Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITION AND METHOD FOR TREATING INFLAMMATORY
DISORDERS
FIELD OF THE INVENTION
The invention is related to cytokine polypeptides and polynucleotides encoding
these polypeptides. The invention is also related to antibodies to cytokine
polypeptides
and to therapeutic compositions that include these antibodies. The
compositions are
useful in treating, e.g., inflammatory states such as arthritis.
BACKGROUND OF THE INVENTION
Inflammatory arthritis represents is a family of arthritic diseases
characterized by
lymphokine-mediated inflammation of the joints. Inflammatory arthritis is
often
autoimmune in origin. Examples of inflammatory arthritis can include
rheumatoid
arthritis, psoriatic arthritis, and lupus-associated arthritis. The most
common form of
inflammatory arthritis is rheumatoid arthritis. Rheumatoid arthritis is
characterized by
persistent inflammation of the joints. Inflammation can eventually lead to
cartilage
destruction and bone erosion.
SUMMARY OF THE INVENTION
The invention is based in part on the discovery that inhibitors of the
cytokine IL-
22 can inhibit symptoms associated with arthritis. More particularly,
antibodies raised
against IL-22 (which is also referred to herein as GIL-19 andAE289) have been
found to
inhibit the development of symptoms associated with collagen-induced arthritis
in a
murine model system.
In one aspect, the invention features an antibody which immunologically reacts
with an IL 22 protein. The antibody can be, e.g., a neutralizing antibody.
In preferred embodiments, the antibody is a monoclonal antibody. Examples of
monoclonal antibodies include a human monoclonal antibody and a humanized
monoclonal antibody.
In some embodiments, the antibody binds specifically to a polypeptide that
includes the amino acid sequence of SEQ ID N0:2, which corresponds to the
amino acid
sequence of a human IL-22 polypeptide.
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Also provided by the invention is a pharmaceutical composition comprising an
IL-22 antibody which immunologically reacts with an IL 22 protein comprising
the
amino acid sequence of SEQ ID N0:2 and a pharmaceutical carrier. The antibody
in the
pharmaceutical composition is preferably a neutralizing antibody. The antibody
is
preferably a monoclonal antibody, 'such as a human monoclonal antibody or a
humanized
monoclonal antibody.
Also provided by the invention is a method of treating a pathological
condition in
a subject associated with IL-22 activity by administering an effective amount
of an agent
that inhibits levels of IL-22 activity, thereby treating the pathological
condition.
The pathological condition can be, e.g., septicemia, and autoimmune disorders.
Suitable autoimmune disorders include, e.g., rheumatoid arthritis,
osteoarthritis, multiple
sclerosis, myasthenia gravis, inflammatory bowel disease, lupus, diabetes and
psoriasis.
These responses can be associated wound healing processes, cholesterol
metabolism,
oxygen free radical injury, ischemia, atherosclerosis and allergies.
Also provided by the invention is a method of treating symptoms associated
with
arthritis, the method by administering to a subject in need thereof a
therapeutically
effective amount of an IL-22 antibody. In some embodiments, the arthritis is
rheumatoid
arthritis.
The IL-22 antibody can be administered therapeutically or prophylactically, or
both.
Also provided by the invention is a method of enhancing a subject's immune
response to an antigen by administering to the subject an immunogenic amount
of the
antigen and an immunogenicity-augmenting amount of IL-22 in concurrent or
sequential
combination with the antigen such that the subject's immune response is
enhanced.
Also provided is a method of treating a pathological condition in a subject in
need of IL-22 modulation by administering an effective amount of an agent that
modulates IL-22 activity, such that the pathological condition in the subject
is treated.
The work described herein reveals that IL-22 is a cytokine involved in acute
phase responses. The invention provides for the use of IL-22 as well as IL-22
modulatory agents (i.e., agents that stimulate or inhibit IL-22 activity) to
alter an immune
response, either through its upregulation or down-regulation, depending on the
clinical
situation.
As used herein, the term "IL-22" molecule includes nucleic acid molecules,
proteins, polypeptides, and fragments or variants thereof having at least one
IL-22
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activity as defined herein. In a preferred embodiment, the IL-22 molecule is a
human IL-
22 molecule (e.g. the human IL-22 nucleic acid and protein molecules set forth
in SEQ
ID NO: 1 and SEQ ID N0:2).
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 65 to nucleotide 601;
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone IL-22 deposited under accession number
ATCC 207231;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone IL-22 deposited under accession number ATCC 207231;
(e) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone IL-22 deposited under accession number ATCC
207231;
(f) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone IL-22 deposited under accession number ATCC 207231;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
(h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:2 having biological activity, the fragment
comprising eight contiguous amino acids of SEQ ID N0:2;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the
protein of (g) or (h) above ;
(k) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(h); and
a polynucleotide that hybridizes under stringent conditions to any one of the
polynucleotides specified in (a)-(h) and that has a length that is at least
25% of the length
of SEQ ID NO:l.
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:1 from nucleotide 65 to nucleotide 601; the nucleotide sequence of the full-
length
protein coding sequence of clone IL-22 deposited under accession number ATCC
207231; or the nucleotide sequence of a mature protein coding sequence of
clone IL-22
deposited under accession number ATCC 207231 (e.g., nucleotides 1-1177 of SEQ
ID
NO: l ). In other preferred embodiments, the polynucleotide encodes the full-
length or a
mature protein encoded by the cDNA insert of clone IL-22 deposited under
accession
number ATCC 207231 (e.g., amino acids 1-179 of SEQ ID NO: 2). In further
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising a fragment of the amino acid sequence of SEQ ID N0:2 having
biological
activity, the fragment preferably comprising eight (more preferably twenty,
most
preferably thirty) contiguous amino acids of SEQ ID N0:2, or a polynucleotide
encoding
a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2
having
biological activity, the fragment comprising the amino acid sequence from
amino acid 84
to amino acid 93 of SEQ ID N0:2.
Other embodiments provide the gene corresponding to the cDNA sequence of
SEQ ID NO:1.
Further embodiments of the invention provide isolated polynucleotides produced
according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize
in 6X SSC at 65 degrees C to a nucleotide sequence selected from the
group consisting of:
(aa) SEQ ID NO:1, but excluding the poly(A) tail at the
3' end of SEQ ID NO:1; and
(ab) the nucleotide sequence of the cDNA insert of
clone IL-22 deposited under.accession number ATCC 207231;
(ii) hybridizing said probes) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 degrees C; and
(iii) isolating the DNA polynucleotides detected with the
probe(s);
and
(b) a process comprising the steps of:
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(i) preparing one or more polynucleotide primers that
hybridize in 6X SSC at 65 degrees C to a nucleotide sequence
selected from the group consisting of:
(ba) SEQ ID NO:1, but excluding the poly(A) tail at the
3' end of SEQ ID NO:1; and
(bb) the nucleotide sequence of the cDNA insert of
clone IL-22 deposited under accession number ATCC 207231;
(ii) hybridizing said primers) to human genomic DNA in
conditions at least as stringent as 4X SSC at 50 degrees C;
~ (iii) amplifying human DNA sequences; and
(iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process
comprises a
nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:1, and
extending contiguously from a nucleotide sequence corresponding to the 5' end
of SEQ
ID NO:1 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:1 ,
but
excluding the poly(A) tail at the 3' end of SEQ ID NO:1. Also preferably the
polynucleotide isolated according to the above process comprises a nucleotide
sequence
corresponding to the cDNA sequence of SEQ ID NO:1 from nucleotide 65 to
nucleotide
601, and extending contiguously from a nucleotide sequence corresponding to
the 5' end
of said sequence of SEQ ID NO:1 from nucleotide 65 to nucleotide 601, to a
nucleotide
sequence corresponding to the 3' end of said sequence of SEQ ID NO:1 from
nucleotide
65 to nucleotide 601.
In other embodiments, the present invention provides a composition comprising
a
protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:2;
(b) a fragment of the amino acid sequence of SEQ ID N0:2, the
fragment comprising eight contiguous amino acids of SEQ ID N0:2; and
(c) the amino acid sequence encoded by the cDNA insert of clone IL-
22 deposited under accession number ATCC 207231;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:2. In further preferred
embodiments, the present invention provides a protein comprising a fragment of
the
amino acid sequence of SEQ ID N0:2 having biological activity, the fragment
preferably
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comprising eight (more preferably twenty, most preferably thirty) contiguous
amino
acids of SEQ ID N0:2.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell,
including bacterial,
yeast, insect and mammalian cells, transformed with such polynucleotide
compositions.
Also provided by the present invention are organisms that have enhanced,
reduced, or
modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present
invention.
Protein compositions of the present invention may further comprise a
pharmaceutically acceptable Garner. Compositions comprising an antibody which
specifically reacts with such protein are also provided by the present
invention.
As used herein, a "modulator", "agent" or "modulating agent" includes any
proteins, polypeptides, nucleic acids, agonists or antagonists or protein,
polypeptide or
nucleic acid fragments or variants thereof that are capable of altering the
activity of IL-
22. For example, such altering of IL-22 activity may include the blocking of
IL-22
activity, down-regulation of IL-22 activity or inhibition of IL-22 activity.
Alternatively,
the alteration of IL-22 activity may include the augmenting of IL-22 activity,
the up-
regulation of IL-22 activity or the enhancing of IL-22 activity.
In another embodiment, modulators that act on the present invention are
provided. In a preferred embodiment, polyclonal and/or monoclonal antibodies
of the
present invention (e.g. neutralizing antibody specific for IL-22) are also
provided to
down-modulate an immune responses (i.e. to treat sepsis and other chronic
inflammatory
disorders). In another preferred embodiment, the present invention is used as
a vaccine
adjuvant to alter the type of immune response achieved by antigen alone.
In another embodiment, a method of treating a pathological condition in a
subject
is provided by modulating the activity of IL-22, such that the pathological
condition is
treated. In a preferred embodiment, the pathological condition treated is an
infectious
disease. More preferably, the infectious disease can be initiated by a
bacteria, virus,
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parasite or fungi. In another preferred embodiment, the pathological condition
is cancer,
more preferably renal cell carcinoma.
In another embodiment, modulatory agents are used to alter inflammatory
pathologies in the kidney, such as the induction of renal proximal tubular
basophilia.
In another embodiment, the present invention will be used for the remodeling
of
tissues, both in vivo and ex vivo. In a preferred embodiment, IL-22, or
agonists of IL-22
is used in the remodeling of epithelial tissue in the kidney.
In another embodiment, a method is provided for studying a disease in a
subject
comprising administering an agent that modulates the activity of IL-22 in
vivo, such that
the disease can be studied. In a preferred embodiment, the subject under study
is a
genetically altered mammal, preferably a genetically altered mouse, most
preferably a
transgenic or gene knock-out mouse.
Methods are also provided for preventing, treating or ameliorating a medical
condition which comprises administering to a mammalian subject a
therapeutically
effective amount of a composition comprising a protein of the present
invention and a
pharmaceutically acceptable carrier.
Unless otherwise defined, all technical and scientific terms used herein have
the
same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Although methods and materials similar or equivalent to
those
described herein can be used in the practice or testing of the present
invention, suitable
methods and materials are described below. All publications, patent
applications,
patents, and other references mentioned herein are incorporated by reference
in their
entirety. In the case of conflict, the present specification, including
definitions, will
control. In addition, the materials, methods, and examples are illustrative
only and not
intended to be limiting.
Other features and advantages of the invention will be apparent from the
following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWAINGS
FIG. 1 is a schematic drawing showing an experimental protocol used to analyze
the effect of an IL-22 antibody on an in vivo murine arthritis model.
FIG. 2 is a graph showing body score following treatment of arthritic mice
with
IL-22 antibody or control using a therapeutic treatment regimen.
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FIG. 3 is a graph showing body score following treatment of arthritic mice
with
IL-22 antibody or control using a prophylactic treatment regimen.
FIG. 4 is a graph showing body score following treatment of severely arthritic
mice with IL-22 antibody or control.
FIG. 5 is graph showing relative percentages of paws showing a given histology
grade following with IL-22 antibody or control.
DETAILED DESCRIPTION
The invention provides inhibitors of IL-22 and therapeutic compositions that
include the antibodies. The inhibitors are useful for treating inflammatory
states, which
include, e.g., autoimmune diseases such as rheumatoid arthritis.
Inhibitors can be prepared using IL-22 polypeptide sequences and nucleic acids
encoding same. IL-22 is produced by activated human and mouse Thl, but not
Th2,
CD4+ cells. The cytokine is a multifunctional molecule whose expression is
stimulated
by LPS, but not IFN-'y. IL-22 shares approximately 20% homology with IL-10 and
is
produced by activated human and mouse Thl, but not Th2, CD4+ cells.
Furthermore, LPS,
but not IFN-7, strongly stimulates IL-22 production from the adherent cell
compartment of
murine PECs, indicating that IL-22 is involved in mediating natural immunity.
The addition of either an adenovirus encoding murine IL-22 or recombinant
purified
murine IL-22 intravenously injected into C57b/6 mice induced numerous systemic
effects,
including decreased red blood cell count, increased platelet count, decreased
serum
albumin, increased serum amyloid A and fibrinogen levels, and decreased body
weight, all
suggestive of an acute phase reaction. Moreover, IL-22 administration also
induces
basophilia in the proximal renal tubules, a finding distinct from an acute
phase reaction,
suggestive of induced cellular proliferation. Identification of these
biological activities of
IL-22 has led to the development of new approaches to and therapeutics useful
for the
treatment of various immune response-related diseases and disorders. Moreover,
the role of
IL-22 in processes including sepsis, chronic inflammation, and autoimmunity
has been
analyzed and new mechanisms for treating such conditions is disclosed herein.
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I. Isolated IL-22 Proteins and Polynucleotides
IL-22 nucleotide and amino acid sequences are provided below. The nucleotide
sequence of each clone can also be determined by sequencing of the deposited
clone in
accordance with known methods. The predicted amino acid sequence (both full-
length
and mature forms) can then be determined from such nucleotide sequence. The
amino
acid sequence of the protein encoded by a particular clone can also be
determined by
expression of the clone in a suitable host cell, collecting the protein and
determining its
sequence.
As used herein a "secreted" protein is one which, when expressed in a suitable
host cell, is transported across or through a membrane, including transport as
a result of
signal sequences in its amino acid sequence. "Secreted" proteins include
without
limitation proteins secreted wholly (e.g., soluble proteins) or partially
(e.g. , receptors)
from the cell in which they are expressed. "Secreted" proteins also include
without
limitation proteins which are transported across the membrane of the
endoplasmic
reticulum.
A polynucleotide of the present invention has been identified initially as
clone
"hTIF/AE289", later renamed and referred to herein also as "IL-22." Clone IL-
22 was
isolated according to the following method. A murine EST was identified from a
murine
cDNA library made from splenocytes activated with both ConA and bone marrow
derived dendritic cells. The EST was identified using methods which are
selective for
cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637). The murine EST
sequence was used to isolate a full-length murine clone from the same cDNA
library
(SEQ ID N0:3, shown below). Analysis of the sequence of the murine clone
revealed a
significant homology to interleukin-10 (IL-10).
In order to isolate a human homolog of the murine clone, PCR primers were
constructed based upon the region of the murine sequence which showed homology
to
IL-10. Use of such primers for amplification in cDNA library derived from
PHA/PMA
stimulated human PBMCs produced a PCR product of significant size. Analysis of
the
sequence of the PCR product confirmed that it was a homolog of the murine
cDNA.
Oligonucleotides were constructed from the sequence of the partial human clone
and
used to isolate a full-length human clone from the PBMC library.
The disclosed human IL-22 nucleotide sequence is a full-length clone,
including
the entire coding sequence of a secreted protein. Analysis of its sequence
confirms its
homology to IL-10 at a level of 20%.
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The nucleotide sequence of the disclosed human IL-22 polynucleotide sequence
is reported below (SEQ ID NO:1), and includes a poly(A) tail. The disclosed
nucleotide
sequence includes an open reading frame and the amino acid sequence of full-
length IL-
22 protein corresponding to the foregoing nucleotide sequence is reported in
SEQ ID
S N0:2. The amino acid sequence of mature IL-22 corresponds to amino acids 34-
179 of
SEQ ID N0:2.
1 GAATTCGGCCAAAGAGGCCTACAGGTTCTCCTTCCCCAGTCACCAGTTGC
51 TCGAGTTAGAATTGTCTGCAATGGCCGCCCTGCAGAAATCTGTGAGCTCT
1O 101 TTCCTTATGGGGACCCTGGCCACCAGCTGCCTCCTTCTCTTGGCCCTCTT
151 GGTACAGGGAGGAGCAGCTGCGCCCATCAGCTCCCACTGCAGGCTTGACA
201 AGTCCAACTTCCAGCAGCCCTATATCACCAACCGCACCTTCATGCTGGCT
251 AAGGAGGCTAGCTTGGCTGATAACAACACAGACGTTCGTCTCATTGGGGA
301 GAAACTGTTCCACGGAGTCAGTATGAGTGAGCGCTGCTATCTGATGAAGC
IS 351 AGGTGCTGAACTTCACCCTTGAAGAAGTGCTGTTCCCTCAATCTGATAGG
401 TTCCAGCCTTATATGCAGGAGGTGGTGCCCTTCCTGGCCAGGCTCAGCAA
451 CAGGCTAAGCACATGTCATATTGAAGGTGATGACCTGCATATCCAGAGGA
501 ATGTGCAAAAGCTGAAGGACACAGTGAAAAAGCTTGGAGAGAGTGGAGAG
551 ATCAAAGCAATTGGAGAACTGGATTTGCTGTTTATGTCTCTGAGAAATGC
2O 601 CTGCATTTGACCAGAGCAAAGCTGAAAAATGAATAACTAACCCCCTTTCC
651 CTGCTAGAAATAACAATTAGATGCCCCAAAGCGATTTTTTTTAACCAAAA
701 GGAAGATGGGAAGCCAAACTCCATCATGATGGGTGGATTCCAAATGAACC
751 CCTGCGTTAGTTACAAAGGAAACCAATGCCACTTTTGTTTATAAGACCAG
801 AAGGTAGACTTTCTAAGCATAGATATTTATTGATAACATTTCATTGTAAC
2S 851 TGGTGTTCTATACACAGAAAACAATTTATTTTTTAAATAATTGTCTTTTT
901 CCATAAAAAAGATTACTTTCCATTCCTTTAGGGGAAAAAACCCCTAAATA
951 GCTTCATGTTTCCATAATCAGTACTTTATATTTATAAATGTATTTATTAT
1001 TATTATAAGACTGCATTTTATTTATATCATTTTATTAATATGGATTTATT
1051 TATAGAAACATCATTCGATATTGCTACTTGAGTGTAAGGCTAATATTGAT
3O 1101 ATTTATGACAATAATTATAGAGCTATAACATGTTTATTTGACCTCAATAA
1151 ACACTTGGATATCCTAAAAAAAAAAAAAAAAAAGCGGCCGC (SEQ ID
N0:1)
The polypeptide sequence of the encoded polypeptide is shown below.
1 MAALQKSVSS FLMGTLATSC LLLLALLVQG GAAAPISSHC RLDKSNFQQP
3S 51 YITNRTFMLA KEASLADNNT DVRLIGEKLF HGVSMSERCY LMKQVLNFTL
101 EEVLFPQSDR FQPYMQEWP FLARLSNRLS TCHIEGDDLH IQRNVQKLKD
151 TVKKLGESGE IKAIGELDLL FMSLRNACI (SEQ ID N0:2)
Clone "IL-22" was deposited on April 28, 1999 with the American Type Culture
40 Collection (10801 University Boulevard, Manassas, Virginia 20110-2209
U.S.A.) as an
original deposit under the Budapest Treaty and were given the accession number
ATCC
207231. All restrictions on the availability to the public of the deposited
material will
be irrevocably removed upon the granting of the patent, except for the
requirements
specified in 37 C.F.R. ~ 1.808(b), and the term of the deposit will comply
with 37 C.F.R.
4S ~ 1.806.
Nucleotide sequences encoding murine IL-22, and the sequence of the encoded
polypeptide, are provided below:
1 GAATTCGGCC AAAGAGGCCT ACCTAAACAG GCTCTCCTCT CAGTTATCAA
SO 51 CTGTTGACAC TTGTGCGATC TCTGATGGCT GTCCTGCAGA AATCTATGAG
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101 TTTTTCCCTTATGGGGACTTTGGCCGCCAGCTGCCTGCTTCTCATTGCCC
151 TGTGGGCCCAGGAGGCAAATGCGCTGCCCGTCAACACCCGGTGCAAGCTT
201 GAGGTGTCCAACTTCCAGCAGCCATACATCGTCAACCGCACCTTTATGCT
251 GGCCAAGGAGGCCAGCCTTGCAGATAACAACACAGATGTCCGGCTCATCG
S 301 GGGAGAAACTGTTCCGAGGAGTCAGTGCTAAGGATCAGTGCTACCTGATG
351 AAGCAGGTGCTCAACTTCACCCTGGAAGACGTTCTGCTCCCCCAGTCAGA
401 CAGGTTCCAGCCCTACATGCAGGAGGTGGTGCCTTTCCTGACCAAACTCA
451 GCAATCAGCTCAGCTCCTGTCACATCAGCGGTGACGACCAGAACATCCAG
501 AAGAATGTCAGAAGGCTGAAGGAGACAGTGAAAAAGCTTGGAGAGAGTGG
1O 551 AGAGATCAAGGCGATTGGGGAACTGGACCTGCTGTTTATGTCTCTGAGAA
601 ATGCTTGCGTCTGAGCGAGAAGAAGCTAGAAAACGAAGAACTGCTCCTTC
651 CTGCCTTCTAAAAAGAACAATAAGATCCCTGAATGGACTTTTTTACTAAA
701 GGAAAGTGAGAAGCTAACGTCCATCATTATTAGAAGATTTCACATGAAAC
751 CTGGCTCAGTTGAAAAAGAAAATAGTGTCAAGTTGTCCATGAGACCAGAG
IS 801 GTAGACTTGATAACCACAAAGATTCATTGACAATATTTTATTGTCACTGA
851 TGATACAACAGAAAAATAATGTACTTTAAAAAATTGTTTGAAAGGAGGTT
901 ACCTCTCATTCCTTTAGAAAAAAAGCTTATGTAACTTCATTTCCATAACC
951 AATATTTTATATATGTAAGTTTATTTATTATAAGTATACATTTTATTTAT
1001 GTCAGTTTATTAATATGGATTTATTTATAGAAACATTATCTGCTATTGAT
2O 1051 ATTTAGTATAAGGCAAATAATATTTATGACAATAACTATGGAAACAAGAT
1101 ATCTTAGGCTTTAATAAACACATGGATATCATAAAAAAAAF~~AP.AAAAAA
1151 AAAAAAAAGCGGCCGC
(SEQ
ID N0:3)
2S
The amino acid sequence of the polypeptide encoded by the above-referenced
polynucleotide sequence is provide below:
1 MAVLQKSMSF SLMGTLAASC LLLIALWAQE ANALPVNTRC KLEVSNFQQP
3O 51 YIVNRTFMLA KEASLADNNT DVRLIGEKLF RGVSAKDQCY LMKQVLNFTL
101 EDVLLPQSDR FQPYMQEWP FLTKLSNQLS SCHISGDDQN IQKNVRRLKE
151 TVKKLGESGE IKAIGELDLL FMSLRNACV* (SEQ ID N0:4)
Fragments of an IL-22 protein (e.g. fragments which are capable of exhibiting
3S biological activity) are also encompassed by the compositions and methods
of the
invention. Fragments of the protein can be in linear form, or they can be
cyclized using
known methods, for example, as described in H.U. Saragovi, et al.,
Bio/Technology 10,
773-778 (1992) and in R.S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-
9253
(1992), both of which are incorporated herein by reference. Such fragments can
be fused
40 to carrier molecules such as immunoglobulins for many purposes, including
increasing
the valency of protein binding sites. For example, fragments of the protein
can be fused
through "linker" sequences to the Fc portion of an immunoglobulin. For a
bivalent form
of the protein, such a fusion can be to the Fc portion of an IgG molecule.
Other
immunoglobulin isotypes may also be used to generate such fusions. For
example, a
4S protein- IgM fusion generates a decavalent form of the protein of the
invention.
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The present invention also provides both full-length and mature forms of the
disclosed proteins. The full-length form of such proteins is identified in the
sequence
listing by translation of the nucleotide sequence of each disclosed clone. The
mature
forms) of such protein can be obtained by expression of the disclosed full-
length
polynucleotide (preferably those deposited with ATCC) in a suitable mammalian
cell or
other host cell. The sequences) of the mature forms) of the protein can also
be
determined from the amino acid sequence of the full-length form. An example of
a
mature IL-22 polypeptide sequence is amino acids 1-179 of SEQ ID N0:2.
The present invention also provides genes corresponding to the polynucleotide
sequences disclosed herein. "Corresponding genes" are the regions of the
genome that
are transcribed to produce the mRNAs from which cDNA polynucleotide sequences
are
derived and may include contiguous regions of the genome necessary for the
regulated
expression of such genes. Corresponding genes may therefore include but are
not
limited to coding sequences, 5' and 3' untranslated regions, alternatively
spliced exons,
introns, promoters, enhancers, and silencer or suppressor elements. The
corresponding
genes can be isolated in accordance with known methods using the sequence
information
disclosed herein. Such methods include the preparation of probes or primers
from the
disclosed sequence information for identification and/or amplification of
genes in
appropriate genomic libraries or other sources of genomic materials. An
"isolated gene"
is a gene that has been separated from the adjacent coding sequences, if any,
present in
the genome of the organism from which the gene was isolated.
The chromosomal location corresponding to the polynucleotide sequences
disclosed herein may also be determined, for example by hybridizing
appropriately
labeled polynucleotides of the present invention to chromosomes in situ. The
corresponding chromosomal location for a disclosed polynucleotide can be
determined
by identifying significantly similar nucleotide sequences in public databases,
such as
expressed sequence tags (ESTs), that have already been mapped to particular
chromosomal locations. For at least some of the polynucleotide sequences
disclosed
herein, public database sequences having at least some similarity to the
polynucleotide of
the present invention have been listed by database accession number. Searches
using the
GenBank accession numbers of these public database sequences can then be
performed
at an Internet site provided by the National Center for Biotechnology
Information having
the address http://www.ncbi.nlm.nih.gov/LJniGene/, in order to identify
"UniGene
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WO 02/068476 PCT/US02/05684
clusters" of overlapping sequences. Many of the "UniGene clusters" so
identified will'
already have been mapped to particular chromosomal sites.
Organisms that have enhanced, reduced,x or modified expression of the genes)
corresponding to the polynucleotide sequences disclosed herein are provided.
The
desired change in gene expression can be achieved through the use of antisense
polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from
the
gene (Albert and Moms, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky
et al.,
1997, Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid
Res.
Mol. Biol. 58: 1-39; all of which are incorporated by reference herein).
Transgenic
animals that have multiple copies of the genes) corresponding to the
polynucleotide
sequences disclosed herein, preferably produced by transformation of cells
with genetic
constructs that are stably maintained within the transformed cells and their
progeny, are
provided. Transgenic animals that have modified genetic control regions that
increase or
reduce gene expression levels, or that change temporal or spatial patterns of
gene
expression, are also provided (see European Patent No. 0 649 464 B 1,
incorporated by
reference herein). In addition, organisms are provided in which the genes)
corresponding to the polynucleotide sequences disclosed herein have been
partially or
completely inactivated, through insertion of extraneous sequences into the
corresponding
genes) or through deletion of all or part of the corresponding gene(s).
Partial or
complete gene inactivation can be accomplished through insertion, preferably
followed
by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays
14(9): 629-
633; Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA 90(16): 7431-7435; Clark
et al.,
1994, Proc. Natl. Acad. Sci. USA 91(2): 719-722; all of which are incorporated
by
reference herein), or through homologous recombination, preferably detected by
positive/negative genetic selection strategies (Mansour et al., 1988, Nature
336: 348-
352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These
organisms with altered gene expression are preferably eukaryotes and more
preferably
are mammals. Such organisms are useful for the development of non-human models
for
the study of disorders involving the corresponding gene(s), and for the
development of
assay systems for the identification of molecules that interact with the
protein products)
of the corresponding gene(s).
Where the protein of the present invention is membrane-bound (e.g., is a
receptor), the present invention also provides for soluble forms of such
protein. In such
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forms, part or all of the intracellular and transmembrane domains of the
protein are
deleted such that the protein is fully secreted fi=om the cell in which it is
expressed. The
intracellular and transmembrane domains of proteins of the invention can be
identified in
accordance with known techniques for determination of such domains from
sequence
information. For example, the TopPredII computer program can be used to
predict the
location of transmembrane domains in an amino acid sequence, domains which are
described by the location of the center of the transmembrane domain, with at
least ten
transmembrane amino acids on each side of the reported central residue(s).
Proteins and protein fragments of the present invention include proteins with
amino acid sequence lengths that are at least 25%(more preferably at least
50%, and
most preferably at least 75%) of the length of a disclosed protein and have at
least 60%
sequence identity (more preferably, at least 75% identity; most preferably at
least 90% or
95% identity) with that disclosed protein, where sequence identity is
determined by
comparing the amino acid sequences of the proteins when aligned so as to
maximize
overlap and identity while minimizing sequence gaps. Also included in the
present
invention are proteins and protein fragments that contain a segment preferably
comprising 8 or more (more preferably 20 or more, most preferably 30 or more)
contiguous amino acids that shares at least 75% sequence identity (more
preferably, at
least 85% identity; most preferably at least 95% identity) with any such
segment of any
of the disclosed proteins.
In another embodiment, proteins, protein fragments, and recombinant proteins
of
the present invention include those which can be identified based on the
presence of at
least one "IL-22 receptor-binding motif." As used herein, the term "IL-22
receptor-
binding motif' includes amino acid sequences or residues which are important
for
binding of IL-22 to its requisite receptor. In a preferred embodiment, a IL-22
protein
contains a IL-22 receptor-binding motif including about amino acids 50-60 of
SEQ ID
N0:2. In another embodiment, an IL-22 protein contains a IL-22 receptor-
binding motif
including about amino acids 63-81 of SEQ ID N0:2. In yet another embodiment,
an IL-
22 protein contains a IL-22 receptor-binding motif including about amino acids
168-177
of SEQ ID N0:2. In a preferred embodiment, an IL-22 protein contains a IL-22
receptor-
binding motif including at least one of amino acids 50-60, amino acids 63-81,
and/or
about amino acids168-177 of SEQ ID N0:2.
In yet another embodiment, a IL-22 receptor binding motif has an amino acid
sequence at least 95%, 96%, 97%, 98%, 99%, or more identical to an amino acid
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sequence selected from the group consisting of amino acids 50-60 of SEQ ID
N0:2,
amino acids 63-81 of SEQ ID N0:2, and amino acids 168-177 of SEQ ID N0:2.
In another embodiment, proteins, protein fragments, and recombinant proteins
of
the present invention include those which can be identified based on the
presence of at
least one, two, three, four or more sites for N-linked glycosylation.
In particular, sequence identity can be determined using WU-BLAST
(Washington University BLAST) version 2.0 software, which builds upon WU-BLAST
version 1.4, which in turn is based on the public domain NCBI-BLAST version
1.4
(Altschul and Gish, 1996, Local alignment statistics, Doolittle ed., Methods
in
Enzymology 266: 460-480; Altschul et al., 1990, Basic local alignment search
tool,
Journal of Molecular Biology 215: 403-410; Gish and States, 1993,
Identification of
protein coding regions by database similarity search, Nature Genetics 3: 266-
272; Karlin
and Altschul, 1993, Applications and statistics for multiple high-scoring
segments in
molecular sequences, Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which
are
incorporated by reference herein). WU-BLAST version 2.0 executable programs
for
several UNIX platforms can be downloaded from
ftp://blast.wustl.edu/blasdexecutables.
The complete suite of search programs (BLASTP, BLASTN, BLASTX, TBLASTN, and
TBLASTX) is provided at that site, in addition to several support programs.
WU-BLAST 2.0 is copyrighted and may not be sold or redistributed in any form
or
manner without the express written consent of the author; but the posted
executables may
otherwise be freely used for commercial, nonprofit, or academic purposes. In
all search
programs in the suite -- BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX --
the gapped alignment routines are integral to the database search itself, and
thus yield
much better sensitivity and selectivity while producing the more easily
interpreted
output. Gapping can optionally be turned off in all of these programs, if
desired. The
default penalty (Q) for a gap of length one is Q=9 for proteins and BLASTP,
and Q=10
for BLASTN, but can be changed to any integer value including zero, one
through eight,
nine, ten, eleven, twelve through twenty, twenty-one through fifty, fifty-one
through one
hundred, etc. The default per-residue penalty for extending a gap (R) is R=2
for proteins
and BLASTP, and R=10 for BLASTN, but can be changed to any integer value
including
zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven,
twelve through
twenty, twenty-one through fifty, fifty-one through one hundred, etc. Any
combination
of values for Q and R can be used in order to align sequences so as to
maximize overlap
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and identity while minimizing sequence gaps. The default amino acid comparison
matrix
is BLOSUM62, but other amino acid comparison matrices such as PAM can be
utilized.
Species homologues of the disclosed polynucleotides and proteins are also
provided by the present invention. As used herein, a "species homologue" is a
protein or
polynucleotide with a different species of origin from that of a given protein
or
polynucleotide, but with significant sequence similarity to the given protein
or
polynucleotide. Preferably, polynucleotide species homologues have at least
60%
sequence identity (more preferably, at least 75%, 80%, 85%, 90%, 95%, 99%)
with the
given polynucleotide, and protein species homologues have at least 30%
sequence
identity (more preferably, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%,
90%) with the given protein, where sequence identity is determined by
comparing the
nucleotide sequences of the polynucleotides or the amino acid sequences of the
proteins
when aligned so as to maximize overlap and identity while minimizing sequence
gaps.
Species homologues can be isolated and identified by making suitable probes or
primers
from the sequences provided herein and screening a suitable nucleic acid
source from the
desired species. Preferably, species homologues are those isolated from
mammalian
species. Most preferably, species homologues are those isolated from certain
mammalian species such as, for example, Pan troglodytes, Gorilla gorilla,
Pongo
pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas,
Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus Oedipus,
Microcebus murinus, Mus musculus, Rattus norvegicus, Cricetulus griseus, Felis
catus,
Mustela vison, Canis familiards, Oryctolagus cuniculus, Bos taurus, Ovis
cries, Sus
scrofa, and Equus caballus, for which genetic maps have been created allowing
the
identification of syntenic relationships between the genomic organization of
genes in one
species and the genomic organization of the related genes in another species
(O'Brien
and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; O'Brien et al., 1993, Nature
Genetics
3:103-112; Johansson et al., 1995, Genomics 25: 682-690; Lyons et al., 1997,
Nature
Genetics 15: 47-56; O'Brien et al., 1997, Trends in Genetics 13(10): 393-399;
Carver
and Stubbs, 1997, Genome Research 7:1123-1137; all of which are incorporated
by
reference herein)
The invention also encompasses allelic variants of the disclosed
polynucleotides
or proteins; that is, naturally-occurring alternative forms of the isolated
polynucleotides
which also encode proteins which are identical or have significantly similar
sequences to
those encoded by the disclosed polynucleotides. Preferably, allelic variants
have at least
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60% sequence identity (more preferably, at least 75%, 80%, 85%, 90%, 95%, 99%)
with
the given polynucleotide, where sequence identity is determined by comparing
the
nucleotide sequences of the polynucleotides when aligned so as to maximize
overlap and
identity while minimizing sequence gaps. Allelic variants can be isolated and
identified
by making suitable probes or primers from the sequences provided herein and
screening
a suitable nucleic acid source from individuals of the appropriate species.
The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides that hybridize under
reduced
stringency conditions, more preferably stringent conditions, and most
preferably highly
stringent conditions, to polynucleotides described herein. Examples of
stringency
conditions are shown in the table below: highly stringent conditions are those
that are at
least as stringent as, for example, conditions A-F; stringent conditions are
at least as
stringent as, for example, conditions G-L; and reduced stringency conditions
are at least
as stringent as, for example, conditions M-R.
Strin PolynucleotideHybridHybridization TemperatureWash
en and
g Hybrid LengthBuffer' Temperature
cy
Condition
(bp)= and Buffer'
A DNA:DNA >_ 65C; lxSSC -or- 65C; 0.3xSSC
50
42C; lxSSC, 50% formamide
B DNA:DNA <50 TB*; lxSSC TB*; lxSSC
C DNA:RNA >_ 67C; lxSSC -or- 67C; 0.3xSSC
50
45C; lxSSC, 50% formamide
D DNA:RNA <50 Tp*; lxSSC Tp*; lxSSC
E RNA:RNA >_ 70C; lxSSC -or- 70C; 0.3xSSC
50
50C; lxSSC, 50% formamide
F RNA:RNA <50 TF*; lxSSC T,.*; lxSSC
G DNA:DNA >_ 65C; 4xSSC -or- 65C; lxSSC
50
42C; 4xSSC, 50% formamide
H DNA:DNA <50 T"*; 4xSSC TH*; 4xSSC
I DNA:RNA >_ 67C; 4xSSC -or- 67C; lxSSC
50
45C; 4xSSC, 50% formamide
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J DNA:RNA <50 T~*; 4xSSC T~*; 4xSSC
K RNA:RNA >_ 70C; 4xSSC -or- 67C; lxSSC
50 50C; 4xSSC, 50% formamide
L RNA:RNA <50 T~*; 2xSSC T~*; 2xSSC
M DNA:DNA >_ 50C; 4xSSC -or- 50C; 2xSSC
50 40C; 6xSSC, 50% formamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA >_ 55C; 4xSSC -or- 55C; 2xSSC
50 42C; 6xSSC, 50% formamide
P DNA:RNA <50 TP*; 6xSSC T~*; 6xSSC
Q RNA:RNA >_ 60C; 4xSSC -or- 60C; 2xSSC
50 45C; 6xSSC, 50% formamide
R RNA:RNA <50 TH*; 4xSSC TR*; 4xSSC
$: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing
polynucleotides. When hybridizing a polynucleotide to a target polynucleotide
of
unknown sequence, the hybrid length is assumed to be that of the hybridizing
polynucleotide. When polynucleotides of known sequence are hybridized, the
hybrid
length can be determined by aligning the sequences of the polynucleotides and
identifying the region or regions of optimal sequence complementarity.
t: SSPE (lxSSPE is O.15M NaCI, IOmM NaH2P04, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC (lxSSC is O.15M NaCI and lSmM sodium citrate) in the
hybridization and wash buffers; washes are performed for 15 minutes after
hybridization
is complete.
*TB - TR: The hybridization temperature for hybrids anticipated to be less
than 50 base
pairs in length should be 5-10°C less than the melting temperature (Tm)
of the hybrid,
where Tm is determined according to the following equations. For hybrids less
than 18
base pairs in length, Tm(°C) = 2(# of A + T bases) + 4(# of G + C
bases). For hybrids
between 18 and 49 base pairs in length, Tm(°C) = 81.5 +
16.6(log,o[Na+]) + 0.41(%G+C)
- (600/N), where N is the number of bases in the hybrid, and [Na+] is the
concentration of
sodium ions in the hybridization buffer ([Na+] for lxSSC = 0.165 M).
Additional examples of stringency conditions for polynucleotide hybridization
are provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F.M.
Ausubel et
al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated
herein by
reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least SO%, and most preferably at least 75%) of the
length of the
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polynucleotide of the present invention to which it hybridizes, and has at
least 60°Io
sequence identity (more preferably, at least 75% identity; most preferably at
least 90% or
95% identity) with the polynucleotide of the present invention to which it
hybridizes,
where sequence identity is determined by comparing the sequences of the
hybridizing
polynucleotides when aligned so as to maximize overlap and identity while
minimizing
sequence gaps.
II. Vectors and Host Cells
The isolated polynucleotide of the invention can be operably linked to an
expression control sequence such as the pMT2 or pED expression vectors
disclosed in
Kaufman et al., Nucleic Acids Res. 19, 4485-4490 ( 1991 ), in order to produce
the
protein recombinantly. Many suitable expression control sequences are known in
the art.
General methods of expressing recombinant proteins are also known and are
exemplified
in R. Kaufman, Methods in Enzymology 185, 537-566 ( 1990). As defined herein
"operably linked" means that the isolated polynucleotide of the invention and
an
expression control sequence are situated within a vector or cell in such a way
that the
protein is expressed by a host.cell which has been transformed (transfected)
with the
ligated polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of
the
protein. Mammalian host cells include, for example, monkey COS cells, Chinese
Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells,
human Co1o205 cells, 3T3 cells, CV-1 cells, other transformed primate cell
lines, normal
diploid cells, cell strains derived from in vitro culture of primary tissue,
primary
explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
Alternatively, it can be possible to produce the protein in lower eukaryotes
such
as yeast or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any yeast strain capable of expressing heterologous proteins.
Potentially
suitable bacterial strains include Escherichia coli, Bacillus subtilis,
Salmonella
typhimurium, or any bacterial strain capable of expressing heterologous
proteins. If the
protein is made in yeast or bacteria, it can be necessary to modify the
protein produced
therein, for example by phosphorylation or glycosylation of the appropriate
sites, in
order to obtain the functional protein. Such covalent attachments can be
accomplished
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using known chemical or enzymatic methods. The protein may also be modified by
covalent modifications including, but not limited, polyethylene glycol
modifications.
The protein may also be produced by operably linking the isolated
polynucleotide
of the invention to suitable control sequences in one or more insect
expression vectors,
and employing an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially available in kit
form from,
e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac~ kit), and such
methods are
well known in the art, as described in Summers and Smith, Texas Agricultural
Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference.
As used
herein, an insect cell capable of expressing a polynucleotide of the present
invention is
"transformed."
The protein of the invention can be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
expressed protein may then be purified from such culture (i.e., from culture
medium or
cell extracts) using known purification processes, such as gel filtration and
ion exchange
chromatography. The purification of the protein may also include an affinity
column
containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography
using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
Alternatively, the protein of the invention may also be expressed in a form
which
will facilitate purification. For example, it can be expressed as a fusion
protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin
(TRX). Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ)
and
Invitrogen Corporation (Carlsbad, CA), respectively. The protein can also be
tagged
with an epitope and subsequently purified by using a specific antibody
directed to such
epitope. One such epitope ("Flag") is commercially available from the Eastman
Kodak
Company (New Haven, CT).
In addition, one or more reverse-phase high performance liquid chromatography
(RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant methyl or other aliphatic groups, can be employed to further purify
the protein.
Some or all of the foregoing purification steps, in various combinations, can
also be
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employed to provide a substantially homogeneous isolated recombinant protein.
The
protein thus purified is substantially free of other mammalian proteins and is
defined in
accordance with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or
sheep which
are characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic
means are
known to those skilled in the art. The synthetically-constructed protein
sequences, by
virtue of sharing primary, secondary or tertiary structural and/or
conformational
characteristics with proteins may possess biological properties in common
therewith,
including protein activity. Thus, they can be employed as biologically active
or
immunological substitutes for natural, purified proteins in screening of
therapeutic
compounds and in immunological processes for the development of antibodies.
The protein may also be produced in a recombinant viral vector through
techniques that are well-known in the art. For example, recombinant
adenoviruses, such
as the Ad5 Ela deleted (d1327) recombinant adenovirus, can be generated
through
homologous recombination in a human kidney embryonic cell line. IL-22 cDNA can
then be ligated into an adenovirus vector such as Adori 1-2. The cloned viral
vector can
then be administered to a subject via subcutaneous or intravenous injection to
allow for
in vivo production of the invention.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification
are naturally
provided or deliberately engineered. For example, modifications in the peptide
or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications of interest in the protein sequences may include the alteration,
substitution, replacement, insertion or deletion of a selected amino acid
residue in the
coding sequence. For example, one or more of the cysteine residues can be
deleted or
replaced with another amino acid to alter the conformation of the molecule.
Techniques
for such alteration, substitution, replacement, insertion or deletion are well
known to
those skilled in the art (see, e.g., U.S. Patent No. 4,518,584). Preferably,
such alteration,
substitution, replacement, insertion or deletion retains the desired activity
of the protein.
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Other fragments and derivatives of the sequences of proteins which would be
expected to retain protein activity in whole or in part and may thus be useful
for
screening or other immunological methodologies may also be easily made by
those
skilled in the art given the disclosures herein. Such modifications are
believed to be
encompassed by the present invention.
III. Methods of Use and Biological Activity
The polynucleotides and proteins of the present invention can exhibit one or
more
of the biological activities (including those associated with assays cited
herein) identified
below and accordingly are useful in a variety of research, pharmaceutical and
therapeutic
methods. Methods, uses or activities described for proteins of the present
invention can
be provided by administration or use of such proteins or by administration or
use of
polynucleotides encoding such proteins (such as, for example, in gene
therapies or
vectors suitable for introduction of DNA).
The structural and functional properties of IL-22 place this protein in the
cytokine
family. Cytokines play important roles both in health and disease and have
multiple
clinical indications. As is described in detail in the Examples, below, IL-22
induces
changes associated with those caused by inflammatory cytokines (such as IL-1
and
TNFoc), and inhibitors of IL-22 ameliorate symptoms of rheumatoid arthritis,
Therefore,
IL-22, and/or agents that increase levels of IL-22 or mimic the actions of IL-
22 (and
other molecules of the present invention) are useful as agonists in certain
clinical
indications, and antagonists of this molecule are useful in other clinical
situations,
particularly in those in which modulation of an inflammatory state is desired.
Whether
the agonist or antagonist is the preferred depends on the particular aspects
of the disease
pathology, such as the cell types involved, the nature of the stimulus and the
cellular
microenvironment.
In a preferred embodiment, IL-22 activity includes induction of at least one
activity indicative of an inflammatory state. Additional activities can
include at least one
or more of the following activities: (1) modulating, for example antagonizing
a signal
transduction pathway (e.g. an IL-22 dependant pathway); (2) modulating
cytokine
production and/or secretion (e.g. production and/or secretion of a
proinflammatory
cytokine); (3) modulating lymphokine production and/or secretion; (4)
modulating
production of adhesion molecules and/or cellular adhesion; (5) modulating
expression or
activity of nuclear transcription factors; (7) modulating secretion of IL-1;
(8) competing
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with receptors for other cytokines; (9) competing with another IL-22 family
member
protein to bind a IL-22 receptor; (10) modulating nuclear translocation of
internalized
receptor for IL-22 or another cytokine or ligand-complexed receptor; ( 11 )
modulating .
cell proliferation, development or differentiation, for example, cytokine-
stimulated or a
IL-22 protein-stimulated proliferation, development or differentiation (e.g.,
of an
epithelial cell, for example, a squamous epithelial cell of the esophagus, or
of a skin cell,
e.g., a keratinocyte); (12) modulating cell proliferation, development or
differentiation of
an osteogenic cell (e.g., of an osteoclast precursor cell, osteoclast and/or
osteoblast); (13)
modulating bone formation, bone metabolism and/or bone homeostasis (e.g.,
inhibiting
bone resorption); (15) modulating cellular immune responses; (16) modulating
cytokine-
mediated proinflammatory actions (e.g., inhibiting acute phase protein
synthesis by
hepatocytes, fever, and/or prostaglandin synthesis, for example PGE2
synthesis); and
(17) promoting and/or potentiating wound healing.
Examples of IL-22 inhibitors include soluble fragments of IL-22 polypeptides.
The soluble fragments can be provided as fusion proteins, e.g., as IgG fusion
proteins.
Inhibitors can additionally include antibodies to IL-22 polypeptides, as well
as small
molecule inhibitors of IL-22 polypeptides. The small molecules can act by
inhibiting the
expression and/or activity of an IL-22 polypeptide.
A. Diagnostic Assays
An exemplary method for detecting the presence or absence of IL-22 protein or
nucleic acid in a biological sample involves obtaining a biological sample
from a test
subject and contacting the biological sample with a compound or an agent
capable of
detecting IL-22 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes
IL-22
protein such that the presence of IL-22 protein or nucleic acid is detected in
the
biological sample. A preferred agent for detecting IL-22 mRNA or genomic DNA
is a
labeled nucleic acid probe capable of hybridizing to IL-22 mRNA or genomic
DNA.
The nucleic acid probe can be, for example, a full-length IL-22 nucleic acid,
such as the
nucleic acid of SEQ ID NO: 1, or a fragment or portion of an IL-22 nucleic
acid such as
an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in
length and
sufficient to specifically hybridize under stringent conditions to IL-22 mRNA
or
genomic DNA. Other suitable probes for use in the diagnostic assays of the
invention
are described herein.
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A preferred agent for detecting IL-22 protein is an antibody capable of
binding to
IL-22 protein, preferably an antibody with a detectable label. Antibodies can
be
polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment
thereof
(e.g., Fab or F(ab')2) can be used. The term "labeled", with regard to the
probe or
antibody, is intended to encompass direct labeling of the probe or antibody by
coupling
(i.e., physically linking) a detectable substance to the probe or antibody, as
well as
indirect labeling of the probe or antibody by reactivity with another reagent
that is
directly labeled. Examples of indirect labeling include detection of a primary
antibody
using a fluorescently labeled secondary antibody and end-labeling of a DNA
probe with
biotin such that it can be detected with fluorescently labeled streptavidin.
The term
"biological sample" is intended to include tissues, cells and biological
fluids isolated
from a subject, as well as tissues, cells and fluids present within a subject.
That is, the
detection method of the invention can be used to detect IL-22 mRNA, protein,
or
genomic.DNA in a biological sample in vitro as well as in vivo. For example,
in vitro
techniques for detection of IL-22 mRNA include Northern hybridizations and in
situ
hybridizations. In vitro techniques for detection of IL-22 protein include
enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. In vitro techniques for detection of IL-22 genomic DNA
include
Southern hybridizations. Furthermore, in vivo techniques for detection of IL-
22 protein
include introducing into a subject a labeled anti-IL-22 antibody. For example,
the
antibody can be labeled with a radioactive marker whose presence and location
in a
subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the
test subject. Alternatively, the biological sample can contain mRNA molecules
from the
test subject or genomic DNA molecules from the test subject. A preferred
biological
sample is a serum sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control
biological sample from a control subject, contacting the control sample with a
compound
or agent capable of detecting IL-22 protein, mRNA, or genomic DNA, such that
the
presence of IL-22 protein, mRNA or genomic DNA is detected in the biological
sample,
and comparing the presence of IL-22 protein, mRNA or genomic DNA in the
control
sample with the presence of IL-22 protein, mRNA or genomic DNA in the test
sample.
The invention also encompasses kits for detecting the presence of IL-22 in a
biological sample. For example, the kit can comprise a labeled compound or
agent (e.g.
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probe or antibody) capable of detecting IL-22 protein or mRNA in a biological
sample;
means for determining the amount of IL-22 in the sample; and means for
comparing the
amount of IL-22 in the sample with a standard. The compound or agent can be
packaged
in a suitable container. The kit can further comprise instructions for using
the kit to
detect IL-22 protein or nucleic acid.
Human IL-22 agonists include without limitation human IL-22 proteins and
fragments, deletion mutants and addition mutants thereof; and peptide and
small
molecule compounds that interact with the receptor or other target to which
human IL-22
is directed. Human IL-22 antagonists include without limitation antibodies
directed to
human IL-22 proteins; soluble forms of the receptor or other target to which
human IL-
22 is directed; antibodies directed to the receptor or other target to which
human IL-22 is
directed; and peptide and small molecule compounds that inhibit or interfere
with the
interaction of human IL-22 with its receptor or other target.
B. Pharmaceutical Compositions
The nucleic acid molecules, proteins, modulatory agents, and/or antibodies and
biosynthetic molecules (also referred to herein as "active compounds") of the
invention
can be incorporated into pharmaceutical compositions suitable for
administration. Such
compositions typically comprise the nucleic acid molecule, protein, modulatory
agents,
and/or antibody and a pharmaceutically acceptable carrier. As used herein the
language
"pharmaceutically acceptable Garner" is intended to include any and all
solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic and
absorption
delaying agents, and the like, compatible with pharmaceutical administration.
The use of
such media and agents for pharmaceutically active substances is well known in
the art.
Except insofar as any conventional media or agent is incompatible with the
active
compound, use thereof in the compositions is contemplated. Supplementary
active
compounds can also be incorporated into the compositions. The pharmaceutical
composition of the invention may also contain additional cytokines,
lymphokines, or
other hematopoietic factors. The pharmaceutical composition may further
contain other
agents which either enhance the activity of the protein or compliment its
activity or use
in treatment.
Thus, the pharmaceutical composition of the invention may also contain
additional cytokines, lymphokines, or other hematopoietic factors such as M-
CSF, G-
CSF, GM-CSF, Meg-GCSF, thrombopoietin, stem cell factor, erythropoietin, TNFa,
IL-
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1 (3, IL-2 through IL-26, IFNa/b, IFNy, as well as inhibitors of all of the
above
cytokines, particularly inhibitors of TNFa, IL-1(3, IL-12 and IL-18.
Such additional factors and/or agents can be included in the pharmaceutical
composition to produce a synergistic effect with protein of the invention, or
to minimize
side effects. Conversely, protein of the present invention can be included in
formulations
of the particular cytokine, lymphokine, other hematopoietic factor,
thrombolytic or anti-
thrombotic factor, or anti-inflammatory agent to minimize side effects of the
cytokine,
lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic
factor, or anti-
inflammatory agent.
A protein of the present invention can be active in multimers (e.g.,
heterodimers
or homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
compositions of the invention may comprise a protein of the invention in such
multimeric or complexed form.
The pharmaceutical composition of the invention can be in the form of a
complex
of the proteins) of present invention along with protein or peptide antigens.
The protein
and/or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface immunoglobulin
receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR)
following
presentation of the antigen by MHC proteins. MHC and structurally related
proteins
including those encoded by class I and class II MHC genes on host cells will
serve to
present the peptide antigens) to T lymphocytes. The antigen components could
also be
supplied as purified MHC-peptide complexes alone or with co-stimulatory
molecules
that can directly signal T cells. Alternatively antibodies able to bind
surface
immunolgobulin and other molecules on B cells as well as antibodies able to
bind the
TCR and other molecules on T cells can be combined with the pharmaceutical
composition of the invention.
The pharmaceutical composition of the invention can be in the form of a
liposome in which protein of the present invention is combined, in addition to
other
pharmaceutically acceptable carriers, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
in aqueous solution. Suitable lipids for liposomal formulation include,
without
limitation, monoglycerides, diglycerides, sulfatides, lysolecithin,
phospholipids, saponin,
bile acids, and the like. Preparation of such liposomal formulations is within
the level of
skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871;
U.S. Patent No.
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4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of
which are
incorporated herein by reference.
A pharmaceutical composition of the invention is formulated to be compatible ,
with its intended route of administration. Examples of routes of
administration include
parenteral, e.g., intravenous, intradermal, subcutaneous; oral (e.g.,
inhalation),
transdermal (topical), transmucosal, and rectal administration. Solutions or
suspensions
used for parenteral, intradermal, or subcutaneous application can include the
following
components: a sterile diluent such as water for injection, saline solution,
fixed oils,
polyethylene glycols, glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants
such as
ascorbic acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents for the
adjustment of
tonicity such as sodium chloride or dextrose. The pH can be adjusted with
acids or
bases, such as hydrochloric acid or sodium hydroxide. The parenteral
preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or dispersion. For
intravenous
administration, suitable Garners include physiological saline, bacteriostatic
water,
Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In
all
cases, the composition must be sterile and should be fluid to the extent that
easy
syringability exists. It must be stable under the conditions of manufacture
and storage
and must be preserved against the contaminating action of microorganisms such
as
bacteria and fungi. The carrier can be a solvent or dispersion medium
containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid
polyetheylene glycol, and the like), and suitable mixtures thereof. The proper
fluidity
can be maintained, for example, by the use of a coating such as lecithin, by
the
maintenance of the required particle size in the case of dispersion and by the
use of
surfactants. Prevention of the action of microorganisms can be achieved by
various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be preferable
to include
isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol,
sodium
chloride in the composition. Prolonged absorption of the injectable
compositions can be
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brought about by including in the composition an agent which delays
absorption, for
example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound
(e.g., a Immunomodulin protein or anti-Immunomodulin antibody) in the required
amount in an appropriate solvent with one or a combination of ingredients
enumerated
above, as required, followed by filtered sterilization. Generally, dispersions
are prepared
by incorporating the active compound into a sterile vehicle which contains a
basic
dispersion medium and the required other ingredients from those enumerated
above. In
the case of sterile powders for the preparation of sterile injectable
solutions, the preferred
methods of preparation are vacuum drying and freeze-drying which yields a
powder of
the active ingredient plus any additional desired ingredient from a previously
sterile-
filtered solution thereof.
Oral compositions generally include an inert diluent or an edible Garner. They
can be enclosed in gelatin capsules or compressed into tablets. For the
purpose of oral
therapeutic administration, the active compound can be incorporated with
excipients and
used in the form of tablets, troches, or capsules. Oral compositions can also
be prepared
using a fluid carrier for use as a mouthwash, wherein the compound in the
fluid carrier is
applied orally and swished and expectorated or swallowed. Pharmaceutically
compatible
binding agents, and/or adjuvant materials can be included as part of the
composition.
The tablets, pills, capsules, troches and the like can contain any of the
following
ingredients, or compounds of a similar nature: a binder such as
microcrystalline
cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose,
a
disintegrating agent such as alginic acid, Primogel, or corn starch; a
lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a
sweetening
agent such as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl
salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of
an
aerosol spray from pressured container or dispenser which contains a suitable
propellant,
e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art,
and include, for example, for transmucosal administration, detergents, bile
salts, and
fusidic acid derivatives. Transmucosal administration can be accomplished
through the
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use of nasal sprays or suppositories. For transdermal administration, the
active
compounds are formulated into ointments, salves, gels, or creams as generally
known in
the art.
The compounds can also be prepared in the form of suppositories (e.g., with
conventional suppository bases such as cocoa butter and other glycerides) or
retention
enemas for rectal delivery.
In one embodiment, the active compounds are prepared with Garners that will
protect the compound against rapid elimination from the body, such as a
controlled
release formulation, including implants and microencapsulated delivery
systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic
acid.
Methods for preparation of such formulations will be apparent to those skilled
in the art.
The materials can also be obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to
infected
cells with monoclonal antibodies to viral antigens) can also be used as
pharmaceutically
acceptable carriers. These can be prepared according to methods known to those
skilled
in the art, for example, as described in U.S. Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit form
as used herein refers to physically discrete units suited as unitary dosages
for the subject
to be treated; each unit containing a predetermined quantity of active
compound
calculated to produce the desired therapeutic effect in association with the
required
pharmaceutical Garner. The specification for the dosage unit forms of the
invention are
dictated by and directly dependent on the unique characteristics of the active
compound
and the particular therapeutic effect to be achieved, and the limitations
inherent in the art
of compounding such an active compound for the treatment of individuals.
Toxicity and therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental animals,
e.g., for
determining the LD50 (the dose lethal to 50°Io of the population) and
the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio between
toxic and
therapeutic effects is the therapeutic index and it can be expressed as the
ratio
LD50/ED50. Compounds which exhibit large therapeutic indices are preferred.
While
compounds that exhibit toxic side effects can be used, care should be taken to
design a
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delivery system that targets such compounds to the site of affected tissue in
order to
minimize potential damage to uninfected cells and, thereby, reduce side
effects.
The data obtained from the cell culture assays and animal studies can be used
in
formulating a range of dosage for use in humans. The dosage of such compounds
lies
preferably within a range of circulating concentrations that include the ED50
with little
or no toxicity. The dosage may vary within this range depending upon the
dosage form
employed and the route of administration utilized. For any compound used in
the
method of the invention, a "therapeutically effective" dose can be estimated
initially
from cell culture assays. A "therapeutically effective" dose can be further
formulated in
animal models to achieve a circulating plasma concentration range that
includes the IC50
(i.e., the concentration of the test compound which achieves a half-maximal
inhibition of
symptoms) as determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can be measured,
for
example, by high performance liquid chromatography.
The nucleic acid molecules of the invention can be inserted into vectors and
used
as gene therapy vectors. Gene therapy vectors can be delivered to a subject
by, for
example, intravenous injection, local administration (see U.S. Patent
5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) PNAS 91:3054-3057). The
pharmaceutical preparation of the gene therapy vector can include the gene
therapy
vector in an acceptable diluent, or can comprise a slow release matrix in
which the gene
delivery vehicle is imbedded. Alternatively, where the complete gene delivery
vector
can be produced intact from recombinant cells, e.g., retroviral vectors, the
pharmaceutical preparation can include one or more cells which produce the
gene
delivery system. ex vivo
The pharmaceutical compositions can be included in a container, pack, or
dispenser together with instructions for administration.
C. Therapeutic Uses
The present invention provides for both prophylactic and therapeutic methods
of
treating subjects (e.g., human subjects). In one aspect, the invention
provides a method
for preventing or treating a disease or a disorder in a subject
prophylactically or
therapeutically. Administration of an agent prophylactically can occur prior
to the
manifestation of symptoms of an undesired disease or disorder, such that the
disease or
disorder is prevented or, alternatively, delayed in its progression. The
prophylactic
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methods of the present invention can be carried out in a similar manner to
therapeutic
methods described herein, although dosage and treatment regimes may differ.
Another aspect of the invention pertains to methods for treating a subject
therapeutically. In one embodiment, the present invention includes methods of
modulating an immune response. In particular, modulation of an immune response
includes, but is not limited to, modulation of cellular toxicity, modulation
of cytokine
expression, production or secretion (e.g., enhancement or inhibition of
cytokine
expression, production or secretion). A preferred embodiment of the invention
involves
modulation of IL-22, in particular, stimulation of IL-22 using a IL-22
stimulatory
modulator or, alternatively, inhibition of IL-22 using a IL-22 inhibitory
modulator.
Accordingly, the present method has therapeutic utility in biasing an immune
response
towards, or away from, a natural immunity-type response depending upon the
desired
therapeutic regimen. Such modulatory methods are particularly useful in
diseases such
as viral and bacterial infection, in particular acute phase responses, sepsis
and
autoimmune disorders (i.e. chronic inflammatory states) . Moreover, the
immunomodulatory methods of the present invention can be used to treat an
immunocompromised individual to enhance immunity. Uses to increase resistance
to
viral infection and enhance the rejection of foreign molecules are also within
the scope of
the present invention. The immunomodulatory methods of the present invention
are
further useful in treating sepsis. For example, an inhibition of cytokines
such as IL-22
and Tumor Necrosis Factor in patients infected with gram negative bacteria can
result in
an attenuated immune response, thus preventing septic shock. The
immunomodulatory
methods of the present invention are further useful in treating acute phase
responses and
chronic inflammatory diseases.
As used herein, the term "therapeutically effective amount" means the total
amount of each active component of the pharmaceutical composition or method
that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
amelioration of the relevant medical condition, or an increase in rate of
treatment,
healing, prevention or amelioration of such conditions. When applied to an
individual
active ingredient, administered alone, the term refers to that ingredient
alone. When
applied to a combination, the term refers to combined amounts of the active
ingredients
that result in the therapeutic effect, whether administered in combination,
serially or
simultaneously.
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As used herein, the term "treatment" includes the application or
administration of
a therapeutic agent to a subject or to an isolated tissue or cell line from a
subject, who is
afflicted with a disease, a symptom of disease or a predisposition toward a
disease, with
the goal of curing, healing, alleviating, relieving, altering, remedying,
ameliorating,
improving or affecting the disease, the symptoms of disease or the
predisposition toward
disease.
In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein of the present invention is
administered to a
mammal having a condition to be treated. Protein of the present invention can
be
administered in accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing cytokines,
lymphokines
or other hematopoietic factors. When co-administered with one or more
cytokines,
lymphokines or other hematopoietic factors, protein of the present invention
can be
administered either simultaneously with the cytokine(s), lymphokine(s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If
administered sequentially, the attending physician will decide on the
appropriate
sequence of administering protein of the present invention in combination with
cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or
anti-
thrombotic factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carned
out in a
variety of conventional ways, such as oral ingestion, inhalation, topical
application or
cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a
tablet,
capsule, powder, solution or elixir. When administered in tablet form, the
pharmaceutical composition of the invention may additionally contain a solid
Garner
such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from
about 5 to
95% protein of the present invention, and preferably from about 25 to 90%
protein of the
present invention. When administered in liquid form, a liquid carrier such as
water,
petroleum, oils of animal or plant origin such as peanut oil, mineral oil,
soybean oil, or
sesame oil, or synthetic oils can be added. The liquid form of the
pharmaceutical
composition may further contain physiological saline solution, dextrose or
other
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saccharide solution, or glycols such as ethylene glycol, propylene glycol or
polyethylene
glycol. When administered in liquid form, the pharmaceutical composition
contains
from about 0.5 to 90°lo by weight of protein of the present invention,
and preferably from
about 1 to 50°lo protein of the present invention.
When a therapeutically effective amount of protein of the present invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of
the present
invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due
regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as
Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose
and Sodium
Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in
the art. The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
response. Larger doses of protein of the present invention can be administered
until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not
increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 wg
to about 100
mg (preferably about O.lng to about 10 mg, more preferably about 0.1 pg to
about 1 mg)
of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
present invention will vary, depending on the severity of the disease being
treated and
the condition and potential idiosyncratic response of each individual patient.
It is
contemplated that the duration of each application of the protein of the
present invention
will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
therapy using the pharmaceutical composition of the present invention.
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In a preferred embodiment, pharmaceutical preparations (e.g. those comprising
neutralizing agents against) of the present invention will be administered 2
to 6 hours
after onset of infection. For example, in vitro assays suggest that adherent
compartment
of peritoneal exudate cells produce IL-22 2 to 6 hours after treatment with
LPS,
suggesting that IL-22 is produced early in an immune response. Accordingly,
early
administration of, for example, neutralizing agents, during the course of
infection may
enhance the therapeutic efficacy of such agents.
Protein of the invention may also be used to immunize animals to obtain
polyclonal and monoclonal antibodies which specifically react with the
protein. As used
herein, the term "antibody" includes without limitation a polyclonal antibody,
a
monoclonal antibody, a chimeric antibody, a single-chain antibody, a CDR-
grafted
antibody, a humanized antibody, or fragments thereof which bind to the
indicated
protein. Such term also includes any other species derived from an antibody or
antibody
sequence which is capable of binding the indicated protein.
Antibodies to a particular protein can be produced by methods well known to
those skilled in the art. For example, monoclonal antibodies can be produced
by
generation of antibody-producing hybridomas in accordance with known methods
(see
for example, Goding, 1983, Monoclonal antibodies: principles and practice,
Academic
Press Inc., New York; and Yokoyama, 1992, "Production of Monoclonal
Antibodies" in
Current Protocols in Immunology, Unit 2.5, Greene Publishing Assoc. and John
Wiley &
Sons). Polyclonal sera and antibodies can be produced by inoculation of a
mammalian
subject with the relevant protein or fragments thereof in accordance with
known
methods. Examples of suitable methods include direct DNA gene or viral (e.g.,
adenovirus or retroviral) administration to an animal. Fragments of
antibodies, receptors,
or other reactive peptides can be produced from the corresponding antibodies
by
cleavage of and collection of the desired fragments in accordance with known
methods
(see for example, Goding, supra; and Andrew et al., 1992, "Fragmentation of
Immunoglobulins" in Current Protocols in Immunology, Unit 2.8, Greene
Publishing
Assoc. and John Wiley & Sons). Chimeric antibodies and single chain antibodies
can
also be produced in accordance with known recombinant methods (see for
example,
5,169,939, 5,194,594, and 5,576,184). Humanized antibodies can also be made
from
corresponding murine antibodies in accordance with well known methods (see for
example, U.S. Patent Nos. 5,530,101, 5,585,089, and 5,693,762). Additionally,
human
antibodies can be produced in non-human animals such as mice that have been
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genetically altered to express human antibody molecules (see for example
Fishwild et
al., 1996, Nature Biotechnology 14: 845-851; Mendez et al., 1997, Nature
Genetics 15:
146-156 (erratum Nature Genetics 16: 410); and U.S. Patents 5,877,397 and
5,625,126).
Such antibodies can be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for
example, as in
R.P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963); J.L. Krstenansky, et
al.,
FEBS Lett. 211, 10 (1987).
Monoclonal antibodies binding to the protein of the invention is useful as
diagnostic agents for the immunodetection of the protein. Neutralizing
monoclonal
antibodies binding to the protein may also be useful therapeutics for both
conditions
associated with the protein and also in the treatment of some forms of cancer
where
abnormal expression of the protein is involved. In the case of cancerous cells
or
leukemic cells, neutralizing monoclonal antibodies against the protein are
useful in
detecting and preventing the metastatic spread of the cancerous cells, which
can be
mediated by the protein.
In a preferred embodiment, monoclonal and polyclonal antibodies are used to
down-modulate an immune response. Examples of immunological conditions which
can
benefit from such treatment include bacterial infections (i.e. induction of
sepsis that can
lead to septic shock and/or septicemia) and other chronic inflammatory
conditions such
as rheumatoid arthritis and osteoarthritis.
Agents which modulate the activity of the present invention are also used to
alter
the inflammatory pathologies of the kidney.
For compositions of the present invention which are useful for bone,
cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the
composition topically, systematically, or locally as an implant or device.
When
administered, the therapeutic composition for use in this invention is, of
course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may
desirably
be encapsulated or injected in a viscous form for delivery to the site of
bone, cartilage or
tissue damage. Topical administration can be suitable for wound healing and
tissue
repair. Therapeutically useful agents other than a protein of the invention
which may
also optionally be included in the composition as described above, may
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
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methods of the invention. Preferably for bone and/or cartilage formation, the
composition would include a matrix capable of delivering the protein-
containing
composition to the site of bone and/or cartilage damage, providing a structure
for the
developing bone and cartilage and optimally capable of being resorbed into the
body.
Such matrices can be formed of materials presently in use for other implanted
medical
applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions can be biodegradable and chemically defined
calcium
sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic
acid and
polyanhydrides. Other potential materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen. Further matrices are comprised of
pure
proteins or extracellular matrix components. Other potential matrices are
nonbiodegradable and chemically defined, such as sintered hydroxapatite,
bioglass,
aluminates, or other ceramics. Matrices can be comprised of combinations of
any of the
above mentioned types of material, such as polylactic acid and hydroxyapatite
or
collagen and tricalciumphosphate. The bioceramics can be altered in
composition, such
as in calcium-aluminate-phosphate and processing to alter pore size, particle
size,
particle shape, and biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic
acid in the form of porous particles having diameters ranging from 150 to 800
microns.
In some applications, it will be useful to utilize a sequestering agent, such
as
carboxymethyl cellulose or autologous blood clot, to prevent the protein
compositions
from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-
methylcellulose, and carboxymethylcellulose, the most preferred being cationic
salts of
carboxymethylcellulose (CMC). Other preferred sequestering agents include
hyaluronic
acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide,
carboxyvinyl
polymer and polyvinyl alcohol). The amount of sequestering agent useful herein
is 0.5-
20 wt%, preferably 1-10 wt% based on total formulation weight, which
represents the
amount necessary to prevent desorbtion of the protein from the polymer matrix
and to
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provide appropriate handling of the composition, yet not so much that the
progenitor
cells are prevented from infiltrating the matrix, thereby providing the
protein the
opportunity to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention can be combined with other
agents beneficial to the treatment of the bone and/or cartilage defect, wound,
or tissue in
question. These agents include various growth factors such as epidermal growth
factor
(EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-
a and
TGF-~3), and insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary
applications. Particularly domestic animals and thoroughbred horses, in
addition to
humans, are desired patients for such treatment with proteins of the present
invention.
The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue regeneration will be determined by the attending physician
considering
various factors which modify the action of the proteins, e.g., amount of
tissue weight
1 S desired to be formed, the site of damage, the condition of the damaged
tissue, the size of
a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and
diet, the severity
of any infection, time of administration and other clinical factors. The
dosage may vary
with the type of matrix used in the reconstitution and with inclusion of other
proteins in
the pharmaceutical composition. For example, the addition of other known
growth
factors, such as IGF I (insulin like growth factor I), to the final
composition, may also
effect the dosage. Progress can be monitored by periodic assessment of
tissue/bone
growth and/or repair, for example, X-rays, histomorphometric determinations
and
tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including,
without limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or
activity in such cells.
Treated cells can then be introduced in vivo for therapeutic purposes.
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D. Screening Assays
The proteins provided by the present invention can be used in assays to
determine
biological activity, including in a panel of multiple proteins for high-
throughput
screening; to raise antibodies or to elicit another immune response; as a
reagent
(including the labeled reagent) in assays designed to quantitatively determine
levels of
the protein (or its receptor) in biological fluids; as markers for tissues in
which the
corresponding protein is preferentially expressed (either constitutively or at
a particular
stage of tissue differentiation or development or in a disease state); and, of
course, to
isolate correlative receptors or ligands. Where the protein binds or
potentially binds to
another protein (such as, for example, in a receptor-ligand interaction), the
protein can be
used to identify the other protein with which binding occurs or to identify
inhibitors of
the binding interaction. Proteins involved in these binding interactions can
also be used
to screen for peptide or small molecule inhibitors or agonists of the binding
interaction.
Any or all of these research utilities are capable of being developed into
reagent
grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in
the art. References disclosing such methods include without limitation
"Molecular
Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press,
Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in
Enzymology:
Guide to Molecular Cloning Techniques", Academic Press, Bergen S.L. and A.R.
Kimmel eds., 1987.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan,
A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene
Publishing
Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse
Lymphocyte
Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al.,
Proc.
Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.
128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et
al., J.
Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988;
Hernnann
et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J.
Immunol.
128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et
al., J.
Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai et
al.,
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J. Immunol. 140:508-512, 1988; Bertagnolli et al., Cellular Immunology 133:327-
34-1,
1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent
antibody
responses and that affect Thl/Th2 profiles) include, without limitation, those
described
in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: In
vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols
in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons,
Toronto.
1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Thl and CTL responses) include, without
limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan,
A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene
Publishing
Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse
Lymphocyte
Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J.
Immunol.
137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli
et al., J.
Immunol. 149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-
260,
1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al.,
Science
264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-
1264,
1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and
Inaba et
al., Journal of Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins that prevent apoptosis after superantigen induction and proteins that
regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993;
Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-
243, 1991;
Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry
14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-
648, 1992.
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Assays for proteins that influence early steps of T-cell commitment and
development include, without limitation, those described in: Antica et al.,
Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et
al.,
Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,
1991.
Modulatory agents identified by the above-described screening assays are
tested
in an appropriate animal model, for example, to determine the efficacy,
toxicity, or side
effects of treatment with such an agent. Alternatively, modulatory agents are
tested in at
least one of the in vitro or in situ assays described herein.
In another aspect of the invention, transgenic and gene knockout animals are
used
to study disease. Specifically, mice that have been genetically altered to
express a
disease phenotype are used to screen agents that modulate IL-22 activity. As
used
herein, the term "genetically altered" means any animal that has manipulated
genetically,
either by the introduction of a heterologous gene encoding a protein (a
transgenic
animal) or by the deletion of a gene by homologous recombination (a gene
knockout
animal). Preferably, the animal that has been genetically altered is a mouse.
In another embodiment, IL-22 molecules (e.g. RNA, DNA, cDNA, protein or
antibodies) are used as diagnostic tools, e.g., to detect the presence of
tissues in an
inflammatory state.
Assayin~ Effects of IL-22 Modulators
The activity of an IL-22 agonist or antagonist can be measure by the following
methods:
Assays for T-cell or thymocyte proliferation include without limitation those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek,
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function
3.1-
3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.
137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990;
Bertagnolli
et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J.
Immunol.
149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node
cells or thymocytes include, without limitation, those described in:
Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology.
J.E. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto.
1994; and
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Measurement of mouse and human Interferon y, Schreiber, R.D. In Current
Protocols in
Immunology. J.E. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons,
Toronto.
1994.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
cells include, without limitation, those described in: Measurement of Human
and Murine
Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In
Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and
Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau
et al.,
Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
80:2931-2938, 1983; Measurement of mouse and human interleukin 6 - Nordan, R.
In
Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5,
John
Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A.
83:1857-1861, 1986; Measurement of human Interleukin 11 - Bennett, F.,
Giannotti, J.,
Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a.
Coligan eds.
Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and
human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner,
K.J. In Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley
and Sons,
Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among
others,
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and
their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al.,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J.
Immunol. 140:508-512, 1988.
Immune Stimulatin o~uppressing'Activity
An IL-22 modulator may also exhibit immune stimulating or immune
suppressing activity, including without limitation the activities for which
assays are
described herein. An IL-22 agonist is useful in the treatment of various
immune
deficiencies and disorders (including severe combined immunodeficiency
(SCID)), e.g.,
in regulating (up or down) growth and proliferation of T and/or B lymphocytes,
as well
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as effecting the cytolytic activity of NK cells and other cell populations.
These immune
deficiencies can be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection can be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal
infections
such as candidiasis. An IL-22 protein is also useful where a boost to the
immune system
generally can be desirable, i.e., in the treatment of cancer.
An IL-22 inhibitor (such as an IL-22 antibody) can be used to treat an
autoimmune disorder. Autoimmune disorders that can be treated using a protein
of the
present invention include, for example, connective tissue disease, multiple
sclerosis,
systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary
inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin
dependent
diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune
inflammatory eye disease. Such a protein of the present invention can also be
used to
treat inflammatory conditions associated with, e.g., pancreatitis. IL-22
inhibitors are
also useful in the treatment of allergic reactions and conditions, such as
asthma
(particularly allergic asthma) or other respiratory problems. Other
conditions, in which
immune suppression is desired (including, for example, organ transplantation),
may also
be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to regulate immune
responses in a number of ways. Down regulation can be in the form of
inhibiting or
blocking an immune response already in progress or may involve preventing the
induction of an immune response. The functions of activated T cells can be
inhibited by
suppressing T cell responses or by inducing specific tolerance in T cells, or
both.
Immunosuppression of T cell responses is generally an active, non-antigen-
specific,
process which requires continuous exposure of the T cells to the suppressive
agent.
Tolerance, which involves inducing non-responsiveness or anergy in T cells, is
distinguishable from immunosuppression in that it is generally antigen-
specific and
persists after exposure to the tolerizing agent has ceased. Operationally,
tolerance can be
demonstrated by the lack of a T cell response upon reexposure to specific
antigen in the
absence of the tolerizing agent.
As used herein, the term "pathological condition" refers to the structural and
functional consequences of injurious stimuli on cells, tissues, and organs and
ultimately
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the consequences on the entire organism. Such injurious stimuli includes, but
is not
limited to, infection with a foreign body (a.e. bacteria, virus, fungi and
parasite),
inflammation, autoimmune disorders (i.e. rheumatoid arthritis, osteoarthritis,
multiple
sclerosis, myasthenia gravis, inflammatory bowel diseases, diabetes, SLE, and
psoriasis),
cancer, necrosis, ischemia, acute phase responses, apoptosis, wound healing
processes,
cholesterol metabolism, oxygen free radical injury, atherosclerosis and
allergies.
In a particular embodiment, the down-regulation or preventing of one or more
activities of the IL-22 molecules of present invention can be helpful in the
treatment of
sepsis. Briefly, sepsis is an out of control inflammatory response, often in
response to
various pus-forming and other pathogenic organisms (i.e. gram negative
bacteria), or
their toxins, in the blood or tissues. Agents that block the activity of IL-22
molecules of
the present invention can, accordingly, attenuate the induction of septic
shock.
In another embodiment, down regulating or preventing one or more antigen
functions (including without limitation B lymphocyte antigen functions (such
as , for
example, B7)), e.g., preventing high level lymphokine synthesis by activated T
cells, will
be useful in situations of tissue, skin and organ transplantation and in graft-
versus-host
disease (GVHD). For example, blockage of T cell function should result in
reduced
tissue destruction in tissue transplantation. Typically, in tissue
transplants, rejection of
the transplant is initiated through its recognition as foreign by T cells,
followed by an
immune reaction that destroys the transplant. The administration of a molecule
which
inhibits or blocks interaction of a B7 lymphocyte antigen with its natural
ligand(s) on
immune cells (such as a soluble, monomeric form of a peptide having B7-2
activity alone
or in conjunction with a monomeric form of a peptide having an activity of
another B
lymphocyte antigen (e.g., B7-l, B7-3) or blocking antibody), prior to
transplantation can
lead to the binding of the molecule to the natural ligand(s) on the immune
cells without
transmitting the corresponding costimulatory signal. Blocking B lymphocyte
antigen
function in this matter prevents cytokine synthesis by immune cells, such as T
cells, and
thus acts as an immunosuppressant. Moreover, the lack of costimulation may
also be
sufficient to anergize the T cells, thereby inducing tolerance in a subject.
Induction of
long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the
necessity
of repeated administration of these blocking reagents. To achieve sufficient
immunosuppression or tolerance in a subject, it may also be necessary to block
the
function of a combination of B lymphocyte antigens.
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The efficacy of particular blocking reagents in preventing organ transplant
rejection or GVHD can be assessed using animal models that are predictive of
efficacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of
which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in
vivo as
described in Lenschow et al., Science 257:789-792 (1992) and Turka et al.,
Proc. Natl.
Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see
Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847)
can be
used to determine the effect of blocking B lymphocyte antigen function in vivo
on the
development of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the
production of cytokines and autoantibodies involved in the pathology of the
diseases.
Preventing the activation of autoreactive T cells may reduce or eliminate
disease
symptoms. Administration of reagents which block costimulation of T cells by
disrupting receptor:ligand interactions of B lymphocyte antigens can be used
to inhibit T
cell activation and prevent production of autoantibodies or T cell-derived
cytokines
which can be involved in the disease process. Additionally, blocking reagents
may
induce antigen-specific tolerance of autoreactive T cells which could lead to
long-term
relief from the disease. The efficacy of blocking reagents in preventing or
alleviating
autoimmune disorders can be determined using a number of well-characterized
animal
models of human autoimmune diseases. Examples include murine experimental
autoimmune encephalitis, systemic lupus erythmatosis in MRLllprllpr mice or
NZB
hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD
mice and
BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental
Immunology, Raven Press, New York, 1989, pp. 840-856).
In a particular embodiment, IL-22 inhibitory agents (e.g. blocking agents) of
the
present invention can be used to treat autoimmune disease, in particular,
lymphocyte-
mediated autoimmune diseases, associated with prolonged acute phase responses
that
persist in the setting of chronic inflammation, such as rheumatoid arthritis,
osteoarthritis,
multiple sclerosis, inflammatory bowel disease, diabetes and Systemic Lupus
Erythomatosis (SLE), atherosclerosis and allergies. In such a situation,
persistent
elevations of serum amyloid A protein may lead to deposition of this protein
in the
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interstitium of tissues, a condition known as amyloidosis. The deposition of
serum
amyloid A in tissues is often in the form of fibrils rich in (3-pleated sheet
structures which
can interfere with normal tissue function (i.e. myocardial contraction,
glomerular
filtration). Thus, inhibiting or blocking IL-22 production, for example, by a
neutralizing
antibody can help stop the acute phase reaction, and therefore prevent the
occurrence of
amyloidosis.
Upregulation of an antigen function (preferably a B lymphocyte antigen
function), as a means of up regulating immune responses, may also be useful in
therapy.
Upregulation of immune responses can be in the form of enhancing an existing
immune
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function is useful in cases
of viral
infection. In addition, systemic viral diseases such as influenza, the common
cold, and
encephalitis might be alleviated by the administration of stimulatory forms of
B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses can be enhanced in an infected
patient
by removing T cells from the patient, costimulating the T cells in vitro with
viral
antigen-pulsed APCs either expressing a peptide of the present invention or
together with
a stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
costimulatory signal to, and thereby activate, T cells in vivo.
In a particular embodiment, the IL-22 molecules and/or modulators of the
present
invention can be used as a vaccine adjuvant due to their ability to modulate
the immune
system. For example, the IL-22 molecules and/or modulators (e.g. positive
modulatory
agents) of the present invention can be co-administered with a potential
vaccine antigen
in order to elicit a nonspecific inflammatory immune response to the potential
vaccine
antigen. Such vaccines can be directed against a foreign organism (i.e.
bacteria, virus,
parasite or fungi) or a tumor antigen. Moreover, such treatments may include,
but are
not limited to, gene transfer with IL-22 DNA.
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As used herein, the term "immunogenicity-augmenting" includes enhancing
and/or increasing the immunogenicity of, for example, a vaccine, as compared
to such
vaccine in the absence of IL-22.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) is useful in the induction of tumor
immunity.
Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma,
carcinoma)
transfected with a nucleic acid encoding at least one peptide of the present
invention can
be administered to a subject to overcome tumor-specific tolerance in the
subject. If
desired, the tumor cell can be transfected to express a combination of
peptides. For
example, tumor cells obtained from a patient can be transfected ex vivo with
an
expression vector directing the expression of a peptide having B7-2-like
activity alone,
or in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
transfected tumor cells are returned to the patient to result in expression of
the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigens) on the surface of the tumor cell provides the necessary
costimulation signal to T cells to induce a T cell mediated immune response
against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class
II molecules, or which fail to reexpress sufficient amounts of MHC class I or
MHC class
II molecules, can be transfected with nucleic acid encoding all or a portion
of (e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
(32
microglobulin protein or an MHC class II a chain protein and an MHC class II
(3 chain
protein to thereby express MHC class I or MHC class II proteins on the cell
surface.
Expression of the appropriate class I or class II MHC in conjunction with a
peptide
having the activity of a B lymphocyte antigen (e.g., B7-l, B7-2, B7-3) induces
a T cell
mediated immune response against the transfected tumor cell. Optionally, a
gene
encoding an antisense construct which blocks expression of an MHC class II
associated
protein, such as the invariant chain, can also be cotransfected with a DNA
encoding a
peptide having the activity of a B lymphocyte antigen to promote presentation
of tumor
associated antigens and induce tumor specific immunity. Thus, the induction of
a T cell
mediated immune response in a human subject can be sufficient to overcome
tumor-
specific tolerance in the subject.
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Hematopoiesis Re ug 1, ating Activity
An IL-22 protein, or modulator can also be used to regulate hematopoiesis and,
consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even
marginal
biological activity in support of colony forming cells or of factor-dependent
cell lines
indicates involvement in regulating hematopoiesis, e.g. in supporting the
growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
thereby indicating utility, for example, in treating various anemias or for
use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid
precursors andlor erythroid cells; in supporting the growth and proliferation
of myeloid
cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity)
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others, proteins that influence embryonic differentiation hematopoiesis)
include, without
limitation, those described in: Johansson et al. Cellular Biology 15:141-151,
1995; Keller
et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,
Blood
81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among
others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
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described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss,
Inc:, New
York, NY: 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911,
1992;
Primitive hematopoietic colony forming cells with high proliferative
potential, McNiece,
LK. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et
al. eds. Vol
pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental
Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher,
R.E.
In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 1-21,
Wiley-Liss,
Inc.., New York, NY. 1994; Long term bone marrow cultures in the presence of
stromal
cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic
Cells. R.I.
Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994;
Long
term culture initiating cell assay, Sutherland, H.J. In Culture of
Hematopoietic Cells. R.I.
Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration,
as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage andlor bone growth
in
circumstances where bone is not normally formed, has application in the
healing of bone
fractures and cartilage damage or defects in humans and other animals. Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
disease, and in other tooth repair processes. Such agents may provide an
environment to
attract bone-forming cells, stimulate growth of bone-forming cells or induce
differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation of
bone and/or cartilage repair or by blocking inflammation or processes of
tissue
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destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
Another category of tissue regeneration activity that can be attributable to
the
protein of the present invention is tendon/ligament formation. A protein of
the present
invention, which induces tendon/ligament-like tissue or other tissue formation
in
circumstances where such tissue is not normally formed, has application in the
healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
other animals. Such a preparation employing a tendon/ligament-like tissue
inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and in
repairing defects to tendon or ligament tissue. De novo tendon/ligament-like
tissue
formation induced by a composition of the present invention contributes to the
repair of
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is
also useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments.
The compositions of the present invention may provide an environment to
attract tendon-
or ligament-forming cells, stimulate growth of tendon- or ligament-forming
cells, induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendonitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also
include an appropriate matrix and/or sequestering agent as a Garner as is well
known in
the art.
The protein of the present invention may also be useful for proliferation of
neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment
of central and
peripheral nervous system diseases and neuropathies, as well as mechanical and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a protein can be used in the treatment of diseases
of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and
Shy-Drager
syndrome. Further conditions which can be treated in accordance with the
present
invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
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from chemotherapy or other medical therapies may also be treatable using a
protein of
the invention.
Proteins of the invention may also be useful to promote better or faster
closure of
non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
A protein of the present invention also exhibits activity for generation or
regeneration of other tissues, such as organs (including, for example,
pancreas, liver,
intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac)
and vascular
(including vascular endothelium) tissue, or for promoting the growth of cells
comprising
such tissues. Part of the desired effects can be by inhibition or modulation
of fibrotic
scarring to allow normal tissue to regenerate. A protein of the invention may
also exhibit
angiogenic activity.
A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
In a preferred embodiment, the present invention is used for the re-modeling
of
kidney tissue, both ex vivo and in vivo. For example, exogenous IL-22 induces
the
generation of epithelial tissue in the proximal tubules of the kidney.
A protein of the present invention is also useful for promoting or inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention can be measured by methods known in
the art. Some of these are described below:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. W091/07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described
in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT,
eds.),
Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and
Mertz, J.
Invest. Dermatol 71:382-84 (1978).
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Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic
activity.
As a result, such a protein is expected to be useful in treatment of various
coagulation
disorders (including hereditary disorders, such as hemophiliac) or to enhance
coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery
or other
causes. A protein of the invention may also be useful for dissolving or
inhibiting
formation of thromboses and for treatment and prevention of conditions
resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels
(e.g., stroke).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et
al.,
Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79
(1991);
Schaub, Prostaglandins 35:467-474, 1988.
Anti-Inflammatory ActivitX
IL-22 antagonists can be used as anit-inflammatory agents. Suitable conditions
including chronic or acute conditions), including without limitation
inflammation
associated with infection (such as septic shock, sepsis or systemic
inflammatory response
syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,
complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-
induced
lung injury, inflammatory bowel disease, Crohn's disease or resulting from
over
production of cytokines such as TNF or IL-1. Additional indications include
anaphylaxis
and hypersensitivity to an antigenic substance or material.
This invention is further illustrated by the non-limiting examples. The
contents
of all references, patents and published patent applications cited throughout
this
application, as well as the Sequence Listing, are incorporated herein by
reference.
EXAMPLES
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Example 1: Identification and characterization of clone "IL-22"
A polynucleotide of the present invention has been identified as clone "IL-
22".
Clone IL-22 was isolated according to the following method. A murine EST was
identified from a murine cDNA library made from splenocytes activated with
both ConA
and bone marrow derived dendritic cells. The EST was identified using methods
which
are selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637). The
murine EST sequence was used to isolate a full-length murine clone from the
same
cDNA library. Analysis of the sequence of the murine clone revealed a
significant
homology to interleukin-10 (IL-10).
In order to isolate a human homolog of the murine clone, PCR primers were
constructed based upon the region of the murine sequence which showed homology
to
IL-10. Use of such primers for amplification in a cDNA library derived from
PHA/PMA-stimulated human PBMCs produced a PCR product of significant size.
Analysis of the sequence of the PCR product confirmed that it was a homolog of
the
murine cDNA. Oligonucleotides were constructed from the sequence of the
partial
human clone and used to isolate a full-length human clone from the PBMC
library.
IL-22 is a full-length human clone, including the entire coding sequence of a
secreted protein (also referred to herein as "IL-22" protein). Analysis of its
amino acid
sequence indicated that it has about 23°Io homology to hIL-10. Based on
the putative
receptor-binding motifs in IL-10, three motifs involved with analogous
function have
been proposed in IL-22 through computer modeling. These are the regions of SEQ
ID
N0:2 from residue 50 to 60, from residue 63 to 81, and from residue 168 to
177.
Analyses of databases revealed that IL-22 also exhibits similar levels of
homology with
IL-10 of other species.
The nucleotide sequence of IL-22 as presently determined is reported in SEQ ID
NO:1, and includes a poly(A) tail. The amino acid sequence of the IL-22
protein
corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:2.
Example 2: Characterization of IL-22 protein
Cell lines which stably express and secrete full length IL-22 protein were
created
by transfecting CHO cells with IL-22 cDNA in appropriate expression vectors.
Transiently transfected COS cells using appropriate IL-22 expression vectors
have been
used to make IL-22 protein for analysis. Transfections were accomplished using
the
commercially available Lipofectamine reagent (Gibco). Interestingly, COS cells
which
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express IL-22 were observed to non-uniformly detach, forming holes in the cell
culture
monolayer. Media conditioned by transfected COS cells was used to demonstrate
cytokine-like activity of IL-22 protein. Western blot analysis of cell lysates
showed that
Stat-3 becomes phosphorylated (activated) in a kidney mesangial tissue-derived
cell line
exhibiting macrophage-like qualities (MES-13; see, Dumoutier et al (2000) J.
of
Immunology 164:1814-1819) upon exposure of that cell to media conditioned by
IL-22-
expressing cells. In addition phosphorylation of Stat-3 is induced in non-
transfected COS
cells that are treated with IL-22 protein.
Electrophoretic analysis of IL-22 protein (derived from the transfected COS
cell
lines described herein) indicated that the expressed protein exists in a range
of sizes.
Treatment of COS-derived IL-22 protein with N-glycanase prior to
electrophoresis
results in a single band corresponding to the highest mobility (e.g. lowest
molecular
weight) species seen in untreated IL-22. This is consistent with proposed
glycosylation
events which may occur at the putative N-linked glycosylation sites identified
in the
amino acid sequence of IL-22 (amino acid residues 54-56, 68-70, 97-99, and 176-
178 of
SEQ ID N0:2).
Edman N-terminal sequencing determined that the N-terminus of the mature IL-
22 protein begins with the residue at position 34 of SEQ ID N0:2 (alanine).
Expression
vectors were created which fuse a "6x histidine" affinity tag and a FLAG
epitope tag to
the N-terminus of the mature IL-22 protein. (The added amino acid tag is given
in SEQ
ID NO:S and has the following amino acid sequence:
MKFLVNVALVFMVVYISYIYAGSGHHHHHHGSGDYKDDDDKAPISSHCR).
These tagged constructs were used to create stably expressing CHO cell lines
and
transiently expressing COS cell lines. The tags provided a convenient means
for
detecting IL-22 (e.g., anti-6xhis antibodies; anti-FLAG antibodies), and for
purifying the
protein from conditioned media (using Ni+2 resin). Human IL-22 protein
purified by this
tag from the IL-22-expressing COS cell lines could used to induce Stat-3
activation in
MES-13 cells.
Comparison of IL-22 mRNA transcripts in activated Thl and Th2 cells (see, for
example, Syrbe et al, (1999) Springer Seminars in Immunopathology, 21:263-85)
indicated a substantially higher level of expression of IL-22 in activated Thl
cells than in
activated Th2 cells. Analysis of IL-22 mRNA was accomplished with RNAse
protection
assays. Therefore, IL-22 is induced during an adaptive immune response,
specifically
by Thl CD4+ T cells.
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Example 3: Establishment of IL-22 recombinant adenovirus vector and in vivo
administration.
The Adori 1-2 murine IL-22 (mIL-22) vector was derived by digesting pED6dpc-
2mIL-22 with EcoRI and NotI, and ligating the 1.1 kb mIL-22 cDNA fragment with
EcoRI and NotI digested adenovirus vector Adori 1-2. Adori 1-1 green
fluorescent
protein (GFP) construct was derived by digesting pEGFP-N1 (CLONTECH
Laboratories, Inc., Palo Alto, CA) with EcoRland Notl and inserting the EGFP
into the
EcoRland Notlsite of Adori 1-1. Both constructs were verified by extensive
restriction
digestion analysis and sequencing of the cDNA inserts within the plasmids.
Expression
of the mIL-22 cDNA and EGFP are driven from cytomegalovirus (CMV) immediate
early promoter and enhancer.
Ad5 Ela deleted (d1327) recombinant adenovirus was generated by homologous
recombination in a human kidney embryonic kidney cell line 293. Recombinant
adenovirus virus was isolated and subsequently amplified on 293 cells. The
virus was
released from infected 293 cells by three cycles of freeze thawing. The virus
was further
purified by two cesium chloride centrifugation gradients and dialyzed against
phosphate
buffered saline (PBS) pH 7.2 at 4°C. Following dialysis, glycerol was
added to a
concentration of 10 % and the virus was stored at - 80 °C until use.
The virus was
characterized by expression of the transgene, plaque forming units on 293
cells,
particles/ml, endotoxin measurements and PCR analysis of the virus and
sequence
analysis of the IL-22 coding region in the virus.
A single dose of 5 X 101° particles of recombinant adenovirus encoding
mIL-22
was injected into the tail vein of female C57B1/6 mice, age 7-8 weeks. Control
mice
received an adenovirus encoding GFP or PBS/10% glycerol. Mice from each
experimental group were sacrificed at various time points post injection. For
hematological and serum chemistry analysis blood was collected by cardiac
puncture.
Blood was collected via retro-orbital sinus and differential counts were
performed on
blood smears. Tissue was harvested, fixed in formalin, and stained with
hematoxylin and
eosin for histopathology.
Example 4: Immunolo~ical effects IL-22
The immunological effects of IL-22 were investigated in a metazoan context by
viral introduction of the cDNA of murine IL-22 into mice. An adenoviral vector
was
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used to express a cDNA of murine IL-22 in 8 week old C57/B6 female mice by
injection
of SxlOt° viral particles either intravenously or subcutaneously. Test
mice were
sacrificed at 7 and 14 days after injection and compared with control mice
injected with
buffer only or with adenovirus expressing green fluorescent protein (GFP). At
days 7 and
14, it was noted that the absolute and relative thymic weights were
significantly
decreased in the mice that expressed the viral murine IL-22. Absolute mean
weight of the
spleen was decreased on day 14 and liver weights were slightly increased on
day 7. A
gross generalized atrophy of the thymus as well as lymphoid depletion
(observed
microscopically) was apparent on days 7 and 14. An increase in kidney weight
as well
as enlargement of the liver were also observed.
In addition, there were a number of hematological effects that were apparent
on
day 7, including decreased red blood cell count, hemoglobin, and hematocrit.
These
effects, taken together, indicated anemia in the animals. Furthermore, there
was an
increase in platelets as well as an increase in the white blood cell count due
to an
increase of neutrophils. In light of these observations there was no evidence
of a
regenerative response, which indicated that the effects can be at the level of
the bone
marrow. A possible cause for this is the loss of small molecules through the
kidney or
gut. Furthermore, there was a slight decrease in Albumin levels at day 7 and
day 14 but
an increase in serum amyloid A and fibrinogen levels, which are indicative of
an acute
phase response. Other clinical observations included loss in body weight,
signs of
minimal dehydrations, increase urine specific gravity, a decrease in urine
output and the
induction of renal proximal tubular basophilia. The basophilia observed is due
to
increased cell division and increased rRNA present in the epithelial cells of
the renal
proximal tube.
Example 5: Preparation and characterization of anti-IL-22 monoclonal and
polyclonal
antibodies.
Monoclonal and polyclonal antibodies were prepared using routine
methodologies also described in the instant specification. The table presented
below
illustrates the binding and neutralizing specificity of monoclonal antibodies
P3/1, P3/2,
P3/3 and P3/5 as well as chicken polyclonal antibody that are directed against
IL-22.
Rat Monoclonal Abs Pol clonal Ab
IL-22 Antibodies P3/1 P3/2 P3/3 P3/5 Chicken Polyclonal
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Rat Pol clonal Ab
Monoclonal
Abs
Binding Mouse Human Mouse/HumanMouse Mouse/Human
S ecificit
Neutralizing Mouse Human Mouse/HumanMouse Mouse/Human
S ecificit
Binding specificity was determined by ELISA using mouse or human h/f tagged
IL-22 microtiter plates. Each antibody showed strong specificity for either
mouse or
human IL-22. The neutralizing specificity was determined by assessing the
ability of the
antibody to inhibit STAT 3 phosphorylation mediated by S ng/ml mouse or human
h/f
tagged IL-22. Enzyme-linked immunosorbant assays (ELISA) using bound murine IL-
22 demonstrate that the mAb P3/1 has ~5 nM EDSO based on ~2 nM for IL-22-Fc
and
~10 nM for H/F IL-22. Moreover, in addition to recognizing recombinant IL-22,
P3/1
mAb also binds native IL-22 secreted from T cells that have been transfected
with an IL-
22 retroviral vector.
The IL-22 antibody P3/1 has been found to have an IDSO of ~ 1 nM, and to work
stoichiometricly to block IL-22 activity when the cytokine is present at just
saturating
conditions ( 1 nM).
Example 6. Expression of IL-22 mRNA and receptor.
Expression of IL-22 and its receptor was examined semi-quantitative reverse-
transcriptase polymerase chain reaction (RT-PCR) in a variety of human and
mouse
tissues. The experiments reveal that IL-22 messenger RNA (mRNA) is present at
very
low levels in human testis, lung, prostate and peripheral blood lymphocytes
(PBL) as
normalized against control actin. Moreover, semi-quantitative RT-PCR shows
that IL-22
receptor is detected at highest levels in the human pancreas, and a lower
levels in the
liver, intestines, skin, thyroid, kidney, heart stomach, testis, salivary
glands, adrenal
glands and prostate. Alternatively, murine IL-22 receptor shows highest
expression in
the liver, small intestine, muscle, skin and ovaries, with lower expression in
kidney and
embryos e8.5 and e19.
Example 7. In situ hybridization and apoptotic stain for IL-22 protein
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In situ hybridization for IL-22 protein and receptor messenger RNA (mRNA) of
mice treated with adenovirus expression IL-22 (AdIL-22) or Lipopolysaccharide
(LPS)
was performed and the results as follows:
A. Detection of IL-22 Cytokine mRNA
Tissue AdIL-22-treated mice LPS-treated mice
Liver Day 1: staining in cytoplasm6 hrs.: staining in cytoplasm
of hepatocytes of
slightly positive hepatocytes slightly positive
Da s 3 and 14: no s ecific
stainin
Spleen Days 1,3 and 14: slight stainingNegative
in
eriarteriolar re ion
Heart N/A Ne ative
Colon N/A Ne ative
KidneysDayl: staining in cytoplasm 2 hrs.: staining in cytoplasm
of proximal and of proximal
distal tubular epithelium, and distal tubular epithelium,
Henle's loop at Henle's
corticomedullary junction, loop at corticomedullary
parietal cells of junction was
Bowman space and some epithelialmildly positive
cells was
mildly positive 6 hrs.: staining in cytoplasm
of the
Day 4: staining in cytoplasmproximal and distal tubular
of proximal and epithelium
distal tubular epithelium and Henle's loop at the
and Henle's loop at corticomedullary
corticomedullary junction junction, glomerular tuft
cells, some
Day 14: staining in cytoplasmparietal cells of the
of proximal Bowman space and
tubular epithelium few endothelial cells
was slightly to
moderatel ositive
PancreasN/A 2 and 6 hrs.: staining
in cytoplasm of
acinar cells sli htl ositive
Lungs N/A 2 and 6 hrs.: staining
in pneumocytes type
II and/or intraaveolar
macrophages was
sli htl to mildl stained
StomachN/A 6 hrs.: staining in cytoplasm
of basal chief
cells was mild
DuodenuN/A 2 and 6 hrs.: staining
in cytoplasm of
m and enterocyte brush border
was moderate to
Jejunum marked and slightly positive
in the
intestine nervous lexus
cells.
B. Detection of the IL-22 Receptor mRNA in LPS-treated mice
Tissue LPS-treated mice
Liver 2 and 6 hrs.: staining in
the cytoplasm of
hepatocytes was slight to
mild, nuclear staining
was observed in heptocytes,
bile duct epithelium
and endothelial cells.
Kidneys2 and 6 hrs.: staining was
slight to moderate in
the cytoplasm and nucleus
of proximal and
distal tubular epithelium,
Henle's loop at the
corticomedullary junction,
glomerular tuft cells,
some parietal cells of Bowman
space and a few
endothelial cells.
Pancreas2 and 6 hrs.: staining in
cytoplasm of acinar
cells sli htl ositive
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Heart 6 hrs.: nuclear staining was moderately positive
in cardiomyocytes and endocardial and
endothelial cells.
IL-22 receptor mRNA is additionally detected in small and large intestine,
stomach, lymph nodes, spleen, and lung. Expression of IL-22 receptor can
additionally
be upregulated by a mediator of an innate immune response, such as LPS.
Finally, TUNEL assays of kidney cells taken from c57BL/6 mice receiving mIL-
22 protein intravenously showed a few apoptotic epithelial sells in several
proximal
convoluted tubules. Mice receiving saline intravenously (control group)
demonstrated
no positive staining.
These data demonstrate that both the cytokine and receptor can be induced
during
an innate immune response, and that the induction is restricted to tissues
that are in an
inflammatory state (LPS). During an adaptive immune response, IL-22 can also
be
induced from Thl CD4+ T cells. Since circulating leukocytes do not appear to
have the
receptor, this result suggests that IL-22 functions as an effector within
tissue downstream
of an adaptive immune response, as is reinforced by the tissue expression of
the receptor,
constitutively and further upregulated by an innate inducer of inflammation.
Example 8. IL-22 mediated changes in eg ne expression
The ability of IL-22 to modulate levels of gene expression in liver cells of
mice
infected with an AdIL-22 or Ad-GFP construct was examined.
Frozen mouse livers from day 1 and day 3 post-infection were pulverized and
RNA was purified using the Promega RNAgents Total RNA Isolation System
(Promega,
Madison, WI). The RNA was further purified using the RNeasy minikit. Total RNA
was
isolated from human PBMC's using the RNeasy minikit (Qiagen, Hidden, Germany).
Total RNA was prepared for hybridization by denaturing 10 ~.g of total RNA for
10 minutes at 70°C with 100 pM T7/T24-tagged oligo-dT primer
(synthesized at
Genetics Institute, Cambridge, MA), and cooled on ice. First strand cDNA
synthesis was
performed under the following buffer conditions: 1X first strand buffer
(Invitrogen Life
Technologies, Carlsbad, CA.), lOmM DTT (GIBCO/Invitrogen), SOO~M of each dNTP
(Invitrogen Life Technologies, Carlsbad, CA)), 400 units of Superscript RT II
(Invitrogen Life Technologies) and 40 units RNAse inhibitor (Ambion, Austin,
TX.).
The reaction proceeded at 47°C for 1 hour. Second strand cDNA was
synthesized with
the addition of the following reagents at the final concentrations listed: 1X
second strand
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WO 02/068476 PCT/US02/05684
buffer (Invitrogen Life Technologies), an additional 200~M of each dNTP
(Invitrogen
Life Technologies), 40 units of E. coli DNA polymerase I (Invitrogen Life
Technologies), 2 units E. coli RNaseH (Invitrogen Life_Technologies), and 10
units of
E.coli DNA ligase. The reaction proceeded at 15.80C for 2 hours. During the
last five
minutes of the reaction 6 units of T4 DNA polymerase (New England Biolabs,
Beverly,
MA) was added.
The resulting double stranded cDNA was purified with the use of BioMag
carboxyl terminated particles as follows: 0.2 mg of BioMag particles
(Polysciences
Inc.,Warrington, PA) were equilibrated by washing three times with 0.5M EDTA
and
resuspended at a concentration of 22.2 mg/ml in 0.5M EDTA. The double stranded
cDNA reaction was diluted to a final concentration of 10%PEG/1.25M NaCI, and
the
bead suspension was added to a final bead concentration of 0.614 mg/ml. The
reaction
was incubated at room temperature for 10 minutes. The cDNA/ bead complexes
were
washed with 300,1 of 70% ethanol, the ethanol was removed and the tubes were
allowed
to air dry. The cDNA was eluted with the addition of 20 ~l of 10 mM Tris-
acetate, pH
7.8, incubated for 2-5 minutes and the cDNA containing supernatate was
removed.
10.1 of purified double stranded cDNA was added to an in vitro transcription
(IVT) solution which contained, 1X IVT buffer (Ambion, Austin, TX) 5,000 units
T7
RNA polymerase (Epicentre Technologies, Madison, WI), 3mM GTP, l.SmM ATP,
l.2mM CTP and 1.2 mM UTP (Amersham/Pharmacia,), 0.4 mM each bio-16 UTP and
bio-11 CTP (Enzo Diagnostics, Farmingdale, NY), and 80 units RNase inhibitor
(Ambion, Austin, TX). The reaction proceeded at 37°C for 16 hours.
Labeled RNA was
purified with the use of an RNeasy (Qiagen). The RNA yield was quantified by
measuring absorbance at 260nm.
12 p,g of the in vitro transcription product was fragmented in 40 mM Tris-
actetate, pH 8.0, 100 mM potassium acetate, and 30 mM magnesium acetate for 35
minutes at 94 °C. The fragmented, labeled RNA probes were diluted in
hybridization
buffer at a final composition of 1X 2-N-Morpholinoethanesulfonic acid (MES
(buffer
(pH 6.5), 50pM Bio948 (control biotinylated oligo that hybridizes to landmark
features
on the probe array (Genetics Institute, Cambridge, MA), 100 ~g/ml herring
sperm DNA
(Promega, Madison,WI), 500 ~.g/ml acetylated BSA (Invitrogen Life
Technologies) and
1 ~1/~g standard curve reagent (Proprietary reagent supplied by Gene Logic,
Gaithersburg, MD). This hybridization solution was pre-hybridized with two
glass beads
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WO 02/068476 PCT/US02/05684
(Fisher Scientific, Pittsburgh, PA) at 45°C overnight. The
hybridization solution was
removed to a clean tube and heated for 1-2 min at 95°C and
microcentrifuged on high for
2 minutes to pellet insoluble debris. Oligonucleotide array cartridges (Murine
74Kv2,
Affymetrix, Santa Clara, CA) were pre-wet with non-stringent wash buffer (0.9M
NaCI,
60mM sodium phosphate, 6mM EDTA and 0.01 %Tween20) and incubated at
45°C with
rotation for 5-10 minutes. Buffer was removed from the cartridges, and the
arrays were
hybridized with 180 u1 of the hybridization solution at 45°C rotating
at 45-60 rpm
overnight. After overnight incubation the hybridization solutions were removed
and the
cartridges were filled with non-stringent wash buffer. The array cartridges
were washed
using a fluidics station according with 10 cycles of 2 mixes/cycle non-
stringent wash
buffer at 25°C followed by 4 cycles of l5mixes/cycle stringent wash
buffer (100mM
MES,O.1M Na+, 0.01%Tween20 and 0.005%antifoam). The probe array was then first
stained for 10 minutes at 25°C in SAPE solution (100mM MES, 1M Na+,
0.05%Tween20, 0.005%antifoam, 2mg/ml acetylated BSA(Invitrogen Life
Technologies) and l0ug/ml R phycoerythrin streptavidin (Molecular Probes,
Eugene,
OR)). After first staining the probe array was washed for 10 cycles of 4
mixes/cycle with
non-stringent wash buffer at 25°C. The probe array was then stained for
10 minutes at
25°C in antibody solution (100mM MES, 1M Na+, 0.05%Tween20, 0.005%
antifoam,
2mg/ml acetylated BSA(Invitrogen Life Technologies), 100p,g/ml Goat IgG
(SIGMA, St.
Louis, MO) and 3pg/ml biotinylated anti-streptavidin antibody(goat) (Vector
Laboratories,). Following the second stain the probe array was stained again
for an
additional 10 minutes at 25°C in SAPE solution. Finally, the probe
array was washed for
15 cycles of 4 mixes/cycle with non-stringent wash buffer at 30°C.
Arrays were scanned using an Affymetrix gene chip scanner (Affymetrix, Santa
Clara, CA). The scanner contained a scanning confocal microscope and used an
argon
ion laser for the excitation source and emission is detected by a
photomultiplier tube at
530 nm bandpass filter (fluorscein0 or 560 longpass filter (phycoerythrin).
mRNA were analyzed on the Murine 74k (Mu74K) chip set. The data was
reduced with the use of GENECHIP 4.0 software. Each experimental sample was
compared to a time matched control in a two-file analysis. The data were
filtered with the
criteria for genes that were called "Present" in one group, and removing all
genes that
were not called either "Increasing" or "Decreasing".
Data for three mice are presented below (AD-GIL-19 Mouse 49, 51, and 52).
Shown are genes whose expression changed relative to Ad-GFP control, with the
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WO 02/068476 PCT/US02/05684
indicated average-fold change shown for each animal. The changes observed in
gene
expression of Ad-IL-22 treated animals are consistent with the induction by IL-
22 of an
acute phase response. The observed changes are also indicative of an
inflammatory state
in the treated animal.
Ad-G Ad-G Ad-G
I I I
L- L- L-
19 19 19
Da 3 Livers - Mouse Mouse Mouse
U74v2
mouse number 49 51 52
Avg Avg Avg
Fold Fold Fold
Identifier Gene Name Chan Chan Chan
a a a
1300017C10RIK RIKEN cDNA 23.4 17.2 19.3
1 300017010 ene
SAA-PS serum amyloid 24.6 13.9 24.3
A,
seudo ene
SAA1 serum am loid 11.9 9.7 12.3
A 1
SAA2 serum am loid 10.0 8.9 10.3
A 2
PRTN3 roteinase 3 15.2 14.3 17.1
SPP1 secreted 10.2 7.8 10.7
hos ho rotein
1
LCN2 l i ocalin 2 13.4 10.3 13.3
SAA3 serum am loid 10.5 5.4 8.2
A 3
GR01 GR01 onco ene 8.2 5.6 7.2
LY6D l ymphocyte antigen6.0 5.5 4.9
6
com lex, locus
D
GR01 GR01 onco ene 7.0 5.6 7.2
RAD51 L1 RAD51 like 1 (S. 4.4 3.7 3.8
cerevisiae
GAS6 growth arrest 4.1 3.5 4.8
specific
6
SP12-2 serine protease 3.7 2.8 3.8
i nhibitor 2-2
GADD45G growth arrest 3.9 2.7 3.4
and
DNA-damage-
i nducible 45 amma
CEBPD CCAAT/enhancer 5.3 3.2 3.9
binding protein
C/EBP , delta
TNFRSF1A t umor necrosis 3.6 2.6 3.0
factor
receptor superfamily,
member 1 a
CISH3 cytokine inducible4.0 3.8
SH2-containing
protein
3
IL1 R1 i nterleukin 1 receptor,5.2 2.6 5.6
t voe I
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WO 02/068476 PCT/US02/05684
type I
SAP serum amyloid 3.1 2.5 3.3
P-
com onent
PEX11 A peroxisomal 4.2 3.2
bio enesis factor
11 a
2310031 E04RIK EST 2.9 2.7 3.3
AA880891 EST 2.7 2.4 2.8
CD14 CD14 anti en 3.4 2.3 2.6
MT1 metallothionein 2.7 2.4 2.9
1
UNK_AW124835 EST 2.2 2.0
TM4SF7 transmembrane 2.6 2.8 2.4
4
su erfamil member
7
DNCLC1 dynein, cytoplasmic,2.5 2.4 2.6
Ii ht chain 1
SAA4 serum am loid 3.2 2.8
A 4
2410006H10RIK RIKEN cDNA 2.2 2.1 2.0
2410006H10 ene
RBM3 RNA binding motif2.7 2.8 2.8
rotein 3
1300003D03RIK RIKEN cDNA 2.2 2.4
1300003D03 ene
CEBPB CCAAT/enhancer 2.0 2.3
binding protein
C/EBP , beta
MT2 metallothionein 2.2 2.1 2.3
2
ORM2 orosomucoid 2 1.7 1.7 2.0
VNN1 vanin 1 2.0 2.1
GTF2A2 general transcription2.2 2.4
factor Ila, 2
(l2kD
subunit
ITIH4 inter alpha-trypsin1.8 1.9
inhibitor, heavy
chain
4
ITIH3 inter-alpha trypsin1.8 1.7 1.9
inhibitor, heavy
chain
3
NPN3 neoplastic progression2.2 2.5
3
U62673 EST -2.4 -3.2
PAPSS2 3'-phosphoadenosine-2.0 -2.3
5'-phosphosulfate
s nthase 2
TEMT thioether S- -2.2 -1.7
meth Itransferase
TTR transth retin -2.0 -1.8
CBG corticosteroid -3.4 -2.8 -2.8
binding
lobulin
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WO 02/068476 PCT/US02/05684
HSD11 B1 hydroxysteroid -2.1 -1.9
11-beta
deh dro enase
1
LIFR leukemia inhibitory-2.5 -2.0 -1.7
factor rece for
LIFR leukemia inhibitory-2.5 -2.0 -1.7
factor rece for
HPGD hydroxyprostaglandin-1.9 -2.5
dehydrogenase
15
NAD
CBG corticosteroid -3.5 -2.8 -2.8
binding
lobulin
HAL histidine ammonia-2.2 -2.0 -2.1
I ase
CYP2F2 c tochrome P450, -2.5 -2.3 -1.7
2f2
KEG 1 kidney expressed -2.9 -2.2
ene 1
AI266885 EST -4.7 -3.1 -2.4
Called Present
in only
one animal
PAP pancreatitis- 9.2
associated rotein
1300007021 RIK RIKEN cDNA 4.7
1300007021 ene
REG2 rat regenerating 9.8
islet-
derived, mouse
homolo 2
UNK_AE000664 EST 9.6
SERINElTHREONINE-SERINE/THREONINE-2.1
PROTEIN KI... PROTEIN KI.
1300007021 RIK RIKEN cDNA 3.8
1300007021 ene
CRAT carnitine 2.6
acet Itransferase
AS2 a Isulfatase A 3.2
2310009M24RIK RIKEN cDNA 2.0
2310009M24 ene
2310004B05RIK RIKEN cDNA 2.8
2310004805 ene
REG1 rat regenerating 2.3
islet-
derived, mouse
homolo 1
AW048468 esterase 31 1.8
PAP pancreatitis- 7.7
associated rotein
SULT-X1 sulfotransferase--2.6
related protein
SULT-
X1
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WO 02/068476 PCT/US02/05684
ES31 esterase 31 -1.8
AW538652 EST -1.9
GAMT guanidinoacetate -2.0
meth Itransferase
SCSD sterol-C5-desaturase -1.9
(fungal ERG3,
delta-5-
desaturase) homolog
S. cerevisae
GHR growth hormone -3.0
rece for
A1839995 EST -1.8
0610025L15RIK RIKEN cDNA -1.9
0610025L15 ene
AGXT alanine-glyoxylate-2.5
aminotransferases
PAH phenylalanine -2.0
h drox lase
IGFBP2 insulin-like growth-2.5
factor bindin
rotein 2
A1647632 EST -2.1
A1647632 EST -2.1
G6PC glucose-6- -2.2
hos hatase, catal
tic
CYP17 c tochrome P450, -3.0
17
GSTA2 glutathione S- -2.3
transferase, alpha
2
Yc2
CYP26 cytochrome P450, -9.0
26,
retinoic acid
THRSP thyroid hormone -2.7
responsive SPOT14
homolo Rattus
FM03 flavin containing-2.6
monoox enase 3
Example 9 Effect of an Anti-IL-22 Antibody in an in vivo Arthritis Model
The ability of the P3/1 monoclonal antibody to ameliorate symptoms in a
collagen-induce arthritis (CIA) murine model was examined. Male DBA/1 (Jackson
Laboratories, Bar Harbor, Maine) mice were used for all experiments. Antibody
was
administered prophylactically or therapeutically to DBA mice. In the
therapeutic
regimen, treatment was initiated if disease was observed for two consecutive
days in a
mouse.
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Arthritis was induced with the use of bovine collagen type II (Chondrex,
Redmond, WA). Bovine collagen type II (Chondrex, Redmond, WA) was dissolved in
0.1 M acetic acid and emulsified in an equal volume of CFA (Sigma) containing
lmg/ml
Mycobacterium tuberculosis (strain H37RA). 100 pg of bovine collagen was
injected
subcutaneously in the base of the tail on day 0. On day 21, mice were injected
subcutaneously, in the base of the tail, with a solution containing 200 ~g of
bovine
collagen in O.1M acetic acid that had been mixed with an equal volume of
Incomplete
Freund's adjuvant (Sigma). Naive animals received the same sets of injections,
minus
collagen. The dosing protocol is shown schematically in FIG. 1.
Mice were monitored at least three times a week for disease progression.
Individual limbs were assigned a clinical score based on the index: 0 =
normal; P =
prearthritic, characterized by focal erythema on the tips of digits.; 1 =
visible erythema
accompanied by 1-2 swollen digits.; 2 = pronounced erythema, characterized by
paw
swelling and/or multi digit swelling.; 3 = massive swelling extending into
ankle or wrist
joint.; 4 = difficulty in use of limb or joint rigidity. Thus, the sum of all
limb scores for
any given mouse yielded a maximum total body score of 16.
At various stages of disease, animals were euthanized, tissues were harvested
and
paws were fixed in 10% formalin for histology or 4% paraformaldeyde, pH 7.47,
decalcified in 20% EDTA (pH 8.0) and embedded in paraffin for in situ
hybridization.
Using light microscopy the paws were scored on a 5-grade scoring method (0-4)
to
characterize the intensity and extent of arthritis. Inflammatory infiltrates
were used for
scoring in addition to other changes related to the inflammation, such as
pannus
formation, fibrous of the synovial membrane, articular cartilage erosin and/or
subchondral bone destruction. Hisotology grades were determined using readings
of
individual paws: NAD=0 or nothing abnormal discovered.; 1=Slight to moderate.;
2:
Mild to moderate.; 3: Marked and 4:Massive.
The effect of the therapeutic administration of IL-22 antibody is shown in
FIG. 2.
Body score is shown as a function of time. Mice administered anti-IL-22
antibody
showed significantly decreased symptoms relative to mice administered control
human
IgG or PBS (data not shown).
The effect of prophylactic administration of neutralizing IL-22 antibody is
shown
in FIGS. 3-5. Body score is shown as a function of time following
administration of
anti-IL-22 or control antibody. Mice administered anti-IL-22 antibody showed
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significantly decreased symptoms relative to mice administered control rat IgG
or PBS
(data not shown).
Body score was also examined in mice subjected to a separate prophylactic
regimen. The results are shown in FIG. 4 as a function of time. Mice treated
with
control antibody demonstrated a significantly higher mean total body score
than mice
treated with anti-IL-22. ice administered anti-IL-22 antibody showed
significantly
decreased symptoms relative to mice administered control rat IgGI or PBS (data
not
shown).
The progression of disease in paws of mice subjected to the prophylactic
regimen
is shown in FIG. 5. Mice at day 36 were sacrificed, and the severity of
disease in their
paws examined. The paws were assigned a histology grade of 0 to 4, with 0
corresponding to no disease and 4 representing most severe disease. For rats
injected
with IL-22 antibody, over 60% had a histology grade of "0", while about 20% of
the
mice had a histology grade of "1". About 15% of the mice showed a histology
grade of
"2", and about 10% of the mice showed a histology grade of "3". A small
percentage of
mice showed a histology grade of "4". For mice injected with control antibody,
about
30% showed a histology grade of "0", and about 5% of the mice showed a
histology
grade of "1". The remaining mice exhibited more severe pathology grades: about
18 %
showed a histology grade of "2", while 20% showed a pathology grade of "3",
and the
remaining mice showed a histology grade of "4". Mice administered anti-IL-22
antibody
showed significantly decreased symptoms relative to mice administered control
rat IgGl
or PBS (data not shown).
These results demonstrate that administration of IL-22 antibody either
prophylactically or therapeutically significantly ameliorates symptoms of
arthritis in an
animal system.
Example 10. In situ hybridization of IL-22 transcripts
The expression of IL-22 and IL-22 receptor sequences in various cell types of
foot pads of arthritic mice was determined. Anti-sense IL-22 and IL-22 murine
receptor
riboprobes were produced by generating 2 independent PCR products from the
corresponding transcripts. The oligonucleotides 5'-
GACTGATAATACGACTCACTATAGGGCGAACAATTTTGACTCCGATATTGTC
CAAG-3' (SEQ ID N0:6) and 5'-AGGATGGAGACATCTGACTGCCCTACG-3'
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WO 02/068476 PCT/US02/05684
(SEQ ID NO:S)were used to generate for a IL-22 receptor sense probe and 5'-
ACAATTTTGACTCCGATATTGTCCAAG (SEQ ID N0:7) and 3'-
GACTGATAATACGACTCACTATAGGGCGAAGGATGGAGACATCTGACTGCCC
TACG-3' (SEQ ID N0:8) were used to generate for a IL-22 receptor antisense
probe.
Following PCR amplification probes were generated using T7 RNA polymerase and
in
vitro transcription.
A probe for IL-22 sequences was constructed by placing the following sequence
in a plasmid and placing the sequence under the control of T7 and SP6
promoters to
produce sense or anti-sense transcripts:
CAGCCATACATCGTCAACCGCACCTTTATGCTGGCCAAGGAGGCCAGCCTTGCAGATAACAACACAGATGT
CCGGCTCATCGGGGAGAAACTGTTCCGAGGAGTCAGTGCTAAGGATCAGTGCTACCTGATGAAGCAGGTGC
TCAACTTCACCCTGGAAGACGTTCTGCTCCCCCAGTCAGACAGGTTCCA (SEQ ID N0:9)
T7 RNA polymerase binding sites were incorporated into the oligonucelotides
to insert T7 binding sites at either the 5'end of the PCR product for sense
riboprobe or
the 3'end of the PCR product for antisense riboprobe. Digoxygenin labeled
probes were
prepared with the use of a DIG RNA labeling mix (Roche Diagnostics,
Mannheim,Germany), as described by the manufacturer, and T7 RNA polymerase
(Roche Diagnostics). IL-22 receptor mRNA-positive cells in the paw of CIA
murine
model were macrophages, fibroblasts, a subpopulation of lymphocytes, activated
osteoblasts, synoviocytes and epidermis. No positive staining was seen in the
control
paws or with sense probes. mIL-22 mRNA positive cells were: neutrophils,
macrophages, fibroblasts and Osteocytes. No staining was seen in the paw
section treated
with the sense probe and the control mouse paw stained with mIL-22 mRNA. In
situ
hybridization showed the presence of both the IL-22 receptor and cytokine in
the paws of
arthritic mice.
Eguivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following
claims.
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