JP2003523209A - FIL-1 theta DNA and polypeptide - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel purified and isolated FIL-1 theta polypeptides and fragments thereof, nucleic acids encoding such polypeptides, methods for the recombinant production of such polypeptides, It relates to the antibodies produced, fragmented peptides derived from these polypeptides, and their use.

Description

Detailed Description of the Invention

[0001] FIELD OF THE INVENTION The present invention is purified and FIL-1 theta polypeptides and fragments thereof have been isolated, nucleic acids encoding such polypeptides, method of production of recombinant forms of such polypeptides, Antibodies raised against these polypeptides, fragmented peptides derived from these polypeptides, as well as their use.

Description of Related Art Interleukin-1 (IL-1) is a member of a large group of cytokines whose primary function is to mediate immune and inflammatory responses. Included in the group of IL-1 family members are IL-1 alpha (IL-1α), IL-1
Beta (IL-1β), IL-1 receptor antagonist (IL-1ra), IL
-1ra beta, FIL-1 delta, IL-1 eta (also called FIL-1 eta), IL-1 zeta (also called FIL-1 zeta), and IL-1
Epsilon (also called FIL-1 epsilon) and IL-18 (formerly IG
IF and sometimes also known as IL-1 gamma). IL-1 secreted by macrophages is primarily a mixture of IL-1β and some IL-1α (Abbas et al., 1994). IL-1α and IL-1β are first produced as 33 kD precursors lacking the signal sequence. These precursors are further processed by proteolytic cleavage to produce the secreted active form of approximately 17 kD each. In addition, the 33 kD precursor of IL-1α is also active.
IL-1α and IL-1β are the products of two different genes located on chromosome 2. The two types are less than 30 percent homologous to each other, but bind to the same receptor and have similar activity.

IL-1α and IL-1β are ligand-binding chains, type I IL-1 receptors (IL
-1RI), and a necessary signaling component, the IL-1R accessory protein (
Binds to a common receptor composed of AcP) (Sims et al., 1998; Gre
eenfeder et al., 1995; Cullinan et al., 1998). Type II IL
The -1 receptor (IL-1RII) binds and sequesters the agonist IL-1 (particularly IL-1β) without inducing any signaling response of its own (McMahan et al., 1991; Sims et al., 1993; Colot.
ta et al., 1993; Colotta et al., 1994). IL-1α and IL-1
β is a naturally occurring soluble proteolytic fragment of IL-1RII (sIL-1
RII) (Colotta et al., 1993).

IL-1ra, a biologically inactive form of IL-1, is structurally homologous to IL-1. IL-1ra is produced containing a signal sequence that allows for efficient secretion into the extracellular domain (Abbas et al., 1994). Furthermore, IL-1ra
Binds to the type I IL-1 receptor but subsequently fails to trigger an interaction with AcP. Thus, IL-1ra blocks IL1-RI and prevents the action of the agonist IL-1 (Hannum et al., 1990; Eise).
nberg et al., 1990).

The major sources of IL-1 are activated macrophages or mononuclear phagocytes.
Other cells that produce IL-1 include epithelial and endothelial cells (Abbas
Et al., 1994). IL-1 secretion from macrophages occurs after macrophages encounter and ingest Gram-negative bacteria. Such bacteria contain a lipopolysaccharide (LPS) molecule, also known as endotoxin, in the bacterial cell wall. LP
The S molecule is the active component that stimulates macrophages to produce tumor necrosis factor (TNF) and IL-1. 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 necessary to clear bacterial infections; at high concentrations, LPS causes severe tissue damage, shock, And can even cause death.

The biological functions of IL-1 include activation of vascular endothelial cells and lymphocytes, local tissue destruction, and fever (Janeway et al., 1996). At low concentration, I
L-1 stimulates macrophages and vascular endothelial cells to produce IL-6, upregulates molecules on the surface of vascular endothelial cells, increases leukocyte adhesion, and mononuclear phagocytes and other cells. Inflammatory leukocytes are indirectly activated by stimulating the production of specific chemokines that activate the inflammatory leukocytes. Furthermore, I
L-1 is involved in inflammatory responses, including induction of prostaglandins, nitric oxide synthetase, and metalloproteinases. These IL-1 functions are very important during low levels of microbial infection. However, as microbial infections spread in stages, IL-1 induces fever, stimulates mononuclear phagocytes to produce IL-1 and IL-6, and increases serum protein production from hepatocytes. And act systemically by activating the coagulation system. Moreover, IL-1 does not cause hemorrhagic necrosis of tumors, does not suppress bone marrow stem cell division, and IL-1 is lethal to humans at high concentrations.

Given the important functions of IL-1, there is a need to identify additional members of the IL-1 ligand family. Furthermore, given the continuing interest in protein research and the immune system, the discovery, identification, and role of novel proteins and their inhibitors is at the forefront of modern molecular biology and biochemistry. Despite increasing knowledge, there remains a need in the art to discover the identities and functions of proteins involved in cellular and immune responses. SUMMARY OF THE INVENTION The present invention provides isolated nucleic acids and polypeptides encoded by the nucleic acids with respect to the IL-1 family of ligands referred to as "FIL-1 theta." In one aspect, the invention provides the DNA of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 14 (FI
L-1 theta), and SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 14
A nucleic acid molecule complementary to. Similarly, in another aspect, the invention encodes an isolated polypeptide comprising the amino acid sequence SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 15, as well as the polypeptides of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 15. Directed to a nucleic acid molecule. The nucleic acids of SEQ ID NO: 1 and SEQ ID NO: 3 have been cloned from human cells and encode the polypeptides of SEQ ID NO: 2 and SEQ ID NO: 4, respectively. SEQ ID NO: 14 has been cloned from mouse cells and encodes the polypeptide of SEQ ID NO: 15.

Nucleic acid molecules that hybridize to denatured, double-stranded DNA, including single or double-stranded RNA and DNA nucleic acid molecules, as well as all or part of SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 14, and / or Alternatively, a DNA encoding the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 15 is included in the present invention.
Also included in the invention is the sequence SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 1.
5, an isolated nucleic acid molecule derived from in vitro mutagenesis of the nucleic acid molecule of 5, and an isolated nucleic acid molecule degenerate from the nucleic acid molecule of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 15, as well as the DNA of the invention. Is an isolated nucleic acid molecule that is an allelic variant of.

The invention also includes a vector or expression vector comprising at least one nucleic acid of the invention and directing the expression of the polypeptide of the invention. Also included is a host cell that has been transformed or transfected with at least one vector of the present invention. Further included in the invention is a recombinant host cell in which the nucleic acid has been integrated into the host cell genome.

The present invention provides a method of producing a polypeptide encoded by the nucleic acid of the present invention. The method comprises culturing a host cell of the invention under conditions that promote expression of the polypeptide. Preferably, the method further comprises purifying the polypeptide.

The present invention further includes methods of using the nucleic acids described above to identify nucleic acids encoding proteins having activities associated with IL-1 family ligands and receptors. Thus, FIL-1 theta nucleic acid molecules can be used to identify FIL-1 theta receptors. Furthermore, these nucleic acids can be used to identify the human chromosome with which they are associated. Accordingly, the FIL-1 nucleic acids of SEQ ID NO: 1 and SEQ ID NO: 3 are used to identify human chromosome 2 and map genes on human chromosome 2 to identify specific diseases, syndromes, or other disorders associated with human chromosome 2. It is possible to identify genes associated with human abnormalities and study cell signaling and immune systems.

The present invention also relates to the use of sense or antisense oligonucleotides derived from the nucleic acids of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 14 for inhibiting the expression of each polynucleotide encoded by the gene of the present invention. Also includes.

The present invention also includes isolated polypeptides and fragments of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 15, and nucleic acid molecules encoding the isolated polypeptides or fragments. The invention further comprises a method of producing these polypeptides, culturing the host cells under conditions that promote expression, and optionally recovering the polypeptides from the medium. In particular, the expression of these polypeptides in bacteria, yeast, plants, insects and animal cells is included in the invention.

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-cell interactions, and inflammatory responses. In addition, these polypeptides can be used to identify proteins associated with FIL-1 theta.

Furthermore, the present invention utilizes these polypeptides to provide polypeptide pair structures (
including assays that screen potential inhibitors of activity associated with the counter-structure molecule, and methods of using these polypeptides as therapeutic agents for the treatment of diseases mediated by the polypeptide versus structural molecules. Furthermore, methods of using these polypeptides for the design of inhibitors (eg engineered receptors that act as inhibitors) are also aspects of the invention.

Further included in the invention is the prediction of FIL-1 theta nucleic acid sequences, polypeptides or fragments thereof for use in searching electronic databases to aid in the identification of sample nucleic acids and / or proteins. Use of a combination of amino acid sequences or predicted amino acid sequences of polypeptides and fragments thereof.

In addition to isolated polyclonal or monoclonal antibodies that bind to these polypeptides, the use of these antibodies to help purify the polypeptides of the invention is also included in the invention. DETAILED DESCRIPTION OF THE INVENTION In one aspect, the invention provides a nucleic acid having the following nucleotide sequence: Name: FIL-1 theta (human)

[0018]

[Chemical 1]

(SEQ ID NO: 1) In another aspect, the present invention includes a polypeptide having the following amino acid sequence, and a nucleic acid molecule encoding the polypeptide.

[0020]   Name: FIL-1 theta (polypeptide)

[0021]

[Chemical 2]

[0022]   (SEQ ID NO: 2)   In another aspect, the invention provides the following nucleic acids:   Name: Full length FIL-1 Theta

[0023]

[Chemical 3]

(SEQ ID NO: 3) In yet another aspect, the invention comprises the following polypeptide and polynucleotide encoding the polypeptide: Name: full length FIL-1 theta

[0025]

[Chemical 4]

(SEQ ID NO: 4) Further included in the invention is the following mouse nucleic acid and encoded mouse polypeptide: Name: Full length mouse FIL-1 theta polynucleotide:

[0027]

[Chemical 5]

[0028]   (SEQ ID NO: 14)   The coding region is nucleotides 29-487.   Name: Full length mouse FIL-1 theta polypeptide:

[0029]

[Chemical 6]

(SEQ ID NO: 15) The discovery of the FIL-1 theta nucleic acid of the present invention enables the construction of an expression vector containing a nucleic acid encoding each polypeptide, and a host cell transfected or transformed with the expression vector. To The present invention also relates to biologically active F
It also allows the isolation and purification of IL-1 theta polypeptides and fragments thereof. In yet another embodiment, the nucleic acids or oligonucleotides of the invention can be used as probes to identify nucleic acids that encode proteins with related activity.
Therefore, FIL-1 theta can be used to identify activities associated with IL-1 family ligands. Furthermore, it is possible to identify human chromosome 2 using the nucleic acids or oligonucleotides of the invention. Similarly, these nucleic acids and oligonucleotides can be used to map genes on human chromosome 2 and identify genes associated with specific diseases, syndromes or other human abnormalities associated with human chromosome 2. Is. Thus, using the nucleic acids and oligonucleotides encoding the polypeptides of the present invention to identify glaucoma, ectodermal dysplasia, insulin-dependent diabetes mellitus, wrinkled skin syndrome, T-cell leukemia / lymphoma, and tibialis dystrophy. Is possible. Finally, single stranded sense or antisense oligonucleotides of the invention can be used to inhibit expression of the polynucleotides of the invention and therapeutically inhibit diseases associated with FIL-1 expression. Or can be treated.

Furthermore, the FIL-1 theta polypeptides and soluble peptide fragments of the invention are used to activate and / or inhibit vascular endothelial cells and lymphocytes, local tissue destruction and fever. , And / or inhibit its induction (
Janeway et al., 1996), macrophages and vascular endothelial cells used IL-6.
To produce and / or stimulate prostaglandins, nitric oxide synthetase, and metalloproteinases, and / or inhibit its induction, and to inhibit molecules on the surface of vascular endothelial cells. It is possible to upregulate and / or inhibit that upregulation. In addition, these polypeptides and fragmented peptides also induce transcription factors NF-κB and AP-1, MAP kinases JNK and p38, COX-2, inflammation mediators such as iNOS, and all of these molecule-stimulated activities. Can also be used to induce and / or inhibit its induction.

The polypeptides and fragments of the invention are useful in the production of antibodies that bind to the polypeptides and / or polypeptide fragments of the invention. Preferably, the antibody is a monoclonal antibody and / or a humanized antibody. The invention further includes, but is not limited to, purification of the polypeptides of the invention and use of antibodies, including inhibiting or activating the activity associated with FIL-1 theta polypeptides.

As described in Example 1, the present invention comprises FIL-1 theta polymorphisms shown in several tissue sources. The set of polymorphisms includes amino acid 44 of SEQ ID NO: 4.
Corresponding to three alleles at nucleotides 130-132 of SEQ ID NO: 3. In one polymorphism, nucleotides 130-132 are ACA,
Amino acid residue 44 then encodes threonine. The second and third polymorphisms are associated with ATA and ATC at nucleotides 130-132, both of which at amino acid residue 44 encode isoleucine. Another polymorphism has been identified at amino acid 51 of SEQ ID NO: 4. More specifically, when nucleotides 151-153 of SEQ ID NO: 3 are GACs, the amino acid encoded by residue 51 of SEQ ID NO: 4 is aspartic acid, and when nucleotides 151-153 are GCC, The encoded amino acid is alanine. Nucleotide 15
Oligonucleotides containing either 1-153 and / or alleles associated with nucleotides 130-132 are useful for detecting disease-associated polymorphisms. Diseases include those directly caused by aberrant forms of FIL-1 theta, or diseases associated with one of these polymorphic markers.

The nucleic acids of the invention relate to isolated compounds that are free of contaminating endogenous components. Nucleic acid
Refers to compounds in the form of discrete fragments or as components of larger nucleic acid constructs. In one embodiment, the nucleic acids of the invention are in substantially pure form, and in standard biochemical methods (Sambrook et al., Molecular Cloning : A Laboratory Manual , 2nd Edition, Cold Spr.
ing Harbor Laboratory, Cold Spring Harbor, NY, such as those outlined in 1989), or DNA that has been isolated at least once in an amount or concentration that allows its constituent nucleotides to be identified, manipulated, and recovered. It is derived from RNA. The present invention further includes nucleic acids having nucleotide sequences that differ due to the degeneracy of the genetic code, but encode the same polypeptide. The nucleotides defining the nucleic acid are preferably provided and / or constructed in the form of an open reading frame uninterrupted by internal untranslated sequences or introns that are typically present in eukaryotic genes. The untranslated DNA sequence may be located 5'or 3'of the open reading frame such that the DNA sequence does not interfere with the manipulation or expression of the coding region.

The nucleic acid of the present invention includes both single-stranded and double-stranded forms of DNA, as well as its RNA complement. DNA includes, for example, cDNA and genomic DN.
A, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA has, for example, SEQ ID NO: 1 and SEQ ID NO: 3
, SEQ ID NO: 14, or a suitable fragment thereof, can be used as a probe and isolated by conventional techniques.

The DNA molecule of the present invention includes the full-length gene as well as nucleic acids and fragments thereof. The full length gene may include an N-terminal signal peptide. The nucleic acids of the invention are preferentially derived from human sources, although the invention also includes those from non-human species.

Preferred polypeptides of the invention are SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 1.
4 nucleic acids are included. The encoded polypeptides are, respectively,
Shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 15. SEQ ID NO: 15 is the native mouse polypeptide and SEQ ID NO: 4 is the human IL-1 theta polypeptide.

SEQ ID NO: 1 identifies FIL-1 theta as a member of the IL-1 family. SEQ ID NO: 3 is the full length sequence of this IL-1 family member, and encodes the full length polypeptide shown in SEQ ID NO: 4. The homology based on this is shown in Table 1.
Shown in.

Table 1

[0040]

[Table 1]

1 Disclosed in WO 00/36108 2 Disclosed in WO 00/71720 3 Disclosed in WO 00/11174 By tissue distribution experiments, tissues positive for human FIL-1 theta were found to have tonsils , Skin, lungs, placenta, and small airway epithelium have been demonstrated. FIL-1
Theta negative tissues include small intestine, liver, grain, fetal liver, lymph nodes, bone marrow, kidney, pancreas, skeletal muscle, heart, prostate, ovary, thymus, spleen, white blood cells, testis, and colon. included.

Due to the known degeneracy of the genetic code, in which more than one codon may code for the same amino acid, the DNA sequence differs from that shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 14. And yet, it may encode polypeptides having the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 15, respectively. Such variant DNA may result from inadvertent mutations (eg, that occur during PCR amplification) or may be the product of deliberate mutagenesis of native sequence.

Accordingly, the invention comprises: (a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1;
(B) DNA containing the nucleotide sequence of SEQ ID NO: 3; (c) DNA encoding the polypeptide of SEQ ID NO: 2; (d) DN encoding the polypeptide of SEQ ID NO: 4.
A; (e) a complement of DNA capable of hybridizing to the DNA of (a), (b), (c) or (d) under conditions of moderate stringency, and DNA encoding a polypeptide of the invention; (f) capable of hybridizing to the DNA of (a) to (d) under conditions of high stringency, and encoding a polypeptide of the invention, DNA; and (g) as a result of the genetic code, degenerate to the DNA defined in (a), (b), (c), (d), (e), or (f), and the invention DNA encoding a polypeptide of
Isolated DNA is provided. Of course, polypeptides encoded by such DNA sequences are included in the invention.

Furthermore, the present invention provides (a) a DNA containing the nucleotide sequence of SEQ ID NO: 14;
b) DNA encoding the polypeptide of SEQ ID NO: 15; (c) a complement of DNA capable of hybridizing to the DNA of (a) or (b) under conditions of moderate stringency, And D, which encodes the polypeptide of the invention
NA; (f) DNA capable of hybridizing under high stringency conditions with the DNA of (a) to (d) and encoding a polypeptide of the invention; and (g) the genetic code. As a result, (a), (b), (c), (d), (
e) or DNA that is degenerate to the DNA defined in (f) and encodes the polypeptide of the present invention. Of course, polypeptides encoded by such DNA sequences are included in the invention.

In the present specification, the condition of moderate stringency can be easily determined by a person having ordinary skill in the art based on, for example, the length of DNA. The basic conditions are described in Sambrook et al., Molecular Cloning : A Laboratory Manual , 2nd edition, Vol. 1
, Pp. 1.101-104, Cold Spring Harbor L
Laboratory Press, 1989, and for nitrocellulose filters, 5X SSC, 0.5% SDS, 1.0 mM EDTA (
pH 8.0) pre-wash solution, about 50% formamide, 6X SSC at about 42 ° C.
(Or Stark's solution (Stark ') in about 50% formamide at about 42 ° C.
other similar hybridization solution) such as S. solution) and about 60 ° C., 0.5 × SSC, 0.1% SDS.
Use of the washing conditions of. Conditions of high stringency also include eg DN
It can be easily determined by those skilled in the art based on the length of A. Generally, such conditions are defined as involving hybridization conditions as described above, and washing at approximately 68 ° C., 0.2X SSC, 0.1% SDS. Those skilled in the art will recognize that the temperature and wash solution salt concentration can be adjusted as needed according to factors such as probe length.

Also included in the invention is a polypeptide fragment containing one or more inactivating N-glycosylation sites, inactivating protease processing sites, or conservative amino acid substitutions, as described below. And D encoding a polypeptide
It is NA.

In another embodiment, the nucleic acid molecule of the present invention also comprises a nucleotide sequence that is at least 80% identical to the native sequence. It is further within the scope of the invention that the nucleic acid molecule has at least 90% of its native sequence.
Identical, at least 95% identical, at least 98% identical, at least 99% identical,
Or an embodiment that includes a sequence that is at least 99.9% identical.

The percent identity can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity between the two nucleic acid sequences is the Devereux.
Nucl. Acids Res. 12: 387, 1984 and available from the University of Wisconsin Genetics Computer Group (UWGCG), GAP computer program, version 6.0, can be determined by comparing sequence information. Preferred default parameters for the GAP program include: (1) unary for nucleotides
A comparison matrix (including values of 1 for identical and 0 for non-identical), and S
chwartz and Dayhoff, Atlas of Protein Sequence and Structure , pp. 353-358
, National Biomedical Research Found
Gribskov and Burg, as described in ation, 1979.
ess, Nucl. Acids Res. 14: 6745, 1986 weighted comparison matrix; (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 those skilled in the art of sequence comparison can also be used.

The present invention also provides isolated nucleic acids useful for the production of polypeptides and fragments, including soluble fragments of FIL-1 theta. Such polypeptides may be prepared by any of several conventional techniques. DNA encoding a polypeptide of the invention, or a desired fragment thereof, can be subcloned into an expression vector to produce the polypeptide or fragment. The DNA is conveniently fused to a sequence encoding a suitable leader or signal peptide. Alternatively, known techniques can be used to chemically synthesize the desired DNA encoding a polypeptide or fragment of the invention. Such coding DNA fragments can also be produced by restriction endonuclease digestion of full length cloned DNA and isolated by electrophoresis on an agarose gel. If desired, oligonucleotides with the 5'or 3'ends reconstructed to the desired point may be ligated to DNA fragments generated by restriction enzyme digestion. Such oligonucleotides may further comprise a desired coding sequence and a restriction endonuclease cleavage site upstream of the N-terminal start codon (ATG) position of the coding sequence.

Known polymerase chain reaction (PCR) methods can also be used to isolate and amplify the DNA encoding the desired protein fragment. Oligonucleotides that define the desired ends of the DNA fragments are used as 5'and 3'primers. The oligonucleotide may further include a recognition site for a restriction endonuclease to facilitate insertion of the amplified DNA fragment into an expression vector. PC
R technology is described by Saiki et al., Science, 239: 487, 1988;
Wu et al., Recombinant DNA Methodology ,
pp. 189-196, Academic Press, Inc. , San Diego, 1989; and Innis et al. Eds, PCR Protocols: A Guide to Methods and Application s, Academic Press, Inc. , 1990.

The present invention includes various types of polypeptides and fragments thereof, including those that are naturally occurring or produced through a variety of techniques such as those involving recombinant DNA technology. Such forms include, but are not limited to, derivatives, variants, and oligomers, as well as fusion proteins or fragments thereof.

The polypeptides of the present invention include partial and full length proteins encoded by the nucleic acid sequences described above. Preferred polypeptides of FIL-1 theta include those having the amino acid sequence of SEQ ID NO: 4 and those having the amino acid sequence of SEQ ID NO: 15.

In general, the use of soluble forms is preferred for certain applications. The soluble polypeptide is secreted from the cell, which facilitates purification of the polypeptide from recombinant host cells. Moreover, soluble polypeptides are generally more suitable for intravenous administration.

The present invention also provides polypeptides and fragments that retain the desired biological activity. Particular aspects include natural cognate, substrate, or paired structure (
"Binding partner") is directed to the polypeptide fragments of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 15 that retain the ability to bind. Such fragments may be soluble polypeptides, as described above. In another aspect, the polypeptides and fragments suitably comprise regions conserved in the IL-1 ligand family, as described above.

Also provided herein is a polypeptide fragment comprising at least 20, or at least 30 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 15. Polypeptide fragments can also be used as immunogens in raising antibodies.

Provided herein are naturally occurring variants, as well as variants derived from polypeptides and fragments. Variants include polypeptides that are at least 80% identical to the polypeptide of SEQ ID NO: 2 and / or the polypeptide of SEQ ID NO: 4 and / or the polypeptide of SEQ ID NO: 15. Also intended is at least 90% identical to at least 90%, at least 95, to the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 15, the fragment of SEQ ID NO: 2, or the fragment of SEQ ID NO: 4, or the fragment of SEQ ID NO: 15. Polypeptides and fragments comprising amino acid sequences that are% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical. The percent identity can be determined by visual inspection and mathematical calculation. Alternatively,
The percent identity between the two protein sequences is Needleman and Wun.
sch (J. Mol. Bio. 48: 443, 1970) and based on the University of Wisconsin Genetics Computer Group (UWG).
It can be determined by comparing the sequence information using the GAP computer program available from CG). Preferred default parameters for the GAP program include: (1) Henikoff et al., Proc. Natl. A
cad. Sci. USA, 89: 10915, 1992, including scoring matrix, blosum62; (2) 12 gap weights; (3) 4 gap length weights; and (4) no penalties for end gaps. Be done. Other programs used by those of skill in the art for sequence comparison also
It can be used.

Variants of the invention include, for example, those that result from alternative mRNA splicing events or proteolytic cleavage. Alternative splicing of mRNA can result, for example, in a truncated but biologically active protein, eg, a naturally occurring soluble form of the protein. Mutations that may be due to proteolysis include, for example, proteolytic removal of one or more terminal amino acids (generally 1-5 terminal amino acids) from the protein upon expression in different types of host cells. N- or C-terminal differences due to Proteins that can result in amino acid sequence differences due to genetic polymorphisms (allelic variations between individuals who produce the protein) are also contemplated herein. See Example 1 for polymorphic variants of the FIL-1 theta molecule.

Further variants within the scope of the invention are modified by forming covalent or aggregate conjugates with other chemical moieties such as glycosyl groups, lipids, phosphates, acetyl groups and the like. Included in the polypeptide, which may produce derivatives. Covalent derivatives can be prepared by linking a chemical moiety to a functional group on the amino acid side chain or at the N- or C-terminus of the polypeptide. Adherent diagnostics (detectable), as discussed in more detail below
Conjugates that include an agent or therapeutic agent are contemplated herein.

Other derivatives include covalent or aggregate conjugates of the polypeptide with other proteins or polypeptides, such as by being synthesized in recombinant culture as N-terminal or C-terminal fusions. Examples of fusion proteins are discussed below in connection with oligomers. In addition, the fusion protein may include peptides added to facilitate purification and identification. Such peptides include, for example, poly-His or US Pat. No. 5,011,912 and Hopp et al.
Bio / Technology, 6: 1204, 1988 is included. One such peptide is FLAG®.
Peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
And the peptide is highly antigenic and provides the epitope to which the specific monoclonal antibody binds reversibly, allowing rapid assay and easy purification of the expressed recombinant protein. The murine hybridoma designated 4E11 has the FLAG® peptide in the presence of certain divalent metal cations as described in US Pat. No. 5,011,912, incorporated herein by reference. Produce a monoclonal antibody that binds to. The 4E11 hybridoma cell line is available under the accession number HB 9259 under American Type Culture Collection (A
It has been deposited with the American Type Culture Collection). The monoclonal antibody that binds to the FLAG® peptide is E
astman Kodak Co. , Scientific Imaging
Available from Systems Division, New Haven, Connecticut.

Among the variant polypeptides provided herein, variants of the native polypeptide that retain the natural biological activity or substantial equivalents thereof.
There is t). One example is a variant that binds with essentially the same binding affinity as the native form. Binding affinity can be measured by conventional methods, eg, as described in US Pat. No. 5,512,457 and as shown below.

Variants include polypeptides that are substantially homologous to the native form but have an amino acid sequence that differs from the native form due to one or more deletions, insertions or substitutions. Specific embodiments include, but are not limited to, when compared to the native sequence:
Included are polypeptides containing 1 to 10 deletions, insertions or substitutions of amino acid residues.

A given amino acid may be replaced, for example, by a residue having similar physicochemical properties. Examples of such conservative substitutions are substitutions of one aliphatic residue for another, for example replacing Ile, Val, Leu, or Ala with each other; Lys
And between Arg, Glu and Asp, or Gln and Asn, 1
Substitution of one polar residue for another; or substitution of an aromatic residue for another,
For example, those in which Phe, Trp, or Tyr are substituted with each other are included. Other conservative substitutions, such as those involving replacement of entire regions with similar hydrophobic properties, are known.

Similarly, the DNA of the present invention includes variants that differ from the native DNA sequence due to one or more deletions, insertions or substitutions, but that encode a biologically active polypeptide. Is included.

The invention further includes polypeptides of the invention with or without associated native pattern glycosylation. A polypeptide expressed in a yeast or mammalian expression system (eg, COS-1 or COS-7 cells) may be similar to the native polypeptide in molecular weight and glycosylation pattern, depending on the choice of expression system. , Or may be significantly different. Expression of a polypeptide of the invention in a bacterial expression system such as E. coli provides a non-glycosylated molecule. In addition, a given preparation may contain a large number of differently glycosylated protein species. Glycosyl groups can be removed through conventional methods, especially those utilizing glycopeptidases. In general, the glycosylated polypeptides of the present invention are treated with a molar excess of glycopeptidase (Boehringer Mannhe).
It may be incubated with im).

Correspondingly, similar DNA constructs encoding various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences are included in the invention. For example, the N-glycosylation site of the polypeptide extracellular domain may be modified to prevent glycosylation, which allows expression of carbohydrate-reducing analogs in mammalian and yeast expression systems. The N-glycosylation site of a eukaryotic polypeptide is characterized by the amino acid triplet Asn-XY, where X can be any amino acid except Pro, and Y is S
er or Thr. Appropriate substitutions, additions or deletions to the nucleotide sequences encoding these triplets will result in the prevention of attachment of carbohydrate residues on the Asn side chain. For example, a single nucleotide modification, where Asn is selected to be replaced by a different amino acid, is sufficient to inactivate the N-glycosylation site. Alternatively, Ser or Thr is replaced by another amino acid, eg A
It may be replaced with la. Known methods for inactivating the N-glycosylation site of a protein include those described in US Pat. No. 5,071,972 and EP 276,846, which are incorporated herein.

In another example of a variant, the sequence encoding a Cys residue that is not essential for biological activity is modified so that the Cys residue is deleted or replaced with another amino acid, During folding or regeneration, it may prevent the formation of false intramolecular disulfide bridges.

Other variants are prepared by modifying flanking 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-directed mutagenesis to inactivate the KEX2 protease processing site of proteins. KEX2
Protease processing sites are Arg-Arg, Arg-Lys, and L.
To modify the ys-Arg pair and remove the occurrence of these flanking basic residues,
It is inactivated by deleting, adding or substituting residues. Ly
The s-Lys pair is considerably less sensitive to KEX2 cleavage, and the conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivate the KEX2 site.

Included in the invention is an oligomeric or fusion protein containing a FIL-1 theta polypeptide. Such oligomers may be in covalent or non-covalent multimeric form, including dimers, trimers, or higher order oligomers.
As mentioned above, preferred polypeptides are soluble, and thus these oligomers may include soluble polypeptides. In one aspect of the invention, the oligomer maintains the binding ability of the polypeptide component, and thus
Provides bivalent, trivalent, etc. binding sites.

One aspect of the invention pertains to oligomers comprising multiple polypeptides linked via covalent or non-covalent interactions between peptide moieties fused to the polypeptides. Such peptides may be peptide linkers (spacers) or may have the property of promoting oligomerization. Among the peptides capable of promoting oligomerization of attached polypeptides are leucine zippers and certain polypeptides derived from antibodies, as described in more detail below.

As an alternative, oligomers are prepared using immunoglobulin-derived polypeptides. Various parts of antibody-derived polypeptide (including Fc domain)
Preparation of a fusion protein containing a particular heterologous polypeptide fused to, eg, Ashkenazi et al., PNAS USA, 88: 10535, 19
91; Byrn et al., Nature 344: 677, 1990; and Ho.
llenbaugh and Aruffo, “Construction of
Immunoglobulin Fusion Proteins ", in Cu rrent Protocols in Immunology, Supp
l. 4, pp. 10.19.1-10.19.11, 1992.

One aspect of the invention relates to a dimer comprising two fusion proteins produced by fusing a polypeptide of the invention to an Fc polypeptide derived from an antibody. The gene fusion encoding the polypeptide / Fc fusion protein is inserted into an appropriate expression vector. Expressing a polypeptide / Fc fusion protein in a host cell transformed with the recombinant expression vector and allowing it to assemble much like an antibody molecule so that interchain disulfide bonds are formed between the Fc moieties, Allows to generate bivalent molecules.

As used herein, the term “Fc polypeptide” refers to native and mutein-type polypeptides composed of the Fc region of an antibody, including any or all of the CH domains of the Fc region. including. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. Preferred polypeptides include Fc polypeptides derived from human IgG1 antibodies.

One suitable Fc polypeptide described in PCT application WO 93/10151, incorporated herein, is a single chain polypeptide spanning the N-terminal hinge region of the Fc region of a human IgG1 antibody to the native C-terminus. It is a peptide. Another useful Fc polypeptide is US Pat. No. 5,457,035 and Baum et al., Incorporated herein by reference.
EMBO J.M. 13: 3992-4001, 1994, Fc.
It is a mutein. The amino acid sequence of this mutein is WO except that amino acid 19 is changed from Leu to Ala, amino acid 20 is changed from Leu to Glu, and amino acid 22 is changed from Gly to Ala.
It is identical to that of the native Fc sequence shown in 93/10151. The mutein exhibits reduced affinity for Fc receptors.

The above-described fusion proteins (and the oligomers formed therefrom) containing the Fc portion provide the advantage of easy purification by affinity chromatography on a Protein A or Protein G column.

In other embodiments, the polypeptides of the invention may be substituted with respect to the variable region of the antibody heavy or light chain. If the fusion protein is made with both heavy and light chains of an antibody, it is possible to form oligomers with as many as four polypeptide extracellular regions.

The oligomers of the invention include fusion proteins of more than one polypeptide described herein, with or without a peptide linker (spacer peptide). In a suitable peptide linker, US Pat.
751,180 and 4,935,233. The DNA sequence encoding the desired peptide linker may be inserted between the DNA sequences of the invention and in the same reading frame as the DNA sequence using any suitable conventional technique. For example, a chemically synthesized oligonucleotide encoding a linker may be linked between the sequences. In a particular embodiment, the fusion protein comprises 2 to 4 soluble polypeptides separated by a peptide linker.

Another method for preparing the oligomers of the invention involves the use of leucine zippers. The leucine zipper domain is a peptide that promotes oligomerization of the protein in which it is found. The leucine zipper originally had several D
Identified in NA-binding proteins (Landschulz et al., Science
240: 1759, 1988), and since then, in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and their derivatives that dimerize or trimerize.

The zipper domain (also referred to herein as the oligomerization, or oligomerization domain) contains repeated 7-residue repeats, often with 4 or 5 leucine residues interspersed with other amino acids. . Examples of zipper domains are the yeast transcription factor GCN4 and a thermostable DNA binding protein found in rat liver (C / EBP; Landschulz et al., Science, 243: 1).
681, 1989). The two nuclear transforming proteins, fos and jun, also show zipper domains, as do the gene products of the murine proto-oncogene, c-myc (Landschulz et al.,
Science, 240: 1759, 1988). The products of the nuclear oncogenes fos and jun contain a zipper domain that preferentially forms a heterodimer (O'Shea et al., Science, 245: 646, 1989; Tur.
ner et al., Science, 243: 1689, 1989). The zipper domain is required for the biological activity (DNA binding) of these proteins.

The fusogenic of several different viruses, including paramyxovirus, coronavirus, measles virus and many retroviruses.
Proteins also possess zipper domains (Buckland et al., Na
Ture, 338: 547, 1989; Britton, Nature,
353: 394, 1991; Delwart and Mosialos, A.
IDS Research and Human Retroviruses,
6: 703, 1990). The zipper domain of these fusion forming viral proteins is near the transmembrane region of the protein; it has been suggested that the zipper domain may contribute to the oligomeric structure of the fusion forming protein. Oligomerization of the fusogenic viral protein is associated with fusion holes (fusi).
on pore) formation (Spruce et al., Proc. Nat)
l. Acad. Sci. U. S. A. , 88: 3523, 1991).
. The zipper domain has also recently been reported to play a role in the oligomerization of heat shock transcription factors (Rabindran et al., Science).
e, 259: 230, 1993).

The zipper domain is a short, parallel coiled coil.
folded as a red coil) (O'Shea et al., Science)
Ce, 254: 539, 1991). The general structure of a parallel coiled coil is
“Knobs into holes (knobs-in, as proposed by Crick in 1953)
well characterized by "to-holes" packing (Cric
k, Acta Crystallogr. 6: 689, 1953). The dimer formed by the zipper domain is McLachlan and Stew.
ert, J .; Mol. Biol. , 98: 293, 1975, and is stabilized by a 7-residue repeat, designated (abcdefg) _n . Where the residues a and d are generally hydrophobic residues, d is leucine,
These line up on the same side of the helix. The oppositely charged residues usually occur at the g and e positions. Thus, in a parallel coiled coil formed from two helical zipper domains, the "knob" formed by the hydrophobic side chain of the first helix becomes a "hole" formed between the side chains of the second helix. Will be packed.

The residue at position d (often leucine) contributes to the enormous hydrophobic stabilization energy and is important for oligomer formation (Krystek et al., Int. J. et al.
Peptide Res. , 38: 229, 1991). Lovejoy et al., Science, 259: 1288, 1993 recently reported triple-stranded α-helix bundle synthesis in which the helix runs up-up-down. Their work confirms that the hydrophobic stabilization energy provides the major driving force for the formation of coiled-coils from helical monomers. These studies also show that electrostatic interactions contribute to the stoichiometry and shape of coiled coils. For a further discussion of the structure of leucine zippers, see Harbury et al., Science, 2
62: 1401, 1993.

An example of a leucine zipper domain suitable for producing soluble oligomeric proteins is described in PCT application WO 94/10308, and the leucine zipper domain from lung surfactant protein D (SPD) is described in Hoppe et al.
FEBS Letters, 344: 191, 1994, which are incorporated herein by reference. Use of the modified leucine zipper, which allows stable trimerization of heterologous proteins fused to the modified leucine zipper, is described in Fan
slow, et al., Semin. Immunol. , 6: 267-278, 1
994. A recombinant fusion protein containing a soluble polypeptide fused to a leucine zipper peptide is expressed in a suitable host cell and the soluble oligomers formed are recovered from the culture supernatant.

Certain leucine zipper moieties preferentially form trimers. One example is Hoppe et al., FEBS Letters, which is incorporated herein by reference in its entirety.
, 344: 191, 1994 and US Pat. No. 5,716,805, which are leucine zippers derived from lung surfactant protein D (SPD). This lung SPD-derived leucine zipper peptide has the amino acid sequence Pro
Asp Val Ala Ser Leu Arg Gln Gln Val
Glu Ala Leu Gln Gly Gln Val Gln His
Includes Leu Gln Ala Ala Phe Ser Gln Tyr.

Another example of a leucine zipper that promotes trimerization is US Pat. No. 5,716,8.
Amino acid sequence Arg Met Lys Gln I as described in No. 05.
le Glu Asp Lys Ile Glu Glu Ile Leu S
er Lys Ile Tyr His Ile Glu Asn Glu I
le Ala Arg Ile Lys Lys Leu Ile Gly G
It is a peptide containing lu Arg (SEQ ID NO: 21). In one alternative, an N-terminal Asp residue is added; in another, the peptide lacks the N-terminal Arg residue.

Fragments of the zipper peptides described above, which retain the property of promoting oligomerization, can also be used. Examples of such fragments include, but are not limited to, peptides lacking one or two N-terminal or C-terminal residues shown in the amino acid sequences above. Leucine zippers can be obtained from naturally occurring leucine zipper peptides, for example via conservative substitutions of one or more of the natural amino acid sequences such that the ability of the peptide to promote oligomerization is retained.

Other peptides derived from naturally occurring trimeric proteins can be used to prepare trimeric oligomers. Alternatively, synthetic peptides that promote oligomerization may be used. In certain embodiments, leucine residues in the leucine zipper moiety may be replaced by isoleucine residues. Such peptides containing isoleucine, which may be referred to as isoleucine zippers, are included herein by the term "leucine zippers".

Production of Polypeptides and Polypeptide Fragments of the Invention Expression, isolation and purification of the polypeptides and fragments of the invention may be accomplished by any suitable technique including, but not limited to: Including.

The isolated nucleic acid of the present invention recombinantly produces a polypeptide, and thus may be used in Kaufma.
n et al., Nucleic Acids Res. 19, 4485-4490
(1991); and Pouwels et al., Cloning Vectors:
It can be operably linked to expression control sequences such as the pDC412 or pDC314 vector, or the pMT2 or pED expression vector disclosed in A Laboratory Manual, Elsevier, New York, (1985). Many suitable expression control sequences are known in the art. General methods for expressing recombinant polypeptides are also known,
And R. Kaufman, Methods in Enzymology
185, 537-566 (1990). In the present specification, “
"Operably linked" means that the nucleic acid of the invention and the expression control sequence are encoded by the nucleic acid when the appropriate molecule (eg, polymerase) is present in the construct, vector, or cell. It is meant to be located in such a way that the peptide is expressed. In one aspect of the invention, at least one expression control sequence is operably linked to a nucleic acid of the invention in a recombinant host cell or its progeny, the nucleic acid and / or the expression control sequence being Has been introduced into the host cell either by, for example, transformation or transfection, or by any other suitable method. In another aspect of the invention, at least one expression control sequence is integrated into the genome of the recombinant host cell so that it is operably linked to the nucleic acid sequence encoding the polypeptide of the invention. . In a further aspect of the invention the at least one expression control sequence is in vitro.
In either ro or recombinant host cells, it is operably linked to the nucleic acid of the invention through the action of trans-acting factors such as transcription factors.

In addition, a sequence encoding a suitable signal peptide (natural or heterologous)
It may be incorporated into an expression vector. The choice of signal peptide or leader is
It may depend on factors such as the type of host cell in which the recombinant polypeptide is to be produced. For example, an example of a heterologous signal peptide that functions in a mammalian host cell is interleukin-7 (I) described in US Pat. No. 4,965,195.
L-7) signal sequence; Cosman et al., Nature 312: 768 (
1984) interleukin-2 receptor signal sequence; EP 3
67,566, interleukin-4 receptor signal peptide; type I interleukin-1 receptor signal peptide, described in US Pat. No. 4,968,607; and II, described in EP 460,846. Type interleukin-1 receptor signal peptide. Signal peptide (secretory leader)
May be fused in-frame to a nucleic acid sequence of the invention such that the DNA is first transcribed and the mRNA is translated into a fusion protein containing a signal peptide. A signal peptide that functions in the intended host cell promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide upon secretion of the polypeptide from the cell. One of skill in the art will also recognize that the position or positions at which the signal peptide is cleaved may be different than predicted by the computer program, and the type of host cell used to express the recombinant polypeptide. It will also be recognized that they may vary depending on factors such as. A polypeptide preparation can include a mixture of polypeptide molecules with different N-terminal amino acids resulting from cleavage of the signal peptide at more than one site.

Established methods for introducing DNA into mammalian cells are described by Kaufman.
, R.K. J. , Large Scale Mammalian Cell C
ultra, 1990, pp. 15-69. Commercially available reagents such as Lipofectamine lipid reagents (Gibco / BR
L) or Lipofectamine-Plus lipid reagents can be used to transfect cells using a further protocol (Fe
lgner et al., Proc. Natl. Acad. Sci. USA 8
4: 7413-7417, 1987). Further, using electroporation, Sambrook et al. (Molecular Cloning: A La).
laboratory Manual, 2nd edition. Vol. 1-3, Cold
Spring Harbor Laboratory Press, 1989.
It is possible to transfect mammalian cells using conventional methods, such as those in). Selection of stable transformants can be performed using methods known in the art, such as resistance to cytotoxic drugs. K
aufman et al., Meth. in Enzymology 185: 487.
-511, 1990 describe some selection strategies such as resistance to dihydrofolate reductase (DHFR). A suitable strain for DHFR selection could be the DHFR deficient CHO strain DX-B11 (Urlaub and Chasin,
Proc. Natl. Acad. Sci. USA 77: 4216-.
4220, 1980). The plasmid expressing the DHFR cDNA was DX
-B11 strain can be introduced and only cells containing the plasmid can be grown in a suitable selection medium. Other examples of selectable markers that can be incorporated into expression vectors include cDNAs that confer resistance to antibiotics such as G418 and hygromycin B. Cells harboring the vector can be selected based on resistance to these compounds.

Alternatively, the gene product can be obtained via homologous recombination, or "gene targeting" techniques. Such techniques use the introduction of exogenous transcriptional regulatory elements (such as the CMV promoter or the like) to induce expression of the endogenous nucleic acid sequence of interest, especially at predetermined sites on the genome (eg, See US Pat. No. 5,272,071). The location of integration into the host chromosome or genome can be readily determined by one of ordinary skill in the art, given the known location and sequence of the gene. In a preferred embodiment, the present invention also introduces exogenous transcriptional regulatory elements in combination with an amplifiable gene to regenerate increased amounts of the gene product without the need to isolate the gene sequence itself from the host cell. It is possible.

Several types of cells can act as suitable host cells for the expression of the polypeptide. Examples of mammalian host cell lines include, for example, monkey kidney cell C.
OS-7 strain (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, BHK (ATCC CRL 10) cell line, McMahan et al. (EMBO J
． 10: 2821, 1991). African green monkeys (A
frican green monkey) kidney cell line CV1 (ATCC CC
L70) -derived CV1 / EBNA cell line, human kidney 293 cell, human epithelial A431 cell, human Colo205 cell, other transformed primate cell line, normal diploid cell, cell derived from in vitro culture of primary tissue Strain, primary explant, HL-6
0, U937, HaK or Jurkat cells are included.

Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes. Commonly used promoter and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus. SV40
Using DNA sequences from the viral genome, such as the SV40 origin, early and late promoters, enhancers, splicing and polyadenylation sites,
It is possible to provide other genetic elements for the expression of the structural gene sequences in mammalian host cells. The viral early and late promoters are particularly useful because both are easily obtained from the viral genome as fragments that may also contain viral origins of replication (Fiers et al., Nature, 273: 113,
1978; and Kaufman et al., Meth. in Enzymolog
y, 1990). Hind III located at the SV40 viral origin of replication
Smaller or larger SV40 fragments may also be used provided they contain approximately 250 bp of sequence spanning the site to the Bgl I site.

Has been shown to improve expression of heterologous genes from mammalian expression vectors,
Additional regulatory sequences include CHO cell-derived increased expression sequence elements (EASE) (Mo
rris et al., Animal Cell Technology , pp. 52
9-534, 1997; and PCT application WO 97/25420) and the tripartite leader (TPL) and VA gene RNA from Adenovirus 2 (Gingeras et al., J. Biol. Chem.,
257: 13475-13491, 1982). Due to the internal ribosome entry site (IRES) sequence of viral origin, dicistronic m
Allows RNA to be translated efficiently (Oh et al., Current O.
pinion in Genetics and Development,
3: 295-300, 1993; and Ramesh et al., Nucleic.
Acids Research, 24: 2697-2700, 1996).
Expression of the heterologous cDNA as part of the bicistronic mRNA, followed by a selectable marker (eg, DHFR) gene, has been shown to improve host transfectability and expression of the heterologous cDNA ( Kaufman,
Meth. in Enzymology, 1990). Bicistronic m
A typical expression vector that uses RNA is Mosser et al., Biotech.
pTR-DC described in Niques, 22: 150-161, 1997.
/ GFP, and Morris et al., Animal Cell Technology.
ology, pp. 529-534, p2A5I described in 1997.

A useful high expression vector, pCAVNOT, is described in Mosley et al., Cell, 5
9: 335-348, 1989. Other expression vectors for use in mammalian host cells are described by Okayama et al., Mol. Cel
l. Biol. , 3: 280, 1983. A system useful for stable high level expression of mammalian cDNA in C127 murine mammary epithelial cells is described essentially by Cosman et al., Mol. Immu
nol. , 23: 935, 1986. A useful high expression vector, PMLSV N1 / N4, described in Cosman et al., Nature, 312: 768, 1984 is ATCC 3989.
Deposited as 0. Additional useful mammalian expression vectors are described in EP-A-0367566 and WO 91/18982, which are incorporated herein. As yet another alternative, the vector may be from a retrovirus.

Another useful expression vector, pFLAG ⁷ , may be used. The FLAG ⁷ technology is centered on fusing a low molecular weight (1 kD) hydrophilic FLAG ⁷ marker peptide to the N-terminus of the recombinant protein expressed by the pFLAG ⁷ expression vector. pDC311 is another specialized vector used to express proteins in CHO cells. pDC311 contains the gene of interest and the dihydrofolate reductase (DHFR) gene, an internal ribosome binding site for DHFR translation,
It is characterized by a dicistronic sequence that includes an enhanced expression sequence element (EASE), a human CMV promoter, a tripartite leader sequence, and a polyadenylation site.

Alternatively, it is possible to produce the polypeptide in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae.
isiae), Schizosaccharom
yces pombe), genus Kluyveromyces
Strains, Candida, or any yeast strain capable of expressing a heterologous polypeptide are included. Potentially suitable bacterial strains include E. coli (Es
cherichia coli), Bacillus subtilis
s), Salmonella typhimurium, or any bacterial strain capable of expressing a heterologous polypeptide. If the polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide produced therein in order to obtain a functional polypeptide, for example by phosphorylation or glycosylation at appropriate sites. There is a nature. Such covalent attachment can be accomplished using known chemical or enzymatic methods.
The polypeptides are also produced by operably linking the isolated nucleic acid of the invention to the appropriate regulatory sequences of one or more insect expression vectors and using an insect expression system. It is also possible. Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form (MaxBac® Kits), eg from Invitrogen, San Diego, Calif., And such methods are described in Summers and Smith,
Texas Agricultural Experiment Status
n Bulletin No. 1555 (1987) and Luckow and Summers, Bio / Technology 6:47 (1988), are known in the art. Insect cells capable of expressing the nucleic acids of the invention are "transformed" herein. Cell-free translation systems can also be used to produce polypeptides using RNA from the nucleic acid constructs disclosed herein. Preferably, a host cell that comprises an isolated nucleic acid of the invention operably linked to at least one expression control sequence is a "recombinant host cell."

The polypeptides of the present invention can be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant polypeptide.
The resulting expressed polypeptide can then be purified from such cultures (ie from media or cell extracts) using known purification methods such as gel filtration and ion exchange chromatography. Polypeptide purification also includes an agent that will bind the polypeptide to an affinity column; concanavalin A-
Agarose, heparin-toyopearl (R) or Cibacro
one or more column steps on an affinity resin such as m blue 3GA Sepharose®; one or more with hydrophobic interaction chromatography using a resin such as phenyl ether, butyl ether, or propyl ether. Alternatively, immunoaffinity chromatography may also be included. Alternatively, the polypeptides of the invention can also be expressed in a form that will facilitate purification. For example, it may be expressed as a fusion polypeptide such as that of maltose binding polypeptide (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion polypeptides are available from New Engla.
commercially available from nd BioLab (Beverly, Mass.), Pharmacia (Piscataway, NJ) and InVitrogen, respectively. Polypeptides can also be tagged with epitopes and subsequently using specific antibodies directed against such epitopes,
It is also possible to purify. One such epitope (FLAG®)
) Is commercially available from Kodak (New Haven, CT). Finally, using one or more reverse phase high performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, such as silica gel with pendant methyl or other aliphatic groups, It is possible to further purify the polypeptide. Several or all of the above purification steps in various combinations can also be used to provide a substantially homogeneous isolated recombinant polypeptide. A polypeptide thus purified is substantially free of other mammalian polypeptides and is defined according to the invention as an "isolated polypeptide"; such an isolated polypeptide of the invention includes FIL. -1 Theta polypeptides, fragments, variants, isolated antibodies that bind to binding partners and the like are included. The polypeptides of the present invention are also characterized as somatic or embryonic cells, comprising a nucleotide sequence encoding the polypeptide, as a product of a transgenic animal, for example the composition of transgenic bovine, goat, porcine or ovine milk. It can be expressed as an element.

It is also possible to affinity purify the expressed polypeptide using an affinity column containing the polypeptide binding polypeptide of the invention, such as a monoclonal antibody raised against the polypeptide of the invention. Is. These polypeptides can be prepared using conventional techniques, for example by dialysis in a high salt elution buffer and then for use in a lower salt buffer, or depending on the affinity matrix utilized. Can be removed from the affinity column by changing the pH or other components, or by competitively removing the affinity moiety with a naturally occurring substrate such as a polypeptide from the invention. Is. In this aspect of the invention, a polypeptide-binding polypeptide, such as an anti-polypeptide antibody of the invention or another polypeptide that may interact with the polypeptide of the invention, is provided on its surface. May be attached to a solid phase support, such as a column chromatography matrix or similar support suitable for identifying, separating, or purifying cells that express. Attachment of the polypeptide-binding polypeptides of the present invention to the solid-phase contact surface may be accomplished by any means, such as coating magnetic microspheres with these polypeptide-binding polypeptides and introducing them into a incubation vessel through a magnetic field. You may keep it. The suspension of cell mixture is contacted with a solid phase having such a polypeptide binding polypeptide thereon. Cells having the polypeptide of the invention on their surface are allowed to bind to the immobilized polypeptide-binding polypeptide, and then the unbound cells 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 include, for example, the use of enzymes. Such enzymes are preferably non-toxic and non-toxic to cells and are preferably directed to cleave cell surface binding partners. Alternatively, a cell mixture suspected of containing cells expressing the polypeptide of the invention may be first incubated with biotinylated polypeptide binding polypeptide of the invention. The resulting mixture is then passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides for binding of polypeptide-bound cells to the beads. The use of avidin coated beads is known in the art. Berenson et al. Cell. Biochem. ，
10D: 239 (1986). Washing of unbound components and release of bound cells is done using conventional methods.

Polypeptides can also be produced by known conventional chemical synthesis. Methods of constructing the polypeptides of the present invention by synthetic means are known to those of skill in the art. Synthetic constructed polypeptide sequences include polypeptide activity because they share primary, secondary or tertiary structure and / or conformational properties with the polypeptide,
It may have biological properties in common with it. Thus, they can be used as biologically active or immunological replacements of natural, purified polypeptides in the screening of therapeutic compounds and in immunological methods of antibody development. is there.

The desired purity depends on the intended use of the polypeptide. For example, a relatively high degree of purity is desirable when the polypeptide is administered in vivo. In such cases, the polypeptide will be subjected to SDS-polyacrylamide gel electrophoresis (SDS-P
Upon analysis by AGE), the polypeptide bands corresponding to other polypeptides are purified such that they are undetectable. One of skill in the art will recognize that multiple bands corresponding to the polypeptide may be visualized by SDS-PAGE due to different glycosylation, different post-translational processing, and the like. . Most preferably, the polypeptide of the invention is SDS-PAGE.
Upon analysis by <RTIgt; A., </ RTI> it is substantially homogeneously purified, as indicated by the single polypeptide band. Polypeptide bands can be visualized by silver staining, Coomassie blue staining, or (if the polypeptide is radiolabeled) by autoradiography.

Useful signal peptides include the natural signal peptide, or the natural signal peptide may be replaced with a heterologous signal peptide or leader sequence.
The choice of signal peptide or leader may depend on such factors as the type of host cell in which the recombinant polypeptide is being produced. For example, examples of heterologous signal peptides that function in mammalian host cells include US Pat. No. 4,965,195.
Signal sequence of interleukin-7 (IL-7) described in No. 7; Cosma
n et al., Nature, 312: 768, 1984, the signal sequence of the interleukin-2 receptor described in EP 367,566; US Pat. No. 4,968,607. Type I interleukin-1 receptor signal peptide described in EP; and EP 460.
, 846 type II interleukin-1 receptor signal peptide.

Ability to bind a purified polypeptide of the invention (including proteins, polypeptides, fragments, variants, oligomers, and other forms) to a binding partner in any suitable assay, including conventional binding assays. May be tested. As an example, the polypeptide may be labeled with a detectable reagent such as a radionuclide, a chromophore, an enzyme that catalyzes a colorimetric or fluorescence spectroscopic reaction, and the like. The labeled polypeptide is contacted with cells expressing the binding partner. The cells are then washed, unbound labeled polypeptide is removed, and the presence of label bound to the cells is determined by a suitable technique selected according to the nature of the label.

One example of a binding assay is as follows. A recombinant expression vector containing the binding partner cDNA is constructed using methods known in the art. 10 c
CV1-EBNA-1 cells in m ² plates are transfected with the recombinant expression vector. CV-1 / EBNA-1 cells (ATCC CRL 1047
8) constitutively expresses EBV nuclear antigen-1 driven from the CMV immediate early enhancer / promoter. CV1-EBNA-1 is derived from McMahan et al. (EMB
OJ. 10: 2821, 1991), and was derived from the African green monkey kidney cell line CV-1 (ATCC CCL 70).

Transfected cells are cultured for 24 hours and then the cells in each plate are split into 24-well plates. After culturing for another 48 hours, the transfected cells (about ⁴ × 10 ⁴ cells / well) are washed with BM-NFDM. BM
-NFDM is a binding medium (25 mg / m2) supplemented with 50 mg / ml skim milk powder.
l Bovine serum albumin, 2 mg / ml sodium azide, 20 mM Hepes
RPMI 1640) containing pH 7.2. The cells are then incubated with various concentrations of soluble polypeptide / Fc fusion protein, eg, prepared as described above, at 37 ° C. for 1 hour. The cells are then washed and incubated with a constant saturating concentration of ¹²⁵ I mouse anti-human IgG in binding medium for 1 hour at 37 ° C. with gentle agitation. After extensive washing, cells are released via trypsinization.

The mouse anti-human IgG used above was directed against the Fc region of human IgG, and the Jackson Immunoresearch Laborato
ries, Inc. , West Grove, Pennsylvania. The antibody is radioiodinated using the standard chloramine-T method. The antibody will bind to the Fc portion of any polypeptide / Fc protein that is bound to cells. In all assays, nonspecific binding of ¹²⁵ I antibody was assayed in the absence of Fc fusion protein / Fc, as well as in the presence of Fc fusion protein and a 200-fold molar excess of unlabeled mouse anti-human IgG antibody. Assay.

Cell-bound ¹²⁵ I antibody is quantified on a Packard Autogamma counter. Affinity calculation (Scatchard, Ann. NY Ac.
ad. Sci. , 51: 660, 1949) is produced on RS / 1 (BBN Software, Boston, MA) running on a Microvax computer.

Another type of suitable binding assay is the competitive binding assay. For example, the biological activity of the variant can be determined by assaying the ability of the variant to compete with the native protein for binding to its binding partner.

Competitive binding assays can be performed by conventional methodologies. Reagents that can be used in competitive binding assays include radiolabeled polypeptides of the invention and intact (int) expressing the binding partner (endogenous or recombinant).
act) cells are included. For example, a radiolabeled soluble FIL-1 theta fragment may be used to compete with a soluble FIL-1 theta variant for binding to the cell surface FIL-1 theta receptor. Instead of intact cells, a soluble binding partner / Fc fusion protein bound to the solid phase through the interaction of the Fc portion with Protein A or Protein G (on the solid phase) may be substituted. Chromatography columns containing Protein A and Protein G include Pharmaci
a Biotech, Inc. , Available from Piscataway, NJ, USA.

Another type of competitive binding assay utilizes radiolabeled soluble binding partners, eg, soluble FIL-1 theta receptor / Fc fusion proteins, and intact cells expressing the binding partners. Qualitative results can be obtained by competitive autoradiographic plate binding assays, while Scatchard plots (Scatchard, An.
n. N. Y. Acad. , Sci. 51: 660, 1949).

Use of FIL-1 Theta Nucleic Acids or Oligonucleotides As described above, in addition to being used to express a polypeptide, DNA, RNA
, Nucleic acids of the invention, including mRNA, and oligonucleotides thereof can be used as follows: As a probe to identify nucleic acids encoding IL-1 ligand and receptor family proteins; human chromosome 2 To map genes on human chromosome 2; to identify genes associated with particular diseases, syndromes, or other abnormalities associated with human chromosome 2; single-stranded sense or antisense oligos As a nucleotide, to inhibit expression of the polypeptide encoded by the FIL-1 theta gene; to help detect defective genes in an individual; and for gene therapy.

The nucleic acids described herein and in particular fragments, oligonucleotides of the nucleic acids of the invention find use as probes or primers. Suitable oligonucleotide primers include those with at least about 17 contiguous nucleotides. In other embodiments, suitable oligonucleotides include at least 30, or at least 60 contiguous nucleotides of a DNA sequence of the invention. Preferably the flanking oligonucleotides are SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 14.
Is a fragment of.

Since a homologue of SEQ ID NO: 1 or SEQ ID NO: 3 from another mammalian species, such as SEQ ID NO: 14, is contemplated herein, a probe based on human DNA of SEQ ID NO: 1 or SEQ ID NO: 3 is used. , It is possible to screen cDNA libraries from other mammalian species using conventional cross-species hybridization techniques.

Knowledge of the genetic code can be used in combination with the amino acid sequences shown above to prepare sets of degenerate oligonucleotides. Such oligonucleotides are used, for example, in the polymerase chain reaction (PCR) to isolate and amplify DNA fragments.
In), it is useful as a primer.

With human chromosome 2 containing the DNA of an IL-1 ligand family member using all or part of the nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 3, including oligonucleotides, using techniques known to those of skill in the art. , It is possible to identify that particular locus. Useful techniques include, but are not limited to, radiation hybrid mapping (high resolution), chromosome extension (
oligonucleotides as probes in a variety of known techniques, such as in situ hybridization (spread) (moderate resolution) and Southern blot hybridization (low resolution) to hybrid cell lines containing individual human chromosomes, Includes the use of arrays or portions.

For example, chromosomes can be mapped by radiation hybridization. Whitehead Institute of 93 Radiant Hybrid
e / MIT Center for Genome Research Gen
PCR is performed using the ebridge4 panel ( http: // www-ge nome.wi.mit.edu/ftp/distribution/hum).
an_STS_releases / july97 / rhmap / genebri
dge4. html). Primers are used that are within the putative exons of the gene of interest and amplify the product from human genomic DNA but not hamster genomic DNA. The PCR result is converted into a data vector, and the Whitehead / MIT radiation mapping site (http: // www-s) on the Internet.
eq. wi. mit. edu). The data is scored and provides chromosomal assignments and placements relative to known sequence tag site (STS) markers on the emission hybrid map. The following websites provide further information on radiative hybrid mapping: http: // www-genome. wi. mit. edu / ftp / dis
tribulation / human_STS_releases / july97 /
07-97. INTRO. html.

As shown below, the DNA of SEQ ID NO: 1 has been mapped to the 2q11-12 region of human chromosome 2 by high throughput shotgun sequencing. Human chromosome 2 is associated with specific diseases including, but not limited to, glaucoma, ectodermal dysplasia, insulin-dependent diabetes mellitus, cutaneous wrinkle syndrome, T-cell leukemia / lymphoma, and tibial muscular dystrophy. There is. Therefore, it is possible for those skilled in the art to analyze the abnormality associated with the gene mapped to the chromosome 2 using the nucleic acid of SEQ ID NO: 1, SEQ ID NO: 3 or a fragment thereof by using a known technique. This allows
It will be possible to distinguish abnormalities in which this marker has been rearranged or deleted. Furthermore, the nucleic acid fragment of SEQ ID NO: 1 or a fragment thereof can be used as a position marker to map other genes at unknown positions.

The DNA can be used to develop a treatment for any disorder mediated (directly or indirectly) by a deficiency or insufficient amount of the gene corresponding to the nucleic acid of the invention. The disclosure herein of native nucleotide sequences allows detection of defective genes and their replacement with normal genes. Defective genes
It can be detected in a tro diagnostic assay and by comparing the native nucleotide sequences disclosed herein with those of human-derived genes suspected of deficient in this gene.

Other useful fragments of the nucleic acids of the invention include target mRNA (sense) or DNA (
Single-stranded nucleic acid sequences (RNA or DN) capable of binding to antisense sequences
Antisense or sense oligonucleotides, including either A).

The antisense RNA and DNA molecules of the invention act to directly block the translation of mRNA by hybridizing to the target mRNA and interfering with polypeptide translation. The antisense approach is based on FIL-1 theta mRNA
With the design of oligonucleotides (either DNA or RNA) complementary to.
The antisense oligonucleotide will bind to the complementary target gene mRNA transcript and prevent translation. In accordance with the present invention, absolute complementarity is desirable but not required. A sequence “complementary” to a portion of a nucleic acid, as referred to herein, is capable of hybridizing to the nucleic acid and forming a stable duplex (or triplex, where appropriate). Means a sequence with sufficient complementarity to Thus, for double-stranded antisense nucleic acids, single strands of double-stranded DNA may be tested or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Preferred oligonucleotides are complementary to the 5'end of the message, eg up to and including the AUG start codon and the 5'untranslated region. However, 5'or 3'untranslated, non-coding regions of FIL-1 theta gene transcripts, or oligonucleotides complementary to the coding regions, can be treated with endogenous FIL-in the antisense approach.
It is useful for inhibiting the translation of 1 theta mRNA. The oligonucleotide complementary to the 5'untranslated region of the mRNA preferably comprises the complement of the AUG start codon. Antisense nucleic acids are at least 6 nucleotides in length, and are preferably oligonucleotides ranging in length from 6 to about 50 nucleotides. In particular aspects, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides. Oligonucleotides are single-stranded or double-stranded DNA or R
It may be NA or a chimeric mixture or a derivative or modified form thereof. The chimeric oligonucleotides, oligonucleosides, or mixed oligonucleotides / oligonucleosides of the invention may be of several different types. These include a first type in which the "gap" portion of the nucleotide is located between the 5'and 3 '"wing" portions of the nucleoside to which it is linked, and the "gap" portion is the 3'or of the oligomeric compound. A second "open end" species located at either the 5'end is included (see, eg, US Pat. No. 5,985,664).
The first class of oligonucleotides is also known in the art as "gapmers" or gapped oligonucleotides. The second type of oligonucleotide is also known in the art as a "hemimer" or "wingmer." Oligonucleotides can be modified with base moieties, sugar moieties, or phosphate backbones to improve, for example, molecular stability, hybridization, and the like. The oligonucleotide may be a peptide (eg, a host cell receptor in vivo).
vo) or agents that promote transport across cell membranes (eg Le
tsinger et al., 1989, Proc Natl Acad Sci U.
． S. A. 86: 6553-6556; Lemaitre et al., 1987 ,.
Proc Natl Acad Sci 84: 648-652; PCT Publication No. WO 88/09810), or a hybridization-inducing cleaving or intercalating agent (eg, Zo.
n, 1988, Pharm. Res. 5: 539-549) and other accompanying groups. The antisense molecule must deliver the FIL-1 theta transcript to cells expressing it in vivo. Several methods have been developed to deliver antisense DNA or RNA; for example, antisense molecules may be injected directly into a tissue or cell-derived site, or may be targeted to a desired cell. Designed modified antisense molecules (eg, antisense linked to a peptide or antibody that specifically binds to a receptor or antigen expressed on the surface of target cells) may be administered systemically. But,
It is often difficult to achieve intracellular concentrations of antisense sufficient to suppress translation of endogenous mRNA. Therefore, the preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. The use of such a construct to transfect target cells in a patient results in the transcription of a sufficient amount of single-stranded RNA that will form complementary base pairs with the endogenous FIL-1 theta gene transcript. , And thereby would prevent translation of FIL-1 theta mRNA. For example, the vector can be introduced in vivo such that the vector is taken up by cells and directs the transcription of antisense RNA.
Such a vector may remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA techniques standard in the art. The vector may be a plasmid, vector, or other known in the art, used for replication and expression in mammalian cells.

Ribozyme molecules designed to catalytically cleave FIL-1 theta mRNA transcripts can also be used to prevent translation of FIL-1 theta mRNA and expression of FIL-1 theta polypeptides ( For example, PCT International Publication WO90
/ 11364, published October 4, 1990; see US Pat. No. 5,824,519). Ribozymes that can be used in the present invention include hammerhead ribozymes (Haseloff and Gerlach, 1988, Natu.
Re, 334: 585-591), Tetrahymena thermophila (Tetra).
hymena Thermophila naturally exists (IVS, or L-
19 IVS RNA), and Thomas Chch and coworkers (International Patent Application No. WO 88/04300; Been and Cec).
h, 1986, Cell, 47: 207-216), including RNA endoribonucleases (herein after referred to as "Cech-type ribozymes"). As in the antisense approach, ribozymes may be composed of modified oligonucleotides (eg, for improved stability, targeting, etc.) and are expressed in vivo in cells expressing the FIL-1 theta polypeptide. Must be transported. A preferred method of delivery is to have strong constitutive pol III or pol II such that the transfected cells will produce sufficient amounts of ribozymes, disrupt the endogenous FIL-1 theta message and inhibit translation. It involves using a ribozyme "coding" DNA construct under the control of a promoter. Ribozymes, unlike antisense molecules, are catalytic and therefore require lower intracellular concentrations to be effective.

Alternatively, endogenous FIL-1 theta gene expression targets a deoxyribonucleotide sequence complementary to the regulatory region of the target gene (ie, target gene promoter and / or enhancer) to direct transcription of the target FIL-1 theta gene. It can be reduced by forming an interfering triple helix structure (generally Helene, 1991, Anticancer Drug De).
s. , 6 (6), 569-584; Helene et al., 1992, Ann.
． N. Y. Acad. Sci. , 660, 27-36; and Mah.
er, 1992, Bioassays 14 (12), 807-815).

Antisense RNA and DNA, ribozymes, and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides known in the art, such as, for example, solid phase phosphoramidite chemical synthesis. Oligonucleotides may be prepared according to standard methods known in the art, for example automated D
Synthesis can be performed by using an NA synthesizer (such as one commercially available from Biosearch, Applied Biosystems, etc.). For example, phosphorothioate oligonucleotides are described by Stein et al., 19
88, Nucl. Acids Res. By the method of 16: 3209,
It is possible to synthesize. Methylphosphonate oligonucleotides are prepared using controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl.
Acad. Sci. U. S. A. 85: 7448-7451). Alternatively, the RNA molecule is an antisense R
It can be produced by in vitro and in vivo transcription of the DNA sequence encoding the NA molecule. Such DNA sequences are T7 or SP
It is possible to incorporate into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the 6 polymerase promoter. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Endogenous target gene expression can also be achieved by inactivating or “knocking out” the target gene or its promoter using targeted homologous recombination.
It can also be reduced (eg Smithies et al., 1985, Na.
317, 230-234; Thomas and Capecchi,
1987, Cell 51, 503-512; Thompson et al., 19
89, Cell 5, 313-321). Target gene in
For transfecting cells expressing in vivo, for example, a mutant non-functional target gene (or not completely related) flanked by DNA homologous to an endogenous target gene (either the coding or regulatory region of the target gene) DNA sequence)
Can be used with or without a selectable marker and / or a negative selectable marker. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. Such an approach is used in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal progeny with inactive target genes (eg Thomas and Capecchi, 19
87, and Thompson, 1989, supra), also "RN
A interference "(" RNAi ") technology (Grishok A, Tabara H, and Mello CC, 2000, Genetic requirements for inheritance of RNAi in nematodes, Science 287 (5462): 2494-2497).
, Or transgene transfer (Dernburg AF, Zalevsky J
, Coliacovo MP, and Villeneuve AM, 20.
00, transgene-mediated co-suppression in the nematode germline, Genes Dev.
14 (13): 1578-1583), which is particularly suitable in model organisms such as Caenorhabditis elegans that inhibit the expression of specific target genes. However, this approach is suitable for use in humans if the recombinant DNA construct is directly administered in vivo with a suitable vector, such as a viral vector, or targeted to the required site. , Can be adapted.

Organisms having enhanced, decreased, or modified expression of the gene (s) corresponding to the nucleic acid sequences disclosed herein are provided. The desired change in gene expression binds to and / or binds to the mRNA transcribed from the gene.
Can be achieved through the use of antisense nucleic acids or ribozymes (Albert and Morris, 1994, Trends
Pharmacol. Sci. 15 (7): 250-254; Lavaro.
sky et al., 1997, Biochem. Mol. Med. 62 (1)
11-22; and Hampel, 1998, Prog. Nuclei
c Acid Res. Mol. Biol. 58: 1-39). Preferably, the gene construct stably maintained in the transformed cell and its progeny, produces a polymorphism of the gene (s) corresponding to the nucleic acid sequences disclosed herein produced by transforming the cell. Transgenic animals having a copy are provided. Transgenic animals having modified gene regulatory regions that increase or decrease gene expression levels or alter temporal or spatial patterns of gene expression are also provided (EP 0 649 464 B1. Please refer). Furthermore, the gene (s) corresponding to the nucleic acid sequences disclosed herein may be deleted through the insertion of an exogenous sequence into the corresponding gene (s) or in the deletion of all or part of the corresponding gene (s). An organism is provided which has been partially or completely inactivated through. Partial or complete gene inactivation is achieved through insertion, preferably by incorrect excision of the transposable element (Plasterk).
, 1992, Bioessays 14 (9): 629-633; Zwaa.
l et al., 1993, Proc. Natl. Acad. Sci. USA
90 (16): 7431-7435; Clark et al., 1994, Proc.
． Natl. Acad. Sci. USA 91 (2): 719-722.
) Or through homologous recombination and preferably can be detected by a positive / negative gene selection strategy (Mansour et al., 1988, N).
ature 336: 348-352; U.S. Pat. No. 5,464,764; 5th
, 487,992; 5,627,059; 5,631,153; 5
, 614, 396; 5,616, 491; and 5,679, 523). These organisms with altered gene expression are preferably eukaryotic, and more preferably mammalian. Such organisms are assayed for the development of non-human models for the study of disorders involving the corresponding gene (s), and for the identification of molecules that interact with the polypeptide product (s) of the corresponding gene (s). It is useful for system development. Uses of FIL-1 Theta Polypeptides and Fragments The polypeptides and polypeptide fragments described herein find many uses, including but not limited to: purification of proteins and their activity. Measurements Delivery agents Therapeutic and research reagents Peptide fragmentation controls Identification of unknown proteins Identification of compounds that alter activity Preparation of antibodies.

The polypeptides of the present invention each find use as protein purification reagents. The polypeptide can be attached to a solid support component and used to purify binding partner proteins by affinity chromatography. In a particular embodiment, the polypeptide (which can be of any type described herein capable of binding a binding partner) is attached to a solid support by conventional methods. As one example, chromatographic columns are available that contain functional groups that will react with functional groups on the amino acid side chains of proteins (Pharma).
cia Biotech, Inc. , Piscataway, NJ).
Alternatively, the polypeptide / Fc protein (as described above) is attached to a Protein A or Protein G containing chromatography column through interaction with the Fc portion.

The polypeptides also find use in purifying or identifying cells that express binding partners on the cell surface. The polypeptide is attached to a solid phase such as a column chromatography matrix or similar suitable support. For example, magnetic microspheres may be coated with the polypeptide and held in the incubation vessel through a magnetic field. A suspension of cell mixture containing binding partner expressing cells is contacted with a solid phase having the polypeptide thereon. Cells expressing the binding partner on the cell surface are allowed to bind to the immobilized polypeptide and then unbound cells are washed away.

Alternatively, the polypeptide may be conjugated to a detectable moiety and then incubated with the cells to be tested for binding partner expression. After incubation, unbound labeled material is removed and the presence or absence of detectable moieties on the cells is determined.

In yet another alternative, a cell mixture suspected of containing binding partner expressing cells is incubated with a biotinylated polypeptide. Incubation time is typically at least 1 hour continuous to ensure sufficient binding. The resulting mixture is then passed through a column loaded with avidin-coated beads, whereby the high affinity of biotin for avidin provides the desired cell binding to the beads. A method using avidin-coated beads is known (Beren)
Son et al. Cell. Biochem. , 10D: 239, 19
86). Washing to remove unbound components and liberation of bound cells is done using conventional methods.

The polypeptides of the invention also find use in measuring the biological activity of a binding partner protein with respect to its binding affinity. Thus, the polypeptides can be used, for example, by those conducting "quality assurance" studies to monitor the shelf life and stability of proteins under different conditions. Therefore,
The polypeptides described herein measure the biological activity of binding partner proteins stored at different temperatures or produced in different cell types,
Useful for binding affinity studies. The polypeptides of the present invention can also be used to determine if biological activity is retained after modification of the binding partner protein (eg, chemical modification, truncation, mutation, etc.). The binding affinity of the modified binding partner protein is compared to that of the unmodified binding partner protein to detect any adverse effects of the modification on the biological activity of the binding partner. Thus, for example, the biological activity of the binding partner protein can be confirmed before use in research studies.

The polypeptides described herein also find use as carriers in methods of delivering a diagnostic or therapeutic agent attached thereto to cells bearing the binding partner of the polypeptide. Therefore, using the polypeptide, in v
In vitro or in vivo methods, the diagnostic or therapeutic agent can be delivered to such cells (or other cell types found to express binding partners on the cell surface).

Detectable agents (diagnostic agents) and therapeutic agents that can be attached to a polypeptide include, but are not limited to, toxins, other cytotoxic agents, agents, radionuclides, chromophores, colorimetric or Enzymes that catalyze fluorescence spectroscopic reactions, and their equivalents, are included with the particular agent selected according to the intended application. Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonas aerug
inosa) exotoxin A, ribosomal inactivating proteins, mycotoxins such as trichocecenes, and their derivatives and fragments (eg single chain). Radionuclides suitable for diagnostic use include, but are not limited to, ¹²³ I, ¹³¹ I, ^99m
There are Tc, ¹¹¹ In, and ⁷⁶ Br. Examples of radionuclides suitable for therapeutic use include ^{1 31} I, ²¹¹ At, ⁷⁷ Br, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Pb, ²¹² Bi, ¹⁰⁹ Pd, ⁶⁴ C.
u and ⁶⁷ Cu.

Such agents may be attached to the polypeptide by any suitable conventional method. Polypeptides include, for example, a functional group on an amino acid side chain that is capable of reacting with a functional group on the desired agent to form a covalent bond. Alternatively, the protein or agent may be derivatized to create or attach desired reactive functional groups. For derivatization, a bifunctional coupling reagent (Pierce Chemical Com) that can be used to attach a variety of molecules to proteins.
pany, Rockford, IL). Several techniques are known for radiolabeling proteins. A radionuclide metal can be attached to the polypeptide by using, for example, a suitable bifunctional chelating agent.

A conjugate (preferably covalently linked) containing the polypeptide and a suitable diagnostic or therapeutic agent is thus prepared. The conjugate is administered or otherwise used in an amount suitable for the particular application.

The polypeptides of the present invention and polypeptide variants of the present invention may be therapeutic agents for deficiencies of the polypeptide or disorders mediated (directly or indirectly) by insufficient amounts. These polypeptides can be administered to mammals suffering from such disorders.

The polypeptides can also be used to inhibit the biological activity of binding partners in in vitro or in vivo methods. For example, using the purified FIL-1 theta polypeptide, fragment or variant of SEQ ID NO: 2, or the polypeptide fragment or variant of SEQ ID NO: 4, or the fragment of SEQ ID NO: 15,
It is possible to inhibit the binding of endogenous FIL-1 theta to its cell surface receptors. The biological effects resulting from the binding of FIL-1 theta to endogenous receptors or receptors are inhibited by blocking the binding and subsequent signaling.

The polypeptides of the present invention can be administered to a mammal to treat disorders mediated by one or more binding partners of the polypeptide. For these binding partner-mediated disorders, depending on the binding partner (directly or indirectly)
Includes abnormalities that are caused or exacerbated.

The compositions of the invention may include any type of polypeptide described herein, including naturally occurring proteins, variants, derivatives, oligomers, and biologically active fragments. In certain embodiments, the composition comprises a soluble polypeptide or oligomer, including a soluble polypeptide of the invention.

For the purposes of this disclosure, the terms “disease”, “disease”, “medical disorder”, “abnormal condition” and the like are used interchangeably with the term “medical disorder”. . As used herein, the terms “treat”, “treating”, and “treatment” include curative, protective (eg, prophylactic) and palliative or ameliorative treatment. For such therapeutic use, FIL-1 theta polypeptides and fragments,
A FIL-1 theta nucleic acid encoding a FIL-1 theta family polypeptide,
And / or agonists or antagonists of FIL-1 theta polypeptides such as antibodies can be administered to the patient in need by known means. The compositions of the invention may include any type of polypeptide described herein, including naturally occurring polypeptides, variants, derivatives, oligomers, and biologically active fragments. In a particular embodiment, the composition comprises an oligomer comprising a soluble polypeptide or a soluble FIL-1 theta polypeptide.

In practicing the treatment or use method of the present invention, a therapeutically effective amount of the therapeutic agent of the present invention is
It is administered to a patient having the disorder to be treated, preferably to treat or ameliorate a disease associated with the activity of FIL-1 theta or IL-1 family polypeptides. "Therapeutic agents" include, without limitation, FIL-1 theta polypeptides, fragments, and variants; nucleic acids encoding FIL-1 theta polypeptides, fragments, and variants; FIL-1 theta polypeptides such as antibodies. Agonists or antagonists of; FIL-1 theta polypeptide binding partners; FIL-1 theta polypeptides, fragments, variants, and complexes formed from binding partners and the like. As used herein, the term “therapeutically effective amount” means a significant patient benefit, ie, the treatment, cure, prevention or amelioration of a corresponding medical condition, or an increase in the rate of treatment, cure, prevention or amelioration of such conditions. Is meant to indicate the total amount of each therapeutic agent or other active component of the pharmaceutical composition or method sufficient to indicate When applied to an individual therapeutic agent or active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to the combined amounts of the components that result in the therapeutic effect either in combination, administered sequentially or simultaneously. The phrase "administering a therapeutically effective amount" of a therapeutic agent, as used herein, refers to the patient's induction of an improvement, and preferably a sustained improvement, in at least one index that reflects the severity of the disorder. Means being treated with the therapeutic agent in a sufficient amount and for a period of time. An improvement is considered "persistent" if the patient exhibits improvement on at least two occasions, one week or more apart. The degree of improvement is determined based on signs or symptoms, and the determination can also use questionnaires conducted on the patient, such as quality of life questionnaires. A variety of indicators that reflect the degree of illness of a patient can be evaluated to determine if the amount and duration of treatment is sufficient. The baseline value for the selected indicator or indicators is confirmed by examining the patient prior to administration of the first dose of therapeutic agent.
Preferably, the baseline test is done within about 60 days of the first dose administration. When a therapeutic agent is administered to treat an acute condition, the first dose is administered on-site, as soon as possible after the injury has occurred. Improvement is induced by administering a therapeutic agent, such as a FIL-1 theta polypeptide or antagonist, until the patient demonstrates an improvement over a selected index or indices over baseline.
When treating a chronic condition, this degree of improvement may be at least 1 month or longer, eg
It is obtained by repeated administration of this drug for a period of 1, 2 or 3 months or longer, or indefinitely. A period of 1 to 6 weeks, or even a single dose is often sufficient to treat an acute condition. For injuries or acute abnormalities, a single dose may be sufficient. The post-treatment patient's degree of illness may appear to improve according to one or more indicators, but treatment is continued indefinitely at the same level or at a reduced dose or frequency. May be. Once the treatment is reduced or interrupted, it may be resumed at its original level later if symptoms reappear.

Dose determination . Those of ordinary skill in the art will appreciate that appropriate dosages will vary depending on such factors as the nature and severity of the disorder being treated, the weight, age, general condition of the patient, and previous illness and / or treatment, and the route of administration. You will recognize that. Preliminary doses can be determined according to animal studies, and dosing for human administration is done according to art-recognized practices, such as standard dosing studies. For example, a therapeutically effective dose can be estimated initially from cell culture assays. The dosage will depend on the particular activity of the compound, and can be readily determined by routine experimentation. The dose is
Minimizes toxicity while including an IC50 (ie, the concentration of test compound that achieves half maximal inhibition of symptoms) as determined in cell culture in an animal model,
It can be formulated to achieve a circulating plasma concentration range. Such information can be used to more accurately determine useful doses in humans.
Ultimately, the attending physician will decide the amount of polypeptide of the invention with which to treat each individual patient. Initially, the attending physician will administer low doses of the polypeptides of the present invention and observe the patient's response. It is possible to administer higher doses of the polypeptide of the invention until the optimal therapeutic effect for the patient is obtained,
And at that point, do not increase the dosage further. A variety of pharmaceutical compositions used to practice the methods of the invention have a composition of the invention of about 0.0
It is contemplated that it should contain from 1 ng to about 100 mg (preferably about 0.1 ng to about 10 mg, more preferably about 0.1 microgram to about 1 mg). In one aspect of the invention, a FIL-1 theta polypeptide or antagonist is administered weekly to treat the various medical disorders disclosed herein, and in another aspect at least twice weekly. Administered, and in another embodiment, at least three times a week. When injected, an effective dose of FI per adult dose
The L-1 theta polypeptide or antagonist is in the range of 1-20 mg / m ² , and preferably about 5-12 mg / m ² . Alternatively, a flat dose may be administered, which amount may range from 5-100 mg / dose. A typical dose range for a flat dose administered by subcutaneous injection is 5-25 mg / dose,
25-50 mg / dose and 50-100 mg / dose. In one aspect of the invention, administering the various indications described below, an injectable preparation comprising FIL-1 theta polypeptide or antagonist at 25 mg / dose, or at 50 mg per dose. Treat by. 25 mg or 5
The 0 mg dose can be given repeatedly, especially for chronic disorders. If a route of administration other than injection is used, the dose is appropriately adjusted in accordance with standard medical practice. Often, improvement in patient status is achieved by administering a dose of about 25 mg of a FIL-1 theta polypeptide or antagonist 1 to 3 times per week for a period of at least 3 weeks, or at least 3 weeks once or twice per week. , FI at a dose of 50 mg
By injecting an L-1 theta polypeptide or antagonist, longer duration of treatment may be required to induce the desired degree of improvement, which would be obtained. In incurable chronic anomalies, measures are taken by the patient's physician.
If you think you need these, adjust the dose and frequency,
You may continue indefinitely. The above doses are examples for adult patients, humans 18 years of age or older. For pediatric patients (age 4-17 years), appropriate measures include a maximum dose of 25 mg of a FIL-1 theta polypeptide or antagonist, administered by subcutaneous injection once or more times per week.
With subcutaneous injection of 4 mg / kg. When an antibody to FIL-1 theta polypeptide is used as a FIL-1 theta polypeptide antagonist, a preferred dose range is 0.1 to 20 mg / kg, and more preferably 1-10 mg / k.
It is g. Another preferred dose range for anti-FIL-1 theta polypeptide antibodies is 0
． 75 to 7.5 mg / kg body weight. Humanized antibodies, ie antibodies in which only the antigen binding portion of the antibody molecule is derived from a non-human source, are preferred. Such antibodies can be injected or administered intravenously.

Prescription . An effective amount of a FIL-1 theta polypeptide of the present invention, which may be derived from any source, including, but not limited to recombinant and non-recombinant sources, is administered in a physiologically acceptable diluent, carrier, or excipient. Provided herein are compositions that include in combination with other components such as excipients. The term "pharmaceutically acceptable" means a non-toxic ingredient that does not interfere with the effectiveness of the biological activity of the active ingredient (s). Formulations suitable for administration include sterile aqueous and non-aqueous injection solutions that may include anti-oxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the recipient; Aqueous and non-aqueous sterile suspensions that may include thickening agents are included. The polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. These include pharmaceutically acceptable diluents (eg, saline, Tris-HCl, acetic acid, and phosphate buffers) as the sole active ingredient, or together with other known active ingredients suitable for the indicated indications. Solutions), preservatives (eg, thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and / or carriers and / or carriers. Suitable formulations for pharmaceutical compositions include Remington's Pharma.
scientific Sciences, 16th Edition, 1980, Mack
Included are those described in Publishing Company, Easton, PA. In addition, such compositions include polyethylene glycol (
PEG), complexed with metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran,
Alternatively, it may be incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable lipids for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, and the like. Preparation of such liposome formulations is described, for example, in US Pat. No. 4,235,871; US Pat. No. 4,501,728; US Pat.
37,028; and, as disclosed in U.S. Pat. No. 4,737,323,
It is within the technical level of the technical field. Such compositions will affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate, and will therefore be selected according to the intended application and thus the carrier properties. Will depend on the route of administration chosen. In one preferred embodiment of the invention, a sustained release form of FIL-1 theta polypeptide is used. Suitable sustained release formulations for use in the disclosed methods include, but are not limited to, slow dissolving biocompatible polymers (US Pat. No. 6,036).
, Alginic acid microparticles as described in US Pat. No. 5,968,961), mixed with such polymers, including topically applied hydrogels, and / or biocompatible semipermeable. Included in the implant.

Combination of therapeutic compounds . The FIL-1 theta polypeptides of the invention can be active in multimers (eg, heterodimers or homodimers), or in complex with itself or other polypeptides. As a result, pharmaceutical compositions of the invention may include the polypeptides of the invention in such multimeric or complexed forms. The pharmaceutical composition of the invention may be in the form of a complex of the polypeptide (s) of the invention with a polypeptide or peptide antigen. The invention further comprises administering the FIL-1 theta polypeptide or antagonist in combination with the FIL-1 theta polypeptide or antagonist, concurrently with one or more other agents administered to the same patient. Each drug is administered according to the procedure appropriate for the drug. "Co-administration" refers to the alternate administration of drugs, with the simultaneous or sequential treatment of the components of the combination, or the chronic administration of one component and the intermittent administration of one or more of the other. Including measures to The components may be administered in the same or separate compositions and by the same or different routes of administration. Examples of components that may be included in the pharmaceutical composition of the present invention are:
Cytokines, lymphokines, or M-CSF, GM-CSF, TNF,
IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL
-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-1
4, IL-15, IL-17, IL-18, IFN, TNF0, TNF1, TN
Other hematopoietic factors such as F2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further comprise other agents which either enhance the activity of the polypeptide or complement its activity or use during treatment. Such additional factors and / or agents may be included in the pharmaceutical composition to produce a synergistic effect with the polypeptides of the invention or to minimize side effects. Conversely, a FIL-1 theta polypeptide or antagonist of the invention may be included in the formulation of certain cytokines, lymphokines, other hematopoietic factors, thrombolytic or antithrombotic factors, or anti-inflammatory agents to induce cytokines, lymphokines, other It is possible to minimize the side effects of hematopoietic factors, thrombolytic or antithrombotic factors, or anti-inflammatory agents. Further examples of agents to be co-administered include, but are not limited to, antiviral agents, antibiotics, analgesics, corticosteroids, inflammatory cytokine antagonists, non-steroidal anti-inflammatory agents. , Pentoxifylline, thalidomide, and azathioprine,
Disease-modifying antirheumatic drugs (DMARDs) such as cyclophosphamide, cyclosporine, hydroxychloroquine sulfate, methotrexate, leflunomide, minocycline, penicillamine, sulfasalazine, and gold compounds such as oral gold, sodium gold thiomalate, and gold thioglucose ) Is included. Furthermore, F
The IL-1 theta polypeptide or antagonist comprises a second FI, comprising an antibody to the FIL-1 theta polypeptide or a peptide derived from the FIL-1 theta polypeptide that acts as a competitive inhibitor of the native FIL-1 theta polypeptide.
It is possible to combine with L-1 theta polypeptides / antagonists.

Route of administration . A FI comprising a composition comprising a nucleic acid using any effective route of administration
The L-1 theta polypeptide or an antagonist thereof may be administered therapeutically. Parenteral administration includes injection via the intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes, for example by bolus injection or by continuous infusion, and also Also included is topical administration, eg, at the site of the disease or injury.
Other suitable means of administration include sustained release from implants; aerosol inhalation and / or insufflation; eye drops; vaginal or rectal suppositories; buccal preparations; oral preparations including pills, syrups, lozenges or chewing gums; And lotions, gels, sprays,
Topical preparations such as ointments or other suitable techniques are included. Alternatively, administering a polypeptide-like FIL-1 theta polypeptide or antagonist by transplanting cultured cells that express the polypeptide, for example, by transplanting cells that express the FIL-1 theta polypeptide or antagonist. Is possible. The cells can also be cultured ex vivo in the presence of the polypeptide of the invention to proliferate or produce a desired effect on or a desired activity on such cells. The treated cells can then be introduced in vivo for therapeutic purposes. In another embodiment, the patient's own cells are induced to produce a FIL-1 theta polypeptide or antagonist by in vivo or ex vivo transfection with DNA encoding the FIL-1 theta polypeptide or antagonist. To do. This DNA may be, for example, naked DNA encoding a FIL-1 theta polypeptide or antagonist or liposome-encapsulated DN.
It can be introduced into the cells of the patient by injecting A or by other means of transfection. The nucleic acids of the invention can also be administered to a patient by other known methods for the introduction of nucleic acids into cells or organisms, without limitation in the form of viral vectors or naked DNA. Is. F
When the IL-1 theta polypeptide or antagonist is administered in combination with one or more other biologically active compounds, they can be administered by the same or different routes, and at the same time, Alternatively, it can be administered separately or sequentially.

Oral administration . Upon oral administration of a therapeutically effective amount of a polypeptide of the invention, the polypeptide of the invention will be in the form of tablets, capsules, powders, solutions or elixirs. When administered in tablet form, the pharmaceutical composition of the present invention may further comprise a solid carrier such as gelatin or an adjuvant. Tablets, capsules, and powders contain about 5 to 95% of the polypeptide of the invention, and preferably about 2% of the polypeptide of the invention.
5 to 90% included. When administered in liquid form, liquid carriers such as water, petroleum, oils of animal or vegetable origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further include physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition comprises about 0.5 to 90% by weight of the polypeptide of the invention, and preferably about 1% of the polypeptide of the invention.
From 50% included. Upon intravenous, cutaneous or subcutaneous injection of a therapeutically effective amount of a polypeptide of the invention, the polypeptide of the invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable polypeptide solutions is within the skill of the art, taking into account pH, isotonicity, stability, and the like. Preferred intravenous, cutaneous, or subcutaneous injection pharmaceutical compositions are:
In addition to the polypeptide of the present invention, it should include sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art. . The pharmaceutical composition of the present invention may also include stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. The duration of intravenous therapy with the pharmaceutical composition of the present invention will vary depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that each application period of the polypeptide of the invention will be within the range of 12 to 24 hours of continuous intravenous administration. Ultimately, the attending physician will decide on the appropriate duration of intravenous therapy with the pharmaceutical composition of the present invention.

Bone and tissue administration . With respect to the compositions of the present invention useful for bone, cartilage, tendon or ligament disorders, the therapy involves administering the composition locally, systemically or locally as an implant or device. When administered, the therapeutic composition for use in the present invention is, of course, in a pyrogen-free, physiologically acceptable form. In addition, the composition may be encapsulated or injected in a viscous form, preferably for delivery to the site of bone, cartilage or tissue injury. Topical administration may be suitable for wound healing and tissue repair. As mentioned above, a therapeutically useful agent other than the polypeptide of the invention, which may also optionally be included in the composition, may alternatively or additionally, simultaneously or sequentially with the composition of the method of the invention, May be administered. Preferably bone and /
Alternatively, for cartilage formation, the composition is capable of delivering the polypeptide-containing composition to the site of bone and / or cartilage injury, providing structure for developing bone and cartilage, and optimally , Will contain a matrix that can be absorbed by the body. Such matrices may be formed of materials currently used for other implanted medical applications. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interfacial properties. The particular application of the composition will define the appropriate formulation. The potential matrix for the composition may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydride. . Other potential materials are those that are biodegradable and biologically well defined, such as bone or skin collagen. The additional matrix is composed of pure polypeptide or extracellular matrix components. Other potential matrices are non-biodegradable, chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. The matrix may be composed of any combination of materials of the types described above, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics can be modified in composition, such as in calcium-aluminate-phosphoric acid, and processed to modify pore size, particle size, particle shape, and biodegradability. . Presently preferred are 50:50 (molar weight) copolymers of lactic acid and glycolic acid in the form of porous particles having diameters in the range of 150 to 800 microns. In some applications, it may be useful to utilize a sequestering agent such as carboxymethyl cellulose or autologous clots to prevent the polypeptide composition from separating from the matrix. A preferred family of sequestrants is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose, most preferably carboxy. It is a cationic salt of methyl cellulose (CMC). Other preferred sequestrants include hyaluronic acid, sodium alginate, poly (ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymers and poly (vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20% by weight, preferably 1-10% by weight, based on the total formulation weight, which provides for the release of the polypeptide from the polymer matrix. So much as to prevent and provide adequate handling of the composition, but not enough to prevent progenitor cells from infiltrating the matrix, thereby providing the polypeptide with the opportunity to assist the osteogenic activity of the progenitor cells. Equivalent to no quantity. In a further composition, the polypeptides of the invention may be combined with other agents that are beneficial in the treatment of the bone and / or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-alpha and TGF-beta), and insulin-like growth factor (IGF). included. Therapeutic compositions are also currently valuable for veterinary applications. In addition to humans, domesticated animals and Thoroughbred horses in particular are desirable patients for such treatment with the polypeptides of the invention. The dosage regimen of the polypeptide-containing pharmaceutical composition to be used for tissue regeneration depends on various factors such as the weight of the tissue desired to be formed, the site of injury, the state of injured tissue, the wound to be treated by the attending physician. Size, type of injured tissue (eg bone), age, sex and diet of the patient, severity of any infection, time of administration and other clinical factors will be considered. The dosage will depend on the type of matrix used for reconstitution and the inclusion of other polypeptides in the pharmaceutical composition.
It will be diverse. For example, addition of other known growth factors such as IGFI (insulin-like growth factor I) to the final composition can also affect dosing. Progress can be monitored by periodic assessment of tissue / bone growth and / or injury, such as X-rays, histomorphometric measurements, and tetracycline labeling.

Veterinary use . In addition to human patients, FIL-1 theta polypeptides and antagonists can be used in pets (dogs, cats, birds, primates, etc.), farm animals (horses, cows, sheep, pigs, birds, etc.), or in TNFα-mediated inflammation or arthritis. It is useful in treating disease disorders in non-human animals, such as any animal suffering from. In such instances, the appropriate dose can be determined according to the weight of the animal. For example,
Doses of 0.2-1 mg / kg can be used. Alternatively, the dose is determined according to the surface area of the animal, a typical dose is 0.1-20 mg / m ^2, or more preferably in the range of 5-12mg / m ^2. For small animals such as dogs and cats, a suitable dose is 0.4 mg / kg. In a preferred embodiment, FIL
-1 theta polypeptide or antagonist, preferably constructed with a gene from the same species as the patient, is administered by injection or other suitable route once or more times weekly until the animal's abnormality is ameliorated Alternatively, it can be administered indefinitely.

Pharmaceutical manufacturing . The present invention also relates to FIL-1 theta polypeptides, fragments and variants in the manufacture of a medicament for the prophylactic or therapeutic treatment of each of the medical disorders disclosed herein; FIL-1 theta family polypeptides, fragments. , And nucleic acids encoding variants; agonists or antagonists of FIL-1 theta polypeptides such as antibodies; FIL-1 theta polypeptide binding partners; FIL-
It also relates to the use of 1-theta family polypeptides, fragments, variants and complexes formed with binding partners.

Provided herein are compositions that include an effective amount of a polypeptide of the invention in combination with other components such as a physiologically acceptable diluent, carrier, or excipient. The polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. These include pharmaceutically acceptable diluents (eg, saline, Tris-HCl, acetic acid, and phosphate buffers) as the sole active ingredient, or together with other known active ingredients suitable for the indicated indications. Solutions), preservatives (eg thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and / or carriers and may be mixed and combined. Suitable formulations for pharmaceutical compositions include Remington's Pharmaceu.
Tal Sciences , 16th Edition, Mack Publish
g Company, Easton, PA, 1980.

Further, such compositions are complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, or liposomes, It may be incorporated into microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such a composition has a physical state,
It will affect solubility, stability, in vivo release rate, and in vivo clearance rate, and is therefore selected according to the intended application.

The compositions of the invention may be administered in any suitable manner, eg topically, parenterally or by inhalation. The term "parenteral" includes, for example, injection by subcutaneous, intravenous, or intramuscular routes, and also includes topical administration, eg, at the site of the disease or injury. Sustained release from the implant is also contemplated. Those skilled in the art
It will be appreciated that the appropriate dose will vary depending on such factors as the nature of the disorder being treated, the weight, age and general condition of the patient, and the route of administration.

Preliminary doses can be determined according to animal studies, and dosing for human administration is done according to art-recognized practices. Compositions containing the nucleic acid in a physiologically acceptable formulation are also contemplated. DNA
Can be formulated for injection, for example.

Another use of the polypeptides of the present invention results from or inhibits these interactions of FIL-1 theta with its binding partner,
It is intended as a research tool for studying biological effects on different cell types. The polypeptides can also be used in in vitro assays to detect FIL-1 theta, their respective binding partners, or their interactions.

Another aspect of the invention relates to the use of the polypeptides of the invention to study cell signaling. IL-1 family ligands play a central role in defense against infection and immune inflammatory response, including cell signaling, activation of vascular endothelial cells and lymphocytes, inflammatory cytokines, acute phase proteins, hematopoiesis. , Fever, bone resorption, induction of prostaglandins, metalloproteinases, and adhesion molecules. With the ever-increasing number of known IL-1 family members, a suitable classification scheme is based on comparing function (activation and regulatory properties) with polypeptide structure. Therefore, FIL-1 theta is associated with other IL-1 family ligands (IL-1α, IL-1β, and IL-1
Like -18), it is involved in many of the functions mentioned above and appears to be involved in promoting the inflammatory response. Therefore, FIL-1 theta appears to be a factor in the induction and maintenance of inflammation and / or autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis. As such, alterations in expression and / or activation of the polypeptides of the invention may be critical to excessive cellular processes, including, but not limited to, cell-specific responses and activation or inhibition of proliferation. May have an impact. Expression of cloned FIL-1 theta or a functionally inactive mutant thereof can be used to identify the role that a particular protein plays in mediating a particular signaling event.

Cellular signaling often involves a cascade of molecular activations during which the receptor activates an intracellular kinase that phosphorylates a target substrate, thereby producing a ligand-receptor mediated signal. To convey. These substrates are themselves kinases, which are activated following phosphorylation. Alternatively, they may be adapter molecules that promote phosphorylation followed by downstream signaling through protein-protein interactions. Substrate molecule (
), The expressed functionally active forms of FIL-1 theta, and their binding partners, are used by the polypeptides of the invention to determine which substrate (
) Are recognized and activated, or can be identified. As such, the novel polypeptides can be used as reagents to identify novel molecules involved in signal transduction pathways.

The polypeptides of the present invention find use in screening assays to identify compounds and small molecules that inhibit (antagonize) or enhance (agonize) the activation of the polypeptides of the present invention. . Thus, for example, the polypeptides described herein can be used to identify antagonists and agonists from cells, cell-free preparations, chemical libraries, and natural product mixtures. Antagonists and agonists may be natural or modified substrates, ligands, enzymes, receptors, etc. of the polypeptides of the invention,
Alternatively, it may be structural or functional mimetics of the polypeptide. Potential antagonists of the polypeptides of the present invention include those that bind to and occupy the binding site of the polypeptide, rendering the binding site unavailable for binding to its ligand and, thus, normal biological activity. Interfering small molecules, peptides, and antibodies may be included. Other potential antagonists are i
An antisense molecule capable of hybridizing to mRNA and blocking translation of mRNA into a polypeptide of the invention in vivo. Potential agonists include small molecules, peptides and antibodies that bind to the polypeptide and elicit the same or enhanced biological effect caused by the binding of the polypeptide of the invention. .

[0157] Typically, small molecule agonists and antagonists have molecular weights less than 10K and enhance some cell penetration, resist degradation and prolong their physiological half-life. May have biological and pharmacological properties (Gi
bbs, J.M. , Pharmaceutical Research in
Molecular Oncology, Cell, Vol. 79 (199
4)). Antibodies comprising fragments, such as Fab and F (ab ′) 2 fragments, with intact molecules are used to bind to a polypeptide of the invention and block the initiation of a signaling cascade to thereby bind the polypeptide. Can be inhibited. It is preferred that the antibody is humanized, and more preferably
The antibody is human. The antibodies of the present invention may be prepared by any of a variety of known methods.

Generally, a) a test compound that is a potential agonist or antagonist is mixed with a FIL-1 theta polypeptide of the invention; and b) the test compound is
By determining whether to modify the FIL-1 theta activity of a polypeptide, the screening methods of the invention are performed to identify compounds that modify the activity associated with FIL-1 theta. The identification of a compound that inhibits the binding activity of the polypeptide of the present invention is carried out by a) mixing a test compound with a polypeptide of the present invention and a binding partner of the polypeptide; Including whether to inhibit.

Several screening techniques are known, including the use of cell-based or in vitro-based specific binding assays or tests (eg, High Throughput Screening: all incorporated herein:
The Discovery of Bioactive Substanc
es, John P. Devlin (supervised), Marcel Dekke
r, New York, 1997, ISBN: 0-8247-0067-8.
, Http: // www. lab-robotics. org / , http: // www. sbsonline. org / )). In combination with integrated robotic systems, high throughput screening techniques can be used to test and screen large collections of chemical compounds and / or natural products for antagonist or agonist activity in a short time. Specific assays or tests include homogeneous assay formats such as fluorescence resonance energy transfer, time-resolved fluorescence resonance energy transfer, fluorescence polarization, scintillation proximity assay, reporter gene assay, fluorescence quencher substrate, chromogenic enzyme substrate and electrochemiluminescence. , More traditional heterogeneous assay formats such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay. Homogeneous assays are mixed and read-only assays that are very amenable to robotic application, while heterogeneous assays bind from free analyte by more complex unit operations such as filtration, centrifugation or washing. Requires separation of analytes. Utilize these assays to detect a wide variety of specific biomolecule interactions and inhibition of specific biomolecule interactions by small organic molecules, drug candidates, antibodies, peptides and other antagonists and / or agonists. It is possible. Specific biomolecule interactions include, but are not limited to, protein-protein interactions, receptor-ligand interactions, enzyme-substrate interactions, and the like.

One embodiment of the method of identifying a molecule that antagonizes or inhibits a polypeptide of the invention is to add the candidate molecule to a medium containing cells expressing a polypeptide of the invention; Apart from its presence, it involves changing the conditions of the medium so that the polypeptide will bind its ligands; and observing stimulation or inhibition of binding and functional responses. The activity of cells contacted with the candidate molecule can then be compared to the same cells not contacted with the candidate molecule and agonists and antagonists of the polypeptides of the invention can be identified. Measurement of biological activity can be done by several known methods, such as measuring the amount of protein present (eg, ELISA) or the amount of activity of the protein. A decrease in biological stimulation or activation would indicate an antagonist. An increase would indicate an agonist. Specifically, one aspect of the invention comprises agonists and antagonists of FIL-1 theta.

Screening assays can further be designed to find molecules that mimic the biological activity of the polypeptides of the invention. Molecules that mimic the biological activity of a polypeptide may be useful in enhancing the biological activity of the polypeptide. To identify a compound for therapeutically active agents that mimic the biological activity of the polypeptide, one must first determine if the candidate molecule binds to the polypeptide. The binding candidate molecule is then added to the biological assay and its biological effect is measured. The biological effect of the candidate molecule is then compared to that of the polypeptide.

Polypeptides or peptide fingerprints can be entered into or compared to a database of known proteins to aid in the identification of unknown proteins using mass spectrometry (WJ. Henzel et al.
Proc. Natl. Acad. Sci. USA, 90: 5011.
-5015, 1993; Fenyo et al., Electrophoresis,
19: 998-1005, 1998). A variety of computer software programs, such as Protein Prospe, that facilitate these comparisons.
ctor (Internet site: prospector.uscf.edu)
, MultiIdent (Internet site: www.expasy.ch
/ Sprot / multiident. html), PeptideSearch
h (Internet site: www.mann.embl-heiedelbe
rg. dedeSearch / FR_PeptideSearchForm. h
tml), and ProFound (Internet site: www.chai
t-sgi. rockefeller. edu / cgi-bin / prot-i
d-frag. , etc.) is accessible via the Internet. These programs allow the user to specify the molecular weight of the cleaving agent and the fragmentation peptides within the indicated tolerances. The program compares observed molecular weights with predicted peptide molecular weights from sequence databases to assist in determining the identity of unknown proteins.

In addition, the polypeptide or peptide digest is tandem mass spectrometric (MS /
MS) and the resulting sequences can be searched against databases (Eng et al., J. Am. Soc.
Mass Spec. 5: 976-989, 1994; Mann et al., Anal. Chem. 66: 4390-4399, 1994; A. Taylor et al., Rapid Comm. Mass Spec
． , 11: 1067-1075, 1997). A search program that can be used for this method is Lutefisk 97 (Internet site: www.lsbc.
． com: 70 / Lutefisk 97. html), and the above-mentioned Prote
in Prospector, Peptide Search and ProFo
It exists on the Internet, such as the und program.

Therefore, adding the sequence of a gene, and its predicted protein sequences and peptide fragments, to a sequence database can aid in the identification of unknown proteins using mass spectrometry.

Provided herein are antibodies that are immunoreactive with the polypeptides of the invention. Such antibodies specifically bind to the polypeptide (as opposed to non-specific binding) through the antigen binding site of the antibody. Thus, the polypeptides, fragments, variants, fusion proteins, etc. of SEQ ID NO: 2 and SEQ ID NO: 4, as described above, can be used as an "immunogen" in producing antibodies immunoreactive therewith. Is. More specifically, polypeptides, fragments, variants, fusion proteins and the like contain antigenic determinants or epitopes that elicit antibody formation.

These antigenic determinants or epitopes can be either linear or conformational.
(international) or either. Linear epitopes are composed of a single portion of amino acids of the polypeptide, while conformational or intermittent epitopes are composed of amino acid portions from different regions of the polypeptide chain that are approached during protein folding. (CA Janeway, Jr.
． And P.C. Travels, Immuno Biology , 3: 9,
Garland Publishing Inc. , 2nd edition, 1996)
. Folded proteins have a complex surface, so the number of available epitopes is very large; however, because of the protein's conformation and steric hindrance, the number of antibodies that actually bind to the epitope is available. a less than the number of epitopes (C. A. Janeway, Jr. and P. Travers, Im muno Biology, 2:14 , Garland Publishi
ng Inc. , 2nd edition, 1996). Epitopes may be identified by any method known in the art.

Accordingly, one aspect of the invention pertains to antigenic epitopes of the polypeptides of the invention. Such epitopes are useful in making antibodies, particularly monoclonal antibodies, as described in more detail below. In addition, the epitopes derived from the polypeptides of the invention can be used in assays and as research reagents to purify antibodies that specifically bind from substances such as supernatants from polyclonal sera or cultured hybridomas. Such epitopes or variants thereof can be produced using techniques known in the art such as solid phase synthesis, chemical or enzymatic cleavage of polypeptides, or using recombinant DNA techniques. is there.

For antibodies that may be elicited by an epitope of a polypeptide of the invention, both polyclonal and monoclonal antibodies, whether the epitope is isolated or remains part of the polypeptide, It can be prepared by conventional techniques. For example, Kennet et al. (Eds.), Mono clonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, P
Lenum Press, New York, 1980; and Harlow and Land (supervised), Antibodies: A Laboratory Manual , Cold Spring Harbor Laborato.
ry Press, Cold Spring Harbor, NY, 1988
Please refer to.

Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the invention are also contemplated herein. Such hybridomas can be produced and identified by conventional techniques. One method for producing such hybridoma cell lines is to immunize an animal with the polypeptide; harvest spleen cells from the immunized animal; fuse the spleen cells to a myeloma cell line, thereby producing hybridoma cells. Producing; and identifying a hybridoma cell line that produces a monoclonal antibody that binds to the polypeptide. Monoclonal antibodies can be recovered by conventional techniques.

Monoclonal antibodies of the invention include chimeric antibodies, eg, humanized versions of murine monoclonal antibodies. Such humanized antibodies may be prepared by known techniques and may provide the advantage of reduced immunogenicity when the antibody is administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or only its antigen binding site) and the constant region derived from a human antibody. Alternatively, a humanized antibody fragment may include the antigen binding site of a murine monoclonal antibody and a variable region fragment derived from a human antibody (lacking the antigen binding site). Methods for producing chimeric and further engineered monoclonal antibodies are described in Riechmann et al., Nat.
ure, 332: 323, 1988; Liu et al., PNAS, 84: 343.
9, 1987; Larrick et al., Bio / Technology, 7:
934, 1989; and Winter et al., TIPS, 14: 139,
Those described in 1993 are included. The method of transgeneously producing antibodies is
GB 2,272,440, US Pat. Nos. 5,569,825 and 5,54.
5,806, as well as related patents claiming priority thereto, all of which are incorporated herein by reference.

Also included in the invention are antigen binding fragments of antibodies, which can be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab and F (ab
') Contains ₂ fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.

In one embodiment, the antibody is specific to a polypeptide of the invention and does not cross-react with other proteins. Screening methods capable of identifying such antibodies are known and may involve, for example, immunoaffinity chromatography.

The antibodies of the present invention can be used in assays to detect the presence of the polypeptides or fragments of the present invention either in vitro or in vivo. Antibodies can also be used in purifying the polypeptides or fragments of the invention by immunoaffinity chromatography.

Furthermore, antibodies capable of blocking the binding of the polypeptides of the invention to their binding partners can be used to inhibit the biological activity resulting from such binding. Such blocking antibodies may be assayed using any suitable assay method, such as by testing the antibodies for their ability to inhibit binding of FIL-1 theta to specific cells expressing the FIL-1 theta receptor. May be identified. Alternatively, blocking antibodies can be identified in an assay for their ability to inhibit a biological effect resulting from a polypeptide of the invention binding to a binding partner of a target cell. Antibodies may be assayed for their ability to inhibit FIL-1 theta-mediated or binding partner-mediated cytolysis, for example.

Such antibodies can be used in in vitro methods or administered in vivo to inhibit the biological activity mediated by the entity that generated the antibody. Therefore, it is possible to treat disorders caused (directly or indirectly) or exacerbated by the interaction of the polypeptides of the invention with binding partners. Therapy involves administering to a mammal an in vivo amount of a blocking antibody effective to inhibit a binding partner-mediated biological activity.
Monoclonal antibodies are generally preferred for use in such therapies. In one embodiment, antigen binding antibody fragments are used.

Antibodies can be screened for agonistic (ie, ligand mimetic) properties. Such antibodies, upon binding to cell surface receptors, have biological effects similar to those induced by IL-1 binding to cell surface IL-1 receptors (eg, of biological signals). Induce). Agonist antibodies are used to activate vascular endothelial cells and lymphocytes to induce local tissue destruction and fever (
(Janeway et al., 1996), macrophages and vascular endothelial cells can be stimulated to produce IL-6 and upregulate molecules on the surface of vascular endothelial cells.

Provided herein is a composition comprising an antibody directed against a polypeptide of the invention and a physiologically acceptable diluent, excipient, or carrier. Suitable components of such compositions relate to compositions comprising a polypeptide of the invention,
As described above.

Also provided herein are conjugates that include a detectable (eg, diagnostic) or therapeutic agent attached to an antibody. Examples of such agents are presented above. The conjugates will find use in in vitro or in vivo methods.

The following examples are provided to further illustrate certain aspects of the invention and should not be construed as limiting the scope of the invention.

[0180]

【Example】

Example 1: Isolation of FIL-1 theta and identification of distribution Two exons of genes with apparent homology to the IL-1 family were found in the genomic database. PCR primers were designed based on the sequences of the two exons. The two exons correspond to typical IL-1 family members exons 5 and 6 (the last two exons). The designed PCR amplification primer had the following sequence: Forward primer: AGA AGA TCT GCA TAC TTC C
TA (from exon 5) SEQ ID NO: 5 Reverse primer: TGA GCA GGA TGA GCT TGG T
The SEQ ID NO: 6 primer (from exon 6) was synthesized, and Clontech human tissue cDNA and Bio
A panel of chain human adult normal skin cDNA was used for PCR amplification. Three
The 96 bp fragment was the expected PCR product. This is based on the size of IL-1 family members and the size of correctly spliced forms of exon 5 and exon 6. The PCR method involved 30 PCR amplification cycles with an annealing temperature of 60 ° C. and 25 picomoles of primer and Ho per reaction.
Included was using tstart Taq polymerase (Qiagen, Valencia, CA). PCR products of the expected size were obtained from skin and tonsil first-strand cDNA and the following Clontech cDNA sources were negative: small intestine, liver, brain, lung, fetal liver, lymph, bone marrow, Kidney, pancreas,
Skeletal muscle, heart, prostate, ovary, thymus, spleen, white blood cells, testis, placenta, and colon.
Following the 30 round PCR method, the same panel was screened again using the 40 round PCR method. The 40-cycle PCR amplification method resulted in positive identification of lung and placenta. Human small airway epithelium c from unstimulated cells and cells stimulated under various conditions
DNA was detected as positive after 40 PCR amplification cycles under the same conditions. Bio
The sequence of the chain human normal skin PCR product is shown in exon 5 and exon 6
Matched the sequence predicted for the correctly spliced product of. Sequences were determined by standard double-stranded sequencing of the composite sequence and additional sequences obtained in the PCR and 5'RACE reactions.

Exon 6 contains a translation stop codon. To extend the cDNA clone in the 5'direction and identify the mature N-terminus, the following primers were used to clone Clontech.
5'RACE was performed on Marathon Ready lung cDNA: Vector entry forward primer: CCA TCC TAA TAC GAC.
TCA CTA TAG GGC (SEQ ID NO: 7) Reverse gene-specific primer: CTC CAG CTG TAG GGA
AGG (SEQ ID NO: 8) (or SEQ ID NO: 6) Nesting reactions were then performed using the first cycle reaction product as a template with a nested vector entry primer as follows: Forward primer: ACT CAC TAT AGG GCT CGA G
CG GC (SEQ ID NO: 9) Reverse gene specific primer: TCT TCT GTC TCC ACA
CAT GC (SEQ ID NO: 10); or CAA GGC TAA AAC
The entire GCA GTT TC (SEQ ID NO: 11) nested PCR reaction was subcloned into Novagen pT7Blue3 and transformants screened to identify potential positives. Two
Miniprep DNA from two transformants was sequenced. The clone, lung 47, resembles the correctly spliced Skin 1 cDNA and further extended the sequence 171 bp upstream until the stop codon was reached. The transformant contained the correctly spliced "exon 4" but was spliced incorrectly upstream of this.

In a further study, from Immunex small airway epithelial RNA, Life T
Custom pCMVSport c created by technologies
The DNA library was screened for the presence of exon 5 and exon 6 by PCR amplification with primers SEQ ID NO: 5 and SEQ ID NO: 6. This library source was positive and therefore a 5'RACE was performed. In the first cycle, the following primers were used: Vector entry forward primer: CAG GAA ACA GCT ATG
ACC AT (SEQ ID NO: 12) Reverse gene specific primer: SEQ ID NO: 6 or SEQ ID NO: 8. Nesting was then performed using the first round reaction product as a template with the following primers: Vector entry forward primer: CTA TTT AGG TGA CAC.
TAT AGA A (SEQ ID NO: 13) Reverse gene specific primer: SEQ ID NO: 10 or SEQ ID NO: 11 The product of the entire nested PCR reaction was subcloned into Novagen pT7Blue3 and the transformants screened for potential positivity. Identified. Miniprep DNA from 12 transformants was sequenced. All 12 clones were identical, including clone SAE48. These are the skin 1
This sequence was homologous to the cDNA and extended 139 bp upstream and contained "exon 3" containing an in-frame initiation methionine, along with exon 4 identified in the clone, lung 47.

The RAC described above, except that the reverse primer was located closer to the N-terminus.
5'RACE was repeated on the SAE library in a manner similar to the E reaction. Nine clones were sequenced and they were identical to SAE48.

Full length FIL-1 theta was amplified from Clontech placental cDNA with primers designed from SAE48. Sequencing data further confirmed that FIL-1 theta exhibits polymorphisms at amino acid 44, which is threonine or isoleucine, and at amino acid 51, which is aspartic acid or alanine. Where start methionine + 1
Called. There are three alleles at the codon for amino acid 44, as shown below. The corresponding nucleotides encoding the polymorphism are: For amino acid 44: threonine (nucleotides 130 to 132) -AC
A; Isoleucine (again nucleotides 130 to 132) ATA or ATC With respect to amino acid 51: Aspartic acid (nucleotides 151 to 153) G
AC; alanine (again nucleotides 151 to 153) -GCC.

Polymorphisms were detected in placental, lung and SAE with the following frequencies: ATC-encoded isoleucine at amino acid 44 is a single placental cDNA.
Detected in clones.

Isoleucine at amino acid 44 encoded by the ATA was detected in four samples, including two lungs, lung 47 and lung 19 (genome), placental cDNA and ac016724 (genome).

Threonine at amino acid 44 encoded by the ACA was detected in 6 placental cDNA samples and small airway epithelial cDNA. Example 2: it was pH9 with serial dilutions (NaOH Purification FIL-1 theta FIL-1 theta use of the polypeptide-specific ELISA, 50mM NaHC
The O _3), 100: 1 / well, Linbro / Titertek 96 well flat bottom E. I. A. Microtitration plate (ICN Biome
dicals Inc. , Aurora, Ohio). After incubation at 4 ° C for 16 hours, PBS containing 0.05% Tween-20 (PB
Wash 6 times with S-Tween at 200: 1. The wells were then incubated with 1 mg / ml FLAG-binding partner in PBS-Tween containing 5% fetal calf serum (FCS) for 90 minutes (100: 1 per well),
Then, it is washed as described above. Next, each well is treated with PBS containing 5% FCS.
-1 mg / ml anti-FLAG (monoclonal antibody M2) in Tween, and
Incubate for 0 minutes (100: 1 per well), then wash as above. Subsequently, the wells were treated with polyclonal goat anti-mIgG1 specific horseradish peroxidase-conjugated antibody (PBS-T containing 5% FCS).
Incubate with commercial stock 1: 5,000 dilution in ween) for 90 minutes (100: 1 per well). HRP-conjugated antibody is S
outern Biotechnology Associates, In
c. , Birmingham, Alabama. The wells are then washed 6 times as above.

For the development of the ELISA, the substrate mixture (TMB peroxidase substrate and peroxidase solution B (Kirkegaard Perry Laborato).
Ries, Gaithersburg, Md.) 100: 1) per well of a 1: 1 premix is added to the wells. After sufficient color reaction, 2N H ₂ SO ₄ (50: 1 per well) is added to terminate the enzyme reaction. Color intensity (indicating ligand-receptor binding) is measured by the V Max plate reader (Molecular D).
devices, Sunnyvale, CA) by measuring the 450 nm extinction.

Example 3: Monoclonal Antibodies that Bind Polypeptides of the Invention This example illustrates a method for preparing monoclonal antibodies that bind FIL-1 theta polypeptides. Suitable immunogens that can be used to generate such antibodies include, but are not limited to, purified FIL-1 theta polypeptide (eg, SEQ ID NO: 2 or SEQ ID NO: 4), or its extracellular domain, such as the extracellular domain. Included are immunogenic fragments or variants, or fusion proteins containing FIL-1 theta (eg, soluble FIL-1 theta / Fc fusion protein).

Using purified FIL-1 theta polypeptide, US Pat. No. 4,411,993
It is possible to produce monoclonal antibodies which are immunoreactive with them using conventional techniques such as those described in US Pat. Briefly, mice are immunized with FIL-theta immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in amounts ranging from 10-100 μg. After 10 to 12 days, immunized animals are boosted with additional FIL-1 theta emulsified in incomplete Freund's adjuvant. Thereafter, the mice are regularly boosted on a weekly to biweekly immunization schedule. Serum samples are taken periodically by retro-orbital bleeding or tail tip excision and tested for FIL-1 theta antibody by dot blot assay, ELISA (enzyme linked immunosorbent assay) or inhibition of FIL-1 theta receptor binding. To do.

Following detection of appropriate antibody titers, positive animals were given one final dose of FIL- in saline.
Inject 1 theta intravenously. After 3 to 4 days, the animals were sacrificed and spleen cells were harvested,
The spleen cells are then transformed into a murine myeloma cell line such as NS1 or preferably p3x6.
3Ag8.653 (ATCC CRL 1580). The fusion produces hybridoma cells which are grown in HAT (hypoxanthine, aminopterin and thymidine) selective medium in a number of microtiter plates in order to inhibit the growth of unfused cells, myeloma hybrids and spleen cell hybrids. Sow.

Engvall et al., Immunochem. , 8: 871, 1971 and US Pat. No. 4,703,004 to adapt the hybridoma cells to EL for reactivity with purified FIL-1 theta.
Screen by ISA. The preferred screening technique is Beck
mann et al. Immunol. , 144: 4212, 1990. Positive hybridoma cells can be injected intraperitoneally into syngeneic BALB / c mice to produce ascites fluid containing high concentrations of anti-FIL-1 theta monoclonal antibody. Alternatively, the hybridoma cells may be cloned in vitro in a flask or roller bottle by a variety of techniques.
It may be grown in vitro. Monoclonal antibodies produced in mouse ascites can be purified by ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on binding of the antibody to protein A or protein G may also be used, FIL-
Affinity chromatography based on binding one theta may also be used.

Example 4: Tissue distribution of FIL-1 theta mRNA Using FIL-1 theta specific primers, multiple human tissue sources (Clon) were used.
tech, Palo Alto, CA) using first-strand cDNA from
The tissue distribution of L-1 theta mRNA was examined. The PCR reaction produced a PCR product with the expected size. FIL-1 theta is associated with tonsils, skin, lungs, placenta,
And small airway epithelium.

Example 5: FIL-1 Theta Binding Assay Full-length FIL-1 theta can be expressed and tested for its ability to bind to the FIL-1 theta receptor. The binding assay can be performed as follows.

In this assay, a fusion protein containing a leucine zipper peptide fused to the N-terminus of a soluble FIL-1 theta polypeptide (LZ-FIL-1 theta).
To use. DNA encoding the FLAG ⁷ peptide, but replacing the sequence encoding a modified leucine zipper that allows for trimerization, essentially, Wiley et al., Which is incorporated herein, Immunity, 3: 673- 682
In 1995, the expression construct is prepared as described for preparing the FLAG ⁷ (FIL-1 theta) expression construct. The construct in the expression vector pDC409 contains a leader sequence from human cytomegalovirus followed by soluble FI.
It encodes the leucine zipper moiety fused to the N-terminus of the L-1 theta polypeptide. LZ-FIL-1 theta is expressed in CHO cells and purified from culture supernatant.

The expression vector designated pDC409 is described in McMah, incorporated herein by reference.
an et al., EMBO J .; , 10: 2821-2832, 1991, which is a mammalian expression vector derived from the pDC406 vector. (PDC406
The features added to pDC409 (compared to
additional unique restriction sites in cs); three stop codons located downstream of mcs (one in each open reading frame); and T7 polymerase downstream of mcs to facilitate sequencing of the DNA inserted in mcs. A promoter is included.

For expression of full length human FIL-1 theta protein, the entire coding region (ie the DNA sequence shown in SEQ ID NO: 3) is amplified by the polymerase chain reaction (PCR). The template used in PCR is as described in Example 1,
(Pancreatic tumor) A cDNA clone isolated from a cDNA library. The isolated and amplified DNA was inserted into expression vector pDC409 to generate pDC
A construct called 409-FIL-1 theta is obtained.

The LZ-FIL-1 theta polypeptides are used to test their ability to bind to host cells expressing recombinant or endogenous FIL-1 theta receptors, as discussed above. Cells expressing the FIL-1 theta receptor are cultured in DMEM supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine. Cells are incubated with LZ-FIL-1 theta (5 mg / ml) for approximately 1 hour. After incubation, cells are washed and unbound LZ-
FIL-1 theta was removed and biotinylated anti-LZ monoclonal antibody (5 m
g / ml), and phycoerythrin-conjugated streptavidin (
Fluorescence-activated cell scan (FACS) after incubation with 1: 400)
). FAC scan (Beckton D
ickinson, San Jose, CA).

Cells expressing the FIL-1 theta receptor will show significantly enhanced FIL-1 theta binding compared to control cells that do not express the FIL-1 theta receptor.

Example 6 Isolation of Mouse FIL-1 Theta This example describes the identification and isolation of mouse FIL-1. Whole human FIL-
The 1 eta amino acid sequence (see WO 00/71720) was used to search a database of translated mouse genomic DNA sequences (tblastn). Among the sequences identified as suitable were those encoding putative exons most closely resembling exons 5 and 6 of human FIL-1 theta. Primers that anneal to these exons were prepared.

Mouse first strand cDNA was screened using the PCR method using the following primers: GTC TGT ATC CTT CCT AAC CGA.
GGC CTA GAC (SEQ ID NO: 16) is located at the 5'end of exon 5
'It is a sense primer. GTA TGG GTG GAG GGT TCA
CTC TCT TTG GTG (SEQ ID NO: 17) is a 3'antisense primer located near the 3'end of exon 6.

Sources of cDNA screened include macrophage cell lines RAW, LP
S-stimulated RAW, testis, liver, lung, skeletal muscle, day 17 embryo, kidney, and LPS-stimulated RAW cell lambda phage libraries were included. The reaction conditions were as follows: 1.0 ng cDNA or 1.0 μl lambda phage library, 12 each.
． 5 pmol primer, 200 μM each dNTP, and 1.25 units H
50μ with otStarTaq (Qiagen Catalog No. 203205)
Reaction volume of l. The PCR temperature cycle was as follows: 95 ° C for 15 minutes, 50
45 seconds at ℃, and 1 cycle at 72 ℃ 1 minute; 95 ℃ 45 seconds, 50 ℃ 45 seconds,
And 1 cycle of 72 ° C. for 1 minute; and 95 ° C. for 45 seconds, 50 ° C. for 45 seconds, and 72 ° C. for 5 minutes.

After the PCR amplification step, 10 μl of each PCR reaction product was analyzed on a 1.5% TAE agarose gel and after staining with ethidium bromide the products were visualized under UV light. A 308 bp product corresponding to the correct splicing of exons 5 and 6 was observed in lung, skeletal muscle, day 17 embryo, and kidney first strand cDNA. This result confirmed the hypothesis that these putative exons are actually expressed as spliced RNA transcripts. The above-mentioned primer is genome D
Amplify the 573 bp target of NA.

Nested PCR primers (3 'antisense) were prepared to amplify exons 3 and 4 using 5'RACE PCR amplification. The outer primer was SEQ ID NO: 17 and the nested primer was: TGT CTT TAC ACA CGC CAG GCA GCA ACT.
TCC (SEQ ID NO: 18), which is located at the end of exon 5. 5'RACE is an adapter cDNA (Marathon-ready c-derived from the 17th day embryo.
DNA, day 17 embryos; Clontech Catalog No. 7460-1). Two nested cycles of PCR were performed.

For 5′RACE Cycle 1, the primer of SEQ ID NO: 17 and SEQ ID NO: 7
Of the mooring primer of. 5'RACE Cycle 1 reaction conditions were as follows: 0.5 ng adapted cDNA, 12.5 pmol each primer, 20 each.
0 μM dNTPs and 1.25 units HotStarTaq (Qiagen
Volume of 50 μl containing Catalog No. 203205). The temperature cycle was as follows: 1 cycle of 95 ° C for 15 minutes, 50 ° C for 45 seconds, and 72 ° C for 1 minute. Then 33 cycles of 95 ° C for 45 seconds, 50 ° C for 45 seconds, and 72 ° C for 1 minute; and 1 cycle of 95 ° C for 45 seconds, 50 ° C for 45 seconds, and 72 ° C for 5 minutes.

The 5'RACE Cycle 2 reaction conditions were as follows: The primer of SEQ ID NO: 18 and the tethered primer of SEQ ID NO: 9 were used. The cycle 2 reaction diluted the cycle 1 product 1:10 in TE and 0.5 ng adapted cDNA, 12.5 pmol each primer, 200 μM each dNTP,
And 1.25 units of HotStarTaq (Qiagen Catalog No. 203
This was done by preparing 50 μl of the mixture containing 205). The temperature cycle was as follows: 95 ° C. 15 minutes, 60 ° C. 45 seconds, and 72 ° C. 1 minute 1 cycle. Then, 33 cycles of 95 ° C. for 45 seconds, 60 ° C. for 45 seconds, and 72 ° C. for 1 minute. Then 1 hour at 95 ° C for 45 seconds, 60 ° C for 45 seconds, and 72 ° C for 5 minutes.
period.

After amplification, 10 μl of products from cycle 1 and cycle 2 were analyzed on a 0.9% TAE gel. A band of approximately 300 bp was observed from the second round of RACE. The entire second round RACE reaction was subcloned into pT7Blue-3 and sequenced for 5 clones. The three clones contained DNA encoding ORFs homologous to exons 3 and 4 of human FIL-1 theta and mouse FIL-
It was shown that the rest of the 1 theta sequence was obtained.

The mouse full-length FIL-1 theta clones prepared primer pairs that spanned the expected mouse FIL-1 theta, and were used to adapt the 17th adapter.
It was amplified by amplifying the embryonic day cDNA. The 5'sense primer is G
CA TTA GAA TCC AGG CAC CAG GAA CTA C
AG (SEQ ID NO: 19) and is at 30nt 5'of the putative ATG. 3'antisense primer is TCC TTA TTC TGC TTT
It was CCA GAG ATG CTG AGC (SEQ ID NO: 20). The reaction conditions were as follows: PCR samples were 0.5 ng adapted cDNA, 12.5 pmol each primer, 200 μM dNTP each, and 0.5 μl 16: 1 KlenTa.
q: Vent was included. The temperature cycle was as follows: 98 ° C. for 3 minutes, 55
One cycle of 45 ° C for 45 seconds and 72 ° C for 1 minute. After that, 98 ℃ 45 seconds, 55 ℃ 4
28 cycles of 5 seconds and 72 ° C. for 45 seconds. After that, 98 ℃ 45 seconds, 55 ℃ 4
One cycle of 5 seconds and 72 ° C. for 5 minutes.

After the amplification reaction, 5 μl of the reaction product was analyzed on a 1.0% TAE agarose gel. A band of 598 bp was observed, which corresponds to the expected size. The product was subcloned into pT7Blue-3 and 4 clones sequenced. Each sequence was identical and was as expected for mouse FIL-1 theta.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/19 C12N 1/21 1/21 C12P 21/08 5/10 C12N 15/00 ZNAA // C12P 21 / 08 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ) , SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, B , BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX , MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F terms (reference) 4B024 AA11 BA26 BA41 CA04 CA12 DA02 DA06 DA12 EA02 EA04 GA01 GA11 GA18 GA19 HA03 HA11 4B064 AG27 CA10 CA20 CC24 DA01 DA13 4B065 AA26X AA72X AA90X AA93Y AB01 AC14 BA10 A41 A41 A41 A41 CA01 CA24 CA24 CA01 A24 CA01 A24 CA01 A24 CA01 CA24 CA41 A4 DA76 DA86 EA22 EA50 FA72 FA74

Claims (24)

[Claims] 1. (a) DNA comprising SEQ ID NO: 1; (b) SEQ ID NO: 3, provided that nucleotides 130-132 are ACA, AT
DNA selected from the group consisting of A and ATC; (c) DNA comprising SEQ ID NO: 3, provided that nucleotides 151-153 are selected from the group consisting of GAC and GCC; (d) SEQ ID NO: 3 DNA containing: (e) DNA containing nucleotides 29-487 of SEQ ID NO: 14; (f) DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 2; (g) Polypeptide containing the amino acid sequence of SEQ ID NO: 4 (H) comprising the amino acid sequence of SEQ ID NO: 4, wherein the amino acid at residue 44 is selected from the group consisting of threonine and isoleucine, and the amino acid at residue 51 consists of aspartic acid and alanine A DNA encoding a polypeptide, selected from: (i) a polypeptide containing the amino acid sequence of SEQ ID NO: 15; DNA encoding tide; (j) DNA containing degenerate DNA from (a) to (i); (k) DNA having at least 80% identity to DNA of (a)-(j) DN including
A; and (l) an isolated DNA selected from the group consisting of DNAs comprising DNAs encoding amino acids 1-152 of SEQ ID NO: 4. 2. An isolated DNA comprising a DNA that is at least 85% identical to the DNA encoding the polypeptide of SEQ ID NO: 4. 3. An isolated nucleic acid molecule comprising DNA encoding the polypeptide of SEQ ID NO: 4. 4. An isolated nucleic acid molecule comprising DNA encoding the polypeptide of SEQ ID NO: 4 except that amino acid 44 is isoleucine. 5. An isolated nucleic acid molecule comprising DNA encoding the polypeptide of SEQ ID NO: 4 except that amino acid 51 is alanine. 6. An isolated DNA which is a fragment of DNA encoding the polypeptide of claim 5, wherein said fragment has IL-1 activity. 7. An isolated DNA comprising a nucleic acid encoding a polypeptide that is at least 85% identical to the polypeptide of SEQ ID NO: 15. 8. An isolated DNA comprising a nucleic acid encoding the polypeptide of SEQ ID NO: 15. 9. An isolated DNA comprising a nucleic acid encoding a fragment of the polypeptide of SEQ ID NO: 4, said fragment having IL-1 activity. 10. A vector containing the DNA according to claim 1. 11. A host cell containing the vector of claim 10. 12. A method for producing a polypeptide, said method comprising culturing the host cell of claim 11 under conditions that promote expression of said polypeptide. 13. An isolated polypeptide comprising a polypeptide encoded by any of the DNAs of claim 1. 14. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 4. 15. The amino acid of SEQ ID NO: 4, except that the amino acid at residue 44 is selected from the group consisting of threonine and isoleucine, and the amino acid at residue 51 is selected from the group consisting of aspartic acid and alanine. An isolated polypeptide comprising a sequence. 16. An isolated polypeptide comprising a polypeptide that is at least 80% identical to the polypeptide of claim 15. 17. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 15. 18. An isolated polypeptide, which is a fragment of the polypeptide of SEQ ID NO: 4, wherein said fragment binds to an IL-1 receptor family member. 19. An isolated polypeptide, which is a fragment of the polypeptide of claim 15 or claim 16, wherein said fragment binds an IL-1 receptor family member. 20. An antibody that binds to the polypeptide of any of claims 13-19. 21. The isolated antibody of claim 20, which is a monoclonal antibody. 22. An oligomer comprising any of the polypeptides of claims 13-19. 23. A method of identifying a compound that alters FIL-1 theta activity, comprising: (a) mixing a polypeptide of any of claims 13-19 with a test compound; and (b) a test compound , Determining whether to alter the FIL-1 theta activity of the polypeptide. 24. A method for identifying a compound that inhibits the binding activity of FIL-1 theta, which comprises: (a) mixing the polypeptide of any one of claims 13-19 and a binding partner of the polypeptide with a test compound. And (b) determining whether the test compound inhibits the binding activity of said polypeptide.