U.S. patent application number 11/896988 was filed with the patent office on 2008-03-13 for isolated nucleic acid molecules which encode a soluble il-tif receptor or binding protein which binds to il-tif/il-22, and uses thereof.
This patent application is currently assigned to WYETH. Invention is credited to Laure Dumoutier, Jean-Christopher Renauld.
Application Number | 20080064096 11/896988 |
Document ID | / |
Family ID | 27398593 |
Filed Date | 2008-03-13 |
United States Patent
Application |
20080064096 |
Kind Code |
A1 |
Renauld; Jean-Christopher ;
et al. |
March 13, 2008 |
Isolated nucleic acid molecules which encode a soluble IL-TIF
receptor or binding protein which binds to IL-TIF/IL-22, and uses
thereof
Abstract
The invention relates to soluble proteins which bind to the
molecule known as IL-TIF/IL-22. The proteins can antagonize the
effect of IL-TIF/IL-22 on target cells. The nucleic acid molecules
encoding the proteins, and uses of the protein, are also
described.
Inventors: |
Renauld; Jean-Christopher;
(Brussels, BE) ; Dumoutier; Laure; (Brussels,
BE) |
Correspondence
Address: |
WYETH/FINNEGAN HENDERSON, LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
WYETH
|
Family ID: |
27398593 |
Appl. No.: |
11/896988 |
Filed: |
September 7, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09919162 |
Jul 31, 2001 |
7268223 |
|
|
11896988 |
Sep 7, 2007 |
|
|
|
60245495 |
Nov 3, 2000 |
|
|
|
60234583 |
Sep 22, 2000 |
|
|
|
Current U.S.
Class: |
435/331 ;
435/326; 530/387.1; 530/387.9; 530/388.1 |
Current CPC
Class: |
C07K 14/7155 20130101;
A61P 11/06 20180101; A61P 35/00 20180101; C07K 2319/30 20130101;
A61P 37/08 20180101; A61K 38/00 20130101; A01K 2217/05
20130101 |
Class at
Publication: |
435/331 ;
435/326; 530/387.1; 530/387.9; 530/388.1 |
International
Class: |
C12N 5/06 20060101
C12N005/06; C07K 16/18 20060101 C07K016/18 |
Claims
1-30. (canceled)
31. An isolated antibody which binds to a soluble binding protein
which binds to IL-TIF/IL-22, wherein the soluble binding protein
has a molecular weight of from about 23 to about 40 kilodaltons as
determined by SDS-PAGE.
32. The isolated antibody of claim 31, wherein the soluble protein
comprises the amino acid sequence set forth in SEQ ID NO: 6 or SEQ
ID NO: 11.
33. The isolated antibody of claim 31, wherein the soluble binding
protein is an antagonist for IL-TIF/IL-22.
34. The isolated antibody of claim 31, wherein said antibody is
monoclonal antibody.
35. A hybridoma cell line which produces the monoclonal antibody of
claim 31.
Description
RELATED APPLICATIONS
[0001] This application claims priority of provisional applications
60/245,495 filed Nov. 3, 2000, and, 60/234,583 filed Sep. 22, 2000,
both of which are incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to newly isolated nucleic acid
molecules, proteins and their uses. More specifically, it relates
to a soluble protein which binds to the molecule referred to as
IL-TIF/IL-22 or as it will be referred to hereafter "IL-22BP" or
"IL-22 binding protein." The proteins of the invention inhibit
TIF/IL-22 by binding thereto, and inhibiting IL-TIF/IL-22's effect
on cells.
BACKGROUND AND PRIOR ART
[0003] The last decade has seen knowledge of the immune system and
its regulation expand tremendously. One area of particular interest
has been that of research on the proteins and glycoproteins which
regulate the immune system. One of the best known families of these
molecules are the cytokines. These are molecules which are involved
in the "communication" of cells with each other. The individual
members of the cytokine family have been found to be involved in a
wide variety of pathological conditions, such as cancer and
allergies. Whereas sometimes the cytokines are involved in the
pathology of the condition, they are also known as being
therapeutically useful.
[0004] Interleukins are one type of cytokine. The literature on
interleukins is vast. An exemplary, but by no means exhaustive
listing of the patents in this area includes U.S. Pat. No.
4,778,879 to Mertelsmann et al.; U.S. Pat. No. 4,490,289 to Stern;
U.S. Pat. No. 4,518,584 to Mark et al.; and U.S. Pat. No. 4,851,512
to Miyaji et al., all of which involve interleukin-2 or "IL-2."
Additional patents have issued which relate to interleukin-1
("IL-1"), such as U.S. Pat. No. 4,808,611 to Cosman. The disclosure
of all of these patents are incorporated by reference herein. More
recent patents on different interleukins include U.S. Pat. No.
5,694,234 (IL-13); U.S. Pat. No. 5,650,492 (IL-12); U.S. Pat. Nos.
5,700,664, 5,371,193 and 5,215,895 (IL-11); U.S. Pat. Nos.
5,728,377, 5,710,251, 5,328,989 (IL-10); U.S. Pat. Nos. 5,580,753,
5,587,302, 5,157,112, 5,208,218 (IL-9); U.S. Pat. Nos. 5,194,375,
4,965,195 (IL-7); U.S. Pat. Nos. 5,723,120, 5,178,856 (IL-6), and
U.S. Pat. No. 5,017,691 (IL-4). Even a cursory review of this
patent literature shows the diversity of the properties of the
members of the interleukin family. One can assume that the larger
cytokine family shows even more diversity. See, e.g., Aggarwal et
al., ed., Human Cytokines: Handbook For Basic And Clinical Research
(Blackwell Scientific Publications, 1992), Paul, ed., Fundamental
Immunology (Raven Press, 1993), pg 763-836, "T-Cell Derived
Cytokines And Their Receptors", and "Proinflammatory Cytokines and
Immunity," and Thomson, ed. "The Cytokine Handbook" (1998, Academic
Press). All cited references are incorporated by reference.
[0005] The relationships between various cytokines are complex. As
will be seen from the references cited herein, as the level of a
particular cytokine increases or decreases, this can affect the
levels of other molecules produced by a subject, either directly or
indirectly. Among the affected molecules are other cytokines.
[0006] The lymphokine IL-9, previously referred to as "P40," is a
T-cell derived molecule which was originally identified as a factor
which sustained permanent antigen independent growth of T4 cell
lines. See, e.g., Uyttenhove et al., Proc. Natl. Acad. Sci. 85:
6934 (1988), and Van Snick et al., J. Exp. Med. 169: 363 (1989),
the disclosures of which are incorporated by reference, as is that
of Simpson et al., Eur. J. Biochem. 183: 715 (1989).
[0007] The activity of IL-9 was at first observed on restricted T4
cell lines, failing to show activity on CTLs or freshly isolated T
cells. See, e.g., Uyttenhove et al., supra, and Schmitt et al.,
Eur. J. Immunol. 19: 2167 (1989). This range of activity was
expanded when experiments showed that IL-9 and the molecule
referred to as T cell growth Factor III ("TCGF III") are identical
to MEA (Mast Cell Growth Enhancing Activity), a factor which
potentiates the proliferative response of bone marrow derived mast
cells to IL-3, as is described by Hultner et al., Eur. J. Immunol.
19: 2167 (1989) and in U.S. Pat. No. 5,164,317, the disclosures of
both being incorporated by reference herein. It was also found that
the human form of IL-9 stimulates proliferation of megakaryoblastic
leukemia. See Yang et al., Blood 74: 1880 (1989). Recent work on
IL-9 has shown that it also supports erythroid colony formation
(Donahue et al., Blood 75(12): 2271-2275 (6-1990)); promotes the
proliferation of myeloid erythroid burst formation (Williams et
al., Blood 76: 306-311 (1990)); and supports clonal maturation of
BFU-E's of adult and fetal origin (Holbrook et al., Blood 77(10):
2129-2134 (1991)). Expression of IL-9 has also been implicated in
Hodgkins's disease and large cell anaplastic lymphoma (Merz et al.,
Blood 78(8): 1311-1317 (1990)). Genetic analyses of mice that were
susceptible or resistant to the development of bronchial
hyperresponsiveness have unraveled a linkage with the IL-9 gene as
well as a correlation between IL-9 production and susceptibility in
this model (Nicolaides et al., Proc. Natl. Acad. Sci. USA, 94,
13175-13180, (1997)). Human genetic studies also point to the IL-9
and IL-9R or "IL-9 receptor" genes as candidates for asthma therapy
(Doull et al., Am. J. Respir. Crit. Care Med., 153, 1280-1284,
(1996); Holroyd et al., Genomics 52, 233-235, (1998)). IL-9
transgenic mice allowed for the demonstration that increased IL-9
expression results in lung mastocytosis, hypereosinophilia,
bronchial hyperresponsiveness and high levels of IgE (Temann et
al., J. Exp. Med. 188, 1307-1320 (1998); Godfraind et al., J.
Immunol. 160, 3989-3996 (1998); McLane et al., Am. J. Resp. Cell.
Mol. 19:713-720 (1999)). Taken together, these observations
strongly suggest that IL-9 plays a major role in this disease
Additional work has implicated IL-9 and muteins of this cytokine in
asthma and allergies. See, e.g. PCT Application US96/12757 (Levitt,
et al), and PCT US97/21992 (Levitt, et al), both of which are
incorporated by reference.
[0008] IL-9 is known to affect the levels of other molecules in
subjects. See Louahed et al., J. Immunol. 154: 5061-5070 (1995),
Demoulin et al., Mol. Cell. Biol. 16: 4710-4716 (1996), both
incorporated by reference. It will be recognized that the molecules
affected have their own functions in biological systems. For
example, Demoulin et al. show that many of the known activities of
IL-9 are mediated by activation of STAT transcription factors. As
such, there is continued interest in trying to identify molecules
whose presence and/or level is affected by other molecules, such as
signal transduction molecules and cytokines.
[0009] A new member of the interleukin family is described in,
e.g., U.S. patent application Ser. No. 09/419,568, filed Oct. 18,
1999, and incorporated by reference in its entirety. Also see
Dumoutier, et al, "Human interleukin-10 related T cell derived
inducible factor molecular cloning and function characterization as
a hepatocyte stimulating factor," Proc. Natl. Acad. Sci. USA
97(18): 10144-10149 (2000), also incorporated by reference. Also
see Dumoutier, et al, J. Immunol 164:1814 (2000), and Dumoutier, et
al, Genes Immunol 1:488 (2000), both of which are incorporated by
reference. Also see Ser. No.09/626, 627 filed Jul. 27, 2000,
incorporated by reference. Dumoutier, et al, Proc. Natl. Acad. Sci.
97(18): 10144-10149 (2000) also suggest that this new molecule,
IL-TIF/IL-22, induces acute phase reactant production by liver
cells, in vitro, and in vivo.
[0010] Xie, et al, J. Biol. Chem 27:31335-31339 (2000), have
suggested that this molecule be renamed as IL-22. Xie et al also
teach that the receptor for this molecule consists of two chains,
each of which bind to the molecule. These chains are referred to as
"CRF 2-4" and "CRF 2-9." The former is also referred to as
"IL-10RB" because it is required for IL-10 signalling. See, e.g.,
Kotenko, et al, EMBO J 16:5894 (1997).
[0011] The second chain, CRF 2-9, was originally considered to be
an orphan receptor. This chain is also known as "ZCYTOR 11," but
Xie, et al., supra, have proposed it be renamed "IL-22R". Due to
their structure, both chains are considered to belong to the class
II cytokine receptor family (Kotenko, et al, Oncogene 19:2557
(2000)), which consists of 8 members of known function (i.e., two
pairs of two receptor subunits for type I interferons (IFN-.alpha.,
IFN-.beta., IFN-w, IFN-t) and type II (IFN-.gamma.) interferon,
IL-10R, tissue factor, and the two chains referred to supra. At
least one orphan receptor, referred to as "CRF 2-8," is also a
member of the family. These receptors are related by their
extracellular domains, which have tandem fibronectin type III
(FNIII) domains. Four of the genes encoding these proteins, i.e.,
"IFNAR1," "IFNAR2," IIL10R2" and "IFNGR2," are located on human
chromosome 21. The IFNGR1 and CRF2-8 genes map to chromosome 6,
IL22R is located on chromosome 1, and IL10R1 is on chromosome
11.
[0012] Additional work on these molecules can be found in, e.g.,
International Patent Application Number PCT/US00/1479 (Publication
Number WO OO/65027) and International Parent Application Number
PCT/US99/11644 (Publication Number WO 99/61667). Also see
International Patent Application Number PCT/US00/32703, publication
number WO/01/40467, describing "ZCYTOR16."
[0013] A nucleic acid molecule has now been identified, and is
referred to as IL-22 binding protein (IL-22BP), which encodes
another molecule which binds IL-TIF/IL-22. The protein which the
nucleic acid molecule encodes serves to inhibit the effect that
IL-TIF/IL-22 has on target cells. Further, a second form of the
nucleic acid molecule has been identified as a splice variant of
the first. This second molecule contains an additional 96
nucleotides, and encodes an additional 32 amino acids.
[0014] These, as well as other features of the invention, will be
seen in the disclosure which follows.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE 1
[0015] This example describes experiments which were carried out to
identify potential new members of the class II cytokine receptor
family. Receptors for the interferons, and for IL-10, are members
of this family.
[0016] The amino acid sequence of the extracellular domain of human
IL-10R was used to screen the database of the Sanger Center
(http://www.sanger.ac.uk/c/cgi-bin/nph-Blast_server; html),
incorporated by reference, using TBLASTN software.
[0017] Two short regions of homology were identified in a BAC clone
from chromosome 6q24 (Genbank Accession Number AL 05337), about 40
kilobases from the known IFNGR-1 gene.
[0018] The first fragment showed 40% amino acid identity with
residues 63-119 of IL-10R, while the other, located 3 kb upstream,
showed 47% identity with residues 29-47.
[0019] Once the BAC sequence was identified, it was analyzed
further, using the NIX analysis program found at
http://www.hgmp.mrc.ac.uk/Registered/Webappp/nix/, incorporated by
reference. The software predicted a gene comprising 5 exons,
stretching over about 16 kilobases, with the last exon
corresponding to several EST sequences.
EXAMPLE 2
[0020] These experiments were designed to determine the pattern of
tissue distribution of the molecule identified in example 1,
supra.
[0021] Total RNA was isolated from samples taken from various
organs using guanidium isothiocyanate lysis and CsCl gradient
centrifugation, following Ausubel, et al., Current Protocols In
Molecular Biology (1993), incorporated by reference. Samples of RNA
(5 ug), were reverse transcribed with an oligo(dT) primer, and the
resulting cDNA was amplified via PCR. Specifically, samples
corresponding to 5 ng of total RNA were amplified using. [0022]
agggtacaat ttcagtcccg a (sense, SEQ ID NO: 1) and [0023] cggcgtcatg
ctccattctg a (antisense, SEQ ID NO: 2). The annealing temperature
was 55.degree. C. Resulting PCR products were analyzed in ethidium
bromide stained, 1% agarose gels.
[0024] Strongest expression was found in breast tissue, and a clear
signal was also detected in lungs and the intestinal tract (i.e.,
stomach and colon). Skin, testis, brain, heart, and thymus tissue
were also positive, at lower levels, and not in all samples tested.
There was no detectable expression in prostate, bladder, kidney,
ovary, muscle, bone marrow, liver or uterine tissue.
[0025] One noteworthy feature of these results was the
identification of a second band in some tissue samples, such as
skin and lungs. The significance of this second band is discussed
infra.
EXAMPLE 3
[0026] These experiments describe work in amplifying full length
cDNA for the materials described supra.
[0027] Breast tissue RNA was prepared, as described supra, and was
amplified via RT-PCR, as described supra, using: TABLE-US-00001
tgaacagtca cactcgagac catgatgc, (sense; SEQ ID NO: 3) and
catcctgttc tcgaggagct ttaga. (antisense; SEQ ID NO: 4)
These primers contain mutations which introduce an XhoI site to
permit direct cloning into pCEP4 plasmid described infra. A cDNA
molecule was identified which consisted of 775 nucleotides, of
which 693 constituted an open reading frame that encoded a protein
of 231 amino acids, with a calculated molecular weight of about 27
kd. The nucleotide sequence, and the predicted amino acid sequence,
are presented as SEQ ID NOS: 5 and 6.
[0028] Analysis of the predicted protein reveals a stretch of
hydrophobic amino acids at the N-terminus, compatible with a signal
peptide. There was significant homology to the extracellular
domains of members of the IL-10 receptor family; however, the
molecule under consideration lacked a hydrophobic transmembrane
domain, suggesting it is a secreted protein.
[0029] When the deduced amino acid sequence was aligned with other
proteins, 33% amino acid identity with the extracellular domain of
IL-22R was found, as was 34% with orphan receptor CRF 2-8. Lower
sequence identity was found with IL-10R (29%), CRF2-4/IL-10.beta.
(30%), tissue factor (26%) and the four interferon receptor chains
(23-25%). The predicted mature form of the protein contains 4
cysteine residues. These are conserved in most members of the class
II cytokine receptors. Additionally, the structure of the gene, as
deduced from the information presented herein, is of one that
contains 5 exons, the first of which encodes the signal peptide,
and the following four of which encode the mature protein.
EXAMPLE 4
[0030] The data developed supra showed that the molecule of
interest had highest homology to the extracellular domain of
IL-22R. Experiments were therefore designed to determine if the
molecule bound to IL-TIF/IL-22.
[0031] A series of IL-22BP fusion proteins were made. The first,
referred to as "IL-TIFR-Ig," was produced by first amplifying the
full length open reading frame of the IL-22BP molecule referred to
supra, using the following, mutated antisense primer:
TABLE-US-00002 (SEQ ID NO. 7) ccaacttcca tgatcaatgg aatttccaca
catctct
[0032] This primer serves to introduce a BclI site into the stop
codon of the ORF. In addition, a region comprising the hinge, CH2
and CH3 domains of murine IgG3 isotype heavy chain was amplified,
using the known IgG3 anti-TNP hybridoma C3110. The following
primers were used: TABLE-US-00003 aagactgagt tgatcaagag aatcgagcct
aga (sense, SEQ ID NO. 8) and aatgtctaga tgctgttctc atttacc
(antisense, SEQ ID NO. 9)
These primers also contain BclI and XbaI sites for cloning.
[0033] Following amplification, both PCR products were digested,
and cloned into pCEP4 plasmid, under control of CMV promoter, as
described supra.
[0034] Clones were sequenced, using standard methodologies. These
were then used to transfect HEK293 cells transiently, also as
described supra. In brief, cells were seeded in 6 well plates, at
3.times.10.sup.5 cells/well, one day prior to transfection.
Standard, lipofectamine methodologies were used, using 2 ug of
plasmid DNA. After transfection, cells were incubated in 1.5 ml of
normal medium for 3 days.
[0035] In similar fashion, a fusion protein of IL-22R and the IgG3
Fc fragment was generated, known as IL-22R-Ig. These two fusion
proteins were used together with a control fusion protein, i.e.
IL-9-Ig, which had been made previously.
[0036] Assays were then carried out by coating polystyrene plates
with either 0.083 mg/ml of recombinant human IL-TIF/IL-22, or 0.2
mg/ul bovine serum albumin, in 20 mm Tris. Glycine buffer
containing 30 mm NaCl, pH 9.2, overnight, at 4.degree. C. Following
washing in PBS buffer plus Tween 20 (10.sup.-4) plates were blocked
with PBS plus 1% BSA for two hours, and then 50 .mu.l of
supernatant from transiently transfected HEK293 cells was added.
Plates were then incubated for 2 hours, at 37.degree. C. Any bound
fusion protein was detected, using murine anti-Ig polyclonal
antibodies coupled to peroxidase. Detection was carried out using
the peroxidase substrate "TMB", or
(3,3',5,5'-tetramethylbenzidine), and stopped by 20 .mu.l
H.sub.2S0.sub.4.
[0037] The results indicated that IL-22BP-Ig and IL-22R-Ig both
bind to IL-TIF/IL-22, but not bovine serum albumin. Supernatants of
mock transfected cells, or IL-9-Ig did not detectably bind
IL-TIF/IL-22.
EXAMPLE 5
[0038] These experiments describe studies designed to assess
whether the protein of the invention was able to block IL-TIF/IL-22
activity.
[0039] To test this, the cell lines H4IIE and HT-29, referred to
supra, were used. It is known that H4IIE responds to IL-TIF/IL-22
by activation of STAT transcription factors, and acute phase
reactant production. The HT-29 cell line shows STAT-3 activation.
STAT activation by IL-TIF/IL-22 can be measured, in both cell
lines, via the use of a luciferase reporter construct which
includes 5 STAT binding sites, plus a minimal TK promoter. See
Dumoutier, et al; Proc. Natl. Acad. Sci. USA, 97:10144 (2000),
incorporated by reference.
[0040] The construct "pGRR5" was used. This construct contains 5
copies of the STAT binding site of the Fc.gamma.R1 gene, upstream
of a luciferase gene under control of a tk promoter. As an internal
control, the vector pRL-TK was used. This construct contains the
renilla luciferase gene under the control of the tk promoter.
[0041] The H4IIE and HT-29 cells were electroporated with 15 .mu.g
of pGRR5 and 1 .mu.g pRL-TK (250V, 192.OMEGA., 1,200 .mu.F), and
were then seeded at 4.times.10.sup.5 cells/well. RAW 264.7 cells
were transfected in the same way, the only difference being the
resistance used (74.OMEGA.). This cell line was used to determine
the effect of the IL-22BP material on IL-10.
[0042] The transfected H4IIE or HT-29 cells were then stimulated
with a preincubated (1 hour) mixture of recombinantly produced
IL-TIF/IL-22, at varying concentrations and 5% supernatant (from
HEK293 cells that had been transfected with the cDNA described
herein), or a preincubated mixture of the IL-TIF and 5% supernatant
from mock transfected cells. After two hours, luciferase activity
was measured either in pelleted or lysed cells, or directly in
plated cells, using a commercially available assay.
[0043] The results indicated that the STAT-activating activity of
IL-TIF/IL-22 (at 4 ng/ml), was blocked completely when combined
with supernatants from cells transfected by constructs encoding the
IL-22BP protein or fusion proteins, described supra. This was the
case for both H4IIE and HT-29 cells. In contrast, when IL-6 was
used in place of IL-TIF/IL-22, there was no effect. Nor was IL-10
activity affected by pre-incubation with IL-IL-22BP protein or
fusion protein.
EXAMPLE 6
[0044] Novick, et al, Cytokine 4:6 (1992), have shown that soluble
IL-6 receptor can increase the sensitivity of cells to subliminal
concentrations of its ligand. Studies were therefore carried out,
in parallel to those presented supra, testing low (<25 ng/ml),
and high (50-200 ng/ml) concentrations of IL-TIF/IL-22 in H4IIE
cells. It was found that, at the low concentrations, STAT
activation was blocked completely by IL-22BP but IL-22BP failed to
block STAT activation in H4IIE cells, when high concentrations of
IL-TIF were used. Decreasing the concentration of IL-22BP led to a
loss of inhibitory effect, but did not reveal any potentiating
activity for IL-TIF/IL-22.
EXAMPLE 7
[0045] Fernandez-Botran, et al, J. Exp. Med 174:673 (1991) have
shown that the soluble and transmembrane forms of the IL-4 receptor
have similar association rates, but the soluble form has a higher
dissociation rate. This indicates that the complexes formed by IL-4
and the IL-4 binding protein ("IL-4BP") must be transient and
reversible allowing the ligand to dissociate from one soluble
receptor and become available for binding to another soluble
receptor or to a membrane receptor from which it would dissociate
more slowly. Experiments were carried out to determine if the
protein of the invention exhibited the same property and thus delay
rather than inhibit IL-TIF/IL-22 action.
[0046] It was found that the effect of IL-TIF/IL-22 on
STAT-activation in HT-29 cells reached its peak after 4-6 hours,
and decreased dramatically at 24 hours but the receptor of the
invention had the same inhibitory effect throughout the assay,
indicating that it could not delay IL-TIF/IL-22 activity in
vitro.
EXAMPLE 8
[0047] Example 2, supra, referred to the identification of a second
band in some tissue samples. The band was excised, and sequenced
using an automated, fluorescence based system, and art recognized
methods. The sequence, set forth at SEQ ID NO. 10, includes an
additional 96 nucleotides, corresponding to 32 additional amino
acids in the predicted protein (SEQ ID NO. 11).
[0048] The preceding examples disclose the aspects of this
invention, including isolated nucleic acid molecules which encode a
soluble, receptor-like antagonist of IL-TIF/IL-22 such as those
with the amino acid sequence of the protein encoded by the
nucleotide sequences set forth in SEQ ID NO: 5 or 10. It will be
appreciated by one of ordinary skill that the degeneracy of the
genetic code facilitates the preparation of nucleic acid molecules
which may not be identical to the nucleotide sequence of SEQ ID NO:
5 or 10, but which encode the same protein. Of course, SEQ ID NO: 5
or 10 are preferred embodiments of this invention, but other
embodiments are also a part of the invention. Genomic DNA,
complementary DNA, and RNA, such as messenger RNA, are all to be
included therein. Isolated nucleic acid molecules from other animal
species, including other mammals, are also a part of the invention.
A preferred aspect of the invention are isolated nucleic acid
molecules whose complements hybridize to SEQ ID NO: 5 or 10 under
stringent conditions. "Stringent conditions," as used herein,
refer, for example, to hybridization at 65.degree. C. in buffer
(3.5.times.SSC), 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02%
bovine serum albumin, 25 mM NaH.sub.2PO.sub.4 (pH 7), 0.1% SDS, 2
mM EDTA, followed by a final wash at 2.times.SSC, room temperature
and then 0.1.times.SSC/0.2.times.SDS at temperatures as high as,
e.g., about 65.degree. C. More stringent conditions, such as
0.1.times.SSC, can also be used. These nucleic acid molecules
encode proteins, such as those with amino acid sequences set forth
at SEQ ID NO: 6 or 11. The soluble, receptor-like antagonist of
this invention may be found in glycosylated or non-glycosylated,
sulfated and non-sulfated forms and so forth. Also a part of the
invention are isolated nucleic acid molecules which encode proteins
having at least 30%, preferably at least 45%, more preferably at
least 60%, and most preferably 90% amino acid identity with an
amino acid sequence of a protein encoded by SEQ ID NO: 5 or 10.
[0049] Also a part of the invention are expression vectors which
include the nucleic acid molecules of the invention, operably
linked to a promoter, so as to facilitate expression of the DNA. It
is well within the skill of the artisan to prepare such
vectors.
[0050] The vectors, as well as the nucleic acid molecules per se,
can be used to prepare recombinant cells, such as isolated
recombinant cells, be these eukaryotic or prokaryotic, wherein
either an expression vector or the nucleic acid molecule itself is
incorporated therein. E. coli cells, COS cells, CHO cells, Sf9
cells etc., are all examples of types of cells which may be used in
accordance with this aspect of the invention.
[0051] Proteins encoded by the above referenced nucleic acid
molecules, preferably in isolated form, are another feature of this
invention. By "protein" is meant both the immediate product of
expression of the nucleic acid molecules, glycosylated forms of it,
forms of the molecule following peptide signal cleavage, such as
mature and/or processed forms of the protein, as well as multimeric
forms, such as dimers, trimers, and so forth. Also a part of the
invention are multimers, such as dimers, which contain at least one
protein molecule of the invention, and at least one, different
protein molecule. These multimers may be homomeric or heteromeric,
such as heteromeric forms that include at least one molecule of a
different soluble receptor, a transmembrane receptor, and so forth.
Such multimers may bind only a single specific ligand. Also a part
of the invention are complexes of the IL-22BP and a ligand, which
then act as heteromeric cytokines in transmembrane receptors. Such
structures parallel, e.g., the structure of IL-12. Also a feature
of this invention is a protein consisting of the sequence set forth
in SEQ ID NO: 6 or 11. Also included as a feature of this invention
are proteins that are essentially identical to the sequence in SEQ
ID NO: 6 or 11 having only conservative amino acid substitutions.
Also included as a feature of the inventions are constructs, such
as fusion proteins, where all or a part of the proteins described
supra are linked in some fashion, e.g., to a "fusion partner" at
least one additional protein or peptide, or amino acid sequence.
The "fusion partner" may be, for example, a molecule which provides
a recognizable signal, either directly or indirectly, such as a
FLAG peptide, .beta.-galactosidase, luciferase, an Fc
immunoglobulin, a fluorescent protein, such as "GFP" (green
fluorescent protein), and so forth. These fusion partners are
preferably joined to the molecule which is described supra at the
N-- and/or C-terminus of the protein; however, it is to be
understood that there are many techniques known for joining
molecules to amino acids, and any and all of these methodologies
can produce constructs which are a part of the invention.
[0052] The individual protein molecules of the invention will
preferably have a molecular weight of from about 23 to about 40
kilodaltons as determined by SDS-PAGE. In multimeric forms, the
molecular weight of the complex will, of course, vary, but the
individual molecules contained therein will each have a molecular
weight of about 23-40 kilodaltons, as determined by SDS-PAGE. These
molecular weights, it is to be understood, refer to monomeric
proteins. Glycosylated monomers will have higher molecular weights,
e.g., up to at least about 40-50 kilodaltons.
[0053] The proteins preferably consist of at least about 180 and no
more than about 300 amino acids. More preferably, the protein
consists of about 230-275, more preferably 230-268, most preferably
231-263 amino acids. Preferably, the amino acids sequences consists
of or comprises all or part of the amino acid sequences encoded by
SEQ ID NO: 6 or 11. Such binding proteins can be produced via,
e.g., transforming host cells with one or more nucleic acid
molecules or expression vectors in accordance with the invention,
culturing the transformant, and then isolating the resulting,
recombinant binding protein.
[0054] It will be appreciated by the skilled artisan that the
proteins encoded by the above recited nucleic acid molecules are a
feature of the invention, and may be used to produce antibodies, in
accordance with standard protocols. Such antibodies, in monoclonal
and polyclonal form, constitute a further feature of the invention
as do fragments of said antibodies, chimeric forms, humanized
forms, recombinant forms, hybridoma cell lines which produce the
antibodies and so forth. Also a feature of the invention are
immunogens, comprising all or a part of the amino acid sequence of
protein molecules of the invention, preferably combined with an
adjuvant, such as Complete or Incomplete Freund's Adjuvant.
Portions of the protein sequences may be linked to other molecules,
such as keyhole limpet hemocyanin, to render them more immunogenic.
These antibodies can be used, e.g., to determine if the proteins of
the invention are present. This is a further feature of the
invention, as is now explained.
[0055] It has been shown, in the examples, that the nucleic acid
molecules of the invention encode proteins that block IL-TIF/IL-22
activity. Hence, a further feature of the invention is a method
inhibiting IL-TIF/IL-22 activity, such as the activation of STAT
transcription factors and acute phase reactant production by
contacting a sample with an amount of the protein of this invention
sufficient to inhibit or block the activity of IL-TIF/IL-22.
[0056] One could also use these molecules to test the efficacy of
IL-9 agonists or antagonists when administered to a subject, such
as a subject suffering from lymphoma, an immune system disorder
such as an allergy, acquired immune deficiency syndrome, autoimmune
diabetes, thyroiditis, or any of the other conditions described in,
e.g, U.S. Pat. Nos. 5,830,454; 5,824,551, and pending application
Ser. No. 08/925,348, filed on Sep. 8, 1997 now allowed, all of
which are incorporated by reference. The molecules can also be used
to modulate the role of IL-9 in these and other conditions. To
elaborate, since IL-9 induces IL-TIF/IL-22 and the proteins of this
invention block the activity of IL-TIF/IL-22, the proteins of this
invention are useful as IL-9 activity modulators. Thus, a further
aspect of the invention is a method to determine activity of
endogenous IL-9, such as in situations where excess IL-9 activity
is implicated, including asthma, allergies, and lymphomas. One can
also block or inhibit IL-9 activity by blocking or inhibiting
IL-TIF/IL-22 or IL-TIF/IL-22 activity, using the receptor-like
antagonist of this invention. Examples of conditions which can be
treated by the use of the protein of this invention are allergies,
asthma, lymphoma, and so forth. The ability to regulate IL-9
activity is important in conditions such as those listed supra, as
well as conditions such as apoptosis, including cortisol induced
apoptosis, conditions involving the nuclear expression of BCL-3,
since IL-9 is known to induce such expression, and so forth.
[0057] IL-TIF/IL-22 type molecules may either promote regeneration
or inhibit differentiation of tissue types in which these molecules
are active. IL-TIF/IL-22 molecules target various cancer and normal
cell lines (i.e., mesangial and neuronal cells, as well as melanoma
and hepatoma cells. See, e.g., U.S. patent application Ser. No.
09/626,617 filed, incorporated by reference). Hence, another
feature of the invention is a method of treatment of a patient in
need thereof wherein the proteins of this invention are used to
inhibit the activity of IL-TIF/IL-22, in, e.g., neoplastic tissue,
such as melanoma or hepatoma.
[0058] It will be clear to the skilled artisan that IL-TIF/IL-22
can regulate the inflammatory response. A preferred aspect of this
regulation is the modulation of the acute phase response by organs,
such as the liver, by administering the receptor-like antagonist of
this invention. See, e.g., Janeway et al., Immunobiology, (4.sup.th
edition), incorporated by reference. Janeway explains that various
cytokines such as IL-1, IL-6 and TNF-.alpha. activate hepatocytes
to synthesize acute phase proteins, such as c-reactive protein, and
mannan binding lectin, as well as those described in the examples,
supra.
[0059] IL-TIF/IL-22 has a role in activating acute phase proteins.
Thus another aspect of this invention is a method for reducing the
production of acute phase proteins, stimulated by IL-TIF/IL-22, by
administering an amount of the receptor-like antagonist of this
invention to a tissue sample or to a patient in need thereof,
wherein said amount is sufficient to reduce production or activity
of acute phase proteins.
[0060] Also a part of the invention are methods for regulating
activity of IL-TIF/IL-22 by administering the receptor-like
antagonist to regulate IL-TIF/IL-22 activity.
[0061] Also a part of this invention is a method for determining
the presence of the receptor-like antagonist of this invention in a
tissue or cell sample comprising contacting said sample with an
antibody specific for said receptor-like antagonist and determining
binding therebetween. Methods for determining the binding of an
antibody and its antigen are well known to those of skill in the
art and need not be elaborated herein.
[0062] The receptor-like antagonist of this invention may also be
used to determine the presence of IL-TIF/IL-22 in a sample by,
e.g., labeling said receptor-like antagonist and then contacting
said sample with said receptor-like antagonist and determining
binding therebetween wherein said binding is indicative of the
presence of IL-TIF. Alternatively, one may determine the presence
of IL-TIF/IL-22 in a sample by treating a cell line that is
responsive to IL-TIF/IL-22 to two aliquots of said sample, one
containing the receptor-like antagonist and one without the
receptor-like antagonist, then measuring and comparing the response
of said responsive cell to the two aliquots wherein a difference in
response to the two aliquots is indicative of the presence of
IL-TIF/IL-22. In the alternative, cells that are responsive to
IL-TIF/IL-22 can be used in such assays. To elaborate, cells which
show some type of response to IL-TIF/IL-22, such as increased STAT
activation or acute phase reactant production, can be used to
screen for presence and/or amount of IL-22BP in a sample. For
example, assuming that the cell is incubated in the sample in
question together with IL-TIF/IL-22, any observed change in the
response, such as a decrease in STAT activation or acute phase
reactant production, is indicative of IL-22 BP in said sample.
[0063] The soluble IL-TIF/IL-22 binding proteins described herein
are further examples of soluble, cytokine receptors generated in
vivo. See, e.g. Rose-John, et al., Biochem J. 300: 281 (1994);
Fernandez-Botran, et al., Adv. Immunol 63:269 (1996). Heaney, et
al., Blood 87: 845 (1996). Soluble cytokine receptors compete with
cell surface receptors for binding to free or unbound cytokine
molecules. With the exception of IL-6R, this binding prevents
cytokines from reaching the cell membrane and generating a signal.
The binding is generally reversible, leading to temporary
sequestration of the cytokine from membrane receptors. Soluble
cytokine receptors also enhance the activity of cytokines by
modifying their stability, decreasing proteolytic degradation, or
reducing clearance. Such functions, i.e., as cytokine carriers in
vivo, are seen to help potentiate the systemic effect of cytokines,
with the antagonistic effect being pertinent to paracrine
activities.
[0064] Other features of the invention will be clear to the artisan
and need not be discussed further.
[0065] The terms and expressions which have been employed are used
as terms of description and not of limitation, and there is no
intention in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, it being recognized that various modifications are
possible within the scope of the invention.
Sequence CWU 1
1
11 1 21 DNA Homo sapiens 1 agggtacaat ttcagtcccg a 21 2 21 DNA Homo
sapiens 2 cggcgtcatg ctccattctg a 21 3 28 DNA Homo sapiens 3
tgaacagtca cactcgagac catgatgc 28 4 25 DNA Homo sapiens 4
catcctgttc tcgaggagct ttaga 25 5 775 DNA Homo sapiens 5 tgaacagtca
cacttgcaac catgatgcct aaacattgct ttctaggctt cctcatcagt 60
ttcttcctta ctggtgtagc aggaactcag tcaacgcatg agtctctgaa gcctcagagg
120 gtacaatttc agtcccgaaa ttttcacaac attttgcaat ggcagcctgg
gagggcactt 180 actggcaaca gcagtgtcta ttttgtgcag tacaaaatat
atggacagag acaatggaaa 240 aataaagaag actgttgggg tactcaagaa
ctctcttgtg accttaccag tgaaacctca 300 gacatacagg aaccttatta
cgggagggtg agggcggcct cggctgggag ctactcagaa 360 tggagcatga
cgccgcggtt cactccctgg tgggaaacaa aaatagatcc tccagtcatg 420
aatataaccc aagtcaatgg ctctttgttg gtaattctcc atgctccaaa tttaccatat
480 agataccaaa aggaaaaaaa tgtatctata gaagattact atgaactact
ataccgagtt 540 tttataatta acaattcact agaaaaggag caaaaggttt
atgaaggggc tcacagagcg 600 gttgaaattg aagctctaac accacactcc
agctactgtg tagtggctga aatatatcag 660 cccatgttag acagaagaag
tcagagaagt gaagagagat gtgtggaaat tccatgactt 720 gtggaatttg
gcattcagca atgtggaaat tctaaagctc cctgagaaca ggatg 775 6 230 PRT
Homo sapiens 6 Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe
Phe Leu Thr 5 10 15 Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu
Lys Pro Gln Arg 20 25 30 Val Gln Phe Gln Ser Arg Asn Phe His Asn
Ile Leu Gln Trp Gln Pro 35 40 45 Gly Arg Ala Leu Thr Gly Asn Ser
Ser Val Tyr Phe Val Gln Tyr Lys 50 55 60 Ile Tyr Gly Gln Arg Gln
Trp Lys Asn Lys Glu Asp Cys Trp Gly Thr 65 70 75 80 Gln Glu Leu Ser
Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln Glu 85 90 95 Pro Tyr
Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser Glu 100 105 110
Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys Ile Asp 115
120 125 Pro Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu Leu Val
Ile 130 135 140 Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu
Lys Asn Val 145 150 155 160 Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr
Arg Val Phe Ile Ile Asn 165 170 175 Asn Ser Leu Glu Lys Glu Gln Lys
Val Tyr Glu Gly Ala His Arg Ala 180 185 190 Val Glu Ile Glu Ala Leu
Thr Pro His Ser Ser Tyr Cys Val Val Ala 195 200 205 Glu Ile Tyr Gln
Pro Met Leu Asp Arg Arg Ser Gln Arg Ser Glu Glu 210 215 220 Arg Cys
Val Glu Ile Pro 225 230 7 37 DNA Homo sapiens 7 ccaacttcca
tgatcaatgg aatttccaca catctct 37 8 33 DNA Homo sapiens 8 aagactgagt
tgatcaagag aatcgagcct aga 33 9 27 DNA Homo sapiens 9 aatgtctaga
tgctgttctc atttacc 27 10 792 DNA Homo sapiens 10 atgatgccta
aacattgctt tctaggcttc ctcatcagtt tcttccttac tggtgtagca 60
ggaactcagt caacgcatga gtctctgaag cctcagaggg tacaatttca gtcccgaaat
120 tttcacaaca ttttgcaatg gcagcctggg agggcactta ctggcaacag
cagtgtctat 180 tttgtgcagt acaaaatcat gttctcatgc agcatgaaaa
gctctcacca gaagccaagt 240 ggatgctggc agcacatttc ttgtaacttc
ccaggctgca gaacattggc taaatatgga 300 cagagacaat ggaaaaataa
agaagactgt tggggtactc aagaactctc ttgtgacctt 360 accagtgaaa
cctcagacat acaggaacct tattacggga gggtgagggc ggcctcggct 420
gggagctact cagaatggag catgacgccg cggttcactc cctggtggga aacaaaaata
480 gatcctccag tcatgaatat aacccaagtc aatggctctt tgttggtaat
tctccatgct 540 ccaaatttac catatagata ccaaaaggaa aaaaatgtat
ctatagaaga ttactatgaa 600 ctactatacc gagtttttat aattaacaat
tcactagaaa aggagcaaaa ggtttatgaa 660 ggggctcaca gagcggttga
aattgaagct ctaacaccac actccagcta ctgtgtagtg 720 gctgaaatat
atcagcccat gttagacaga agaagtcaga gaagtgaaga gagatgtgtg 780
gaaattccat ga 792 11 263 PRT Homo sapiens 11 Met Met Pro Lys His
Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu 5 10 15 Thr Gly Val Ala
Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln 20 25 30 Arg Val
Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln 35 40 45
Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr 50
55 60 Lys Ile Met Phe Ser Cys Ser Met Lys Ser Ser His Gln Lys Pro
Ser 65 70 75 80 Gly Cys Trp Gln His Ile Ser Cys Asn Phe Pro Gly Cys
Arg Thr Leu 85 90 95 Ala Lys Tyr Gly Gln Arg Gln Trp Lys Asn Lys
Glu Asp Cys Trp Gly 100 105 110 Thr Gln Glu Leu Ser Cys Asp Leu Thr
Ser Glu Thr Ser Asp Ile Gln 115 120 125 Glu Pro Tyr Tyr Gly Arg Val
Arg Ala Ala Ser Ala Gly Ser Tyr Ser 130 135 140 Glu Trp Ser Met Thr
Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys Ile 145 150 155 160 Asp Pro
Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu Leu Val 165 170 175
Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn 180
185 190 Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe Ile
Ile 195 200 205 Asn Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly
Ala His Arg 210 215 220 Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser
Ser Tyr Cys Val Val 225 230 235 240 Ala Glu Ile Tyr Gln Pro Met Leu
Asp Arg Arg Ser Gln Arg Ser Glu 245 250 255 Glu Arg Cys Val Glu Ile
Pro 260
* * * * *
References