U.S. patent application number 11/675913 was filed with the patent office on 2008-02-21 for interleukin-1 related gene and protein.
Invention is credited to Vadim Iourgenko, Mark A. Labow.
Application Number | 20080045699 11/675913 |
Document ID | / |
Family ID | 22976589 |
Filed Date | 2008-02-21 |
United States Patent
Application |
20080045699 |
Kind Code |
A1 |
Labow; Mark A. ; et
al. |
February 21, 2008 |
Interleukin-1 Related Gene and Protein
Abstract
Disclosed is an interleukin-1 related gene and gene product. In
particular, the invention relates to a protein that is highly
homologous to known interleukin-1 cytokines, nucleic acid molecules
that encode such a protein, antibodies that recognize the protein,
and methods for diagnosing conditions related to host inflammatory
and immune responses.
Inventors: |
Labow; Mark A.; (Westfield,
NJ) ; Iourgenko; Vadim; (Bridgewater, NJ) |
Correspondence
Address: |
NOVARTIS INSTITUTES FOR BIOMEDICAL RESEARCH, INC.
400 TECHNOLOGY SQUARE
CAMBRIDGE
MA
02139
US
|
Family ID: |
22976589 |
Appl. No.: |
11/675913 |
Filed: |
July 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10451315 |
Jul 10, 2003 |
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PCT/EP01/15125 |
Dec 20, 2001 |
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11675913 |
Jul 12, 2007 |
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60257509 |
Dec 21, 2000 |
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Current U.S.
Class: |
530/387.9 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/545 20130101 |
Class at
Publication: |
530/387.9 |
International
Class: |
C07K 16/00 20060101
C07K016/00 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
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10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
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15. (canceled)
16. (canceled)
17. An antibody or a fragment thereof which specifically binds to a
polypeptide that comprises the amino acid sequence set forth in SEQ
ID NO:1 or to a fragment of said polypeptide.
18. An antibody fragment according to claim 17 which is an Fab or
F(ab').sub.2 fragment.
19. An antibody according to claim 17 which is a polyclonal
antibody.
20. An antibody according to claim 17 which is a monoclonal
antibody.
21. A method for producing a polypeptide as defined in claim 1 or
2, which method comprises: culturing a host cell having
incorporated therein an expression vector comprising an
exogenously-derived polynucleotide encoding a polypeptide
comprising an amino acid sequence as set forth in SEQ ID NO:1 under
conditions sufficient for expression of the polypeptide in the host
cell, thereby causing the production of the expressed
polypeptide.
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
Description
FIELD OF THE INVENTION
[0001] This invention relates to an interleukin-1 related gene and
gene product. In particular, the invention relates to a protein
that is highly homologous to known interleukin-1 cytokines, nucleic
acid molecules that encode such a protein, antibodies that
recognize the protein, and methods for diagnosing conditions
related to host inflammatory and immune responses.
BACKGROUND OF THE INVENTION
[0002] The proinflammatory cytokine interleukin-1 (IL-1) elicits a
wide array of biological activities that initiate and promote the
host response to pathophysiological states including infection,
fever, sleep, loss of appetite, acute phase protein synthesis,
chemokine production, adhesion molecule upregulation, vasodilation,
the coagulant state, increased hematopoiesis, and production and
release of matrix metalloproteinases and growth factors. Until
recently IL-1 activity was thought to reside in each of two
molecules, IL-1 alpha (IL-1.alpha.) and IL-1 beta (IL-1.beta.),
which are potent inflammatory cytokines that play important roles
in host immune responses and in the development of inflammatory and
autoimmune diseases. While only being about 25% identical, the two
cytokines interact with and activate the same receptor complex,
composed of the IL-1 Type-I receptor (IL-1RI) and IL-1 Receptor
Accessory Protein (IL-1RAP) subunits. Upon binding and receptor
activation, a number of signal transduction pathways are activated
including that controlled by NF-kappa B. In fact, IL-1.alpha. and
IL-1.beta. are thought to positively or negatively modulate NF-kB
and AP-1 signaling stimulated through IL-IR1-associated kinase/TNF
receptor-associated factor-like complexes recruited through the
IL-1 receptor family. IL-1 induces expression of a large number of
genes including cytokines, growth factors, cell adhesion molecules
transcription factors and proteases. In addition to the agonist
peptides, a third IL-1, IL-1 receptor antagonist protein (IL-1RA)
can bind to IL-1 receptors and block activity of IL-1.alpha. and
IL-1.beta.. The IL-1 system has been shown to effect a number of
inflammatory diseases in animals and in humans. Blockade of IL-1
signalling with an antibody to either of IL-1.alpha., IL-1.beta. or
the IL-1RI blocks the development of the disease processes in
animal models of arthritis, encephalitis, contact sensitivity,
graft rejection, endotoxic shock and inflammatory bowel disease
among others. In addition, the recombinant IL-1 receptor antagonist
protein has been shown to block the progression of rheumatoid
arthritis in human clinical trials.
[0003] Recent studies have shown the IL-1 system is represented by
a family of related genes. One IL-1 related gene is IL-18. This
cytokine was originally cloned by its ability to induce gamma
interferon expression. It was later shown that this molecule had
significant structural homology to IL-1 and subsequently shown to
bind to receptors highly related, but distinct from the IL-1RI and
IL-1 RAP. Another member of the family, the IL-1 receptor
antagonist (IL-1RA), also binds to IL-1RI but fails to induce the
subsequent interaction with IL-1 RAP, thus not only not signaling
itself, but also, by blocking the receptor, preventing the action
of agonist IL-1s. Recently, 4 new IL-1 related molecules have been
identified. These proteins share between 13 and 50% identify to the
characterized IL-1 molecules. In this respect, two reports have
identified overlapping but nonidentical sets of IL-1 related genes
including IL-1H1, IL-1H2, IL-1H3 and IL-H4 (Kumar et al., J. Biol.
Chem. 275 (2000), 10308-10314), and FIL.delta., FIL.epsilon.,
FIL.nu. and FIL.zeta. (Smith et al., J. Biol. Chem. 275 (2000),
1169-1175). Several of these genes are either identical or clearly
derived from the same gene including IL-1H2 and FIL.nu., IL-1H3 and
FIL.delta., and IL-1H4 and FIL.zeta.. IL-1H4 and FIL.zeta. are 88%
identical with changes only at the extreme ends of cDNAs. Thus,
these two sequences are derived from alternative splicing of the
same gene. Interestingly, most of these molecules are relatively
more related to IL-1RA than to either IL-1.alpha., IL-1.beta. or
IL-18, implying that common, IL-1 antagonizing, effect of these
molecules may coincide with separate, unique activities.
Importantly, while related at the amino acid level, none of the
IL-1 related proteins appears to bind to the known IL-1
receptors.
SUMMARY OF THE INVENTION
[0004] The present invention relates to interleukin-1 cytokines, in
particular to a novel interleukin-1 related polypeptide 1
(IL-1RP1).
[0005] In a first aspect, the invention provides an isolated
polypeptide comprising an amino acid sequence as set forth in SEQ
ID NO:1. Furthermore, the invention provides an isolated
polypeptide consisting of an amino acid sequence as set forth in
SEQ ID NO:1. The amino acid sequence as set forth in SEQ ID NO:1
shows a considerable degree of homology to that of known members of
the family of interleukin-1 polypeptides. For convenience, the
polypeptide consisting of the amino acid sequence as set forth in
SEQ ID NO:1 will be designated as interleukin-1 related polypeptide
1, or IL-1RP1. Such a polypeptide, or a fragment thereof, is
expressed in various tissues, predominantly in skin, like another
known member of the interleukin-1 polypeptide family. Fragments of
the isolated polypeptide having an amino acid sequence as set forth
in SEQ ID NO:1 will comprise polypeptides comprising from about 5
to 152 amino acids, preferably from about 10 to about 152 amino
acids, more preferably from about 20 to about 100 amino acids, and
most preferably from about 20 to about 50 amino acids. Such
fragments also form a part of the present invention. In accordance
with this aspect of the invention there are provided novel
polypeptides of human origin as well as biologically,
diagnostically or therapeutically useful fragments, variants and
derivatives thereof, variants and derivatives of the fragments, and
analogs of the foregoing.
[0006] In a second aspect, the invention provides an isolated DNA
comprising a nucleotide sequence that encodes a polypeptide as
mentioned above. In particular, the invention provides (1) an
isolated DNA comprising the nucleotide sequence as set forth in SEQ
ID NO:2; (2) an isolated DNA comprising the nucleotide sequence set
forth in SEQ ID NO:3; (3) an isolated DNA capable of hybridizing
under high stringency conditions to the nucleotide sequence set
forth in SEQ ID NO:3; and (4) an isolated DNA comprising the
nucleotide sequence set forth in SEQ ID NO:4. Also provided are
nucleic acid sequences comprising at least about 15 bases,
preferably at least about 20 bases, more preferably a nucleic acid
sequence comprising about 30 contiguous bases of SEQ ID NO:2 or SEQ
ID NO:3. Also within the scope of the present invention are nucleic
acids that are substantially similar to the nucleic acid with the
nucleotide sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3. In
a preferred embodiment, the isolated DNA takes the form of a vector
molecule comprising at least a fragment of a DNA of the present
invention, in particular comprising the DNA consisting of a
nucleotide sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3.
[0007] A third aspect of the present invention encompasses a method
for the diagnosis of conditions associated with host inflammatory
or immune responses in a human which includes detecting elevated
transcription of messenger RNA transcribed from the natural
endogenous human gene encoding the novel polypeptide consisting of
the amino acid sequence set forth in SEQ ID NO:1 in an appropriate
tissue or cell from a human, where such elevated transcription is
diagnostic of the human's affliction with such a condition. In
particular, the said natural endogenous human gene encoding the
novel polypeptide consisting of the amino acid sequence set forth
in SEQ ID NO:1 comprises the genomic nucleotide sequence set forth
in SEQ ID NO:4. In one embodiment of the present invention, the
diagnostic method comprises contacting a sample of said appropriate
tissue or cell or contacting an isolated RNA or DNA molecule
derived from that tissue or cell with an isolated nucleotide
sequence of at least about 15-20 nucleotides in length that
hybridizes under high stringency conditions with the isolated
nucleotide sequence encoding the novel polypeptide having an amino
acid sequence set forth in SEQ ID NO:1. Another embodiment of the
assay aspect of the invention provides a method for the diagnosis
of certain diseases associated with host inflammatory or immune
responses in a human which requires measuring the amount of the
polypeptide of SEQ ID NO:1 or fragments thereof in a certain tissue
or cell from a human suffering from such a disease, where the
presence of an elevated amount of the polypeptide or fragments
thereof, relative to the amount of the polypeptide or fragments
thereof in the respective tissue or cell of a healthy individual,
is diagnostic of the human's suffering from conditions associated
with host inflammatory or immune responses.
[0008] In accordance with one embodiment of this aspect of the
invention there are provided anti-sense polynucleotides that can
regulate transcription of the gene encoding the novel interleukin-1
related polypeptide 1; in another embodiment, double stranded RNA
is provided that can regulate the transcription of the gene
encoding the novel IL-1RP1.
[0009] Another aspect of the invention provides a process for
producing the aforementioned polypeptides, polypeptide fragments,
variants and derivatives, fragments of the variants and
derivatives, and analogs of the foregoing. In a preferred
embodiment of this aspect of the invention there are provided
methods for producing the aforementioned interleukin-1 related
peptide 1 comprising culturing host cells having incorporated
therein an expression vector containing an exogenously-derived
nucleotide sequence encoding such a polynucleotide under conditions
sufficient for expression of the polypeptide in the host cell,
thereby causing expression of the polypeptide, and optionally
recovering the expressed polypeptide. In a preferred embodiment of
this aspect of the present invention, there is provided a method
for producing polypeptides comprising or consisting of an amino
acid sequence as set forth in SEQ ID NO:1, which comprises
culturing a host cell having incorporated therein an expression
vector containing an exogenously-derived polynucleotide encoding a
polypeptide comprising or consisting of an amino acid sequence as
set forth in SEQ ID NO:1, under conditions sufficient for
expression of such a polypeptide in the host cell, thereby causing
the production of an expressed polypeptide, and optionally
recovering the expressed polypeptide. Preferably, in any of such
methods the exogenously derived polynucleotide comprises or
consists of the nucleotide sequence set forth in SEQ ID NO:2, the
nucleotide sequence set forth in SEQ ID NO:3, or the nucleotide
sequence set forth in SEQ ID NO:4. In accordance with another
aspect of the invention there are provided products, compositions,
processes and methods that utilize the aforementioned polypeptides
and polynucleotides for, inter alia, research, biological, clinical
and therapeutic purposes.
[0010] In certain additional preferred embodiments of this aspect
of the invention there is provided an antibody or a fragment
thereof which specifically binds to a polypeptide that comprises
the amino acid sequence set forth in SEQ ID NO:1, i.e., IL-1RP1. In
certain particularly preferred embodiments in this regard, the
antibodies are highly selective for human IL-1RP1-polypeptides or
portions of human IL-1RP1 polypeptides.
[0011] In a further aspect, an antibody or fragment thereof is
provided that binds to a fragment or portion of the amino acid
sequence set forth in SEQ ID NO:1.
[0012] In another aspect, methods of treating a disease in a
subject, where the disease is mediated by or associated with an
increase or decrease in IL-1RP1 gene expression or an increase or
decrease in the presence of IL-1RP1 polypeptide in a certain tissue
or cell by the administration of an effective amount of an antibody
that binds to a polypeptide with the amino acid sequence set out in
SEQ ID NO:1, or a fragment or portion thereof to the subject are
provided. Also provided are methods for the diagnosis of a disease
or condition associated with an increase or decrease in IL-1RP1
gene expression or an increase or decrease in the presence of the
IL-1RP1 polypeptide in a subject, which comprises utilizing an
antibody that binds to a polypeptide with the amino acid sequence
set out in SEQ ID NO:1, or a fragment or portion thereof, in an
immunoassay.
[0013] In yet another aspect, the invention provides host cells
which can be propagated in vitro, preferably vertebrate cells, in
particular mammalian cells, or bacterial cells, which are capable
upon growth in culture of producing a polypeptide that comprises
the amino acid sequence set forth in SEQ ID NO:1 or fragments
thereof, where the cells contain transcriptional control DNA
sequences, preferably other than human IL-1RP1 transcriptional
control sequences, where the transcriptional control sequences
control transcription of DNA encoding a polypeptide with the amino
acid sequence according to SEQ ID NO:1 or fragments thereof.
[0014] In yet another aspect of the present invention there are
provided assay methods and kits comprising the components necessary
to detect above-normal expression of polynucleotides encoding a
polypeptide comprising an amino acid sequence as set forth in SEQ
ID NO:1, or polypeptides comprising an amino acid sequence set
forth in SEQ ID NO:1, or fragments thereof, in body tissue samples
derived from a patient, such kits comprising e.g., antibodies that
bind to a polypeptide comprising an amino acid sequence set forth
in SEQ ID NO:1, or to fragments thereof, or oligonucleotide probes
that hybridize with polynucleotides of the invention. In a
preferred embodiment, such kits also comprise instructions
detailing the procedures by which the kit components are to be
used.
[0015] In another aspect, the invention is directed to use of a
polypeptide comprising an amino acid sequence set forth in SEQ ID
NO:1 or fragment thereof, polynucleotide encoding such a
polypeptide or a fragment thereof, or antibody that binds to said
polypeptide comprising an amino acid sequence set forth in SEQ ID
NO:1 or a fragment thereof in the manufacture of a medicament to
treat diseases associated with host inflammatory or immune
responses.
[0016] Another aspect is directed to pharmaceutical compositions
comprising a polypeptide comprising or consisting of an amino acid
sequence set forth in SEQ ID NO:1 or fragment thereof, a
polynucleotide encoding such a polypeptide or a fragment thereof,
or antibody that binds to such a polypeptide or a fragment thereof,
in conjunction with a suitable pharmaceutical carrier, excipient or
diluent, for the treatment of diseases associated with host
inflammatory or immune responses.
[0017] In another aspect, the invention is directed to methods for
the identification of molecules that can bind to a polypeptide
comprising an amino acid sequence set forth in SEQ ID NO:1 and/or
modulate the activity of a polypeptide comprising an amino acid
sequence set forth in SEQ ID NO:1 or molecules that can bind to
nucleic acid sequences that modulate the transcription or
translation of a polynucleotide encoding a polypeptide comprising
an amino acid sequence set forth in SEQ ID NO:1. Such methods are
disclosed in, e.g., U.S. Pat. Nos. 5,541,070; 5,567,317; 5,593,853;
5,670,326; 5,679,582; 5,856,083; 5,858,657; 5,866,341; 5,876,946;
5,989,814; 6,010,861; 6,020,141; 6,030,779; and 6,043,024, all of
which are incorporated by reference herein in their entirety.
Molecules identified by such methods also fall within the scope of
the present invention.
[0018] In a related aspect, the invention is directed to methods
for identification of a receptor to which the novel IL-1RP1 of the
invention can bind. In this regard a number of technologies allow
for the identification of receptors and binding proteins for
molecules such as IL-1RP1 including but not limited to
yeast-two-Hybrid analysis (S. Fields and O. Song, Nature, 1989 340:
245-6) or techniques of expression cloning in mammalian cells
involving using labeled or tagged ligands to find cells expressing
a receptor after transfection of cDNA libraries.
[0019] In yet another aspect, the invention is directed to methods
for the introduction of nucleic acids of the invention into one or
more tissues of a subject in need of treatment with the result that
one or more proteins encoded by the nucleic acids are expressed and
or secreted by cells within the tissue.
[0020] Other objects, features, advantages and aspects of the
present invention will become apparent to those of skill from the
following description. It should be understood, however, that the
following description and the specific examples, while indicating
preferred embodiments of the invention, are given by way of
illustration only. Various changes and modifications within the
spirit and scope of the disclosed invention will become readily
apparent to those skilled in the art from reading the following
description and from reading the other parts of the present
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1A is a depiction of the full length cDNA sequence (SEQ
ID No:2), encoding the novel IL-1RP1. Start and stop codons of the
ORF are noted in bold underline; the sequence of the ORF contained
within (SEQ ID NO:3) starts at nucleotide position no. 60 (atg) and
ends at nucleotide position no. 516 i.e. before the stop codon
(tag). FIG. 1B depicts the amino acid sequence (SEQ ID NO:1) of the
novel IL-1RP1 which comprises 152 amino acids. The asterix (*)
refers to the stop codon.
[0022] FIG. 2 is a depiction of the genomic DNA sequence (SEQ ID
NO:4), encoding the novel interleukin-1 related polypeptide 1. The
amino acids of the corresponding ORF are indicated. Also shown are
restriction sites to allow for precise physical mapping of the
genomic region.
[0023] FIG. 3 is an amino acid sequence comparison of the IL-1
family of proteins.
[0024] FIG. 4 depicts the expression of IL-1RP1 in different human
tissues determined by RT-PCR.
[0025] FIG. 5 depicts results of Northern blot analysis of human
IL-1RP1 mRNA expression in various tissues.
[0026] FIG. 6 depicts the primary sequence of IL-1 RP1.
[0027] FIG. 7 depicts the exon/intron structure of human
IL-1RP1.
[0028] FIG. 8 depicts the double stranded nucleotide sequence of
IL-1RP1 cDNA.
DETAILED DESCRIPTION OF THE INVENTION
[0029] All patent applications, patents and literature references
cited herein are hereby incorporated by reference in their
entirety.
[0030] In practicing the present invention, many conventional
techniques in molecular biology, microbiology, and recombinant DNA
are used. These techniques are well known and are explained in, for
example, Current Protocols in Molecular Biology, Volumes I, II, and
III, 1997 (F. M. Ausubel ed.); Sambrook et al., 1989, Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A
Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.);
Oligonucleotide Synthesis, 1984 (M. L. Gait ed.); Nucleic Acid
Hybridization, 1985, (Hames and Higgins); Transcription and
Translation, 1984 (Hames and Higgins eds.); Animal Cell Culture,
1986 (R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL
Press); Perbal, 1984, A Practical Guide to Molecular Cloning; the
series, Methods in Enzymology (Academic Press, Inc.); Gene Transfer
Vectors for Mammalian Cells, 1987 (J. H. Miller and M. P. Calos
eds., Cold Spring Harbor Laboratory); and Methods in Enzymology
Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds.,
respectively).
[0031] In its broadest sense, the term "substantially similar",
when used herein with respect to a nucleotide sequence, means a
nucleotide sequence corresponding to a reference nucleotide
sequence, wherein the corresponding sequence encodes a polypeptide
having substantially the same structure and function as the
polypeptide encoded by the reference nucleotide sequence, e.g.
where only changes in amino acids not affecting the polypeptide
function occur. Desirably the substantially similar nucleotide
sequence encodes the polypeptide encoded by the reference
nucleotide sequence. The percentage of identity between the
substantially similar nucleotide sequence and the reference
nucleotide sequence desirably is at least 80%, more desirably at
least 85%, preferably at least 90%, more preferably at least 95%,
still more preferably at least 99%. Sequence comparisons are
carried out using a Smith-Waterman sequence alignment algorithm
(see e.g. Waterman, M. S. Introduction to Computational Biology:
Maps, sequences and genomes. Chapman & Hall. London: 1995. ISBN
0-412-99391-0, or at
http://www-hto.usc.edu/software/seqaln/index.html). The localS
program, version 1.16, is used with following parameters: match: 1,
mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty:
2.
[0032] A nucleotide sequence "substantially similar" to reference
nucleotide sequence hybridizes to the reference nucleotide sequence
in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at
50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree.
C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC,
0.1% SDS at 50.degree. C., more desirably still in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C.,
preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1
mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at
50.degree. C., more preferably in 7% sodium dodecyl sulfate (SDS),
0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in
0.1.times.SSC, 0.1% SDS at 65.degree. C., yet still encodes a
functionally equivalent gene product.
[0033] "Elevated transcription of mRNA" refers to a greater amount
of messenger RNA transcribed from the natural endogenous human gene
encoding the novel polypeptide of the present invention present in
an appropriate tissue or cell of an individual suffering from a
condition associated with host inflammatory or immune response in a
larger amount, in particular at least about twice, preferably at
least about five times, more preferably at least about ten times,
most preferably at least about 100 times the amount of mRNA found
in corresponding tissues in humans who do not suffer from such a
condition. Such elevated level of mRNA may eventually lead to
increased levels of protein translated from such mRNA in an
individual suffering from a condition associated with host
inflammatory or immune response as compared with a healthy
individual.
[0034] A "host cell," as used herein, refers to a prokaryotic or
eukaryotic cell that contains heterologous DNA that has been
introduced into the cell by any means, e.g., electroporation,
calcium phosphate precipitation, microinjection, transformation,
viral infection, and the like.
[0035] "Heterologous" as used herein means "of different natural
origin" or represents a non-natural state. For example, if a host
cell is transformed with a DNA or gene derived from another
organism, particularly from another species, that gene is
heterologous with respect to that host cell and also with respect
to descendants of the host cell which carry that gene. Similarly,
heterologous refers to a nucleotide sequence derived from and
inserted into the same natural, original cell type, but which is
present in a non-natural state, e.g. a different copy number, or
under the control of different regulatory elements.
[0036] A "vector" molecule is a nucleic acid molecule into which
heterologous nucleic acid may be inserted which can then be
introduced into an appropriate host cell. Vectors preferably have
one or more origin of replication, and one or more site into which
the recombinant DNA can be inserted. Vectors often have convenient
means by which cells with vectors can be selected from those
without, e.g., they encode drug resistance genes. Common vectors
include plasmids, viral genomes, and (primarily in yeast and
bacteria) "artificial chromosomes." "Plasmids" generally are
designated herein by a lower case p preceded and/or followed by
capital letters and/or numbers, in accordance with standard naming
conventions that are familiar to those of skill in the art.
Starting plasmids disclosed herein are either commercially
available, publicly available on an unrestricted basis, or can be
constructed from available plasmids by routine application of well
known, published procedures. Many plasmids and other cloning and
expression vectors that can be used in accordance with the present
invention are well known and readily available to those of skill in
the art. Moreover, those of skill readily may construct any number
of other plasmids suitable for use in the invention. The
properties, construction and use of such plasmids, as well as other
vectors, in the present invention will be readily apparent to those
of skill from the present disclosure.
[0037] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated, even if subsequently reintroduced into the natural
system. Such polynucleotides could be part of a vector and/or such
polynucleotides or polypeptides could be part of a composition, and
still be isolated in that such vector or composition is not part of
its natural environment.
[0038] As used herein, the term "transcriptional control sequence"
refers to DNA sequences, such as initiator sequences, enhancer
sequences, and promoter sequences, which induce, repress, or
otherwise control the transcription of protein encoding nucleic
acid sequences to which they are operably linked.
[0039] As used herein, "human transcriptional control sequences"
are any of those transcriptional control sequences normally found
associated with the human gene encoding the novel interleukin-1
related polypeptide of the present invention as it is found in the
respective human chromosome.
[0040] As used herein, "non-human transcriptional control sequence"
is any transcriptional control sequence not found in the human
genome.
[0041] The term "polypeptide" is used interchangeably herein with
the terms "polypeptides" and "protein(s)".
[0042] As used herein, a "chemical derivative" of a polypeptide of
the invention is a polypeptide of the invention that contains
additional chemical moieties not normally a part of the molecule.
Such moieties may improve the molecule's solubility, absorption,
biological half-life, etc. The moieties may alternatively decrease
the toxicity of the molecule, eliminate or attenuate any
undesirable side effect of the molecule, etc. Moieties capable of
mediating such effects are disclosed, for example, in Remington's
Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa.
(1980).
[0043] The invention generally relates to a novel nucleotide
sequence which uniquely encodes a novel interleukin. The new gene
encodes a polypeptide designated herein as interleukin-1 related
polypeptide, or IL-1RP1, belonging to the interleukin-1 cytokine
family, as will be outlined in detail herein.
[0044] In order to determine if additional members of the IL-1
family of polypeptides exist in the human genome, in-house and
public human genomic databases are searched with the human IL-1 and
IL-1H1 peptide sequences. IL-1H1 is an IL-1 related molecule that
was recently shown to be constitutively expressed only in placenta
and squamous epithelium of esophagus, and could be induced in vitro
in keratinocytes. Using these two sequences as a bait and tBlastN
program one novel sequence, not previously reported, is found
corresponding to a certain clone of the in-house database.
Translation of this genomic sequence shows 33% identity and 52%
similarity to IL-1H1. This sequence is then used to find additional
overlapping genomic sequences using BlastN thereby identifying a
large segment of genomic DNA from the public database.
Approximately 1 kb of genomic sequence flanking the sequence of the
in-house database is translated in all six reading frames and
compared by ClustalW to IL-1H1 and IL-1.beta. peptide sequences. An
additional sequence of 127 nucleotides is found. Based on
identified primary sequence PCR primers are designed to amplify the
coding region from fetal human small intestine Marathon.RTM. cDNA
(Clonetech, Palo Alto, Calif.) and the sequence is found to be
identical with that predicted by the genomic region. Finally a 5'
RACE is used to identify the 5' end of the cDNA. The sequence of
the complete coding region for IL-1RP1 is shown in FIG. 1. Similar
to all of the IL-1 family members except for IL-1RA, IL-1RP1 does
not contain a hydrophobic secretion signal. Thus IL-1RP1 is likely
produced as an intracellular protein and released upon cell damage
as are IL-1.alpha. and the intracellular forms of IL-RA to gain its
biological function.
[0045] The full length cDNA for IL-1RP1 predicts a protein of 152
amino acids. The identity profile of the family of IL-1 related
molecules is shown in the following Table 1. TABLE-US-00001 TABLE 1
IL1RP1 IL-1RA IL-1.beta. IL-1.alpha. IL1H1 IL1H2/.nu. FIL.delta./H3
FIL.epsilon. FIL.zeta./H4 IL-18 IL1RP1 38 22 20 28 17 42 27 27 18
IL-1RA 31 20 29 26 21 23 21 21 IL-1.beta. 24 20 32 32 27 24 17 IL-1
25 26 20 23 21 21 IL1H1 26 29 60 30 24 IL- 21 46 44 21 1H2/.nu.
FIL.delta./H3 31 35 27 FIL.epsilon. 36 21 FIL.zeta./H4 21 IL-18
Table 1 shows the percentage of identity of IL-1 family of
proteins, including the novel IL-1RP1. Note that FIL.zeta. and
IL1H4 are listed as the same gene as they are 88% identical and
differ only at the very amino and carboxyl terminal ends and thus
likely represent alternate spliced forms of the same gene. Also
IL-1H2 and FIL.nu. as well as IL-1H3 and FIL.delta. are listed as
the same gene. The mature forms of IL-1.alpha., IL-1, IL-1RA and
IL-18 are used for comparison. Percent identifies were determined
using a global alignment as described by Myers and Miller, CABIOS
1989 4:11-17 and using a BLOSUM50 scoring matrix with gap penalties
of -12/-2.
[0046] The predicted IL-1RP1 peptide is most homologous to
IL-1H3/FIL.delta. and IL-1RA, being 42% and 38% identical,
respectively.
[0047] As thus being qualified as a member of the IL-1 polypeptide
family, the novel polypeptide IL-1RP1 will have similar
physiological functions. It is likely to be involved in
inflammation or host immune response due to its similarity to the
IL-1 related polypeptides of known function, all of which have
major impact in human disease. The sequence and predicted structure
of IL-1RP1 will be useful for designing antinflammatory agents
either through the use of small molecules or proteins (e.g.
antibodies) directed against it or its receptor. In addition,
protein derived from the IL-1RP1 sequence may also be used as a
therapeutic to modify host immune or inflammatory responses.
[0048] To determine the expression pattern of the novel
polypeptide, a panel of cDNAs from a variety of human tissues is
subjected to PCR analysis using PCR primers that could identify an
IL-1RP1 transcript. PCR primers are chosen that are present on
separate exons of the predicted IL-1RP1 gene so that PCR products
from a transcribed, processed mRNA can readily be distinguished
from any originating genomic DNA. As a result, IL-1RP1 is
differentially expressed in human tissues, being detectable in
brain, heart, liver, lung, stomach, testis, placenta, prostate and
skin. Most notably IL-1RP1 is highly expressed in skin. Thus IL-1RP
represents a transcribed gene. Further its constitutive expression
is very high in skin suggesting it may play a role in cutaneous
inflammatory responses. See FIG. 4.
[0049] Therefore, in one aspect, the present invention relates to a
novel interleukin-1 related polypeptide (IL-1RP1). As outlined
above, IL-1RP1 is clearly a member of the IL-1 family since it is
highly similar to other IL-1 proteins as discussed above. It also
shares many similarities with the IL-1 family. The genomic
sequences for IL-1RP1 is found on chromosome 2 on the same human
genomic BAC clone as FIL.epsilon. and IL-1H1. To date all of the
IL-1 related molecules are on the same portion of chromosome 2
suggesting a relatively recent evolutionary divergence. Finally,
analysis of genomic sequences and cDNA sequences shows that the
intron exon organization for IL-1RP1 is almost identical to IL-1RA
and FIL.delta.. These observations are consistent with the
assumption that all are derived from the same ancestral gene.
[0050] The present invention relates to an isolated polypeptide
comprising the amino acid sequence set forth in SEQ ID NO:1. Such a
polypeptide may for example be a fusion protein including the amino
acid sequence of the novel interleukin-1 related polypeptide 1. In
another aspect the present invention relates to an isolated
polypeptide consisting of the amino acid sequence set forth in SEQ
ID NO:1, which is, in particular, the novel IL-1RP1.
[0051] The invention includes nucleic acid or nucleotide molecules,
preferably DNA molecules, in particular encoding the novel IL-1RP1.
Preferably, an isolated nucleic acid molecule, preferably a DNA
molecule, of the present invention encodes a polypeptide comprising
the amino acid sequence set forth in SEQ ID NO:1. Likewise
preferred is an isolated nucleic acid molecule, preferably a DNA
molecule, encoding a polypeptide consisting of the amino acid
sequence set forth in SEQ ID NO:1. Such a nucleic acid or
nucleotide, in particular such a DNA molecule, preferably comprises
a nucleotide sequence selected from the group consisting of (1) the
nucleotide sequence as set forth in SEQ ID NO:2, which is the
complete cDNA sequence encoding the polypeptide consisting of the
amino acid sequence set forth in SEQ ID NO:1: (2) the nucleotide
sequence set forth in SEQ ID NO:3, which corresponds to the open
reading frame of the cDNA sequence set forth in SEQ ID NO:2; (3) a
nucleotide sequence capable of hybridizing under high stringency
conditions to a nucleotide sequence set forth in SEQ ID NO:3; and
(4) the nucleotide sequence set forth in SEQ ID NO:4, which
corresponds to the endogenous human genomic DNA encoding the
polypeptide consisting of the amino acid sequence set forth in SEQ
ID NO:1. Such hybridization conditions may be highly stringent or
less highly stringent, as described above. In instances wherein the
nucleic acid molecules are deoxyoligonucleotides ("oligos"), highly
stringent conditions may refer, e.g., to washing in
6.times.SSC/0.05% sodium pyrophosphate at 37.degree. C. (for
14-base oligos), 48.degree. C. (for 17-base oligos), 55.degree. C.
(for 20-base oligos), and 60.degree. C. (for 23-base oligos).
Suitable ranges of such stringency conditions for nucleic acids of
varying compositions are described in Krause and Aaronson (1991),
Methods in Enzymology, 200:546-556 in addition to Maniatis et al.,
cited above.
[0052] These nucleic acid molecules may act as target gene
antisense molecules, useful, for example, in target gene regulation
and/or as antisense primers in amplification reactions of target
gene nucleic acid sequences. Further, such sequences may be used as
part of ribozyme and/or triple helix sequences, also useful for
target gene regulation. Still further, such molecules may be used
as components of diagnostic methods whereby the presence of an
allele causing diseases associated with host inflammatory or immune
response may be detected.
[0053] The invention also encompasses (a) vectors that contain at
least a fragment of any of the foregoing nucleotide sequences
and/or their complements (i.e., antisense); (b) vector molecules,
preferably vector molecules comprising transcriptional control
sequences, in particular expression vectors, which contain any of
the foregoing coding sequences operatively associated with a
regulatory element that directs the expression of the coding
sequences; and (c) genetically engineered host cells that contain a
vector molecule as mentioned herein or at least a fragment of any
of the foregoing nucleotide sequences operatively associated with a
regulatory element that directs the expression of the coding
sequences in the host cell. As used herein, regulatory elements
include, but are not limited to, inducible and non-inducible
promoters, enhancers, operators and other elements known to those
skilled in the art that drive and regulate expression. Preferably,
host cells can be vertebrate host cells, preferably mammalian host
cells, like human cells or rodent cells, such as CHO or BHK cells.
Likewise preferred, host cells can be bacterial host cells, in
particular E. coli cells.
[0054] Particularly preferred is a host cell, in particular of the
above described type, which can be propagated in vitro and which is
capable upon growth in culture of producing an IL-1RP1 polypeptide,
in particular a polypeptide comprising or consisting of an amino
acid sequence set forth in SEQ ID NO:1, wherein said cell comprises
at least one transcriptional control sequence that is not a
transcriptional control sequence of the natural endogenous human
gene encoding said polypeptide, wherein said one or more
transcriptional control sequences control transcription of a DNA
encoding said polypeptide.
[0055] The invention includes fragments of any of the nucleic acid
sequences disclosed herein. Fragments of the nucleic acid sequences
encoding the novel IL-1RP1 polypeptide may be used as a
hybridization probe for a cDNA library to isolate the full length
gene and to isolate other genes which have a high sequence
similarity to the IL-1RP1 gene or similar biological activity.
Probes of this type preferably have at least about 30 bases and may
contain, for example, from about 30 to about 50 bases, about 50 to
about 100 bases, about 100 to about 200 bases, or more than 200
bases. The probe may also be used to identify a cDNA clone
corresponding to a full length transcript and a genomic clone or
clones that contain the complete IL-1RP1 gene including regulatory
and promoter regions, exons, and introns. An example of a screen
comprises isolating the coding region of the IL-1RP1 gene by using
the known DNA sequence to synthesize an oligonucleotide probe.
Labeled oligonucleotides having a sequence complementary to that of
the gene of the present invention are used to screen a library of
human cDNA, genomic DNA or mRNA to determine to which members of
the library the probe hybridizes.
[0056] In addition to the gene sequences described above, homologs
of such sequences, as may, for example, be present in other
species, may be identified and may be readily isolated, without
undue experimentation, by molecular biological techniques well
known in the art. Further, there may exist genes at other genetic
loci within the genome that encode proteins which have extensive
homology to one or more domains of such gene products. These genes
may also be identified via similar techniques.
[0057] For example, the isolated nucleotide sequence of the present
invention encoding the novel IL-1RP1 polypeptide may be labeled and
used to screen a cDNA library constructed from mRNA obtained from
the organism of interest. Hybridization conditions will be of a
lower stringency when the cDNA library was derived from an organism
different from the type of organism from which the labeled sequence
was derived. Alternatively, the labeled fragment may be used to
screen a genomic library derived from the organism of interest,
again, using appropriately stringent conditions. Such low
stringency conditions will be well known to those of skill in the
art, and will vary predictably depending on the specific organisms
from which the library and the labeled sequences are derived. For
guidance regarding such conditions see, for example, Sambrook et
al. cited above.
[0058] Further, a previously unknown differentially expressed
gene-type sequence may be isolated by performing PCR using two
degenerate oligonucleotide primer pools designed on the basis of
amino acid sequences within the gene of interest. The template for
the reaction may be cDNA obtained by reverse transcription of mRNA
prepared from human or non-human cell lines or tissue known or
suspected to express a differentially expressed gene allele.
[0059] The PCR product may be subcloned and sequenced to ensure
that the amplified sequences represent the sequences of a
differentially expressed gene-like nucleic acid sequence. The PCR
fragment may then be used to isolate a full length cDNA clone by a
variety of methods. For example, the amplified fragment may be
labeled and used to screen a bacteriophage cDNA library.
Alternatively, the labeled fragment may be used to screen a genomic
library.
[0060] PCR technology may also be utilized to isolate full length
cDNA sequences. For example, RNA may be isolated, following
standard procedures, from an appropriate cellular or tissue source.
A reverse transcription reaction may be performed on the RNA using
an oligonucleotide primer specific for the most 5' end of the
amplified fragment for the priming of first strand synthesis. The
resulting RNA/DNA hybrid may then be "tailed" with guanines using a
standard terminal transferase reaction, the hybrid may be digested
with RNAase H, and second strand synthesis may then be primed with
a poly-C primer. Thus, cDNA sequences upstream of the amplified
fragment may easily be isolated. For a review of cloning strategies
which may be used, see e.g., Sambrook et al., 1989, supra.
[0061] In cases where the gene identified is the normal, or wild
type, gene, this gene may be used to isolate mutant alleles of the
gene. Such an isolation is preferable in processes and disorders
which are known or suspected to have a genetic basis. Mutant
alleles may be isolated from individuals either known or suspected
to have a genotype which contributes to disease symptoms related to
inflammation or immune response. Mutant alleles and mutant allele
products may then be utilized in the diagnostic assay systems
described below.
[0062] A cDNA of the mutant gene may be isolated, for example, by
using PCR, a technique which is well known to those of skill in the
art. In this case, the first cDNA strand may be synthesized by
hybridizing an oligo-dT oligonucleotide to mRNA isolated from
tissue known or suspected to be expressed in an individual
putatively carrying the mutant allele, and by extending the new
strand with reverse transcriptase. The second strand of the cDNA is
then synthesized using an oligonucleotide that hybridizes
specifically to the 5' end of the normal gene. Using these two
primers, the product is then amplified via PCR, cloned into a
suitable vector, and subjected to DNA sequence analysis through
methods well known to those of skill in the art. By comparing the
DNA sequence of the mutant gene to that of the normal gene, the
mutation(s) responsible for the loss or alteration of function of
the mutant gene product can be ascertained.
[0063] Alternatively, a genomic or cDNA library can be constructed
and screened using DNA or RNA, respectively, from a tissue known to
or suspected of expressing the gene of interest in an individual
suspected of or known to carry the mutant allele. The normal gene
or any suitable fragment thereof may then be labeled and used as a
probed to identify the corresponding mutant allele in the library.
The clone containing this gene may then be purified through methods
routinely practiced in the art, and subjected to sequence analysis
as described above.
[0064] Additionally, an expression library can be constructed
utilizing DNA isolated from or cDNA synthesized from a tissue known
to or suspected of expressing the gene of interest in an individual
suspected of or known to carry the mutant allele. In this manner,
gene products made by the putatively mutant tissue may be expressed
and screened using standard antibody screening techniques in
conjunction with antibodies raised against the normal gene product,
as described, below. (For screening techniques, see, for example,
Harlow, E. and Lane, eds., 1988, "Antibodies: A Laboratory Manual",
Cold Spring Harbor Press, Cold Spring Harbor.) In cases where the
mutation results in an expressed gene product with altered function
(e.g., as a result of a missense mutation), a polyclonal set of
antibodies are likely to cross-react with the mutant gene product.
Library clones detected via their reaction with such labeled
antibodies can be purified and subjected to sequence analysis as
described above.
[0065] The present invention includes those proteins encoded by
nucleotide sequences set forth in any of SEQ ID NOs:2, 3 or 4, in
particular, a polypeptide that is or includes the amino acid
sequence set out in SEQ ID NO:1, or fragments thereof.
[0066] Furthermore, the present invention includes proteins that
represent functionally equivalent gene products. Such an equivalent
differentially expressed gene product may contain deletions,
additions or substitutions of amino acid residues within the amino
acid sequence encoded by the differentially expressed gene
sequences described, above, but which result in a silent change,
thus producing a functionally equivalent differentially expressed
gene product. Amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues
involved.
[0067] For example, nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and methionine; polar neutral amino acids include
glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine; positively charged (basic) amino acids include arginine,
lysine, and histidine; and negatively charged (acidic) amino acids
include aspartic acid and glutamic acid. "Functionally equivalent,"
as utilized herein, may refer to a protein or polypeptide capable
of exhibiting a substantially similar in vivo or in vitro activity
as the endogenous differentially expressed gene products encoded by
the differentially expressed gene sequences described above.
"Functionally equivalent" may also refer to proteins or
polypeptides capable of interacting with other cellular or
extracellular molecules in a manner substantially similar to the
way in which the corresponding portion of the endogenous
differentially expressed gene product would. For example, a
"functionally equivalent" peptide would be able, in an immunoassay,
to diminish the binding of an antibody to the corresponding peptide
(i.e., the peptide the amino acid sequence of which was modified to
achieve the "functionally equivalent" peptide) of the endogenous
protein, or to the endogenous protein itself, where the antibody
was raised against the corresponding peptide of the endogenous
protein. An equimolar concentration of the functionally equivalent
peptide will diminish the aforesaid binding of the corresponding
peptide by at least about 5%, preferably between about 5% and 10%,
more preferably between about 10% and 25%, even more preferably
between about 25% and 50%, and most preferably between about 40%
and 50%.
[0068] The polypeptides of the present invention may be produced by
recombinant DNA technology using techniques well known in the art.
Therefore, there is provided a method of producing a polypeptide of
the present invention, which method comprises culturing a host cell
having incorporated therein an expression vector containing an
exogenously-derived polynucleotide encoding a polypeptide
comprising an amino acid sequence as set forth in SEQ ID NO:1 under
conditions sufficient for expression of the polypeptide in the host
cell, thereby causing the production of the expressed polypeptide.
Optionally, said method further comprises recovering the
polypeptide produced by said cell. In a preferred embodiment of
such a method, said exogenously-derived polynucleotide encodes a
polypeptide consisting of an amino acid sequence set forth in SEQ
ID NO:1. Preferably, said exogenously-derived polynucleotide
comprises the nucleotide sequence as set forth in any of SEQ ID
NO:2, SEQ ID NO:3 or SEQ ID NO:4. In case of using the nucleotide
sequence set forth in SEQ ID NO:2, i.e. the open reading frame, the
sequence, when inserted into a vector, may be followed by one or
more appropriate translation stop codons, preferably by the natural
endogenous stop codon as shown in FIG. 1.
[0069] Thus, methods for preparing the polypeptides and peptides of
the invention by expressing nucleic acid encoding respective
nucleotide sequences are described herein. Methods that are well
known to those skilled in the art can be used to construct
expression vectors containing protein-coding sequences and
appropriate transcriptional/translational control signals. These
methods include, for example, in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. See, for example, the techniques described in
Sambrook et al., 1989, supra, and Ausubel et al., 1989, supra.
Alternatively, RNA capable of encoding differentially expressed
gene protein sequences may be chemically synthesized using, for
example, synthesizers. See, for example, the techniques described
in "Oligonucleotide Synthesis", 1984, Gait, M. J. ed., IRL Press,
Oxford, which is incorporated by reference herein in its
entirety.
[0070] A variety of host-expression vector systems may be utilized
to express the differentially expressed gene coding sequences of
the invention. Such host-expression systems represent vehicles by
which the coding sequences of interest may be produced and
subsequently purified, but also represent cells which may, when
transformed or transfected with the appropriate nucleotide coding
sequences, exhibit the differentially expressed gene protein of the
invention in situ. These include but are not limited to
microorganisms such as bacteria (e.g., E. coli, B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DNA or
cosmid DNA expression vectors containing differentially expressed
gene protein coding sequences; yeast (e.g. Saccharomyces, Pichia)
transformed with recombinant yeast expression vectors containing
the differentially expressed gene protein coding sequences; insect
cell systems infected or transfected with recombinant virus
expression vectors (e.g., baculovirus) containing the
differentially expressed gene protein coding sequences; plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant vectors, including plasmids, (e.g., Ti
plasmid) containing protein coding sequences; or mammalian cell
systems (e.g. COS, CHO, BHK, 293, 3T3) harboring recombinant
expression constructs containing promoters derived from the genome
of mammalian cells (e.g., metallothioneine promoter) or from
mammalian viruses (e.g., the adenovirus late promoter; the vaccinia
virus 7.5K promoter, or the CMV promoter).
[0071] Expression of the interleukin-1 related polypeptide 1 of the
present invention by a cell from an IL-1RP1-encoding gene that is
native to the cell can also be performed. Methods for such
expression are detailed in, e.g., U.S. Pat. Nos. 5,641,670;
5,733,761; 5,968,502; and 5,994,127, all of which are expressly
incorporated by reference herein in their entirety. Cells that have
been induced to express IL-1RP1 by the methods of any of U.S. Pat.
Nos. 5,641,670; 5,733,761; 5,968,502; and 5,994,127 can be
implanted into a desired tissue in a living animal in order to
increase the local concentration of IL-1RP1 in the tissue.
[0072] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
protein being expressed. For example, when a large quantity of such
a protein is to be produced, for the generation of antibodies or to
screen peptide libraries, for example, vectors which direct the
expression of high levels of fusion protein products that are
readily purified may be desirable. In this respect, fusion proteins
comprising hexahistidine tags may be used (Sisk et al., 1994: J.
Virol 68: 766-775) as provided by a number of vendors (e.g. Qiagen,
Valencia, Calif.). Such vectors include, but are not limited, to
the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
2:1791), in which the protein-encoding sequence may be ligated
individually into the vector in frame with the lac Z coding region
so that a fusion protein is produced; pIN vectors (Inouye &
Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke &
Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX
vectors may also be used to express foreign polypeptides as fusion
proteins with glutathione S-transferase (GST). In general, such
fusion proteins are soluble and can easily be purified from lysed
cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. The pGEX vectors are
designed to include thrombin or factor Xa protease cleavage sites
so that the cloned target gene protein can be released from the GST
moiety.
[0073] Promoter regions can be selected from any desired gene using
vectors that contain a reporter transcription unit lacking a
promoter region, such as a chloramphenicol acetyl transferase
("CAT"), or the luciferase transcription unit, downstream of
restriction site or sites for introducing a candidate promoter
fragment; i.e., a fragment that may contain a promoter. For
example, introduction into the vector of a promoter-containing
fragment at the restriction site upstream of the CAT gene engenders
production of CAT activity, which can be detected by standard CAT
assays. Vectors suitable to this end are well known and readily
available. Two such vectors are pKK232-8 and pCM7. Thus, promoters
for expression of polynucleotides of the present invention include
not only well-known and readily available promoters, but also
promoters that readily may be obtained by the foregoing technique,
using a reporter gene.
[0074] Among known bacterial promoters suitable for expression of
polynucleotides and polypeptides in accordance with the present
invention are the E. coli lacI and lacZ promoters, the T3 and T7
promoters, the T5 tac promoter, the lambda PR, PL promoters and the
trp promoter. Among known eukaryotic promoters suitable in this
regard are the CMV immediate early promoter, the HSV thymidine
kinase promoter, the early and late SV40 promoters, the promoters
of retroviral LTRs, such as those of the Rous sarcoma virus
("RSV"), and metallothionein promoters, such as the mouse
metallothionein-I promoter.
[0075] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is one of several insect systems that
can be used as a vector to express foreign genes. The virus grows
in Spodoptera frugiperda cells. The coding sequence may be cloned
individually into non-essential regions (for example the polyhedrin
gene) of the virus and placed under control of an AcNPV promoter
(for example the polyhedrin promoter). Successful insertion of the
coding sequence will result in inactivation of the polyhedrin gene
and production of non-occluded recombinant virus (i.e., virus
lacking the proteinaceous coat coded for by the polyhedrin gene).
These recombinant viruses are then used to infect Spodoptera
frugiperda cells in which the inserted gene is expressed. (E.g.,
see Smith et al., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No.
4,215,051).
[0076] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the coding sequence of interest may be ligated
to an adenovirus transcription/translation control complex, e.g.,
the late promoter and tripartite leader sequence. This chimeric
gene may then be inserted in the adenovirus genome by in vitro or
in vivo recombination. Insertion in a non-essential region of the
viral genome (e.g., region E1 or E3) will result in a recombinant
virus that is viable and capable of expressing the desired protein
in infected hosts. (E.g., See Logan & Shenk, 1984, Proc. Natl.
Acad. Sci. USA 81:3655-3659). Specific initiation signals may also
be required for efficient translation of inserted gene coding
sequences. These signals include the ATG initiation codon and
adjacent sequences. In cases where an entire gene, including its
own initiation codon and adjacent sequences, is inserted into the
appropriate expression vector, no additional translational control
signals may be needed. However, in cases where only a portion of
the gene coding sequence is inserted, exogenous translational
control signals, including, perhaps, the ATG initiation codon, must
be provided. Furthermore, the initiation codon must be in phase
with the reading frame of the desired coding sequence to ensure
translation of the entire insert. These exogenous translational
control signals and initiation codons can be of a variety of
origins, both natural and synthetic. The efficiency of expression
may be enhanced by the inclusion of appropriate transcription
enhancer elements, transcription terminators, etc. (see Bittner et
al., 1987, Methods in Enzymol. 153:516-544). Other common systems
are based on SV40, retrovirus or adeno-associated virus. Selection
of appropriate vectors and promoters for expression in a host cell
is a well-known procedure and the requisite techniques for
expression vector construction, introduction of the vector into the
host and expression in the host per se are routine skills in the
art. Generally, recombinant expression vectors will include origins
of replication, a promoter derived from a highly expressed gene to
direct transcription of a downstream structural sequence, and a
selectable marker to permit isolation of vector containing cells
after exposure to the vector.
[0077] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins. Appropriate cell lines or host systems can be chosen
to ensure the correct modification and processing of the foreign
protein expressed. To this end, eukaryotic host cells which possess
the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the gene product
may be used. Such mammalian host cells include but are not limited
to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, etc.
[0078] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the differentially expressed gene protein may
be engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines that express the differentially expressed gene protein. Such
engineered cell lines may be particularly useful in screening and
evaluation of compounds that affect the endogenous activity of the
expressed protein.
[0079] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for dhfr, which confers resistance to
methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567;
O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt,
which confers resistance to mycophenolic acid (Mulligan & Berg,
1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers
resistance to the aminoglycoside G-418 (Colberre-Garapin, et al.,
1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to
hygromycin (Santerre, et al., 1984, Gene 30:147) genes.
[0080] An alternative fusion protein system allows for the ready
purification of non-denatured fusion proteins expressed in human
cell lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:
8972-8976). In this system, the gene of interest is subcloned into
a vaccinia recombination plasmid such that the gene's open reading
frame is translationally fused to an amino-terminal tag consisting
of six histidine residues. Extracts from cells infected with
recombinant vaccinia virus are loaded onto Ni.sup.2+ nitriloacetic
acid-agarose columns and histidine-tagged proteins are selectively
eluted with imidazole-containing buffers.
[0081] When used as a component in assay systems such as those
described below, a protein of the present invention may be labeled,
either directly or indirectly, to facilitate detection of a complex
formed between the protein and a test substance. Any of a variety
of suitable labeling systems may be used including but not limited
to radioisotopes such as .sup.125I; enzyme labeling systems that
generate a detectable calorimetric signal or light when exposed to
substrate; and fluorescent labels.
[0082] Where recombinant DNA technology is used to produce a
protein of the present invention for such assay systems, it may be
advantageous to engineer fusion proteins that can facilitate
labeling, immobilization, detection and/or isolation
[0083] Indirect labeling involves the use of a protein, such as a
labeled antibody, which specifically binds to a polypeptide of the
present invention. Such antibodies include but are not limited to
polyclonal, monoclonal, chimeric, single chain, Fab fragments and
fragments produced by a Fab expression library.
[0084] In another embodiment, nucleic acids comprising a sequence
encoding an IL-1RP1 protein or functional derivative thereof are
administered to promote normal immune system function, by way of
gene therapy. Gene therapy refers to therapy performed by the
administration of a nucleic acid to a subject. In this embodiment
of the invention, the nucleic acid produces its encoded protein
that mediates a therapeutic effect by promoting normal immune
system function.
[0085] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0086] In a preferred aspect, the therapeutic comprises an IL-1RP1
nucleic acid that is part of an expression vector that expresses an
IL-1RP1 protein or fragment or chimeric protein thereof in a
suitable host. In particular, such a nucleic acid has a promoter
operably linked to the IL-1RP1 coding region, said promoter being
inducible or constitutive, and, optionally, tissue-specific. In
another particular embodiment, a nucleic acid molecule is used in
which the IL-1RP1 coding sequences and any other desired sequences
are flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the IL-1RP1 nucleic acid (Koller and Smithies, 1989,
Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989,
Nature 342:435-438).
[0087] Delivery of the nucleic acid into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vector, or indirect, in which
case, cells are first transformed with the nucleic acid in vitro,
then transplanted into the patient. These two approaches are known,
respectively, as in vivo or ex vivo gene therapy.
[0088] In a specific embodiment, the nucleic acid is directly
administered in vivo, where it is expressed to produce the encoded
product. This can be accomplished by any of numerous methods known
in the art, e.g., by constructing it as part of an appropriate
nucleic acid expression vector and administering it so that it
becomes intracellular, e.g., by infection using a defective or
attenuated retroviral or other viral vector (see, e.g., U.S. Pat.
No. 4,980,286 and others mentioned infra), or by direct injection
of naked DNA, or by use of microparticle bombardment (e.g., a gene
gun; Biolistic, Dupont), or coating with lipids or cell-surface
receptors or transfecting agents, encapsulation in liposomes,
microparticles, or microcapsules, or by administering it in linkage
to a peptide which is known to enter the nucleus, by administering
it in linkage to a ligand subject to receptor-mediated endocytosis
(see e.g., U.S. Pat. Nos. 5,166,320; 5,728,399; 5,874,297; and
6,030,954, all of which are incorporated by reference herein in
their entirety) (which can be used to target cell types
specifically expressing the receptors), etc. In another embodiment,
a nucleic acid-ligand complex can be formed in which the ligand
comprises a fusogenic viral peptide to disrupt endosomes, allowing
the nucleic acid to avoid lysosomal degradation. In yet another
embodiment, the nucleic acid can be targeted in vivo for cell
specific uptake and expression, by targeting a specific receptor
(see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316;
WO93/14188; and WO 93/20221). Alternatively, the nucleic acid can
be introduced intracellularly and incorporated within host cell DNA
for expression, by homologous recombination (see, e.g., U.S. Pat.
Nos. 5,413,923; 5,416,260; and 5,574,205; and Zijlstra et al.,
1989, Nature 342:435-438).
[0089] In a specific embodiment, a viral vector that contains the
IL-1RP1 nucleic acid is used. For example, a retroviral vector can
be used (see, e.g., U.S. Pat. Nos. 5,219,740; 5,604,090; and
5,834,182). These retroviral vectors have been modified to delete
retroviral sequences that are not necessary for packaging of the
viral genome and integration into host cell DNA. The IL-1RP1
nucleic acid to be used in gene therapy is cloned into the vector,
which facilitates delivery of the gene into a patient.
[0090] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Methods for conducting adenovirus-based gene therapy are
described in, e.g., U.S. Pat. Nos. 5,824,544; 5,868,040; 5,871,722;
5,880,102; 5,882,877; 5,885,808; 5,932,210; 5,981,225; 5,994,106;
5,994,132; 5,994,134; 6,001,557; and 6,033,8843, all of which are
incorporated by reference herein in their entirety.
[0091] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy. Methods for producing and utilizing AAV are
described, e.g., in U.S. Pat. Nos. 5,173,414; 5,252,479; 5,552,311;
5,658,785; 5,763,416; 5,773,289; 5,843,742; 5,869,040; 5,942,496;
and 5,948,675, all of which are incorporated by reference herein in
their entirety.
[0092] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0093] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells and may be
used in accordance with the present invention, provided that the
necessary developmental and physiological functions of the
recipient cells are not disrupted. The technique should provide for
the stable transfer of the nucleic acid to the cell, so that the
nucleic acid is expressible by the cell and preferably heritable
and expressible by its cell progeny.
[0094] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. In a preferred
embodiment, epithelial cells are injected, e.g., subcutaneously. In
another embodiment, recombinant skin cells may be applied as a skin
graft onto the patient. Recombinant blood cells (e.g.,
hematopoietic stem or progenitor cells) are preferably administered
intravenously. The amount of cells envisioned for use depends on
the desired effect, patient state, etc., and can be determined by
one skilled in the art.
[0095] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0096] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0097] In an embodiment in which recombinant cells are used in gene
therapy, an IL-1RP1 nucleic acid is introduced into the cells such
that it is expressible by the cells or their progeny, and the
recombinant cells are then administered in vivo for therapeutic
effect. In a specific embodiment, stem or progenitor cells are
used. Any stem- and/or progenitor cells that can be isolated and
maintained in vitro can potentially be used in accordance with this
embodiment of the present invention. Such stem cells include but
are not limited to hematopoietic stem cells (HSC), stem cells of
epithelial tissues such as the skin and the lining of the gut,
embryonic heart muscle cells, liver stem cells (see, e.g., WO
94/08598), and neural stem cells (Stemple and Anderson, 1992, Cell
71:973-985).
[0098] Epithelial stem cells (ESCs) or keratinocytes can be
obtained from tissues such as the skin and the lining of the gut by
known procedures (Rheinwald, 1980, Meth. Cell Bio. 21A:229). In
stratified epithelial tissue such as the skin, renewal occurs by
mitosis of stem cells within the germinal layer, the layer closest
to the basal lamina. Stem cells within the lining of the gut
provide for a rapid renewal rate of this tissue. ESCs or
keratinocytes obtained from the skin or lining of the gut of a
patient or donor can be grown in tissue culture (Pittelkow and
Scott, 1986, Mayo Clinic Proc. 61:771). If the ESCs are provided by
a donor, a method for suppression of host versus graft reactivity
(e.g., irradiation, drug or antibody administration to promote
moderate immunosuppression) can also be used.
[0099] With respect to hematopoietic stem cells (HSC), any
technique that provides for the isolation, propagation, and
maintenance in vitro of HSC can be used in this embodiment of the
invention. Techniques by which this may be accomplished include (a)
the isolation and establishment of HSC cultures from bone marrow
cells isolated from the future host, or a donor, or (b) the use of
previously established long-term HSC cultures, which may be
allogeneic or xenogeneic. Non-autologous HSC are used preferably in
conjunction with a method of suppressing transplantation immune
reactions of the future host/patient. In a particular embodiment of
the present invention, human bone marrow cells can be obtained from
the posterior iliac crest by needle aspiration (see, e.g., Kodo et
al., 1984, J. Clin. Invest. 73:1377-1384). In a preferred
embodiment of the present invention, the HSCs can be made highly
enriched or in substantially pure form. This enrichment can be
accomplished before, during, or after long-term culturing, and can
be done by any techniques known in the art. Long-term cultures of
bone marrow cells can be established and maintained by using, for
example, modified Dexter cell culture techniques (Dexter et al.,
1977, J. Cell Physiol. 91:335) or Witlock-Witte culture techniques
(Witlock and Witte, 1982, Proc. Natl. Acad. Sci. USA
79:3608-3612).
[0100] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[0101] A further embodiment of the present invention relates to a
purified antibody or a fragment thereof which specifically binds to
a polypeptide that comprises the amino acid sequence set forth in
SEQ ID NO:1 or to a fragment of said polypeptide. A preferred
embodiment relates to a fragment of such an antibody, which
fragment is an Fab or F(ab').sub.2 fragment. In particular, the
antibody can be a polyclonal antibody or a monoclonal antibody.
[0102] Described herein are methods for the production of
antibodies capable of specifically recognizing one or more
differentially expressed gene epitopes. Such antibodies may
include, but are not limited to polyclonal antibodies, monoclonal
antibodies (mAbs), humanized or chimeric antibodies, single chain
antibodies, Fab fragments, F(ab').sub.2 fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies, and epitope-binding fragments of any of the above. Such
antibodies may be used, for example, in the detection of a
fingerprint, target, gene in a biological sample, or,
alternatively, as a method for the inhibition of abnormal target
gene activity. Thus, such antibodies may be utilized as part of
cardiovascular disease treatment methods, and/or may be used as
part of diagnostic techniques whereby patients may be tested for
abnormal levels of the IL-1RP1 polypeptide, or for the presence of
abnormal forms of the IL-1RP1 polypeptide.
[0103] For the production of antibodies to the IL-1RP1 polypeptide,
various host animals may be immunized by injection with the IL-1RP1
polypeptide, or a portion thereof. Such host animals may include
but are not limited to rabbits, mice, and rats, to name but a few.
Various adjuvants may be used to increase the immunological
response, depending on the host species, including but not limited
to Freund's (complete and incomplete), mineral gels such as
aluminum hydroxide, surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet hemocyanin, dinitrophenol, and potentially useful human
adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium
parvum.
[0104] Polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of animals immunized with
an antigen, such as target gene product, or an antigenic functional
derivative thereof. For the production of polyclonal antibodies,
host animals such as those described above, may be immunized by
injection with the IL-1RP1 polypeptide, or a portion thereof,
supplemented with adjuvants as also described above.
[0105] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, may be obtained by any
technique that provides for the production of antibody molecules by
continuous cell lines in culture. These include, but are not
limited to the hybridoma technique of Kohler and Milstein, (1975,
Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell
hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72;
Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and
the EBV-hybridoma technique (Cole et al., 1985, Monoclonal
Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such
antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. The hybridoma producing the
mAb of this invention may be cultivated in vitro or in vivo.
Production of high titers of mAbs in vivo makes this the presently
preferred method of production.
[0106] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608;
Takeda et al., 1985, Nature, 314:452-454) by splicing the genes
from a mouse antibody molecule of appropriate antigen specificity
together with genes from a human antibody molecule of appropriate
biological activity can be used. A chimeric antibody is a molecule
in which different portions are derived from different animal
species, such as those having a variable or hypervariable region
derived from a murine mAb and a human immunoglobulin constant
region.
[0107] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci.
USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be
adapted to produce differentially expressed gene-single chain
antibodies. Single chain antibodies are formed by linking the heavy
and light chain fragments of the Fv region via an amino acid
bridge, resulting in a single chain polypeptide.
[0108] Most preferably, techniques useful for the production of
"humanized antibodies" can be adapted to produce antibodies to the
polypeptides, fragments, derivatives, and functional equivalents
disclosed herein. Such techniques are disclosed in U.S. Pat. Nos.
5,932,448; 5,693,762; 5,693,761; 5,585,089; 5,530,101; 5,910,771;
5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,545,580; 5,661,016;
and 5,770,429, the disclosures of all of which are incorporated by
reference herein in their entirety.
[0109] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, such fragments include
but are not limited to: the F(ab').sub.2 fragments which can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments. Alternatively, Fab expression
libraries may be constructed (Huse et al., 1989, Science,
246:1275-1281) to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity.
[0110] An antibody of the present invention can be preferably used
in a method for the diagnosis of a condition associated with host
inflammatory or immune response in a human which comprises:
measuring the amount of a polypeptide comprising the amino acid
sequence set forth in SEQ ID NO:1, or fragments thereof, in an
appropriate tissue or cell from a human suffering from a condition
associated with host inflammatory or immune response, wherein the
presence of an elevated amount of said polypeptide or fragments
thereof, relative to the amount of said polypeptide or fragments
thereof in the respective tissue from a human not suffering from a
condition associated with host inflammatory or immune response is
diagnostic of said human's suffering from a condition associated
with host inflammatory or immune response. Such a method forms a
further embodiment of the present invention. Preferably, said
detecting step comprises contacting said appropriate tissue or cell
with an antibody which specifically binds to a polypeptide that
comprises the amino acid sequence set forth in SEQ ID NO:1 or a
fragment thereof and detecting specific binding of said antibody
with a polypeptide in said appropriate tissue or cell, wherein
detection of specific binding to a polypeptide indicates the
presence of a polypeptide that comprises the amino acid sequence
set forth in SEQ ID NO:1 or a fragment thereof.
[0111] Particularly preferred, for ease of detection, is the
sandwich assay, of which a number of variations exist, all of which
are intended to be encompassed by the present invention.
[0112] For example, in a typical forward assay, unlabeled antibody
is immobilized on a solid substrate and the sample to be tested
brought into contact with the bound molecule. After a suitable
period of incubation, for a period of time sufficient to allow
formation of an antibody-antigen binary complex. At this point, a
second antibody, labeled with a reporter molecule capable of
inducing a detectable signal, is then added and incubated, allowing
time sufficient for the formation of a ternary complex of
antibody-antigen-labeled antibody. Any unreacted material is washed
away, and the presence of the antigen is determined by observation
of a signal, or may be quantitated by comparing with a control
sample containing known amounts of antigen. Variations on the
forward assay include the simultaneous assay, in which both sample
and antibody are added simultaneously to the bound antibody, or a
reverse assay in which the labeled antibody and sample to be tested
are first combined, incubated and added to the unlabeled surface
bound antibody. These techniques are well known to those skilled in
the art, and the possibility of minor variations will be readily
apparent. As used herein, "sandwich assay" is intended to encompass
all variations on the basic two-site technique. For the
immunoassays of the present invention, the only limiting factor is
that the labeled antibody be an antibody that is specific for the
IL-1RP1 polypeptide or a fragment thereof.
[0113] The most commonly used reporter molecules in this type of
assay are either enzymes, fluorophore- or radionuclide-containing
molecules. In the case of an enzyme immunoassay an enzyme is
conjugated to the second antibody, usually by means of
glutaraldehyde or periodate. As will be readily recognized,
however, a wide variety of different ligation techniques exist,
which are well-known to the skilled artisan. Commonly used enzymes
include horseradish peroxidase, glucose oxidase, beta-galactosidase
and alkaline phosphatase, among others. The substrates to be used
with the specific enzymes are generally chosen for the production,
upon hydrolysis by the corresponding enzyme, of a detectable color
change. For example, p-nitrophenyl phosphate is suitable for use
with alkaline phosphatase conjugates; for peroxidase conjugates,
1,2-phenylenediamine or toluidine are commonly used. It is also
possible to employ fluorogenic substrates, which yield a
fluorescent product rather than the chromogenic substrates noted
above. A solution containing the appropriate substrate is then
added to the tertiary complex. The substrate reacts with the enzyme
linked to the second antibody, giving a qualitative visual signal,
which may be further quantitated, usually spectrophotometrically,
to give an evaluation of the amount of IL-1RP1 which is present in
the serum sample.
[0114] Alternately, fluorescent compounds, such as fluorescein and
rhodamine, may be chemically coupled to antibodies without altering
their binding capacity. When activated by illumination with light
of a particular wavelength, the fluorochrome-labeled antibody
absorbs the light energy, inducing a state of excitability in the
molecule, followed by emission of the light at a characteristic
longer wavelength. The emission appears as a characteristic color
visually detectable with a light microscope. Immunofluorescence and
EIA techniques are both very well established in the art and are
particularly preferred for the present method. However, other
reporter molecules, such as radioisotopes, chemiluminescent or
bioluminescent molecules may also be employed. It will be readily
apparent to the skilled artisan how to vary the procedure to suit
the required use.
[0115] This invention also relates to the use of polynucleotides of
the present invention as diagnostic reagents. In particular, the
invention relates to a method for the diagnosis of a condition
associated with host inflammatory or immune response in a human
which comprises: detecting elevated transcription of messenger RNA
transcribed from the natural endogenous human gene encoding the
polypeptide consisting of an amino acid sequence set forth in SEQ
ID NO: 1 in an appropriate tissue or cell from a human, wherein
said elevated transcription is diagnostic of said human's suffering
from the condition associated with host inflammatory or immune
response. In particular, said natural endogenous human gene
comprises the nucleotide sequence set forth in SEQ ID NO:4. In a
preferred embodiment such a method comprises contacting a sample of
said appropriate tissue or cell or contacting an isolated RNA or
DNA molecule derived from that tissue or cell with an isolated
nucleotide sequence of at least about 20 nucleotides in length that
hybridizes under high stringency conditions with the isolated
nucleotide sequence encoding a polypeptide consisting of an amino
acid sequence set forth in SEQ ID NO:1.
[0116] Detection of a mutated form of the gene characterized by the
polynucleotide of SEQ ID NO:4 which is associated with a
dysfunction will provide a diagnostic tool that can add to, or
define, a diagnosis of a disease, or susceptibility to a disease,
which results from under-expression, over-expression or altered
spatial or temporal expression of the gene. Individuals carrying
mutations in the gene may be detected at the DNA level by a variety
of techniques.
[0117] Nucleic acids, in particular mRNA, for diagnosis may be
obtained from a subject's cells, such as from blood, urine, saliva,
tissue biopsy or autopsy material. The genomic DNA may be used
directly for detection or may be amplified enzymatically by using
PCR or other amplification techniques prior to analysis. RNA or
cDNA may also be used in similar fashion. Deletions and insertions
can be detected by a change in size of the amplified product in
comparison to the normal genotype. Hybridizing amplified DNA to
labeled nucleotide sequences encoding the IL-1RP1 polypeptide of
the present invention can identify point mutations. Perfectly
matched sequences can be distinguished from mismatched duplexes by
RNase digestion or by differences in melting temperatures. DNA
sequence differences may also be detected by alterations in
electrophoretic mobility of DNA fragments in gels, with or without
denaturing agents, or by direct DNA sequencing (e.g., Myers et al.,
Science (1985) 230:1242). Sequence changes at specific locations
may also be revealed by nuclease protection assays, such as RNase
and S1 protection or the chemical cleavage method (see Cotton et
al., Proc Natl Acad Sci USA (1985) 85: 4397-4401). In another
embodiment, an array of oligonucleotides probes comprising
nucleotide sequence encoding the IL-1RP1 polypeptide of the present
invention or fragments of such a nucleotide sequence can be
constructed to conduct efficient screening of e.g., genetic
mutations. Array technology methods are well known and have general
applicability and can be used to address a variety of questions in
molecular genetics including gene expression, genetic linkage, and
genetic variability (see for example: M. Chee et al., Science, Vol
274, pp 610-613 (1996)).
[0118] The diagnostic assays offer a process for diagnosing or
determining a susceptibility to disease through detection of
mutation in the IL-1RP1 gene by the methods described. In addition,
such diseases may be diagnosed by methods comprising determining
from a sample derived from a subject an abnormally decreased or
increased level of polypeptide or mRNA. Decreased or increased
expression can be measured at the RNA level using any of the
methods well known in the art for the quantitation of
polynucleotides, such as, for example, nucleic acid amplification,
for instance PCR, RT-PCR, RNase protection, Northern blotting and
other hybridization methods. Assay techniques that can be used to
determine levels of a protein, such as a polypeptide of the present
invention, in a sample derived from a host are well known to those
of skill in the art. Such assay methods include radioimmunoassays,
competitive-binding assays, Western Blot analysis and ELISA
assays.
[0119] Thus in another aspect, the present invention relates to a
diagnostic kit which comprises:
[0120] (a) a polynucleotide of the present invention, preferably
the nucleotide sequence of SEQ ID NO:2, 3 or 4, or a fragment
thereof;
[0121] (b) a nucleotide sequence complementary to that of (a);
[0122] (c) a polypeptide of the present invention, preferably the
polypeptide of SEQ ID NO:1 or a fragment thereof; or
[0123] (d) an antibody to a polypeptide of the present invention,
preferably to the polypeptide of SEQ ID NO:1.
[0124] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component. Such a kit will be of
use in diagnosing a disease or susceptibility to a disease,
particularly to a disease or condition associated with inflammation
or immune response.
[0125] The nucleotide sequences of the present invention are also
valuable for chromosome localization. The sequence is specifically
targeted to, and can hybridize with, a particular location on an
individual human chromosome. The mapping of relevant sequences to
chromosomes according to the present invention is an important
first step in correlating those sequences with gene associated
disease. Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found in,
for example, V. McKusick, Mendelian Inheritance in Man (available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between genes and diseases that have been mapped
to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0126] The differences in the cDNA or genomic sequence between
affected and unaffected individuals can also be determined. If a
mutation is observed in some or all of the affected individuals but
not in any normal individuals, then the mutation is likely to be
the causative agent of the disease.
[0127] An additional embodiment of the invention relates to the
administration of a pharmaceutical composition, in conjunction with
a pharmaceutically acceptable carrier, excipient or diluent, for
any of the therapeutic effects discussed above. Such pharmaceutical
compositions may consist of the interleukin-1 related polypeptide
1, antibodies to that polypeptide, mimetics, agonists, antagonists,
or inhibitors of IL-1RP1 function. The compositions may be
administered alone or in combination with at least one other agent,
such as stabilizing compound, which may be administered in any
sterile, biocompatible pharmaceutical carrier, including, but not
limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs or hormones.
[0128] The pharmaceutical compositions encompassed by the invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-articular,
intra-arterial, intramedullary, intrathecal, intraventricular,
transdermal, subcutaneous, intraperitoneal, intranasal, enteral,
topical, sublingual, or rectal means.
[0129] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries that facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.).
[0130] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0131] Pharmaceutical preparations for oral use can be obtained
through combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers, such as sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums including arabic and tragacanth; and proteins such as gelatin
and collagen. If desired, disintegrating or solubilizing agents may
be added, such as the cross-linked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate.
[0132] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0133] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with a
filler or binders, such as lactose or starches, lubricants, such as
talc or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0134] Pharmaceutical formulations suitable for parenteral
administration may be formulated m aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances that increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents that
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions.
[0135] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0136] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0137] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
etc. Salts tend to be more soluble in aqueous or other protonic
solvents than are the corresponding free base forms. In other
cases, the preferred preparation may be a lyophilized powder that
may contain any or all of the following: 1-50 mM histidine, 0.1%-2%
sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is
combined with buffer prior to use.
[0138] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of the
interleukin-1-related polypeptide 1, such labeling would include
amount, frequency, and method of administration.
[0139] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0140] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually mice, rabbits, dogs,
or pigs. The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0141] A therapeutically effective dose refers to that amount of
active ingredient, for example IL-1RP1 or fragments thereof,
antibodies of IL-1RP1, agonists, antagonists or inhibitors of
IL-1RP1, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g ED50 (the
dose therapeutically effective in 50% of the population) and LD50
(the dose lethal to 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. Pharmaceutical compositions
that exhibit large therapeutic indices are preferred. The data
obtained from cell culture assays and animal studies is used in
formulating a range of dosage for human use. The dosage contained
in such compositions is preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage varies within this range depending upon the dosage form
employed, sensitivity of the patient, and the route of
administration.
[0142] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active moiety or to maintain the desired effect. Factors
that may be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0143] Normal dosage amounts may vary from 0.1 to 100,000
micrograms, up to a total dose of about 1 g, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc. Pharmaceutical formulations
suitable for oral administration of proteins are described, e.g.,
in U.S. Pat. Nos. 5,008,114; 5,505,962; 5,641,515; 5,681,811;
5,700,486; 5,766,633; 5,792,451; 5,853,748; 5,972,387; 5,976,569;
and 6,051,561.
[0144] The following Examples illustrate the present invention,
without in any way limiting the scope thereof.
EXAMPLES
Example 1
Identification of a Novel Interleukin 1 Related Human DNA Sequence
Using Bioinformatics
[0145] Public and in-house databases are initially searched with
the predicted amino acid sequences for human IL-1 beta (IL-1>)
(Genbank Accession Number AAC03536) and a novel human IL-1 homolog
50795, later published as IL-1H1, (Genbank Accession Number
AAF69248.1). TBlastN algorithm is used and the hits are aligned
with ClustalW. One 229-nucleotide sequence is found to be unique
within the in-house database. This sequence is then used to
identify additional genomic sequences from the public and in-house
databases (Genbank Accession number AF200492). A one 1 kb genomic
sequence flanking the identified genomic fragment is translated
into 6 reading frames and aligned with IL-1.beta. and IL-1H1 amino
acid sequence using ClustalW. Additional 127 nucleotides encoding a
protein homologous to IL-1.beta. and IL-1H1 is found 298
nucleotides upstream of the 5' end of the original genomic
sequence.
[0146] Further analyses of isolated cDNA clones is done using
ClustalW, BlastN, tBlastN according to conventional methods.
Example 2
Preparation of Full-Length cDNA Encoding the Novel Interleukin-1
Related Polypeptide 1 Consisting of SEQ ID NO:1
[0147] Polymerase chain reaction (PCR) is used to isolate cDNA
encoding the novel interleukin-1 related polypeptide (IL-1RP1). In
all experiments AdvanTaq.TM. DNA polymerase (Clontech) is used. Two
primers vio#3: 5'-GTCCCCATTTTCCTGGGGATCCAGGG-3' corresponding to
predicted AA sequence VPIFLGIQG and vio#4:
5'-ACCAGCTCTGTTCAAAGTAAAACTTGG-3' corresponding to predicted AA
sequence RTKFYFEQSW are synthesised (FIG. 1) and a two step PCR
(95.degree. C.-2 min.; [95.degree. C.-30 sec., 68.degree. C.-1
min.].times.35; 72.degree. C.-3 min) is performed in the volume of
25 .mu.l. Each reaction contains 1.times. Advantage 2 PCR buffer
and dNTP mix (Clontech). A panel of Clontech Marathon-Ready.TM.
double-stranded cDNA (3 .mu.l per reaction) from a variety of human
tissues is used as a source of template cDNA. Amplified 295 and 595
nt PCR products correspond to IL-1RP1 cDNA and genomic DNA,
respectively. The 295 nt PCR product amplified from Clontech
Marathon-Ready.TM. double-stranded cDNA from Fetal Human Intestine
where IL-1RP cDNA is the most abundant, is purified using Gel
Extraction Kit (Qiagen) and cloned into pCR-Blunt 1'-TOPO vector
(Invitrogen, Carlsbad, Calif.) according to manufacturer supplied
protocol. Plasmid DNA minipreps are prepared using Plasmid DNA
Miniprep Kit (Qiagen) and sequenced in both strains with T7 and M13
reverse primers (ACGT Inc, Bethesda, Md.). A Clone (#6) is found to
be free of PCR introduced mutations compared to human genomic
sequence and is used to reconstruct a full-length cDNA.
[0148] A 5'RACE procedure is performed using two sets of primers:
vio#4 and AP1 (Clontech); and vio#7:
5'-CTCCAGCTGTAGGGAAGGCCCCTC-3', corresponding to predicted AA
sequence EEGPSLQLE (FIG. 1) and AP2 according to protocol provided
by the supplier. The first PCR is performed on Clontech
Marathon-Ready.TM. double-stranded cDNA from Fetal Human Intestine
using vio#4 and AP1 primers in three identical 25-.mu.l reactions
in a two step PCR (95.degree. C.-2 min.; [95.degree. C.-30 sec.,
68.degree. C.-3 min.].times.35; 72.degree. C.-3 min.). PCR mixes
are pooled and extracted once with 75 .mu.l of
Phenol:Chloroform:Isoamyl alcohol--25:24:1 (Gibco BRL) and once
with 75 .mu.l of Chloroform:Isoamyl alcohol--24:1. Total PCR
products are precipitated with equal volume of Isopropanol for 30
min at -20 C. Precipitated material is centrifuged at 13 000 rpm
for 30 min and the pellet is washed once with ice-cold 70% Ethanol
and resuspended in 100 mls of TE buffer, 1.times. (Boehringer
Mannheim, Ridgefield, Conn.). Second PCR is performed with vio#7
and AP2 primers using 2 .mu.l of resuspended first PCR products as
described. A major 360 nt PCR product is purified and cloned into
pCR-Blunt II-TOPO vector (Invitrogen). Plasmid DNA minipreps are
sequenced with T7 and M13 reverse primer. 5'RACE clone #4 is used
to reconstruct the full-length cDNA.
[0149] To reconstruct IL-1RP1 full-length cDNA Clone#6 is cut with
Bam HI and Xho I and 5'RACE Clone#4 is cut with Not I and Bam HI.
Both cDNA fragments are purified and ligated into pFastBac (Gibco
BRL, Rockville, Md.) vector pre-digested with Ho I and Not I.
Clones with correct IL-1RP cDNA are selected by BAM HI digest and
the primary sequence is verified by sequencing with custom made
primer #17A 5'-TAT AGT TCT AGT GGT TGG CT-3'.
[0150] Preparation of genomic DNA containing the IL-1RP1 gene can
be accomplished by PCR using primers flanking the coding region
according to conventional methods. Alternatively, a genomic library
in lambda or P1 bacteriophage, BAC or plasmid libraries can be
screened using PCR or hybridization technologies as known to one of
skill in the art. As disclosed herein, information regarding the
genomic sequence of the IL-1RP1 protein of the present invention is
depicted in FIGS. 2 and 7 and is obtained by searching a public
database, for example, the Human Genome database, according to
conventional methods.
Example 3
mRNA Expression of IL-1RP1
[0151] To determine the relative abundance of IL-1RP1 in different
human tissues, and hence its potential utility, an RT-PCR
experiment (as described in Example 2) is carried out using a large
panel of cDNAs derived from 24 different human tissue RNA. The
panel is a premade panel obtained from OriGene (OriGene Inc.,
Rockville, Md.) wherein each cDNA is present in exact amounts. The
equivalency of RNA amounts is verified by a control PCR carried out
with primers to human beta-actin, a housekeeping gene present in
roughly equal amounts in all tissues. Two primers for IL-1RP1,
vio#3: 5'-GTCCCCATTTTCCTGGGGATCCAGGG-3' corresponding to predicted
AA sequence VPIFLGIQ and vio#4: 5'-ACCAGCTCTGTTCAAAGTAAAACTTGG-3'
corresponding to predicted AA sequence RTKFYFEQSW are synthesized
and a two step PCR (95 C-2'; [95 C-30'':68 C-1].times.35; 72 C-3')
is performed from the panel of 24 human tissues. The 295 nt PCR
product is again cloned into pCR-Blunt 1'-TOPO vector (Invitrogen
Inc., Carlsbad, Calif.) and sequenced to verify that it indeed
corresponds to the IL-1RP1 gene. In addition, after PCR the
products are analyzed by electrophoresis, blotted to a Nylon
membrane (a Southern blot) and hybridized to a P.sup.32 labeled
probe derived from the IL-1RP1 cDNA described in Example 2. This
blot is washed at high stringency (01.times.SSC 0.1% SDS for 1
hour) to ensure specific hybridization. The resulting hybridization
signal is revealed by autoradiography using exposure to Kodak XAR-5
film. All the above procedures are performed using conventional
methods.
[0152] As shown in FIG. 4, IL-1RP1 is present in low amounts in a
variety of tissues. However, skin expressed very high levels of
IL-1RP1 mRNA. This level is at least 10-fold higher than in other
tissues. This high level of expression in skin indicates that
IL-1RP1 may be of particular relevance to inflammation in the skin
and is may be important in the regulation of diseases such as
psoriasis and contact dermatitis.
Sequence CWU 0
0
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 15 <210>
SEQ ID NO 1 <211> LENGTH: 152 <212> TYPE: PRT
<213> ORGANISM: Human <400> SEQUENCE: 1 Met Cys Ser Leu
Pro Met Ala Arg Tyr Tyr Ile Ile Lys Tyr Ala Asp 1 5 10 15 Gln Lys
Ala Leu Tyr Thr Arg Asp Gly Gln Leu Leu Val Gly Asp Pro 20 25 30
Val Ala Asp Asn Cys Cys Ala Glu Lys Ile Cys Ile Leu Pro Asn Arg 35
40 45 Gly Leu Ala Arg Thr Lys Val Pro Ile Phe Leu Gly Ile Gln Gly
Gly 50 55 60 Ser Arg Cys Leu Ala Cys Val Glu Thr Glu Glu Gly Pro
Ser Leu Gln 65 70 75 80 Leu Glu Asp Val Asn Ile Glu Glu Leu Tyr Lys
Gly Gly Glu Glu Ala 85 90 95 Thr Arg Phe Thr Phe Phe Gln Ser Ser
Ser Gly Ser Ala Phe Arg Leu 100 105 110 Glu Ala Ala Ala Trp Pro Gly
Trp Phe Leu Cys Gly Pro Ala Glu Pro 115 120 125 Gln Gln Pro Val Gln
Leu Thr Lys Glu Ser Glu Pro Ser Ala Arg Thr 130 135 140 Lys Phe Tyr
Phe Glu Gln Ser Trp 145 150 <210> SEQ ID NO 2 <211>
LENGTH: 534 <212> TYPE: DNA <213> ORGANISM: Human
<400> SEQUENCE: 2 gatcagggtt ccaggaactc aggatctgca gtgaggacca
gacaccactg attgcaggaa 60 tgtgttccct ccccatggca agatactaca
taattaaata tgcagaccag aaggctctat 120 acacaagaga tggccagctg
ctggtgggag atcctgttgc agacaactgc tgtgcagaga 180 agatctgcat
acttcctaac agaggcttgg cccgcaccaa ggtccccatt ttcctgggga 240
tccagggagg gagccgctgc ctggcatgtg tggagacaga agaggggcct tccctacagc
300 tggaggatgt gaacattgag gaactgtaca aaggtggtga agaggccaca
cgcttcacct 360 tcttccagag cagctcaggc tccgccttca ggcttgaggc
tgctgcctgg cctggctggt 420 tcctgtgtgg cccggcagag ccccagcagc
cagtacagct caccaaggag agtgagccct 480 cagcccgtac caagttttac
tttgaacaga gctggtaggg agacaggaaa ctgc 534 <210> SEQ ID NO 3
<211> LENGTH: 459 <212> TYPE: DNA <213> ORGANISM:
Human <400> SEQUENCE: 3 atgtgttccc tccccatggc aagatactac
ataattaaat atgcagacca gaaggctcta 60 tacacaagag atggccagct
gctggtggga gatcctgttg cagacaactg ctgtgcagag 120 aagatctgca
tacttcctaa cagaggcttg gcccgcacca aggtccccat tttcctgggg 180
atccagggag ggagccgctg cctggcatgt gtggagacag aagaggggcc ttccctacag
240 ctggaggatg tgaacattga ggaactgtac aaaggtggtg aagaggccac
acgcttcacc 300 ttcttccaga gcagctcagg ctccgccttc aggcttgagg
ctgctgcctg gcctggctgg 360 ttcctgtgtg gcccggcaga gccccagcag
ccagtacagc tcaccaagga gagtgagccc 420 tcagcccgta ccaagtttta
ctttgaacag agctggtag 459 <210> SEQ ID NO 4 <211>
LENGTH: 7660 <212> TYPE: DNA <213> ORGANISM: Human
<400> SEQUENCE: 4 atgcccaggc tcttcttcct tcatgtcctg cagccaatta
tagagattgg tgcaggcctg 60 acccacctgt accagacggt ataaacacag
cgcaatgccc tggagaaatc agttggagtc 120 tccagggatc agggttccag
gaactcagga tctgcaggtc agtgatggac aggcaatatt 180 ctctctctct
tttctttctt ttctactctc tcctgtcaat atctctggta tgccctctat 240
cccttcactc ctcctggcaa gctgtctagg taacaaggta gtcccctcat atgacatgat
300 gggacatcaa aaacatttag gagcacataa agagatttga ggagaaggaa
taggttaagc 360 caaatcaaga tttctgaact acttagaaca actgacttta
gaaagtaatc tcaaaagaaa 420 ccatctgggc caatggtatt caaaccgtgt
ttggtggaag cttaagagat tttttgtttt 480 cgtttttttt ttttttgtgc
tccttggtag ctcctcagtg tcttctttgt aggcaagaga 540 aatactagca
gggttccaaa gtctccattg cttcctgtgt cagcaaagca gctgcccttt 600
atccttttta caatcattag aattctgcat aaaattttat tgtgaaactt aaaacagttt
660 tgaaagatac cttactcaat ctttccatcc aatgttgata agttgaaggc
ccagggataa 720 aaggtggcaa aataaattgg tagcagagct aggtctagga
ctcagccttt ctattcaaag 780 tctgctctgt gtgtttctat gtcctggcca
caaggaccag ggtagttcaa ttagggtggt 840 tggagagagc ttcaagtgag
ctgtaagcca tgcttcatga cttcaagatc ttataaactt 900 ttataaggga
aacaaatatc tttatgtaca acaataaata atacagtaaa atttaatagg 960
agttcagagt agagttacaa tagtaaggga aagattcatt aaactctgag tcatggaatt
1020 ataaatagtt tggcctagaa agaaccttca taaccacata atctaatccc
atgaatgtat 1080 agtgctgaaa ctgagggttg gaggttaaaa gacataaccc
tgtatgtcta agctgtaact 1140 ctacactggt aattaatttg atagaactag
atactgaacc catctatact gatactatct 1200 ccagtgatct taatatcaaa
agatctgaat ggatgacaat gagaagggat gacttttgag 1260 gcagggagaa
caatgggaca cctgtgagta cgtccactga gaagataaga aaggaattat 1320
gtcgccagga ggtgtggttt agtgtccatg gagaagtgag atgctatcac acaagaaaat
1380 cagtgccaga cggtgacaaa acagtcagtc actgtagatt ctagagctgg
gcagggatgt 1440 ggtgaaaaaa atctttagaa cgtgggtcca cattcctcac
ataggttgga atgaatgaaa 1500 ggcagaaaaa ccgggaatac aagtacagta
gtaatggcaa ttaaaaacac aaagttatat 1560 tttaaagaaa taacctgtag
ggacttgata agttagacac tgagtcagga gaagtaggag 1620 atattgtcaa
tgagtctcag gttttgaata tcactggtga gaggaatatt atgacagtga 1680
gagaaatgaa attgggagga atgtgtgggc tgggaggaga agataattaa cttaacctta
1740 aatatatttg gttggatatc agcaggatgt ccaaagtggg ctgtcctgta
ggctgcagga 1800 ggtaaagtgg gatgtgcagt ctacatttgg gagccattag
cccaagataa tcattgcagc 1860 tgtagaagtc aatgaattgc agcttaatat
gatgtgaaat agaagtgctt aggaccttgt 1920 tagccttagg ggtggcccca
gaaggaggtg aaatctacag tgggagaaag aggtgtgaag 1980 ggagtcattc
aatgtcaagg gttccaagga ggcaatgact aaatatcaaa ggaatcaaaa 2040
gaatctctgg atcttgctcc acagaagtta atattggtct tggacagaac atcccacata
2100 gatactgtgc aggagaatga tgaaggatag gacagggtgc tgtgcaaaag
aacatgggga 2160 aagcccaagt ttagctgatg gcagtgtttg agccaggaca
ggtgaattgc ggaggtctga 2220 gagccagatg ccatgtccag ggagaaatga
gaggaagaaa agagggatat agactgaagt 2280 tcagtgaaaa gtcatttgag
gaaaataggg aggggaagct ttgggggtga ggcatgtggc 2340 acactgggag
gggcttggca cacagcagag gttcagcacc aagacccagg ctctctgatg 2400
gaccagacgc tagcttccta cccttactca cttcatcaca atctatcaga acccaggcgg
2460 agggagccga ataggggagc ctttgggaaa gacactgtac attttggctg
tgccagaatg 2520 ggaggtttct agggcccatg ggatccagct ggactggacc
agcattgaat ttcttccagc 2580 tctttgagct gacactgacc cagagtggga
gtcatcagct tgctatccac cttcacccag 2640 ggccctccac tttgttgccc
cacctagatc tgggcacagc taccacactg cccactgtcc 2700 tgctgctaca
accaaagaag ccccagtggt ttggccaagg ggagcccatc atcaagtggg 2760
cttgcattga ggccatgatg ctgttgagtt atctgtactg ggggattgtc tagtccttta
2820 ggactcaaag tgctggccag gaggaaccag cagcattgac atcacctggt
tgcatatttg 2880 aaatgtacag tctcaggccc caccccaggc ctgaaaaacc
agaatctgtt attttaacaa 2940 gaactgcagg tggtttatat atttattaat
aagtgtgaag aatggaatga aagtacacca 3000 gttcccaagc agcatggctg
attgctggaa tcactccaag tcctactgaa ttagaacctt 3060 cggcccagga
aatagtaatt atacagagtc ccccaggtga tgcagatggg caggcacatt 3120
taggagccaa tgactttaac tgaacacttc atttaaaaaa tgttgaaact tacttgatac
3180 tacaaaggaa attcatgttc attataggaa aatgttgata tgtttaaaaa
attactcata 3240 aagccatagg taagtggtgc aacaacacga gtaacatttc
tatgtatgtg tctctatgtg 3300 tggatttaaa tagaattaca gtgtacactt
gatttataat ctgcattttt cacctaatat 3360 attttgaaaa tttttatgtc
ctaaaacaag cttctataat atcatcttta acaaacacat 3420 acatccttat
ttattgaatt ttgctataat ttcttagcca attacctatt actgaaaatt 3480
cagatttttt tcaacttctt gctattgtaa aaaattatgc agtgaacatt tttgtaagta
3540 aacatttggg caatccgtta tttttcctaa gagtaaggga aacacatgca
atcacaaagt 3600 atacagaatg ctttaagact ttcattcaca gcaccaacat
ccctccagaa tttgcacttg 3660 ttagtcccta ttatccttca ctctaagtct
caaagtcata ccccaaggcc tggggacaga 3720 aaatgacttg tccaaagtga
cagtgacaga cccagtacta aaagccacct tggctacagc 3780 cctgtttctg
gaacttgaga gctgaggtgg ttggaagccg tatcctcagc acccacctgt 3840
tccttctcac ctgcctcccc agggtccctc agcatctctc tattcctccc tgagccctat
3900 tactttcttc cacctgcctt cttcctttct cttctctcat tttctgcttt
cttatatttt 3960 ttcttctcta ttcccttctt atttggtgag aatcagatct
actcggtaaa cctcagccct 4020 agtcatactt gcgttacttt cctgagctaa
tttccaactc ctgattagct ctgggtttat 4080 ttccatgcta aattctggac
tggcctttcc aatgggtgtt cattttaggg aagagctcta 4140 ggacaggata
acccatcggg aaggagcaga gtcatgtgag gctgtgtggc ctggcattta 4200
tacagggcca ctatcttcac tgtgccattt tccatctgga acagaatggg ggagtttgga
4260 tgggctgttt tcggcagtct tggccaagca cttctagtca ctaggaatga
tgttttccaa 4320 ctctctgggg agaccccacc agcctcactg ctgctggaga
ccccttctag ttgtgctctc 4380 ttctttcact ctgggctcta gttatctaac
ccttggctag ttatgggggc gggggtgtgg 4440
tgccctgttg gccaacaggg cagtgggact gggtttgagc tgggcttatc ctccaactgt
4500 gagggaggct acagcacact ccaccccact ctcagggctg ggaattgttg
tggctcagct 4560 atttggggga atctgttttc cagtttctca gaaccagcgc
aagcacacac atcccaggct 4620 cacacccctg gtggctggac ttgctcccgg
atagcctcag tcagggagag gcagagctgc 4680 ctggagcctg ctgggctggt
ggaagccttg gtggattctg gcaggccaat tatagacgaa 4740 tggcctgggg
aacccgtgca gcccttggct gagtggttct aagccccagc acgtctgcct 4800
ctggcttcac ccagcctcct tttctaactg cccttctctc ctccccatca gtgaggacca
4860 gacaccactg attgcaggaa tgtgttccct ccccatggca agatactaca
tgtaagttgt 4920 cctggcatgt ccctgctttc caagccaggg ggtcagggtg
ggaagaggaa aggaatgctg 4980 agtcagagga tgaggctcct tctcacctta
gaaattgcaa gtgccccata attaagcttc 5040 atcatcacca cagtagcaac
agctctttcc tgaacgtctg caagatgcca gccaatctac 5100 tgcctcatct
ctgttccaaa aagtctgtaa gtggagtgtt attaaaccca ttttacagat 5160
ctggaagctg aggctcaaag agggtaaata acttccccca tgtcacacag ctaccaaaag
5220 gcagagccag gaatcagact tcatgtcctc tgtgctgctc catccgcctc
tctgaaatgt 5280 cagaaagttt tgaatctcaa tgacagcatc ttgatggtgg
tccctgtggc ctttactccc 5340 agtgtgggct tctaacactt acttacattt
catctcattt gagatttgca tccttcctta 5400 tcttttacta ctttgttgtc
tgtgattttg tcataagctc ctttcaggaa ggaggtgagg 5460 cataagaaaa
atcaaagagg actctgggat gcatttcctc tgcccctccc atggaccctg 5520
taatgtccag ggctgtgtcc tggacaaggt gggtggggag cagtcctggt ctcaaggagg
5580 tgacagcctg gctgggaagc aagacacata cataggaagc acataaatga
caaagcagat 5640 gtcagcactt cagggcatct aatctgggtt ctggtctcca
aatagaatgc tgctggcatg 5700 tgagttgtca catctgggtt gtcaaggtgg
caaggggaat gccagataac acgcccagga 5760 tctttccgga agtttatttt
tattgtacaa gtgaacctgc tttaaatatg tacagtcatt 5820 agctaagggt
attatcgtta gctgttattg agatagaaaa atcccctgga ggtggtggaa 5880
tttgtccaga ggttctgccc taaaaggtta atgagagctc tccagccctg acagcagctg
5940 acaggcatct ttgaaaccaa ctaggtgact gagctaatac cctgcatgac
tttgaagcct 6000 ttaaaatatc tgaaaagcaa atcacacttc agtatacact
caatctctgt actaaagaga 6060 ataaacattt ataaacaatt agggcaggcc
caaaaaattt aagataaggt ccactgtatc 6120 ccaaagtcat ctgagcctca
ctaagaaatt tctcaggaag ccaggaacat tttctttacc 6180 cctctgtcag
agggcattgg ctctccgttc tcctctgaag gcctccccaa gccatgagaa 6240
ggcaggaagc acagcctctg aaaagcaaga acacaggaga ccttccttgc tttaaggctg
6300 gcctggtctt tacctgctct tgggagtgac cattcccctc ttaccacctg
tgaaggagag 6360 aaaatcgccc aaatgctcaa ggtggtgatt cagagcatgg
aagtggaagg gcttgggggc 6420 cagtggtgca taaagggaat gggccatcag
cactgtcata ctgtttcaga attaaatatg 6480 cagaccagaa ggctctatac
acaagagatg gccagctgct ggtgggagat cctgttgcag 6540 acaactgctg
tgcaggtgag cttctggggc ctccacccca tgctccatct gccataggcc 6600
ctcccttctc ttcttccctt tcctccccag cagagggtca gcagctgccc ccagtgacag
6660 tgagaagggc cagagagcag ctgtggcctc tcctagcgag gggacatgac
tcctgcagaa 6720 gtcctggctc accgtccagt ctgcatgcag ggccaggcca
ggtgtgccca tgtccagttc 6780 cttcctgcct gagcctttac ctgccaagag
cctgcaacat ggggttccct tgtcccttga 6840 ctcttctctc tcttccctcc
tagagaagat ctgcatactt cctaacagag gcttggcccg 6900 caccaaggtc
cccattttcc tggggatcca gggagggagc cgctgcctgg catgtgtgga 6960
gacagaagag gggccttccc tacagctgga ggtgagaggc ctctccccat tctaggggac
7020 actgcagacc tggcctgacc cctgggatgc tctggcatct ttgtgcctat
ctgtggattc 7080 ccagccaggt ccacatgtcc tacttcctca ggtttccacc
atctccctct gcacctagca 7140 ccaagaccct tgccctctag aatctgcaga
aggcagtccc ttgggtaaaa accagccctg 7200 tcaggtcctt ttttggccaa
gccccagagg cctccagggc taacacctcc atcagcactc 7260 tcattctgca
gccatccacc ttgcccccac aggatgtgaa cattgaggaa ctgtacaaag 7320
gtggtgaaga ggccacacgc ttcaccttct tccagagcag ctcaggctcc gccttcaggc
7380 ttgaggctgc tgcctggcct ggctggttcc tgtgtggccc ggcagagccc
cagcagccag 7440 tacagctcac caaggagagt gagccctcag cccgtaccaa
gttttacttt gaacagagct 7500 ggtagggaga caggaaactg cgttttagcc
ttgtgccccc aaaccaagct catcctgctc 7560 agggtctatg gtaggcagaa
taatgtcccc cgaaatatgt ccacatccta atcccaagat 7620 ctgtgcatat
gttaccatac atgtccaaag aggttttgca 7660 <210> SEQ ID NO 5
<400> SEQUENCE: 5 000 <210> SEQ ID NO 6 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
predicted amino acid sequence, chemically synthesized <400>
SEQUENCE: 6 Val Pro Ile Phe Leu Gly Ile Gln Gly 1 5 <210> SEQ
ID NO 7 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: primer <400> SEQUENCE: 7 accagctctg
ttcaaagtaa aacttgg 27 <210> SEQ ID NO 8 <211> LENGTH:
10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: predicted amino
acid sequence, chemically synthesized <400> SEQUENCE: 8 Arg
Thr Lys Phe Tyr Phe Glu Gln Ser Trp 1 5 10 <210> SEQ ID NO 9
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: primer <400> SEQUENCE: 9 ctccagctgt agggaaggcc
cctc 24 <210> SEQ ID NO 10 <211> LENGTH: 9 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: predicted amino acid
sequence, chemically synthesized <400> SEQUENCE: 10 Glu Glu
Gly Pro Ser Leu Gln Leu Glu 1 5 <210> SEQ ID NO 11
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: primer <400> SEQUENCE: 11 tatagttcta gtggttggct
20 <210> SEQ ID NO 12 <211> LENGTH: 26 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: primer <400>
SEQUENCE: 12 gtccccattt tcctggggat ccaggg 26 <210> SEQ ID NO
13 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Homo Sapiens <220> FEATURE: <223> OTHER
INFORMATION: peptide <400> SEQUENCE: 13 Val Pro Ile Phe Leu
Gly Ile Gln 1 5 <210> SEQ ID NO 14 <211> LENGTH: 27
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: primer
<400> SEQUENCE: 14 accagctctg ttcaaagtaa aacttgg 27
<210> SEQ ID NO 15 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Homo Sapiens <220> FEATURE:
<223> OTHER INFORMATION: peptide <400> SEQUENCE: 15 Arg
Thr Lys Phe Tyr Phe Glu Gln Ser Trp 1 5 10
* * * * *
References