U.S. patent application number 10/011548 was filed with the patent office on 2003-03-20 for human receptor proteins; related reagents and methods.
Invention is credited to Antonius Debets, Johannes Eduard Maria, Bazan, J. Fernando, Kastelein, Robert A., Sana, Theodore R., Timans, Jacqueline C..
Application Number | 20030055218 10/011548 |
Document ID | / |
Family ID | 27568197 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030055218 |
Kind Code |
A1 |
Timans, Jacqueline C. ; et
al. |
March 20, 2003 |
Human receptor proteins; related reagents and methods
Abstract
Nucleic acids encoding mammalian, e.g., human receptors,
purified receptor proteins and fragments thereof. Antibodies, both
polyclonal and monoclonal, are also provided. Methods of using the
compositions for both diagnostic and therapeutic utilities are
provided.
Inventors: |
Timans, Jacqueline C.;
(Mountain View, CA) ; Antonius Debets, Johannes Eduard
Maria; (Mountain View, CA) ; Sana, Theodore R.;
(East Palo Alto, CA) ; Bazan, J. Fernando; (Menlo
Park, CA) ; Kastelein, Robert A.; (Redwood City,
CA) |
Correspondence
Address: |
DNAX Research Institute
901 California Avenue
Palo Alto
CA
94304-1104
US
|
Family ID: |
27568197 |
Appl. No.: |
10/011548 |
Filed: |
October 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10011548 |
Oct 22, 2001 |
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09173151 |
Oct 14, 1998 |
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60065776 |
Nov 17, 1997 |
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60078008 |
Mar 12, 1998 |
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60081883 |
Apr 15, 1998 |
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60095987 |
Aug 10, 1998 |
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60078416 |
Mar 18, 1998 |
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60062066 |
Oct 15, 1997 |
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Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/69.1; 536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/7155 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
530/350 ;
435/69.1; 435/325; 435/320.1; 536/23.5 |
International
Class: |
C07K 014/715; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated or recombinant IL-1RD9 polypeptide: a) consisting of
SEQ ID NO: 6, 8, 10, 12, 14, or 16; b) encoded by a polynucleotide
comprising the open reading frame of SEQ ID NO: 5, 7, 9, 11, 13, or
15; or c) encoded by a naturally occurring allelic variant of a
polynucleotide comprising the open reading frame of SEQ ID NO: 5,
7, 9, 11, 13, or 15.
2. The polypeptide of claim 1, encoded by a naturally occurring
allelic variant of a polynucleotide comprising the open reading
frame of SEQ ID NO: 5, 7, 9, 11, 13, or 15.
3. An isolated or recombinant IL-1RD9 polypeptide which: a) has an
apparent molecular weight of approximately 68.3 kD as determined by
calculation from sequence and an estimated pI of approximately
9.04; and b) is found on T cells; wherein said polypeptide has at
least one of the following properties: i) is a heterodimer; iii) is
an IL-1 receptor .alpha. subunit type, or iii) when brought into
contact with IL-1RD5 and IL-1.alpha., for a sufficient time, forms
a functional high affinity receptor complex that activates an
NF.kappa.B transcription factor reporter construct.
4. An isolated or recombinant polypeptide comprising a segment of
contiguous amino acid residues selected from the following group:
a) 15 contiguous amino acid residues of said polypeptide of claim
2; b) 20 contiguous amino acid residues of said polypeptide of
claim 2; c) 25 contiguous amino acid residues of said polypeptide
of claim 2; d) 30 contiguous amino acid residues of said
polypeptide of claim 2; e) 35 contiguous amino acid residues of
said polypeptide of claim 2; or f) 40 contiguous amino acid
residues of said polypeptide of claim 2.
5. The polypeptide of claim 1 which is immunogenic.
6. An isolated or recombinant polypeptide comprising an immunogenic
peptide of said polypeptide of claim 3.
7. An isolated or recombinant polypeptide comprising an immunogenic
polypeptide of claim 4.
8. A fusion protein comprising said polypeptide of claim 4 and: a)
a detection or purification tag selected from the group consisting
of a FLAG, His6, and immunoglobulin peptide; b) a carrier protein
selected from the group consisting of keyhole limpet hemocyanin,
bovine serum albumin, and tetanus toxoid; or c) another peptide
selected from the group consisting of luciferase, bacterial
.beta.-galactosidase, trpE, protein A, .beta.-lactamase, alpha
amylase, alcohol dehydrogenase, and yeast alpha mating factor.
9. A fusion protein comprising said polypeptide of claim 5 and: a)
a detection or purification tag selected from the group consisting
of a FLAG, His6, and immunoglobulin peptide; b) a carrier protein
selected from the group consisting of keyhole limpet hemocyanin,
bovine serum albumin, the tetanus toxoid; or c) another peptide
selected from the group consisting of luciferase, bacterial
.beta.-galactosidase, trpE, protein A, .beta.-lactamase, alpha
amylase, alcohol dehydrogenase, and yeast alpha mating factor.
10. A composition comprising said polypeptide of claim 1, that is:
a) in a pharmaceutically acceptable carrier; b) in a sterile
composition; c) in a buffered solution; d) in an aqueous
suspension; or e) in combination with IL-1RD5 and/or
IL-1.alpha..
11. A composition comprising said polypeptide of claim 4, that is:
a) in a pharmaceutically acceptable carrier; b) in a sterile
composition; c) in a buffered solution; or d) in an aqueous
suspension.
12. A polypeptide of claim 4, that is: a) denatured; b)
immunopurified; c) attached to a solid substrate; d) detectably
labeled; or e) chemically synthesized.
13. A polypeptide of claim 5, that is: a) denatured; b)
immunopurified; c) attached to a solid substrate; d) detectably
labeled; or e) chemically synthesized.
14. A kit comprising said polypeptide of claim 1, and: a) a
compartment comprising said protein; or b) instructions for use or
disposal of reagents in said kit.
15. A kit comprising said polypeptide of claim 4, and: a) a
compartment comprising said protein; or b) instructions for use or
disposal of reagents in said kit.
16. A method of raising an antibody, comprising immunizing an
animal with a polypeptide of claim 5.
17. A method of producing an antibody:antigen complex, comprising
contacting a polypeptide of claim 5 with an antibody which
specifically binds said polypeptide, thereby forming said
complex.
18. A composition of matter selected from the group consisting of:
a) a substantially pure or recombinant IL-1RD8 polypeptide
exhibiting identity over a length of at least about 12 amino acids
to SEQ ID NO: 4; b) a natural sequence IL-1RD8 comprising SEQ ID
NO: 4; c) a fusion polypeptide comprising IL-1RD8 sequence; d) a
substantially pure or recombinant IL-1RD10 polypeptide exhibiting
identity over a length of at least about 12 amino acids to SEQ ID
NO: 35; e) a natural sequence IL-1RD10 comprising SEQ ID NO: 35;
and f) a fusion protein comprising IL-1RD10 sequence.
19. A substantially pure or isolated polypeptide comprising a
segment exhibiting sequence identity to a corresponding portion of
an: a) IL-1RD8 of claim 18, wherein: i) said polypeptide further
exhibits identity to a distinct segment of 9 amino acids; ii) said
length of identity is at least 17 amino acids; iii) said length of
identity is at least about 25 amino acids; or b) IL-1RD10 of claim
18, wherein: i) said polypeptide further exhibits identity to a
distinct segment of 9 amino acids; ii) said length of identity is
at least 17 amino acids; iii) said length of identity is at least
about 25 amino acids.
20. The composition of matter of claim 18, wherein said: a) IL-1RD8
comprises a mature sequence of Table 1; b) IL-1RD10 comprises a
mature sequence of Table 3; or c) polypeptide: i) is from a warm
blooded animal selected from a primate, such as a human; ii)
comprises at least one polypeptide segment of SEQ ID NO: 4 or 35;
iii) exhibits a plurality of portions exhibiting said identity; iv)
is a natural allelic variant of a primate or rodent IL-1RD8 or
primate IL-1RD10; v) has a length at least about 30 amino acids;
vi) exhibits at least two non-overlapping epitopes which are
specific for a primate or rodent IL-1RD8 or primate IL-1RD10; vii)
exhibits sequence identity over a length of at least about 20 amino
acids to a primate IL-1RD8 or IL-1RD10; viii) has a molecular
weight of at least 100 kD with natural glycosylation; ix) is a
synthetic polypeptide; x) is attached to a solid substrate; xi) is
conjugated to another chemical moiety; xii) is a 5-fold or less
substitution from natural sequence; or xiii) is a deletion or
insertion variant from a natural sequence.
21. A composition comprising: a) a sterile IL-1RD8 polypeptide of
claim 18; b) said IL-1RD8 polypeptide of claim 18 and a carrier,
wherein said carrier is: i) an aqueous compound, including water,
saline, and/or buffer; and/or ii) formulated for oral, rectal,
nasal, topical, or parenteral administration; c) a sterile IL-1RD10
polypeptide of claim 18; or d) said IL-1RD10 polypeptide of claim
18 and a carrier, wherein said carrier is: i) an aqueous compound,
including water, saline, and/or buffer; and/or ii) formulated for
oral, rectal, nasal, topical, or parenteral administration.
22. A fusion protein of claim 18, comprising: a) mature protein
sequence of Table 1 or 3; b) a detection or purification tag,
including a FLAG, His6, or Ig sequence; or c) sequence of another
receptor protein.
23. A kit comprising a polypeptide of claim 18, and: a) a
compartment comprising said polypeptide; and/or b) instructions for
use or disposal of reagents in said kit.
24. A binding compound comprising an antigen binding site from an
antibody, which specifically binds to a natural: A) IL-1RD8
polypeptide of claim 18, wherein: a) said polypeptide is a primate
or rodent protein; b) said binding compound is an Fv, Fab, or Fab2
fragment; c) said binding compound is conjugated to another
chemical moiety; or d) said antibody: i) is raised against a
polypeptide sequence of a mature polypeptide of Table 1; ii) is
raised against a mature primate or rodent IL-1RD8; iii) is raised
to a purified human IL-1RD8; iv) is raised to a purified mouse
IL-1RD8; v) is immunoselected; vi) is a polyclonal antibody; vii)
binds to a denatured IL-1RD8; viii) exhibits a Kd to antigen of at
least 30 .mu.M; ix) is attached to a solid substrate, including a
bead or plastic membrane; x) is in a sterile composition; or xi) is
detectably labeled, including a radioactive or fluorescent label;
or B) IL-1RD10 polypeptide of claim 18, wherein: a) said
polypeptide is a primate polypeptide; b) said binding compound is
an Fv, Fab, or Fab2 fragment; c) said binding compound is
conjugated to another chemical moiety; d) said polypeptide is
associated with an IL-1 receptor alpha type subunit; or e) said
antibody: i) is raised against a peptide sequence of a mature
polypeptide of Table 3; ii) is raised against a mature primate
IL-1RD10; iii) is raised to a purified human IL-1RD10; iv) is
immunoselected; v) is a polyclonal antibody; vi) binds to a
denatured IL-1RD10; vii) exhibits a Kd to antigen of at least 30
.mu.M; viii) is attached to a solid substrate, including a bead or
plastic membrane; ix) is in a sterile composition; or x) is
detectably labeled, including a radioactive or fluorescent
label
25. A kit comprising said binding compound of claim 24, and: a) a
compartment comprising said binding compound; and/or b)
instructions for use or disposal of reagents in said kit.
26. A method of: A) making an antibody of claim 23, comprising
immunizing an immune system with an immunogenic amount of: a) a
primate IL-1RD8 polypeptide; or b) a primate IL-1RD10 polypeptide;
thereby causing said antibody to be produced; or B) producing an
antigen:antibody complex, comprising contacting: a) a primate
IL-1RD8 polypeptide with an antibody of claim 23A; or b) a primate
IL-1RD10 polypeptide with an antibody of claim 23B; thereby
allowing said complex to form.
27. A composition comprising: a) a sterile binding compound of
claim 23, or b) said binding compound of claim 23 and a carrier,
wherein said carrier is: i) an aqueous compound, including water,
saline, and/or buffer; and/or ii) formulated for oral, rectal,
nasal, topical, or parenteral administration.
28. An isolated or recombinant nucleic acid encoding a protein or
peptide or fusion protein of claim 18, wherein: a) said IL-1RD8 or
IL-1RD10 is from a mammal; or b) said nucleic acid: i) encodes an
antigenic polypeptide sequence of Table 1 or 3; ii) encodes a
plurality of antigenic polypeptide sequences of Table 1 or 3; iii)
exhibits identity to a natural cDNA encoding said segment; iv) is
an expression vector; v) further comprises an origin of
replication; vi) is from a natural source; vii) comprises a
detectable label; viii) comprises synthetic nucleotide sequence;
ix) is less than 6 kb, preferably less than 3 kb; x) is from a
mammal, including a primate, such as a human; xi) comprises a
natural full length coding sequence; xii) is a hybridization probe
for a gene encoding said IL-1RD8 or IL-1RD10; xiii) comprises a
plurality of nonoverlapping segments of at least 15 nucleotides
from Table 1 or 3; or xiv) is a PCR primer, PCR product, or
mutagenesis primer.
29. A cell transfected or transformed with a recombinant nucleic
acid of claim 28.
30. The cell of claim 29, wherein said cell is: a) a prokaryotic
cell; b) a eukaryotic cell; c) a bacterial cell; d) a yeast cell;
e) an insect cell; f) a mammalian cell; g) a mouse cell; h) a
primate cell; or i) a human cell.
31. A kit comprising said nucleic acid of claim 28, and: a) a
compartment comprising said nucleic acid; b) a compartment further
comprising a primate or rodent IL-1RD8 or primate IL-1RD10
polypeptide; and/or c) instructions for use or disposal of reagents
in said kit.
32. A method of: A) making a polypeptide, comprising expressing
said nucleic acid of claim 28, thereby producing said polypeptide;
or B) making a duplex nucleic acid, comprising contacting said
nucleic acid of claim 28 with a hybridizing nucleic acid, thereby
allowing said duplex to form.
33. A nucleic acid which: a) hybridizes under wash conditions of
40.degree. C. and less than 2M salt to SEQ ID NO: 3, 19, or 34; or
b) exhibits identity over a stretch of at least about 30
nucleotides to a primate IL-1RD8 or IL-1RD10.
34. The nucleic acid of claim 33, wherein: a) said wash conditions
are at 55.degree. C. and/or 500 mM salt; or b) said stretch is at
least 55 nucleotides.
35. The nucleic acid of claim 34, wherein: a) said wash conditions
are at 65.degree. C. and/or 150 mM salt; or b) said stretch is at
least 75 nucleotides.
36. A method of modulating physiology or development of a cell or
tissue culture cells comprising contacting said cell with an
agonist or antagonist of a primate IL-1RD8 or IL-1RD10.
37. The method of claim 36, wherein said cell is transformed with a
nucleic acid encoding either an IL-1RD8 or IL-1RD10, and another
IL-1R.
Description
[0001] This filing is a conversion Utility Patent Application which
claims priority to U.S. Ser. No. 60/065,776 filed Nov. 17, 1997;
U.S. Ser. No. 60/078,008 filed Mar. 12, 1998; U.S. Ser. No.
60/081,883 filed Apr. 15, 1998; U.S. Ser. No. 60/095,987 filed Aug.
10, 1998; U.S. Ser. No. 60/078,416 filed Mar. 18, 1998; and U.S.
Ser. No. 60/062,066 filed Oct. 15, 1997; each of which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
for affecting mammalian physiology, including, e.g., morphogenesis
or immune system function. In particular, it provides nucleic
acids, proteins, and antibodies, e.g., which regulate development
and/or the immune system along with related reagents and methods.
Diagnostic and therapeutic uses of these materials are also
disclosed.
BACKGROUND OF THE INVENTION
[0003] Recombinant DNA technology refers generally to techniques of
integrating genetic information from a donor source into vectors
for subsequent processing, such as through introduction into a
host, whereby the transferred genetic information is copied and/or
expressed in the new environment. Commonly, the genetic information
exists in the form of complementary DNA (cDNA) derived from
messenger RNA (mRNA) coding for a desired polypeptide product. The
carrier is frequently a plasmid having the capacity to incorporate
cDNA for later replication and/or expression in a host and, in some
cases, actually to control expression of the cDNA and thereby
direct synthesis of the encoded product in the host.
[0004] For some time, it has been known that the mammalian immune
response is based on a series of complex cellular interactions,
called the "immune network". Recent research has provided new
insights into the inner workings of this network. While it remains
clear that much of the immune response does, in fact, revolve
around the network-like interactions of lymphocytes, macrophages,
granulocytes, and other cells, immunologists now generally hold the
opinion that soluble proteins, known as lymphokines, cytokines, or
monokines, play critical roles in controlling these cellular
interactions. Thus, there is considerable interest in the
isolation, characterization, and mechanisms of action of cell
modulatory factors, an understanding of which will lead to
significant advancements in the diagnosis and therapy of numerous
medical abnormalities, e.g., immune system disorders.
[0005] Lymphokines apparently mediate cellular activities in a
variety of ways. They have been shown to support the proliferation,
growth, and/or differentiation of pluripotential hematopoietic stem
cells into vast numbers of progenitors comprising diverse cellular
lineages which make up a complex immune system. Proper and balanced
interactions between the cellular components are necessary for a
healthy immune response. The different cellular lineages often
respond in a different manner when lymphokines are administered in
conjunction with other agents.
[0006] Cell lineages especially important to the immune response
include two classes of lymphocytes: B-cells, which can produce and
secrete immunoglobulins (proteins with the capability of
recognizing and binding to foreign matter to effect its removal),
and T-cells of various subsets that secrete lymphokines and induce
or suppress the B-cells and various other cells (including other
T-cells) making up the immune network. These lymphocytes interact
with many other cell types.
[0007] Another important cell lineage is the mast cell (which has
not been positively identified in all mammalian species), which is
a granule-containing connective tissue cell located proximal to
capillaries throughout the body. These cells are found in
especially high concentrations in the lungs, skin, and
gastrointestinal and genitourinary tracts. Mast cells play a
central role in allergy-related disorders, particularly anaphylaxis
as follows: when selected antigens crosslink one class of
immunoglobulins bound to receptors on the mast cell surface, the
mast cell degranulates and releases mediators, e.g., histamine,
serotonin, heparin, and prostaglandins, which cause allergic
reactions, e.g., anaphylaxis.
[0008] Research to better understand and treat various immune
disorders has been hampered by the general inability to maintain
cells of the immune system in vitro. Immunologists have discovered
that culturing many of these cells can be accomplished through the
use of T-cell and other cell supernatants, which contain various
growth factors, including many of the lymphokines.
[0009] The interleukin-1 family of proteins includes the
IL-1.alpha., the IL-1.beta., the IL-1RA, and recently the
IL-1.gamma. (also designated Interferon-Gamma Inducing Factor,
IGIF). This related family of genes has been implicated in a broad
range of biological functions. See Dinarello (1994) FASEB J.
8:1314-1325; Dinarello (1991) Blood 77:1627-1652; and Okamura, et
al. (1995) Nature 378:88-91.
[0010] From the foregoing, it is evident that the discovery and
development of new soluble proteins and their receptors, including
ones similar to lymphokines, should contribute to new therapies. A
number of degenerative or abnormal conditions directly or
indirectly involve development, differentiation, or function, e.g.,
of the immune system and/or hematopoietic cells. In particular, the
discovery and understanding of novel receptors for lymphokine-like
molecules which enhance or potentiate the beneficial activities of
other lymphokines, would be highly advantageous. The present
invention provides new receptors for ligands exhibiting similarity
to interleukin-1 like compositions and related compounds, and
methods for their use.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to novel receptors related
to IL-1 receptors and their biological activities. These receptors,
e.g., primate or rodent, are designated IL-1 receptor like
molecular structures, IL-1 Receptor DNAX designation 8 (IL-1RD8),
IL-1 Receptor DNAX designation 9 (IL-1RD9) and IL-1 Receptor DNAX
designation 10 (IL-1RD10). The invention includes nucleic acids
coding for the polypeptides themselves and methods for their
production and use. The nucleic acids of the invention are
characterized, in part, by their homology to cloned complementary
DNA (cDNA) sequences enclosed herein.
[0012] In certain embodiments, the invention provides a composition
of matter selected from the group of: an isolated or recombinant
IL-1RD8 polypeptide comprising a segment of at least 12 contiguous
amino acids of SEQ ID NO: 2 or 4, a natural sequence IL-1RD8
polypeptide comprising SEQ ID NO: 2 or 4, a fusion protein
comprising IL-1RD8 sequence; an isolated or recombinant IL-1RD9
polypeptide comprising at least 12 contiguous amino acids of SEQ ID
NO: 6, 8, 10, 12, 14, or 16; a natural sequence IL-1RD9 comprising
SEQ ID NO: 6, 8, 10, 12, 14, or 16; a fusion protein comprising
IL-1RD9 sequence; an isolated or recombinant IL-1RD10 polypeptide
comprising at least 12 contiguous amino acids of SEQ ID NO: 18, 20,
or 35; a natural sequence IL-1RD10 comprising SEQ ID NO: 18, 20, or
35; and a fusion protein comprising IL-1RD10 sequence. In various
embodiments, the recombinant or isolated polypeptide comprises a
segment identical to a corresponding portion of an IL-1RD8, as
described, wherein: the number of contiguous amino acid residues
is: at least 17 amino acids; at least 21 amino acids; or at least
25 amino acids; or to a corresponding portion of an IL-1RD9, as
described, wherein the number of identical contiguous amino acid
residues is: at least 17 amino acids; at least 21 amino acids; or
at least 25 amino acids; or of an IL-1RD10, as described, wherein
the number of identical contiguous amino acid residues is: at least
17 amino acids; at least 21 amino acids;or at least 25 amino
acids.
[0013] In polypeptide embodiments, the invention provides a
composition of matter wherein the IL-1RD8 comprises a mature
sequence of Table 1; an IL-1RD9 that comprises a mature sequence of
Table 2; an IL-1RD10 that comprises a mature sequence of Table 3;
or the IL-1RD8, IL-1RD9, or IL-1RD10 polypeptide: is from a warm
blooded animal, e.g., a primate, such as a human; comprises at
least one polypeptide segment of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, or 35; exhibits a plurality of portions having segments
identical to specific sequence identifiers; is a natural allelic
variant of a primate IL-1RD8; a primate or rodent IL-1RD9; or a
primate IL-1RD10; has a length at least about 30 amino acids;
exhibits at least two non-overlapping epitopes that are specific
for: a primate IL-1RD8, a primate or rodent IL-1RD9, or primate
IL-1RD10; exhibits a sequence identity over a length of at least
about 20 amino acids to: a primate IL-1RD8, a primate or rodent
IL-1RD9, or a primate IL-1RD10; has a molecular weight of at least
100 kD with natural glycosylation; is a synthetic polypeptide; is
attached to a solid substrate; is conjugated to another chemical
moiety; is a 5-fold or less substitution from natural sequence; or
is a deletion or insertion variant from a natural sequence. Certain
preferred embodiments include compositions comprising: a sterile
IL-1RD8, IL-1RD9, or IL-1RD10 polypeptide; or the IL-1RD8, IL-1RD9,
or IL-1RD10 polypeptide and a carrier, wherein the carrier is: an
aqueous compound, including water, saline, and/or buffer; and/or
formulated for oral, rectal, nasal, topical, or parenteral
administration; a sterile IL-1RD8, IL-1RD9, or IL-1RD10
polypeptide; or the IL-1RD8, IL-1RD9, or IL-1RD10 polypeptide, as
described, and a carrier, wherein the carrier is: an aqueous
compound, including water, saline, and/or buffer; and/or formulated
for oral, rectal, nasal, topical, or parenteral administration.
[0014] Certain fusion proteins are provided, e.g., comprising:
mature polypeptide sequence of Table 1, 2, or 3; a detection or
purification tag, including a FLAG, His6, or Ig sequence; or
sequence of another receptor protein. Kit embodiments include a kit
comprising such a polypeptide, and: a compartment comprising the
polypeptide; and/or instructions for use or disposal of reagents in
the kit.
[0015] In binding compound embodiments, the invention provides a
binding compound comprising an antigen binding site from an
antibody, which specifically binds to a natural: IL-1RD8, IL-1RD9,
or IL-1RD10 polypeptide, wherein: the polypeptide is a primate or
rodent protein; the binding compound is an Fv, Fab, or Fab2
fragment; the binding compound is conjugated to another chemical
moiety; or the antibody: is raised to a polypeptide sequence of a
mature polypeptide comprising sequence of Table 1, 2, or 3; is
raised to a mature primate or rodent IL-1RD8; is raised to a
purified human IL-1RD8; is raised to a purified mouse IL-1RD9; is
immunoselected; is a polyclonal antibody; binds to a denatured
IL-1RD8, IL-1RD9, or IL-1RD10; exhibits a Kd to antigen of at least
30 .mu.M; is attached to a solid substrate, including a bead or
plastic membrane; is in a sterile composition; or is detectably
labeled, including a radioactive or fluorescent label; IL-1RD9
protein, wherein: the polypeptide is a primate or rodent protein;
the binding compound is an Fv, Fab, or Fab2 fragment; the binding
compound is conjugated to another chemical moiety; or the antibody:
is raised against a polypeptide sequence of a mature polypeptide
comprising sequence of Table 1, 2, or 3; is raised against a mature
primate IL-1RD9; is raised to a purified human IL-1RD9; is
immunoselected; is a polyclonal antibody; binds to a denatured
IL-1RD9; exhibits a Kd to antigen of at least 30 .mu.M; is attached
to a solid substrate, including a bead or plastic membrane; is in a
sterile composition; or is detectably labeled, including a
radioactive or fluorescent label; IL-1RD10 protein, wherein: the
polypeptide is a primate or rodent protein; the binding compound is
an Fv, Fab, or Fab2 fragment; the binding compound is conjugated to
another chemical moiety; or the antibody: is raised against a
polypeptide sequence of a mature polypeptide comprising sequence of
Table 1, 2, or 3; is raised against a mature primate IL-1RD10; is
raised to a purified human IL-1RD10; is immunoselected; is a
polyclonal antibody; binds to a denatured IL-1RD10; exhibits a Kd
to antigen of at least 30 .mu.M; is attached to a solid substrate,
including a bead or plastic membrane; is in a sterile composition;
or is detectably labeled, including a radioactive or fluorescent
label. Kits are provided, e.g., those comprising the binding
compound, and: a compartment comprising the binding compound;
and/or instructions for use or disposal of reagents in the kit.
Preferably, the kit is capable of making a qualitative or
quantitative analysis.
[0016] Other embodiments include a composition comprising: a
sterile binding compound, or the binding compound and a carrier,
wherein the carrier is: an aqueous compound, including water,
saline, and/or buffer; and/or formulated for oral, rectal, nasal,
topical, or parenteral administration.
[0017] Nucleic acid embodiments include an isolated or recombinant
nucleic acid encoding a polypeptide or fusion protein, wherein: the
IL-1RD8, IL-1RD9, or 1L-1RD10 is from a mammal; said nucleic acid:
encodes an antigenic polypeptide sequence of Table 1, 2, or 3;
encodes a plurality of antigenic polypeptide sequences of Table 1,
2, or 3; exhibits at least about 30 nucleotides to a natural cDNA
encoding the segment; is an expression vector; further comprises an
origin of replication; is from a natural source; comprises a
detectable label; comprises synthetic nucleotide sequence; is less
than 6 kb, preferably less than 3 kb; is from a mammal, including a
primate; comprises a natural full length coding sequence; is a
hybridization probe for a gene encoding said IL-1RD8, IL-1RD9, or
IL-1RD10; comprises a plurality of nonoverlapping segments of at
least 15, 18, 21, or 25 nucleotides from Table 1, 2, or 3; or is a
PCR primer, PCR product, or mutagenesis primer. The invention
further provides a cell comprising such a recombinant nucleic acid,
e.g., where the cell is: a prokaryotic cell; a eukaryotic cell; a
bacterial cell; a yeast cell; an insect cell; a mammalian cell; a
mouse cell; a primate cell; or a human cell. Certain kit
embodiments include a comprising the nucleic acid, and: a
compartment comprising the nucleic acid; a compartment further
comprising: a primate IL-1RD8, a primate or rodent IL-1RD9, or a
primate IL-1RD10 polypeptide; and/or instructions for use or
disposal of reagents in the kit. Preferably, the kit is capable of
making a qualitative or quantitative analysis.
[0018] In other nucleic acid embodiments, the nucleic acid is one
which: hybridizes under wash conditions of 40.degree. C. and less
than 2M salt to either SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, or 34; or exhibits identity over a stretch of at least about 30
nucleotides to a primate IL-1RD8, a primate or rodent IL-1RD9, or a
primate IL-1RD10. In various preferred embodiments: the wash
conditions are: at 45.degree. C. and/or 500 mM salt; at 55.degree.
C. and/or 150 mM salt; or the stretch is at least 55 nucleotides;
or at least 75 nucleotides.
[0019] Methods of modulating physiology or development of a cell or
tissue culture cells are provided, e.g., comprising contacting the
cell with an agonist or antagonist of a primate IL-1RD8, a primate
or rodent IL-1RD9, or a primate IL-1RD10. Preferably, the cell is
transformed with a nucleic acid encoding either IL-1RD8, IL-1RD9,
or IL-1RD10, and another IL-1R.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0020] OUTLINE
[0021] I. General
[0022] II. Activities
[0023] III. Nucleic acids
[0024] A. encoding fragments, sequence, probes
[0025] B. mutations, chimeras, fusions
[0026] C. making nucleic acids
[0027] D. vectors, cells comprising
[0028] IV. Proteins, Peptides
[0029] A. fragments, sequence, immunogens, antigens
[0030] B. muteins
[0031] C. agonists/antagonists, functional equivalents
[0032] D. making proteins
[0033] V. Making nucleic acids, proteins
[0034] A. synthetic
[0035] B. recombinant
[0036] C. natural sources
[0037] VI. Antibodies
[0038] A. polyclonals
[0039] B. monoclonal
[0040] C. fragments; Kd
[0041] D. anti-idiotypic antibodies
[0042] E. hybridoma cell lines
[0043] VII. Kits and Methods to quantify IL-1Rs
[0044] A. ELISA
[0045] B. assay mRNA encoding
[0046] C. qualitative/quantitative
[0047] D. kits
[0048] VIII. Therapeutic compositions, methods
[0049] A. combination compositions
[0050] B. unit dose
[0051] C. administration
[0052] IX. Ligands
[0053] I. General
[0054] The present invention provides the amino acid sequence and
DNA sequence of mammalian, herein, e.g., primate and rodent IL-1
receptor-like molecules, these molecules IL-1 Receptor DNAX
designation 8 (IL-1RD8), IL-1 Receptor DNAX designation 9 (IL-1RD9)
and IL-1 Receptor DNAX designation 10 (IL-1RD10) having particular
defined properties, both structural and/or biological. These
embodiments increase the number of members of the human IL-1
receptor-like family from 7 to at least 10. These receptors have
been numbered internally as DNAX designations D1, D2, D3, D4, D5,
D6, and now D8, D9, and D10, and are referred to as IL-1RD1 through
D10. Various cDNAs encoding these molecules were obtained from
primate, e.g., human, or rodent, e.g., mouse, cDNA sequence
libraries. Other primate, rodent, or other mammalian counterparts
would also be desired.
[0055] Some of the standard methods applicable are described or
referenced, e.g., in Maniatis, et al. (1982) Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor Press; Sambrook, et al. (1989) Molecular Cloning: A
Laboratory Manual, (2d ed.), vols. 1-3, CSH Press, NY; Ausubel, et
al. Biology, Greene Publishing Associates, Brooklyn, N.Y.; or
Ausubel, et al. (1987 and periodic supplements) Current Protocols
in Molecular Biology, Greene/Wiley, New York; each of which is
incorporated herein by reference.
[0056] A partial nucleotide (SEQ ID NO: 1) and corresponding amino
acid sequence (SEQ ID NO: 2) of a human IL-1RD8 coding segment is
shown in Table 1. Supplemental human IL-1RD8 sequence is provided
in SEQ ID NO: 3 and 4.
[0057] Similarly for primate IL-1RD9, partial nucleotide (SEQ ID
NO: 5) and corresponding amino acid sequence (SEQ ID NO: 6) of a
primate IL-1RD9 coding segment are provided. Supplemental primate
IL-1RD9 is provided in SEQ ID NO: 7, 8, 9, and 10. Rodent
embodiments of IL-1RD9 are provided in SEQ ID NO: 11, 12, with
supplemental IL-1RD9 rodent sequence in SEQ ID NO: 13, 14, 15, and
16.
[0058] For an embodiment of primate, e.g., human, IL-1RD10, a
partial nucleotide (SEQ ID NO: 17) and corresponding partial amino
acid sequence (SEQ ID NO: 18) are provided in Table 3, with
supplemental primate IL-1RD10 sequence provided in SEQ ID NO: 19,
20, 34, and 35.
[0059] Some sequences provided lack some portions of these
receptors, as suggested by alignment of sequences (see Table 4).
Note the alignment of IL-1RD10 with IL-1RD8 and D3s, which are
alpha type receptor subunits, in Table 4. Table 4 also exhibits
alignment of primate and rodent IL-1RD9.
[0060] It is to be understood that this invention is not limited to
the particular methods, compositions and receptors specifically
embodied herein, as such methods, compositions and receptors may,
of course, vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which is only limited by the appended claims.
[0061] As used herein, including the appended claims, singular
forms of words such as "a," "an," and "the" include their
corresponding plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "an organism" includes
one or more different organisms, reference to "a cell" includes one
or more of such cells, and reference to "a method" includes
reference to equivalent steps and methods known to a person of
ordinary skill in the art, and so forth.
[0062] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by a
person of ordinary skill in the art to which this invention
belongs. Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references discussed above are provided solely for their disclosure
prior to the filing date of the present application. Nothing herein
is to be construed as an admission that the invention is not
entitled to antedate any such disclosure by virtue of its prior
invention. All publications, patent applications, patents, and
other references mentioned herein are incorporated by reference in
their entirety including all figures, graphs, and drawings.
1TABLE 1 Nucleotide and amino acid sequences (see SEQ ID NO: 1 and
2) of a primate, e.g., human, IL-1 receptor like embodiment DNAX
designated 8 (IL-1RD8). TTA CTG CTC ACA CTA TTA GTG TCA ACA ATG CTC
ACT GTA TCT TAT ACC 48 Leu Leu Leu Thr Leu Leu Val Ser Thr Met Leu
Thr Val Ser Tyr Thr 1 5 10 15 TCT TCT GAT TTT CTT TCA GTG GAT GGC
TGC ATT GAC TGG TCA GTG GAT 96 Ser Ser Asp Phe Leu Ser Val Asp Gly
Cys Ile Asp Trp Ser Val Asp 20 25 30 CTC AAG ACA TAC ATG GCT TTG
GCA GGT GAA CCA GTC CGA GTG AAA TGT 144 Leu Lys Thr Tyr Met Ala Leu
Ala Gly Glu Pro Val Arg Val Lys Cys 35 40 45 GCC CTT TTC TAC AGT
TAT ATT CGT ACC AAC TAT AGC ACG GCC CAG AGC 192 Ala Leu Phe Tyr Ser
Tyr Ile Arg Thr Asn Tyr Ser Thr Ala Gln Ser 50 55 60 ACT GGG CTC
AGG CTT ATG TGG TAC AAA AAC AAA GGT GAT TTG GAA GAG 240 Thr Gly Leu
Arg Leu Met Trp Tyr Lys Asn Lys Gly Asp Leu Glu Glu 65 70 75 80 CCC
ATC ATC TTT TCA GAG GTC AGG ATG AGC AAA GAG GAA GAT TCA ATA 288 Pro
Ile Ile Phe Ser Glu Val Arg Met Ser Lys Glu Glu Asp Ser Ile 85 90
95 TGG TTT CAC TCA GCT GAG GCA CAA GAC AGT GGA TTC TAC ACT TGT GTT
336 Trp Phe His Ser Ala Glu Ala Gln Asp Ser Gly Phe Tyr Thr Cys Val
100 105 110 TTA AGG AAC TCA ACA TAT TGC ATG AAG GTG TCA ATG TCC TTG
ACT GTT 384 Leu Arg Asn Ser Thr Tyr Cys Met Lys Val Ser Met Ser Leu
Thr Val 115 120 125 GCA GAG AAT GAA TCA GGC CTG TGC TAC AAC AGC AGG
ATC CGC TAT TTA 432 Ala Glu Asn Glu Ser Gly Leu Cys Tyr Asn Ser Arg
Ile Arg Tyr Leu 130 135 140 GAA AAA TCT GAA GTC ACT AAA AGA AAG GAG
ATC TCC TGT CCA GAC ATG 480 Glu Lys Ser Glu Val Thr Lys Arg Lys Glu
Ile Ser Cys Pro Asp Met 145 150 155 160 GAT GAC TTT AAA AAG TCC GAT
CAG GAG CCT GAT GTT GTG TGG TAT AAG 528 Asp Asp Phe Lys Lys Ser Asp
Gln Glu Pro Asp Val Val Trp Tyr Lys 165 170 175 GAA TGC AAG CCA AAA
ATG TGG AGA AGC ATA ATA ATA CAG AAA GGA AAT 576 Glu Cys Lys Pro Lys
Met Trp Arg Ser Ile Ile Ile Gln Lys Gly Asn 180 185 190 GCT CTT CTG
ATC CAA GAA GTT CAA GAA GAA GAT GGA GGA AAT TAC ACA 624 Ala Leu Leu
Ile Gln Glu Val Gln Glu Glu Asp Gly Gly Asn Tyr Thr 195 200 205 TGT
GAA CTT AAA TAT GAA GGA AAA CTT GTA AGA CGA ACA ACT GAA TTG 672 Cys
Glu Leu Lys Tyr Glu Gly Lys Leu Val Arg Arg Thr Thr Glu Leu 210 215
220 AAA GTT ACA GCT TTA CTC ACA GAC AAG CCT CCC AAG CCA TTG TTC CCC
720 Lys Val Thr Ala Leu Leu Thr Asp Lys Pro Pro Lys Pro Leu Phe Pro
225 230 235 240 ATG GAG AAT CAG CGA AGT GTT ATA GAT GTC GAG CTG GGT
AAG CCT CTG 768 Met Glu Asn Gln Pro Ser Val Ile Asp Val Gln Leu Gly
Lys Pro Leu 245 250 255 AAC ATC CCC TGC AAA GCA TTC TTC GGA TTC AGT
GGA GAG TCT GGG CCA 816 Asn Ile Pro Cys Lys Ala Phe Phe Gly Phe Ser
Gly Glu Ser Gly Pro 260 265 270 ATG ATC TAC TGG ATG AAA GGA GAA AAG
TTT ATT GAA GAA CTG GCA GGT 864 Met Ile Tyr Trp Met Lys Gly Glu Lys
Phe Ile Glu Glu Leu Ala Gly 275 280 285 CAC ATT AGA GAA GGT GAA ATA
AGG CTT CTC AAA GAG CAT CTT GGA GAA 912 His Ile Arg Glu Gly Glu Ile
Arg Leu Leu Lys Glu His Leu Gly Glu 290 295 300 AAA GAA GTT GAA TTG
GCA CTC ATC TTT GAC TCA GTT GTG GAA GCT GAC 960 Lys Glu Val Glu Leu
Ala Leu Ile Phe Asp Ser Val Val Glu Ala Asp 305 310 315 320 CTG GCG
AAT TAT ACC TGC CAT GTT GAA AAC CGA AAT GGA CGG AAA CAT 1008 Leu
Ala Asn Tyr Thr Cys His Val Glu Asn Arg Asn Gly Arg Lys His 325 330
335 GCC AGT GTT TTG CTG CGT AAA AAG GAT TTA ATC TAT AAA ATT GAG CTT
1056 Ala Ser Val Leu Leu Arg Lys Lys Asp Leu Ile Tyr Lys Ile Glu
Leu 340 345 350 GCA GGG GGC CTG GGA GCA ATC TTC CTC CTC CTT GTA GTG
CTG GTG GTC 1104 Ala Gly Gly Leu Gly Ala Ile Phe Leu Leu Leu Val
Leu Leu Val Val 355 360 365 ATT TAC AAA TGC TAC AAC ATT GAA TTG ATG
CTC TTC TAC AGG CAG CAC 1152 Ile Tyr Lys Cys Tyr Asn Ile Glu Leu
Met Leu Phe Tyr Arg Gln His 370 375 380 TTT GGA GCT GAT GAA ACT AAT
GAT GAC AAC AAG GAA TAT GAT GCC TAT 1200 Phe Gly Ala Asp Glu Thr
Asn Asp Asp Asn Lys Glu Tyr Asp Ala Tyr 385 390 395 400 CTC TCT TAC
ACA AAA GTG GAC CAA GAT ACT TTA GAC TGT GAC AAT CCT 1248 Leu Ser
Tyr Thr Lys Val Asp Gln Asp Thr Leu Asp Cys Asp Asn Pro 405 410 415
GAA GAA GAG CAG TTT GCT CTT GAA GTA CTG CCA GAT GTC CTG GAA AAA
1296 Glu Glu Glu Gln Phe Ala Leu Glu Val Leu Pro Asp Val Leu Glu
Lys 420 425 430 CAC TAT GGA TAT AAA CTC TTC ATC CCA GAA AGA GAC CTG
ATT CCA AGT 1344 His Tyr Gly Tyr Lys Leu Phe Ile Pro Glu Arg Asp
Leu Ile Pro Ser 435 440 445 GGA AGT GCA TAC ATG GAA GAT CTC ACA AGA
TAT GTT GAA CAA AGC AGA 1392 Gly Ser Ala Tyr Met Glu Asp Leu Thr
Arg Tyr Val Glu Gln Ser Arg 450 455 460 AGA CTT ATT ATC GTG CTA ACT
CCA GAC TAT ATT CTC AGA CGG GGA TGG 1440 Arg Leu Ile Ile Val Leu
Thr Pro Asp Tyr Ile Leu Arg Arg Gly Trp 465 470 475 480 AGT ATT TTC
GAA CTG GAA AGC AGA CTC CAT AAC ATG CTA GTC AGT GGA 1488 Ser Ile
Phe Glu Leu Glu Ser Arg Leu His Asn Met Leu Val Ser Gly 485 490 495
GAA ATC AAA GTG ATT TTG ATT GAG TGT ACA GAA TTA AAA GGG AAA GTG
1536 Glu Ile Lys Val Ile Leu Ile Glu Cys Thr Glu Leu Lys Gly Lys
Val 500 505 510 AAT TGC CAG GAA GTG GAA TCA CTA AAG CGT AGC ATC AAA
CTT CTG TCC 1584 Asn Cys Gln Glu Val Glu Ser Leu Lys Arg Ser Ile
Lys Leu Leu Ser 515 520 525 CTG ATC AAG TGG AAG GGA TCC AAA AGC AGC
AAA TTA AAT TCT AAG TTT 1632 Leu Ile Lys Trp Lys Gly Ser Lys Ser
Ser Lys Leu Asn Ser Lys Phe 530 535 540 TGG AAG CAC TTA GTA TAT GAA
ATG CCC ATC AAG AAA AAA GAA ATG CTA 1680 Trp Lys His Leu Val Tyr
Glu Met Pro Ile Lys Lys Lys Glu Met Leu 545 550 555 560 CCT CGG TGC
CAT GTT CTG GAC TCC GCA GAA CAA GGA CTT TTT GGA GAA 1728 Pro Arg
Cys His Val Leu Asp Ser Ala Glu Gln Gly Leu Phe Gly Glu 565 570 575
CTC CAG CCT 1737 Leu Gln Pro Updated and corrected nucleotide and
amino acid sequences of primate, e.g., human, IL-1RD8 (SEQ ID NO: 3
and 4). ATG AAG CCA CCA TTT CTT TTG GCC CTT GTG GTC TGT TCT GTA GTC
AGC 48 Met Lys Pro Pro Phe Leu Leu Ala Leu Val Val Cys Ser Val Val
Ser 1 5 10 15 ACA AAT CTG AAG ATG GTG TCA AAG AGA AAT TCT GTG GAT
GGC TGC ATT 96 Thr Asn Leu Lys Met Val Ser Lys Arg Asn Ser Val Asp
Gly Cys Ile 20 25 30 GAC TGG TCA GTG GAT CTC AAG ACA TAC ATG GCT
TTG GCA GGT GAA CCA 144 Asp Trp Ser Val Asp Leu Lys Thr Tyr Met Ala
Leu Ala Gly Glu Pro 35 40 45 GTC CGA GTG AAA TGT GCC CTT TTC TAC
AGT TAT ATT CGT ACC AAC TAT 192 Val Arg Val Lys Cys Ala Leu Phe Tyr
Ser Tyr Ile Arg Thr Asn Tyr 50 55 60 AGC ACG GCC CAG AGC ACT GGG
CTC AGG CTT ATG TGG TAC AAA AAC AAA 240 Ser Thr Ala Gln Ser Thr Gly
Leu Arg Leu Met Trp Tyr Lys Asn Lys 65 70 75 80 GGT GAT TTG GAA GAG
CCC ATC ATC TTT TCA GAG GTC AGG ATG AGC AAA 288 Gly Asp Leu Glu Glu
Pro Ile Ile Phe Ser Glu Val Arg Met Ser Lys 85 90 95 GAG GAA GAT
TCA ATA TGG TTT CAC TCA GCT GAG GCA CAA GAC AGT GGA 336 Glu Glu Asp
Ser Ile Trp Phe His Ser Ala Glu Ala Gln Asp Ser Gly 100 105 110 TTC
TAC ACT TGT GTT TTA AGG AAC TCA ACA TAT TGC ATG AAG GTG TCA 384 Phe
Tyr Thr Cys Val Leu Arg Asn Ser Thr Tyr Cys Met Lys Val Ser 115 120
125 ATG TCC TTG ACT GTT GCA GAG AAT GAA TCA GGC CTG TGC TAC AAC AGC
432 Met Ser Leu Thr Val Ala Glu Asn Glu Ser Gly Leu Cys Tyr Asn Ser
130 135 140 AGG ATC CGC TAT TTA GAA AAA TCT GAA GTC ACT AAA AGA AAG
GAG ATC 480 Arg Ile Arg Tyr Leu Glu Lys Ser Glu Val Thr Lys Arg Lys
Glu Ile 145 150 155 160 TCC TGT CCA GAC ATG GAT GAC TTT AAA AAG TCC
GAT CAG GAG CCT GAT 528 Ser Cys Pro Asp Met Asp Asp Phe Lys Lys Ser
Asp Gln Glu Pro Asp 165 170 175 GTT GTG TGG TAT AAG GAA TGC AAG CCA
AAA ATG TGG AGA AGC ATA ATA 576 Val Val Trp Tyr Lys Glu Cys Lys Pro
Lys Met Trp Arg Ser Ile Ile 180 185 190 ATA CAG AAA GGA AAT GCT CTT
CTG ATC CAA GAA GTT CAA GAA GAA GAT 624 Ile Gln Lys Gly Asn Ala Leu
Leu Ile Gln Glu Val Gln Glu Glu Asp 195 200 205 GGA GGA AAT TAC ACA
TGT GAA CTT AAA TAT GAA GGA AAA CTT GTA AGA 672 Gly Gly Asn Tyr Thr
Cys Glu Leu Lys Tyr Glu Gly Lys Leu Val Arg 210 215 220 CGA ACA ACT
GAA TTG AAA GTT ACA GCT TTA CTC ACA GAC AAG CCT CCC 720 Arg Thr Thr
Glu Leu Lys Val Thr Ala Leu Leu Thr Asp Lys Pro Pro 225 230 235 240
AAG CCA TTG TTC CCC ATG GAG AAT CAG CCA AGT GTT ATA GAT GTC CAG 768
Lys Pro Leu Phe Pro Met Glu Asn Gln Pro Ser Val Ile Asp Val Gln 245
250 255 CTG GGT AAG CCT CTG AAC ATC CCC TGC AAA GCA TTC TTC GGA TTC
AGT 816 Leu Gly Lys Pro Leu Asn Ile Pro Cys Lys Ala Phe Phe Gly Phe
Ser 260 265 270 GGA GAG TCT GGG CCA ATG ATC TAC TGG ATG AAA GGA GAA
AAG TTT ATT 864 Gly Glu Ser Gly Pro Met Ile Tyr Trp Met Lys Gly Glu
Lys Phe Ile 275 280 285 GAA GAA CTG GCA GGT CAC ATT AGA GAA GGT GAA
ATA AGG CTT CTC AAA 912 Glu Glu Leu Ala Gly His Ile Arg Glu Gly Glu
Ile Arg Leu Leu Lys 290 295 300 GAG CAT CTT GGA GAA AAA GAA GTT GAA
TTG GCA CTC ATC TTT GAC TCA 960 Glu His Leu Gly Glu Lys Glu Val Glu
Leu Ala Leu Ile Phe Asp Ser 305 310 315 320 GTT GTG GAA GCT GAC CTG
GCG AAT TAT ACC TGC CAT GTT GAA AAC CGA 1008 Val Val Glu Ala Asp
Leu Ala Asn Tyr Thr Cys His Val Glu Asn Arg 325 330 335 AAT GGA CGG
AAA CAT GCC AGT GTT TTG CTG CGT AAA AAG GAT TTA ATC 1056 Asn Gly
Arg Lys His Ala Ser Val Leu Leu Arg Lys Lys Asp Leu Ile 340 345 350
TAT AAA ATT GAG CTT GCA GGG GGC CTG GGA GCA ATC TTC CTC CTC CTT
1104 Tyr Lys Ile Glu Leu Ala Gly Gly Leu Gly Ala Ile Phe Leu Leu
Leu 355 360 365 GTA CTG CTG GTG GTC ATT TAC AAA TGC TAC AAC ATT GAA
TTG ATG CTC 1152 Val Leu Leu Val Val Ile Tyr Lys Cys Tyr Asn Ile
Glu Leu Met Leu 370 375 380 TTC TAC AGG CAG CAC TTT GGA GCT GAT GAA
ACT AAT GAT GAC AAC AAG 1200 Phe Tyr Arg Gln His Phe Gly Ala Asp
Glu Thr Asn Asp Asp Asn Lys 385 390 395 400 GAA TAT GAT GCC TAT CTC
TCT TAC ACA AAA GTG GAC CAA GAT ACT TTA 1248 Glu Tyr Asp Ala Tyr
Leu Ser Tyr Thr Lys Val Asp Gln Asp Thr Leu 405 410 415 GAC TGT GAC
AAT CCT GAA GAA GAG CAG TTT GCT CTT GAA GTA CTG CCA 1296 Asp Cys
Asp Asn Pro Glu Glu Glu Gln Phe Ala Leu Glu Val Leu Pro 420 425 430
GAT GTC CTG GAA AAA CAC TAT GGA TAT AAA CTC TTC ATC CCA GAA AGA
1344 Asp Val Leu Glu Lys His Tyr Gly Tyr Lys Leu Phe Ile Pro Glu
Arg 435 440 445 GAC CTG ATT CCA AGT GGA ACA TAC ATG GAA GAT CTC ACA
AGA TAT GTT 1392 Asp Leu Ile Pro Ser Gly Thr Tyr Met Glu Asp Leu
Thr Arg Tyr Val 450 455 460 GAA CAA AGC AGA AGA CTT ATT ATC GTG CTA
ACT CCA GAC TAT ATT CTC 1440 Glu Gln Ser Arg Arg Leu Ile Ile Val
Leu Thr Pro Asp Tyr Ile Leu 465 470 475 480 AGA CGG GGA TGG AGT ATT
TTC GAA CTG GAA AGC AGA CTC CAT AAC ATG 1488 Arg Arg Gly Trp Ser
Ile Phe Glu Leu Glu Ser Arg Leu His Asn Met 485 490 495 CTA GTC AGT
GGA GAA ATC AAA GTG ATT TTG ATT GAG TGT ACA GAA TTA 1536 Leu Val
Ser Gly Glu Ile Lys Val Ile Leu Ile Glu Cys Thr Glu Leu 500 505 510
AAA GGG AAA GTG AAT TGC CAG GAA GTG GAA TCA CTA AAG CGT AGC ATC
1584 Lys Gly Lys Val Asn Cys Gln Glu Val Glu Ser Leu Lys Arg Ser
Ile 515 520 525 AAA CTT CTG TCC CTG ATC AAG TGG AAG GGA TCC AAA AGC
AGC AAA TTA 1632 Lys Leu Leu Ser Leu Ile Lys Trp Lys Gly Ser Lys
Ser Ser Lys Leu 530 535 540 AAT TCT AAG TTT TGG AAG CAC TTA GTA TAT
GAA ATG CCC ATC AAG AAA 1680 Asn Ser Lys Phe Trp Lys His Leu Val
Tyr Glu Met Pro Ile Lys Lys 545 550 555 560 AAA GAA ATG CTA CCT CGG
TGC CAT GTT CTG GAC TCC GCA GAA CAA GGA 1728 Lys Glu Met Leu Pro
Arg Cys His Val Leu Asp Ser Ala Glu Gln Gly 565 570 575 CTT TTT GGA
GAA CTC CAG CCT ATA CCC TCT ATT GCC ATG ACC AGT ACT 1776 Leu Phe
Gly Glu Leu Gln Pro Ile Pro Ser Ile Ala Met Thr Ser Thr 580 585 590
TCA GCC ACT CTG GTG TCA TCT CAG GCT GAT CTC CCT GAA TTC CAC CCT
1824 Ser Ala Thr Leu Val Ser Ser Gln Ala Asp Leu Pro Glu Phe His
Pro 595 600 605 TCA GAT TCA ATG CAA ATC AGG CAC TGT TGC AGA GGT TAT
AAA CAT GAG 1872 Ser Asp Ser Met Gln Ile Arg His Cys Cys Arg Gly
Tyr Lys His Glu 610 615 620 ATA CCA GCC ACG ACC TTG CCA GTA CCT TCC
TTA GGC AAC CAC CAT ACT 1920 Ile Pro Ala Thr Thr Leu Pro Val Pro
Ser Leu Gly Asn His His Thr 625 630 635 640 TAT TGT AAC CTG CCT CTG
ACG CTA CTC AAC GGA CAG CTA CCC CTT AAT 1968 Tyr Cys Asn Leu Pro
Leu Thr Leu Leu Asn Gly Gln Leu Pro Leu Asn 645 650 655 AAC ACC CTG
AAA GAT ACC CAG GAA TTT CAC AGG AAC AGT TCT TTG CTG 2016 Asn Thr
Leu Lys Asp Thr Gln Glu Phe His Arg Asn Ser Ser Leu Leu 660 665 670
CCT TTA TCC TCC AAA GAG CTT AGC TTT ACC AGT GAT ATT TGG 2058 Pro
Leu Ser Ser Lys Glu Leu Ser Phe Thr Ser Asp Ile Trp 675 680 685 TAG
2061
[0063]
2TABLE 2 Nucleotide and amino acid sequences (see SEQ ID NO: 5 and
6) of primate, e.g., human, IL-1 receptor like embodiment DNAX
designation 9 (IL-1RD9). Nucleotides 9, 459, 462, 469, and 474 are
designated C, but may be A, C, G, or T. Nucleotide 246 is
designated C, but may be C or G. Nucleotides 321, 336, 360, and 423
are designated C, but may be C or T. Nucleotide 426 is designated
C, but may be A or C. AAA TAT GGC TAT AGC CTG TTT TTC CTT GAA AGA
AAT GTG GCT CCA GGA 48 Lys Tyr Gly Tyr Ser Leu Phe Phe Leu Glu Arg
Asn Val Ala Pro Gly 1 5 10 15 GGA GTG TAT GCA GAA GAC ATT GTA AGC
ATT ATT AAG AGA AGC AGA AGA 96 Gly Val Tyr Ala Glu Asp Ile Val Ser
Ile Ile Lys Arg Ser Arg Arg 20 25 30 GGA ATA TTT ATC TTA ACC CCC
AAC TAT GTC AAT GGA CCC AGT ATC TTT 144 Gly Ile Phe Ile Leu Thr Pro
Asn Tyr Val Asn Gly Pro Ser Ile Phe 35 40 45 GAA CTA GAA GCA GCA
GTG AAT CTT GCC TTG GAT GAT CAA ACA CTG AAA 192 Glu Leu Gln Ala Ala
Val Asn Leu Ala Leu Asp Asp Gln Thr Leu Lys 50 55 60 CTC ATT TTA
ATT AAG TTC TGT TAC TTC CAA GAG CCA GAG TCT CTA CCT 240 Leu Ile Leu
Ile Lys Phe Cys Tyr Phe Gln Glu Pro Glu Ser Leu Pro 65 70 75 80 CAT
CTC GTG AAA AAA GCT CTC AGG GTT TTG CCC ACA GTT ACT TGG AGA 288 His
Leu Val Lys Lys Ala Leu Arg Val Leu Pro Thr Val Thr Trp Arg 85 90
95 GGC TTA AAA TCA GTT CCT CCC AAT TCT AGG TTC TGG GCC AAA ATG CGC
336 Gly Leu Lys Ser Val Pro Pro Asn Ser Arg Phe Trp Ala Lys Met Arg
100 105 110 TAC CAC ATG CCT GTG AAA AAT CTC TCA GGG ATT CAC GTG GGA
ACC AGC 384 Tyr His Met Pro Val Lys Asn Leu Ser Gly Ile His Val Gly
Thr Ser 115 120 125 TCC AGA ATT ACC TCT AGG GAT TTT TTC AGT GGA AAG
GAC TCC GTA GAA 432 Ser Arg Ile Thr Ser Arg Asp Phe Phe Ser Gly Lys
Asp Ser Val Glu 130 135 140 CAG AAA CCA TGG GGA GGA GCT CCC AGC CTC
AAG GGA CGG TGC AAT GAG 480 Gln Lys Pro Trp Gly Gly Ala Pro Ser Leu
Lys Gly Arg Cys Asn Glu 145 150 155 160 CC 482
KYGYSLFFLERNVAPGGVYAEDIVSIIKRSRRGIFILTPNYVNGPSIFELQAAVNLALDDQTL-
KLILIK FCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNL-
SGIHVGTSSRITSRDFFS GKDSVEQKPWGGAP?LKE Supplemental sequence of
primate, e.g., human, IL-1RD9 (SEQ ID NO: 7 and 8). TTT CCT AGG AGC
CCC TAT GAT GTA GCC TGT TGT GTC AAG ATG ATT TTA 48 Phe Pro Arg Ser
Pro Tyr Asp Val Ala Cys Cys Val Lys Met Ile Leu 1 5 10 15 GAA GTT
AAG CCC CAG ACA AAT GCA TCC TGT GAG TAT TCC GCA TCA CAT 96 Glu Val
Lys Pro Gln Thr Asn Ala Ser Cys Glu Tyr Ser Ala Ser His 20 25 30
AAG CAA GAC CTA CTT CTT GGG AGC ACT GGC TCT ATT TCT TGC CCC AGT 144
Lys Gln Asp Leu Leu Leu Gly Ser Thr Gly Ser Ile Ser Cys Pro Ser 35
40 45 CTC AGC TGC CAA AGT GAT GCA CAA AGT CCA GCG GTA ACC TGG TAC
AAG 192 Leu Ser Cys Gln Ser Asp Ala Gln Ser Pro Ala Val Thr Trp Tyr
Lys 50 55 60 AAT GGA AAA CTC CTC TCT GTG GAA AGG AGC AAC CGA ATC
GTA GTG GAT 240 Asn Gly Lys Leu Leu Ser Val Glu Arg Ser Asn Arg Ile
Val Val Asp 65 70 75 80 GAA GTT TAT GAC TAT CAC CAG GGC ACA TAT GTA
TGT GAT TAC ACT CAG 288 Glu Val Tyr Asp Tyr His Gln Gly Thr Tyr Val
Cys Asp Tyr Thr Gln 85 90 95 TCG GAT ACT GTG AGT TCG TGG ACA GTC
AGA GCT GTT GTT CAA GTG AGA 336 Ser Asp Thr Val Ser Ser Trp Thr Val
Arg Ala Val Val Gln Val Arg 100 105 110 ACC ATT GTG GGA GAC ACT AAA
CTC AAA CCA GAT ATT CTG GAT CCT GTC 384 Thr Ile Val Gly Asp Thr Lys
Leu Lys Pro Asp Ile Leu Asp Pro Val 115 120 125 GAG GAC ACA CTG GAA
GTA GAA CTT GGA AAG CCT TTA ACT ATT AGC TGC 432 Glu Asp Thr Leu Glu
Val Glu Leu Gly Lys Pro Leu Thr Ile Ser Cys 130 135 140 AAA GCA CGA
TTT GGC TTT GAA AGG GTC TTT AAC CCT GTC ATA AAA TGG 480 Lys Ala Arg
Phe Gly Phe Glu Arg Val Phe Asn Pro Val Ile Lys Trp 145 150 155 160
TAC ATC AAA GAT TCT GAC CTA GAG TGG GAA GTC TCA GTA CCT GAG GCG 528
Tyr Ile Lys Asp Ser Asp Leu Glu Trp Glu Val Ser Val Pro Glu Ala 165
170 175 AAA AGT ATT AAA TCC ACT TTA AAG GAT GAA ATC ATT GAG CGT AAT
ATC 576 Lys Ser Ile Lys Ser Thr Leu Lys Asp Glu Ile Ile Glu Arg Asn
Ile 180 185 190 ATC TTG GAA AAA GTC ACT CAG CGT GAT CTT CGC AGG AAG
TTT GTT TGC 624 Ile Leu Glu Lys Val Thr Gln Arg Asp Leu Arg Arg Lys
Phe Val Cys 195 200 205 TTT GTC CAG AAC TCC ATT GGA AAC ACA ACC CAG
TCC GTC CAA CTG AAA 672 Phe Val Gln Asn Ser Ile Gly Asn Thr Thr Gln
Ser Val Gln Leu Lys 210 215 220 GAA AAG AGA GGA GTG GTG CTC CTG TAC
ATC CTG CTT GGC ACC ATC GGG 720 Glu Lys Arg Gly Val Val Leu Leu Tyr
Ile Leu Leu Gly Thr Ile Gly 225 230 235 240 ACC CTG GTG GCC GTG CTG
GCG GCG AGT GCC CTC CTC TAC AGG CAC TGG 768 Thr Leu Val Ala Val Leu
Ala Ala Ser Ala Leu Leu Tyr Arg His Trp 245 250 255 ATT GAA ATA GTG
CTG CTG TAC CGG ACC TAC CAG AGC AAG GAT CAG ACG 816 Ile Glu Ile Val
Leu Leu Tyr Arg Thr Tyr Gln Ser Lys Asp Gln Thr 260 265 270 CTT GGG
GAT AAA AAG GAT TTT GAT GCT TTC GTA TCC TAT GCA AAA TGG 864 Leu Gly
Asp Lys Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala Lys Trp 275 280 285
AGC TCT TTT CCA AGT GAG GCC ACT TCA TCT CTG AGT GAA GAA CAC TTG 912
Ser Ser Phe Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu Glu His Leu 290
295 300 GCC CTG AGC CTA TTT CCT GAT GTT TTA GAA AAC AAA TAT GGA TAT
AGC 960 Ala Leu Ser Leu Phe Pro Asp Val Leu Glu Asn Lys Tyr Gly Tyr
Ser 305 310 315 320 CTG TGT TTG CTT GAA AGA GAT GTG GCT CCA GGA GGA
GTG TAT GCA GAA 1008 Leu Cys Leu Leu Glu Arg Asp Val Ala Pro Gly
Gly Val Tyr Ala Glu 325 330 335 GAC ATT GTG AGC ATT ATT AAG AGA AGC
AGA GAG GTA ATA TTT ATC TTG 1056 Asp Ile Val Ser Ile Ile Lys Arg
Ser Arg Glu Val Ile Phe Ile Leu 340 345 350 AGC CCC AAC TAT GTC AAT
GGA CCC AGT ATC TTT GAA CTA CAA GCA GCA 1104 Ser Pro Asn Tyr Val
Asn Gly Pro Ser Ile Phe Glu Leu Gln Ala Ala 355 360 365 GTG AAT CTT
GCC TTG GAT GAT CAA ACA CTG AAA CTC ATT TTA ATT AAG 1152 Val Asn
Leu Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu Ile Lys 370 375 380
TTC TGT TAC TTC CAA GAG CCA GAG TCT CTA CCT CAT CTC GTG AAA AAA
1200 Phe Cys Tyr Phe Gln Glu Pro Glu Ser Leu Pro His Leu Val Lys
Lys 385 390 395 400 GCT CTC AGG GTT TTG CCC ACA GTT ACT TGG AGA GGC
TTA AAA TCA GTT 1248 Ala Leu Arg Val Leu Pro Thr Val Thr Trp Arg
Gly Leu Lys Ser Val 405 410 415 CCT CCC AAT TCT AGG TTC TGG GCC AAA
ATG CGC TAC CAC ATG CCT GTG 1296 Pro Pro Asn Ser Arg Phe Trp Ala
Lys Met Arg Tyr His Met Pro Val 420 425 430 AAA AAC TCT CAG GGA TTC
ACG TGG AAC CAG CTC AGA ATT ACC TCT AGG 1344 Lys Asn Ser Gln Gly
Phe Thr Trp Asn Gln Leu Arg Ile Thr Ser Arg 435 440 445 ATT TTT CAG
TGG AAA GGA CTC AGT AGA ACA GAA ACC ACT GGG GAG GAG 1392 Ile Phe
Gln Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly Glu Glu 450 455 460
CTC CCA GCC TAA 1404 Leu Pro Ala 465 Supplemental sequence of
primate, e.g., human, IL-1RD9 (SEQ ID NO: 9 and 10). CCAGCGTGGT
GGAATTCGGA TACTCAGGGC AGAGTTCTGA ATCTCAAAAC ACTTTAATCT 60
GGCAAAGGAA TGAAGTTATT GGAGTGATGA CAGGAACACG GGAGAACA ATG CTC TGT
117 Met Leu Cys 1 TTG GGC TGG ATA TTT CTT TGG CTT GTT GCA GGA GAG
CGA ATT AAA GGA 165 Leu Gly Trp Ile Phe Leu Trp Leu Val Ala Gly Glu
Arg Ile Lys Gly 5 10 15 TTT AAT ATT TCA GGT TGT TCC ACA AAA AAA CTC
CTT TGG ACA TAT TCT 213 Phe Asn Ile Ser Gly Cys Ser Thr Lys Lys Leu
Leu Trp Thr Tyr Ser 20 25 30 35 ACA AGG AGT GAA GAG GAA TTT GTC TTA
TTT TGT GAT TTA CCA GAG CCA 261 Thr Arg Ser Glu Glu Glu Phe Val Leu
Phe Cys Asp Leu Pro Glu Pro 40 45 50 CAG AAA TCA CAT TTC TGC CAC
AGA AAT CGA CTC TCA CCA AAA CAA GTC 309 Gln Lys Ser His Phe Cys His
Arg Asn Arg Leu Ser Pro Lys Gln Val 55 60 65 CCT GAG CAC CTG CCC
TTC ATG GGT AGT AAC GAC CTA TCT GAT GTC CAA 357 Pro Glu His Leu Pro
Phe Met Gly Ser Asn Asp Leu Ser Asp Val Gln 70 75 80 TGG TAC CAA
CAA CCT TCG AAT GGA GAT CCA TTA GAG GAC ATT AGG AAA 405 Trp Tyr Gln
Gln Pro Ser Asn Gly Asp Pro Leu Glu Asp Ile Arg Lys 85 90 95 AGC
TAT CCT CAC ATC ATT CAG GAC AAA TGT ACC CTT CAC TTT TTG ACC 453 Ser
Tyr Pro His Ile Ile Gln Asp Lys Cys Thr Leu His Phe Leu Thr 100 105
110 115 CCA GGG GTG AAT AAT TCT GGG TCA TAT ATT TGT AGA CCC AAG ATG
ATT 501 Pro Gly Val Asn Asn Ser Gly Ser Tyr Ile Cys Arg Pro Lys Met
Ile 120 125 130 AAG AGC CCC TAT GAT GTA GCC TGT TGT GTC AAG ATG ATT
TTA GAA GTT 549 Lys Ser Pro Tyr Asp Val Ala Cys Cys Val Lys Met Ile
Leu Glu Val 135 140 145 AAG CCC CAG ACA AAT GCA TCC TGT GAG TAT TCC
GCA TCA CAT AAG CAA 597 Lys Pro Gln Thr Asn Ala Ser Cys Glu Tyr Ser
Ala Ser His Lys Gln 150 155 160 GAC CTA CTT CTT GGG AGC ACT GGC TCT
ATT TCT TGC CCC AGT CTC AGC 645 Asp Leu Leu Leu Gly Ser Thr Gly Ser
Ile Ser Cys Pro Ser Leu Ser 165 170 175 TGC CAA AGT GAT GCA CAA AGT
CCA GCG GTA ACC TGG TAC AAG AAT GGA 693 Cys Gln Ser Asp Ala Gln Ser
Pro Ala Val Thr Trp Tyr Lys Asn Gly 180 185 190 195 AAA CTC CTC TCT
GTG GAA AGG AGC AAC CGA ATC GTA GTG GAT GAA GTT 741 Lys Leu Leu Ser
Val Glu Arg Ser Asn Arg Ile Val Val Asp Glu Val 200 205 210 TAT GAC
TAT CAC CAG GGC ACA TAT GTA TGT GAT TAC ACT CAG TCG GAT 789 Tyr Asp
Tyr His Gln Gly Thr Tyr Val Cys Asp Tyr Thr Gln Ser Asp 215 220 225
ACT GTG AGT TCG TGG ACA GTC AGA GCT GTT GTT CAA GTG AGA ACC ATT 837
Thr Val Ser Ser Trp Thr Val Arg Ala Val Val Gln Val Arg Thr Ile 230
235 240 GTG GGA GAC ACT AAA CTC AAA CCA GAT ATT CTG GAT CCT GTC GAG
GAC 885 Val Gly Asp Thr Lys Leu Lys Pro Asp Ile Leu Asp Pro Val Glu
Asp 245 250 255 ACA CTG GAA GTA GAA CTT GGA AAG CCT TTA ACT ATT AGC
TGC AAA GCA 933 Thr Leu Glu Val Glu Leu Gly Lys Pro Leu Thr Ile Ser
Cys Lys Ala 260 265 270 275 CGA TTT GGC TTT GAA AGG GTC TTT AAC CCT
GTC ATA AAA TGG TAC ATC 981 Arg Phe Gly Phe Glu Arg Val Phe Asn Pro
Val Ile Lys Trp Tyr Ile 280 285 290 AAA GAT TCT GAC CTA GAG TGG GAA
GTC TCA GTA CCT GAG GCG AAA AGT 1029 Lys Asp Ser Asp Leu Glu Trp
Glu Val Ser Val Pro Glu Ala Lys Ser 295 300 305 ATT AAA TCC ACT TTA
AAG GAT GAA ATC ATT GAG CGT AAT ATC ATC TTG 1077 Ile Lys Ser Thr
Leu Lys Asp Glu Ile Ile Glu Arg Asn Ile Ile Leu 310 315 320 GAA AAA
GTC ACT CAG CGT GAT CTT CGC AGG AAG TTT GTT TGC TTT GTC 1125 Glu
Lys Val Thr Gln Arg Asp Leu Arg Arg Lys Phe Val Cys Phe Val 325 330
335 CAG AAC TCC ATT GGA AAC ACA ACC CAG TCC GTC CAA CTG AAA GAA AAG
1173 Gln Asn Ser Ile Gly Asn Thr Thr Gln Ser Val Gln Leu Lys Glu
Lys 340 345 350 355 AGA GGA GTG GTG CTC CTG TAC ATC CTG CTT GGC ACC
ATC GGG ACC CTG 1221 Arg Gly Val Val Leu Leu Tyr Ile Leu Leu Gly
Thr Ile Gly Thr Leu 360 365 370 GTG GCC GTG CTG GCG GCG AGT GCC CTC
CTC TAC AGG CAC TGG ATT GAA 1269 Val Ala Val Leu Ala Ala Ser Ala
Leu Leu Tyr Arg His Trp Ile Glu 375 380 385 ATA GTG CTG CTG TAC CGG
ACC TAC CAG AGC AAG GAT CAG ACG CTT GGG 1317 Ile Val Leu Leu Tyr
Arg Thr Tyr Gln Ser Lys Asp Gln Thr Leu Gly 390 395 400 GAT AAA AAG
GAT TTT GAT GCT TTC GTA TCC TAT GCA AAA TGG AGC TCT 1365 Asp Lys
Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala Lys Trp Ser Ser 405 410 415
TTT CCA AGT GAG GCC ACT TCA TCT CTG AGT GAA GAA CAC TTG GCC CTG
1413 Phe Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu Glu His Leu Ala
Leu 420 425 430 435 AGC CTA TTT CCT GAT GTT TTA GAA AAC AAA TAT GGA
TAT AGC CTG TGT 1461 Ser Leu Phe Pro Asp Val Leu Glu Asn Lys Tyr
Gly Tyr Ser Leu Cys 440 445 450 TTG CTT GAA AGA GAT GTG GCT CCA GGA
GGA GTG TAT GCA GAA GAC ATT 1509 Leu Leu Glu Arg Asp Val Ala Pro
Gly Gly Val Tyr Ala Glu Asp Ile 455 460 465 GTG AGC ATT ATT AAG AGA
AGC AGA AGA GGA ATA TTT ATC TTG AGC CCC 1557 Val Ser Ile Ile Lys
Arg Ser Arg Arg Gly Ile Phe Ile Leu Ser Pro 470 475 480 AAC TAT GTC
AAT GGA CCC AGT ATC TTT GAA CTA CAA GCA GCA GTG AAT 1605 Asn Tyr
Val Asn Gly Pro Ser Ile Phe Glu Leu Gln Ala Ala Val Asn 485 490 495
CTT GCC TTG GAT GAT CAA ACA CTG AAA CTC ATT TTA ATT AAG TTC TGT
1653 Leu Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu Ile Lys Phe
Cys 500 505 510 515 TAC TTC CAA GAG CCA GAG TCT CTA CCT CAT CTC GTG
AAA AAA GCT CTC 1701 Tyr Phe Gln Glu Pro Glu Ser Leu Pro His Leu
Val Lys Lys Ala Leu 520 525 530 AGG GTT TTG CCC ACA GTT ACT TGG AGA
GGC TTA AAA TCA GTT CCT CCC 1749 Arg Val Leu Pro Thr Val Thr Trp
Arg Gly Leu Lys Ser Val Pro Pro 535 540 545 AAT TCT AGG TTC TGG GCC
AAA ATG CGC TAC CAC ATG CCT GTG AAA AAC 1797 Asn Ser Arg Phe Trp
Ala Lys Met Arg Tyr His Met Pro Val Lys Asn 550 555 560 TCT CAG GGA
TTC ACG TGG AAC CAG CTC AGA ATT ACC TCT AGG ATT TTT 1845 Ser Gln
Gly Phe Thr Trp Asn Gln Leu Arg Ile Thr Ser Arg Ile Phe 565 570 575
CAG TGG AAA GGA CTC AGT AGA ACA GAA ACC ACT GGG AGG AGC TCC CAG
1893 Gln Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly Arg Ser Ser
Gln 580 585 590 595 CCT AAG GAA TGG TGAAATGAGC CCTGGAGCCC
CCTCCAGTCC AGTCCCTGGG 1945 Pro Lys Glu Trp ATAGAGATGT TGCTGGACAG
AACTCACAGC TCTGTGTGTG TGTGTTCAGG CTGATAGGAA 2005 ATTCAAAGAG
TCTCCTGCCA GCACCAAGCA AGCTTGATGG ACAATGGAAT GGGATTGAGA 2065
CTGTGGTTTA GAGCCTTTGA TTTCCTGGAC TGGACAGACG GCGAGTGAAT TCTCTAGACC
2125 TTGGGTACTT
TCAGTACACA ACACCCCTAA GATTTCCCAG TGGTCCGAGC AGAATCAGAA 2185
AATACAGCTA CTTCTGCCTT ATGGCTAGGG AACTGTCATG TCTACCATGT ATTGTACATA
2245 TGACTTTATG TATACTTGCA ATCAAATAAA TATTATTTTA TTAGAAAAAA
AAAAAAAAAG 2305 GGCGGCCGC 2314
MLCLGWIFLWLVAGERIKGFNISGCSTKKLLWTYSTRSEEEFVLFCDLPEPQKSHFCHRNRLSPKQVPE
HLPFMGSNDLSDVQWYQQPSNGDPLEDIRKSYPHIIQDKCTLHFLTPGVNNSGSYICRPKM-
IKSPYDVA CCVKMILEVKPQTNASCEYSASHKQDLLLGSTGSISCPSLSCQSDAQSP-
AVTWYKNGKLLSVERSNRIV VDEVYDYHQGTYVCDYTQSDTVSSWTVRAVVQVRTIV-
GDTKLKPDILDPVEDTLEVELGKPLTISCKAR FGFERVFNPVIKWYIKDSDLEWEVS-
VPEAKSIKSTLKDEIIERNIILEKVTQRDLRRKFVCFVQNSIGN
TTQSVQLKEKRGVVLLYILLGTIGTLVAVLAASALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSY
AKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYSLCLLERDVAPGGVYAEDIVSIIKRSR-
RGIFILSP NYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALR-
VLPTVTWRGLKSVPPNSRFW AKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTET-
TGRSSQPKEW Nucleotide and amino acid sequences (see SEQ ID NO: 11
and 12) of a rodent, e.g., mouse, embodiment of IL-1RD9. Single
clone sequence from thymus from 4 week old male C57BL/6J. GCA GCA
GTG AAT CTT GCC TTG GTT GAT CAG ACA CTG AAG TTG ATT TTA 48 Ala Ala
Val Asn Leu Ala Leu Val Asp Gln Thr Leu Lys Leu Ile Leu 1 5 10 15
ATT AAG TTC TGT TCC TTC CAA GAG CCA GAA TCT CTT CCT TAC CTT GTC 96
Ile Lys Phe Cys Ser Phe Gln Glu Pro Glu Ser Leu Pro Tyr Leu Val 20
25 30 AAA AAG GCT CTG CGG GTT CTC CCC ACA GTC ACA TGG AAA GGC TTG
AAG 144 Lys Lys Ala Leu Arg Val Leu Pro Thr Val Thr Trp Lys Gly Leu
Lys 35 40 45 TCG GTC CAC GCC AGT TCC AGG TTC TGG ACC CAA ATT CGT
TAC CAC ATG 192 Ser Val His Ala Ser Ser Arg Phe Trp Thr Gln Ile Arg
Tyr His Met 50 55 60 CCT GTG AAG AAC TCC AAC AGG TTT ATG TTC AAC
GGG CTC AGA ATT TTC 240 Pro Val Lys Asn Ser Asn Arg Phe Met Phe Asn
Gly Leu Arg Ile Phe 65 70 75 80 CTG AAG GGG TTT TCC CCT GAA AAG GAC
CTA GTG ACA CAG AAA CCC CTG 288 Leu Lys Gly Phe Ser Pro Glu Lys Asp
Leu Val Thr Gln Lys Pro Leu 85 90 95 GAA GGA ATG CCC AAG TCT GGG
AAT GAC CAC GGA GCT CAG AAC CTC CTT 336 Glu Gly Met Pro Lys Ser Gly
Asn Asp His Gly Ala Gln Asn Leu Leu 100 105 110 CTC TAC AGT GAC CAG
AAG AGG TGC TGATGGGTAG AACTTGCTGT GTGGATCAGG 390 Leu Tyr Ser Asp
Gln Lys Arg Cys 115 120 CTGATAGAAA TTGAGCCTTT CTGCTCTCAG TGCCAAGCAA
GCTTGACAGG CAGTGGAATG 450 AAGCGGCATC TGTGGTTTTA GGGTCTGGGT
TCCTGGAACA GACACAGAGC AATACTCCAG 510 ACCTCTGCCG TGTGCTTAGC
ACACATTTCC CTGAGAGTTC CCAAGTAGCC TGAACAGAAT 570 CAAGAGAAAT
AGCTCCATGG GCTGTCCAAC ATTCATGCAC GCATGCCTGT TTTGCACTAT 630
ATATATGAAT TTATCATACG TTTGTGTGTG TATATGCATT CAGATAAATA GGATTTTATT
690 TTGTTCGATA CGAGTGATTG AAACTCCATT TAAAGCCCTT CTGTAAAGAA
ATTTTGCTGC 750 AAAAAAAAAA AAAAAAAA 768
AAVNLALVDQTLKLILTKFCSFQEPESLPYLVKKALRVLPTVTWKGLKSVHAS-
SRFWTQIRYHMPVKNS NRFMFNGLRIFLKGFSPEKDLVTQKPLDRMPKSGNDHGAQN- LLLYSD
Supplemental sequence of rodent, e.g., mouse, IL-1RD9 (SEQ ID NO:
13 and 14). Putative signal processing site is indicated, but
actual may depend upon cell type, and may be at a different nearby
site. ATG TCT GTT TGG CTG GTG TTC TTG GTT TGT GCA GGA GAG AAG ACC
ACA 48 Met Ser Val Trp Leu Val Phe Leu Val Cys Ala Gly Glu Lys Thr
Thr -17 -15 -10 -5 GGA TTT AAT CAT TCA GCT TGT GCC ACC AAA AAT TCT
GTG GAC ATA TTC 96 Gly Phe Asn His Ser Ala Cys Ala Thr Lys Asn Ser
Val Asp Ile Phe 1 5 10 15 GCA AGG GGT GCA GAG AAT TTT GTC TAT TTT
GTG ACT TAC AAG AGC TTC 144 Ala Arg Gly Ala Glu Asn Phe Val Tyr Phe
Val Thr Tyr Lys Ser Phe 20 25 30 AGG AGC AAA AAT TCT CCC ATG CAA
GTC AAC TGT CAC CAA CAC AAA GTC 192 Arg Ser Lys Asn Ser Pro Met Gln
Val Asn Cys His Gln His Lys Val 35 40 45 TGC TCA CAA ACT TGC AGT
GGC AGT CAG AAG GAC TTA TCT GAT GTC CAG 240 Cys Ser Gln Thr Cys Ser
Gly Ser Gln Lys Asp Leu Ser Asp Val Gln 50 55 60 TGG TAC ATG CAA
CCT CGG AGT GGA AGT CCA CTA GAG GAG ATC AGT AGA 288 Trp Tyr Met Gln
Pro Arg Ser Gly Ser Pro Leu Glu Glu Ile Ser Arg 65 70 75 AAC TCT
CCC CAT ATG CAG AGT GAA GGC ATG CTG CAT ATA TTG GCC CCA 336 Asn Ser
Pro His Met Gln Ser Glu Gly Met Leu His Ile Leu Ala Pro 80 85 90 95
CAG ACG AAC AGC ATT TGG TCA TAT ATT TGT AGA CCC AGA ATT AGG AGC 384
Gln Thr Asn Ser Ile Trp Ser Tyr Ile Cys Arg Pro Arg Ile Arg Ser 100
105 110 CCC CAG GAT ATG GCC TGT TGT ATC AAG ACA GTC TTA GAA GTT AAG
CCT 432 Pro Gln Asp Met Ala Cys Cys Ile Lys Thr Val Leu Glu Val Lys
Pro 115 120 125 CAG AGA AAC GTG TCC TGT GGG AAC ACA GCA CAA GAT GAA
CAA GTC CTA 480 Gln Arg Asn Val Ser Cys Gly Asn Thr Ala Gln Asp Glu
Gln Val Leu 130 135 140 CTT CTT GGC AGT ACT GGC TCC ATT CAT TGT CCC
AGT CTC AGC TGC CAA 528 Leu Leu Gly Ser Thr Gly Ser Ile His Cys Pro
Ser Leu Ser Cys Gln 145 150 155 AGT GAT GTA CAG AGT CCA GAG ATG ACC
TGG TAC AAG GAT GGA AGA CTA 576 Ser Asp Val Gln Ser Pro Glu Met Thr
Trp Tyr Lys Asp Gly Arg Leu 160 165 170 175 CTT CCT GAG CAC AAG AAA
AAT CCA ATT GAG ATG GCA GAT ATT TAT GTT 624 Leu Pro Glu His Lys Lys
Asn Pro Ile Glu Met Ala Asp Ile Tyr Val 180 185 190 TTT AAT CAA GGC
TTG TAT GTA TGT GAT TAC ACA CAG TCA GAT AAT GTG 672 Phe Asn Gln Gly
Leu Tyr Val Cys Asp Tyr Thr Gln Ser Asp Asn Val 195 200 205 AGT TCC
TGG ACA GTC CGA GCT GTG GTT AAA GTG AGA ACC ATT GGT AAG 720 Ser Ser
Trp Thr Val Arg Ala Val Val Lys Val Arg Thr Ile Gly Lys 210 215 220
GAC ATC AAT GTG AAG CCG GAA ATT CTG GAT CCC ATT ACA GAT ACA CTG 768
Asp Ile Asn Val Lys Pro Glu Ile Leu Asp Pro Ile Thr Asp Thr Leu 225
230 235 GAC GTA GAG CTT GGA AAG CCT TTA ACT CTC CCC TGC AGA GTA CAG
TTT 816 Asp Val Glu Leu Gly Lys Pro Leu Thr Leu Pro Cys Arg Val Gln
Phe 240 245 250 255 GGC TTC CAA AGA CTT TCA AAG CCT GTG ATA AAG TGG
TAT GTC AAA GAA 864 Gly Phe Gln Arg Leu Ser Lys Pro Val Ile Lys Trp
Tyr Val Lys Glu 260 265 270 TCT ACA CAG GAG TGG GAA ATG TCA GTA TTT
GAG GAG AAA AGA ATT CAA 912 Ser Thr Gln Glu Trp Glu Met Ser Val Phe
Glu Glu Lys Arg Ile Gln 275 280 285 TCC ACT TTC AAG AAT GAA GTC ATT
GAA CGT ACC ATC TTC TTG AGA GAA 960 Ser Thr Phe Lys Asn Glu Val Ile
Glu Arg Thr Ile Phe Leu Arg Glu 290 295 300 GTT ACC CAG AGA GAT CTC
AGC AGA AAG TTT GTT TGC TTT GCC CAG AAC 1008 Val Thr Gln Arg Asp
Leu Ser Arg Lys Phe Val Cys Phe Ala Gln Asn 305 310 315 TCC ATT GGG
AAC ACA ACA CGG ACC ATA CGG CTG AGG AAG AAG GAA GAG 1056 Ser Ile
Gly Asn Thr Thr Arg Thr Ile Arg Leu Arg Lys Lys Glu Glu 320 325 330
335 GTG GTG TTT GTA TAC ATC CTT CTC GGC ACG GCC TTG ATG CTG GTG GGC
1104 Val Val Phe Val Tyr Ile Leu Leu Gly Thr Ala Leu Met Leu Val
Gly 340 345 350 GTT CTG GTG GCA GCT GCT TTC CTC TAC TGG TAC TGG ATT
GAA GTT GTC 1152 Val Leu Val Ala Ala Ala Phe Leu Tyr Trp Tyr Trp
Ile Glu Val Val 355 360 365 CTG CTC TGT CGA ACC TAC AAG AAC AAA GAT
GAG ACT CTG GGG GAT AAG 1200 Leu Leu Cys Arg Thr Tyr Lys Asn Lys
Asp Glu Thr Leu Gly Asp Lys 370 375 380 AAG GAA TTC GAT GCA TTT GTA
TCC TAC TCG AAT TGG AGC TCT CCT GAG 1248 Lys Glu Phe Asp Ala Phe
Val Ser Tyr Ser Asn Trp Ser Ser Pro Glu 385 390 395 ACT GAC GCC GTG
GGA TCT CTG AGT GAG GAA CAC CTG GCT CTG AAT CTT 1296 Thr Asp Ala
Val Gly Ser Leu Ser Glu Glu His Leu Ala Leu Asn Leu 400 405 410 415
TTC CCG GAA GTG CTA GAA GAC ACC TAT GGG TAC AGA TTG TGT TTG CTT
1344 Phe Pro Glu Val Leu Glu Asp Thr Tyr Gly Tyr Arg Leu Cys Leu
Leu 420 425 430 GAC CGA GAT GTG ACC CCA GGA GGA GTG TAT GCA GAT GAC
ATT GTG AGC 1392 Asp Arg Asp Val Thr Pro Gly Gly Val Tyr Ala Asp
Asp Ile Val Ser 435 440 445 ATC ATT AAG AAA AGC CGA AGA GGA ATA TTT
ATC CTG AGT CCC AGC TAC 1440 Ile Ile Lys Lys Ser Arg Arg Gly Ile
Phe Ile Leu Ser Pro Ser Tyr 450 455 460 CTC AAT GGA CCC CGT GTC TTT
GAG CTA CAA GCA GCA GTG AAT CTT GCC 1488 Leu Asn Gly Pro Arg Val
Phe Glu Leu Gln Ala Ala Val Asn Leu Ala 465 470 475 TTG GTT GAT CAG
ACA CTG AAG TTG ATT TTA ATT AAG TTC TGT TCC TTC 1536 Leu Val Asp
Gln Thr Leu Lys Leu Ile Leu Ile Lys Phe Cys Ser Phe 480 485 490 495
CAA GAG CCA GAA TCT CTT CCT TAC CTT GTC AAA AAG GCT CTG CGG GTT
1584 Gln Glu Pro Glu Ser Leu Pro Tyr Leu Val Lys Lys Ala Leu Arg
Val 500 505 510 CTC CCC ACA GTC ACA TGG AAA GGC TTG AAG TCG GTC CAC
GCC AGT TCC 1632 Leu Pro Thr Val Thr Trp Lys Gly Leu Lys Ser Val
His Ala Ser Ser 515 520 525 AGG TTC TGG ACC CAA ATT CGT TAC CAC ATG
CCT GTG AAG AAC TCC AAC 1680 Arg Phe Trp Thr Gln Ile Arg Tyr His
Met Pro Val Lys Asn Ser Asn 530 535 540 AGG TTT ATG TTC AAC GGG CTC
AGA ATT TTC CTG AAG GGC TTT TCC CCT 1728 Arg Phe Met Phe Asn Gly
Leu Arg Ile Phe Leu Lys Gly Phe Ser Pro 545 550 555 GAA AAG GAC CTA
GTG ACA CAG AAA CCC CTG GAA GGA ATG CCC AAG TCT 1776 Glu Lys Asp
Leu Val Thr Gln Lys Pro Leu Glu Gly Met Pro Lys Ser 560 565 570 575
GGG AAT GAC CAC GGA GCT CAG AAC CTC CTT CTC TAC AGT GAC CAG AAG
1824 Gly Asn Asp His Gly Ala Gln Asn Leu Leu Leu Tyr Ser Asp Gln
Lys 580 585 590 AGG TGC TGA 1833 Arg Cys Supplemental sequence of
rodent, e.g., mouse, IL-1RD9 (SEQ ID NO: 15 and 16). TGACAGGAGC
AAAGGGGAAC C ATG CTC TGT TTG GGC TGG GTG TTT CTT TGG 51 Met Leu Cys
Leu Gly Trp Val Phe Leu Trp 1 5 10 TTT GTT GCA GGA GAG AAG ACC ACA
GGA TTT AAT CAT TCA GCT TGT GCC 99 Phe Val Ala Gly Glu Lys Thr Thr
Gly Phe Asn His Ser Ala Cys Ala 15 20 25 ACC AAA AAA CTT CTG TGG
ACA TAT TCT GCA AGG GGT GCA GAG AAT TTT 147 Thr Lys Lys Leu Leu Trp
Thr Tyr Ser Ala Arg Gly Ala Glu Asn Phe 30 35 40 GTC CTA TTT TGT
GAC TTA CAA GAG CTT CAG GAG CAA AAA TTC TCC CAT 195 Val Leu Phe Cys
Asp Leu Gln Glu Leu Gln Glu Gln Lys Phe Ser His 45 50 55 GCA AGT
CAA CTG TCA CCA ACA CAA AGT CCT GCT CAC AAA CCT TGC AGT 243 Ala Ser
Gln Leu Ser Pro Thr Gln Ser Pro Ala His Lys Pro Cys Ser 60 65 70
GGC AGT CAG AAG GAC CTA TCT GAT GTC CAG TGG TAC ATG CAA CCT CGG 291
Gly Ser Gln Lys Asp Leu Ser Asp Val Gln Trp Tyr Met Gln Pro Arg 75
80 85 90 AGT GGA AGT CCA CTA GAG GAG ATC AGT AGA AAC TCT CCC CAT
ATG CAG 339 Ser Gly Ser Pro Leu Glu Glu Ile Ser Arg Asn Ser Pro His
Met Gln 95 100 105 AGT GAA GGC ATG CTG CAT ATA TTG GCC CCA CAG ACG
AAC AGC ATT TGG 387 Ser Glu Gly Met Leu His Ile Leu Ala Pro Gln Thr
Asn Ser Ile Trp 110 115 120 TCA TAT ATT TGT AGA CCC AGA ATT AGG AGC
CCC CAG GAT ATG GCC TGT 435 Ser Tyr Ile Cys Arg Pro Arg Ile Arg Ser
Pro Gln Asp Met Ala Cys 125 130 135 TGT ATC AAG ACA GTC TTA GAA GTT
AAG CCT CAG AGA AAC GTG TCC TGT 483 Cys Ile Lys Thr Val Leu Glu Val
Lys Pro Gln Arg Asn Val Ser Cys 140 145 150 GGG AAC ACA GCA CAA GAT
GAA CAA GTC CTA CTT CTT GGC AGT ACT GGC 531 Gly Asn Thr Ala Gln Asp
Glu Gln Val Leu Leu Leu Gly Ser Thr Gly 155 160 165 170 TCC ATT CAT
TGT CCC AGT CTC AGC TGC CAA AGT GAT GTA CAG AGT CCA 579 Ser Ile His
Cys Pro Ser Leu Ser Cys Gln Ser Asp Val Gln Ser Pro 175 180 185 GAG
ATG ACC TGG TAC AAG GAT GGA AGA CTA CTT CCT GAG CAC AAG AAA 627 Glu
Met Thr Trp Tyr Lys Asp Gly Arg Leu Leu Pro Glu His Lys Lys 190 195
200 AAT CCA ATT GAG ATG GCA GAT ATT TAT GTT TTT AAT CAA GGC TTG TAT
675 Asn Pro Ile Glu Met Ala Asp Ile Tyr Val Phe Asn Gln Gly Leu Tyr
205 210 215 GTA TGT GAT TAC ACA CAG TCA GAT AAT GTG AGT TCC TGG ACA
GTC CGA 723 Val Cys Asp Tyr Thr Gln Ser Asp Asn Val Ser Ser Trp Thr
Val Arg 220 225 230 GCT GTG GTT AAA GTG AGA ACC ATT GGT AAG GAC ATC
AAT GTG AAG CCG 771 Ala Val Val Lys Val Arg Thr Ile Gly Lys Asp Ile
Asn Val Lys Pro 235 240 245 250 GAA ATT CTG GAT CCC ATT ACA GAT ACA
CTG GAC GTA GAG CTT GGA AAG 819 Glu Ile Leu Asp Pro Ile Thr Asp Thr
Leu Asp Val Glu Leu Gly Lys 255 260 265 CCT TTA ACT CTC CCC TGC AGA
GTA CAG TTT GGC TTC CAA AGA CTT TCA 867 Pro Leu Thr Leu Pro Cys Arg
Val Gln Phe Gly Phe Gln Arg Leu Ser 270 275 280 AAG CCT GTG ATA AAG
TGG TAT GTC AAA GAA TCT ACA CAG GAG TGG GAA 915 Lys Pro Val Ile Lys
Trp Tyr Val Lys Glu Ser Thr Gln Glu Trp Glu 285 290 295 ATG TCA GTA
TTT GAG GAG AAA AGA ATT CAA TCC ACT TTC AAG AAT GAA 963 Met Ser Val
Phe Glu Glu Lys Arg Ile Gln Ser Thr Phe Lys Asn Glu 300 305 310 GTC
ATT GAA CGT ACC ATC TTC TTG AGA GAA GTT ACC CAG AGA GAT CTC 1011
Val Ile Glu Arg Thr Ile Phe Leu Arg Glu Val Thr Gln Arg Asp Leu 315
320 325 330 AGC AGA AAG TTT GTT TGC TTT GCC CAG AAC TCC ATT GGG AAC
ACA ACA 1059 Ser Arg Lys Phe Val Cys Phe Ala Gln Asn Ser Ile Gly
Asn Thr Thr 335 340 345 CGG ACC ATA CGG CTG AGG AAG AAG GAA GAG GTG
GTG TTT GTA TAC ATC 1107 Arg Thr Ile Arg Leu Arg Lys Lys Glu Glu
Val Val Phe Val Tyr Ile 350 355 360 CTT CTC GGC ACG GCC TTG ATG CTG
GTG GGC GTT CTG GTG GCA GCT GCT 1155 Leu Leu Gly Thr Ala Leu Met
Leu Val Gly Val Leu Val Ala Ala Ala 365 370 375 TTC CTC TAC TGG TAC
TGG ATT GAA GTT GTC CTG CTC TGT CGA ACC TAC 1203 Phe Leu Tyr Trp
Tyr Trp Ile Glu Val Val Leu Leu Cys Arg Thr Tyr 380 385 390 AAG AAC
AAA GAT GAG ACT CTG GGG GAT AAG AAG GAA TTC GAT GCA TTT 1251 Lys
Asn Lys Asp Glu Thr Leu Gly Asp Lys Lys Glu Phe Asp Ala Phe 395 400
405 410 GTA TCC TAC TCG AAT TGG AGC TCT CCT GAG ACT GAC GCC GTG GGA
TCT 1299 Val Ser Tyr Ser Asn Trp Ser Ser Pro Glu Thr Asp Ala Val
Gly Ser 415 420 425 CTG AGT GAG GAA CAC CTG GCT
CTG AAT CTT TTC CCG GAA GTG CTA GAA 1347 Leu Ser Glu Glu His Leu
Ala Leu Asn Leu Phe Pro Glu Val Leu Glu 430 435 440 GAC ACC TAT GGG
TAC AGA TTG TGT TTG CTT GAC CGA GAT GTG ACC CCA 1395 Asp Thr Tyr
Gly Tyr Arg Leu Cys Leu Leu Asp Arg Asp Val Thr Pro 445 450 455 GGA
GGA GTG TAT GCA GAT GAC ATT GTG AGC ATC ATT AAG AAA AGC CGA 1443
Gly Gly Val Tyr Ala Asp Asp Ile Val Ser Ile Ile Lys Lys Ser Arg 460
465 470 AGA GGA ATA TTT ATC CTG AGT CCC AGC TAC CTC AAT GGA CCC CGT
GTC 1491 Arg Gly Ile Phe Ile Leu Ser Pro Ser Tyr Leu Asn Gly Pro
Arg Val 475 480 485 490 TTT GAG CTA CAA GCA GCA GTG AAT CTT GCC TTG
GTT GAT CAG ACA CTG 1539 Phe Glu Leu Gln Ala Ala Val Asn Leu Ala
Leu Val Asp Gln Thr Leu 495 500 505 AAG TTG ATT TTA ATT AAG TTC TGT
TCC TTC CAA GAG CCA GAA TCT CTT 1587 Lys Leu Ile Leu Ile Lys Phe
Cys Ser Phe Gln Glu Pro Glu Ser Leu 510 515 520 CCT TAC CTT GTC AAA
AAG GCT CTG CGG GTT CTC CCC ACA GTC ACA TGG 1635 Pro Tyr Leu Val
Lys Lys Ala Leu Arg Val Leu Pro Thr Val Thr Trp 525 530 535 AAA GGC
TTG AAG TGG GTC CAC GCC AGT TCC AGG TTC TGG ACC CAA ATT 1683 Lys
Gly Leu Lys Ser Val His Ala Ser Ser Arg Phe Trp Thr Gln Ile 540 545
550 CGT TAC CAC ATG CCT GTG AAG AAC TCC AAC AGG TTT ATG TTC AAC GGG
1731 Arg Tyr His Met Pro Val Lys Asn Ser Asn Arg Phe Met Phe Asn
Gly 555 560 565 570 CTC AGA ATT TTC CTG AAG GGC TTT TCC CCT GAA AAG
GAC CTA GTG ACA 1779 Leu Arg Ile Phe Leu Lys Gly Phe Ser Pro Glu
Lys Asp Leu Val Thr 575 580 585 CAG AAA CCC CTG GAA GGA ATG CCC AAG
TCT GGG AAT GAC CAC GGA GCT 1827 Gln Lys Pro Leu Glu Gly Met Pro
Lys Ser Gly Asn Asp His Gly Ala 590 595 600 CAG AAC CTC CTT CTC TAC
AGT GAC CAG AAG AGG TGC TGATGGGTAG 1873 Gln Asn Leu Leu Leu Tyr Ser
Asp Gln Lys Arg Cys 605 610 AACTTGCTGT GTGGATCAGG CTGATAGAAA
TTGAGCCTTT CTGCTCTCAG TGCCAAGCAA 1933 GCTTGACAGG CAGTGGAATG
AAGCGGCATC TGTGGTTTTA GGGTCTGGGT TCCTGGAACA 1993 GACACAGAGC
AATACTCCAG ACCTCTGCCG TGTGCTTAGC ACACATTTCC CTGAGAGTTC 2053
CCAAGTAGCC TGAACAGAAT CAACAGAAAT AGCTCCATGG GCTGTCCAAC ATTCATGCAC
2113 GCATGCCTGT TTTGCACTAT ATATATGAAT TTATCATACG TTTGTGTGTG
TATATGCATT 2173 CAGATAAATA GGATTTTATT TTGTTCGATA CGAGTGATTG
AAACTCCATC TAAAGCCCTT 2233 CTGTAAAGAA AAAAAAAAAA AAAAAA 2259
MLCLGWVFLWFVAGEKTTGFNHSACATKKLLWTYSARGAENFVLFCDLQELQEQKFSHASQLSPT-
QSPA HKPCSGSQKDLSDVQWYMQPRSGSPLEEISRNSPHMQSEGMLHILAPQTNSIW-
SYICRPRIRSPQDMAC CIKTVLEVKPQRNVSCGNTAQDEQVLLLGSTGSIHCPSLSC-
QSDVQSPEMTWYKDGRLLPEHKKNPIEM ADIYVFNQGLYVCDYTQSDNVSSWTVRAV-
VKVRTIGKDINVKPEILDPITDTLDVELGKPLTLPCRVQF
GFQRLSKPVIKWYVKESTQEWEMSVFEEKRIQSTFKNEVIERTIFLREVTQRDLSRKFVCFAQNSIGNT
TRTIRLRKKEEVVFVYILLGTALMLVGVLVAAAFLYWYWIEVVLLCRTYKNKDETLGDKKE-
FDAFVSYS NWSSPETDAVGSLSEEHLALNLFPEVLEDTYGYRLCLLDRDVTPGGVYA-
DDIVSIIKKSRRGIFILSPS YLNGPRVFELQAAVNLALVDQTLKLILIKFCSFQEPE-
SLPYLVKKALRVLPTVTWKGLKSVHASSRFWT QIRYHMPVKNSNRFMFNGLRIFLKG-
FSPEKDLVTQKPLEGMPKSGNDHGAQNLLLYSDQKRC
[0064]
3TABLE 3 Nucleotide and amino acid sequences (see SEQ ID NO: 17 and
18) of primate, e.g., human, embodiment of IL-1RD10. Single
sequence derived from human brain frontal cortex, epileptic;
re-excision. Nucleotides 374, 383, 396, 403, 433, 458, 459, 483,
and 515 are indicated as C, each may be A, C, G, or T. C TGT GAA
TTA AAA TAT GGA GGC TTT GTT GTG AGA AGA ACT ACT GAA 46 Cys Glu Leu
Lys Tyr Gly Gly Phe Val Val Arg Arg Thr Thr Glu 1 5 10 15 15 TTA
ACT GTT ACA GCC CCT CTG ACT GAT AAG CCA CCC AAG CTT TTG TAT 94 Leu
Thr Val Thr Ala Pro Leu Thr Asp Lys Pro Pro Lys Leu Leu Tyr 20 25
30 CCT ATG GAA AGT AAA CTG ACA ATT CAG GAG ACC CAG CTG GGT GAC TCT
142 Pro Met Glu Ser Lys Leu Thr Ile Gln Glu Thr Gln Leu Gly Asp Ser
35 40 45 GCT AAT CTA ACC TGC AGA GCT TTC TTT GGG TAC AGC GGA GAT
GTC AGT 190 Ala Asn Leu Thr Cys Arg Ala Phe Phe Gly Tyr Ser Gly Asp
Val Ser 50 55 60 CCT TTA ATT TAC TGG ATG AAA GGA GAA AAA TTT ATT
GAA GAT CTG GAT 238 Pro Leu Ile Tyr Trp Met Lys Gly Glu Lys Phe Ile
Glu Asp Leu Asp 65 70 75 GAA AAT CGA GTT TGG GAA AGT GAC ATT AGA
ATT CTT AAG GAG CAT CTT 286 Glu Asn Arg Val Trp Glu Ser Asp Ile Arg
Ile Leu Lys Glu His Leu 80 85 90 95 GGG GAA CAG GAA GTT TCC ATC TCA
TTA ATT GTG GAC TCT GTG GAA GAA 334 Gly Glu Gln Glu Val Ser Ile Ser
Leu Ile Val Asp Ser Val Glu Glu 100 105 110 GGT GAC TTG GGA AAT TAC
TCC TGT TAT GTT GAA AAA TGG CAA TGG ACG 382 Gly Asp Leu Gly Asn Tyr
Ser Cys Tyr Val Glu Lys Trp Gln Trp Thr 115 120 125 CCG ACA CGC CAG
CCG TCC CCC TTC ATA AAC GAG AGC CTA ATG TAC ACA 430 Pro Thr Arg Gln
Pro Ser Pro Phe Ile Asn Glu Ser Leu Met Tyr Thr 130 135 140 GTC GGA
ACT TGC CTG GAG GCC CTT GGG CCA AAA CCT TGG TGG TTG AAT 478 Val Gly
Thr Cys Leu Glu Ala Leu Gly Pro Lys Pro Trp Trp Leu Asn 145 150 155
GTT TCG GGA CCA CCT TCA AAG TGT ACC AAG GTT GGA CC 516 Val Ser Gly
Pro Pro Ser Lys Cys Thr Lys Val Gly 160 165 170
CELKYGGFVVRRTTELTVTAPLTDKPPKLLYPMESKLTIQETQLGDSA-
NLTCRAFFGYSGDVSPLIYWM KGEKFIEDLDENRVWESDIRILKEHLGEQEVSISLI-
VDSVEEGDLGNYSCYVEKWXWTXTRQXSXFINE SLMYTXGTCLEALGXKPWWLNVXG-
PPSKCTKVG Supplemental sequence of primate, e.g., human, IL-1RD10
(SEQ ID NO: 19 and 20). Note nucleotides 1501, 1775, 1777, 1820,
1832, 1841, and 1844 are designated C; each may be A, C, G, or T.
GAA TTC GGC ACG AGC TGT GAA TTA AAA TAT GGA GGC TTT GTT GTG AGA 48
Glu Phe Gly Thr Ser Cys Glu Leu Lys Tyr Gly Gly Phe Val Val Arg 1 5
10 15 AGA ACT ACT GAA TTA ACT GTT ACA GCC CCT CTG ACT GAT AAG CCA
CCC 96 Arg Thr Thr Glu Leu Thr Val Thr Ala Pro Leu Thr Asp Lys Pro
Pro 20 25 30 AAG CTT TTG TAT CCT ATG GAA AGT AAA CTG ACA ATT CAG
GAG ACC CAG 144 Lys Leu Leu Tyr Pro Met Glu Ser Lys Leu Thr Ile Gln
Glu Thr Gln 35 40 45 CTG GGT GAC TCT GCT AAT CTA ACC TGC AGA GCT
TTC TTT GGG TAC AGC 192 Leu Gly Asp Ser Ala Asn Leu Thr Cys Arg Ala
Phe Phe Gly Tyr Ser 50 55 60 GGA GAT GTC AGT CCT TTA ATT TAC TGG
ATG AAA GGA GAA AAA TTT ATT 240 Gly Asp Val Ser Pro Leu Ile Tyr Trp
Met Lys Gly Glu Lys Phe Ile 65 70 75 80 GAA GAT CTG GAT GAA AAT CGA
GTT TGG GAA AGT GAC ATT AGA ATT CTT 288 Glu Asp Leu Asp Glu Asn Arg
Val Trp Glu Ser Asp Ile Arg Ile Leu 85 90 95 AAG GAG CAT CTT GGG
GAA CAG GAA GTT TCC ATC TCA TTA ATT GTG GAC 336 Lys Glu His Leu Gly
Glu Gln Glu Val Ser Ile Ser Leu Ile Val Asp 100 105 110 TCT GTG GAA
GAA GGT GAC TTG GGA AAT TAC TCC TGT TAT GTT GAA AAT 384 Ser Val Glu
Glu Gly Asp Leu Gly Asn Tyr Ser Cys Tyr Val Glu Asn 115 120 125 GGA
AAT GGA CGT CGA CAC GCC AGC GTT CTC CTT CAT AAA CGA GAG CTA 432 Gly
Asn Gly Arg Arg His Ala Ser Val Leu Leu His Lys Arg Glu Leu 130 135
140 ATG TAC ACA GTG GAA CTT GCT GGA GGC CTT GGT GCT ATA CTC TTG CTG
480 Met Tyr Thr Val Glu Leu Ala Gly Gly Leu Gly Ala Ile Leu Leu Leu
145 150 155 160 CTT GTA TGT TTG GTG ACC ATC TAC AAG TGT TAC AAG ATA
GAA ATC ATG 528 Leu Val Cys Leu Val Thr Ile Tyr Lys Cys Tyr Lys Ile
Glu Ile Met 165 170 175 CTC TTC TAC AGG AAT CAT TTT GGA GCT GAA GAA
CTC GAT GGA GAC AAT 576 Leu Phe Tyr Arg Asn His Phe Gly Ala Glu Glu
Leu Asp Gly Asp Asn 180 185 190 AAA GAT TAT GAT GCA TAC TTA TCA TAC
ACC AAA GTG GAT CCT GAC CAG 624 Lys Asp Tyr Asp Ala Tyr Leu Ser Tyr
Thr Lys Val Asp Pro Asp Gln 195 200 205 TGG AAT CAA GAG ACT GGG GAA
GAA GAA CGT TTT GCC CTT GAA ATC CTA 672 Trp Asn Gln Glu Thr Gly Glu
Glu Glu Arg Phe Ala Leu Glu Ile Leu 210 215 220 CCT GAT ATG CTT GAA
AAG CAT TAT GGA TAT AAG TTG TTT ATA CCA GAT 720 Pro Asp Met Leu Glu
Lys His Tyr Gly Tyr Lys Leu Phe Ile Pro Asp 225 230 235 240 AGA GAT
TTA ATC CCA ACT GGA ACA TAC ATT GAA GAT GTG GCA AGA TGT 768 Arg Asp
Leu Ile Pro Thr Gly Thr Tyr Ile Glu Asp Val Ala Arg Cys 245 250 255
GTA GAT CAA AGC AAG CGG CTG ATT ATT GTC ATG ACC CCA AAT TAC GTA 816
Val Asp Gln Ser Lys Arg Leu Ile Ile Val Met Thr Pro Asn Tyr Val 260
265 270 GTT AGA AGG GGC TGG AGC ATC TTT GAG CTG GAA ACC ACA CTT CTA
AAT 864 Val Arg Arg Gly Trp Ser Ile Phe Glu Leu Glu Thr Thr Leu Leu
Asn 275 280 285 ATG CTT GTG ACT GGA GAA ATT AAA GTG ATT CTA ATT GAA
TGC AGT GAA 912 Met Leu Val Thr Gly Glu Ile Lys Val Ile Leu Ile Glu
Cys Ser Glu 290 295 300 CTG AGA GGA ATT ATG AAC TAC CAC GAG GTG GAC
GCC CTG AAG CAC ACC 960 Leu Arg Gly Ile Met Asn Tyr His Glu Val Asp
Ala Leu Lys His Thr 305 310 315 320 ATC AAG CTC CTG ACG GTC ATT AAA
TGG CAT GGA CCA AAA TGC AAC AAG 1008 Ile Lys Leu Leu Thr Val Ile
Lys Trp His Gly Pro Lys Cys Asn Lys 325 330 335 TTG AAC TCC AAG TTC
TGG AAA CGT TTA CAG TAT GAA ATG CCT TTT AAG 1056 Leu Asn Ser Lys
Phe Trp Lys Arg Leu Gln Tyr Glu Met Pro Phe Lys 340 345 350 AGG ATA
GAA CCC ATT ACA CAT GAG CAG GCT TTA GAT GTC AGT GAG CAA 1104 Arg
Ile Glu Pro Ile Thr His Glu Gln Ala Leu Asp Val Ser Glu Gln 355 360
365 GGG CCT TTT GGG GAG CTG CAG ACT GTC TCG GCC ATT TCC ATG GCC GCG
1152 Gly Pro Phe Gly Glu Leu Gln Thr Val Ser Ala Ile Ser Met Ala
Ala 370 375 380 GCC ACC TCC ACA GCT CTA GCC ACT GCC CAT CCA GAT CTC
CGT TGT ACC 1200 Ala Thr Ser Thr Ala Leu Ala Thr Ala His Pro Asp
Leu Arg Cys Thr 385 390 395 400 TTT CAC AAC ACG TAC CAT TCA CAA ATG
CGT CAG AAA CAC TAC TAC CGA 1248 Phe His Asn Thr Tyr His Ser Gln
Met Arg Gln Lys His Tyr Tyr Arg 405 410 415 AGC TAT GAG TAC GAC GTA
CCT CCT ACC GGC ACC CTG CCT CTT ACC TCC 1296 Ser Tyr Glu Tyr Asp
Val Pro Pro Thr Gly Thr Leu Pro Leu Thr Ser 420 425 430 ATA GGC AAT
CAG CAT ACC TAC TGT AAC ATC CCT ATG ACA CTC ATC AAC 1344 Ile Gly
Asn Gln His Thr Tyr Cys Asn Ile Pro Met Thr Leu Ile Asn 435 440 445
GGG CAG CGG CCA CAG ACA AAA TCG AGC AGG GAG CAG AAT CCA GAT GAG
1392 Gly Gln Arg Pro Gln Thr Lys Ser Ser Arg Glu Gln Asn Pro Asp
Glu 450 455 460 GCC CAC ACA AAC AGT GCC ATC CTG CCG CTG TTG CCA AGG
GAG ACC AGT 1440 Ala His Thr Asn Ser Ala Ile Leu Pro Leu Leu Pro
Arg Glu Thr Ser 465 470 475 480 ATA TCC AGT GTG ATA TGG TGACAGAAAA
GCAAGGGACA TCCCGTCCCT 1488 Ile Ser Ser Val Ile Trp 485 GGGAGGTTGA
GTCGGAATCT GCAGTCCAGT GCCTGGAACT AAATCCTCGA CTGCTGCTGT 1548
TAAAAAACAT GCATTAGAAT CTTTAGAACA CGAGGAAAAA CAGGGTCTTG TACATATGTT
1608 TTTTGGAATT TCTTTGTAGC ATCAGTGTCC TCCTGTTTTA CCATGTCTTT
TACCATTACA 1668 TTTTTTGACT TTGTTTTATA TGTCGTTGGA ATTTGTAAAT
TTACATTTTT TTTAAAGAAG 1728 AGACTGATGT GTAGATAGAA AACCCTTTTT
TTGCTTCATT AGTTTACGCT TTTAGAATGG 1788 GTTTTTATTT TATTTCCTTT
TTTAAAATTT TCACTTTGCT TTTCAACATT TCCCTCTGGG 1848 GTGCTTGAAC
AAATCTATCC GATGGGACAA GGAGCACCGG ATTCTTTCTC GGGTTCTGCC 1908
TAGCATCAAC TGGGCCACGT CGGCCTTCAG AGAACAGTGC AACAAATGCC AGCATTGCCA
1968 TTCGGGGGGA AAAAAAAAAA AAAAAAAAAA CTCGAG 2004 Supplemented
sequence of primate IL-1RD10 (SEQ ID NO: 34 and 35): GAT GGA TGC
ACT GAC TGG TCT ATC GAT ATC AAG AAA TAT CAA GTT TTG 48 Asp Gly Cys
Thr Asp Trp Ser Ile Asp Ile Lys Lys Tyr Gln Val Leu 1 5 10 15 GTG
GGA GAG CCT GTT CGA ATC AAA TGT GCA CTC TTT TAT GGT TAT ATC 96 Val
Gly Glu Pro Val Arg Ile Lys Cys Ala Leu Phe Tyr Gly Tyr Ile 20 25
30 AGA ACA AAT TAC TCC CTT GCC CAA AGT GCT GGA CTC AGT TTG ATG TGG
144 Arg Thr Asn Tyr Ser Leu Ala Gln Ser Ala Gly Leu Ser Leu Met Trp
35 40 45 TAC AAA AGT TCT GGT CCT GGA GAC TTT GAA GAG CCA ATA GCC
TTT GAC 192 Tyr Lys Ser Ser Gly Pro Gly Asp Phe Glu Glu Pro Ile Ala
Phe Asp 50 55 60 GGA AGT AGA ATG AGC AAA GAA GAA GAC TCC ATT TGG
TTC CGG CCA ACA 240 Gly Ser Arg Met Ser Lys Glu Glu Asp Ser Ile Trp
Phe Arg Pro Thr 65 70 75 80 TTG CTA CAG GAC AGT GGT CTC TAC GCC TGT
GTC ATC AGG AAC TCC ACT 288 Leu Leu Gln Asp Ser Gly Leu Tyr Ala Cys
Val Ile Arg Asn Ser Thr 85 90 95 TAC TGT ATG AAA GTA TCC ATC TCA
CTG ACA GTG GGT GAA AAT GAC ACT 336 Tyr Cys Met Lys Val Ser Ile Ser
Leu Thr Val Gly Glu Asn Asp Thr 100 105 110 GGA CTC TGC TAT AAT TCC
AAG ATG AAG TAT TTT GAA AAA GCT GAA CTT 384 Gly Leu Cys Tyr Asn Ser
Lys Met Lys Tyr Phe Glu Lys Ala Glu Leu 115 120 125 AGC AAA AGC AAG
GAA ATT TCA TGC CGT GAC ATA GAG GAT TTT CTA CTG 432 Ser Lys Ser Lys
Glu Ile Ser Cys Arg Asp Ile Glu Asp Phe Leu Leu 130 135 140 CCA ACC
AGA GAA CCT GAA ATC CTT TGG TAC AAG GAA TGC AGG ACA AAA 480 Pro Thr
Arg Glu Pro Glu Ile Leu Trp Tyr Lys Glu Cys Arg Thr Lys 145 150 155
160 ACA TGG AGG CCA AGT ATT GTA TTC AAA AGA GAT ACT CTG CTT ATA AGA
528 Thr Trp Arg Pro Ser Ile Val Phe Lys Arg Asp Thr Leu Leu Ile Arg
165 170 175 GAA GTC AGA GAA GAT GAC ATT GGA AAT TAT ACC TGT GAA TTA
AAA TAT 576 Glu Val Arg Glu Asp Asp Ile Gly Asn Tyr Thr Cys Glu Leu
Lys Tyr 180 185 190 GGA GGC TTT GTT GTG AGA AGA ACT ACT GAA TTA ACT
GTT ACA GCC CCT 624 Gly Gly Phe Val Val Arg Arg Thr Thr Glu Leu Thr
Val Thr Ala Pro 195 200 205 CTG ACT GAT AAG CCA CCC AAG CTT TTG TAT
CCT ATG GAA AGT AAA CTG 672 Leu Thr Asp Lys Pro Pro Lys Leu Leu Tyr
Pro Met Glu Ser Lys Leu 210 215 220 ACA ATT CAG GAG ACC CAG CTG GGT
GAC TCT GCT AAT CTA ACC TGC AGA 720 Thr Ile Gln Glu Thr Gln Leu Gly
Asp Ser Ala Asn Leu Thr Cys Arg 225 230 235 240 GCT TTC TTT GGG TAC
AGC GGA GAT GTC AGT CCT TTA ATT TAC TGG ATG 768 Ala Phe Phe Gly Tyr
Ser Gly Asp Val Ser Pro Leu Ile Tyr Trp Met 245 250 255 AAA GGA GAA
AAA TTT ATT GAA GAT CTG GAT GAA AAT CGA GTT TGG GAA 816 Lys Gly Glu
Lys Phe Ile Glu Asp Leu Asp Glu Asn Arg Val Trp Glu 260 265 270 AGT
GAC ATT AGA ATT CTT AAG GAG CAT CTT GGG GAA CAG GAA GTT TCC 864 Ser
Asp Ile Arg Ile Leu Lys Glu His Leu Gly Glu Gln Glu Val Ser 275 280
285 ATC TCA TTA ATT GTG GAC TCT GTG GAA GAA GGT GAC TTG GGA AAT TAC
912 Ile Ser Leu Ile Val Asp Ser Val Glu Glu Gly Asp Leu Gly Asn Tyr
290 295 300 TCC TGT TAT GTT GAA AAT GGA AAT GGA CGT CGA CAC GCC AGC
GTT CTC 960 Ser Cys Tyr Val Glu Asn Gly Asn Gly Arg Arg His Ala Ser
Val Leu 305 310 315 320 CTT CAT AAA CGA GAG CTA ATG TAC ACA GTG GAA
CTT GCT GGA GGC CTT 1008 Leu His Lys Arg Glu Leu Met Tyr Thr Val
Glu Leu Ala Gly Gly Leu 325 330 335 GGT GCT ATA CTC TTG CTG CTT GTA
TGT TTG GTG ACC ATC TAC AAG TGT 1056 Gly Ala Ile Leu Leu Leu Leu
Val Cys Leu Val Thr Ile Tyr Lys Cys 340 345 350 TAC AAG ATA GAA ATC
ATG CTC TTC TAC AGG AAT CAT TTT GGA GCT GAA 1104 Tyr Lys Ile Glu
Ile Met Leu Phe Tyr Arg Asn His Phe Gly Ala Glu 355 360 365 GAG CTC
GAT GGA GAC AAT AAA GAT TAT GAT GCA TAC TTA TCA TAC ACC 1152 Glu
Leu Asp Gly Asp Asn Lys Asp Tyr Asp Ala Tyr Leu Ser Tyr Thr 370 375
380 AAA GTG GAT CCT GAC CAG TGG AAT CAA GAG ACT GGG GAA GAA GAA CGT
1200 Lys Val Asp Pro Asp Gln Trp Asn Gln Glu Thr Gly Glu Glu Glu
Arg 385 390 395 400 TTT GCC CTT GAA ATC CTA CCT GAT ATG CTT GAA AAG
CAT TAT GGA TAT 1248 Phe Ala Leu Glu Ile Leu Pro Asp Met Leu Glu
Lys His Tyr Gly Tyr 405 410 415 AAG TTG TTT ATA CCA GAT AGA GAT TTA
ATC CCA ACT GGA ACA TAC ATT 1296 Lys Leu Phe Ile Pro Asp Arg Asp
Leu Ile Pro Thr Gly Thr Tyr Ile 420 425 430 GAA GAT GTG GCA AGA TGT
GTA GAT CAA AGC AAG CGG CTG ATT ATT GTC 1344 Glu Asp Val Ala Arg
Cys Val Asp Gln Ser Lys Arg Leu Ile Ile Val 435 440 445 ATG ACC CCA
AAT TAC GTA GTT AGA AGG GGC TGG AGC ATC TTT GAG CTG 1392 Met Thr
Pro Asn Tyr Val Val Arg Arg Gly Trp Ser Ile Phe Glu Leu 450 455 460
GAA ACC AGA CTT CGA AAT ATG CTT GTG ACT GGA GAA ATT AAA GTG ATT
1440 Glu Thr Arg Leu Arg Asn Met Leu Val Thr Gly Glu Ile Lys Val
Ile 465 470 475 480 CTA ATT GAA TGC AGT GAA CTG AGA GGA ATT ATG AAC
TAC CAG GAG GTG 1488 Leu Ile Glu Cys Ser Glu Leu Arg Gly Ile Met
Asn Tyr Gln Glu Val 485 490 495 GAG GCC CTG AAG CAC ACC ATC AAG CTC
CTG ACG GTC ATT AAA TGG CAT 1536 Glu Ala Leu Lys His Thr Ile Lys
Leu Leu Thr Val Ile Lys Trp His 500 505 510 GGA CCA AAA TGC AAC AAG
TTG AAC TCC AAG TTC TGG AAA CGT TTA CAG 1584 Gly Pro Lys Cys Asn
Lys Leu Asn Ser Lys Phe Trp Lys Arg Leu Gln 515 520 525 TAT GAA ATG
CCT TTT AAG AGG ATA GAA CCC ATT ACA CAT GAG CAG GCT 1632 Tyr Glu
Met Pro Phe Lys Arg Ile Glu Pro Ile Thr His Glu Gln Ala 530 535 540
TTA GAT GTC AGT GAG CAA GGG CCT TTT GGG GAG CTG CAG ACT GTC TCG
1680 Leu Asp Val Ser Glu Gln Gly Pro Phe Gly Glu Leu Gln Thr Val
Ser 545
550 555 560 GCC ATT TCC ATG GCC GCG GCC ACC TCC ACA GCT CTA GCC ACT
GCC CAT 1728 Ala Ile Ser Met Ala Ala Ala Thr Ser Thr Ala Leu Ala
Thr Ala His 565 570 575 CCA GAT CTC CGT TCT ACC TTT CAC AAC ACG TAC
CAT TCA CAA ATG CGT 1776 Pro Asp Leu Arg Ser Thr Phe His Asn Thr
Tyr His Ser Gln Met Arg 580 585 590 CAG AAA CAC TAC TAC CGA AGC TAT
GAG TAC GAC GTA CCT CCT ACC GGC 1824 Gln Lys His Tyr Tyr Arg Ser
Tyr Glu Tyr Asp Val Pro Pro Thr Gly 595 600 605 ACC CTG CCT CTT ACC
TCC ATA GGC AAT CAG CAT ACC TAC TGT AAC ATC 1872 Thr Leu Pro Leu
Thr Ser Ile Gly Asn Gln His Thr Tyr Cys Asn Ile 610 615 620 CCT ATG
ACA CTC ATC AAC GGG CAG CGG CCA CAG ACA AAA TCG AGC AGG 1920 Pro
Met Thr Leu Ile Asn Gly Gln Arg Pro Gln Thr Lys Ser Ser Arg 625 630
635 640 GAG CAG AAT CCA GAT GAG GCC CAC ACA AAC AGT GCC ATC CTG CCG
CTG 1968 Glu Gln Asn Pro Asp Glu Ala His Thr Asn Ser Ala Ile Leu
Pro Leu 645 650 655 TTG CCA AGG GAG ACC AGT ATA TCC AGT GTG ATA TGG
TGACAGAAAA 2014 Leu Pro Arg Glu Thr Ser Ile Ser Ser Val Ile Trp 660
665 GCAAGGGACA TCCCGTCCCT GGGAGGTTGA GTGGAATCTG CAGTCCAGTG
CCTGGAACTA 2074 AATCCTCGAC TGCTGCTGTT AAAAAACATG CATTAGAATC
TTTAGAACAC GAGGAAAAAC 2134 AGGGTCTTGT ACATATGTTT TTTGGAATTT
CTTTGTAGCA TCAGTGTCCT CCTGTTTTAC 2194 CATGTCTTTT ACCATTACAT
TTTTTGACTT TGTTTTATAT GTCGTTGGAA TTTGTAAATT 2254 TACATTTTTT
TTAAAGAAGA GACTGATGTG TAGATAGAAA ACCCTTTTTT TGCTTCATTA 2314
GTTTAGTTTT AGAATGGGTT TTTATTTTAT TTCCTTTTTT AAAATTTTAC TTTGCTTTTA
2374 ACATTTCCTT GGGGTGCTTG AACAAATCTA TCCGATGGGA CAAGGAGCAC
CGGATTCTTT 2434 CTCGGGTTCT GCCTAGCATC AACTGGGCCA CGTCGGCCTT
CAGAGAACAG TGCAACAAAT 2494 GCCAGCATTG CCATTCGGGG GGAAAAAAAA
AAAAAAAAAA AAA 2537
[0065]
4TABLE 4 Alignment of the extracellular domains of various IL-1Rs.
hIL-1RD10 is SEQ ID NO: 20; hIL-1RD8 is SEQ ID NO: 3; mIL-1RD3 is
GenBank X85999; hIL-1RD6 is GenBank U49065; rIL-1RD6 is GenBank
U49066; mIL-1RD4 is GenBank Y07519 and GenBank D13695; hTL-1RD4 is
GenBank D12763; hIL-1RD2 is GenBank X59770; mIL-1RD2 is GenBank
X59769; hIL-1RD5 is GenBank U43672; mIL-1RD5 is GenBank U43673;
mIL- 1RD1 is GenBank M20658, M29752; hIL-1RD1 is GenBank X16896;
cIL-1RD1 is GenBank 86325; and hFGR4 is GenBank P22455. Other
species counterparts may be obtained from public sequence
databases. mIL-1RD3 .......... ......MGLL WYLMSLSFYG ILQSHASERC
DDWLDTMR.. hIL-1RDG .......... .........M WSLLLCGLSI ALPLSVTADG
CKDIFMKN.. rIL-1RDG .......... .......MGM PPLLFCWVSF VLPLFVAAGN
CTDVYMHH.. mIL-1RD4 .......... ........MI DRQRMGLWAL AILTLPMYLT
VTEGSKSS.. hIL-1RD4 .......... ........MG FWILAILTIL MYSTAAKFSK
QS........ hIL-1RD2 .......... ....MLRLYV LVMGVSAFTL QPAAHTGAAR
SCRFRGRHYK mIL-1RD2 MFILLVLVTG VSAFTTPTVV HTGKVSESPI TSEKPTVHGD
NCQFRGREFK hIL-1RD10 .......... .......... .......... ..........
.......... hIL-1RD5 .......... .....MNCRE LPLTLWVLIS VSTAESCTSR
PHITVVE... mIL-1RD5 .......... .....MHHEE LILTLCILIV KSASKSCIHR
SQIHVVE... mIL-1RD1 .......... .....MENMK VLLGLICLMV PLLSLEIDVC
TEYPNQIVLF hIL-1RD1 .......... ........MK VLLRLICFIA LLISSLEADK
CKEREEKIIL cIL-1RD1 .......... .... MHKMT STFLLIGHLI LLIPLFSAEE
CVICNYFVLV hIL-1RD8 .........M KPPFLLALVV CSVVSTNLKM VSKRNSVDGC
IDWSVDLKTY hFGR4 ...MRLLLAL LGVLLSVPGP PVLSLEASEE VELEPCLAPS
LEQQEQELTV mIL-1RD3 QIQVFEDEPA RIKCPLFEHF LKYNYSTAHS SGLTLLWYWT
RQDRDLEEPI hIL-1RD6 .EILSASQPF AFNCTFPPI. ........TS GEVSVTWYKN
....SSKIPV rIL-1RD6 .EMISEGQPF PFNCTYPPV. ........TN GAVNLTWHRT
....PSKSPI mIL-1RD4 ..WGLENEAL IVRCPQRG.. .........R STYPVEWYYS
....DTNESI hIL-1RD4 ..WGLENEAL IVRCPRQG.. .........K PSYTVDWYYS
....QTNKSI hIL-1RD2 REFRLEGEPV ALRCPQVPYW LWA....SVS PRINLTWHKN
....DSARTV mIL-1RD2 SELRLEGEPV VLRCPLAPHS DIS.....SS SHSFLTWSKL
....DSSQLI hIL-1RD10 .......... .......... .......... ..........
.......... hIL-1RD5 .....GEPFY LKHCSCSLAH ........EI ETTTKSWYKS
...SGSQEHV mIL-1RD5 .....GEPFY LKPCGISAPV .......HRN ETATMRWFKG
...SASHEYR mIL-1RD1 LSV...NEID IRKCPLTPN. ........KM HGDTIIWYKN
....DSKTPI hIL-1RD1 VSS..ANEID VRPCPLNPN. .........E HKGTITWYKD
....DSKTPV cIL-1RD1 ......GEPT AISCPVITL. ......PMLH SDYNLTWYRN
....GSNMPI hIL-1RD8 ..MALAGEPV RVKCALFYSY IRTNYSTAQS TGLRLMWYKN
..KGDLEEPI hFGR4 ....ALGQPV RLCCGRAERG G......... .....HWYKE
....GSRLAP mIL-1RD3 NFRLP.ENRI SKEKDVLWFR PTLLNDTGNY TCMLRNTTYC
SKVAFPLEVV hIL-1RD6 SKII..QSRI HQDETWILFL PMEWGDSGVY QCVIKGRDSC
HRIHVNLTVF rIL-1RD6 SINR..HVRI HQDQSWILFL PLALEDSGIY QCVIKDAHSC
YRIAINLTVF mIL-1RD4 PTQK..RNRI FVSRDRLKFL PARVEDSGIY ACVIRSPNLN
KTGYLNVTIH hIL-1RD4 PTQE..RNRV FASGQLLKFL PAEVADSGIY TCIVRSPTFN
RTGYANVTIY hIL-1RD2 PGEE..ETRM WAQDGALWLL PALQEDSGTY VCTTRNASYC
DKMSIELRVF mIL-1RD2 PRDEP...RM WVKGNILWIL PAVQQDSGTY ICTFRNASHC
EQMSVELKVF hIL-1RD10 .......... .......... .......... ..........
.......... hIL-1RD5 ELNPRSSSRI ALHDCVLEFW PVELNDTGSY FFQMKN..YT
QKWKLNVIRR mIL-1RD5 ELNNRSSPRV TFHDHTLEFW PVEMEDEGTY ISQVGN..DR
RNWTLNVTKR mIL-1RD1 SADR..DSRI HQQNEHLWFV PAKVEDSGYY YCIVRNSTYC
LKTKVTVTVL hIL-1RD1 STEQ..ASRI HQHKEKLWFV PAKVEDSGHY YCVVRNSSYC
LRIKISAKFV cIL-1RD1 TTER..RARI HQRKGLLWFI PAALEDSGLY ECEVRSLNRS
KQKIINLKVF hIL-1RD8 IFS...EVRN SKEEDSIWFH SAEAQDSGFY TCVLRNSTYC
MKVSMSLTVA hFGR4 AG......RV RGWRGRLEIA SFLPEDAGRY LCLARGSMIV
LQNLTLITGD mIL-1RD3 QK........ .......... .......DSC FNSANRFPVH
KMYIEHGIHK hIL-1RD6 EK........ .......... .HWCDTSIGG LP.NLSDEYK
QILHLGKDDS rIL-1RD6 RK........ .......... .HWCDSSNEE SSINSSDEYQ
QWLPIGKSGS mIL-1RD4 KK........ .......... .....PPSCN .IPDY.LNYS
TVRGSDKNFK hIL-1RD4 KK........ .......... .....QSDCN .VPDY.LMYS
TVSGSEKNSK hIL-1RD2 EN........ .......... .......TDA FLPFI..SYP
QILTLSTSGV mIL-1RD2 KN........ .......... .......TEA SLPHV..SYL
QISALSTTGL hIL-1RD10 .......... .......... .......... ..........
.......... hIL-1RD5 NK........ .......... .......HSC FTERQ..VTS
KIVEVKKFFQ mIL-1RD5 NK........ .......... .......HSC FSDKL..VTS
RDVEVNKSLH mIL-1RD1 EN........ .......... .....DPGIC .YSTQ.ATFP
QRLHIAGDGS hIL-1RD1 EN........ .......... .....EPNLC .YNAQ.AIFK
QKLPVAGDGG cIL-1RD1 KN........ .......... .....DNGLC .FNGE.MKYD
QIVKSANAGK hIL-1RD8 EN........ .......... .....ESGLC .YNSR.IRYL
EKSEVTKRKE hFGR4 SLTSSNDDED PKSHRDPSNR HSYPQQAPYW THPQRMEKKL
HAVPAGNTVK mIL-1RD3 ITCPNVDGYF P.SSVKPSVT WYKGCTEIVD FHN...VLPE
GMNLSFFIPL hIL-1RD6 LTCHLHFPKS ...CVLGPIK WYKDCNEIKG E......RFT
VLETRLLVSN rIL-1RD6 LTCHLYFPES ...CVLDSIK WYKGCEEIKV S.....KKFC
PTGTKLLVNN mIL-1RD4 ITCPTIDLY ...NWTAPVQ WFKNCKALQE P......RFR
AHRSYLFIDN hIL-1RD4 IYCPTIDLY ...NWTAPLE WFKNCQALQG S......RYR
AHKSFLVIDN hIL-1RD2 LVCPDLSEFT R.DKTDVKIQ WYKDSLLLDK DNEK..FLSV
RGTTHLLVHD mIL-1RD2 LVCPDLKEFI S.SNADGKIQ WYKGAILLDK GNKE..FLSA
GDPTRLLISN hIL-1RD10 .......... .......... .......... ..........
.......... hIL-1RD5 ITCENEYYQ ...TLVNSTS LYKNCKKLLL ENN....KNP
TIKKNAEF.. mIL-1RD5 ITCKNPNYE ...ELIQDTW LYKNCKEISK TPRI...LKD
AEFGDAEF.. mIL-1RD1 LVCPYVSYFK DENNELPEVQ WYKNCKPLLL DN....VSFF
GVKDKLLVRN hIL-1RD1 LVCPYMEFFK NENNELPKLQ WYKDCKPLLL DN....IHFS
GVKDRLTVMN cIL-1RD1 IICPDLENFK DEDNINPEIH WYKECKSGFL EDKR..LVLA
EGENAILILN hIL-1RD8 ISCPDMDDFK KED.QEPDVV WYKECKPKMW R.....SIII
QKGNALLIQE hFGR4 FRCPAAG... ...NPTPTIR WLKDGQAFHG ENRIGGIRLR
HQHWSLVMES mIL-1RD3 VSNN..GNYT CVVTYPENGR LFHLTRTVTV KVVGS.PKDA
LPPQIYSPND hIL-1RD6 VSAEDRGNYA CQAILTHSGK QYEVLNGITV SITERAGYGG
SVP.KIIYPK rIL-1RD6 IDVEDSGSYA CSARLTHLGR IFTVRNYIAV NTKE.VGSGG
RIP.NITYPK mIL-1RD4 VTHDDEGDYT CQFTHAENGT NYIVTATRSF TVE.EKGFS
MFPVITNPPY hIL-1RD4 VMTEDAGDYT CKFIHNENGA NYSVTATRSF TVKDEQGFS
LFPVIGAPAQ hIL-1RD2 VALEDAGYYR CVLTFAHEGQ QYNITRSIEL RIKKK..KEE
TIPVIISP.. mIL-1RD2 TSMDDAGYYR CVMTFTYNGQ EYNITRNIEL RVKGT..TTE
PIPVIISP.. hIL-1RD10 ...EFG..TS CEL..KYGGF V..VRRTTEL TVTAPLTDKP
PKLLYPMESK hIL-1RD5 ...EDQGYYS CVHFLNHNGK LFNITKTFNI TIVED..RSN
IVPVLLGP.K mIL-1RD5 ...GDEGYYS CVFSVHHNGT RYNITKTVNI TVIEG..RSK
VTPAILGP.K mIL-1RD1 VAEEHRGDYI CRMSYTFRGK QYPVIRVTQF ITIDE..NKR
DRPVILSP.R hIL-1RD1 VAEKHRGNYT CHASYTYLGK QYPITRVIEF ITLEE..NKP
TRPVIVSP.A cIL-1RD1 VTIQDKGNYT CRMVYTYMGK QYNVSRTMNL EVKES..PLK
MRPEFIYP.N hIL-1RD8 VQEEDGGNYT CEL..KYEGK L..VRRTTEL KVTALLTDKP
PKPLFPMENQ hFGR4 VVPSDRGTYT CLVENAVGSI RYNYLLDVLE RSPH..RPIL
QAGLPANTT. mIL-1RD3 RVVYEKEPGE ELVIPCKVYF SFIMD.SHNE VWWTIDGKKP
.DDVTVDITI hTL-1RD6 NHSTEVQLGT TLIVDCNVTD TK..D.NTNL RCWRVNNTLV
DDYYDESKRI rIL-1RD6 NNSIEVQLGS TLIVDCNITD TK..E.NTNL RCWRVNNTLV
DDYYNDFKRI mIL-1RD4 NHTMEVEIGK PASIACSACF GKGSH.FLAD VLWQINKTVV
GNFGEARIQE hIL-1RD4 NEIKEVEIGK NANLTCSACF GKGTQ.FLAA VLWQLNGTKI
TDFGEPRIQQ hIL-1RD2 LKTTSASLGS RLTTPCKVFL GTGTP.LTTM LWWTANDTHI
.ESAYPGGRV mIL-1RD2 LETIPASLGS RLIVPCKVFL GTGTS.SNTI VWWLANSTFI
.SAAYPRGRV hIL-1RD10 LTIQETQLGD SANLTCRAFF GYSGD.VSPL IYWMKGEKFI
EDLDENRVWE hTL-1RD5 LNHVAVELGK NVRLNCSALL N.....EEDV IYWNFGEENG
...SDPNIHE mIL-1RD5 CEKVGVELGK DVELNCSASL N.....KDDL FYWSIRKEDS
...SDPNVQE mIL-1RD1 NETIEADPGS MIQLICNVTG Q.....FSDL VYWKWNGSEI
.EWNDPFLAE hIL-1RD1 NETMEVDLGS QIQLICNVTG Q.....LSDI AYWKWNGSVI
.DEDDPVLGE cIL-1RD1 NNTIEVELGS HVVMECNVSS GV....YGLL PYWQVNDEDV
.DSFDSTYRE hIL-1RD8 PSVIDVQLGK PLNIPCKAFF GFSGE.SGPM IYWMKGEKFI
.EELAGHIRE hFGR4 .....AVVGS DVELLCKVYS DA...QPHIQ ..WLKHIVIN
GSSFGA..DG mIL-1RD3 NESVSYSSTE D..ETRTQIL SIKKVTPEDL RRNYVCHARN
TKGEAEQAAK hIL-1RD6 REGVETHVSF REHNLYTVNI TFLEVKMEDY GLPFMCHAG
...VSTAYII rIL-1RD6 QEGTETNLSL RNHILYTVNI TFLEVKMEDY GHPFTCHAA
...VSAAYII mIL-1RD4 EEGRNESSSN D.MDCLTSVL RITGVTEKDL SLEYDCLALN
LHGMIRHTIR hIL-1RD4 EEGQNQSFSN G.LACLDMVL RIADVKEEDL LLQYDCLALN
LHGLRRHTVR hIL-1RD2 TEGPRQEYSE NNENYIEVPL IFDPVTREDL HMDFKCVVHN
TLSFQTLRTT mIL-1RD2 TEGLHHQYSE NDENYVEVSL IFDPVTREDL HTDFKCVASN
PRSSQSLHTT hIL-1RD10 SDIRILKEHL G.EQEVSISL IVDSVEEGDL .GNYSCYVEN
GNGRRHASVL hIL-1RD5 EKEMRIMTPE G.KWHASKVL RIENIGESNL NVLYNCTVAS
TGGTDTKSFI mIL-1RD5 DRKETTTWIS EGKLHASKIL RFQKITENYL NVLYNCTVAN
EEAIDTKSFV mIL-1RD1 DYQFVEHPST KRKYTLITTL NISEVKSQFY RYPFICVVKN
TNIFESAHVQ hIL-1RD1 DYYSVENPAN KRRSTLITVL NISEIESRFY KHPFTCFAKN
THGIDAAYIQ cIL-1RD1 QFYEEGMPHG ..IAVSGTKF NISEVKLKDY AYKFFCHFIY
DSQEFTSYIK hIL-1RD8 GEIRLLKEHL G.EKEVELAL IFDSVVEADL AN.YTCHVEN
RNGRKHASVL hFGR4 FPYVQVLKTA DINSSEVEVL YLRNVSAED AGEYTCLAGN
SIGLSYQSAW mIL-1RD3 VKQKV....I PPRYTVELAC GFGATVFLVV VLIVVY
hIL-1RD6 LQLP.....A PDFRAYLIGG LIALVAVAVS VVYIYNIFKI DIVLWY
rIL-1RD6 LKRP.....A PDFRAYLIGG LMAFLLLAVS ILYIYNTFKV DIVLWY
mIL-1RD4 LRRK.....Q PSKECPSHIA IYYIVAGCSL LLMFINVLVI VL hIL-1RD4
LSRK.....N PSKEC hIL-1RD2 VKEASS .TFSWGIVLA PLSLAFLVLG GIWM
mIL-1RD2 VKEVSS .TFSWSIALA PLSLIILVVG AIW. hIL-1RD10 LHKREL
.MYTVELAGG LGATLLLLVC LVTIYKCY hIL-1RD5 LVRKADMADI P..GHVFTRG
MIIAVLILVA VVCLVTVCVI Y mIL-1RD5 LVRKEIPDIP ...GHVFTGG VTVLVLASVA
AVCIVILCVI Y mIL-1RD1 LTYP.....V PDFKNYLIGG FIILTATIVC CVCIY
hIL-1RD1 LIYP.....V TNFQKHMIGI CVTLTVIIVC SVFIY cIL-1RD1 LEHP.....V
QNIRGYLIGG GISLIFLLFL ILIVY hIL-1RD8 LRKKDL .TYKIELAGG LGAIFLLLVL
LVVIYKCY hFGR4 LTVLP....E EDPTWTAAAP EARYTDIILY ASGSLALAVL LLLAGLY
Alignment of the intracellular domains of various IL-1Rs. hIL-1RD9
is SEQ ID NO: 8; mIL-1RD9 is SEQ ID NO: 14; hIL-1RD1 is GenBank
X16896; hIL-1RD6 is GenBank U49065; mIL-1RD3 is GenBank X85999;
huIL-1RD8 is SEQ ID NO: 3; and mIL-1RD4 is GenBank Y07519.
HuIL-1RD1 SDGKTYDAYI LYPKTVGEG. ..STSDCDIF VFKVLPEVLE KQCGYKLFIY
HuIL-1RD6 VDGKLYDAYV LYPKPHKES. ..QRHAVDAL VLNILPEVLE RQCGYKLFIF
MoIL-1RD3 LDGKEYDIYV SYAR...... ...NVEEEEF VLLTLRGVLE NEFGYKLCIF
HuIL-1RD8 DDNKEYDAYL SYTKVDQDTL DCDNPEEEQF ALEVLPDVLE KHYGYKLFIP
HuIL-1RD5 TDGKTYDAFV SYLKECRP.. ..ENGEEHTF AVEILPRVLE KHFGYKLCIF
MoIL-1RD9 .......... .......... .......... .......... ..........
HuIL-1RD9 .......... .......... .......... .......... .KYGYSLCLL
MoIL-1RD4 NDGKLYDAYI IYPRVFRGS AAGTHSVEYF VHETLPDVLE NKCGYKLCIY
HuIL-1RD1 GRDDYV.GED IVEVINENVK KSRRLIIILV RETSGFSWLG GSSEEQIAMY
HuIL-1RD6 GRDEFP.GQA VANVIDENVK LCRRLIVIVV PESLGFGLLK NLSEEQIAVY
MoIL-1RD3 DRDSLPGGIV TDETLS.FIQ KSRRLLVVLS PNYVLQG.TQ ALLELKAGLE
HuIL-1RD8 ERDLIPSG.T YMEDLTRYVE QSRRLIIVLT PDYILRR.GW SIFELESRLH
HuIL-1RD5 ERDVVPGGAV VDEIHS.LIE KSRRLIIVLS KSYMSN...E VRYELESGLH
MoIL-1RD9 DRDVTP.GGV YADDIVSIIK KSRRGIFILS PSYLNG...P RVFELQAAVN
HuIL-1RD9 ERDVAP.GGV YAEDIVSIIK RSRRGIFILS PNYVNG...P SIFELQAAVN
MoIL-1RD4 GRDLLP.GQD AATVVESSIQ NSRRQVFVLA PHNMHSK..E FAYEQEIALH
HuIL-1RD1 NALVQDGIKV VLLELEKIQ. .....DYEKM PESIKFIKQK HGAIRWSGDF
HuIL-1RD6 SALIQDGMKV ILIELEKIE. .....DYTVM PESIQYIKQK HGAIRWHGDF
MoIL-1RD3 NMASRGNINV ILVQYKAVK. ...DMKVKEL KRAKTVLT.. ..VIKWKGEK
HuIL-1RD8 NMLVSGEIKV ILIECTELKG KVNCQEVESL KRSIKLLS.. ..LIKWKGSK
HuIL-1RD5 EALVERKIKI ILIEFTPVT. .....DFTFL PQSLKLLKSH R.VLKWKADK
MoIL-1RD9 LALVDQTLKL ILIKFCSFQ. .....EPESL PYLVKKALRV LPTVTWKGLK
HuIL-1RD9 LALDDQTLKL ILIKFCYFQ. .....EPESL PHLVKKALRV LPTVTWRGLK
MoIL-1RD4 SALIQNNSKV ILIEMEPLG. EASRLQVGDL QDSLQHLVKI QGTIKWREDH
HuIL-1RD1 TQGPQSAKTR FWKNVRYHMP VQRRSPSSKH HuIL-1RD6 TEQSQCMKTK
FWKTVRYHMP PRRCRPFLRS MoIL-1RD3 SKYPQ...GR FWKQLQVAMP VKKSPRWSSN
HuIL-1RD8 SSKLN...SK FWKHLVYEMP IKKKEMLPRC HuIL-1RD5 SLSYN...SR
FWKNLLYLMP AKTVKPGRDE MoIL-1RD9 SVHAS...SR FWTQIRYHMP VKNSNRFMFN
HuIL-1RD9 SVPPN...SR FWAKMRYHMP VKNSQGFTWN MoIL-1RD4 VADKQSLSSK
FWKHVRYQMP VPERASKTAS hRD8 MKPPFLLALVVCSVVSTNLKMVSKRNSVDG-
CIDWSVD-LKTYMALAGEPV hRD10
----------------------------DGCTDWSTD-IK- KYQVLVGEPV hRD3
------MTLLWC-VVSLYFYGILQSDASERCDDWGLDTMRQIQVFEDEPA mRD3
------MGLLWY-LMSLSFYGILQSHASERCDDWGLDTMRQIQVFEDEPA : * **.:* ::
.:.**. hRD8 RVKCALFYSYIRTNYSTAQSTGLRLMWYKNKG--DLEEPIIFS--EVRMS
hRD10 RIKCALFYGYIRTNYSLAQSAGLSLMWYKSSGPGDFEEPIAFD--GSRMS hRD3
RIKCPLFEHFLKFNYSTAHSAGLTLIWYWTRQDRDLEEPINFRLPENRIS mRD3
RIKCPLFEHFLKYNYSTAHSSGLTLIWYWTRQDRDLEEPINFRLPENRIS *:**.**
:::****:*:***:**. *:**** * *:* hRD8
KEEDSIWFHSAEAQDSGFYTCVLRNSTYCMKVSMSLTVAENESGLCYNSR hRD10
KEEDSIWFRPTLLQDSGLYACVIRNSTYCMKVSISLTVGENDTGLCYNSK hRD3
KEKDVLWFRPTLLNDTGNYTCMLRNTTYCSKVAFPLEVVQKDS--CFNSP mRD3
KEKDVLWFRPTLLNDTGNYTCMLRNTTYCSKVAFPLEVVQKDS--CFNSA **:*.:**:.: :*:*
*:*::**:*** **::.*.*.:::: *:** hRD8
IRY-LEKSEVTK-RKEISCPDMDDFKKSDQEPDVVWYKECKPKMWRSIII hRD10
MKY-FEKAELSK-SKEISCRDIEDFLLPTREPEILWYKECRTKTWRPSIV hRD3
MKLPVHKLYIEYGIQRITCPNVDGYFPSSVKPTITWYMGCYKIQNFNNVI mRD3
MRFPVHKMYIEHGIHKITCPNVDGYFPSSVKPSVTWYKGCTEIVDFHNVL :: * :
:.*:*.:::.: . :*:** * :: hRD8
QKGN--ALLIQEVQEEDGGNYTCELKY--EGKLVRRTTELKVTALLTDK hRD10
FKRD--TLLIREVREDDIGNYTCELKY--GGFVVRRTTELTVTAPLTDK hRD3
PEGMNLSFLIALISNN--GNYTCVVTYPENGRTFHLTRTLTVKVVGSPKN mRD3
PEGMNLSFFIPLVSNN--GNYTCVVTYPENGRLFHLTRTVTVKVVGSPKD : :::* :::
*****:.* * .:.* :.*.. :* hRD8
--PPKPLFPMENQPSVIDVQLGKPLNIPCKAFFGFSGESGPMIYWMKGEK hRD10
--PPKLLYPMESKLTTQETQLGDSANLTCRAFFGYSGDVSPLIYWMKGEK hRD3
AVPPVIHSPNDH--VVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTIDGK mRD3
ALPPQIYSPNDR--VVYEKEPGEELVIPCKVYFSFIMDSHNEVWWTIDGK ** *.: :.:.:.*.
:.* :*.: : ::* . * hRD8
FIEEL-AGHIREGEIRLLKEHLGEKEVELALIFDSVVEADLA-NYTCHVE hRD10
FIEDLDENRVWESDIRILKEHLGEQEVSISLIVDSVEEGDLG-NYSCYVE hRD3
KPDDI-TIDVTINESISHSRTEDETRTQI-LSIKKVTSEDLKRSYVCHAR mRD3
KPDDV-TVDITINESVSYSSTEDETRTQI-LSIKKVTPEDLRRNYVCHAR ::: : : . . *
...: * .* ** .* *:.. hRD8
NRNGR--KHASVLLRKKDLIYKIELAGGLGAIFLLLVLLVVIYKCYNIEL hRD10
NGNGR--RHASVLLHKRELMYTVELAGGLGAILLLLVCLVTIYKCYKIEI hRD3
SAKGEVAKAAKVKQKVPAPRYTVELACGFGATVLLVVILIVVYHVYWLEM mRD3
NTKGEAEQAAKVKQKVIPPRYTVELACGFGATVFLVVVLIVVYHVYWLEM . :*. : *.* :
*.:*** *:** .:*:* *: :*: * :*: hRD8
MLFYRQHFGADETNDDNKEYDAYLSYTKVDQDTLDCDNPEEEQFALEVLP hRD10
MLFYRNHFGAEELDGDNKDYDAYLSYTKVDPDQWNQETGEEERFALEILP hRD3
VLFYRAHFGTDETILDGKEYDIYVSYAR---------NAEEEEFVLLTLR mRD3
VLFYRAHFGTDETILDGKEYDIYVSYAR---------NVEEEEFVLLTLR :**** ***::*
*.*:** *:**:: . ***.*.* * hRD8
DVLEKHYGYKLFIPERDLIPSGTYMEDLTRYVEQSRRLIIVLTPDYILRR hRD10
DMLEKHYGYKLFIPDRDLIPTGTYIEDVARCVDQSKRLIIVMTPNYVVRR hRD3
GVLENEFGYKLCIFDRDSLPGGIVTDETLSFIQKSRRLLVVLSPNYVLQG mRD3
GVLENEFGYKLCIFDRDSLPGGIVTDETLSFIQKSRRLLVVLSPNYVLQG .:**:.:**** *
:** :* * :: :::*:**::*::*:*::: hRD8
GWSIFELESRLHNMLVSGEIKVILIECTELKGKVNCQEVESLKRSIKLLS hRD10
GWSIFELETRLRNMLVTGEIKVILIECSELRGIMNYQEVEALKHTIKLLT hRD3
TQALLELKAGLENMASRGNINVILVQYKAVK----ETKVKELKRAKTVLT mRD3
TQALLELKAGLENMASRGNINVILVQYKAVK----DMKVKELKRAKTVLT :::**:: *.**
*:*:***:: . :: :*: **:: .:*: hRD8
LIKWKGSKSSKLNSKFWKHLVYEMPIKKKEMLPRCHVLDSAEQGL-FGEL hRD10
VIKWHGPKCNKLNSKFWKRLQYEMPFKRIEPITHEQALDVSEQGP-FGEL hRD3
VIKWKGEKSKYPQGRFWKQLQVAMPVKKS---PRRSSSD--EQGLSYSSL mRD3
VIKWKGEKSKYPQGRFWKQLQVAMPVKKS---PRWSSND--KQGLSYSSL :***:* *..
:.:***:* **.*: .: * :** :..* hRD8
QPIPSIAMTS-TSATLVSSQADLP-EFHPS--DSMQIRHCCRGYKHEIPA hRD10
QTVSAISMAAATSTALATAHPDLRSTFHNTYHSQMRQKHYYRSYEYDVPP hRD3
KNV----------------------------------------------- mRD3
KNV----------------------------------------------- : : hRD8
T-TLPVPSLGNHHTYCNLPLTLLNGQLPLNNTLKDT--QEFHRNSSLLPL hRD10
TGTLPLTSIGNQHTYCNIPMTLINGQRPQTKSSREQNPDEAHTNSAILPL hRD3
-------------------------------------------------- mRD3
-------------------------------------------------- hRD8
SSKELSFTSDTW hRD10 LPRETSISSVIW hRD3 ------------ mRD3 ------------
Alignment and comparison of primate and rodent IL-1RD9. hIL-1RD9
MLCLGWIFLWLVAGERIKGFNISGCSTKKLLWT- YSTRSEEEFVLFCDLPE mIL-1RD9
MLCLGWVFLWFVAGEKTTGFNHSACATKKLLWTYSARGAE- NFVLFCDLQE ******.***
****. *** * *.*********.* * ******* * hIL-1RD9
PQKSHFCHRNRLSPKQVPEHLPFMGSN-DLSDVQWYQQPSNGDPLEDIRK mIL-1RD9
LQEQKFSHASQLSPTQSPAHKPCSGSQKDLSDVQWYMQPRSGSPLEEISR
* .*.* .*** * * * * **. ******** ** * ***.* . hIL-1RD9
SYPHIIQDKCTLHFLTPGVNNSGSYICRPKMIKSPYDVACCVKMILEVKP mIL-1RD9
NSPHMQSE-GMLHILAPQTNSIWSYICRPR-IRSPQDMACCIKTVLEVKP **. . ** *.* *
******. *.** *.***.* .***** hIL-1RD9
QTNASCEYSASHKQDLLLGSTGSISCPSLSCQSDAQSPAVTWYKNGKLLS mIL-1RD9
QRNVSCGNTAQDEQVLLLGSTGSIHCPSLSCQSDVQSPEMTWYKDGRLLP * * ** .* *
********* ********* *** .**** *.** hIL-1RD9
VERSNRIVVDEVYDYHQGTYVCDYTQSDTVSSWTVRAVVQVRTIVGDTKL mIL-1RD9
EHKKNPIEMADIYVFNQGLYVCDYTQSDNVSSWTVRAVVKVRTIGKDINV . * * . ..* ..**
*********.**********.**** * . hIL-1RD9
KPDILDPVEDTLEVELGKPLTISCKARFGFERVFNPVIKWYIKDSDLEWE mIL-1RD9
KPEILDPITDTLDVELGKPLTLPCRVQFGFQRLSKPVIKWYVKESTQEWE **.****.
***.********. *. .***.*. ******.*.* *** hIL-1RD9
VSVPEAKSIKSTLKDEIIERNIILEKVTQRDLRRKFVCFVQNSIGNTTQS mIL-1RD9
MSVFEEKRIQSTFKNEVIERTIFLREVTQRDLSRKFVCFAQNSIGNTTRT .** * * *.** *
*.***.* * ****** ****** ********.. hIL-1RD9
VQLKEKRGVVLLYILLGTIGTLVAVLAASALLYRHWIEIVLLYRTYQSKD mIL-1RD9
IRLRKKEEVVFVYILLGTALMLVGVLVAAAFLYWYWIEVVLLCRTYKNKD ..*. * **
.****** ** ** *.* ** ***.*** ***. ** hIL-1RD9
QTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYS mIL-1RD9
ETLGDKKEFDAFVSYSNWSSPETDAVGSLSEEHLALNLFPEVLEDTYGYR .******.*******.
***. * ********* ***.*** *** hIL-1RD9
LCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVN mIL-1RD9
LCLLDRDVTPGGVYADDIVSIIKKSRRGIFILSPSYLNGPRVFELQAAVN
****.***.******.*******.********** *.*** .******** hIL-1RD9
LALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNS mIL-1RD9
LALVDQTLKLILIKFCSFQEPESLPYLVKKALRVLPTVTWKGLKSVHASS *** ************
******** **************.***** * hIL-1RD9
RFWAKMRYHMPVKNSQGFTWNQLRITSRIFQ-------WKGLSRTETTGR mIL-1RD9
RFWTQIRYHMPVKNSNRFMFNGLRIFLKGFSPEKDLVTQKPLEGMPKSGN ***...*********.
* * *** . * * * . * hIL-1RD9 ----------SSQPKEW mlL-1RD9
DHGAQNLLLYSDQKRC * * .
[0066] Structural analysis of the primate IL-1RD10 sequence (SEQ ID
NO: 18, 20, and 35), in comparison with other IL-1Rs, shows
characteristic features exist, which are conserved with the
IL-1RD10 embodiment described herein. For example, there are
characteristic Ig domains, and subdomains therein. The
corresponding regions of the IL-1RD10 (SEQ ID NO: 18 and 20) are
about: f2 to gly7; g2 from val10 to thr23; a3 from leu30 to met33;
a3' from thr38 to gln40; b3 from ala48 to ala54; c3 from pro64 to
lys70; c3' from glu72 to phe74; d3 from val83 to lys92; e3 from
gln98 to val106; and f3 from tyr117 to trp126.
[0067] Structural analysis of the rodent IL-1RD9 sequence (SEQ ID
NO: 12, 14, and 16), in comparison with other IL-1Rs, shows
characteristic features exist (see Table 4). For example, there are
characteristic Ig domains, and subdomains therein. The
corresponding regions of the IL-1RD9 (SEQ ID NO: 12, 14, and 16)
are about: Ig1 domain from gly18 to pro127, with cys105 probably
linked to cys52 (or possibly cys48); Ig2 domain from gly128 to
pro229, with cys153 probably linked to cys199; and the Ig3 domain
from glu230 to lys333, with cys251 probably linked to cys315;
transmembrane segment from val336 to tyr360; THD domain from gly381
to val539; conserved trp residues probably correspond to residues
64, 169, and 267. Alignment of the IL-1RD9 embodiments is shown in
Table 4. There are characteristic beta strand sections, and alpha
helical structures, as described above for IL-1RD10. The
corresponding segments of the human IL-1RD9 sequence (SEQ ID NO: 6,
8, and 10) are roughly: .beta.B from gly3 to val13; .alpha.2 from
pro15 to lys28; .beta.c from ser30 to ser46; .alpha.3 from ile47 to
gln61; .beta.D from lys64 to glu75; .alpha.4 from glu77 to leu87;
.beta.E from val93 to leu98; and .alpha.5 from arg106 to val117.
The corresponding segments of the mouse IL-1RD9 sequence (SEQ ID
NO: 12, 14, and 16) are roughly: .alpha.3 to gln10; .beta.D from
lys13 to glu24; .alpha.4 from glu26 to leu36; .beta.E from va42 to
leu47; and .alpha.5 from arg55 to val66.
[0068] As used herein, the terms IL-1 like receptor D8 (IL-1RD8),
IL-1 like receptor D9 (IL-1RD9), or IL-1 like receptor D10
(IL-1RD10) shall be used to describe a polypeptide comprising a
segment having or sharing the amino acid sequence shown in Tables
1, 2, or 3, or a substantial fragment thereof. The invention also
includes a polypeptide variation of the respective IL-1RD8,
IL-1RD9, IL-1RD10 alleles whose sequences are provided, e,g., a
mutein or soluble extracellular or intracellular construct.
Typically, such agonists or antagonists will exhibit less than
about 10% sequence differences, and thus will often have between 1-
and 11-fold substitutions, e.g., 2-, 3-, 5-, 7-fold, and others. It
also encompasses allelic and other variants, e.g., natural
polymorphic, of the polypeptide described. Typically, it will bind
to its corresponding biological ligand, perhaps in a dimerized
state with an alpha receptor subunit, with high affinity, e.g., at
least about 100 nM, usually better than about 30 nM, preferably
better than about 10 nM, and more preferably at better than about 3
nM. The term shall also be used herein to refer to related
naturally occurring forms, e.g., alleles, polymorphic variants, and
metabolic variants of the mammalian protein.
[0069] This invention also encompasses polypeptides having
substantial amino acid sequence identity with the amino acid
sequences in Tables 1-3, preferably having segments of contiguous
amino acid residues identical to segments of SEQ ID NO: 4, 10, or
35. It will include sequence variants with relatively few
substitutions, e.g., typically less than about 25, ordinarily less
than about 15, preferably less than about 3-5. Other embodiments
include forms in association with an alpha subunit, e.g., an
IL-1RD4, IL-1RD5, or IL-1RD6.
[0070] A substantial polypeptide "fragment", or "segment", is a
stretch of amino acid residues of at least about 8 contiguous amino
acids, generally at least 10 contiguous amino acids, more generally
at least 12 contiguous amino acids, often at least 14 contiguous
amino acids, more often at least 16 contiguous amino acids,
typically at least 18 contiguous amino acids, more typically at
least 20 contiguous amino acids, usually at least 22 contiguous
amino acids, more usually at least 24 contiguous amino acids,
preferably at least 26 contiguous amino acids, more preferably at
least 28 contiguous amino acids, and, in particularly preferred
embodiments, at least about 30 or more contiguous amino acids,
usually 40, 50, 70, 90, 110, etc. Sequences of segments of
different polypeptides can be compared to one another over
appropriate length stretches. In many cases, the matching will
involve a plurality of distinct, e.g., nonoverlapping, segments of
the specified length. Typically, the plurality will be at least
two, more usually at least three, and preferably 5, 7, or even
more. While the length minima are provided, longer lengths, of
various sizes, may be appropriate, e.g., one of length 7, and two
of length 12. Similar features apply to segments of nucleic
acid.
[0071] Amino acid sequence homology, or sequence identity, is
determined by optimizing residue matches, if necessary, by
introducing gaps as required. See, e.g., Needleham, et al. (1970)
J. Mol. Biol. 48:443-453; Sankoff, et al. (1983) chapter one in
Time Warps, String Edits, and Macromolecules: The Theory and
Practice of Sequence Comparison, Addison-Wesley, Reading, Mass.;
and software packages from IntelliGenetics, Mountain View, Ca.; and
the University of Wisconsin Genetics Computer Group (GCG), Madison,
Wis.; each of which is incorporated herein by reference. This
changes when considering conservative substitutions as matches.
Conservative substitutions typically include substitutions within
the following groups: glycine, alanine; valine, isoleucine,
leucine; aspartic acid, glutamic acid; asparagine, glutamine;
serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
Homologous amino acid sequences are intended to include natural
allelic and interspecies variations in the cytokine sequence.
Typical homologous polypeptides will have from 50-100% homology (if
gaps can be introduced), to 60-100% homology (if conservative
substitutions are included) with an amino acid sequence segment of
Table 1, 2, or 3. Homology measures will be at least about 70%,
generally at least 76%, more generally at least 81%, often at least
85%, more often at least 88%, typically at least 90%, more
typically at least 92%, usually at least 94%, more usually at least
95%, preferably at least 96%, and more preferably at least 97%, and
in particularly preferred embodiments, at least 98% or more. The
degree of homology will vary with the length of the compared
segments. Homologous polypeptides, such as the allelic variants,
will share most biological activities with the embodiments
described in Table 1, 2, or 3.
[0072] As used herein, the term "biological activity" is used to
describe, without limitation, effects on inflammatory responses,
innate immunity, and/or morphogenic development by respective
ligands. For example, these receptors should, like IL-1 receptors,
mediate phosphatase or phosphorylase activities, which activities
are easily measured by standard procedures. See, e.g., Hardie, et
al. (eds. 1995) The Protein Kinase FactBook vols. I and II,
Academic Press, San Diego, Calif.; Hanks, et al. (1991) Meth.
Enzymol. 200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin
(1990) Cell 61:743-752; Pines, et al. (1991) Cold Spring Harbor
Symp. Quant. Biol. 56:449-463; and Parker, et al. (1993) Nature
363:736-738. Other activities include antigenic or immunogenic
functions. The receptors exhibit biological activities much like
regulatable enzymes, regulated by ligand binding. However, the
enzyme turnover number is more close to an enzyme than a receptor
complex. Moreover, the numbers of occupied receptors necessary to
induce such enzymatic activity is less than most receptor systems,
and may number closer to dozens per cell, in contrast to most
receptors which will trigger at numbers in the thousands per cell.
The receptors, or portions thereof, may be useful as phosphate
labeling enzymes to label general or specific substrates.
[0073] The terms ligand, agonist, antagonist, and analog of, e.g.,
an IL-1RD8, IL-1RD9, or IL-1RD10, include molecules that modulate
the characteristic cellular responses to IL-1 ligand proteins, as
well as molecules possessing the more standard structural binding
competition features of ligand-receptor interactions, e.g., where
the receptor is a natural receptor or an antibody. The cellular
responses likely are mediated through binding of various IL-1
ligands to cellular receptors related to, but possibly distinct
from, the type I or type II IL-1 receptors. See, e.g., Belvin and
Anderson (1996) Ann. Rev. Cell Dev. Biol. 12:393-416; Morisato and
Anderson (1995) Ann. Rev. Genetics 29:371-3991 and Hultmark (1994)
Nature 367:116-117.
[0074] Also, a ligand is a molecule which serves either as a
natural ligand to which said receptor, or an analog thereof, binds,
or a molecule which is a functional analog of the natural ligand.
The functional analog may be a ligand with structural
modifications, or may be a wholly unrelated molecule which has a
molecular shape which interacts with the appropriate ligand binding
determinants. The ligands may serve as agonists or antagonists,
see, e.g., Goodman, et al. (eds. 1990) Goodman & Gilman's: The
Pharmacological Bases of Therapeutics, Pergamon Press, New
York.
[0075] Rational drug design may also be based upon structural
studies of the molecular shapes of a receptor or antibody and other
effectors or ligands. Effectors may be other proteins which mediate
other functions in response to ligand binding, or other proteins
which normally interact with the receptor. One means for
determining which sites interact with specific other proteins is a
physical structure determination, e.g., x-ray crystallography or 2
dimensional NMR techniques. These will provide guidance as to which
amino acid residues form molecular contact regions. For a detailed
description of protein structural determination, see, e.g.,
Blundell and Johnson (1976) Protein Crystallography, Academic
Press, New York, which is hereby incorporated herein by
reference.
[0076] II. Activities
[0077] The IL-1 receptor-like polypeptides will have a number of
different biological activities, e.g., in phosphate metabolism,
being added to or removed from specific substrates, typically
proteins. Such will generally result in modulation of an
inflammatory function, other innate immunity response, or a
morphological effect. For example, a human IL-1RD9 gene coding
sequence probably has about 60-80% identity with the nucleotide
coding sequence of mouse IL-1RD9. At the amino acid level, there is
also likely to be reasonable identity.
[0078] The receptors will also exhibit immunogenic activity, e.g.,
in being capable of eliciting a selective immune response.
Antiserum or antibodies resulting therefrom will exhibit both
selectivity and affinity of binding. The polypeptides will also be
antigenic, in binding antibodies raised thereto, in the native
state, or in denatured.
[0079] The biological activities of the IL-1RDs will generally be
related to addition or removal of phosphate moieties to substrates,
typically in a specific manner, but occasionally in a non specific
manner. Substrates may be identified, or conditions for enzymatic
activity may be assayed by standard methods, e.g., as described in
Hardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and
II, Academic Press, San Diego, Calif.; Hanks, et al. (1991) Meth.
Enzymol. 200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin
(1990) Cell 61:743-752; Pines, et al. (1991) Cold Spring Harbor
Symp. Quant. Biol. 56:449-463; and Parker, et al. (1993) Nature
363:736-738.
[0080] III. Nucleic Acids
[0081] This invention contemplates use of isolated nucleic acid or
fragments, e.g., which encode these or closely related proteins, or
fragments thereof, e.g., to encode a corresponding polypeptide,
preferably one which is biologically active. In addition, this
invention covers isolated or recombinant DNA which encodes such
polypeptides or polypeptides having characteristic sequences of the
respective IL-1RDs, individually or as a group. Typically, the
nucleic acid is capable of hybridizing, under appropriate
conditions, with a nucleic acid coding sequence segment shown in
Table 1, 2, or 3 but preferably not with a corresponding segment of
other receptors. Said biologically active polypeptide can be a full
length polypeptide, or fragment, and will typically have a segment
of amino acid sequence highly homologous to one shown in Table 1,
2, or 3. Further, this invention covers the use of isolated or
recombinant nucleic acid, or fragments thereof, which encode
polypeptides having fragments which are equivalent to the IL-1RD9
proteins. The isolated nucleic acids can have the respective
regulatory sequences in the 5' and 3' flanks, e.g., promoters,
enhancers, poly-A addition signals, and others from the natural
gene.
[0082] An "isolated" nucleic acid is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially pure, e.g.,
separated from other components which naturally accompany a native
sequence, e.g., ribosomes, polymerases, and flanking genomic
sequences from the originating species. The term embraces a nucleic
acid sequence which has been removed from its naturally occurring
environment, and includes recombinant or cloned DNA isolates, which
are thereby distinguishable from naturally occurring compositions,
and chemically synthesized analogs or analogs biologically
synthesized by heterologous systems. A substantially pure molecule
includes isolated forms of the molecule, either completely or
substantially pure.
[0083] An isolated nucleic acid will generally be a homogeneous
composition of molecules, but will, in some embodiments, contain
heterogeneity, preferably minor. This heterogeneity is typically
found at the polymer ends or portions not critical to a desired
biological function or activity.
[0084] A "recombinant" nucleic acid is typically defined either by
its method of production or its structure. In reference to its
method of production, e.g., a product made by a process, the
process is use of recombinant nucleic acid techniques, e.g.,
involving human intervention in the nucleotide sequence. Typically
this intervention involves in vitro manipulation, although under
certain circumstances it may involve more classical animal breeding
techniques. Alternatively, it can be a nucleic acid made by
generating a sequence comprising fusion of two fragments which are
not naturally contiguous to each other, but is meant to exclude
products of nature, e.g., naturally occurring mutants as found in
their natural state. Thus, for example, products made by
transforming cells with an unnaturally occurring vector is
encompassed, as are nucleic acids comprising sequence derived using
any synthetic oligonucleotide process. Such a process is often done
to replace a codon with a redundant codon encoding the same or a
conservative amino acid, while typically introducing or removing a
restriction enzyme sequence recognition site. Alternatively, the
process is performed to join together nucleic acid segments of
desired functions to generate a single genetic entity comprising a
desired combination of functions not found in the commonly
available natural forms, e.g., encoding a fusion protein.
Restriction enzyme recognition sites are often the target of such
artificial manipulations, but other site specific targets, e.g.,
promoters, DNA replication sites, regulation sequences, control
sequences, or other useful features may be incorporated by design.
A similar concept is intended for a recombinant, e.g., fusion,
polypeptide. This will include a dimeric repeat. Specifically
included are synthetic nucleic acids which, by genetic code
redundancy, encode equivalent polypeptides to fragments of, e.g,
IL-1RD9, and fusions of sequences from various different related
molecules, e.g., other IL-1 receptor family members.
[0085] A "fragment" in a nucleic acid context is a contiguous
segment of at least about 17 contiguous nucleotides, generally at
least 21 contiguous nucleotides, more generally at least 25
contiguous nucleotides, ordinarily at least 30 contiguous
nucleotides, more ordinarily at least 35 contiguous nucleotides,
often at least 39 contiguous nucleotides, more often at least 45
contiguous nucleotides, typically at least 50 contiguous
nucleotides, more typically at least 55 contiguous nucleotides,
usually at least 60 contiguous nucleotides, more usually at least
66 contiguous nucleotides, preferably at least 72 contiguous
nucleotides, more preferably at least 79 contiguous nucleotides,
and in particularly preferred embodiments will be at least 85 or
more contiguous nucleotides, e.g., 100, 120, 140, etc. Typically,
fragments of different genetic sequences can be compared to one
another over appropriate length stretches, particularly defined
segments such as the domains described below.
[0086] A nucleic acid which codes for an IL-1RD8, IL-1RD9, or
IL-1RD10 will be particularly useful to identify genes, mRNA, and
cDNA species which code for itself or closely related proteins, as
well as DNAs which code for polymorphic, allelic, or other genetic
variants, e.g., from different individuals or related species.
Preferred probes for such screens are those regions of the
interleukin which are conserved between different polymorphic
variants or which contain nucleotides which lack specificity, and
will preferably be full length or nearly so. In other situations,
polymorphic variant specific sequences will be more useful.
[0087] This invention further covers recombinant nucleic acid
molecules and fragments having a nucleic acid sequence identical to
or highly homologous to the isolated DNA set forth herein. In
particular, the sequences will often be operably linked to DNA
segments which control transcription, translation, and DNA
replication. These additional segments typically assist in
expression of the desired nucleic acid segment.
[0088] Homologous, or highly identical, nucleic acid sequences,
when compared to one another, e.g., IL-1RD9 sequences, exhibit
significant similarity. The standards for homology in nucleic acids
are either measures for homology generally used in the art by
sequence comparison or based upon hybridization conditions.
Comparative hybridization conditions are described in greater
detail below.
[0089] Substantial identity in the nucleic acid sequence comparison
context means either that the segments, or their complementary
strands, when compared, are identical when optimally aligned, with
appropriate nucleotide insertions or deletions, in at least about
60% of the nucleotides, generally at least 66%, ordinarily at least
71%, often at least 76%, more often at least 80%, usually at least
84%, more usually at least 88%, typically at least 91%, more
typically at least about 93%, preferably at least about 95%, more
preferably at least about 96 to 98% or more, and in particular
embodiments, as high at about 99% or more of the nucleotides,
including, e.g., segments encoding structural domains such as the
segments described below. Alternatively, substantial identity will
exist when the segments will hybridize under selective
hybridization conditions, to a strand or its complement, typically
using a sequence derived from Table 1, 2, or 3. Typically,
selective hybridization will occur when there is at least about 55%
homology over a stretch of at least about 14 nucleotides, more
typically at least about 65%, preferably at least about 75%, and
more preferably at least about 90%. See, Kanehisa (1984) Nuc. Acids
Res. 12:203-213, which is incorporated herein by reference. The
length of homology comparison, as described, may be over longer
stretches, and in certain embodiments will be over a stretch of at
least about 17 nucleotides, generally at least about 20
nucleotides, ordinarily at least about 24 nucleotides, usually at
least about 28 nucleotides, typically at least about 32
nucleotides, more typically at least about 40 nucleotides,
preferably at least about 50 nucleotides, and more preferably at
least about 75 to 100 or more nucleotides.
[0090] Stringent conditions, in referring to homology in the
hybridization context, will be stringent combined conditions of
salt, temperature, organic solvents, and other parameters typically
controlled in hybridization reactions. Stringent temperature
conditions will usually include temperatures in excess of about
30.degree. C., more usually in excess of about 37.degree. C.,
typically in excess of about 45.degree. C., more typically in
excess of about 55.degree. C., preferably in excess of about
65.degree. C., and more preferably in excess of about 70.degree. C.
Stringent salt conditions will ordinarily be less than about 500
mM, usually less than about 400 mM, more usually less than about
300 mM, typically less than about 200 mM, preferably less than
about 100 mM, and more preferably less than about 80 mM, even down
to less than about 20 mM. However, the combination of parameters is
much more important than the measure of any single parameter. See,
e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370, which is
hereby incorporated herein by reference. The signal should be at
least 2.times. over background, generally at least 5-10.times. over
background, and preferably even more.
[0091] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequent coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0092] Optical alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity method of Pearson and Lipman (1988) Proc.
Nat'l Acad. Sci. USA 85:2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by visual inspection (see generally Ausubel
et al., supra).
[0093] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendrogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng and Doolittle (1987) J. Mol. Evol. 35:351-360. The method used
is similar to the method described by Higgins and Sharp (1989)
CABIOS 5:151-153. The program can align up to 300 sequences, each
of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. For example, a reference sequence can be
compared to other test sequences to determine the percent sequence
identity relationship using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps.
[0094] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described Altschul, et al. (1990) J.
Mol. Biol. 215:403-410. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http:www.ncbi.nlm.nih.gov/- ). This algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match
or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to
as the neighborhood word score threshold (Altschul, et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
then extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Extension of the
word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLAST program uses as defaults a
wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and
Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915) alignments (B)
of 50, expectation (E) of 10, M=5, N=4, and a comparison of both
strands.
[0095] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and Altschul
(1993) Proc. Nat'l Acad. Sci. USA 90:5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0096] A further indication that two nucleic acid sequences of
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the polypeptide encoded by the second nucleic acid, as
described below. Thus, a polypeptide is typically substantially
identical to a second polypeptide, e.g., where the two peptides
differ only by conservative substitutions. Another indication that
two nucleic acid sequences are substantially identical is that the
two molecules hybridize to each other under stringent conditions,
as described below.
[0097] The isolated DNA can be readily modified by nucleotide
substitutions, nucleotide deletions, nucleotide insertions, and
inversions of nucleotide stretches. These modifications result in
novel DNA sequences which encode this polypeptide or its
derivatives. These modified sequences can be used to produce mutant
proteins (muteins) or to enhance the expression of variant species.
Enhanced expression may involve gene amplification, increased
transcription, increased translation, and other mechanisms. Such
mutant IL-1RD9-like derivatives include predetermined or
site-specific mutations of the polypeptide or its fragments,
including silent mutations using genetic code degeneracy. "Mutant
IL-1RD9" as used herein encompasses a polypeptide otherwise falling
within the homology definition of the IL-1R9 as set forth above,
but having an amino acid sequence which differs from that of other
IL-1RD-like polypeptides as found in nature, whether by way of
deletion, substitution, or insertion. In particular, "site specific
mutant IL-1RD9" encompasses a polypeptide having substantial
homology with a polypeptide of Table 2, and typically shares most
of the biological activities or effects of the forms disclosed
herein.
[0098] Although site specific mutation sites are predetermined,
mutants need not be site specific. Mammalian IL-1RD9 mutagenesis
can be achieved by making amino acid insertions or deletions in the
gene, coupled with expression. Substitutions, deletions,
insertions, or many combinations may be generated to arrive at a
final construct. Insertions include amino- or carboxy-terminal
fusions. Random mutagenesis can be conducted at a target codon and
the expressed mammalian IL-1RD9 mutants can then be screened for
the desired activity, providing some aspect of a structure-activity
relationship. Methods for making substitution mutations at
predetermined sites in DNA having a known sequence are well known
in the art, e.g., by M13 primer mutagenesis. See also Sambrook, et
al. (1989) and Ausubel, et al. (1987 and periodic Supplements).
[0099] The mutations in the DNA normally should not place coding
sequences out of reading frames and preferably will not create
complementary regions that could hybridize to produce secondary
mRNA structure such as loops or hairpins.
[0100] The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable
synthetic DNA fragments. A double stranded fragment will often be
obtained either by synthesizing the complementary strand and
annealing the strand together under appropriate conditions or by
adding the complementary strand using DNA polymerase with an
appropriate primer sequence.
[0101] Polymerase chain reaction (PCR) techniques can often be
applied in mutagenesis. Alternatively, mutagenesis primers are
commonly used methods for generating defined mutations at
predetermined sites. See, e.g., Innis, et al. (eds. 1990) PCR
Protocols: A Guide to Methods and Applications Academic Press, San
Diego, Calif.; and Dieffenbach and Dveksler (1995; eds.) PCR
Primer: A Laboratory Manual Cold Spring Harbor Press, CSH, NY.
Appropriate primers of length, e.g., 15, 20, 25, or longer can be
made using sequence provided.
[0102] IV. Proteins, Peptides
[0103] As described above, the present invention encompasses
primate IL-1RD8, primate or rodent IL-1RD9, and primate IL-1RD10,
e.g., whose sequences are disclosed, e.g., in Tables 1-3, and
described herein. Descriptions of features of IL-1RD9 are
applicable in most cases, with appropriate modifications, also to
IL-1RD8 and/or to IL-1RD10. Allelic and other variants are also
contemplated, including, e.g., fusion proteins combining portions
of such sequences with others, including epitope tags and
functional domains. Particularly interesting constructs will be
intact extracellular or intracellular domains.
[0104] The present invention also provides recombinant
polypeptides, e.g., heterologous fusion proteins using segments
from these rodent proteins. A heterologous fusion protein is a
fusion of proteins or segments which are naturally not normally
fused in the same manner. Thus, the fusion product of, e.g., an
IL-1RD9 with another IL-1 receptor is a continuous protein molecule
having sequences fused in a typical polypeptide linkage, typically
made as a single translation product and exhibiting properties,
e.g., sequence or antigenicity, derived from each source peptide. A
similar concept applies to heterologous nucleic acid sequences.
[0105] In addition, new constructs may be made from combining
similar functional or structural domains from other related
proteins, e.g., IL-1 receptors or Toll-like receptors, including
species variants. For example, ligand-binding or other segments may
be "swapped" between different new fusion polypeptides or
fragments. See, e.g., Cunningham, et al. (1989) Science
243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem.
263:15985-15992, each of which is incorporated herein by reference.
Thus, new chimeric polypeptides exhibiting new combinations of
specificities will result from the functional linkage of
receptor-binding specificities. For example, the ligand binding
domains from other related receptor molecules may be added or
substituted for other domains of this or related proteins. The
resulting protein will often have hybrid function and properties.
For example, a fusion protein may include a targeting domain which
may serve to provide sequestering of the fusion protein to a
particular subcellular organelle.
[0106] Candidate fusion partners and sequences can be selected from
various sequence data bases, e.g., GenBank, c/o NCBI, and BCG,
University of Wisconsin Biotechnology Computing Group, Madison,
Wis., which are each incorporated herein by reference.
[0107] The present invention particularly provides muteins which
bind IL-1-like ligands, and/or which are affected in signal
transduction. Structural alignment of human IL-1RD9 with other
members of the IL-1R family show conserved features/residues. See
Table 4. Alignment of the human IL-1RD9 sequence with other members
of the IL-1R family indicates various structural and functionally
shared features. See also, Bazan, et al. (1996) Nature 379:591;
Lodi, et al. (1994) Science 263:1762-1766; Sayle and Milner-White
(1995) TIBS 20:374-376; and Gronenberg, et al. (1991) Protein
Engineering 4:263-269.
[0108] The IL-1.alpha. and IL-1.beta. ligands bind an IL-1 receptor
type I (IL-1RD1) as the primary receptor and this complex then
forms a high affinity receptor complex with the IL-1 receptor type
III (IL-1RD3). Such receptor subunits are probably shared with the
receptors for the new IL-1 ligand family members. See, e.g., U.S.
Ser. No. 60/044,165 and U.S. Ser. No. 60/055,111. It is likely that
the IL-1.gamma. ligand signals through a receptor comprising the
association of IL-1RD9 (alpha component) with IL-1RD5 (beta
component). The IL-1.delta. and IL-1.epsilon. ligands each probably
signal through a receptor comprising the association of one of
IL-1RD4, IL-1RD6, or IL-1RD9 (alpha components) with one of
IL-1RD3, IL-1RD5, IL-1RD7, IL-1RD8, or IL-1RD10 (beta
components).
[0109] Similar variations in other species counterparts of IL-1R
sequences, e.g., receptors D1-D6, D8, D9, or D10, in the
corresponding regions, should provide similar interactions with
ligand or substrate. Substitutions with either rodent or primate,
e.g., mouse sequences or human sequences, are particularly
preferred. Conversely, conservative substitutions away from the
ligand binding interaction regions will probably preserve most
signaling activities; and conservative substitutions away from the
intracellular domains will probably preserve most ligand binding
properties.
[0110] "Derivatives" of the primate or mouse IL-1RD9 include amino
acid sequence mutants, glycosylation variants, metabolic
derivatives and covalent or aggregative conjugates with other
chemical moieties. Covalent derivatives can be prepared by linkage
of functionalities to groups which are found in the IL-1RD9 amino
acid side chains or at the N- or C-termini, e.g., by means which
are well known in the art. These derivatives can include, without
limitation, aliphatic esters or amides of the carboxyl terminus, or
of residues containing carboxyl side chains, O-acyl derivatives of
hydroxyl group-containing residues, and N-acyl derivatives of the
amino terminal amino acid or amino-group containing residues, e.g.,
lysine or arginine. Acyl groups are selected from the group of
alkyl-moieties including C3 to C18 normal alkyl, thereby forming
alkanoyl aroyl species.
[0111] In particular, glycosylation alterations are included, e.g.,
made by modifying the glycosylation patterns of a polypeptide
during its synthesis and processing, or in further processing
steps. Particularly preferred means for accomplishing this are by
exposing the polypeptide to glycosylating enzymes derived from
cells which normally provide such processing, e.g., mammalian
glycosylation enzymes. Deglycosylation enzymes are also
contemplated. Also embraced are versions of the same primary amino
acid sequence which have other minor modifications, including
phosphorylated amino acid residues, e.g., phosphotyrosine,
phosphoserine, or phosphothreonine.
[0112] A major group of derivatives are covalent conjugates of the
receptors or fragments thereof with other polypeptides. These
derivatives can be synthesized in recombinant culture such as N- or
C-terminal fusions or by the use of agents known in the art for
their usefulness in cross-linking proteins through reactive side
groups. Preferred derivatization sites with cross-linking agents
are at free amino groups, carbohydrate moieties, and cysteine
residues.
[0113] Fusion polypeptides between the receptors and other
homologous or heterologous proteins are also provided. Homologous
polypeptides may be fusions between different receptors, resulting
in, for instance, a hybrid protein exhibiting binding specificity
for multiple different IL-1 ligands, or a receptor which may have
broadened or weakened specificity of substrate effect. Likewise,
heterologous fusions may be constructed which would exhibit a
combination of properties or activities of the derivative proteins.
Typical examples are fusions of a reporter polypeptide, e.g.,
luciferase, with a segment or domain of a receptor, e.g., a
ligand-binding segment, so that the presence or location of a
desired ligand may be easily determined. See, e.g., Dull, et al.,
U.S. Pat. No. 4,859,609, which is hereby incorporated herein by
reference. Other gene fusion partners include
glutathione-S-transferase (GST), bacterial .beta.-galactosidase,
trpE, Protein A, .beta.-lactamase, alpha amylase, alcohol
dehydrogenase, and yeast alpha mating factor. See, e.g., Godowski,
et al. (1988) Science 241:812-816.
[0114] The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable
synthetic DNA fragments. A double stranded fragment will often be
obtained either by synthesizing the complementary strand and
annealing the strand together under appropriate conditions or by
adding the complementary strand using DNA polymerase with an
appropriate primer sequence.
[0115] Such polypeptides may also have amino acid residues which
have been chemically modified by phosphorylation, sulfonation,
biotinylation, or the addition or removal of other moieties,
particularly those which have molecular shapes similar to phosphate
groups. In some embodiments, the modifications will be useful
labeling reagents, or serve as purification targets, e.g., affinity
ligands.
[0116] Fusion proteins will typically be made by either recombinant
nucleic acid methods or by synthetic polypeptide methods.
Techniques for nucleic acid manipulation and expression are
described generally, e.g., in Sambrook, et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed.), Vols. 1-3, Cold Spring
Harbor Laboratory, and Ausubel, et al. (eds. 1987 and periodic
supplements) Current Protocols in Molecular Biology, Greene/Wiley,
New York, which are each incorporated herein by reference.
Techniques for synthesis of polypeptides are described, e.g., in
Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield
(1986) Science 232: 341-347; and Atherton, et al. (1989) Solid
Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford;
each of which is incorporated herein by reference. See also Dawson,
et al. (1994) Science 266:776-779 for methods to make larger
polypeptides.
[0117] This invention also contemplates the use of derivatives of
an IL-1RD8, IL-1RD9, or IL-1RD10 other than variations in amino
acid sequence or glycosylation. Such derivatives may involve
covalent or aggregative association with chemical moieties. These
derivatives generally fall into three classes: (1) salts, (2) side
chain and terminal residue covalent modifications, and (3)
adsorption complexes, for example with cell membranes. Such
covalent or aggregative derivatives are useful as immunogens, as
reagents in immunoassays, or in purification methods such as for
affinity purification of a receptor or other binding molecule,
e.g., an antibody. For example, an IL-1 ligand can be immobilized
by covalent bonding to a solid support such as cyanogen
bromide-activated Sepharose, by methods which are well known in the
art, or adsorbed onto polyolefin surfaces, with or without
glutaraldehyde cross-linking, for use in the assay or purification
of an IL-1 receptor, antibodies, or other similar molecules. The
ligand can also be labeled with a detectable group, e.g.,
radioiodinated by the chloramine T procedure, covalently bound to
rare earth chelates, or conjugated to another fluorescent moiety
for use in diagnostic assays.
[0118] An IL-1RD8, IL-1RD9, or IL-1RD10 of this invention can be
used as an immunogen for the production of antisera or antibodies
specific, e.g., capable of distinguishing between other IL-1
receptor family members, for the IL-1RD8, IL-1RD9, or IL-1RD10 or
various fragments thereof. The purified IL-1RD8, IL-1RD9, or
IL-1RD10 can be used to screen monoclonal antibodies or
antigen-binding fragments prepared by immunization with various
forms of impure preparations containing the protein. In particular,
the term "antibodies" also encompasses antigen binding fragments of
natural antibodies, e.g., Fab, Fab2, Fv, etc. The purified IL-1RD9
can also be used as a reagent to detect antibodies generated in
response to the presence of elevated levels of expression, or
immunological disorders which lead to antibody production to the
endogenous receptor. Additionally, IL-1RD8, IL-1RD9, or IL-1RD10
fragments may also serve as immunogens to produce the antibodies of
the present invention, as described immediately below. For example,
this invention contemplates antibodies having binding affinity to
or being raised against the amino acid sequences shown, e.g., in
Tables 1, 2, or 3, fragments thereof, or various homologous
peptides. In particular, this invention contemplates antibodies
having binding affinity to, or having been raised against, specific
fragments which are predicted to be, or actually are, exposed at
the exterior polypeptide surface of the native IL-1RD8, IL-1RD9, or
IL-1RD10. Various preparations of desired selectivity in binding
can be prepared by appropriate cross absorptions, etc.
[0119] The blocking of physiological response to the receptor
ligands may result from the inhibition of binding of the ligand to
the receptor, likely through competitive inhibition. Thus, in vitro
assays of the present invention will often use antibodies or
antigen binding segments of these antibodies, or fragments attached
to solid phase substrates. These assays will also allow for the
diagnostic determination of the effects of either ligand binding
region mutations and modifications, or other mutations and
modifications, e.g., which affect signaling or enzymatic
function.
[0120] This invention also contemplates the use of competitive drug
screening assays, e.g., where neutralizing antibodies to the
receptor or fragments compete with a test compound for binding to a
ligand or other antibody. In this manner, the neutralizing
antibodies or fragments can be used to detect the presence of a
polypeptide which shares one or more binding sites to a receptor
and can also be used to occupy binding sites on a receptor that
might otherwise bind a ligand.
[0121] V. Making Nucleic Acids and Protein
[0122] DNA which encodes the polypeptides or fragments thereof can
be obtained by chemical synthesis, screening cDNA libraries, or by
screening genomic libraries prepared from a wide variety of cell
lines or tissue samples. Natural sequences can be isolated using
standard methods and the sequences provided herein, e.g., in Tables
1-3. Other species counterparts can be identified by hybridization
techniques, or by various PCR techniques, combined with or by
searching in sequence databases, e.g., GenBank.
[0123] This DNA can be expressed in a wide variety of host cells
for the synthesis of a full-length receptor or fragments which can
in turn, e.g., be used to generate polyclonal or monoclonal
antibodies; for binding studies; for construction and expression of
modified ligand binding or kinase/phosphatase domains; and for
structure/function studies. Variants or fragments can be expressed
in host cells that are transformed or transfected with appropriate
expression vectors. These molecules can be substantially free of
protein or cellular contaminants, other than those derived from the
recombinant host, and therefore are particularly useful in
pharmaceutical compositions when combined with a pharmaceutically
acceptable carrier and/or diluent. The protein, or portions
thereof, may be expressed as fusions with other proteins.
[0124] Expression vectors are typically self-replicating DNA or RNA
constructs containing the desired receptor gene or its fragments,
usually operably linked to suitable genetic control elements that
are recognized in a suitable host cell. These control elements are
capable of effecting expression within a suitable host. The
specific type of control elements necessary to effect expression
will depend upon the eventual host cell used. Generally, the
genetic control elements can include a prokaryotic promoter system
or a eukaryotic promoter expression control system, and typically
include a transcriptional promoter, an optional operator to control
the onset of transcription, transcription enhancers to elevate the
level of mRNA expression, a sequence that encodes a suitable
ribosome binding site, and sequences that terminate transcription
and translation. Expression vectors also usually contain an origin
of replication that allows the vector to replicate independently of
the host cell.
[0125] The vectors of this invention include those which contain
DNA which encodes a protein, as described, or a fragment thereof
encoding a biologically active equivalent polypeptide. The DNA can
be under the control of a viral promoter and can encode a selection
marker. This invention further contemplates use of such expression
vectors which are capable of expressing eukaryotic cDNA coding for
such a polypeptide in a prokaryotic or eukaryotic host, where the
vector is compatible with the host and where the eukaryotic cDNA
coding for the receptor is inserted into the vector such that
growth of the host containing the vector expresses the cDNA in
question. Usually, expression vectors are designed for stable
replication in their host cells or for amplification to greatly
increase the total number of copies of the desirable gene per cell.
It is not always necessary to require that an expression vector
replicate in a host cell, e.g., it is possible to effect transient
expression of the polypeptide or its fragments in various hosts
using vectors that do not contain a replication origin that is
recognized by the host cell. It is also possible to use vectors
that cause integration of the polypeptide encoding portion or its
fragments into the host DNA by recombination.
[0126] Vectors, as used herein, comprise plasmids, viruses,
bacteriophage, integratable DNA fragments, and other vehicles which
enable the integration of DNA fragments into the genome of the
host. Expression vectors are specialized vectors which contain
genetic control elements that effect expression of operably linked
genes. Plasmids are the most commonly used form of vector but all
other forms of vectors which serve an equivalent function and which
are, or become, known in the art are suitable for use herein. See,
e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors: A
Laboratory Manual, Elsevier, N.Y., and Rodriquez, et al. (eds.
1988) Vectors: A Survey of Molecular Cloning Vectors and Their
Uses, Buttersworth, Boston, which are incorporated herein by
reference.
[0127] Transformed cells are cells, preferably mammalian, that have
been transformed or transfected with receptor vectors constructed
using recombinant DNA techniques. Transformed host cells usually
express the desired polypeptide or its fragments, but for purposes
of cloning, amplifying, and manipulating its DNA, do not need to
express the subject protein. This invention further contemplates
culturing transformed cells in a nutrient medium, thus permitting
the receptor to accumulate in the cell membrane. The polypeptide
can be recovered, either from the culture or, in certain instances,
from the culture medium.
[0128] For purposes of this invention, nucleic sequences are
operably linked when they are functionally related to each other.
For example, DNA for a presequence or secretory leader is operably
linked to a polypeptide if it is expressed as a preprotein or
participates in directing the polypeptide to the cell membrane or
in secretion of the polypeptide. A promoter is operably linked to a
coding sequence if it controls the transcription of the
polypeptide; a ribosome binding site is operably linked to a coding
sequence if it is positioned to permit translation. Usually,
operably linked means contiguous and in reading frame, however,
certain genetic elements such as repressor genes are not
contiguously linked but still bind to operator sequences that in
turn control expression.
[0129] Suitable host cells include prokaryotes, lower eukaryotes,
and higher eukaryotes. Prokaryotes include both gram negative and
gram positive organisms, e.g., E. coli and B. subtilis. Lower
eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and
species of the genus Dictyostelium. Higher eukaryotes include
established tissue culture cell lines from animal cells, both of
non-mammalian origin, e.g., insect cells, and birds, and of
mammalian origin, e.g., human, primates, and rodents.
[0130] Prokaryotic host-vector systems include a wide variety of
vectors for many different species. As used herein, E. coli and its
vectors will be used generically to include equivalent vectors used
in other prokaryotes. A representative vector for amplifying DNA is
pBR322 or many of its derivatives. Vectors that can be used to
express the receptor or its fragments include, but are not limited
to, such vectors as those containing the lac promoter (pUC-series);
trp promoter (pBR322-trp); Ipp promoter (the pIN-series); lambda-pP
or pR promoters (pOTS); or hybrid promoters such as ptac (pDR540).
See Brosius, et al. (1988) "Expression Vectors Employing Lambda-,
trp-, lac-, and Ipp-derived Promoters", in Vectors: A Survey of
Molecular Cloning Vectors and Their Uses, (eds. Rodriguez and
Denhardt), Buttersworth, Boston, Chapter 10, pp. 205-236, which is
incorporated herein by reference.
[0131] Lower eukaryotes, e.g., yeasts and Dictyostelium, may be
transformed with IL-1RD9 sequence containing vectors. For purposes
of this invention, the most common lower eukaryotic host is the
baker's yeast, Saccharomyces cerevisiae. It will be used to
generically represent lower eukaryotes although a number of other
strains and species are also available. Yeast vectors typically
consist of a replication origin (unless of the integrating type), a
selection gene, a promoter, DNA encoding the receptor or its
fragments, and sequences for translation termination,
polyadenylation, and transcription termination. Suitable expression
vectors for yeast include such constitutive promoters as
3-phosphoglycerate kinase and various other glycolytic enzyme gene
promoters or such inducible promoters as the alcohol dehydrogenase
2 promoter or metallothionine promoter. Suitable vectors include
derivatives of the following types: self-replicating low copy
number (such as the YRp-series), self-replicating high copy number
(such as the YEp-series); integrating types (such as the
YIp-series), or mini-chromosomes (such as the YCp-series).
[0132] Higher eukaryotic tissue culture cells are normally the
preferred host cells for expression of the functionally active
interleukin protein. In principle, many higher eukaryotic tissue
culture cell lines are workable, e.g., insect baculovirus
expression systems, whether from an invertebrate or vertebrate
source. However, mammalian cells are preferred. Transformation or
transfection and propagation of such cells has become a routine
procedure. Examples of useful cell lines include HeLa cells,
Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell
lines, insect cell lines, bird cell lines, and monkey (COS) cell
lines. Expression vectors for such cell lines usually include an
origin of replication, a promoter, a translation initiation site,
RNA splice sites (if genomic DNA is used), a polyadenylation site,
and a transcription termination site. These vectors also usually
contain a selection gene or amplification gene. Suitable expression
vectors may be plasmids, viruses, or retroviruses carrying
promoters derived, e.g., from such sources as from adenovirus,
SV40, parvoviruses, vaccinia virus, or cytomegalovirus.
Representative examples of suitable expression vectors include
pCDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol.
5:1136-1142; pMC1neo PolyA, see Thomas, et al. (1987) Cell
51:503-512; and a baculovirus vector such as pAC 373 or pAC
610.
[0133] For secreted proteins, an open reading frame usually encodes
a polypeptide that consists of a mature or secreted product
covalently linked at its N-terminus to a signal peptide. The signal
peptide is cleaved prior to secretion of the mature, or active,
polypeptide. The cleavage site can be predicted with a high degree
of accuracy from empirical rules, e.g., von-Heijne (1986) Nucleic
Acids Research 14:4683-4690 and Nielsen, et al. (1997) Protein Eng.
10:1-12, and the precise amino acid composition of the signal
peptide often does not appear to be critical to its function, e.g.,
Randall, et al. (1989) Science 243:1156-1159; Kaiser, et al. (1987)
Science 235:312-317.
[0134] It will often be desired to express these polypeptides in a
system which provides a specific or defined glycosylation pattern.
In this case, the usual pattern will be that provided naturally by
the expression system. However, the pattern will be modifiable by
exposing the polypeptide, e.g., an unglycosylated form, to
appropriate glycosylating proteins introduced into a heterologous
expression system. For example, the receptor gene may be
co-transformed with one or more genes encoding mammalian or other
glycosylating enzymes. Using this approach, certain mammalian
glycosylation patterns will be achievable in prokaryote or other
cells.
[0135] The source of IL-1RD8, IL-1RD9, or IL-1RD10 can be a
eukaryotic or prokaryotic host expressing recombinant IL-1RD8,
IL-1RD9, or IL-1RD10 such as is described above. The source can
also be a cell line such as mouse Swiss 3T3 fibroblasts, but other
mammalian cell lines are also contemplated by this invention, with
the preferred cell line being from the human species.
[0136] Now that the sequences are known, the primate IL-1Rs,
fragments, or derivatives thereof can be prepared by conventional
processes for synthesizing peptides. These include processes such
as are described in Stewart and Young (1984) Solid Phase Peptide
Synthesis, Pierce Chemical Co., Rockford, Ill.; Bodanszky and
Bodanszky (1984) The Practice of Peptide Synthesis,
Springer-Verlag, New York; and Bodanszky (1984) The Principles of
Peptide Synthesis, Springer-Verlag, New York; all of each which are
incorporated herein by reference. For example, an azide process, an
acid chloride process, an acid anhydride process, a mixed anhydride
process, an active ester process (e.g., p-nitrophenyl ester,
N-hydroxysuccinimide ester, or cyanomethyl ester), a
carbodiimidazole process, an oxidative-reductive process, or a
dicyclohexylcarbodiimide (DCCD)/additive process can be used. Solid
phase and solution phase syntheses are both applicable to the
foregoing processes. Similar techniques can be used with partial
IL-1RD9 sequences.
[0137] The IL-1RD8, IL-1RD9, or IL-1RD10 proteins, polypeptides,
fragments, or derivatives are suitably prepared in accordance with
the above processes as typically employed in peptide synthesis,
generally either by a so-called stepwise process which comprises
condensing an amino acid to the terminal amino acid, one by one in
sequence, or by coupling peptide fragments to the terminal amino
acid. Amino groups that are not being used in the coupling reaction
typically must be protected to prevent coupling at an incorrect
location.
[0138] If a solid phase synthesis is adopted, the C-terminal amino
acid is bound to an insoluble carrier or support through its
carboxyl group. The insoluble carrier is not particularly limited
as long as it has a binding capability to a reactive carboxyl
group. Examples of such insoluble carriers include halomethyl
resins, such as chloromethyl resin or bromomethyl resin,
hydroxymethyl resins, phenol resins,
tert-alkyloxycarbonylhydrazidated resins, and the like.
[0139] An amino group-protected amino acid is bound in sequence
through condensation of its activated carboxyl group and the
reactive amino group of the previously formed peptide or chain, to
synthesize the peptide step by step. After synthesizing the
complete sequence, the peptide is split off from the insoluble
carrier to produce the peptide. This solid-phase approach is
generally described by Merrifield, et al. (1963) in J. Am. Chem.
Soc. 85:2149-2156, which is incorporated herein by reference.
[0140] The prepared protein and fragments thereof can be isolated
and purified from the reaction mixture by means of peptide
separation, e.g., by extraction, precipitation, electrophoresis,
various forms of chromatography, and the like. The receptors of
this invention can be obtained in varying degrees of purity
depending upon desired uses. Purification can be accomplished by
use of the protein purification techniques disclosed herein, see
below, or by the use of the antibodies herein described in methods
of immunoabsorbant affinity chromatography. This immunoabsorbant
affinity chromatography is carried out by first linking the
antibodies to a solid support and then contacting the linked
antibodies with solubilized lysates of appropriate cells, lysates
of other cells expressing the receptor, or lysates or supernatants
of cells producing the polypeptide as a result of DNA techniques,
see below.
[0141] Generally, the purified protein will be at least about 40%
pure, ordinarily at least about 50% pure, usually at least about
60% pure, typically at least about 70% pure, more typically at
least about 80% pure, preferable at least about 90% pure and more
preferably at least about 95% pure, and in particular embodiments,
97%-99% or more. Purity will usually be on a weight basis, but can
also be on a molar basis. Different assays will be applied as
appropriate. Similar concepts apply to polynucleotides and
antibodies.
[0142] VI. Antibodies
[0143] Antibodies can be raised to the various mammalian IL-1RD8,
IL-1RD9, or IL-1RD10 described herein, e.g., primate IL-1RD9
polypeptides and fragments thereof, both in naturally occurring
native forms and in their recombinant forms, the difference being
that antibodies to the active receptor are more likely to recognize
epitopes which are only present in the native conformations.
Denatured antigen detection can also be useful in, e.g., Western
analysis. Anti-idiotypic antibodies are also contemplated, which
would be useful as agonists or antagonists of a natural receptor or
an antibody.
[0144] Antibodies, including binding fragments and single chain
versions, against predetermined fragments of the polypeptide can be
raised by immunization of animals with conjugates of the fragments
with immunogenic proteins. Monoclonal antibodies are prepared from
cells secreting the desired antibody. These antibodies can be
screened for binding to normal or defective protein, or screened
for agonistic or antagonistic activity. These monoclonal antibodies
will usually bind with at least a K.sub.D of about 1 mM, more
usually at least about 300 .mu.M, typically at least about 100
.mu.M, more typically at least about 30 .mu.M, preferably at least
about 10 .mu.M, and more preferably at least about 3 .mu.M or
better.
[0145] The antibodies, including antigen binding fragments, of this
invention can have significant diagnostic or therapeutic value.
They can be potent antagonists that bind to the receptor and
inhibit binding to ligand or inhibit the ability of the receptor to
elicit a biological response, e.g., act on its substrate. They also
can be useful as non-neutralizing antibodies and can be coupled to
toxins or radionuclides to bind producing cells, or cells localized
to the source of the interleukin. Further, these antibodies can be
conjugated to drugs or other therapeutic agents, either directly or
indirectly by means of a linker.
[0146] The antibodies of this invention can also be useful in
diagnostic applications. As capture or non-neutralizing antibodies,
they might bind to the receptor without inhibiting ligand or
substrate binding. As neutralizing antibodies, they can be useful
in competitive binding assays. They will also be useful in
detecting or quantifying ligand. They may be used as reagents for
Western blot analysis, or for immunoprecipitation or
immunopurification of the respective protein.
[0147] Protein fragments may be joined to other materials,
particularly polypeptides, as fused or covalently joined
polypeptides to be used as immunogens. Mammalian IL-1Rs and
fragments may be fused or covalently linked to a variety of
immunogens, such as keyhole limpet hemocyanin, bovine serum
albumin, tetanus toxoid, etc. See Microbiology, Hoeber Medical
Division, Harper and Row, 1969; Landsteiner (1962) Specificity of
Serological Reactions, Dover Publications, New York; and Williams,
et al. (1967) Methods in Immunology and Immunochemistry, Vol. 1,
Academic Press, New York; each of which are incorporated herein by
reference, for descriptions of methods of preparing polyclonal
antisera. A typical method involves hyperimmunization of an animal
with an antigen. The blood of the animal is then collected shortly
after the repeated immunizations and the gamma globulin is
isolated.
[0148] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies may be found in, e.g., Stites, et al. (eds.)
Basic and Clinical Immunology (4th ed.), Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.) Academic Press, New York; and particularly in Kohler and
Milstein (1975) in Nature 256:495-497, which discusses one method
of generating monoclonal antibodies. Each of these references is
incorporated herein by reference. Summarized briefly, this method
involves injecting an animal with an immunogen. The animal is then
sacrificed and cells taken from its spleen, which are then fused
with myeloma cells. The result is a hybrid cell or "hybridoma" that
is capable of reproducing in vitro. The population of hybridomas is
then screened to isolate individual clones, each of which secrete a
single antibody species to the immunogen. In this manner, the
individual antibody species obtained are the products of
immortalized and cloned single B cells from the immune animal
generated in response to a specific site recognized on the
immunogenic substance.
[0149] Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively to
selection of libraries of antibodies in phage or similar vectors.
See, Huse, et al. (1989) "Generation of a Large Combinatorial
Library of the Immunoglobulin Repertoire in Phage Lambda," Science
246:1275-1281; and Ward, et al. (1989) Nature 341:544-546, each of
which is hereby incorporated herein by reference. The polypeptides
and antibodies of the present invention may be used with or without
modification, including chimeric or humanized antibodies.
Frequently, the polypeptides and antibodies will be labeled by
joining, either covalently or non-covalently, a substance which
provides for a detectable signal. A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, chemiluminescent moieties, magnetic
particles, and the like. Patents, teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant or chimeric
immunoglobulins may be produced, see Cabilly, U.S. Pat. No.
4,816,567; or made in transgenic mice, see Mendez, et al. (1997)
Nature Genetics 15:146-156. These references are incorporated
herein by reference.
[0150] The antibodies of this invention can also be used for
affinity chromatography in isolating the IL-1Rs. Columns can be
prepared where the antibodies are linked to a solid support, e.g.,
particles, such as agarose, Sephadex, or the like, where a cell
lysate may be passed through the column, the column washed,
followed by increasing concentrations of a mild denaturant, whereby
the purified protein will be released. The protein may be used to
purify antibody.
[0151] The antibodies may also be used to screen expression
libraries for particular expression products. Usually the
antibodies used in such a procedure will be labeled with a moiety
allowing easy detection of presence of antigen by antibody
binding.
[0152] Antibodies raised against an IL-1R will also be used to
raise anti-idiotypic antibodies. These will be useful in detecting
or diagnosing various immunological conditions related to
expression of the protein or cells which express the protein. They
also will be useful as agonists or antagonists of the ligand, which
may be competitive inhibitors or substitutes for naturally
occurring ligands.
[0153] An IL-1R polypeptide that specifically binds to or that is
specifically immunoreactive with an antibody generated against a
defined immunogen, such as an immunogen consisting of the amino
acid sequence of, e.g., SEQ ID NO: 4, 10, or 35, is typically
determined in an immunoassay. The immunoassay typically uses a
polyclonal antiserum which was raised, e.g., to a polypeptide of
SEQ ID NO: 4, 10, or 35. This antiserum is selected to have low
crossreactivity against other IL-1R family members, e.g., IL-1Rs D1
through D8, preferably from the same species, and any such
crossreactivity is removed by immunoabsorption prior to use in the
immunoassay.
[0154] To produce antisera for use in an immunoassay, the
polypeptide of, e.g., SEQ ID NO: 4, 10, or 35, is isolated as
described herein. For example, recombinant polypeptide may be
produced in a mammalian cell line. An appropriate host, e.g., an
inbred strain of mice such as Balb/c, is immunized with the
selected protein, typically using a standard adjuvant, such as
Freund's adjuvant, and a standard mouse immunization protocol (see
Harlow and Lane, supra). Alternatively, a synthetic peptide derived
from the sequences disclosed herein and conjugated to a carrier
polypeptide can be used an immunogen. Polyclonal sera are collected
and titered against the immunogen polypeptide in an immunoassay,
e.g., a solid phase immunoassay with the immunogen immobilized on a
solid support. Polyclonal antisera with a titer of 10.sup.4 or
greater are selected and tested for their cross reactivity against
other IL-1R family members, e.g., IL-1RD1 through IL-1RD6, using a
competitive binding immunoassay such as the one described in Harlow
and Lane, supra, at pages 570-573. Preferably at least two IL-1R
family members are used in this determination. These IL-1R family
members can be produced as recombinant polypeptides and isolated
using standard molecular biology and protein chemistry techniques
as described herein.
[0155] Immunoassays in the competitive binding format can be used
for the crossreactivity determinations. For example, the
polypeptide of SEQ ID NO: 4, 10, or 35 can be immobilized to a
solid support. Polypeptides added to the assay compete with the
binding of the antisera to the immobilized antigen. The ability of
the above polypeptides to compete with the binding of the antisera
to the immobilized polypeptide is compared to the polypeptides of
IL-1RD1 through IL-1RD6. The percent crossreactivity for the above
polypeptides is calculated, using standard calculations. Those
antisera with less than 10% crossreactivity with each of the
polypeptides listed above are selected and pooled. The
cross-reacting antibodies are then removed from the pooled antisera
by immunoabsorption with the above-listed proteins.
[0156] The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second polypeptide to the immunogen polypeptide (e.g., the IL-1RD8,
IL-1RD9, or IL-1RD10 like polypeptide of SEQ ID NO: 4, 10, or 35).
To make this comparison, the two polypeptides are each assayed at a
wide range of concentrations and the amount of each polypeptide
required to inhibit 50% of the binding of the antisera to the
immobilized polypeptide is determined. If the amount of the second
polypeptide required is less than twice the amount of the
polypeptide of the selected polypeptide or polypeptides that is
required, then the second polypeptide is said to specifically bind
to an antibody generated to the immunogen.
[0157] It is understood that these IL-1R polypeptides are members
of a family of homologous polypeptides that comprise at least 7
genes previously identified. For a particular gene product, such
as, e.g., IL-1RD9, the term refers not only to the amino acid
sequences disclosed herein, but also to other polypeptides that are
allelic, non-allelic, or species variants. It is also understood
that the terms include nonnatural mutations introduced by
deliberate mutation using conventional recombinant technology such
as single site mutation, or by excising short sections of DNA
encoding the respective proteins, or by substituting new amino
acids, or adding new amino acids. Such minor alterations typically
will substantially maintain the immunoidentity of the original
molecule and/or its biological activity. Thus, these alterations
include polypeptides that are specifically immunoreactive with a
designated naturally occurring IL-1RD8, IL-1RD9, or IL-1RD10
protein. The biological properties of the altered polypeptides can
be determined by expressing the polypeptide in an appropriate cell
line and measuring the appropriate effect, e.g., upon transfected
lymphocytes. Particular polypeptide modifications considered minor
would include conservative substitution of amino acids with similar
chemical properties, as described above for the IL-1R family as a
whole. By aligning a polypeptide optimally with the polypeptide of
the IL-1Rs and by using the conventional immunoassays described
herein to determine immunoidentity, one can determine the
polypeptide compositions of the invention.
[0158] VII. Kits and Quantitation
[0159] Both naturally occurring and recombinant forms of the IL-1R
like molecules of this invention are particularly useful in kits
and assay methods. For example, these methods would also be applied
to screening for binding activity, e.g., ligands for these
proteins. Several methods of automating assays have been developed
in recent years so as to permit screening of tens of thousands of
compounds per year. See, e.g., a BIOMEK automated workstation,
Beckman Instruments, Palo Alto, Calif., and Fodor, et al. (1991)
Science 251:767-773, which is incorporated herein by reference. The
latter describes means for testing binding by a plurality of
defined polymers synthesized on a solid substrate. The development
of suitable assays to screen for a ligand or agonist/antagonist
homologous polypeptides can be greatly facilitated by the
availability of large amounts of purified, soluble IL-1Rs in an
active state such as is provided by this invention.
[0160] Purified IL-1RD8, IL-1RD9, or IL-1RD10 can be coated
directly onto plates for use in the aforementioned ligand screening
techniques. However, non-neutralizing antibodies to these
polypeptides can be used as capture antibodies to immobilize the
respective receptor on the solid phase, useful, e.g., in diagnostic
uses.
[0161] This invention also contemplates use of IL-1RD8, IL-1RD9, or
IL-1RD10 fragments thereof, peptides, and their fusion products in
a variety of diagnostic kits and methods for detecting the presence
of the protein or its ligand. Alternatively, or additionally,
antibodies against the molecules may be incorporated into the kits
and methods. Typically the kit will have a compartment containing,
e.g., either an IL-1RD9 peptide or gene segment or a reagent which
recognizes one or the other. Typically, recognition reagents, in
the case of peptide, would be a ligand or antibody, or in the case
of a gene segment, would usually be a hybridization probe.
[0162] A preferred kit for determining the concentration of
IL-1RD8, IL-1RD9, or IL-1RD10 in a sample would typically comprise
a labeled compound, e.g., ligand or antibody, having known binding
affinity for IL-1RD9, a source of IL-1RD9 (naturally occurring or
recombinant) as a positive control, and a means for separating the
bound from free labeled compound, for example a solid phase for
immobilizing the IL-1RD9 in the test sample. Compartments
containing reagents, and instructions, will normally be
provided.
[0163] Antibodies, including antigen binding fragments, specific
for mammalian IL-1RD8 or a peptide fragment, or receptor fragments
are useful in diagnostic applications to detect the presence of
elevated levels of ligand and/or its fragments. Diagnostic assays
may be homogeneous (without a separation step between free reagent
and antibody-antigen complex) or heterogeneous (with a separation
step). Various commercial assays exist, such as radioimmunoassay
(RIA), enzyme-linked immunosorbent assay (ELISA), enzyme
immunoassay (EIA), enzyme-multiplied immunoassay technique (EMIT),
substrate-labeled fluorescent immunoassay (SLFIA) and the like. For
example, unlabeled antibodies can be employed by using a second
antibody which is labeled and which recognizes the antibody to an
IL-1R or to a particular fragment thereof. These assays have also
been extensively discussed in the literature. See, e.g., Harlow and
Lane (1988) Antibodies: A Laboratory Manual, CSH., and Coligan (ed.
1991) and periodic supplements, Current Protocols In Immunology
Greene/Wiley, New York.
[0164] Anti-idiotypic antibodies may have similar use to serve as
agonists or antagonists of IL-1Rs. These should be useful as
therapeutic reagents under appropriate circumstances.
[0165] Frequently, the reagents for diagnostic assays are supplied
in kits, so as to optimize the sensitivity of the assay. For the
subject invention, depending upon the nature of the assay, the
protocol, and the label, either labeled or unlabeled antibody, or
labeled ligand is provided. This is usually in conjunction with
other additives, such as buffers, stabilizers, materials necessary
for signal production such as substrates for enzymes, and the like.
Preferably, the kit will also contain instructions for proper use
and disposal of the contents after use. Typically the kit has
compartments for each useful reagent, and will contain instructions
for proper use and disposal of reagents. Desirably, the reagents
are provided as a dry lyophilized powder, where the reagents may be
reconstituted in an aqueous medium having appropriate
concentrations for performing the assay.
[0166] The aforementioned constituents of the diagnostic assays may
be used without modification or may be modified in a variety of
ways. For example, labeling may be achieved by covalently or
non-covalently joining a moiety which directly or indirectly
provides a detectable signal. In many of these assays, a test
compound, IL-1R, or antibodies thereto can be labeled either
directly or indirectly. Possibilities for direct labeling include
label groups: radiolabels such as .sup.125I, enzymes (U.S. Pat. No.
3,645,090) such as peroxidase and alkaline phosphatase, and
fluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring
the change in fluorescence intensity, wavelength shift, or
fluorescence polarization. Both of the patents are incorporated
herein by reference. Possibilities for indirect labeling include
biotinylation of one constituent followed by binding to avidin
coupled to one of the above label groups.
[0167] There are also numerous methods of separating the bound from
the free ligand, or alternatively the bound from the free test
compound. The IL-1R can be immobilized on various matrixes followed
by washing. Suitable matrices include plastic such as an ELISA
plate, filters, and beads. Methods of immobilizing the receptor to
a matrix include, without limitation, direct adhesion to plastic,
use of a capture antibody, chemical coupling, and biotin-avidin.
The last step in this approach involves the precipitation of
antibody/antigen complex by any of several methods including those
utilizing, e.g., an organic solvent such as polyethylene glycol or
a salt such as ammonium sulfate. Other suitable separation
techniques include, without limitation, the fluorescein antibody
magnetizable particle method described in Rattle, et al. (1984)
Clin. Chem. 30(9):1457-1461, and the double antibody magnetic
particle separation as described in U.S. Pat. No. 4,659,678, each
of which is incorporated herein by reference.
[0168] The methods for linking protein or fragments to various
labels have been extensively reported in the literature and do not
require detailed discussion here. Many of the techniques involve
the use of activated carboxyl groups either through the use of
carbodiimide or active esters to form peptide bonds, the formation
of thioethers by reaction of a mercapto group with an activated
halogen such as chloroacetyl, or an activated olefin such as
maleimide, for linkage, or the like. Fusion polypeptides will also
find use in these applications.
[0169] Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence
of an IL-1R. These sequences can be used as probes for detecting
levels of the respective IL-1R in patients suspected of having an
immunological disorder. The preparation of both RNA and DNA
nucleotide sequences, the labeling of the sequences, and the
preferred size of the sequences has received ample description and
discussion in the literature. Normally an oligonucleotide probe
should have at least about 14 nucleotides, usually at least about
18 nucleotides, and the polynucleotide probes may be up to several
kilobases. Various labels may be employed, most commonly
radionuclides, particularly .sup.32P. However, other techniques may
also be employed, such as using biotin modified nucleotides for
introduction into a polynucleotide. The biotin then serves as the
site for binding to avidin or antibodies, which may be labeled with
a wide variety of labels, such as radionuclides, fluorescers,
enzymes, or the like. Alternatively, antibodies may be employed
which can recognize specific duplexes, including DNA duplexes, RNA
duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes. The
antibodies in turn may be labeled and the assay carried out where
the duplex is bound to a surface, so that upon the formation of
duplex on the surface, the presence of antibody bound to the duplex
can be detected. The use of probes to the novel anti-sense RNA may
be carried out in conventional techniques such as nucleic acid
hybridization, plus and minus screening, recombinational probing,
hybrid released translation (HRT), and hybrid arrested translation
(HART). This also includes amplification techniques such as
polymerase chain reaction (PCR).
[0170] Diagnostic kits which also test for the qualitative or
quantitative presence of other markers are also contemplated.
Diagnosis or prognosis may depend on the combination of multiple
indications used as markers. Thus, kits may test for combinations
of markers. See, e.g., Viallet, et al. (1989) Progress in Growth
Factor Res. 1:89-97.
[0171] VIII. Therapeutic Utility
[0172] This invention provides reagents with significant
therapeutic value. The IL-1Rs (naturally occurring or recombinant),
fragments thereof, mutein receptors, and antibodies, along with
compounds identified as having binding affinity to the receptors or
antibodies, should be useful in the treatment of conditions
exhibiting abnormal expression of the receptors of their ligands.
Such abnormality will typically be manifested by immunological
disorders. Additionally, this invention should provide therapeutic
value in various diseases or disorders associated with abnormal
expression or abnormal triggering of response to the ligand. The
IL-1 ligands have been suggested to be involved in morphologic
development, e.g., dorso-ventral polarity determination, and immune
responses, particularly the primitive innate responses. See, e.g.,
Sun, et al. (1991) Eur. J. Biochem. 196:247-254; Hultmark (1994)
Nature 367:116-117.
[0173] Recombinant IL-1Rs, muteins, agonist or antagonist
antibodies thereto, or antibodies can be purified and then
administered to a patient. These reagents can be combined for
therapeutic use with additional active ingredients, e.g., in
conventional pharmaceutically acceptable carriers or diluents,
along with physiologically innocuous stabilizers and excipients.
These combinations can be sterile, e.g., filtered, and placed into
dosage forms as by lyophilization in dosage vials or storage in
stabilized aqueous preparations. This invention also contemplates
use of antibodies or binding fragments thereof which are not
complement binding.
[0174] Ligand screening using IL-1R or fragments thereof can be
performed to identify molecules having binding affinity to the
receptors. Subsequent biological assays can then be utilized to
determine if a putative ligand can provide competitive binding,
which can block intrinsic stimulating activity. Receptor fragments
can be used as a blocker or antagonist in that it blocks the
activity of ligand. Likewise, a compound having intrinsic
stimulating activity can activate the receptor and is thus an
agonist in that it simulates the activity of ligand, e.g., inducing
signaling. This invention further contemplates the therapeutic use
of antibodies to IL-1Rs as antagonists.
[0175] The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, reagent physiological life,
pharmacological life, physiological state of the patient, and other
medicants administered. Thus, treatment dosages should be titrated
to optimize safety and efficacy. Typically, dosages used in vitro
may provide useful guidance in the amounts useful for in situ
administration of these reagents. Animal testing of effective doses
for treatment of particular disorders will provide further
predictive indication of human dosage. Various considerations are
described, e.g., in Gilman, et al. (eds. 1990) Goodman and
Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed.,
Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed.
(1990), Mack Publishing Co., Easton, Pa.; each of which is hereby
incorporated herein by reference. Methods for administration are
discussed therein and below, e.g., for oral, intravenous,
intraperitoneal, or intramuscular administration, transdermal
diffusion, and others. Pharmaceutically acceptable carriers will
include water, saline, buffers, and other compounds described,
e.g., in the Merck Index, Merck & Co., Rahway, N.J. Because of
the likely high affinity binding, or turnover numbers, between a
putative ligand and its receptors, low dosages of these reagents
would be initially expected to be effective. And the signaling
pathway suggests extremely low amounts of ligand may have effect.
Thus, dosage ranges would ordinarily be expected to be in amounts
lower than 1 mM concentrations, typically less than about 10 .mu.M
concentrations, usually less than about 100 nM, preferably less
than about 10 pM (picomolar), and most preferably less than about 1
fM (femtomolar), with an appropriate carrier. Slow release
formulations, or slow release apparatus will often be utilized for
continuous administration.
[0176] IL-1Rs, fragments thereof, and antibodies or its fragments,
antagonists, and agonists, may be administered directly to the host
to be treated or, depending on the size of the compounds, it may be
desirable to conjugate them to carrier proteins such as ovalbumin
or serum albumin prior to their administration. Therapeutic
formulations may be administered in many conventional dosage
formulations. While it is possible for the active ingredient to be
administered alone, it is preferable to present it as a
pharmaceutical formulation. Formulations typically comprise at
least one active ingredient, as defined above, together with one or
more acceptable carriers thereof. However, combinations of the
compositions of the inventions with each other and with other
compositions or reagents are also contemplated and encompassed by
the present specification e.g., IL-1RD5 combined with IL-1RD9,
Additionally, both agonists or antagonists, are contemplated in
combination with compositions of the invention. Every carrier
should be both pharmaceutically and physiologically acceptable in
the sense of being compatible with the other ingredients and not
injurious to the patient. Formulations comprise at least one active
ingredient, as defined above, together with one or more acceptable
carriers thereof. Formulations include those suitable for oral,
rectal, nasal, or parenteral (including subcutaneous,
intramuscular, intravenous and intradermal) administration. The
formulations may conveniently be presented in unit dosage form and
may be prepared by methods well known in the art of pharmacy. See,
e.g., Gilman, et al. (eds. 1990) Goodman and Gilman's: The
Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and
Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack
Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993)
Pharmaceutical Dosage Forms: Parenteral Medications Dekker, N.Y.;
Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets
Dekker, N.Y.; and Lieberman, et al. (eds. 1990) Pharmaceutical
Dosage Forms: Disperse Systems Dekker, N.Y. The therapy of this
invention may be combined with or used in association with other
therapeutic agents, particularly agonists or antagonists of other
IL-1 family members.
[0177] IX. Ligands
[0178] The description of the IL-1 receptors herein provide means
to identify ligands, as described above. Such ligand should bind
specifically to the respective receptor with reasonably high
affinity. Typical ligand receptor binding constants will be at
least about 30 mM, e.g., generally at least about 3 mM, more
generally at least about 300 .mu.M, typically at least about 30
.mu.M, 3 .mu.M, 300 nM, 30 nM, etc. Various constructs are made
available which allow either labeling of the receptor to detect its
ligand. For example, directly labeling IL-1R, fusing onto it
markers for secondary labeling, e.g., FLAG or other epitope tags,
etc., will allow detection of receptor. This can be histological,
as an affinity method for biochemical purification, or labeling or
selection in an expression cloning approach. A two-hybrid selection
system may also be applied making appropriate constructs with the
available IL-1R sequences. See, e.g., Fields and Song (1989) Nature
340:245-246.
[0179] Generally, descriptions of IL-1Rs will be analogously
applicable to individual specific embodiments directed to IL-1RD8,
IL-1RD9, or IL-1RD10 reagents and compositions.
[0180] The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the inventions to the specific embodiments.
EXAMPLES
[0181] I. General Methods
[0182] Some of the standard methods are described or referenced,
e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press;
Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, (2d
ed.), vols. 1-3, CSH Press, NY; Ausubel, et al. Biology Greene
Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987 and
Supplements) Current Protocols in Molecular Biology, Greene/Wiley,
New York. Methods for protein purification include such methods as
ammonium sulfate precipitation, column chromatography,
electrophoresis, centrifugation, crystallization, and others. See,
e.g., Ausubel, et al. (1987 and periodic supplements); Coligan, et
al. (ed. 1996 and periodic supplements) Current Protocols In
Protein Science Greene/Wiley, New York; Deutscher (1990) "Guide to
Protein Purification" in Methods in Enzymology, vol. 182, and other
volumes in this series; and manufacturer's literature on use of
protein purification products, e.g., Pharmacia, Piscataway, N.J.,
or Bio-Rad, Richmond, Calif. Combination with recombinant
techniques allow fusion to appropriate segments, e.g., to a FLAG
sequence or an equivalent which can be fused via a
protease-removable sequence. See, e.g., Hochuli (1989) Chemische
Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant
Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic
Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;
and Crowe, et al. (1992) OIAexpress: The High Level Expression
& Protein Purification System QUIAGEN, Inc., Chatsworth,
Calif.
[0183] Computer sequence analysis is performed, e.g., using
available software programs, including those from the GCG (U.
Wisconsin) and GenBank sources. Public sequence databases were also
used, e.g., from GenBank, NCBI, SWISSPROT, and others.
[0184] Many techniques applicable to IL-10 receptors may be applied
to IL-1Rs, as described, e.g., in U.S. Ser. No. 08/110,683 (IL-10
receptor), which is incorporated herein by reference for all
purposes. Also, while many of the techniques described are directed
to the IL-1RD9 reagents, corresponding methods will typically be
applicable with the IL-1RD8, and IL-1RD10 reagents. See also, U.S.
Ser. No. 60/065,776, filed Nov. 17, 1997, and U.S. Ser. No.
60/078,008, filed Mar. 12, 1998, both of which are incorporated
herein by reference.
[0185] II. Computational Analysis.
[0186] Human sequences related to IL-1Rs were identified from
various EST databases using, e.g., the BLAST server (Altschul, et
al. (1994) Nature Genet. 6:119-129). More sensitive pattern- and
profile-based methods (Bork and Gibson (1996) Meth. Enzymol.
266:162-184) were used to identify a fragment of a gene which
exhibited certain homology to the IL-1Rs.
[0187] III. Cloning of Full-Length Human IL-1R cDNAs.
[0188] PCR primers derived from the IL-1RD8, IL-1RD9, or IL-1RD10
sequences are used (Nomura, et al. (1994) DNA Res. 1:27-35) to
probe an appropriate human cDNA library to yield a full length
IL-1RD9 or IL-1RD10 cDNA sequence or to probe a human
erythroleukemic, TF-1 cell line-derived cDNA library (Kitamura, et
al. (1989) Blood 73:375-380) to yield the IL-1R8 cDNA sequence.
Full length cDNAs for human IL-1RD9 are cloned, e.g., by DNA
hybridization screening of .lambda.gt10 phage. PCR reactions were
conducted using T. aquaticus Taqplus DNA polymerase (Stratagene)
under appropriate conditions.
[0189] IV. Localization of IL-1RD8, IL-1RD9, and IL-1RD10 mRNA
[0190] Human multiple tissue (Cat# 1, 2) and cancer cell line blots
(Cat# 7757-1), containing approximately 2 .mu.g of poly(A).sup.+
RNA per lane, are purchased from Clontech (Palo Alto, Calif.).
Probes are radiolabeled with [.alpha.-.sup.32P] dATP, e.g., using
the Amersham Rediprime random primer labeling kit (RPN1633).
Prehybridization and hybridizations are performed at 65.degree. C.
in 0.5 M Na.sub.2HPO.sub.4, 7% SDS, 0.5 M EDTA (pH 8.0). High
stringency washes are conducted, e.g., at 65.degree. C. with two
initial washes in 2.times. SSC, 0.1% SDS for 40 min followed by a
subsequent wash in 0.1.times. SSC, 0.1% SDS for 20 min. Membranes
are then exposed at -70.degree. C. to X-Ray film (Kodak) in the
presence of intensifying screens. More detailed studies by cDNA
library Southerns are performed with selected human IL-1RD9 clones
to examine their expression in hemopoietic or other cell
subsets.
[0191] Two prediction algorithms that take advantage of the
patterns of conservation and variation in multiply aligned
sequences, PHD (Rost and Sander (1994) Proteins 19:55-72) and DSC
(King and Sternberg (1996) Protein Sci. 5:2298-2310), are used.
[0192] Alternatively, two appropriate primers are selected from
Tables 1, 2, or 3. RT-PCR is used on an appropriate mRNA sample
selected for the presence of message to produce a cDNA, e.g., a
sample which expresses the gene.
[0193] Full length clones may be isolated by hybridization of cDNA
libraries from appropriate tissues pre-selected by PCR signal.
Northern blots can be performed.
[0194] Message for genes encoding, e.g., IL-1RD9 will be assayed by
appropriate technology, e.g., PCR, immunoassay, hybridization, or
otherwise. Tissue and organ cDNA preparations are available, e.g.,
from Clontech, Mountain View, Calif. Identification of sources of
natural expression are useful, as described. And the identification
of functional receptor subunit pairings will allow for prediction
of what cells express the combination of receptor subunits which
will result in a physiological responsiveness to each of the IL-1
ligands.
[0195] The message for IL-1RD9 is quite rare, as it is not found
with a degree of frequency in the available sequence databases.
This suggests, e.g., a very rare message, or a highly restricted
distribution. IL-1R9 is expressed predominantly on T cells, NK
cells, monocytes and dendritic cells.
[0196] Southern Analysis on cDNA libraries can be performed: DNA (5
.mu.g) from a primary amplified cDNA library is digested with
appropriate restriction enzymes to release the inserts, run on a 1%
agarose gel and transferred to a nylon membrane (Schleicher and
Schuell, Keene, N.H.).
[0197] Samples for human mRNA isolation may include, e.g.:
peripheral blood mononuclear cells (monocytes, T cells, NK cells,
granulocytes, B cells), resting (T100); peripheral blood
mononuclear cells, activated with anti-CD3 for 2, 6, 12 h pooled
(T101); T cell, TH0 clone Mot 72, resting (T102); T cell, TH0 clone
Mot 72, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled
(T103); T cell, TH0 clone Mot 72, anergic treated with specific
peptide for 2, 7, 12 h pooled (T104); T cell, TH1 clone HY06,
resting (T107); T cell, TH1 clone HY06, activated with anti-CD28
and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06,
anergic treated with specific peptide for 2, 6, 12 h pooled (T109);
T cell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935,
activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111);
T cells CD4+CD45RO- T cells polarized 27 days in anti-CD28, IL-4,
and anti IFN-.gamma., TH2 polarized, activated with anti-CD3 and
anti-CD28 4 h (T116); T cell tumor lines Jurkat and Hut78, resting
(T117); T cell clones, pooled AD130.2, Tc783.12, Tc783.13,
Tc783.58, Tc782.69, resting (T118); T cell random .gamma..delta.
cell clones, resting (T119); Splenocytes, resting (B100);
Splenocytes, activated with anti-CD40 and IL-4 (B101); B cell EBV
lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting
(B102); B cell line JY, activated with PMA and ionomycin for 1, 6 h
pooled (B103); NK 20 clones pooled, resting (K100); NK 20 clones
pooled, activated with PMA and ionomycin for 6 h (K101); NKL clone,
derived from peripheral blood of LGL leukemia patient, IL-2 treated
(K106); NK cytotoxic clone 640-A30-1, resting (K107); hematopoietic
precursor line TF1, activated with PMA and ionomycin for 1, 6 h
pooled (C100); U937 premonocytic line, resting (M100); U937
premonocytic line, activated with PMA and ionomycin for 1, 6 h
pooled (M101); elutriated monocytes, activated with LPS,
IFN.gamma., anti-IL-10 for 1, 2, 6, 12, 24 h pooled (M102);
elutriated monocytes, activated with LPS, IFN.gamma., IL-10 for 1,
2, 6, 12, 24 h pooled (M103); elutriated monocytes, activated with
LPS, IFN.gamma., anti-IL-10 for 4, 16 h pooled (M106); elutriated
monocytes, activated with LPS, IFN.gamma., IL-10 for 4, 16 h pooled
(M107); elutriated monocytes, activated LPS for 1 h (M108);
elutriated monocytes, activated LPS for 6 h (M109); DC 70% CD1a+,
from CD34+ GM-CSF, TNF.alpha. 12 days, resting (D101); DC 70%
CD1a+, from CD34+ GM-CSF, TNF.alpha. 12 days, activated with PMA
and ionomycin for 1 h (D102); DC 70% CD1a+, from CD34+ GM-CSF,
TNF.alpha. 12 days, activated with PMA and ionomycin for 6 h
(D103); DC 95% CD1a+, from CD34+ GM-CSF, TNF.alpha. 12 days FACS
sorted, activated with PMA and ionomycin for 1, 6 h pooled (D104);
DC 95% CD14+, ex CD34+ GM-CSF, TNF.alpha. 12 days FACS sorted,
activated with PMA and ionomycin 1, 6 h pooled (D105); DC CD1a+
CD86+, from CD34+ GM-CSF, TNF.alpha. 12 days FACS sorted, activated
with PMA and ionomycin for 1, 6 h pooled (D106); DC from monocytes
GM-CSF, IL-4 5 days, resting (D107); DC from monocytes GM-CSF, IL-4
5 days, resting (D 108); DC from monocytes GM-CSF, IL-4 5 days,
activated LPS 4, 16 h pooled (D109); DC from monocytes GM-CSF, IL-4
5 days, activated TNF.alpha., monocyte supe for 4, 16 h pooled
(D110); leiomyoma L11 benign tumor (X101); normal myometrium M5
(O115); malignant leiomyosarcoma GS1 (X103); lung fibroblast
sarcoma line MRC5, activated with PMA and ionomycin for 1, 6 h
pooled (C101); kidney epithelial carcinoma cell line CHA, activated
with PMA and ionomycin for 1, 6 h pooled (C102); kidney fetal 28 wk
male (O100); lung fetal 28 wk male (O101); liver fetal 28 wk male
(O102); heart fetal 28 wk male (O103); brain fetal 28 wk male
(O104); gallbladder fetal 28 wk male (O106); small intestine fetal
28 wk male (O107); adipose tissue fetal 28 wk male (O108); ovary
fetal 25 wk female (O109); uterus fetal 25 wk female (O110); testes
fetal 28 wk male (O111); spleen fetal 28 wk male (O112); adult
placenta 28 wk (O113); tonsil inflamed, from 12 year old (X100);
psoriasis human skin sample; normal human skin sample; pool of
rheumatioid arthritis human; Hashimoto's thryroiditis thryroid;
normal human throid; ulceratived colitis human colon; normal human
colon; normal weight monkey colon; pheumocysitc carnii pneumonia
lung; allergic lung; poll of three heavy smoker human lung; pool of
two normal human lung; Ascaris-challenged monkey lung, 24 hr;
Ascaris-challenged monkey lung, 4 hr; normal weight monkey
lung.
[0198] IL-1RD8 message is described below in Table 5. There appears
to be a correlation between developmental stage of tissues and the
levels of messages: fetal and transformed tissues express high
levels, whereas normal, adult tissues express low levels (with the
exception of skeletal muscle). Further insights into this
phenomenon will need further experiments.
[0199] Message for genes encoding IL-1RD8 will be assayed by
appropriate technology, e.g., PCR, immunoassay, hybridization, or
otherwise. Tissue and organ cDNA preparations are available, e.g.,
from Clontech, Mountain View, Calif. Identification of sources of
natural expression are useful, as described. And the identification
of functional receptor subunit pairings will allow for prediction
of what cells express the combination of receptor subunits which
will result in a physiological responsiveness to each of the IL-1
ligands.
[0200] Table 5: Multiple Tissue Northern Blots were screened with a
radiolabeled probe, encompassing the cytoplasmic region of
Interleukin-1 receptor R8 (IL-1RD8). The results are summarized
below:
[0201] In all cases listed there is a smaller band at 3.4 Kb and in
a few cases a larger band at 4.0 Kb as well.
5 Tissue 3.4 kb 4.0 kb Spleen weak Thymus weak Prostate weak Testis
weak Ovary weak Small Intestine weak Colon (mucosal lining) weak
Peripheral Blood Leukocyte weak Heart moderate Brain weak Placenta
moderate Lung weak Liver weak Skeletal Muscle strong Kidney weak
Pancreas weak Fetal brain strong weak Fetal lung strong weak Fetal
Liver strong weak Fetal Kidney strong weak proleukocytic leukemia
HL-60 strong HeLa Cell S3 very strong weak Chronic myelogenous
leukemia, K-562 very strong weak Lymphoblastic leukemia, MOLT-4
weak Burkitt's lymphoma Rajii moderate Colorectal adenocarcinoma
SW40 very strong strong Lung carcinoma A549 strong strong Melanoma
very strong weak
[0202] V. Cloning of Species Counterparts of IL-1RDs
[0203] Various strategies are used to obtain species counterparts
of IL-1RD8, IL-1RD9, and IL-1RD10 preferably from other primates.
One method is by cross hybridization using closely related species
DNA probes. It may be useful to go into evolutionarily similar
species as intermediate steps. Another method is by using specific
PCR primers based on the identification of blocks of similarity or
difference between genes, e.g., areas of highly conserved or
nonconserved polypeptide or nucleotide sequence. In addition, gene
sequence databases may be screened for related sequences from other
species.
[0204] VI. Production of Mammalian IL-1RD Protein
[0205] An appropriate, e.g., GST, fusion construct is engineered
for expression, e.g., in E. coli. For example, a mouse IGIF pGex
plasmid is constructed and transformed into E. coli. Freshly
transformed cells are grown, e.g., in LB medium containing 50
.mu.g/ml ampicillin and induced with IPTG (Sigma, St. Louis, Mo.).
After overnight induction, the bacteria are harvested and the
pellets containing, e.g., the IL-1R8 polypeptide are isolated. The
pellets are homogenized, e.g., in TE buffer (50 mM Tris-base pH
8.0, 10 mM EDTA and 2 mM pefabloc) in 2 liters. This material is
passed through a microfluidizer (Microfluidics, Newton, Mass.)
three times. The fluidized supernatant is spun down on a Sorvall
GS-3 rotor for 1 h at 13,000 rpm. The resulting supernatant
containing the IL-1R polypeptide is filtered and passed over a
glutathione-SEPHAROSE column equilibrated in 50 mM Tris-base pH
8.0. The fractions containing the IL-1RD9-GST fusion protein are
pooled and cleaved, e.g., with thrombin (Enzyme Research
Laboratories, Inc., South Bend, Ind.). The cleaved pool is then
passed over a Q-SEPHAROSE column equilibrated in 50 mM Tris-base.
Fractions containing IL-1RD9 are pooled and diluted in cold
distilled H.sub.2O, to lower the conductivity, and passed back over
a fresh Q-Sepharose column, alone or in succession with an
immunoaffinity antibody column. Fractions containing the IL-1RD9
polypeptide are pooled, aliquoted, and stored in the -70.degree. C.
freezer.
[0206] Comparison of the CD spectrum with IL-1R polypeptide may
suggest that the protein is correctly folded. See Hazuda, et al.
(1969) J. Biol. Chem. 264:1689-1693.
[0207] VII. Determining Physiological Forms of Receptors
[0208] The IL-1.alpha. and IL-1.beta. ligands bind an IL-1RD1 as
the primary receptor and this complex then forms a high affinity
receptor complex with the IL-1RD3. Such receptor subunits are
probably shared with the receptors for the new IL-1 ligand family
members. See, e.g., U.S. Ser. No. 60/044,165 and U.S. Ser. No.
60/055,111. Combination of the IL-1RD9 (.alpha. subunit type, based
upon sequence analysis) will combine with the IL-1RD5 (.beta.
subunit type, based upon sequence analysis) to form a heterodimer
receptor. The IL-1.delta. and IL-1.epsilon. ligands each probably
signal through a receptor comprising the association of IL-1RD4,
IL-1RD6, or IL-1RD9 (alpha components) with IL-1RD3, IL-1RD8, or
IL-1RD10 (beta components).
[0209] These defined subunit combinations can be tested now with
the provided reagents. In particular, appropriate constructs can be
made for transformation or transfection of subunits into cells.
Constructs for the alpha chains, e.g., IL-1RD1, IL-1RD4, IL-1RD6,
and IL-1RD9 forms can be made. Likewise for the beta subunits
IL-1RD3, IL-1RD5, IL-1RD7, and IL-1RD8. Structurally, the IL-1RD10
is most similar to the IL-1RD8, suggesting that it may also be a
beta receptor subunit. Combinatorial transfections of
transformations can make cells expressing defined subunits, which
can be tested for response to each of the IL-1 ligands. Appropriate
cell types can be used, e.g., 293 T cells, Jurkat cells, with,
e.g., a nuclear kappa B (NF.kappa.b) controlled luciferase reporter
construct such as described e.g., in Otieno et al., (1997) Am J
Physiol 273:F136-F143.
[0210] Such combinations of various IL-1 ligands and receptors were
tested to determine if a functional signaling complex had been
formed using an NF.kappa.b-controlled luciferase reporter construct
to indicate formation of a functional signaling complex (+) or
failure to form a functional signaling complex (-). The results,
presented below,
[0211] IL-1.alpha.+IL-1.beta.+IL-1RD1+IL-1RD3=+;
[0212] IL-1.alpha.+IL-1.beta.+IL-1RD1+IL-1RD5=+;
[0213] IL-1.alpha.+IL-1.beta.+IL-1RD1+IL-1RD8=+;
[0214] IL-1.alpha.+IL-1.beta.+IL-1RD1+IL-1RD10 may=+/?;
[0215] suggest that IL-1RD3, IL-1RD5, IL-1RD8, and IL-1RD10 may
functionally substitute for each other when in combination with
IL-1.alpha.+IL-1.beta.+IL-1RD1.
[0216] Other combinations (below) demonstrate a failure of
functional substitution; suggesting the importance of contextual
dependence on substitution e.g., IL-1RD3, and IL-1RD8 cannot
functionally replace IL-1RD5 in the following combination:
IL-1.gamma.+IL-1RD9+IL-1RD5.
[0217] IL-1.gamma.+IL-1RD9+IL-1RD5=+;
[0218] IL-1.gamma.+IL-1RD9+IL-1RD3=-;
[0219] IL-1.gamma.+IL-1RD9+IL-1RD8=-;
[0220] A further series of experiments tested the ability of mouse
(m) and human (h) homologues to functionally substitute for each
other. The results, shown below,
[0221] mIL-1.gamma.+mIL-1RD5+mIL-1RD9=+;
[0222] mIL-1.gamma.+mIL-1RD5+hIL-1RD9=-;
[0223] mIL-1.gamma.+hIL-1RD5+hIL-1RD9=-;
[0224] mIL-1.gamma.+hIL-1RD5+mIL-1RD9=-;
[0225] hIL-1.gamma.+mIL-1RD5+mIL-1RD9=-;
[0226] hIL-1.gamma.+mIL-1RD5+hIL-1RD9=-;
[0227] hIL-1.gamma.+hIL-1RD5+mIL-1RD9=-;
[0228] hIL-1.gamma.+hIL-1RD5+hIL-1RD9=+;
[0229] suggest that species homogeneity is required to form a
functioning complex in this particular constellation of ligand and
receptor units.
[0230] Biological assays will generally be directed to the ligand
binding feature of the protein or to the kinase/phosphatase
activity of the receptor. The activity will typically be
reversible, as are many other enzyme actions that mediate
phosphatase or phosphorylase activities, which activities are
easily measured by standard procedures. See, e.g., Hardie, et al.
(eds. 1995) The Protein Kinase FactBook vols. I and II, Academic
Press, San Diego, Calif.; Hanks, et al. (1991) Meth. Enzymol.
200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin (1990) Cell
61:743-752; Pines, et al. (1991) Cold Spring Harbor Symp. Quant.
Biol. 56:449-463; and Parker, et al. (1993) Nature 363:736-738.
[0231] The family of interleukins 1 contains molecules, each of
which is an important mediator of inflammatory disease. For a
comprehensive review, see Dinarello (1996) "Biologic basis for
interleukin-1 in disease" Blood 87:2095-2147. There are suggestions
that the various IL-1 ligands may play important roles in the
initiation of disease, particularly inflammatory responses. The
finding of novel polypeptides related to the IL-1 family furthers
the identification of molecules that provide the molecular basis
for initiation of disease and allow for the development of
therapeutic strategies of increased range and efficacy.
[0232] VIII. Preparation of Antibodies Specific for IL-1Rs
[0233] Inbred Balb/c mice are immunized intraperitoneally with
recombinant forms of the polypeptide, e.g., purified IL-1RD8,
IL-1RD9, or IL-1RD10, or stable transfected NIH-3T3 cells. Animals
are boosted at appropriate time points with protein, with or
without additional adjuvant, to further stimulate antibody
production. Serum is collected, or hybridomas produced with
harvested spleens.
[0234] Alternatively, Balb/c mice are immunized with cells
transformed with the gene or fragments thereof, either endogenous
or exogenous cells, or with isolated membranes enriched for
expression of the antigen. Serum is collected at the appropriate
time, typically after numerous further administrations. Various
gene therapy techniques may be useful, e.g., in producing protein
in situ, for generating an immune response.
[0235] Monoclonal antibodies may be made. For example, splenocytes
are fused with an appropriate fusion partner and hybridomas are
selected in growth medium by standard procedures. Hybridoma
supernatants are screened for the presence of antibodies which bind
to the desired IL-1R, e.g., by ELISA or other assay. Antibodies
which selectively recognize specific IL-1R embodiments may also be
selected or prepared.
[0236] In another method, synthetic peptides or purified protein
are presented to an immune system to generate monoclonal or
polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols
in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies:
A Laboratory Manual Cold Spring Harbor Press. In appropriate
situations, the binding reagent is either labeled as described
above, e.g., fluorescence or otherwise, or immobilized to a
substrate for panning methods. Nucleic acids may also be introduced
into cells in an animal to produce the antigen, which serves to
elicit an immune response. See, e.g., Wang, et al. (1993) Proc.
Nat'l. Acad. Sci. 90:4156-4160; Barry, et al. (1994) BioTechniques
16:616-619; and Xiang, et al. (1995) Immunity 2:129-135.
[0237] Moreover, antibodies which may be useful to determine the
combination of the IL-1RD8, IL-1RD9, or IL-1RD10 with a functional
beta subunit may be generated. Thus, e.g., epitopes characteristic
of a particular functional alpha/beta combination may be identified
with appropriate antibodies.
[0238] IX. Production of Fusion Proteins with IL-1Rs
[0239] Various fusion constructs are made with IL-1Rs. A portion of
the appropriate gene is fused to an epitope tag, e.g., a FLAG tag,
or to a two hybrid system construct. See, e.g., Fields and Song
(1989) Nature 340:245-246.
[0240] The epitope tag may be used in an expression cloning
procedure with detection with anti-FLAG antibodies to detect a
binding partner, e.g., ligand for the respective IL-1R. The two
hybrid system may also be used to isolate proteins which
specifically bind, e.g., to IL-1RD9.
[0241] X. Mapping of Human or Mouse Genes
[0242] Chromosome spreads are prepared. In situ hybridization is
performed on chromosome preparations obtained from
phytohemagglutinin-stimulated human lymphocytes cultured for 72 h.
5-bromodeoxyuridine was added for the final seven hours of culture
(60 .mu.g/ml of medium), to ensure a posthybridization chromosomal
banding of good quality.
[0243] A PCR fragment, amplified with the help of primers, is
cloned into an appropriate vector. The vector is labeled by
nick-translation with .sup.3H. The radiolabeled probe is hybridized
to metaphase spreads at final concentration of 200 ng/ml of
hybridization solution as described in Mattei, et al. (1985) Hum.
Genet. 69:327-331.
[0244] After coating with nuclear track emulsion (KODAK NTB.sub.2),
slides are exposed. To avoid any slipping of silver grains during
the banding procedure, chromosome spreads are first stained with
buffered Giemsa solution and metaphase photographed. R-banding is
then performed by the fluorochrome-photolysis-Giemsa (FPG) method
and metaphases rephotographed before analysis.
[0245] The IL-1RD10 has been localized to the X chromosome.
[0246] XI. Structure Activity Relationship
[0247] Information on the criticality of particular residues is
determined using standard procedures and analysis. Standard
mutagenesis analysis is performed, e.g., by generating many
different variants at determined positions, e.g., at the positions
identified above, and evaluating biological activities of the
variants. This may be performed to the extent of determining
positions which modify activity, or to focus on specific positions
to determine the residues which can be substituted to either
retain, block, or modulate biological activity.
[0248] Alternatively, analysis of natural variants can indicate
what positions tolerate natural mutations. This may result from
population analysis of variation among individuals, or across
strains or species. Samples from selected individuals are analyzed,
e.g., by PCR analysis and sequencing. This allows evaluation of
population polymorphisms.
[0249] XII. Isolation of a Ligand for IL-1Rs
[0250] An IL-1R can be used as a specific binding reagent to
identify its binding partner, by taking advantage of its
specificity of binding, much like an antibody would be used.
Typically, the binding receptor is a heterodimer of receptor
subunits. A binding reagent is either labeled as described above,
e.g., fluorescence or otherwise, or immobilized to a substrate for
panning methods.
[0251] The binding composition is used to screen an expression
library made from a cell line which expresses a binding partner,
i.e., ligand, preferably membrane associated. Standard staining
techniques are used to detect or sort surface expressed ligand, or
surface expressing transformed cells are screened by panning.
Screening of intracellular expression is performed by various
staining or immunofluorescence procedures. See also McMahan, et al.
(1991) EMBO J. 10:2821-2832.
[0252] For example, on day 0, precoat 2-chamber permanox slides
with 1 ml per chamber of fibronectin, 10 ng/ml in PBS, for 30 min
at room temperature. Rinse once with PBS. Then plate COS cells at
2-3.times.10.sup.5 cells per chamber in 1.5 ml of growth media.
Incubate overnight at 37.degree. C.
[0253] On day 1 for each sample, prepare 0.5 ml of a solution of 66
.mu.g/ml DEAE-dextran, 66 .mu.M chloroquine, and 4 .mu.g DNA in
serum free DME. For each set, a positive control is prepared, e.g.,
of IL-1R-FLAG cDNA at 1 and 1/200 dilution, and a negative mock.
Rinse cells with serum free DME. Add the DNA solution and incubate
5 h at 37.degree. C. Remove the medium and add 0.5 ml 10% DMSO in
DME for 2.5 min. Remove and wash once with DME. Add 1.5 ml growth
medium and incubate overnight.
[0254] On day 2, change the medium. On days 3 or 4, the cells are
fixed and stained. Rinse the cells twice with Hank's Buffered
Saline Solution (HBSS) and fix in 4% paraformaldehyde (PFA)/glucose
for 5 min. Wash 3.times. with HBSS. The slides may be stored at
-80.degree. C. after all liquid is removed. For each chamber, 0.5
ml incubations are performed as follows. Add HBSS/saponin (0.1%)
with 32 .mu.l/ml of 1 M NaN.sub.3 for 20 min. Cells are then washed
with HBSS/saponin 1.times.. Add appropriate IL-1R or IL-1R/antibody
complex to cells and incubate for 30 min. Wash cells twice with
HBSS/saponin. If appropriate, add first antibody for 30 min. Add
second antibody, e.g., Vector anti-mouse antibody, at 1/200
dilution, and incubate for 30 min. Prepare ELISA solution, e.g.,
Vector Elite ABC horseradish peroxidase solution, and preincubate
for 30 min. Use, e.g., 1 drop of solution A (avidin) and 1 drop
solution B (biotin) per 2.5 ml HBSS/saponin. Wash cells twice with
HBSS/saponin. Add ABC HRP solution and incubate for 30 min. Wash
cells twice with HBSS, second wash for 2 min, which closes cells.
Then add Vector diaminobenzoic acid (DAB) for 5 to 10 min. Use 2
drops of buffer plus 4 drops DAB plus 2 drops of H.sub.2O.sub.2 per
5 ml of glass distilled water. Carefully remove chamber and rinse
slide in water. Air dry for a few minutes, then add 1 drop of
Crystal Mount and a cover slip. Bake for 5 min at 85-90.degree.
C.
[0255] Evaluate positive staining of pools and progressively
subclone to isolation of single genes responsible for the
binding.
[0256] Alternatively, IL-1R reagents are used to affinity purify or
sort out cells expressing a putative ligand. See, e.g., Sambrook,
et al. or Ausubel, et al.
[0257] Another strategy is to screen for a membrane bound receptor
by panning. The receptor cDNA is constructed as described above.
The ligand can be immobilized and used to immobilize expressing
cells. Immobilization may be achieved by use of appropriate
antibodies which recognize, e.g., a FLAG sequence of an IL-1R
fusion construct, or by use of antibodies raised against the first
antibodies. Recursive cycles of selection and amplification lead to
enrichment of appropriate clones and eventual isolation of receptor
expressing clones.
[0258] Phage expression libraries can be screened by mammalian
IL-1Rs. Appropriate label techniques, e.g., anti-FLAG antibodies,
will allow specific labeling of appropriate clones.
[0259] All citations herein are incorporated herein by reference to
the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
[0260] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited by the terms of the appended claims,
along with the full scope of equivalents to which such claims are
entitled; and the invention is not to be limited by the specific
embodiments that have been presented herein by way of example.
Sequence CWU 1
1
* * * * *