Leu Ser His Phe Glu Lys Leu Glu Pro Gln Ala Ala
Val Val Lys 145 150 155 160 Ala Leu Gly Glu Leu Asp Ile Leu Leu Gln
Trp Met Glu Glu Thr Glu 165 170 175 <210> SEQ ID NO 9
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Peptide Linker <400> SEQUENCE: 9 Gly Gly Ser Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
<210> SEQ ID NO 10 <211> LENGTH: 1050 <212> TYPE:
DNA <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (5)...(589)
<400> SEQUENCE: 10 aaca ggc tct cct ctc act tat caa ctt ttg
aca ctt gtg cga tcg gtg 49 Gly Ser Pro Leu Thr Tyr Gln Leu Leu Thr
Leu Val Arg Ser Val 1 5 10 15 atg gct gtc ctg cag aaa tct atg agt
ttt tcc ctt atg ggg act ttg 97 Met Ala Val Leu Gln Lys Ser Met Ser
Phe Ser Leu Met Gly Thr Leu 20 25 30 gcc gcc agc tgc ctg ctt ctc
att gcc ctg tgg gcc cag gag gca aat 145 Ala Ala Ser Cys Leu Leu Leu
Ile Ala Leu Trp Ala Gln Glu Ala Asn 35 40 45 gcg ctg ccc atc aac
acc cgg tgc aag ctt gag gtg tcc aac ttc cag 193 Ala Leu Pro Ile Asn
Thr Arg Cys Lys Leu Glu Val Ser Asn Phe Gln 50 55 60 cag ccg tac
atc gtc aac cgc acc ttt atg ctg gcc aag gag gcc agc 241 Gln Pro Tyr
Ile Val Asn Arg Thr Phe Met Leu Ala Lys Glu Ala Ser 65 70 75 ctt
gca gat aac aac aca gac gtc cgg ctc atc ggg gag aaa ctg ttc 289 Leu
Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys Leu Phe 80 85
90 95 cga gga gtc agt gct aag gat cag tgc tac ctg atg aag cag gtg
ctc 337 Arg Gly Val Ser Ala Lys Asp Gln Cys Tyr Leu Met Lys Gln Val
Leu 100 105 110 aac ttc acc ctg gaa gac att ctg ctc ccc cag tca gac
agg ttc cgg 385 Asn Phe Thr Leu Glu Asp Ile Leu Leu Pro Gln Ser Asp
Arg Phe Arg 115 120 125 ccc tac atg cag gag gtg gtg cct ttc ctg acc
aaa ctc agc aat cag 433 Pro Tyr Met Gln Glu Val Val Pro Phe Leu Thr
Lys Leu Ser Asn Gln 130 135 140 ctc agc tcc tgt cac atc agt ggt gac
gac cag aac atc cag aag aat 481 Leu Ser Ser Cys His Ile Ser Gly Asp
Asp Gln Asn Ile Gln Lys Asn 145 150 155 gtc aga agg ctg aag gag aca
gtg aaa aag ctt gga gag agc gga gag 529 Val Arg Arg Leu Lys Glu Thr
Val Lys Lys Leu Gly Glu Ser Gly Glu 160 165 170 175 atc aaa gcg atc
ggg gaa ctg gac ctg ctg ttt atg tct ctg aga aat 577 Ile Lys Ala Ile
Gly Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn 180 185 190 gct tgc
gtc tga gcgagaagaa gctagaaaac gaagaactgc tccttcctgc 629 Ala Cys Val
* cttctaaaaa gaacaataag atccctgaat ggactttttt actaaaggaa agtgagaagc
689 taacgtccac catcattaga agatttcaca tgaaacctgg ctcagttgaa
agagaaaata 749 gtgtcaagtt gtccatgaga ccagaggtag acttgataac
cacaaagatt cattgacaat 809 attttattgt cattgataat gcaacagaaa
aagtatgtac tttaaaaaat tgtttgaaag 869 gaggttacct ctcattcctc
tagaagaaaa gcctatgtaa cttcatttcc ataaccaata 929 ctttatatat
gtaagtttat ttattataag tatacatttt atttatgtca gtttattaat 989
atggatttat ttatagaaaa attatctgat gttgatattt gagtataaag caaataatat
1049 t 1050 <210> SEQ ID NO 11 <211> LENGTH: 194
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 11 Gly Ser Pro Leu Thr Tyr Gln Leu Leu Thr
Leu Val Arg Ser Val Met 1 5 10 15 Ala Val Leu Gln Lys Ser Met Ser
Phe Ser Leu Met Gly Thr Leu Ala 20 25 30 Ala Ser Cys Leu Leu Leu
Ile Ala Leu Trp Ala Gln Glu Ala Asn Ala 35 40 45 Leu Pro Ile Asn
Thr Arg Cys Lys Leu Glu Val Ser Asn Phe Gln Gln 50 55 60 Pro Tyr
Ile Val Asn Arg Thr Phe Met Leu Ala Lys Glu Ala Ser Leu 65 70 75 80
Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys Leu Phe Arg 85
90 95 Gly Val Ser Ala Lys Asp Gln Cys Tyr Leu Met Lys Gln Val Leu
Asn 100 105 110 Phe Thr Leu Glu Asp Ile Leu Leu Pro Gln Ser Asp Arg
Phe Arg Pro 115 120 125 Tyr Met Gln Glu Val Val Pro Phe Leu Thr Lys
Leu Ser Asn Gln Leu 130 135 140 Ser Ser Cys His Ile Ser Gly Asp Asp
Gln Asn Ile Gln Lys Asn Val 145 150 155 160 Arg Arg Leu Lys Glu Thr
Val Lys Lys Leu Gly Glu Ser Gly Glu Ile 165 170 175 Lys Ala Ile Gly
Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn Ala 180 185 190 Cys Val
<210> SEQ ID NO 12 <211> LENGTH: 2149 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)...(693)
<400> SEQUENCE: 12 atg atg cct aaa cat tgc ttt cta ggc ttc
ctc atc agt ttc ttc ctt 48 Met Met Pro Lys His Cys Phe Leu Gly Phe
Leu Ile Ser Phe Phe Leu 1 5 10 15 act ggt gta gca gga act cag tca
acg cat gag tct ctg aag cct cag 96 Thr Gly Val Ala Gly Thr Gln Ser
Thr His Glu Ser Leu Lys Pro Gln 20 25 30 agg gta caa ttt cag tcc
cga aat ttt cac aac att ttg caa tgg cag 144 Arg Val Gln Phe Gln Ser
Arg Asn Phe His Asn Ile Leu Gln Trp Gln 35 40 45 cct ggg agg gca
ctt act ggc aac agc agt gtc tat ttt gtg cag tac 192 Pro Gly Arg Ala
Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr 50 55 60 aaa ata
tat gga cag aga caa tgg aaa aat aaa gaa gac tgt tgg ggt 240 Lys Ile
Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly 65 70 75 80
act caa gaa ctc tct tgt gac ctt acc agt gaa acc tca gac ata cag 288
Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln 85
90 95 gaa cct tat tac ggg agg gtg agg gcg gcc tcg gct ggg agc tac
tca 336 Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr
Ser 100 105 110 gaa tgg agc atg acg ccg cgg ttc act ccc tgg tgg gaa
aca aaa ata 384 Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu
Thr Lys Ile 115 120 125 gat cct cca gtc atg aat ata acc caa gtc aat
ggc tct ttg ttg gta 432 Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn
Gly Ser Leu Leu Val 130 135 140 att ctc cat gct cca aat tta cca tat
aga tac caa aag gaa aaa aat 480 Ile Leu His Ala Pro Asn Leu Pro Tyr
Arg Tyr Gln Lys Glu Lys Asn 145 150 155 160 gta tct ata gaa gat tac
tat gaa cta cta tac cga gtt ttt ata att 528 Val Ser Ile Glu Asp Tyr
Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile 165 170 175 aac aat tca cta
gaa aag gag caa aag gtt tat gaa ggg gct cac aga 576 Asn Asn Ser Leu
Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg 180 185 190 gcg gtt
gaa att gaa gct cta aca cca cac tcc agc tac tgt gta gtg 624 Ala Val
Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val 195 200 205
gct gaa ata tat cag ccc atg tta gac aga aga agt cag aga agt gaa 672
Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser Glu 210
215 220 gag aga tgt gtg gaa att cca tgacttgtgg aatttggcat
tcagcaatgt 723 Glu Arg Cys Val Glu Ile Pro 225 230 ggaaattcta
aagctccctg agaacaggat gactcgtgtt tgaaggatct tatttaaaat 783
tgtttttgta ttttcttaaa gcaatattca ctgttacacc ttggggactt ctttgtttat
843 ccattctttt atcctttata tttcatttta aactatattt gaacgacatt
ccccccgaaa 903 aattgaaatg taaagatgag gcagagaata aagtgttcta
tgaaattcag aactttattt 963 ctgaatgtaa catccctaat aacaaccttc
attcttctaa tacagcaaaa taaaaattta 1023 acaaccaagg aatagtattt
aagaaaatgt tgaaataatt tttttaaaat agcattacag 1083 actgaggcgg
tcctgaagca atggtttttc actctcttat tgagccaatt aaattgacat 1143
tgctttgaca atttaaaact tctataaagg tgaatatttt tcatacattt ctattttata
1203 tgaatatact ttttatatat ttattattat taaatatttc tacttaatga
atcaaaattt 1263 tgttttaaag tctactttat gtaaataaga acaggttttg
gggaaaaaaa tcttatgatt 1323 tctggattga tatctgaatt aaaactatca
acaacaagga agtctactct gtacaattgt 1383 ccctcattta aaagatatat
taagcttttc ttttctgttt gtttttgttt tgtttagttt 1443 ttaatcctgt
cttagaagaa cttatcttta ttctcaaaat taaatgtaat ttttttagtg 1503
acaaagaaga aaggaaacct cattactcaa tccttctggc caagagtgtc ttgcttgtgg
1563 cgccttcctc atctctatat aggaggatcc catgaatgat ggtttattgg
gaactgctgg 1623 ggtcgacccc atacagagaa ctcagcttga agctggaagc
acacagtggg tagcaggaga 1683 aggaccggtg ttggtaggtg cctacagaga
ctatagagct agacaaagcc ctccaaactg 1743 gcccctcctg ctcactgcct
ctcctgagta gaaatctggt gacctaaggc tcagtgcggt 1803 caacagaaag
ctgccttctt cacttgaggc taagtcttca tatatgttta aggttgtctt 1863
tctagtgagg agatacatat cagagaacat ttgtacaatt ccccatgaaa attgctccaa
1923 agttgataac aatatagtcg gtgcttctag ttatatgcaa gtactcagtg
ataaatggat 1983 taaaaaatat tcagaaatgt attggggggt ggaggagaat
aagaggcaga gcaagagcta 2043 gagaattggt ttccttgctt ccctgtatgc
tcagaaaaca ttgatttgag catagacgca 2103 gagactgaaa aaaaaaaaat
gctcgagcgg ccgccatatc cttggt 2149 <210> SEQ ID NO 13
<211> LENGTH: 231 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 13 Met Met Pro Lys His Cys Phe
Leu Gly Phe Leu Ile Ser Phe Phe Leu 1 5 10 15 Thr Gly Val Ala Gly
Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln 20 25 30 Arg Val Gln
Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln 35 40 45 Pro
Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr 50 55
60 Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly
65 70 75 80 Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp
Ile Gln 85 90 95 Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala
Gly Ser Tyr Ser 100 105 110 Glu Trp Ser Met Thr Pro Arg Phe Thr Pro
Trp Trp Glu Thr Lys Ile 115 120 125 Asp Pro Pro Val Met Asn Ile Thr
Gln Val Asn Gly Ser Leu Leu Val 130 135 140 Ile Leu His Ala Pro Asn
Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn 145 150 155 160 Val Ser Ile
Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile 165 170 175 Asn
Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg 180 185
190 Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val
195 200 205 Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg
Ser Glu 210 215 220 Glu Arg Cys Val Glu Ile Pro 225 230 <210>
SEQ ID NO 14 <211> LENGTH: 699 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: C-Terminal Fc4 tag <400>
SEQUENCE: 14 gagcccagat cttcagacaa aactcacaca tgcccaccgt gcccagcacc
tgaagccgag 60 ggggcaccgt cagtcttcct cttcccccca aaacccaagg
acaccctcat gatctcccgg 120 acccctgagg tcacatgcgt ggtggtggac
gtgagccacg aagaccctga ggtcaagttc 180 aactggtacg tggacggcgt
ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240 tacaacagca
cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300
ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc catcctccat cgagaaaacc
360 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc
cccatcccgg 420 gatgagctga ccaagaacca ggtcagcctg acctgcctgg
tcaaaggctt ctatcccagc 480 gacatcgccg tggagtggga gagcaatggg
cagccggaga acaactacaa gaccacgcct 540 cccgtgctgg actccgacgg
ctccttcttc ctctacagca agctcaccgt ggacaagagc 600 aggtggcagc
aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660
tacacgcaga agagcctctc cctgtctccg ggtaaataa 699 <210> SEQ ID
NO 15 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Glu-Glu (CEE) Peptide Tag <400> SEQUENCE:
15 Glu Tyr Met Pro Met Glu 1 5 <210> SEQ ID NO 16 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Glu-Glu (CEE) Peptide Tag with spacer <400> SEQUENCE: 16 Gly
Ser Gly Gly Glu Tyr Met Pro Met Glu 1 5 10 <210> SEQ ID NO 17
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Oligonucleotide primer ZC39289 <400> SEQUENCE:
17 tccgaggagt caatgctaag 20 <210> SEQ ID NO 18 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Oligonucleotide Primer ZC39290 <400> SEQUENCE: 18 tccaagcttt
ttcactgtct 20 <210> SEQ ID NO 19 <211> LENGTH: 16
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide
Primer ZC39776 <400> SEQUENCE: 19 gggcccgcta gcacct 16
<210> SEQ ID NO 20 <211> LENGTH: 16 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide Primer ZC39777
<400> SEQUENCE: 20 gggtgatccg ctggca 16 <210> SEQ ID NO
21 <211> LENGTH: 36 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: IL-20 FAM/TAMRA labeled TaqMan probe ZC38752
<400> SEQUENCE: 21 ccagccactt tctctctccg tatttcttat attcca 36
<210> SEQ ID NO 22 <211> LENGTH: 16 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: forward primer, ZC42459 <400>
SEQUENCE: 22 tggccaggct cagcaa 16 <210> SEQ ID NO 23
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: reverse primer, ZC42458 <400> SEQUENCE: 23
gcacattcct ctggatatgc a 21 <210> SEQ ID NO 24 <211>
LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: IL-22
TaqMan probe, ZC42460 <400> SEQUENCE: 24 aggctaagca
catgtcatat tgaaggtgat g 31 <210> SEQ ID NO 25 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
forward primer, ZC40541 <400> SEQUENCE: 25 tcgccaattc
ctttcttacc a 21 <210> SEQ ID NO 26 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: reverse primer,
ZC40542 <400> SEQUENCE: 26 cccacaatgg catgtcatgt 20
<210> SEQ ID NO 27 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: IL-20 TaqMan? probe ZC40544
<400> SEQUENCE: 27 agaaggacct ccggctctgt catgc 25 <210>
SEQ ID NO 28 <211> LENGTH: 57 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide primer ZC45,593
<400> SEQUENCE: 28 caggaaatcc atgccgagtt gagacgcttc
cgtagacacg cccctgagga cccctcg 57 <210> SEQ ID NO 29
<211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Oligonucleotide primer ZC45,592 <400> SEQUENCE:
29 tctgggctca ccgcttccag acccgcttcc agacccgctt cctgtccggt
ctggcagtgt 60 ctt 63 <210> SEQ ID NO 30 <211> LENGTH:
63 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide
primer ZC45,591 <400> SEQUENCE: 30 gaccggacag gaagcgggtc
tggaagcggg tctggaagcg gtgagcccag aggccccaca 60 atc 63 <210>
SEQ ID NO 31 <211> LENGTH: 57 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide primer ZC45,594
<400> SEQUENCE: 31 agagctgttt taaggcgcgc ctctagatta
tttttattta cccggagtcc gggagaa 57 <210> SEQ ID NO 32
<211> LENGTH: 531 <212> TYPE: DNA <213> ORGANISM:
Mus musculus <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (1)...(531) <400> SEQUENCE: 32 atg aaa
ggc ttt ggt ctt gcc ttt gga ctg ttc tcc gct gtg ggt ttt 48 Met Lys
Gly Phe Gly Leu Ala Phe Gly Leu Phe Ser Ala Val Gly Phe 1 5 10 15
ctt ctc tgg act cct tta act ggg ctc aag acc ctc cat ttg gga agc 96
Leu Leu Trp Thr Pro Leu Thr Gly Leu Lys Thr Leu His Leu Gly Ser 20
25 30 tgt gtg att act gca aac cta cag gca ata caa aag gaa ttt tct
gag 144 Cys Val Ile Thr Ala Asn Leu Gln Ala Ile Gln Lys Glu Phe Ser
Glu 35 40 45 att cgg gat agt gtg caa gct gaa gat aca aat att gac
atc aga att 192 Ile Arg Asp Ser Val Gln Ala Glu Asp Thr Asn Ile Asp
Ile Arg Ile 50 55 60 tta agg acg act gag tct ttg aaa gac ata aag
tct ttg gat agg tgc 240 Leu Arg Thr Thr Glu Ser Leu Lys Asp Ile Lys
Ser Leu Asp Arg Cys 65 70 75 80 tgc ttc ctt cgt cat cta gtg aga ttc
tat ctg gac agg gta ttc aaa 288 Cys Phe Leu Arg His Leu Val Arg Phe
Tyr Leu Asp Arg Val Phe Lys 85 90 95 gtc tac cag acc cct gac cac
cat acc ctg aga aag atc agc agc ctc 336 Val Tyr Gln Thr Pro Asp His
His Thr Leu Arg Lys Ile Ser Ser Leu 100 105 110 gcc aac tcc ttt ctt
atc atc aag aag gac ctc tca gtc tgt cat tct 384 Ala Asn Ser Phe Leu
Ile Ile Lys Lys Asp Leu Ser Val Cys His Ser 115 120 125 cac atg gca
tgt cat tgt ggg gaa gaa gca atg gag aaa tac aac caa 432 His Met Ala
Cys His Cys Gly Glu Glu Ala Met Glu Lys Tyr Asn Gln 130 135 140 att
ctg agt cac ttc ata gag ttg gaa ctt cag gca gcg gtg gta aag 480 Ile
Leu Ser His Phe Ile Glu Leu Glu Leu Gln Ala Ala Val Val Lys 145 150
155 160 gct ttg gga gaa cta ggc att ctt ctg aga tgg atg gag gag atg
cta 528 Ala Leu Gly Glu Leu Gly Ile Leu Leu Arg Trp Met Glu Glu Met
Leu 165 170 175 tag 531 * <210> SEQ ID NO 33 <211>
LENGTH: 176 <212> TYPE: PRT <213> ORGANISM: Mus
musculus <400> SEQUENCE: 33 Met Lys Gly Phe Gly Leu Ala Phe
Gly Leu Phe Ser Ala Val Gly Phe 1 5 10 15 Leu Leu Trp Thr Pro Leu
Thr Gly Leu Lys Thr Leu His Leu Gly Ser 20 25 30 Cys Val Ile Thr
Ala Asn Leu Gln Ala Ile Gln Lys Glu Phe Ser Glu 35 40 45 Ile Arg
Asp Ser Val Gln Ala Glu Asp Thr Asn Ile Asp Ile Arg Ile 50 55 60
Leu Arg Thr Thr Glu Ser Leu Lys Asp Ile Lys Ser Leu Asp Arg Cys 65
70 75 80 Cys Phe Leu Arg His Leu Val Arg Phe Tyr Leu Asp Arg Val
Phe Lys 85 90 95 Val Tyr Gln Thr Pro Asp His His Thr Leu Arg Lys
Ile Ser Ser Leu 100 105 110 Ala Asn Ser Phe Leu Ile Ile Lys Lys Asp
Leu Ser Val Cys His Ser 115 120 125 His Met Ala Cys His Cys Gly Glu
Glu Ala Met Glu Lys Tyr Asn Gln 130 135 140 Ile Leu Ser His Phe Ile
Glu Leu Glu Leu Gln Ala Ala Val Val Lys 145 150 155 160 Ala Leu Gly
Glu Leu Gly Ile Leu Leu Arg Trp Met Glu Glu Met Leu 165 170 175
<210> SEQ ID NO 34 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide primer ZC22901
<400> SEQUENCE: 34 catcaaaccg cctgatgtga c 21 <210> SEQ
ID NO 35 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Oligonucleotide primer ZC45039 <400>
SEQUENCE: 35 attaggcttg ggagggaatg g 21 <210> SEQ ID NO 36
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Oligonucleotide primer ZC38573 <400> SEQUENCE:
36 tggcgatgcc tgcttgccga ata 23 <210> SEQ ID NO 37
<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Oligonucleotide primer ZC25223 <400> SEQUENCE:
37 gtcttcctca catctgttat cg 22 <210> SEQ ID NO 38 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Oligonucleotide primer ZC40128 <400> SEQUENCE: 38 ggcttgaact
ttgagaaagg cagt 24 <210> SEQ ID NO 39 <211> LENGTH:
1473 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
IL-22RA Extracellular domain with tPA leader and fused to murine
gamma 2a heavy chain Fc region (mG2a) <221> NAME/KEY: CDS
<222> LOCATION: (1)...(1473) <400> SEQUENCE: 39 atg gat
gca atg aag aga ggg ctc tgc tgt gtg ctg ctg ctg tgt ggc 48 Met Asp
Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15
gcc gtc ttc gtt tcg ctc agc cag gaa atc cat gcc gag ttg aga cgc 96
Ala Val Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu Leu Arg Arg 20
25 30 ttc cgt aga cac gcc cct gag gac ccc tcg gat ctg ctc cag cac
gtg 144 Phe Arg Arg His Ala Pro Glu Asp Pro Ser Asp Leu Leu Gln His
Val 35 40 45 aaa ttc cag tcc agc aac ttt gaa aac atc ctg acg tgg
gac agc ggg 192 Lys Phe Gln Ser Ser Asn Phe Glu Asn Ile Leu Thr Trp
Asp Ser Gly 50 55 60 cca gag ggc acc cca gac acg gtc tac agc atc
gag tat aag acg tac 240 Pro Glu Gly Thr Pro Asp Thr Val Tyr Ser Ile
Glu Tyr Lys Thr Tyr 65 70 75 80 gga gag agg gac tgg gtg gca aag aag
ggc tgt cag cgg atc acc cgg 288 Gly Glu Arg Asp Trp Val Ala Lys Lys
Gly Cys Gln Arg Ile Thr Arg 85 90 95 aag tcc tgc aac ctg acg gtg
gag acg ggc aac ctc acg gag ctc tac 336 Lys Ser Cys Asn Leu Thr Val
Glu Thr Gly Asn Leu Thr Glu Leu Tyr 100 105 110 tat gcc agg gtc acc
gct gtc agt gcg gga ggc cgg tca gcc acc aag 384 Tyr Ala Arg Val Thr
Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys 115 120 125 atg act gac
agg ttc agc tct ctg cag cac act acc ctc aag cca cct 432 Met Thr Asp
Arg Phe Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro 130 135 140 gat
gtg acc tgt atc tcc aaa gtg aga tcg att cag atg att gtt cat 480 Asp
Val Thr Cys Ile Ser Lys Val Arg Ser Ile Gln Met Ile Val His 145 150
155 160 cct acc ccc acg cca atc cgt gca ggc gat ggc cac cgg cta acc
ctg 528 Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr
Leu 165 170 175 gaa gac atc ttc cat gac ctg ttc tac cac tta gag ctc
cag gtc aac 576 Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu Glu Leu
Gln Val Asn 180 185 190 cgc acc tac caa atg cac ctt gga ggg aag cag
aga gaa tat gag ttc 624 Arg Thr Tyr Gln Met His Leu Gly Gly Lys Gln
Arg Glu Tyr Glu Phe 195 200 205 ttc ggc ctg acc cct gac aca gag ttc
ctt ggc acc atc atg att tgc 672 Phe Gly Leu Thr Pro Asp Thr Glu Phe
Leu Gly Thr Ile Met Ile Cys 210 215 220 gtt ccc acc tgg gcc aag gag
agt gcc ccc tac atg tgc cga gtg aag 720 Val Pro Thr Trp Ala Lys Glu
Ser Ala Pro Tyr Met Cys Arg Val Lys 225 230 235 240 aca ctg cca gac
cgg aca gga agc ggg tct gga agc ggg tct gga agc 768 Thr Leu Pro Asp
Arg Thr Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 245 250 255 ggt gag
ccc aga ggc ccc aca atc aag ccc tgt cct cca tgc aaa tgc 816 Gly Glu
Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys 260 265 270
cca gca cct aac ctc ttg ggt gga cca tcc gtc ttc atc ttc cct cca 864
Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 275
280 285 aag atc aag gat gta ctc atg atc tcc ctg agc ccc ata gtc aca
tgt 912 Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr
Cys 290 295 300 gtg gtg gtg gat gtg agc gag gat gac cca gat gtc cag
atc agc tgg 960 Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln
Ile Ser Trp 305 310 315 320 ttt gtg aac aac gtg gaa gta cac aca gct
cag aca caa acc cat aga 1008 Phe Val Asn Asn Val Glu Val His Thr
Ala Gln Thr Gln Thr His Arg 325 330 335 gag gat tac aac agt act ctc
cgg gtg gtc agt gcc ctc ccc atc cag 1056 Glu Asp Tyr Asn Ser Thr
Leu Arg Val Val Ser Ala Leu Pro Ile Gln 340 345 350 cac cag gac tgg
atg agt ggc aag gag ttc aaa tgc aag gtc aac aac 1104 His Gln Asp
Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn 355 360 365 aaa
gac ctc cca gcg ccc atc gag aga acc atc tca aaa ccc aaa ggg 1152
Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly 370
375 380 tca gta aga gct cca cag gta tat gtc ttg cct cca cca gaa gaa
gag 1200 Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu
Glu Glu 385 390 395 400 atg act aag aaa cag gtc act ctg acc tgc atg
gtc aca gac ttc atg 1248 Met Thr Lys Lys Gln Val Thr Leu Thr Cys
Met Val Thr Asp Phe Met 405 410 415 cct gaa gac att tac gtg gag tgg
acc aac aac ggg aaa aca gag cta 1296 Pro Glu Asp Ile Tyr Val Glu
Trp Thr Asn Asn Gly Lys Thr Glu Leu 420 425 430 aac tac aag aac act
gaa cca gtc ctg gac tct gat ggt tct tac ttc 1344 Asn Tyr Lys Asn
Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe 435 440 445 atg tac
agc aag ctg aga gtg gaa aag aag aac tgg gtg gaa aga aat 1392 Met
Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn 450 455
460 agc tac tcc tgt tca gtg gtc cac gag ggt ctg cac aat cac cac acg
1440 Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His
Thr 465 470 475 480 act aag agc ttc tcc cgg act ccg ggt aaa taa
1473 Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys * 485 490 <210>
SEQ ID NO 40 <211> LENGTH: 490 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: IL-22RA Extracellular domain with
tPA leader and fused to murine gamma 2a heavy chain Fc region
(mG2a) <400> SEQUENCE: 40 Met Asp Ala Met Lys Arg Gly Leu Cys
Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Leu Ser
Gln Glu Ile His Ala Glu Leu Arg Arg 20 25 30 Phe Arg Arg His Ala
Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val 35 40 45 Lys Phe Gln
Ser Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly 50 55 60 Pro
Glu Gly Thr Pro Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr 65 70
75 80 Gly Glu Arg Asp Trp Val Ala Lys Lys Gly Cys Gln Arg Ile Thr
Arg 85 90 95 Lys Ser Cys Asn Leu Thr Val Glu Thr Gly Asn Leu Thr
Glu Leu Tyr 100 105 110 Tyr Ala Arg Val Thr Ala Val Ser Ala Gly Gly
Arg Ser Ala Thr Lys 115 120 125 Met Thr Asp Arg Phe Ser Ser Leu Gln
His Thr Thr Leu Lys Pro Pro 130 135 140 Asp Val Thr Cys Ile Ser Lys
Val Arg Ser Ile Gln Met Ile Val His 145 150 155 160 Pro Thr Pro Thr
Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu 165 170 175 Glu Asp
Ile Phe His Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn 180 185 190
Arg Thr Tyr Gln Met His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe 195
200 205 Phe Gly Leu Thr Pro Asp Thr Glu Phe Leu Gly Thr Ile Met Ile
Cys 210 215 220 Val Pro Thr Trp Ala Lys Glu Ser Ala Pro Tyr Met Cys
Arg Val Lys 225 230 235 240 Thr Leu Pro Asp Arg Thr Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser 245 250 255 Gly Glu Pro Arg Gly Pro Thr Ile
Lys Pro Cys Pro Pro Cys Lys Cys 260 265 270 Pro Ala Pro Asn Leu Leu
Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 275 280 285 Lys Ile Lys Asp
Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys 290 295 300 Val Val
Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp 305 310 315
320 Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
325 330 335 Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro
Ile Gln 340 345 350 His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys
Lys Val Asn Asn 355 360 365 Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr
Ile Ser Lys Pro Lys Gly 370 375 380 Ser Val Arg Ala Pro Gln Val Tyr
Val Leu Pro Pro Pro Glu Glu Glu 385 390 395 400 Met Thr Lys Lys Gln
Val Thr Leu Thr Cys Met Val Thr Asp Phe Met 405 410 415 Pro Glu Asp
Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu 420 425 430 Asn
Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe 435 440
445 Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
450 455 460 Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His
His Thr 465 470 475 480 Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 485
490 <210> SEQ ID NO 41 <211> LENGTH: 1834 <212>
TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (43)...(1788)
<400> SEQUENCE: 41 ttggtccaga gccgaggccc gaaggggccc
tggagggacc ca atg aag aca cta 54 Met Lys Thr Leu 1 ctg acc atc ctg
acg gtg gga tcc ctg gcc gct cac acc act gtg gac 102 Leu Thr Ile Leu
Thr Val Gly Ser Leu Ala Ala His Thr Thr Val Asp 5 10 15 20 aca tcc
ggt ctc ctt caa cac gtg aaa ttc cag tcc agc aac ttt gag 150 Thr Ser
Gly Leu Leu Gln His Val Lys Phe Gln Ser Ser Asn Phe Glu 25 30 35
aac atc ttg acg tgg gat ggt ggg ccc gct agc acc tct gac acc gtc 198
Asn Ile Leu Thr Trp Asp Gly Gly Pro Ala Ser Thr Ser Asp Thr Val 40
45 50 tac agt gtg gaa tat aag aaa tac gga gag aga aag tgg ctg gcc
aag 246 Tyr Ser Val Glu Tyr Lys Lys Tyr Gly Glu Arg Lys Trp Leu Ala
Lys 55 60 65 gcg ggc tgc cag cgg atc acc cag aag ttc tgc aac ctg
act atg gag 294 Ala Gly Cys Gln Arg Ile Thr Gln Lys Phe Cys Asn Leu
Thr Met Glu 70 75 80 acc cgc aac cac act gag ttt tac tac gcc aag
gtc acg gca gtc agc 342 Thr Arg Asn His Thr Glu Phe Tyr Tyr Ala Lys
Val Thr Ala Val Ser 85 90 95 100 gca gga ggc cca cca gtc aca aag
atg act gat cgt ttc agc tcg ctg 390 Ala Gly Gly Pro Pro Val Thr Lys
Met Thr Asp Arg Phe Ser Ser Leu 105 110 115 cag cac act acc atc aaa
ccg cct gat gtg acc tgt atc ccc aaa gtg 438 Gln His Thr Thr Ile Lys
Pro Pro Asp Val Thr Cys Ile Pro Lys Val 120 125 130 agg tcc att cag
atg ctg gtc cac ccc aca ctc aca ccg gtc ctc tcg 486 Arg Ser Ile Gln
Met Leu Val His Pro Thr Leu Thr Pro Val Leu Ser 135 140 145 gaa gat
ggc cac cag cta acc ctg gag gag att ttc cat gac ctg ttc 534 Glu Asp
Gly His Gln Leu Thr Leu Glu Glu Ile Phe His Asp Leu Phe 150 155 160
tac cgc tta gag ctc cac gtc aac cac acc tac cag atg cac ctt gaa 582
Tyr Arg Leu Glu Leu His Val Asn His Thr Tyr Gln Met His Leu Glu 165
170 175 180 ggc aaa cag aga gaa tac gag ttc ctt ggc ctg act ccc gac
aca gag 630 Gly Lys Gln Arg Glu Tyr Glu Phe Leu Gly Leu Thr Pro Asp
Thr Glu 185 190 195 ttc ctc ggc tcc atc aca att ttg act ccg ata ttg
tcc aag gaa agt 678 Phe Leu Gly Ser Ile Thr Ile Leu Thr Pro Ile Leu
Ser Lys Glu Ser 200 205 210 gcc ccc tac gtg tgc cga gtg aag acg ctg
ccc gat cgg acg tgg gcc 726 Ala Pro Tyr Val Cys Arg Val Lys Thr Leu
Pro Asp Arg Thr Trp Ala 215 220 225 tac tcc ttc tcg ggc gcc gtg ctc
ttt tcc atg ggt ttc ctc gtc ggc 774 Tyr Ser Phe Ser Gly Ala Val Leu
Phe Ser Met Gly Phe Leu Val Gly 230 235 240 ttg ctc tgt tat ctg ggc
tac aaa tac atc acc aag cca cct gta cct 822 Leu Leu Cys Tyr Leu Gly
Tyr Lys Tyr Ile Thr Lys Pro Pro Val Pro 245 250 255 260 cct aac tcc
ctg aac gtc caa cgt gtc ctg acc ttt caa ccc cta cgc 870 Pro Asn Ser
Leu Asn Val Gln Arg Val Leu Thr Phe Gln Pro Leu Arg 265 270 275 ttc
atc caa gaa cac gta ctg atc cct gtc ttg gac ctc agt ggc ccc 918 Phe
Ile Gln Glu His Val Leu Ile Pro Val Leu Asp Leu Ser Gly Pro 280 285
290 agc agt ctg cct cag ccc atc cag tac tcc caa gtg gtg gtg tct ggg
966 Ser Ser Leu Pro Gln Pro Ile Gln Tyr Ser Gln Val Val Val Ser Gly
295 300 305 ccc agg gag cct cct gga gct gtg tgg cgg cag agc ctg tct
gac ctc 1014 Pro Arg Glu Pro Pro Gly Ala Val Trp Arg Gln Ser Leu
Ser Asp Leu 310 315 320 acc tac gta ggg cag tca gat gtc tcc atc ctg
caa cct acc aac gtg 1062 Thr Tyr Val Gly Gln Ser Asp Val Ser Ile
Leu Gln Pro Thr Asn Val 325 330 335 340 cca gct cag cag aca ctg tcc
cca cca tcc tac gct ccg aag gct gtc 1110 Pro Ala Gln Gln Thr Leu
Ser Pro Pro Ser Tyr Ala Pro Lys Ala Val 345 350 355 cct gag gtc cag
ccc cct tcc tat gcg cct cag gta gcc tcg gat gcc 1158 Pro Glu Val
Gln Pro Pro Ser Tyr Ala Pro Gln Val Ala Ser Asp Ala 360 365 370 aaa
gct ctg ttc tac tca cca caa cag ggg atg aag acc agg cct gcc 1206
Lys Ala Leu Phe Tyr Ser Pro Gln Gln Gly Met Lys Thr Arg Pro Ala 375
380 385 acc tat gac ccg cag gac att ctg gac agc tgc cct gct tct tat
gct 1254 Thr Tyr Asp Pro Gln Asp Ile Leu Asp Ser Cys Pro Ala Ser
Tyr Ala 390 395 400 gtg tgt gtg gaa gac tct ggc aaa gac tct acc cca
ggc atc ctc tcc 1302 Val Cys Val Glu Asp Ser Gly Lys Asp Ser Thr
Pro Gly Ile Leu Ser 405 410 415 420 act ccc aaa tac ctc aag aca aaa
ggt cag ctc cag gaa gac aca ctt 1350 Thr Pro Lys Tyr Leu Lys Thr
Lys Gly Gln Leu Gln Glu Asp Thr Leu 425 430 435 gtt aga agc tgt ctc
cca ggg gac ctt tct cta cag aaa gtc acc tcc 1398 Val Arg Ser Cys
Leu Pro Gly Asp Leu Ser Leu Gln Lys Val Thr Ser 440 445 450 tta ggt
gaa ggg gag aca cag aga cca aaa tca ctc ccc tca cct ctg 1446 Leu
Gly Glu Gly Glu Thr Gln Arg Pro Lys Ser Leu Pro Ser Pro Leu 455 460
465 gga ttt tgc aca gac aga gga cct gac ctt cac aca ctg cgc agt gag
1494 Gly Phe Cys Thr Asp Arg Gly Pro Asp Leu His Thr Leu Arg Ser
Glu 470 475 480 gaa cca gag aca cca cgg tac ctg aag ggg gcg ctg tct
ctc ctg tcc 1542 Glu Pro Glu Thr Pro Arg Tyr Leu Lys Gly Ala Leu
Ser Leu Leu Ser 485 490 495 500 tct gtg cag atc gag ggc cac cct gtc
tcc ctc cct ttg cac gtc cat 1590 Ser Val Gln Ile Glu Gly His Pro
Val Ser Leu Pro Leu His Val His 505 510 515 tct gtc tca tgt tcc ccc
tca gac gag gga cca agt ccc tgg ggc ctg 1638 Ser Val Ser Cys Ser
Pro Ser Asp Glu Gly Pro Ser Pro Trp Gly Leu 520 525 530 ctg gac tcc
ctt gtg tgt cca aag gat gag ggt ccc gcg gtt gag act 1686 Leu Asp
Ser Leu Val Cys Pro Lys Asp Glu Gly Pro Ala Val Glu Thr 535 540 545
gag gcc atg tgc ccc agt gct gca gcc tct gag ctg gag cag tcc aca
1734 Glu Ala Met Cys Pro Ser Ala Ala Ala Ser Glu Leu Glu Gln Ser
Thr 550 555 560 gaa ctg gac tct ctt ttc aaa ggc ttg gcc ctg act gtg
cag tgg gaa 1782 Glu Leu Asp Ser Leu Phe Lys Gly Leu Ala Leu Thr
Val Gln Trp Glu 565 570 575 580 tcc tga agggagatcg gagcaagcag
gcctaagttt cctcccgccc caccta 1834 Ser * <210> SEQ ID NO 42
<211> LENGTH: 581 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <400> SEQUENCE: 42 Met Lys Thr Leu Leu Thr Ile
Leu Thr Val Gly Ser Leu Ala Ala His 1 5 10 15 Thr Thr Val Asp Thr
Ser Gly Leu Leu Gln His Val Lys Phe Gln Ser 20 25 30 Ser Asn Phe
Glu Asn Ile Leu Thr Trp Asp Gly Gly Pro Ala Ser Thr 35 40 45 Ser
Asp Thr Val Tyr Ser Val Glu Tyr Lys Lys Tyr Gly Glu Arg Lys 50 55
60 Trp Leu Ala Lys Ala Gly Cys Gln Arg Ile Thr Gln Lys Phe Cys Asn
65 70 75 80 Leu Thr Met Glu Thr Arg Asn His Thr Glu Phe Tyr Tyr Ala
Lys Val 85 90 95 Thr Ala Val Ser Ala Gly Gly Pro Pro Val Thr Lys
Met Thr Asp Arg 100 105 110 Phe Ser Ser Leu Gln His Thr Thr Ile Lys
Pro Pro Asp Val Thr Cys 115 120 125 Ile Pro Lys Val Arg Ser Ile Gln
Met Leu Val His Pro Thr Leu Thr 130 135 140 Pro Val Leu Ser Glu Asp
Gly His Gln Leu Thr Leu Glu Glu Ile Phe 145 150 155 160 His Asp Leu
Phe Tyr Arg Leu Glu Leu His Val Asn His Thr Tyr Gln 165 170 175 Met
His Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu Gly Leu Thr 180 185
190 Pro Asp Thr Glu Phe Leu Gly Ser Ile Thr Ile Leu Thr Pro Ile Leu
195 200 205 Ser Lys Glu Ser Ala Pro Tyr Val Cys Arg Val Lys Thr Leu
Pro Asp 210 215 220 Arg Thr Trp Ala Tyr Ser Phe Ser Gly Ala Val Leu
Phe Ser Met Gly 225 230 235 240 Phe Leu Val Gly Leu Leu Cys Tyr Leu
Gly Tyr Lys Tyr Ile Thr Lys 245 250 255 Pro Pro Val Pro Pro Asn Ser
Leu Asn Val Gln Arg Val Leu Thr Phe 260 265 270 Gln Pro Leu Arg Phe
Ile Gln Glu His Val Leu Ile Pro Val Leu Asp 275 280 285 Leu Ser Gly
Pro Ser Ser Leu Pro Gln Pro Ile Gln Tyr Ser Gln Val 290 295 300 Val
Val Ser Gly Pro Arg Glu Pro Pro Gly Ala Val Trp Arg Gln Ser 305 310
315 320 Leu Ser Asp Leu Thr Tyr Val Gly Gln Ser Asp Val Ser Ile Leu
Gln 325 330 335 Pro Thr Asn Val Pro Ala Gln Gln Thr Leu Ser Pro Pro
Ser Tyr Ala 340 345 350 Pro Lys Ala Val Pro Glu Val Gln Pro Pro Ser
Tyr Ala Pro Gln Val 355 360 365 Ala Ser Asp Ala Lys Ala Leu Phe Tyr
Ser Pro Gln Gln Gly Met Lys 370 375 380 Thr Arg Pro Ala Thr Tyr Asp
Pro Gln Asp Ile Leu Asp Ser Cys Pro 385 390 395 400 Ala Ser Tyr Ala
Val Cys Val Glu Asp Ser Gly Lys Asp Ser Thr Pro 405 410 415 Gly Ile
Leu Ser Thr Pro Lys Tyr Leu Lys Thr Lys Gly Gln Leu Gln 420 425 430
Glu Asp Thr Leu Val Arg Ser Cys Leu Pro Gly Asp Leu Ser Leu Gln 435
440 445 Lys Val Thr Ser Leu Gly Glu Gly Glu Thr Gln Arg Pro Lys Ser
Leu 450 455 460 Pro Ser Pro Leu Gly Phe Cys Thr Asp Arg Gly Pro Asp
Leu His Thr 465 470 475 480 Leu Arg Ser Glu Glu Pro Glu Thr Pro Arg
Tyr Leu Lys Gly Ala Leu 485 490 495 Ser Leu Leu Ser Ser Val Gln Ile
Glu Gly His Pro Val Ser Leu Pro 500 505 510 Leu His Val His Ser Val
Ser Cys Ser Pro Ser Asp Glu Gly Pro Ser 515 520 525 Pro Trp Gly Leu
Leu Asp Ser Leu Val Cys Pro Lys Asp Glu Gly Pro 530 535 540 Ala Val
Glu Thr Glu Ala Met Cys Pro Ser Ala Ala Ala Ser Glu Leu 545 550 555
560 Glu Gln Ser Thr Glu Leu Asp Ser Leu Phe Lys Gly Leu Ala Leu Thr
565 570 575 Val Gln Trp Glu Ser 580 <210> SEQ ID NO 43
<211> LENGTH: 660 <212> TYPE: DNA <213> ORGANISM:
Homo Sapiens <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (1)...(660) <400> SEQUENCE: 43 atg gcg
tgg agt ctt ggg agc tgg ctg ggt ggc tgc ctg ctg gtg tca 48 Met Ala
Trp Ser Leu Gly Ser Trp Leu Gly Gly Cys Leu Leu Val Ser 1 5 10 15
gca ttg gga atg gta cca cct ccc gaa aat gtc aga atg aat tct gtt 96
Ala Leu Gly Met Val Pro Pro Pro Glu Asn Val Arg Met Asn Ser Val 20
25 30 aat ttc aag aac att cta cag tgg gag tca cct gct ttt gcc aaa
ggg 144 Asn Phe Lys Asn Ile Leu Gln Trp Glu Ser Pro Ala Phe Ala Lys
Gly 35 40 45 aac ctg act ttc aca gct cag tac cta agt tat agg ata
ttc caa gat 192 Asn Leu Thr Phe Thr Ala Gln Tyr Leu Ser Tyr Arg Ile
Phe Gln Asp 50 55 60 aaa tgc atg aat act acc ttg acg gaa tgt gat
ttc tca agt ctt tcc 240 Lys Cys Met Asn Thr Thr Leu Thr Glu Cys Asp
Phe Ser Ser Leu Ser 65 70 75 80 aag tat ggt gac cac acc ttg aga gtc
agg gct gaa ttt gca gat gag 288 Lys Tyr Gly Asp His Thr Leu Arg Val
Arg Ala Glu Phe Ala Asp Glu 85 90 95 cat tca gac tgg gta aac atc
acc ttc tgt cct gtg gat gac acc att 336 His Ser Asp Trp Val Asn Ile
Thr Phe Cys Pro Val Asp Asp Thr Ile 100 105 110 att gga ccc cct gga
atg caa gta gaa gta ctt gat gat tct tta cat 384 Ile Gly Pro Pro Gly
Met Gln Val Glu Val Leu Asp Asp Ser Leu His 115 120 125 atg cgt ttc
tta gcc cct aaa att gag aat gaa tac gaa act tgg act 432 Met Arg Phe
Leu Ala Pro Lys Ile Glu Asn Glu Tyr Glu Thr Trp Thr 130 135 140 atg
aag aat gtg tat aac tca tgg act tat aat gtg caa tac tgg aaa 480 Met
Lys Asn Val Tyr Asn Ser Trp Thr Tyr Asn Val Gln Tyr Trp Lys 145 150
155 160 aac ggt act gat gaa aag ttt caa att act ccc cag tat gac ttt
gag 528 Asn Gly Thr Asp Glu Lys Phe Gln Ile Thr Pro Gln Tyr Asp Phe
Glu 165 170 175 gtc ctc aga aac ctg gag cca tgg aca act tat tgt gtt
caa gtt cga 576 Val Leu Arg Asn Leu Glu Pro Trp Thr Thr Tyr Cys Val
Gln Val Arg 180 185 190 ggg ttt ctt cct gat cgg aac aaa gct ggg gaa
tgg agt gag cct gtc 624 Gly Phe Leu Pro Asp Arg Asn Lys Ala Gly Glu
Trp Ser Glu Pro Val 195 200 205 tgt gag caa aca acc cat gac gaa acg
gtc ccc tcc 660 Cys Glu Gln Thr Thr His Asp Glu Thr Val Pro Ser 210
215 220 <210> SEQ ID NO 44 <211> LENGTH: 220
<212> TYPE: PRT <213> ORGANISM: Homo Sapiens
<400> SEQUENCE: 44 Met Ala Trp Ser Leu Gly Ser Trp Leu Gly
Gly Cys Leu Leu Val Ser 1 5 10 15 Ala Leu Gly Met Val Pro Pro Pro
Glu Asn Val Arg Met Asn Ser Val 20 25 30 Asn Phe Lys Asn Ile Leu
Gln Trp Glu Ser Pro Ala Phe Ala Lys Gly 35 40 45 Asn Leu Thr Phe
Thr Ala Gln Tyr Leu Ser Tyr Arg Ile Phe Gln Asp 50 55 60 Lys Cys
Met Asn Thr Thr Leu Thr Glu Cys Asp Phe Ser Ser Leu Ser 65 70 75 80
Lys Tyr Gly Asp His Thr Leu Arg Val Arg Ala Glu Phe Ala Asp Glu 85
90 95 His Ser Asp Trp Val Asn Ile Thr Phe Cys Pro Val Asp Asp Thr
Ile 100 105 110 Ile Gly Pro Pro Gly Met Gln Val Glu Val Leu Asp Asp
Ser Leu His 115 120 125 Met Arg Phe Leu Ala Pro Lys Ile Glu Asn Glu
Tyr Glu Thr Trp Thr 130 135 140 Met Lys Asn Val Tyr Asn Ser Trp Thr
Tyr Asn Val Gln Tyr Trp Lys 145 150 155 160 Asn Gly Thr Asp Glu Lys
Phe Gln Ile Thr Pro Gln Tyr Asp Phe Glu 165 170 175 Val Leu Arg Asn
Leu Glu Pro Trp Thr Thr Tyr Cys Val Gln Val Arg 180 185 190 Gly Phe
Leu Pro Asp Arg Asn Lys Ala Gly Glu Trp Ser Glu Pro Val 195 200 205
Cys Glu Gln Thr Thr His Asp Glu Thr Val Pro Ser 210 215 220
<210> SEQ ID NO 45 <211> LENGTH: 199 <212> TYPE:
PRT <213> ORGANISM: homo sapiens <400> SEQUENCE: 45 Met
Val Pro Pro Pro Glu Asn Val Arg Met Asn Ser Val Asn Phe Lys 1 5 10
15 Asn Ile Leu Gln Trp Glu Ser Pro Ala Phe Ala Lys Gly Asn Leu Thr
20 25 30 Phe Thr Ala Gln Tyr Leu Ser Tyr Arg Ile Phe Gln Asp Lys
Cys Met 35 40 45 Asn Thr Thr Leu Thr Glu Cys Asp Phe Ser Ser Leu
Ser Lys Tyr Gly 50 55 60 Asp His Thr Leu Arg Val Arg Ala Glu Phe
Ala Asp Glu His Ser Asp 65 70 75 80 Trp Val Asn Ile Thr Phe Cys Pro
Val Asp Asp Thr Ile Ile Gly Pro 85 90 95 Pro Gly Met Gln Val Glu
Val Leu Ala Asp Ser Leu His Met Arg Phe 100 105 110 Leu Ala Pro Lys
Ile Glu Asn Glu Tyr Glu Thr Trp Thr Met Lys Asn 115 120 125 Val Tyr
Asn Ser Trp Thr Tyr Asn Val Gln Tyr Trp Lys Asn Gly Thr 130 135 140
Asp Glu Lys Phe Gln Ile Thr Pro Gln Tyr Asp Phe Glu Val Leu Arg 145
150 155 160 Asn Leu Glu Pro Trp Thr Thr Tyr Cys Val Gln Val Arg Gly
Phe Leu 165 170 175 Pro Asp Arg Asn Lys Ala Gly Glu Trp Ser Glu Pro
Val Cys Glu Gln 180 185 190 Thr Thr His Asp Glu Thr Val 195
<210> SEQ ID NO 46 <211> LENGTH: 211 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 46 Ser
Asp Ala His Gly Thr Glu Leu Pro Ser Pro Pro Ser Val Trp Phe 1 5 10
15 Glu Ala Glu Phe Phe His His Ile Leu His Trp Thr Pro Ile Pro Asn
20 25 30 Gln Ser Glu Ser Thr Cys Tyr Glu Val Ala Leu Leu Arg Tyr
Gly Ile 35 40 45 Glu Ser Trp Asn Ser Ile Ser Asn Cys Ser Gln Thr
Leu Ser Tyr Asp 50 55 60 Leu Thr Ala Val Thr Leu Asp Leu Tyr His
Ser Asn Gly Tyr Arg Ala 65 70 75 80 Arg Val Arg Ala Val Asp Gly Ser
Arg His Ser Asn Trp Thr Val Thr 85 90 95 Asn Thr Arg Phe Ser Val
Asp Glu Val Thr Leu Thr Val Gly Ser Val 100 105 110 Asn Leu Glu Ile
His Asn Gly Phe Ile Leu Gly Lys Ile Gln Leu Pro 115 120 125 Arg Pro
Lys Met Ala Pro Ala Asn Asp Thr Tyr Glu Ser Ile Phe Ser 130 135 140
His Phe Arg Glu Tyr Glu Ile Ala Ile Arg Lys Val Pro Gly Asn Phe 145
150 155 160 Thr Phe Thr His Lys Lys Val Lys His Glu Asn Phe Ser Leu
Leu Thr 165 170 175 Ser Gly Glu Val Gly Glu Phe Cys Val Gln Val Lys
Pro Ser Val Ala 180 185 190 Ser Arg Ser Asn Lys Gly Met Trp Ser Lys
Glu Glu Cys Ile Ser Leu 195 200 205 Thr Arg Gln 210 <210> SEQ
ID NO 47 <211> LENGTH: 201 <212> TYPE: PRT <213>
ORGANISM: homo sapiens <400> SEQUENCE: 47 Asp Glu Val Ala Ile
Leu Pro Ala Pro Gln Asn Leu Ser Val Leu Ser 1 5 10 15 Thr Asn Met
Lys His Leu Leu Met Trp Ser Pro Val Ile Ala Pro Gly 20 25 30 Glu
Thr Val Tyr Tyr Ser Val Glu Tyr Gln Gly Glu Tyr Glu Ser Leu 35 40
45 Tyr Thr Ser His Ile Trp Ile Pro Ser Ser Trp Cys Ser Leu Thr Glu
50 55 60 Gly Pro Glu Cys Asp Val Thr Asp Asp Ile Thr Ala Thr Val
Pro Tyr 65 70 75 80 Asn Leu Arg Val Arg Ala Thr Leu Gly Ser Gln Thr
Ser Ala Trp Ser 85 90 95 Ile Leu Lys His Pro Phe Asn Arg Asn Ser
Thr Ile Leu Thr Arg Pro 100 105 110 Gly Met Glu Ile Thr Lys Asp Gly
Phe His Leu Val Ile Glu Leu Glu 115 120 125 Asp Leu Gly Pro Gln Phe
Glu Phe Leu Val Ala Tyr Trp Arg Arg Glu 130 135 140 Pro Gly Ala Glu
Glu His Val Lys Met Val Arg Ser Gly Gly Ile Pro 145 150 155 160 Val
His Leu Glu Thr Met Glu Pro Gly Ala Ala Tyr Cys Val Lys Ala 165 170
175 Gln Thr Phe Val Lys Ala Ile Gly Arg Tyr Ser Ala Phe Ser Gln Thr
180 185 190 Glu Cys Val Glu Val Gln Gly Glu Ala 195 200 <210>
SEQ ID NO 48 <211> LENGTH: 68 <212> TYPE: PRT
<213> ORGANISM: Mus musculus <400> SEQUENCE: 48 His Thr
Thr Val Asp Thr Ser Gly Leu Leu Gln His Val Lys Phe Gln 1 5 10 15
Ser Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Gly Gly Pro Ala Ser 20
25 30 Thr Ser Asp Thr Val Tyr Ser Val Glu Tyr Lys Lys Tyr Gly Glu
Arg 35 40 45 Lys Trp Leu Ala Lys Ala Gly Cys Gln Arg Ile Thr Gln
Lys Phe Cys 50 55 60 Asn Leu Thr Met 65 <210> SEQ ID NO 49
<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:
mus musculus <400> SEQUENCE: 49 Glu Thr Arg Asn His Thr Glu
Phe Tyr Tyr Ala Lys Val Thr Ala Val 1 5 10 15 Ser Ala Gly Gly Pro
Pro Val Thr Lys Met 20 25 <210> SEQ ID NO 50 <211>
LENGTH: 28 <212> TYPE: PRT <213> ORGANISM: mus musculus
<400> SEQUENCE: 50 Thr Asp Arg Phe Ser Ser Leu Gln His Thr
Thr Ile Lys Pro Pro Asp 1 5 10 15 Val Thr Cys Ile Pro Lys Val Arg
Ser Ile Gln Met 20 25 <210> SEQ ID NO 51 <211> LENGTH:
40 <212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 51 Leu Val His Pro Thr Leu Thr Pro Val Leu
Ser Glu Asp Gly His Gln 1 5 10 15 Leu Thr Leu Glu Glu Ile Phe His
Asp Leu Phe Tyr Arg Leu Glu Leu 20 25 30 His Val Asn His Thr Tyr
Gln Met 35 40 <210> SEQ ID NO 52 <211> LENGTH: 50
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 52 His Leu Glu Gly Lys Gln Arg Glu Tyr Glu
Phe Leu Gly Leu Thr Pro 1 5 10 15 Asp Thr Glu Phe Leu Gly Ser Ile
Thr Ile Leu Thr Pro Ile Leu Ser 20 25 30 Lys Glu Ser Ala Pro Tyr
Val Cys Arg Val Lys Thr Leu Pro Leu Val 35 40 45 Pro Arg 50
<210> SEQ ID NO 53 <211> LENGTH: 70 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 53 His
Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu Gly Leu Thr Pro 1 5 10
15 Asp Thr Glu Phe His Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu
20 25 30 Gly Leu Thr Pro Asp Thr Glu Phe Leu Gly Ser Ile Thr Ile
Leu Thr 35 40 45 Pro Ile Leu Ser Lys Glu Ser Ala Pro Tyr Val Cys
Arg Val Lys Thr 50 55 60 Leu Pro Leu Val Pro Arg 65 70 <210>
SEQ ID NO 54 <211> LENGTH: 46 <212> TYPE: PRT
<213> ORGANISM: Mus musculus <400> SEQUENCE: 54 Glu Thr
Arg Asn His Thr Glu Phe Tyr Tyr Ala Lys Val Thr Ala Val 1 5 10 15
Ser Ala Gly Gly Glu Thr Arg Asn His Thr Glu Phe Tyr Tyr Ala Lys 20
25 30 Val Thr Ala Val Ser Ala Gly Gly Pro Pro Val Thr Lys Met 35 40
45 <210> SEQ ID NO 55 <211> LENGTH: 48 <212>
TYPE: PRT <213> ORGANISM: mus musculus <220> FEATURE:
<221> NAME/KEY: VARIANT <222> LOCATION: 6, 11, 13, 15,
17, 18, and 19 <223> OTHER INFORMATION: Xaa = Any Amino Acid
<400> SEQUENCE: 55 Thr Asp Arg Phe Ser Xaa Leu Gln His Thr
Xaa Ile Xaa Pro Xaa Asp 1 5 10 15 Xaa Xaa Xaa Ile Thr Asp Arg Phe
Ser Ser Leu Gln His Thr Thr Ile 20 25 30 Lys Pro Pro Asp Val Thr
Cys Ile Pro Lys Val Arg Ser Ile Gln Met 35 40 45 <210> SEQ ID
NO 56 <211> LENGTH: 92 <212> TYPE: PRT <213>
ORGANISM: homo sapiens <400> SEQUENCE: 56 Pro Glu Asp Pro Ser
Asp Leu Leu Gln His Val Lys Phe Gln Ser Ser 1 5 10 15 Asn Phe Glu
Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr Pro 20 25 30 Asp
Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg Asp Trp 35 40
45 Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn Leu
50 55 60 Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala Arg
Val Thr 65 70 75 80 Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys Met
85 90 <210> SEQ ID NO 57 <211> LENGTH: 28 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
57 Thr Asp Arg Phe Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro Asp
1 5 10 15 Val Thr Cys Ile Ser Lys Val Arg Ser Ile Gln Met 20 25
<210> SEQ ID NO 58 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 58 Ile
Val His Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly His Arg 1 5 10
15 Leu Thr Leu Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu Glu Leu
20 25 30 Gln Val Asn Arg Thr Tyr Gln Met 35 40 <210> SEQ ID
NO 59 <211> LENGTH: 25 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 59 His Leu Gly Gly Lys
Gln Arg Glu Tyr Glu Phe Phe Gly Leu Thr Pro 1 5 10 15 Asp Thr Glu
Phe Leu Gly Thr Ile Met 20 25 <210> SEQ ID NO 60 <211>
LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 60 Ile Cys Val Pro Thr Trp Ala Lys Glu Ser
Ala Pro Tyr Met 1 5 10 <210> SEQ ID NO 61 <211> LENGTH:
12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 61 Cys Arg Val Lys Thr Leu Pro Asp Arg Thr
Trp Thr 1 5 10 <210> SEQ ID NO 62 <211> LENGTH: 212
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A murine
IL-22RA soluble receptor with cleavage site (Leu Val Pro Arg)
remaining on C-Terminus <400> SEQUENCE: 62 His Thr Thr Val
Asp Thr Ser Gly Leu Leu Gln His Val Lys Phe Gln 1 5 10 15 Ser Ser
Asn Phe Glu Asn Ile Leu Thr Trp Asp Gly Gly Pro Ala Ser 20 25 30
Thr Ser Asp Thr Val Tyr Ser Val Glu Tyr Lys Lys Tyr Gly Glu Arg 35
40 45 Lys Trp Leu Ala Lys Ala Gly Cys Gln Arg Ile Thr Gln Lys Phe
Cys 50 55 60 Asn Leu Thr Met Glu Thr Arg Asn His Thr Glu Phe Tyr
Tyr Ala Lys 65 70 75 80 Val Thr Ala Val Ser Ala Gly Gly Pro Pro Val
Thr Lys Met Thr Asp 85 90 95 Arg Phe Ser Ser Leu Gln His Thr Thr
Ile Lys Pro Pro Asp Val Thr 100 105 110 Cys Ile Pro Lys Val Arg Ser
Ile Gln Met Leu Val His Pro Thr Leu 115 120 125 Thr Pro Val Leu Ser
Glu Asp Gly His Gln Leu Thr Leu Glu Glu Ile 130 135 140 Phe His Asp
Leu Phe Tyr Arg Leu Glu Leu His Val Asn His Thr Tyr 145 150 155 160
Gln Met His Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu Gly Leu 165
170 175 Thr Pro Asp Thr Glu Phe Leu Gly Ser Ile Thr Ile Leu Thr Pro
Ile 180 185 190 Leu Ser Lys Glu Ser Ala Pro Tyr Val Cys Arg Val Lys
Thr Leu Pro 195 200 205 Leu Val Pro Arg 210
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 62 <210>
SEQ ID NO 1 <211> LENGTH: 2831 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (34)...(1755) <400>
SEQUENCE: 1 tagaggccaa gggagggctc tgtgccagcc ccg atg agg acg ctg
ctg acc atc 54 Met Arg Thr Leu Leu Thr Ile 1 5 ttg act gtg gga tcc
ctg gct gct cac gcc cct gag gac ccc tcg gat 102 Leu Thr Val Gly Ser
Leu Ala Ala His Ala Pro Glu Asp Pro Ser Asp 10 15 20 ctg ctc cag
cac gtg aaa ttc cag tcc agc aac ttt gaa aac atc ctg 150 Leu Leu Gln
His Val Lys Phe Gln Ser Ser Asn Phe Glu Asn Ile Leu 25 30 35 acg
tgg gac agc ggg cca gag ggc acc cca gac acg gtc tac agc atc 198 Thr
Trp Asp Ser Gly Pro Glu Gly Thr Pro Asp Thr Val Tyr Ser Ile 40 45
50 55 gag tat aag acg tac gga gag agg gac tgg gtg gca aag aag ggc
tgt 246 Glu Tyr Lys Thr Tyr Gly Glu Arg Asp Trp Val Ala Lys Lys Gly
Cys 60 65 70 cag cgg atc acc cgg aag tcc tgc aac ctg acg gtg gag
acg ggc aac 294 Gln Arg Ile Thr Arg Lys Ser Cys Asn Leu Thr Val Glu
Thr Gly Asn 75 80 85 ctc acg gag ctc tac tat gcc agg gtc acc gct
gtc agt gcg gga ggc 342 Leu Thr Glu Leu Tyr Tyr Ala Arg Val Thr Ala
Val Ser Ala Gly Gly 90 95 100 cgg tca gcc acc aag atg act gac agg
ttc agc tct ctg cag cac act 390 Arg Ser Ala Thr Lys Met Thr Asp Arg
Phe Ser Ser Leu Gln His Thr 105 110 115 acc ctc aag cca cct gat gtg
acc tgt atc tcc aaa gtg aga tcg att 438 Thr Leu Lys Pro Pro Asp Val
Thr Cys Ile Ser Lys Val Arg Ser Ile 120 125 130 135 cag atg att gtt
cat cct acc ccc acg cca atc cgt gca ggc gat ggc 486 Gln Met Ile Val
His Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly 140 145 150 cac cgg
cta acc ctg gaa gac atc ttc cat gac ctg ttc tac cac tta 534 His Arg
Leu Thr Leu Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu 155 160 165
gag ctc cag gtc aac cgc acc tac caa atg cac ctt gga ggg aag cag 582
Glu Leu Gln Val Asn Arg Thr Tyr Gln Met His Leu Gly Gly Lys Gln 170
175 180 aga gaa tat gag ttc ttc ggc ctg acc cct gac aca gag ttc ctt
ggc 630 Arg Glu Tyr Glu Phe Phe Gly Leu Thr Pro Asp Thr Glu Phe Leu
Gly 185 190 195 acc atc atg att tgc gtt ccc acc tgg gcc aag gag agt
gcc ccc tac 678 Thr Ile Met Ile Cys Val Pro Thr Trp Ala Lys Glu Ser
Ala Pro Tyr 200 205 210 215 atg tgc cga gtg aag aca ctg cca gac cgg
aca tgg acc tac tcc ttc 726 Met Cys Arg Val Lys Thr Leu Pro Asp Arg
Thr Trp Thr Tyr Ser Phe 220 225 230 tcc gga gcc ttc ctg ttc tcc atg
ggc ttc ctc gtc gca gta ctc tgc 774 Ser Gly Ala Phe Leu Phe Ser Met
Gly Phe Leu Val Ala Val Leu Cys 235 240 245 tac ctg agc tac aga tat
gtc acc aag ccg cct gca cct ccc aac tcc 822 Tyr Leu Ser Tyr Arg Tyr
Val Thr Lys Pro Pro Ala Pro Pro Asn Ser 250 255 260 ctg aac gtc cag
cga gtc ctg act ttc cag ccg ctg cgc ttc atc cag 870 Leu Asn Val Gln
Arg Val Leu Thr Phe Gln Pro Leu Arg Phe Ile Gln 265 270 275 gag cac
gtc ctg atc cct gtc ttt gac ctc agc ggc ccc agc agt ctg 918 Glu His
Val Leu Ile Pro Val Phe Asp Leu Ser Gly Pro Ser Ser Leu 280 285 290
295 gcc cag cct gtc cag tac tcc cag atc agg gtg tct gga ccc agg gag
966 Ala Gln Pro Val Gln Tyr Ser Gln Ile Arg Val Ser Gly Pro Arg Glu
300 305 310 ccc gca gga gct cca cag cgg cat agc ctg tcc gag atc acc
tac tta 1014 Pro Ala Gly Ala Pro Gln Arg His Ser Leu Ser Glu Ile
Thr Tyr Leu 315 320 325 ggg cag cca gac atc tcc atc ctc cag ccc tcc
aac gtg cca cct ccc 1062 Gly Gln Pro Asp Ile Ser Ile Leu Gln Pro
Ser Asn Val Pro Pro Pro 330 335 340 cag atc ctc tcc cca ctg tcc tat
gcc cca aac gct gcc cct gag gtc 1110 Gln Ile Leu Ser Pro Leu Ser
Tyr Ala Pro Asn Ala Ala Pro Glu Val 345 350 355 ggg ccc cca tcc tat
gca cct cag gtg acc ccc gaa gct caa ttc cca 1158 Gly Pro Pro Ser
Tyr Ala Pro Gln Val Thr Pro Glu Ala Gln Phe Pro 360 365 370 375 ttc
tac gcc cca cag gcc atc tct aag gtc cag cct tcc tcc tat gcc 1206
Phe Tyr Ala Pro Gln Ala Ile Ser Lys Val Gln Pro Ser Ser Tyr Ala 380
385 390 cct caa gcc act ccg gac agc tgg cct ccc tcc tat ggg gta tgc
atg 1254 Pro Gln Ala Thr Pro Asp Ser Trp Pro Pro Ser Tyr Gly Val
Cys Met 395 400 405 gaa ggt tct ggc aaa gac tcc ccc act ggg aca ctt
tct agt cct aaa 1302 Glu Gly Ser Gly Lys Asp Ser Pro Thr Gly Thr
Leu Ser Ser Pro Lys 410 415 420 cac ctt agg cct aaa ggt cag ctt cag
aaa gag cca cca gct gga agc 1350 His Leu Arg Pro Lys Gly Gln Leu
Gln Lys Glu Pro Pro Ala Gly Ser 425 430 435 tgc atg tta ggt ggc ctt
tct ctg cag gag gtg acc tcc ttg gct atg 1398 Cys Met Leu Gly Gly
Leu Ser Leu Gln Glu Val Thr Ser Leu Ala Met 440 445 450 455 gag gaa
tcc caa gaa gca aaa tca ttg cac cag ccc ctg ggg att tgc 1446 Glu
Glu Ser Gln Glu Ala Lys Ser Leu His Gln Pro Leu Gly Ile Cys 460 465
470 aca gac aga aca tct gac cca aat gtg cta cac agt ggg gag gaa ggg
1494 Thr Asp Arg Thr Ser Asp Pro Asn Val Leu His Ser Gly Glu Glu
Gly 475 480 485 aca cca cag tac cta aag ggc cag ctc ccc ctc ctc tcc
tca gtc cag 1542 Thr Pro Gln Tyr Leu Lys Gly Gln Leu Pro Leu Leu
Ser Ser Val Gln 490 495 500 atc gag ggc cac ccc atg tcc ctc cct ttg
caa cct cct tcc ggt cca 1590 Ile Glu Gly His Pro Met Ser Leu Pro
Leu Gln Pro Pro Ser Gly Pro 505 510 515 tgt tcc ccc tcg gac caa ggt
cca agt ccc tgg ggc ctg ctg gag tcc 1638 Cys Ser Pro Ser Asp Gln
Gly Pro Ser Pro Trp Gly Leu Leu Glu Ser 520 525 530 535 ctt gtg tgt
ccc aag gat gaa gcc aag agc cca gcc cct gag acc tca 1686 Leu Val
Cys Pro Lys Asp Glu Ala Lys Ser Pro Ala Pro Glu Thr Ser 540 545 550
gac ctg gag cag ccc aca gaa ctg gat tct ctt ttc aga ggc ctg gcc
1734 Asp Leu Glu Gln Pro Thr Glu Leu Asp Ser Leu Phe Arg Gly Leu
Ala 555 560 565 ctg act gtg cag tgg gag tcc tgaggggaat gggaaaggct
tggtgcttcc 1785 Leu Thr Val Gln Trp Glu Ser 570 tccctgtccc
tacccagtgt cacatccttg gctgtcaatc ccatgcctgc ccatgccaca 1845
cactctgcga tctggcctca gacgggtgcc cttgagagaa gcagagggag tggcatgcag
1905 ggcccctgcc atgggtgcgc tcctcaccgg aacaaagcag catgataagg
actgcagcgg 1965 gggagctctg gggagcagct tgtgtagaca agcgcgtgct
cgctgagccc tgcaaggcag 2025 aaatgacagt gcaaggagga aatgcaggga
aactcccgag gtccagagcc ccacctccta 2085 acaccatgga ttcaaagtgc
tcagggaatt tgcctctcct tgccccattc ctggccagtt 2145 tcacaatcta
gctcgacaga gcatgaggcc cctgcctctt ctgtcattgt tcaaaggtgg 2205
gaagagagcc tggaaaagaa ccaggcctgg aaaagaacca gaaggaggct gggcagaacc
2265 agaacaacct gcacttctgc caaggccagg gccagcagga cggcaggact
ctagggaggg 2325 gtgtggcctg cagctcattc ccagccaggg caactgcctg
acgttgcacg atttcagctt 2385 cattcctctg atagaacaaa gcgaaatgca
ggtccaccag ggagggagac acacaagcct 2445 tttctgcagg caggagtttc
agaccctatc ctgagaatgg ggtttgaaag gaaggtgagg 2505 gctgtggccc
ctggacgggt acaataacac actgtactga tgtcacaact ttgcaagctc 2565
tgccttgggt tcagcccatc tgggctcaaa ttccagcctc accactcaca agctgtgtga
2625 cttcaaacaa atgaaatcag tgcccagaac ctcggtttcc tcatctgtaa
tgtggggatc 2685 ataacaccta cctcatggag ttgtggtgaa gatgaaatga
agtcatgtct ttaaagtgct 2745 taatagtgcc tggtacatgg gcagtgccca
ataaacggta gctatttaaa aaaaaaaaaa 2805 aaaaaaaaaa atagcggccg cctcga
2831 <210> SEQ ID NO 2 <211> LENGTH: 574 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
2 Met Arg Thr Leu Leu Thr Ile Leu Thr Val Gly Ser Leu Ala Ala His 1
5 10 15 Ala Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val Lys Phe Gln
Ser 20 25 30 Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro
Glu Gly Thr 35 40 45 Pro Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr
Tyr Gly Glu Arg Asp 50 55 60 Trp Val Ala Lys Lys Gly Cys Gln Arg
Ile Thr Arg Lys Ser Cys Asn 65 70 75 80 Leu Thr Val Glu Thr Gly Asn
Leu Thr Glu Leu Tyr Tyr Ala Arg Val 85 90 95 Thr Ala Val Ser Ala
Gly Gly Arg Ser Ala Thr Lys Met Thr Asp Arg 100 105 110 Phe Ser Ser
Leu Gln His Thr Thr Leu Lys Pro Pro Asp Val Thr Cys 115 120 125 Ile
Ser Lys Val Arg Ser Ile Gln Met Ile Val His Pro Thr Pro Thr 130 135
140 Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu Glu Asp Ile Phe
145 150 155 160 His Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn Arg
Thr Tyr Gln 165 170 175 Met His Leu Gly Gly Lys Gln Arg Glu Tyr Glu
Phe Phe Gly Leu Thr 180 185 190 Pro Asp Thr Glu Phe Leu Gly Thr Ile
Met Ile Cys Val Pro Thr Trp 195 200 205 Ala Lys Glu Ser Ala Pro Tyr
Met Cys Arg Val Lys Thr Leu Pro Asp 210 215 220
Arg Thr Trp Thr Tyr Ser Phe Ser Gly Ala Phe Leu Phe Ser Met Gly 225
230 235 240 Phe Leu Val Ala Val Leu Cys Tyr Leu Ser Tyr Arg Tyr Val
Thr Lys 245 250 255 Pro Pro Ala Pro Pro Asn Ser Leu Asn Val Gln Arg
Val Leu Thr Phe 260 265 270 Gln Pro Leu Arg Phe Ile Gln Glu His Val
Leu Ile Pro Val Phe Asp 275 280 285 Leu Ser Gly Pro Ser Ser Leu Ala
Gln Pro Val Gln Tyr Ser Gln Ile 290 295 300 Arg Val Ser Gly Pro Arg
Glu Pro Ala Gly Ala Pro Gln Arg His Ser 305 310 315 320 Leu Ser Glu
Ile Thr Tyr Leu Gly Gln Pro Asp Ile Ser Ile Leu Gln 325 330 335 Pro
Ser Asn Val Pro Pro Pro Gln Ile Leu Ser Pro Leu Ser Tyr Ala 340 345
350 Pro Asn Ala Ala Pro Glu Val Gly Pro Pro Ser Tyr Ala Pro Gln Val
355 360 365 Thr Pro Glu Ala Gln Phe Pro Phe Tyr Ala Pro Gln Ala Ile
Ser Lys 370 375 380 Val Gln Pro Ser Ser Tyr Ala Pro Gln Ala Thr Pro
Asp Ser Trp Pro 385 390 395 400 Pro Ser Tyr Gly Val Cys Met Glu Gly
Ser Gly Lys Asp Ser Pro Thr 405 410 415 Gly Thr Leu Ser Ser Pro Lys
His Leu Arg Pro Lys Gly Gln Leu Gln 420 425 430 Lys Glu Pro Pro Ala
Gly Ser Cys Met Leu Gly Gly Leu Ser Leu Gln 435 440 445 Glu Val Thr
Ser Leu Ala Met Glu Glu Ser Gln Glu Ala Lys Ser Leu 450 455 460 His
Gln Pro Leu Gly Ile Cys Thr Asp Arg Thr Ser Asp Pro Asn Val 465 470
475 480 Leu His Ser Gly Glu Glu Gly Thr Pro Gln Tyr Leu Lys Gly Gln
Leu 485 490 495 Pro Leu Leu Ser Ser Val Gln Ile Glu Gly His Pro Met
Ser Leu Pro 500 505 510 Leu Gln Pro Pro Ser Gly Pro Cys Ser Pro Ser
Asp Gln Gly Pro Ser 515 520 525 Pro Trp Gly Leu Leu Glu Ser Leu Val
Cys Pro Lys Asp Glu Ala Lys 530 535 540 Ser Pro Ala Pro Glu Thr Ser
Asp Leu Glu Gln Pro Thr Glu Leu Asp 545 550 555 560 Ser Leu Phe Arg
Gly Leu Ala Leu Thr Val Gln Trp Glu Ser 565 570 <210> SEQ ID
NO 3 <211> LENGTH: 211 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 3 Pro Glu Asp Pro Ser
Asp Leu Leu Gln His Val Lys Phe Gln Ser Ser 1 5 10 15 Asn Phe Glu
Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr Pro 20 25 30 Asp
Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg Asp Trp 35 40
45 Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn Leu
50 55 60 Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala Arg
Val Thr 65 70 75 80 Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys Met
Thr Asp Arg Phe 85 90 95 Ser Ser Leu Gln His Thr Thr Leu Lys Pro
Pro Asp Val Thr Cys Ile 100 105 110 Ser Lys Val Arg Ser Ile Gln Met
Ile Val His Pro Thr Pro Thr Pro 115 120 125 Ile Arg Ala Gly Asp Gly
His Arg Leu Thr Leu Glu Asp Ile Phe His 130 135 140 Asp Leu Phe Tyr
His Leu Glu Leu Gln Val Asn Arg Thr Tyr Gln Met 145 150 155 160 His
Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe Phe Gly Leu Thr Pro 165 170
175 Asp Thr Glu Phe Leu Gly Thr Ile Met Ile Cys Val Pro Thr Trp Ala
180 185 190 Lys Glu Ser Ala Pro Tyr Met Cys Arg Val Lys Thr Leu Pro
Asp Arg 195 200 205 Thr Trp Thr 210 <210> SEQ ID NO 4
<211> LENGTH: 541 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: A Soluble IL-22RA-Fc Fusion Polypeptide <400>
SEQUENCE: 4 Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val Lys Phe Gln
Ser Ser 1 5 10 15 Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro
Glu Gly Thr Pro 20 25 30 Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr
Tyr Gly Glu Arg Asp Trp 35 40 45 Val Ala Lys Lys Gly Cys Gln Arg
Ile Thr Arg Lys Ser Cys Asn Leu 50 55 60 Thr Val Glu Thr Gly Asn
Leu Thr Glu Leu Tyr Tyr Ala Arg Val Thr 65 70 75 80 Ala Val Ser Ala
Gly Gly Arg Ser Ala Thr Lys Met Thr Asp Arg Phe 85 90 95 Ser Ser
Leu Gln His Thr Thr Leu Lys Pro Pro Asp Val Thr Cys Ile 100 105 110
Ser Lys Val Arg Ser Ile Gln Met Ile Val His Pro Thr Pro Thr Pro 115
120 125 Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu Glu Asp Ile Phe
His 130 135 140 Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn Arg Thr
Tyr Gln Met 145 150 155 160 His Leu Gly Gly Lys Gln Arg Glu Tyr Glu
Phe Phe Gly Leu Thr Pro 165 170 175 Asp Thr Glu Phe Leu Gly Thr Ile
Met Ile Cys Val Pro Thr Trp Ala 180 185 190 Lys Glu Ser Ala Pro Tyr
Met Cys Arg Val Lys Thr Leu Pro Asp Arg 195 200 205 Thr Trp Thr Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 210 215 220 Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 225 230 235
240 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
245 250 255 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly 260 265 270 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly 275 280 285 Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys 290 295 300 Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys 305 310 315 320 Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 325 330 335 Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 340 345 350 Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 355 360
365 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
370 375 380 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu 385 390 395 400 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys 405 410 415 Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys 420 425 430 Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser 435 440 445 Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 450 455 460 Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 465 470 475 480
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 485
490 495 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln 500 505 510 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn 515 520 525 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 530 535 540 <210> SEQ ID NO 5 <211> LENGTH:
1116 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (21)...(557) <400> SEQUENCE: 5 tcgagttaga
attgtctgca atg gcc gcc ctg cag aaa tct gtg agc tct ttc 53 Met Ala
Ala Leu Gln Lys Ser Val Ser Ser Phe 1 5 10 ctt atg ggg acc ctg gcc
acc agc tgc ctc ctt ctc ttg gcc ctc ttg 101 Leu Met Gly Thr Leu Ala
Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu 15 20 25 gta cag gga gga
gca gct gcg ccc atc agc tcc cac tgc agg ctt gac 149 Val Gln Gly Gly
Ala Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp 30 35 40
aag tcc aac ttc cag cag ccc tat atc acc aac cgc acc ttc atg ctg 197
Lys Ser Asn Phe Gln Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu 45
50 55 gct aag gag gct agc ttg gct gat aac aac aca gac gtt cgt ctc
att 245 Ala Lys Glu Ala Ser Leu Ala Asp Asn Asn Thr Asp Val Arg Leu
Ile 60 65 70 75 ggg gag aaa ctg ttc cac gga gtc agt atg agt gag cgc
tgc tat ctg 293 Gly Glu Lys Leu Phe His Gly Val Ser Met Ser Glu Arg
Cys Tyr Leu 80 85 90 atg aag cag gtg ctg aac ttc acc ctt gaa gaa
gtg ctg ttc cct caa 341 Met Lys Gln Val Leu Asn Phe Thr Leu Glu Glu
Val Leu Phe Pro Gln 95 100 105 tct gat agg ttc cag cct tat atg cag
gag gtg gtg ccc ttc ctg gcc 389 Ser Asp Arg Phe Gln Pro Tyr Met Gln
Glu Val Val Pro Phe Leu Ala 110 115 120 agg ctc agc aac agg cta agc
aca tgt cat att gaa ggt gat gac ctg 437 Arg Leu Ser Asn Arg Leu Ser
Thr Cys His Ile Glu Gly Asp Asp Leu 125 130 135 cat atc cag agg aat
gtg caa aag ctg aag gac aca gtg aaa aag ctt 485 His Ile Gln Arg Asn
Val Gln Lys Leu Lys Asp Thr Val Lys Lys Leu 140 145 150 155 gga gag
agt gga gag atc aaa gca att gga gaa ctg gat ttg ctg ttt 533 Gly Glu
Ser Gly Glu Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe 160 165 170
atg tct ctg aga aat gcc tgc att tgaccagagc aaagctgaaa aatgaataac
587 Met Ser Leu Arg Asn Ala Cys Ile 175 taaccccctt tccctgctag
aaataacaat tagatgcccc aaagcgattt tttttaacca 647 aaaggaagat
gggaagccaa actccatcat gatgggtgga ttccaaatga acccctgcgt 707
tagttacaaa ggaaaccaat gccacttttg tttataagac cagaaggtag actttctaag
767 catagatatt tattgataac atttcattgt aactggtgtt ctatacacag
aaaacaattt 827 attttttaaa taattgtctt tttccataaa aaagattact
ttccattcct ttaggggaaa 887 aaacccctaa atagcttcat gtttccataa
tcagtacttt atatttataa atgtatttat 947 tattattata agactgcatt
ttatttatat cattttatta atatggattt atttatagaa 1007 acatcattcg
atattgctac ttgagtgtaa ggctaatatt gatatttatg acaataatta 1067
tagagctata acatgtttat ttgacctcaa taaacacttg gatatccta 1116
<210> SEQ ID NO 6 <211> LENGTH: 179 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 6 Met
Ala Ala Leu Gln Lys Ser Val Ser Ser Phe Leu Met Gly Thr Leu 1 5 10
15 Ala Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu Val Gln Gly Gly Ala
20 25 30 Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp Lys Ser Asn
Phe Gln 35 40 45 Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu Ala
Lys Glu Ala Ser 50 55 60 Leu Ala Asp Asn Asn Thr Asp Val Arg Leu
Ile Gly Glu Lys Leu Phe 65 70 75 80 His Gly Val Ser Met Ser Glu Arg
Cys Tyr Leu Met Lys Gln Val Leu 85 90 95 Asn Phe Thr Leu Glu Glu
Val Leu Phe Pro Gln Ser Asp Arg Phe Gln 100 105 110 Pro Tyr Met Gln
Glu Val Val Pro Phe Leu Ala Arg Leu Ser Asn Arg 115 120 125 Leu Ser
Thr Cys His Ile Glu Gly Asp Asp Leu His Ile Gln Arg Asn 130 135 140
Val Gln Lys Leu Lys Asp Thr Val Lys Lys Leu Gly Glu Ser Gly Glu 145
150 155 160 Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu
Arg Asn 165 170 175 Ala Cys Ile <210> SEQ ID NO 7 <211>
LENGTH: 926 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (45)...(575) <221> NAME/KEY: variation <222>
LOCATION: (188)...(188) <223> OTHER INFORMATION: Nucleotide
may be C or G at position 188 <400> SEQUENCE: 7 ctttgaattc
ctagctcctg tggtctccag atttcaggcc taag atg aaa gcc tct 56 Met Lys
Ala Ser 1 agt ctt gcc ttc agc ctt ctc tct gct gcg ttt tat ctc cta
tgg act 104 Ser Leu Ala Phe Ser Leu Leu Ser Ala Ala Phe Tyr Leu Leu
Trp Thr 5 10 15 20 cct tcc act gga ctg aag aca ctc aat ttg gga agc
tgt gtg atc gcc 152 Pro Ser Thr Gly Leu Lys Thr Leu Asn Leu Gly Ser
Cys Val Ile Ala 25 30 35 aca aac ctt cag gaa ata cga aat gga ttt
tct gas ata cgg ggc agt 200 Thr Asn Leu Gln Glu Ile Arg Asn Gly Phe
Ser Xaa Ile Arg Gly Ser 40 45 50 gtg caa gcc aaa gat gga aac att
gac atc aga atc tta agg agg act 248 Val Gln Ala Lys Asp Gly Asn Ile
Asp Ile Arg Ile Leu Arg Arg Thr 55 60 65 gag tct ttg caa gac aca
aag cct gcg aat cga tgc tgc ctc ctg cgc 296 Glu Ser Leu Gln Asp Thr
Lys Pro Ala Asn Arg Cys Cys Leu Leu Arg 70 75 80 cat ttg cta aga
ctc tat ctg gac agg gta ttt aaa aac tac cag acc 344 His Leu Leu Arg
Leu Tyr Leu Asp Arg Val Phe Lys Asn Tyr Gln Thr 85 90 95 100 cct
gac cat tat act ctc cgg aag atc agc agc ctc gcc aat tcc ttt 392 Pro
Asp His Tyr Thr Leu Arg Lys Ile Ser Ser Leu Ala Asn Ser Phe 105 110
115 ctt acc atc aag aag gac ctc cgg ctc tgt cat gcc cac atg aca tgc
440 Leu Thr Ile Lys Lys Asp Leu Arg Leu Cys His Ala His Met Thr Cys
120 125 130 cat tgt ggg gag gaa gca atg aag aaa tac agc cag att ctg
agt cac 488 His Cys Gly Glu Glu Ala Met Lys Lys Tyr Ser Gln Ile Leu
Ser His 135 140 145 ttt gaa aag ctg gaa cct cag gca gca gtt gtg aag
gct ttg ggg gaa 536 Phe Glu Lys Leu Glu Pro Gln Ala Ala Val Val Lys
Ala Leu Gly Glu 150 155 160 cta gac att ctt ctg caa tgg atg gag gag
aca gaa tag gaggaaagtg 585 Leu Asp Ile Leu Leu Gln Trp Met Glu Glu
Thr Glu * 165 170 175 atgctgctgc taagaatatt cgaggtcaag agctccagtc
ttcaatacct gcagaggagg 645 catgacccca aaccaccatc tctttactgt
actagtcttg tgctggtcac agtgtatctt 705 atttatgcat tacttgcttc
cttgcatgat tgtctttatg catccccaat cttaattgag 765 accatacttg
tataagattt ttgtaatatc tttctgctat tggatatatt tattagttaa 825
tatatttatt tattttttgc tattaatgta tttaattttt tacttgggca tgaaacttta
885 aaaaaaattc acaagattat atttataacc tgactagagc a 926 <210>
SEQ ID NO 8 <211> LENGTH: 176 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: VARIANT <222> LOCATION: (48)...(48) <223>
OTHER INFORMATION: Amino acid at position 48 can be a D (Asp) or E
(Glu) <221> NAME/KEY: VARIANT <222> LOCATION: 48
<223> OTHER INFORMATION: Xaa = Any Amino Acid <400>
SEQUENCE: 8 Met Lys Ala Ser Ser Leu Ala Phe Ser Leu Leu Ser Ala Ala
Phe Tyr 1 5 10 15 Leu Leu Trp Thr Pro Ser Thr Gly Leu Lys Thr Leu
Asn Leu Gly Ser 20 25 30 Cys Val Ile Ala Thr Asn Leu Gln Glu Ile
Arg Asn Gly Phe Ser Xaa 35 40 45 Ile Arg Gly Ser Val Gln Ala Lys
Asp Gly Asn Ile Asp Ile Arg Ile 50 55 60 Leu Arg Arg Thr Glu Ser
Leu Gln Asp Thr Lys Pro Ala Asn Arg Cys 65 70 75 80 Cys Leu Leu Arg
His Leu Leu Arg Leu Tyr Leu Asp Arg Val Phe Lys 85 90 95 Asn Tyr
Gln Thr Pro Asp His Tyr Thr Leu Arg Lys Ile Ser Ser Leu 100 105 110
Ala Asn Ser Phe Leu Thr Ile Lys Lys Asp Leu Arg Leu Cys His Ala 115
120 125 His Met Thr Cys His Cys Gly Glu Glu Ala Met Lys Lys Tyr Ser
Gln 130 135 140 Ile Leu Ser His Phe Glu Lys Leu Glu Pro Gln Ala Ala
Val Val Lys 145 150 155 160 Ala Leu Gly Glu Leu Asp Ile Leu Leu Gln
Trp Met Glu Glu Thr Glu 165 170 175 <210> SEQ ID NO 9
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Peptide Linker <400> SEQUENCE: 9 Gly Gly Ser Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
<210> SEQ ID NO 10 <211> LENGTH: 1050 <212> TYPE:
DNA <213> ORGANISM: Mus musculus <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (5)...(589)
<400> SEQUENCE: 10 aaca ggc tct cct ctc act tat caa ctt ttg
aca ctt gtg cga tcg gtg 49 Gly Ser Pro Leu Thr Tyr Gln Leu Leu Thr
Leu Val Arg Ser Val 1 5 10 15 atg gct gtc ctg cag aaa tct atg agt
ttt tcc ctt atg ggg act ttg 97 Met Ala Val Leu Gln Lys Ser Met Ser
Phe Ser Leu Met Gly Thr Leu
20 25 30 gcc gcc agc tgc ctg ctt ctc att gcc ctg tgg gcc cag gag
gca aat 145 Ala Ala Ser Cys Leu Leu Leu Ile Ala Leu Trp Ala Gln Glu
Ala Asn 35 40 45 gcg ctg ccc atc aac acc cgg tgc aag ctt gag gtg
tcc aac ttc cag 193 Ala Leu Pro Ile Asn Thr Arg Cys Lys Leu Glu Val
Ser Asn Phe Gln 50 55 60 cag ccg tac atc gtc aac cgc acc ttt atg
ctg gcc aag gag gcc agc 241 Gln Pro Tyr Ile Val Asn Arg Thr Phe Met
Leu Ala Lys Glu Ala Ser 65 70 75 ctt gca gat aac aac aca gac gtc
cgg ctc atc ggg gag aaa ctg ttc 289 Leu Ala Asp Asn Asn Thr Asp Val
Arg Leu Ile Gly Glu Lys Leu Phe 80 85 90 95 cga gga gtc agt gct aag
gat cag tgc tac ctg atg aag cag gtg ctc 337 Arg Gly Val Ser Ala Lys
Asp Gln Cys Tyr Leu Met Lys Gln Val Leu 100 105 110 aac ttc acc ctg
gaa gac att ctg ctc ccc cag tca gac agg ttc cgg 385 Asn Phe Thr Leu
Glu Asp Ile Leu Leu Pro Gln Ser Asp Arg Phe Arg 115 120 125 ccc tac
atg cag gag gtg gtg cct ttc ctg acc aaa ctc agc aat cag 433 Pro Tyr
Met Gln Glu Val Val Pro Phe Leu Thr Lys Leu Ser Asn Gln 130 135 140
ctc agc tcc tgt cac atc agt ggt gac gac cag aac atc cag aag aat 481
Leu Ser Ser Cys His Ile Ser Gly Asp Asp Gln Asn Ile Gln Lys Asn 145
150 155 gtc aga agg ctg aag gag aca gtg aaa aag ctt gga gag agc gga
gag 529 Val Arg Arg Leu Lys Glu Thr Val Lys Lys Leu Gly Glu Ser Gly
Glu 160 165 170 175 atc aaa gcg atc ggg gaa ctg gac ctg ctg ttt atg
tct ctg aga aat 577 Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met
Ser Leu Arg Asn 180 185 190 gct tgc gtc tga gcgagaagaa gctagaaaac
gaagaactgc tccttcctgc 629 Ala Cys Val * cttctaaaaa gaacaataag
atccctgaat ggactttttt actaaaggaa agtgagaagc 689 taacgtccac
catcattaga agatttcaca tgaaacctgg ctcagttgaa agagaaaata 749
gtgtcaagtt gtccatgaga ccagaggtag acttgataac cacaaagatt cattgacaat
809 attttattgt cattgataat gcaacagaaa aagtatgtac tttaaaaaat
tgtttgaaag 869 gaggttacct ctcattcctc tagaagaaaa gcctatgtaa
cttcatttcc ataaccaata 929 ctttatatat gtaagtttat ttattataag
tatacatttt atttatgtca gtttattaat 989 atggatttat ttatagaaaa
attatctgat gttgatattt gagtataaag caaataatat 1049 t 1050 <210>
SEQ ID NO 11 <211> LENGTH: 194 <212> TYPE: PRT
<213> ORGANISM: Mus musculus <400> SEQUENCE: 11 Gly Ser
Pro Leu Thr Tyr Gln Leu Leu Thr Leu Val Arg Ser Val Met 1 5 10 15
Ala Val Leu Gln Lys Ser Met Ser Phe Ser Leu Met Gly Thr Leu Ala 20
25 30 Ala Ser Cys Leu Leu Leu Ile Ala Leu Trp Ala Gln Glu Ala Asn
Ala 35 40 45 Leu Pro Ile Asn Thr Arg Cys Lys Leu Glu Val Ser Asn
Phe Gln Gln 50 55 60 Pro Tyr Ile Val Asn Arg Thr Phe Met Leu Ala
Lys Glu Ala Ser Leu 65 70 75 80 Ala Asp Asn Asn Thr Asp Val Arg Leu
Ile Gly Glu Lys Leu Phe Arg 85 90 95 Gly Val Ser Ala Lys Asp Gln
Cys Tyr Leu Met Lys Gln Val Leu Asn 100 105 110 Phe Thr Leu Glu Asp
Ile Leu Leu Pro Gln Ser Asp Arg Phe Arg Pro 115 120 125 Tyr Met Gln
Glu Val Val Pro Phe Leu Thr Lys Leu Ser Asn Gln Leu 130 135 140 Ser
Ser Cys His Ile Ser Gly Asp Asp Gln Asn Ile Gln Lys Asn Val 145 150
155 160 Arg Arg Leu Lys Glu Thr Val Lys Lys Leu Gly Glu Ser Gly Glu
Ile 165 170 175 Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu
Arg Asn Ala 180 185 190 Cys Val <210> SEQ ID NO 12
<211> LENGTH: 2149 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (1)...(693) <400> SEQUENCE: 12 atg
atg cct aaa cat tgc ttt cta ggc ttc ctc atc agt ttc ttc ctt 48 Met
Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu 1 5 10
15 act ggt gta gca gga act cag tca acg cat gag tct ctg aag cct cag
96 Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
20 25 30 agg gta caa ttt cag tcc cga aat ttt cac aac att ttg caa
tgg cag 144 Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln
Trp Gln 35 40 45 cct ggg agg gca ctt act ggc aac agc agt gtc tat
ttt gtg cag tac 192 Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr
Phe Val Gln Tyr 50 55 60 aaa ata tat gga cag aga caa tgg aaa aat
aaa gaa gac tgt tgg ggt 240 Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn
Lys Glu Asp Cys Trp Gly 65 70 75 80 act caa gaa ctc tct tgt gac ctt
acc agt gaa acc tca gac ata cag 288 Thr Gln Glu Leu Ser Cys Asp Leu
Thr Ser Glu Thr Ser Asp Ile Gln 85 90 95 gaa cct tat tac ggg agg
gtg agg gcg gcc tcg gct ggg agc tac tca 336 Glu Pro Tyr Tyr Gly Arg
Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser 100 105 110 gaa tgg agc atg
acg ccg cgg ttc act ccc tgg tgg gaa aca aaa ata 384 Glu Trp Ser Met
Thr Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys Ile 115 120 125 gat cct
cca gtc atg aat ata acc caa gtc aat ggc tct ttg ttg gta 432 Asp Pro
Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu Leu Val 130 135 140
att ctc cat gct cca aat tta cca tat aga tac caa aag gaa aaa aat 480
Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn 145
150 155 160 gta tct ata gaa gat tac tat gaa cta cta tac cga gtt ttt
ata att 528 Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe
Ile Ile 165 170 175 aac aat tca cta gaa aag gag caa aag gtt tat gaa
ggg gct cac aga 576 Asn Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu
Gly Ala His Arg 180 185 190 gcg gtt gaa att gaa gct cta aca cca cac
tcc agc tac tgt gta gtg 624 Ala Val Glu Ile Glu Ala Leu Thr Pro His
Ser Ser Tyr Cys Val Val 195 200 205 gct gaa ata tat cag ccc atg tta
gac aga aga agt cag aga agt gaa 672 Ala Glu Ile Tyr Gln Pro Met Leu
Asp Arg Arg Ser Gln Arg Ser Glu 210 215 220 gag aga tgt gtg gaa att
cca tgacttgtgg aatttggcat tcagcaatgt 723 Glu Arg Cys Val Glu Ile
Pro 225 230 ggaaattcta aagctccctg agaacaggat gactcgtgtt tgaaggatct
tatttaaaat 783 tgtttttgta ttttcttaaa gcaatattca ctgttacacc
ttggggactt ctttgtttat 843 ccattctttt atcctttata tttcatttta
aactatattt gaacgacatt ccccccgaaa 903 aattgaaatg taaagatgag
gcagagaata aagtgttcta tgaaattcag aactttattt 963 ctgaatgtaa
catccctaat aacaaccttc attcttctaa tacagcaaaa taaaaattta 1023
acaaccaagg aatagtattt aagaaaatgt tgaaataatt tttttaaaat agcattacag
1083 actgaggcgg tcctgaagca atggtttttc actctcttat tgagccaatt
aaattgacat 1143 tgctttgaca atttaaaact tctataaagg tgaatatttt
tcatacattt ctattttata 1203 tgaatatact ttttatatat ttattattat
taaatatttc tacttaatga atcaaaattt 1263 tgttttaaag tctactttat
gtaaataaga acaggttttg gggaaaaaaa tcttatgatt 1323 tctggattga
tatctgaatt aaaactatca acaacaagga agtctactct gtacaattgt 1383
ccctcattta aaagatatat taagcttttc ttttctgttt gtttttgttt tgtttagttt
1443 ttaatcctgt cttagaagaa cttatcttta ttctcaaaat taaatgtaat
ttttttagtg 1503 acaaagaaga aaggaaacct cattactcaa tccttctggc
caagagtgtc ttgcttgtgg 1563 cgccttcctc atctctatat aggaggatcc
catgaatgat ggtttattgg gaactgctgg 1623 ggtcgacccc atacagagaa
ctcagcttga agctggaagc acacagtggg tagcaggaga 1683 aggaccggtg
ttggtaggtg cctacagaga ctatagagct agacaaagcc ctccaaactg 1743
gcccctcctg ctcactgcct ctcctgagta gaaatctggt gacctaaggc tcagtgcggt
1803 caacagaaag ctgccttctt cacttgaggc taagtcttca tatatgttta
aggttgtctt 1863 tctagtgagg agatacatat cagagaacat ttgtacaatt
ccccatgaaa attgctccaa 1923 agttgataac aatatagtcg gtgcttctag
ttatatgcaa gtactcagtg ataaatggat 1983 taaaaaatat tcagaaatgt
attggggggt ggaggagaat aagaggcaga gcaagagcta 2043 gagaattggt
ttccttgctt ccctgtatgc tcagaaaaca ttgatttgag catagacgca 2103
gagactgaaa aaaaaaaaat gctcgagcgg ccgccatatc cttggt 2149 <210>
SEQ ID NO 13 <211> LENGTH: 231 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 13 Met Met
Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu 1 5 10 15
Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln 20
25 30 Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp
Gln 35 40 45 Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe
Val Gln Tyr 50 55 60 Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys
Glu Asp Cys Trp Gly 65 70 75 80 Thr Gln Glu Leu Ser Cys Asp Leu Thr
Ser Glu Thr Ser Asp Ile Gln 85 90 95
Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser 100
105 110 Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys
Ile 115 120 125 Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser
Leu Leu Val 130 135 140 Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr
Gln Lys Glu Lys Asn 145 150 155 160 Val Ser Ile Glu Asp Tyr Tyr Glu
Leu Leu Tyr Arg Val Phe Ile Ile 165 170 175 Asn Asn Ser Leu Glu Lys
Glu Gln Lys Val Tyr Glu Gly Ala His Arg 180 185 190 Ala Val Glu Ile
Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val 195 200 205 Ala Glu
Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser Glu 210 215 220
Glu Arg Cys Val Glu Ile Pro 225 230 <210> SEQ ID NO 14
<211> LENGTH: 699 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: C-Terminal Fc4 tag <400> SEQUENCE: 14 gagcccagat
cttcagacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgag 60
ggggcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg
120 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga
ggtcaagttc 180 aactggtacg tggacggcgt ggaggtgcat aatgccaaga
caaagccgcg ggaggagcag 240 tacaacagca cgtaccgtgt ggtcagcgtc
ctcaccgtcc tgcaccagga ctggctgaat 300 ggcaaggagt acaagtgcaa
ggtctccaac aaagccctcc catcctccat cgagaaaacc 360 atctccaaag
ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc
480 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa
gaccacgcct 540 cccgtgctgg actccgacgg ctccttcttc ctctacagca
agctcaccgt ggacaagagc 600 aggtggcagc aggggaacgt cttctcatgc
tccgtgatgc atgaggctct gcacaaccac 660 tacacgcaga agagcctctc
cctgtctccg ggtaaataa 699 <210> SEQ ID NO 15 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Glu-Glu (CEE) Peptide Tag <400> SEQUENCE: 15 Glu Tyr Met Pro
Met Glu 1 5 <210> SEQ ID NO 16 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Glu-Glu (CEE)
Peptide Tag with spacer <400> SEQUENCE: 16 Gly Ser Gly Gly
Glu Tyr Met Pro Met Glu 1 5 10 <210> SEQ ID NO 17 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Oligonucleotide primer ZC39289 <400> SEQUENCE: 17 tccgaggagt
caatgctaag 20 <210> SEQ ID NO 18 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide
Primer ZC39290 <400> SEQUENCE: 18 tccaagcttt ttcactgtct 20
<210> SEQ ID NO 19 <211> LENGTH: 16 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide Primer ZC39776
<400> SEQUENCE: 19 gggcccgcta gcacct 16 <210> SEQ ID NO
20 <211> LENGTH: 16 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Oligonucleotide Primer ZC39777 <400>
SEQUENCE: 20 gggtgatccg ctggca 16 <210> SEQ ID NO 21
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: IL-20 FAM/TAMRA labeled TaqMan probe ZC38752
<400> SEQUENCE: 21 ccagccactt tctctctccg tatttcttat attcca 36
<210> SEQ ID NO 22 <211> LENGTH: 16 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: forward primer, ZC42459 <400>
SEQUENCE: 22 tggccaggct cagcaa 16 <210> SEQ ID NO 23
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: reverse primer, ZC42458 <400> SEQUENCE: 23
gcacattcct ctggatatgc a 21 <210> SEQ ID NO 24 <211>
LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: IL-22
TaqMan probe, ZC42460 <400> SEQUENCE: 24 aggctaagca
catgtcatat tgaaggtgat g 31 <210> SEQ ID NO 25 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
forward primer, ZC40541 <400> SEQUENCE: 25 tcgccaattc
ctttcttacc a 21 <210> SEQ ID NO 26 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: reverse primer,
ZC40542 <400> SEQUENCE: 26 cccacaatgg catgtcatgt 20
<210> SEQ ID NO 27 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: IL-20 TaqMan? probe ZC40544
<400> SEQUENCE: 27 agaaggacct ccggctctgt catgc 25 <210>
SEQ ID NO 28 <211> LENGTH: 57 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide primer ZC45,593
<400> SEQUENCE: 28 caggaaatcc atgccgagtt gagacgcttc
cgtagacacg cccctgagga cccctcg 57 <210> SEQ ID NO 29
<211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Oligonucleotide primer ZC45,592 <400> SEQUENCE:
29 tctgggctca ccgcttccag acccgcttcc agacccgctt cctgtccggt
ctggcagtgt 60 ctt 63 <210> SEQ ID NO 30 <211> LENGTH:
63 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence
<220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide
primer ZC45,591 <400> SEQUENCE: 30 gaccggacag gaagcgggtc
tggaagcggg tctggaagcg gtgagcccag aggccccaca 60 atc 63 <210>
SEQ ID NO 31 <211> LENGTH: 57 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide primer ZC45,594
<400> SEQUENCE: 31 agagctgttt taaggcgcgc ctctagatta
tttttattta cccggagtcc gggagaa 57 <210> SEQ ID NO 32
<211> LENGTH: 531 <212> TYPE: DNA <213> ORGANISM:
Mus musculus <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (1)...(531) <400> SEQUENCE: 32 atg aaa
ggc ttt ggt ctt gcc ttt gga ctg ttc tcc gct gtg ggt ttt 48 Met Lys
Gly Phe Gly Leu Ala Phe Gly Leu Phe Ser Ala Val Gly Phe 1 5 10 15
ctt ctc tgg act cct tta act ggg ctc aag acc ctc cat ttg gga agc 96
Leu Leu Trp Thr Pro Leu Thr Gly Leu Lys Thr Leu His Leu Gly Ser 20
25 30 tgt gtg att act gca aac cta cag gca ata caa aag gaa ttt tct
gag 144 Cys Val Ile Thr Ala Asn Leu Gln Ala Ile Gln Lys Glu Phe Ser
Glu 35 40 45 att cgg gat agt gtg caa gct gaa gat aca aat att gac
atc aga att 192 Ile Arg Asp Ser Val Gln Ala Glu Asp Thr Asn Ile Asp
Ile Arg Ile 50 55 60 tta agg acg act gag tct ttg aaa gac ata aag
tct ttg gat agg tgc 240 Leu Arg Thr Thr Glu Ser Leu Lys Asp Ile Lys
Ser Leu Asp Arg Cys 65 70 75 80 tgc ttc ctt cgt cat cta gtg aga ttc
tat ctg gac agg gta ttc aaa 288 Cys Phe Leu Arg His Leu Val Arg Phe
Tyr Leu Asp Arg Val Phe Lys 85 90 95 gtc tac cag acc cct gac cac
cat acc ctg aga aag atc agc agc ctc 336 Val Tyr Gln Thr Pro Asp His
His Thr Leu Arg Lys Ile Ser Ser Leu 100 105 110 gcc aac tcc ttt ctt
atc atc aag aag gac ctc tca gtc tgt cat tct 384 Ala Asn Ser Phe Leu
Ile Ile Lys Lys Asp Leu Ser Val Cys His Ser 115 120 125 cac atg gca
tgt cat tgt ggg gaa gaa gca atg gag aaa tac aac caa 432 His Met Ala
Cys His Cys Gly Glu Glu Ala Met Glu Lys Tyr Asn Gln 130 135 140 att
ctg agt cac ttc ata gag ttg gaa ctt cag gca gcg gtg gta aag 480 Ile
Leu Ser His Phe Ile Glu Leu Glu Leu Gln Ala Ala Val Val Lys 145 150
155 160 gct ttg gga gaa cta ggc att ctt ctg aga tgg atg gag gag atg
cta 528 Ala Leu Gly Glu Leu Gly Ile Leu Leu Arg Trp Met Glu Glu Met
Leu 165 170 175 tag 531 * <210> SEQ ID NO 33 <211>
LENGTH: 176 <212> TYPE: PRT <213> ORGANISM: Mus
musculus <400> SEQUENCE: 33 Met Lys Gly Phe Gly Leu Ala Phe
Gly Leu Phe Ser Ala Val Gly Phe 1 5 10 15 Leu Leu Trp Thr Pro Leu
Thr Gly Leu Lys Thr Leu His Leu Gly Ser 20 25 30 Cys Val Ile Thr
Ala Asn Leu Gln Ala Ile Gln Lys Glu Phe Ser Glu 35 40 45 Ile Arg
Asp Ser Val Gln Ala Glu Asp Thr Asn Ile Asp Ile Arg Ile 50 55 60
Leu Arg Thr Thr Glu Ser Leu Lys Asp Ile Lys Ser Leu Asp Arg Cys 65
70 75 80 Cys Phe Leu Arg His Leu Val Arg Phe Tyr Leu Asp Arg Val
Phe Lys 85 90 95 Val Tyr Gln Thr Pro Asp His His Thr Leu Arg Lys
Ile Ser Ser Leu 100 105 110 Ala Asn Ser Phe Leu Ile Ile Lys Lys Asp
Leu Ser Val Cys His Ser 115 120 125 His Met Ala Cys His Cys Gly Glu
Glu Ala Met Glu Lys Tyr Asn Gln 130 135 140 Ile Leu Ser His Phe Ile
Glu Leu Glu Leu Gln Ala Ala Val Val Lys 145 150 155 160 Ala Leu Gly
Glu Leu Gly Ile Leu Leu Arg Trp Met Glu Glu Met Leu 165 170 175
<210> SEQ ID NO 34 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide primer ZC22901
<400> SEQUENCE: 34 catcaaaccg cctgatgtga c 21 <210> SEQ
ID NO 35 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Oligonucleotide primer ZC45039 <400>
SEQUENCE: 35 attaggcttg ggagggaatg g 21 <210> SEQ ID NO 36
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Oligonucleotide primer ZC38573 <400> SEQUENCE:
36 tggcgatgcc tgcttgccga ata 23 <210> SEQ ID NO 37
<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Oligonucleotide primer ZC25223 <400> SEQUENCE:
37 gtcttcctca catctgttat cg 22 <210> SEQ ID NO 38 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Oligonucleotide primer ZC40128 <400> SEQUENCE: 38 ggcttgaact
ttgagaaagg cagt 24 <210> SEQ ID NO 39 <211> LENGTH:
1473 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
IL-22RA Extracellular domain with tPA leader and fused to murine
gamma 2a heavy chain Fc region (mG2a) <221> NAME/KEY: CDS
<222> LOCATION: (1)...(1473) <400> SEQUENCE: 39 atg gat
gca atg aag aga ggg ctc tgc tgt gtg ctg ctg ctg tgt ggc 48 Met Asp
Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15
gcc gtc ttc gtt tcg ctc agc cag gaa atc cat gcc gag ttg aga cgc 96
Ala Val Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu Leu Arg Arg 20
25 30 ttc cgt aga cac gcc cct gag gac ccc tcg gat ctg ctc cag cac
gtg 144 Phe Arg Arg His Ala Pro Glu Asp Pro Ser Asp Leu Leu Gln His
Val 35 40 45 aaa ttc cag tcc agc aac ttt gaa aac atc ctg acg tgg
gac agc ggg 192 Lys Phe Gln Ser Ser Asn Phe Glu Asn Ile Leu Thr Trp
Asp Ser Gly 50 55 60 cca gag ggc acc cca gac acg gtc tac agc atc
gag tat aag acg tac 240 Pro Glu Gly Thr Pro Asp Thr Val Tyr Ser Ile
Glu Tyr Lys Thr Tyr 65 70 75 80 gga gag agg gac tgg gtg gca aag aag
ggc tgt cag cgg atc acc cgg 288 Gly Glu Arg Asp Trp Val Ala Lys Lys
Gly Cys Gln Arg Ile Thr Arg 85 90 95 aag tcc tgc aac ctg acg gtg
gag acg ggc aac ctc acg gag ctc tac 336 Lys Ser Cys Asn Leu Thr Val
Glu Thr Gly Asn Leu Thr Glu Leu Tyr 100 105 110 tat gcc agg gtc acc
gct gtc agt gcg gga ggc cgg tca gcc acc aag 384 Tyr Ala Arg Val Thr
Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys 115 120 125 atg act gac
agg ttc agc tct ctg cag cac act acc ctc aag cca cct 432 Met Thr Asp
Arg Phe Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro 130 135 140 gat
gtg acc tgt atc tcc aaa gtg aga tcg att cag atg att gtt cat 480 Asp
Val Thr Cys Ile Ser Lys Val Arg Ser Ile Gln Met Ile Val His 145 150
155 160 cct acc ccc acg cca atc cgt gca ggc gat ggc cac cgg cta acc
ctg 528 Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr
Leu 165 170 175 gaa gac atc ttc cat gac ctg ttc tac cac tta gag ctc
cag gtc aac 576 Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu Glu Leu
Gln Val Asn 180 185 190 cgc acc tac caa atg cac ctt gga ggg aag cag
aga gaa tat gag ttc 624 Arg Thr Tyr Gln Met His Leu Gly Gly Lys Gln
Arg Glu Tyr Glu Phe 195 200 205 ttc ggc ctg acc cct gac aca gag ttc
ctt ggc acc atc atg att tgc 672 Phe Gly Leu Thr Pro Asp Thr Glu Phe
Leu Gly Thr Ile Met Ile Cys 210 215 220 gtt ccc acc tgg gcc aag gag
agt gcc ccc tac atg tgc cga gtg aag 720
Val Pro Thr Trp Ala Lys Glu Ser Ala Pro Tyr Met Cys Arg Val Lys 225
230 235 240 aca ctg cca gac cgg aca gga agc ggg tct gga agc ggg tct
gga agc 768 Thr Leu Pro Asp Arg Thr Gly Ser Gly Ser Gly Ser Gly Ser
Gly Ser 245 250 255 ggt gag ccc aga ggc ccc aca atc aag ccc tgt cct
cca tgc aaa tgc 816 Gly Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro
Pro Cys Lys Cys 260 265 270 cca gca cct aac ctc ttg ggt gga cca tcc
gtc ttc atc ttc cct cca 864 Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser
Val Phe Ile Phe Pro Pro 275 280 285 aag atc aag gat gta ctc atg atc
tcc ctg agc ccc ata gtc aca tgt 912 Lys Ile Lys Asp Val Leu Met Ile
Ser Leu Ser Pro Ile Val Thr Cys 290 295 300 gtg gtg gtg gat gtg agc
gag gat gac cca gat gtc cag atc agc tgg 960 Val Val Val Asp Val Ser
Glu Asp Asp Pro Asp Val Gln Ile Ser Trp 305 310 315 320 ttt gtg aac
aac gtg gaa gta cac aca gct cag aca caa acc cat aga 1008 Phe Val
Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg 325 330 335
gag gat tac aac agt act ctc cgg gtg gtc agt gcc ctc ccc atc cag
1056 Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile
Gln 340 345 350 cac cag gac tgg atg agt ggc aag gag ttc aaa tgc aag
gtc aac aac 1104 His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys
Lys Val Asn Asn 355 360 365 aaa gac ctc cca gcg ccc atc gag aga acc
atc tca aaa ccc aaa ggg 1152 Lys Asp Leu Pro Ala Pro Ile Glu Arg
Thr Ile Ser Lys Pro Lys Gly 370 375 380 tca gta aga gct cca cag gta
tat gtc ttg cct cca cca gaa gaa gag 1200 Ser Val Arg Ala Pro Gln
Val Tyr Val Leu Pro Pro Pro Glu Glu Glu 385 390 395 400 atg act aag
aaa cag gtc act ctg acc tgc atg gtc aca gac ttc atg 1248 Met Thr
Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met 405 410 415
cct gaa gac att tac gtg gag tgg acc aac aac ggg aaa aca gag cta
1296 Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu
Leu 420 425 430 aac tac aag aac act gaa cca gtc ctg gac tct gat ggt
tct tac ttc 1344 Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp
Gly Ser Tyr Phe 435 440 445 atg tac agc aag ctg aga gtg gaa aag aag
aac tgg gtg gaa aga aat 1392 Met Tyr Ser Lys Leu Arg Val Glu Lys
Lys Asn Trp Val Glu Arg Asn 450 455 460 agc tac tcc tgt tca gtg gtc
cac gag ggt ctg cac aat cac cac acg 1440 Ser Tyr Ser Cys Ser Val
Val His Glu Gly Leu His Asn His His Thr 465 470 475 480 act aag agc
ttc tcc cgg act ccg ggt aaa taa 1473 Thr Lys Ser Phe Ser Arg Thr
Pro Gly Lys * 485 490 <210> SEQ ID NO 40 <211> LENGTH:
490 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: IL-22RA
Extracellular domain with tPA leader and fused to murine gamma 2a
heavy chain Fc region (mG2a) <400> SEQUENCE: 40 Met Asp Ala
Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala
Val Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu Leu Arg Arg 20 25
30 Phe Arg Arg His Ala Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val
35 40 45 Lys Phe Gln Ser Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp
Ser Gly 50 55 60 Pro Glu Gly Thr Pro Asp Thr Val Tyr Ser Ile Glu
Tyr Lys Thr Tyr 65 70 75 80 Gly Glu Arg Asp Trp Val Ala Lys Lys Gly
Cys Gln Arg Ile Thr Arg 85 90 95 Lys Ser Cys Asn Leu Thr Val Glu
Thr Gly Asn Leu Thr Glu Leu Tyr 100 105 110 Tyr Ala Arg Val Thr Ala
Val Ser Ala Gly Gly Arg Ser Ala Thr Lys 115 120 125 Met Thr Asp Arg
Phe Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro 130 135 140 Asp Val
Thr Cys Ile Ser Lys Val Arg Ser Ile Gln Met Ile Val His 145 150 155
160 Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu
165 170 175 Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu Glu Leu Gln
Val Asn 180 185 190 Arg Thr Tyr Gln Met His Leu Gly Gly Lys Gln Arg
Glu Tyr Glu Phe 195 200 205 Phe Gly Leu Thr Pro Asp Thr Glu Phe Leu
Gly Thr Ile Met Ile Cys 210 215 220 Val Pro Thr Trp Ala Lys Glu Ser
Ala Pro Tyr Met Cys Arg Val Lys 225 230 235 240 Thr Leu Pro Asp Arg
Thr Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 245 250 255 Gly Glu Pro
Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys 260 265 270 Pro
Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 275 280
285 Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys
290 295 300 Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile
Ser Trp 305 310 315 320 Phe Val Asn Asn Val Glu Val His Thr Ala Gln
Thr Gln Thr His Arg 325 330 335 Glu Asp Tyr Asn Ser Thr Leu Arg Val
Val Ser Ala Leu Pro Ile Gln 340 345 350 His Gln Asp Trp Met Ser Gly
Lys Glu Phe Lys Cys Lys Val Asn Asn 355 360 365 Lys Asp Leu Pro Ala
Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly 370 375 380 Ser Val Arg
Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu 385 390 395 400
Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met 405
410 415 Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu
Leu 420 425 430 Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly
Ser Tyr Phe 435 440 445 Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn
Trp Val Glu Arg Asn 450 455 460 Ser Tyr Ser Cys Ser Val Val His Glu
Gly Leu His Asn His His Thr 465 470 475 480 Thr Lys Ser Phe Ser Arg
Thr Pro Gly Lys 485 490 <210> SEQ ID NO 41 <211>
LENGTH: 1834 <212> TYPE: DNA <213> ORGANISM: Mus
musculus <220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (43)...(1788) <400> SEQUENCE: 41 ttggtccaga
gccgaggccc gaaggggccc tggagggacc ca atg aag aca cta 54 Met Lys Thr
Leu 1 ctg acc atc ctg acg gtg gga tcc ctg gcc gct cac acc act gtg
gac 102 Leu Thr Ile Leu Thr Val Gly Ser Leu Ala Ala His Thr Thr Val
Asp 5 10 15 20 aca tcc ggt ctc ctt caa cac gtg aaa ttc cag tcc agc
aac ttt gag 150 Thr Ser Gly Leu Leu Gln His Val Lys Phe Gln Ser Ser
Asn Phe Glu 25 30 35 aac atc ttg acg tgg gat ggt ggg ccc gct agc
acc tct gac acc gtc 198 Asn Ile Leu Thr Trp Asp Gly Gly Pro Ala Ser
Thr Ser Asp Thr Val 40 45 50 tac agt gtg gaa tat aag aaa tac gga
gag aga aag tgg ctg gcc aag 246 Tyr Ser Val Glu Tyr Lys Lys Tyr Gly
Glu Arg Lys Trp Leu Ala Lys 55 60 65 gcg ggc tgc cag cgg atc acc
cag aag ttc tgc aac ctg act atg gag 294 Ala Gly Cys Gln Arg Ile Thr
Gln Lys Phe Cys Asn Leu Thr Met Glu 70 75 80 acc cgc aac cac act
gag ttt tac tac gcc aag gtc acg gca gtc agc 342 Thr Arg Asn His Thr
Glu Phe Tyr Tyr Ala Lys Val Thr Ala Val Ser 85 90 95 100 gca gga
ggc cca cca gtc aca aag atg act gat cgt ttc agc tcg ctg 390 Ala Gly
Gly Pro Pro Val Thr Lys Met Thr Asp Arg Phe Ser Ser Leu 105 110 115
cag cac act acc atc aaa ccg cct gat gtg acc tgt atc ccc aaa gtg 438
Gln His Thr Thr Ile Lys Pro Pro Asp Val Thr Cys Ile Pro Lys Val 120
125 130 agg tcc att cag atg ctg gtc cac ccc aca ctc aca ccg gtc ctc
tcg 486 Arg Ser Ile Gln Met Leu Val His Pro Thr Leu Thr Pro Val Leu
Ser 135 140 145 gaa gat ggc cac cag cta acc ctg gag gag att ttc cat
gac ctg ttc 534 Glu Asp Gly His Gln Leu Thr Leu Glu Glu Ile Phe His
Asp Leu Phe 150 155 160 tac cgc tta gag ctc cac gtc aac cac acc tac
cag atg cac ctt gaa 582 Tyr Arg Leu Glu Leu His Val Asn His Thr Tyr
Gln Met His Leu Glu 165 170 175 180 ggc aaa cag aga gaa tac gag ttc
ctt ggc ctg act ccc gac aca gag 630 Gly Lys Gln Arg Glu Tyr Glu Phe
Leu Gly Leu Thr Pro Asp Thr Glu 185 190 195 ttc ctc ggc tcc atc aca
att ttg act ccg ata ttg tcc aag gaa agt 678 Phe Leu Gly Ser Ile Thr
Ile Leu Thr Pro Ile Leu Ser Lys Glu Ser 200 205 210 gcc ccc tac gtg
tgc cga gtg aag acg ctg ccc gat cgg acg tgg gcc 726 Ala Pro Tyr Val
Cys Arg Val Lys Thr Leu Pro Asp Arg Thr Trp Ala 215 220 225 tac tcc
ttc tcg ggc gcc gtg ctc ttt tcc atg ggt ttc ctc gtc ggc 774 Tyr Ser
Phe Ser Gly Ala Val Leu Phe Ser Met Gly Phe Leu Val Gly 230 235 240
ttg ctc tgt tat ctg ggc tac aaa tac atc acc aag cca cct gta cct 822
Leu Leu Cys Tyr Leu Gly Tyr Lys Tyr Ile Thr Lys Pro Pro Val Pro 245
250 255 260
cct aac tcc ctg aac gtc caa cgt gtc ctg acc ttt caa ccc cta cgc 870
Pro Asn Ser Leu Asn Val Gln Arg Val Leu Thr Phe Gln Pro Leu Arg 265
270 275 ttc atc caa gaa cac gta ctg atc cct gtc ttg gac ctc agt ggc
ccc 918 Phe Ile Gln Glu His Val Leu Ile Pro Val Leu Asp Leu Ser Gly
Pro 280 285 290 agc agt ctg cct cag ccc atc cag tac tcc caa gtg gtg
gtg tct ggg 966 Ser Ser Leu Pro Gln Pro Ile Gln Tyr Ser Gln Val Val
Val Ser Gly 295 300 305 ccc agg gag cct cct gga gct gtg tgg cgg cag
agc ctg tct gac ctc 1014 Pro Arg Glu Pro Pro Gly Ala Val Trp Arg
Gln Ser Leu Ser Asp Leu 310 315 320 acc tac gta ggg cag tca gat gtc
tcc atc ctg caa cct acc aac gtg 1062 Thr Tyr Val Gly Gln Ser Asp
Val Ser Ile Leu Gln Pro Thr Asn Val 325 330 335 340 cca gct cag cag
aca ctg tcc cca cca tcc tac gct ccg aag gct gtc 1110 Pro Ala Gln
Gln Thr Leu Ser Pro Pro Ser Tyr Ala Pro Lys Ala Val 345 350 355 cct
gag gtc cag ccc cct tcc tat gcg cct cag gta gcc tcg gat gcc 1158
Pro Glu Val Gln Pro Pro Ser Tyr Ala Pro Gln Val Ala Ser Asp Ala 360
365 370 aaa gct ctg ttc tac tca cca caa cag ggg atg aag acc agg cct
gcc 1206 Lys Ala Leu Phe Tyr Ser Pro Gln Gln Gly Met Lys Thr Arg
Pro Ala 375 380 385 acc tat gac ccg cag gac att ctg gac agc tgc cct
gct tct tat gct 1254 Thr Tyr Asp Pro Gln Asp Ile Leu Asp Ser Cys
Pro Ala Ser Tyr Ala 390 395 400 gtg tgt gtg gaa gac tct ggc aaa gac
tct acc cca ggc atc ctc tcc 1302 Val Cys Val Glu Asp Ser Gly Lys
Asp Ser Thr Pro Gly Ile Leu Ser 405 410 415 420 act ccc aaa tac ctc
aag aca aaa ggt cag ctc cag gaa gac aca ctt 1350 Thr Pro Lys Tyr
Leu Lys Thr Lys Gly Gln Leu Gln Glu Asp Thr Leu 425 430 435 gtt aga
agc tgt ctc cca ggg gac ctt tct cta cag aaa gtc acc tcc 1398 Val
Arg Ser Cys Leu Pro Gly Asp Leu Ser Leu Gln Lys Val Thr Ser 440 445
450 tta ggt gaa ggg gag aca cag aga cca aaa tca ctc ccc tca cct ctg
1446 Leu Gly Glu Gly Glu Thr Gln Arg Pro Lys Ser Leu Pro Ser Pro
Leu 455 460 465 gga ttt tgc aca gac aga gga cct gac ctt cac aca ctg
cgc agt gag 1494 Gly Phe Cys Thr Asp Arg Gly Pro Asp Leu His Thr
Leu Arg Ser Glu 470 475 480 gaa cca gag aca cca cgg tac ctg aag ggg
gcg ctg tct ctc ctg tcc 1542 Glu Pro Glu Thr Pro Arg Tyr Leu Lys
Gly Ala Leu Ser Leu Leu Ser 485 490 495 500 tct gtg cag atc gag ggc
cac cct gtc tcc ctc cct ttg cac gtc cat 1590 Ser Val Gln Ile Glu
Gly His Pro Val Ser Leu Pro Leu His Val His 505 510 515 tct gtc tca
tgt tcc ccc tca gac gag gga cca agt ccc tgg ggc ctg 1638 Ser Val
Ser Cys Ser Pro Ser Asp Glu Gly Pro Ser Pro Trp Gly Leu 520 525 530
ctg gac tcc ctt gtg tgt cca aag gat gag ggt ccc gcg gtt gag act
1686 Leu Asp Ser Leu Val Cys Pro Lys Asp Glu Gly Pro Ala Val Glu
Thr 535 540 545 gag gcc atg tgc ccc agt gct gca gcc tct gag ctg gag
cag tcc aca 1734 Glu Ala Met Cys Pro Ser Ala Ala Ala Ser Glu Leu
Glu Gln Ser Thr 550 555 560 gaa ctg gac tct ctt ttc aaa ggc ttg gcc
ctg act gtg cag tgg gaa 1782 Glu Leu Asp Ser Leu Phe Lys Gly Leu
Ala Leu Thr Val Gln Trp Glu 565 570 575 580 tcc tga agggagatcg
gagcaagcag gcctaagttt cctcccgccc caccta 1834 Ser * <210> SEQ
ID NO 42 <211> LENGTH: 581 <212> TYPE: PRT <213>
ORGANISM: Mus musculus <400> SEQUENCE: 42 Met Lys Thr Leu Leu
Thr Ile Leu Thr Val Gly Ser Leu Ala Ala His 1 5 10 15 Thr Thr Val
Asp Thr Ser Gly Leu Leu Gln His Val Lys Phe Gln Ser 20 25 30 Ser
Asn Phe Glu Asn Ile Leu Thr Trp Asp Gly Gly Pro Ala Ser Thr 35 40
45 Ser Asp Thr Val Tyr Ser Val Glu Tyr Lys Lys Tyr Gly Glu Arg Lys
50 55 60 Trp Leu Ala Lys Ala Gly Cys Gln Arg Ile Thr Gln Lys Phe
Cys Asn 65 70 75 80 Leu Thr Met Glu Thr Arg Asn His Thr Glu Phe Tyr
Tyr Ala Lys Val 85 90 95 Thr Ala Val Ser Ala Gly Gly Pro Pro Val
Thr Lys Met Thr Asp Arg 100 105 110 Phe Ser Ser Leu Gln His Thr Thr
Ile Lys Pro Pro Asp Val Thr Cys 115 120 125 Ile Pro Lys Val Arg Ser
Ile Gln Met Leu Val His Pro Thr Leu Thr 130 135 140 Pro Val Leu Ser
Glu Asp Gly His Gln Leu Thr Leu Glu Glu Ile Phe 145 150 155 160 His
Asp Leu Phe Tyr Arg Leu Glu Leu His Val Asn His Thr Tyr Gln 165 170
175 Met His Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu Gly Leu Thr
180 185 190 Pro Asp Thr Glu Phe Leu Gly Ser Ile Thr Ile Leu Thr Pro
Ile Leu 195 200 205 Ser Lys Glu Ser Ala Pro Tyr Val Cys Arg Val Lys
Thr Leu Pro Asp 210 215 220 Arg Thr Trp Ala Tyr Ser Phe Ser Gly Ala
Val Leu Phe Ser Met Gly 225 230 235 240 Phe Leu Val Gly Leu Leu Cys
Tyr Leu Gly Tyr Lys Tyr Ile Thr Lys 245 250 255 Pro Pro Val Pro Pro
Asn Ser Leu Asn Val Gln Arg Val Leu Thr Phe 260 265 270 Gln Pro Leu
Arg Phe Ile Gln Glu His Val Leu Ile Pro Val Leu Asp 275 280 285 Leu
Ser Gly Pro Ser Ser Leu Pro Gln Pro Ile Gln Tyr Ser Gln Val 290 295
300 Val Val Ser Gly Pro Arg Glu Pro Pro Gly Ala Val Trp Arg Gln Ser
305 310 315 320 Leu Ser Asp Leu Thr Tyr Val Gly Gln Ser Asp Val Ser
Ile Leu Gln 325 330 335 Pro Thr Asn Val Pro Ala Gln Gln Thr Leu Ser
Pro Pro Ser Tyr Ala 340 345 350 Pro Lys Ala Val Pro Glu Val Gln Pro
Pro Ser Tyr Ala Pro Gln Val 355 360 365 Ala Ser Asp Ala Lys Ala Leu
Phe Tyr Ser Pro Gln Gln Gly Met Lys 370 375 380 Thr Arg Pro Ala Thr
Tyr Asp Pro Gln Asp Ile Leu Asp Ser Cys Pro 385 390 395 400 Ala Ser
Tyr Ala Val Cys Val Glu Asp Ser Gly Lys Asp Ser Thr Pro 405 410 415
Gly Ile Leu Ser Thr Pro Lys Tyr Leu Lys Thr Lys Gly Gln Leu Gln 420
425 430 Glu Asp Thr Leu Val Arg Ser Cys Leu Pro Gly Asp Leu Ser Leu
Gln 435 440 445 Lys Val Thr Ser Leu Gly Glu Gly Glu Thr Gln Arg Pro
Lys Ser Leu 450 455 460 Pro Ser Pro Leu Gly Phe Cys Thr Asp Arg Gly
Pro Asp Leu His Thr 465 470 475 480 Leu Arg Ser Glu Glu Pro Glu Thr
Pro Arg Tyr Leu Lys Gly Ala Leu 485 490 495 Ser Leu Leu Ser Ser Val
Gln Ile Glu Gly His Pro Val Ser Leu Pro 500 505 510 Leu His Val His
Ser Val Ser Cys Ser Pro Ser Asp Glu Gly Pro Ser 515 520 525 Pro Trp
Gly Leu Leu Asp Ser Leu Val Cys Pro Lys Asp Glu Gly Pro 530 535 540
Ala Val Glu Thr Glu Ala Met Cys Pro Ser Ala Ala Ala Ser Glu Leu 545
550 555 560 Glu Gln Ser Thr Glu Leu Asp Ser Leu Phe Lys Gly Leu Ala
Leu Thr 565 570 575 Val Gln Trp Glu Ser 580 <210> SEQ ID NO
43 <211> LENGTH: 660 <212> TYPE: DNA <213>
ORGANISM: Homo Sapiens <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (1)...(660) <400> SEQUENCE: 43 atg
gcg tgg agt ctt ggg agc tgg ctg ggt ggc tgc ctg ctg gtg tca 48 Met
Ala Trp Ser Leu Gly Ser Trp Leu Gly Gly Cys Leu Leu Val Ser 1 5 10
15 gca ttg gga atg gta cca cct ccc gaa aat gtc aga atg aat tct gtt
96 Ala Leu Gly Met Val Pro Pro Pro Glu Asn Val Arg Met Asn Ser Val
20 25 30 aat ttc aag aac att cta cag tgg gag tca cct gct ttt gcc
aaa ggg 144 Asn Phe Lys Asn Ile Leu Gln Trp Glu Ser Pro Ala Phe Ala
Lys Gly 35 40 45 aac ctg act ttc aca gct cag tac cta agt tat agg
ata ttc caa gat 192 Asn Leu Thr Phe Thr Ala Gln Tyr Leu Ser Tyr Arg
Ile Phe Gln Asp 50 55 60 aaa tgc atg aat act acc ttg acg gaa tgt
gat ttc tca agt ctt tcc 240 Lys Cys Met Asn Thr Thr Leu Thr Glu Cys
Asp Phe Ser Ser Leu Ser 65 70 75 80 aag tat ggt gac cac acc ttg aga
gtc agg gct gaa ttt gca gat gag 288 Lys Tyr Gly Asp His Thr Leu Arg
Val Arg Ala Glu Phe Ala Asp Glu 85 90 95 cat tca gac tgg gta aac
atc acc ttc tgt cct gtg gat gac acc att 336 His Ser Asp Trp Val Asn
Ile Thr Phe Cys Pro Val Asp Asp Thr Ile 100 105 110 att gga ccc cct
gga atg caa gta gaa gta ctt gat gat tct tta cat 384 Ile Gly Pro Pro
Gly Met Gln Val Glu Val Leu Asp Asp Ser Leu His 115 120 125 atg cgt
ttc tta gcc cct aaa att gag aat gaa tac gaa act tgg act 432 Met Arg
Phe Leu Ala Pro Lys Ile Glu Asn Glu Tyr Glu Thr Trp Thr 130 135 140
atg aag aat gtg tat aac tca tgg act tat aat gtg caa tac tgg aaa 480
Met Lys Asn Val Tyr Asn Ser Trp Thr Tyr Asn Val Gln Tyr Trp Lys
145 150 155 160 aac ggt act gat gaa aag ttt caa att act ccc cag tat
gac ttt gag 528 Asn Gly Thr Asp Glu Lys Phe Gln Ile Thr Pro Gln Tyr
Asp Phe Glu 165 170 175 gtc ctc aga aac ctg gag cca tgg aca act tat
tgt gtt caa gtt cga 576 Val Leu Arg Asn Leu Glu Pro Trp Thr Thr Tyr
Cys Val Gln Val Arg 180 185 190 ggg ttt ctt cct gat cgg aac aaa gct
ggg gaa tgg agt gag cct gtc 624 Gly Phe Leu Pro Asp Arg Asn Lys Ala
Gly Glu Trp Ser Glu Pro Val 195 200 205 tgt gag caa aca acc cat gac
gaa acg gtc ccc tcc 660 Cys Glu Gln Thr Thr His Asp Glu Thr Val Pro
Ser 210 215 220 <210> SEQ ID NO 44 <211> LENGTH: 220
<212> TYPE: PRT <213> ORGANISM: Homo Sapiens
<400> SEQUENCE: 44 Met Ala Trp Ser Leu Gly Ser Trp Leu Gly
Gly Cys Leu Leu Val Ser 1 5 10 15 Ala Leu Gly Met Val Pro Pro Pro
Glu Asn Val Arg Met Asn Ser Val 20 25 30 Asn Phe Lys Asn Ile Leu
Gln Trp Glu Ser Pro Ala Phe Ala Lys Gly 35 40 45 Asn Leu Thr Phe
Thr Ala Gln Tyr Leu Ser Tyr Arg Ile Phe Gln Asp 50 55 60 Lys Cys
Met Asn Thr Thr Leu Thr Glu Cys Asp Phe Ser Ser Leu Ser 65 70 75 80
Lys Tyr Gly Asp His Thr Leu Arg Val Arg Ala Glu Phe Ala Asp Glu 85
90 95 His Ser Asp Trp Val Asn Ile Thr Phe Cys Pro Val Asp Asp Thr
Ile 100 105 110 Ile Gly Pro Pro Gly Met Gln Val Glu Val Leu Asp Asp
Ser Leu His 115 120 125 Met Arg Phe Leu Ala Pro Lys Ile Glu Asn Glu
Tyr Glu Thr Trp Thr 130 135 140 Met Lys Asn Val Tyr Asn Ser Trp Thr
Tyr Asn Val Gln Tyr Trp Lys 145 150 155 160 Asn Gly Thr Asp Glu Lys
Phe Gln Ile Thr Pro Gln Tyr Asp Phe Glu 165 170 175 Val Leu Arg Asn
Leu Glu Pro Trp Thr Thr Tyr Cys Val Gln Val Arg 180 185 190 Gly Phe
Leu Pro Asp Arg Asn Lys Ala Gly Glu Trp Ser Glu Pro Val 195 200 205
Cys Glu Gln Thr Thr His Asp Glu Thr Val Pro Ser 210 215 220
<210> SEQ ID NO 45 <211> LENGTH: 199 <212> TYPE:
PRT <213> ORGANISM: homo sapiens <400> SEQUENCE: 45 Met
Val Pro Pro Pro Glu Asn Val Arg Met Asn Ser Val Asn Phe Lys 1 5 10
15 Asn Ile Leu Gln Trp Glu Ser Pro Ala Phe Ala Lys Gly Asn Leu Thr
20 25 30 Phe Thr Ala Gln Tyr Leu Ser Tyr Arg Ile Phe Gln Asp Lys
Cys Met 35 40 45 Asn Thr Thr Leu Thr Glu Cys Asp Phe Ser Ser Leu
Ser Lys Tyr Gly 50 55 60 Asp His Thr Leu Arg Val Arg Ala Glu Phe
Ala Asp Glu His Ser Asp 65 70 75 80 Trp Val Asn Ile Thr Phe Cys Pro
Val Asp Asp Thr Ile Ile Gly Pro 85 90 95 Pro Gly Met Gln Val Glu
Val Leu Ala Asp Ser Leu His Met Arg Phe 100 105 110 Leu Ala Pro Lys
Ile Glu Asn Glu Tyr Glu Thr Trp Thr Met Lys Asn 115 120 125 Val Tyr
Asn Ser Trp Thr Tyr Asn Val Gln Tyr Trp Lys Asn Gly Thr 130 135 140
Asp Glu Lys Phe Gln Ile Thr Pro Gln Tyr Asp Phe Glu Val Leu Arg 145
150 155 160 Asn Leu Glu Pro Trp Thr Thr Tyr Cys Val Gln Val Arg Gly
Phe Leu 165 170 175 Pro Asp Arg Asn Lys Ala Gly Glu Trp Ser Glu Pro
Val Cys Glu Gln 180 185 190 Thr Thr His Asp Glu Thr Val 195
<210> SEQ ID NO 46 <211> LENGTH: 211 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 46 Ser
Asp Ala His Gly Thr Glu Leu Pro Ser Pro Pro Ser Val Trp Phe 1 5 10
15 Glu Ala Glu Phe Phe His His Ile Leu His Trp Thr Pro Ile Pro Asn
20 25 30 Gln Ser Glu Ser Thr Cys Tyr Glu Val Ala Leu Leu Arg Tyr
Gly Ile 35 40 45 Glu Ser Trp Asn Ser Ile Ser Asn Cys Ser Gln Thr
Leu Ser Tyr Asp 50 55 60 Leu Thr Ala Val Thr Leu Asp Leu Tyr His
Ser Asn Gly Tyr Arg Ala 65 70 75 80 Arg Val Arg Ala Val Asp Gly Ser
Arg His Ser Asn Trp Thr Val Thr 85 90 95 Asn Thr Arg Phe Ser Val
Asp Glu Val Thr Leu Thr Val Gly Ser Val 100 105 110 Asn Leu Glu Ile
His Asn Gly Phe Ile Leu Gly Lys Ile Gln Leu Pro 115 120 125 Arg Pro
Lys Met Ala Pro Ala Asn Asp Thr Tyr Glu Ser Ile Phe Ser 130 135 140
His Phe Arg Glu Tyr Glu Ile Ala Ile Arg Lys Val Pro Gly Asn Phe 145
150 155 160 Thr Phe Thr His Lys Lys Val Lys His Glu Asn Phe Ser Leu
Leu Thr 165 170 175 Ser Gly Glu Val Gly Glu Phe Cys Val Gln Val Lys
Pro Ser Val Ala 180 185 190 Ser Arg Ser Asn Lys Gly Met Trp Ser Lys
Glu Glu Cys Ile Ser Leu 195 200 205 Thr Arg Gln 210 <210> SEQ
ID NO 47 <211> LENGTH: 201 <212> TYPE: PRT <213>
ORGANISM: homo sapiens <400> SEQUENCE: 47 Asp Glu Val Ala Ile
Leu Pro Ala Pro Gln Asn Leu Ser Val Leu Ser 1 5 10 15 Thr Asn Met
Lys His Leu Leu Met Trp Ser Pro Val Ile Ala Pro Gly 20 25 30 Glu
Thr Val Tyr Tyr Ser Val Glu Tyr Gln Gly Glu Tyr Glu Ser Leu 35 40
45 Tyr Thr Ser His Ile Trp Ile Pro Ser Ser Trp Cys Ser Leu Thr Glu
50 55 60 Gly Pro Glu Cys Asp Val Thr Asp Asp Ile Thr Ala Thr Val
Pro Tyr 65 70 75 80 Asn Leu Arg Val Arg Ala Thr Leu Gly Ser Gln Thr
Ser Ala Trp Ser 85 90 95 Ile Leu Lys His Pro Phe Asn Arg Asn Ser
Thr Ile Leu Thr Arg Pro 100 105 110 Gly Met Glu Ile Thr Lys Asp Gly
Phe His Leu Val Ile Glu Leu Glu 115 120 125 Asp Leu Gly Pro Gln Phe
Glu Phe Leu Val Ala Tyr Trp Arg Arg Glu 130 135 140 Pro Gly Ala Glu
Glu His Val Lys Met Val Arg Ser Gly Gly Ile Pro 145 150 155 160 Val
His Leu Glu Thr Met Glu Pro Gly Ala Ala Tyr Cys Val Lys Ala 165 170
175 Gln Thr Phe Val Lys Ala Ile Gly Arg Tyr Ser Ala Phe Ser Gln Thr
180 185 190 Glu Cys Val Glu Val Gln Gly Glu Ala 195 200 <210>
SEQ ID NO 48 <211> LENGTH: 68 <212> TYPE: PRT
<213> ORGANISM: Mus musculus <400> SEQUENCE: 48 His Thr
Thr Val Asp Thr Ser Gly Leu Leu Gln His Val Lys Phe Gln 1 5 10 15
Ser Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Gly Gly Pro Ala Ser 20
25 30 Thr Ser Asp Thr Val Tyr Ser Val Glu Tyr Lys Lys Tyr Gly Glu
Arg 35 40 45 Lys Trp Leu Ala Lys Ala Gly Cys Gln Arg Ile Thr Gln
Lys Phe Cys 50 55 60 Asn Leu Thr Met 65 <210> SEQ ID NO 49
<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:
mus musculus <400> SEQUENCE: 49 Glu Thr Arg Asn His Thr Glu
Phe Tyr Tyr Ala Lys Val Thr Ala Val 1 5 10 15 Ser Ala Gly Gly Pro
Pro Val Thr Lys Met 20 25 <210> SEQ ID NO 50
<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM:
mus musculus <400> SEQUENCE: 50 Thr Asp Arg Phe Ser Ser Leu
Gln His Thr Thr Ile Lys Pro Pro Asp 1 5 10 15 Val Thr Cys Ile Pro
Lys Val Arg Ser Ile Gln Met 20 25 <210> SEQ ID NO 51
<211> LENGTH: 40 <212> TYPE: PRT <213> ORGANISM:
Mus musculus <400> SEQUENCE: 51 Leu Val His Pro Thr Leu Thr
Pro Val Leu Ser Glu Asp Gly His Gln 1 5 10 15 Leu Thr Leu Glu Glu
Ile Phe His Asp Leu Phe Tyr Arg Leu Glu Leu 20 25 30 His Val Asn
His Thr Tyr Gln Met 35 40 <210> SEQ ID NO 52 <211>
LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 52 His Leu Glu Gly Lys Gln Arg Glu Tyr Glu
Phe Leu Gly Leu Thr Pro 1 5 10 15 Asp Thr Glu Phe Leu Gly Ser Ile
Thr Ile Leu Thr Pro Ile Leu Ser 20 25 30 Lys Glu Ser Ala Pro Tyr
Val Cys Arg Val Lys Thr Leu Pro Leu Val 35 40 45 Pro Arg 50
<210> SEQ ID NO 53 <211> LENGTH: 70 <212> TYPE:
PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 53 His
Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu Gly Leu Thr Pro 1 5 10
15 Asp Thr Glu Phe His Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu
20 25 30 Gly Leu Thr Pro Asp Thr Glu Phe Leu Gly Ser Ile Thr Ile
Leu Thr 35 40 45 Pro Ile Leu Ser Lys Glu Ser Ala Pro Tyr Val Cys
Arg Val Lys Thr 50 55 60 Leu Pro Leu Val Pro Arg 65 70 <210>
SEQ ID NO 54 <211> LENGTH: 46 <212> TYPE: PRT
<213> ORGANISM: Mus musculus <400> SEQUENCE: 54 Glu Thr
Arg Asn His Thr Glu Phe Tyr Tyr Ala Lys Val Thr Ala Val 1 5 10 15
Ser Ala Gly Gly Glu Thr Arg Asn His Thr Glu Phe Tyr Tyr Ala Lys 20
25 30 Val Thr Ala Val Ser Ala Gly Gly Pro Pro Val Thr Lys Met 35 40
45 <210> SEQ ID NO 55 <211> LENGTH: 48 <212>
TYPE: PRT <213> ORGANISM: mus musculus <220> FEATURE:
<221> NAME/KEY: VARIANT <222> LOCATION: 6, 11, 13, 15,
17, 18, and 19 <223> OTHER INFORMATION: Xaa = Any Amino Acid
<400> SEQUENCE: 55 Thr Asp Arg Phe Ser Xaa Leu Gln His Thr
Xaa Ile Xaa Pro Xaa Asp 1 5 10 15 Xaa Xaa Xaa Ile Thr Asp Arg Phe
Ser Ser Leu Gln His Thr Thr Ile 20 25 30 Lys Pro Pro Asp Val Thr
Cys Ile Pro Lys Val Arg Ser Ile Gln Met 35 40 45 <210> SEQ ID
NO 56 <211> LENGTH: 92 <212> TYPE: PRT <213>
ORGANISM: homo sapiens <400> SEQUENCE: 56 Pro Glu Asp Pro Ser
Asp Leu Leu Gln His Val Lys Phe Gln Ser Ser 1 5 10 15 Asn Phe Glu
Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr Pro 20 25 30 Asp
Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg Asp Trp 35 40
45 Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn Leu
50 55 60 Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala Arg
Val Thr 65 70 75 80 Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys Met
85 90 <210> SEQ ID NO 57 <211> LENGTH: 28 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
57 Thr Asp Arg Phe Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro Asp
1 5 10 15 Val Thr Cys Ile Ser Lys Val Arg Ser Ile Gln Met 20 25
<210> SEQ ID NO 58 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 58 Ile
Val His Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly His Arg 1 5 10
15 Leu Thr Leu Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu Glu Leu
20 25 30 Gln Val Asn Arg Thr Tyr Gln Met 35 40 <210> SEQ ID
NO 59 <211> LENGTH: 25 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 59 His Leu Gly Gly Lys
Gln Arg Glu Tyr Glu Phe Phe Gly Leu Thr Pro 1 5 10 15 Asp Thr Glu
Phe Leu Gly Thr Ile Met 20 25 <210> SEQ ID NO 60 <211>
LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 60 Ile Cys Val Pro Thr Trp Ala Lys Glu Ser
Ala Pro Tyr Met 1 5 10 <210> SEQ ID NO 61 <211> LENGTH:
12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 61 Cys Arg Val Lys Thr Leu Pro Asp Arg Thr
Trp Thr 1 5 10 <210> SEQ ID NO 62 <211> LENGTH: 212
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: A murine
IL-22RA soluble receptor with cleavage site (Leu Val Pro Arg)
remaining on C-Terminus <400> SEQUENCE: 62 His Thr Thr Val
Asp Thr Ser Gly Leu Leu Gln His Val Lys Phe Gln 1 5 10 15 Ser Ser
Asn Phe Glu Asn Ile Leu Thr Trp Asp Gly Gly Pro Ala Ser 20 25 30
Thr Ser Asp Thr Val Tyr Ser Val Glu Tyr Lys Lys Tyr Gly Glu Arg 35
40 45 Lys Trp Leu Ala Lys Ala Gly Cys Gln Arg Ile Thr Gln Lys Phe
Cys 50 55 60 Asn Leu Thr Met Glu Thr Arg Asn His Thr Glu Phe Tyr
Tyr Ala Lys 65 70 75 80 Val Thr Ala Val Ser Ala Gly Gly Pro Pro Val
Thr Lys Met Thr Asp 85 90 95 Arg Phe Ser Ser Leu Gln His Thr Thr
Ile Lys Pro Pro Asp Val Thr 100 105 110 Cys Ile Pro Lys Val Arg Ser
Ile Gln Met Leu Val His Pro Thr Leu 115 120 125 Thr Pro Val Leu Ser
Glu Asp Gly His Gln Leu Thr Leu Glu Glu Ile 130 135 140 Phe His Asp
Leu Phe Tyr Arg Leu Glu Leu His Val Asn His Thr Tyr 145 150 155 160
Gln Met His Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu Gly Leu 165
170 175 Thr Pro Asp Thr Glu Phe Leu Gly Ser Ile Thr Ile Leu Thr Pro
Ile 180 185 190
Leu Ser Lys Glu Ser Ala Pro Tyr Val Cys Arg Val Lys Thr Leu Pro 195
200 205 Leu Val Pro Arg 210
* * * * *