U.S. patent application number 11/548249 was filed with the patent office on 2007-02-22 for il-1 related polypeptides.
Invention is credited to Audrey Goddard, Guohua James Pan.
Application Number | 20070042466 11/548249 |
Document ID | / |
Family ID | 27381318 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070042466 |
Kind Code |
A1 |
Goddard; Audrey ; et
al. |
February 22, 2007 |
IL-1 RELATED POLYPEPTIDES
Abstract
The present invention is directed to novel polypeptides having
homology to the IL-1-like family of proteins and to nucleic acid
molecules encoding those polypeptides. Also provided herein are
vectors and host cells comprising those nucleic acid sequences,
chimeric polypeptide molecules comprising the polypeptides of the
present invention fused to heterologous polypeptide sequences,
antibodies which bind to the polypeptides of the present invention,
and methods for producing the polypeptides of the present
invention.
Inventors: |
Goddard; Audrey; (San
Francisco, CA) ; Pan; Guohua James; (Etobicoke,
CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Family ID: |
27381318 |
Appl. No.: |
11/548249 |
Filed: |
October 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09869566 |
Feb 19, 2002 |
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PCT/US99/30720 |
Dec 22, 1999 |
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11548249 |
Oct 10, 2006 |
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60113430 |
Dec 23, 1998 |
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60116843 |
Jan 22, 1999 |
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60129122 |
Apr 13, 1999 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 530/388.22; 536/23.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
C07K 14/545 20130101 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/325; 530/350; 536/023.5; 530/388.22 |
International
Class: |
C12P 21/06 20070101
C12P021/06; C07K 14/715 20070101 C07K014/715; C07K 16/28 20070101
C07K016/28; C07H 21/04 20060101 C07H021/04 |
Claims
1. An isolated DNA molecule selected from the group consisting of:
(1) a DNA molecule encoding an hIL-1Ra1 polypeptide comprising the
amino acid sequence of amino acid residues from about 37 to about
203 of FIG. 2 (SEQ ID NO:5); (2) a DNA molecule encoding an
hIL-1Ra1 polypeptide comprising the amino acid sequence of amino
acid residues from about 15 to about 193 of FIG. 3 (SEQ ID NO:7);
(3) a DNA molecule encoding an hIL-1Ra2 polypeptide comprising the
amino acid sequence of amino acid residues from about 1 to about
134 of FIG. 5 (SEQ ID NO:10); (4) a DNA molecule encoding an
hIL-1Ra3 polypeptide comprising the amino acid sequence of amino
acid residues from about 95 to about 134 of FIG. 7 (SEQ ID NO:13);
(5) a DNA molecule encoding a mIL-1Ra3 polypeptide comprising the
amino acid sequence of amino acid residues from about 95 to about
134 of FIG. 9 (SEQ ID NO:16); (6) a DNA molecule encoding an
hIL-1Ra1L polypeptide comprising the amino acid sequence of amino
acid residues from about 26 to about 207 of FIG. 15 (SEQ ID NO:19);
(7) a DNA molecule encoding an hIL-1Ra1S polypeptide comprising the
amino acid sequence of amino acid residues from about 26 to about
167 of FIG. 16 (SEQ ID NO:21); (8) a DNA molecule encoding an
hIL-1Ra1V polypeptide comprising the amino acid sequence of amino
acid residues from about 46 to about 218 of FIG. 19 (SEQ ID NO:25);
and (9) the complement of any of the DNA molecules of (1)-(8).
2. The isolated DNA molecule of claim 1 selected from the group
consisting of: (1) a DNA molecule encoding an hIL-1Ra1 polypeptide
comprising the amino acid sequence of amino acid residues from
about 37 to about 203 of FIG. 2 (SEQ ID NO:5); (2) a DNA molecule
encoding an hIL-1Ra1 polypeptide comprising the amino acid sequence
of amino acid residues from about 15 to about 193 of FIG. 3 (SEQ ID
NO:7); (3) a DNA molecule encoding an hIL-1Ra2 polypeptide
comprising the amino acid sequence of amino acid residues from
about 1 to about 134 of FIG. 5 (SEQ ID NO:10); (4) a DNA molecule
encoding an hIL-1Ra3 polypeptide comprising the amino acid sequence
of amino acid residues from about 95 to about 134 of FIG. 7 (SEQ ID
NO:13); (5) a DNA molecule encoding a mIL-1Ra3 polypeptide
comprising the amino acid sequence of amino acid residues from
about 95 to about 134 of FIG. 9 (SEQ ID NO:16); and (6) the
complement of any of the DNA molecules of (1)-(5).
3. The isolated DNA molecule of claim 1 selected from the group
consisting of: (1) a DNA molecule encoding an hIL-1Ra1L polypeptide
comprising the amino acid sequence of amino acid residues from
about 26 to about 207 of FIG. 15 (SEQ ID NO:19); (2) a DNA molecule
encoding an hIL-1Ra1S polypeptide comprising the amino acid
sequence of amino acid residues from about 26 to about 167 of FIG.
16 (SEQ ID NO:21); (3) a DNA molecule encoding an hIL-1Ra1V
polypeptide comprising the amino acid sequence of amino acid
residues from about 46 to about 218 of FIG. 19 (SEQ ID NO:25); and
(4) the complement of any of the DNA molecules of (1)-(3).
4. The isolated DNA molecule of claim 3 selected from the group
consisting of: (1) a DNA molecule encoding an hIL-1Ra1L polypeptide
comprising the amino acid sequence of amino acid residues from
about 1 to about 207 of FIG. 15 (SEQ ID NO:19); (2) a DNA molecule
encoding an hIL-1Ra1S polypeptide comprising the amino acid
sequence of amino acid residues from about 1 to about 167 of FIG.
16 (SEQ ID NO:21); (3) a DNA molecule encoding an hIL-1Ra1V
polypeptide comprising the amino acid sequence of amino acid
residues from about 1 to about 218 of FIG. 19 (SEQ ID NO:25); and
(4) the complement of any of the DNA molecules of (1)-(2).
5. The isolated DNA molecule of claim 2 selected from the group
consisting of: (1) a DNA molecule encoding an hIL-1Ra3 polypeptide
comprising the amino acid sequence of amino acid residues from
about 34 to about 155 of FIG. 7 (SEQ ID NO:13); (2) a DNA molecule
encoding a mIL-1Ra3 polypeptide comprising the amino acid sequence
of amino acid residues from about 34 to about 155 of FIG. 9 (SEQ ID
NO:16); and (3) the complement of any of the DNA molecules of
(1)-(2).
6. The isolated DNA molecule of claim 5 selected from the group
consisting of: (1) a DNA molecule encoding an hIL-1Ra3 polypeptide
comprising the amino acid sequence of amino acid residues from
about 2 to about 155 of FIG. 7 (SEQ ID NO:13); (2) a DNA molecule
encoding a mIL-1Ra3 polypeptide comprising the amino acid sequence
of amino acid residues from about 2 to about 155 of FIG. 9 (SEQ ID
NO:16); and (3) the complement of any of the DNA molecules of
(1)-(2).
7. The isolated DNA molecule of claim 1 selected from the group
consisting of: (1) a DNA molecule which encodes an hIL-1Ra1
polypeptide, and which comprises the nucleic acid sequence of
nucleotide positions from about 118 to about 618 in the sense
strand of FIG. 2 (SEQ ID NO:4); (2) a DNA molecule which encodes an
hIL-1Ra1 polypeptide, and which comprises the nucleic acid sequence
of nucleotide positions from about 145 to about 681 in the sense
strand of FIG. 3 (SEQ ID NO:6); (3) a DNA molecule which encodes an
hIL-1Ra2 polypeptide, and which comprises the nucleic acid sequence
of nucleotide positions from about 96 to about 497 in the sense
strand of FIG. 5 (SEQ ID NO:9); (4) a DNA molecule which encodes an
hIL-1Ra3 polypeptide, and which comprises the nucleic acid sequence
of nucleotide positions from about 283 to about 402 in the sense
strand of FIG. 7 (SEQ ID NO:12); (5) a DNA molecule which encodes a
mIL-1Ra3 polypeptide, and which comprises the nucleic acid sequence
of nucleotide positions from about 427 to about 546 in the sense
strand of FIG. 9 (SEQ ID NO:15); (6) a DNA molecule which encodes
an hIL-1Ra1L polypeptide, and which comprises the nucleic acid
sequence of nucleotide positions from about 79 to about 624 in the
sense strand of FIG. 15 (SEQ ID NO:18); (7) a DNA molecule which
encodes an hIL-1Ra1S polypeptide, and which comprises the nucleic
acid sequence of nucleotide positions from about 79 to about 504 in
the sense strand of FIG. 16 (SEQ ID NO:20); (8) a DNA molecule
which encodes an hIL-1Ra1V polypeptide, and which comprises the
nucleic acid sequence of nucleotide positions from about 208 to
about 726 in the sense strand of FIG. 19 (SEQ ID NO:24); and (9)
the complement of any of the DNA molecules of (1)-(8).
8. The isolated DNA molecule of claim 7 selected from the group
consisting of: (1) a DNA molecule which encodes an hIL-1Ra1
polypeptide, and which comprises the nucleic acid sequence of
nucleotide positions from about 118 to about 618 in the sense
strand of FIG. 2 (SEQ ID NO:4); (2) a DNA molecule which encodes an
hIL-1Ra1 polypeptide, and which comprises the nucleic acid sequence
of nucleotide positions from about 145 to about 681 in the sense
strand of FIG. 3 (SEQ ID NO:6); (3) a DNA molecule which encodes an
hIL-1Ra2 polypeptide, and which comprises the nucleic acid sequence
of nucleotide positions from about 96 to about 497 in the sense
strand of FIG. 5 (SEQ ID NO:9); (4) a DNA molecule which encodes an
hIL-1Ra3 polypeptide, and which comprises the nucleic acid sequence
of nucleotide positions from about 283 to about 402 in the sense
strand of FIG. 7 (SEQ ID NO:12); (5) a DNA molecule which encodes a
mIL-1Ra3 polypeptide, and which comprises the nucleic acid sequence
of nucleotide positions from about 427 to about 546 in the sense
strand of FIG. 9 (SEQ ID NO:15); and (6) the complement of any of
the DNA molecules of (1)-(5).
9. The isolated DNA molecule of claim 7 selected from the group
consisting of: (1) a DNA molecule which encodes an hIL-1Ra1L
polypeptide, and which comprises the nucleic acid sequence of
nucleotide positions from about 79 to about 624 in the sense strand
of FIG. 15 (SEQ ID NO:18); (2) a DNA molecule which encodes an
hIL-1Ra1S polypeptide, and which comprises the nucleic acid
sequence of nucleotide positions from about 79 to about 504 in the
sense strand of FIG. 16 (SEQ ID NO:20); (3) a DNA molecule which
encodes an hIL-1Ra1V polypeptide, and which comprises the nucleic
acid sequence of nucleotide positions from about 208 to about 726
in the sense strand of FIG. 19 (SEQ ID NO:24); and (4) the
complement of any of the DNA molecules of (1)-(3).
10. The isolated DNA molecule of claim 9 selected from the group
consisting of: (1) a DNA molecule which encodes an hIL-1Ra1L
polypeptide, and which comprises the nucleic acid sequence of
nucleotide positions from about 4 to about 624 in the sense strand
of FIG. 15 (SEQ ID NO:18); (2) a DNA molecule which encodes an
hIL-1Ra1S polypeptide, and which comprises the nucleic acid
sequence of nucleotide positions from about 4 to about 504 in the
sense strand of FIG. 16 (SEQ ID NO:20); (3) a DNA molecule which
encodes an hIL-1Ra1V polypeptide, and which comprises the nucleic
acid sequence of nucleotide positions from about 73 to about 726 in
the sense strand of FIG. 19 (SEQ ID NO:24); and (4) the complement
of any of the DNA molecules of (1)-(3).
11. The isolated nucleic acid molecule of claim 8 selected from the
group consisting of: (1) a DNA molecule comprising the nucleic acid
sequence of nucleotide positions from about 103 to about 681 in the
sense strand of FIG. 3 (SEQ ID NO:6); (2) a DNA molecule comprising
the nucleic acid sequence of nucleotide positions from about 100 to
about 465 in the sense strand of FIG. 7 (SEQ ID NO:12); (3) a DNA
molecule comprising the nucleic acid sequence of nucleotide
positions from about 244 to about 609 in the sense strand of FIG. 9
(SEQ ID NO:15); and (4) the complement of any of the DNA molecules
of (1)-(3).
12. The isolated nucleic acid molecule of claim 8 comprising (a)
the complete DNA sequence in the sense strand of FIG. 2 (SEQ ID
NO:4), FIG. 3 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:9), FIG. 7 (SEQ ID
NO:12), or FIG. 9 (SEQ ID NO:15), or (b) the complement of (a).
13. An isolated nucleic acid molecule encoding an IL-1lp
polypeptide, comprising DNA hybridizing to the complement of a
nucleic acid sequence selected from the group consisting of: (1)
the nucleic acid sequence consisting of nucleotide positions from
about 238 to about 465 in the sense strand of FIG. 7 (SEQ ID
NO:12); (2) the nucleic acid sequence consisting of nucleotide
positions from about 427 to about 609 in the sense strand of FIG. 9
(SEQ ID NO:15); and (3) the nucleic acid sequence consisting of
nucleotide positions from about 114 to about 135 in the sense
strand of FIG. 15 (SEQ ID NO:18).
14. An isolated nucleic acid molecule comprising (a) a DNA molecule
encoding a polypeptide selected from the group consisting of: (1) a
polypeptide comprising the entire amino acid sequence encoded by
the longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203588; (2) a polypeptide comprising
the entire amino acid sequence, or the entire amino acid sequence
excluding the 36 N-terminal amino acid residues of such sequence,
encoded by the longest open reading frame in the cDNA insert in the
vector deposited as ATCC Deposit No. 203587; (3) a polypeptide
comprising the entire amino acid sequence encoded by the longest
open reading frame in the cDNA insert in the vector deposited as
ATCC Deposit No. 203586; (4) a polypeptide comprising the entire
amino acid sequence, or the entire amino acid sequence excluding
the N-terminal amino acid residue of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203589; (5) a polypeptide comprising
the entire amino acid sequence, or the entire amino acid sequence
excluding the N-terminal amino acid residue of such sequence,
encoded by the cDNA insert in the vector deposited as ATCC Deposit
No. 203590; (6) a polypeptide comprising the entire amino acid
sequence, or the entire amino acid sequence excluding the 34
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203846; (7) a polypeptide comprising
the entire amino acid sequence encoded by the longest open reading
frame in the cDNA insert in the vector deposited as ATCC Deposit
No. 203855; and (8) a polypeptide comprising the entire amino acid
sequence, or the entire amino acid sequence excluding the 45
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203973; or (b) the complement of the
DNA molecule of (a).
15. An isolated nucleic acid molecule comprising (a) DNA encoding
the IL-1lp polypeptide of claim 21, or (b) the complement of the
DNA of (a).
16. A vector comprising the nucleic acid of claim 1.
17. The vector of claim 16 operably linked to control sequences
recognized by a host cell transfected with the vector.
18. A host cell comprising the vector of claim 16.
19. A process for producing an IL-1lp polypeptide comprising the
steps of: (1) culturing a host cell comprising the DNA molecule of
claim 15 under conditions suitable for expression of the IL-1lp
polypeptide encoded by the DNA molecule; and (2) recovering said
IL-1lp polypeptide from the cell culture.
20. An isolated IL-1lp polypeptide encoded by the nucleic acid
molecule of claim 1.
21. An isolated IL-1lp polypeptide selected from the group
consisting of: (1) an hIL-1Ra1V polypeptide consisting of an amino
acid sequence having at least an 80% sequence identity to the
sequence of amino acid residues from about 46 to about 218 of FIG.
19 (SEQ ID NO:25); (2) an hIL-1Ra3 polypeptide consisting of an
amino acid sequence having at least an 80% sequence identity to the
sequence of amino acid residues from about 95 to about 134 of FIG.
7 (SEQ ID NO:13); and (3) a mIL-1Ra3 polypeptide consisting of an
amino acid sequence having at least an 80% sequence identity to the
sequence of amino acid residues from about 95 to about 134 of FIG.
9 (SEQ ID NO:16).
22. An isolated IL-1lp polypeptide selected from the group
consisting of: (1) an hIL-1Ra1 polypeptide comprising amino acid
residues from about 37 to about 203 of FIG. 2 (SEQ ID NO:5); (2) an
hIL-1Ra1 polypeptide comprising amino acid residues from about 15
to about 193 of FIG. 3 (SEQ ID NO:7); (3) an hIL-1Ra2 polypeptide
comprising amino acid residues from about 1 to about 134 of FIG. 5
(SEQ ID NO:10); (4) an hIL-1Ra3 polypeptide comprising amino acid
residues from about 95 to about 134 of FIG. 7 (SEQ ID NO:13); (5) a
mIL-1Ra3 polypeptide comprising amino acid residues from about 95
to about 134 of FIG. 9 (SEQ ID NO:16); (6) an hIL-1Ra1L polypeptide
comprising amino acid residues from about 26 to about 207 of FIG.
15 (SEQ ID NO:19); (7) an hIL-1Ra1S polypeptide comprising amino
acid residues from about 26 to about 167 of FIG. 16 (SEQ ID NO:21);
and (8) an hIL-1Ra1V polypeptide comprising amino acid residues
from about 46 to about 218 of FIG. 19 (SEQ ID NO:25).
23. The isolated IL-1lp polypeptide of claim 22 selected from the
group consisting of: (1) an hIL-1Ra1L polypeptide comprising amino
acid residues from about 1 to about 207 of FIG. 15 (SEQ ID NO:19);
(2) an hIL-1Ra1S polypeptide comprising amino acid residues from
about 1 to about 167 of FIG. 16 (SEQ ID NO:21); and (3) an
hIL-1Ra1V polypeptide comprising amino acid residues from about 1
to about 218 of FIG. 19 (SEQ ID NO:25).
24. The isolated polypeptide of claim 22 selected from the group
consisting of: (1) an hIL-1Ra3 polypeptide comprising amino acid
residues from about 34 to about 155 of FIG. 7 (SEQ ID NO:13); and
(2) a mIL-1Ra3 polypeptide comprising amino acid residues from
about 34 to about 155 of FIG. 9 (SEQ ID NO:16).
25. The polypeptide encoded by the DNA molecule of (a) in claim
14.
26. The IL-1lp polypeptide of claim 22 that comprises a native
amino acid sequence of the IL-1lp fused at its C-terminus or
N-terminus to a heterologous amino acid sequence.
27. The IL-1lp polypeptide of claim 26, wherein said heterologous
amino acid sequence is an epitope tag sequence.
28. The IL-1lp polypeptide of claim 26, wherein said heterologous
amino acid sequence is a Fc region of an immunoglobulin.
29. An antibody which specifically binds to the IL-1lp polypeptide
of claim 22.
30. The antibody of claim 29, wherein said antibody is a monoclonal
antibody.
Description
RELATED APPLICATIONS
[0001] This is a continuation application of U.S. Ser. No.
09/869,566 filed Feb. 19, 2002, now pending, which is a National
Phase application of PCT/US99/30720 filed Dec. 22, 1999, now
national, which claims the benefit of U.S. Ser. No. 60/129,122
filed Apr. 13, 1999, U.S. Ser. No. 60/116,843 filed Jan. 22, 1999,
and U.S. Ser. No. 60/113,430 filed Dec. 23, 1998, all of which are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the
identification and isolation of novel DNAs having homology to
interleukin-1 (IL-1) or interleukin-1 receptor antagonist (IL-1Ra)
polypeptides, and to the recombinant production of novel
polypeptides, designated herein as interleukin-1-like polypeptides
("IL-1lp").
BACKGROUND OF THE INVENTION
[0003] Interleukin-1 refers to two proteins (IL-1.alpha. and
IL-1.beta.) which play a key role early in the inflammatory
response (for a review, see Dinarello, Blood, 87: 2095-2147 (1996)
and references therein). both proteins are made as intracellular
precursor proteins which are cleaved upon secretion to yield mature
carboxy-terminal 17 kDa fragments which are biologically active. In
the case of IL-1.beta., this cleavage involves an intracellular
cysteine protease, known as ICE, which is required to release the
active fragment from the inactive precursor. The precursor of
IL-1.alpha. is active.
[0004] These two proteins act by binding to cell surface receptors
found on almost all cell types and triggering a range of responses
either alone or in concert with other secreted factors. These range
from effects on proliferation (e.g. fibroblasts, T cells) apoptosis
(e.g. A375 melanoma cells), cytokine induction (e.g. of TNF, IL-1,
IL-8), receptor activation (e.g. E-selectin), eicosanoid production
(e.g. PGE2) and the secretion of degradative enzymes (e.g.
collagenase). To achieve these effects, IL-1 activates
transcription factors such as NF-KB and AP-1. Several of the
activities of IL-1 action on target cells are believed to be
mediated through activation of kinase cascades that have also been
associated with cellular stresses, such as the stress activated MAP
kinase JNK/SAPK and p38.
[0005] A third member of the IL-1 family was subsequently
discovered which acts as a natural antagonist of IL-1.alpha. and
IL-1.beta. by binding to the IL-1 receptor but not transducing an
intracellular signal or a biological response. The protein is
called IL-1Ra (for IL-1 receptor antagonist) or IRAP (for IL-1
receptor antagonist protein). At least three alternatively spliced
forms of IL-1Ra exist: one encodes a secreted protein, also known
as secretory IL-1Ra ("sIL-1Ra") (described in Eisenberg et al.,
Nature, 343: 341-346 (1990)), and the other two encode
intracellular proteins. IL-1.alpha., IL-1.beta. and IL-1Ra exhibit
approximately 25-30% sequence identity with each other and share a
similar three dimensional structure consisting of twelve
.beta.-strands folded into a .beta.-barrel, with an internal thrice
repeated structural motif.
[0006] There are three known IL-1 receptor subunits. The active
receptor complex consists of the type I receptor and IL-1 accessory
protein (IL-1RAcP). The type I receptor is responsible for binding
of the IL-1.alpha., IL-1.beta. and IL-1Ra ligands, and is able to
do so in the absence of the IL-1RAcP. However, signal transduction
requires the interaction of IL-1.alpha. or IL-1.beta. with the
IL-1RAcP. IL-1Ra does not interact with the IL-1RAcP and hence
cannot induce signal transduction. A third receptor subunit, the
type II receptor, binds IL-1.alpha. and IL-1.beta. but cannot
transduce signal due its lack of an intracellular domain. Instead,
the type II receptor either acts as a decoy in its membrane bound
form or as an IL-1 antagonist in a processed, secreted form, and
hence inhibits IL-1 activity. The type II receptor weakly binds to
IL-1Ra.
[0007] Many studies using IL-1Ra, soluble IL-1R derived from the
extracellular domain of the type I IL-1 receptor, antibodies to
IL-1.alpha. or IL-1.beta., and transgenic knockout mice for these
genes have shown that IL-1 plays a role in a number of
pathophysiologies (for a review, see Dinarello, Blood, 87:
2095-2147 (1996)). For example, IL-1Ra has been shown to be
effective in animal models of septic shock, rheumatoid arthritis,
graft-versus-host disease (GVHD), stroke, cardiac ischemia,
psoriasis, inflammatory bowel disease, and asthma. In addition,
IL-1Ra has demonstrated efficacy in clinical trials for rheumatoid
arthritis and GVHD, and is also in clinical trials for inflammatory
bowel disease, asthma and psoriasis.
[0008] More recently, interleukin-18 (IL-18) was placed in the IL-1
family (for a review, see Dinarello et al, J. Leukocyte Biol., 63:
658-664 (1998)). IL-18 shares the .beta.-pleated, barrel-like form
of IL-1.alpha. and IL-1.beta.. In addition, IL-18 is the natural
ligand for the IL-1 receptor family member formerly known as
IL-1R-related protein (IL-1Rrp) (now known as the IL-18 receptor
(IL-18R)). IL-18 has been shown to initiate the inflammatory
cytokine cascade in a mixed population of peripheral blood
mononuclear cells (PBMCs) by triggering the constitutive IL-18
receptors on lymphocytes and NK cells, inducing TNF production in
the activated cells. TNF, in turn, stimulates IL-1 and IL-8
production in CD14+ cells. Because of its ability to induce TNF,
IL-1, and both C--C and C--X--C chemokines, and because IL-18
induces Fas ligand as well as nuclear translocation of nuclear
factor .kappa.B (NF-.kappa.B), IL-18 ranks with other
pro-inflammatory cytokines as a likely contributor to systemic and
local inflammation.
SUMMARY OF THE INVENTION
[0009] A family of cDNA clones (DNA85066, DNA96786, DNA94618,
DNA102043, DNA114876, DNA102044, DNA92929, DNA96787, and DNA92505)
has been identified, having homology to interleukin-1, that encode
novel polypeptides. The novel polypeptides and variants thereof are
collectively designated in the present application as
"interleukin-1-like polypeptides" or "IL-1lp", as further defined
herein. Accordingly, one aspect of the invention is an isolated
IL-1lp polypeptide.
[0010] In another embodiment, the invention provides an isolated
nucleic acid molecule encoding an IL-1lp polypeptide.
[0011] In another embodiment, the invention provides a method for
producing an IL-1lp comprising culturing a host cell comprising a
heterologous nucleic acid sequence encoding an IL-1lp polypeptide,
under conditions wherein the IL-1lp polypeptide is expressed, and
recovering the IL-1lp polypeptide from the host cell.
[0012] In another embodiment, the invention provides an anti-IL-1lp
antibody.
[0013] In another embodiment, the invention provides chimeric
molecules comprising an IL-1lp polypeptide fused to a heterologous
polypeptide or amino acid sequence. An example of such a chimeric
molecule comprises an IL-1lp polypeptide fused to an epitope tag
sequence or a Fc region of an immunoglobulin.
[0014] In another embodiment, the invention provides an antibody
which specifically binds to an IL-1lp polypeptide. Optionally, the
antibody is a monoclonal antibody.
[0015] In yet another embodiment, the invention concerns agonists
and antagonists of a native IL-1lp polypeptide. In a particular
embodiment, the agonist or antagonist is an anti-IL-1lp
antibody.
[0016] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists of a native IL-1lp polypeptide,
by contacting the native IL-1lp polypeptide with a candidate
molecule and monitoring a biological activity mediated by said
polypeptide.
[0017] In a still further embodiment, the invention concerns a
composition comprising an IL-1lp polypeptide, or an agonist or
antagonist as hereinabove defined, in combination with a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) and derived
amino acid sequences (SEQ ID NOS:2-3) related to a native sequence
hIL-1Ra1. The nucleotide sequence (SEQ ID NO:1) contains an intron
believed to extend from nucleotide positions 181 to 432, with a
splice donor site at nucleotide positions 181 to 186 and splice
acceptor site at nucleotide positions 430 to 432. The amino acid
sequences (SEQ ID NOS:2 and 3) are derived from the exonic
sequences that are believed to make up the processed (intron-free)
coding sequence.
[0019] FIG. 2 shows the nucleotide sequence (SEQ ID NO:4) and
derived amino acid sequence (SEQ ID NO:5) of a native sequence
hIL-1Ra1 polypeptide fused at its N-terminus to a heterologous
signal peptide (amino acid positions 1-15), flag peptide affinity
handle (amino acid positions 16-23) and peptide linker (amino acid
positions 24-36).
[0020] FIG. 3 shows the nucleotide sequence (SEQ ID NO:6) and
derived amino acid sequence (SEQ ID NO:7) of a native sequence
hIL-1Ra1 polypeptide. The nucleotide sequence (SEQ ID NO:6) and
derived amino acid sequence (SEQ ID NO:7) are believed to represent
the processed (intron-free) form and intact hIL-1Ra1 polypeptide,
respectively, of the nucleotide sequence (SEQ ID NO:1) and amino
acid sequences (SEQ ID NOS:2-3) of FIG. 1. The start and stop
codons in the coding sequence are located at nucleotide positions
103-105 and 682-684, respectively. The putative signal sequence
extends from amino acid positions 1 to 14. A putative cAMP- and
cGMP-dependent protein kinase phosphorylation site is located at
amino acid positions 33-36. Putative N-myristoylation sites are
located at amino acid positions 50-55 and 87-92.
[0021] FIG. 4 shows the nucleotide sequence (SEQ ID NO:8) of EST
AI014548.
[0022] FIG. 5 shows the nucleotide sequence (SEQ ID NO:9) and
derived amino acid sequence (SEQ ID NO:10) of a native sequence
hIL-1Ra2 polypeptide. The start and stop codons in the coding
sequence are located at nucleotide positions 96-98 and 498-500,
respectively. The putative signal sequence extends from amino acid
positions 1-26.
[0023] FIG. 6 shows the nucleotide sequence (SEQ ID NO:11) of EST
1433156.
[0024] FIG. 7 shows the nucleotide sequence (SEQ ID NO:12) and
derived amino acid sequence (SEQ ID NO:13) of a native sequence
hIL-1Ra3 polypeptide. The start and stop codons in the coding
sequence are located at nucleotide positions 1-3 and 466-468,
respectively. The putative signal sequence extends from amino acid
positions 1-33. Putative N-myristoylation sites are located at
amino acid positions 29-34, 30-35, 60-65, 63-68, 73-78, 91-96 and
106-111. An interleukin-1-like sequence is located at amino acid
positions 111-131.
[0025] FIG. 8 shows the nucleotide sequence (SEQ ID NO:14) of EST
5120028.
[0026] FIG. 9 shows the nucleotide sequence (SEQ ID NO:15) and
derived amino acid sequence (SEQ ID NO:16) of a native sequence
mIL-1Ra3 polypeptide. The start and stop codons in the coding
sequence are located at nucleotide positions 145-147 and 610-612,
respectively. The putative signal sequence extends from amino acid
positions 1-33. Putative N-myristoylation sites are located at
amino acid positions 29-34, 60-65, 63-68, 91-96 and 106-111. An
interleukin-1-like sequence is located at amino acid positions
111-131.
[0027] FIG. 10 shows the nucleotide sequence (SEQ ID NO:17) of EST
W08205.
[0028] FIG. 11 is an autoradiograph of Northern blots depicting
expression of hIL-1Ra3 mRNA in placental tissue and expression of
mIL-1Ra3 mRNA in day-17 mouse embryo tissue.
[0029] FIG. 12 is an amino acid sequence alignment of native
sequence hIL-1Ra1L (SEQ ID NO:19), hIL-1Ra1V (SEQ ID NO:25),
hIL-1Ra1S (SEQ ID NO:21), hIL-1Ra2 (SEQ ID NO:10), hIL-1Ra3 (SEQ ID
NO:13) and mIL-1Ra3 (SEQ ID NO:16) polypeptides with secretory
hIL-1Ra (also referred to as "sIL-1Ra" and "hIL-1Ra") (SEQ ID
NO:26), hIL-1Ra.beta. (SEQ ID NO:27) and TANGO-77 (SEQ ID
NO:28).
[0030] FIG. 13A is a Western blot depicting the interleukin-18
receptor (IL-18R) binding activity of hIL-1Ra1. In the top panel
(depicting a protein band at approximately 22 kD), a conditioned
medium containing FLAGhIL-1Ra1 and FLAGIL-1R-ECD-Fc (shown in the
left lane) and a conditioned medium containing FLAGhIL-1Ra1 and
FLAGIL-18R-ECD-Fc (shown in the right lane) were each
immunoprecipitated with protein G-sepharose, and the resulting
precipitates were resolved by gel electrophoresis and Western
blotting with anti-FLAG monoclonal antibody. In the middle and
bottom panels (depicting protein bands at approximately 22 kD and
85 kD), a second aliquot from the FLAGhIL-1Ra1 and FLAGIL-1R-ECD-Fc
conditioned medium used in the top panel (shown in the left lane)
and a second aliquot from the FLAGhIL-1Ra1 and FLAGIL-18R-ECD-Fc
conditioned medium used in the top panel (shown in the right lane)
were each immunoprecipitated with anti-FLAG monoclonal antibody,
and the resulting precipitates were resolved by gel electrophoresis
and Western blotting with anti-FLAG monoclonal antibody.
[0031] FIG. 13B is a Western blot depicting the IL-1R binding
activity of hIL-1Ra3. In the top panel (depicting a protein band at
approximately 20 kD), a conditioned medium containing hIL-1Ra3-FLAG
and FLAGDR6-Fc (shown in the left lane), a conditioned medium
containing hIL-1Ra3-FLAG and FLAGIL-1R-ECD-Fc (shown in the middle
lane), and conditioned medium containing hIL-1Ra3-FLAG and
FLAGIL-18R-ECD-Fc (shown in the right lane) were each
immunoprecipitated with protein G sepharose, and the resulting
precipitates were resolved by gel electrophoresis and Western
blotting with anti-FLAG monoclonal antibody. In the middle and
bottom panels (depicting protein bands at approximately 20 kD and
85 kD), a second aliquot from the hIL-1Ra3-FLAG and FLAGDR6-Fc
conditioned medium used in the top panel (shown in the left lane),
a second aliquot from the hIL-1Ra3-FLAG and FLAGIL-1R-ECD-Fc
conditioned medium used in the top panel (shown in the middle lane)
and a second aliquot from the hIL-1Ra3-FLAG and FLAGIL-18R-ECD-Fc
conditioned medium used in the top panel (shown in the right lane)
were each immunoprecipitated with anti-FLAG monoclonal antibody,
and the resulting precipitates were resolved by gel electrophoresis
and Western blotting with anti-FLAG monoclonal antibody.
[0032] FIG. 14 is a Western blot depicting the interleukin-1
receptor (IL-1R) binding activity of mIL-1Ra3. In the top panel
(depicting a protein band at approximately 21 kD) and the bottom
panel (depicting protein bands at approximately 85 kD) the
FLAGIL-1R-ECD-Fc in conditioned medium (shown in the left lane) and
the FLAGIL-18R-ECD-Fc in conditioned medium (shown in the right
lane) were immobilized with protein G-agarose, the resulting solid
phase was contacted with conditioned medium containing
FLAGmIL-1Ra3, and the resulting bound complexes were resolved by
gel electrophoresis and Western blotting with anti-FLAG monoclonal
antibody.
[0033] FIG. 15 shows the nucleotide sequence (SEQ ID NO:18) and
derived amino acid sequence (SEQ ID NO:19) of a native sequence
hIL-1Ra1L polypeptide. The start and stop codons in the coding
sequence are located at nucleotide positions 4-6 and 625-627,
respectively. The putative signal sequence extends from amino acid
positions 1 to 34. A putative cAMP- and cGMP-dependent protein
kinase phosphorylation site is located at amino acid positions
47-50. Putative N-myristoylation sites are located at amino acid
positions 64-69 and 101-106.
[0034] FIG. 16 shows the nucleotide sequence (SEQ ID NO:20) and
derived amino acid sequence (SEQ ID NO:21) of a native sequence
hIL-1Ra1S polypeptide. The start and stop codons in the coding
sequence are located at nucleotide positions 4-6 and 505-507,
respectively. A putative signal sequence extends from amino acid
positions 1 to 46. A putative N-myristoylation site is located at
amino acid positions 61-66.
[0035] FIG. 17 shows the single stranded nucleotide sequence (SEQ
ID NO:23) of EST AI343258 (lower strand) along with its
complementary nucleotide sequence (SEQ ID NO:22) (upper
strand).
[0036] FIG. 18 is an amino acid sequence alignment of native
sequence hIL-1Ra1 (SEQ ID NO:3), hIL-1Ra1L (SEQ ID NO:19),
hIL-1Ra1V (SEQ ID NO:25) and hIL-1Ra1S (SEQ ID NO:21)
polypeptides.
[0037] FIG. 19 shows the nucleotide sequence (SEQ ID NO:24) and
derived amino acid sequence (SEQ ID NO:25) of a native sequence
hIL-1Ra1V polypeptide. The start and stop codons in the coding
sequence are located at nucleotide positions 73-75 and 727-729,
respectively. An alternate start codon is located at nucleotide
positions 106-108. A putative signal sequence extends from amino
acid positions 1 to 48.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions:
[0038] The terms "interleukin-1-like polypeptide",
"interleukin-1-like protein", "IL-1lp", "IL-1lp polypeptide", and
"IL-1lp protein" encompass any native sequence IL-1lp, and further
encompass IL-1lp variants (which are further defined herein). The
IL-1lp may be isolated from a variety of sources, such as from
human tissue types or from another source, or prepared by
recombinant and/or synthetic methods.
[0039] A "native sequence IL-1lp" comprises a polypeptide having
the same amino acid sequence as a native sequence hIL-1Ra1,
hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, or mIL-1Ra3,
(which are further defined herein). Such native sequence IL-1lp can
be isolated from nature or can be produced by recombinant and/or
synthetic means. The term "native sequence IL-1lp" specifically
encompasses naturally-occurring truncated or secreted forms (e.g.,
a processed, mature sequence) and naturally-occurring allelic
variants of the IL-1lp.
[0040] The terms "naturally-occurring amino acid sequence" and
"native amino acid sequence" mean any amino acid sequence found in
a polypeptide existing in nature, i.e. present in a
naturally-occurring polypeptide.
[0041] The terms "non-naturally-occurring amino acid sequence" and
"non-native amino acid sequence" mean any amino acid sequence not
found in a polypeptide existing in nature, i.e. not present in a
naturally-occurring polypeptide.
[0042] "IL-1lp variant" is defined as any polypeptide that
comprises a variant of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S,
hIL-1Ra2, hIL-1Ra3, or mIL-1Ra3 (which are further defined
herein).
[0043] Human interleukin-1 receptor antagonist analog 1
("hIL-1Ra1"), hIL-1Ra1 polypeptide, and hIL-1Ra1 protein are
defined as any native sequence hIL-1Ra1 or variant hIL-1Ra1.
[0044] A "native sequence hIL-1Ra1" means a polypeptide comprising
a naturally-occurring amino acid sequence selected from the group
consisting of: (1) the amino acid sequence of amino acid residues
from at or about 37 to at or about 63 of FIG. 2 (SEQ ID NO:5); (2)
the amino acid sequence of amino acid residues from at or about 37
to at or about 203 of FIG. 2 (SEQ ID NO:5); (3) the amino acid
sequence of amino acid residues from at or about 15 to about 53 of
FIG. 3 (SEQ ID NO:7); (4) the amino acid sequence of amino acid
residues from at or about 15 to at or about 193 of FIG. 3 (SEQ ID
NO:7); and (5) the amino acid sequence of any naturally-occurring
truncated or secreted form or any naturally-occurring allelic
variant of a polypeptide comprising the amino acid sequence of (1)
or (2) or (3) or (4). In one embodiment of the invention, the
native sequence hIL-1Ra1 comprises amino acids from at or about 37
to at or about 203 of FIG. 2 (SEQ ID NO:5) or amino acids from at
or about 15 to at or about 193 of FIG. 3 (SEQ ID NO:7).
[0045] "hIL-1Ra1 variant" is defined as any hIL-1Ra1 N-terminal
variant or hIL-1Ra1 full sequence variant (which are further
defined herein).
[0046] "hIL-1Ra1 N-terminal variant" means any hIL-1Ra1 other than
a native sequence hIL-1Ra1, which variant is an active hIL-1Ra1, as
defined below, having at least about 80% amino acid sequence
identity with an amino acid sequence selected from the group
consisting of: (1) the amino acid sequence of amino acid residues
from at or about 37 to at or about 63 of FIG. 2 (SEQ ID NO:5); and
(2) the amino acid sequence of amino acid residues from at or about
15 to at or about 53 of FIG. 3 (SEQ ID NO:7). Such hIL-1Ra1
N-terminal variants include, for instance, hIL-1Ra1 polypeptides
wherein one or more amino acid residues are added, or deleted,
internally or at the N- or C-terminus, in the sequence of amino
acid residues from at or about 37 to at or about 63 of FIG. 2 (SEQ
ID NO:5) or in the sequence of amino acid residues from at or about
15 to at or about 53 of FIG. 3 (SEQ ID NO:7). Ordinarily, an
hIL-1Ra1 N-terminal variant will have at least about 80% amino acid
sequence identity, or at least about 85% amino acid sequence
identity, or at least about 90% amino acid sequence identity, or at
least about 95% amino acid sequence identity with an amino acid
sequence selected from the group consisting of: (1) the amino acid
sequence of amino acid residues from at or about 37 to at or about
63 of FIG. 2 (SEQ ID NO:5); and (2) the amino acid sequence of
amino acid residues from at or about 15 to at or about 53 of FIG. 3
(SEQ ID NO:7).
[0047] "hIL-1Ra1 full sequence variant" means any hIL-1Ra1 other
than a native sequence hIL-1Ra1, which variant retains at least one
biologic activity of a native sequence hIL-1Ra1, such as the
ability to bind IL-18R, and which variant has at least about 80%
amino acid sequence identity, or at least about 85% amino acid
sequence identity, or at least about 90% amino acid sequence
identity, or at least about 95% amino acid sequence identity with
an amino acid sequence selected from the group consisting of: (1)
the amino acid sequence of amino acid residues from at or about 37
to at or about 203 of FIG. 2 (SEQ ID NO:5); and (2) the amino acid
sequence of amino acid residues from at or about 15 to at or about
193 of FIG. 3 (SEQ ID NO:7). Such hIL-1Ra1 full sequence variants
include, for instance, hIL-1Ra1 polypeptides wherein one or more
amino acid residues are added, or deleted, internally or at the N-
or C-terminus, in the sequence of amino acid residues from at or
about 37 to at or about 203 of FIG. 2 (SEQ ID NO:5) or in the
sequence of amino acid residues from at or about 15 to at or about
193 of FIG. 3 (SEQ ID NO:7).
[0048] Human interleukin-1 receptor antagonist analog 1 long
("hIL-1Ra1L"), hIL-1Ra1L polypeptide, and hIL-1Ra1L protein are
defined as any native sequence hIL-1Ra1L or hIL-1Ra1L variant
(which are further defined herein).
[0049] A "native sequence hIL-1Ra1L" means a polypeptide comprising
a naturally-occurring amino acid sequence selected from the group
consisting of: (1) the amino acid sequence of amino acid residues
from at or about 26 to at or about 44 of FIG. 15 (SEQ ID NO:19);
(2) the amino acid sequence of amino acid residues from at or about
26 to at or about 207 of FIG. 15 (SEQ ID NO:19); and (3) the amino
acid sequence of any naturally-occurring truncated or secreted form
or any naturally-occurring allelic variant of a polypeptide
comprising the amino acid sequence of (1) or (2). In one embodiment
of the invention, the native sequence hIL-1Ra1L comprises amino
acids from at or about 26 to at or about 207 of FIG. 15 (SEQ ID
NO:19).
[0050] "hIL-1Ra1L variant" is defined as any hIL-1Ra1L N-terminal
variant or hIL-1Ra1L full sequence variant or hIL-1Ra1L fusion
variant (which are further defined herein).
[0051] "hIL-1Ra1L N-terminal variant" means any hIL-1Ra1L other
than a native sequence hIL-1Ra1L, which variant is an active
hIL-1Ra1L, as defined below, having at least about 80% amino acid
sequence identity with the amino acid sequence of amino acid
residues from at or about 26 to at or about 44 of FIG. 15 (SEQ ID
NO:19). Such hIL-1Ra1L N-terminal variants include, for instance,
hIL-1Ra1L polypeptides wherein one or more amino acid residues are
added, or deleted, internally or at the N- or C-terminus, in the
sequence of amino acid residues from at or about 26 to at or about
44 of FIG. 15 (SEQ ID NO:19). Ordinarily, an hIL-1Ra1L N-terminal
variant will have at least about 80% amino acid sequence identity,
or at least about 85% amino acid sequence identity, or at least
about 90% amino acid sequence identity, or at least about 95% amino
acid sequence identity with the amino acid sequence of amino acid
residues from at or about 26 to at or about 44 of FIG. 15 (SEQ ID
NO:19).
[0052] "hIL-1Ra1L full sequence variant" means any hIL-1Ra1L other
than a native sequence hIL-1Ra1L, which variant retains at least
one biologic activity of a native sequence hIL-1Ra1L, such as the
ability to bind IL-18R, and which variant has at least about 80%
amino acid sequence identity, or at least about 85% amino acid
sequence identity, or at least about 90% amino acid sequence
identity, or at least about 95% amino acid sequence identity with
the amino acid sequence of amino acid residues from at or about 26
to at or about 207 of FIG. 15 (SEQ ID NO:19). Such hIL-1Ra1L full
sequence variants include, for instance, hIL-1Ra1L polypeptides
wherein one or more amino acid residues are added, or deleted,
internally or at the N- or C-terminus, in the sequence of amino
acid residues from at or about 26 to at or about 207 of FIG. 15
(SEQ ID NO:19).
[0053] "hIL-1Ra1L fusion variant" means a chimeric hIL-1Ra1L
consisting of a native sequence hIL-1Ra1L fused at its N- or
C-terminus to a heterologous amino acid or amino acid sequence. In
one embodiment, the hIL-1Ra1L fusion variant polypeptide consists
of a native sequence of hIL-1Ra1L fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence,
wherein the heterologous amino acid or amino acid sequence is
heterologous to the native sequence, i.e. the resulting chimeric
sequence is non-naturally occurring. In another embodiment, the
hIL-1Ra1L fusion variant consists of the amino acid sequence of
amino acids from at or about 26 to at or about 207, inclusive of
FIG. 15 (SEQ ID NO:19), or the amino acid sequence of amino acid
residues from at or about 1 to at or about 207, inclusive of FIG.
15 (SEQ ID NO:19), fused at its N-terminus or C-terminus to a
heterologous amino acid or amino acid sequence to form a
non-naturally occurring fusion protein. Such hIL-1Ra1L fusion
variants include, for instance, hIL-1Ra1L polypeptides wherein a
heterologous secretion leader sequence is fused to the N-terminus
of the sequence of amino acid residues from at or about 26 to at or
about 207 of FIG. 15 (SEQ ID NO:19), or amino acid residues from at
or about 1 to at or about 207 of FIG. 15 (SEQ ID NO:19).
[0054] Human interleukin-1 receptor antagonist analog 1 long
allelic variant ("hIL-1Ra1V"), hIL-1Ra1V polypeptide, and hIL-1Ra1V
protein are defined as any native sequence hIL-1Ra1V or hIL-1Ra1V
variant (which are further defined herein).
[0055] A "native sequence hIL-1Ra1V" means a polypeptide comprising
a naturally-occurring amino acid sequence selected from the group
consisting of: (1) the amino acid sequence of amino acid residues
from at or about 46 to at or about 55 of FIG. 19 (SEQ ID NO:25);
(2) the amino acid sequence of amino acid residues from at or about
46 to at or about 218 of FIG. 19 (SEQ ID NO:25); (3) the amino acid
sequence of amino acid residues from at or about 37 to at or about
218 of FIG. 19 (SEQ ID NO:25); (4) the amino acid sequence of amino
acid residues from at or about 12 to at or about 218 of FIG. 19
(SEQ ID NO:25); and (5) the amino acid sequence of any
naturally-occurring truncated or secreted form or any
naturally-occurring allelic variant of a polypeptide comprising the
amino acid sequence of (1) or (2) or (3) or (4). In one embodiment
of the invention, the native sequence hIL-1Ra1V comprises amino
acids from at or about 46 to at or about 218 of FIG. 19 (SEQ ID
NO:25), or amino acids from at or about 37 to at or about 218 of
FIG. 19 (SEQ ID NO:25), or amino acids from at or about 12 to at or
about 218 of FIG. 19 (SEQ ID NO:25), or amino acids from at or
about 1 to at or about 218 of FIG. 19 (SEQ ID NO:25).
[0056] "hIL-1Ra1V variant" is defined as any hIL-1Ra1V N-terminal
variant or hIL-1Ra1V full sequence variant or hIL-1Ra1V fusion
variant (which are further defined herein).
[0057] "hIL-1Ra1V N-terminal variant" is defined as any hIL-1Ra1V
other than a native sequence hIL-1Ra1V, which variant is an active
hIL-1Ra1V, as defined below, having at least about 80% amino acid
sequence identity with the amino acid sequence of amino acid
residues from at or about 46 to at or about 89 of FIG. 19 (SEQ ID
NO:25). Such hIL-1Ra1V N-terminal variants include, for instance,
hIL-1Ra1V polypeptides wherein one or more amino acid residues are
added, internally or at the N- or C-terminus, in the sequence of
amino acid residues from at or about 46 to at or about 89 of FIG.
19 (SEQ ID NO:25). Ordinarily, an hIL-1Ra1V N-terminal variant will
have at least about 80% amino acid sequence identity, or at least
about 85% amino acid sequence identity, or at least about 90% amino
acid sequence identity, or at least about 95% amino acid sequence
identity with the sequence of amino acid residues from at or about
46 to at or about 89 of FIG. 19 (SEQ ID NO:25).
[0058] "hIL-1Ra1V full sequence variant" means any hIL-1Ra1V other
than a native sequence hIL-1Ra1V, which variant retains at least
one biologic activity of a native sequence hIL-1Ra1V, such as the
ability to bind IL-118R, and which variant has at least about 80%
amino acid sequence identity, or at least about 85% amino acid
sequence identity, or at least about 90% amino acid sequence
identity, or at least about 95% amino acid sequence identity with
the sequence of amino acid residues from at or about 46 to at or
about 218 of FIG. 19 (SEQ ID NO:25).
[0059] "hIL-1Ra1V fusion variant" means a chimeric hIL-1Ra1V
consisting of a native sequence hIL-1Ra1V fused at its N- or
C-terminus to a heterologous amino acid or amino acid sequence. In
one embodiment, the hIL-1Ra1V fusion variant polypeptide consists
of a native sequence of hIL-1Ra1V fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence,
wherein the heterologous amino acid or amino acid sequence is
heterologous to the native sequence, i.e. the resulting chimeric
sequence is non-naturally occurring. In another embodiment, the
hIL-1Ra1V fusion variant consists of the amino acid sequence of
amino acids from at or about 46 to at or about 218 of FIG. 19 (SEQ
ID NO:25), or the amino acid sequence of amino acids from at or
about 37 to at or about 218 of FIG. 19 (SEQ ID NO:25), or the amino
acid sequence of amino acids from at or about 12 to at or about 218
of FIG. 19 (SEQ ID NO:25), or the amino acid sequence of amino
acids from at or about 1 to at or about 218 of FIG. 19 (SEQ ID
NO:25), fused at its N-terminus or C-terminus to a heterologous
amino acid sequence to form a non-naturally occurring fusion
protein. Such hIL-1Ra1V fusion variants include, for instance,
hIL-1Ra1V polypeptides wherein a heterologous secretion leader
sequence is fused to the N-terminus of the sequence of amino acid
residues from at or about 46 to at or about 218 of FIG. 19 (SEQ ID
NO:25), or amino acid residues from at or about 37 to at or about
218 of FIG. 19 (SEQ ID NO:25), or amino acid residues from at or
about 12 to at or about 218 of FIG. 19 (SEQ ID NO:25), or amino
acid residues from at or about 1 to at or about 218 of FIG. 19 (SEQ
ID NO:25).
[0060] Human interleukin-1 receptor antagonist analog 1 short
("hIL-1Ra1S"), hIL-1Ra1S polypeptide, and hIL-1Ra1S protein are
defined as any native sequence hIL-1Ra1S or hIL-1Ra1S variant
(which are further defined herein).
[0061] A "native sequence hIL-1Ra1S" means a polypeptide comprising
a naturally-occurring amino acid sequence selected from the group
consisting of: (1) the amino acid sequence of amino acid residues
from at or about 1 to at or about 38 of FIG. 16 (SEQ ID NO:21); (2)
the amino acid sequence of amino acid residues from at or about 26
to at or about 167 of FIG. 16 (SEQ ID NO:21); (3) the amino acid
sequence of amino acid residues from at or about 39 to at or about
167 of FIG. 16 (SEQ ID NO:21); (4) the amino acid sequence of amino
acid residues from at or about 47 to at or about 167 of FIG. 16
(SEQ ID NO:21); and (5) the amino acid sequence of any
naturally-occurring truncated or secreted form or any
naturally-occurring allelic variant of a polypeptide comprising the
amino acid sequence of (1) or (2) or (3) or (4). In one embodiment
of the invention, the native sequence hIL-1Ra1S comprises amino
acids from at or about 26 to at or about 167 of FIG. 16 (SEQ ID
NO:21), or amino acids from at or about 1 to at or about 167 of
FIG. 16 (SEQ ID NO:21). In another embodiment, the native sequence
hIL-1Ra1S consists of amino acids from at or about 47 to at or
about 167 of FIG. 16 (SEQ ID NO:21) or amino acids from at or about
39 to at or about 167 of FIG. 16 (SEQ ID NO:21).
[0062] "hIL-1Ra1S fusion variant" and "hIL-1Ra1S variant" mean a
chimeric hIL-1Ra1S consisting of a native sequence hIL-1Ra1S fused
at its N-terminus or C-terminus to a heterologous amino acid or
amino acid sequence. In one embodiment, the hIL-1Ra1S fusion
variant polypeptide consists of a native sequence of hIL-1Ra1S
fused at its N-terminus or C-terminus to a heterologous amino acid
or amino acid sequence, wherein the heterologous amino acid or
amino acid sequence is heterologous to the native sequence, i.e.
the resulting chimeric sequence is non-naturally occurring. In
another embodiment, the hIL-1Ra1S fusion variant consists of the
amino acid sequence of amino acids from at or about 47 to at or
about 167 of FIG. 16 (SEQ ID NO:21), or the amino acid sequence of
amino acids from at or about 39 to at or about 167 of FIG. 16 (SEQ
ID NO:21), fused at its N-terminus or C-terminus to a heterologous
amino acid or amino acid sequence to form a non-naturally occurring
fusion protein. Such hIL-1Ra1S fusion variants include, for
instance, hIL-1Ra1S polypeptides wherein a heterologous secretion
leader sequence is fused to the N-terminus of the sequence of amino
acid residues from at or about 47 to at or about 167 of FIG. 16
(SEQ ID NO:21), or amino acid residues from at or about 39 to at or
about 167 of FIG. 16 (SEQ ID NO:21).
[0063] Human interleukin-1 receptor antagonist analog 2
("hIL-1Ra2"), hIL-1Ra2 polypeptide, and hIL-1Ra2 protein are
defined as any native sequence hIL-1Ra2 or hIL-1Ra2 fusion variant
(which are further defined herein).
[0064] A "native sequence hIL-1Ra2" means (1) a polypeptide
comprising the amino acid sequence of amino acid residues from at
or about 1 to at or about 134 of FIG. 5 (SEQ ID NO:10) or (2) a
polypeptide consisting of a naturally-occurring truncated or
secreted form of the polypeptide of (1). In one embodiment of the
invention, the native sequence hIL-1Ra2 consists of amino acids
from at or about 27 to at or about 134 of FIG. 5 (SEQ ID NO:10), or
amino acids from at or about 1 to at or about 134 of FIG. 5 (SEQ ID
NO:10).
[0065] "hIL-1Ra2 fusion variant" and "hIL-1Ra2 variant" mean a
chimeric hIL-1Ra2 consisting of a native sequence hIL-1Ra2 fused at
its N-terminus or C-terminus to a heterologous amino acid or amino
acid sequence. In one embodiment, the hIL-1Ra2 fusion variant
polypeptide consists of a native sequence of hIL-1Ra2 fused at its
N-terminus or C-terminus to a heterologous amino acid or amino acid
sequence, wherein the heterologous amino acid or amino acid
sequence is heterologous to the native sequence, i.e. the resulting
chimeric sequence is non-naturally occurring. In another
embodiment, the hIL-1Ra2 variant consists of the amino acid
sequence of amino acids from at or about 27 to at or about 134 of
FIG. 5 (SEQ ID NO:10), or amino acids from at or about 1 to at or
about 134 of FIG. 5 (SEQ ID NO:10), fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence to
form a non-naturally occurring fusion protein. Such hIL-1Ra2 fusion
variants include, for instance, hIL-1Ra2 polypeptides wherein a
heterologous secretion leader sequence is fused to the N-terminus
of the sequence of amino acids from at or about 27 to at or about
134 of FIG. 5 (SEQ ID NO:10), or amino acids from at or about 1 to
at or about 134 of FIG. 5 (SEQ ID NO:10).
[0066] Human interleukin-1 receptor antagonist analog 3
("hIL-1Ra3"), hIL-1Ra3 polypeptide, and hIL-1Ra3 protein are
defined as any native sequence hIL-1Ra3 or variant hIL-1Ra3 (which
are further defined herein).
[0067] A "native sequence hIL-1Ra3" means a polypeptide comprising
an amino acid sequence selected from the group consisting of: (1)
the amino acid sequence of amino acid residues from at or about 95
to at or about 134 of FIG. 7 (SEQ ID NO:13); (2) the amino acid
sequence of amino acid residues from at or about 34 to at or about
155 of FIG. 7 (SEQ ID NO:13); and (3) the amino acid sequence of
any naturally-occurring truncated or secreted form or any
naturally-occurring allelic variant of a polypeptide comprising the
amino acid sequence of (1) or (2). In one embodiment of the
invention, the native sequence hIL-1Ra3 comprises amino acids from
at or about 34 to at or about 155 of FIG. 7 (SEQ ID NO:13), or
amino acids from at or about 2 to at or about 155 of FIG. 7 (SEQ ID
NO:13).
[0068] "hIL-1Ra3 variant" is defined as any hIL-1Ra3 C-terminal
variant or hIL-1Ra3 full sequence variant (which are further
defined herein).
[0069] "hIL-1Ra3 C-terminal variant" means any hIL-1Ra3 other than
a native sequence hIL-1Ra3, which variant is an active hIL-1Ra3, as
defined below, having at least about 80% amino acid sequence
identity with the amino acid sequence of amino acid residues from
at or about 95 to at or about 134 of FIG. 7 (SEQ ID NO:13) or the
amino acid sequence of amino acid residues from at or about 80 to
at or about 155 of FIG. 7 (SEQ ID NO:13). Such hIL-1Ra3 C-terminal
variants include, for instance, hIL-1Ra3 polypeptides wherein one
or more amino acid residues are added, or deleted, internally or at
the N- or C-terminus, in the sequence of amino acid residues from
at or about 95 to at or about 134 of FIG. 7 (SEQ ID NO:13) or in
the sequence of amino acid residues from at or about 80 to at or
about 155 of FIG. 7 (SEQ ID NO:13). Ordinarily, an hIL-1Ra3
C-terminal variant will have at least about 80% amino acid sequence
identity, or at least about 85% amino acid sequence identity, or at
least about 90% amino acid sequence identity, or at least about 95%
amino acid sequence identity with the amino acid sequence of amino
acid residues from at or about 95 to at or about 134 of FIG. 7 (SEQ
ID NO:13) or the amino acid sequence of amino acid residues from at
or about 80 to at or about 155 of FIG. 7 (SEQ ID NO:13).
[0070] "hIL-1Ra3 full sequence variant" means any hIL-1Ra3 other
than a native sequence hIL-1Ra3, which variant retains at least one
biologic activity of a native sequence hIL-1Ra3, such as the
ability to bind IL-1R, and which variant has at least about 80%
amino acid sequence identity, or at least about 85% amino acid
sequence identity, or at least about 90% amino acid sequence
identity, or at least about 95% amino acid sequence identity with
the amino acid sequence of amino acid residues from at or about 34
to at or about 155 of FIG. 7 (SEQ ID NO:13) or the amino acid
sequence of amino acid residues from at or about 2 to at or about
155 of FIG. 7 (SEQ ID NO:13). Such hIL-1Ra3 full sequence variants
include, for instance, hIL-1Ra3 polypeptides wherein one or more
amino acid residues are added, or deleted, internally or at the N-
or C-terminus, in the sequence of amino acid residues from at or
about 34 to at or about 155 of FIG. 7 (SEQ ID NO:13) or the amino
acid sequence of amino acid residues from at or about 2 to at or
about 155 of FIG. 7 (SEQ ID NO:13).
[0071] Murine interleukin-1 receptor antagonist analog 3
("mIL-1Ra3"), mIL-1Ra3 polypeptide, and mIL-1Ra3 protein are
defined as any native sequence mIL-1Ra3 or variant mIL-1Ra3.
[0072] A "native sequence mIL-1Ra3" means a polypeptide comprising
an amino acid sequence selected from the group consisting of: (1)
the amino acid sequence of amino acid residues from at or about 95
to at or about 134 of FIG. 9 (SEQ ID NO:16); (2) the amino acid
sequence of amino acid residues from at or about 34 to at or about
155 of FIG. 9 (SEQ ID NO:16); and (3) the amino acid sequence of
any naturally-occurring truncated or secreted form or
naturally-occurring allelic variant of a polypeptide comprising the
amino acid sequence of (1) or (2). In one embodiment of the
invention, the native sequence mIL-1Ra3 comprises amino acids from
at or about 34 to at or about 155 of FIG. 9 (SEQ ID NO:16).
[0073] "mIL-1Ra3 variant" is defined as any mIL-1Ra3 C-terminal
variant or mIL-1Ra3 full sequence variant (which are further
defined herein).
[0074] "mIL-1Ra3 C-terminal variant" means any mIL-1Ra3 other than
a native sequence mIL-1Ra3, which variant is an active mIL-1Ra3, as
defined below, having at least about 80% amino acid sequence
identity with the amino acid sequence of amino acids from at or
about 95 to at or about 134 of FIG. 9 (SEQ ID NO:16). Such mIL-1Ra3
C-terminal variants include, for instance, mIL-1Ra3 polypeptides
wherein one or more amino acid residues are added, or deleted,
internally or at the N- or C-terminus, in the sequence of amino
acids from at or about 95 to at or about 134 of FIG. 9 (SEQ ID
NO:16). Ordinarily, an mIL-1Ra3 C-terminal variant will have at
least about 80% amino acid sequence identity, or at least about 85%
amino acid sequence identity, or at least about 90% amino acid
sequence identity, and or at least about 95% amino acid sequence
identity with the amino acid sequence of amino acids 95 to 134 of
FIG. 9 (SEQ ID NO:16).
[0075] "mIL-1Ra3 full sequence variant" means any mIL-1Ra3 other
than a native sequence mIL-1Ra3, which variant retains at least one
biologic activity of a native sequence mIL-1Ra3, such as the
ability to bind IL-1R, and which variant has at least about 85%
amino acid sequence identity, or at least about 90% amino acid
sequence identity, or at least about 95% sequence identity with the
amino acid sequence of amino acid residues from at or about 34 to
at or about 155 of FIG. 9 (SEQ ID NO:16) or the amino acid sequence
of amino acid residues from at or about 2 to at or about 155 of
FIG. 9 (SEQ ID NO:16). Such mIL-1Ra3 full sequence variants
include, for instance, mIL-1Ra3 polypeptides wherein one or more
amino acid residues are added, or deleted, internally or at the N-
or C-terminus, in the sequence of amino acid residues from at or
about 34 to at or about 155 of FIG. 9 (SEQ ID NO:16) or in the
sequence of amino acid residues from at or about 2 to at or about
155 of FIG. 9 (SEQ ID NO:16).
[0076] "Human interleukin-1-like polypeptide", "hIL-1lp", "hIL-1lp
polypeptide", "hIL-1lp protein", "human interleukin-1 receptor
antagonist analog", "hIL-1Raa", "hIL-1Raa polypeptide", and
"hIL-1Raa protein" are defined as any hIL-1Ra1, hIL-1Ra2 or
hIL-1Ra3 polypeptide.
[0077] "Native sequence hIL-1lp" and "native sequence hIL-1Raa" are
defined as any polypeptide that comprises a native sequence
hIL-1Ra1, hIL-1Ra2, or hIL-1Ra3.
[0078] "hIL-1lp variant" is defined as any polypeptide that
comprises a variant of hIL-1Ra1, hIL-1Ra2, or hIL-1Ra3.
[0079] "Interleukin-1 receptor", "interleukin-1 receptor
polypeptide", "interleukin-1 receptor protein", "IL-1 receptor",
"IL-1R", "IL-1R polypeptide", and "IL-1R protein", are defined as
the family of cell surface proteins that bind to interleukin-1
(IL-1) and/or function in IL-1-induced signal transduction in a
given species, such as human or mouse. IL-1R includes the human T
cell-expressed IL-1 receptor disclosed in Sims, et al., Proc. Natl.
Acad. Sci. (USA), 86: 8946-8950 (1989).
[0080] "Interleukin-18 receptor", "interleukin-18 receptor
polypeptide", "interleukin-18 receptor protein", "IL-18 receptor",
"IL-18R", "IL-18R polypeptide", and "IL-18R protein", are defined
as the family of cell surface proteins that bind to interleukin-18
(IL-18) and/or function in IL-18-induced signal transduction in a
given species, such as human or mouse. IL-18R includes the IL-1
receptor related protein (IL-1Rrp) described in Torigoe et al., J.
Biol. Chem., 272: 25737-25742 (1997) and the IL-18 receptor
accessory protein-like molecule (IL-18RAcPL) described in Born et
al., J. Biol. Chem., 273: 29445-29450 (1998).
[0081] "Interleukin-1-like family" and "IL-1-like family" are used
to indicate the family of polypeptides related to the ligands of
IL-1R or IL-18R. The IL-1-like family includes IL-1 receptor
agonists and antagonists and related polypeptides such as
IL-1.alpha. (described in Bazan et al., Nature, 379: 591 (1996),
IL-1.beta. (Bazan et al.), IL-18 (interferon-.gamma. inducing
factor)(IGIF)(Bazan et al.), IL-1 receptor antagonist polypeptides
such as secretory IL-1Ra (sIL-1Ra)(described in Eisenberg et al.,
Nature, 343: 341-346 (1990)) and intracellular IL-1Ra (icIL-1Ra)
(described in Haskill et al., Proc. Natl. Acad. Sci. (USA), 88:
3681-3685 (1991)), and the IL-1lp polypeptides of the
invention.
[0082] "Percent (%) amino acid sequence identity" with respect to
the IL-1lp sequences identified herein is defined as the percentage
of amino acid residues in a candidate sequence that are identical
with the amino acid residues in an IL-1lp sequence, after aligning
the sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity, and not considering any
conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign
(DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full-length
of the sequences being compared. For purposes herein, however, %
amino acid sequence identity values are obtained as described below
by using the sequence comparison computer program ALIGN-2, wherein
the complete source code for the ALIGN-2 program is provided in
Tables 3A-3Q. The ALIGN-2 sequence comparison computer program was
authored by Genentech, Inc. and the source code shown in Tables
3A-3Q has been filed with user documentation in the U.S. Copyright
Office, Washington D.C., 20559, where it is registered under U.S.
Copyright Registration No. TXU510087. The ALIGN-2 program is
publicly available through Genentech, Inc., South San Francisco,
Calif. or may be compiled from the source code provided in Tables
3A-3Q. The ALIGN-2 program should be compiled for use on a UNIX
operating system, preferably digital UNIX V4.0D. All sequence
comparison parameters are set by the ALIGN-2 program and do not
vary.
[0083] For purposes herein, the % amino acid sequence identity of a
given amino acid sequence A to, with, or against a given amino acid
sequence B (which also can be phrased as a given amino acid
sequence A that has or comprises a certain % amino acid sequence
identity to, with, or against a given amino acid sequence B) is
calculated as follows: 100 times the fraction X/Y where X is the
number of amino acid residues scored as identical matches by the
sequence alignment program ALIGN-2 in that program's alignment of A
and B, and where Y is the total number of amino acid residues in B.
It will be appreciated that where the length of amino acid sequence
A is not equal to the length of amino acid sequence B, the % amino
acid sequence identity of A to B will not equal the % amino acid
sequence identity of B to A. As examples of % amino acid sequence
identity calculations, Tables 2A-2B demonstrate how to calculate
the % amino acid sequence identity of the amino acid sequence
designated "Comparison Protein" to the amino acid sequence
designated "PRO".
[0084] Unless specifically stated otherwise, all % amino acid
sequence identity values used herein are obtained as described
above using the ALIGN-2 sequence comparison computer program.
However, % amino acid sequence identity may also be determined
using the sequence comparison program NCBI-BLAST2 (Altschul et al.,
Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence
comparison program may be downloaded from
http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search
parameters, wherein all of those search parameters are set to
default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5,
multi-pass e-value=0.01, constant for multi-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[0085] In situations where NCBI-BLAST2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which also can be phrased as a given amino acid
sequence A that has or comprises a certain % amino acid sequence
identity to, with, or against a given amino acid sequence B) is
calculated as follows: 100 times the fraction X/Y where X is the
number of amino acid residues scored as identical matches by the
sequence alignment program NCBI-BLAST2 in that program's alignment
of A and B, and where Y is the total number of amino acid residues
in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the
% amino acid sequence identity of A to B will not equal the % amino
acid sequence identity of B to A.
[0086] "Percent (%) nucleic acid sequence identity" with respect to
the IL-1lp polypeptide-encoding nucleic acid sequences identified
herein is defined as the percentage of nucleotides in a candidate
sequence that are identical with the nucleotides in an IL-1lp
polypeptide-encoding nucleic acid sequence, after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity. Alignment for purposes of
determining percent nucleic acid sequence identity can be achieved
in various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the
art can determine appropriate parameters for measuring alignment,
including any algorithms needed to achieve maximal alignment over
the full-length of the sequences being compared. For purposes
herein, however, % nucleic acid sequence identity values are
obtained as described below by using the sequence comparison
computer program ALIGN-2, wherein the complete source code for the
ALIGN-2 program is provided in Tables 3A-3Q. The ALIGN-2 sequence
comparison computer program was authored by Genentech, Inc. and the
source code shown in Tables 3A-3Q has been filed with user
documentation in the U.S. Copyright Office, Washington D.C., 20559,
where it is registered under U.S. Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through
Genentech, Inc., South San Francisco, Calif. or may be compiled
from the source code provided in Tables 3A-3Q. The ALIGN-2 program
should be compiled for use on a UNIX operating system, preferably
digital UNIX V4.0D. All sequence comparison parameters are set by
the ALIGN-2 program and do not vary.
[0087] For purposes herein, the % nucleic acid sequence identity of
a given nucleic acid sequence C to, with, or against a given
nucleic acid sequence D (which also can be phrased as a given
nucleic acid sequence C that has or comprises a certain % nucleic
acid sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows: 100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of C and D, and where Z is the total number of nucleotides in D. It
will be appreciated that where the length of nucleic acid sequence
C is not equal to the length of nucleic acid sequence D, the %
nucleic acid sequence identity of C to D will not equal the %
nucleic acid sequence identity of D to C. As examples of % nucleic
acid sequence identity calculations, Tables 2C-2D demonstrate how
to calculate the % nucleic acid sequence identity of the nucleic
acid sequence designated "Comparison DNA" to the nucleic acid
sequence designated "PRO-DNA".
[0088] Unless specifically stated otherwise, all % nucleic acid
sequence identity values used herein are obtained as described
above using the ALIGN-2 sequence comparison computer program.
However, % nucleic acid sequence identity may also be determined
using the sequence comparison program NCBI-BLAST2 (Altschul et al.,
Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence
comparison program may be downloaded from
http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search
parameters, wherein all of those search parameters are set to
default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5,
multi-pass e-value=0.01, constant for multi-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[0089] In situations where NCBI-BLAST2 is employed for sequence
comparisons, the % nucleic acid sequence identity of a given
nucleic acid sequence C to, with, or against a given nucleic acid
sequence D (which also can be phrased as a given nucleic acid
sequence C that has or comprises a certain % nucleic acid sequence
identity to, with, or against a given nucleic acid sequence D) is
calculated as follows: 100 times the fraction W/Z where W is the
number of nucleotides scored as identical matches by the sequence
alignment program NCBI-BLAST2 in that program's alignment of C and
D, and where Z is the total number of nucleotides in D. It will be
appreciated that where the length of nucleic acid sequence C is not
equal to the length of nucleic acid sequence D, the % nucleic acid
sequence identity of C to D will not equal the % nucleic acid
sequence identity of D to C.
[0090] The term "positives", in the context of the amino acid
sequence identity comparisons performed as described above,
includes amino acid residues in the sequences compared that are not
only identical, but also those that have similar properties. Amino
acid residues that score a positive value to an amino acid residue
of interest are those that are either identical to the amino acid
residue of interest or are a preferred substitution (as defined in
Table 1 below) of the amino acid residue of interest.
[0091] For purposes herein, the % value of positives of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which also can be phrased as a given amino acid
sequence A that has or comprises a certain % positives to, with, or
against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y where X is the number of amino acid
residues scoring a positive value as defined above by the sequence
alignment program ALIGN-2 in that program's alignment of A and B,
and where Y is the total number of amino acid residues in B. It
will be appreciated that where the length of amino acid sequence A
is not equal to the length of amino acid sequence B, the %
positives of A to B will not equal the % positives of B to A.
[0092] "Isolated," when used to describe the various polypeptides
disclosed herein, means polypeptide that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. In
preferred embodiments, the polypeptide will be purified (1) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator,
or (2) to homogeneity by SDS-PAGE under non-reducing or reducing
conditions using Coomassie blue or, preferably, silver stain.
Isolated polypeptide includes polypeptide in situ within
recombinant cells, since at least one component of the IL-1lp
natural environment will not be present. Ordinarily, however,
isolated polypeptide will be prepared by at least one purification
step.
[0093] An "isolated" nucleic acid molecule encoding a IL-1lp
polypeptide is a nucleic acid molecule that is identified and
separated from at least one contaminant nucleic acid molecule with
which it is ordinarily associated in the natural source of the
IL-1lp-encoding nucleic acid. An isolated IL-1lp-encoding nucleic
acid molecule is other than in the form or setting in which it is
found in nature. Isolated nucleic acid molecules therefore are
distinguished from the IL-1lp-encoding nucleic acid molecule as it
exists in natural cells. However, an isolated nucleic acid molecule
encoding a IL-1lp polypeptide includes IL-1lp-encoding nucleic acid
molecules contained in cells that ordinarily express IL-1lp where,
for example, the nucleic acid molecule is in a chromosomal location
different from that of natural cells.
[0094] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0095] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0096] The term "antibody" is used in the broadest sense and
specifically covers single anti-IL-1lp monoclonal antibodies
(including agonist, antagonist, and neutralizing antibodies) and
anti-IL-1lp antibody compositions with polyepitopic specificity.
The term "monoclonal antibody" as used herein refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical except for possible naturally-occurring mutations that
may be present in minor amounts.
[0097] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of desired homology between the probe and
hybridizable sequence, the higher the relative temperature which
can be used. As a result, it follows that higher relative
temperatures would tend to make the reaction conditions more
stringent, while lower temperatures less so. For additional details
and explanation of stringency of hybridization reactions, see
Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, (1995).
[0098] "Stringent conditions" or "high stringency conditions", as
defined herein, may be identified by those that: (1) employ low
ionic strength and high temperature for washing, for example 0.015
M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl
sulfate at 50.degree. C.; (2) employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or
(3) employ 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium
citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times. Denhardt's solution, sonicated salmon sperm
DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree.
C., with washes at 42.degree. C. in 0.2.times.SSC (sodium
chloride/sodium citrate) and 50% formamide at 55.degree. C.,
followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA at 55.degree. C.
[0099] "Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength and % SDS) less stringent that those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0100] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising an IL-1lp polypeptide fused to a
"tag polypeptide". The tag polypeptide has enough residues to
provide an epitope against which an antibody can be made, yet is
short enough such that it does not interfere with activity of the
polypeptide to which it is fused. The tag polypeptide preferably
also is fairly unique so that the antibody does not substantially
cross-react with other epitopes. Suitable tag polypeptides
generally have at least six amino acid residues and usually between
about 8 and 50 amino acid residues (preferably, between about 10
and 20 amino acid residues).
[0101] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired
binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as
IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM.
[0102] "Active" or "activity" for the purposes herein refers to
form(s) of IL-1lp which retain one or more of the biologic
activities of native or naturally-occurring IL-1lp, or which
exhibit immunological cross-reactivity with a native or
naturally-occurring IL-1lp.
[0103] As used herein, a "biologic activity" or "biological
activity" of an IL-1lp means any effector function exhibited by the
IL-1lp in the physiology or pathophysiology of a mammal, excluding
any immunogenic or antigenic functions of the IL-1lp. Immunogenic
and antigenic functions of an IL-1lp refer to the ability of the
IL-1lp to generate a humoral or cell-mediated immune response
specific to the IL-1lp, and the ability of the IL-1lp to
specifically recognize and interact with anti-IL-1lp antibodies, B
cells or T cells, respectively, in a mammal.
[0104] As used herein, "immunological cross-reactivity" with an
IL-1lp means that the candidate polypeptide is capable of
competitively inhibiting the binding of the IL-1lp to polyclonal or
monoclonal antibodies raised against the IL-1lp.
[0105] In one embodiment, IL-1lp activity includes the ability to
agonize or antagonize one or more biological activities of any
IL-1-like family member, e.g. an IL-1lp activity that antagonizes
an IL-1-mediated or IL-18-mediated inflammatory response. In
another embodiment, IL-1lp activity includes the ability to bind to
the IL-18 receptor and/or IL-1 receptor.
[0106] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or
neutralizes a biological activity of a native IL-1lp polypeptide
disclosed herein. In a similar manner, the term "agonist" is used
in the broadest sense and includes any molecule that mimics a
biological activity of a native IL-1lp polypeptide disclosed
herein. Suitable agonist or antagonist molecules specifically
include agonist or antagonist antibodies or antibody fragments,
fragments or amino acid sequence variants of native IL-1lp
polypeptides, peptides, small organic molecules, etc.
[0107] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in
whom the disorder is to be prevented.
[0108] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption, but rather is cyclic
in nature.
[0109] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, cats,
cattle, horses, sheep, pigs, etc. Preferably, the mammal is
human.
[0110] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0111] The terms "inflammatory disorders" and "inflammatory
diseases" are used interchangeably herein and refer to pathological
states resulting in inflammation. Examples of such disorders
include inflammatory skin diseases such as psoriasis and atopic
dermatitis; systemic scleroderma and sclerosis; responses
associated with inflammatory bowel disease (such as Crohn's disease
and ulcerative colitis); ischemic reperfusion disorders including
surgical tissue reperfusion injury, myocardial ischemic conditions
such as myocardial infarction, cardiac arrest, reperfusion after
cardiac surgery and constriction after percutaneous transluminal
coronary angioplasty, stroke, and abdominal aortic aneurysms;
cerebral edema secondary to stroke; cranial trauma; hypovolemic
shock; asphyxia; adult respiratory distress syndrome; acute lung
injury; Behcet's Disease; dermatomyositis; polymyositis; multiple
sclerosis; dermatitis; meningitis; encephalitis; uveitis;
osteoarthritis; autoimmune diseases such as rheumatoid arthritis,
Sjorgen's syndrome, vasculitis, and insulin-dependent diabetes
mellitus (IDDM); diseases involving leukocyte diapedesis; central
nervous system (CNS) inflammatory disorder; meningitis; multiple
organ injury syndrome secondary to septicaemia or trauma;
inflammatory diseases of the liver, including alcoholic hepatitis
and hepatic fibrosis; pathologic host responses to infection,
including pathologic inflammation in granulomatous diseases,
hepatitis, and bacterial pneumonia; antigen-antibody complex
mediated diseases including glomerulonephritis; sepsis;
sarcoidosis; immunopathologic responses to tissue/organ
transplantation, including graft-versus host disease (GVHD);
inflammations of the lung, including pleurisy, alveolitis,
vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse
panbronchiolitis, hypersensitivity pneumonitis, idiopathic
pulmonary fibrosis (IPF), and cystic fibrosis; inflammation in
renal diseases, including acute or chronic nephritic conditions
such as lupus nephritis; pancreatitis; etc. The preferred
indications include rheumatoid arthritis, osteoarthritis, sepsis,
acute lung injury, adult respiratory distress syndrome, idiopathic
pulmonary fibrosis, ischemic reperfusion (including surgical tissue
reperfusion injury, stroke, myocardial ischemia, and acute
myocardial infarction), asthma, psoriasis, graft-versus-host
disease (GVHD), and inflammatory bowel disease such as ulcerative
colitis.
[0112] As used herein, the terms "asthma", "asthmatic disorder",
"asthmatic disease", and "bronchial asthma" refer to a condition of
the lungs in which there is widespread narrowing of lower airways.
"Atopic asthma" and "allergic asthma" refer to asthma that is a
manifestation of an IgE-mediated hypersensitivity reaction in the
lower airways, including, e.g., moderate or severe chronic asthma,
such as conditions requiring the frequent or constant use of
inhaled or systemic steroids to control the asthma symptoms. A
preferred indication is allergic asthma.
II. Detailed Description of the Invention
[0113] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as IL-1lp. In particular, cDNAs encoding IL-1lp
polypeptides have been identified and isolated, as disclosed in
further detail in the Examples below.
[0114] Using NCBI-BLAST2 sequence alignment computer programs, it
has been found that a full-length native sequence hIL-1Ra1 (shown
in FIG. 3 and SEQ ID NO:7) has some amino acid sequence identity
with human IL-1 receptor antagonist beta (hIL-1Ra.beta.) and
TANGO-77 protein, a full-length native sequence hIL-1Ra1L (shown in
FIG. 15 and SEQ ID NO:19) has some amino acid sequence identity
with human IL-1 receptor antagonist beta (hIL-1Ra.beta.) and
TANGO-77 protein, a full-length native sequence hIL-1Ra1V (shown in
FIG. 19 and SEQ ID NO:25) has some amino acid sequence identity
with human IL-1 receptor antagonist beta (hIL-1Ra.beta.) and
TANGO-77 protein, a full-length native sequence hIL-1Ra1S (shown in
FIG. 16 and SEQ ID NO:21) appears to be an allelic variant of
TANGO-77 protein and has some amino acid sequence identity with
human IL-1 receptor antagonist beta (hIL-1Ra.beta.), a full-length
native sequence hIL-1Ra2 (shown in FIG. 5 and SEQ ID NO:10) has
some amino acid sequence identity with hIL-1Ra.beta., a full-length
native sequence hIL-1Ra3 (shown in FIG. 7 and SEQ ID NO:13) has
some amino acid sequence identity with human intracellular IL-1
receptor antagonist (hicIL-1Ra), and a full-length native sequence
mIL-1Ra3 (shown in FIG. 9 and SEQ ID NO:16) has some amino acid
sequence identity with mouse IL-1 receptor antagonist (mIL-1Ra) and
has some amino acid sequence identity with hicIL-1Ra. hIL-1Ra.beta.
is described in EP 0855404 published Jul. 29, 1998. TANGO-77
protein is described in WO 99/06426 published Feb. 11, 1999.
hicIL-1Ra is described in WO 95/10298 published Apr. 20, 1995 and
in Haskill et al., Proc. Natl. Acad. Sci. (USA), 88: 3681-3685
(1991). mIL-1Ra is described in Zahedi et al., J. Immunol., 146:
4228-4233 (1991), Matsushime et al., Blood, 78: 616-623 (1991),
Zahedi et al., Cytokine, 6: 1-9 (1994), Eisenberg et al., Proc.
Natl. Acad. Sci. (USA), 88: 5232-5236 (1991) and Shuck et al., Eur.
J. Immunol., 21: 2775-2780 (1991). Accordingly, it is presently
believed that the IL-1lp polypeptides disclosed in the present
application are newly identified members of the interleukin-1-like
family and possess inflammatory or anti-inflammatory activities, or
other cellular response activating or inhibiting activities,
typical of the IL-1-like family.
[0115] In addition to the full-length native sequence IL-1lp
polypeptides described herein, it is contemplated that IL-1lp
variants can be prepared. Such embodiments of the invention include
all IL-1lp polypeptides that are IL-1lp variants as defined herein,
such as hIL-1Ra1 variants, hIL-1Ra1L variants, hIL-1Ra1S variants,
hIL-1Ra2 variants, hIL-1Ra3 variants, and mIL-1Ra3 variants.
[0116] IL-1lp variants can be prepared by introducing appropriate
nucleotide changes into the IL-1lp DNA, and/or by synthesis of the
desired IL-1lp polypeptide. Those skilled in the art will
appreciate that amino acid changes may alter post-translational
processes of the IL-1lp, such as changing the number or position of
glycosylation sites or altering the membrane anchoring
characteristics.
[0117] Variations in the native full-length sequence IL-1lp or in
various domains of the IL-1lp described herein, can be made, for
example, using any of the techniques and guidelines for
conservative and non-conservative mutations set forth, for
instance, in U.S. Pat. No. 5,364,934. Variations may be a
substitution, deletion or insertion of one or more codons encoding
the IL-1lp that results in a change in the amino acid sequence of
the IL-1lp as compared with the native sequence IL-1lp. Optionally
the variation is by substitution of at least one amino acid with
any other amino acid in one or more of the domains of the IL-1lp.
Guidance in determining which amino acid residue may be inserted,
substituted or deleted without adversely affecting the desired
activity may be found by comparing the sequence of the IL-1lp with
that of homologous known protein molecules and minimizing the
number of amino acid sequence changes made in regions of high
homology. Amino acid substitutions can be the result of replacing
one amino acid with another amino acid having similar structural
and/or chemical properties, such as the replacement of a leucine
with a serine, i.e., conservative amino acid replacements.
Insertions or deletions may optionally be in the range of 1 to 5
amino acids. The variation allowed may be determined by
systematically making insertions, deletions or substitutions of
amino acids in the sequence and testing the resulting variants for
activity in the in vitro assay described in the Examples below.
[0118] Table 1 below lists conservative amino acid substitutions
(under the heading of "Preferred Substitutions") that are useful in
generating variants of the native sequence IL-1lp. If such
substitutions result in alteration of biological activity, it is
useful to introduce more substantial changes, such as the
"Exemplary Substitutions" denoted in Table 1 or the substantial
changes described below in reference to amino acid classes, at the
active site in question. TABLE-US-00001 TABLE 1 Original Exemplary
Preferred Residue Substitutions Substitutions Ala (A) val; leu; ile
val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp
(D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G)
pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met;
ala; phe; leu norleucine Leu (L) norleucine; ile; val; ile met;
ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe
(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr
(T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val
(V) ile; leu; met; phe; leu ala; norleucine
[0119] Substantial modifications in function or immunological
identity of the IL-1lp polypeptide are accomplished by selecting
substitutions that differ significantly in their effect on
maintaining (a) the structure of the polypeptide backbone in the
area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common
side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
[0120] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, more preferably, into the remaining (non-conserved)
sites.
[0121] The variations can be made using methods known in the art
such as oligonucleotide-mediated (site-directed) mutagenesis,
alanine scanning, and PCR mutagenesis. Site-directed mutagenesis
(Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al.,
Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et
al., Gene, 34:315 (1985)), restriction selection mutagenesis [Wells
et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or
other known techniques can be performed on the cloned DNA to
produce the IL-1lp variant DNA.
[0122] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence. Among
the preferred scanning amino acids are relatively small, neutral
amino acids. Such amino acids include alanine, glycine, serine, and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant [Cunningham and Wells, Science, 244: 1081-1085
(1989)]. Alanine is also typically preferred because it is the most
common amino acid. Further, it is frequently found in both buried
and exposed positions [Creighton, The Proteins, (W.H. Freeman &
Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine
substitution does not yield adequate amounts of variant, an
isoteric amino acid can be used.
[0123] Covalent modifications of IL-1lp are included within the
scope of this invention. One type of covalent modification includes
reacting targeted amino acid residues of an IL-1lp polypeptide with
an organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues of the
IL-1lp. Derivatization with bifunctional agents is useful, for
instance, for crosslinking IL-1lp to a water-insoluble support
matrix or surface for use in the method for purifying anti-IL-1lp
antibodies, and vice-versa. Commonly used crosslinking agents
include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0124] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0125] Another type of covalent modification of the IL-1lp
polypeptide included within the scope of this invention comprises
altering the native glycosylation pattern of the polypeptide.
"Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence IL-1lp (either by removing the underlying
glycosylation site or by deleting the glycosylation by chemical
and/or enzymatic means), and/or adding one or more glycosylation
sites that are not present in the native sequence IL-1lp. In
addition, the phrase includes qualitative changes in the
glycosylation of the native proteins, involving a change in the
nature and proportions of the various carbohydrate moieties
present.
[0126] Addition of glycosylation sites to the IL-1lp polypeptide
may be accomplished by altering the amino acid sequence. The
alteration may be made, for example, by the addition of, or
substitution by, one or more serine or threonine residues to the
native sequence IL-1lp (for O-linked glycosylation sites). The
IL-1lp amino acid sequence may optionally be altered through
changes at the DNA level, particularly by mutating the DNA encoding
the IL-1lp polypeptide at preselected bases such that codons are
generated that will translate into the desired amino acids.
[0127] Another means of increasing the number of carbohydrate
moieties on the IL-1lp polypeptide is by chemical or enzymatic
coupling of glycosides to the polypeptide. Such methods are
described in the art, e.g., in WO 87/05330 published 11 Sep. 1987,
and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981).
[0128] Removal of carbohydrate moieties present on the IL-1lp
polypeptide may be accomplished chemically or enzymatically or by
mutational substitution of codons encoding for amino acid residues
that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and described, for instance, by
Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by
Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties on polypeptides can be achieved by the use of
a variety of endo- and exo-glycosidases as described by Thotakura
et al., Meth. Enzymol., 138:350 (1987).
[0129] Another type of covalent modification of IL-1lp comprises
linking the IL-1lp polypeptide to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol (PEG),
polypropylene glycol, or polyoxyalkylenes, in the manner set forth
in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337.
[0130] The IL-1lp of the present invention may also be modified in
a way to form a chimeric molecule comprising IL-1lp fused to
another, heterologous polypeptide or amino acid sequence.
[0131] In one embodiment, such a chimeric molecule comprises a
fusion of the IL-1lp with a tag polypeptide which provides an
epitope to which an anti-tag antibody can selectively bind. The
epitope tag is generally placed at the amino- or carboxyl-terminus
of the IL-1lp. The presence of such epitope-tagged forms of the
IL-1lp can be detected using an antibody against the tag
polypeptide. Also, provision of the epitope tag enables the IL-1lp
to be readily purified by affinity purification using an anti-tag
antibody or another type of affinity matrix that binds to the
epitope tag. Various tag polypeptides and their respective
antibodies are well known in the art. Examples include
poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly)
tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et
al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the
8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,
Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes
Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et
al., Protein Engineering, 3(6):547-553 (1990)]. Other tag
polypeptides include the Flag-peptide [Hopp et al., BioTechnology,
6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al.,
Science, 255:192-194 (1992)]; an .alpha.-tubulin epitope peptide
[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the
T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990)].
[0132] In an alternative embodiment, the chimeric molecule may
comprise a fusion of the IL-1lp with an immunoglobulin or a
particular region of an immunoglobulin. For a bivalent form of the
chimeric molecule (also referred to as an immunoadhesin), such a
fusion could be to the Fc region of an IgG molecule. The Ig fusions
preferably include the substitution of a soluble form of an IL-1lp
polypeptide in place of at least one variable region within an Ig
molecule. In a particularly preferred embodiment, the
immunoglobulin fusion includes the hinge, CH2 and CH3, or the
hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the
production of immunoglobulin fusions see also U.S. Pat. No.
5,428,130 issued Jun. 27, 1995.
[0133] In one aspect, the invention provides an isolated nucleic
acid comprising DNA encoding an IL-1lp polypeptide that retains at
least one biologic activity of a native sequence IL-1lp, such as
the IL-1R binding activity of a native sequence hIL-1Ra3 or
mIL-1Ra3, or the IL-18R binding activity of a native sequence
hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, which DNA has at least at or
about 80% sequence identity, or at least at or about 85% sequence
identity, or at least at or about 90% sequence identity, or at
least at or about 95% sequence identity to (a) a DNA molecule
selected from the group consisting of: (1) a DNA molecule encoding
an IL-1lp polypeptide comprising amino acid residues from at or
about 37 to at or about 203, inclusive of FIG. 2 (SEQ ID NO:5), (2)
a DNA molecule encoding an IL-1lp polypeptide comprising amino acid
residues from at or about 15 to at or about 193, inclusive of FIG.
3 (SEQ ID NO:7), (3) a DNA molecule encoding an IL-1lp polypeptide
comprising amino acid residues from at or about 34 to at or about
155, inclusive of FIG. 7 (SEQ ID NO:13), (4) a DNA molecule
encoding an IL-1lp polypeptide comprising amino acid residues from
at or about 34 to at or about 155, inclusive of FIG. 9 (SEQ ID
NO:16), (5) a DNA molecule encoding an IL-1lp polypeptide
comprising amino acid residues from at or about 26 to at or about
207, inclusive of FIG. 15 (SEQ ID NO:19), and (6) a DNA molecule
encoding an IL-1lp polypeptide comprising amino acid residues from
at or about 46 to at or about 218 of FIG. 19 (SEQ ID NO:25), or (b)
the complement of the DNA molecule of (a).
[0134] In another aspect, the invention provides an isolated
nucleic acid comprising DNA encoding an IL-1lp polypeptide that
retains at least one biologic activity of a native sequence IL-1lp,
such as the IL-18R binding activity of a native sequence hIL-1Ra1,
hIL-1Ra1L, or hIL-1Ra1V, which DNA has at least at or about 80%
sequence identity, or at least at or about 85% sequence identity,
or at least at or about 90% sequence identity, or at least at or
about 95% sequence identity to (a) a DNA molecule selected from the
group consisting of: (1) a DNA molecule encoding an IL-1lp
polypeptide comprising amino acid residues from at or about 1 to at
or about 207, inclusive of FIG. 15 (SEQ ID NO:19), and (2) a DNA
molecule encoding an IL-1lp polypeptide comprising amino acid
residues from at or about 1 to at or about 218 of FIG. 19 (SEQ ID
NO:25), or (b) the complement of the DNA molecule of (a).
[0135] In another aspect, the invention provides an isolated
nucleic acid comprising DNA encoding an IL-1lp polypeptide that
retains at least one biologic activity of a native sequence IL-1lp,
such as the IL-18R binding activity of a native sequence hIL-1Ra1,
or the IL-1R binding activity of a native sequence hIL-1Ra3 or
mIL-1Ra3, which DNA has at least at or about 80% sequence identity,
or at least at or about 85% sequence identity, or at least at or
about 90% sequence identity, or at least at or about 95% sequence
identity to (a) a DNA molecule selected from the group consisting
of: (1) a DNA molecule encoding an IL-1lp polypeptide comprising
amino acid residues from at or about 95 to at or about 134,
inclusive of FIG. 7 (SEQ ID NO:13), and (2) a DNA molecule encoding
an IL-1lp polypeptide comprising amino acid residues from at or
about 95 to at or about 134, inclusive of FIG. 9 (SEQ ID NO:16), or
(b) the complement of the DNA molecule of (a).
[0136] In another aspect, the invention provides an isolated
nucleic acid comprising DNA encoding an IL-1lp polypeptide that
retains at least one biologic activity of a native sequence IL-1lp,
such as the IL-18R binding activity of a native sequence hIL-1Ra1,
or the IL-1R binding activity of a native sequence hIL-1Ra3 or
mIL-1Ra3, which DNA has at least at or about 80% sequence identity,
or at least at or about 85% sequence identity, or at least at or
about 90% sequence identity, or at least at or about 95% sequence
identity to (a) a DNA molecule encoding an IL-1lp polypeptide
comprising amino acid residues from at or about 80 to at or about
155, inclusive of FIG. 7 (SEQ ID NO:13), or (b) the complement of
the DNA molecule of (a).
[0137] In another aspect, the invention provides an isolated
nucleic acid comprising DNA encoding an IL-1lp polypeptide that
retains at least one biologic activity of a native sequence IL-1lp,
such as the IL-18R binding activity of a native sequence hIL-1Ra1,
or the IL-1R binding activity of a native sequence hIL-1Ra3 or
mIL-1Ra3, which DNA has at least at or about 80% sequence identity,
or at least at or about 85% sequence identity, or at least at or
about 90% sequence identity, or at least at or about 95% sequence
identity to (a) a DNA molecule selected from the group consisting
of: (1) a DNA molecule encoding an IL-1lp polypeptide comprising
amino acid residues from at or about 2 to at or about 155,
inclusive of FIG. 7 (SEQ ID NO:13), and (2) a DNA molecule encoding
an IL-1lp polypeptide comprising amino acid residues from at or
about 2 to at or about 155, inclusive of FIG. 9 (SEQ ID NO:16), or
(b) the complement of the DNA molecule of (a).
[0138] In another aspect, the invention provides an isolated
nucleic acid comprising DNA having at least at or about 80%
sequence identity, or at least at or about 85% sequence identity,
or at least at or about 90% sequence identity, or at least at or
about 95% sequence identity to (a) a DNA molecule selected from the
group consisting of: (1) a DNA molecule encoding an IL-1lp
polypeptide comprising amino acid residues from at or about 95 to
at or about 134, inclusive of FIG. 7 (SEQ ID NO:13), and (2) a DNA
molecule encoding an IL-1lp polypeptide comprising amino acid
residues from at or about 95 to at or about 134, inclusive of FIG.
9 (SEQ ID NO:16), or (b) the complement of the DNA molecule of
(a).
[0139] In another aspect, the invention provides an isolated
nucleic acid comprising DNA having at least at or about 80%
sequence identity, or at least at or about 85% sequence identity,
or at least at or about 90% sequence identity, or at least at or
about 95% sequence identity to (a) a DNA molecule encoding an
IL-1lp polypeptide comprising amino acid residues from at or about
80 to at or about 155, inclusive of FIG. 7 (SEQ ID NO:13), or (b)
the complement of the DNA molecule of (a).
[0140] In another aspect, the invention provides an isolated
nucleic acid comprising DNA having at least at or about 80%
sequence identity, or at least at or about 85% sequence identity,
or at least at or about 90% sequence identity, or at least at or
about 95% sequence identity to (a) a DNA molecule selected from the
group consisting of: (1) a DNA molecule encoding an IL-1lp
polypeptide comprising amino acid residues from at or about 2 to at
or about 155, inclusive of FIG. 7 (SEQ ID NO:13), and (2) a DNA
molecule encoding an IL-1lp polypeptide comprising amino acid
residues from at or about 2 to at or about 155, inclusive of FIG. 9
(SEQ ID NO:16), or (b) the complement of the DNA molecule of
(a).
[0141] In another aspect, the invention concerns an isolated
nucleic acid molecule encoding an IL-1lp polypeptide, comprising
DNA hybridizing to the complement of a nucleic acid sequence
selected from the group consisting of: (1) the nucleic acid
sequence consisting of nucleotide positions from at or about 238 to
at or about 465 in the sense strand of FIG. 7 (SEQ ID NO:12); (2)
the nucleic acid sequence consisting of nucleotide positions from
at or about 427 to at or about 609 in the sense strand of FIG. 9
(SEQ ID NO:15); and (3) the nucleic acid sequence consisting of
nucleotide positions from at or about 79 to at or about 135 in the
sense strand of FIG. 15 (SEQ ID NO:18). Preferably, hybridization
occurs under stringent hybridization and wash conditions.
[0142] In another aspect, the invention concerns an isolated
nucleic acid molecule, comprising DNA that is at least 90
nucleotides in length and that hybridizes to the complement of a
nucleic acid sequence selected from the group consisting of: (1)
the nucleic acid sequence consisting of nucleotide positions from
at or about 238 to at or about 465 in the sense strand of FIG. 7
(SEQ ID NO:12); (2) the nucleic acid sequence consisting of
nucleotide positions from at or about 427 to at or about 609 in the
sense strand of FIG. 9 (SEQ ID NO:15); and (3) the nucleic acid
sequence consisting of nucleotide positions from at or about 115 to
at or about 135 in the sense strand of FIG. 15 (SEQ ID NO:18).
Preferably, hybridization occurs under stringent hybridization and
wash conditions.
[0143] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA encoding an IL-1lp polypeptide
that retains at least one biologic activity of a native sequence
IL-1lp, such as the IL-1R binding activity of a native sequence
hIL-1Ra3 or mIL-1Ra3, or the IL-18R binding activity of a native
sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, which DNA hybridizes to
the complement of a nucleic acid sequence selected from the group
consisting of: (1) the nucleic acid sequence consisting of
nucleotide positions from at or about 118 to at or about 231 in the
sense strand of FIG. 2 (SEQ ID NO:4); (2) the nucleic acid sequence
consisting of nucleotide positions from at or about 100 to at or
about 465 in the sense strand of FIG. 7 (SEQ ID NO:12); (3) the
nucleic acid sequence consisting of nucleotide positions from at or
about 244 to at or about 609 in the sense strand of FIG. 9 (SEQ ID
NO:15); and (4) the nucleic acid sequence consisting of nucleotide
positions from at or about 208 to at or about 339 in the sense
strand of FIG. 19 (SEQ ID NO:24). Preferably, hybridization occurs
under stringent hybridization and wash conditions.
[0144] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA encoding an IL-1lp polypeptide
that retains at least one biologic activity of a native sequence
IL-1lp, such as the IL-1R binding activity of a native sequence
hIL-1Ra3 or mIL-1Ra3, which DNA hybridizes to the complement of a
nucleic acid sequence selected from the group consisting of: (1)
the nucleic acid sequence consisting of nucleotide positions from
at or about 4 to at or about 465 in the sense strand of FIG. 7 (SEQ
ID NO:12); and (2) the nucleic acid sequence consisting of
nucleotide positions from at or about 148 to at or about 609 in the
sense strand of FIG. 9 (SEQ ID NO:15). Preferably, hybridization
occurs under stringent hybridization and wash conditions.
[0145] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA encoding an IL-1lp polypeptide
that retains at least one biologic activity of a native sequence
IL-1lp, such as the IL-1R binding activity of a native sequence
hIL-1Ra3 or mIL-1Ra3, or the IL-18R binding activity of a native
sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, which DNA has at least
at or about 80% sequence identity, or at least at or about 85%
sequence identity, or at least at or about 90% sequence identity,
or at least at or about 95% sequence identity to (a) a DNA encoding
an IL-1lp, such as a mature IL-1lp polypeptide, encoded by the cDNA
insert in the vector deposited as ATCC Deposit No. 203588
(DNA85066-2534), ATCC Deposit No. 203587 (DNA96786-2534), ATCC
Deposit No. 203589 (DNA96787-2534), ATCC Deposit No. 203590
(DNA92505-2534), ATCC Deposit No. 203846 (DNA102043-2534), or ATCC
Deposit No. 203973 (DNA114876-2534), or (b) the complement of the
DNA molecule of (a). In a preferred embodiment, the nucleic acid
comprises a DNA encoding an IL-1lp polypeptide, such as a mature
IL-1lp polypeptide, encoded by the cDNA insert in the vector
deposited as ATCC Deposit No. 203588 (DNA85066-2534), ATCC Deposit
No. 203587 (DNA96786-2534), ATCC Deposit No. 203586
(DNA92929-2534), ATCC Deposit No. 203589 (DNA96787-2534), ATCC
Deposit No. 203590 (DNA92505-2534), ATCC Deposit No. 203846
(DNA102043-2534), ATCC Deposit No. 203973 (DNA1 14876-2534), or
ATCC Deposit No. 203855 (DNA102044-2534).
[0146] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA encoding an IL-1lp polypeptide
that retains at least one biologic activity of a native sequence
IL-1lp, such as the IL-1R binding activity of a native sequence
hIL-1Ra3 or mIL-1Ra3, or the IL-18R binding activity of a native
sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, which DNA has at least
at or about 80% sequence identity, or at least at or about 85%
sequence identity, or at least at or about 90% sequence identity,
or at least at or about 95% sequence identity to (a) a DNA encoding
the entire amino acid sequence encoded by the longest open reading
frame in the cDNA insert of a vector selected from the group
consisting of the vectors deposited as ATCC Deposit No. 203588
(DNA85066-2534), ATCC Deposit No. 203587 (DNA96786-2534), ATCC
Deposit No. 203589 (DNA96787-2534), ATCC Deposit No. 203590
(DNA92505-2534), ATCC Deposit No. 203846 (DNA102043-2534), and ATCC
Deposit No. 203973 (DNA114876-2534), or (b) the complement of the
DNA molecule of (a). In a preferred embodiment, the nucleic acid
comprises (a) DNA encoding the entire amino acid sequence encoded
by the longest open reading frame in the cDNA insert of a vector
selected from the group consisting of the vectors deposited as ATCC
Deposit No. 203588 (DNA85066-2534), ATCC Deposit No. 203587
(DNA96786-2534), ATCC Deposit No. 203586 (DNA92929-2534), ATCC
Deposit No. 203589 (DNA96787-2534), and ATCC Deposit No. 203590
(DNA92505-2534), or (b) the complement of the DNA of (a). In
another preferred embodiment, the nucleic acid comprises (a) DNA
encoding the entire amino acid sequence encoded by the longest open
reading frame in the cDNA insert of a vector selected from the
group consisting of the vectors deposited as ATCC Deposit No.
203846 (DNA102043-2534), ATCC Deposit No. 203855 (DNA102044-2534),
and ATCC Deposit No. 203973 (DNA114876-2534), or (b) the complement
of the DNA of (a).
[0147] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA encoding an IL-1lp polypeptide
that retains at least one biologic activity of a native sequence
IL-1lp, such as the IL-1R binding activity of a native sequence
IL-1Ra3 or mIL-1Ra3, or the IL-18R binding activity of a native
sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, which DNA has at least
at or about 80% sequence identity, or at least at or about 85%
sequence identity, or at least at or about 90% sequence identity,
or at least at or about 95% sequence identity to (a) DNA encoding
an amino acid sequence selected from the group consisting of: (1)
the entire amino acid sequence encoded by the longest open reading
frame in the cDNA insert in the vector deposited as ATCC Deposit
No. 203588, (2) the entire amino acid sequence, or the entire amino
acid sequence excluding the 36 N-terminal amino acid residues of
such sequence, encoded by the longest open reading frame in the
cDNA insert in the vector deposited as ATCC Deposit No. 203587, (3)
the entire amino acid sequence, or the entire amino acid sequence
excluding the N-terminal amino acid residue of such sequence,
encoded by the longest open reading frame in the cDNA insert in the
vector deposited as ATCC Deposit No. 203589, (4) the entire amino
acid sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203590, (5) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, or the entire amino
acid sequence excluding the 34 N-terminal amino acid residues of
such sequence, encoded by the longest open reading frame in the
cDNA insert in the vector deposited as ATCC Deposit No. 203846, and
(6) the entire amino acid sequence, or the entire amino acid
sequence excluding the N-terminal amino acid residue of such
sequence, or the entire amino acid sequence excluding the 45
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203973, or (b) the complement of the
DNA of (a).
[0148] In a preferred embodiment, the nucleic acid comprises (a)
DNA encoding an amino acid sequence selected from the group
consisting of: (1) the entire amino acid sequence encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203588, (2) the entire amino acid
sequence, or the entire amino acid sequence excluding the 36
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203587, (3) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203589, (4) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203590, (5) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, or the entire amino
acid sequence excluding the 34 N-terminal amino acid residues of
such sequence, encoded by the longest open reading frame in the
cDNA insert in the vector deposited as ATCC Deposit No. 203846, and
(6) the entire amino acid sequence, or the entire amino acid
sequence excluding the N-terminal amino acid residue of such
sequence, or the entire amino acid sequence excluding the 45
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203973, or (b) the complement of the
DNA of (a).
[0149] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-18R binding activity of a
native sequence hIL-1Ra1, or the IL-1R binding activity of a native
sequence hIL-1Ra3 or mIL-1Ra3, and which IL-1lp polypeptide has at
least at or about 80% sequence identity, or at least at or about
85% sequence identity, or at least at or about 90% sequence
identity, or at least at or about 95% sequence identity to an amino
acid sequence selected from the group consisting of: (1) amino acid
residues from at or about 37 to at or about 203, inclusive of FIG.
2 (SEQ ID NO:5), (2) amino acid residues from at or about 15 to at
or about 193, inclusive of FIG. 3 (SEQ ID NO:7), (3) amino acid
residues from at or about 34 to at or about 155, inclusive of FIG.
7 (SEQ ID NO:13), (4) amino acid residues from at or about 34 to at
or about 155, inclusive of FIG. 9 (SEQ ID NO:16), (5) amino acid
residues from at or about 26 to at or about 207, inclusive of FIG.
15 (SEQ ID NO:19), and (6) amino acid residues from at or about 46
to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25), or (b) the
complement of the DNA of (a).
[0150] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-18R binding activity of a
native sequence hIL-1Ra1, or the IL-1R binding activity of a native
sequence hIL-1Ra3 or mIL-1Ra3, and which IL-1lp polypeptide has at
least at or about 80% sequence identity, or at least at or about
85% sequence identity, or at least at or about 90% sequence
identity, or at least at or about 95% sequence identity to an amino
acid sequence selected from the group consisting of: (1) amino acid
residues from at or about 95 to at or about 134, inclusive of FIG.
7 (SEQ ID NO:13), and (2) amino acid residues from at or about 95
to at or about 134, inclusive of FIG. 9 (SEQ ID NO:16), or (b) the
complement of the DNA of (a).
[0151] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-18R binding activity of a
native sequence hIL-1Ra1, or the IL-1R binding activity of a native
sequence hIL-1Ra3 or mIL-1Ra3, and which IL-1lp polypeptide has at
least at or about 80% sequence identity, or at least at or about
85% sequence identity, or at least at or about 90% sequence
identity, or at least at or about 95% sequence identity to the
amino acid sequence of amino acid residues from at or about 80 to
at or about 155, inclusive of FIG. 7 (SEQ ID NO:13), or (b) the
complement of the DNA of (a).
[0152] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-18R binding activity of a
native sequence hIL-1Ra1, or the IL-1R binding activity of a native
sequence hIL-1Ra3 or mIL-1Ra3, and which IL-1lp polypeptide has at
least at or about 80% sequence identity, or at least at or about
85% sequence identity, or at least at or about 90% sequence
identity, or at least at or about 95% sequence identity to an amino
acid sequence selected from the group consisting of: (1) amino acid
residues from at or about 2 to at or about 155, inclusive of FIG. 7
(SEQ ID NO:13), and (2) amino acid residues from at or about 2 to
at or about 155, inclusive of FIG. 9 (SEQ ID NO:16), or (b) the
complement of the DNA of (a).
[0153] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide having at least at or about 80% sequence
identity, or at least at or about 85% sequence identity, or at
least at or about 90% sequence identity, or at least at or about
95% sequence identity to an amino acid sequence selected from the
group consisting of: (1) amino acid residues from at or about 95 to
at or about 134, inclusive of FIG. 7 (SEQ ID NO:13), and (2) amino
acid residues from at or about 95 to at or about 134, inclusive of
FIG. 9 (SEQ ID NO:16), or (b) the complement of the DNA of (a).
[0154] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide having at least at or about 80% sequence
identity, or at least at or about 85% sequence identity, or at
least at or about 90% sequence identity, or at least at or about
95% sequence identity to the amino acid sequence of amino acid
residues from at or about 80 to at or about 155, inclusive of FIG.
7 (SEQ ID NO:13), or (b) the complement of the DNA of (a).
[0155] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide having at least at or about 80% sequence
identity, or at least at or about 85% sequence identity, or at
least at or about 90% sequence identity, or at least at or about
95% sequence identity to an amino acid sequence selected from the
group consisting of: (1) amino acid residues from at or about 2 to
at or about 155, inclusive of FIG. 7 (SEQ ID NO:13), and (2) amino
acid residues from at or about 2 to at or about 155, inclusive of
FIG. 9 (SEQ ID NO:16), or (b) the complement of the DNA of (a).
[0156] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-18R binding activity of a
native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and which IL-1lp
polypeptide has at least at or about 80% sequence identity, or at
least at or about 85% sequence identity, or at least at or about
90% sequence identity, or at least at or about 95% sequence
identity to an amino acid sequence selected from the group
consisting of: (1) amino acid residues from at or about 1 to at or
about 207, inclusive of FIG. 15 (SEQ ID NO:19), and (2) amino acid
residues from at or about 1 to at or about 218, inclusive of FIG.
19 (SEQ ID NO:25), or (b) the complement of the DNA of (a).
[0157] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA encoding an IL-1lp that
retains at least one biologic activity of a native sequence IL-1lp,
such as the IL-1R binding activity of a native sequence hIL-1Ra3 or
mIL-1Ra3, or the IL-18R binding activity of a native sequence
hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1S, or hIL-1Ra1V, which DNA is produced
by hybridizing a test DNA molecule under stringent conditions with
(a) a DNA molecule encoding an IL-1lp polypeptide selected from the
group consisting of: (1) an IL-1lp polypeptide comprising the
sequence of amino acid residues from at or about 37 to at or about
203, inclusive of FIG. 2 (SEQ ID NO:5), (2) an IL-1lp polypeptide
comprising the sequence of amino acid residues from at or about 15
to at or about 193, inclusive of FIG. 3 (SEQ ID NO:7), (3) an
IL-1lp polypeptide comprising the sequence of amino acid residues
from at or about 34 to at or about 155, inclusive of FIG. 7 (SEQ ID
NO:13), (4) an IL-1lp polypeptide comprising the sequence of amino
acid residues from at or about 34 to at or about 155, inclusive of
FIG. 9 (SEQ ID NO:16), (5) an IL-1lp polypeptide comprising the
sequence of amino acid residues from at or about 26 to at or about
207, inclusive of FIG. 15 (SEQ ID NO:19), and (6) an IL-1lp
polypeptide comprising the sequence of amino acid residues from at
or about 46 to at or about 218, inclusive of FIG. 19 (SEQ ID
NO:25), or (b) the complement of the DNA molecule of (a), and, if
the test DNA molecule encodes an IL-1lp that retains at least one
biologic activity of a native sequence IL-1lp, such as the IL-1R
binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, or the
IL-18R binding activity of a native sequence hIL-1Ra1, hIL-1Ra1L,
or hIL-1Ra1V, and if the test DNA molecule has at least at or about
an 80% sequence identity, or at least at or about an 85% sequence
identity, or at least at or about a 90% sequence identity, or at
least at or about a 95% sequence identity to the DNA molecule of
(a) or (b), isolating the test DNA molecule.
[0158] In another aspect, the invention provides an isolated
nucleic acid molecule comprising (a) DNA encoding an a polypeptide,
such as IL-1lp polypeptide, selected from the group consisting of:
(1) a polypeptide, such as an hIL-1Ra1 polypeptide, comprising
amino acid residues from at or about 37 to at or about 63,
inclusive of FIG. 2 (SEQ ID NO:5); (2) a polypeptide, such as an
hIL-1Ra1 polypeptide, comprising amino acid residues from at or
about 15 to at or about 53, inclusive of FIG. 3 (SEQ ID NO:7); (3)
a polypeptide, such as an hIL-1Ra2 polypeptide, comprising amino
acid residues from at or about 1 to at or about 134, inclusive of
FIG. 5 (SEQ ID NO:10); (4) a polypeptide comprising amino acid
residues from at or about 10 to at or about 134, inclusive of FIG.
5 (SEQ ID NO:10); (5) a polypeptide, such as an hIL-1Ra2
polypeptide, consisting of amino acid residues from at or about 27
to at or about 134, inclusive of FIG. 5 (SEQ ID NO:10); (6) a
polypeptide, such as an hIL-1Ra2 fusion variant polypeptide,
consisting of a native amino acid sequence of hIL-1Ra2 consisting
of amino acid residues from at or about 27 to at or about 134,
inclusive of FIG. 5 (SEQ ID NO:10) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; (7)
a polypeptide, such as an hIL-1Ra3 polypeptide, comprising amino
acid residues from at or about 95 to at or about 134, inclusive of
FIG. 7 (SEQ ID NO:13); and (8) a polypeptide, such as a mIL-1Ra3
polypeptide, comprising amino acid residues from at or about 95 to
at or about 134, inclusive of FIG. 9 (SEQ ID NO:16); or (b) the
complement of the DNA of (a).
[0159] In another aspect, the invention provides an isolated
nucleic acid molecule comprising (a) DNA encoding a polypeptide,
such as an IL-1lp polypeptide, selected from the group consisting
of: (1) a polypeptide, such as an hIL-1Ra1L polypeptide, comprising
amino acid residues from at or about 26 to at or about 44,
inclusive of FIG. 15 (SEQ ID NO:19); (2) a polypeptide, such as an
hIL-1Ra1L polypeptide, comprising amino acid residues from at or
about 1 to at or about 44, inclusive of FIG. 15 (SEQ ID NO:19); (3)
a polypeptide, such as an hIL-1Ra1L polypeptide, comprising amino
acid residues from at or about 26 to at or about 78, inclusive of
FIG. 15 (SEQ ID NO:19); (4) a polypeptide, such as an hIL-1Ra1L
polypeptide, comprising amino acid residues from at or about 1 to
at or about 78, inclusive of FIG. 15 (SEQ ID NO:19); (5) a
polypeptide, such as an hIL-1Ra1S polypeptide, comprising amino
acid residues from at or about 1 to at or about 38, inclusive of
FIG. 16 (SEQ ID NO:21); (6) a polypeptide, such as an hIL-1Ra1V
polypeptide, comprising amino acid residues from at or about 37 to
at or about 55, inclusive of FIG. 19 (SEQ ID NO:25); (7) a
polypeptide, such as an hIL-1Ra1V polypeptide, comprising amino
acid residues from at or about 12 to at or about 55, inclusive of
FIG. 19 (SEQ ID NO:25); (8) a polypeptide, such as an hIL-1Ra1V
polypeptide, comprising amino acid residues from at or about 1 to
at or about 55, inclusive of FIG. 19 (SEQ ID NO:25); (9) a
polypeptide, such as an hIL-1Ra1V polypeptide, comprising amino
acid residues from at or about 46 to at or about 55, inclusive of
FIG. 19 (SEQ ID NO:25); (10) a polypeptide, such as an hIL-1Ra1V
polypeptide, comprising amino acid residues from at or about 46 to
at or about 89, inclusive of FIG. 19 (SEQ ID NO:25); (11) a
polypeptide, such as an hIL-1Ra1V polypeptide, comprising amino
acid residues from at or about 37 to at or about 89, inclusive of
FIG. 19 (SEQ ID NO:25); (12) a polypeptide, such as an hIL-1Ra1V
polypeptide, comprising amino acid residues from at or about 12 to
at or about 89, inclusive of FIG. 19 (SEQ ID NO:25); and (13) a
polypeptide, such as an hIL-1Ra1V polypeptide, comprising amino
acid residues from at or about 1 to at or about 89, inclusive of
FIG. 19 (SEQ ID NO:25); or (b) the complement of the DNA of
(a).
[0160] In another aspect, the invention provides an isolated
nucleic acid molecule comprising (a) DNA encoding a polypeptide,
such as an hIL-1lp polypeptide, selected from the group consisting
of: (1) a polypeptide, such as an hIL-1Ra1L polypeptide, consisting
of a native amino acid sequence of hIL-1Ra1L consisting of amino
acid residues from at or about 26 to at or about 207, inclusive of
FIG. 15 (SEQ ID NO:19) fused at its N-terminus or C-terminus to a
heterologous amino acid or amino acid sequence; (2) a polypeptide,
such as an hIL-1Ra1L polypeptide, consisting of a native amino acid
sequence of hIL-1Ra1L consisting of amino acid residues from at or
about 1 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19)
fused at its N-terminus or C-terminus to a heterologous amino acid
or amino acid sequence; (3) a polypeptide, such as an hIL-1Ra1L
polypeptide, consisting of amino acid residues from at or about 26
to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19); (4) a
polypeptide, such as an hIL-1Ra1L polypeptide, consisting of amino
acid residues from at or about 1 to at or about 207, inclusive of
FIG. 15 (SEQ ID NO:19); (5) a polypeptide, such as an hIL-1Ra1S
fusion variant polypeptide, consisting of a native amino acid
sequence of hIL-1Ra1S consisting of amino acid residues from at or
about 26 to at or about 167, inclusive of FIG. 16 (SEQ ID NO:21)
fused at its N-terminus or C-terminus to a heterologous amino acid
or amino acid sequence; (6) a polypeptide, such as an hIL-1Ra1S
polypeptide, consisting of amino acid residues from at or about 26
to at or about 167, inclusive of FIG. 16 (SEQ ID NO:21); (7) a
polypeptide, such as an hIL-1Ra1S fusion variant polypeptide,
consisting of a native amino acid sequence of hIL-1Ra1S consisting
of amino acid residues from at or about 1 to at or about 167,
inclusive of FIG. 16 (SEQ ID NO:21) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; (8)
a polypeptide, such as an hIL-1Ra1S polypeptide, consisting of
amino acid residues from at or about 1 to at or about 167,
inclusive of FIG. 16 (SEQ ID NO:21); (9) a polypeptide, such as an
hIL-1Ra1S fusion variant polypeptide, consisting of a native amino
acid sequence of hIL-1Ra1S consisting of amino acid residues from
at or about 39 to at or about 167, inclusive of FIG. 16 (SEQ ID
NO:21) fused at its N-terminus or C-terminus to a heterologous
amino acid or amino acid sequence; (10) a polypeptide, such as an
hIL-1Ra1S fusion variant polypeptide, consisting of a native amino
acid sequence of hIL-1Ra1S consisting of amino acid residues from
at or about 47 to at or about 167, inclusive of FIG. 16 (SEQ ID
NO:21) fused at its N-terminus or C-terminus to a heterologous
amino acid or amino acid sequence; (11) a polypeptide, such as an
hIL-1Ra1S polypeptide, consisting of amino acid residues from at or
about 39 to at or about 167, inclusive of FIG. 16 (SEQ ID NO:21);
(12) a polypeptide, such as an hIL-1Ra1S polypeptide, consisting of
amino acid residues from at or about 47 to at or about 167,
inclusive of FIG. 16 (SEQ ID NO:21); (13) a polypeptide, such as an
hIL-1Ra1V polypeptide, consisting of a native amino acid sequence
of hIL-1Ra1V consisting of amino acid residues from at or about 1
to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25) fused at
its N-terminus or C-terminus to a heterologous amino acid or amino
acid sequence; (14) a polypeptide, such as an hIL-1Ra1V
polypeptide, consisting of amino acid residues from at or about 1
to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25); (15) a
polypeptide, such as an hIL-1Ra1V polypeptide, consisting of a
native amino acid sequence of hIL-1Ra1V consisting of amino acid
residues from at or about 12 to at or about 218, inclusive of FIG.
19 (SEQ ID NO:25) fused at its N-terminus or C-terminus to a
heterologous amino acid or amino acid sequence; (16) a polypeptide,
such as an hIL-1Ra1V polypeptide, consisting of amino acid residues
from at or about 12 to at or about 218, inclusive of FIG. 19 (SEQ
ID NO:25); (17) a polypeptide, such as an hIL-1Ra1V fusion variant
polypeptide, consisting of a native amino acid sequence of
hIL-1Ra1V consisting of amino acid residues from at or about 37 to
at or about 218, inclusive of FIG. 19 (SEQ ID NO:25) fused at its
N-terminus or C-terminus to a heterologous amino acid or amino acid
sequence; (18) a polypeptide, such as an hIL-1Ra1V fusion variant
polypeptide, consisting of a native amino acid sequence of
hIL-1Ra1V consisting of amino acid residues from at or about 46 to
at or about 218, inclusive of FIG. 19 (SEQ ID NO:25) fused at its
N-terminus or C-terminus to a heterologous amino acid or amino acid
sequence; (19) a polypeptide, such as an hIL-1Ra1V polypeptide,
consisting of amino acid residues from at or about 37 to at or
about 218, inclusive of FIG. 19 (SEQ ID NO:25); and (20) a
polypeptide, such as an hIL-1Ra1V polypeptide, consisting of amino
acid residues from at or about 46 to at or about 218, inclusive of
FIG. 19 (SEQ ID NO:25); or (b) the complement of the DNA of
(a).
[0161] In another aspect, the invention provides an isolated DNA
molecule selected from the group consisting of: (1) a DNA molecule
encoding a polypeptide, such as an hIL-1Ra1 polypeptide, comprising
the amino acid sequence of amino acid residues from at or about 37
to at or about 203, inclusive of FIG. 2 (SEQ ID NO:5); (2) a DNA
molecule encoding a polypeptide, such as an hIL-1Ra1 polypeptide,
comprising the amino acid sequence of amino acid residues from at
or about 15 to at or about 193, inclusive of FIG. 3 (SEQ ID NO:7);
(3) a DNA molecule encoding a polypeptide, such as an hIL-1Ra2
polypeptide, comprising the amino acid sequence of amino acid
residues from at or about 1 to at or about 134, inclusive of FIG. 5
(SEQ ID NO:10); (4) a DNA molecule encoding a polypeptide
comprising the amino acid sequence of amino acid residues from at
or about 10 to at or about 134, inclusive of FIG. 5 (SEQ ID NO:10);
(5) a DNA molecule encoding a polypeptide, such as an hIL-1Ra2
fusion variant polypeptide, consisting of a native amino acid
sequence of hIL-1Ra2 consisting of amino acid residues from at or
about 27 to at or about 134, inclusive of FIG. 5 (SEQ ID NO:10)
fused at its N-terminus or C-terminus to a heterologous amino acid
or amino acid sequence; (6) a DNA molecule encoding a polypeptide,
such as an hIL-1Ra2 polypeptide, consisting of the amino acid
sequence of amino acid residues from at or about 27 to at or about
134, inclusive of FIG. 5 (SEQ ID NO:10); (7) a DNA molecule
encoding a polypeptide, such as an hIL-1Ra3 polypeptide, comprising
the amino acid sequence of amino acid residues from at or about 95
to at or about 134, inclusive of FIG. 7 (SEQ ID NO:13); (8) a DNA
molecule encoding a polypeptide, such as a mIL-1Ra3 polypeptide,
comprising the amino acid sequence of amino acid residues from
about 95 to at or about 134, inclusive of FIG. 9 (SEQ ID NO:16);
and (9) the complement of any of the DNA molecules of (1)-(8).
[0162] In another aspect, the invention provides an isolated DNA
molecule selected from the group consisting of: (1) a DNA molecule
encoding a polypeptide, such as an hIL-1Ra1L polypeptide,
comprising the amino acid sequence of amino acid residues from at
or about 26 to at or about 207, inclusive of FIG. 15 (SEQ ID
NO:19); (2) a DNA molecule encoding a polypeptide, such as an
hIL-1Ra1S polypeptide, comprising the amino acid sequence of amino
acid residues from at or about 26 to at or about 167, inclusive of
FIG. 16 (SEQ ID NO:21); (3) a DNA molecule encoding a polypeptide,
such as an hIL-1Ra1V polypeptide, comprising the amino acid
sequence of amino acid residues from at or about 37 to at or about
218, inclusive of FIG. 19 (SEQ ID NO:25); (4) a DNA molecule
encoding a polypeptide, such as an hIL-1Ra1V polypeptide,
comprising the amino acid sequence of amino acid residues from at
or about 46 to at or about 218, inclusive of FIG. 19 (SEQ ID
NO:25); and (5) the complement of any of the DNA molecules of
(1)-(4).
[0163] In another aspect, the invention provides an isolated DNA
molecule selected from the group consisting of: (1) a DNA molecule
encoding a polypeptide, such as an hIL-1Ra1L polypeptide,
comprising the amino acid sequence of amino acid residues from at
or about 1 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19);
(2) a DNA molecule encoding a polypeptide, such as an hIL-1Ra1S
polypeptide, comprising the amino acid sequence of amino acid
residues from at or about 1 to at or about 167, inclusive of FIG.
16 (SEQ ID NO:21); (3) a DNA molecule encoding a polypeptide, such
as an hIL-1Ra1V polypeptide, comprising the amino acid sequence of
amino acid residues from at or about 12 to at or about 218,
inclusive of FIG. 19 (SEQ ID NO:25); (4) a DNA molecule encoding a
polypeptide, such as an hIL-1Ra1V polypeptide, comprising the amino
acid sequence of amino acid residues from at or about 1 to at or
about 218, inclusive of FIG. 19 (SEQ ID NO:25); and (5) the
complement of any of the DNA molecules of (1)-(4).
[0164] In another aspect, the invention provides an isolated DNA
molecule selected from the group consisting of: (1) a DNA molecule
encoding a polypeptide, such as an hIL-1Ra3 polypeptide, comprising
amino acid residues from at or about 80 to at or about 155,
inclusive of FIG. 7 (SEQ ID NO:13); and (2) the complement of the
DNA molecule of (1).
[0165] In another aspect, the invention provides an isolated DNA
molecule selected from the group consisting of: (1) a DNA molecule
encoding a polypeptide, such as an hIL-1Ra1 polypeptide, comprising
the amino acid sequence of amino acid residues from at or about 1
to at or about 203, inclusive of FIG. 2 (SEQ ID NO:5); (2) a DNA
molecule encoding a polypeptide, such as an hIL-1Ra1 polypeptide,
comprising the amino acid sequence of amino acid residues from at
or about 1 to at or about 193, inclusive of FIG. 3 (SEQ ID NO:7);
(3) a DNA molecule encoding a polypeptide, such as an hIL-1Ra3
polypeptide, comprising the amino acid sequence of amino acid
residues from at or about 34 to at or about 155, inclusive of FIG.
7 (SEQ ID NO:13); (4) a DNA molecule encoding a polypeptide, such
as a mIL-1Ra3 polypeptide, comprising the amino acid sequence of
amino acid residues from at or about 34 to at or about 155,
inclusive of FIG. 9 (SEQ ID NO:16); and (5) the complement of any
of the DNA molecules of (1)-(4).
[0166] In another aspect, the invention provides an isolated DNA
molecule selected from the group consisting of: (1) a DNA molecule
encoding a polypeptide, such as an hIL-1Ra3 polypeptide, comprising
the amino acid sequence of amino acid residues from at or about 1
to at or about 155, inclusive of FIG. 7 (SEQ ID NO:13); (2) a DNA
molecule encoding a polypeptide, such as a mIL-1Ra3 polypeptide,
comprising the amino acid sequence of amino acid residues from at
or about 1 to at or about 155, inclusive of FIG. 9 (SEQ ID NO:16);
and (3) the complement of any of the DNA molecules of (1)-(2).
[0167] In another aspect, the invention provides an isolated DNA
molecule selected from the group consisting of: (1) a DNA molecule
encoding a polypeptide, such as an hIL-1Ra3 polypeptide, comprising
the amino acid sequence of amino acid residues from at or about 2
to at or about 155, inclusive of FIG. 7 (SEQ ID NO:13); (2) a DNA
molecule encoding a polypeptide, such as a mIL-1Ra3 polypeptide,
comprising the amino acid sequence of amino acid residues from at
or about 2 to at or about 155, inclusive of FIG. 9 (SEQ ID NO:16);
and (3) the complement of any of the DNA molecules of (1)-(2).
[0168] In another aspect, the invention provides an isolated
nucleic acid molecule comprising (a) a DNA molecule encoding a
polypeptide selected from the group consisting of: (1) a
polypeptide comprising an hIL-1Ra1 polypeptide, such as a mature
hIL-1Ra1 polypeptide, encoded by the cDNA insert in the vector
deposited as ATCC Deposit No. 203588; (2) a polypeptide comprising
an hIL-1Ra1 polypeptide, such as a mature hIL-1Ra1 polypeptide,
encoded by the cDNA insert in the vector deposited as ATCC Deposit
No. 203587; (3) a polypeptide consisting of an hIL-1Ra2
polypeptide, such as a mature hIL-1Ra2 polypeptide, encoded by the
cDNA insert in the vector deposited as ATCC Deposit No. 203586,
which hIL-1Ra2 polypeptide is fused at its N-terminus or C-terminus
to a heterologous amino acid or amino acid sequence; (4) a
polypeptide consisting of an hIL-1Ra2 polypeptide, such as a mature
hIL-1Ra2 polypeptide, encoded by the cDNA insert in the vector
deposited as ATCC Deposit No. 203586; (5) a polypeptide comprising
an hIL-1Ra3 polypeptide, such as a mature hIL-1Ra3 polypeptide,
encoded by the cDNA insert in the vector deposited as ATCC Deposit
No. 203589; and (6) a polypeptide comprising a mIL-1Ra3
polypeptide, such as a mature mIL-1Ra3 polypeptide, encoded by the
cDNA insert in the vector deposited as ATCC Deposit No. 203590; or
(b) the complement of the DNA molecule of (a).
[0169] In another aspect, the invention provides an isolated
nucleic acid molecule comprising (a) a DNA molecule encoding a
polypeptide selected from the group consisting of: (1) a
polypeptide comprising an hIL-1Ra1L polypeptide, such as a mature
hIL-1Ra1L polypeptide, encoded by the cDNA insert in the vector
deposited as ATCC Deposit No. 203846; (2) a polypeptide consisting
of an hIL-1Ra1S polypeptide, such as a mature hIL-1Ra1S
polypeptide, encoded by the cDNA insert in the vector deposited as
ATCC Deposit No. 203855, which hIL-1Ra1S polypeptide is fused at
its N-terminus or C-terminus to a heterologous amino acid or amino
acid sequence; (3) a polypeptide consisting of an hIL-1Ra1S
polypeptide, such as a mature hIL-1Ra1S polypeptide, encoded by the
cDNA insert in the vector deposited as ATCC Deposit No. 203855; (4)
a polypeptide comprising an hIL-1Ra1V polypeptide, such as a mature
hIL-1Ra1V polypeptide, encoded by the cDNA insert in the vector
deposited as ATCC Deposit No. 203973; or (b) the complement of the
DNA molecule of (a).
[0170] In another aspect, the invention provides an isolated
nucleic acid molecule comprising (a) a DNA molecule encoding a
polypeptide comprising an hIL-1Ra1S polypeptide, such as a mature
hIL-1Ra1S polypeptide, encoded by the cDNA insert in the vector
deposited as ATCC Deposit No. 203855; or (b) the complement of the
DNA molecule of (a).
[0171] In another aspect, the invention provides an isolated
nucleic acid molecule comprising (a) a DNA encoding a polypeptide
selected from the group consisting of: (1) a polypeptide comprising
the entire amino acid sequence encoded by the longest open reading
frame in the cDNA insert in the vector deposited as ATCC Deposit
No. 203588; (2) a polypeptide comprising the entire amino acid
sequence, or the entire amino acid sequence excluding the 36
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203587; (3) a polypeptide comprising
the entire amino acid sequence, or the entire amino acid sequence
excluding the N-terminal amino acid residue of such sequence, or
the entire amino acid sequence excluding the 9 N-terminal amino
acid residues of such sequence, encoded by the longest open reading
frame in the cDNA insert in the vector deposited as ATCC Deposit
No. 203586; (4) a polypeptide comprising the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203589; and (5) a polypeptide
comprising the entire amino acid sequence, or the entire amino acid
sequence excluding the N-terminal amino acid residue of such
sequence, encoded by the longest open reading frame in the cDNA
insert in the vector deposited as ATCC Deposit No. 203590; or (b)
the complement of the DNA of (a).
[0172] In another aspect, the invention provides an isolated
nucleic acid molecule comprising (a) a DNA encoding a polypeptide
comprising the entire amino acid sequence encoded by the longest
open reading frame in the cDNA insert of a vector selected from the
group consisting of the vectors deposited as ATCC Deposit Nos.
203588, 203586, 203589, 203590, and 203973, or (b) the complement
of the DNA of (a).
[0173] In another aspect, the invention provides an isolated
nucleic acid molecule comprising (a) a DNA encoding a polypeptide
selected from the group consisting of: (1) a polypeptide comprising
the entire amino acid sequence, or the entire amino acid sequence
excluding the N-terminal amino acid residue of such sequence, or
the entire amino acid sequence excluding the 34 N-terminal amino
acid residues of such sequence, encoded by the longest open reading
frame in the cDNA insert in the vector deposited as ATCC Deposit
No. 203846; (2) a polypeptide comprising the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, or the entire amino
acid sequence excluding the 25 N-terminal amino acid residues of
such sequence, encoded by the longest open reading frame in the
cDNA insert in the vector deposited as ATCC Deposit No. 204855; and
(3) a polypeptide comprising the entire amino acid sequence, or the
entire amino acid sequence excluding the N-terminal amino acid
residue of such sequence, or the entire amino acid sequence
excluding the 11 N-terminal amino acid residues of such sequence,
or the entire amino acid sequence excluding the 36 N-terminal amino
acid residues of such sequence, or the entire amino acid sequence
excluding the 45 N-terminal amino acid residues of such sequence,
encoded by the longest open reading frame in the cDNA insert in the
vector deposited as ATCC Deposit No. 203973; or (b) the complement
of the DNA of (a).
[0174] In another aspect, the invention provides an isolated
nucleic acid molecule comprising (a) a DNA encoding a non-naturally
occurring, chimeric polypeptide formed by fusing the entire amino
acid sequence excluding the 38 N-terminal amino acid residues of
such sequence, or the entire amino acid sequence excluding the 46
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203855, at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; or
(b) the complement of the DNA of (a).
[0175] In another aspect, the invention provides an isolated
nucleic acid molecule comprising (a) a DNA encoding a polypeptide
consisting of the entire amino acid sequence excluding the 38
N-terminal amino acid residues of such sequence, or the entire
amino acid sequence excluding the 46 N-terminal amino acid residues
of such sequence, encoded by the longest open reading frame in the
cDNA insert in the vector deposited as ATCC Deposit No. 203855; or
(b) the complement of the DNA of (a).
[0176] In another aspect, the invention provides an isolated
nucleic acid molecule comprising (a) a DNA molecule encoding a
polypeptide comprising the entire amino acid sequence encoded by
the longest open reading frame in the cDNA insert of a vector
selected from the group consisting of the vectors deposited as ATCC
Deposit Nos. 203846, 203855 and 203973, or (b) the complement of
the DNA of (a).
[0177] In another aspect, the invention provides an isolated DNA
molecule selected from the group consisting of: (1) a DNA molecule
which encodes a polypeptide, such as an hIL-1Ra1V fusion variant
polypeptide, consisting of a native amino acid sequence of
hIL-1Ra1V consisting of amino acid residues from at or about 37 to
at or about 218, inclusive of FIG. 19 (SEQ ID NO:25) fused at its
N-terminus or C-terminus to a heterologous amino acid or amino acid
sequence, and which DNA molecule comprises the nucleic acid
sequence in the sense strand of FIG. 19 (SEQ ID NO:24) that encodes
the native amino acid sequence; (2) a DNA molecule which encodes a
polypeptide, such as an hIL-1Ra1V fusion variant polypeptide,
consisting of a native amino acid sequence of hIL-1Ra1V consisting
of amino acid residues from at or about 46 to at or about 218,
inclusive of FIG. 19 (SEQ ID NO:25) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence, and
which DNA molecule comprises the nucleic acid sequence in the sense
strand of FIG. 19 (SEQ ID NO:24) that encodes the native amino acid
sequence; (3) a DNA molecule which encodes a polypeptide, such as
an hIL-1Ra1V polypeptide, consisting of a native amino acid
sequence of hIL-1Ra1V consisting of amino acid residues from at or
about 37 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25),
and which DNA molecule comprises the nucleic acid sequence in the
sense strand of FIG. 19 (SEQ ID NO:24) that encodes the native
amino acid sequence; (4) a DNA molecule which encodes a
polypeptide, such as an hIL-1Ra1V polypeptide, consisting of a
native amino acid sequence of hIL-1Ra1V consisting of amino acid
residues from at or about 46 to at or about 218, inclusive of FIG.
19 (SEQ ID NO:25), and which DNA molecule comprises the nucleic
acid sequence in the sense strand of FIG. 19 (SEQ ID NO:24) that
encodes the native amino acid sequence; and (5) the complement of
any of the DNA molecules of (1)-(4).
[0178] In another aspect, the invention provides an isolated DNA
molecule selected from the group consisting of: (1) a DNA molecule
which encodes a polypeptide, such as an hIL-1Ra1 polypeptide, and
which DNA molecule comprises the nucleic acid sequence of
nucleotide positions from at or about 118 to at or about 618,
inclusive in the sense strand of FIG. 2 (SEQ ID NO:4); (2) a DNA
molecule which encodes a polypeptide, such as an hIL-1Ra1
polypeptide, and which DNA molecule comprises the nucleic acid
sequence of nucleotide positions from at or about 145 to at or
about 681, inclusive in the sense strand of FIG. 3 (SEQ ID NO:6);
(3) a DNA molecule which encodes a polypeptide, such as an hIL-1Ra2
polypeptide, and which DNA molecule comprises the nucleic acid
sequence of nucleotide positions from at or about 96 to at or about
497, inclusive in the sense strand of FIG. 5 (SEQ ID NO:9); (4) a
DNA molecule which comprises the nucleic acid sequence of
nucleotide positions from at or about 123 to at or about 497,
inclusive in the sense strand of FIG. 5 (SEQ ID NO:9); (5) a DNA
molecule which encodes a polypeptide, such as an hIL-1Ra2 fusion
variant polypeptide, consisting of a native amino acid sequence of
hIL-1Ra2 consisting of amino acid residues from at or about 27 to
at or about 134, inclusive of FIG. 5 (SEQ ID NO:10) fused at its
N-terminus or C-terminus to a heterologous amino acid or amino acid
sequence, and which DNA molecule comprises the nucleic acid
sequence in the sense strand of FIG. 5 (SEQ ID NO:9) that encodes
the native amino acid sequence; (6) a DNA molecule which encodes a
polypeptide, such as an hIL-1Ra2 polypeptide, consisting of a
native amino acid sequence of hIL-1Ra2 consisting of amino acid
residues from at or about 27 to at or about 134, inclusive of FIG.
5 (SEQ ID NO:10), and which DNA molecule comprises the nucleic acid
sequence in the sense strand of FIG. 5 (SEQ ID NO:9) that encodes
the native amino acid sequence; (7) a DNA molecule which encodes a
polypeptide, such as an hIL-1Ra3 polypeptide, and which DNA
molecule comprises the nucleic acid sequence of nucleotide
positions from at or about 283 to at or about 402, inclusive in the
sense strand of FIG. 7 (SEQ ID NO:12); (8) a DNA molecule which
encodes a polypeptide, such as a mIL-1Ra3 polypeptide, and which
DNA molecule comprises the nucleic acid sequence of nucleotide
positions from at or about 427 to at or about 546, inclusive in the
sense strand of FIG. 9 (SEQ ID NO:15); and (9) the complement of
any of the DNA molecules of (1)-(8).
[0179] In another aspect, the invention provides an isolated DNA
molecule selected from the group consisting of: (1) a DNA molecule
which encodes a polypeptide, such as an hIL-1Ra1L polypeptide, and
which DNA molecule comprises the nucleic acid sequence of
nucleotide positions from at or about 79 to at or about 624,
inclusive in the sense strand of FIG. 15 (SEQ ID NO:18); (2) a DNA
molecule which encodes a polypeptide, such as an hIL-1Ra1S
polypeptide, and which DNA molecule comprises the nucleic acid
sequence of nucleotide positions from at or about 79 to at or about
504, inclusive in the sense strand of FIG. 16 (SEQ ID NO:20); (3) a
DNA molecule which encodes a polypeptide, such as an hIL-1Ra1S
fusion variant polypeptide, consisting of a native amino acid
sequence of hIL-1Ra1S consisting of amino acid residues from at or
about 39 to at or about 167, inclusive of FIG. 16 (SEQ ID NO:21)
fused at its N-terminus or C-terminus to a heterologous amino acid
or amino acid sequence, and which DNA molecule comprises the
nucleic acid sequence in the sense strand of FIG. 16 (SEQ ID NO:20)
that encodes the native amino acid sequence; (4) a DNA molecule
which encodes a polypeptide, such as an hIL-1Ra1S fusion variant
polypeptide, consisting of a native amino acid sequence of
hIL-1Ra1S consisting of amino acid residues from at or about 47 to
at or about 167, inclusive of FIG. 16 (SEQ ID NO:21) fused at its
N-terminus or C-terminus to a heterologous amino acid or amino acid
sequence, and which DNA molecule comprises the nucleic acid
sequence in the sense strand of FIG. 16 (SEQ ID NO:20) that encodes
the native amino acid sequence; (5) a DNA molecule which encodes a
polypeptide, such as an hIL-1Ra1S polypeptide, consisting of a
native amino acid sequence of hIL-1Ra1S consisting of amino acid
residues from at or about 39 to at or about 167, inclusive of FIG.
16 (SEQ ID NO:21), and which DNA molecule comprises the nucleic
acid sequence in the sense strand of FIG. 16 (SEQ ID NO:20) that
encodes the native amino acid sequence; (6) a DNA molecule which
encodes a polypeptide, such as an hIL-1Ra1S polypeptide, consisting
of a native amino acid sequence of hIL-1Ra1S consisting of amino
acid residues from at or about 47 to at or about 167, inclusive of
FIG. 16 (SEQ ID NO:21), and which DNA molecule comprises the
nucleic acid sequence in the sense strand of FIG. 16 (SEQ ID NO:20)
that encodes the native amino acid sequence; (7) a DNA molecule
which encodes a polypeptide, such as an hIL-1Ra1V polypeptide, and
which DNA molecule comprises the nucleic acid sequence of
nucleotide positions from at or about 181 to at or about 729,
inclusive in the sense strand of FIG. 19 (SEQ ID NO:24); (8) a DNA
molecule which encodes a polypeptide, such as an hIL-1Ra1V
polypeptide, and which DNA molecule comprises the nucleic acid
sequence of nucleotide positions from at or about 208 to at or
about 729, inclusive in the sense strand of FIG. 19 (SEQ ID NO:24);
and (9) the complement of any of the DNA molecules of (1)-(8).
[0180] In another aspect, the invention provides an isolated DNA
molecule selected from the group consisting of: (1) a DNA molecule
which encodes a polypeptide, such as an hIL-1Ra1L polypeptide, and
which DNA molecule comprises the nucleic acid sequence of
nucleotide positions from at or about 4 to at or about 624,
inclusive in the sense strand of FIG. 15 (SEQ ID NO:18); (2) a DNA
molecule which encodes a polypeptide, such as an hIL-1Ra1S
polypeptide, and which DNA molecule comprises the nucleic acid
sequence of nucleotide positions from at or about 4 to at or about
504, inclusive in the sense strand of FIG. 16 (SEQ ID NO:20); (3) a
DNA molecule which encodes a polypeptide, such as an hIL-1Ra1V
polypeptide, and which DNA molecule comprises the nucleic acid
sequence of nucleotide positions from at or about 106 to at or
about 729, inclusive in the sense strand of FIG. 19 (SEQ ID NO:24);
(4) a DNA molecule which encodes a polypeptide, such as an
hIL-1Ra1V polypeptide, and which comprises the nucleic acid
sequence of nucleotide positions from at or about 73 to at or about
729, inclusive in the sense strand of FIG. 19 (SEQ ID NO:24); and
(5) the complement of any of the DNA molecules of (1)-(4).
[0181] In another aspect, the invention provides an isolated DNA
molecule selected from the group consisting of: (1) a DNA molecule
comprising the nucleic acid sequence of nucleotide positions from
at or about 103 to at or about 681, inclusive in the sense strand
of FIG. 3 (SEQ ID NO:6); (2) a DNA molecule comprising the nucleic
acid sequence of nucleotide positions from at or about 100 to at or
about 465, inclusive in the sense strand of FIG. 7 (SEQ ID NO:12);
(3) a DNA molecule comprising the nucleic acid sequence of
nucleotide positions from at or about 244 to at or about 609,
inclusive in the sense strand of FIG. 9 (SEQ ID NO:15); and (4) the
complement of any of the DNA molecules of (1)-(3).
[0182] In another aspect, the invention provides an isolated DNA
molecule comprising (a) the complete DNA sequence in the sense
strand of FIG. 2 (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6), FIG. 5 (SEQ
ID NO:9), FIG. 7 (SEQ ID NO:12), or FIG. 9 (SEQ ID NO:15), or (b)
the complement of (a).
[0183] In another aspect, the invention provides an isolated DNA
molecule comprising (a) the complete DNA sequence in the sense
strand of FIG. 15 (SEQ ID NO:18), FIG. 16 (SEQ ID NO:20), or FIG.
19 (SEQ ID NO:24), or (b) the complement of (a).
[0184] In a specific aspect, the invention provides an isolated
nucleic acid molecule comprising DNA encoding an IL-1lp
polypeptide, with or without the N-terminal signal sequence and/or
the initiating methionine, or is complementary to such IL-1lp
encoding nucleic acid molecule. The signal peptide has been
tentatively identified as extending from amino acid position 1 to
at or about amino acid position 14 in the IL-1lp sequence of FIG. 3
(SEQ ID NO:7), from amino acid position 1 to at or about amino acid
position 26 in the IL-1lp sequence of FIG. 5 (SEQ ID NO:10), from
amino acid position 1 to at or about amino acid position 33 in the
IL-1lp sequence of FIG. 7 (SEQ ID NO:13), and from amino acid
position 1 to at or about amino acid position 33 in the IL-1lp
sequence of FIG. 9 (SEQ ID NO:16).
[0185] The IL-1lp sequence of amino acids from at or about 1 to at
or about 207 of FIG. 15 (SEQ ID NO:19) is believed to behave as a
mature sequence (without a presequence that is removed in
post-translational processing) in certain animal cells. In
addition, it is believed that other animal cells recognize and
remove in post-translational processing one or more signal
peptide(s) contained in the sequence of amino acid positions 1 to
about 34 of FIG. 15 (SEQ ID NO:19).
[0186] The IL-1lp sequence of amino acids from at or about 1 to at
or about 167 of FIG. 16 (SEQ ID NO:21) is believed to behave as a
mature sequence (without a presequence that is removed in
post-translational processing) in certain animal cells. In
addition, it is believed that other animal cells recognize and
remove in post-translational processing one or more signal
peptide(s) contained in the sequence of amino acid positions 1 to
about 46 in the IL-1lp sequence of FIG. 16 (SEQ ID NO:21).
[0187] The IL-1lp sequence of amino acids from at or about 1 to at
or about 218 of FIG. 19 (SEQ ID NO:25) is believed to behave as a
mature sequence (without a presequence that is removable in
post-translational processing) in certain animal cells. The IL-1lp
sequence of amino acids from at or about 12 to at or about 218 of
FIG. 19 (SEQ ID NO:25) that results from initiation of translation
at the start codon occurring at nucleotide positions 106-108 is
also believed to behave as mature sequence in certain animal cells.
It is further believed that other animal cells recognize and remove
in post-translational processing one or more signal peptide(s)
contained in the sequence of amino acid positions 1 to 45 in the
IL-1lp polypeptide of amino acid positions 1 to 218 of FIG. 19 (SEQ
ID NO:25) or contained in the sequence of amino acid positions 12
to 45 in the IL-1lp polypeptide of amino acid positions 12 to 218
of FIG. 19 (SEQ ID NO:25).
[0188] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-18R binding activity of a
native sequence hIL-1Ra1, or the IL-1R binding activity of a native
sequence hIL-1Ra3 or mIL-1Ra3, and which IL-1lp polypeptide has at
least at or about 80% positives, or at least at or about 85%
positives, or at least at or about 90% positives, or at least at or
about 95% positives to an amino acid sequence selected from the
group consisting of: (1) amino acid residues from at or about 37 to
at or about 203, inclusive of FIG. 2 (SEQ ID NO:5), (2) amino acid
residues from at or about 15 to at or about 193, inclusive of FIG.
3 (SEQ ID NO:7), (3) amino acid residues from at or about 34 to at
or about 155, inclusive of FIG. 7 (SEQ ID NO:13), (4) amino acid
residues from at or about 34 to at or about 155, inclusive of FIG.
9 (SEQ ID NO:16), (5) amino acid residues from at or about 26 to at
or about 207, inclusive of FIG. 15 (SEQ ID NO:19), and (6) amino
acid residues from at or about 46 to at or about 218, inclusive of
FIG. 19 (SEQ ID NO:25), or (b) the complement of the DNA of
(a).
[0189] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-18R binding activity of a
native sequence hIL-1Ra1, or the IL-1R binding activity of a native
sequence hIL-1Ra3 or mIL-1Ra3, and which IL-1lp polypeptide has at
least at or about 80% positives, or at least at or about 85%
positives, or at least at or about 90% positives, or at least at or
about 95% positives to an amino acid sequence selected from the
group consisting of: (1) amino acid residues from at or about 95 to
at or about 134, inclusive of FIG. 7 (SEQ ID NO:13), and (2) amino
acid residues from at or about 95 to at or about 134, inclusive of
FIG. 9 (SEQ ID NO:16), or (b) the complement of the DNA of (a).
[0190] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-18R binding activity of a
native sequence hIL-Ra1, or the IL-1R binding activity of a native
sequence hIL-1Ra3 or mIL-1Ra3, and which IL-1lp polypeptide has at
least at or about 80% positives, or at least at or about 85%
positives, or at least at or about 90% positives, or at least at or
about 95% positives to the amino acid sequence of amino acid
residues from at or about 80 to at or about 155, inclusive of FIG.
7 (SEQ ID NO:13), or (b) the complement of the DNA of (a).
[0191] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-18R binding activity of a
native sequence hIL-1Ra1, or the IL-1R binding activity of a native
sequence hIL-1Ra3 or mIL-1Ra3, and which IL-1lp polypeptide has at
least at or about 80% positives, or at least at or about 85%
positives, or at least at or about 90% positives, or at least at or
about 95% positives to an amino acid sequence selected from the
group consisting of: (1) amino acid residues from at or about 2 to
at or about 155, inclusive of FIG. 7 (SEQ ID NO:13), and (2) amino
acid residues from at or about 2 to at or about 155, inclusive of
FIG. 9 (SEQ ID NO:16), or (b) the complement of the DNA of (a).
[0192] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide having at least at or about 80% positives, or at
least at or about 85% positives, or at least at or about 90%
positives, or at least at or about 95% positives to an amino acid
sequence selected from the group consisting of: (1) amino acid
residues from at or about 95 to at or about 134, inclusive of FIG.
7 (SEQ ID NO:13), and (2) amino acid residues from at or about 95
to at or about 134, inclusive of FIG. 9 (SEQ ID NO:16), or (b) the
complement of the DNA of (a).
[0193] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide having at least at or about 80% positives, or at
least at or about 85% positives, or at least at or about 90%
positives, or at least at or about 95% positives to the amino acid
sequence of amino acid residues from at or about 80 to at or about
155, inclusive of FIG. 7 (SEQ ID NO:13), or (b) the complement of
the DNA of (a).
[0194] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide having at least at or about 80% positives, or at
least at or about 85% positives, or at least at or about 90%
positives, or at least at or about 95% positives to an amino acid
sequence selected from the group consisting of: (1) amino acid
residues from at or about 2 to at or about 155, inclusive of FIG. 7
(SEQ ID NO:13), and (2) amino acid residues from at or about 2 to
at or about 155, inclusive of FIG. 9 (SEQ ID NO:16), or (b) the
complement of the DNA of (a).
[0195] In a still further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding an
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-18R binding activity of a
native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and which IL-1lp
polypeptide has at least at or about 80% positives, or at least at
or about 85% positives, or at least at or about 90% positives, or
at least at or about 95% positives to an amino acid sequence
selected from the group consisting of: (1) amino acid residues from
at or about 1 to at or about 207, inclusive of FIG. 15 (SEQ ID
NO:19), and (2) amino acid residues from at or about 1 to at or
about 218, inclusive of FIG. 19 (SEQ ID NO:25), or (b) the
complement of the DNA of (a).
[0196] In another embodiment, the invention provides a vector
comprising DNA encoding IL-1lp or its variants. The vector may
comprise any of the isolated nucleic acid molecules hereinabove
defined.
[0197] A host cell comprising such a vector is also provided. By
way of example, the host cells may be CHO cells, E. coli, or yeast.
A process for producing IL-1lp polypeptides is further provided and
comprises culturing host cells under conditions suitable for
expression of IL-1lp and recovering IL-1lp from the cell
culture.
[0198] In another embodiment, the invention provides isolated
IL-1lp polypeptide encoded by any of the isolated nucleic acid
sequences hereinabove defined.
[0199] In another aspect, the invention provides isolated native
sequence IL-1lp polypeptide, which in one embodiment, comprises an
amino acid sequence selected from the group consisting of: (1) the
amino acid sequence of residues from at or about 37 to at or about
203, inclusive of FIG. 2 (SEQ ID NO:5), (2) the amino acid sequence
of residues from at or about 15 to at or about 193, inclusive of
FIG. 3 (SEQ ID NO:7), (3) the amino acid sequence of residues from
at or about 34 to at or about 155, inclusive of FIG. 7 (SEQ ID
NO:13), and (4) the amino acid sequence of residues from at or
about 34 to at or about 155, inclusive of FIG. 9 (SEQ ID
NO:16).
[0200] In another aspect, the invention provides isolated native
sequence IL-1lp polypeptide, which in one embodiment, comprises an
amino acid sequence selected from the group consisting of: (1) the
amino acid sequence of residues from at or about 1 to at or about
207, inclusive of FIG. 15 (SEQ ID NO:19), (2) the amino acid
sequence of residues from at or about 26 to at or about 207,
inclusive of FIG. 15 (SEQ ID NO:19), (3) the amino acid sequence of
residues from at or about 1 to at or about 167, inclusive of FIG.
16 (SEQ ID NO:21), (4) the amino acid sequence of residues from at
or about 26 to at or about 167, inclusive of FIG. 16 (SEQ ID
NO:21), (5) the amino acid sequence of residues from at or about 1
to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25), (6) the
amino acid sequence of residues from at or about 12 to at or about
218, inclusive of FIG. 19 (SEQ ID NO:25), (7) the amino acid
sequence of residues from at or about 37 to at or about 218,
inclusive of FIG. 19 (SEQ ID NO:25), and (8) the amino acid
sequence of residues from at or about 46 to at or about 218,
inclusive of FIG. 19 (SEQ ID NO:25).
[0201] In another aspect, the invention concerns an isolated IL-1lp
polypeptide that retains at least one biologic activity of a native
sequence IL-1lp, such as the IL-1R binding activity of a native
sequence hIL-1Ra3 or mIL-1Ra3, or the IL18R binding activity of a
native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and that
consists of an amino acid sequence having at least at or about 80%
sequence identity, or at least at or about 85% sequence identity,
or at least at or about 90% sequence identity, or at least at or
about 95% sequence identity to the sequence of amino acid residues
from at or about 37 to at or about 203, inclusive of FIG. 2 (SEQ ID
NO:5), the sequence of amino acid residues from at or about 15 to
at or about 193, inclusive of FIG. 3 (SEQ ID NO:7), the sequence of
amino acid residues from at or about 34 to at or about 155,
inclusive of FIG. 7 (SEQ ID NO:13), the sequence of amino acid
residues from at or about 34 to at or about 155, inclusive of FIG.
9 (SEQ ID NO:16), the sequence of amino acid residues from at or
about 26 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19),
or the sequence of amino acid residues from at or about 46 to at or
about 218, inclusive of FIG. 19 (SEQ ID NO:25).
[0202] In another aspect, the invention concerns an isolated IL-1lp
polypeptide that retains at least one biologic activity of a native
sequence IL-1lp, such as the IL-18R binding activity of a native
sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and that consists of an
amino acid sequence having at least at or about 80% sequence
identity, or at least at or about 85% sequence identity, or at
least at or about 90% sequence identity, or at least at or about
95% sequence identity to the sequence of amino acid residues from
at or about 1 to at or about 207, inclusive of FIG. 15 (SEQ ID
NO:19), or the sequence of amino acid residues from at or about 1
to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25).
[0203] In another aspect, the invention provides an isolated IL-1lp
selected from the group consisting of: (1) a polypeptide, such as
an hIL-1Ra1 polypeptide, consisting of an amino acid sequence
having a sequence identity of at least at or about 80%, or at least
at or about 85%, or at least at or about 90%, or at least at or
about 95%, to the sequence of amino acid residues from at or about
37 to at or about 63, inclusive of FIG. 2 (SEQ ID NO:5); (2) a
polypeptide, such as an hIL-1Ra1 polypeptide, consisting of an
amino acid sequence having a sequence identity of at least at or
about 80%, or at least at or about 85%, or at least at or about
90%, or at least at or about 95%, to the sequence of amino acid
residues from at or about 15 to at or about 53, inclusive of FIG. 3
(SEQ ID NO:7); (3) a polypeptide, such as an hIL-1Ra2 polypeptide,
comprising the amino acid sequence of amino acid residues from at
or about 1 to at or about 134, inclusive of FIG. 5 (SEQ ID NO:10);
(4) a polypeptide comprising the amino acid sequence of amino acid
residues from at or about 10 to at or about 134, inclusive of FIG.
5 (SEQ ID NO:10); (5) a polypeptide, such as an hIL-1Ra2
polypeptide, consisting of the amino acid sequence of amino acid
residues from at or about 27 to at or about 134, inclusive of FIG.
5 (SEQ ID NO:10); (6) a polypeptide, such as an hIL-1Ra2 fusion
variant polypeptide, consisting of a native amino acid sequence of
hIL-1Ra2 consisting of amino acid residues from at or about 27 to
at or about 134, inclusive of FIG. 5 (SEQ ID NO:10) fused at its
N-terminus or C-terminus to a heterologous amino acid or amino acid
sequence; (7) a polypeptide, such as an hIL-1Ra3 polypeptide,
consisting of an amino acid sequence having a sequence identity of
at least at or about 80%, or at least at or about 85%, or at least
at or about 90%, or at least at or about 95%, to the sequence of
amino acid residues from at or about 95 to at or about 134,
inclusive of FIG. 7 (SEQ ID NO:13); and (8) a polypeptide, such as
a mIL-1Ra3 polypeptide, consisting of an amino acid sequence having
a sequence identity of at least at or about 80%, or at least at or
about 85%, or at least at or about 90%, or at least at or about
95%, to the sequence of amino acid residues from at or about 95 to
at or about 134, inclusive of FIG. 9 (SEQ ID NO:16).
[0204] In another aspect, the invention provides an isolated IL-1lp
selected from the group consisting of: (1) a polypeptide, such as
an hIL-1Ra1L polypeptide, consisting of an amino acid sequence
having a sequence identity of at least at or about 80%, or at least
at or about 85%, or at least at or about 90%, or at least at or
about 95%, to the sequence of amino acid residues from at or about
27 to at or about 44, inclusive of FIG. 15 (SEQ ID NO:19); (2) a
polypeptide, such as an hIL-1Ra1S fusion variant polypeptide,
consisting of a native amino acid sequence of hIL-1Ra1S consisting
of amino acid residues from at or about 39 to at or about 167,
inclusive of FIG. 16 (SEQ ID NO:21) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; (3)
a polypeptide, such as an hIL-1Ra1S fusion variant polypeptide,
consisting of a native amino acid sequence of h[L-1Ra1S consisting
of amino acid residues from at or about 47 to at or about 167,
inclusive of FIG. 16 (SEQ ID NO:21) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; (4)
a polypeptide, such as an hIL-1Ra1S polypeptide, consisting of
amino acid residues from at or about 39 to at or about 167,
inclusive of FIG. 16 (SEQ ID NO:21); (5) a polypeptide, such as an
hIL-1Ra1S polypeptide, consisting of amino acid residues from at or
about 47 to at or about 167, inclusive of FIG. 16 (SEQ ID NO:21);
and (6) a polypeptide, such as an hIL-1Ra1V polypeptide, consisting
of an amino acid sequence having a sequence identity of at least at
or about 80%, or at least at or about 85%, or at least at or about
90%, or at least at or about 95%, to the sequence of amino acid
residues from at or about 37 to at or about 55, inclusive of FIG.
19 (SEQ ID NO:25).
[0205] In another aspect, the invention concerns an isolated IL-1lp
polypeptide that consists of an amino acid sequence having at least
at or about 80% sequence identity, or at least at or about 85%
sequence identity, or at least at or about 90% sequence identity,
or at least at or about 95% sequence identity to the sequence of
amino acid residues from at or about 37 to at or about 203,
inclusive of FIG. 2 (SEQ ID NO:5), the sequence of amino acid
residues from at or about 15 to at or about 193, inclusive of FIG.
3 (SEQ ID NO:7), the sequence of amino acid residues from at or
about 34 to at or about 155, inclusive of FIG. 7 (SEQ ID NO:13),
the sequence of amino acid residues from at or about 34 to at or
about 155, inclusive of FIG. 9 (SEQ ID NO:16), the sequence of
amino acid residues from at or about 26 to at or about 207,
inclusive of FIG. 15 (SEQ ID NO:19), or the sequence of amino acid
residues from at or about 46 to at or about 218, inclusive of FIG.
19 (SEQ ID NO:25).
[0206] In another aspect, the invention concerns an isolated IL-1lp
polypeptide that consists of an amino acid sequence having at least
at or about 80% sequence identity, or at least at or about 85%
sequence identity, or at least at or about 90% sequence identity,
or at least at or about 95% sequence identity to the sequence of
amino acid residues from at or about 1 to at or about 207,
inclusive of FIG. 15 (SEQ ID NO:19), or the sequence of amino acid
residues from at or about 1 to at or about 218, inclusive of FIG.
19 (SEQ ID NO:25).
[0207] In another aspect, the invention provides an isolated
polypeptide, such as an hIL-1Ra3 polypeptide, consisting of an
amino acid sequence having a sequence identity of at least at or
about 80%, or at least at or about 85%, or at least at or about
90%, or at least at or about 95%, to the sequence of amino acid
residues from at or about 80 to at or about 155, inclusive of FIG.
7 (SEQ ID NO:13).
[0208] In a further aspect, the invention concerns an isolated
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-1R binding activity of a
native sequence hIL-1Ra3 or mIL-1Ra3, or the IL-18R binding
activity of a native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V,
and that consists of an amino acid sequence having at least at or
about 80% sequence identity, or at least at or about 85% sequence
identity, or at least at or about 90% sequence identity, or at
least at or about 95% sequence identity to the amino acid sequence
of an IL-1lp, such as a mature IL-1lp polypeptide, encoded by the
cDNA insert in the vector deposited as ATCC Deposit No. 203588
(DNA85066-2534), ATCC Deposit No. 203587 (DNA96786-2534), ATCC
Deposit No. 203589 (DNA96787-2534), ATCC Deposit No. 203590
(DNA92505-2534), ATCC Deposit No. 203846 (DNA 102043-2534), or ATCC
Deposit No. 203973 (DNA114876-2534). In a preferred embodiment, the
IL-1lp polypeptide comprises the amino acid sequence of an IL-1lp,
such as a mature IL-1lp polypeptide, encoded by the cDNA insert in
the vector deposited as ATCC Deposit No. 203588 (DNA85066-2534),
ATCC Deposit No. 203587 (DNA96786-2534), ATCC Deposit No. 203586
(DNA92929-2534), ATCC Deposit No. 203589 (DNA96787-2534), ATCC
Deposit No. 203590 (DNA92505-2534), ATCC Deposit No. 203846
(DNA102043-2534), ATCC Deposit No. 203973 (DNA1 14876-2534), or
ATCC Deposit No. 203855 (DNA102044-2534).
[0209] In a further aspect, the invention concerns an isolated
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-1R binding activity of a
native sequence hIL-1Ra3 or mIL-1Ra3, or the IL-18R binding
activity of a native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V,
and that consists of an amino acid sequence having at least at or
about 80% sequence identity, or at least at or about 85% sequence
identity, or at least at or about 90% sequence identity, or at
least at or about 95% sequence identity to the entire amino acid
sequence encoded by the longest open reading frame in the cDNA
insert of a vector selected from the group consisting of the
vectors deposited as ATCC Deposit No. 203588 (DNA85066-2534), ATCC
Deposit No. 203587 (DNA96786-2534), ATCC Deposit No. 203589
(DNA96787-2534), ATCC Deposit No. 203590 (DNA92505-2534), ATCC
Deposit No. 203846 (DNA102043-2534), and ATCC Deposit No. 203973
(DNA114876-2534). In a preferred embodiment, the IL-1lp polypeptide
comprises the entire amino acid sequence encoded by the longest
open reading frame in the cDNA insert of a vector selected from the
group consisting of the vectors deposited as ATCC Deposit No.
203588 (DNA85066-2534), ATCC Deposit No. 203587 (DNA96786-2534),
ATCC Deposit No. 203586 (DNA92929-2534), ATCC Deposit No. 203589
(DNA96787-2534), and ATCC Deposit No. 203590 (DNA92505-2534). In
another preferred embodiment, the IL-1lp polypeptide comprises the
entire amino acid sequence encoded by the longest open reading
frame in the cDNA insert of a vector selected from the group
consisting of the vectors deposited as ATCC Deposit No. 203846
(DNA102043-2534), ATCC Deposit No. 203855 (DNA102044-2534), and
ATCC Deposit No. 203973 (DNA114876-2534).
[0210] In another aspect, the invention concerns an isolated IL-1lp
polypeptide that retains at least one biologic activity of a native
sequence IL-1lp, such as the IL-1R binding activity of a native
sequence IL-1Ra3 or mIL-1Ra3, or the IL-18R binding activity of a
native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and that
consists of an amino acid sequence having at least at or about 80%
sequence identity, or at least at or about 85% sequence identity,
or at least at or about 90% sequence identity, or at least at or
about 95% sequence identity to an amino acid sequence selected from
the group consisting of: (1) the entire amino acid sequence encoded
by the longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203588, (2) the entire amino acid
sequence, or the entire amino acid sequence excluding the 36
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203587, (3) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203589, (4) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203590, (5) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, or the entire amino
acid sequence excluding the 34 N-terminal amino acid residues of
such sequence, encoded by the longest open reading frame in the
cDNA insert in the vector deposited as ATCC Deposit No. 203846, and
(6) the entire amino acid sequence, or the entire amino acid
sequence excluding the N-terminal amino acid residue of such
sequence, or the entire amino acid sequence excluding the 45
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203973.
[0211] In a further aspect, the invention concerns an isolated
IL-1lp polypeptide that consists of an amino acid sequence having
at least at or about 80% sequence identity, or at least at or about
85% sequence identity, or at least at or about 90% sequence
identity, or at least at or about 95% sequence identity to the
amino acid sequence of an IL-1lp, such as a mature IL-1lp
polypeptide, encoded by the cDNA insert in the vector deposited as
ATCC Deposit No. 203588 (DNA85066-2534), ATCC Deposit No. 203587
(DNA96786-2534), ATCC Deposit No. 203589 (DNA96787-2534), ATCC
Deposit No. 203590 (DNA92505-2534), ATCC Deposit No. 203846
(DNA102043-2534), or ATCC Deposit No. 203973 (DNA114876-2534).
[0212] In a further aspect, the invention concerns an isolated
IL-1lp polypeptide that consists of an amino acid sequence having
at least at or about 80% sequence identity, or at least at or about
85% sequence identity, or at least at or about 90% sequence
identity, or at least at or about 95% sequence identity to the
entire amino acid sequence encoded by the longest open reading
frame in the cDNA insert of a vector selected from the group
consisting of the vectors deposited as ATCC Deposit No. 203588
(DNA85066-2534), ATCC Deposit No. 203587 (DNA96786-2534), ATCC
Deposit No. 203589 (DNA96787-2534), ATCC Deposit No. 203590
(DNA92505-2534), ATCC Deposit No. 203846 (DNA 102043-2534), and
ATCC Deposit No. 203973 (DNA114876-2534).
[0213] In another aspect, the invention concerns an isolated IL-1lp
polypeptide that consists of an amino acid sequence having at least
at or about 80% sequence identity, or at least at or about 85%
sequence identity, or at least at or about 90% sequence identity,
or at least at or about 95% sequence identity to an amino acid
sequence selected from the group consisting of: (1) the entire
amino acid sequence encoded by the longest open reading frame in
the cDNA insert in the vector deposited as ATCC Deposit No. 203588,
(2) the entire amino acid sequence, or the entire amino acid
sequence excluding the 36 N-terminal amino acid residues of such
sequence, encoded by the longest open reading frame in the cDNA
insert in the vector deposited as ATCC Deposit No. 203587, (3) the
entire amino acid sequence, or the entire amino acid sequence
excluding the N-terminal amino acid residue of such sequence,
encoded by the longest open reading frame in the cDNA insert in the
vector deposited as ATCC Deposit No. 203589, (4) the entire amino
acid sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203590, (5) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, or the entire amino
acid sequence excluding the 34 N-terminal amino acid residues of
such sequence, encoded by the longest open reading frame in the
cDNA insert in the vector deposited as ATCC Deposit No. 203846, and
(6) the entire amino acid sequence, or the entire amino acid
sequence excluding the N-terminal amino acid residue of such
sequence, or the entire amino acid sequence excluding the 45
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203973.
[0214] In a preferred embodiment, the IL-1lp polypeptide comprises
an amino acid sequence selected from the group consisting of: (1)
the entire amino acid sequence encoded by the longest open reading
frame in the cDNA insert in the vector deposited as ATCC Deposit
No. 203588, (2) the entire amino acid sequence, or the entire amino
acid sequence excluding the 36 N-terminal amino acid residues of
such sequence, encoded by the longest open reading frame in the
cDNA insert in the vector deposited as ATCC Deposit No. 203587, (3)
the entire amino acid sequence, or the entire amino acid sequence
excluding the N-terminal amino acid residue of such sequence,
encoded by the longest open reading frame in the cDNA insert in the
vector deposited as ATCC Deposit No. 203589, (4) the entire amino
acid sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203590, (5) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, or the entire amino
acid sequence excluding the 34 N-terminal amino acid residues of
such sequence, encoded by the longest open reading frame in the
cDNA insert in the vector deposited as ATCC Deposit No. 203846, and
(6) the entire amino acid sequence, or the entire amino acid
sequence excluding the N-terminal amino acid residue of such
sequence, or the entire amino acid sequence excluding the 45
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203973.
[0215] In another aspect, the invention concerns an isolated IL-1lp
polypeptide that retains at least one biologic activity of a native
sequence IL-1lp, such as the IL-1R binding activity of a native
sequence hIL-1Ra3 or mIL-1Ra3, or the IL-18R binding activity of a
native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and that
consists of an amino acid sequence having at least at or about 80%
positives, or at least at or about 85% positives, or at least at or
about 90% positives, or at least at or about 95% positives to the
sequence of amino acid residues from at or about 37 to at or about
203, inclusive of FIG. 2 (SEQ ID NO:5), the sequence of amino acid
residues from at or about 15 to at or about 193, inclusive of FIG.
3 (SEQ ID NO:7), the sequence of amino acid residues from at or
about 34 to at or about 155, inclusive of FIG. 7 (SEQ ID NO:13),
the sequence of amino acid residues from at or about 34 to at or
about 155, inclusive of FIG. 9 (SEQ ID NO:16), or the sequence of
amino acid residues from at or about 46 to at or about 218,
inclusive of FIG. 19 (SEQ ID NO:25).
[0216] In another aspect, the invention concerns an isolated IL-1lp
polypeptide that retains at least one biologic activity of a native
sequence IL-1lp, such as the IL-18R binding activity of a native
sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and that consists of an
amino acid sequence having at least at or about 80% positives, or
at least at or about 85% positives, or at least at or about 90%
positives, or at least at or about 95% positives to the sequence of
amino acid residues from at or about 1 to at or about 207,
inclusive of FIG. 15 (SEQ ID NO:19), or the sequence of amino acid
residues from at or about 1 to at or about 218, inclusive of FIG.
19 (SEQ ID NO:25).
[0217] In another aspect, the invention provides an isolated IL-1lp
selected from the group consisting of: (1) a polypeptide, such as
an hIL-1Ra1 polypeptide, consisting of an amino acid sequence
having a % positives value of at least at or about 80%, or at least
at or about 85%, or at least at or about 90%, or at least at or
about 95%, to the sequence of amino acid residues from at or about
37 to at or about 63, inclusive of FIG. 2 (SEQ ID NO:5); (2) a
polypeptide, such as an hIL-1Ra1 polypeptide, consisting of an
amino acid sequence having a % positives value of at least at or
about 80%, or at least at or about 85%, or at least at or about
90%, or at least at or about 95%, to the sequence of amino acid
residues from at or about 15 to at or about 53, inclusive of FIG. 3
(SEQ ID NO:7); (3) a polypeptide, such as an hIL-1Ra2 polypeptide,
comprising the amino acid sequence of amino acid residues from at
or about 1 to at or about 134, inclusive of FIG. 5 (SEQ ID NO:10);
(4) a polypeptide comprising the amino acid sequence of amino acid
residues from at or about 10 to at or about 134, inclusive of FIG.
5 (SEQ ID NO:10); (5) a polypeptide, such as an hIL-1Ra2
polypeptide, consisting of the amino acid sequence of amino acid
residues from at or about 27 to at or about 134, inclusive of FIG.
5 (SEQ ID NO:10); (6) a polypeptide, such as an hIL-1Ra2 fusion
variant polypeptide, consisting of a native amino acid sequence of
hIL-1Ra2 consisting of amino acid residues from at or about 27 to
at or about 134, inclusive of FIG. 5 (SEQ ID NO:10) fused at its
N-terminus or C-terminus to a heterologous amino acid or amino acid
sequence; (7) a polypeptide, such as an hIL-1Ra3 polypeptide,
consisting of an amino acid sequence having a % positives value of
at least at or about 80%, or at least at or about 85%, or at least
at or about 90%, or at least at or about 95%, to the sequence of
amino acid residues from at or about 95 to at or about 134,
inclusive of FIG. 7 (SEQ ID NO:13); and (8) a polypeptide, such as
a mIL-1Ra3 polypeptide, consisting of an amino acid sequence having
a % positives value of at least at or about 80%, or at least at or
about 85%, or at least at or about 90%, or at least at or about
95%, to the sequence of amino acid residues from at or about 95 to
at or about 134, inclusive of FIG. 9 (SEQ ID NO:16).
[0218] In another aspect, the invention provides an isolated IL-1lp
selected from the group consisting of: (1) a polypeptide, such as
an hIL-1Ra1L polypeptide, consisting of an amino acid sequence
having a % positives value of at least at or about 80%, or at least
at or about 85%, or at least at or about 90%, or at least at or
about 95%, to the sequence of amino acid residues from at or about
27 to at or about 44, inclusive of FIG. 15 (SEQ ID NO:19); (2) a
polypeptide, such as an hIL-1Ra1S fusion variant polypeptide,
consisting of a native amino acid sequence of hIL-1Ra1S consisting
of amino acid residues from at or about 39 to at or about 167,
inclusive of FIG. 16 (SEQ ID NO:21) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; (3)
a polypeptide, such as an hIL-1Ra1S fusion variant polypeptide,
consisting of a native amino acid sequence of hIL-1Ra1S consisting
of amino acid residues from at or about 47 to at or about 167,
inclusive of FIG. 16 (SEQ ID NO:21) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; (4)
a polypeptide, such as an hIL-1Ra1S polypeptide, consisting of
amino acid residues from at or about 39 to at or about 167,
inclusive of FIG. 16 (SEQ ID NO:21); (5) a polypeptide, such as an
hIL-1Ra1S polypeptide, consisting of amino acid residues from at or
about 47 to at or about 167, inclusive of FIG. 16 (SEQ ID NO:21);
and (6) a polypeptide, such as an hIL-1Ra1V polypeptide, consisting
of an amino acid sequence having a % positives value of at least at
or about 80%, or at least at or about 85%, or at least at or about
90%, or at least at or about 95%, to the sequence of amino acid
residues from at or about 37 to at or about 55, inclusive of FIG.
19 (SEQ ID NO:25).
[0219] In another aspect, the invention concerns an isolated IL-1lp
polypeptide that consists of an amino acid sequence having at least
at or about 80% positives, or at least at or about 85% positives,
or at least at or about 90% positives, or at least at or about 95%
positives to the sequence of amino acid residues from at or about
37 to at or about 203, inclusive of FIG. 2 (SEQ ID NO:5), the
sequence of amino acid residues from at or about 15 to at or about
193, inclusive of FIG. 3 (SEQ ID NO:7), the sequence of amino acid
residues from at or about 34 to at or about 155, inclusive of FIG.
7 (SEQ ID NO:13), the sequence of amino acid residues from at or
about 34 to at or about 155, inclusive of FIG. 9 (SEQ ID NO:16), or
the sequence of amino acid residues from at or about 46 to at or
about 218, inclusive of FIG. 19 (SEQ ID NO:25).
[0220] In another aspect, the invention concerns an isolated IL-1lp
polypeptide that consists of an amino acid sequence having at least
at or about 80% positives, or at least at or about 85% positives,
or at least at or about 90% positives, or at least at or about 95%
positives to the sequence of amino acid residues from at or about 1
to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19), or the
sequence of amino acid residues from at or about 1 to at or about
218, inclusive of FIG. 19 (SEQ ID NO:25).
[0221] In a further aspect, the invention concerns an isolated
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-1R binding activity of a
native sequence hIL-1Ra3 or mIL-1Ra3, or the IL-18R binding
activity of a native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V,
and that consists of an amino acid sequence that has at least at or
about 80% positives, or at least at or about 85% positives, or at
least at or about 90% positives, or at least at or about 95%
positives to the amino acid sequence of an IL-1lp, such as a mature
IL-1lp polypeptide, encoded by the cDNA insert in the vector
deposited as ATCC Deposit No. 203588 (DNA85066-2534), ATCC Deposit
No. 203587 (DNA96786-2534), ATCC Deposit No. 203589
(DNA96787-2534), ATCC Deposit No. 203590 (DNA92505-2534), ATCC
Deposit No. 203846 (DNA102043-2534), or ATCC Deposit No. 203973
(DNA114876-2534).
[0222] In a further aspect, the invention concerns an isolated
IL-1lp polypeptide that retains at least one biologic activity of a
native sequence IL-1lp, such as the IL-1R binding activity of a
native sequence hIL-1Ra3 or mIL-1Ra3, or the IL-18R binding
activity of a native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V,
and that consists of an amino acid sequence that has at least at or
about 80% positives, or at least at or about 85% positives, or at
least at or about 90% positives, or at least at or about 95%
positives to the entire amino acid sequence encoded by the longest
open reading frame in the cDNA insert of a vector selected from the
group consisting of the vectors deposited as ATCC Deposit No.
203588 (DNA85066-2534), ATCC Deposit No. 203587 (DNA96786-2534),
ATCC Deposit No. 203589 (DNA96787-2534), ATCC Deposit No. 203590
(DNA92505-2534), ATCC Deposit No. 203846 (DNA102043-2534), and ATCC
Deposit No. 203973 (DNA114876-2534).
[0223] In another aspect, the invention concerns an isolated IL-1lp
polypeptide that retains at least one biologic activity of a native
sequence IL-1lp, such as the IL-1R binding activity of a native
sequence IL-1Ra3 or mIL-1Ra3, or the IL-18R binding activity of a
native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and that
consists of an amino acid sequence having at least at or about 80%
positives, or at least at or about 85% positives, or at least at or
about 90% positives, or at least at or about 95% positives to an
amino acid sequence selected from the group consisting of: (1) the
entire amino acid sequence encoded by the longest open reading
frame in the cDNA insert in the vector deposited as ATCC Deposit
No. 203588, (2) the entire amino acid sequence, or the entire amino
acid sequence excluding the 36 N-terminal amino acid residues of
such sequence, encoded by the longest open reading frame in the
cDNA insert in the vector deposited as ATCC Deposit No. 203587, (3)
the entire amino acid sequence, or the entire amino acid sequence
excluding the N-terminal amino acid residue of such sequence,
encoded by the longest open reading frame in the cDNA insert in the
vector deposited as ATCC Deposit No. 203589, (4) the entire amino
acid sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203590, (5) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, or the entire amino
acid sequence excluding the 34 N-terminal amino acid residues of
such sequence, encoded by the longest open reading frame in the
cDNA insert in the vector deposited as ATCC Deposit No. 203846, and
(6) the entire amino acid sequence, or the entire amino acid
sequence excluding the N-terminal amino acid residue of such
sequence, or the entire amino acid sequence excluding the 45
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203973.
[0224] In a further aspect, the invention concerns an isolated
IL-1lp polypeptide that consists of an amino acid sequence that has
at least at or about 80% positives, or at least at or about 85%
positives, or at least at or about 90% positives, or at least at or
about 95% positives to the amino acid sequence of an IL-1lp, such
as a mature IL-1lp polypeptide, encoded by the cDNA insert in the
vector deposited as ATCC Deposit No. 203588 (DNA85066-2534), ATCC
Deposit No. 203587 (DNA96786-2534), ATCC Deposit No. 203589
(DNA96787-2534), ATCC Deposit No. 203590 (DNA92505-2534), ATCC
Deposit No. 203846 (DNA102043-2534), or ATCC Deposit No. 203973
(DNA114876-2534).
[0225] In a further aspect, the invention concerns an isolated
IL-1lp polypeptide that consists of an amino acid sequence that has
at least at or about 80% positives, or at least at or about 85%
positives, or at least at or about 90% positives, or at least at or
about 95% positives to the entire amino acid sequence encoded by
the longest open reading frame in the cDNA insert of a vector
selected from the group consisting of the vectors deposited as ATCC
Deposit No. 203588 (DNA85066-2534), ATCC Deposit No. 203587
(DNA96786-2534), ATCC Deposit No. 203589 (DNA96787-2534), ATCC
Deposit No. 203590 (DNA92505-2534), ATCC Deposit No. 203846
(DNA102043-2534), and ATCC Deposit No. 203973 (DNA114876-2534).
[0226] In another aspect, the invention concerns an isolated IL-1lp
polypeptide that consists of an amino acid sequence having at least
at or about 80% positives, or at least at or about 85% positives,
or at least at or about 90% positives, or at least at or about 95%
positives to an amino acid sequence selected from the group
consisting of: (1) the entire amino acid sequence encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203588, (2) the entire amino acid
sequence, or the entire amino acid sequence excluding the 36
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203587, (3) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203589, (4) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203590, (5) the entire amino acid
sequence, or the entire amino acid sequence excluding the
N-terminal amino acid residue of such sequence, or the entire amino
acid sequence excluding the 34 N-terminal amino acid residues of
such sequence, encoded by the longest open reading frame in the
cDNA insert in the vector deposited as ATCC Deposit No. 203846, and
(6) the entire amino acid sequence, or the entire amino acid
sequence excluding the N-terminal amino acid residue of such
sequence, or the entire amino acid sequence excluding the 45
N-terminal amino acid residues of such sequence, encoded by the
longest open reading frame in the cDNA insert in the vector
deposited as ATCC Deposit No. 203973.
[0227] In another aspect, the invention provides an isolated
polypeptide, such as an hIL-1Ra3 polypeptide, consisting of an
amino acid sequence having a % positives value of at least at or
about 80%, or at least at or about 85%, or at least at or about
90%, or at least at or about 95%, to the sequence of amino acid
residues from at or about 80 to at or about 155, inclusive of FIG.
7 (SEQ ID NO:13).
[0228] In yet another aspect, the invention concerns an isolated
IL-1lp polypeptide comprising an amino acid sequence selected from
the group consisting of: (1) amino acid residues from at or about
37 to at or about 63, inclusive of FIG. 2 (SEQ ID NO:5); (2) amino
acid residues from at or about 15 to at or about 53, inclusive of
FIG. 3 (SEQ ID NO:7); (3) amino acid residues from at or about 80
to at or about 155, inclusive of FIG. 7 (SEQ ID NO:13); and (4)
amino acid residues from at or about 95 to at or about 155,
inclusive of FIG. 9 (SEQ ID NO:16), or a fragment of such IL-1lp
polypeptide that coincides with a stretch of at least about 10
contiguous amino acids in such amino acid sequence, wherein the
IL-1lp polypeptide or fragment thereof is sufficient to provide a
binding site for an anti-IL-1lp antibody. Preferably, the IL-1lp
fragment retains at least one biologic activity of a native
sequence IL-1lp polypeptide.
[0229] In yet another aspect, the invention concerns an isolated
IL-1lp polypeptide comprising an amino acid sequence selected from
the group consisting of: (1) amino acid residues from at or about
26 to at or about 44, inclusive of FIG. 15 (SEQ ID NO:19), (2)
amino acid residues from at or about 26 to at or about 78,
inclusive of FIG. 19 (SEQ ID NO:25), and (3) amino acid residues
from at or about 46 to at or about 89, inclusive of FIG. 19 (SEQ ID
NO:25), or a fragment of such IL-1lp polypeptide that coincides
with a stretch of at least about 10 contiguous amino acids in such
amino acid sequence, wherein the IL-1lp polypeptide or fragment
thereof is sufficient to provide a binding site for an anti-IL-1lp
antibody. Preferably, the IL-1lp fragment retains at least one
biologic activity of a native sequence IL-1lp polypeptide.
[0230] In a further aspect, the invention provides an isolated
IL-1lp polypeptide selected from the group consisting of: (1) a
polypeptide, such as an hIL-1Ra1 polypeptide, comprising amino acid
residues from at or about 37 to at or about 63, inclusive of FIG. 2
(SEQ ID NO:5); (2) a polypeptide, such as an hIL-1Ra1 polypeptide,
comprising amino acid residues from at or about 15 to at or about
53, inclusive of FIG. 3 (SEQ ID NO:7); (3) a polypeptide, such as
an hIL-1Ra3 polypeptide, comprising amino acid residues from at or
about 95 to at or about 134, inclusive of FIG. 7 (SEQ ID NO:13);
and (4) a polypeptide, such as a mIL-1Ra3 polypeptide, comprising
amino acid residues from at or about 95 to at or about 134,
inclusive of FIG. 9 (SEQ ID NO:16).
[0231] In a further aspect, the invention provides an isolated
IL-1lp polypeptide selected from the group consisting of: (1) a
polypeptide, such as an hIL-1Ra1 polypeptide, comprising amino acid
residues from at or about 37 to at or about 203, inclusive of FIG.
2 (SEQ ID NO:5); (2) a polypeptide, such as an hIL-1Ra1
polypeptide, comprising amino acid residues from at or about 15 to
at or about 193, inclusive of FIG. 3 (SEQ ID NO:7); (3) a
polypeptide, such as an hIL-1Ra3 polypeptide, comprising amino acid
residues from at or about 34 to at or about 155, inclusive of FIG.
7 (SEQ ID NO:13); and (3) a polypeptide, such as a mIL-1Ra3
polypeptide, comprising amino acid residues from at or about 34 to
at or about 155, inclusive of FIG. 9 (SEQ ID NO:16).
[0232] In a further aspect, the invention provides an isolated
polypeptide, such as an hIL-1Ra3 polypeptide, comprising amino acid
residues from at or about 80 to at or about 155, inclusive of FIG.
7 (SEQ ID NO:13).
[0233] In a further aspect, the invention provides an isolated
IL-1lp polypeptide selected from the group consisting of: (1) a
polypeptide, such as an hIL-1Ra1L polypeptide, comprising amino
acid residues from at or about 26 to at or about 44, inclusive of
FIG. 15 (SEQ ID NO:19); (2) a polypeptide, such as an hIL-1Ra1L
polypeptide, comprising amino acid residues from at or about 1 to
at or about 44, inclusive of FIG. 15 (SEQ ID NO:19); (3) a
polypeptide, such as an hIL-1Ra1S polypeptide, comprising amino
acid residues from at or about 1 to at or about 38, inclusive of
FIG. 16 (SEQ ID NO:21); (4) a polypeptide, such as an hIL-1Ra1V
polypeptide, comprising amino acid residues from at or about 37 to
at or about 55, inclusive of FIG. 19 (SEQ ID NO:25); (5) a
polypeptide, such as an hIL-1Ra1V polypeptide, comprising amino
acid residues from at or about 12 to at or about 55, inclusive of
FIG. 19 (SEQ ID NO:25); and (6) a polypeptide, such as an hIL-1Ra1V
polypeptide, comprising amino acid residues from at or about 1 to
at or about 55, inclusive of FIG. 19 (SEQ ID NO:25).
[0234] In a further aspect, the invention provides an isolated
IL-1lp polypeptide selected from the group consisting of: (1) a
polypeptide, such as an hIL-1Ra1L fusion variant polypeptide,
consisting of a native amino acid sequence of hIL-1Ra1L consisting
of amino acid residues from at or about 1 to at or about 207,
inclusive of FIG. 15 (SEQ ID NO:19) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; (2)
a polypeptide, such as an hIL-1Ra1L fusion variant polypeptide,
consisting of a native amino acid sequence of hIL-1Ra1L consisting
of amino acid residues from at or about 26 to at or about 207,
inclusive of FIG. 15 (SEQ ID NO:19) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; (3)
a polypeptide, such as an hIL-1Ra1L polypeptide, consisting of
amino acid residues from at or about 1 to at or about 207,
inclusive of FIG. 15 (SEQ ID NO:19); (4) a polypeptide, such as an
hIL-1Ra1L polypeptide, consisting of amino acid residues from at or
about 26 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19);
(5) a polypeptide, such as an hIL-1Ra1V fusion variant polypeptide,
consisting of a native amino acid sequence of hIL-1Ra1V consisting
of amino acid residues from at or about 1 to at or about 218,
inclusive of FIG. 19 (SEQ ID NO:25) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; (6)
a polypeptide, such as an hIL-1Ra1V fusion variant polypeptide,
consisting of a native amino acid sequence of hIL-1Ra1V consisting
of amino acid residues from at or about 12 to at or about 218,
inclusive of FIG. 19 (SEQ ID NO:25) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; (7)
a polypeptide, such as an hIL-1Ra1V fusion variant polypeptide,
consisting of a native amino acid sequence of hIL-1Ra1V consisting
of amino acid residues from at or about 37 to at or about 218,
inclusive of FIG. 19 (SEQ ID NO:25) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; (8)
a polypeptide, such as an hIL-1Ra1V fusion variant polypeptide,
consisting of a native amino acid sequence of hIL-1Ra1V consisting
of amino acid residues from at or about 46 to at or about 218,
inclusive of FIG. 19 (SEQ ID NO:25) fused at its N-terminus or
C-terminus to a heterologous amino acid or amino acid sequence; (9)
a polypeptide, such as an hIL-1Ra1V polypeptide, consisting of the
amino acid sequence of amino acid residues from at or about 1 to at
or about 218, inclusive of FIG. 19 (SEQ ID NO:25); (10) a
polypeptide, such as an hIL-1Ra1V polypeptide, consisting of the
amino acid sequence of amino acid residues from at or about 12 to
at or about 218, inclusive of FIG. 19 (SEQ ID NO:25); (11) a
polypeptide, such as an hIL-1Ra1V polypeptide, consisting of the
amino acid sequence of amino acid residues from at or about 37 to
at or about 218, inclusive of FIG. 19 (SEQ ID NO:25); and (12) a
polypeptide, such as an hIL-1Ra1V polypeptide, consisting of the
amino acid sequence of amino acid residues from at or about 46 to
at or about 218, inclusive of FIG. 19 (SEQ ID NO:25).
[0235] In a further aspect, the invention provides an isolated
IL-1lp polypeptide selected from the group consisting of: (1) a
polypeptide, such as an hIL-1Ra1L polypeptide, comprising amino
acid residues from at or about 1 to at or about 207, inclusive of
FIG. 15 (SEQ ID NO:19); (2) a polypeptide, such as an hIL-1Ra1L
polypeptide, comprising amino acid residues from at or about 26 to
at or about 207, inclusive of FIG. 15 (SEQ ID NO:19); (3) a
polypeptide, such as an hIL-1Ra1S polypeptide, comprising amino
acid residues from at or about 1 to at or about 167, inclusive of
FIG. 16 (SEQ ID NO:21); (4) a polypeptide, such as an hIL-1Ra1S
polypeptide, comprising amino acid residues from at or about 26 to
at or about 167, inclusive of FIG. 16 (SEQ ID NO:21); (5) a
polypeptide, such as an hIL-1Ra1V polypeptide, comprising amino
acid residues from at or about 1 to at or about 218, inclusive of
FIG. 19 (SEQ ID NO:25); and (6) a polypeptide, such as an hIL-1Ra1V
polypeptide, comprising amino acid residues from at or about 12 to
at or about 218, inclusive of FIG. 19 (SEQ ID NO:25).
[0236] In a further aspect, the invention provides an isolated
IL-1lp polypeptide selected from the group consisting of: (1) a
polypeptide, such as an hIL-1Ra1V polypeptide, comprising amino
acid residues from at or about 37 to at or about 218, inclusive of
FIG. 19 (SEQ ID NO:25); and (2) a polypeptide, such as an hIL-1Ra1V
polypeptide, comprising amino acid residues from at or about 46 to
at or about 218, inclusive of FIG. 19 (SEQ ID NO:25).
[0237] In a further aspect, the invention provides an isolated
IL-1lp polypeptide selected from the group consisting of: (1) a
polypeptide consisting of the amino acid sequence of amino acid
residues 10 to 134, inclusive of FIG. 5 (SEQ ID NO:10) fused at its
N-terminus or C-terminus to a heterologous amino acid or amino acid
sequence; (2) a polypeptide consisting of the amino acid sequence
of amino acid residues 10 to 134, inclusive of FIG. 5 (SEQ ID
NO:10); (3) a polypeptide, such as an hIL-1Ra3 polypeptide,
consisting of a native amino acid sequence of hIL-1Ra3 consisting
of amino acid residues 2 to 155, inclusive of FIG. 7 (SEQ ID NO:13)
fused at its N-terminus or C-terminus to a heterologous amino acid
or amino acid sequence; (4) a polypeptide, such as an hIL-1Ra3
polypeptide, consisting of the amino acid sequence of amino acid
residues from 2 to 155, inclusive of FIG. 7 (SEQ ID NO:13); (5) a
polypeptide, such as a mIL-1Ra3 polypeptide, consisting of a native
amino acid sequence of mIL-1Ra3 consisting of amino acid residues 2
to 155, inclusive of FIG. 9 (SEQ ID NO:16) fused at its N-terminus
or C-terminus to a heterologous amino acid or amino acid sequence;
(6) a polypeptide, such as a mIL-1Ra3 polypeptide, consisting of
the amino acid sequence of amino acid residues 2 to 155, inclusive
of FIG. 9 (SEQ ID NO:16).
[0238] In a further aspect, the invention provides an isolated
IL-1lp polypeptide selected from the group consisting of: (1) a
polypeptide comprising the amino acid sequence of amino acid
residues 10 to 134, inclusive of FIG. 5 (SEQ ID NO:10); (2) a
polypeptide, such as an hIL-1Ra3 polypeptide, comprising the amino
acid sequence of amino acid residues 2 to 155, inclusive of FIG. 7
(SEQ ID NO:13); and (3) a polypeptide, such as a mIL-1Ra3
polypeptide, comprising the amino acid sequence of amino acid
residues from 2 to 155, inclusive of FIG. 9 (SEQ ID NO:16).
[0239] In a still further aspect, the invention provides an
isolated IL-1lp polypeptide that is the same as a mature
polypeptide encoded by the cDNA insert of a vector selected from
the group consisting of the vectors deposited as ATCC Deposit Nos.
203588, 203587, 203586, 203589, and 203590.
[0240] In a still further aspect, the invention provides an
isolated IL-1lp polypeptide that is the same as a mature
polypeptide encoded by the cDNA insert of a vector selected from
the group consisting of the vectors deposited as ATCC Deposit Nos.
203846, 203855 and 203973.
[0241] In a still further aspect, the invention provides an
isolated polypeptide comprising the entire amino acid sequence
encoded by the longest open reading frame in the cDNA insert of a
vector selected from the group consisting of the vectors deposited
as ATCC Deposit Nos. 203588, 203586, 203589 and 203590.
[0242] In a still further aspect, the invention provides an
isolated polypeptide comprising the entire amino acid sequence
encoded by the longest open reading frame in the cDNA insert of a
vector selected from the group consisting of the vectors deposited
as ATCC Deposit Nos. 203846, 203855 and 203973.
[0243] In another aspect, the invention provides a polypeptide that
is produced by (i) hybridizing a test DNA molecule under stringent
conditions with (a) a DNA molecule encoding an amino acid sequence
selected from the group consisting of: (1) amino acid residues from
at or about 37 to at or about 203, inclusive of FIG. 2 (SEQ ID
NO:5), (2) amino acid residues from at or about 15 to at or about
193, inclusive of FIG. 3 (SEQ ID NO:7), (3) amino acid residues
from at or about 2 to at or about 155, inclusive of FIG. 7 (SEQ ID
NO:13), and (4) amino acid residues from at or about 2 to at or
about 155, inclusive of FIG. 9 (SEQ ID NO:16), or (b) the
complement of the DNA molecule of (a), and if the test DNA molecule
encodes an IL-1lp polypeptide that retains at least one biologic
activity of a native sequence IL-1lp, such as the IL-1R binding
activity of a native sequence hIL-1Ra3 or mIL-1Ra3, or the IL-18R
binding activity of a native sequence hIL-1Ra1, hIL-1Ra1L, or
hIL-1Ra1V, and if the test DNA molecule has at least at or about an
80% sequence identity, or at least at or about an 85% sequence
identity, or at least at or about a 90% sequence identity, or at
least at or about a 95% sequence identity to the DNA molecule of
(a) or (b), (ii) culturing a host cell comprising the test DNA
molecule under conditions suitable for expression of the IL-1lp
polypeptide, and (iii) recovering the IL-1lp polypeptide from the
cell culture.
[0244] In a still further aspect, the invention provides a
polypeptide that is produced by (i) hybridizing a test DNA molecule
under stringent conditions with (a) a DNA molecule encoding an
amino acid sequence selected from the group consisting of: (1)
amino acid residues from at or about 26 to at or about 207,
inclusive of FIG. 15 (SEQ ID NO:19), (2) amino acid residues from
at or about 1 to at or about 218, inclusive of FIG. 19 (SEQ ID
NO:25), (3) amino acid residues from at or about 12 to at or about
218, inclusive of FIG. 19 (SEQ ID NO:25), (4) amino acid residues
from at or about 37 to at or about 218, inclusive of FIG. 19 (SEQ
ID NO:25), and (5) amino acid residues from at or about 46 to at or
about 218, inclusive of FIG. 19 (SEQ ID NO:25), or (b) the
complement of the DNA molecule of (a), and if the test DNA molecule
encodes an IL-1lp polypeptide that retains at least one biologic
activity of a native sequence IL-1lp, such as the IL-18R binding
activity of a native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V,
and if the test DNA molecule has at least at or about an 80%
sequence identity, or at least at or about an 85% sequence
identity, or at least at or about a 90% sequence identity, or at
least at or about a 95% sequence identity to the DNA molecule of
(a) or (b), (ii) culturing a host cell comprising the test DNA
molecule under conditions suitable for expression of the IL-1lp
polypeptide, and (iii) recovering the IL-1lp polypeptide from the
cell culture.
[0245] A. Preparation of IL-1lp
[0246] The description below relates primarily to production of
IL-1lp by culturing cells transformed or transfected with a vector
containing IL-1lp nucleic acid. It is, of course, contemplated that
alternative methods, which are well known in the art, may be
employed to prepare IL-1lp. For instance, the IL-1lp sequence, or
portions thereof, may be produced by direct peptide synthesis using
solid-phase techniques [see, e.g., Stewart et al., Solid-Phase
Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969);
Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro
protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be accomplished, for instance,
using an Applied Biosystems Peptide Synthesizer (Foster City,
Calif.) using manufacturer's instructions. Various portions of the
IL-1lp may be chemically synthesized separately and combined using
chemical or enzymatic methods to produce the full-length
IL-1lp.
[0247] 1. Isolation of DNA Encoding IL-1lp
[0248] DNA encoding IL-1lp may be obtained from a cDNA library
prepared from tissue believed to possess the IL-1lp mRNA and to
express it at a detectable level. Accordingly, human IL-1lp DNA can
be conveniently obtained from a cDNA library prepared from human
tissue, such as described in the Examples. The IL-1lp-encoding gene
may also be obtained from a genomic library or by oligonucleotide
synthesis.
[0249] Libraries can be screened with probes (such as antibodies to
the IL-1lp or oligonucleotides of at least about 20-80 bases)
designed to identify the gene of interest or the protein encoded by
it. Screening the cDNA or genomic library with the selected probe
may be conducted using standard procedures, such as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual (New York:
Cold Spring Harbor Laboratory Press, 1989). An alternative means to
isolate the gene encoding IL-1lp is to use PCR methodology
[Sambrook et al., supra; Dieffenbach et al., PCR Primer: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
[0250] The Examples below describe techniques for screening a cDNA
library. The oligonucleotide sequences selected as probes should be
of sufficient length and sufficiently unambiguous that false
positives are minimized. The oligonucleotide is preferably labeled
such that it can be detected upon hybridization to DNA in the
library being screened. Methods of labeling are well known in the
art, and include the use of radiolabels like .sup.32P-labeled ATP,
biotinylation or enzyme labeling. Hybridization conditions,
including moderate stringency and high stringency, are provided in
Sambrook et al., supra.
[0251] Sequences identified in such library screening methods can
be compared and aligned to other known sequences deposited and
available in public databases such as GenBank or other private
sequence databases. Sequence identity (at either the amino acid or
nucleotide level) within defined regions of the molecule or across
the full-length sequence can be determined through sequence
alignment using computer software programs such as BLAST, BLAST2,
ALIGN-2, DNAstar, and INHERIT which employ various algorithms to
measure homology.
[0252] Nucleic acid having protein coding sequence may be obtained
by screening selected cDNA or genomic libraries using the deduced
amino acid sequence disclosed herein for the first time, and, if
necessary, using conventional primer extension procedures as
described in Sambrook et al., supra, to detect precursors and
processing intermediates of mRNA that may not have been
reverse-transcribed into cDNA.
[0253] 2. Selection and Transformation of Host Cells
[0254] Host cells are transfected or transformed with expression or
cloning vectors described herein for IL-1lp production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences. The culture conditions, such as
media, temperature, pH and the like, can be selected by the skilled
artisan without undue experimentation. In general, principles,
protocols, and practical techniques for maximizing the productivity
of cell cultures can be found in Mammalian Cell Biotechnology: A
Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook
et al., supra.
[0255] Methods of transfection are known to the ordinarily skilled
artisan, for example, CaPO.sub.4 and electroporation. Depending on
the host cell used, transformation is performed using standard
techniques appropriate to such cells. The calcium treatment
employing calcium chloride, as described in Sambrook et al., supra,
or electroporation is generally used for prokaryotes or other cells
that contain substantial cell-wall barriers. Infection with
Agrobacterium tumefaciens is used for transformation of certain
plant cells, as described by Shaw et al., Gene, 23:315 (1983) and
WO 89/05859 published 29 Jun. 1989. For mammalian cells without
such cell walls, the calcium phosphate precipitation method of
Graham and van der Eb, Virology, 52:456-457 (1978) can be employed.
General aspects of mammalian cell host system transformations have
been described in U.S. Pat. No. 4,399,216. Transformations into
yeast are typically carried out according to the method of Van
Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc.
Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for
introducing DNA into cells, such as by nuclear microinjection,
electroporation, bacterial protoplast fusion with intact cells, or
polycations, e.g., polybrene, polyornithine, may also be used. For
various techniques for transforming mammalian cells, see Keown et
al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,
Nature, 336:348-352 (1988).
[0256] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly available, such as E. coli K12 strain MM294
(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5 772 (ATCC 53,635).
[0257] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for IL-1lp-encoding vectors. Saccharomyces cerevisiae is a commonly
used lower eukaryotic host microorganism.
[0258] Suitable host cells for the expression of glycosylated
IL-1lp are derived from multicellular organisms. Examples of
invertebrate cells include insect cells such as Drosophila S2 and
Spodoptera Sf9, as well as plant cells. Examples of useful
mammalian host cell lines include Chinese hamster ovary (CHO) and
COS cells. More specific examples include monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary
cells/-DHFR(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,
77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,
23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human
liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562,
ATCC CCL51). The selection of the appropriate host cell is deemed
to be within the skill in the art.
[0259] 3. Selection and Use of a Replicable Vector
[0260] The nucleic acid (e.g., cDNA or genomic DNA) encoding IL-1lp
may be inserted into a replicable vector for cloning (amplification
of the DNA) or for expression. Various vectors are publicly
available. The vector may, for example, be in the form of a
plasmid, cosmid, viral particle, or phage. The appropriate nucleic
acid sequence may be inserted into the vector by a variety of
procedures. In general, DNA is inserted into an appropriate
restriction endonuclease site(s) using techniques known in the art.
Vector components generally include, but are not limited to, one or
more of a signal sequence, an origin of replication, one or more
marker genes, an enhancer element, a promoter, and a transcription
termination sequence. Construction of suitable vectors containing
one or more of these components employs standard ligation
techniques which are known to the skilled artisan.
[0261] The IL-1lp may be produced recombinantly not only directly,
but also as a fusion polypeptide with a heterologous polypeptide,
which may be a signal sequence or other polypeptide having a
specific cleavage site at the N-terminus of the mature protein or
polypeptide. In general, the signal sequence may be a component of
the vector, or it may be a part of the IL-1lp-encoding DNA that is
inserted into the vector. The signal sequence may be a prokaryotic
signal sequence selected, for example, from the group of the
alkaline phosphatase, penicillinase, 1 pp, or heat-stable
enterotoxin II leaders. For yeast secretion the signal sequence may
be, e.g., the yeast invertase leader, alpha factor leader
(including Saccharomyces and Kluyveromyces .alpha.-factor leaders,
the latter described in U.S. Pat. No. 5,010,182), or acid
phosphatase leader, the C. albicans glucoamylase leader (EP 362,179
published 4 Apr. 1990), or the signal described in WO 90/13646
published 15 Nov. 1990. In mammalian cell expression, mammalian
signal sequences may be used to direct secretion of the protein,
such as signal sequences from secreted polypeptides of the same or
related species, as well as viral secretory leaders.
[0262] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the
2.mu. plasmid origin is suitable for yeast, and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
[0263] Expression and cloning vectors will typically contain a
selection gene, also termed a selectable marker. Typical selection
genes encode proteins that (a) confer resistance to antibiotics or
other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline, (b) complement auxotrophic deficiencies, or (c)
supply critical nutrients not available from complex media, e.g.,
the gene encoding D-alanine racemase for Bacilli.
[0264] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the IL-1lp-encoding nucleic acid, such as DHFR or
thymidine kinase. An appropriate host cell when wild-type DHFR is
employed is the CHO cell line deficient in DHFR activity, prepared
and propagated as described by Urlaub et al., Proc. Natl. Acad.
Sci. USA, 77:4216 (1980). A suitable selection gene for use in
yeast is the trp1 gene present in the yeast plasmid YRp7
[Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene,
7:141 (1979); Tschemper et al., Gene, 10: 157 (1980)]. The trp1
gene provides a selection marker for a mutant strain of yeast
lacking the ability to grow in tryptophan, for example, ATCC No.
44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
[0265] Expression and cloning vectors usually contain a promoter
operably linked to the IL-1lp-encoding nucleic acid sequence to
direct mRNA synthesis. Promoters recognized by a variety of
potential host cells are well known. Promoters suitable for use
with prokaryotic hosts include the .beta.-lactamase and lactose
promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et
al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan
(trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980);
EP 36,776], and hybrid promoters such as the tac promoter [deBoer
et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for
use in bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA encoding IL-1lp.
[0266] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic
enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0267] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657.
[0268] IL-1lp transcription from vectors in mammalian host cells is
controlled, for example, by promoters obtained from the genomes of
viruses such as polyoma virus, fowlpox virus (UK 2,211,504
published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine
papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from
heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin promoter, and from heat-shock promoters, provided
such promoters are compatible with the host cell systems.
[0269] Transcription of a DNA encoding the IL-1lp by higher
eukaryotes may be increased by inserting an enhancer sequence into
the vector. Enhancers are cis-acting elements of DNA, usually about
from 10 to 300 bp, that act on a promoter to increase its
transcription. Many enhancer sequences are now known from mammalian
genes (globin, elastase, albumin, .alpha.-fetoprotein, and
insulin). Typically, however, one will use an enhancer from a
eukaryotic cell virus. Examples include the SV40 enhancer on the
late side of the replication origin (bp 100-270), the
cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus enhancers.
The enhancer may be spliced into the vector at a position 5' or 3'
to the IL-1lp coding sequence, but is preferably located at a site
5' from the promoter.
[0270] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding
IL-1lp.
[0271] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of IL-1lp in recombinant vertebrate
cell culture are described in Gething et al., Nature, 293:620-625
(1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP
117,058.
[0272] 4. Detecting Gene Amplification/Expression
[0273] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA [Thomas,
Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can recognize specific duplexes,
including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and
the assay may be 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.
[0274] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared
in any mammal. Conveniently, the antibodies may be prepared against
a native sequence IL-1lp polypeptide or against a synthetic peptide
based on the DNA sequences provided herein or against exogenous
sequence fused to IL-1lp DNA and encoding a specific antibody
epitope.
[0275] 5. Purification of Polypeptide
[0276] Forms of IL-1lp may be recovered from culture medium or from
host cell lysates. If membrane-bound, it can be released from the
membrane using a suitable detergent solution (e.g. Triton-X 100) or
by enzymatic cleavage. Cells employed in expression of IL-1lp can
be disrupted by various physical or chemical means, such as
freeze-thaw cycling, sonication, mechanical disruption, or cell
lysing agents. It may be desired to purify IL-1lp from recombinant
cell proteins or polypeptides. The following procedures are
exemplary of suitable purification procedures: by fractionation on
an ion-exchange column; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation-exchange resin such as
DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
gel filtration using, for example, Sephadex G-75; protein A
Sepharose columns to remove contaminants such as IgG; and metal
chelating columns to bind epitope-tagged forms of the IL-1lp.
Various methods of protein purification may be employed and such
methods are known in the art and described for example in
Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein
Purification: Principles and Practice, Springer-Verlag, New York
(1982). The purification step(s) selected will depend, for example,
on the nature of the production process used and the particular
IL-1lp produced.
[0277] B. Activity Assays for IL-1lp Variants
[0278] The biological activity or activities of a particular IL-1lp
variant polypeptide can be characterized using a variety of in
vitro assays known in the art. For example, the ability of an
hIL-1Ra3 variant polypeptide or a mIL-1Ra3 variant polypeptide to
bind IL-1R can be assayed using a radioimmunoprecipitation assay
wherein IL-1R extracellular domain (ECD) fused to the Fc region of
human immunoglobulin G (IL-1R ECD-Fc) (which can be prepared, e.g.,
as described in Examples 9 and 10 below) is incubated in solution
with radiolabeled hIL-1Ra3 variant polypeptide or mIL-1Ra3 variant
polypeptide to form labeled complexes, followed by
immunoprecipitation of the labeled complexes with goat anti-human
IgG Fc and quantitation of radioactivity in the precipitate. In
another example, an hIL-1Ra3 variant polypeptide-FLAG tag fusion
protein-encoding DNA and an IL-1R ECD-Fc encoding DNA can be
coexpressed in a host cell and secreted into the cell's culture
medium, followed by immunoprecipitation of culture supernatant with
protein G-sepharose and identification of bound hIL-1Ra3 variant
polypeptide-FLAG tag fusion protein by immunoblotting with
anti-FLAG monoclonal antibody, essentially as described in Example
9 below.
[0279] In another embodiment, the ability of an hIL-1Ra3 variant
polypeptide or a mIL-1Ra3 variant polypeptide to inhibit the
binding of IL-1 to IL-1R can be assayed using a competitive binding
assay. For example, a radioimmunoprecipitation assay can be
employed wherein IL-1R ECD-Fc is incubated in solution of
radiolabeled IL-1 with or without unlabeled hIL-1Ra3 variant
polypeptide or unlabeled mIL-1Ra3 variant polypeptide to form
labeled or unlabeled complexes, followed by immunoprecipitation of
complexes with anti-human IgG Fc and quantitation of radioactivity
in the precipitate. If the presence of unlabeled hIL-1Ra3 variant
polypeptide or unlabeled mIL-1Ra3 variant polypeptide in the
incubation solution diminishes the radioactivity measured in the
resulting immunoprecipitate, the hIL-1Ra3 variant polypeptide or
mIL-1Ra3 variant polypeptide in question qualifies as an inhibitor
of IL-1 binding to IL-1R. In yet another embodiment, IL-1R ECD-Fc
and an hIL-1Ra3 variant-FLAG tag fusion protein or mIL-1Ra3
variant-FLAG tag fusion protein are obtained by recombinant
expression in separate cell cultures (essentially as described in
Example 10 below), IL-1 and IL-1R ECD-Fc are admixed together with
or without the hIL-1Ra3 variant-FLAG tag fusion protein or mIL-1Ra3
variant-FLAG tag fusion protein and incubated in solution, the
incubation solution is immunoprecipitated with protein G-sepharose,
and the bound hIL-1Ra3 variant-FLAG tag fusion protein or mIL-1Ra3
variant-FLAG tag fusion protein is identified by immunoblotting
with anti-FLAG monoclonal antibody. If the presence of IL-1 in the
incubation solution diminishes the signal detected by anti-FLAG
immunoblotting, the hIL-1Ra3 variant polypeptide or mIL-1Ra3
variant polypeptide in question qualifies as an inhibitor of IL-1
binding to IL-1R.
[0280] Similarly, the biological activity or activities of a
particular hIL-1Ra1 variant polypeptide can be determined by using
a variety of in vitro assays known in the art. For example, the
ability of an hIL-1Ra1 variant polypeptide to bind IL-18R can be
assayed using a radioimmunoprecipitation assay wherein IL-18R
extracellular domain (ECD) fused to the Fc region of human
immunoglobulin G (IL-18R ECD-Fc) (which can be prepared, e.g., as
described in Examples 9 and 10 below) is incubated in solution with
radiolabeled hIL-1Ra1 variant polypeptide to form labeled complex,
followed by immunoprecipitation of the labeled complex with goat
anti-human IgG Fc and quantitation of radioactivity in the
precipitate. In another example, an hIL-1Ra1 variant
polypeptide-FLAG tag fusion protein-encoding DNA and an IL-18R
ECD-Fc encoding DNA can be coexpressed in a host cell and secreted
into the cell's culture medium, followed by immunoprecipitation of
culture supernatant with protein G-sepharose and identification of
bound hIL-1Ra1 variant polypeptide-FLAG tag fusion protein by
immunoblotting with anti-FLAG monoclonal antibody, essentially as
described in Example 9 below.
[0281] In another embodiment, the ability of an hIL-1Ra1 variant
polypeptide to inhibit the binding of IL-18 to IL-18R can be
assayed using a competitive binding assay. For example, a
radioimmunoprecipitation assay can be employed wherein IL-18R
ECD-Fc is incubated in solution of radiolabeled IL-18 with or
without unlabeled hIL-1Ra1 variant polypeptide to form labeled or
unlabeled complexes, followed by immunoprecipitation of complexes
with anti-human IgG Fc and quantitation of radioactivity in the
precipitate. If the presence of unlabeled hIL-1Ra1 variant
polypeptide in the incubation solution diminishes the radioactivity
measured in the resulting immunoprecipitate, the hIL-1Ra1 variant
polypeptide in question qualifies as an inhibitor of IL-18 binding
to IL-18R. In yet another embodiment, IL-18R ECD-Fc and an hIL-1Ra1
variant-FLAG tag fusion protein are obtained by recombinant
expression in separate cell cultures (essentially as described in
Example 10 below), IL-18 and IL-18R ECD-Fc are admixed together
with or without the hIL-1Ra1 variant-FLAG tag fusion protein and
incubated in solution, the incubation solution is
immunoprecipitated with protein G-sepharose, and the bound hIL-1Ra1
variant-FLAG tag fusion protein is identified by immunoblotting
with anti-FLAG monoclonal antibody. If the presence of IL-18 in the
incubation solution diminishes the signal detected by anti-FLAG
immunoblotting, the hIL-1Ra1 variant polypeptide in question
qualifies as an inhibitor of IL-18 binding to IL-18R.
[0282] C. Uses for IL-1lp
[0283] Nucleotide sequences (or their complement) encoding IL-1lp
have various applications in the art of molecular biology,
including uses as hybridization probes, in chromosome and gene
mapping and in the generation of anti-sense RNA and DNA. IL-1lp
nucleic acid will also be useful for the preparation of IL-1lp
polypeptides by the recombinant techniques described herein.
[0284] The full-length native sequence IL-1lp genes of FIG. 1 (SEQ
ID NO:1), FIG. 2 (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6), FIG. 5 (SEQ
ID NO:9), FIG. 7 (SEQ ID NO:12), FIG. 9 (SEQ ID NO:15), FIG. 15
(SEQ ID NO:18), and FIG. 16 (SEQ ID NO:20), and FIG. 19 (SEQ ID
NO:24), or portions thereof, may be used as hybridization probes
for a cDNA library to isolate the full-length IL-1lp gene or to
isolate still other genes (for instance, those encoding
naturally-occurring variants of IL-1lp or IL-1lp from other
species) which have a desired sequence identity to the IL-1lp
sequence disclosed in FIG. 1 (SEQ ID NO:1), FIG. 2 (SEQ ID NO:4),
FIG. 3 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:9), FIG. 7 (SEQ ID NO:12),
FIG. 9 (SEQ ID NO:15), FIG. 15 (SEQ ID NO:18), FIG. 16 (SEQ ID
NO:20), or FIG. 19 (SEQ ID NO:24). Optionally, the length of the
probes will be about 20 to about 50 bases. The hybridization probes
may be derived from the nucleotide sequence of FIG. 1 (SEQ ID
NO:1), FIG. 2 (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6), FIG. 5 (SEQ ID
NO:9), FIG. 7 (SEQ ID NO:12), FIG. 9 (SEQ ID NO:15), FIG. 15 (SEQ
ID NO:18), FIG. 16 (SEQ ID NO:20) or FIG. 19 (SEQ ID NO:24), or
from genomic sequences including promoters, enhancer elements and
introns of native sequence IL-1lp. By way of example, a screening
method will comprise isolating the coding region of the IL-1lp gene
using the known DNA sequence to synthesize a selected probe of
about 40 bases. Hybridization probes may be labeled by a variety of
labels, including radionucleotides such as .sup.32P or .sup.35S, or
enzymatic labels such as alkaline phosphatase coupled to the probe
via avidin/biotin coupling systems. Labeled probes having a
sequence complementary to that of the IL-1lp gene of the present
invention can be used to screen libraries of human cDNA, genomic
DNA or mRNA to determine which members of such libraries the probe
hybridizes to. Hybridization techniques are described in further
detail in the Examples below.
[0285] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
IL-1lp coding sequences.
[0286] Nucleotide sequences encoding an IL-1lp can also be used to
construct hybridization probes for mapping the gene which encodes
that IL-1lp and for the genetic analysis of individuals with
genetic disorders. The nucleotide sequences provided herein may be
mapped to a chromosome and specific regions of a chromosome using
known techniques, such as in situ hybridization, linkage analysis
against known chromosomal markers, and hybridization screening with
libraries.
[0287] When the coding sequences for IL-1lp encode a protein which
binds to another protein (example, where the IL-1lp binds to an
IL-1 receptor or IL-18 receptor), the IL-1lp can be used in assays
to identify the other proteins or molecules involved in the binding
interaction. By such methods, inhibitors of the receptor/ligand
binding interaction can be identified. Proteins involved in such
binding interactions can also be used to screen for peptide or
small molecule inhibitors or agonists of the binding interaction.
Screening assays can be designed to find lead compounds that mimic
the biological activity of a native IL-1lp or a receptor for
IL-1lp. Such screening assays will include assays amenable to
high-throughput screening of chemical libraries, making them
particularly suitable for identifying small molecule drug
candidates. Small molecules contemplated include synthetic organic
or inorganic compounds. The assays can be performed in a variety of
formats, including protein-protein binding assays, biochemical
screening assays, immunoassays and cell based assays, which are
well characterized in the art.
[0288] Nucleic acids which encode IL-1lp or its modified forms can
also be used to generate either transgenic animals or "knock out"
animals which, in turn, are useful in the development and screening
of therapeutically useful reagents. A transgenic animal (e.g., a
mouse or rat) is an animal having cells that contain a transgene,
which transgene was introduced into the animal or an ancestor of
the animal at a prenatal, e.g., an embryonic stage. A transgene is
a DNA which is integrated into the genome of a cell from which a
transgenic animal develops. In one embodiment, cDNA encoding IL-1lp
can be used to clone genomic DNA encoding IL-1lp in accordance with
established techniques and the genomic sequences used to generate
transgenic animals that contain cells which express DNA encoding
IL-1lp. Methods for generating transgenic animals, particularly
animals such as mice or rats, have become conventional in the art
and are described, for example, in U.S. Pat. Nos. 4,736,866 and
4,870,009. Typically, particular cells would be targeted for IL-1lp
transgene incorporation with tissue-specific enhancers. Transgenic
animals that include a copy of a transgene encoding IL-1lp
introduced into the germ line of the animal at an embryonic stage
can be used to examine the effect of increased expression of DNA
encoding IL-1lp. Such animals can be used as tester animals for
reagents thought to confer protection from, for example,
pathological conditions associated with its overexpression. In
accordance with this facet of the invention, an animal is treated
with the reagent and a reduced incidence of the pathological
condition, compared to untreated animals bearing the transgene,
would indicate a potential therapeutic intervention for the
pathological condition.
[0289] Alternatively, non-human homologues of IL-1lp can be used to
construct an IL-1lp "knock out" animal which has a defective or
altered gene encoding IL-1lp as a result of homologous
recombination between the endogenous gene encoding IL-1lp and
altered genomic DNA encoding IL-1lp introduced into an embryonic
cell of the animal. For example, cDNA encoding IL-1lp can be used
to clone genomic DNA encoding IL-1lp in accordance with established
techniques. A portion of the genomic DNA encoding IL-1lp can be
deleted or replaced with another gene, such as a gene encoding a
selectable marker which can be used to monitor integration.
Typically, several kilobases of unaltered flanking DNA (both at the
5' and 3' ends) are included in the vector [see e.g., Thomas and
Capecchi, Cell, 51:503 (1987) for a description of homologous
recombination vectors]. The vector is introduced into an embryonic
stem cell line (e.g., by electroporation) and cells in which the
introduced DNA has homologously recombined with the endogenous DNA
are selected [see e.g., Li et al., Cell, 69:915 (1992)]. The
selected cells are then injected into a blastocyst of an animal
(e.g., a mouse or rat) to form aggregation chimeras [see e.g.,
Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A
chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term
to create a "knock out" animal. Progeny harboring the homologously
recombined DNA in their germ cells can be identified by standard
techniques and used to breed animals in which all cells of the
animal contain the homologously recombined DNA. Knockout animals
can be characterized for instance, for their ability to defend
against certain pathological conditions and for their development
of pathological conditions due to absence of the IL-1lp
polypeptide.
[0290] Nucleic acid encoding the IL-1lp polypeptides may also be
used in gene therapy. In gene therapy applications, genes are
introduced into cells in order to achieve in vivo synthesis of a
therapeutically effective genetic product, for example for
replacement of a defective gene. "Gene therapy" includes both
conventional gene therapy where a lasting effect is achieved by a
single treatment, and the administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can
be used as therapeutic agents for blocking the expression of
certain genes in vivo. It has already been shown that short
antisense oligonucleotides can be imported into cells where they
act as inhibitors, despite their low intracellular concentrations
caused by their restricted uptake by the cell membrane. (Zamecnik
et al., Proc. Natl. Acad. Sci. USA 83, 4143-4146 [1986]). The
oligonucleotides can be modified to enhance their uptake, e.g. by
substituting their negatively charged phosphodiester groups by
uncharged groups.
[0291] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in Biotechnology 11, 205-210
[1993]). In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414
(1990). For review of gene marking and gene therapy protocols see
Anderson et al., Science 256, 808-813 (1992).
[0292] The IL-1lp polypeptides of the present invention can be
formulated according to known methods to prepare pharmaceutically
useful compositions, whereby the IL-1lp product hereof is combined
in admixture with a pharmaceutically acceptable carrier vehicle.
Therapeutic formulations are prepared for storage by mixing the
active ingredient having the desired degree of purity with optional
physiologically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)), in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients or stabilizers are
nontoxic to recipients at the dosages and concentrations employed,
and include buffers such as phosphate, citrate and other organic
acids; antioxidants including ascorbic acid; low molecular weight
(less than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone, amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as Tween, Pluronics or PEG.
[0293] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes, prior to or following lyophilization
and reconstitution.
[0294] Therapeutic compositions herein generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0295] The route of administration is in accord with known methods,
e.g. injection or infusion by intravenous, intraperitoneal,
intracerebral, intramuscular, intraocular, intraarterial or
intralesional routes, topical administration, or by sustained
release systems.
[0296] Dosages and desired drug concentrations of pharmaceutical
compositions of the present invention may vary depending on the
particular use envisioned. The determination of the appropriate
dosage or route of administration is well within the skill of an
ordinary physician. Animal experiments provide reliable guidance
for the determination of effective doses for human therapy.
Interspecies scaling of effective doses can be performed following
the principles laid down by Mordenti, J. and Chappell, W. "The use
of interspecies scaling in toxicokinetics" In Toxicokinetics and
New Drug Development, Yacobi et al., Eds., Pergamon Press, New York
1989, pp. 42-96.
[0297] An "effective amount" of the IL-1lp to be employed
therapeutically will depend, for example, upon the therapeutic
objectives, the route of administration, and the condition of the
patient. Accordingly, it will be necessary for the therapist to
titer the dosage and modify the route of administration as required
to obtain the optimal therapeutic effect. Typically, the clinician
will administer the IL-1lp until a dosage is reached that achieves
the desired effect. The progress of this therapy is easily
monitored by conventional assays.
[0298] In one embodiment, the invention provides a method for
treating an IL-1-mediated disorder comprising administering to a
mammal, such as human, in need of such treatment an effective
amount of an IL-1lp, such as a native sequence IL-1lp.
[0299] In another embodiment, the invention provides a method for
treating an IL-1-mediated disorder comprising administering to a
mammal, such as human, in need of such treatment an effective
amount of an IL-1lp selected from the group consisting of hIL-1Ra1,
hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and
mIL-1Ra3.
[0300] In another embodiment, the invention provides a method for
treating an IL-1-mediated disorder comprising administering to a
mammal, such as human, in need of such treatment an effective
amount of an hIL-1lp, such as a native sequence hIL-1lp, e.g.
native sequence hIL-1Ra3.
[0301] In another embodiment, the invention provides a method for
treating an IL-18-mediated disorder comprising administering to a
mammal, such as human, in need of such treatment an effective
amount of an IL-1lp, such as a native sequence IL-1lp.
[0302] In another embodiment, the invention provides a method for
treating an IL-18-mediated disorder comprising administering to a
mammal, such as human, in need of such treatment an effective
amount of an IL-1lp selected from the group consisting of hIL-1Ra1,
hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and
mIL-1Ra3.
[0303] In another embodiment, the invention provides a method for
treating an IL-18-mediated disorder comprising administering to a
mammal, such as human, in need of such treatment an effective
amount of an hIL-1lp, such as a native sequence hIL-1lp, e.g.
native sequence hIL-1Ra1.
[0304] In another embodiment, the invention provides a method for
treating an IL-18-mediated disorder comprising administering to a
mammal, such as human, in need of such treatment an effective
amount of an hIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an
effective amount of an hIL-1Ra1V, such as a native sequence
hIL-1Ra1V, or an effective amount of an hIL-1Ra1S, such as a native
sequence hIL-1Ra1S.
[0305] In one embodiment, the invention provides a method for
treating an inflammatory disorder comprising administering to a
mammal, such as human, in need of such treatment an effective
amount of an IL-1lp, such as a native sequence IL-1lp.
[0306] In another embodiment, the invention provides a method for
treating an inflammatory disorder comprising administering to a
mammal, such as human, in need of such treatment an effective
amount of an IL-1lp selected from the group consisting of hIL-1Ra1,
hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and
mIL-1Ra3.
[0307] In another embodiment, the invention provides a method for
treating an inflammatory disorder comprising administering to a
mammal, such as human, in need of such treatment an effective
amount of an hIL-1lp, such as a native sequence hIL-1lp, e.g.
native sequence hIL-1Ra1 or hIL-1Ra3.
[0308] In another embodiment, the invention provides a method for
treating an inflammatory disorder comprising administering to a
mammal, such as human, in need of such treatment an effective
amount of an hIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an
effective amount of an hIL-1Ra1V, such as a native sequence
hIL-1Ra1V, or an effective amount of an hIL-1Ra1S, such as a native
sequence hIL-1Ra1S.
[0309] In another embodiment, the invention provides a method for
treating asthma comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an IL-1lp,
such as a native sequence IL-1lp.
[0310] In another embodiment, the invention provides a method for
treating asthma comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an IL-1lp
selected from the group consisting of hIL-1Ra1, hIL-1Ra1L,
hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.
[0311] In another embodiment, the invention provides a method for
treating asthma comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an hIL-1lp,
such as a native sequence hIL-1lp, e.g. native sequence hIL-1Ra1 or
hIL-1Ra3.
[0312] In another embodiment, the invention provides a method for
treating asthma comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an
hIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an effective
amount of an hIL-1Ra1V, such as a native sequence hIL-1Ra1V, or an
effective amount of an hIL-1Ra1S, such as a native sequence
hIL-1Ra1S.
[0313] In another embodiment, the invention provides a method for
treating rheumatoid arthritis comprising administering to a mammal,
such as human, in need of such treatment an effective amount of an
IL-1lp, such as a native sequence IL-1lp.
[0314] In another embodiment, the invention provides a method for
treating rheumatoid arthritis comprising administering to a mammal,
such as human, in need of such treatment an effective amount of an
IL-1lp selected from the group consisting of hIL-1Ra1, hIL-1Ra1L,
hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.
[0315] In another embodiment, the invention provides a method for
treating rheumatoid arthritis comprising administering to a mammal,
such as human, in need of such treatment an effective amount of an
hIL-1lp, such as a native sequence hIL-1lp, e.g. native sequence
hIL-1Ra1 or hIL-1Ra3.
[0316] In another embodiment, the invention provides a method for
treating rheumatoid arthritis comprising administering to a mammal,
such as human, in need of such treatment an effective amount of an
hIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an effective
amount of an hIL-1Ra1V, such as a native sequence hIL-1Ra1V, or an
effective amount of an hIL-1Ra1S, such as a native sequence
hIL-1Ra1S.
[0317] In another embodiment, the invention provides a method for
treating osteoarthritis comprising administering to a mammal, such
as human, in need of such treatment an effective amount of an
IL-1lp, such as a native sequence IL-1lp.
[0318] In another embodiment, the invention provides a method for
treating osteoarthritis comprising administering to a mammal, such
as human, in need of such treatment an effective amount of an
IL-1lp selected from the group consisting of hIL-1Ra1, hIL-1Ra1L,
hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.
[0319] In another embodiment, the invention provides a method for
treating osteoarthritis comprising administering to a mammal, such
as human, in need of such treatment an effective amount of an
hIL-1lp, such as a native sequence hIL-1lp, e.g. native sequence
hIL-1Ra1 or hIL-1Ra3.
[0320] In another embodiment, the invention provides a method for
treating osteoarthritis comprising administering to a mammal, such
as human, in need of such treatment an effective amount of an
hIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an effective
amount of an hIL-1Ra1V, such as a native sequence hIL-1Ra1V, or an
effective amount of an hIL-1Ra1S, such as a native sequence
hIL-1Ra1S.
[0321] In another embodiment, the invention provides a method for
treating sepsis comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an IL-1lp,
such as a native sequence IL-1lp.
[0322] In another embodiment, the invention provides a method for
treating sepsis comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an IL-1lp
selected from the group consisting of hIL-1Ra1, hIL-1Ra1L,
hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.
[0323] In another embodiment, the invention provides a method for
treating sepsis comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an hIL-1lp,
such as a native sequence hIL-1lp, e.g. native sequence hIL-1Ra1 or
hIL-1Ra3.
[0324] In another embodiment, the invention provides a method for
treating sepsis comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an
hIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an effective
amount of an hIL-1Ra1V, such as a native sequence hIL-1Ra1V, or an
effective amount of an hIL-1Ra1S, such as a native sequence
hIL-1Ra1S.
[0325] In another embodiment, the invention provides a method for
treating acute lung injury comprising administering to a mammal,
such as human, in need of such treatment an effective amount of an
IL-1lp, such as a native sequence IL-1lp.
[0326] In another embodiment, the invention provides a method for
treating acute lung injury comprising administering to a mammal,
such as human, in need of such treatment an effective amount of an
IL-1lp selected from the group consisting of hIL-1Ra1, hIL-1Ra1L,
hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.
[0327] In another embodiment, the invention provides a method for
treating acute lung injury comprising administering to a mammal,
such as human, in need of such treatment an effective amount of an
hIL-1lp, such as a native sequence hIL-1lp, e.g. native sequence
hIL-1Ra1 or hIL-1Ra3.
[0328] In another embodiment, the invention provides a method for
treating acute lung injury comprising administering to a mammal,
such as human, in need of such treatment an effective amount of an
hIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an effective
amount of an hIL-1Ra1V, such as a native sequence hIL-1Ra1V, or an
effective amount of an hIL-1Ra1S, such as a native sequence
hIL-1Ra1S.
[0329] In another embodiment, the invention provides a method for
treating adult respiratory distress syndrome comprising
administering to a mammal, such as human, in need of such treatment
an effective amount of an IL-1lp, such as a native sequence
IL-1lp.
[0330] In another embodiment, the invention provides a method for
treating adult respiratory distress syndrome comprising
administering to a mammal, such as human, in need of such treatment
an effective amount of an IL-1lp selected from the group consisting
of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3,
and mIL-1Ra3.
[0331] In another embodiment, the invention provides a method for
treating adult respiratory distress syndrome comprising
administering to a mammal, such as human, in need of such treatment
an effective amount of an hIL-1lp, such as a native sequence
hIL-1lp, e.g. native sequence hIL-1Ra1 or hIL-1Ra3.
[0332] In another embodiment, the invention provides a method for
treating adult respiratory distress syndrome comprising
administering to a mammal, such as human, in need of such treatment
an effective amount of an hIL-1Ra1L, such as a native sequence
hIL-1Ra1L, or an effective amount of an hIL-1Ra1V, such as a native
sequence hIL-1Ra1V, or an effective amount of an hIL-1Ra1S, such as
a native sequence hIL-1Ra1S.
[0333] In another embodiment, the invention provides a method for
treating idiopathic pulmonary fibrosis comprising administering to
a mammal, such as human, in need of such treatment an effective
amount of an IL-1lp, such as a native sequence IL-1lp.
[0334] In another embodiment, the invention provides a method for
treating idiopathic pulmonary fibrosis comprising administering to
a mammal, such as human, in need of such treatment an effective
amount of an IL-1lp selected from the group consisting of hIL-1Ra1,
hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and
mIL-1Ra3.
[0335] In another embodiment, the invention provides a method for
treating idiopathic pulmonary fibrosis comprising administering to
a mammal, such as human, in need of such treatment an effective
amount of an hIL-1lp, such as a native sequence hIL-1lp, e.g.
native sequence hIL-1Ra1 or hIL-1Ra3.
[0336] In another embodiment, the invention provides a method for
treating idiopathic pulmonary fibrosis comprising administering to
a mammal, such as human, in need of such treatment an effective
amount of an hIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an
effective amount of an hIL-1Ra1V, such as a native sequence
hIL-1Ra1V, or an effective amount of an hIL-1Ra1S, such as a native
sequence hIL-1Ra1S.
[0337] In another embodiment, the invention provides a method for
treating an ischemic reperfusion disease, such as surgical tissue
reperfusion injury, stroke, myocardial ischemia, or acute
myocardial infarction, comprising administering to a mammal, such
as human, in need of such treatment an effective amount of an
IL-1lp, such as a native sequence IL-1lp.
[0338] In another embodiment, the invention provides a method for
treating an ischemic reperfusion disease, such as surgical tissue
reperfusion injury, stroke, myocardial ischemia, or acute
myocardial infarction, comprising administering to a mammal, such
as human, in need of such treatment an effective amount of an
IL-1lp selected from the group consisting of hIL-1Ra1, hIL-1Ra1L,
hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.
[0339] In another embodiment, the invention provides a method for
treating an ischemic reperfusion disease, such as surgical tissue
reperfusion injury, stroke, myocardial ischemia, or acute
myocardial infarction, comprising administering to a mammal, such
as human, in need of such treatment an effective amount of an
hIL-1lp, such as a native sequence hIL-1lp, e.g. native sequence
hIL-1Ra1 or hIL-1Ra3.
[0340] In another embodiment, the invention provides a method for
treating an ischemic reperfusion disease, such as surgical tissue
reperfusion injury, stroke, myocardial ischemia, or acute
myocardial infarction, comprising administering to a mammal, such
as human, in need of such treatment an effective amount of an
hIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an effective
amount of an hIL-1Ra1V, such as a native sequence hIL-1Ra1V, or an
effective amount of an hIL-1Ra1S, such as a native sequence
hIL-1Ra1S.
[0341] In another embodiment, the invention provides a method for
treating psoriasis comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an IL-1lp,
such as a native sequence IL-1lp.
[0342] In another embodiment, the invention provides a method for
treating psoriasis comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an IL-1lp
selected from the group consisting of hIL-1Ra1, hIL-1Ra1L,
hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.
[0343] In another embodiment, the invention provides a method for
treating psoriasis comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an hIL-1lp,
such as a native sequence hIL-1lp, e.g. native sequence hIL-1Ra1 or
hIL-1Ra3.
[0344] In another embodiment, the invention provides a method for
treating psoriasis comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an
hIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an effective
amount of an hIL-1Ra1V, such as a native sequence hIL-1Ra1V, or an
effective amount of an hIL-1Ra1S, such as a native sequence
hIL-1Ra1S.
[0345] In another embodiment, the invention provides a method for
treating graft-versus-host disease (GVHD) comprising administering
to a mammal, such as human, in need of such treatment an effective
amount of an IL-1lp, such as a native sequence IL-1lp.
[0346] In another embodiment, the invention provides a method for
treating graft-versus-host disease (GVHD) comprising administering
to a mammal, such as human, in need of such treatment an effective
amount of an IL-1lp selected from the group consisting of hIL-1Ra1,
hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and
mIL-1Ra3.
[0347] In another embodiment, the invention provides a method for
treating graft-versus-host disease (GVHD) comprising administering
to a mammal, such as human, in need of such treatment an effective
amount of an hIL-1lp, such as a native sequence hIL-1lp, e.g.
native sequence hIL-1Ra1 or hIL-1Ra3.
[0348] In another embodiment, the invention provides a method for
treating graft-versus-host disease (GVHD) comprising administering
to a mammal, such as human, in need of such treatment an effective
amount of an hIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an
effective amount of an hIL-1Ra1V, such as a native sequence
hIL-1Ra1V, or an effective amount of an hIL-1Ra1S, such as a native
sequence hIL-1Ra1S.
[0349] In another embodiment, the invention provides a method for
treating an inflammatory bowel disease such as ulcerative colitis,
comprising administering to a mammal, such as human, in need of
such treatment an effective amount of an IL-1lp, such as a native
sequence IL-1lp.
[0350] In another embodiment, the invention provides a method for
treating an inflammatory bowel disease such as ulcerative colitis,
comprising administering to a mammal, such as human, in need of
such treatment an effective amount of an IL-1lp selected from the
group consisting of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S,
hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.
[0351] In another embodiment, the invention provides a method for
treating an inflammatory bowel disease such as ulcerative colitis,
comprising administering to a mammal, such as human, in need of
such treatment an effective amount of an hIL-1lp, such as a native
sequence hIL-1lp, e.g. native sequence hIL-1Ra1 or hIL-1Ra3.
[0352] In another embodiment, the invention provides a method for
treating an inflammatory bowel disease such as ulcerative colitis,
comprising administering to a mammal, such as human, in need of
such treatment an effective amount of an hIL-1Ra1L, such as a
native sequence hIL-1Ra1L, or an effective amount of an hIL-1Ra1V,
such as a native sequence hIL-1Ra1V, or an effective amount of an
hIL-1Ra1S, such as a native sequence hIL-1Ra1S.
[0353] D. Anti-IL-1lp Antibodies
[0354] The present invention further provides anti-IL-1lp
antibodies. Exemplary antibodies include polyclonal, monoclonal,
humanized, bispecific, and heteroconjugate antibodies.
[0355] 1. Polyclonal Antibodies
[0356] The anti-IL-1lp antibodies may comprise polyclonal
antibodies. Methods of preparing polyclonal antibodies are known to
the skilled artisan. Polyclonal antibodies can be raised in a
mammal, for example, by one or more injections of an immunizing
agent and, if desired, an adjuvant. Typically, the immunizing agent
and/or adjuvant will be injected in the mammal by multiple
subcutaneous or intraperitoneal injections. The immunizing agent
may include the IL-1lp polypeptide or a fusion protein thereof. It
may be useful to conjugate the immunizing agent to a protein known
to be immunogenic in the mammal being immunized. Examples of such
immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin inhibitor. Examples of adjuvants which may be employed
include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The
immunization protocol may be selected by one skilled in the art
without undue experimentation.
[0357] 2. Monoclonal Antibodies
[0358] The anti-IL-1lp antibodies may, alternatively, be monoclonal
antibodies. Monoclonal antibodies may be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes may be immunized in
vitro.
[0359] The immunizing agent will typically include the IL-1lp
polypeptide or a fusion protein thereof. Generally, either
peripheral blood lymphocytes ("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell [Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-103]. Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridoma cells may be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0360] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Rockville, Md. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies [Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63].
[0361] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against IL-1lp. Preferably, the binding specificity of
monoclonal antibodies produced by the hybridoma cells is determined
by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunosorbant assay
(ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal.
Biochem., 107:220 (1980).
[0362] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods [Goding, supra]. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0363] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0364] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences [U.S.
Pat. No. 4,816,567; Morrison et al., supra] or by covalently
joining to the immunoglobulin coding sequence all or part of the
coding sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant
domains of an antibody of the invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody
of the invention to create a chimeric bivalent antibody.
[0365] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
[0366] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art.
[0367] 3. Human and Humanized Antibodies
[0368] The anti-IL-1lp antibodies of the invention may further
comprise humanized antibodies or human antibodies. Humanized forms
of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Humanized antibodies may also comprise residues
which are found neither in the recipient antibody nor in the
imported CDR or framework sequences. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature, 321: 522-525 (1986);
Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr.
Op. Struct. Biol., 2: 593-596 (1992)].
[0369] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers [Jones et al.,
Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327
(1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0370] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et
al., J. Mol. Biol., 222: 581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol., 147(1): 86-95 (1991)]. Similarly, human antibodies can be
made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10, 779-783 (1992);
Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368,
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[0371] 4. Bispecific Antibodies
[0372] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for the IL-1lp, the other one is for any other
antigen, and preferably for a cell-surface protein or receptor or
receptor subunit.
[0373] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities [Milstein and Cuello, Nature, 305:537-539
(1983)]. Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May
1993, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).
[0374] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0375] 5. Heteroconjugate Antibodies
[0376] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells [U.S.
Pat. No. 4,676,980], and for treatment of HIV infection [WO
91/00360; WO 92/200373; EP 03089]. It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0377] E. Uses for anti-IL-1lp Antibodies
[0378] The anti-IL-1lp antibodies of the invention have various
utilities. For example, anti-IL-1lp antibodies may be used in
diagnostic assays for IL-1lp, e.g., detecting its expression in
specific cells, tissues, or serum. Various diagnostic assay
techniques known in the art may be used, such as competitive
binding assays, direct or indirect sandwich assays and
immunoprecipitation assays conducted in either heterogeneous or
homogeneous phases [Zola, Monoclonal Antibodies: A Manual of
Techniques, CRC Press, Inc. (1987) pp. 147-158]. The antibodies
used in the diagnostic assays can be labeled with a detectable
moiety. The detectable moiety should be capable of producing,
either directly or indirectly, a detectable signal. For example,
the detectable moiety may be a radioisotope, such as .sup.3H,
.sup.14C, .sup.32P, .sup.35S, or .sup.125I, a fluorescent or
chemiluminescent compound, such as fluorescein isothiocyanate,
rhodamine, or luciferin, or an enzyme, such as alkaline
phosphatase, beta-galactosidase or horseradish peroxidase. Any
method known in the art for conjugating the antibody to the
detectable moiety may be employed, including those methods
described by Hunter et al., Nature, 144: 945 (1962); David et al.,
Biochemistry, 13: 1014 (1974); Pain et al., J. Immunol. Meth., 40:
219 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407
(1982).
[0379] Anti-IL-1lp antibodies also are useful for the affinity
purification of IL-1lp from recombinant cell culture or natural
sources. In this process, the antibodies against IL-1lp are
immobilized on a suitable support, such a Sephadex resin or filter
paper, using methods well known in the art. The immobilized
antibody then is contacted with a sample containing the IL-1lp to
be purified, and thereafter the support is washed with a suitable
solvent that will remove substantially all the material in the
sample except the IL-1lp, which is bound to the immobilized
antibody. Finally, the support is washed with another suitable
solvent that will release the IL-1lp from the antibody.
[0380] In addition, anti-IL-1lp antibodies are useful as
therapeutic agents for targeting of native IL-1lp in
IL-1lp-mediated disease conditions, e.g. disease states
characterized by pathologic IL-1 or IL-18 agonist or agonist-like
activity of the native IL-1lp. In the treatment and prevention of a
native IL-1lp-mediated disorder with the anti-IL-1lp antibody of
the invention, the antibody composition will be formulated, dosed,
and administered in a fashion consistent with good medical
practice. Factors for consideration in this context include the
particular disorder being treated, the particular mammal being
treated, the clinical condition of the individual patient, the
cause of the disorder, the site of delivery of the antibody, the
particular type of antibody, the method of administration, the
scheduling of administration, and other factors known to medical
practitioners. The "effective amount" or "therapeutically effective
amount" of antibody to be administered will be governed by such
considerations, and is the minimum amount necessary to prevent,
ameliorate, or treat the native IL-1lp-mediated disorder, including
treating inflammatory diseases and reducing inflammatory responses.
Such amount is preferably below the amount that is toxic to the
host or renders the host significantly more susceptible to
infections.
[0381] As a general proposition, the initial pharmaceutically
effective amount of the antibody or antibody fragment administered
parenterally per dose will be in the range of about 0.1 to 50 mg/kg
of patient body weight per day, with the typical initial range of
antibody used being 0.3 to 20 mg/kg/day, more preferably 0.3 to 15
mg/kg/day.
[0382] In one embodiment, using systemic administration, the
initial pharmaceutically effective amount will be in the range of
about 2 to 5 mg/kg/day.
[0383] For methods of the invention using administration by
inhalation, the initial pharmaceutically effective amount will be
in the range of about 1 microgram (.mu.g)/kg/day to 100
mg/kg/day.
[0384] In one embodiment, the invention provides a method for
treating an IL-1lp-mediated inflammatory disorder comprising
administering to a mammal, such as human, in need of such treatment
an effective amount of an anti-IL-1lp antibody.
[0385] In another embodiment, the invention provides a method for
treating an hIL-1lp-mediated asthmatic disorder comprising
administering to a human in need of such treatment an effective
amount of an anti-hIL-1lp antibody.
[0386] In another embodiment, the invention provides a method for
treating an hIL-1Ra1-mediated asthmatic disorder comprising
administering to a human in need of such treatment an effective
amount of an anti-hIL-1Ra1 antibody.
[0387] In another embodiment, the invention provides a method for
treating an IL-1lp-mediated rheumatoid arthritic disorder
comprising administering to a mammal, such as human, in need of
such treatment an effective amount of an anti-IL-1lp antibody.
[0388] In another embodiment, the invention provides a method for
treating an hIL-1lp-mediated rheumatoid arthritic disorder
comprising administering to a human in need of such treatment an
effective amount of an anti-hIL-1lp antibody.
[0389] In another embodiment, the invention provides a method for
treating an hIL-1Ra1-mediated rheumatoid arthritic disorder
comprising administering to a human in need of such treatment an
effective amount of an anti-hIL-1Ra1 antibody.
[0390] In another embodiment, the invention provides a method for
treating an IL-1lp-mediated osteoarthritic disorder comprising
administering to a mammal, such as human, in need of such treatment
an effective amount of an anti-IL-1lp antibody.
[0391] In another embodiment, the invention provides a method for
treating an hIL-1lp-mediated osteoarthritic disorder comprising
administering to a human in need of such treatment an effective
amount of an anti-hIL-1lp antibody.
[0392] In another embodiment, the invention provides a method for
treating an hIL-1Ra1-mediated osteoarthritic disorder comprising
administering to a human in need of such treatment an effective
amount of an anti-hIL-1Ra1 antibody.
[0393] In another embodiment, the invention provides a method for
treating an IL-1lp-mediated septic disorder comprising
administering to a mammal, such as human, in need of such treatment
an effective amount of an anti-IL-1lp antibody.
[0394] In another embodiment, the invention provides a method for
treating an hIL-1lp-mediated septic disorder comprising
administering to a human in need of such treatment an effective
amount of an anti-hIL-1lp antibody.
[0395] In another embodiment, the invention provides a method for
treating an hIL-1Ra1-mediated septic disorder comprising
administering to a human in need of such treatment an effective
amount of an anti-hIL-1Ra1 antibody.
[0396] In another embodiment, the invention provides a method for
treating IL-1lp-mediated acute lung injury comprising administering
to a mammal, such as human, in need of such treatment an effective
amount of an anti-IL-1lp antibody.
[0397] In another embodiment, the invention provides a method for
treating hIL-1lp-mediated acute lung injury comprising
administering to a human in need of such treatment an effective
amount of an anti-hIL-1lp antibody.
[0398] In another embodiment, the invention provides a method for
treating hIL-1Ra1-mediated acute lung injury comprising
administering to a human in need of such treatment an effective
amount of an anti-hIL-1Ra1 antibody.
[0399] In another embodiment, the invention provides a method for
treating IL-1lp-mediated adult respiratory distress syndrome
comprising administering to a mammal, such as human, in need of
such treatment an effective amount of an anti-IL-1lp antibody.
[0400] In another embodiment, the invention provides a method for
treating hIL-1lp-mediated adult respiratory distress syndrome
comprising administering to a human in need of such treatment an
effective amount of an anti-hIL-1lp antibody.
[0401] In another embodiment, the invention provides a method for
treating hIL-1Ra1-mediated adult respiratory distress syndrome
comprising administering to a human in need of such treatment an
effective amount of an anti-hIL-1Ra1 antibody.
[0402] In another embodiment, the invention provides a method for
treating IL-1lp-mediated idiopathic pulmonary fibrosis comprising
administering to a mammal, such as human, in need of such treatment
an effective amount of an anti-IL-1lp antibody.
[0403] In another embodiment, the invention provides a method for
treating hIL-1lp-mediated idiopathic pulmonary fibrosis comprising
administering to a human in need of such treatment an effective
amount of an anti-hIL-1lp antibody.
[0404] In another embodiment, the invention provides a method for
treating hIL-1Ra1-mediated idiopathic pulmonary fibrosis comprising
administering to a human in need of such treatment an effective
amount of an anti-hIL-1Ra1 antibody.
[0405] In another embodiment, the invention provides a method for
treating an IL-1lp-mediated ischemic reperfusion disease, such as
surgical tissue reperfusion injury, stroke, myocardial ischemia, or
acute myocardial infarction, comprising administering to a mammal,
such as human, in need of such treatment an effective amount of an
anti-IL-1lp antibody.
[0406] In another embodiment, the invention provides a method for
treating an hIL-1lp-mediated ischemic reperfusion disease, such as
surgical tissue reperfusion injury, stroke, myocardial ischemia, or
acute myocardial infarction, comprising administering to a human in
need of such treatment an effective amount of an anti-hIL-1lp
antibody.
[0407] In another embodiment, the invention provides a method for
treating an hIL-1Ra1-mediated ischemic reperfusion disease, such as
surgical tissue reperfusion injury, stroke, myocardial ischemia, or
acute myocardial infarction, comprising administering to a human in
need of such treatment an effective amount of an anti-hIL-1Ra1
antibody.
[0408] In another embodiment, the invention provides a method for
treating an IL-1lp-mediated psoriatic disorder comprising
administering to a mammal, such as human, in need of such treatment
an effective amount of an anti-IL-1lp antibody.
[0409] In another embodiment, the invention provides a method for
treating an hIL-1lp-mediated psoriatic disorder comprising
administering to a human in need of such treatment an effective
amount of an anti-hIL-1lp antibody.
[0410] In another embodiment, the invention provides a method for
treating an hIL-1Ra1-mediated psoriatic disorder comprising
administering to a human in need of such treatment an effective
amount of an anti-hIL-1Ra1 antibody.
[0411] In another embodiment, the invention provides a method for
treating an IL-1lp-mediated graft-versus-host disease (GVHD)
comprising administering to a mammal, such as human, in need of
such treatment an effective amount of an anti-IL-1lp antibody.
[0412] In another embodiment, the invention provides a method for
treating an hIL-1lp-mediated graft-versus-host disease (GVHD)
comprising administering to a human in need of such treatment an
effective amount of an anti-hIL-1lp antibody.
[0413] In another embodiment, the invention provides a method for
treating an hIL-1Ra1-mediated graft-versus-host disease (GVHD)
comprising administering to a human in need of such treatment an
effective amount of an anti-hIL-1Ra1 antibody.
[0414] In another embodiment, the invention provides a method for
treating an IL-1lp-mediated inflammatory bowel disease such as
ulcerative colitis, comprising administering to a mammal, such as
human, in need of such treatment an effective amount of an
anti-IL-1lp antibody.
[0415] In another embodiment, the invention provides a method for
treating an hIL-1lp-mediated inflammatory bowel disease such as
ulcerative colitis, comprising administering to a human in need of
such treatment an effective amount of an anti-hIL-1lp antibody.
[0416] In another embodiment, the invention provides a method for
treating an hIL-1Ra1-mediated inflammatory bowel disease such as
ulcerative colitis, comprising administering to a human in need of
such treatment an effective amount of an anti-hIL-1Ra1
antibody.
[0417] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0418] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0419] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Example 1
Isolation of DNA encoding hIL-1Ra1 and mIL-1Ra3
[0420] A public expressed sequence tag (EST) DNA database (Genbank)
was searched with human interleukin-1 receptor antagonist (hIL-1Ra)
sequence, also known as secretory human interleukin-1 receptor
antagonist ("sIL-1Ra") sequence, and a human EST designated
AI014548 (FIG. 4, SEQ ID NO:8), and a murine EST designated W08205
(FIG. 10, SEQ ID NO:17), were identified, which showed homology
with the known protein hIL-1Ra (sIL-1Ra).
[0421] EST clones AI014548 and W08205 were purchased from Research
Genetics (Huntsville, Ala.) and the cDNA inserts were obtained and
sequenced in their entireties.
[0422] The entire nucleotide sequence of the clone AI014548,
designated DNA85066, is shown in FIG. 1 (SEQ ID NO:1). Clone
DNA85066 contains a single open reading frame that is interrupted
by an apparent intronic sequence. The intron is bounded by splice
junctions at nucleotide positions 181 to 186 (splice donor site)
and nucleotide positions 430 to 432 (splice acceptor site) (FIG. 1;
SEQ ID NO:1).
[0423] A virtual processed nucleotide sequence (FIG. 3; SEQ ID
NO:6), designated DNA94618, was derived by removing the apparent
intronic sequence from clone DNA85066. Clone DNA94618 contains a
single open reading frame with an apparent translational initiation
site at nucleotide positions 103-105, and a stop codon at
nucleotide positions 682-684 (FIG. 3; SEQ ID NO:6). The predicted
polypeptide precursor (hIL-1Ra1) (FIG. 3; SEQ ID NO:7) is 193 amino
acids long. The putative signal sequence extends from amino acid
positions 1 to 14. A putative cAMP- and cGMP-dependent protein
kinase phosphorylation site is located at amino acid positions
33-36. Putative N-myristoylation sites are located at amino acid
positions 50-55 and 87-92.
[0424] Clone DNA85066 (designated as DNA85066-2534) has been
deposited with ATCC and was assigned ATCC deposit no. 203588. The
full-length hIL-1Ra1 protein shown in FIG. 3 (SEQ ID NO:7) has an
estimated molecular weight of about 21,822 daltons and a pI of
about 8.9.
[0425] Based on a sequence alignment analysis of the full-length
sequence (SEQ ID NO:7), hIL-1Ra1 shows significant amino acid
sequence identity to hIL-1Ra (sIL-1Ra) and hIL-1Ra.beta.
proteins.
[0426] The entire nucleotide sequence of the clone W08205,
designated DNA92505, is shown in FIG. 9 (SEQ ID NO:15). Clone
DNA92505 contains a single open reading frame with an apparent
translational initiation site at nucleotide positions 145-147, and
a stop codon at nucleotide positions 610-612 (FIG. 9; SEQ ID
NO:15). The predicted polypeptide precursor (mIL-1Ra3) (FIG. 9; SEQ
ID NO:16) is 155 amino acids long. The putative signal sequence
extends from amino acid positions 1-33. Putative N-myristoylation
sites are located at amino acid positions 29-34, 60-65, 63-68,
91-96 and 106-111. An interleukin-1-like sequence is located at
amino acid positions 111-131.
[0427] Clone DNA92505 (designated as DNA92505-2534) was deposited
with ATCC and was assigned ATCC deposit no. 203590. The full length
mIL-1Ra3 protein shown in FIG. 9 (SEQ ID NO:16) has an estimated
molecular weight of about 17,134 daltons and a pI of about 4.8.
[0428] Based on a sequence alignment analysis of the full-length
sequence (SEQ ID NO:16), mIL-1Ra3 shows significant amino acid
sequence identity to mIL-1Ra, hicIL-1Ra, hIL-1Ra (sIL-1Ra) and
hIL-1Ra.beta. proteins.
Example 2
Isolation of DNA encoding hIL-1ra2 and hIL-1Ra3
[0429] A expressed sequence tag (EST) DNA database (LIFESEQ.RTM.,
Incyte Pharmaceuticals, Palo Alto, Calif.) was searched with human
interleukin-1 receptor antagonist (hIL-1Ra) sequence, also known as
secretory human interleukin-1 receptor antagonist ("sIL-1Ra")
sequence, and the ESTs, designated 1433156 (FIG. 5, SEQ ID NO:9)
and 5120028 (FIG. 7, SEQ ID NO:12), were identified, which showed
homology with the hIL-1Ra known protein.
[0430] EST clones 1433156 and 5120028 were purchased from Incyte
Pharmaceuticals (Palo Alto, Calif.) and the cDNA inserts were
obtained and sequenced in their entireties.
[0431] The entire nucleotide sequence of the clone 1433156,
designated DNA92929, is shown in FIG. 5 (SEQ ID NO:9). Clone
DNA92929 contains a single open reading frame with an apparent
translational initiation site at nucleotide positions 96-98, and a
stop codon at nucleotide positions 498-500 (FIG. 5; SEQ ID NO:9).
The predicted polypeptide precursor (hIL-1Ra2) (FIG. 5; SEQ ID
NO:10) is 134 amino acids long. A putative signal sequence extends
from amino acid positions 1-26.
[0432] Clone DNA92929 (designated as DNA92929-2534) was deposited
with ATCC and was assigned ATCC deposit no. 203586. The full-length
hIL-1Ra2 protein shown in FIG. 5 (SEQ ID NO:10) has an estimated
molecular weight of about 14,927 daltons and a pI of about 4.8.
[0433] Based on a sequence alignment analysis of the full-length
sequence (SEQ ID NO:10), hIL-1Ra2 shows significant amino acid
sequence identity to hIL-1Ra.beta. protein. hIL-1Ra2 is believed to
be a splice variant of hIL-1Ra.beta..
[0434] The entire nucleotide sequence of the clone 5120028,
designated DNA96787, is shown in FIG. 7 (SEQ ID NO:12). Clone
DNA96787 contains a single open reading frame with an apparent
translational initiation site at nucleotide positions 1-3, and a
stop codon at nucleotide positions 466-468 (FIG. 7; SEQ ID NO:12).
The predicted polypeptide precursor (hIL-1Ra3) (FIG. 7; SEQ ID
NO:13) is 155 amino acids long. A putative signal sequence extends
from amino acid positions 1-33. Putative N-myristoylation sites are
located at amino acid positions 29-34, 60-65, 63-68, 73-78, 91-96
and 106-111. An interleukin-1-like sequence is located at amino
acid positions 111-131.
[0435] It is believed that the predicted 155 amino acid polypeptide
of hIL-1Ra3 behaves as a mature sequence (without a presequence
that is removed in post-translational processing) in certain animal
cells. It is also believed that other animal cells recognize and
remove one or more signal peptide(s) extending from amino acid
positions 1 to about 33. As shown in Example 14 below, transiently
transfected CHO host cells secrete a form of hIL-1Ra3 that only
lacks the N-terminal methionine in the sequence of FIG. 7 (SEQ ID
NO:13).
[0436] Clone DNA96787 (designated as DNA96787-2534) was deposited
with ATCC and was assigned ATCC deposit no. 203589. The full length
hIL-1Ra3 protein shown in FIG. 7 (SEQ ID NO:13) has an estimated
molecular weight of about 16,961 daltons and a pI of about 4.9.
[0437] Based on a sequence alignment analysis of the full-length
sequence (SEQ ID NO:13), hIL-1Ra3 shows significant amino acid
sequence identity to hicIL-1Ra and hIL-1Ra (sIL-1Ra) proteins.
Example 3
Northern Blot Analysis
[0438] Expression of hIL-1Ra3 mRNA in human tissues and mIL-1Ra3
mRNA in mouse tissues was examined by Northern blot analysis. Human
and mouse multiple tissue northern (RNA) blots and mouse embryo
blots were purchased from Clontech and probed with corresponding
cDNA according to the manufacturer's instructions.
[0439] As shown in FIG. 11, hIL-1Ra3 mRNA (2.7 kb) were detected
only in human placenta and mIL-1Ra3 mRNA transcripts (1.4 kb and
2.5 kb) were detected only in the day-17 mouse embryo.
Example 4
Use of IL-1lp as a Hybridization Probe
[0440] The following method describes use of a nucleotide sequence
encoding IL-1lp as a hybridization probe.
[0441] DNA comprising the coding sequence of full-length IL-1lp (as
shown in FIGS. 3, 5, 7, 9, 15, 16 and 19; SEQ ID NOS:6, 9, 12, 15,
18, 20 and 24) is employed as a probe to screen for homologous DNAs
(such as those encoding naturally-occurring variants of IL-1lp) in
human tissue cDNA libraries or human tissue genomic libraries.
[0442] Hybridization and washing of filters containing either
library DNAs is performed under the following high stringency
conditions. Hybridization of radiolabeled IL-1lp-derived probe to
the filters is performed in a solution of 50% formamide,
5.times.SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium
phosphate, pH 6.8, 2.times. Denhardt's solution, and 10% dextran
sulfate at 42.degree. C. for 20 hours. Washing of the filters is
performed in an aqueous solution of 0.1.times.SSC and 0.1% SDS at
42.degree. C.
[0443] DNAs having a desired sequence identity with the DNA
encoding full-length native sequence IL-1lp can then be identified
using standard techniques known in the art.
Example 5
Expression of IL-1lp in E. coli
[0444] This example illustrates preparation of an unglycosylated
form of IL-1lp by recombinant expression in E. coli.
[0445] The DNA sequence encoding an IL-1lp is initially amplified
using selected PCR primers. The primers should contain restriction
enzyme sites which correspond to the restriction enzyme sites on
the selected expression vector. A variety of expression vectors may
be employed. An example of a suitable vector is pBR322 (derived
from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains
genes for ampicillin and tetracycline resistance. The vector is
digested with restriction enzyme and dephosphorylated. The PCR
amplified sequences are then ligated into the vector. The vector
will preferably include sequences which encode for an antibiotic
resistance gene, a trp promoter, a polyhis leader (including the
first six STII codons, polyhis sequence, and enterokinase cleavage
site), the IL-1lp coding region, lambda transcriptional terminator,
and an argU gene.
[0446] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates
and antibiotic resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[0447] Selected clones can be grown overnight in liquid culture
medium such as LB broth supplemented with antibiotics. The
overnight culture may subsequently be used to inoculate a larger
scale culture. The cells are then grown to a desired optical
density, during which the expression promoter is turned on.
[0448] After culturing the cells for several more hours, the cells
can be harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the
art, and the solubilized IL-1lp protein can then be purified using
a metal chelating column under conditions that allow tight binding
of the protein.
Example 6
Expression of IL-1lp in Mammalian Cells
[0449] This example illustrates preparation of a potentially
glycosylated form of IL-1lp by recombinant expression in mammalian
cells.
[0450] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the IL-1lp DNA is
ligated into pRK5 with selected restriction enzymes to allow
insertion of the IL-1lp DNA using ligation methods such as
described in Sambrook et al., supra. The resulting vector is called
pRK5-IL-1lp.
[0451] In one embodiment, the selected host cells may be 293 cells.
Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue
culture plates in medium such as DMEM supplemented with fetal calf
serum and optionally, nutrient components and/or antibiotics. About
10 .mu.g pRK5-IL-1lp DNA is mixed with about 1 .mu.g DNA encoding
the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and
dissolved in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M
CaCl.sub.2. To this mixture is added, dropwise, 500 .mu.l of 50 mM
HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate
is allowed to form for 10 minutes at 25.degree. C. The precipitate
is suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for about 5 days.
[0452] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200
.mu.Ci/ml .sup.35S-methionine. After a 12 hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of IL-1lp polypeptide. The cultures containing transfected
cells may undergo further incubation (in serum free medium) and the
medium is tested in selected bioassays.
[0453] In an alternative technique, IL-1lp may be introduced into
293 cells transiently using the dextran sulfate method described by
Somparyrac et al., Proc. Natl. Acad. Sci. USA, 12: 7575 (1981). 293
cells are grown to maximal density in a spinner flask and 700 .mu.g
pRK5-IL-1lp DNA is added. The cells are first concentrated from the
spinner flask by centrifugation and washed with PBS. The
DNA-dextran precipitate is incubated on the cell pellet for four
hours. The cells are treated with 20% glycerol for 90 seconds,
washed with tissue culture medium, and re-introduced into the
spinner flask containing tissue culture medium, 5 .mu.g/ml bovine
insulin and 0.1 .mu.g/ml bovine transferrin. After about four days,
the conditioned media is centrifuged and filtered to remove cells
and debris. The sample containing expressed IL-1lp can then be
concentrated and purified by any selected method, such as dialysis
and/or column chromatography.
[0454] In another embodiment, IL-1lp can be expressed in CHO cells.
The pRK5-IL-1lp can be transfected into CHO cells using known
reagents such as CaPO.sub.4 or DEAE-dextran. As described above,
the cell cultures can be incubated, and the medium replaced with
culture medium (alone) or medium containing a radiolabel such as
.sup.35S-methionine. After determining the presence of IL-1lp
polypeptide, the culture medium may be replaced with serum free
medium. Preferably, the cultures are incubated for about 6 days,
and then the conditioned medium is harvested. The medium containing
the expressed IL-1lp can then be concentrated and purified by any
selected method.
[0455] Epitope-tagged IL-1lp may also be expressed in host CHO
cells. The IL-1lp may be subcloned out of the pRK5 vector. The
subclone insert can undergo PCR to fuse in frame with a selected
epitope tag such as a poly-his tag into a Baculovirus expression
vector. The poly-his tagged IL-1lp insert can then be subcloned
into a SV40 driven vector containing a selection marker such as
DHFR for selection of stable clones. Finally, the CHO cells can be
transfected (as described above) with the SV40 driven vector.
Labeling may be performed, as described above, to verify
expression. The culture medium containing the expressed poly-His
tagged IL-1lp can then be concentrated and purified by any selected
method, such as by Ni.sup.2+-chelate affinity chromatography.
Example 7
Expression of IL-1lp in Yeast
[0456] The following method describes recombinant expression of
IL-1lp in yeast.
[0457] First, yeast expression vectors are constructed for
intracellular production or secretion of IL-1lp from the ADH2/GAPDH
promoter. DNA encoding IL-1lp and the promoter is inserted into
suitable restriction enzyme sites in the selected plasmid to direct
intracellular expression of IL-1lp. For secretion, DNA encoding
IL-1lp can be cloned into the selected plasmid, together with DNA
encoding the ADH2/GAPDH promoter, a native IL-1lp signal peptide or
other mammalian signal peptide, or, for example, a yeast
alpha-factor or invertase secretory signal/leader sequence, and
linker sequences (if needed) for expression of IL-1lp.
[0458] Yeast cells, such as yeast strain AB110, can then be
transformed with the expression plasmids described above and
cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with 10%
trichloroacetic acid and separation by SDS-PAGE, followed by
staining of the gels with Coomassie Blue stain.
[0459] Recombinant IL-1lp can subsequently be isolated and purified
by removing the yeast cells from the fermentation medium by
centrifugation and then concentrating the medium using selected
cartridge filters. The concentrate containing IL-1lp may further be
purified using selected column chromatography resins.
Example 8
Expression of IL-1lp in Baculovirus-Infected Insect Cells
[0460] The following method describes recombinant expression of
IL-1lp in Baculovirus-infected insect cells.
[0461] The sequence coding for IL-1lp is fused upstream of an
epitope tag contained within a baculovirus expression vector. Such
epitope tags include poly-his tags and immunoglobulin tags (like Fc
regions of IgG). A variety of plasmids may be employed, including
plasmids derived from commercially available plasmids such as
pVL1393 (Novagen). Briefly, the sequence encoding IL-1lp or the
desired portion of the coding sequence of IL-1lp (such as the
sequence encoding the mature protein) is amplified by PCR with
primers complementary to the 5' and 3' regions. The 5' primer may
incorporate flanking (selected) restriction enzyme sites. The
product is then digested with those selected restriction enzymes
and subcloned into the expression vector.
[0462] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BaculoGold.TM. virus DNA (Pharmingen) into
Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from GIBCO-BRL). After 4-5 days
of incubation at 28.degree. C., the released viruses are harvested
and used for further amplifications. Viral infection and protein
expression are performed as described by O'Reilley et al.,
Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994).
[0463] Expressed poly-his tagged IL-1lp can then be purified, for
example, by Ni.sup.2+-chelate affinity chromatography as follows.
Extracts are prepared from recombinant virus-infected Sf9 cells as
described by Rupert et al., Nature, 362: 175-179 (1993). Briefly,
Sf9 cells are washed, resuspended in sonication buffer (25 mL
Hepes, pH 7.9; 12.5 mM MgCl.sub.2; 0.1 mM EDTA; 10% glycerol; 0.1%
NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The
sonicates are cleared by centrifugation, and the supernatant is
diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl,
10% glycerol, pH 7.8) and filtered through a 0.45 .mu.m filter. A
Ni.sup.2+-NTA agarose column (commercially available from Qiagen)
is prepared with a bed volume of 5 mL, washed with 25 mL of water
and equilibrated with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column at 0.5 mL per minute. The column
is washed to baseline A.sub.280 with loading buffer, at which point
fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol,
pH 6.0), which elutes nonspecifically bound protein. After reaching
A.sub.280 baseline again, the column is developed with a 0 to 500
mM Imidazole gradient in the secondary wash buffer. One mL
fractions are collected and analyzed by SDS-PAGE and silver
staining or Western blot with Ni.sup.2+-NTA-conjugated to alkaline
phosphatase (Qiagen). Fractions containing the eluted
His.sub.10-tagged IL-1lp are pooled and dialyzed against loading
buffer.
[0464] Alternatively, purification of the IgG tagged (or Fc tagged)
IL-1lp can be performed using known chromatography techniques,
including for instance, Protein A or protein G column
chromatography.
Example 9
IL-18 Receptor and IL-1 Receptor Binding of hIL-1Ra1
[0465] To facilitate the characterization of hIL-1Ra1, a PCR
fragment containing the partial ORF of clone DNA85066 (FIG. 1; SEQ
ID NO:3) was cloned into pCMV1FLAG (IBI Kodak, described in Pan et
al., Science, 276: 111-113) as an in-frame fusion to a
NH.sub.2-terminal preprotrypsin leader sequence and FLAG tag
encoded by the vector. The entire cDNA insert of the recombinant
pCMV1FLAG vector clone (designated clone DNA96786) was sequenced
(FIG. 2; SEQ ID NO:4). The cDNAs encoding the extracellular domain
of hIL1R and hIL18R (formerly known as hIL1Rrp) were obtained by
polymerase chain reaction (PCR) and cloned into a modified
pCMV1FLAG vector that allowed for in-frame fusion with the Fc
portion of human immunoglobulin G.
[0466] Human embryonic kidney 293 cells were grown in high glucose
DMEM (Genentech, Inc). The cells were seeded at 3-4.times.10.sup.6
per plate (100 mm) and co-transfected with pCMV 1FLAG-hIL-1Ra1 and
pCMV1FLAG-IL1R-ECD-Fc or pCMV1FLAG-IL18R-ECD-Fc by means of calcium
phosphate precipitation. The media were changed 12 hours post
transfection. The resultant conditioned media (10 ml each) were
harvested after a further 70-74 hour incubation, clarified by
centrifugation, aliquoted and stored at -70.degree. C. The
receptor-Fc and ligand complex from 1.5 ml conditioned medium was
immunoprecipitated with protein G-Sepharose, washed three times
with buffer containing 50 mM Hepes, pH 7.0, 150 mM NaCl, 1 mM EDTA,
1% NP-40, and a protease inhibitor cocktail (BMB) and resolved on a
10-20% SDS-PAGE gel. The bound ligand was identified by
immunoblotting using anti-FLAG monoclonal antibody (BMB).
[0467] As shown in FIG. 13A, the secreted FLAGhIL-1Ra1 fusion
protein bound to IL-18R ECD and did not bind to IL-1R ECD, which
indicates that hIL-1Ra1 could be an agonist or antagonist of
IL-18R.
Example 10
IL-1 Receptor and IL-18 Receptor Binding of mIL-1Ra3 and
hIL-1Ra3
[0468] cDNA encoding mIL-1Ra3 (DNA92505 shown in FIG. 9; SEQ ID
NO:15) was cloned into pRK7 with a carboxy-terminal FLAG-tag. The
resulting expression construct was transfected into human embryonic
kidney 293 cells by means of calcium phosphate precipitation. 84-90
hours post transfection, the conditioned media containing secreted
FLAGmIL-1Ra3 fusion protein was harvested. Conditioned media
containing secreted IL-18R-Fc and IL-1R-Fc proteins were prepared
as described in Example 9 above, with the exception that the 293
cells were transfected with either pCMV1FLAG-IL1R-ECD-Fc or
pCMV1FLAG-IL18R-ECD-Fc alone (without pCMV1FLAG-IL-1Ra1
cotransfection).
[0469] For in vitro binding assays, IL-1R-Fc or IL-18R-Fc from 0.5
ml of the conditioned medium was immobilized to protein G-agarose
and then mixed with 1.2 ml conditioned medium containing
FLAGmIL-1Ra3. The receptor-ligand complexes were washed and
resolved on an 10-20% SDS-PAGE gel and the bound ligand was
detected by immunoblotting using anti-FLAG monoclonal antibody
(Boehringer Mannheim).
[0470] As shown in FIG. 14, FLAGmIL-1Ra3 fusion protein bound to
IL-1R ECD and did not bind to IL-18R ECD. Since the amino acid
sequence of mIL-1Ra3 is related to that of the known interleukin-1
receptor antagonist protein (IL-1Ra), mIL-3Ra3 is believed to be a
novel IL-1 receptor antagonist.
[0471] cDNA encoding hIL-1Ra3 (DNA96787 shown in FIG. 7; SEQ ID
NO:12) was cloned into pRK7 with a carboxy-terminal FLAG tag to
form pRK7hIL-1Ra3-FLAG. pCMV1FLAG-IL1R-ECD-Fc and
pCMV1FLAG-IL18R-ECD-Fc were obtained as described in Example 9
above. Similarly, cDNA encoding DR6 was cloned into the modified
pCMV1FLAG vector of Example 9 to form pCMV1FLAG-DR6-Fc, encoding
DR6 fused to the Fc portion of human immunoglobulin G. Conditioned
media containing (1) a combination of secreted FLAGhIL-1Ra3 and
FLAG-DR6-Fc (2) a combination of secreted FLAGhIL-1Ra3 and
FLAG-IL1R-ECD-Fc or (3) a combination of secreted FLAGhIL-1Ra3 and
FLAG-IL18R-ECD-Fc were prepared by cotransfecting Human 293 cells
with (1) pRK7hIL-1Ra3-FLAG and pCMV1FLAG-DR6-Fc (2)
pRK7hIL-1Ra3-FLAG and pCMV1FLAG-IL1R-ECD-Fc or (3)
pRK7hIL-1Ra3-FLAG and pCMV1FLAG-IL18R-ECD-Fc, culturing the
transfectant cells and harvesting culture media essentially as
described in Example 9 above. The receptor-Fc and ligand complex
from each conditioned medium was immunoprecipitated with protein
G-Sepharose or anti-FLAG monoclonal antibody, and
immunoprecipitates were resolved by gel electrophoresis and
immunoblotting with anti-FLAG monoclonal antibody essentially as
described in Example 9 above.
[0472] As shown in FIG. 13B, FLAGhIL-1Ra3 fusion protein bound to
IL-1R-ECD-Fc and did not bind to IL-18R-ECD-Fc or DR6-Fc. Since the
amino acid sequence of hIL-1Ra3 is related to that of the known
interleukin-1 receptor antagonist protein (IL-1Ra), hIL-3Ra3 is
believed to be a novel IL-1 receptor antagonist.
Example 11
Preparation of Antibodies that Bind IL-1lp
[0473] This example illustrates preparation of monoclonal
antibodies which can specifically bind IL-1lp.
[0474] Techniques for producing the monoclonal antibodies are known
in the art and are described, for instance, in Goding, supra.
Immunogens that may be employed include purified IL-1lp, fusion
proteins containing IL-1lp, and cells expressing recombinant IL-1lp
on the cell surface. Selection of the immunogen can be made by the
skilled artisan without undue experimentation.
[0475] Mice, such as Balb/c, are immunized with the IL-1lp
immunogen emulsified in complete Freund's adjuvant and injected
subcutaneously or intraperitoneally in an amount from 1-100
micrograms. Alternatively, the immunogen is emulsified in MPL-TDM
adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and
injected into the animal's hind foot pads. The immunized mice are
then boosted 10 to 12 days later with additional immunogen
emulsified in the selected adjuvant. Thereafter, for several weeks,
the mice may also be boosted with additional immunization
injections. Serum samples may be periodically obtained from the
mice by retro-orbital bleeding for testing in ELISA assays to
detect anti-IL-1lp antibodies.
[0476] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of IL-1lp. Three to four days later, the mice
are sacrificed and the spleen cells are harvested. The spleen cells
are then fused (using 35% polyethylene glycol) to a selected murine
myeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL
1597. The fusions generate hybridoma cells which can then be plated
in 96 well tissue culture plates containing HAT (hypoxanthine,
aminopterin, and thymidine) medium to inhibit proliferation of
non-fused cells, myeloma hybrids, and spleen cell hybrids.
[0477] The hybridoma cells will be screened in an ELISA for
reactivity against IL-1lp. Determination of "positive" hybridoma
cells secreting the desired monoclonal antibodies against IL-1lp is
within the skill in the art.
[0478] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-IL-1lp monoclonal antibodies. Alternatively,
the hybridoma cells can be grown in tissue culture flasks or roller
bottles. Purification of the monoclonal antibodies produced in the
ascites can be accomplished using ammonium sulfate precipitation,
followed by gel exclusion chromatography. Alternatively, affinity
chromatography based upon binding of antibody to protein A or
protein G can be employed.
Example 12
Isolation of DNA encoding hIL-1Ra1L, hIL-1Ra1V and hIL-1Ra1S
[0479] Several intron-containing cDNA clones related to the
hIL-1Ra1 intron-containing clone DNA85066 (FIG. 2) (SEQ ID NO:4)
were isolated from a human testis cDNA library and fully sequenced.
The intron-containing cDNA sequences were used to determine a
full-length open reading frame (ORF) with the GENESCAN program
(http://CCR-081.mit.edu/GENESCAN.html). The ORF-encoding sequence
was used to design two DNA primers, ggc gga tcc aaa atg ggc tct gag
gac tgg g (SEQ ID NO:29) (1Ra1016) and gcg gaa ttc taa tcg ctg acc
tca ctg ggg (SEQ ID NO:30) (1Ra1017). The 1Ra1016 and 1Ra1017
primers were synthesized and used to clone cDNA from human fetal
skin and SK-lu-1 cell cDNA libraries using polymerase chain
reaction (PCR). Several PCR products were isolated and sequenced.
Two full length cDNA clones (designated DNA102043 and DNA 102044)
from PCR products were found to encode hIL-1Ra1 isoforms.
[0480] The entire nucleotide sequence of clone DNA102043 is shown
in FIG. 15 (SEQ ID NO:18). Clone DNA102043 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 4-6, and a stop codon at nucleotide positions
625-627 (FIG. 15; SEQ ID NO:18). The predicted polypeptide
precursor (designated hIL-1Ra1L) (FIG. 15; SEQ ID NO:19) is 207
amino acids long. The putative signal sequence extends from amino
acid positions 1-34.
[0481] Clone DNA102043 (designated DNA102043-2534) was deposited
with ATCC and was assigned ATCC deposit no. 203846. The full-length
hIL-1Ra1L protein shown in FIG. 15 (SEQ ID NO:19) has an estimated
molecular weight of about 23,000 daltons and a pI of about
6.08.
[0482] Based on a sequence alignment analysis of the full length
sequence (SEQ ID NO:19), hIL-1Ra1L shows significant amino acid
sequence identity to hIL-1Ra.beta. and TANGO-77 protein. In
addition, a portion of the DNA sequence of clone DNA102043 (FIG.
15) (SEQ ID NO:18) was found to coincide with the DNA sequence of
EST AI014548 (FIG. 4) (SEQ ID NO:8) and with the complement of the
DNA sequence of EST AI323258 (FIG. 17) (SEQ ID NO:23).
[0483] The entire nucleotide sequence of clone DNA102044 is shown
in FIG. 16 (SEQ ID NO:20). Clone DNA102044 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 4-6, and a stop codon at nucleotide positions
505-507 (FIG. 16; SEQ ID NO:20). The predicted polypeptide
(designated hIL-1Ra1S) (FIG. 16; SEQ ID NO:21) is 167 amino acids
long, and it is believed to behave as a mature sequence (without a
presequence that is removed in post-translational processing) in
certain animal cells. In addition, it is believed that other animal
cells recognize and remove in post-translational processing one or
more signal peptide(s) contained in the sequence extending from
amino acid positions 1 to about 46.
[0484] Clone DNA102044 (designated DNA102044-2534) was deposited
with ATCC and was assigned ATCC deposit no. 203855. The full-length
hIL-1Ra1S protein shown in FIG. 16 (SEQ ID NO:21) has an estimated
molecular weight of about 18,478 daltons and a pI of about 5.5.
[0485] Based on a sequence alignment analysis of the full length
sequence (SEQ ID NO:21), hIL-1Ra1S appears to be an allelic variant
of TANGO-77 protein and also shows significant amino acid sequence
identity to hIL-1Ra.beta.. In addition, a portion of the DNA
sequence of clone DNA102044 (FIG. 16) (SEQ ID NO:20) was found to
coincide with the DNA sequence of EST AI014548 (FIG. 4) (SEQ ID
NO:8) and with the complement of the DNA sequence of EST AI323258
(FIG. 17) (SEQ ID NO:23).
[0486] EST clone AI323258 was purchased from Research Genetics
(Huntsville, Ala.) and the cDNA insert was obtained and sequenced
in its entirety. The entire sequence of the clone AI323258,
designated DNA114876, is shown in FIG. 19 (SEQ ID NO:24). Clone
DNA114876 contains a single open reading frame (ORF) with an
apparent translation initiation site at nucleotide positions 73-75
and a stop codon at nucleotide positions 726-728 (FIG. 19; SEQ ID
NO:24), encoding a predicted polypeptide precursor (hIL-1Ra1V)
(FIG. 19; SEQ ID NO:25) that is 218 amino acids long. In addition,
the ORF contains an alternate translation initiation site at
nucleotide positions 106-108. The predicted polypeptide (also
designated hIL-1Ra1V) for translation initiated at the alternate
start codon is 207 amino acids in length (lacking the first eleven
residues at the N-terminus of the 218 amino acid polypeptide). It
is believed that the predicted 218 amino acid and 207 amino acid
polypeptides behave as mature sequences (without a presequence that
is removed in post-translational processing) in certain animal
cells. It is also believed that other animal cells recognize and
remove one or more signal peptide(s) extending from amino acid
positions 1 to about 48 (a putative leader sequence in the 218
amino acid polypeptide) or from amino acid positions 12 to 36 (a
putative leader sequence in the 207 amino acid polypeptide) in the
amino acid sequence of FIG. 19 (SEQ ID NO:25). As shown in Example
14 below, transiently transfected CHO host cells secrete
unprocessed forms of hIL-1Ra1V and hIL-1Ra1L and a single processed
form that results from the removal of a signal peptide extending
from amino acid positions 1 to 45 in FIG. 19 (SEQ ID NO:25) or the
removal of a signal peptide extending from amino acid positions 1
to 34 of FIG. 15 (SEQ ID NO:19). The processed form of hIL-1Ra1V
and hIL-1Ra1L secreted by transiently transfected CHO host cells
has the amino acid sequence of amino acid residues 35 to 207 of
FIG. 15 (SEQ ID NO:19) and amino acid residues 46 to 218 of FIG. 19
(SEQ ID NO:25).
[0487] Clone DNA114876 (designated DNA114876-2534) was deposited
with ATCC and was assigned ATCC deposit no. 203973. The full length
hIL-1Ra1V protein shown in FIG. 19 (SEQ ID NO:25) has an estimated
molecular weight of about 24,124 and a pI of about 6.1.
[0488] Based on a sequence alignment analysis of the full length
sequence (SEQ ID NO:25), hIL-1Ra1V shows significant amino acid
sequence identity to hIL-1Ra.beta.. hIL-1Ra1V is believed to be an
allelic variant of hIL-1Ra1L.
Example 13
IL-18 Receptor and IL-1 Receptor Binding of hIL-1Ra1S
[0489] To facilitate the characterization of hIL-1Ra1S, a PCR
fragment encoding amino acid residues 39-167 in the ORF of clone
DNA102044 (FIG. 16; SEQ ID NO:21) was cloned into pCMV1FLAG (IBI
Kodak, described in Pan et al., Science, 276: 111-113) as an
in-frame fusion to a NH.sub.2-terminal preprotrypsin leader
sequence and FLAG tag encoded by the vector to form plasmid
pCMV1FLAG-IL-1Ra1S. Plasmid pCMV1FLAG-IL18R-ECD-Fc was obtained as
described in Example 9 above.
[0490] Human embryonic kidney 293 cells were grown in high glucose
DMEM (Genentech, Inc). The cells were seeded at 3-4.times.10.sup.6
per plate (100 mm) and co-transfected with pCMV1FLAG-hIL-1Ra1S and
pCMV1FLAG-IL18R-ECD-Fc by means of calcium phosphate precipitation.
The media were changed 12 hours post transfection. The resultant
conditioned media (10 ml each) were harvested after a further 70-74
hour incubation, clarified by centrifugation, aliquoted and stored
at -70.degree. C. The receptor-Fc and ligand complex from 1.5 ml
conditioned medium was immunoprecipitated with protein G-Sepharose,
washed three times with buffer containing 50 mM Hepes, pH7.0, 150
mM NaCl, 1 mM EDTA, 1% NP-40, and a protease inhibitor cocktail
(BMB) and resolved on a 10-20% SDS-PAGE gel. The bound ligand was
identified by immunoblotting using anti-FLAG monoclonal antibody
(BMB).
[0491] The immunoblotting results indicated that the secreted
FLAGhIL-1Ra1S fusion protein bound to IL-18R ECD. These data show
that hIL-1Ra1S could be an agonist or antagonist of IL-18R.
Example 14
hIL-1Ra1V, hIL-1Ra1L and hIL-1Ra3 Processing
[0492] cDNAs encoding full-length hIL-1Ra1V (amino acids 1-218 in
the ORF of clone DNA114876 shown in FIG. 19 (SEQ ID NO:25)), full
length hIL-1Ra1L (amino acids 1-207 in the ORF of clone DNA102043
shown in FIG. 15 (SEQ ID NO:19)), and full length hIL-1Ra3 (amino
acids 1-155 in the ORF of clone DNA96787 shown in FIG. 7 (SEQ ID
NO:13)) were each cloned into a pRK7 expression vector as an
in-frame fusion with a carboxy-terminal FLAG-tag sequence. In
preparation for mammalian cell transient transfections, CHO DP12
cells were seeded at 4.times.10.sup.6 cells per plate (100 mm petri
dish) in growth medium (PS20, 5% FBS, 1.times.GHT, 1.times.
pen/strep, 1.times. L-glutamine) the day before transfection. On
the day of transfection, cells were washed with PBS and fed with 10
ml serum-free transfection medium (PS20, 1.times.GHT). DNA-lipid
transfection mixtures were prepared by adding stepwise into
eppendorf tubes (1) 400 .mu.l transfection medium (PS20,
1.times.GHT); (2) 12 .mu.g DNA; (3) 10 .mu.g poly-lysine; and (4)
50 .mu.l Dosper liposomal transfection reagent (Boehringer
Mannheim). The DNA-lipid mixtures were incubated for 15 minutes at
room temperature and then added dropwise to cell culture plates.
Cells were incubated overnight at 37.degree. C. On the day after
transfection, cells were washed with PBS, fed with 10 ml serum-free
production medium (PS24, 10 mg/L insulin, 1.times. trace elements,
1.4 mg/L lipid EtOH), and placed in a 32.degree. C. incubator.
After 5 days, the culture media containing the expressed proteins
were harvested and cleared by centrifugation. For peptide
sequencing of each expressed protein, 5-10 ml of the conditioned
medium containing the expressed protein was incubated with
monoclonal anti-FLAG antibody (Boehringer Mannheim) coupled to
agarose beads. The immunoprecipitated FLAG-tag proteins were
extensively washed with 1% NP-40 buffer (125 mM NaCl, 1 mM EDTA and
50 mM Tris-HCl, pH 7.4). The immunoprecipitates were run on a SDS
polyacrylamide gel, the separated polypeptides on the gel were
transferred to a PVDF membrane, the PVDF membrane was stained with
Coomassie blue, and the corresponding protein bands were excised
from the membrane. The amino-terminal protein sequences were
obtained by conventional methods.
[0493] The processed N-terminal sequence of both of the hIL-1Ra1L
and hIL-1Ra1V polypeptides was determined to be VHTSPKVKN (SEQ ID
NO:31). Approximately 50% of hIL-1Ra1L and hIL-1Ra1V material
recovered from conditioned media exhibited the processed N-terminal
sequence, indicating that the CHO host cells secreted a processed
form corresponding to amino acid residues 35 to 207 in the amino
acid sequence of FIG. 15 (SEQ ID NO:19) and amino acid residues 46
to 218 in the amino acid sequence of FIG. 19 (SEQ ID NO:25). The
remaining 50% of the hIL-1Ra1L and hIL-1Ra1V material recovered
from conditioned media exhibited an unprocessed N-terminus,
indicating that the CHO host cells also secreted unprocessed forms
of hIL-1Ra1L and hIL-1Ra1V corresponding to amino acid residues 1
to 207 in the amino acid sequence of FIG. 15 (SEQ ID NO:19) and to
amino acid residues 1 to 218 in the amino acid sequence of FIG. 19
(SEQ ID NO:25), respectively.
[0494] The processed N-terminal sequence of both of the hIL-1Ra3
and mIL-1Ra3 polypeptides was determined to be VLSGALCFRM (SEQ ID
NO:33). Approximately 100% of the hIL-1Ra3 and mIL-1Ra3 material
recovered from conditioned media exhibited the processed N-terminal
sequence, indicating that the CHO host cells secreted processed
forms of hIL-1Ra3 and mIL-1Ra3 that lack the N-terminal methionine
and correspond to amino acid residues 2 to 155 in the amino acid
sequence of FIG. 7 (SEQ ID NO:13) and amino acid residues 2 to 155
in the amino acid sequence of FIG. 9 (SEQ ID NO:16),
respectively.
Deposit of Material
[0495] The following materials have been deposited with the
American Type Culture Collection, 10801 University Blvd., Manassas,
Va. 20110-2209, USA (ATCC): TABLE-US-00002 Material ATCC Dep. No.
Deposit Date pSPORT1-based plasmid 203586 Jan. 12, 1999
DNA92929-2534 pCMV-1Flag-pcmv5 plasmid 203587 Jan. 12, 1999
DNA96786-2534 pT7T3D-Pac plasmid 203588 Jan. 12, 1999 DNA85066-2534
pINCY-based plasmid 203589 Jan. 12, 1999 DNA96787-2534 pT7T3D-Pac
plasmid 203590 Jan. 12, 1999 DNA92505-2534 pRK7-based plasmid
203846 Mar. 16, 1999 DNA102043-2534 pRK7-based plasmid 203855 Mar.
16, 1999 DNA102044-2534 pRK7-based plasmid 203973 Apr. 27, 1999
DNA114876-2534
[0496] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of viable cultures of the deposits for 30 years from the date of
deposit. The deposits will be made available by ATCC under the
terms of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the cultures of the deposits to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn.122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn.1.14
with particular reference to 886OG 638).
[0497] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0498] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the constructs deposited, since the deposited embodiment is
intended as a single illustration of certain aspects of the
invention and any constructs that are functionally equivalent are
within the scope of this invention. The deposit of material herein
does not constitute an admission that the written description
herein contained is inadequate to enable the practice of any aspect
of the invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
TABLE-US-00003 TABLE 2A PRO XXXXXXXXXXXXXXX (Length = 15 amino
acids) Comparison XXXXXYYYYYYY (Length = 12 amino acids) Protein %
amino acid sequence identity = (the number of identically matching
amino acid residues between the two polypeptide sequences as
determined by ALIGN-2) divided by (the total number of amino acid
residues of the PRO polypeptide) = 5 divided by 15 = 33.3%
[0499] TABLE-US-00004 TABLE 2B PRO XXXXXXXXXX (Length = 10 amino
acids) Comparison XXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein
% amino acid sequence identity = (the number of identically
matching amino acid residues between the two polypeptide sequences
as determined by ALIGN-2) divided by (the total number of amino
acid residues of the PRO polypeptide) = 5 divided by 10 = 50%
[0500] TABLE-US-00005 TABLE 2C PRO-DNA NNNNNNNNNNNNNN (Length = 14
nucleotides) Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
DNA % nucleic acid sequence identity = (the number of identically
matching nucleotides between the two nucleic acid sequences as
determined by ALIGN-2) divided by (the total number of nucleotides
of the PRO-DNA nucleic acid sequence) = 6 divided by 14 = 42.9%
[0501] TABLE-US-00006 TABLE 2D PRO-DNA NNNNNNNNNNNN (Length = 12
nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides) %
nucleic acid sequence identity = (the number of identically
matching nucleotides between the two nucleic acid sequences as
determined by ALIGN-2) divided by (the total number of nucleotides
of the PRO-DNA nucleic acid sequence) = 4 divided by 12 = 33.3%
[0502] TABLE-US-00007 TABLE 3A /* * * C--C increased from 12 to 15
* Z is average of EQ * B is average of ND * match with stop is _M;
stop-stop = 0; J (joker) match = 0 */ #define _M -8 /* value of a
match with a stop */ int _day[26][26] = { /* A B C D E F G H I J K
L M N O P Q R S T U V W X Y Z */ /* A */ { 2, 0,-2, 0, 0,-4,
1,-1,-1, 0,-1,-2,-1, 0,_M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3, 0}, /* B
*/ { 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2,_M,-1, 1, 0, 0, 0,
0,-2,-5, 0,-3, 1}, /* C */ {-2,-4,15,-5,-5,-4,-3,-3,-2,
0,-5,-6,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0, 0,-5}, /* D */ { 0,
3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7,
0,-4, 2}, /* E */ { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1,_M,-1,
2,-1, 0, 0, 0,-2,-7, 0,-4, 3}, /* F */ {-4,-5,-4,-6,-5, 9,-5,-2, 1,
0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5}, /* G */ { 1,
0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,_M,-1,-1,-3, 1, 0, 0,-1,-7,
0,-5, 0}, /* H */ {-1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2,_M, 0,
3, 2,-1,-1, 0,-2,-3, 0, 0, 2}, /* I */ {-1,-2,-2,-2,-2, 1,-3,-2, 5,
0,-2, 2, 2,-2,_M,-2,-2,-2,-1, 0, 0, 4,-5, 0,-1,-2}, /* J */ { 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0}, /* K */ {-1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1,_M,-1, 1,
3, 0, 0, 0,-2,-3, 0,-4, 0}, /* L */ {-2,-3,-6,-4,-3, 2,-4,-2, 2,
0,-3, 6, 4,-3,_M,-3,-2,-3,-3,-1, 0, 2,-2, 0,-1,-2}, /* M */
{-1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2,_M,-2,-1, 0,-2,-1, 0,
2,-4, 0,-2,-1}, /* N */ { 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2,
2,_M,-1, 1, 0, 1, 0, 0,-2,-4, 0,-2, 1}, /* O */
{_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,
0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M}, /* P */ { 1,-1,-3,-1,-1,-5,-1,
0,-2, 0,-1,-3,-2,-1,_M, 6, 0, 0, 1, 0, 0,-1,-6, 0,-5, 0}, /* Q */ {
0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1,_M, 0, 4, 1,-1,-1,
0,-2,-5, 0,-4, 3}, /* R */ {-2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0,
0,_M, 0, 1, 6, 0,-1, 0,-2, 2, 0,-4, 0}, /* S */ { 1, 0, 0, 0, 0,-3,
1,-1,-1, 0, 0,-3,-2, 1,_M, 1,-1, 0, 2, 1, 0,-1,-2, 0,-3, 0}, /* T
*/ { 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, 0,_M, 0,-1,-1, 1, 3, 0,
0,-5, 0,-3, 0}, /* U */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* V */ {
0,-2,-2,-2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2,_M,-1,-2,-2,-1, 0, 0,
4,-6, 0,-2,-2}, /* W */ {-6,-5,-8,-7,-7, 0,-7,-3,-5,
0,-3,-2,-4,-4,_M,-6,-5, 2,-2,-5, 0,-6,17, 0, 0,-6}, /* X */ { 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0}, /* Y */ {-3,-3, 0,-4,-4, 7,-5, 0,-1,
0,-4,-1,-2,-2,_M,-5,-4,-4,-3,-3, 0,-2, 0, 0,10,-4}, /* Z */ { 0,
1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1,_M, 0, 3, 0, 0, 0, 0,-2,-6,
0,-4, 4} };
[0503] TABLE-US-00008 TABLE 3B /* */ #include <stdio.h>
#include <ctype.h> #define MAXJMP 16 /* max jumps in a diag
*/ #define MAXGAP 24 /* don't continue to penalize gaps larger than
this */ #define JMPS 1024 /* max jmps in an path */ #define MX 4 /*
save if there's at least MX-1 bases since last jmp */ #define DMAT
3 /* value of matching bases */ #define DMIS 0 /* penalty for
mismatched bases */ #define DINS0 8 /* penalty for a gap */ #define
DINS1 1 /* penalty per base */ #define PINS0 8 /* penalty for a gap
*/ #define PINS1 4 /* penalty per residue */ struct jmp { short
n[MAXJMP]; /* size of jmp (neg for dely) */ unsigned short
x[MAXJMP]; /* base no. of jmp in seq x */ }; /* limits seq to
2{circumflex over ( )}16 -1 */ struct diag { int score; /* score at
last jmp */ long offset; /* offset of prev block */ short ijmp; /*
current jmp index */ struct jmp jp; /* list of jmps */ }; struct
path { int spc; /* number of leading spaces */ short n[JMPS];/*
size of jmp (gap) */ int x[JMPS];/* loc of jmp (last elem before
gap) */ }; char *ofile; /* output file name */ char *namex[2]; /*
seq names: getseqs( ) */ char *prog; /* prog name for err msgs */
char *seqx[2]; /* seqs: getseqs( ) */ int dmax; /* best diag: nw( )
*/ int dmax0; /* final diag */ int dna; /* set if dna: main( ) */
int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /*
total gaps in seqs */ int len0, len1; /* seq lens */ int ngapx,
ngapy; /* total size of gaps */ int smax; /* max score: nw( ) */
int *xbm; /* bitmap for matching */ long offset; /* current offset
in jmp file */ struct diag *dx; /* holds diagonals */ struct path
pp[2]; /* holds path for seqs */ char *calloc( ), *malloc( ),
*index( ), *strcpy( ); char *getseq( ), *g_calloc( );
[0504] TABLE-US-00009 TABLE 3C /* Needleman-Wunsch alignment
program * * usage: progs file1 file2 * where file1 and file2 are
two dna or two protein sequences. * The sequences can be in upper-
or lower-case an may contain ambiguity * Any lines beginning with
`;`, `>` or `<` are ignored * Max file length is 65535
(limited by unsigned short x in the jmp struct) * A sequence with
1/3 or more of its elements ACGTU is assumed to be DNA * Output is
in the file "align.out" * * The program may create a tmp file in
/tmp to hold info about traceback. * Original version developed
under BSD 4.3 on a vax 8650 */ #include "nw.h" #include "day.h"
static _dbval[26] = {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static
_pbval[26] = { 1, 2|(1 < < (`D`-`A`))|(1 < <
(`N`-`A`)), 4, 8, 16, 32, 64, 128, 256, 0xFFFFFFF, 1 < < 10,
1 < < 11, 1 < < 12, 1 < < 13, 1 < < 14, 1
< < 15, 1 < < 16, 1 < < 17, 1 < < 18, 1
< < 19, 1 < < 20, 1 < < 21, 1 < < 22, 1
< < 23, 1 < < 24, 1 < < 25|(1 < <
(`E`-`A`))|(1 < < (`Q`-`A`)) }; main main(ac, av) int ac;
char *av[ ]; { prog = av[0]; if (ac != 3) { fprintf(stderr,"usage:
%s file1 file2\n", prog); fprintf(stderr,"where file1 and file2 are
two dna or two protein sequences.\n"); fprintf(stderr,"The
sequences can be in upper- or lower-case\n"); fprintf(stderr,"Any
lines beginning with `;` or `<` are ignored\n");
fprintf(stderr,"Output is in the file \"align.out\"\n"); exit(1); }
namex[0] = av[1]; namex[1] = av[2]; seqx[0] = getseq(namex[0],
&len0); seqx[1] = getseq(namex[1], &len1); xbm = (dna)?
_dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ ofile =
"align.out"; /* output file */ nw( ); /* fill in the matrix, get
the possible jmps */ readjmps( ); /* get the actual jmps */ print(
); /* print stats, alignment */ cleanup(0); /* unlink any tmp files
*/ }
[0505] TABLE-US-00010 TABLE 3D /* do the alignment, return best
score: main( ) * dna: values in Fitch and Smith, PNAS, 80,
1382-1386, 1983 * pro: PAM 250 values * When scores are equal, we
prefer mismatches to any gap, prefer * a new gap to extending an
ongoing gap, and prefer a gap in seqx * to a gap in seq y. */ nw( )
nw { char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /* keep
track of dely */ int ndelx, delx; /* keep track of delx */ int
*tmp; /* for swapping row0, row1 */ int mis; /* score for each type
*/ int ins0, ins1; /* insertion penalties */ register id; /*
diagonal index */ register ij; /* jmp index */ register *col0,
*col1; /* score for curr, last row */ register xx, yy; /* index
into seqs */ dx = (struct diag *)g_calloc("to get diags",
len0+len1+1, sizeof(struct diag)); ndely = (int *)g_calloc("to get
ndely", len1+1, sizeof(int)); dely = (int *)g_calloc("to get dely",
len1+1, sizeof(int)); col0 = (int *)g_calloc("to get col0", len1+1,
sizeof(int)); col1 = (int *)g_calloc("to get col1", len1+1,
sizeof(int)); ins0 = (dna)? DINS0 : PINS0; ins1 = (dna)? DINS1 :
PINS1; smax = -10000; if (endgaps) { for (col0[0] = dely[0] =
-ins0, yy = 1; yy <= len1; yy++) { col0[yy] = dely[yy] =
col0[yy-1] - ins1; ndely[yy] = yy; } col0[0] = 0; /* Waterman Bull
Math Biol 84 */ } else for (yy = 1; yy <= len1; yy++) dely[yy] =
-ins0; /* fill in match matrix */ for (px = seqx[0], xx = 1; xx
<= len0; px++, xx++) { /* initialize first entry in col */ if
(endgaps) { if (xx == 1) col1[0] = delx = -(ins0+ins1); else
col1[0] = delx = col0[0] - ins1; ndelx = xx; } else { col1[0] = 0;
delx = -ins0; ndelx = 0; }
[0506] TABLE-US-00011 TABLE 3E ...nw for (py = seqx[1], yy = 1; yy
<= len1; py++, yy++) { mis = col0[yy-1]; if (dna) mis +=
(xbm[*px-`A`]&xbm[*py-`A`])? DMAT : DMIS; else mis +=
_day[*px-`A`][*py-`A`]; /* update penalty for del in x seq; * favor
new del over ongong del * ignore MAXGAP if weighting endgaps */ if
(endgaps || ndely[yy] < MAXGAP) { if (col0[yy] - ins0 >=
dely[yy]) { dely[yy] = col0[yy] - (ins0+ins1); ndely[yy] = 1; }
else { dely[yy] -= ins1; ndely[yy]++; } } else { if (col0[yy] -
(ins0+ins1) >= dely[yy]) { dely[yy] = col0[yy] - (ins0+ins1);
ndely[yy] = 1; } else ndely[yy]++; } /* update penalty for del in y
seq; * favor new del over ongong del */ if (endgaps || ndelx <
MAXGAP) { if (col1[yy-1] - ins0 >= delx) { delx = col1[yy-1] -
(ins0+ins1); ndelx = 1; } else { delx -= ins1; ndelx++; } } else {
if (col1[yy-1] - (ins0+ins1) >= delx) { delx = col1[yy-1] -
(ins0+ins1); ndelx = 1; } else ndelx++; } /* pick the maximum
score; we're favoring * mis over any del and delx over dely */
[0507] TABLE-US-00012 TABLE 3F ..nw id = xx - yy + len1 - 1; if
(mis >= delx && mis >= dely[yy]) col1[yy] = mis; else
if (delx >= dely[yy]) { col1[yy] = delx; ij = dx[id].ijmp; if
(dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP &&
xx > dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) {
dx[id].ijmp++; if (++ij >= MAXJMP) { writejmps(id); ij =
dx[id].ijmp = 0; dx[id].offset = offset; offset += sizeof(struct
jmp) + sizeof(offset); } } dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij]
= xx; dx[id].score = delx; } else { col1[yy] = dely[yy]; ij =
dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndely[yy]
>= MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >
dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {
writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset
+= sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] =
-ndely[yy]; dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx
== len0 && yy < len1) { /* last col */ if (endgaps)
col1[yy] - = ins0+ins1*(len1-yy); if (col1[yy] > smax) { smax =
col1[yy]; dmax = id; } } } if (endgaps && xx < len0)
col1[yy-1] -= ins0+ins1*(len0-xx); if (col1[yy-1] > smax) { smax
= col1[yy-1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; }
(void) free((char *)ndely); (void) free((char *)dely); (void)
free((char *)col0); (void) free((char *)col1); }
[0508] TABLE-US-00013 TABLE 3G /* * * print( ) -- only routine
visible outside this module * * static: * getmat( ) -- trace back
best path, count matches: print( ) * pr_align( ) -- print alignment
of described in array p[ ]: print( ) * dumpblock( ) -- dump a block
of lines with numbers, stars: pr_align( ) * nums( ) -- put out a
number line: dumpblock( ) * putline( ) -- put out a line (name,
[num], seq, [num]): dumpblock( ) * stars( ) - - put a line of
stars: dumpblock( ) * stripname( ) -- strip any path and prefix
from a seqname */ #include "nw.h" #define SPC 3 #define P_LINE 256
/* maximum output line */ #define P_SPC 3 /* space between name or
num and seq */ extern _day[26][26]; int olen; /* set output line
length */ FILE *fx; /* output file */ print( ) print { int lx, ly,
firstgap, lastgap; /* overlap */ if ((fx = fopen(ofile, "w")) == 0)
{ fprintf(stderr,"%s: can't write %s\n", prog, ofile); cleanup(1);
} fprintf(fx, "<first sequence: %s (length = %d)\n"., namex[0],
len0); fprintf(fx, "<second sequence: %s (length = %d)\n",
namex[1], len1); olen = 60; lx = len0; ly = len1; firstgap =
lastgap = 0; if (dmax < len1 - 1) { /* leading gap in x */
pp[0].spc = firstgap = len1 - dmax - 1; ly -= pp[0].spc; } else if
(dmax > len1 - 1) { /* leading gap in y */ pp[1].spc = firstgap
= dmax - (len1 - 1); lx -= pp[1].spc; } if (dmax0 < len0 - 1) {
/* trailing gap in x */ lastgap = len0 - dmax0 -1; lx -= lastgap; }
else if (dmax0 > len0 - 1) { /* trailing gap in y */ lastgap =
dmax0 - (len0 - 1); ly -= lastgap; } getmat(lx, ly, firstgap,
lastgap); pr_align( ); }
[0509] TABLE-US-00014 TABLE 3H /* * trace back the best path, count
matches */ static getmat(lx, ly, firstgap, lastgap) getmat int lx,
ly; /* "core" (minus endgaps) */ int firstgap, lastgap; /* leading
trailing overlap */ { int nm, i0, i1, siz0, siz1; char outx[32];
double pct; register n0, n1; register char *p0, *p1; /* get total
matches, score */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] +
pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =
pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if (siz0) {
p1++; n1++; siz0--; } else if (siz1) { p0++; n0++; siz1--; } else {
if (xbm[*p0-`A`]&xbm[*p1-`A`]) nm++; if (n0++ == pp[0].x[i0])
siz0 = pp[0].n[i0++]; if (n1++ == pp[1].x[i1]) siz1 =
pp[1].n[i1++]; p0++; p1++; } } /* pct homology: * if penalizing
endgaps, base is the shorter seq * else, knock off overhangs and
take shorter core */ if (endgaps) lx = (len0 < len1)? len0 :
len1; else lx = (lx < ly)? lx : ly; pct =
100.*(double)nm/(double)lx; fprintf(fx, "\n"); fprintf(fx, "< %d
match%s in an overlap of %d: %.2f percent similarity\n", nm, (nm ==
1)? "" : "es", lx, pct);
[0510] TABLE-US-00015 TABLE 3I fprintf(fx, "<gaps in first
sequence: %d", gapx); ...getmat if (gapx) { (void) sprintf(outx, "
(%d %s%s)", ngapx, (dna)? "base":"residue", (ngapx == 1)? "":"s");
fprintf(fx,"%s", outx); fprintf(fx, ", gaps in second sequence:
%d", gapy); if (gapy) { (void) sprintf(outx, " (%d %s%s)", ngapy,
(dna)? "base":"residue", (ngapy == 1)? "":"s"); fprintf(fx,"%s",
outx); } if (dna) fprintf(fx, "\n<score: %d (match = %d,
mismatch = %d, gap penalty = %d + %d per base)\n", smax, DMAT,
DMIS, DINS0, DINS1); else fprintf(fx, "\n<score: %d (Dayhoff PAM
250 matrix, gap penalty = %d + %d per residue)\n", smax, PINS0,
PINS1); if (endgaps) fprintf(fx, "<endgaps penalized. left
endgap: %d %s%s, right endgap: %d %s%s\n", firstgap, (dna)? "base"
: "residue", (firstgap == 1)? "" : "s", lastgap, (dna)? "base" :
"residue", (lastgap == 1)? "" : "s"); else fprintf(fx, "<endgaps
not penalized\n"); } static nm; /* matches in core -- for checking
*/ static lmax; /* lengths of stripped file names */ static ij[2];
/* jmp index for a path */ static nc[2]; /* number at start of
current line */ static ni[2]; /* current elem number -- for gapping
*/ static siz[2]; static char *ps[2]; /* ptr to current element */
static char *po[2]; /* ptr to next output char slot */ static char
out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* set
by stars( ) */ /* * print alignment of described in struct path pp[
] */ static pr_align( ) pr_align { int nn; /* char count */ int
more; register i; for (i = 0, lmax = 0; i < 2; i++) { nn =
stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i]
= 1; siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i];
[0511] TABLE-US-00016 TABLE 3J for (nn = nm = 0, more = 1; more; )
{ ...pr_align for (i = more = 0; i < 2; i++) { /* * do we have
more of this sequence? */ if (!*ps[i]) continue; more++; if
(pp[i].spc) { /* leading space */ *po[i]++ = ` `; pp[i].spc--; }
else if (siz[i]) { /* in a gap */ *po[i]++ = `-`; siz[i]--; } else
{ /* we're putting a seq element */ *po[i] = *ps[i]; if
(islower(*ps[i])) *ps[i] = toupper(*ps[i]); po[i]++; ps[i]++; /* *
are we at next gap for this seq? */ if (ni[i] == pp[i].x[ij[i]]) {
/* * we need to merge all gaps * at this location */ siz[i] =
pp[i].n[ij[i]++]; while (ni[i] == pp[i].x[ij[i]]) siz[i] +=
pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn == olen || !more
&& nn) { dumpblock( ); for (i = 0; i < 2; i++) po[i] =
out[i]; nn = 0; } } } /* * dump a block of lines, including
numbers, stars: pr_align( ) */ static dumpblock( ) dumpblock {
register i; for (i = 0; i < 2; i++) *po[i]-- = `\0`;
[0512] TABLE-US-00017 TABLE 3K ...dumpblock (void) putc(`\n`, fx);
for(i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ` `
|| *(po[i]) != ` `)) { if (i == 0) nums(i); if (i == 0 &&
*out[1]) stars( ); putline(i); if (i == 0 && *out[1])
fprintf(fx, star); if (i == 1) nums(i); } } } /* * put out a number
line: dumpblock( ) */ static nums(ix) nums int ix; /* index in out[
] holding seq line */ { char nline[P_LINE]; register i, j; register
char *pn, *px, *py; for (pn = nline, i = 0; i < lmax+P_SPC; i++,
pn++) *pn = ` `; for (i = nc[ix], py = out[ix]; *py; py++, pn++) {
if (*py == ` ` || *py == `-`) *pn = ` `; else { if (i%10 == 0 || (i
== 1 && nc[ix] != 1)) { j = (i < 0)? -i : i; for (px =
pn; j; j /= 10, px--) *px = j%10 + `0`; if (i < 0) *px = `-`; }
else *pn = ` `; i++; } } *pn = `\0`; nc[ix] = i; for (pn = nline;
*pn; pn++) (void) putc(*pn, fx); (void) putc(`\n`, fx); } /* * put
out a line (name, [num], seq, [num]): dumpblock( ) */ static
putline(ix) putline int ix; {
[0513] TABLE-US-00018 TABLE 3L ...putline int i; register char *px;
for (px = namex[ix], i = 0; *px && *px != `:`; px++, i++)
(void) putc(*px, fx); for (; i < lmax+P_SPC; i++) (void) putc(`
`, fx); /* these count from 1: * ni[ ] is current element (from 1)
* nc[ ] is number at start of current line */ for (px = out[ix];
*px; px++) (void) putc(*px&0x7F, fx); (void) putc(`\n`, fx); }
/* * put a line of stars (seqs always in out[0], out[1]):
dumpblock( ) */ static stars( ) stars { int i; register char *p0,
*p1, cx, *px; if (!*out[0] || (*out[0] == ` ` && *(po[0])
== ` `) || !*out[1] || (*out[1] == ` ` && *(po[1]) == ` `))
return; px = star; for (i = lmax+P_SPC; i; i--) *px++ = ` `; for
(p0 = out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if
(isalpha(*p0) && isalpha(*p1)) { if
(xbm[*p0-`A`]&xbm[*p1-`A`]) { cx = `*`; nm++; } else if (!dna
&& _day[*p0-`A`][*p1-`A`] > 0) cx = `.`; else cx = ` `;
} else cx = ` `; *px++ = cx; } *px++ = `\n`; *px = `\0`; }
[0514] TABLE-US-00019 TABLE 3M /* * strip path or prefix from pn,
return len: pr_align( ) */ static stripname(pn) stripname char *pn;
/* file name (may be path) */ { register char *px, *py; py = 0; for
(px = pn; *px; px++) if(*px == `/`) py = px + 1; if (py) (void)
strcpy(pn, py); return(strlen(pn)); }
[0515] TABLE-US-00020 TABLE 3N /* * cleanup( ) -- cleanup any tmp
file * getseq( ) -- read in seq, set dna, len, maxlen * g_calloc( )
-- calloc( ) with error checkin * readjmps( ) -- get the good jmps,
from tmp file if necessary * writejmps( ) -- write a filled array
of jmps to a tmp file: nw( ) */ #include "nw.h" #include
<sys/file.h> char *jname = "/tmp/homgXXXXXX"; /* tmp file for
jmps */ FILE *fj; int cleanup( ); /* cleanup tmp file */ long
lseek( ); /* * remove any tmp file if we blow */ cleanup(i) cleanup
int i; { if(fj) (void) unlink(jname); exit(i); } /* * read, return
ptr to seq, set dna, len, maxlen * skip lines starting with `;`,
`<`, or `>` * seq in upper or lower case */ char *
getseq(file, len) getseq char *file; /* file name */ int *len; /*
seq len */ { char line[1024], *pseq; register char *px, *py; int
natgc, tlen; FILE *fp; if ((fp = fopen(file,"r")) == 0) {
fprintf(stderr, "%s: can't read %s\n", prog, file); exit(1); } tlen
= natgc = 0; while (fgets(line, 1024, fp)) { if (*line == `;` ||
*line == `<` || *line == `>`) continue; for (px = line; *px
!= `\n`; px++) if (isupper(*px) || islower(*px)) tlen++; } if
((pseq = malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr, "%s:
malloc( ) failed to get %d bytes for %s\n", prog, tlen+6, file);
exit(1); } pseq[0] = pseq[1] = pseq[2] = pseq[3] = `\0`;
[0516] TABLE-US-00021 TABLE 3O ...getseq py = pseq + 4; *len =
tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line == `;`
|| *line == `<` || *line == `>`) continue; for (px = line;
*px != `\n`; px++) { if (isupper(*px)) *py++ = *px; else if
(islower(*px)) *py++ = toupper(*px); if (index("ATGCU",*(py-1)))
natgc++; } } *py++ = `\0`; *py = `\0`; (void) fclose(fp); dna =
natgc > (tlen/3); return(pseq+4); } char * g_calloc(msg, nx, sz)
g_calloc char *msg; /* program, calling routine */ int nx, sz; /*
number and size of elements */ { char *px, *calloc( ); if ((px =
calloc((unsigned)nx, (unsigned)sz)) = = 0) { if (*msg) {
fprintf(stderr, "%s: g_calloc( ) failed %s (n = %d, sz = %d, prog,
msg, nx, sz); exit(1); } } return(px); } /* * get final jmps from
dx[ ] or tmp file, set pp[ ], reset dmax: main( ) */ readjmps( )
readjmps { int fd = -1; int siz, i0, i1; register i, j, xx; if (fj)
{ (void) fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) {
fprintf(stderr, "%s: can't open( ) %s\n", prog, jname); cleanup(1);
} } for (i = i0 = i1 = 0, dmax0 = dmax, xx = len0; ; i++) { while
(1) { for (j = dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j]
>= xx; j--) ;
[0517] TABLE-US-00022 TABLE 3P ...readjmps if (j < 0 &&
dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset,
0); (void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax].offset,
sizeof(dx[dmax].offset)); dx[dmax].ijmp = MAXJMP-1; } else break; }
if (i >= JMPS) { fprintf(stderr, "%s: too many gaps in
alignment\n", prog); cleanup(1); } if (j >= 0) { siz =
dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax += siz; if (siz <
0) { /* gap in second seq */ pp[1].n[i1] = -siz; xx += siz; /* id =
xx - yy + len1 - 1 */ pp[1].x[i1] = xx - dmax + len1 - 1; gapy++;
ngapy -= siz; /* ignore MAXGAP when doing endgaps */ siz = (-siz
< MAXGAP || endgaps)? -siz : MAXGAP; i1++; } else if (siz >
0) { /* gap in first seq */ pp[0].n[i0] = siz; pp[0].x[i0] = xx;
gapx++; ngapx += siz; /* ignore MAXGAP when doing endgaps */ siz =
(siz < MAXGAP || endgaps)? siz : MAXGAP; i0++; } } else break; }
/* reverse the order of jmps */ for (j = 0, i0--; j < i0; j++,
i0--) { i = pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i;
i = pp[0].x[j]; pp[0].x[j] = pp[0].x[i0]; pp[0].x[i0] = i; } for (j
= 0, i1--; j < i1; j++, i1--) { i = pp[1].n[j]; pp[1].n[j] =
pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j]; pp[1].x[j] =
pp[1].x[i1]; pp[1].x[i1] = i; } if (fd >= 0) (void) close(fd);
if (fj) { (void) unlink(jname); fj = 0; offset = 0;
[0518] TABLE-US-00023 TABLE 3Q /* * write a filled jmp struct
offset of the prev one (if any): nw( ) */ writejmps(ix) writejmps
int ix; { char *mktemp( ); if (!fj) { if (mktemp(jname) < 0) {
fprintf(stderr, "%s: can't mktemp( ) %s\n", prog, jname);
cleanup(1); } if ((fj = fopen(jname, "w")) == 0) { fprintf(stderr,
"%s: can't write %s\n", prog, jname); exit(1); } } (void)
fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)
fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj);
}
[0519]
Sequence CWU 1
1
32 1 1006 DNA Homo sapiens 1 ggcacgaggc aagccttcca ggttatcgtg
acgcaccttg aaagtctgag 50 agctactgcc ctacagaaag ttactagtgc
cctaaagctg gcgctggcac 100 tgatgttact gctgctgttg gagtacaact
tccctataga aaacaactgc 150 cagcacctta agaccactca caccttcaga
gtggccttga gaaagatttg 200 gggtcaagga tcatgagcga gaacaccact
taagaggata gtgaactagt 250 ctgcatgtga gacgctgaga tcctatgtca
ggctgtgata ggagggaaac 300 agaaaccaaa ggaaagaaca gctttaagaa
gcgcttaaga gccacccacc 350 cattcttgac agtcactggc ccagcctggg
ggcccctgtt ctttatcaaa 400 caagtgcctg agctctttgc agaggtccaa
aggtgaagaa cttaaacccg 450 aagaaattca gcattcatga ccaggatcac
aaagtactgg tcctggactc 500 tgggaatctc atagcagttc cagataaaaa
ctacatacgc ccagagatct 550 tctttgcatt agcctcatcc ttgagctcag
cctctgcgga gaaaggaagt 600 ccgattctcc tgggggtctc taaaggggag
ttttgtctct actgtgacaa 650 ggataaagga caaagtcatc catcccttca
gctgaagaag gagaaactga 700 tgaagctggc tgcccaaaag gaatcagcac
gccggccctt catcttttat 750 agggctcagg tgggctcctg gaacatgctg
gagtcggcgg ctcaccccgg 800 atggttcatc tgcacctcct gcaattgtaa
tgagcctgtt ggggtgacag 850 ataaatttga gaacaggaaa cacattgaat
tttcatttca accagtttgc 900 aaagctgaaa tgagccccag tgaggtcagc
gattaggaaa ctgccccatt 950 gaacgccttc ctcgctaatt tgaactaatt
gtataaaaac accaaacctg 1000 ctcact 1006 2 26 PRT Homo sapiens 2 Met
Leu Leu Leu Leu Leu Glu Tyr Asn Phe Pro Ile Glu Asn Asn 1 5 10 15
Cys Gln His Leu Lys Thr Thr His Thr Phe Arg 20 25 3 167 PRT Homo
sapiens 3 Val Lys Asn Leu Asn Pro Lys Lys Phe Ser Ile His Asp Gln
Asp 1 5 10 15 His Lys Val Leu Val Leu Asp Ser Gly Asn Leu Ile Ala
Val Pro 20 25 30 Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala
Leu Ala Ser 35 40 45 Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser
Pro Ile Leu Leu 50 55 60 Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr
Cys Asp Lys Asp Lys 65 70 75 Gly Gln Ser His Pro Ser Leu Gln Leu
Lys Lys Glu Lys Leu Met 80 85 90 Lys Leu Ala Ala Gln Lys Glu Ser
Ala Arg Arg Pro Phe Ile Phe 95 100 105 Tyr Arg Ala Gln Val Gly Ser
Trp Asn Met Leu Glu Ser Ala Ala 110 115 120 His Pro Gly Trp Phe Ile
Cys Thr Ser Cys Asn Cys Asn Glu Pro 125 130 135 Val Gly Val Thr Asp
Lys Phe Glu Asn Arg Lys His Ile Glu Phe 140 145 150 Ser Phe Gln Pro
Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val 155 160 165 Ser Asp 4
650 DNA Artificial Sequence recombinant DNA 4 taattcacca tgtctgcact
tctgatccta gctcttgttg gagctgcagt 50 tgctgactac aaagacgatg
acgacaagct tgcggccgcg aattcagctc 100 tttgcagagg tccaaaggtg
aagaacttaa acccgaagaa attcagcatt 150 catgaccagg atcacaaagt
actggtcctg gactctggga atctcatagc 200 agttccagat aaaaactaca
tacgcccaga gatcttcttt gcattagcct 250 catccttgag ctcagcctct
gcggagaaag gaagtccgat tctcctgggg 300 gtctctaaag gggagttttg
tctctactgt gacaaggata aaggacaaag 350 tcatccatcc cttcagctga
agaaggagaa actgatgaag ctggctgccc 400 aaaaggaatc agcacgccgg
cccttcatct tttatagggc tcaggtgggc 450 tcctggaaca tgctggagtc
ggcggctcac cccggatggt tcatctgcac 500 ctcctgcaat tgtaatgagc
ctgttggggt gacagataaa tttgagaaca 550 ggaaacacat tgaattttca
tttcaaccag tttgcaaagc tgaaatgagc 600 cccagtgagg tcagcgatta
gggtaccagt cgactctaga ggatcccggg 650 5 203 PRT Artificial Sequence
recombinant protein 5 Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly
Ala Ala Val Ala 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Lys Leu Ala
Ala Ala Asn Ser Ala 20 25 30 Leu Cys Arg Gly Pro Lys Val Lys Asn
Leu Asn Pro Lys Lys Phe 35 40 45 Ser Ile His Asp Gln Asp His Lys
Val Leu Val Leu Asp Ser Gly 50 55 60 Asn Leu Ile Ala Val Pro Asp
Lys Asn Tyr Ile Arg Pro Glu Ile 65 70 75 Phe Phe Ala Leu Ala Ser
Ser Leu Ser Ser Ala Ser Ala Glu Lys 80 85 90 Gly Ser Pro Ile Leu
Leu Gly Val Ser Lys Gly Glu Phe Cys Leu 95 100 105 Tyr Cys Asp Lys
Asp Lys Gly Gln Ser His Pro Ser Leu Gln Leu 110 115 120 Lys Lys Glu
Lys Leu Met Lys Leu Ala Ala Gln Lys Glu Ser Ala 125 130 135 Arg Arg
Pro Phe Ile Phe Tyr Arg Ala Gln Val Gly Ser Trp Asn 140 145 150 Met
Leu Glu Ser Ala Ala His Pro Gly Trp Phe Ile Cys Thr Ser 155 160 165
Cys Asn Cys Asn Glu Pro Val Gly Val Thr Asp Lys Phe Glu Asn 170 175
180 Arg Lys His Ile Glu Phe Ser Phe Gln Pro Val Cys Lys Ala Glu 185
190 195 Met Ser Pro Ser Glu Val Ser Asp 200 6 754 DNA Homo sapiens
6 ggcacgaggc aagccttcca ggttatcgtg acgcaccttg aaagtctgag 50
agctactgcc ctacagaaag ttactagtgc cctaaagctg gcgctggcac 100
tgatgttact gctgctgttg gagtacaact tccctataga aaacaactgc 150
cagcacctta agaccactca caccttcaga gtgaagaact taaacccgaa 200
gaaattcagc attcatgacc aggatcacaa agtactggtc ctggactctg 250
ggaatctcat agcagttcca gataaaaact acatacgccc agagatcttc 300
tttgcattag cctcatcctt gagctcagcc tctgcggaga aaggaagtcc 350
gattctcctg ggggtctcta aaggggagtt ttgtctctac tgtgacaagg 400
ataaaggaca aagtcatcca tcccttcagc tgaagaagga gaaactgatg 450
aagctggctg cccaaaagga atcagcacgc cggcccttca tcttttatag 500
ggctcaggtg ggctcctgga acatgctgga gtcggcggct caccccggat 550
ggttcatctg cacctcctgc aattgtaatg agcctgttgg ggtgacagat 600
aaatttgaga acaggaaaca cattgaattt tcatttcaac cagtttgcaa 650
agctgaaatg agccccagtg aggtcagcga ttaggaaact gccccattga 700
acgccttcct cgctaatttg aactaattgt ataaaaacac caaacctgct 750 cact 754
7 193 PRT Homo sapiens 7 Met Leu Leu Leu Leu Leu Glu Tyr Asn Phe
Pro Ile Glu Asn Asn 1 5 10 15 Cys Gln His Leu Lys Thr Thr His Thr
Phe Arg Val Lys Asn Leu 20 25 30 Asn Pro Lys Lys Phe Ser Ile His
Asp Gln Asp His Lys Val Leu 35 40 45 Val Leu Asp Ser Gly Asn Leu
Ile Ala Val Pro Asp Lys Asn Tyr 50 55 60 Ile Arg Pro Glu Ile Phe
Phe Ala Leu Ala Ser Ser Leu Ser Ser 65 70 75 Ala Ser Ala Glu Lys
Gly Ser Pro Ile Leu Leu Gly Val Ser Lys 80 85 90 Gly Glu Phe Cys
Leu Tyr Cys Asp Lys Asp Lys Gly Gln Ser His 95 100 105 Pro Ser Leu
Gln Leu Lys Lys Glu Lys Leu Met Lys Leu Ala Ala 110 115 120 Gln Lys
Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr Arg Ala Gln 125 130 135 Val
Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro Gly Trp 140 145 150
Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val Thr 155 160
165 Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro 170
175 180 Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp 185 190
8 629 DNA Homo sapiens unsure 13 unknown base 8 ccaggcccaa
gcntccccac catgaatttt gttcacacaa gtcgaaaggt 50 gaagagctta
aacccgaaga aattcagcat tcatgaccag gatcacaaag 100 tactggcctg
gactctggga atctcatagc agttccagat aaaaactaca 150 tacgcccaga
gatcttcttt gcattagcct catccttgag ctcagcctct 200 gcggagaaag
gaagtccgat tctcctgggg gtctctaaag gggagttttg 250 tctctactgt
gacaaggata aaggacaaag tcatccatcc cttcagctga 300 agaaggagaa
actgatgaag ctggctgccc aaaaggaatc agcacgccgg 350 cccttcatct
tttatagggc tcaggtgggc tcctggaaca tgctggagtc 400 ggcggctcac
cccggatggt tcatctgcac ctcctgcaat tgtaatgagc 450 ctgttggggt
gacagataaa tttgagaaca ggaaacacat tgaattttca 500 tttcaaccag
tttgcaaagc tgaaatgagc cccagtgagg tcagcgatta 550 ggaaactgcc
ccattgaacg ccttcctcgc taatttgaac taattgtata 600 aaaaccccaa
acctgctcac taaaaaaaa 629 9 1321 DNA Homo sapiens 9 gtcgacccac
gcgtccgaag ctgctggagc cacgattcag tcccctggac 50 tgtagataaa
gaccctttct tgccaggtgc tgagacaacc acactatgag 100 aggcactcca
ggagacgctg atggtggagg aagggccgtc tatcaatcaa 150 tcactgttgc
tgttatcaca tgcaagtatc cagaggctct tgagcaaggc 200 agaggggatc
ccatttattt gggaatccag aatccagaaa tgtgtttgta 250 ttgtgagaag
gttggagaac agcccacatt gcagctaaaa gagcagaaga 300 tcatggatct
gtatggccaa cccgagcccg tgaaaccctt ccttttctac 350 cgtgccaaga
ctggtaggac ctccaccctt gagtctgtgg ccttcccgga 400 ctggttcatt
gcctcctcca agagagacca gcccatcatt ctgacttcag 450 aacttgggaa
gtcatacaac actgcctttg aattaaatat aaatgactga 500 actcagccta
gaggtggcag cttggtcttt gtcttaaagt ttctggttcc 550 caatgtgttt
tcgtctacat tttcttagtg tcattttcac gctggtgctg 600 agacaggagc
aaggctgctg ttatcatctc attttataat gaagaagaag 650 caattacttc
atagcaactg aagaacagga tgtggcctca gaagcaggag 700 agctgggtgg
tataaggctg tcctctcaag ctggtgctgt gtaggccaca 750 aggcatctgc
atgagtgact ttaagactca aagaccaaac actgagcttt 800 cttctagggg
tgggtatgaa gatgcttcag agctcatgcg cgttacccac 850 gatggcatga
ctagcacaga gctgatctct gtttctgttt tgctttattc 900 cctcttggga
tgatatcatc cagtctttat atgttgccaa tatacctcat 950 tgtgtgtaat
agaaccttct tagcattaag accttgtaaa caaaaataat 1000 tcttggggtg
ggtatgaaga tgcttcagag ctcatgcgcg ttacccacga 1050 tggcatgact
agcacagagc tgatctctgt ttctgttttg ctttattccc 1100 tcttgggatg
atatcatcca gtctttatat gttgccaata tacctcattg 1150 tgtgtaatag
aaccttctta gcattaagac cttgtaaaca aaaataattc 1200 ttgtgttaag
ttaaatcatt tttgtcctaa ttgtaatgtg taatcttaaa 1250 gttaaataaa
ctttgtgtat ttatataata ataaagctaa aactgatata 1300 aaataaagaa
agagtaaact g 1321 10 134 PRT Homo sapiens 10 Met Arg Gly Thr Pro
Gly Asp Ala Asp Gly Gly Gly Arg Ala Val 1 5 10 15 Tyr Gln Ser Ile
Thr Val Ala Val Ile Thr Cys Lys Tyr Pro Glu 20 25 30 Ala Leu Glu
Gln Gly Arg Gly Asp Pro Ile Tyr Leu Gly Ile Gln 35 40 45 Asn Pro
Glu Met Cys Leu Tyr Cys Glu Lys Val Gly Glu Gln Pro 50 55 60 Thr
Leu Gln Leu Lys Glu Gln Lys Ile Met Asp Leu Tyr Gly Gln 65 70 75
Pro Glu Pro Val Lys Pro Phe Leu Phe Tyr Arg Ala Lys Thr Gly 80 85
90 Arg Thr Ser Thr Leu Glu Ser Val Ala Phe Pro Asp Trp Phe Ile 95
100 105 Ala Ser Ser Lys Arg Asp Gln Pro Ile Ile Leu Thr Ser Glu Leu
110 115 120 Gly Lys Ser Tyr Asn Thr Ala Phe Glu Leu Asn Ile Asn Asp
125 130 11 249 DNA Homo sapiens 11 aagctgctgg agccacgatt cagtcccctg
gactgtagat aaagaccctt 50 tcttgccagg tgctgagaca accacactat
gagaggcact ccaggagacg 100 ctgatggtgg aggaagggcc gtctatcaat
caatcactgt tgctgttatc 150 acatgcaagt atccagaggc tcttgagcaa
ggcagagggg atcccattta 200 tttgggaatc cagaatccag aaatgtgttt
gtattgtgag aaggttgga 249 12 468 DNA Homo sapiens 12 atggtcctga
gtggggcgct gtgcttccga atgaaggact cggcattgaa 50 ggtgctttat
ctgcataata accagcttct agctggaggg ctgcatgcag 100 ggaaggtcat
taaaggtgaa gagatcagcg tggtccccaa tcggtggctg 150 gatgccagcc
tgtcccccgt catcctgggt gtccagggtg gaagccagtg 200 cctgtcatgt
ggggtggggc aggagccgac tctaacacta gagccagtga 250 acatcatgga
gctctatctt ggtgccaagg aatccaagag cttcaccttc 300 taccggcggg
acatggggct cacctccagc ttcgagtcgg ctgcctaccc 350 gggctggttc
ctgtgcacgg tgcctgaagc cgatcagcct gtcagactca 400 cccagcttcc
cgagaatggt ggctggaatg cccccatcac agacttctac 450 ttccagcagt gtgactag
468 13 155 PRT Homo sapiens 13 Met Val Leu Ser Gly Ala Leu Cys Phe
Arg Met Lys Asp Ser Ala 1 5 10 15 Leu Lys Val Leu Tyr Leu His Asn
Asn Gln Leu Leu Ala Gly Gly 20 25 30 Leu His Ala Gly Lys Val Ile
Lys Gly Glu Glu Ile Ser Val Val 35 40 45 Pro Asn Arg Trp Leu Asp
Ala Ser Leu Ser Pro Val Ile Leu Gly 50 55 60 Val Gln Gly Gly Ser
Gln Cys Leu Ser Cys Gly Val Gly Gln Glu 65 70 75 Pro Thr Leu Thr
Leu Glu Pro Val Asn Ile Met Glu Leu Tyr Leu 80 85 90 Gly Ala Lys
Glu Ser Lys Ser Phe Thr Phe Tyr Arg Arg Asp Met 95 100 105 Gly Leu
Thr Ser Ser Phe Glu Ser Ala Ala Tyr Pro Gly Trp Phe 110 115 120 Leu
Cys Thr Val Pro Glu Ala Asp Gln Pro Val Arg Leu Thr Gln 125 130 135
Leu Pro Glu Asn Gly Gly Trp Asn Ala Pro Ile Thr Asp Phe Tyr 140 145
150 Phe Gln Gln Cys Asp 155 14 295 DNA Homo sapiens unsure 283
unknown base 14 gctcccgcca ggagaaagga acattctgag gggagtctac
accctgtgga 50 gctcaagatg gtcctgagtg gggcgctgtg cttccgaatg
aaggactcgg 100 cattgaaggt gctttatctg cataataacc agcttctagc
tggagggctg 150 catgcaggga aggtcattaa aggtgaagag atcagcgtgg
tccccaatcg 200 gtggctggat gccagcctgt cccccgtcat cctgggtgtc
cagggtggaa 250 gccagtgcct gtcatgtggg gtggggcagg agncgactct aacat
295 15 1385 DNA Mus musculus 15 atagggaatt tggccctcga ggccaagaat
tcggcacgag gggagcctgc 50 tttctactta ggtctcaaat tttccagcct
tgtctttgcc taaaatttcc 100 tgctgtttat ttcaaaatag ggtctacata
ctgtggagct catgatggtt 150 ctgagtgggg cactatgctt ccgaatgaag
gattcagcct tgaaggtact 200 gtatctgcac aataaccagc tgctggctgg
aggactgcac gcagagaagg 250 tcattaaagg tgaggagatc agtgttgtcc
caaatcgggc actggatgcc 300 agtctgtccc ctgtcatcct gggcgttcaa
ggaggaagcc agtgcctatc 350 ttgtgggaca gagaaagggc caattctgaa
acttgagcca gtgaacatca 400 tggagctcta cctcggggcc aaggaatcaa
agagcttcac cttctaccgg 450 cgggatatgg gtcttacctc cagcttcgaa
tccgctgcct acccaggctg 500 gttcctctgc acctcaccgg aagctgacca
gcctgtcagg ctcactcaga 550 tccctgagga ccccgcctgg gatgctccca
tcacagactt ctactttcag 600 cagtgtgact agggctgcgt ggtccccaaa
actccataag cagaggcaga 650 gtaggcagtg gcggctcctg atagaggata
gagagacaga ggagctccac 700 agtaggtggc ttactcctct ccttccctac
tggactcccg cttctgacct 750 aaggcacaca gacactctct tctcctgcat
cccagtgctg gtaaatcttc 800 tggtatttgg agctcaatgt gtagattctt
tcagattgga tggtactacc 850 tctggtgtgg aacccaatag aaaccacgta
ggaccaacaa agagcaacat 900 aaaagattct tgggtgaaga agaggtggga
actgttcata catagtaaga 950 tctgacacag tacctcagaa gtcctgccat
tccttatgtt ctggagaaag 1000 tggagggggg gtcaccaaga ctttctctgg
ctggctgggc cctttccctc 1050 aacctttctg acatctgcag cctctctcat
tcttgccttc attctctggc 1100 cctgaaccga gagggtgata tcaggatagc
tgacagaaga tgaccaggca 1150 cactgtcctg gtttgaaacc agaggggaca
ataaaaaacc ctgattctgg 1200 tctctactca cataaaaaga agcttgtgaa
cattaagtgg gaagagattg 1250 ctactaaata acataccttg taatttcatc
ttaattaaaa tatacttctc 1300 tatattatat attttaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1350 aaaaacatgc ggccgcaagc ttattccatt tagga
1385 16 155 PRT Mus musculus 16 Met Val Leu Ser Gly Ala Leu Cys Phe
Arg Met Lys Asp Ser Ala 1 5 10 15 Leu Lys Val Leu Tyr Leu His Asn
Asn Gln Leu Leu Ala Gly Gly 20 25 30 Leu His Ala Glu Lys Val Ile
Lys Gly Glu Glu Ile Ser Val Val 35 40 45 Pro Asn Arg Ala Leu Asp
Ala Ser Leu Ser Pro Val Ile Leu Gly 50 55 60 Val Gln Gly Gly Ser
Gln Cys Leu Ser Cys Gly Thr Glu Lys Gly 65 70 75 Pro Ile Leu Lys
Leu Glu Pro Val Asn Ile Met Glu Leu Tyr Leu 80 85
90 Gly Ala Lys Glu Ser Lys Ser Phe Thr Phe Tyr Arg Arg Asp Met 95
100 105 Gly Leu Thr Ser Ser Phe Glu Ser Ala Ala Tyr Pro Gly Trp Phe
110 115 120 Leu Cys Thr Ser Pro Glu Ala Asp Gln Pro Val Arg Leu Thr
Gln 125 130 135 Ile Pro Glu Asp Pro Ala Trp Asp Ala Pro Ile Thr Asp
Phe Tyr 140 145 150 Phe Gln Gln Cys Asp 155 17 382 DNA Mus musculus
17 ggagcctgct ttctacttag gtctcaaatt ttccagcctt gtctttgcct 50
aaaatttcct gctgtttatt tcaaaatagg gtctacatac tgtggagctc 100
atgatggttc tgagtggggc actatgcttc cgaatgaagg attcagcctt 150
gaaggtactg tatctgcaca ataaccagct gctggctgga ggactgcacg 200
cagagaaggt cattaaaggt gaggagatca gtgttgtccc aaatcgggca 250
ctggatgcca gtctgtcccc tgtcatcctg ggcgttcaag gaggaagcca 300
gtgcctatct tgtgggacag agaaagggcc aattctgaaa cttgagccag 350
tgaacatcat ggagctctac ctcggggcca ag 382 18 626 DNA Homo sapiens 18
aaaatgggct ctgaggactg ggaaaaagat gaaccccagt gctgcttaga 50
agacccggct gtaagccccc tggaaccagg cccaagcctc cccgccatga 100
attttgttca cacaagtcca aaggtgaaga acttaaaccc gaagaaattc 150
agcattcatg accaggatca caaagtactg gtcctggact ctgggaatct 200
catagcagtt ccagataaaa actacatacg cccagagatc ttctttgcat 250
tagcctcatc cttgagctca gcctctgcgg agaaaggaag tccgattctc 300
ctgggggtct ctaaagggga gttttgtctc tactgtgaca aggataaagg 350
acaaagtcat ccatcccttc agctgaagaa ggagaaactg atgaagctgg 400
ctgcccaaaa ggaatcagca cgccggccct tcatctttta tagggctcag 450
gtgggctcct ggaacatgct ggagtcggcg gctcaccccg gatggttcat 500
ctgcacctcc tgcaattgta atgagcctgt tggggtgaca gataaatttg 550
agaacaggaa acacattgaa ttttcatttc aaccagtttg caaagctgaa 600
atgagcccca gtgaggtcag cgatta 626 19 207 PRT Homo sapiens 19 Met Gly
Ser Glu Asp Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu 1 5 10 15 Glu
Asp Pro Ala Val Ser Pro Leu Glu Pro Gly Pro Ser Leu Pro 20 25 30
Ala Met Asn Phe Val His Thr Ser Pro Lys Val Lys Asn Leu Asn 35 40
45 Pro Lys Lys Phe Ser Ile His Asp Gln Asp His Lys Val Leu Val 50
55 60 Leu Asp Ser Gly Asn Leu Ile Ala Val Pro Asp Lys Asn Tyr Ile
65 70 75 Arg Pro Glu Ile Phe Phe Ala Leu Ala Ser Ser Leu Ser Ser
Ala 80 85 90 Ser Ala Glu Lys Gly Ser Pro Ile Leu Leu Gly Val Ser
Lys Gly 95 100 105 Glu Phe Cys Leu Tyr Cys Asp Lys Asp Lys Gly Gln
Ser His Pro 110 115 120 Ser Leu Gln Leu Lys Lys Glu Lys Leu Met Lys
Leu Ala Ala Gln 125 130 135 Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe
Tyr Arg Ala Gln Val 140 145 150 Gly Ser Trp Asn Met Leu Glu Ser Ala
Ala His Pro Gly Trp Phe 155 160 165 Ile Cys Thr Ser Cys Asn Cys Asn
Glu Pro Val Gly Val Thr Asp 170 175 180 Lys Phe Glu Asn Arg Lys His
Ile Glu Phe Ser Phe Gln Pro Val 185 190 195 Cys Lys Ala Glu Met Ser
Pro Ser Glu Val Ser Asp 200 205 20 506 DNA Homo sapiens 20
aaaatgggct ctgaggactg ggaaaaagat gaaccccagt gctgcttaga 50
agacccggct gtaagccccc tggaaccagg cccaagcctc cccgccatga 100
attttgttca cacaaagatc ttctttgcat tagcctcatc cttgagctca 150
gcctctgcgg agaaaggaag tccgattctc ctgggggtct ctaaagggga 200
gttttgtctc tactgtgaca aggataaagg acaaagtcat ccatcccttc 250
agctgaagaa ggagaaactg atgaagctgg ctgcccaaaa ggaatcagca 300
cgccggccct tcatctttta tagggctcag gtgggctcct ggaacatgct 350
ggagtcggcg gctcaccccg gatggttcat ctgcacctcc tgcaattgta 400
atgagcctgt tggggtgaca gataaatttg agaacaggaa acacattgaa 450
ttttcatttc aaccagtttg caaagctgaa atgagcccca gtgaggtcag 500 cgatta
506 21 167 PRT Homo sapiens 21 Met Gly Ser Glu Asp Trp Glu Lys Asp
Glu Pro Gln Cys Cys Leu 1 5 10 15 Glu Asp Pro Ala Val Ser Pro Leu
Glu Pro Gly Pro Ser Leu Pro 20 25 30 Ala Met Asn Phe Val His Thr
Lys Ile Phe Phe Ala Leu Ala Ser 35 40 45 Ser Leu Ser Ser Ala Ser
Ala Glu Lys Gly Ser Pro Ile Leu Leu 50 55 60 Gly Val Ser Lys Gly
Glu Phe Cys Leu Tyr Cys Asp Lys Asp Lys 65 70 75 Gly Gln Ser His
Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 80 85 90 Lys Leu Ala
Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe 95 100 105 Tyr Arg
Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala 110 115 120 His
Pro Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro 125 130 135
Val Gly Val Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe 140 145
150 Ser Phe Gln Pro Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val 155
160 165 Ser Asp 22 561 DNA Homo sapiens 22 aacccgaaga aattcagcat
tcatgaccag gatcacaaag tactggtcct 50 ggactctggg aatctcatag
cagttccaga taaaaactac atacgcccag 100 agatcttctt tgcattagcc
tcatccttga gctcagcctc tgcggagaaa 150 ggaagtccga ttctcctggg
ggtctctaaa ggggagtttt gtctctactg 200 tgacaaggat aaaggacaaa
gtcatccatc ccttcagctg aagaaggaga 250 aactgatgaa gctggctgcc
caaaaggaat cagcacgccg gcccttcatc 300 ttttataggg ctcaggtggg
ctcctggaac atgctggagt cggcggctca 350 ccccggatgg ttcatctgca
cctcctgcaa ttgtaatgag cctgttgggg 400 tgacagataa atttgagaac
aggaaacaca ttgaattttc atttcaacca 450 gtttgcaaag ctgaaatgag
ccccagtgag gtcagcgatt aggaaactgc 500 cccattgaac gccttcctcg
ctaatttgaa ctaattgtat aaaaacacca 550 aacctgctca c 561 23 561 DNA
Homo sapiens 23 ttgggcttct ttaagtcgta agtactggtc ctagtgtttc
atgaccagga 50 cctgagaccc ttagagtatc gtcaaggtct atttttgatg
tatgcgggtc 100 tctagaagaa acgtaatcgg agtaggaact cgagtcggag
acgcctcttt 150 ccttcaggct aagaggaccc ccagagattt cccctcaaaa
cagagatgac 200 actgttccta tttcctgttt cagtaggtag ggaagtcgac
ttcttcctct 250 ttgactactt cgaccgacgg gttttcctta gtcgtgcggc
cgggaagtag 300 aaaatatccc gagtccaccc gaggaccttg tacgacctca
gccgccgagt 350 ggggcctacc aagtagacgt ggaggacgtt aacattactc
ggacaacccc 400 actgtctatt taaactcttg tcctttgtgt aacttaaaag
taaagttggt 450 caaacgtttc gactttactc ggggtcactc cagtcgctaa
tcctttgacg 500 gggtaacttg cggaaggagc gattaaactt gattaacata
tttttgtggt 550 ttggacgagt g 561 24 839 DNA Homo sapiens 24
ggccctcgag gccaagaatt cggcacgagg cttcattcca ttttctgttg 50
agtaataaac tcaacgttga aaatgtcctt tgtgggggag aactcaggag 100
tgaaaatggg ctctgaggac tgggaaaaag atgaacccca gtgctgctta 150
gaagacccgg ctggaagccc cctggaacca ggcccaagcc tccccaccat 200
gaattttgtt cacacaagtc caaaggtgaa gaacttaaac ccgaagaaat 250
tcagcattca tgaccaggat cacaaagtac tggtcctgga ctctgggaat 300
ctcatagcag ttccagataa aaactacata cgcccagaga tcttctttgc 350
attagcctca tccttgagct cagcctctgc ggagaaagga agtccgattc 400
tcctgggggt ctctaaaggg gagttttgtc tctactgtga caaggataaa 450
ggacaaagtc atccatccct tcagctgaag aaggagaaac tgatgaagct 500
ggctgcccaa aaggaatcag cacgccggcc cttcatcttt tatagggctc 550
aggtgggctc ctggaacatg ctggagtcgg cggctcaccc cggatggttc 600
atctgcacct cctgcaattg taatgagcct gttggggtga cagataaatt 650
tgagaacagg aaacacattg aattttcatt tcaaccagtt tgcaaagctg 700
aaatgagccc cagtgaggtc agcgattagg aaactgcccc attgaacgcc 750
ttcctcgcta atttgaacta attgtataaa aacaccaaac ctgctcacta 800
aaaaaaaaaa aaaaaaacgt ttgcggccgc aagcttatt 839 25 218 PRT Homo
sapiens 25 Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser
Glu 1 5 10 15 Asp Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu Glu Asp
Pro Ala 20 25 30 Gly Ser Pro Leu Glu Pro Gly Pro Ser Leu Pro Thr
Met Asn Phe 35 40 45 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn
Pro Lys Lys Phe 50 55 60 Ser Ile His Asp Gln Asp His Lys Val Leu
Val Leu Asp Ser Gly 65 70 75 Asn Leu Ile Ala Val Pro Asp Lys Asn
Tyr Ile Arg Pro Glu Ile 80 85 90 Phe Phe Ala Leu Ala Ser Ser Leu
Ser Ser Ala Ser Ala Glu Lys 95 100 105 Gly Ser Pro Ile Leu Leu Gly
Val Ser Lys Gly Glu Phe Cys Leu 110 115 120 Tyr Cys Asp Lys Asp Lys
Gly Gln Ser His Pro Ser Leu Gln Leu 125 130 135 Lys Lys Glu Lys Leu
Met Lys Leu Ala Ala Gln Lys Glu Ser Ala 140 145 150 Arg Arg Pro Phe
Ile Phe Tyr Arg Ala Gln Val Gly Ser Trp Asn 155 160 165 Met Leu Glu
Ser Ala Ala His Pro Gly Trp Phe Ile Cys Thr Ser 170 175 180 Cys Asn
Cys Asn Glu Pro Val Gly Val Thr Asp Lys Phe Glu Asn 185 190 195 Arg
Lys His Ile Glu Phe Ser Phe Gln Pro Val Cys Lys Ala Glu 200 205 210
Met Ser Pro Ser Glu Val Ser Asp 215 26 177 PRT Homo sapiens 26 Met
Glu Ile Cys Arg Gly Leu Arg Ser His Leu Ile Thr Leu Leu 1 5 10 15
Leu Phe Leu Phe His Ser Glu Thr Ile Cys Arg Pro Ser Gly Arg 20 25
30 Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln 35
40 45 Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu
50 55 60 Gln Gly Pro Asn Val Asn Leu Glu Glu Lys Ile Asp Val Val
Pro 65 70 75 Ile Glu Pro His Ala Leu Phe Leu Gly Ile His Gly Gly
Lys Met 80 85 90 Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr Arg
Leu Gln Leu 95 100 105 Glu Ala Val Asn Ile Thr Asp Leu Ser Glu Asn
Arg Lys Gln Asp 110 115 120 Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser
Gly Pro Thr Thr Ser 125 130 135 Phe Glu Ser Ala Ala Cys Pro Gly Trp
Phe Leu Cys Thr Ala Met 140 145 150 Glu Ala Asp Gln Pro Val Ser Leu
Thr Asn Met Pro Asp Glu Gly 155 160 165 Val Met Val Thr Lys Phe Tyr
Phe Gln Glu Asp Glu 170 175 27 169 PRT Homo sapiens 27 Met Arg Gly
Thr Pro Gly Asp Ala Asp Gly Gly Gly Arg Ala Val 1 5 10 15 Tyr Gln
Ser Met Cys Lys Pro Ile Thr Gly Thr Ile Asn Asp Leu 20 25 30 Asn
Gln Gln Val Trp Thr Leu Gln Gly Gln Asn Leu Val Ala Val 35 40 45
Pro Arg Ser Asp Ser Val Thr Pro Val Thr Val Ala Val Ile Thr 50 55
60 Cys Lys Tyr Pro Glu Ala Leu Glu Gln Gly Arg Gly Asp Pro Ile 65
70 75 Tyr Leu Gly Ile Gln Asn Pro Glu Met Cys Leu Tyr Cys Glu Lys
80 85 90 Val Gly Glu Gln Pro Thr Leu Gln Leu Lys Glu Gln Lys Ile
Met 95 100 105 Asp Leu Tyr Gly Gln Pro Glu Pro Val Lys Pro Phe Leu
Phe Tyr 110 115 120 Arg Ala Lys Thr Gly Arg Thr Ser Thr Leu Glu Ser
Val Ala Phe 125 130 135 Pro Asp Trp Phe Ile Ala Ser Ser Lys Arg Asp
Gln Pro Ile Ile 140 145 150 Leu Thr Ser Glu Leu Gly Lys Ser Tyr Asn
Thr Ala Phe Glu Leu 155 160 165 Asn Ile Asn Asp 28 167 PRT Homo
sapiens 28 Met Gly Ser Glu Asp Trp Glu Lys Asp Glu Pro Gln Cys Cys
Leu 1 5 10 15 Glu Asp Pro Ala Gly Ser Pro Leu Glu Pro Gly Pro Ser
Leu Pro 20 25 30 Thr Met Asn Phe Val His Thr Lys Ile Phe Phe Ala
Leu Ala Ser 35 40 45 Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser
Pro Ile Leu Leu 50 55 60 Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr
Cys Asp Lys Asp Lys 65 70 75 Gly Gln Ser His Pro Ser Leu Gln Leu
Lys Lys Glu Lys Leu Met 80 85 90 Lys Leu Ala Ala Gln Lys Glu Ser
Ala Arg Arg Pro Phe Ile Phe 95 100 105 Tyr Arg Ala Gln Val Gly Ser
Trp Asn Met Leu Glu Ser Ala Ala 110 115 120 His Pro Gly Trp Phe Ile
Cys Thr Ser Cys Asn Cys Asn Glu Pro 125 130 135 Val Gly Val Thr Asp
Lys Phe Glu Asn Arg Lys His Ile Glu Phe 140 145 150 Ser Phe Gln Pro
Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val 155 160 165 Ser Asp 29
31 DNA Homo sapiens 29 ggcggatcca aaatgggctc tgaggactgg g 31 30 30
DNA Homo sapiens 30 gcggaattct aatcgctgac ctcactgggg 30 31 9 PRT
Homo sapiens 31 Val His Thr Ser Pro Lys Val Lys Asn 1 5 32 10 PRT
Homo sapiens 32 Val Leu Ser Gly Ala Leu Cys Phe Arg Met 1 5 10
* * * * *
References