U.S. patent application number 12/534821 was filed with the patent office on 2010-02-25 for il-1 zeta, il-1 zeta splice variants and xrec2 dnas and polypeptides.
This patent application is currently assigned to Immunex Corporation. Invention is credited to Teresa L. Born, John E. Sims, Dirk E. Smith.
Application Number | 20100047867 12/534821 |
Document ID | / |
Family ID | 27381114 |
Filed Date | 2010-02-25 |
United States Patent
Application |
20100047867 |
Kind Code |
A1 |
Sims; John E. ; et
al. |
February 25, 2010 |
IL-1 ZETA, IL-1 ZETA SPLICE VARIANTS AND XREC2 DNAS AND
POLYPEPTIDES
Abstract
The invention is directed to novel, purified and isolated IL-1
zeta and Xrec2 polypeptides and fragments thereof, the nucleic
acids encoding such polypeptides, processes for production of
recombinant forms of such polypeptides, antibodies generated
against these polypeptides, fragmented peptides derived from these
polypeptides, and uses thereof.
Inventors: |
Sims; John E.; (Seattle,
WA) ; Smith; Dirk E.; (Bainbridge Island, WA)
; Born; Teresa L.; (Kenmore, WA) |
Correspondence
Address: |
IMMUNEX CORPORATION
PATENT OPERATIONS/MS 28-2-C, ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Assignee: |
Immunex Corporation
Thousand Oaks
CA
|
Family ID: |
27381114 |
Appl. No.: |
12/534821 |
Filed: |
August 3, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11717859 |
Mar 13, 2007 |
7585949 |
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12534821 |
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10888867 |
Jul 9, 2004 |
7217540 |
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11717859 |
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09876790 |
Jun 6, 2001 |
7033783 |
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10888867 |
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PCT/US99/29549 |
Dec 14, 1999 |
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09876790 |
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60164675 |
Nov 10, 1999 |
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60112163 |
Dec 14, 1998 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 530/387.9; 536/23.1 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 16/245 20130101; G01N 2500/02 20130101; G01N 33/6863 20130101;
C07K 14/545 20130101; C07K 14/7155 20130101; C07K 2319/00 20130101;
C07K 2317/34 20130101; C07K 16/244 20130101 |
Class at
Publication: |
435/69.1 ;
536/23.1; 435/320.1; 435/325; 530/350; 530/387.9 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C07H 21/00 20060101 C07H021/00; C12N 15/63 20060101
C12N015/63; C12N 5/00 20060101 C12N005/00; C07K 14/00 20060101
C07K014/00; C07K 16/00 20060101 C07K016/00 |
Claims
1. An isolated polynucleotide comprising a polynucleotide of SEQ ID
NO:2.
2. An isolated polynucleotide comprising a nucleic acid molecule
that encodes a polypeptide comprising SEQ ID NO:4.
3. A vector comprising the polynucleotide of claim 1.
4. A vector comprising the polynucleotide of claim 2.
5. A host cell transformed or transfected with the vector of claim
3.
6. A host cell transformed or transfected with the vector of claim
4.
7. A method for preparing a polypeptide, the method comprising
culturing the host cell of claim 5 under conditions promoting
expression of the polypeptide.
8. A method for preparing a polypeptide, the method comprising
culturing the host cell of claim 6 under conditions promoting
expression of the polypeptide.
9. An isolated polypeptide comprising SEQ ID NO:4.
10. An oligomeric polypeptide comprising a polypeptide of claim
9.
11. An isolated antibody that binds the polypeptide of claim 9
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/888,867, filed Jul. 9, 2004, now allowed, which is a
divisional of U.S. patent application Ser. No. 09/876,790, filed
Jun. 6, 2001, now allowed, which is a continuation-in-part of
International Application PCT/US99/29549, with an international
filing date of Dec. 14, 1999 and published in English on Jun. 22,
2000; and claims the benefit of U.S. provisional Application
60/164,675, filed on Nov. 10, 1999, and U.S. Provisional
Application 60/112, 163, filed Dec. 14, 1998.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention is directed to novel, purified and isolated
IL-1 zeta, IL-1 zeta splice variants and Xrec2 polypeptides and
fragments thereof, the nucleic acids encoding such polypeptides,
processes for production of recombinant forms of such polypeptides,
antibodies generated against these polypeptides, fragmented
peptides derived from these polypeptides, and uses thereof.
[0004] 2. Description of Related Art
[0005] Interleukin-1 (IL-1) is a member of a large group of
cytokines whose primary function is to mediate immune and
inflammatory responses. There are five known IL-1 family members
which include IL-1 alpha (IL-1.alpha.), IL-1 beta (IL-1.beta.),
IL-1 receptor antagonist (IL-1ra), IL-1 delta (IL-1.delta.) as
disclosed in US/99/00514), and IL-18 (previously known as IGIF and
sometimes IL-1 gamma). IL-1 that is secreted by macrophages is
actually a mixture of mostly IL-1.beta. and some IL-1.alpha. (Abbas
et al., 1994). IL-1.alpha. and IL-1.beta., which are first produced
as 33 kD precursors that lack a signal sequence, are further
processed by proteolytic cleavage to produce secreted active forms,
each about 17 kD. Additionally, the 33 kD precursor of IL-1.alpha.
is also active. Both forms of IL-1 are the products of two
different genes located on chromosome 2. Although the two forms are
less than 30 percent homologous to each other, they both bind to
the same receptors and have similar activities.
[0006] IL-1ra, a biologically inactive form of IL-1, is
structurally homologous to IL-1 and binds to the same receptors.
Additionally, IL-1ra is produced with a signal sequence which
allows for efficient secretion into the extracellular region where
it competitively competes with IL-1 (Abbas et al., 1994).
[0007] The IL-1 family of ligands binds to a family of two IL-1
receptors, which are members of the Ig superfamily. IL-1 receptors
include the 80 kDa type I receptor (IL-1RI) and a 68 kDa type II
receptor (IL-1RII). IL-1 ligands can also bind to a soluble
proteolytic fragment of IL-1RII (sIL-1RII) (Colotta et al.,
1993).
[0008] The major source of IL-1 is the activated macrophage or
mononuclear phagocyte. Other cells that produce IL-1 include
epithelial and endothelial cells (Abbas et al., 1994). IL-1
secretion from macrophages occurs after the macrophage encounters
and ingests gram-negative bacteria. Such bacteria contain
lipopolysaccharide (LPS) molecules, also known as endotoxin, in the
bacterial cell wall. LPS molecules are the active components that
stimulate macrophages to produce tumor necrosis factor (TNF) and
IL-1. In this case, IL-1 is produced in response to LPS and TNF
production. At low concentrations, LPS stimulates macrophages and
activates B-cells and other host responses needed to eliminate the
bacterial infection; however, at high concentrations, LPS can cause
severe tissue damage, shock, and even death.
[0009] The biological functions of IL-1 include activating vascular
endothelial cells and lymphocytes, local tissue destruction, and
fever (Janeway et al., 1996). At low levels, IL-1 stimulates
macrophages and vascular endothelial cells to produce IL-6,
upregulates molecules on the surface of vascular endothelial cells
to increase leukocyte adhesion, and indirectly activates
inflammatory leukocytes by stimulating mononuclear phagocytes and
other cells to produce certain chemokines that activate
inflammatory leukocytes. Additionally, IL-1 is involved in other
inflammatory responses such as induction of prostaglandins, nitric
oxide synthetase, and metalloproteinases. These IL-1 functions are
crucial during low level microbial infections. However, if the
microbial infection escalates, IL-1 acts systemically by inducing
fever, stimulating mononuclear phagocytes to produce IL-1 and IL-6,
increasing the production of serum proteins from hepatocytes, and
activating the coagulation system. Additionally, IL-1 does not
cause hemorrhagic necrosis of tumors, suppress bone marrow stem
cell division, and IL-1 is lethal to humans at high
concentrations.
[0010] Given the important function of IL-1, there is a need to
identify additional members of the IL-1 ligand family and the IL-1
receptor family. In addition, in view of the continuing interest in
protein research and the immune system, the discovery,
identification, and roles of new proteins and their inhibitors, are
at the forefront of modern molecular biology and biochemistry.
Despite the growing body of knowledge, there is still a need in the
art to discover the identity and function of proteins involved in
cellular and immune responses.
[0011] In another aspect, the identification of the primary
structure, or sequence, of an unknown protein is the culmination of
an arduous process of experimentation. In order to identify an
unknown protein, the investigator can rely upon a comparison of the
unknown protein to known peptides using a variety of techniques
known to those skilled in the art. For instance, proteins are
routinely analyzed using techniques such as electrophoresis,
sedimentation, chromatography, sequencing and mass
spectrometry.
[0012] In particular, comparison of an unknown protein to
polypeptides of known molecular weight allows a determination of
the apparent molecular weight of the unknown protein (T. D. Brock
and M. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall,
6d ed. 1991)). Protein molecular weight standards are commercially
available to assist in the estimation of molecular weights of
unknown protein (New England Biolabs Inc. Catalog: 130-131, 1995;
J. L. Hartley, U.S. Pat. No. 5,449,758). However, the molecular
weight standards may not correspond closely enough in size to the
unknown protein to allow an accurate estimation of apparent
molecular weight. The difficulty in estimation of molecular weight
is compounded in the case of proteins that are subjected to
fragmentation by chemical or enzymatic means, modified by
post-translational modification or processing, and/or associated
with other proteins in non-covalent complexes.
[0013] In addition, the unique nature of the composition of a
protein with regard to its specific amino acid constituents results
in unique positioning of cleavage sites within the protein.
Specific fragmentation of a protein by chemical or enzymatic
cleavage results in a unique "peptide fingerprint" (D. W. Cleveland
et al., J. Biol. Chem. 252:1102-1106, 1977; M. Brown et al., J.
Gen. Virol. 50:309-316, 1980). Consequently, cleavage at specific
sites results in reproducible fragmentation of a given protein into
peptides of precise molecular weights. Furthermore, these peptides
possess unique charge characteristics that determine the
isoelectric pH of the peptide. These unique characteristics can be
exploited using a variety of electrophoretic and other techniques
(T. D. Brock and M. T. Madigan, Biology of Microorganisms 76-77
(Prentice Hall, 6d ed. 1991)).
[0014] Fragmentation of proteins is further employed for amino acid
composition analysis and protein sequencing (P. Matsudiara, J.
Biol. Chem. 262:10035-10038, 1987; C. Eckerskorn et al.,
Electrophoresis 1988, 9:830-838, 1988), particularly the production
of fragments from proteins with a "blocked" N-terminus. In
addition, fragmented proteins can be used for immunization, for
affinity selection (R. A. Brown, U.S. Pat. No. 5,151,412), for
determination of modification sites (e.g. phosphorylation), for
generation of active biological compounds (T. D. Brock and M. T.
Madigan, Biology of Microorganisms 300-301 (Prentice Hall, 6d ed.
1991)), and for differentiation of homologous proteins (M. Brown et
al., J. Gen. Virol. 50:309-316, 1980).
[0015] In addition, when a peptide fingerprint of an unknown
protein is obtained, it can be compared to a database of known
proteins to assist in the identification of the unknown protein
using mass spectrometry (W. J. Henzel et al., Proc. Natl. Acad.
Sci. USA 90:5011-5015, 1993; D. Fenyo et al., Electrophoresis
19:998-1005, 1998). A variety of computer software programs to
facilitate these comparisons are accessible via the Internet, such
as Protein Prospector (Internet site: prospector.uscf.edu),
MultiIdent (Internet site: www.expasy.ch/sprot/multiident.html),
PeptideSearch (Internet site:
www.mann.embl-heiedelberg.de...deSearch/FR_PeptideSearch
Form.html), and ProFound (Internet site:
www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These
programs allow the user to specify the cleavage agent and the
molecular weights of the fragmented peptides within a designated
tolerance. The programs compare these molecular weights to protein
molecular weight information stored in databases to assist in
determining the identity of the unknown protein. Accurate
information concerning the number of fragmented peptides and the
precise molecular weight of those peptides is required for accurate
identification. Therefore, increasing the accuracy in determining
the number of fragmented peptides and their molecular weight should
result in enhanced likelihood of success in the identification of
unknown proteins.
[0016] In addition, peptide digests of unknown proteins can be
sequenced using tandem mass spectrometry (MS/MS) and the resulting
sequence searched against databases (J. K. Eng, et al., J. Am. Soc.
Mass Spec. 5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem.
66:4390-4399 (1994); J. A. Taylor and R. S. Johnson, Rapid Comm.
Mass Spec. 11:1067-1075 (1997)). Searching programs that can be
used in this process exist on the Internet, such as Lutefisk 97
(Internet site: www.lsbc.com:70/Lutefisk97.html), and the Protein
Prospector, Peptide Search and ProFound programs described above.
Therefore, adding the sequence of a gene and its predicted protein
sequence and peptide fragments to a sequence database can aid in
the identification of unknown proteins using tandem mass
spectrometry.
[0017] Thus, there also exists a need in the art for polypeptides
suitable for use in peptide fragmentation studies, for use in
molecular weight measurements, and for use in protein sequencing
using tandem mass spectrometry.
SUMMARY OF THE INVENTION
[0018] The present invention provides isolated nucleic acids and
polypeptides encoded by the nucleic acids for an IL-1 family ligand
termed "IL-1 zeta" and three splice variants of IL-1 zeta, termed
TDZ.1, TDZ.2, and TDZ.3. The present invention also provides
isolated nucleic acid molecules and polypeptides encoded by the
nucleic acid molecules for an IL-1 family receptor termed "Xrec2."
Thus, in one aspect, the invention is directed to isolated nucleic
acid molecules of IL-1 zeta, TDZ.1, TDZ.2, and TDZ.3 comprising the
DNA sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID
NO:7, respectively, and nucleic acid molecules complementary to SEQ
ID NO:1, 5, 6, and 7. Similarly, the invention is directed to
isolated nucleic acid molecules of Xrec2 comprising the nucleic
acid molecule of SEQ ID NO:2 and nucleic acid molecules
complementary to SEQ ID NO:2. In another aspect, the invention is
directed to isolated IL-1 zeta, TDZ.1, TDZ.2, and TDZ.3
polypeptides having the amino acid sequences SEQ ID NO:3 SEQ ID
NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively, and nucleic acid
molecules encoding the polypeptides of SEQ ID NO:3, 8, 9, and 10.
Further included in the invention are isolated Xrec2 polypeptides
comprising the amino acid sequence of SEQ ID NO:4 and nucleic acid
molecules that encode the polypeptide of SEQ ID NO:4
[0019] Both single-stranded and double-stranded RNA and DNA nucleic
acid molecules are encompassed by the invention, as well as nucleic
acid molecules that hybridize to a denatured, double-stranded DNA
comprising all or a portion of SEQ ID NOs:1, 2, 5, 6, and 7 and/or
a DNA that encodes the amino acid sequences set forth in SEQ ID
NOs:3, 4, 8, 9, and 10. Also encompassed are isolated nucleic acid
molecules that are derived by in vitro mutagenesis of nucleic acid
molecules comprising sequences of SEQ ID NOs:1, 2, 5, 6, and 7 that
are degenerate from nucleic acid molecules comprising sequences of
SEQ ID NOs:1, 2, 5, 6, and 7, and that are allelic variants of DNA
of the invention. The invention also encompasses recombinant
vectors that direct the expression of these nucleic acid molecules
and host cells transformed or transfected with these vectors.
[0020] In addition, the invention encompasses methods of using the
nucleic acids noted above to identify nucleic acids encoding
proteins having activities associated with IL-1 family ligands and
receptors. Thus, the IL-1 zeta nucleic acid molecules can be used
to identify the IL-1 zeta receptor while the Xrec2 nucleic acid
molecule can be used to identify the Xrec2 ligand.
[0021] In addition, these nucleic acids can be used to identify the
human chromosomes with which the nucleic acids are associated.
Thus, the IL-1 zeta, TDZ.1, TDZ.2, and TDZ.3 nucleic acids can be
used to identify human chromosome 2 while the Xrec2 nucleic acids
can be used to identify human chromosome X. Accordingly, these
nucleic acids can also be used to map genes on human chromosomes 2
and X, respectively; to identify genes associated with certain
diseases, syndromes, or other human conditions associated with
human chromosomes 2 and X, respectively; and to study cell signal
transduction and the immune system.
[0022] The invention also encompasses the use of sense or antisense
oligonucleotides from the nucleic acids of SEQ ID NOs:1, 2, 5, 6,
and 7 to inhibit the expression of the respective polynucleotide
encoded by the genes of the invention.
[0023] The invention also encompasses isolated polypeptides and
fragments of IL-1 zeta and Xrec2 as encoded by these nucleic acid
molecules, including soluble polypeptide portions of SEQ ID NOs:3
4, 8, 9, and 10, respectively. The invention further encompasses
methods for the production of these polypeptides, including
culturing a host cell under conditions promoting expression and
recovering the polypeptide from the culture medium. Especially, the
expression of these polypeptides in bacteria, yeast, plant, insect,
and animal cells is encompassed by the invention.
[0024] In general, the polypeptides of the invention can be used to
study cellular processes such as immune regulation, cell
proliferation, cell death, cell migration, cell-to-cell
interaction, and inflammatory responses. In addition, these
polypeptides can be used to identify proteins associated with IL-1
zeta, TDZ.1, TDZ.2, and TDZ.3 ligands and with Xrec2 receptors.
[0025] In addition, the invention includes assays utilizing these
polypeptides to screen for potential inhibitors or enhancers of
activity associated with the polypeptides of this invention. The
present invention also includes assays and screening methods for
identifying inhibitors or enhancers of activities associated with
counter-structure molecules of the polypeptides of this invention.
Further, methods of using these polypeptides in the design of
inhibitors (e.g., engineered receptors that act as inhibitors)
thereof are also an aspect of the invention.
[0026] The present invention further encompasses therapeutic
methods utilizing antagonist and/or agonists of the polypeptides of
this invention and antagonists or agonists discovered in accordance
with the screening methods of this invention. For example, IL-1
zeta polypeptides of the present invention enhance the secretion of
IL-12 from isolated primary human monocytes. In view of IL-12
activity associated with stimulating and enhancing immune responses
and IL-12 activity in promoting Th1 mediated diseases, IL-1 zeta
polypeptide agonists, together with IL-1 zeta antagonists are
useful for treating disease or medical conditions associated with
immune system imbalances, particularly imbalances involving
cell-mediated immune responses. For example, inhibitors or
antagonists of IL-1 zeta polypeptides can be used to treat disease
associated with abnormal Th1 immune responses, including the
deleterious effects of inflammation. Agonists of IL-1 zeta
polypeptide activity are useful in treating disease responsive to
IL-12 stimulation such as certain infectious diseases, including
Leishmania, parasitic diseases and diseases preferentially
inhibited by a Th1 immune response. Additionally Il-1 zeta
polypeptides upregulate TNF expression and thus antagonists of IL-1
zeta polypeptides are useful in treating inflammatory conditions
including rheumatoid arthritis, SLE, myasthenia gravis,
insulin-dependent diabetes mellitus, thyroiditis, etc. and diseases
preferentially inhibited by a Th1 immune response.
[0027] The invention further provides a method for using these
polypeptides as molecular weight markers that allow the estimation
of the molecular weight of a protein or a fragmented protein, as
well as a method for the visualization of the molecular weight
markers of the invention thereof using electrophoresis. The
invention further encompasses methods for using the polypeptides of
the invention as markers for determining the isoelectric point of
an unknown protein, as well as controls for establishing the extent
of fragmentation of a protein.
[0028] Further encompassed by this invention are kits to aid in
these determinations.
[0029] Further encompassed by this invention is the use of the IL-1
zeta and Xrec2 nucleic acid sequences, predicted amino acid
sequences of the polypeptide or fragments thereof, or a combination
of the predicted amino acid sequences of the polypeptide and
fragments thereof for use in searching an electronic database to
aid in the identification of sample nucleic acids and/or
proteins.
[0030] Isolated polyclonal or monoclonal antibodies that bind to
these polypeptides are also encompassed by the invention, in
addition the use of these antibodies to aid in purifying the
polypeptides of the invention.
BRIEF DESCRIPTION OF THE FIGURE
[0031] FIG. 1 diagrams the genomic structure of the IL-1 zeta
locus.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The sequence listing, electronically filed as text file
entitled 2008USDIV7SEQ.txt, which is 21 KB and which was renamed on
Aug. 3, 2009, is hereby incorporated by reference in its
entirety.
[0033] The nucleic acid molecules encompassed in the invention
include the following nucleotide sequences:
TABLE-US-00001 Name: IL-1 zeta (SEQ ID NO: 1) 1 ATGTCAGGCT
GTGATAGGAG GGAAACAGAA ACCAAAGGAA AGAACAGCTT 51 TAAGAAGCGC
TTAAGAGGTC CAAAGGTGAA GAACTTAAAC CCGAAGAAAT 101 TCAGCATTCA
TGACCAGGAT CACAAAGTAC TGGTCCTGGA CTCTGGGAAT 151 CTCATAGCAG
TTCCAGATAA AAACTACATA CGCCCAGAGA TCTTCTTTGC 201 ATTAGCCTCA
TCCTTGAGCT CAGCCTCTGC GGAGAAAGGA AGTCCGATTC 251 TCCTGGGGGT
CTCTAAAGGG GAGTTTTGTC TCTACTGTGA CAAGGATAAA 301 GGACAAAGTC
ATCCATCCCT TCAGCTGAAG AAGGAGAAAC TGATGAAGCT 351 GGCTGCCCAA
AAGGAATCAG CACGCCGGCC CTTCATCTTT TATAGGGCTC 401 AGGTGGGCTC
CTGGAACATG CTGGAGTCGG CGGCTCACCC CGGATGGTTC 451 ATCTGCACCT
CCTGCAATTG TAATGAGCCT GTTGGGGTGA CAGATAAATT 501 TGAGAACAGG
AAACACATTG AATTTTCATT TCAACCAGTT TGCAAAGCTG 551 AAATGAGCCC
CAGTGAGGTC AGCGATTAG Name: Xrec2 (SEQ ID NO: 2) 1 ATGAAAGCTC
CGATTCCACA CTTGATTCTC TTATACGCTA CTTTTACTCA 51 GAGTTTGAAG
GTTGTGACCA AAAGAGGCTC CGCCGATGGA TGCACTGACT 101 GGTCTATCGA
TATCAAGAAA TATCAAGTTT TGGTGGGAGA GCCTGTTCGA 151 ATCAAATGTG
CACTCTTTTA TGGTTATATC AGAACAAATT ACTCCCTTGC 201 CCAAAGTGCT
GGACTCAGTT TGATGTGGTA CAAAAGTTCT GGTCCTGGAG 251 ACTTTGAAGA
GCCAATAGCC TTTGACGGAA GTAGAATGAG CAAAGAAGAA 301 GACTCCATTT
GGTTCCGGCC AACATTGCTA CAGGACAGTG GTCTCTACGC 351 CTGTGTCATC
AGAAACTCCA CTTACTGTAT GAAAGTATCC ATCTCACTGA 401 CAGTGGGTGA
AAATGACACT GGACTCTGCT ATAATTCCAA GATGAAGTAT 451 TTTGAAAAAG
CTGAACTTAG CAAAAGCAAG GAAATTTCAT GCCGTGACAT 501 AGAGGATTTT
CTACTGCCAA CCAGAGAACC TGAAATCCTT TGGTACAAGG 551 AATGCAGGAC
AAAAACATGG AGGCCAAGTA TTGTATTCAA AAGAGATACT 601 CTGCTTATAA
GAGAAGTCAG AGAAGATGAC ATTGGAAATT ATACCTGTGA 651 ATTAAAATAT
GGAGGCTTTG TTGTGAGAAG AACTACTGAA TTAACTGTTA 701 CAGCCCCTCT
GACTGATAAG CCACCCAAGC TTTTGTATCC TATGGAAAGT 751 AAACTGACAA
TTCAGGAGAC CCAGCTGGGT GACTCTGCTA ATCTAACCTG 801 CAGAGCTTTC
TTTGGGTACA GCGGAGATGT CAGTCCTTTA ATTTACTGGA 851 TGAAAGGAGA
AAAATTTATT GAAGATCTGG ATGAAAATCG AGTTTGGGAA 901 AGTGACATTA
GAATTCTTAA GGAGCATCTT GGGGAACAGG AAGTTTCCAT 951 CTCATTAATT
GTGGACTCTG TGGAAGAAGG TGACTTGGGA AATTACTCCT 1001 GTTATGTTGA
AAATGGAAAT GGACGTCGAC ACGCCAGCGT TCTCCTTCAT 1051 AAACGAGAGC
TAATGTACAC AGTGGAACTT GCTGGAGGCC TTGGTGCTAT 1101 ACTCTTGCTG
CTTGTATGTT TGGTGACCAT CTACAAGTGT TACAAGATAG 1151 AAATCATGCT
CTTCTACAGG AATCATTTTG GAGCTGAAGA GCTCGATGGA 1201 GACAATAAAG
ATTATGATGC ATACTTATCA TACACCAAAG TGGATCCTGA 1251 CCAGTGGAAT
CAAGAGACTG GGGAAGAAGA ACGTTTTGCC CTTGAAATCC 1301 TACCTGATAT
GCTTGAAAAG CATTATGGAT ATAAGTTGTT TATACCAGAT 1351 AGAGATTTAA
TCCCAACTGG AACATACATT GAAGATGTGG CAAGATGTGT 1401 AGATCAAAGC
AAGCGGCTGA TTATTGTCAT GACCCCAAAT TACGTAGTTA 1451 GAAGGGGCTG
GAGCATCTTT GAGCTGGAAA CCAGACTTCG AAATATGCTT 1501 GTGACTGGAG
AAATTAAAGT GATTCTAATT GAATGCAGTG AACTGAGAGG 1551 AATTATGAAC
TACCAGGAGG TGGAGGCCCT GAAGCACACC ATCAAGCTCC 1601 TGACGGTCAT
TAAATGGCAT GGACCAAAAT GCAACAAGTT GAACTCCAAG 1651 TTCTGGAAAC
GTTTACAGTA TGAAATGCCT TTTAAGAGGA TAGAACCCAT 1701 TACACATGAG
CAGGCTTTAG ATGTCAGTGA GCAAGGGCCT TTTGGGGAGC 1751 TGCAGACTGT
CTCGGCCATT TCCATGGCCG CGGCCACCTC CACAGCTCTA 1801 GCCACTGCCC
ATCCAGATCT CCGTTCTACC TTTCACAACA CGTACCATTC 1851 ACAAATGCGT
CAGAAACACT ACTACCGAAG CTATGAGTAC GACGTACCTC 1901 CTACCGGCAC
CCTGCCTCTT ACCTCCATAG GCAATCAGCA TACCTACTGT 1951 AACATCCCTA
TGACACTCAT CAACGGGCAG CGGCCACAGA CAAAATCGAG 2001 CAGGGAGCAG
AATCCAGATG AGGCCCACAC AAACAGTGCC ATCCTGCCGC 2051 TGTTGCCAAG
GGAGACCAGT ATATCCAGTG TGATATGGTG A Name: TDZ.1 (SEQ ID NO: 5) 1
ATGTCCTTTG TGGGGGAGAA CTCAGGAGTG AAAATGGGCT CTGAGGACTG 51
GGAAAAAGAT GAACCCCAGT GCTGCTTAGA AGACCCGGCT GTAAGCCCCC 101
TGGAACCAGG CCCAAGCCTC CCCACCATGA ATTTTGTTCA CACAAGTCCA 151
AAGGTGAAGA ACTTAAACCC GAAGAAATTC AGCATTCATG ACCAGGATCA 201
CAAAGTACTG GTCCTGGACT CTGGGAATCT CATAGCAGTT CCAGATAAAA 251
ACTACATACG CCCAGAGATC TTCTTTGCAT TAGCCTCATC CTTGAGCTCA 301
GCCTCTGCGG AGAAAGGAAG TCCGATTCTC CTGGGGGTCT CTAAAGGGGA 351
GTTTTGTCTC TACTGTGACA AGGATAAAGG ACAAAGTCAT CCATCCCTTC 401
AGCTGAAGAA GGAGAAACTG ATGAAGCTGG CTGCCCAAAA GGAATCAGCA 451
CGCCGGCCCT TCATCTTTTA TAGGGCTCAG GTGGGCTCCT GGAACATGCT 501
GGAGTCGGCG GCTCACCCCG GATGGTTCAT CTGCACCTCC TGCAATTGTA 551
ATGAGCCTGT TGGGGTGACA GATAAATTTG AGAACAGGAA ACACATTGAA 601
TTTTCATTTC AACCAGTTTG CAAAGCTGAA ATGAGCCCCA GTGAGGTCAG 651 CGATTAG
Name: TDZ.2 (SEQ ID NO: 6) 1 ATGTCCTTTG TGGGGGAGAA CTCAGGAGTG
AAAATGGGCT CTGAGGACTG 51 GGAAAAAGAT GAACCCCAGT GCTGCTTAGA
AGGTCCAAAG GTGAAGAACT 101 TAAACCCGAA GAAATTCAGC ATTCATGACC
AGGATCACAA AGTACTGGTC 151 CTGGACTCTG GGAATCTCAT AGCAGTTCCA
GATAAAAACT ACATACGCCC 201 AGAGATCTTC TTTGCATTAG CCTCATCCTT
GAGCTCAGCC TCTGCGGAGA 251 AAGGAAGTCC GATTCTCCTG GGGGTCTCTA
AAGGGGAGTT TTGTCTCTAC 301 TGTGACAAGG ATAAAGGACA AAGTCATCCA
TCCCTTCAGC TGAAGAAGGA 351 GAAACTGATG AAGCTGGCTG CCCAAAAGGA
ATCAGCACGC CGGCCCTTCA 401 TCTTTTATAG GGCTCAGGTG GGCTCCTGGA
ACATGCTGGA GTCGGCGGCT 451 CACCCCGGAT GGTTCATCTG CACCTCCTGC
AATTGTAATG AGCCTGTTGG 501 GGTGACAGAT AAATTTGAGA ACAGGAAACA
CATTGAATTT TCATTTCAAC 551 CAGTTTGCAA AGCTGAAATG AGCCCCAGTG
AGGTCAGCGA TTAG Name: TDZ.3 (SEQ ID NO: 7) 1 ATGTCCTTTG TGGGGGAGAA
CTCAGGAGTG AAAATGGGCT CTGAGGACTG 51 GGAAAAAGAT GAACCCCAGT
GCTGCTTAGA AGAGATCTTC TTTGCATTAG 101 CCTCATCCTT GAGCTCAGCC
TCTGCGGAGA AAGGAAGTCC GATTCTCCTG 151 GGGGTCTCTA AAGGGGAGTT
TTGTCTCTAC TGTGACAAGG ATAAAGGACA 201 AAGTCATCCA TCCCTTCAGC
TGAAGAAGGA GAAACTGATG AAGCTGGCTG 251 CCCAAAAGGA ATCAGCACGC
CGGCCCTTCA TCTTTTATAG GGCTCAGGTG 301 GGCTCCTGGA ACATGCTGGA
GTCGGCGGCT CACCCCGGAT GGTTCATCTG 351 CACCTCCTGC AATTGTAATG
AGCCTGTTGG GGTGACAGAT AAATTTGAGA 401 ACAGGAAACA CATTGAATTT
TCATTTCAAC CAGTTTGCAA AGCTGAAATG 451 AGCCCCAGTG AGGTCAGCGA TTAG
[0034] The amino acid sequences of the polypeptides encoded by the
nucleotide sequence of the invention include:
TABLE-US-00002 Name: IL-1 zeta (polypeptide) (SEQ ID NO: 3) 1
MSGCDRRETE TKGKNSFKKR LRGPKVKNLN PKKFSIHDQD HKVLVLDSGN 51
LIAVPDKNYI RPEIFFALAS SLSSASAEKG SPILLGVSKG EFCLYCDKDK 101
GQSHPSLQLK KEKLMKLAAQ KESARRPFIF YRAQVGSWNM LESAAHPGWF 151
ICTSCNCNEP VGVTDKFENR KHIEFSFQPV CKAEMSPSEV SD* Name: Xrec2
(polypeptide) (SEQ ID NO: 4) 1 MKAPIPHLIL LYATFTQSLK VVTKRGSADG
CTDWSIDIKK YQVLVGEPVR 51 IKCALFYGYI RTNYSLAQSA GLSLMWYKSS
GPGDFEEPIA FDGSRMSKEE 101 DSIWFRPTLL QDSGLYACVI RNSTYCMKVS
ISLTVGENDT GLCYNSKMKY 151 FEKAELSKSK EISCRDIEDF LLPTREPEIL
WYKECRTKTW RPSIVFKRDT 201 LLIREVREDD IGNYTCELKY GGFVVRRTTE
LTVTAPLTDK PPKLLYPMES 251 KLTIQETQLG DSANLTCRAF FGYSGDVSPL
IYWMKGEKFI EDLDENRVWE 301 SDIRILKEHL GEQEVSISLI VDSVEEGDLG
NYSCYVENGN GRRHASVLLH 351 KRELMYTVEL AGGLGAILLL LVCLVTIYKC
YKIEIMLFYR NHFGAEELDG 401 DNKDYDAYLS YTKVDPDQWN QETGEEERFA
LEILPDMLEK HYGYKLFIPD 451 RDLIPTGTYI EDVARCVDQS KRLIIVMTPN
YVVRRGWSIF ELETRLRNML 501 VTGEIKVILI ECSELRGIMN YQEVEALKHT
IKLLTVIKWH GPKCNKLNSK 551 FWKRLQYEMP FKRIEPITHE QALDVSEQGP
FGELQTVSAI SMAAATSTAL 601 ATAHPDLRST FHNTYHSQMR QKHYYRSYEY
DVPPTGTLPL TSIGNQHTYC 651 NIPMTLINGQ RPQTKSSREQ NPDEAHTNSA
ILPLLPRETS ISSVIW* TDZ.1 polypeptide (SEQ ID NO: 8) 1 MSFVGENSGV
KMGSEDWEKD EPQCCLEDPA VSPLEPGPSL PTMNFVHTSP 51 KVKNLNPKKF
SIHDQDHKVL VLDSGNLIAV PDKNYIRPEI FFALASSLSS 101 ASAEKGSPIL
LGVSKGEFCL YCDKDKGQSH PSLQLKKEKL MKLAAQKESA 151 RRPFIFYRAQ
VGSWNMLESA AHPGWFICTS CNCNEPVGVT DKFENRKHIE 201 FSFQPVCKAE
MSPSEVSD* Name: TDZ.2 polypeptide (SEQ ID NO: 9) 1 MSFVGENSGV
KMGSEDWEKD EPQCCLEGPK VKNLNPKKFS IHDQDHKVLV 51 LDSGNLIAVP
DKNYIRPEIF FALASSLSSA SAEKGSPILL GVSKGEFCLY 101 CDKDKGQSHP
SLQLKKEKLM KLAAQKESAR RPFIFYRAQV GSWNMLESAA 151 HPGWFICTSC
NCNEPVGVTD KFENRKHIEF SFQPVCKAEM SPSEVSD* Name: TDZ.3 polypeptide
(SEQ ID NO: 10) 1 MSFVGENSGV KMGSEDWEKD EPQCCLEEIF FALASSLSSA
SAEKGSPILL 51 GVSKGEFCLY CDKDKGQSHP SLQLKKEKLM KLAAQKESAR
RPFIFYRAQV 101 GSWNMLESAA HPGWFICTSC NCNEPVGVTD KFENRKHIEF
SFQPVCKAEM 151 SPSEVSD*
[0035] The discovery of the IL-1 zeta, the IL-1 zeta splice
variants (TDZ.1, TDZ.2, and TDZ.3) and the Xrec2 nucleic acids of
the invention enables the construction of expression vectors
comprising nucleic acid sequences encoding the respective
polypeptides and host cells transfected or transformed with the
expression vectors. The invention also enables the isolation and
purification of biologically active IL-1 zeta, the IL-1 zeta splice
variants, and Xrec2 polypeptides and fragments thereof. In yet
another embodiment, the nucleic acids or oligonucleotides thereof
can be used as probes to identify nucleic acid encoding proteins
having associated activities. Thus, IL-1 zeta and the IL-1 splice
variants can be used to identify activities associated with IL-1
family ligands and Xrec2 can be used to identify activities
associated with IL-1 family receptors. In addition, the nucleic
acids or oligonucleotides thereof of IL-1 zeta can be used to
identify human chromosomes 2 while those of Xrec2 can be used to
identify human chromosome X. Similarly, these nucleic acids or
oligonucleotides thereof can be used to map genes on human
chromosomes 2 and X, respectively, and to identify genes associated
with certain diseases, syndromes or other human conditions
associated with human chromosomes 2 and X. Thus, the nucleic acids
or oligonucleotides thereof of IL-1 zeta, TDZ.1, TDZ.2, and TDZ.3
can be used to identify glaucoma, ectodermal dysplasia,
insulin-dependent diabetes mellitus, wrinkly skin syndrome, T-cell
leukemia/lymphoma, and tibial muscular dystrophy while the nucleic
acids or oligonucleotides thereof of Xrec2 can be used to identify
retinoschisis, lissencephaly, subcortical laminalheteropia, mental
retardation, cowchock syndrome, bazex syndrome, hypertrichosis,
lymphoproliferative syndrome, immunodeficiency, Langer mesomelic
dysplasia, and leukemia. Finally, single-stranded sense or
antisense oligonucleotides from these nucleic acids can be used to
inhibit expression of polynucleotides encoded by the IL-1 zeta and
Xrec2 genes, respectively.
[0036] Further, the IL-1 zeta, TDZ.1, TDZ.2, TDZ.3 and Xrec2
polypeptides and soluble fragments thereof can be used to activate
and/or inhibit the activation of vascular endothelial cells and
lymphocytes, induce and/or inhibit the induction of local tissue
destruction and fever (Janeway et al., 1996), inhibit and/or
stimulate macrophages and vascular endothelial cells to produce
IL-6, induce and/or inhibit the induction of prostaglandins, nitric
oxide synthetase, and metalloproteinases, and upregulate and/or
inhibit the upregulation of molecules on the surface of vascular
endothelial cells. In addition these polypeptides and fragmented
peptides can also be used to induce and/or inhibit the induction of
inflammatory mediators such as transcription factors NF-.kappa.B
and AP-1, MAP kinases JNK and p38, COX-2, iNOS, and all of the
activities stimulated by these molecules.
[0037] In addition, these polypeptides and fragmented peptides can
be used as molecular weight markers and as controls for peptide
fragmentation, and the invention includes the kits comprising these
reagents. Finally, these polypeptides and fragments thereof can be
used to generate antibodies, and the invention includes the use of
such antibodies to purify IL-1 zeta and Xrec2 polypeptides.
Nucleic Acid Molecules
[0038] In a particular embodiment, the invention relates to certain
isolated nucleotide sequences that are free from contaminating
endogenous material. A "nucleotide sequence" refers to a
polynucleotide molecule in the form of a separate fragment or as a
component of a larger nucleic acid construct. The nucleic acid
molecule has been derived from DNA or RNA isolated at least once in
substantially pure form and in a quantity or concentration enabling
identification, manipulation, and recovery of its component
nucleotide sequences by standard biochemical methods (such as those
outlined in Sambrook et al., Molecular Cloning. A Laboratory
Manual, 2nd sed., Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1989)). Such sequences are preferably provided and/or
constructed in the form of an open reading frame uninterrupted by
internal non-translated sequences, or introns, that are typically
present in eukaryotic genes. Sequences of non-translated DNA can be
present 5' or 3' from an open reading frame, where the same do not
interfere with manipulation or expression of the coding region.
[0039] Nucleic acid molecules of the invention include DNA in both
single-stranded and double-stranded form, as well as the RNA
complement thereof. DNA includes, for example, cDNA, genomic DNA,
chemically synthesized DNA, DNA amplified by PCR, and combinations
thereof. Genomic DNA may be isolated by conventional techniques,
e.g., using the cDNA of SEQ ID NOs:1, 2, 5, 6, 7 or a suitable
fragment thereof, as a probe.
[0040] The DNA molecules of the invention include full length genes
as well as polynucleotides and fragments thereof. The full length
gene may include the N-terminal signal peptide. Other embodiments
include DNA encoding a soluble form, e.g., encoding the
extracellular domain of the protein, either with or without the
signal peptide.
[0041] The nucleic acids of the invention are preferentially
derived from human sources, but the invention includes those
derived from non-human species, as well.
[0042] Preferred Sequences
[0043] The particularly preferred nucleic acid molecules of the
invention are those shown in SEQ ID NOs:1, 5, 6, 7 for IL-1 zeta,
TDZ.1, TDZ.2, and TDZ.3, respectively, and SEQ ID NO:2 for Xrec2.
cDNA clones having the nucleic acid sequence of SEQ ID NOs:1 and 2
were isolated as described in Example 1. The sequences of the amino
acids of IL-1 zeta and Xrec2 encoded by the DNAs of SEQ ID NOs:1
and 2 are shown in SEQ ID NOs:3 and 4, respectively. CDNA clones
having the nucleic acid sequence of SEQ ID NOs:5, 6, and 7 were
isolated as described in Example 8. The sequences of the amino
acids of TDZ.1, TDZ.2, and TDZ.3 encoded by the DNAs of SEQ ID
NOs:5, 6, and 7 are shown in SEQ ID NOs:8, 9, and 10,
respectively.
[0044] SEQ ID NOs:1-4 identify the IL-1 zeta of SEQ ID NO:3 as a
member of the IL-1 family and the Xrec2 of SEQ ID NO:4 as a member
of the IL-1 receptor family. The homologies on which this is based
is set forth at Table I below:
TABLE-US-00003 TABLE I Protein Source Percent identity to IL-1 zeta
IL-1 alpha Human LOW IL-1 beta Human 22% IL-1 delta Human 34% IL-1
epsilon Human 34% IL-18 Human LOW IL-1ra Human 29% Percent identity
to Xrec2 TIGIRR (IL-1R family member) Human 63% TIGIRR (IL-1R
family member) Murine 61% SIGIRR Human 22% IL-1R-AcP Human 35%
IL-1R-AcPL Human 26% IL-1R Human 29% RP1 Human 31% RP2 Human 28%
ST2 Human 26%
Percent identity of IL-1 zeta and Xrec2 to human and murine
proteins.
[0045] As described in Example 8, the IL-1 zeta splice variants
were discovered in a stretch of genomic DNA sequence (X22304.gbn).
This genomic sequence also contains the different IL-1 zeta exons
and another splice variant known as Tango-77 (WO 99/06426).
Comparing the cDNA sequences of the cloned IL-1 zeta, TDZ.1, TDZ.2,
TDZ.3 and Tango-77 with the genomic sequence provides insight into
the generation of the splicing events. FIG. 1 shows the genomic
structure of the IL-1 zeta locus and the cDNA generated by
alternative splicing. The numbered boxes indicate individual exons
1-6 and the approximate size of the intervening introns is
indicated at the top. The asterisk (*) indicates the presence of a
stop codon, at the end of the coding sequence (exon 6) or as an
in-frame stop codon (exon 3). "M" indicates potential initiating
methionine originating either from exon 1 or exon 3. Tango-77 is
the cDNA structure disclosed in WO 99/06426. A significant feature
of IL-1 zeta and its splice variants is the presence or the absence
of exon 4. Exon 4 is present in IL-1 zeta, TDZ.1 and TDZ.2. It is
not present in Tango-77. The amino acid sequence encoded by exon 4
aligns well with the amino acid sequences of other IL-1 family
members in the first few beta strands of the mature peptides. By
contrast, the amino acid sequence encoded by Tango-77 cDNA and by
TDZ.3 cDNA aligns well with other IL-1 family members in the
regions encoded by exons 5 and 6. Exons 5 and 6 align well with
amino acid sequences of other IL-1 family members in the C-terminal
2/3 of the mature peptide, but does not align well in the
N-terminal 1/3. Thus, the "mature peptide" encoded by IL1zeta,
TDZ.1 and TDZ.2 DNAs is likely to represent a functional IL-1 like
molecule. This contrasts with the polypeptide encoded by Tango-77
or TDZ.3 DNAS which are less likely to represent a functional IL-1
like molecule.
[0046] It is probable that all of the splice isoforms, except
TDZ.3, encode proforms of an IL-1 like cytokine, since in the
N-terminal direction the DNAs extend well beyond the N-terminus of
mature IL-1s. This observation predicts that IL-1zeta, TDZ.1 and
TDZ.2 encode the same mature peptide. In connection with this
observation it is the pro-domains (as well as 5' UTRs) that differs
between IL-1 zeta, TDZ.1 and TDZ.2.
[0047] Table II, which details the tissue distribution of IL-1
zeta, TDZ.1, TDZ.2, TDZ.3 and Tango-77, shows that the expression
of Tango-77 is more widespread than that of IL-1 zeta. Table II
also shows that the TDZ.1 expression is comparable, and almost
entirely overlapping, with that of Tango-77. The tissue
distribution data combined with the alignment information of FIG. 1
shows that TDZ.1 is the only member of the splice variants that
aligns well with other IL-1 family members, and is widespread in
its expression. These observations suggest that TDZ.1 may be the
most significant of the splice variants in terms of group in terms
of relevance to biological responses.
[0048] Additional Sequences
[0049] Due to the known degeneracy of the genetic code, wherein
more than one codon can encode the same amino acid, a DNA sequence
can vary from that shown in SEQ ID NOs:1, 2, 5, 6, and 7 and still
encode a polypeptide having the amino acid sequence of SEQ ID
NOs:3, 4, 8, 9, and 10, respectively. Such variant DNA sequences
can result from silent mutations (e.g., occurring during PCR
amplification), or can be the product of deliberate mutagenesis of
a native sequence.
[0050] The invention thus provides isolated DNA sequences encoding
polypeptides of the invention, selected from: (a) DNA comprising
the nucleotide sequences of SEQ ID NOs:1, 2; 5, 6, and 7 (b) DNA
encoding the polypeptides of SEQ ID NOs:3, 4; 8, 9, and 10 (c) DNA
capable of hybridization to a DNA of (a) or (b) under conditions of
moderate stringency and which encodes polypeptides of the
invention; (d) DNA capable of hybridization to a DNA of (a) or (b)
under conditions of high stringency and which encodes polypeptides
of the invention, and (e) DNA which is degenerate, as a result of
the genetic code, to a DNA defined in (a), (b), (c), or (d) and
which encode polypeptides of the invention. Of course, polypeptides
encoded by such DNA sequences are encompassed by the invention.
[0051] As used herein, conditions of moderate stringency can be
readily determined by those having ordinary skill in the art based
on, for example, the length of the DNA. The basic conditions are
set forth by Sambrook et al. Molecular Cloning: A Laboratory
Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory
Press, (1989), and include use of a prewashing solution for the
nitrocellulose filters 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0),
hybridization conditions of about 50% formamide, 6.times.SSC at
about 42.degree. C. (or other similar hybridization solution, such
as Stark's solution, in about 50% formamide at about 42.degree.
C.), and washing conditions of about 60.degree. C., 0.5.times.SSC,
0.1% SDS. Conditions of high stringency can also be readily
determined by the skilled artisan based on, for example, the length
of the DNA. Generally, such conditions are defined as hybridization
conditions as above, and with washing at approximately 68.degree.
C., 0.2.times.SSC, 0.1% SDS. The skilled artisan will recognize
that the temperature and wash solution salt concentration can be
adjusted as necessary according to factors such as the length of
the probe.
[0052] Also included as an embodiment of the invention is DNA
encoding polypeptide fragments and polypeptides comprising
inactivated N-glycosylation site(s), inactivated protease
processing site(s), or conservative amino acid substitution(s), as
described below.
[0053] In another embodiment, the nucleic acid molecules of the
invention also comprise nucleotide sequences that are at least 80%
identical to a native sequence. Also contemplated are embodiments
in which a nucleic acid molecule comprises a sequence that is at
least 90% identical, at least 95% identical, at least 98%
identical, at least 99% identical, or at least 99.9% identical to a
native sequence.
[0054] The percent identity may be determined by visual inspection
and mathematical calculation. Alternatively, the percent identity
of two nucleic acid sequences can be determined by comparing
sequence information using the GAP computer program, version 6.0
described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and
available from the University of Wisconsin Genetics Computer Group
(UWGCG). The preferred default parameters for the GAP program
include: (1) a unary comparison matrix (containing a value of 1 for
identities and 0 for non-identities) for nucleotides, and the
weighted comparison matrix of Gribskov and Burgess, Nucl. Acids
Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds.,
Atlas of Protein Sequence and Structure, National Biomedical
Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for
each gap and an additional 0.10 penalty for each symbol in each
gap; and (3) no penalty for end gaps. Other programs used by one
skilled in the art of sequence comparison may also be used.
[0055] The invention provides isolated nucleic acids useful in the
production of polypeptides. Such polypeptides may be prepared by
any of a number of conventional techniques. A DNA sequence encoding
a polypeptide of the invention, or desired fragment thereof may be
subcloned into an expression vector for production of the
polypeptide or fragment. The DNA sequence advantageously is fused
to a sequence encoding a suitable leader or signal peptide.
Alternatively, the desired fragment may be chemically synthesized
using known techniques. DNA fragments also may be produced by
restriction endonuclease digestion of a full length cloned DNA
sequence, and isolated by electrophoresis on agarose gels. If
necessary, oligonucleotides that reconstruct the 5' or 3' terminus
to a desired point may be ligated to a DNA fragment generated by
restriction enzyme digestion. Such oligonucleotides may
additionally contain a restriction endonuclease cleavage site
upstream of the desired coding sequence, and position an initiation
codon (ATG) at the N-terminus of the coding sequence.
[0056] The well-known polymerase chain reaction (PCR) procedure
also may be employed to isolate and amplify a DNA sequence encoding
a desired protein fragment. Oligonucleotides that define the
desired termini of the DNA fragment are employed as 5' and 3'
primers. The oligonucleotides may additionally contain recognition
sites for restriction endonucleases, to facilitate insertion of the
amplified DNA fragment into an expression vector. PCR techniques
are described in Saiki et al., Science 239:487 (1988); Recombinant
DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego
(1989), pp. 189-196; and PCR Protocols: A Guide to Methods and
Applications, Innis et al., eds., Academic Press, Inc. (1990).
[0057] Polypeptides and Fragments Thereof
[0058] The invention encompasses polypeptides and fragments thereof
in various forms, including those that are naturally occurring or
produced through various techniques such as procedures involving
recombinant DNA technology. Such forms include, but are not limited
to, derivatives, variants, and oligomers, as well as fusion
proteins or fragments thereof.
[0059] The polypeptides of the invention include full length
proteins encoded by the nucleic acid sequences set forth above.
Particularly preferred polypeptides of IL-1 zeta, TDZ.1, TDZ.2
TDZ.3 and Xrec2 comprise the amino acid sequence of SEQ ID NOs:3,
4, 8, 9, and 10 respectively. For TDZ.1 and TDZ.2 the N-terminus
does not encode a classical signal peptide but the extra length
relative to the mature form other family members is suggestive that
it may act as a prodomain. A predicted cleavage site is the point
where the conserved structural portion of the protein begins.
Structural modeling data supports this assumption. For IL-1 zeta
and the TDZ.1 and TDZ.2 variants site is somewhere immediately
upstream of the last three exons. Thus, the polypeptide of IL-1
zeta, as set forth in SEQ ID NO:3, includes a putative pro-domain
that extends from amino acids 1 to x, where x is an integer from 20
to 50. Similarly, TDZ.1 of SEQ ID NO:8 includes a putative
prodomain that extends from amino acids 1 to x' where x' is an
integer from 40-50 and most preferably x' is about 48. TDZ.2 of SEQ
ID NO:9 includes a putative prodomain that extends from amino acids
1 to x'', where x'' is an integer from 25-30 and most preferable
x'' is 27.
[0060] Unlike IL-1 zeta and its splice variants, the polypeptide of
Xrec2, as set forth in SEQ ID NO:4, includes an N-terminal
hydrophobic region that functions as a signal peptide, followed by
an extracellular domain comprising amino acids 19 to 359, a
transmembrane region comprising amino acids 360 through 378, and a
C-terminal cytoplasmic domain comprising amino acids 379 to 696.
Computer analysis predicts that the signal peptide corresponds to
residues 1 to 19 of SEQ ID NO:4 (although the next most likely
computer-predicted signal peptide cleavage sites (in descending
order) occur after amino acids 20 and 16 of SEQ ID NO:4.)).
Cleavage of the signal peptide thus would yield a mature protein
comprising amino acids 19 through 696 of SEQ ID NO:4.
[0061] The skilled artisan will recognize that the above-described
boundaries of such regions of the polypeptide are approximate. To
illustrate, the boundaries of the transmembrane region (which may
be predicted by using computer programs available for that purpose)
may differ from those described above.
[0062] The polypeptides of the invention may be membrane bound or
they may be secreted and, thus, soluble. Soluble polypeptides are
capable of being secreted from the cells in which they are
expressed. In general, soluble polypeptides may be identified (and
distinguished from non-soluble membrane-bound counterparts) by
separating intact cells which express the desired polypeptide from
the culture medium, e.g., by centrifugation, and assaying the
medium (supernatant) for the presence of the desired polypeptide.
The presence of polypeptide in the medium indicates that the
polypeptide was secreted from the cells and thus is a soluble form
of the protein.
[0063] In one embodiment, the soluble polypeptides and fragments
thereof comprise all or part of the extracellular domain, but lack
the transmembrane region that would cause retention of the
polypeptide on a cell membrane. A soluble polypeptide may include
the cytoplasmic domain, or a portion thereof, as long as the
polypeptide is secreted from the cell in which it is produced.
[0064] In general, the use of soluble forms is advantageous for
certain applications. Purification of the polypeptides from
recombinant host cells is facilitated, since the soluble
polypeptides are secreted from the cells. Further, soluble
polypeptides are generally more suitable for intravenous
administration.
[0065] The invention also provides polypeptides and fragments of
the extracellular domain that retain a desired biological activity.
Particular embodiments are directed to polypeptide fragments of SEQ
ID NOs:3, 4, 8, 9, and 10 that retain the ability to bind the
native cognates, substrates, or counter-structure ("binding
partner"). Such a fragment may be a soluble polypeptide, as
described above. In another embodiment, the polypeptides and
fragments advantageously include regions that are conserved in the
IL-1 ligand and IL-1 receptor family as described above.
[0066] Also provided herein are polypeptide fragments comprising at
least 20, or at least 30, contiguous amino acids of the sequences
of SEQ ID NOs:3 4, 8, 9, and 10. In one aspect, fragments derived
from the cytoplasmic domain of Xrec2 of SEQ ID NO:4 find use in
studies of signal transduction, and in regulating cellular
processes associated with transduction of biological signals.
Polypeptide fragments also may be employed as immunogens, in
generating antibodies.
[0067] Variants
[0068] Naturally occurring variants as well as derived variants of
the polypeptides and fragments are provided herein.
[0069] Variants may exhibit amino acid sequences that are at least
80% identical. Also contemplated are embodiments in which a
polypeptide or fragment comprises an amino acid sequence that is at
least 90% identical, at least 95% identical, at least 98%
identical, at least 99% identical, or at least 99.9% identical to
the preferred polypeptide or fragment thereof. Percent identity may
be determined by visual inspection and mathematical calculation.
Alternatively, the percent identity of two protein sequences can be
determined by comparing sequence information using the GAP computer
program, based on the algorithm of Needleman and Wunsch (J. Mol.
Bio. 48:443, 1970) and available from the University of Wisconsin
Genetics Computer Group (UWGCG). The preferred default parameters
for the GAP program include: (1) a scoring matrix, blosum62, as
described by Henikoff and Henikoff (Proc. Natl. Acad. Sci. USA
89:10915, 1992); (2) a gap weight of 12; (3) a gap length weight of
4; and (4) no penalty for end gaps. Other programs used by one
skilled in the art of sequence comparison may also be used.
[0070] The variants of the invention include, for example, those
that result from alternate mRNA splicing events or from proteolytic
cleavage. Alternate splicing of mRNA may, for example, yield a
truncated but biologically active protein, such as a naturally
occurring soluble form of the protein. Variations attributable to
proteolysis include, for example, differences in the N- or
C-termini upon expression in different types of host cells, due to
proteolytic removal of one or more terminal amino acids from the
protein (generally from 1-5 terminal amino acids). Proteins in
which differences in amino acid sequence are attributable to
genetic polymorphism (allelic variation among individuals producing
the protein) are also contemplated herein.
[0071] Additional variants within the scope of the invention
include polypeptides that may be modified to create derivatives
thereof by forming covalent or aggregative conjugates with other
chemical moieties, such as glycosyl groups, lipids, phosphate,
acetyl groups and the like. Covalent derivatives may be prepared by
linking the chemical moieties to functional groups on amino acid
side chains or at the N-terminus or C-terminus of a polypeptide.
Conjugates comprising diagnostic (detectable) or therapeutic agents
attached thereto are contemplated herein, as discussed in more
detail below.
[0072] Other derivatives include covalent or aggregative conjugates
of the polypeptides with other proteins or polypeptides, such as by
synthesis in recombinant culture as N-terminal or C-terminal
fusions. Examples of fusion proteins are discussed below in
connection with oligomers. Further, fusion proteins can comprise
peptides added to facilitate purification and identification. Such
peptides include, for example, poly-His or the antigenic
identification peptides described in U.S. Pat. No. 5,011,912 and in
Hopp et al., Bio/Technology 6:1204, 1988. One such peptide is the
FLAG.sup.7 peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, which is
highly antigenic and provides an epitope reversibly bound by a
specific monoclonal antibody, enabling rapid assay and facile
purification of expressed recombinant protein. A murine hybridoma
designated 4E11 produces a monoclonal antibody that binds the
FLAG.sup.7 peptide in the presence of certain divalent metal
cations, as described in U.S. Pat. No. 5,011,912, hereby
incorporated by reference. The 4E11 hybridoma cell line has been
deposited with the American Type Culture Collection under accession
no. HB 9259. Monoclonal antibodies that bind the FLAG.sup.7 peptide
are available from Eastman Kodak Co., Scientific Imaging Systems
Division, New Haven, Conn.
[0073] Among the variant polypeptides provided herein are variants
of native polypeptides that retain the native biological activity
or the substantial equivalent thereof. One example is a variant
that binds with essentially the same binding affinity as does the
native form. Binding affinity can be measured by conventional
procedures, e.g., as described in U.S. Pat. No. 5,512,457 and as
set forth below.
[0074] Variants include polypeptides that are substantially
homologous to the native form, but which have an amino acid
sequence different from that of the native form because of one or
more deletions, insertions or substitutions. Particular embodiments
include, but are not limited to, polypeptides that comprise from
one to ten deletions, insertions or substitutions of amino acid
residues, when compared to a native sequence.
[0075] A given amino acid may be replaced, for example, by a
residue having similar physiochemical characteristics. Examples of
such conservative substitutions include substitution of one
aliphatic residue for another, such as Ile, Val, Leu, or Ala for
one another; substitutions of one polar residue for another, such
as between Lys and Arg, Glu and Asp, or Gln and Asn; or
substitutions of one aromatic residue for another, such as Phe,
Trp, or Tyr for one another. Other conservative substitutions,
e.g., involving substitutions of entire regions having similar
hydrophobicity characteristics, are well known.
[0076] Similarly, the DNAs of the invention include variants that
differ from a native DNA sequence because of one or more deletions,
insertions or substitutions, but that encode a biologically active
polypeptide.
[0077] The invention further includes polypeptides of the invention
with or without associated native-pattern glycosylation.
Polypeptides expressed in yeast or mammalian expression systems
(e.g., COS-1 or COS-7 cells) can be similar to or significantly
different from a native polypeptide in molecular weight and
glycosylation pattern, depending upon the choice of expression
system. Expression of polypeptides of the invention in bacterial
expression systems, such as E. coli, provides non-glycosylated
molecules. Further, a given preparation may include multiple
differentially glycosylated species of the protein. Glycosyl groups
can be removed through conventional methods, in particular those
utilizing glycopeptidase. In general, glycosylated polypeptides of
the invention can be incubated with a molar excess of
glycopeptidase (Boehringer Mannheim).
[0078] Correspondingly, similar DNA constructs that encode various
additions or substitutions of amino acid residues or sequences, or
deletions of terminal or internal residues or sequences are
encompassed by the invention. For example, N-glycosylation sites in
the polypeptide extracellular domain can be modified to preclude
glycosylation, allowing expression of a reduced carbohydrate analog
in mammalian and yeast expression systems. N-glycosylation sites in
eukaryotic polypeptides are characterized by an amino acid triplet
Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or
Thr. Appropriate substitutions, additions, or deletions to the
nucleotide sequence encoding these triplets will result in
prevention of attachment of carbohydrate residues at the Asn side
chain. Alteration of a single nucleotide, chosen so that Asn is
replaced by a different amino acid, for example, is sufficient to
inactivate an N-glycosylation site. Alternatively, the Ser or Thr
can by replaced with another amino acid, such as Ala. Known
procedures for inactivating N-glycosylation sites in proteins
include those described in U.S. Pat. No. 5,071,972 and EP 276,846,
hereby incorporated by reference.
[0079] In another example of variants, sequences encoding Cys
residues that are not essential for biological activity can be
altered to cause the Cys residues to be deleted or replaced with
other amino acids, preventing formation of incorrect intramolecular
disulfide bridges upon folding or renaturation.
[0080] Other variants are prepared by modification of adjacent
dibasic amino acid residues, to enhance expression in yeast systems
in which KEX2 protease activity is present. EP 212,914 discloses
the use of site-specific mutagenesis to inactivate KEX2 protease
processing sites in a protein. KEX2 protease processing sites are
inactivated by deleting, adding or substituting residues to alter
Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of
these adjacent basic residues. Lys-Lys pairings are considerably
less susceptible to KEX2 cleavage, and conversion of Arg-Lys or
Lys-Arg to Lys-Lys represents a conservative and preferred approach
to inactivating KEX2 sites.
[0081] Oligomers
[0082] Encompassed by the invention are oligomers or fusion
proteins that contain IL-1 zeta, TDZ.1, TDZ.2, TDZ.3 or Xrec2
polypeptides. When the polypeptide of the invention is a type I
membrane protein, such as Xrec2, the fusion partner is linked to
the C terminus of the type I membrane protein. Such oligomers may
be in the form of covalently-linked or non-covalently-linked
multimers, including dimers, trimers, or higher oligomers. As noted
above, preferred polypeptides are soluble and thus these oligomers
may comprise soluble polypeptides. In one aspect of the invention,
the oligomers maintain the binding ability of the polypeptide
components and provide therefor, bivalent, trivalent, etc., binding
sites.
[0083] One embodiment of the invention is directed to oligomers
comprising multiple polypeptides joined via covalent or
non-covalent interactions between peptide moieties fused to the
polypeptides. Such peptides may be peptide linkers (spacers), or
peptides that have the property of promoting oligomerization.
Leucine zippers and certain polypeptides derived from antibodies
are among the peptides that can promote oligomerization of the
polypeptides attached thereto, as described in more detail
below.
[0084] Immunoglobulin-Based Oligomers
[0085] As one alternative, an oligomer is prepared using
polypeptides derived from immunoglobulins. Preparation of fusion
proteins comprising certain heterologous polypeptides fused to
various portions of antibody-derived polypeptides (including the Fc
domain) has been described, e.g., by Ashkenazi et al. (PNAS USA
88:10535, 1991); Byrn et al. (Nature 344:677, 1990); and
Hollenbaugh and Aruffo ("Construction of Immunoglobulin Fusion
Proteins", in Current Protocols in Immunology, Suppl. 4, pages
10.19.1-10.19.11, 1992).
[0086] One embodiment of the present invention is directed to a
dimer comprising two fusion proteins created by fusing a
polypeptide of the invention to an Fc polypeptide derived from an
antibody. A gene fusion encoding the polypeptide/Fc fusion protein
is inserted into an appropriate expression vector. Polypeptide/Fc
fusion proteins are expressed in host cells transformed with the
recombinant expression vector, and allowed to assemble much like
antibody molecules, whereupon interchain disulfide bonds form
between the Fc moieties to yield divalent molecules.
[0087] The term "Fc polypeptide" as used herein includes native and
mutein forms of polypeptides made up of the Fc region of an
antibody comprising any or all of the CH domains of the Fc region.
Truncated forms of such polypeptides containing the hinge region
that promotes dimerization are also included. Preferred
polypeptides comprise an Fc polypeptide derived from a human IgG1
antibody.
[0088] One suitable Fc polypeptide, described in PCT application WO
93/10151, hereby incorporated by reference, is a single chain
polypeptide extending from the N-terminal hinge region to the
native C-terminus of the Fc region of a human IgG1 antibody.
Another useful Fc polypeptide is the Fc mutein described in U.S.
Pat. No. 5,457,035 and in Baum et al., (EMBO J. 13:3992-4001, 1994)
incorporated herein by reference. The amino acid sequence of this
mutein is identical to that of the native Fc sequence presented in
WO 93/10151, except that amino acid 19 has been changed from Leu to
Ala, amino acid 20 has been changed from Leu to Glu, and amino acid
22 has been changed from Gly to Ala. The mutein exhibits reduced
affinity for Fc receptors.
[0089] The above-described fusion proteins comprising Fc moieties
(and oligomers formed therefrom) offer the advantage of facile
purification by affinity chromatography over Protein A or Protein G
columns.
[0090] In other embodiments, the polypeptides of the invention may
be substituted for the variable portion of an antibody heavy or
light chain. If fusion proteins are made with both heavy and light
chains of an antibody, it is possible to form an oligomer with as
many as four polypeptide extracellular regions.
[0091] Peptide-Linker Based Oligomers
[0092] Alternatively, the oligomer is a fusion protein comprising
multiple polypeptides, with or without peptide linkers (spacer
peptides). Among the suitable peptide linkers are those described
in U.S. Pat. Nos. 4,751,180 and 4,935,233, which are hereby
incorporated by reference. A DNA sequence encoding a desired
peptide linker may be inserted between, and in the same reading
frame as, the DNA sequences of the invention, using any suitable
conventional technique. For example, a chemically synthesized
oligonucleotide encoding the linker may be ligated between the
sequences. In particular embodiments, a fusion protein comprises
from two to four soluble polypeptides of the invention, separated
by peptide linkers.
[0093] Leucine-Zippers
[0094] Another method for preparing the oligomers of the invention
involves use of a leucine zipper. Leucine zipper domains are
peptides that promote oligomerization of the proteins in which they
are found. Leucine zippers were originally identified in several
DNA-binding proteins (Landschulz et al., Science 240:1759, 1988),
and have since been found in a variety of different proteins. Among
the known leucine zippers are naturally occurring peptides and
derivatives thereof that dimerize or trimerize.
[0095] The zipper domain (also referred to herein as an
oligomerizing, or oligomer-forming, domain) comprises a repetitive
heptad repeat, often with four or five leucine residues
interspersed with other amino acids. Examples of zipper domains are
those found in the yeast transcription factor GCN4 and a
heat-stable DNA-binding protein found in rat liver (C/EBP;
Landschulz et al., Science 243:1681, 1989). Two nuclear
transforming proteins, fos and jun, also exhibit zipper domains, as
does the gene product of the murine proto-oncogene, c-myc
(Landschulz et al., Science 240:1759, 1988). The products of the
nuclear oncogenes fos and jun comprise zipper domains that
preferentially form heterodimers (O'Shea et al., Science 245:646,
1989, Turner and Tjian, Science 243:1689, 1989). The zipper domain
is necessary for biological activity (DNA binding) in these
proteins.
[0096] The fusogenic proteins of several different viruses,
including paramyxovirus, coronavirus, measles virus and many
retroviruses, also possess zipper domains (Buckland and Wild,
Nature 338:547, 1989; Britton, Nature 353:394, 1991; Delwart and
Mosialos, AIDS Research and Human Retroviruses 6:703, 1990). The
zipper domains in these fusogenic viral proteins are near the
transmembrane region of the proteins; it has been suggested that
the zipper domains could contribute to the oligomeric structure of
the fusogenic proteins. Oligomerization of fusogenic viral proteins
is involved in fusion pore formation (Spruce et al, Proc. Natl.
Acad. Sci. U.S.A. 88:3523, 1991). Zipper domains have also been
recently reported to play a role in oligomerization of heat-shock
transcription factors (Rabindran et al., Science 259:230,
1993).
[0097] Zipper domains fold as short, parallel coiled coils. (O'Shea
et al., Science 254:539; 1991) The general architecture of the
parallel coiled coil has been well characterized, with a
"knobs-into-holes" packing as proposed by Crick in 1953 (Acta
Crystallogr. 6:689). The dimer formed by a zipper domain is
stabilized by the heptad repeat, designated (abcdefg).sub.n
according to the notation of McLachlan and Stewart (J. Mol. Biol.
98:293; 1975), in which residues a and d are generally hydrophobic
residues, with d being a leucine, which line up on the same face of
a helix. Oppositely-charged residues commonly occur at positions g
and e. Thus, in a parallel coiled coil formed from two helical
zipper domains, the "knobs" formed by the hydrophobic side chains
of the first helix are packed into the "holes" formed between the
side chains of the second helix.
[0098] The residues at position d (often leucine) contribute large
hydrophobic stabilization energies, and are important for oligomer
formation (Krystek: et al., Int. J. Peptide Res. 38:229, 1991).
Lovejoy et al. (Science 259:1288, 1993) recently reported the
synthesis of a triple-stranded .alpha.-helical bundle in which the
helices run up-up-down. Their studies confirmed that hydrophobic
stabilization energy provides the main driving force for the
formation of coiled coils from helical monomers. These studies also
indicate that electrostatic interactions contribute to the
stoichiometry and geometry of coiled coils. Further discussion of
the structure of leucine zippers is found in Harbury et al.
(Science 262:1401, 26 Nov. 1993)
[0099] Examples of leucine zipper domains suitable for producing
soluble oligomeric proteins are described in PCT application WO
94/10308, and the leucine zipper derived from lung surfactant
protein D (SPD) described in Hoppe et al. (FEBS Letters 344:191,
1994), hereby incorporated by reference. The use of a modified
leucine zipper that allows for stable trimerization of a
heterologous protein fused thereto is described in Fanslow et al.
(Semin. Immunol. 6:267-278, 1994). Recombinant fusion proteins
comprising a soluble polypeptide fused to a leucine zipper peptide
are expressed in suitable host cells, and the soluble oligomer that
forms is recovered from the culture supernatant.
[0100] Certain leucine zipper moieties preferentially form trimers.
One example is a leucine zipper derived from lung surfactant
protein D (SPD), as described in Hoppe et al. (FEBS Letters
344:191, 1994) and in U.S. Pat. No. 5,716,805, hereby incorporated
by reference in their entirety. This lung SPD-derived leucine
zipper peptide comprises the amino acid sequence Pro Asp Val Ala
Ser Leu Arg Gln Gln Val Glu Ala Leu Gln Gly Gln Val Gln His Leu Gln
Ala Ala Phe Ser Gln Tyr.
[0101] Another example of a leucine zipper that promotes
trimerization is a peptide comprising the amino acid sequence Arg
Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His
Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Arg, as
described in U.S. Pat. No. 5,716,805. In one alternative
embodiment, an N-terminal Asp residue is added; in another, the
peptide lacks the N-terminal Arg residue.
[0102] Fragments of the foregoing zipper peptides that retain the
property of promoting oligomerization may be employed as well.
Examples of such fragments include, but are not limited to,
peptides lacking one or two of the N-terminal or C-terminal
residues presented in the foregoing amino acid sequences. Leucine
zippers may be derived from naturally occurring leucine zipper
peptides, e.g., via conservative substitution(s) in the native
amino acid sequence, wherein the peptide's ability to promote
oligomerization is retained.
[0103] Other peptides derived from naturally occurring trimeric
proteins may be employed in preparing trimeric oligomers.
Alternatively, synthetic peptides that promote oligomerization may
be employed. In particular embodiments, leucine residues in a
leucine zipper moiety are replaced by isoleucine residues. Such
peptides comprising isoleucine may be referred to as isoleucine
zippers, but are encompassed by the term "leucine zippers" as
employed herein.
Production of Polypeptides and Fragments Thereof
[0104] Expression, isolation and purification of the polypeptides
and fragments of the invention may be accomplished by any suitable
technique, including but not limited to the following:
[0105] Expression Systems
[0106] The present invention also provides recombinant cloning and
expression vectors containing DNA, as well as host cell containing
the recombinant vectors. Expression vectors comprising DNA may be
used to prepare the polypeptides or fragments of the invention
encoded by the DNA. A method for producing polypeptides comprises
culturing host cells transformed with a recombinant expression
vector encoding the polypeptide, under conditions that promote
expression of the polypeptide, then recovering the expressed
polypeptides from the culture. The skilled artisan will recognize
that the procedure for purifying the expressed polypeptides will
vary according to such factors as the type of host cells employed,
and whether the polypeptide is membrane-bound or a soluble form
that is secreted from the host cell.
[0107] Any suitable expression system may be employed. The vectors
include a DNA encoding a polypeptide or fragment of the invention,
operably linked to suitable transcriptional or translational
regulatory nucleotide sequences, such as those derived from a
mammalian, microbial, viral, or insect gene. Examples of regulatory
sequences include transcriptional promoters, operators, or
enhancers, an mRNA ribosomal binding site, and appropriate
sequences which control transcription and translation initiation
and termination. Nucleotide sequences are operably linked when the
regulatory sequence functionally relates to the DNA sequence. Thus,
a promoter nucleotide sequence is operably linked to a DNA sequence
if the promoter nucleotide sequence controls the transcription of
the DNA sequence. An origin of replication that confers the ability
to replicate in the desired host cells, and a selection gene by
which transformants are identified, are generally incorporated into
the expression vector.
[0108] In addition, a sequence encoding an appropriate signal
peptide (native or heterologous) can be incorporated into
expression vectors. A DNA sequence for a signal peptide (secretory
leader) may be fused in frame to the nucleic acid sequence of the
invention so that the DNA is initially transcribed, and the mRNA
translated, into a fusion protein comprising the signal peptide. A
signal peptide that is functional in the intended host cells
promotes extracellular secretion of the polypeptide. The signal
peptide is cleaved from the polypeptide upon secretion of
polypeptide from the cell.
[0109] The skilled artisan will also recognize that the position(s)
at which the signal peptide is cleaved may differ from that
predicted by computer program, and may vary according to such
factors as the type of host cells employed in expressing a
recombinant polypeptide. A protein preparation may include a
mixture of protein molecules having different N-terminal amino
acids, resulting from cleavage of the signal peptide at more than
one site. Particular embodiments of mature proteins provided herein
include, but are not limited to, proteins having the residue at
position 6, 23, 25, 26, 39, 41, or 48 of SEQ ID NO:3 and at
position 1 or 19 of SEQ ID NO:4 as the N-terminal amino acid.
[0110] Suitable host cells for expression of polypeptides include
prokaryotes, yeast or higher eukaryotic cells. Mammalian or insect
cells are generally preferred for use as host cells. Appropriate
cloning and expression vectors for use with bacterial, fungal,
yeast, and mammalian cellular hosts are described, for example, in
Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New
York, (1985). Cell-free translation systems could also be employed
to produce polypeptides using RNAs derived from DNA constructs
disclosed herein.
[0111] Prokaryotic Systems
[0112] Prokaryotes include gram-negative or gram-positive
organisms. Suitable prokaryotic host cells for transformation
include, for example, E. coli, Bacillus subtilis, Salmonella
typhimurium, and various other species within the genera
Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic
host cell, such as E. coli, a polypeptide may include an N-terminal
methionine residue to facilitate expression of the recombinant
polypeptide in the prokaryotic host cell. The N-terminal Met may be
cleaved from the expressed recombinant polypeptide.
[0113] Expression vectors for use in prokaryotic host cells
generally comprise one or more phenotypic selectable marker genes.
A phenotypic selectable marker gene is, for example, a gene
encoding a protein that confers antibiotic resistance or that
supplies an autotrophic requirement. Examples of useful expression
vectors for prokaryotic host cells include those derived from
commercially available plasmids such as the cloning vector pBR322
(ATCC 37017). pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides simple means for identifying
transformed cells. An appropriate promoter and a DNA sequence are
inserted into the pBR322 vector. Other commercially available
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,
USA).
[0114] Promoter sequences commonly used for recombinant prokaryotic
host cell expression vectors include .beta.-lactamase
(penicillinase), lactose promoter system (Chang et al., Nature
275:615, 1978; and Goeddel et al., Nature 281:544, 1979),
tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res.
8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p.
412, 1982). A particularly useful prokaryotic host cell expression
system employs a phage .lamda.P.sub.L promoter and a cI857ts
thermolabile repressor sequence. Plasmid vectors available from the
American Type Culture Collection which incorporate derivatives of
the .lamda.P.sub.L promoter include plasmid pHUB2 (resident in E.
coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1,
ATCC 53082).
[0115] Yeast Systems
[0116] Alternatively, the polypeptides may be expressed in yeast
host cells, preferably from the Saccharomyces genus (e.g., S.
cerevisiae). Other genera of yeast, such as Pichia or
Kluyveromyces, may also be employed. Yeast vectors will often
contain an origin of replication sequence from a 2 .mu.L yeast
plasmid, an autonomously replicating sequence (ARS), a promoter
region, sequences for polyadenylation, sequences for transcription
termination, and a selectable marker gene. Suitable promoter
sequences for yeast vectors include, among others, promoters for
metallothionein, 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; and Holland et al., Biochem.
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, phospho-glucose isomerase, and glucokinase. Other
suitable vectors and promoters for use in yeast expression are
further described in Hitzeman, EPA-73,657. Another alternative is
the glucose-repressible ADH2 promoter described by Russell et al.
(J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724,
1982). Shuttle vectors replicable in both yeast and E. coli may be
constructed by inserting DNA sequences from pBR322 for selection
and replication in E. coli (Amp.sup.r gene and origin of
replication) into the above-described yeast vectors.
[0117] The yeast .alpha.-factor leader sequence may be employed to
direct secretion of the polypeptide. The .alpha.-factor leader
sequence is often inserted between the promoter sequence and the
structural gene sequence. See, e.g., Kurjan et al., Cell 30:933,
1982 and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984.
Other leader sequences suitable for facilitating secretion of
recombinant polypeptides from yeast hosts are known to those of
skill in the art. A leader sequence may be modified near its 3' end
to contain one or more restriction sites. This will facilitate
fusion of the leader sequence to the structural gene.
[0118] Yeast transformation protocols are known to those of skill
in the art. One such protocol is described by Hinnen et al., Proc.
Natl. Acad. Sci. USA 75:1929, 1978. The Hinnen et al. protocol
selects for Trp.sup.+ transformants in a selective medium, wherein
the selective medium consists of 0.67% yeast nitrogen base, 0.5%
casamino acids, 2% glucose, 10 mg/ml adenine and 20 mg/ml
uracil.
[0119] Yeast host cells transformed by vectors containing an ADH2
promoter sequence may be grown for inducing expression in a "rich"
medium. An example of a rich medium is one consisting of 1% yeast
extract, 2% peptone, and 1% glucose supplemented with 80 mg/ml
adenine and 80 mg/ml uracil. Derepression of the ADH2 promoter
occurs when glucose is exhausted from the medium.
[0120] Mammalian or Insect Systems
[0121] Mammalian or insect host cell culture systems also may be
employed to express recombinant polypeptides. Bacculovirus systems
for production of heterologous proteins in insect cells are
reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).
Established cell lines of mammalian origin also may be employed.
Examples of suitable mammalian host cell lines include the COS-7
line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell
23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163),
Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL
10) cell lines, and the CV1/EBNA cell line derived from the African
green monkey kidney cell line CV1 (ATCC CCL 70) as described by
McMahan et al. (EMBO J. 10: 2821, 1991).
[0122] Established methods for introducing DNA into mammalian cells
have been described (Kaufman, R. J., Large Scale Mammalian Cell
Culture, 1990, pp. 15-69). Additional protocols using commercially
available reagents, such as Lipofectamine lipid reagent (Gibco/BRL)
or Lipofectamine-Plus lipid reagent, can be used to transfect cells
(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987). In
addition, electroporation can be used to transfect mammalian cells
using conventional procedures, such as those in Sambrook et al.
(Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold
Spring Harbor Laboratory Press, 1989). Selection of stable
transformants can be performed using methods known in the art, such
as, for example, resistance to cytotoxic drugs. Kaufman et al.,
Meth. in Enzymology 185:487-511, 1990, describes several selection
schemes, such as dihydrofolate reductase (DHFR) resistance. A
suitable host strain for DHFR selection can be CHO strain DX-B11,
which is deficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad.
Sci. USA 77:4216-4220, 1980). A plasmid expressing the DHFR cDNA
can be introduced into strain DX-B11, and only cells that contain
the plasmid can grow in the appropriate selective media. Other
examples of selectable markers that can be incorporated into an
expression vector include cDNAs conferring resistance to
antibiotics, such as G418 and hygromycin B. Cells harboring the
vector can be selected on the basis of resistance to these
compounds.
[0123] Transcriptional and translational control sequences for
mammalian host cell expression vectors can be excised from viral
genomes. Commonly used promoter sequences and enhancer sequences
are derived from polyoma virus, adenovirus 2, simian virus 40
(SV40), and human cytomegalovirus. DNA sequences derived from the
SV40 viral genome, for example, SV40 origin, early and late
promoter, enhancer, splice, and polyadenylation sites can be used
to provide other genetic elements for expression of a structural
gene sequence in a mammalian host cell. Viral early and late
promoters are particularly useful because both are easily obtained
from a viral genome as a fragment, which can also contain a viral
origin of replication (Fiers et al., Nature 273:113, 1978; Kaufman,
Meth. in Enzymology, 1990). Smaller or larger SV40 fragments can
also be used, provided the approximately 250 bp sequence extending
from the Hind III site toward the Bgl I site located in the SV40
viral origin of replication site is included.
[0124] Additional control sequences shown to improve expression of
heterologous genes from mammalian expression vectors include such
elements as the expression augmenting sequence element (EASE)
derived from CHO cells (Morris et al., Animal Cell Technology,
1997, pp. 529-534 and PCT Application WO 97/25420) and the
tripartite leader (TPL) and VA gene RNAs from Adenovirus 2
(Gingeras et al., J. Biol. Chem. 257:13475-13491, 1982). The
internal ribosome entry site (IRES) sequences of viral origin
allows dicistronic mRNAs to be translated efficiently (Oh and
Sarnow, Current Opinion in Genetics and Development 3:295-300,
1993; Ramesh et al., Nucleic Acids Research 24:2697-2700, 1996).
Expression of a heterologous cDNA as part of a dicistronic mRNA
followed by the gene for a selectable marker (e.g. DHFR) has been
shown to improve transfectability of the host and expression of the
heterologous cDNA (Kaufman, Meth. in Enzymology, 1990). Exemplary
expression vectors that employ dicistronic mRNAs are pTR-DC/GFP
described by Mosser et al., Biotechniques 22:150-161, 1997, and
p2A5I described by Morris et al., Animal Cell Technology, 1997, pp.
529-534.
[0125] A useful high expression vector, pCAVNOT, has been described
by Mosley et al., Cell 59:335-348, 1989. Other expression vectors
for use in mammalian host cells can be constructed as disclosed by
Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A useful system
for stable high level expression of mammalian cDNAs in C127 murine
mammary epithelial cells can be constructed substantially as
described by Cosman et al. (Mol. Immunol. 23:935, 1986). A useful
high expression vector, PMLSV N1/N4, described by Cosman et al.,
Nature 312:768, 1984, has been deposited as ATCC 39890. Additional
useful mammalian expression vectors are described in EP-A-0367566,
and in WO 91/18982, incorporated by reference herein. In yet
another alternative, the vectors can be derived from
retroviruses.
[0126] Another useful expression vector, pFLAG.sup.7, can be used.
FLAG.sup.7 technology is centered on the fusion of a low molecular
weight (1 kD), hydrophilic, FLAG.sup.7 marker peptide to the
N-terminus of a recombinant protein expressed by pFLAG.sup.7
expression vectors.
[0127] Regarding signal peptides that may be employed, the native
signal peptide may be replaced by a heterologous signal peptide or
leader sequence, if desired. The choice of signal peptide or leader
may depend on factors such as the type of host cells in which the
recombinant polypeptide is to be produced. To illustrate, examples
of heterologous signal peptides that are functional in mammalian
host cells include the signal sequence for interleukin-7 (IL-7)
described in U.S. Pat. No. 4,965,195; the signal sequence for
interleukin-2 receptor described in Cosman et al., Nature 312:768
(1984); the interleukin-4 receptor signal peptide described in EP
367,566; the type I interleukin-1 receptor signal peptide described
in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor
signal peptide described in EP 460,846.
[0128] Purification
[0129] The invention also includes methods of isolating and
purifying the polypeptides and fragments thereof.
[0130] Isolation and Purification
[0131] The "isolated" polypeptides or fragments thereof encompassed
by this invention are polypeptides or fragments that are not in an
environment identical to an environment in which it or they can be
found in nature. The "purified" polypeptides or fragments thereof
encompassed by this invention are essentially free of association
with other proteins or polypeptides, for example, as a purification
product of recombinant expression systems such as those described
above or as a purified product from a non-recombinant source such
as naturally occurring cells and/or tissues.
[0132] In one preferred embodiment, the purification of recombinant
polypeptides or fragments can be accomplished using fusions of
polypeptides or fragments of the invention to another polypeptide
to aid in the purification of polypeptides or fragments of the
invention. Such fusion partners can include the poly-His or other
antigenic identification peptides described above as well as the Fc
moieties described previously.
[0133] With respect to any type of host cell, as is known to the
skilled artisan, procedures for purifying a recombinant polypeptide
or fragment will vary according to such factors as the type of host
cells employed and whether or not the recombinant polypeptide or
fragment is secreted into the culture medium.
[0134] In general, the recombinant polypeptide or fragment can be
isolated from the host cells if not secreted, or from the medium or
supernatant if soluble and secreted, followed by one or more
concentration, salting-out, ion exchange, hydrophobic interaction,
affinity purification or size exclusion chromatography steps. As to
specific ways to accomplish these steps, the culture medium first
can be concentrated using a commercially available protein
concentration filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. Following the concentration step, the
concentrate can be applied to a purification matrix such as a gel
filtration medium. Alternatively, an anion exchange resin can be
employed, for example, a matrix or substrate having pendant
diethylaminoethyl (DEAE) groups. The matrices can be acrylamide,
agarose, dextran, cellulose or other types commonly employed in
protein purification. Alternatively, a cation exchange step can be
employed. Suitable cation exchangers include various insoluble
matrices comprising sulfopropyl or carboxymethyl groups. In
addition, a chromatofocusing step can be employed. Alternatively, a
hydrophobic interaction chromatography step can be employed.
Suitable matrices can be phenyl or octyl moieties bound to resins.
In addition, affinity chromatography with a matrix which
selectively binds the recombinant protein can be employed. Examples
of such resins employed are lectin columns, dye columns, and
metal-chelating columns. Finally, one or more reverse-phase high
performance liquid chromatography (RP-HPLC) steps employing
hydrophobic RP-HPLC media, (e.g., silica gel or polymer resin
having pendant methyl, octyl, octyldecyl or other aliphatic groups)
can be employed to further purify the polypeptides. Some or all of
the foregoing purification steps, in various combinations, are well
known and can be employed to provide an isolated and purified
recombinant protein.
[0135] It is also possible to utilize an affinity column comprising
a polypeptide-binding protein of the invention, such as a
monoclonal antibody generated against polypeptides of the
invention, to affinity-purify expressed polypeptides. These
polypeptides can be removed from an affinity column using
conventional techniques, e.g., in a high salt elution buffer and
then dialyzed into a lower salt buffer for use or by changing pH or
other components depending on the affinity matrix utilized, or be
competitively removed using the naturally occurring substrate of
the affinity moiety, such as a polypeptide derived from the
invention.
[0136] In this aspect of the invention, polypeptide-binding
proteins, such as the anti-polypeptide antibodies of the invention
or other proteins that may interact with the polypeptide of the
invention, can be bound to a solid phase support such as a column
chromatography matrix or a similar substrate suitable for
identifying, separating, or purifying cells that express
polypeptides of the invention on their surface. Adherence of
polypeptide-binding proteins of the invention to a solid phase
contacting surface can be accomplished by any means. For example,
magnetic microspheres can be coated with these polypeptide-binding
proteins and held in the incubation vessel through a magnetic
field. Suspensions of cell mixtures are contacted with the solid
phase that has such polypeptide-binding proteins thereon. Cells
having polypeptides of the invention on their surface bind to the
fixed polypeptide-binding protein and unbound cells then are washed
away. This affinity-binding method is useful for purifying,
screening, or separating such polypeptide-expressing cells from
solution. Methods of releasing positively selected cells from the
solid phase are known in the art and encompass, for example, the
use of enzymes. Such enzymes are preferably non-toxic and
non-injurious to the cells and are preferably directed to cleaving
the cell-surface binding partner.
[0137] Alternatively, mixtures of cells suspected of containing
polypeptide-expressing cells of the invention first can be
incubated with a biotinylated polypeptide-binding protein of the
invention. Incubation periods are typically at least one hour in
duration to ensure sufficient binding to polypeptides of the
invention. The resulting mixture then is passed through a column
packed with avidin-coated beads, whereby the high affinity of
biotin for avidin provides the binding of the polypeptide-binding
cells to the beads. Use of avidin-coated beads is known in the art.
See Berenson, et al. J. Cell. Biochem., 10D:239 (1986). Wash of
unbound material and the release of the bound cells is performed
using conventional methods.
[0138] The desired degree of purity depends on the intended use of
the protein. A relatively high degree of purity is desired when the
polypeptide is to be administered in vivo, for example. In such a
case, the polypeptides are purified such that no protein bands
corresponding to other proteins are detectable upon analysis by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be
recognized by one skilled in the pertinent field that multiple
bands corresponding to the polypeptide may be visualized by
SDS-PAGE, due to differential glycosylation, differential
post-translational processing, and the like. Most preferably, the
polypeptide of the invention is purified to substantial
homogeneity, as indicated by a single protein band upon analysis by
SDS-PAGE. The protein band may be visualized by silver staining,
Coomassie blue staining, or (if the protein is radiolabeled) by
autoradiography.
[0139] Assays
[0140] The purified polypeptides of the invention (including
proteins, polypeptides, fragments, variants, oligomers, and other
forms) may be tested for the ability to bind the binding partner in
any suitable assay, such as a conventional binding assay. To
illustrate, the polypeptide may be labeled with a detectable
reagent (e.g., a radionuclide, chromophore, enzyme that catalyzes a
calorimetric or fluorometric reaction, and the like). The labeled
polypeptide is contacted with cells expressing the binding partner.
The cells then are washed to remove unbound labeled polypeptide,
and the presence of cell-bound label is determined by a suitable
technique, chosen according to the nature of the label.
[0141] One example of a binding assay procedure is as follows. A
recombinant expression vector containing the binding partner cDNA
is constructed using methods well known in the art. CV1-EBNA-1
cells in 10 cm.sup.2 dishes are transfected with the recombinant
expression vector. CV-1/EBNA-1 cells (ATCC CRL 10478)
constitutively express EBV nuclear antigen-1 driven from the CMV
immediate-early enhancer/promoter. CV1-EBNA-1 was derived from the
African Green Monkey kidney cell line CV-1 (ATCC CCL 70), as
described by McMahan et al. (EMBO J. 10:2821, 1991).
[0142] The transfected cells are cultured for 24 hours, and the
cells in each dish then are split into a 24-well plate. After
culturing an additional 48 hours, the transfected cells (about
4.times.10.sup.4 cells/well) are washed with BM-NFDM, which is
binding medium (RPMI 1640 containing 25 mg/ml bovine serum albumin,
2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50 mg/ml nonfat
dry milk has been added. The cells then are incubated for 1 hour at
37.degree. C. with various concentrations of, for example, a
soluble polypeptide/Fc fusion protein made as set forth above.
Cells then are washed and incubated with a constant saturating
concentration of a .sup.125I-mouse anti-human IgG in binding
medium, with gentle agitation for 1 hour at 37.degree. C. After
extensive washing, cells are released via trypsinization.
[0143] The mouse anti-human IgG employed above is directed against
the Fc region of human IgG and can be obtained from Jackson
Immunoresearch Laboratories, Inc., West Grove, Pa. The antibody is
radioiodinated using the standard chloramine-T method. The antibody
will bind to the Fc portion of any polypeptide/Fc protein that has
bound to the cells. In all assays, non-specific binding of
.sup.125I-antibody is assayed in the absence of the Fc fusion
protein/Fc, as well as in the presence of the Fc fusion protein and
a 200-fold molar excess of unlabeled mouse anti-human IgG
antibody.
[0144] Cell-bound .sup.125I-antibody is quantified on a Packard
Autogamma counter. Affinity calculations (Scatchard, Ann. N.Y.
Acad. Sci. 51:660, 1949) are generated on RS/1 (BBN Software,
Boston, Mass.) run on a Microvax computer.
[0145] Another type of suitable binding assay is a competitive
binding assay. To illustrate, biological activity of a variant may
be determined by assaying for the variant's ability to compete with
the native protein for binding to the binding partner.
[0146] Competitive binding assays can be performed by conventional
methodology. Reagents that may be employed in competitive binding
assays include radiolabeled polypeptides of the invention and
intact cells expressing the binding partner (endogenous or
recombinant). For example, a radiolabeled soluble IL-1 zeta
fragment can be used to compete with a soluble IL-1 zeta variant
for binding to cell surface IL-1 zeta receptors. Instead of intact
cells, one could substitute a soluble binding partner/Fc fusion
protein bound to a solid phase through the interaction of Protein A
or Protein G (on the solid phase) with the Fc moiety.
Chromatography columns that contain Protein A and Protein G include
those available from Pharmacia Biotech, Inc., Piscataway, N.J.
[0147] Another type of competitive binding assay utilizes
radiolabeled soluble binding partner, such as a soluble IL-1 zeta
receptor/Fc fusion or Xrec2 ligand/Fc fusion protein, and intact
cells expressing the binding partner. Qualitative results can be
obtained by competitive autoradiographic plate binding assays,
while Scatchard plots (Scatchard, Ann. N.Y. Acad. Sci. 51:660,
1949) may be utilized to generate quantitative results.
Use of IL-1 Zeta, TDZ.1, TDZ.2, TDZ.3 and Xrec2 Nucleic Acid or
Oligonucleotides
[0148] In addition to being used to express polypeptides as
described above, the nucleic acids of the invention, including DNA,
and oligonucleotides thereof can be used: [0149] as probes to
identify nucleic acid encoding proteins of the IL-1 ligand and
receptor families; [0150] to identify human chromosomes 2 and X;
[0151] to map genes on human chromosomes 2 and X; [0152] to
identify genes associated with certain diseases, syndromes, or
other conditions associated with human chromosomes 2 and X; [0153]
as single-stranded sense or antisense oligonucleotides, to inhibit
expression of polypeptides encoded by the IL-1 zeta, TDZ.1, TDZ.2,
TDZ.3 and Xrec2 genes; [0154] to help detect defective genes in an
individual; and [0155] for gene therapy.
[0156] Probes
[0157] Among the uses of nucleic acids of the invention is the use
of fragments as probes or primers. Such fragments generally
comprise at least about 17 contiguous nucleotides of a DNA
sequence. In other embodiments, a DNA fragment comprises at least
30, or at least 60, contiguous nucleotides of a DNA sequence.
[0158] Because homologs of SEQ ID NOs:1, 2, 5, 6 and 7, from other
mammalian species, are contemplated herein, probes based on the
human DNA sequences of SEQ ID NOs: 1, 2, 5, 6 and 7 may be used to
screen cDNA libraries derived from other mammalian species, using
conventional cross-species hybridization techniques.
[0159] Using knowledge of the genetic code in combination with the
amino acid sequences set forth above, sets of degenerate
oligonucleotides can be prepared. Such oligonucleotides are useful
as primers, e.g., in polymerase chain reactions (PCR), whereby DNA
fragments are isolated and amplified.
[0160] Chromosome Mapping
[0161] All or a portion of the nucleic acids of IL-1 zeta of SEQ ID
NO:1 and of Xrec2 of SEQ ID NO:2, including oligonucleotides, can
be used by those skilled in the art using well-known techniques to
identify the human chromosomes 2 and X, respectively, as well as
the specific locus thereof, that contains the DNA of IL-1 ligand
and IL-1 receptor family members. Useful techniques include, but
are not limited to, using the sequence or portions, including
oligonucleotides, as a probe in various well-known techniques such
as radiation hybrid mapping (high resolution), in situ
hybridization to chromosome spreads (moderate resolution), and
Southern blot hybridization to hybrid cell lines containing
individual human chromosomes (low resolution).
[0162] For example, chromosomes can be mapped by radiation
hybridization. PCR is performed using the Whitehead Institute/MIT
Center for Genome Research Genebridge4 panel of 93 radiation
hybrids
(http://www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/-
rhmap/genebridge4.html). Primers are used which lie within a
putative exon of the gene of interest and which amplify a product
from human genomic DNA, but do not amplify hamster genomic DNA. The
results of the PCRs are converted into a data vector that is
submitted to the Whitehead/MIT Radiation Mapping site on the
internet (http://www-seq.wi.mit.edu). The data is scored and the
chromosomal assignment and placement relative to known Sequence Tag
Site (STS) markers on the radiation hybrid map is provided. The
following web site provides additional information about radiation
hybrid mapping:
http://www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/0-
7-97.INTRO.html).
Identifying Associated Diseases
[0163] As set forth below, IL-1 zeta of SEQ ID NO:1, IL-1 zeta
splice variants, and Xrec2 of SEQ ID NO:2 have been mapped by
radiation hybridization and high-throughput-shotgun sequencing to
the 2q11-12 and Xp22 regions of human chromosomes 2 and X,
respectively. Human chromosome 2 is associated with specific
diseases which include but are not limited to glaucoma, ectodermal
dysplasia, insulin-dependent diabetes mellitus, wrinkly skin
syndrome, T-cell leukemia/lymphoma, and tibial muscular dystrophy
while human chromosome X is associated with retinoschisis,
lissencephaly, subcortical laminalheteropia, mental retardation,
cowchock syndrome, bazex syndrome, hypertrichosis,
lymphoproliferative syndrome, immunodeficiency, Langer mesomelic
dysplasia, and leukemia. Thus, the nucleic acids of SEQ ID NOs:1
and 2 or a fragment thereof can be used by one skilled in the art
using well-known techniques to analyze abnormalities associated
with gene mapping to chromosomes 2 and X. This enables one to
distinguish conditions in which this marker is rearranged or
deleted. In addition, nucleotides of SEQ ID NOs: 1, 2, 5, 6 and 7
or a fragment thereof can be used as a positional marker to map
other genes of unknown location.
[0164] The DNA may be used in developing treatments for any
disorder mediated (directly or indirectly) by defective, or
insufficient amounts of, the genes corresponding to the nucleic
acids of the invention. Disclosure herein of native nucleotide
sequences permits the detection of defective genes, and the
replacement thereof with normal genes. Defective genes may be
detected in in vitro diagnostic assays, and by comparison of a
native nucleotide sequence disclosed herein with that of a gene
derived from a person suspected of harboring a defect in this
gene.
[0165] Sense-Antisense
[0166] Other useful fragments of the nucleic acids include
antisense or sense oligonucleotides comprising a single-stranded
nucleic acid sequence (either RNA or DNA) capable of binding to
target mRNA (sense) or DNA (antisense) sequences. Antisense or
sense oligonucleotides according to the present invention comprise
a fragment of DNA (SEQ ID NOs: 1, 2, 5, 6 and 7). Such a fragment
generally comprises at least about 14 nucleotides, preferably from
about 14 to about 30 nucleotides. The ability to derive an
antisense or a sense oligonucleotide, based upon a cDNA sequence
encoding a given protein is described in, for example, Stein and
Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.
(BioTechniques 6:958, 1988).
[0167] Binding of antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of duplexes that
block or inhibit protein expression by one of several means,
including enhanced degradation of the mRNA by RNAseH, inhibition of
splicing, premature termination of transcription or translation, or
by other means. The antisense oligonucleotides thus may be used to
block expression of proteins. Antisense or sense oligonucleotides
further comprise oligonucleotides having modified
sugar-phosphodiester backbones (or other sugar linkages, such as
those described in WO91/06629) and wherein such sugar linkages are
resistant to endogenous nucleases. Such oligonucleotides with
resistant sugar linkages are stable in vivo (i.e., capable of
resisting enzymatic degradation) but retain sequence specificity to
be able to bind to target nucleotide sequences.
[0168] Other examples of sense or antisense oligonucleotides
include those oligonucleotides which are covalently linked to
organic moieties, such as those described in WO 90/10448, and other
moieties that increases affinity of the oligonucleotide for a
target nucleic acid sequence, such as poly-(L-lysine). Further
still, intercalating agents, such as ellipticine, and alkylating
agents or metal complexes may be attached to sense or antisense
oligonucleotides to modify binding specificities of the antisense
or sense oligonucleotide for the target nucleotide sequence.
[0169] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, lipofection,
CaPO.sub.4-mediated DNA transfection, electroporation, or by using
gene transfer vectors such as Epstein-Barr virus.
[0170] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or
block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
[0171] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0172] IL-1 zeta anti-sense are useful as therapeutics to treat
medical conditions and disease associated with immune system
dysfunction and IL-12 production. Such medical conditions and
disease are described below and include the deleterious effects of
inflammation and auto-immune diseases. Accordingly, IL-1 zeta
anti-sense are IL-12 antagonists and are useful in treating disease
and medical conditions that are benefited by IL-12 expression
downregulation.
Use of IL-1 Zeta TDZ.1, TDZ.2 TDZ.3 and Xrec2 Polypeptides and
Fragmented Polypeptides
[0173] Uses include, but are not limited to, the following: [0174]
Purifying proteins and measuring activity thereof [0175] Delivery
Agents [0176] Therapeutic and Research Reagents [0177] Molecular
weight and Isoelectric focusing markers [0178] Controls for peptide
fragmentation [0179] Identification of unknown proteins [0180]
Preparation of Antibodies
[0181] Purification Reagents
[0182] Each of the polypeptides of the invention finds use as a
protein purification reagent. The polypeptides may be attached to a
solid support material and used to purify the binding partner
proteins by affinity chromatography. In particular embodiments, a
polypeptide (in any form described herein that is capable of
binding the binding partner) is attached to a solid support by
conventional procedures. As one example, chromatography columns
containing functional groups that will react with functional groups
on amino acid side chains of proteins are available (Pharmacia
Biotech, Inc., Piscataway, N.J.). In an alternative, a
polypeptide/Fc protein (as discussed above) is attached to Protein
A- or Protein G-containing chromatography columns through
interaction with the Fc moiety.
[0183] The polypeptide also finds use in purifying or identifying
cells that express the binding partner on the cell surface.
Polypeptides are bound to a solid phase such as a column
chromatography matrix or a similar suitable substrate. For example,
magnetic microspheres can be coated with the polypeptides and held
in an incubation vessel through a magnetic field. Suspensions of
cell mixtures containing the binding partner expressing cells are
contacted with the solid phase having the polypeptides thereon.
Cells expressing the binding partner on the cell surface bind to
the fixed polypeptides, and unbound cells then are washed away.
[0184] Alternatively, the polypeptides can be conjugated to a
detectable moiety, then incubated with cells to be tested for
binding partner expression. After incubation, unbound labeled
matter is removed and the presence or absence of the detectable
moiety on the cells is determined.
[0185] In a further alternative, mixtures of cells suspected of
containing cells expressing the binding partner are incubated with
biotinylated polypeptides. Incubation periods are typically at
least one hour in duration to ensure sufficient binding. The
resulting mixture then is passed through a column packed with
avidin-coated beads, whereby the high affinity of biotin for avidin
provides binding of the desired cells to the beads. Procedures for
using avidin-coated beads are known (see Berenson, et al. J. Cell.
Biochem., 10D:239, 1986). Washing to remove unbound material, and
the release of the bound cells, are performed using conventional
methods.
[0186] Measuring Activity
[0187] Polypeptides also find use in measuring the biological
activity of the binding partner protein in terms of their binding
affinity. The polypeptides thus may be employed by those conducting
"quality assurance" studies, e.g., to monitor shelf life and
stability of protein under different conditions. For example, the
polypeptides may be employed in a binding affinity study to measure
the biological activity of a binding partner protein that has been
stored at different temperatures, or produced in different cell
types. The proteins also may be used to determine whether
biological activity is retained after modification of a binding
partner protein (e.g., chemical modification, truncation, mutation,
etc.). The binding affinity of the modified binding partner protein
is compared to that of an unmodified binding partner protein to
detect any adverse impact of the modifications on biological
activity of the binding partner. The biological activity of a
binding partner protein thus can be ascertained before it is used
in a research study, for example.
[0188] Delivery Agents
[0189] The polypeptides also find use as carriers for delivering
agents attached thereto to cells bearing the binding partner. The
polypeptides thus can be used to deliver diagnostic or therapeutic
agents to such cells (or to other cell types found to express the
binding partner on the cell surface) in in vitro or in vivo
procedures.
[0190] Detectable (diagnostic) and therapeutic agents that may be
attached to a polypeptide include, but are not limited to, toxins,
other cytotoxic agents, drugs, radionuclides, chromophores, enzymes
that catalyze a colorimetric or fluorometric reaction, and the
like, with the particular agent being chosen according to the
intended application. Among the toxins are ricin, abrin, diphtheria
toxin, Pseudomonas aeruginosa exotoxin A, ribosomal inactivating
proteins, mycotoxins such as trichothecenes, and derivatives and
fragments (e.g., single chains) thereof. Radionuclides suitable for
diagnostic use include, but are not limited to, .sup.123I,
.sup.131I, .sup.99mTc, .sup.111In, and .sup.76Br. Examples of
radionuclides suitable for therapeutic use are .sup.131I,
.sup.211At, .sup.77Br, .sup.186Re, .sup.188Re, .sup.212Pb,
.sup.212Bi, .sup.109Pd, .sup.64Cu, and .sup.67Cu.
[0191] Such agents may be attached to the polypeptide by any
suitable conventional procedure. The polypeptide comprises
functional groups on amino acid side chains that can be reacted
with functional groups on a desired agent to form covalent bonds,
for example. Alternatively, the protein or agent may be derivatized
to generate or attach a desired reactive functional group. The
derivatization may involve attachment of one of the bifunctional
coupling reagents available for attaching various molecules to
proteins (Pierce Chemical Company, Rockford, Ill.). A number of
techniques for radiolabeling proteins are known. Radionuclide
metals may be attached to polypeptides by using a suitable
bifunctional chelating agent, for example.
[0192] Conjugates comprising polypeptides and a suitable diagnostic
or therapeutic agent (preferably covalently linked) are thus
prepared. The conjugates are administered or otherwise employed in
an amount appropriate for the particular application.
[0193] Therapeutic Agents
[0194] Polypeptides of the invention may be used in developing
treatments for any disorder mediated (directly or indirectly) by
defective, or insufficient amounts of the polypeptides. These
polypeptides may be administered to a mammal afflicted with such a
disorder.
[0195] The polypeptides may also be employed in inhibiting a
biological activity of the binding partner, in in vitro or in vivo
procedures. For example, a purified Xrec2 receptor polypeptide may
be used to inhibit binding of Xrec2 ligand to endogenous cell
surface Xrec2 receptor, or a purified IL-1 zeta polypeptide, or any
of its splice variants can be used to inhibit binding of endogenous
IL-1 zeta or splice variants to its cell surface receptor.
Biological effects that result from the binding of Xrec2 ligand to
endogenous Xrec2 receptors thus are inhibited. In particular, IL-1
zeta polypeptides and fragments of these polypeptides that induce
IL-12 expression are useful to upregulate IL-12 expression in
individuals who can benefit from increased IL-12 production,
including individuals who benefit from enhanced cell mediated
immunity. Diseases and medical conditions treatable with agonists
of IL-1 zeta polypeptide, as described below, may be suitably
treated using IL-1 zeta polypeptides and fragments of this
invention.
[0196] Polypeptides of the invention may be administered to a
mammal to treat a binding partner-mediated disorder. Such binding
partner-mediated disorders include conditions caused (directly or
indirectly) or exacerbated by the binding partner.
[0197] Compositions of the present invention may contain a
polypeptide in any form described herein, such as native proteins,
variants, derivatives, oligomers, and biologically active
fragments. In particular embodiments, the composition comprises a
soluble polypeptide or an oligomer comprising soluble polypeptides
of the invention.
[0198] Compositions comprising an effective amount of a polypeptide
of the present invention, in combination with other components such
as a physiologically acceptable diluent, carrier, or excipient, are
provided herein. The polypeptides can be formulated according to
known methods used to prepare pharmaceutically useful compositions.
They can be combined in admixture, either as the sole active
material or with other known active materials suitable for a given
indication, with pharmaceutically acceptable diluents (e.g.,
saline, Tris-HCl, acetate, and phosphate buffered solutions),
preservatives (e.g., thimerosal, benzyl alcohol, parabens),
emulsifiers, solubilizers, adjuvants and/or carriers. Suitable
formulations for pharmaceutical compositions include those
described in Remington's Pharmaceutical Sciences, 16th ed. 1980,
Mack Publishing Company, Easton, Pa.
[0199] In addition, such compositions can be complexed with
polyethylene glycol (PEG), metal ions, or incorporated into
polymeric compounds such as polyacetic acid, polyglycolic acid,
hydrogels, dextran, etc., or incorporated into liposomes,
microemulsions, micelles, unilamellar or multilamellar vesicles,
erythrocyte ghosts or spheroblasts. Such compositions will
influence the physical state, solubility, stability, rate of in
vivo release, and rate of in vivo clearance, and are thus chosen
according to the intended application.
[0200] The compositions of the invention can be administered in any
suitable manner, e.g., topically, parenterally, or by inhalation.
The term "parenteral" includes injection, e.g., by subcutaneous,
intravenous, or intramuscular routes, also including localized
administration, e.g., at a site of disease or injury. Sustained
release from implants is also contemplated. One skilled in the
pertinent art will recognize that suitable dosages will vary,
depending upon such factors as the nature of the disorder to be
treated, the patient's body weight, age, and general condition, and
the route of administration. Preliminary doses can be determined
according to animal tests, and the scaling of dosages for human
administration is performed according to art-accepted
practices.
[0201] Compositions comprising nucleic acids in physiologically
acceptable formulations are also contemplated. DNA may be
formulated for injection, for example.
[0202] Research Agents
[0203] Another use of the polypeptide of the present invention is
as a research tool for studying the biological effects that result
from the interactions of IL-1 zeta, or any of its splice variants,
with its binding partner, and of Xrec2 with its binding partner, or
from inhibiting these interactions, on different cell types.
Polypeptides also may be employed in in vitro assays for detecting
IL-1 zeta, Xrec2, the respective binding partners or the
interactions thereof.
[0204] Another embodiment of the invention relates to uses of the
polypeptides of the invention to study cell signal transduction.
IL-1 family ligands and receptors play a central role in protection
against infection and immune inflammatory responses which includes
cellular signal transduction, activating vascular endothelial cells
and lymphocytes, induction of inflammatory cytokines, acute phase
proteins, hematopoiesis, fever, bone resorption, prostaglandins,
metalloproteinases, and adhesion molecules. With the continued
increase in the number of known IL-1 family members, a suitable
classification scheme is one based on comparing polypeptide
structure as well as function (activation and regulatory
properties). Thus, IL-1 zeta, TDZ.1, TDZ.2, and TDZ.3, like other
IL-1 family ligands (IL-1.alpha., IL-1.beta., and IL-18) and Xrec2,
like other IL-1R family receptors (IL-1RI, IL-1RII, IL-1Rrp1, and
AcPL), would likely be involved in many of the functions noted
above as well as promote inflammatory responses and therefore
perhaps be involved in the causation and maintenance of
inflammatory and/or autoimmune diseases such as rheumatoid
arthritis, inflammatory bowel disease, and psoriasis. As such,
alterations in the expression and/or activation of the polypeptides
of the invention can have profound effects on a plethora of
cellular processes, including, but not limited to, activation or
inhibition of cell specific responses and proliferation. Expression
of cloned IL-1 zeta, Xrec2, or of functionally inactive mutants
thereof can be used to identify the role a particular protein plays
in mediating specific signaling events.
[0205] IL-1 mediated cellular signaling often involves a molecular
activation cascade, during which a receptor propagates a
ligand-receptor mediated signal by specifically activating
intracellular kinases which phosphorylate target substrates. These
substrates can themselves be kinases which become activated
following phosphorylation. Alternatively, they can be adapter
molecules that facilitate down stream signaling through
protein-protein interaction following phosphorylation. Regardless
of the nature of the substrate molecule(s), expressed functionally
active versions of Xrec2, IL-1 zeta, its splice variants, and their
binding partners can be used to identify what substrate(s) were
recognized and activated by the polypeptides of the invention. As
such, these novel polypeptides can be used as reagents to identify
novel molecules involved in signal transduction pathways.
[0206] Molecular Weight, Isoelectric Point Markers
[0207] The polypeptides of the present invention can be subjected
to fragmentation into smaller peptides by chemical and enzymatic
means, and the peptide fragments so produced can be used in the
analysis of other proteins or polypeptides. For example, such
peptide fragments can be used as peptide molecular weight markers,
peptide isoelectric point markers, or in the analysis of the degree
of peptide fragmentation. Thus, the invention also includes these
polypeptides and peptide fragments, as well as kits to aid in the
determination of the apparent molecular weight and isoelectric
point of an unknown protein and kits to assess the degree of
fragmentation of an unknown protein.
[0208] Although all methods of fragmentation are encompassed by the
invention, chemical fragmentation is a preferred embodiment, and
includes the use of cyanogen bromide to cleave under neutral or
acidic conditions such that specific cleavage occurs at methionine
residues (E. Gross, Methods in Enz. 11:238-255, 1967). This can
further include additional steps, such as a carboxymethylation step
to convert cysteine residues to an unreactive species.
[0209] Enzymatic fragmentation is another preferred embodiment, and
includes the use of a protease such as Asparaginylendo-peptidase,
Arginylendo-peptidase, Achromobacter protease I, Trypsin,
Staphylococcus aureus V8 protease, Endoproteinase Asp-N, or
Endoproteinase Lys-C under conventional conditions to result in
cleavage at specific amino acid residues. Asparaginylendo-peptidase
can cleave specifically on the carboxyl side of the asparagine
residues present within the polypeptides of the invention.
Arginylendo-peptidase can cleave specifically on the carboxyl side
of the arginine residues present within these polypeptides.
Achromobacter protease I can cleave specifically on the carboxyl
side of the lysine residues present within the polypeptides
(Sakiyama and Nakat, U.S. Pat. No. 5,248,599; T. Masaki et al.,
Biochim. Biophys. Acta 660:44-50, 1981; T. Masaki et al., Biochim.
Biophys. Acta 660:51-55, 1981). Trypsin can cleave specifically on
the carboxyl side of the arginine and lysine residues present
within polypeptides of the invention. Enzymatic fragmentation may
also occur with a protease that cleaves at multiple amino acid
residues. For example, Staphylococcus aureus V8 protease can cleave
specifically on the carboxyl side of the aspartic and glutamic acid
residues present within polypeptides (D. W. Cleveland, J. Biol.
Chem. 3:1102-1106, 1977). Endoproteinase Asp-N can cleave
specifically on the amino side of the asparagine residues present
within polypeptides. Endoproteinase Lys-C can cleave specifically
on the carboxyl side of the lysine residues present within
polypeptides of the invention. Other enzymatic and chemical
treatments can likewise be used to specifically fragment these
polypeptides into a unique set of specific peptides.
[0210] Of course, the peptides and fragments of the polypeptides of
the invention can also be produced by conventional recombinant
processes and synthetic processes well known in the art. With
regard to recombinant processes, the polypeptides and peptide
fragments encompassed by invention can have variable molecular
weights, depending upon the host cell in which they are expressed.
Glycosylation of polypeptides and peptide fragments of the
invention in various cell types can result in variations of the
molecular weight of these pieces, depending upon the extent of
modification. The size of these pieces can be most heterogeneous
with fragments of polypeptide derived from the extracellular
portion of the polypeptide. Consistent polypeptides and peptide
fragments can be obtained by using polypeptides derived entirely
from the transmembrane and cytoplasmic regions, pretreating with
N-glycanase to remove glycosylation, or expressing the polypeptides
in bacterial hosts.
[0211] The molecular weight of these polypeptides can also be
varied by fusing additional peptide sequences to both the amino and
carboxyl terminal ends of polypeptides of the invention. Fusions of
additional peptide sequences at the amino and carboxyl terminal
ends of polypeptides of the invention can be used to enhance
expression of these polypeptides or aid in the purification of the
protein. In addition, fusions of additional peptide sequences at
the amino and carboxyl terminal ends of polypeptides of the
invention will alter some, but usually not all, of the fragmented
peptides of the polypeptides generated by enzymatic or chemical
treatment. Of course, mutations can be introduced into polypeptides
of the invention using routine and known techniques of molecular
biology. For example, a mutation can be designed so as to eliminate
a site of proteolytic cleavage by a specific enzyme or a site of
cleavage by a specific chemically induced fragmentation procedure.
The elimination of the site will alter the peptide fingerprint of
polypeptides of the invention upon fragmentation with the specific
enzyme or chemical procedure.
[0212] The polypeptides and the resultant fragmented peptides can
be analyzed by methods including sedimentation, electrophoresis,
chromatography, and mass spectrometry to determine their molecular
weights. Because the unique amino acid sequence of each piece
specifies a molecular weight, these pieces can thereafter serve as
molecular weight markers using such analysis techniques to assist
in the determination of the molecular weight of an unknown protein,
polypeptides or fragments thereof. The molecular weight markers of
the invention serve particularly well as molecular weight markers
for the estimation of the apparent molecular weight of proteins
that have similar apparent molecular weights and, consequently,
allow increased accuracy in the determination of apparent molecular
weight of proteins.
[0213] When the invention relates to the use of fragmented peptide
molecular weight markers, those markers are preferably at least 10
amino acids in size. More preferably, these fragmented peptide
molecular weight markers are between 10 and 100 amino acids in
size. Even more preferable are fragmented peptide molecular weight
markers between 10 and 50 amino acids in size and especially
between 10 and 35 amino acids in size. Most preferable are
fragmented peptide molecular weight markers between 10 and 20 amino
acids in size.
[0214] Among the methods for determining molecular weight are
sedimentation, gel electrophoresis, chromatography, and mass
spectrometry. A particularly preferred embodiment is denaturing
polyacrylamide gel electrophoresis (U. K. Laemmli, Nature
227:680-685, 1970). Conventionally, the method uses two separate
lanes of a gel containing sodium dodecyl sulfate and a
concentration of acrylamide between 6-20%. The ability to
simultaneously resolve the marker and the sample under identical
conditions allows for increased accuracy. It is understood, of
course, that many different techniques can be used for the
determination of the molecular weight of an unknown protein using
polypeptides of the invention, and that this embodiment in no way
limits the scope of the invention.
[0215] Each unglycosylated polypeptide or fragment thereof has a pI
that is intrinsically determined by its unique amino acid sequence
(which pI can be estimated by the skilled artisan using any of the
computer programs designed to predict pI values currently
available, calculated using any well-known amino acid pKa table, or
measured empirically). Therefore these polypeptides and fragments
thereof can serve as specific markers to assist in the
determination of the isoelectric point of an unknown protein,
polypeptide, or fragmented peptide using techniques such as
isoelectric focusing. These polypeptide or fragmented peptide
markers serve particularly well for the estimation of apparent
isoelectric points of unknown proteins that have apparent
isoelectric points close to that of the polypeptide or fragmented
peptide markers of the invention.
[0216] The technique of isoelectric focusing can be further
combined with other techniques such as gel electrophoresis to
simultaneously separate a protein on the basis of molecular weight
and charge. The ability to simultaneously resolve these polypeptide
or fragmented peptide markers and the unknown protein under
identical conditions allows for increased accuracy in the
determination of the apparent isoelectric point of the unknown
protein. This is of particular interest in techniques, such as two
dimensional electrophoresis (T. D. Brock and M. T. Madigan, Biology
of Microorganisms 76-77 (Prentice Hall, 6d ed. 1991)), where the
nature of the procedure dictates that any markers should be
resolved simultaneously with the unknown protein. In addition, with
such methods, these polypeptides and fragmented peptides thereof
can assist in the determination of both the isoelectric point and
molecular weight of an unknown protein or fragmented peptide.
[0217] Polypeptides and fragmented peptides can be visualized using
two different methods that allow a discrimination between the
unknown protein and the molecular weight markers. In one
embodiment, the polypeptide and fragmented peptide molecular weight
markers of the invention can be visualized using antibodies
generated against these markers and conventional immunoblotting
techniques. This detection is performed under conventional
conditions that do not result in the detection of the unknown
protein. It is understood that it may not be possible to generate
antibodies against all polypeptide fragments of the invention,
since small peptides may not contain immunogenic epitopes. It is
further understood that not all antibodies will work in this assay;
however, those antibodies which are able to bind polypeptides and
fragments of the invention can be readily determined using
conventional techniques.
[0218] The unknown protein is also visualized by using a
conventional staining procedure. The molar excess of unknown
protein to polypeptide or fragmented peptide molecular weight
markers of the invention is such that the conventional staining
procedure predominantly detects the unknown protein. The level of
these polypeptide or fragmented peptide molecular weight markers is
such as to allow little or no detection of these markers by the
conventional staining method. The preferred molar excess of unknown
protein to polypeptide molecular weight markers of the invention is
between 2 and 100,000 fold. More preferably, the preferred molar
excess of unknown protein to these polypeptide molecular weight
markers is between 10 and 10,000 fold and especially between 100
and 1,000 fold.
[0219] It is understood of course that many techniques can be used
for the determination and detection of molecular weight and
isoelectric point of an unknown protein, polypeptides, and
fragmented peptides thereof using these polypeptide molecular
weight markers and peptide fragments thereof and that these
embodiments in no way limit the scope of the invention.
[0220] In another embodiment, the analysis of the progressive
fragmentation of the polypeptides of the invention into specific
peptides (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106,
1977), such as by altering the time or temperature of the
fragmentation reaction, can be used as a control for the extent of
cleavage of an unknown protein. For example, cleavage of the same
amount of polypeptide and unknown protein under identical
conditions can allow for a direct comparison of the extent of
fragmentation. Conditions that result in the complete fragmentation
of the polypeptide can also result in complete fragmentation of the
unknown protein.
[0221] As to the specific use of the polypeptides and fragmented
peptides of the invention as molecular weight markers, the
fragmentation of the IL-1 zeta polypeptide of SEQ ID NO:3 with
cyanogen bromide generates a unique set of fragmented peptide
molecular weight markers with molecular weights of approximately
701.7, 2955.4, 5101.8 and 12688.5 Daltons. Additionally,
fragmentation of the Xrec2 polypeptide of SEQ ID NO:4 with cyanogen
bromide generates the following fragmented peptide molecular weight
markers with molecular weights of approximately 2216.7, 2259.6,
2376.6, 2738.1, 2901.1, 3417.2, 3627.1, 3656.1, 4042.5, 4144.6,
4668.1, 4710.5, 4916.8, 5288.1, 6089.5, 8199.1, and 11919.7 Daltons
in the absence of glycosylation. In the fragmentation of both SEQ
ID NOs:3 and 4, an additional fragment of 149.2 Daltons results if
the initiating methionine is present. The distribution of
methionine residues determines the number of amino acids in each
peptide and the unique amino acid composition of each peptide
determines its molecular weight.
[0222] In addition, the preferred purified polypeptides of the
invention (SEQ ID NOs:3 and 4) have a calculated molecular weight
of approximately 21542.56 and 79967.85 Daltons, respectively. Thus,
where an intact protein is used, the use of these polypeptide
molecular weight markers allows increased accuracy in the
determination of apparent molecular weight of proteins that have
apparent molecular weights close to 21542.56 and 79967.85 Daltons.
Where fragments are used, there is increased accuracy in
determining molecular weight over the range of the molecular
weights of the fragment.
[0223] Finally, as to the kits that are encompassed by the
invention, the constituents of such kits can be varied, but
typically contain the polypeptide and fragmented peptide molecular
weight markers. Also, such kits can contain the polypeptides
wherein a site necessary for fragmentation has been removed.
Furthermore, the kits can contain reagents for the specific
cleavage of the polypeptide and the unknown protein by chemical or
enzymatic cleavage. Kits can further contain antibodies directed
against polypeptides or fragments thereof of the invention.
[0224] Identification of Unknown Proteins
[0225] As set forth above, a polypeptide or peptide fingerprint can
be entered into or compared to a database of known proteins to
assist in the identification of the unknown protein using mass
spectrometry (W. J. Henzel et al., Proc. Natl. Acad. Sci. USA
90:5011-5015, 1993; D. Fenyo et al., Electrophoresis 19:998-1005,
1998). A variety of computer software programs to facilitate these
comparisons are accessible via the Internet, such as Protein
Prospector (Internet site: prospector.uscf.edu), MultiIdent
(Internet site: www.expasy.ch/sprot/multiident.html), PeptideSearch
(Internet site:
www.mann.embl-heiedelberg.de...deSearch/FR_PeptideSearch
Form.html), and ProFound (Internet site:
www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These
programs allow the user to specify the cleavage agent and the
molecular weights of the fragmented peptides within a designated
tolerance. The programs compare observed molecular weights to
predicted peptide molecular weights derived from sequence databases
to assist in determining the identity of the unknown protein.
[0226] In addition, a polypeptide or peptide digest can be
sequenced using tandem mass spectrometry (MS/MS) and the resulting
sequence searched against databases (J. K. Eng, et al., J. Am. Soc.
Mass Spec. 5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem.
66:4390-4399 (1994); J. A. Taylor and R. S. Johnson, Rapid Comm.
Mass Spec. 11:1067-1075 (1997)). Searching programs that can be
used in this process exist on the Internet, such as Lutefisk 97
(Internet site: www.lsbc.com:70/Lutefisk97.html), and the Protein
Prospector, Peptide Search and ProFound programs described
above.
[0227] Therefore, adding the sequence of a gene and its predicted
protein sequence and peptide fragments to a sequence database can
aid in the identification of unknown proteins using mass
spectrometry.
[0228] Antibodies
[0229] Antibodies that are immunoreactive with the polypeptides of
the invention are provided herein. Such antibodies specifically
bind to the polypeptides via the antigen-binding sites of the
antibody (as opposed to non-specific binding). Thus, the
polypeptides, fragments, variants, fusion proteins, etc., as set
forth above may be employed as "immunogens" in producing antibodies
immunoreactive therewith. More specifically, the polypeptides,
fragment, variants, fusion proteins, etc. contain antigenic
determinants or epitopes that elicit the formation of
antibodies.
[0230] These antigenic determinants or epitopes can be either
linear or conformational (discontinuous). Linear epitopes are
composed of a single section of amino acids of the polypeptide,
while conformational or discontinuous epitopes are composed of
amino acids sections from different regions of the polypeptide
chain that are brought into close proximity upon protein folding
(C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland
Publishing Inc., 2nd ed. 1996)). Because folded proteins have
complex surfaces, the number of epitopes available is quite
numerous; however, due to the conformation of the protein and
steric hindrances, the number of antibodies that actually bind to
the epitopes is less than the number of available epitopes (C. A.
Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland
Publishing Inc., 2nd ed. 1996)). Epitopes may be identified by any
of the methods known in the art.
[0231] Thus, one aspect of the present invention relates to the
antigenic epitopes of the polypeptides of the invention. Such
epitopes are useful for raising antibodies, in particular
monoclonal antibodies, as described in more detail below.
Additionally, epitopes from the polypeptides of the invention can
be used as research reagents, in assays, and to purify specific
binding antibodies from substances such as polyclonal sera or
supernatants from cultured hybridomas. Such epitopes or variants
thereof can be produced using techniques well known in the art such
as solid-phase synthesis, chemical or enzymatic cleavage of a
polypeptide, or using recombinant DNA technology.
[0232] As to the antibodies that can be elicited by the epitopes of
the polypeptides of the invention, whether the epitopes have been
isolated or remain part of the polypeptides, both polyclonal and
monoclonal antibodies may be prepared by conventional techniques.
See, for example, Monoclonal Antibodies, Hybridomas: A New
Dimension in Biological Analyses, Kennet et al. (eds.), Plenum
Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow
and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1988).
[0233] Hybridoma cell lines that produce monoclonal antibodies
specific for the polypeptides of the invention are also
contemplated herein. Such hybridomas may be produced and identified
by conventional techniques. One method for producing such a
hybridoma cell line comprises immunizing an animal with a
polypeptide; harvesting spleen cells from the immunized animal;
fusing said spleen cells to a myeloma cell line, thereby generating
hybridoma cells; and identifying a hybridoma cell line that
produces a monoclonal antibody that binds the polypeptide. The
monoclonal antibodies may be recovered by conventional
techniques.
[0234] The monoclonal antibodies of the present invention include
chimeric antibodies, e.g., humanized versions of murine monoclonal
antibodies. Such humanized antibodies may be prepared by known
techniques and offer the advantage of reduced immunogenicity when
the antibodies are administered to humans. In one embodiment, a
humanized monoclonal antibody comprises the variable region of a
murine antibody (or just the antigen binding site thereof) and a
constant region derived from a human antibody. Alternatively, a
humanized antibody fragment may comprise the antigen binding site
of a murine monoclonal antibody and a variable region fragment
(lacking the antigen-binding site) derived from a human antibody.
Procedures for the production of chimeric and further engineered
monoclonal antibodies include those described in Riechmann et al.
(Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et
al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS
14:139, May, 1993). Procedures to generate antibodies
transgenically can be found in GB 2,272,440, U.S. Pat. Nos.
5,569,825 and 5,545,806 and related patents claiming priority
therefrom, all of which are incorporated by reference herein.
[0235] Antigen-binding fragments of the antibodies, which may be
produced by conventional techniques, are also encompassed by the
present invention. Examples of such fragments include, but are not
limited to, Fab and F(ab').sub.2 fragments. Antibody fragments and
derivatives produced by genetic engineering techniques are also
provided.
[0236] In one embodiment, the antibodies are specific for the
polypeptides of the present invention and do not cross-react with
other proteins. Screening procedures by which such antibodies may
be identified are well known, and may involve immunoaffinity
chromatography, for example.
[0237] Uses Thereof
[0238] The antibodies of the invention can be used in assays to
detect the presence of the polypeptides or fragments of the
invention, either in vitro or in vivo. The antibodies also may be
employed in purifying polypeptides or fragments of the invention by
immunoaffinity chromatography. Those antibodies that additionally
can block binding of the polypeptides of the invention to the
binding partner may be used to inhibit a biological activity that
results from such binding. Such blocking antibodies may be
identified using any suitable assay procedure, such as by testing
antibodies for the ability to inhibit binding of IL-1 zeta to
certain cells expressing the IL-1 zeta receptors. Alternatively,
blocking antibodies may be identified in assays for the ability to
inhibit a biological effect that results from polypeptides of the
invention binding to their binding partners to target cells.
Antibodies may be assayed for the ability to inhibit IL-1
zeta-mediated, Xrec2-mediated, or binding partner-mediated cell
lysis, for example. Antibodies that are antagonistic or block IL-1
zeta activity are useful as therapeutic agents for down-regulating
IL-12 expression and TNF expression. Thus, such antagonists are
useful in treating deleterious affects of inflammation and disease
associated with adverse immune responses as described herein.
Similarly, agonistic antibodies to IL-1 zeta polypeptide are useful
in upregulating IL-12 expression and are useful in enhancing the
effects of Th1 mediated immune response as described herein.
[0239] Such an antibody may be employed in an in vitro procedure,
or administered in vivo to inhibit a biological activity mediated
by the entity that generated the antibody. Disorders caused or
exacerbated (directly or indirectly) by the interaction of the
polypeptides of the invention with the binding partner thus may be
treated. A therapeutic method involves in vivo administration of a
blocking antibody to a mammal in an amount effective in inhibiting
a binding partner-mediated biological activity or a biological
activity such as the inhibition of IL-12 and TNF expression.
Monoclonal antibodies are generally preferred for use in such
therapeutic methods. In one embodiment, an antigen-binding antibody
fragment is employed.
[0240] Antibodies may be screened for agonistic (i.e.,
ligand-mimicking) properties. Such antibodies, upon binding to cell
surface receptor, induce biological effects (e.g., transduction of
biological signals) similar to the biological effects induced when
IL-1 binds to cell surface IL-1 receptors. Agonistic antibodies may
be used to activate IL-12 expression and treat disease associated
with Th1 mediated pathways.
[0241] Compositions comprising an antibody that is directed against
polypeptides of the invention, and a physiologically acceptable
diluent, excipient, or carrier, are provided herein. Suitable
components of such compositions are as described above for
compositions containing polypeptides of the invention.
[0242] Also provided herein are conjugates comprising a detectable
(e.g., diagnostic) or therapeutic agent, attached to the antibody.
Examples of such agents are presented above. The conjugates find
use in in vitro or in vivo procedures.
[0243] Because the IL-1 zeta polypeptides, and particularly the
TDZ1 isoform, are active in IL-12 regulation and TNF regulation,
inhibitors such as small molecule inhibitors of its function or its
protein associations (or antisense or other inhibitors of its
synthesis) will be useful in treating autoimmune and/or
inflammatory disorders. Accordingly, IL-1 zeta polypeptides and
fragments of IL-1 zeta polypeptides that are capable of
upregulating IL-12 production or TNF production as described below,
for example, are useful in screening assays to identify compounds
and small molecules which inhibit (antagonize) functions and
activities of IL-1 zeta polypeptide and described herein.
Similarly, IL-1 zeta polypeptides and fragments of IL-1 zeta
polypeptides that are capable of upregulating IL-12 production are
useful in screening assays to identify compounds and small
molecules which agonize or enhance IL-12 expression. Such compounds
are useful as therapeutics for the herein described uses associated
with enhanced IL-12 expression. (U.S. Pat. No. 5,674,483 and U.S.
Pat. No. 5,928,636 which are incorporated herein by reference).
[0244] Thus, for example, polypeptides and polypeptide fragments of
the invention may be used to identify antagonists and agonists from
cells, cell-free preparations, chemical libraries, and natural
product mixtures. The antagonists and agonists may be natural or
modified substrates, ligands, enzymes, receptors, etc. of the
polypeptides of the instant invention, or may be structural or
functional mimetics of the polypeptides. Potential antagonists of
the instant invention may include small molecules, peptides and
antibodies that bind to and occupy a binding site of the inventive
polypeptides or a binding partner thereof, causing them to be
unavailable to bind to their natural binding partners and therefore
preventing normal biological activity. Potential agonists include
small molecules, peptides and antibodies which bind to the instant
polypeptides or binding partners thereof, and elicit the same or
enhanced biologic effects as those caused by the binding of the
polypeptides of the instant invention.
[0245] Small molecule agonists and antagonists are usually less
than 10K molecular weight and may possess a number of
physicochemical and pharmacological properties which enhance cell
penetration, resist degradation and prolong their physiological
half-lives (Gibbs, J., Pharmaceutical Research in Molecular
Oncology, Cell, Vol. 79 (1994)). Antibodies, which include intact
molecules as well as fragments such as Fab and F(ab')2 fragments,
as well as recombinant molecules derived therefrom, may be used to
bind to and inhibit the polypeptides of the instant invention by
blocking the propagation of a signaling cascade. It is preferable
that the antibodies are humanized, and more preferable that the
antibodies are human. The antibodies of the present invention may
be prepared by any of a variety of well-known methods.
[0246] Screening methods are known in the art and along with
integrated robotic systems and collections of chemical
compounds/natural products are extensively incorporated in high
throughput screening so that large numbers of test compounds can be
tested for antagonist or agonist activity within a short amount of
time. These methods include homogeneous assay formats such as
fluorescence resonance energy transfer, fluorescence polarization,
time-resolved fluorescence resonance energy transfer, scintillation
proximity assays, reporter gene assays, fluorescence quenched
enzyme substrate, chromogenic enzyme substrate and
electrochemiluminescence, as well as more traditional heterogeneous
assay formats such as enzyme-linked immunosorbant assays (ELISA) or
radioimmunoassays.
[0247] Homogeneous assays are mix-and-read style assays that are
very amenable to robotic application, whereas heterogeneous assays
require separation of free from bound analyte by more complex unit
operations such as filtration, centrifugation or washing. These
assays are utilized to detect a wide variety of specific
biomolecular interactions and the inhibition thereof by small
organic molecules, including protein-protein, receptor-ligand,
enzyme-substrate, and so on. These assay methods and techniques are
well known in the art (see, e.g., High Throughput Screening: The
Discovery of Bioactive Substances, John P. Devlin (ed.), Marcel
Dekker, New York, 1997 ISBN: 0-8247-0067-8). The screening assays
of the present invention are amenable to high throughput screening
of chemical libraries and are suitable for screening test compounds
in order to identify small molecule drug candidates, antibodies,
peptides, and other antagonists and/or agonists, natural or
synthetic.
[0248] Thus, a method of the present invention includes screening a
test compound to determine its effect on the ability of a
polypeptide of this invention to increase or decrease IL-12
expression and/or TNF expression. Such a method involves
co-culturing an IL-1 zeta polypeptide of this invention,
particularly the TDZ1 isoform, and cells capable of expressing
IL-12 and/or TNF (e.g. monocytes, PBMC) and analyzing the culture
for IL-12 and/or TNF levels. If the level of expression differs
from that level of expression that is observed in the absence of
test compound, a test compound that affects IL-12 and/or TNF
expression is identified. Polypeptides that are useful in the
screening methods include the IL-1 zeta polypeptides of this
invention and fragments of the IL-1 zeta polypeptides that
upregulate IL-12 expression and/or TNF expression, particularly the
TDZ1 isoform.
[0249] In one embodiment of a method for identifying molecules
which inhibit or antagonize the polypeptides of this invention
involves adding a test compound to a medium which contains cells
that express the polypeptides of the instant invention; changing
the conditions of the medium so that, but for the presence of the
test compound, the polypeptides would be bound to their natural
ligands, substrates or effector molecules, and observing the
binding and stimulation or inhibition of a functional response. The
activity of the cells which were contacted with the test compound
may then be compared with the identical cells which were not
contacted and antagonists and agonists of the polypeptides of the
instant invention may be identified. The measurement of biological
activity may be performed by a number of well-known methods such as
measuring the amount of protein present (e.g. an ELISA) or
measuring the protein's activity. A decrease in biological
stimulation or activation indicates an antagonist. An increase
indicates an agonist.
[0250] Another embodiment of the invention relates to uses of
polypeptides of this invention to study cell signal transduction.
Cellular signaling often involves a molecular activation cascade,
during which a receptor propagates a ligand-receptor mediated
signal by specifically activating intracellular kinases which
phosphorylate target substrates. These substrates can themselves be
kinases which become activated following phosphorylation.
Alternatively, they can be adapter molecules that facilitate down
stream signaling through protein-protein interaction following
phosphorylation. Accordingly, these polypeptides and active
fragments can be used as reagents to identify novel molecules
involved in signal transduction pathways.
[0251] As therapeutics, inhibitors or agonists of IL-1 zeta
activity can be administered to agonize or antagonize IL-1 zeta
activity, thus providing useful immunoregulators. Various
liposome-based compositions of the inventive polypeptides are
envisioned herein.
[0252] Inhibitors and enhancers of the polypeptides or polypeptide
fragments having biological activity are useful in treating a
variety of medical conditions. IL-1 zeta polypeptides are
associated with IL-12 production and dysregulation of IL-12
production, and thus agonists of IL-1 zeta polypeptides are useful
for treating diseases and medical conditions that are
therapeutically responsive to IL-12 expression. Such diseases and
medical conditions include infectious diseases, such as HIV,
Hepatitis B and Hepatitis C, papilloma, etc.; and, bacterial
infections, including tuberculosis, salmonellosis, listeriousis;
and, parasitic infections such as malaria, leishmaniasis and
schistosomiasis. Agonists are also useful for treating dysregulated
immune response, e.g. use as a vaccine (e.g. for use in connection
with antigen such as for measles vaccination) or vaccine adjuvant,
increased response to bacterial and viral infection, as just
discussed, and as therapeutic immunotherapies including anticancer
immunotherapy treatments. (See U.S. Pat. Nos. 6,086,876, and
6,168,923 both of which are incorporated herein by reference) In
another embodiment, agonists of IL-1 zeta polypeptides can be
administered in combination with other agents or cytokines for
treating disease and medical conditions. For example, agonists can
be administered in combination with IFN or IFN alpha. Antagonists
of IL-1 zeta polypeptides are useful in treating certain types of
immune system dysfunction associated with IL-12 dysregulation such
as autoimmune diseases, inflammatory conditions, complications that
are associated with bacterial infections that occur with increased
IL-12 activity and conditions associated with increased expression
or activity of IL-12. Thus, therapeutics discovered by screening
IL-1 zeta polypeptides, the TDZ1 isoform and active fragments for
agonistic or antagonistic activity have properties that make them
suitable for use as: anti-inflammatory, anti-tumor or anti-cancer,
anti-bacterial, and anti-viral.
[0253] Compositions of the present invention may contain a
polypeptide or and antagonist or agonist in any form described
herein, such as native proteins, variants, derivatives, oligomers,
biologically active fragments of the compounds described herein,
small molecules, antibodies, etc. In particular embodiments, the
composition comprises peptides, small molecules, antibodies or
oligomers comprising soluble polypeptides.
[0254] Compositions comprising an effective amount of a polypeptide
of the present invention, in combination with other components such
as a physiologically acceptable diluent, carrier, or excipient, are
provided herein. The polypeptides can be formulated according to
known methods used to prepare pharmaceutically useful compositions.
They can be combined in admixture, either as the sole active
material or with other known active materials suitable for a given
indication, with pharmaceutically acceptable diluents (e.g.,
saline, Tris-HCl, acetate, and phosphate buffered solutions),
preservatives (e.g., thimerosal, benzyl alcohol, parabens),
emulsifiers, solubilizers, adjuvants and/or carriers. Suitable
formulations for pharmaceutical compositions include those
described in Remington's Pharmaceutical Sciences, 16th ed. 1980,
Mack Publishing Company, Easton, Pa.
[0255] In addition, such compositions can be complexed with
polyethylene glycol (PEG), metal ions, or incorporated into
polymeric compounds such as polyacetic acid, polyglycolic acid,
hydrogels, dextran, etc., or incorporated into liposomes,
microemulsions, micelles, unilamellar or multilamellar vesicles,
erythrocyte ghosts or spheroblasts. Such compositions will
influence the physical state, solubility, stability, rate of in
vivo release, and rate of in vivo clearance, and are thus chosen
according to the intended application.
[0256] The compositions of the invention can be administered in any
suitable manner, e.g., topically, parenterally, orally,
intracranially or by inhalation. The term "parenteral" includes
injection, e.g., by subcutaneous, intravenous, or intramuscular
routes, also including localized administration, e.g., at a site of
disease or injury (for example, intracoronary or intra tumor
administration or injection into a joint undergoing an inflammatory
reaction). Sustained release from implants is also contemplated.
One skilled in the pertinent art will recognize that suitable
dosages will vary, depending upon such factors as the nature of the
disorder to be treated, the patient's body weight, age, and general
condition, and the route of administration. Preliminary doses can
be determined according to animal tests, and the scaling of dosages
for human administration is performed according to art-accepted
practices.
[0257] Moreover, it has been found that DNA encoding a polypeptide
can be administered to a mammal in such a way that it is taken up
by cells, and expressed. The resultant protein will then be
available to exert a therapeutic effect. Accordingly, compositions
comprising nucleic acids in physiologically acceptable formulations
are also contemplated. DNA may be formulated for injection, for
example.
[0258] The following examples are provided to further illustrate
particular embodiments of the invention, and are not to be
construed as limiting the scope of the present invention.
Example 1
Isolation of the IL-1 Zeta and Xrec2 Nucleic Acids
[0259] Human IL-1 zeta nucleic acid sequence was obtained by
sequencing EST IMAGE clone 1628761, accession #AI014548, which
encoded a partial open reading frame (ORF). A number of cDNA
libraries were screened with internal primers to determine the
expression pattern of the polypeptide. After performing PCR using
two internal primers of human IL-1 zeta sequence, the following
cDNA libraries were positive for IL-1 zeta sequences: bone marrow
stromal, human pancreatic tumor, and Raji. IL-1 zeta clones were
isolated from human genomic DNA sequences, bone marrow stromal and
human pancreatic tumor libraries, and sequenced.
[0260] Human Xrec2 sequences were obtained by high-throughput
sequencing, PCR, and 5' RACE reactions. High-throughput shotgun
sequencing of chromosome region Xp11 yielded sequences for exons
4-6 of Xrec2 (Genbank accession numbers AL031466 and AL031575).
Similarly, sequence of chromosome region Xp22-164-166 (Genbank
accession number AC005748) yielded sequences for exons 10-12 of
Xrec2.
[0261] PCR performed on human brain first strand cDNA using primers
within exons 5 and 11 generated sequence for exons 7-9. 5' RACE
reactions were then performed using testis cDNA and nested primers
within exon 4 to obtain exon 3 sequences which contained the
predicted initiator methionine. Both PCR and the 5' RACE reactions
were performed using standard protocols.
Example 2
Use of Purified IL-1 Zeta and Xrec2 Polypeptides
Polypeptide-Specific ELISA:
[0262] Serial dilutions of IL-1 zeta- or Xrec2-containing samples
(in 50 mM NaHCO.sub.3, brought to pH 9 with NaOH) are coated onto
Linbro/Titertek 96 well flat bottom E.I.A. microtitration plates
(ICN Biomedicals Inc., Aurora, Ohio) at 100:1/well. After
incubation at 4EC for 16 hours, the wells are washed six times with
200:1 PBS containing 0.05% Tween-20 (PBS-Tween). The wells are then
incubated with FLAG7-binding partner at 1 mg/ml in PBS-Tween with
5% fetal calf serum (FCS) for 90 minutes (100:1 per well), followed
by washing as above. Next, each well is incubated with the
anti-FLAG7 (monoclonal antibody M2 at 1 mg/ml in PBS-Tween
containing 5% FCS for 90 minutes (100:1 per well), followed by
washing as above. Subsequently, wells are incubated with a
polyclonal goat anti-mIgG1-specific horseradish
peroxidase-conjugated antibody (a 1:5000 dilution of the commercial
stock in PBS-Tween containing 5% FCS) for 90 minutes (100:1 per
well). The HRP-conjugated antibody is obtained from Southern
Biotechnology Associates, Inc., Birmingham, Ala. Wells then are
washed six times, as above.
[0263] For development of the ELISA, a substrate mix [100:1 per
well of a 1:1 premix of the TMB Peroxidase Substrate and Peroxidase
Solution B (Kirkegaard Perry Laboratories, Gaithersburg, Md.)] is
added to the wells. After sufficient color reaction, the enzymatic
reaction is terminated by addition of 2 NH.sub.2SO.sub.4 (50:1 per
well). Color intensity (indicating ligand receptor binding) is
determined by measuring extinction at 450 nm on a V Max plate
reader (Molecular Devices, Sunnyvale, Calif.).
Example 3
Amino Acid Sequence
[0264] The amino acid sequence of IL-1 zeta and Xrec2 were
determined by translation of the complete nucleotide sequences of
SEQ ID NOs:1 and 2, respectively.
Example 4
DNA and Amino Acid Sequences
[0265] The IL-1 zeta and Xrec2 nucleic acid sequences were
determined by standard double stranded sequencing of the composite
sequence of EST IMAGE clones (accession #AI014548 (IL-1 zeta) and #
AL031575 and #AC005748 (Xrec2)), and of additional sequences
obtained from PCR and 5' RACE reactions.
[0266] The nucleotide sequence of the isolated IL-1 zeta and Xrec2
DNA and the amino acid sequence encoded thereby, are presented in
SEQ ID NOs:1-4. The sequence of the IL-1 zeta DNA fragment isolated
by PCR corresponds to nucleotides 1 to 579 of SEQ ID NO:1, which
encode amino acids 1 to 192 of SEQ ID NO:3; and the sequence of the
Xrec2 DNA fragment also isolated by PCR corresponds to nucleotides
1 to 2088 of SEQ ID NO:2, which encode amino acids 1 to 698 of SEQ
ID NO:4.
[0267] The amino acid sequences of SEQ ID NOs:3 and 4 bear
significant homology to other known IL-1 ligand and receptor family
members, respectively.
Example 5
Monoclonal Antibodies that Bind Polypeptides of the Invention
[0268] This example illustrates a method for preparing monoclonal
antibodies that bind IL-1 zeta. The same protocol can be used to
produce monoclonal antibodies that bind Xrec2. Suitable immunogens
that may be employed in generating such antibodies include, but are
not limited to, purified IL-1 zeta polypeptide or an immunogenic
fragment thereof such as the extracellular domain, or fusion
proteins containing IL-1 zeta (e.g., a soluble IL-1 zeta/Fc fusion
protein).
[0269] Purified IL-1 zeta can be used to generate monoclonal
antibodies immunoreactive therewith, using conventional techniques
such as those described in U.S. Pat. No. 4,411,993. Briefly, mice
are immunized with IL-1 zeta immunogen emulsified in complete
Freund's adjuvant, and injected in amounts ranging from 10-100 g
subcutaneously or intraperitoneally. Ten to twelve days later, the
immunized animals are boosted with additional IL-1 zeta emulsified
in incomplete Freund's adjuvant. Mice are periodically boosted
thereafter on a weekly to bi-weekly immunization schedule. Serum
samples are periodically taken by retro-orbital bleeding or
tail-tip excision to test for IL-1 zeta antibodies by dot blot
assay, ELISA (Enzyme-Linked Immunosorbent Assay) or inhibition of
IL-1 zeta receptor binding.
[0270] Following detection of an appropriate antibody titer,
positive animals are provided one last intravenous injection of
IL-1 zeta in saline. Three to four days later, the animals are
sacrificed, spleen cells harvested, and spleen cells are fused to a
murine myeloma cell line, e.g., NS1 or preferably P3x63Ag8.653
(ATCC CRL 1580). Fusions generate hybridoma cells, which are plated
in multiple microtiter plates in a HAT (hypoxanthine, aminopterin
and thymidine) selective medium to inhibit proliferation of
non-fused cells, myeloma hybrids, and spleen cell hybrids.
[0271] The hybridoma cells are screened by ELISA for reactivity
against purified IL-1 zeta by adaptations of the techniques
disclosed in Engvall et al., (Immunochem. 8:871, 1971) and in U.S.
Pat. No. 4,703,004. A preferred screening technique is the antibody
capture technique described in Beckmann et al., (J. Immunol.
144:4212, 1990). Positive hybridoma cells can be injected
intraperitoneally into syngeneic BALB/c mice to produce ascites
containing high concentrations of anti-IL-1 zeta monoclonal
antibodies. Alternatively, hybridoma cells can be grown in vitro in
flasks or roller bottles by various techniques. Monoclonal
antibodies produced in mouse ascites can be purified by ammonium
sulfate precipitation, followed by gel exclusion chromatography.
Alternatively, affinity chromatography based upon binding of
antibody to Protein A or Protein G can also be used, as can
affinity chromatography based upon binding to IL-1 zeta.
Example 6
Northern Blot Analysis
[0272] The tissue distribution of IL-1 zeta and Xrec2 mRNA is
investigated by Northern blot analysis, as follows. An aliquot of a
radiolabeled probe is added to two different human multiple tissue
Northern blots (Clontech, Palo Alto, Calif.; Biochain, Palo Alto,
Calif.). The blots are hybridized in 10.times.Denhardts, 50 mM Tris
pH 7.5, 900 mM NaCl, 0.1% Na pyrophosphate, 1% SDS, 200 .mu.g/mL
salmon sperm DNA. Hybridization is conducted overnight at 63EC in
50% formamide as previously described (March et al., Nature
315:641-647, 1985). The blots are then washed with 2.times.SSC,
0.1% SDS at 68EC for 30 minutes. The cells and tissues with the
highest levels of IL-1 zeta and Xrec2 mRNA are determined by
comparison to control probing with a .beta.-actin-specific
probe.
Example 7
Binding Assay
[0273] Full length IL-1 zeta can be expressed and tested for the
ability to bind IL-1 zeta receptors. The binding assay can be
conducted as follows.
[0274] A fusion protein comprising a leucine zipper peptide fused
to the N-terminus of a soluble IL-1 zeta polypeptide (LZ-IL-1 zeta)
is employed in the assay. An expression construct is prepared,
essentially as described for preparation of the FLAG.sup.7 (IL-1
zeta) expression construct in Wiley et al. (Immunity, 3:673-682,
1995; hereby incorporated by reference), except that DNA encoding
the FLAG.sup.7 peptide was replaced with a sequence encoding a
modified leucine zipper that allows for trimerization. The
construct, in expression vector pDC409, encodes a leader sequence
derived from human cytomegalovirus, followed by the leucine zipper
moiety fused to the N-terminus of a soluble IL-1 zeta polypeptide.
The LZ-IL-1 zeta is expressed in CHO cells, and purified from the
culture supernatant.
[0275] The expression vector designated pDC409 is a mammalian
expression vector derived from the pDC406 vector described in
McMahan et al. (EMBO J. 10:2821-2832, 1991; hereby incorporated by
reference). Features added to pDC409 (compared to pDC406) include
additional unique restriction sites in the multiple cloning site
(mcs); three stop codons (one in each reading frame) positioned
downstream of the mcs; and a T7 polymerase promoter, downstream of
the mcs, that facilitates sequencing of DNA inserted into the
mcs.
[0276] For expression of full length human IL-1 zeta protein, the
entire coding region (i.e., the DNA sequence presented in SEQ ID
NO:1) is amplified by polymerase chain reaction (PCR). The template
employed in the PCR is the cDNA clone isolated from a (pancreatic
tumor) cDNA library, as described in example 1. The isolated and
amplified DNA is inserted into the expression vector pDC409, to
yield a construct designated pDC409-IL-1 zeta.
[0277] LZ-IL-1 zeta polypeptide is employed to test the ability to
bind to host cells expressing recombinant or endogenous IL-1 zeta
receptors, as discussed above. Cells expressing IL-1 zeta receptor
are cultured in DMEM supplemented with 10% fetal bovine serum,
penicillin, streptomycin, and glutamine. Cells are incubated with
LZ-IL-1 zeta (5 mg/ml) for about 1 hour. Following incubation, the
cells are washed to remove unbound LZ-IL-1 zeta and incubated with
a biotinylated anti-LZ monoclonal antibody (5 mg/ml), and
phycoerythrin-conjugated streptavidin (1:400), before analysis by
fluorescence-activated cell scanning (FACS). The cytometric
analysis was conducted on a FACscan (Beckton Dickinson, San Jose,
Calif.).
[0278] The cells expressing IL-1 zeta receptors showed
significantly enhanced binding of LZ-IL-1 zeta, compared to the
control cells not expressing IL-1 zeta receptors.
Example 8
Obtaining TDZ.1, TDZ.2, and TDZ.3 and Tissue Distribution
[0279] In order to determine and study the relative abundance and
tissue distribution of Tango-77 (WO 99/06426), an alternatively
spliced form of IL-1 zeta, and IL-1 zeta, RT-PCR was performed. The
primers used in the RT PCR were 5' primers specific for either
Tango-77 exon #1 (see FIG. 1) or IL-1 zeta exon #1 (exons #3 in
FIG. 1) in combination with a common 3' primer from the common
final exon (exon #6 in FIG. 1). The PCR reactions were performed
using first strand cDNA from multiple human tissue sources
purchased from Clontech, Palo Alto, Calif. The PCR reaction
generated PCR products that included the predicted size product and
additional bands. In particular, three different sized PCR products
were isolated and used to obtain sequence information from multiple
tissue cDNAs. The sequences of these three products, SEQ ID NOs:5,
6, 7 and encoded amino acids of SEQ ID NO:8, 9, and 10, are splice
variants. The organization the relationship of these splice
variants are shown in FIG. 1 and discussed above. The splice
variants are TDZ.1, TDZ.2, and TDZ.3 (Testis-Derived Zeta variants)
because all three of them are expressed in testis. Testis is a
common expression tissue. However, it is not the only expression
tissue. Table II describes the results of the tissue expression
study for Tango-77, IL-1 zeta, TDZ.1, TDZ.2, and TDZ.3. TDZ.1 and
TDZ.2 contain exons 4, 5 and 6 which correspond to the last three
exons of IL-1 zeta and correspond to the conserved structural
domain of the molecule. As discussed above, when aligned with other
members of the IL-1 family, exons 4, 5 and 6 are shown to contain
many conserved residues within conserved structural motifs.
[0280] A polymorphism of Tango 77 in exon #2 of FIG. 1 is noted. In
the isolated cDNAS a valine occurs in lieu of a glycine at the
third residue of exon #2. In the Tango-77 sequence, the amino acid
sequence is PAGSPLEP. In the polymorphism the sequence is
PAVSPLEP.
TABLE-US-00004 TABLE II TISSUE DISTRIBUTION OF FIL-1Z SPLICE
VARIANTS Tissue IL-1z Tango-77 TDZ.1 TDZ.2 TDZ.3 kidney - - + - -
pancreas - - - - - skeletal muscle - - + - - heart - + - - - testis
+ + + + + prostrate + - + - - spleen - - - - - ovary - + + - -
thymus - - - - - colon + + + - - leukocytes - - - - - small
intestine - + + - - liver - + + - - brain + - - - - placenta + + +
- + lung + + + - + tonsil - + + - - fetal liver + + + - - lymph
node + + + - - bone marrow - + + + +
Example 9
IL-1 Zeta Polypeptide Induces TNF and IL-12 Secretion
[0281] The following assays were performed to study cytokine
induction by IL-1 Zeta polypeptides. A protein of IL-1 zeta, TDZ.1
isoform, fused to a FLAG-poly His polypeptide at its C-terminus,
was prepared and co-cultured with human monocytes. Varying
concentrations of the TDZ.1 isoform were used with a lower level
concentration of 5 nM. The culture was analyzed for cytokines and
found to have increased levels of TNF-alpha and IL-12. This
cytokine inducing activity was dose dependent.
[0282] The references cited herein are incorporated by reference
herein in their entirety.
Sequence CWU 1
1
151579DNAHomo sapiens 1atgtcaggct gtgataggag ggaaacagaa accaaaggaa
agaacagctt taagaagcgc 60ttaagaggtc caaaggtgaa gaacttaaac ccgaagaaat
tcagcattca tgaccaggat 120cacaaagtac tggtcctgga ctctgggaat
ctcatagcag ttccagataa aaactacata 180cgcccagaga tcttctttgc
attagcctca tccttgagct cagcctctgc ggagaaagga 240agtccgattc
tcctgggggt ctctaaaggg gagttttgtc tctactgtga caaggataaa
300ggacaaagtc atccatccct tcagctgaag aaggagaaac tgatgaagct
ggctgcccaa 360aaggaatcag cacgccggcc cttcatcttt tatagggctc
aggtgggctc ctggaacatg 420ctggagtcgg cggctcaccc cggatggttc
atctgcacct cctgcaattg taatgagcct 480gttggggtga cagataaatt
tgagaacagg aaacacattg aattttcatt tcaaccagtt 540tgcaaagctg
aaatgagccc cagtgaggtc agcgattag 57922091DNAHomo sapiens 2atgaaagctc
cgattccaca cttgattctc ttatacgcta cttttactca gagtttgaag 60gttgtgacca
aaagaggctc cgccgatgga tgcactgact ggtctatcga tatcaagaaa
120tatcaagttt tggtgggaga gcctgttcga atcaaatgtg cactctttta
tggttatatc 180agaacaaatt actcccttgc ccaaagtgct ggactcagtt
tgatgtggta caaaagttct 240ggtcctggag actttgaaga gccaatagcc
tttgacggaa gtagaatgag caaagaagaa 300gactccattt ggttccggcc
aacattgcta caggacagtg gtctctacgc ctgtgtcatc 360agaaactcca
cttactgtat gaaagtatcc atctcactga cagtgggtga aaatgacact
420ggactctgct ataattccaa gatgaagtat tttgaaaaag ctgaacttag
caaaagcaag 480gaaatttcat gccgtgacat agaggatttt ctactgccaa
ccagagaacc tgaaatcctt 540tggtacaagg aatgcaggac aaaaacatgg
aggccaagta ttgtattcaa aagagatact 600ctgcttataa gagaagtcag
agaagatgac attggaaatt atacctgtga attaaaatat 660ggaggctttg
ttgtgagaag aactactgaa ttaactgtta cagcccctct gactgataag
720ccacccaagc ttttgtatcc tatggaaagt aaactgacaa ttcaggagac
ccagctgggt 780gactctgcta atctaacctg cagagctttc tttgggtaca
gcggagatgt cagtccttta 840atttactgga tgaaaggaga aaaatttatt
gaagatctgg atgaaaatcg agtttgggaa 900agtgacatta gaattcttaa
ggagcatctt ggggaacagg aagtttccat ctcattaatt 960gtggactctg
tggaagaagg tgacttggga aattactcct gttatgttga aaatggaaat
1020ggacgtcgac acgccagcgt tctccttcat aaacgagagc taatgtacac
agtggaactt 1080gctggaggcc ttggtgctat actcttgctg cttgtatgtt
tggtgaccat ctacaagtgt 1140tacaagatag aaatcatgct cttctacagg
aatcattttg gagctgaaga gctcgatgga 1200gacaataaag attatgatgc
atacttatca tacaccaaag tggatcctga ccagtggaat 1260caagagactg
gggaagaaga acgttttgcc cttgaaatcc tacctgatat gcttgaaaag
1320cattatggat ataagttgtt tataccagat agagatttaa tcccaactgg
aacatacatt 1380gaagatgtgg caagatgtgt agatcaaagc aagcggctga
ttattgtcat gaccccaaat 1440tacgtagtta gaaggggctg gagcatcttt
gagctggaaa ccagacttcg aaatatgctt 1500gtgactggag aaattaaagt
gattctaatt gaatgcagtg aactgagagg aattatgaac 1560taccaggagg
tggaggccct gaagcacacc atcaagctcc tgacggtcat taaatggcat
1620ggaccaaaat gcaacaagtt gaactccaag ttctggaaac gtttacagta
tgaaatgcct 1680tttaagagga tagaacccat tacacatgag caggctttag
atgtcagtga gcaagggcct 1740tttggggagc tgcagactgt ctcggccatt
tccatggccg cggccacctc cacagctcta 1800gccactgccc atccagatct
ccgttctacc tttcacaaca cgtaccattc acaaatgcgt 1860cagaaacact
actaccgaag ctatgagtac gacgtacctc ctaccggcac cctgcctctt
1920acctccatag gcaatcagca tacctactgt aacatcccta tgacactcat
caacgggcag 1980cggccacaga caaaatcgag cagggagcag aatccagatg
aggcccacac aaacagtgcc 2040atcctgccgc tgttgccaag ggagaccagt
atatccagtg tgatatggtg a 20913192PRTHomo sapiens 3Met Ser Gly Cys
Asp Arg Arg Glu Thr Glu Thr Lys Gly Lys Asn Ser1 5 10 15Phe Lys Lys
Arg Leu Arg Gly Pro Lys Val Lys Asn Leu Asn Pro Lys 20 25 30Lys Phe
Ser Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser 35 40 45Gly
Asn Leu Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile 50 55
60Phe Phe Ala Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly65
70 75 80Ser Pro Ile Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr
Cys 85 90 95Asp Lys Asp Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys
Lys Glu 100 105 110Lys Leu Met Lys Leu Ala Ala Gln Lys Glu Ser Ala
Arg Arg Pro Phe 115 120 125Ile Phe Tyr Arg Ala Gln Val Gly Ser Trp
Asn Met Leu Glu Ser Ala 130 135 140Ala His Pro Gly Trp Phe Ile Cys
Thr Ser Cys Asn Cys Asn Glu Pro145 150 155 160Val Gly Val Thr Asp
Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser 165 170 175Phe Gln Pro
Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp 180 185
1904696PRTHomo sapiens 4Met Lys Ala Pro Ile Pro His Leu Ile Leu Leu
Tyr Ala Thr Phe Thr1 5 10 15Gln Ser Leu Lys Val Val Thr Lys Arg Gly
Ser Ala Asp Gly Cys Thr 20 25 30Asp Trp Ser Ile Asp Ile Lys Lys Tyr
Gln Val Leu Val Gly Glu Pro 35 40 45Val Arg Ile Lys Cys Ala Leu Phe
Tyr Gly Tyr Ile Arg Thr Asn Tyr 50 55 60Ser Leu Ala Gln Ser Ala Gly
Leu Ser Leu Met Trp Tyr Lys Ser Ser65 70 75 80Gly Pro Gly Asp Phe
Glu Glu Pro Ile Ala Phe Asp Gly Ser Arg Met 85 90 95Ser Lys Glu Glu
Asp Ser Ile Trp Phe Arg Pro Thr Leu Leu Gln Asp 100 105 110Ser Gly
Leu Tyr Ala Cys Val Ile Arg Asn Ser Thr Tyr Cys Met Lys 115 120
125Val Ser Ile Ser Leu Thr Val Gly Glu Asn Asp Thr Gly Leu Cys Tyr
130 135 140Asn Ser Lys Met Lys Tyr Phe Glu Lys Ala Glu Leu Ser Lys
Ser Lys145 150 155 160Glu Ile Ser Cys Arg Asp Ile Glu Asp Phe Leu
Leu Pro Thr Arg Glu 165 170 175Pro Glu Ile Leu Trp Tyr Lys Glu Cys
Arg Thr Lys Thr Trp Arg Pro 180 185 190Ser Ile Val Phe Lys Arg Asp
Thr Leu Leu Ile Arg Glu Val Arg Glu 195 200 205Asp Asp Ile Gly Asn
Tyr Thr Cys Glu Leu Lys Tyr Gly Gly Phe Val 210 215 220Val Arg Arg
Thr Thr Glu Leu Thr Val Thr Ala Pro Leu Thr Asp Lys225 230 235
240Pro Pro Lys Leu Leu Tyr Pro Met Glu Ser Lys Leu Thr Ile Gln Glu
245 250 255Thr Gln Leu Gly Asp Ser Ala Asn Leu Thr Cys Arg Ala Phe
Phe Gly 260 265 270Tyr Ser Gly Asp Val Ser Pro Leu Ile Tyr Trp Met
Lys Gly Glu Lys 275 280 285Phe Ile Glu Asp Leu Asp Glu Asn Arg Val
Trp Glu Ser Asp Ile Arg 290 295 300Ile Leu Lys Glu His Leu Gly Glu
Gln Glu Val Ser Ile Ser Leu Ile305 310 315 320Val Asp Ser Val Glu
Glu Gly Asp Leu Gly Asn Tyr Ser Cys Tyr Val 325 330 335Glu Asn Gly
Asn Gly Arg Arg His Ala Ser Val Leu Leu His Lys Arg 340 345 350Glu
Leu Met Tyr Thr Val Glu Leu Ala Gly Gly Leu Gly Ala Ile Leu 355 360
365Leu Leu Leu Val Cys Leu Val Thr Ile Tyr Lys Cys Tyr Lys Ile Glu
370 375 380Ile Met Leu Phe Tyr Arg Asn His Phe Gly Ala Glu Glu Leu
Asp Gly385 390 395 400Asp Asn Lys Asp Tyr Asp Ala Tyr Leu Ser Tyr
Thr Lys Val Asp Pro 405 410 415Asp Gln Trp Asn Gln Glu Thr Gly Glu
Glu Glu Arg Phe Ala Leu Glu 420 425 430Ile Leu Pro Asp Met Leu Glu
Lys His Tyr Gly Tyr Lys Leu Phe Ile 435 440 445Pro Asp Arg Asp Leu
Ile Pro Thr Gly Thr Tyr Ile Glu Asp Val Ala 450 455 460Arg Cys Val
Asp Gln Ser Lys Arg Leu Ile Ile Val Met Thr Pro Asn465 470 475
480Tyr Val Val Arg Arg Gly Trp Ser Ile Phe Glu Leu Glu Thr Arg Leu
485 490 495Arg Asn Met Leu Val Thr Gly Glu Ile Lys Val Ile Leu Ile
Glu Cys 500 505 510Ser Glu Leu Arg Gly Ile Met Asn Tyr Gln Glu Val
Glu Ala Leu Lys 515 520 525His Thr Ile Lys Leu Leu Thr Val Ile Lys
Trp His Gly Pro Lys Cys 530 535 540Asn Lys Leu Asn Ser Lys Phe Trp
Lys Arg Leu Gln Tyr Glu Met Pro545 550 555 560Phe Lys Arg Ile Glu
Pro Ile Thr His Glu Gln Ala Leu Asp Val Ser 565 570 575Glu Gln Gly
Pro Phe Gly Glu Leu Gln Thr Val Ser Ala Ile Ser Met 580 585 590Ala
Ala Ala Thr Ser Thr Ala Leu Ala Thr Ala His Pro Asp Leu Arg 595 600
605Ser Thr Phe His Asn Thr Tyr His Ser Gln Met Arg Gln Lys His Tyr
610 615 620Tyr Arg Ser Tyr Glu Tyr Asp Val Pro Pro Thr Gly Thr Leu
Pro Leu625 630 635 640Thr Ser Ile Gly Asn Gln His Thr Tyr Cys Asn
Ile Pro Met Thr Leu 645 650 655Ile Asn Gly Gln Arg Pro Gln Thr Lys
Ser Ser Arg Glu Gln Asn Pro 660 665 670Asp Glu Ala His Thr Asn Ser
Ala Ile Leu Pro Leu Leu Pro Arg Glu 675 680 685Thr Ser Ile Ser Ser
Val Ile Trp 690 6955657DNAHomo sapiens 5atgtcctttg tgggggagaa
ctcaggagtg aaaatgggct ctgaggactg ggaaaaagat 60gaaccccagt gctgcttaga
agacccggct gtaagccccc tggaaccagg cccaagcctc 120cccaccatga
attttgttca cacaagtcca aaggtgaaga acttaaaccc gaagaaattc
180agcattcatg accaggatca caaagtactg gtcctggact ctgggaatct
catagcagtt 240ccagataaaa actacatacg cccagagatc ttctttgcat
tagcctcatc cttgagctca 300gcctctgcgg agaaaggaag tccgattctc
ctgggggtct ctaaagggga gttttgtctc 360tactgtgaca aggataaagg
acaaagtcat ccatcccttc agctgaagaa ggagaaactg 420atgaagctgg
ctgcccaaaa ggaatcagca cgccggccct tcatctttta tagggctcag
480gtgggctcct ggaacatgct ggagtcggcg gctcaccccg gatggttcat
ctgcacctcc 540tgcaattgta atgagcctgt tggggtgaca gataaatttg
agaacaggaa acacattgaa 600ttttcatttc aaccagtttg caaagctgaa
atgagcccca gtgaggtcag cgattag 6576594DNAHomo sapiens 6atgtcctttg
tgggggagaa ctcaggagtg aaaatgggct ctgaggactg ggaaaaagat 60gaaccccagt
gctgcttaga aggtccaaag gtgaagaact taaacccgaa gaaattcagc
120attcatgacc aggatcacaa agtactggtc ctggactctg ggaatctcat
agcagttcca 180gataaaaact acatacgccc agagatcttc tttgcattag
cctcatcctt gagctcagcc 240tctgcggaga aaggaagtcc gattctcctg
ggggtctcta aaggggagtt ttgtctctac 300tgtgacaagg ataaaggaca
aagtcatcca tcccttcagc tgaagaagga gaaactgatg 360aagctggctg
cccaaaagga atcagcacgc cggcccttca tcttttatag ggctcaggtg
420ggctcctgga acatgctgga gtcggcggct caccccggat ggttcatctg
cacctcctgc 480aattgtaatg agcctgttgg ggtgacagat aaatttgaga
acaggaaaca cattgaattt 540tcatttcaac cagtttgcaa agctgaaatg
agccccagtg aggtcagcga ttag 5947474DNAHomo sapiens 7atgtcctttg
tgggggagaa ctcaggagtg aaaatgggct ctgaggactg ggaaaaagat 60gaaccccagt
gctgcttaga agagatcttc tttgcattag cctcatcctt gagctcagcc
120tctgcggaga aaggaagtcc gattctcctg ggggtctcta aaggggagtt
ttgtctctac 180tgtgacaagg ataaaggaca aagtcatcca tcccttcagc
tgaagaagga gaaactgatg 240aagctggctg cccaaaagga atcagcacgc
cggcccttca tcttttatag ggctcaggtg 300ggctcctgga acatgctgga
gtcggcggct caccccggat ggttcatctg cacctcctgc 360aattgtaatg
agcctgttgg ggtgacagat aaatttgaga acaggaaaca cattgaattt
420tcatttcaac cagtttgcaa agctgaaatg agccccagtg aggtcagcga ttag
4748218PRTHomo sapiens 8Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys
Met Gly Ser Glu Asp1 5 10 15Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu
Glu Asp Pro Ala Val Ser 20 25 30Pro Leu Glu Pro Gly Pro Ser Leu Pro
Thr Met Asn Phe Val His Thr 35 40 45Ser Pro Lys Val Lys Asn Leu Asn
Pro Lys Lys Phe Ser Ile His Asp 50 55 60Gln Asp His Lys Val Leu Val
Leu Asp Ser Gly Asn Leu Ile Ala Val65 70 75 80Pro Asp Lys Asn Tyr
Ile Arg Pro Glu Ile Phe Phe Ala Leu Ala Ser 85 90 95Ser Leu Ser Ser
Ala Ser Ala Glu Lys Gly Ser Pro Ile Leu Leu Gly 100 105 110Val Ser
Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp Lys Gly Gln 115 120
125Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met Lys Leu Ala
130 135 140Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr Arg
Ala Gln145 150 155 160Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala
His Pro Gly Trp Phe 165 170 175Ile Cys Thr Ser Cys Asn Cys Asn Glu
Pro Val Gly Val Thr Asp Lys 180 185 190Phe Glu Asn Arg Lys His Ile
Glu Phe Ser Phe Gln Pro Val Cys Lys 195 200 205Ala Glu Met Ser Pro
Ser Glu Val Ser Asp 210 2159197PRTHomo sapiens 9Met Ser Phe Val Gly
Glu Asn Ser Gly Val Lys Met Gly Ser Glu Asp1 5 10 15Trp Glu Lys Asp
Glu Pro Gln Cys Cys Leu Glu Gly Pro Lys Val Lys 20 25 30Asn Leu Asn
Pro Lys Lys Phe Ser Ile His Asp Gln Asp His Lys Val 35 40 45Leu Val
Leu Asp Ser Gly Asn Leu Ile Ala Val Pro Asp Lys Asn Tyr 50 55 60Ile
Arg Pro Glu Ile Phe Phe Ala Leu Ala Ser Ser Leu Ser Ser Ala65 70 75
80Ser Ala Glu Lys Gly Ser Pro Ile Leu Leu Gly Val Ser Lys Gly Glu
85 90 95Phe Cys Leu Tyr Cys Asp Lys Asp Lys Gly Gln Ser His Pro Ser
Leu 100 105 110Gln Leu Lys Lys Glu Lys Leu Met Lys Leu Ala Ala Gln
Lys Glu Ser 115 120 125Ala Arg Arg Pro Phe Ile Phe Tyr Arg Ala Gln
Val Gly Ser Trp Asn 130 135 140Met Leu Glu Ser Ala Ala His Pro Gly
Trp Phe Ile Cys Thr Ser Cys145 150 155 160Asn Cys Asn Glu Pro Val
Gly Val Thr Asp Lys Phe Glu Asn Arg Lys 165 170 175His Ile Glu Phe
Ser Phe Gln Pro Val Cys Lys Ala Glu Met Ser Pro 180 185 190Ser Glu
Val Ser Asp 19510157PRTHomo sapiens 10Met Ser Phe Val Gly Glu Asn
Ser Gly Val Lys Met Gly Ser Glu Asp1 5 10 15 Trp Glu Lys Asp Glu
Pro Gln Cys Cys Leu Glu Glu Ile Phe Phe Ala 20 25 30Leu Ala Ser Ser
Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 35 40 45Leu Leu Gly
Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 50 55 60Lys Gly
Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met65 70 75
80Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr
85 90 95Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His
Pro 100 105 110Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro
Val Gly Val 115 120 125Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu
Phe Ser Phe Gln Pro 130 135 140Val Cys Lys Ala Glu Met Ser Pro Ser
Glu Val Ser Asp145 150 155118PRTArtificial sequenceantigenic
peptide used in fusion proteins 11Asp Tyr Lys Asp Asp Asp Asp Lys1
51227PRTArtificial sequenceleucine zipper polypeptide 12Pro Asp Val
Ala Ser Leu Arg Gln Gln Val Glu Ala Leu Gln Gly Gln1 5 10 15Val Gln
His Leu Gln Ala Ala Phe Ser Gln Tyr 20 251333PRTArtificial
sequenceleucine zipper polypeptide 13Arg Met Lys Gln Ile Glu Asp
Lys Ile Glu Glu Ile Leu Ser Lys Ile1 5 10 15Tyr His Ile Glu Asn Glu
Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu 20 25 30Arg148PRTArtificial
sequencepolymorphic sequence from exon 2 of Tango 77 14Pro Ala Gly
Ser Pro Leu Glu Pro1 5158PRTArtificial sequencepolymorphic sequence
from exon 2 of Tango 77 15Pro Ala Val Ser Pro Leu Glu Pro1 5
* * * * *
References