JP2003520590A - Chondromodulin I-related peptides - Google Patents

Abstract

(57) [Summary] The present invention relates to novel polynucleotides encoding polypeptides structurally related to chondromodulin I, as well as the purified proteins themselves. The present invention also relates to vectors, host cells, antibodies and recombinant methods for producing the polypeptides. One aspect of the present invention is a process for producing a chondromodulin I-related peptide, comprising culturing the host cells described above under conditions suitable for expressing the polypeptide, and, optionally, isolating the polypeptide from the culture. Furthermore, the present invention discloses therapeutic, diagnostic and research utilities for these and related products.

Description

Detailed Description of the Invention

RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 09/724, filed Nov. 28, 2000.
,310 and U.S. Provisional Patent Application No. 60/1, filed January 19, 2000.
Priority is claimed under US Pat. No. 76,898.

FIELD OF THEINVENTION The present invention relates to novel polypeptides related to chondromodulin-I (termed ChMIrp), and nucleic acid molecules encoding the peptides. The invention also relates to vectors, host cells, selective binding agents (e.g., antibodies, and methods for the preparation of ChMIrp.
The present invention relates to methods for producing the rp polypeptide. Also provided are methods for the use of ChMIrp, including methods for the diagnosis of and disorders associated with ChMIrp.

BACKGROUND OF THEINVENTION Technological advances in the identification, cloning, expression and manipulation of nucleic acid molecules have greatly accelerated the discovery of new therapeutics based on the deciphering of the human genome. Rapid nucleic acid sequencing technologies can now generate sequence information at unprecedented rates and, coupled with computer-based analysis, allow the assembly of overlapping sequences throughout the genome and the identification of polypeptide coding regions. Comparison of predicted amino acid sequences to database compilations of known amino acid sequences allows the degree of homology to previously identified sequences and/or structural hallmarks to be determined. Cloning and expression of polypeptide coding regions of nucleic acid molecules provides polypeptide products for structural and functional analysis. Genetic engineering to produce variants and derivatives thereof.
Manipulation of nucleic acid molecules and the encoded polypeptides can impart advantageous properties to the product for use as a therapeutic agent.

Despite considerable technological advances in genomic research over the past decade, the ability to develop novel therapeutic agents based on the human genome remains largely unrealized. Numerous genes encoding potentially beneficial protein therapeutic agents, or genes encoding polypeptides that can act as "targets" for therapeutic molecules, have been isolated using recombinant DNA technology.
Although identified using NA techniques, the structure and function of many genes in mammalian genomes are still unknown.

[0005] It is therefore an object of the present invention to identify novel polypeptides and nucleic acid molecules encoding novel polypeptides that have diagnostic or therapeutic advantages.

Chondromodulin-I (ChM-I) was first identified as a protein component in fetal bovine cartilage extracts in the presence of fibroblast growth factor-2 (FGF-2) which stimulates DNA synthesis in cultured rabbit growth plate chondrocytes. The growth stimulating factor was subsequently isolated and the amino acid sequence determined. Based on this amino acid sequence, degenerate primers were used in PCR to clone a fragment of the bovine gene. This DNA fragment was used to identify a 1.7 kb band in bovine epiphyseal cartilage mRNA, which was then used to clone the bovine epiphyseal cartilage cDNA.
The bovine chondromodulin-I gene was isolated from a DNA library.
aki et al., Biochem. Biophys. Res. Com. , 175:97
1-974, 1999). Subsequently, ChM-I orthologs from human, mouse, rat, rabbit, and chicken were isolated; for the isolation of the human and rabbit orthologs, respectively, Hiraki et al.
J. Biochemistry, 260:869-878, 1999) and Shukunami and Hiraki (Biochem. Biophys. R
es. Com., 249:885-890, 1998).
I was found to be expressed by chondrocytes (Hiraki et al., Eur.
J. Biochemistry, 260:869-878, 1999).

Expression of the human ChM-I gene in CHO cells revealed that the secreted mature protein was larger than the polypeptide isolated from cartilage extracts (Hir et al., 2003).
aki et al., Eur. J. Biochemistry, 260:869-878,
1999). This proteolytic processing was also demonstrated when the ChM-I rabbit ortholog was expressed in monkey COS cells (Shukunami and Hiraki, Biochem. Biophys.
Res. Com., 249:885-890, 1998). Alignment of the amino acid sequences of the mature and precursor proteins revealed that the mature protein sequence begins immediately after the RERR amino acid sequence present in the precursor form. The ChM-I precursor contains a single hydrophobic region near its amino terminus that is thought to be involved in membrane insertion. The RERR sequence acts as a processing signal that mediates proteolytic cleavage resulting in secretion of the mature form. This processing leaves the majority of the amino-terminal portion of the protein inserted into the plasma membrane of chondrocytes. Thus, these results indicate that ChM-I is expressed as a larger transmembrane precursor protein, which is cleaved into the carboxy-terminal mature form and deposited in cartilage (Suzuki, B
iochem. Biophys. Res. Comm. 259:1-7, 1999
(As outlined in ).

[0008] Expression of ChM-I mRNA has been detected only in human and bovine cartilage during embryonic development. In particular, high levels of expression were detected in chondrocytes present in the proliferative cartilage zone, and lower levels in chondrocytes present in the adjacent telogenous zone and upper hyperplastic zone. No discernible expression was detected in chondrocytes present in the articular zone and lower calcified hyperplastic zone. ChM-I polypeptide was localized to the inter-territorial regions of the proliferative zone, telogenous zone, and upper hyperplastic zone (Hirak et al., 2003).
Eur. J. Biochemistry, 260: 869-878, 19
99).

[0009] ChM-I polypeptide can stimulate DNA and proteoglycan synthesis in rabbit cultured growth plate chondrocytes in the presence or absence of FGF-2. ChM-I inhibits proliferation and tubular morphogenesis in bovine carotid artery endothelial cells (in vitro) and inhibits capillary formation in the chick chorioallantoic membrane assay (in vivo) (Hiraki et al., Eur. J. Biochemistry, 1999, 143:131-132).
, 260:869-878, 1999). The ChM-I polypeptide is a polypeptide that binds FGF-
2 to induce soft agar colony formation in cultured growth plate chondrocytes (Inoue et al., Biochem. Biophys. Res. Com., 24
1:395-400, 1999). Primary osteoblasts and MC3T3-e1 osteoblast-like cells proliferate when stimulated with ChM-I (Mori et al., FEBS
Lett. , 406:310-314, 1999).

[0010] Thus, the identification of chondromodulin-I has led to a better understanding of the processes involved in mediating the development of skeletal components, including inhibition of bone growth, cartilage formation and cartilage angiogenesis. The identification of chondromodulin-I related genes and polypeptides, as well as other chondromodulin-related polypeptides, as described herein, will further clarify the understanding of these processes and facilitate the development of treatments for pathological conditions involving the degradation of skeletal components and increased angiogenesis.

SUMMARY OF THEINVENTION The present invention relates to a novel serine/threonine kinase family and uses thereof. More particularly, the present invention relates to novel ChMIrp nucleic acid molecules and encoded polypeptides, and uses thereof.

The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID NO:1; (b) a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO:2; (c) a nucleotide sequence that hybridizes to the complement of (a) or (b) under moderate or high stringency conditions, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2; and (d) a nucleotide sequence complementary to any of (a)-(c).

The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a sequence that is at least about 70%, about 70%, or more similar to the polypeptide set forth in SEQ ID NO:2.
5%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%
99% or about 99% identical to the sequence of the nucleotides in the sequence of the present invention, wherein the polypeptide is a nucleotide sequence that can be analyzed by GAP, BLASTP, BLASTN, FA
STA, BLASTA, BLASTX, BestFit, and Smith-W
(b) a nucleotide sequence encoding an allelic variant or splice variant of the nucleotide sequence set forth in SEQ ID NO: 1, wherein the encoded polypeptide has the activity of the encoded polypeptide set forth in SEQ ID NO: 2, as determined using a computer program selected from the group consisting of the Aterman algorithm; (c) a nucleotide sequence of SEQ ID NO: 1, (a), or (b), encoding a polypeptide fragment of at least about 25 amino acid residues, wherein the polypeptide has the activity of the encoded polypeptide set forth in SEQ ID NO: 2; (d) a nucleotide sequence encoding a polypeptide having substitutions and/or deletions of 1 to 250 amino acid residues set forth in any of SEQ ID NOs: 1-2,
(e) comprising the nucleotide sequence of SEQ ID NO:1, or a fragment of at least about 16 nucleotides (a)-(d); (f) under moderately or highly stringent conditions, (a)-(e)
(g) a nucleotide sequence that hybridizes to the complementary strand of any of (a)-(e), wherein the encoded polypeptide has an activity of the encoded polypeptide shown in SEQ ID NO:2; and

The present invention further provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO:2 with at least one conservatively substituted amino acid, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2; (b) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO:2 with at least one conservatively inserted amino acid, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2; (c) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO:2 with at least one conservatively deleted amino acid, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2; (d) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO:2 with a C-terminal and/or N-terminal deletion, wherein the encoded polypeptide has an activity of the encoded polypeptide set forth in SEQ ID NO:2; (e) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO:2 with at least one modification selected from the group consisting of an amino acid substitution, an amino acid insertion, an amino acid deletion, a C-terminal deletion, and an N-terminal deletion.
(f) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO:2, wherein the polypeptide has an activity of the encoded polypeptide as set forth in SEQ ID NO:2; (c) a nucleotide sequence encoding a polypeptide as set forth in SEQ ID NO:3, wherein the polypeptide has an activity of the encoded polypeptide as set forth in SEQ ID NO:4;
(g) a nucleotide sequence of (a) to (f) under moderately or highly stringent conditions;
(h) a nucleotide sequence that hybridizes to the complementary strand of any of (a)-(e), wherein the encoded polypeptide has an activity of the encoded polypeptide shown in SEQ ID NO:2; and

The present invention also provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a mature amino acid sequence set forth in SEQ ID NO:2, including a mature amino acid terminus at residue 1, and optionally further including a methionine amino terminus; (b) an amino acid sequence for an orthologue of SEQ ID NO:2, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2; (c) an amino acid sequence for an orthologue of SEQ ID NO:2, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2, and wherein the activity of the polypeptide is at least about 70%, about 70%, or more preferably about 70%.
5%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%
99% or about 99% identical to the sequence of the nucleotides in the sequence of the present invention, wherein the polypeptide is a nucleotide sequence that can be analyzed by GAP, BLASTP, BLASTN, FA
STA, BLASTA, BLASTX, BestFit, and Smith-W
(d) an amino acid sequence fragment of SEQ ID NO:2 comprising at least about 25 amino acid residues, wherein said polypeptide has the activity of the polypeptide shown in SEQ ID NO:2; (e) an amino acid sequence which is either an allelic variant or a splice variant of the amino acid sequence shown in SEQ ID NO:2, or of at least one of (a)-(e), wherein said polypeptide has the activity of the polypeptide shown in SEQ ID NO:2; The present invention further provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2 with at least one conservatively substituted amino acid, wherein said polypeptide has the activity of the polypeptide shown in SEQ ID NO:2; (b) an amino acid sequence shown in SEQ ID NO:2 with at least one inserted amino acid, wherein said polypeptide has the activity of the polypeptide shown in SEQ ID NO:2; (c) an amino acid sequence shown in SEQ ID NO:2 with at least one missing amino acid, wherein said polypeptide has the activity of the polypeptide shown in SEQ ID NO:2; (d) an amino acid sequence as set forth in SEQ ID NO:2 having a C-terminal and/or N-terminal deletion, wherein the encoded polypeptide has the activity of the polypeptide as set forth in SEQ ID NO:2; (e) an amino acid sequence as set forth in SEQ ID NO:2 having at least one modification selected from the group consisting of an amino acid substitution, an amino acid insertion, an amino acid deletion, a C-terminal deletion, and an N-terminal deletion.
wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2; Also provided is a fusion polypeptide comprising the polypeptide sequence of (a)-(e) in the preceding paragraph.

The present invention also provides expression vectors comprising the isolated nucleic acid molecules set forth herein, recombinant host cells comprising the recombinant nucleic acid molecules set forth herein, and methods of producing a ChMIrp polypeptide comprising culturing the host cell and, optionally, isolating the polypeptide so produced.

[0017] Transgenic non-human animals comprising a nucleic acid molecule encoding a ChMIrp polypeptide are also encompassed by the present invention. The ChMIrp nucleic acid molecule is introduced into the animal in a manner that allows for expression and increased levels of the ChMIrp polypeptide, which may include increased circulating levels. The transgenic non-human animal is preferably a mammal.

[0018] Derivatives of the ChMIrp polypeptide of the present invention are also provided.

[0019] Provided herein are analogs of ChMIrp resulting from conservative or non-conservative amino acid substitutions of the ChMIrp polypeptide of SEQ ID NO: 2. Such analogs include ChMIrp polypeptides in which the amino acid at position 276 is selected from the group consisting of cysteine, serine, or alanine, in which the amino acid at position 280 is selected from the group consisting of cysteine, serine, or alanine, in which the amino acid at position 281 is selected from the group consisting of glutamic acid or aspartic acid, in which the amino acid at position 285 is selected from the group consisting of glycine, proline, or alanine, in which the amino acid at position 297 is selected from the group consisting of arginine, lysine, glutamine, or asparagine, and in which the amino acid at position 300 is selected from the group consisting of cysteine, serine, or alanine.
a ChMIrp polypeptide, wherein the amino acid at position 306 is selected from the group consisting of cysteine, serine, or alanine, and the amino acid at position 310 is selected from the group consisting of valine, isoleucine, methionine, leucine,
C selected from the group consisting of phenylalanine, alanine, or norleucine
The invention comprises a hMIrp polypeptide.

Further provided are selective binding agents, such as antibodies and peptides capable of specifically binding to the ChMIrp polypeptides of the invention. Such antibodies and peptides may be repulsive or antagonistic.

[0021] Pharmaceutical compositions containing the nucleotides, polypeptides or selective binding agents of the invention and one or more pharma- ceutically acceptable formulating agents are also included by the invention. The pharmaceutical compositions are used to provide a therapeutically effective amount of the nucleotides or polypeptides of the invention. The invention also relates to methods of using the polypeptides, nucleic acid molecules and selective binding agents. The invention also provides a device for administering the ChMIrp polypeptide encapsulated in a membrane.

The ChMIrp polypeptides of the present invention and biologically active variants thereof,
Analogs, homologues and fragments may be used for therapeutic and/or diagnostic purposes to treat, prevent and/or detect conditions resulting from abnormal levels of ChMIrp polypeptide or susceptibility to pathological conditions, which conditions include chondr
Host hyperreaction to omodulin family members or skeletal conditions (
Deficiencies in the autoregulatory network controlled by these proteins, as frequently observed in diseases such as osteoporosis and dwarfism, and in pathological conditions involving angiogenesis, including cancer, inflammatory diseases (e.g., psoriasis and cirrhosis of the liver) and vascular diseases (e.g., atherosclerosis), coronary heart disease and hypertension.

The present invention provides for the treatment, prevention or amelioration of diseases resulting from abnormal levels of ChMIrp polypeptide in a mammal by biologically active ChMIrp polypeptide and/or one or more biologically active variants, fragments, homologues or mutants thereof, other pharmaceutical agents, either alone or in combination. The present invention also provides methods for diagnosing such disorders/diseases or for detecting C in ChMIrp polypeptide and/or antibodies and/or body fluids.
The present invention provides a method for diagnosing a disorder/disease resulting from a mutation in the ChMIrp gene using the nucleic acid molecule of the present invention as a probe, the animal being preferably a mammal, more preferably a human.

The present invention also provides methods for testing molecular collisions upon expression of ChMIrp polypeptide. Expression and modulating (i.e., increasing or decreasing) levels of ChMIrp polypeptide are also encompassed by the present invention. One method involves administering the nucleic acid molecule to an animal in vivo. In another method, the nucleic acid molecule is administered to an animal in vivo.
A nucleic acid molecule containing elements which regulate the expression of the rp polypeptide can be administered to the animal.
Examples of these methods include gene therapy and antisense therapy.

[0025] Methods of regulating expression and modulating (i.e., increasing or decreasing) levels of ChMIrp polypeptide are also encompassed by the present invention. One method involves administering to an animal a nucleic acid molecule encoding a ChMIrp polypeptide. In another method, a nucleic acid molecule can be administered that contains elements that regulate or modulate expression of a ChMIrp polypeptide. Examples of these methods include gene therapy, cell therapy, and antisense therapy, as further described herein.

[0026] The ChMIrp polypeptide was highly expressed in a wide range of primary human tumors, thus demonstrating the utility of the present polypeptide and its useful nucleic acid intermediates in differentiating transformed cells from background.

In another aspect of the invention, the ChMIrp polypeptide can be used to identify its receptor or binding partner ("ChMIrp receptor" or "ChMIrp binding partner"). Various forms of "expression cloning" have been used extensively to clone receptors for proteins or cofactors. See, e.g., Simonsen and Lodish, Tre
nds in Pharmacological Sciences, 15:4
37-441, 1994 and Tartaglia et al., Cell, 83:12
63-1271, 1995. ChMIrp receptor or ChM
Isolation of Irp binding partners is useful for identifying or developing novel agonists and antagonists of the ChMIrp polypeptide signaling pathway.

In another aspect of the invention, the ChMIrp polypeptide is capable of binding to its binding partner (such as a ChMIrp receptor or other ChMIrp cofactor)
The Irp binding partners can be identified using the
oc. Natl. Acad. Sci. USA, 88:9578-9583, 19
91) Isolation of ChMIrp binding partners is useful for identifying or developing novel agonists and antagonists of ChMIrp activation.

Such agonists and antagonists include soluble ChMIrp ligands, anti-h2520-109 selective binding agents (e.g., ChMIrp antibodies and derivatives thereof), small molecules, peptides or derivatives thereof capable of binding ChMIrp polypeptides, or antisense oligonucleotides;
Any of these may be useful in the treatment of one or more diseases or disorders, including those disclosed herein.

In certain embodiments, ChMIrp polynucleotide agonists or antagonists can be proteins, peptides, carbohydrates, lipids, or small molecular weight molecules that interact with a ChMIrp polypeptide to modulate its activation.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references cited in this application are expressly incorporated herein by reference.

DEFINITIONS The term "nucleic acid molecule encoding ChMIrp" refers to a nucleic acid molecule or polynucleotide that comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO:1 and/or that comprises or consists essentially of a nucleotide sequence that encodes the polynucleotide set forth in SEQ ID NO:2. Related nucleic acid molecules have a similar structure to the nucleotide sequence as set forth in SEQ ID NO:1, but may differ from the nucleotide sequence in any of the following ways:
In a preferred embodiment, the nucleotide sequence comprises or essentially consists of a nucleotide sequence that is about 75 percent identical to the nucleotide sequence as set forth in SEQ ID NO: 1, or that encodes a polypeptide that is about 70 percent identical to the nucleotide sequence as set forth in SEQ ID NO: 2. In a preferred embodiment, the nucleotide sequence comprises or essentially consists of a nucleotide sequence that is about 75 percent identical to the nucleotide sequence as set forth in SEQ ID NO: 1, or that encodes a polypeptide that is about 80 percent identical to the nucleotide sequence as set forth in SEQ ID NO: 2.
percent, or about 90 percent, or about 95, 96, 97, 98,
or 99 percent identical, or the nucleotide sequence is SEQ ID NO:2
about 75 percent, or about 8 percent, of the polypeptide sequence as shown in
0 percent, or about 85 percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percent identical to the polypeptide set forth in SEQ ID NO:2. Related nucleic acid molecules encode polypeptides that retain at least one activity of the polypeptide set forth in SEQ ID NO:2.

Related nucleic acid molecules also include fragments of the above ChMIrp nucleic acid molecules, which fragments are at least about 10 contiguous nucleotides, or about 15, or about 20, or about 25, or about 50, or about 75, or about 100, or more than about 100 contiguous nucleotides. Related nucleic acid molecules also include fragments of the above ChMIrp nucleic acid molecules, which fragments encode polypeptides of at least about 25 amino acid residues, or about 50, or about 75, or about 100, or more than about 100 amino acid residues. Related nucleic acid molecules also include nucleotide sequences that encode polypeptides that contain, or consist essentially of, substitutions and/or deletions of 1-317 amino acid residues compared to the polypeptide of SEQ ID NO: 2. Related Fhm ligand nucleic acid molecules include such molecules that contain a nucleotide sequence that hybridizes to any of the above nucleic acid molecules under moderate or high stringency conditions as defined herein. In preferred embodiments, the related nucleic acid molecules comprise sequences which hybridize under moderately or highly stringent conditions to the sequence set forth in SEQ ID NO:1, or to a molecule encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, or to the nucleic acid fragments described above, or to a nucleic acid fragment encoding a polypeptide as defined above. It is also understood that the related nucleic acid molecules include allelic or splice variants of any of the above nucleic acids, and include sequences complementary to any of the above nucleotide sequences.

The term "isolated nucleic acid molecule" refers to (1) a nucleic acid molecule of the invention that is separated from at least about 50 percent of the proteins, lipids, carbohydrates, or other materials with which it is naturally found when the total DNA is isolated from the source cell;
(2) an "isolated nucleic acid molecule" refers to a nucleic acid molecule of the invention that is not linked to all or a portion of a polynucleotide with which it is naturally linked, (3) a nucleic acid molecule of the invention that is operably linked to a polynucleotide with which it is not naturally linked, or (4) a nucleic acid molecule of the invention that is not naturally occurring as part of a larger polynucleotide sequence. Preferably, an isolated nucleic acid molecule of the invention is substantially free of any other contaminating nucleic acid molecules or other contaminants found in its natural environment that would interfere with its use in polypeptide production, or for therapeutic, diagnostic, prophylactic, or research uses.

As used herein, the term "nucleic acid" sequence or nucleic acid molecule refers to a DNA or RNA sequence. The term includes molecules formed from any of the known base analogs of DNA and RNA, including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, and the like.
e), 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylamino-methyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-
Methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, β-D-
β-D-mannosylqueosine, 5'
-Methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxosin
e), pseudouracil, queusine, 2-thiocytosine, 5-methyl-2-
Thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N
-Uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0036] The term "operably linked" is used herein to refer to the manner in which flanking sequences are positioned such that the flanking sequences so described are constructed or assembled to perform their useful function. Thus, a flanking sequence operably linked to a coding sequence can effect the replication, transcription and/or translation of the coding sequence. For example, a coding sequence is operably linked to a promoter if it is capable of directing the transcription of the coding sequence. A flanking sequence need not be contiguous with the coding sequence so long as it functions correctly. Thus, for example, intervening sequences that are not translated but are transcribed can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0037] The term "ChMIrp polypeptide allelic variant" refers to one of several possible naturally occurring alternative forms of a gene occupying a given locus on a chromosome of an organism or population of organisms.

The term "ChMIrp polypeptide splice variant" refers to a nucleic acid molecule (usually R
This nucleic acid molecule is produced by the selective processing of intron sequences in an RNA transcript.

The term "expression vector" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control the expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing (if introns are present).

The term "vector" is used to refer to any molecule (eg, nucleic acid, plasmid, or virus) used to transfer coding information into a host cell.

As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell, and a cell is transformed when it has been modified to contain new DNA. For example, a cell is transformed when it is genetically modified from its native state. Following transfection or transduction, transformation of DNA involves the physical integration of the DNA into the cell's chromosomes.
The transformed DNA may recombine with the NA, may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to be stably transformed when the DNA is replicated with cell division.

The term "transfection" is used to refer to the uptake of heterologous or exogenous DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced into the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. See, for example, Grah, et al.
Am et al., Virology 52:456, 1973; Sambrook et al., M
Olecular Cloning, A Laboratory Manual
, Cold Spring Harbor Laboratories (Col.
d Spring Harbor Laboratories, New York
K, 1989); Davis et al., Basic Methods in Mole
Neurophysiology, Elsevier, 1986; and Chu et al.,
See Gene 13:197, 1981. Using such techniques,
One or more exogenous DNA moieties can be introduced into a suitable host cell.

The term "transduction" is used to refer to the transfer of genes from one bacterium to another, usually by a phage. "Transduction" also refers to the acquisition and transfer of eukaryotic cell sequences by retroviruses.

The term "host cell" is used to refer to a cell that has been transformed, or can be transformed with a nucleic acid sequence, by a vector containing a selected gene of interest, which is then expressed by the cell. The term includes the progeny of a parent cell, whether or not the progeny is identical in morphology or genetic make-up to the original parent, so long as the selected gene is present.

The term "vector" is used to refer to any molecule (eg, nucleic acid, plasmid, or virus) used to transfer coding information to a host cell.

The term "highly stringent conditions" refers to conditions in which DNA sequences are highly complementary.
High stringency refers to conditions designed to permit hybridization of A strands and exclude hybridization of significantly mismatched DNA. Hybridization stringency is determined primarily by temperature, ionic strength, and the concentration of denaturing agents (e.g., formamide). An example of a "highly stringent condition" for hybridization and washing is 0.015 to 100 ng/mL at 65-68°C.
M sodium chloride, 0.0015 M sodium citrate, or 0.
0.015 M sodium chloride, 0.0015 M sodium citrate and 50% formamide. Sambrook et al., Molecular Cloning:
A Laboratory Manual, 2nd Edition, Cold Spring
Harbor Laboratory, (Cold Spring Harbor
r Laboratory,N. Y. 1989); Anderson et al., Nuc
leic Acid Hybridization:A Practical
Approach Chapter 4, IRL Press Limited (Oxford
(D, England).

More stringent conditions (e.g., higher temperature, lower ionic strength,
Higher concentrations of formamide or other denaturing agents may also be used, but the rate of hybridization will be affected. Other agents may be included in the hybridization and washing buffers for the purpose of reducing non-specific and/or background hybridization. Examples include 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% glycerol ...
% sodium dodecyl sulfate, NaDodSO ₄ , (SDS), Ficoll (ficoll
Coll), Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, although other suitable agents may also be used. The concentration and type of these additives may be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4. However, under typical ionic strength conditions, the rate of hybridization is nearly independent of pH. Anderson et al., Nucleic Acid Hybridization
n: A Practical Approach, Chapter 4, IRL Press
Limited, Oxford, England.

Factors that affect the stability of a DNA duplex include base composition, length, and the degree of base pair mismatch. Hybridization conditions can be adjusted by one of skill in the art to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation: T _m (° C.)=81.5+16.6(log[Na+])+0.41(% G+C)
)-600/N-0.72 (% formamide), where N is the length of the duplex formed, [Na+] is the molar concentration of sodium ions in the hybridization solution or wash solution, and G+C
% is the percentage of (guanine + cytosine) bases in the hybrid. For imperfectly matched hybrids, the melting temperature is reduced by approximately 1°C for every 1% mismatch.

The term "moderately stringent conditions" refers to conditions under which DNA duplexes having a higher degree of base pair mismatch than can occur under "highly stringent conditions" can be formed. Representative examples of "moderately stringent conditions" include the 5
0.015 M sodium chloride, 0.0015 M sodium citrate at 0-65° C. or 0.015 M sodium chloride, 0.0015 M sodium citrate and 20% formamide at 37-50° C. By way of example, “moderately stringent conditions” of 0.015 M sodium ion at 50° C. is approximately 2
Allow for 1% discrepancy.

It will be appreciated by those skilled in the art that there is no absolute distinction between "highly" stringent conditions and "moderately" stringent conditions.
At 0.015 M sodium ion (without formamide), the melting temperature of perfectly matched lengths of DNA is about 71° C. With a wash at 65° C. (at the same ionic strength), this allows for about 6% mismatches. To capture more distantly related sequences, one may simply lower the temperature or increase the ionic strength.

1M Na for oligonucleotide probes up to about 20 nucleotides
A good estimate of the melting temperature in Cl ^* is given by: Tm = 2°C per A-T base pair + 4°C per G-C base pair ^* The sodium ion concentration in 6x salt sodium citrate (SSC) is 1M. Suggs et al., Developmental Biology Usin
Purified Genes, p. 683, Brown and Fox (eds.)
(1981).

High stringency washing conditions for oligonucleotides are typically 6x
SSC, 0.1% SDS at 0°C to 5°C above the Tm of the oligonucleotide
At temperatures 100°C lower.

[0053] The term "ChMIrp polypeptide" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO:2, and related polypeptides described herein. Related polypeptides include ChMIrp polypeptide allelic variants, ChMIrp polypeptide analogs, ChMIrp polypeptide splice variants, ChMIrp polypeptide variants, ChMIrp polypeptide fragments, ChMIrp polypeptide derivatives, substitution variants, deletion variants, and/or insertion variants, ChMIrp fusion polypeptides, and ChMIrp polypeptide orthologs.

[0054] The term "ChMIrp polypeptide analog" refers to related polypeptides having an amino acid sequence similar to the ChMIrp amino acid sequence shown in SEQ ID NO:2, with conservative and non-conservative amino acid substitutions.

[0055] The term "ChMIrp polypeptide ortholog" refers to a polypeptide from another species that corresponds to the ChMIrp polypeptide amino acid sequence set forth in SEQ ID NO: 2. For example, mouse and human ChMIrp polypeptides are considered to be orthologs of each other.

[0056] ChMIrp polypeptides, as defined herein, may be mature polypeptides, which may or may not have an amino-terminal methionine residue, depending on the method by which these polypeptides are prepared.
"Polypeptide fragment" refers to a peptide or polypeptide that contains less than the full-length amino acid sequence of a ChMIrp polypeptide as set forth in SEQ ID NO: 2. Such fragments include, for example, truncations at the amino terminus.
ChMIrp fragments can arise by alternative RNA splicing or by protease activity in vivo.

The term "ChMIrp polypeptide fragment" refers to a polypeptide that contains less than the full-length amino acid sequence of a ChMIrp polypeptide as set forth in SEQ ID NO: 2. Such ChMIrp fragments can be six or more amino acids in length, and can include, for example, an amino-terminal (with or without a leader sequence) fragment.
truncation at the carboxyl terminus and/or truncation of a portion of the amino acid sequence
The ChMIrp polypeptide fragment may be generated by internal deletion of 10 or more residues. The ChMIrp fragment may be generated by alternative RNA splicing or by protease activity in vivo. Membrane-bound forms of the ChMIrp polypeptide are also contemplated by the present invention. In a preferred embodiment, the truncation and/or deletion comprises about 10 amino acids, or about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than about 100 amino acids. The polypeptide fragment thus generated comprises about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids. Such ChMIrp polypeptide fragments may optionally include an amino-terminal methionine residue. It is understood that such fragments may be used, for example, to generate antibodies against the ChMIrp polypeptide.

[0058] The term "ChMIrp polypeptide variant" refers to a ChMIrp polypeptide that includes an amino acid sequence having one or more amino acid sequence substitutions, deletions, and/or additions compared to the ChMIrp polypeptide amino acid sequence. Variants may be naturally occurring or artificially constructed using recombinant DNA technology. Such ChMIrp polypeptide variants may be prepared from the corresponding nucleic acid molecule that encodes the variant. Thus, the variants may be prepared from DNA sequences that are modified from the DNA sequence for the wild-type ChMIrp polypeptide as set forth in SEQ ID NO:1.
It has an array.

[0059] One of skill in the art can determine suitable variants of a native polypeptide using well-known techniques. For example, one of skill in the art can predict suitable regions of the molecule that can be altered without destroying biological activity. One of skill in the art will also recognize that regions that may be important for activity or for structure may even be subject to conservative amino acid substitutions that do not destroy biological activity or adversely affect the structure of the polypeptide.

To predict suitable regions of the molecule that may be altered without destroying activity, one of skill in the art may target regions not believed to be important for activity. For example, if a similar polypeptide with similar activity from the same or another species is known, one of skill in the art may compare the amino acid sequence of the ChMIrp polypeptide to
Such a comparison can be used to determine residues and portions of the molecules that are conserved between similar polypeptides.
It is known that changes in regions of the ChMIrp molecule that are not conserved are unlikely to adversely affect the biological activity and/or structure of the ChMIrp polypeptide. Those skilled in the art will also recognize that even in relatively conserved regions,
It is known that chemically similar amino acids can be substituted for naturally occurring residues while retaining activity (conservative amino acid residue substitutions).

[0061] Additionally, one skilled in the art can review structure-function studies which identify residues in similar polypeptides that are important for activity or structure. In view of such comparisons, one can predict the importance of amino acid residues in the ChMIrp polypeptide that correspond to amino acid residues important for activity or structure in the similar polypeptides. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues in the ChMIrp polypeptide.

Where possible, the skilled artisan can also analyze the three-dimensional structure and amino acid sequence associated with that structure in similar polypeptides. In view of such information,
One skilled in the art can predict the alignment of amino acid residues of a ChMIrp polypeptide with respect to its three-dimensional structure, and can choose not to make drastic changes to amino acid residues predicted to be on the surface of the protein.
This is because such residues may be involved in important interactions with other molecules.

ChMIrp polypeptide analogs of the present invention can be determined by comparing the amino acid sequence of a ChMIrp polypeptide with related family members. Specific ChMIrp polypeptide-related family members include, but are not limited to, human chondromodulin (chondromodulin).
chondromodulin I, mouse ChMIrp, mouse chondromodulin I, rat chondromodulin I, bovine chondromodulin I, and rabbit chondromodulin I. This comparison was performed using the Pileup alignment (Wisconsin GCG
This can be accomplished by using the nucleotide sequence comparison tool (GenBank Accession Number (NAT) Program Package) or equivalence (overlap) comparison of multiple family members in conserved and non-conserved regions.

As shown in Figure 4, the deduced amino acid sequence of the human ChMIrp polypeptide (SEQ ID NO: 2) aligns with mouse ChMIrp, mouse chondromodulin I, rat chondromodulin I, bovine chondromodulin I, human chondromodulin I, and rabbit chondromodulin I (SEQ ID NOs: 4-9).
hMIrp polypeptide analogs can be determined using these or other methods well known to those of skill in the art. These overlapping sequences provide guidance for conservative and non-conservative amino acid substitutions that will generate additional ChMIrp analogs.
It is understood that these amino acid substitutions may consist of naturally occurring or non-naturally occurring amino acids. For example, as shown in FIG. 4, an alignment of these related polypeptides shows that potential ChMIrp analogs contain a Cy2A4 residue substituted with a Ser or Ala residue at position 276 in SEQ ID NO:2 (position 296 in FIG. 4).
s residue, a Cys residue substituted for a Ser or Ala residue at position 280 in SEQ ID NO:2 (position 300 in FIG. 4 ); or
The glutamic acid residue at position 281 in SEQ ID NO:2 (position 30 in FIG.
In addition, the glycine residue at position 285 in SEQ ID NO:2 (position 305 in FIG. 4) can be substituted with Pro and Ala, and the Arg residue at position 297 in SEQ ID NO:2 (position 317 in FIG. 4) can be substituted with Lys, Gly, or Arg.
The Cys residue at position 300 in SEQ ID NO:2 (position 320 in FIG. 4 ) may be replaced with a Ser or Ala residue, and the Cys residue at position 306 in SEQ ID NO:2 (position 320 in FIG. 4 ) may be replaced with a Ser or Ala residue.
4 ) may be substituted with a Ser or Ala residue, and the Val residue at position 310 in SEQ ID NO:2 (position 33 in FIG. 4 ) may be substituted with a Ser or Ala residue.
0) may be substituted with Ile, Met, Leu, Phe, Ala, or norleucine.

[0065] Additionally, one skilled in the art can generate test mutants containing a single amino acid substitution at each amino acid residue. The mutants can be screened using the activity assays described herein. Such mutants can be used to gather information on suitable mutants. For example, if a change to a particular amino acid is found that results in a mutant that is destroyed, undesirably reduced, or has inappropriate activity,
Variants with such changes are avoided. In other words, based on the information gathered by such routine experimentation, one skilled in the art can readily determine amino acids whose substitutions should be avoided, either alone or in combination with other mutations.

In a similar vein, in making such changes, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. The hydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine (+
3.8); Phenylalanine (+2.8); Cysteine/Cystine (+2.5)
;Methionine (+1.9);Alanine (+1.8);Glycine (-0.4);Threonine (-0.7);Serine (-0.8);Tryptophan (-0.9);Tyrosine (-1.3);Proline (-1.6);Histidine (-3.2);Glutamic acid (-3.5);Glutamine (-3.5);Aspartic acid (-3.5);
Asparagine (-3.5); Lysine (-3.9); and Arginine (-4.5
).

The importance of the hydropathic amino acid index in identifying interactive biological function for a protein is generally understood in the art (Kyte et al., J. Am. Chem. Soc., 1999, 113:1311-1325).
(E. et al., 1982, J. Mol. Biol. 157:105-31). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still maintain similar biological activity. In making changes based on hydropathic index, substitutions of amino acids whose hydropathic indexes are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that substitutions of similar amino acids can be made effectively on the basis of hydrophilicity, particularly when, as in the present invention, the biologically functional proteins or peptides they produce are contemplated for use in immunological embodiments.

[0069] U.S. Patent No. 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., a biological property of the protein. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.
0); Lysine (+3.0); Aspartic acid (+3.0±1); Glutamic acid (
+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5
±1); alanine (-0.5); histidine (-0.5); cysteine (-1.
0); methionine (-1.3); valine (-1.5); leucine (-1.8);
Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.
5); and tryptophan (-3.4).

In making changes based on similar hydrophilicity values, substitutions of amino acids whose hydrophilicity values are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. U.S. Patent No. 4,554,101 also teaches the identification and preparation of epitopes from a primary amino acid sequence based on hydrophilicity. Through the methods described in U.S. Patent No. 4,554,101, one of skill in the art can identify epitopes within a given amino acid sequence. These regions are also referred to as "epitope regions."

Numerous scientific publications have been devoted to the prediction of secondary structure and the identification of epitopes from amino acid analysis. Chou et al., 1974, Biochemistry, vol.
13:222-45; Chou et al., 1974, Biochemistry 11
3:211-22; Chou et al., 1978, Adv. Enzymol. Rela
t. Area Mol. Biol. 47:45-48; Chou et al., 1978
, Ann. Rev. Biochem. 47:251-276; and Chou et al., 1979, Biophys. J. 26:367-84.
Computer programs are available to aid in the prediction of antigenic sites and epitopic core regions in proteins.
n-Wolf analysis (Jameson et al., Compt. Appl. Biosci.
4(1):181-186, 1998 and Wolf et al., Comput.
osci., 4(1):187-191, 1988); the PepPlot program (Brutlag et al., CABS, 6:237-245, 1990, and We
Inberger et al., Science, 228:740-742, 1985) and a new program for protein tertiary structure prediction (Fetrow et al., Biol.
Technology, 11:479-483, 1993).

Computer programs are now available to aid in the prediction of secondary structure. One method of predicting secondary structure is based on homology modeling. For example, two polypeptides or proteins with greater than 30% sequence identity or greater than 40% similarity often have similar structural topologies. The recent growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the number of possible folds in a polypeptide's or protein's structure. Holm et al., 1999, Nucleic Acids Replicativ 1999, ...
s. 27:244-47. It has been suggested that there are a limited number of folds in a given polypeptide or protein, and that once a critical number of structures have been solved, structure prediction becomes dramatically more accurate (Breier, 1999).
Nerner et al., 1997, Curr. Opin. Struct. Biol. 7:3
69-376).

A further method for predicting secondary structure is called "threading".
)” (Jones, 1997, Curr. Opin. Struct. Biol.
7(3):377-87; Sippl et al., 1996, Structure 4(
1):15-19), "Profile Analysis" (Bowie et al., 1991, Sci.
ence, 253:164-170; Gribskov et al., 1990, Meth
ods Enzymol. 183:146-59; Gribskov et al., 198
7, Proc. Nat. Acad. Sci. U. S. A. 84(3):4355
~4358), and "evolutionary link
ge" (see Holm et al., supra, and Brenner et al., supra).

In a preferred embodiment, the variants are 1 to 3, or 1 to 5, or 1 to 10
or 1-15, or 1-20, or 1-25, or 1-50, or 1-75, or 1-100, or 100 or more amino acid substitutions, insertions, additions and/or deletions, where the substitutions are conservative or non-conservative, or any combination thereof, as indicated herein. Additionally, these variants may have additions of amino acid residues at the carboxyl or amino terminus (with or without leader sequences).

Preferred ChMIrp polypeptide variants include glycosylation variants in which the number and/or type of glycosylation sites have been altered compared to the native amino acid sequence. In one embodiment, the ChMIrp polypeptide variant contains a greater or lesser number of N-linked glycosylation sites than the amino acid sequence shown in SEQ ID NO: 2. The N-linked glycosylation sites are of the sequence Asn-X-S
The ChMIrp polypeptide variants are characterized by Asn-X-Thr or Asn-X-Thr, where the amino acid residue marked with X can be any amino acid residue except proline. Substitution of amino acid residues to create this sequence provides a potential new site for the addition of N-linked glycans. Alternatively, substitution to eliminate this sequence removes an existing N-linked glycan. Rearrangements of N-linked glycans are also provided in which one or more N-linked glycosylation sites (typically naturally occurring glycosylation sites) are eliminated and one or more new N-linked sites are created. Additional preferred ChMIrp polypeptide variants include cysteine variants in which one or more cysteine residues are deleted or replaced with another amino acid (e.g., serine) compared to the amino acid sequence shown in SEQ ID NO:2. Cysteine variants include:
They are useful when ChMIrp polypeptides must be refolded into a biologically active conformation, for example after isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number of cysteine residues to minimize interactions resulting from unpaired cysteines.

The term "ChMIrp fusion polynucleotide" refers to a fusion of a heterologous peptide or heterologous polynucleotide with a ChMIrp polypeptide, fragment and/or variant thereof. Heterologous peptides and polypeptides include, but are not limited to: epitopes that allow for detection and/or isolation of the ChMIrp fusion polypeptide; transmembrane receptor proteins or portions thereof (e.g., extracellular domains or transmembrane and intracellular domains);
Ligands or portions thereof that bind to transmembrane receptor proteins; enzymes or portions thereof that are catalytically active; polypeptides or peptides that promote oligomerization (e.g., leucine zipper domains); polypeptides or peptides that increase stability (e.g., immunoglobulin constant regions); and polypeptides that include an amino acid sequence as set forth in SEQ ID NO:2 or have a therapeutic activity that is distinct from a ChMIrp polypeptide variant.

[0077] Furthermore, the ChMIrp polypeptide may be fused to itself or to fragments, variants, or derivatives thereof. Fusions may be made at either the amino or carboxyl terminus of the ChMIrp polypeptide. Fusions may be direct without a linker or adapter molecule, or may be via a linker or adapter molecule (e.g., one or more up to about 20 amino acid residues to about 50 amino acid residues). The linker or adapter molecule may also be designed with a cleavage site for a DNA restriction endonuclease or protease to allow separation of the fused moieties. It is understood that once constructed, the fusion polypeptide may be derivatized according to the methods described herein.

In a further embodiment of the invention, the ChMIrp polypeptide, including fragments, variants and/or derivatives, is fused to the Fc region of human IgG.
Antibodies contain two functionally independent portions: a variable domain known as "Fab," which binds antigen, and a constant domain known as "Fc," which is involved in effector functions such as complement activation and phagocytic attack. Fc has a long serum half-life, while Fab is short-lived (Capon et al., Nature 337:525-31, 1989). When constructed with a therapeutic protein, the Fc domain may provide a longer half-life or may even incorporate functions such as Fc receptor binding, protein binding, complement fixation, and perhaps placental transfer. Ibid. Table I summarizes the uses of certain Fc fusions known in the art, including materials and methods applicable to the production of fused ChMIrp polypeptides.

[0079]

Table 1 In one example, a human IgG hinge region, CH2 and CH3 regions can be fused to either the N-terminus or C-terminus of a ChMIrp polypeptide using methods known to those skilled in the art. As another example, a portion of the hinge and the CH2 and CH3 regions can be fused. The resulting ChMIrpFc-fusion polypeptide can be purified by use of a Protein A affinity column.
It has been found that peptides and proteins fused to the Fc region exhibit substantially longer half-lives in vivo than their unfused counterparts. Fusion to an Fc region also allows for dimerization/multimerization of the fusion polypeptide. The Fc region may be a naturally occurring Fc region or may be altered to improve certain qualities (e.g., therapeutic qualities, circulation time, reduced aggregation, etc.).

The term "ChMIrp polypeptide derivative" refers to a ChMIrp polypeptide,
The term "covalently modified" refers to variants, or fragments thereof, that have been chemically modified, for example, by the covalent attachment of one or more water soluble polymers, N-linked carbohydrates, O-linked carbohydrates, sugars, phosphates, and/or other such molecules. Such modifications can be introduced into the molecule by reacting targeted amino acid residues of the purified or crude protein with an organic derivatizing agent that can react with selected side chains or terminal residues. The resulting covalent derivatives are also useful in programs directed at identifying residues important for biological activity. The derivatives can be used to identify, for example, the functional groups of the polypeptides, which are important for the identification of the functional groups.
It is modified in a manner that differs from the naturally occurring ChMIrp polypeptide, either in terms of the type or location of the molecules attached to the ChMIrp polypeptide. Derivatives further include deletion of one or more chemical groups naturally attached to the ChMIrp polypeptide.

For example, a polypeptide may be modified by the covalent attachment of one or more polymers, including, but not limited to, water soluble polymers, N-linked carbohydrates, O-linked carbohydrates, sugars, phosphates, and/or other such molecules. For example, the polymer selected is typically water soluble, such that the protein to which it is linked does not precipitate in an aqueous environment, such as a physiological environment. The polymer may be of any molecular weight, and may be branched or unbranched. Mixtures of polymers are included within the scope of ChMIrp polypeptide polymers. Preferably, for therapeutic use in the preparation of the final product, the polymer is pharma- ceutical acceptable.

Suitable water-soluble polymers or mixtures thereof include, but are not limited to, polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran (e.g., low molecular weight (e.g., about 6 kD dextran), cellulose, or other carbohydrate-based polymers, poly-(
N-vinylpyrrolidine) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), and vinyl alcohol. Also included in the present invention are bifunctional PEG cross-linking molecules that can be used to prepare covalently linked ChMIrp polypeptide multimers.

For acylation reactions, the polymer selected has a single reactive ester group. For reductive alkylation, the polymer selected has a single reactive aldehyde group. For example, the reactive aldehyde is polyethylene glycol propionaldehyde, which is water soluble, or mono _C1 - _C10 alkoxy or aryloxy derivatives thereof (see U.S. Pat. No. 5,252,714).

Pegylation of the ChMIrp polypeptide can be specifically carried out using any pegylation reaction known in the art. Such reactions are described, for example, in the following references: Francis et al., 1992,
Focus on Growth Factors 3:4-10; European Patent Nos. 0154316 and 0401384; and U.S. Pat. No. 4,179,323.
No. 37. For example, pegylation can be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer) as described herein.
) are suitable water-soluble polymers for use herein. As used herein, the terms "polyethylene glycol" and "PEG" refer to the polysaccharides that are capable of binding to proteins (
The term "PEG" is intended to include any form of PEG used to derivatize the PEG-containing copolymer (including mono ( _C1 - _C10 ) alkoxypolyethylene glycol or aryloxypolyethylene glycol).

In general, chemical derivatization can be carried out under any suitable condition used to react a biologically active substrate with an activated polymer molecule. Methods for preparing chemical derivatives of pegylated polypeptides generally include the following steps:
(b) reacting the polypeptide with an activated polymer molecule (e.g., a reactive ester or aldehyde derivative of a polymer molecule) under conditions such that the ChMIrp polypeptide variant is conjugated to one or more polymer molecules; and
Obtaining a reaction product. In general, optimal reaction conditions are determined based on known parameters and desired results. For example, the higher the ratio of polymer molecules:protein, the greater the proportion of polymer molecules that are bound. In one embodiment, the ChMIrp polypeptide derivative can have a single polymer molecule moiety at its amino terminus (see, for example, U.S. Patent No. 5,234,784).

[0086] Generally, conditions that may be alleviated or modulated by administration of the ChMIrp polypeptide derivatives of the invention include those described herein. However, the ChMIrp polypeptide derivatives disclosed herein may have additional activity, enhanced or decreased biological activity, or other properties (e.g., increased or decreased half-life) when compared to the underivatized molecule.

The term "biologically active ChMIrp polypeptide", "biologically active C
"ChMIrp polypeptide fragment,""biologically active ChMIrp polypeptide variant," and "biologically active ChMIrp polypeptide derivative" refer to a ChMIrp polypeptide having at least one activity characteristic of a ChMIrp polypeptide.
An immunogenic fragment of a ChMIrp polypeptide is a fragment that is capable of inducing antibodies in a host animal to be raised against the ChMIrp fragment.

[0088] "Naturally-occurring" or "native," when used in reference to biological material such as nucleic acid molecules, polypeptides, host cells, and the like, refers to a material that is found in nature and has not been manipulated by man. Similarly, "non-naturally occurring" or "non-native," as used herein, refers to a material that is not found in nature or that has been structurally modified or synthesized by man.

The term "isolated polypeptide" refers to (1) a polypeptide of the invention that, when isolated from a source cell, is separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally found; (2) a polypeptide of the invention that is not linked (by covalent or non-covalent interactions) to all or a portion of a polypeptide with which it is naturally associated; (3) a polypeptide of the invention that is operably linked (by covalent or non-covalent interactions) to a polypeptide with which it is not naturally associated; or (4) a polypeptide of the invention that is operably linked (by covalent or non-covalent interactions) to a polypeptide with which it is not naturally associated.
) refers to a polypeptide of the invention that is not naturally occurring. Preferably, the isolated polypeptide is substantially free of any other contaminating polypeptides or other contaminants found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, or research use.

The term "mature ChMIrp polypeptide" refers to a polypeptide that lacks a leader sequence, and may also include other modifications of the polypeptide, such as proteolytic processing of the amino terminus (with or without a leader sequence) and/or the carboxy terminus, cleavage of smaller polypeptides from larger precursors, N-linked and/or O-linked glycosylation, etc.

[0091] The term "mutein" refers to a mutant protein, polypeptide, variant, analog, or fragment of a ChMIrp polypeptide. Muteins of ChMIrp may be prepared by deletion, insertion, substitution, point mutation, truncation, addition, rearrangement, PCR amplification, site-directed mutagenesis, or other methods known in the art.

The terms "effective amount" and "therapeutically effective amount" refer to the amount of a ChMIrp polypeptide (or a ChMIrp antagonist) necessary to support an observable change in the level of one or more biological activities of the ChMIrp polypeptide described above, to result in a meaningful patient benefit (i.e., treatment, cure, prevention, or amelioration of a condition). When applied to an individual active ingredient, the term administered alone means:
It refers to the component alone. When applied to a combination, the term refers to the combined amount of active components that produces a therapeutic effect when administered in combination, either sequentially or simultaneously. The ChMIrp polypeptides having use in carrying out the present invention can be naturally occurring full-length polypeptides, or truncated polypeptides or modified homologs or analogs or derivatives or peptide fragments. Exemplary analogs include those in which one or more different amino acids between two species are replaced with different amino acids from another species. The different amino acids can also be replaced with any other amino acid, whether it is a conservative or non-conservative amino acid.

[0093] The term "pharmaceutical acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation substances suitable for achieving or enhancing the delivery of a ChMIrp polypeptide, ChMIrp nucleic acid molecule, or ChMIrp selective binding agent as a pharmaceutical composition.

The term "selective binding agent" refers to a molecule that has specificity for a ChMIrp molecule.
Selective binding agents include antibodies (e.g., polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, CDR-grafted antibodies, anti-idiotypic (anti-Id) antibodies against antibodies that may be labeled in soluble or bound form), as well as fragments, regions, or derivatives thereof provided by known techniques, including, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant techniques. Anti-ChMIrp selective binding agents of the present invention can be used to bind, for example, ChMIrp.
It is possible to bind a portion of the molecule that inhibits the binding of the ChMIrp molecule to the ChMIrp receptor.

As used herein, the terms "specific" and "specificity" refer to the ability of a selective binding agent to bind to a human ChMIrp polypeptide and to bind to a human non-ChMIrp polypeptide.
However, selective binding agents may also bind to ChMI polypeptides.
It will be appreciated that the present invention may also bind to orthologues of the ChMIrp polypeptide (i.e., interspecies forms of the ChMIrp polypeptide, such as mouse ChMIrp polypeptide and rat ChMIrp polypeptide). A preferred embodiment is
It is contemplated that antibodies that are highly specific for a polypeptide do not specifically cross-react with (ie, do not bind to) non-ChMIrp polypeptides.

The term "antigen" refers to a molecule or a portion of a molecule that can be bound by a selective binding agent, such as an antibody, and that can further induce an animal to produce an antibody capable of binding to an epitope of that antigen. An antigen can have one or more epitopes. The specific binding reaction referred to above means that the antigen reacts in a highly selective manner with its corresponding antibody and not with the multitude of other antibodies that may be elicited by other antigens.

[0097] ChMIrp polypeptides, fragments, variants, and derivatives can be used to prepare ChMIrp selective binding agents using methods known in the art. Thus, antibodies and antibody fragments that bind to ChMIrp polypeptides are within the scope of the present invention. Antibody fragments include portions of antibodies that bind to epitopes of ChMIrp polypeptides. Examples of such fragments include Fab and F(ab') fragments generated by enzymatic cleavage of full-length antibodies. Other binding fragments include fragments generated by recombinant DNA techniques (e.g., expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions). These antibodies can be, for example, polyclonal, polyclonal monospecific, monoclonal, recombinant, chimeric, humanized, human, single-chain, and bispecific antibodies.

Relatedness of Nucleic Acid Molecules and/or Polypeptides The term "identity," as known in the art, refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or nucleic acid molecule sequences, in this case perhaps as determined by the match between strings of nucleotide or amino acid sequences. "Identity"
measures the percent identity match between two or more sequences, with gapped alignments, positioned by a particular mathematical model computer program (i.e., an "algorithm").

The term "similarity" refers to a measure of similarity that includes both identical matches and conservative substitution matches, as opposed to "identity." Identity and similarity of related nucleic acid molecules and polypeptides can be readily calculated by known methods. Such methods include the Computational Molecular
lar Biology, Lesk, A. M. ed., Oxford University
City Press, New York, 1988;
g:Informatics and Genome Projects, Sm
Ith, D. W., Academic Press, New York, 19
93;Computer Analysis of Sequence Dat
a, Part 1, Griffin, A. M. and Griffin, H. G.
ed., Humana Press, New Jersey, 1994;
nce Analysis in Molecular Biology, vol.
Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M. Stockton Press, New York.
York, 1991); and Carillo and Lipman, SIA
M J. Applied Math., 48:1073, 1998. When conservative substitutions are applied to peptides and not to nucleic acid molecules, similarity only relates to the comparison of peptide sequences. Then, if two polypeptide sequences have, for example, 10/20 identical amino acids and the rest are all non-conservative substitutions, the percent identity and similarity are both 50%. In the same example, if there are five more positions that are conservative substitutions, the percent identity remains 50%, but the percent similarity is 75% (15/20). Thus, when conservative substitutions exist, the degree of similarity between two polypeptide sequences is higher than the percent identity between these two sequences.

Identity and similarity of related nucleic acid molecules and polypeptides can be readily calculated by known methods. Such methods include Computational Methods.
al Molecular Biology (ed. A.M. Lesk, Oxford
d University Press 1988);
g: Informatics and Genome Projects (D.
W. Smith, Academic Press 1993); Compute
Analysis of Sequence Data (Part 1,
In A. M. Griffin and H. G. Griffin, eds., Humana Pr
ess 1994);G. von Heinle, Sequence Anal
ysis in Molecular Biology (Academic P
ress 1987); Sequence Analysis Primer (
In M. Gribskov and J. Devereux, eds., M. Stockton
Press 1991); and Carillo et al., 1988, SIAM J.
Applied Math., 48:1073.

[0101] Differences in the nucleic acid sequence can result in conservative and/or non-conservative modifications of the amino acid sequence relative to the amino acid sequence of SEQ ID NO:2.

The term "conservative amino acid substitution" refers to the substitution of a natural amino acid residue with a non-natural residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. For example, a conservative substitution would result in the replacement of a non-polar residue in a polypeptide with any other non-polar residue. Additionally, any natural residue in a polypeptide can also be replaced with a non-polar residue, as described above as "alanine scanning mutagenesis."
The general rules for amino acid substitutions are shown in Table II below.

[0103] (Table II)

[0104]

Table 2 Conservative modifications to this amino acid sequence (and corresponding modifications to the encoding nucleotides) are predicted to generate ChMIrp with functional and chemical characteristics similar to those of naturally occurring ChMIrp.
Substantial alterations in the functional and/or chemical characteristics of rp can be achieved by selecting substitutions that differ significantly in (a) the structure of the backbone of the molecule in the region of the substitution (e.g., sheet or helix structure), (b) the charge or hydrophobicity of the molecule at the target site, or (c) its effect of maintaining the bulk of the side chain. Naturally occurring residues are classified based on common side chain attributes:
They can be divided into the following categories: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the human ChMIrp molecule that are homologous to the non-human ChMIrp, or into non-homologous regions of the molecule.

Conservative amino acid substitutions also include non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptidomimetics and other forms of reverse or inverse amino acid components.

Preferred methods to determine identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer programs for determining identity and similarity between two sequences include GAP (Devereux et al., Nucleic Acids Res.
12(1):387, 1984); Genetics Comput
er Group, University of Wisconsin, Med.
, BLASTP, BLASTN, and FASTA (Atsc
Examples of suitable GCG programs include, but are not limited to, the GCG program package, which includes the GCG program package (B. Hul et al., J. Molec. Biol. 215:403-410.1990).
The LAST X program is a joint project of the National Center for Biosciences.
The National Cancer Institute (NCBI) and other sources (BLAST Manual, Altschul et al., NCB NLM NIH
Bethesda, MD 20894; Altschul et al., J.; Molec.
Biol. 215:403-410.1990). The well-known Smith Waterman algorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences may result in matching of only short regions of the two sequences, and the aligned small regions may have very high sequence identity even if there is no significant relatedness between the two full-length sequences. Thus, in a preferred embodiment, the alignment method selected (GAP program) produces an alignment over at least 50 contiguous amino acids of the claimed polypeptide.

For example, the computer algorithm GAP (Genetics Computational Algorithm)
ter Group, University of Wisconsin, Me.
The algorithm (Dison, WI) is used to align the two polypeptides for which the percent sequence identity is to be determined for optimal matching of their respective amino acids (the "match range" as determined by the algorithm).
Disadvantage) (calculated as the average diagonal x 3;
"Average diagonal" is the average of the diagonals of the comparison matrix used; this "diagonal" is the score or number assigned to each perfect amino acid match by a particular comparison matrix) and gap extension penalty (usually 1/10 of the gap opening penalty), as well as PAM 25
A comparison matrix such as BLOSUM 62 or BLOSUM 63 is used with this algorithm. A standard comparison matrix (see Dayhoff et al., Atlas of Protein Sequencing for the PAM250 comparison matrix) is used with this algorithm.
In BLO and Structure, vol. 5, Supplement 3, 1978,
For the SUM 62 comparison matrix, see Henikoff et al., Proc. Na
lt. Acad. Sci USA, 89:10915-10919, 1992.) is also used by this algorithm.

Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol.
l. 48:443-453, 1970; Comparison matrix: Henikoff and Henikoff, Proc.
alt. Acad. Sci USA, 89:10915-10919, 1992
to BLOSUM 62; gap penalty: 12; gap length penalty: 4; similarity threshold: 0.

The GAP program is useful with these aforementioned parameters, which are the default parameters for polypeptide comparisons (no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol.
I. 48:443-453, 1970; comparison matrix: match = +10, mismatch = 0 gap penalty: 50 gap length penalty: 3.

The GAP program is also useful with these above parameters.
These above parameters are the default parameters for nucleic acid molecule comparisons.

Program Manual, Wisconsin Package, Ve
Other exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices, similarity thresholds, etc., including those set forth in rsion9 (September 1997), can be used by one of skill in the art. The particular choice to be made will depend on the particular comparison to be made (e.g., DNA to DNA, protein to protein, protein to DNA); and further on whether the comparison is between a set of sequences (in which case GAP or BestFit are generally preferred) or between one sequence and a large database of sequences (in which case FASTA or BLASTA are preferred).

SYNTHETIC It will be understood by those of skill in the art that the nucleic acid and polypeptide molecules described herein can be produced by recombinant and other means.

Nucleic Acid Molecules Nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of a ChMIrp polypeptide can be prepared in a variety of ways, including chemical synthesis, screening of cDNA or genomic libraries, expression library screening, and/or PCR of cDNA.
The amplification methods can be easily obtained by PCR amplification.

The recombinant DNA techniques used herein are generally as described in Sambrook,
(Molecular Cloning: A Laboratory Man
ual(Cold Spring Harbor Laboratory Pr
ess, Cold Spring Harbor, NY, 1989) and/or Ausubel et al. (Current Protocols in Mol.
ecular Biology, Green Publishers Inc.
and Wiley and Sons 1994), but are not limited to these.

The present invention provides nucleic acid molecules as described herein and methods for obtaining the molecules. Genes or cDNAs encoding "CHMIrp polypeptides" or fragments thereof can be obtained by hybridization screening of genomic or cDNA libraries or by PCR amplification. Probes or primers useful for screening libraries by hybridization can be made based on sequence information for other known genes or gene fragments from the same or related families of genes, such as, for example, conserved sites.

When a gene encoding a ChMIrp polypeptide is identified from one species, all or portions of the gene can be used as a probe to identify corresponding genes from other species (orthologs) or related genes from the same species (homologs). The probes or primers can be used to screen cDNA libraries from various tissue sources thought to express the ChMIrp gene.

[0120] Furthermore, part or all of the nucleic acid molecule having the sequence as shown in SEQ ID NO: 1 can be used to screen a genomic library to identify and isolate the gene encoding ChMIrp. Typically, moderate or high stringency conditions are used for screening to minimize the number of false positives obtained from the screen. Availability of the cDNA encoding ChMIrp or its function is a prerequisite for obtaining genomic DNA. Under stringent conditions, a DNA library is screened and the resulting clones are examined to see whether they contain, in addition to the coding region, the necessary regulatory sequence elements for gene expression (e.g., by fusing with the coding region of an appropriate reporter gene to check promoter function). Methods for screening DNA libraries under stringent conditions are taught, for example, in published European Patent Application No. EPA 0 174 143. Obtaining the genomic sequence allows the identification and characterization of ChMIrp for any possible interactions with known substances that regulate gene expression (e.g., transcription factors or steroids).
This allows the investigation of regulatory sequences located in non-coding regions of Irp (especially in the 5' flanking region) or, perhaps, to discover new substances that may have a specific effect on the expression of this gene. The results of such investigations will be used to investigate the mechanism of action of ChMIr
p expression and thus chondromodulin
n) provide the basis for the targeted use of such agents to directly affect the ability of cells to interact with receptors, so that specific reactions with the receptor and the resulting effects can be suppressed.

The scope of the present invention also includes DNA encoding subtypes of ChMIrp that may have properties different from those of the ChMIrp of the present invention, which are expression products formed by alternative splicing and have modified structures in specific regions, such as structures that may result in changes in affinity and specificity for receptors or changes in signal transduction properties and efficiency.

With the aid of the cDNA encoding ChMIrp, the cDNA was synthesized under low, medium or high stringency conditions.
It is possible to obtain a nucleic acid which encodes a polypeptide capable of hybridizing to the NA or a fragment thereof and binding the receptor, or which contains a sequence which encodes such a polypeptide.

Nucleic acid molecules encoding ChMIrp polypeptides may also be isolated by expression cloning using detection of positive clones based on the properties of the expressed protein.
Typically, nucleic acid libraries are screened by binding an antibody or other binding partner (e.g., a receptor or ligand) to the clone protein expressed and displayed on the host cell surface. The antibody or binding partner is modified with a detectable label to identify cells expressing the desired clone.

[0124] These polynucleotides can be produced, and the encoded polypeptides can be expressed, in accordance with recombinant expression techniques carried out in accordance with the instructions set forth below.
For example, by inserting a nucleic acid sequence encoding the amino acid sequence of a ChMIrp polypeptide into a suitable vector, one skilled in the art can easily generate large quantities of the desired nucleotide sequence. These sequences can then be used to generate detection probes or amplification primers. Alternatively, a polynucleotide encoding the amino acid sequence of a ChMIrp polypeptide can be inserted into an expression vector. By introducing the expression vector into a suitable host, the encoded ChMIrp polypeptide can be produced in large quantities.

Another method for obtaining suitable nucleic acid sequences is the polymerase chain reaction (PCR). In this method, cDNA is synthesized using the enzyme reverse transcriptase to produce poly(
A) is prepared from +RNA or total RNA. Two primers, typically complementary to two separate regions of a cDNA (oligonucleotides) that encode the amino acid sequence of a ChMIrp polypeptide, are then added to the cDNA using a polymerase such as Taq polymerase, which amplifies the region of the cDNA between the two primers.

Another means for preparing nucleic acid molecules encoding the amino acid sequence of a ChMIrp polypeptide is the method described by Engels et al. (Angew. Chem. Intl. Ed. 28:7).
Chemical synthesis using methods well known to those skilled in the art, such as those described by G. K. Schmidt, 16-34, 1989. These methods include, among others, the phosphate triester, phosphoramidite, and H-phosphonate methods for nucleic acid synthesis. The preferred method for such chemical synthesis is polymer-supported synthesis using standard phosphoramidite chemistry. Typically,
DNA encoding the amino acid sequence of the hMIrp polypeptide is several hundred nucleotides long. Nucleic acids longer than about 100 nucleotides can be synthesized using these methods.
The ChMIrp polypeptide may be synthesized as several fragments that can then be ligated together to form the full-length nucleotide sequence of the ChMIrp polynucleotide. Usually, the DNA fragment that encodes the amino terminus of the polypeptide has an ATG, which encodes a methionine residue. This methionine may or may not be present in the mature form of the ChMIrp polypeptide, depending on whether the polypeptide produced in the host cell is designed to be secreted from the cell.

In some cases, it may be desirable to prepare nucleic acid molecules encoding ChMIrp polypeptide variants or muteins.
The nucleic acid molecules encoding the variants can be prepared by site-directed mutagenesis, transposition, deletion, addition, truncation, PCR with desired point mutations, provided that the nucleic acid encodes a polypeptide capable of binding one or more members of the chondromodulin family.
Mutagenesis may be performed using R amplification, or other suitable methods (see Sambrook et al., supra, and Ausubel et al., supra, for a description of mutagenesis techniques). Chemical synthesis using the methods described by Engels et al., supra, may also be used to prepare such variants. Other methods known to those of skill in the art may be used as well.

In certain embodiments, the nucleic acid variants are capable of expressing ChMIr in a given host cell.
The codons may be altered for optimal expression of the ChMIrp polypeptide. The particular codon alterations will depend on the host cell and the ChMIrp polypeptide selected for expression. Such "codon optimization" may be performed by a variety of methods, such as by selecting codons that are preferred for use in genes that are highly expressed in a given host cell. Computer algorithms incorporating codon frequency tables such as "Eco_high.Cod" for codon preferences of highly expressed bacterial genes may be used, and may be implemented using the techniques of the University of Wisconsin.
onsin Package Version 9.0 (Genetics C
Other useful codon frequency tables include those provided by the Computer Group, Madison, WI.
Celegans_low. cod”, “Drosophila_high.c
od”, “Human_high.cod”, “Maize_high.cod”
", and "Yeast_high.cod.".

In other embodiments, the nucleic acid molecule encodes a ChMIrp variant having conservative amino acid substitutions as defined above, a ChMIrp variant including the addition and/or deletion of one or more N-linked or O-linked glycosylation sites, or a ChMIrp polypeptide fragment as described above. Furthermore, the nucleic acid molecule may encode any combination of ChMIrp variants, fragments, and fusion polypeptides described herein, provided that DNA modified in this manner may encode a polypeptide that may be found to be one or more members of the chondromodulin family of ligands and receptors.

Vectors and Host Cells A nucleic acid molecule encoding the amino acid sequence of a ChMIrp polypeptide can be inserted into an appropriate expression vector using standard ligation techniques. Vectors typically contain
Selected to be functional in the particular host cell to be used (i.e.
The vector is compatible with the host cell machinery so that gene amplification and/or gene expression occurs. Nucleic acid molecules encoding the amino acid sequence of the ChMIrp polypeptide can be expressed in prokaryotic host cells, yeast host cells, insect (baculovirus systems)
The ChMIrp polypeptide may be amplified/expressed in a host cell and/or a eukaryotic host cell. The choice of host cell will depend in part on whether the ChMIrp polypeptide is post-translationally modified (e.g., glycosylated and/or phosphorylated). If so, yeast host cells, insect host cells, or mammalian host cells are preferred. For a review of expression vectors, see Meth. Enz., vol. 185 (D.V. Goeddel,
ed., Academic Press 1990).

Typically, expression vectors used in any host cell will contain sequences for plasmid maintenance and for cloning and expression of foreign nucleotide sequences. Such sequences (collectively referred to as "flanking sequences"), in certain embodiments, will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

Optionally, the vector may include a "tag" coding sequence (i.e., an oligonucleotide molecule located at the 5' or 3' end of the ChMIrp polypeptide coding sequence); the oligonucleotide sequence may be polyHis (e.g., he
The tag typically encodes a 6His tag, another "tag" (e.g., FLAG, HA (influenza virus hemagglutinin) or myc (for which commercially available antibodies exist). This tag is typically fused to the polypeptide upon expression of the polypeptide and can serve as a means for affinity purification of the ChMIrp polypeptide from host cells. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix (6His
This can be achieved by using a suitable means, such as a nickel column, that has an affinity for the tag.
If desired, the tag can be subsequently removed from the purified ChMIrp polypeptide by various means, such as using specific peptidases for cleavage.

The flanking sequences may be homologous (i.e., from the same species and/or lineage as the host cell), heterologous (i.e., from a species other than the host cell species or lineage), hybrid (i.e., a combination of flanking sequences from more than one source), or synthetic, or the flanking sequences may be from any suitable source, including, but not limited to, C.
The flanking sequences may be native sequences that normally function to regulate hMIrp polypeptide expression. Thus, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences are functional in and can be activated by the host cell's machinery.

Flanking sequences useful in the vectors of the present invention may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein, other than the ChMIrp gene flanking sequences, have been previously identified by mapping and/or restriction endonuclease digestion and may therefore be isolated from a suitable tissue source using the appropriate restriction endonuclease. In some cases, the full-length nucleotide sequence of one or more flanking sequences may be known, where:
The flanking sequences may be synthesized using the methods described herein for nucleic acid synthesis or cloning.

[0135] When all or only part of a flanking sequence is known, the flanking sequence can be obtained using PCR and/or by screening a genomic library with appropriate oligonucleotides and/or flanking sequence fragments from the same or another species.

[0136] If the flanking sequences are not known, a fragment of DNA containing the flanking sequences can be isolated from a larger DNA fragment which may contain, for example, coding sequences or another gene.
Isolation may be accomplished by restriction endonuclease digestion to generate the appropriate DNA fragment, followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, Calif.), or other methods known to those of skill in the art. Selection of appropriate enzymes to achieve this end may be accomplished by:
This will be readily apparent to one skilled in the art.

[0137] An origin of replication is typically part of commercially available prokaryotic expression vectors, which aids in the amplification of the vector in the host cell. Amplification of the vector to a certain copy number may in some cases be important for optimal expression of the ChMIrp polypeptide. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence and ligated into the vector. For example,
Plasmid pBR322 (New England Biolabs, Beve
Origins of replication from Escherichia coli (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomaviruses such as HPV or BPV) are suitable for most gram-negative bacteria, and various origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (e.g., SV
The 40 origin is often used only because it contains the early promoter.
is not required.

A transcription termination sequence is typically located 3' to the end of a polypeptide coding region and serves to terminate transcription. Usually, a transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly-T sequence. This sequence can be easily cloned from a library or is commercially available as part of a vector, but it can also be easily synthesized using methods for nucleic acid synthesis as described herein above.

A selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Exemplary selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin) on the prokaryotic host cell; (b) complement an auxotrophic deficiency of the cell; or (c) alternatively supply critical nutrients not available from complex media. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. A neomycin resistance gene may also be used for selection in prokaryotic and eukaryotic host cells.

Other selection genes can be used to amplify the expressed gene. Amplification is the process by which a gene in greater demand for the production of a protein important for growth is repeated in tandem in successive generations of the chromosome of the recombinant cell. Examples of suitable selection markers for mammalian cells include dihydrofolate reductase (DHF) and IFN-glucose (IFN-glucose)ase.
HFR) and thymidine kinase. Mammalian cell transformants are placed under selection pressure, where only the transformants are uniquely adapted to survive by virtue of the selection gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is continuously changed, thereby leading to the amplification of both the selection gene and the DNA encoding the ChMIrp polypeptide. As a result, increased amounts of the ChMIrp polypeptide are synthesized from the amplified DNA.

A ribosome binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located 3' to the promoter and 5' to the coding sequence of the expressed ChMIrp polypeptide. Shine-Dalgarno sequences are variable but are typically polypurines (i.e., high A-G content). Many Shine-Dalgarno sequences have been identified,
Each can be readily synthesized using the methods described herein and used in a prokaryotic vector.

A leader sequence, or signal sequence, can be used to direct a ChMIrp polypeptide from the host cell in which it is synthesized. Typically, the signal sequence is located in the coding region of the ChMIrp nucleic acid molecule or
or directly 5' to the ChMIrp polypeptide coding region. Many signal sequences have been identified, and any signal sequence that is functional in a selected host cell may be used in combination with the ChMIrp gene or cDNA. Thus, the signal sequence may be homologous (native) or heterologous to the ChMIrp gene or cDNA. Additionally, the signal sequence may be chemically synthesized using the methods described herein. For the most part, secretion of the ChMIrp polypeptide from the host cell by the presence of a signal peptide is achieved by the secretion of the ChMIrp polypeptide from the host cell.
This results in the removal of the signal peptide from the rp polypeptide.

The signal sequence may be a component of the vector or may be part of the ChMIrp DNA inserted into the vector. Native ChMIrp DNA encodes a signal sequence at the amino terminus of the protein that is cleaved during post-translational processing of the molecule to form the mature ChMIrp polypeptide product. Included within the scope of the present invention are ChMIrp nucleotides with the native signal sequence as well as ChMIrp nucleotides in which the native signal sequence has been deleted and replaced with a heterologous signal sequence. The heterologous signal sequence selected should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native ChMIrp signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected from the group of, for example, alkaline phosphatase, penicillinase, or heat-stable enterotoxin II leaders. For yeast secretion, the native ChMIrp signal sequence may be selected from the group of yeast invertase,
The ChMIrp polypeptide may be substituted with the α-factor, α-factor protein, or acid phosphatase signal sequence. In mammalian cell expression, the native signal sequence of the ChMIrp polypeptide is sufficient, although other mammalian signal sequences may be suitable.

In some cases where glycosylation is desired in a eukaryotic host cell expression system, various nucleic acid or polypeptide sequences can be engineered to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be altered or a pro-sequence can be added,
This may also affect glycosylation. The final protein product may have one or more additional amino acids at position 1 (relative to the first amino acid of the mature protein) associated with expression, which may not be completely removed. For example, the final protein product may have one or two amino acid residues found in a peptidase cleavage site attached to the N-terminus. Alternatively, the use of some enzyme cleavage sites may result in a slightly truncated form of the desired ChMIrp polypeptide if the enzyme cleaves such a region in the mature polypeptide.

In many cases, transcription of a nucleic acid molecule is increased by the presence of one or more introns in the vector; this is especially true when the polypeptide is produced in a eukaryotic host cell, particularly a mammalian host cell. The intron used may be naturally present in the ChMIrp gene, especially if the gene used is a full-length genomic sequence or a fragment thereof. If the intron does not naturally occur in the gene (as is the case for most cDNAs), the intron may be obtained from another source. The location of the intron with respect to the 5'-flanking sequence and the ChMIrp gene is generally important, since the intron must be efficiently transcribed. Thus, when the ChMIrp cDNA molecule is transcribed, the preferred location of the intron is 3' to the transcription start site and 5' to the poly-A transcription termination sequence. Preferably, the intron is placed on one side or the other (i.e., 5' or 3') of the cDNA so as not to interfere with the coding sequence. The invention may be practiced with any intron from any source, including viral, prokaryotic and eukaryotic (plant or animal), provided that the intron is compatible with the host cell into which it is inserted. Synthetic introns are also included herein.
Optionally, more than one intron can be used in the vector.

[0146] Expression and cloning vectors of the present invention will typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding the ChMIrp polypeptide.

A promoter is a molecule that controls the transcription of a structural gene (generally about 10
A promoter is a non-transcribed sequence located upstream (i.e., 5') to the start codon of a gene (within 0-1000 bp). Promoters are usually grouped into one of two classes: inducible and constitutive. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions (e.g., the presence or absence of a nutrient or a change in temperature). Constitutive promoters, on the other hand, initiate the production of a gene product continuously; i.e., they exert little or no control over gene expression. A large number of promoters (recognized by a variety of potential host cells)
are well known. A suitable promoter is operably linked to the DNA encoding the ChMIrp polypeptide by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into a vector. The native ChMIrp promoter sequence may be used to direct amplification and/or expression of the ChMIrp nucleic acid molecule. However, heterologous promoters are preferred if they allow for greater transcription and higher production of expressed protein compared to the native promoter and are compatible with the host cell system selected for use.

Suitable promoters for use with prokaryotic hosts include the β-lactamase and lactose promoter systems; alkaline phosphatase; tryptophan (tr
p) promoter systems; and hybrid promoters, such as the tac promoter. Other known bacterial promoters are also suitable. These sequences have been published so that one of skill in the art can ligate them to the desired DNA using linkers or adapters as needed to provide any useful restriction sites.

Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2),
These include promoters derived from the genomes of viruses such as bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, herpes viruses and, most preferably, Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and the actin promoter.

Additional promoters that may be of interest in controlling ChMIrp gene transcription include, but are not limited to, the SV40 early promoter region (B
Ernoist and Chambon, Nature 290:304-1
0, 1980); CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 2
2:787-97, 1980); herpes thymidine kinase promoter (W
Agner et al. , Proc. Natl. Acad. Sci. U. S.
A. 78:1444-45, 1981); the regulatory sequence of the metallothionein gene (
Brinster et al. , Nature 296:39-42, 198
2); prokaryotic expression vectors such as the β-lactamase promoter (Vill
a-Kamaroff et al. , Proc. Natl. Acad. Sci
. U.S.A., 75:3727-31, 1978); or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. U.S.A., 75:3727-31, 1978);
Also of interest are the following animal transcriptional control regions which exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region, active in pancreatic acinar cells (Swift, et al., J. Med. 1999, 80:21-25, 1983);
et al. , Cell 38:639-46, 1984;
tal. , Cold Spring Harbor Symp. Quant.
Biol. 50:399-409, 1986; MacDonald, Hepat.
logy 7:425-515, 1987); the insulin gene regulatory region active in pancreatic β cells (Hanahan, Nature 315:115-2
2, 1985); Immunoglobulin gene control region active in lymphocytes (Gr
Osschedl et al. , Cell 38:647-58, 1984;
Adams et al. , Nature 318:533-38, 1985
; Alexander et al. , Mol. Cell. Biol. ,7:1
436-44, 1987); mouse mammary tumor virus control region active in testis, breast, lymphocytes, and mast cells (Leder et al., Cell 4
5:485-95, 1986); the albumin gene control region active in the liver (Pinkert et al., Genes and Devel. 1:26
8-76, 1987); the α-fetoprotein gene control region active in the liver (Krumlauf et al., Mol. Cell. Biol., 5:1
639-48, 1985; Hammer et al. ,Science 23
5:53-58, 1987); the α1-antitrypsin gene control region active in the liver (Kelsey et al., Genes and Devel. 1:
161-71, 1987); the β-globin gene control region active in myeloid cells (Mogram et al., Nature 315:338-40, 1987);
985; Kollias et al. , Cell 46:89-94, 198
6); myelin basic protein gene regulatory region active in brain oligodendrocytes (Readhead et al., Cell 48:703-12, 1
987); the myosin light chain-2 gene regulatory region active in skeletal muscle (Sani,
Nature 314:283-86, 1985); as well as the gonadotropin releasing hormone gene control region active in the hypothalamus (Mason et al.
, Science 234:1372-78, 1986).

An enhancer sequence can be inserted into the vector to increase transcription of the DNA encoding the ChMIrp polypeptide of the present invention by higher eukaryotes. Enhancers act on a promoter to increase transcription, usually at about 1 nucleotide.
Enhancers are cis-acting elements of DNA ranging from 0 to 300 bp in length.
The relative orientation and position are independent. They are found 5' and 3' to the transcription unit. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, α-fetoprotein, and insulin). However, typically enhancers from viruses are used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer are exemplary enhancing elements for the activation of eukaryotic promoters. The enhancer can be spliced into the vector at the 5' or 3' position to the ChMIrp nucleic acid molecule, but is typically located at the 5' position from the promoter.

[0152] The expression vectors of the present invention can be constructed from a starting vector, such as a commercially available vector. Such a vector may or may not contain all of the desired flanking sequences. If one or more of the desired flanking sequences are not already within the vector being used, they can be obtained individually and ligated into the vector. The methods used to obtain each of the flanking sequences are well known to those of skill in the art.

Preferred vectors for carrying out the present invention are those that are suitable for use in bacterial host cells, insect host cells,
and vectors compatible with mammalian host cells. Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen).
en, Company, Carsbad, CA), pBSII (Stratag
ene Company, La Jolla, CA), pET15 (Novag
en, Madison, WI), pGEX (Pharmacia Biotec
h, Piscataway, NJ), pEGFP-N2 (Clontech, P
alo Alto, CA), pETL (BlueBacII, Invitrog
pFastBac1, pFastBacHT, and pFastBacDu
al (Gibco/BRL, Grand Island, NY).

[0154] Further suitable vectors include, but are not limited to, cosmids, plasmids, or
These vector systems include vectors that are either recombinant or modified viruses, but these vector systems are suitable for use in the host cell of choice.
It is understood that the vector must be compatible with
Bluescript® plasmid derivatives (high copy
Several ColEl-based phagemids, Stratagene Cloning
Systems, La Jolla CA), and the Taq amplified PCR products were cloned.
PCR cloning plasmids (e.g., TOPO ^T.M. TA Cloning Kit, PCR2.1 Pro
(plasmid derivatives, Invitrogen, Carlsbad, CA)
Plasmids, as well as mammalian vectors, yeast vectors or baculovirus vectors.
Viral vectors such as the current system (pBacPAK plasmid derivatives, Clontech
ech, Palo Alto, Calif.

After the vector has been constructed and the nucleic acid molecule encoding the ChMIrp polypeptide has been inserted into the appropriate site of the vector, the complete vector can be inserted into a suitable host cell for amplification and/or polypeptide expression. Transformation of the expression vector for the ChMIrp polypeptide into a selected host cell can be accomplished by well-known methods, including such methods as transfection, infection, calcium chloride, electroporation, microinjection, lipofectin, DEAE-dextran methods, or other known techniques. The selected method may, in part, be
Depending on the type of host cell used, these and other suitable methods are well known to those of skill in the art and are described, for example, in Sambrook et al., supra.

[0156] The host cell may be a prokaryotic host cell (e.g., E. coli) or a eukaryotic host cell (e.g., yeast cell, insect cell, or vertebrate cell). When the host cell is cultured under appropriate conditions, it synthesizes a ChMIrp polypeptide, which can then be collected from the culture medium (if the host cell secretes the polypeptide into the medium) or directly from the host cell that produces the polypeptide (if the polypeptide is not secreted). The selection of an appropriate host cell depends on various factors, such as the desired expression level, the polypeptide modifications desired or necessary for activity (e.g., glycosylation or phosphorylation), and the ease with which it folds into a biologically active molecule.

Yeast and mammalian cells are preferred hosts for the present invention. The use of such hosts offers the substantial advantage that they can also perform post-translational peptide modifications, including glycosylation. Many recombinant DNA strategies exist which utilize strong promoter sequences and high copy number plasmids for use in the production of the desired proteins in these hosts.

Yeast-recognized leader sequences on cloned mammalian gene products produce and secrete peptide-bearing leader sequences (i.e., pre-peptides). Mammalian cells provide post-translational modifications to the protein molecule, including correct folding or glycosylation at the correct sites.

Mammalian cells that may be useful as hosts include cells of fibroblast origin (e.g., VERO or CHO-K1) and their derivatives. For mammalian hosts, several possible vector systems are available for the expression of the desired ChMIrp polypeptide. A wide variety of transcriptional and translational regulatory sequences may be utilized, depending on the nature of the host. Transcriptional and translational regulatory signals may be derived from viral sources such as adenovirus, bovine papilloma virus, simian virus, etc., in which the regulatory signals are associated with specific genes that have high levels of expression. Alternatively, promoters from mammalian expression products (e.g., actin, collagen, myosin, etc.) may be utilized. Transcriptional initiation regulatory signals that allow repression or activation may be selected so that expression of the gene may be regulated.
Useful signals are regulatory signals that are temperature sensitive (so that expression can be repressed or inhibited by changing the temperature) or chemically regulated (
For example, metabolic products.

As is widely known, translation of eukaryotic mRNA is initiated at a codon that encodes an initiating methionine. For this reason, eukaryotic promoters and
It is preferable to ensure that the link between the DNA sequence encoding the desired receptor molecule does not contain any intervening codons which may code for methionine (i.e., AUG). The presence of such codons may result in the formation of a fusion protein (AU
A frameshift mutation (when an AUG codon is present in the same reading frame as the DNA sequence encoding the desired receptor molecule) or a frameshift mutation (when an AUG codon is present in the same reading frame as the DNA sequence encoding the desired receptor molecule).
(if not in the same reading frame as the desired ChMIrp polypeptide coding sequence).

[0161] Expression of ChMIrp polypeptides can also be achieved in prokaryotic cells. Preferred prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc. The most preferred eukaryotic host is E. coli. Bacterial hosts of particular interest include E. coli K12 strain 294 (ATCC 31446), E. coli
X1776 (ATCC 31537), E. coli W3110 (F￣, Λ￣
, prototrophic strain (ATCC 27325)), and other enterobacteria (e.g., Sa
Imonella typhimurium or Serratia marc
Prokaryotic hosts include Pseudomonas escens, as well as various Pseudomonas species. The prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.

Prokaryotic cells (e.g., E. coli, Bacillus subtilis, Pseudomonas sp., Streptococcus aureus,
In order to express the desired ChMIrp polypeptide in a desired Bacillus subtilis,
The sequence encoding the receptor molecule is operably linked to a functional prokaryotic promoter.
Such a promoter may be constitutive or, more preferably,
or can be regulatable (i.e., inducible or derepressible).
An example of a constitutive promoter is the int promoter of bacteriophage λ.
and the bla promoter of the β-lactamase gene of pBR322.
Examples of inducible prokaryotic promoters include the bacteriophage λ (P _L and P_R), and the major right and left promoters of E. c
The trp, recA, lacZ, lacI, gal and tac promoters of oli
α-amylase (Ulmane et al., J. Bacteriol. 162:1
76-182, 1985), the σ28-specific promoter of Bacillus subtilis (Gilma
et al., Gene 32:11-20, 1984), bacteriophage of Bacillus
(Gryczan, T. J., The Molecular B
iology of the Bacilli, Academic Press
, Inc., New York, 1982), and Streptomyces sp.
(Ward et al., Mol. Gen. Genet. 203:468-478
Prokaryotic promoters include those described by Glick (J. Ind. 1986).
Microbiol. 1:277-282, 1987);
, Biochimie 68:505-516, 1986); and Gotte
Sman, Ann. Rev. Genet. 18:415-442 1984)
More outlined below.

Proper expression in prokaryotic cells also requires the presence of a ribosome binding site upstream from the gene coding sequence. Such ribosome binding sites include, for example,
Gold et al. (Ann. Rev. Microbiol. 35:365-404, 1
981).

The desired ChMIrp polypeptide coding sequence and an operably linked promoter can be introduced into a recipient prokaryotic or eukaryotic cell as a non-replicating DNA (or RNA) molecule, which can be linear or, more preferably, a closed covalently linked circular molecule. Because such molecules are incapable of autonomous replication, expression of the desired receptor molecule can occur through transient expression of the introduced sequence. Alternatively, persistent expression can occur through integration of the introduced sequence into the host chromosome.

In one embodiment, vectors are used that can integrate the desired gene sequences into the host cell chromosome. Cells that have stably integrated the introduced DNA into their chromosomes can also be selected by introducing one or more markers that allow for selection of host cells that contain the expression vector. Markers can include those that express auxotrophy in the host (e.g., common yeast auxotrophic markers leu21 or ura3), biocide (biocid), or other similar markers.
e) Complementing resistance (e.g., antibiotics) or heavy metals (e.g., copper), etc. The selectable marker gene can be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection.

In a preferred embodiment, the introduced sequence is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors can be used for this purpose. Important factors in selecting a particular plasmid or viral vector, such as the convenience of the recipient cells containing the vector (the number of copies of the vector desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species), can be recognized and selected from recipient cells that do not contain these vectors.

Any of a range of yeast gene expression systems may also be used. Examples of such expression vectors include the yeast 2 micron circle, expression plasmids (YEP13, YV
Such plasmids include plasmids 1001-1005 (such as 1001-1005, ...
mp. 19:265-274 (1982); Breach, The Molec
ular Biology of the Yeast Saccharomy
ces: Life Cycle and Inheritance, Cold
Spring Harbor Laboratory, Cold Spring
Harbor, N. Y. , pp. 445-470 (1981); Broach, C.
ell 28:203-204 1982).

For mammalian hosts, several possible vector systems are available for expression. One class of vectors that uses DNA elements providing autonomous replication of an extrachromosomal plasmid is derived from animal viruses (e.g., bovine papilloma virus,
Polyomavirus, adenovirus, or SV40 virus).
The second class of vectors relies on the integration of the desired gene sequences into the host chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can also be selected by incorporating one or more markers which allow for the selection of host cells which contain the expression vector. The markers can provide prototropy for auxotrophic hosts, such as resistance to biocides (e.g., antibiotics), or heavy metals (e.g., copper), etc.
The selectable marker gene may be operably linked to the DNA sequence to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be required for optimal synthesis of mRNA. These elements include:
It may contain splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements are described in Okayama, H. (Mol. Cell. Biol. 3:280 1999).
Preferred eukaryotic vectors include those described by
PWLNEO, PSV2CAT, POG44, PXTI, pSG, pSVK3,
Examples include pBPV, pMSG, and pSVL (Pharmacia).

Preferred prokaryotic vectors include plasmids such as those capable of replicating in E. coli (e.g., pBR322, ColEl, pSC101, p
ACYC 184, πVX, pQE70, pQE60, pQE9, pBG, pD
10, Phage script, psixl74, pbmescript S
K, pbsks, pNH8A, pNHIBa, pNH18A, pNH46A (S
L rare gone), ptrc99a, pKK223-3, pKK233
For example, such plasmids include those described in Maniatis, T. et al. (Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor
Press, Cold Spring Harbor, N.Press. Y. (1982)
Bacillus plasmids include pC194, pC
221, pT127, etc. Such plasmids are
n,T. (The Molecular Biology of the Ba
cilli, Academic Press, New York (1982)
Suitable Streptomyces plasmids include pISJ101 (Kendall et al., J. Bacteriol. 169).
: 4177-4183 1987) and Streptomyces bacteriophages such as ΦC31 (Chater et al., Sixth International
Symposium on Actinomycetales Biolo
gy, Akademiai Kaido, Budapest, Hungary,
1986, pp. 45-541. Streptomyces plasmids include:
John et al. (Rev. Infect. Dis. 8:693-704, 1986,
and Izaki, K. (Jpn. J. Bacteriol 33:729-74
2 1978). However, any other plasmid or vector can be used as long as they are replicable and viable in the host cell.

Once a DNA sequence containing vector or construct is prepared for expression,
The DNA construct can be introduced into a suitable host. A variety of techniques can be used, such as protoplast fusion, calcium phosphate precipitation, electroporation or other conventional techniques. After fusion, the cells are grown in culture and screened for the appropriate activity. Expression of the sequence results in the production of the ChMIrp polypeptide.

Suitable host cells or cell lines include mammalian cells, such as Chinese hamster ovary cells (CHO; ATCC No. CCL61), CHO DHFR cells (U
rlaub et al., Proc. Natl. Acad. Sci. U. S. A, 97:4
216-4220, 1980), human embryonic kidney (HEK), 293 cells or 293T cells (ATCC number CRL 1573), or 3T3 cells (ATCC
No. CCL92).

The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening, product production and purification are known in the art. Other suitable mammalian cell lines include monkey COS-1 (ATCC No. CRL 1650) and CO
S-7 (ATCC No. CEL 1651) and CV-1 (ATCC No. CCL70) cell lines. Further exemplary mammalian host cells include primate and rodent cell lines, including transformed cell lines. Normal diploid cells, cell lines derived from in vitro culture of primary tissues, and primary explants are also suitable. Candidate cells may be genotypically deficient in the selection gene or may contain a dominantly acting selection gene. Other suitable mammalian cell lines include, but are not limited to, Swi
Mouse neuroblastoma cell line N2A derived from ss, Balb-c or NIH mice
cells, HeLa cells, mouse L-929 cells, 3T3 cells, or the BHK or HaK hamster cell lines, which are available from the ATCC. Each of these cell lines is known to and available to those of skill in the art of protein expression.

Similarly, also useful as host cells suitable for the present invention are bacterial cells, such as various strains of E. coli (e.g., HB101 (ATCC No.
33694), DH5α, DH10, and MC1061 (ATCC No. 5
3338) are well known as host cells in the field of biotechnology. B. subtilis, Pseudomonas spp., other Bacillus subtilis,
Various strains of Streptomyces spp., Streptomyces spp., and the like may also be used in the present methods.

Many strains of yeast cells known to those of skill in the art are also available as host cells for expression of the polypeptides of the present invention (e.g., Saccharomyces, i.
chia, Candida, Hansenula, and Torulopsis
) (Bitter, G., Heterologous Gene Express
sion in Yeast In: Berger, S. L. and Kimme
l, A. R., 152:673-684, 1987). Preferred yeast strains include, for example, Saccharomyces cerevisiae, which is
It can be easily transformed with DNA either by preparation of spheroplasts or by treatment with alkaline salts (e.g., LiCl) (Itoh et al., 2001).
(E. et al., J. Bactriol., 153:163, 1983). Some proteins expressed in yeast cells are efficiently screened into the culture medium, while others accumulate intracellularly.

Additionally, insect cell systems can be utilized in the methods of the present invention. Such systems are described, for example, in Kitts et al. (Biotechniques 14:810-817).
, 1993), Lucklow (Curr. Opin. Biotechnol.
4:564-572, 1993) and Lucklow et al. (J. Virol. 6
7:4566-4579, 1993). A preferred insect cell is Sf
-9 and Hi5 (Invitrogen, Carlsbad, Calif.).
Preferably, the insect cells are infected with recombinant baculovirus particles that have the ChMIrp polypeptide sequence integrated into their genome. The baculovirus infection leads to efficient eukaryotic expression of the recombinant ChMIrp. Such baculovirus expression systems include the Bac-to-Bac® system (Life Technologies) and the BacPAK® system (Clontech).

Polypeptide Production Host cells containing the ChMIrp polypeptide expression vector can be cultured using standard media well known to those skilled in the art. The media usually contains all the nutrients necessary for the growth and survival of the cells. Suitable media for culturing E. coli cells include, for example, Luria Broth (LB) and/or Terrificial Broth (TBB).
A suitable medium for culturing eukaryotic cells is Ros Broth (TB).
ewell Park Memorial Media 1640 (RPMI
1640), Minimum Essential Medium (MEM), and/or Dulbecco's
and Modified Eagle Media (DMEM), all of which may be supplemented with serum and/or growth factors as indicated by the particular cell line being cultured. A suitable medium for insect culture is Grace's medium, supplemented with yeastolate, lactalbumin hydrolysate and/or fetal bovine serum as needed.

Typically, an antibiotic or other compound useful for the selective growth of transformed cells is added as a supplement to the medium. The compound used is determined by the selection marker element present on the plasmid with which the host cell was transformed. For example, if the selection marker element is kanamycin resistance, the compound added to the culture medium is kanamycin. Other compounds for selective growth medium include ampicillin, tetracycline and neomycin.

The amount of ChMIrp polypeptide produced by a host cell can be assessed using standard methods known in the art, including, but not limited to, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, HPLC separation, immunoprecipitation, and/or activity assays.

If the ChMIrp polypeptide is designed to be secreted from the host cell,
The majority of the polypeptide can be found in the cell culture medium.
If the p polypeptide is secreted from the host cell, the ChMIrp polypeptide is present in the cytoplasm and/or nucleus (for eukaryotic host cells) or in the cytosol (for bacterial host cells).

For ChMIrp polypeptides present in the cytoplasm and/or nucleus (for eukaryotic hosts) or cytosol (for bacterial host cells) of a host cell,
The host cells are typically first disrupted mechanically or with detergent to release the intracellular contents into a buffer solution.
It can then be isolated from the solution.

Purification of ChMIrp polypeptides from solution can be accomplished using a variety of techniques. The polypeptide may be tagged at either its carboxy or amino terminus with a tag such as hexahistidine (ChMIrp polypeptide/hexaHis
), or other small peptides, such as FLAG (Eastman Koda
k Co., New Haven, CT) or myc (Invitrogen
If the polypeptide has been synthesized to contain a tag such as ChMIrp polypeptide (e.g., a monoclonal antibody that specifically recognizes the ChMIrp polypeptide), or a fused IgG Fc fragment, the polypeptide can be essentially purified in one step by passing the solution over an affinity column (i.e., a monoclonal antibody that specifically recognizes the ChMIrp polypeptide) where the column matrix has high affinity for the tag or for the polypeptide directly. For example, polyhistidine binds with great affinity and specificity to nickel, and thus a nickel affinity column (e.g., a Qiagen® nickel column) can be used to purify the ChMIrp polypeptide/polyHis. (See, e.g., Ausubel et al., eds., Current Protocols in Protein Sciences, 1999, 144:131-132, 1999).
ls in Molecular Biology, Section 10.11.8, Joh
(See, e.g., John Wiley & Sons, New York, 1993.) Additionally, ChMIrp polypeptides can be purified through the use of monoclonal antibodies capable of specifically recognizing and binding to ChMIrp polypeptides.

[0182] If the ChMIrp polypeptide is prepared without an attached tag and if an antibody is not available, other well-known procedures for purification can be used, including, but not limited to, ion exchange chromatography, molecular sieve chromatography, HPLC, native gel electrophoresis coupled with gel elution, and preparative isoelectric focusing ("Isoprime" machine/technique,
Examples of purification techniques include those available from the National Institute of Standards and Technology (NIS), Hoefer Scientific. In some cases, two or more of these purification techniques may be combined to achieve increased purity.

[0183] If the ChMIrp polypeptide is produced intracellularly, the intracellular material (including inclusion bodies for gram-negative bacteria) can be extracted from the host cell using any standard method known to those of skill in the art. For example, host cells can be lysed by lysis with detergent (e.g., Triton X-100), French press, homogenization, and/or sonication followed by centrifugation to release the periplasmic/cytoplasmic contents.

When ChMIrp polypeptides form inclusion bodies within the cytoplasm, the inclusion bodies may often be associated with the inner and/or outer surface of the cell membrane and thus are found primarily in the pellet material after centrifugation. The pellet material is then
Treatment with extreme pH or chaotropic agents (e.g., detergents, guanidine, guanidine derivatives, urea, or urea derivatives) in the presence of reducing agents (e.g., dithiothreitol at alkaline pH, or carboxyethylphosphine at acidic pH) releases, disrupts, and solubilizes the inclusion bodies. The ChMIrp polypeptide in solubilized form can then be analyzed using gel electrophoresis, immunoprecipitation, and the like. If it is desired to isolate the ChMIrp polypeptide, isolation can be performed as described herein or by methods such as those described in Marston et al. (Meth. Enz.
This can be accomplished using standard methods, such as those described in (J. Am. Chem. Soc., 182:264-275, 1990).

In some cases, the ChMIrp polypeptide may not be biologically active upon isolation. Various methods for "refolding" or converting the polypeptide into its tertiary structure and generating disulfide bonds can be used to restore biological activity. Such methods include solubilizing the solubilized polypeptide at pH 7, usually above pH 7, and in the presence of a particular concentration of a chaotropic agent.
The refolding/oxidation solution includes exposure to a 2000 ng/l 2000 ng/l 1000 ng/l 15 ...
Co-solvents include b-mercaptoethanol (bME)/dithio-b(ME). Co-solvents can be used to increase the efficiency of refolding, and the more common reagents used for this purpose include glycerol, polyethylene glycols of various molecular weights, arginine, etc.

If inclusion bodies are not formed to a significant extent upon expression of the ChMIrp polypeptide, the polypeptide will be found primarily in the supernatant following centrifugation of the cell homogenate, from which it may be further isolated using methods as described herein.

In situations where it is desirable to partially or completely purify the ChMIrp polypeptide so that it is partially or completely free of contaminants, standard methods known to those skilled in the art may be used. Such methods include, but are not limited to, electrophoretic separation followed by electroelution, various types of chromatography (affinity, immunoaffinity, molecular sieve, and/or ion exchange), and/or high pressure liquid chromatography. In some cases, it is preferable to use one or more of these methods for complete purification.

[0188] ChMIrp polypeptides, fragments, and/or derivatives thereof can also be prepared by chemical synthesis methods (e.g., solid phase peptide synthesis) using techniques known in the art, such as those described in, for example, Merrifield et al. (J.A.
m. Chem. Soc. 85:2149, 193), Houghton et al.
oc Natl Acad. Sci. USA 82:5132, 1985), and Stewart and Young (Solid Phase Peptide
de Synthesis, Pierce Chemical Co. , Roc
Such polypeptides may be synthesized with or without a methionine at the amino terminus. Chemically synthesized ChMIrp polypeptides or fragments may be oxidized to form disulfide bridges using the methods set forth in these references. Chemically synthesized ChMIrp polypeptides, fragments or derivatives may be synthesized using the techniques set forth in the corresponding C5H1 polypeptides, either recombinantly produced or purified from natural sources.
They are predicted to have biological activity comparable to the hMIrp polypeptides, fragments or derivatives, and therefore may be used interchangeably with recombinant or naturally occurring ChMIrp polypeptides.

Another means of obtaining the ChMIrp polypeptide is by purification from biological samples, such as tissues and/or fluids from sources in which the ChMIrp polypeptide is naturally found. Such purification can be carried out using methods for protein purification as described herein. During purification, the presence of the ChMIrp polypeptide can be monitored, for example, using antibodies prepared against recombinantly produced ChMIrp polypeptide or peptide fragments thereof.

Many additional methods for producing nucleic acids and polypeptides are known in the art and can be used to produce polypeptides with specificity for ChMIrp. See, for example, Roberts et al., Proc. N
atl. Acad. Sci. U. S. A. 94:12297-12303, 19
See, e.g., 97, which describes the production of a fusion protein between an mRNA and its encoded peptide. Roberts, R., Curr. Opin. C
hem. Biol 3:268-73, 1999. Additionally, U.S. Patent No. 5,824,469 describes a method for obtaining oligonucleotides capable of performing a specific biological function. The procedure involves generating a heterogeneous pool of oligonucleotides, each having a 5' randomized sequence, a central preselection sequence, and a 3' randomized sequence. The resulting heterogeneous pool is introduced into a population of cells that does not exhibit the desired biological function. Subpopulations of cells are then screened for those that exhibit the predetermined biological function. From the subpopulation, oligonucleotides capable of performing the desired biological function are isolated.

U.S. Patent Nos. 5,763,192; 5,814,476;
Nos. 3,323 and 5,817,483 describe a process for producing peptides or polypeptides. This is accomplished by producing stochastic genes or fragments thereof and then introducing these genes into a host cell that produces one or more proteins encoded by the stochastic genes. The host is then screened to identify those clones that produce peptides or polypeptides with the desired activity.

Another method for producing peptides or polypeptides is the Athersys
PCT/US98/20094 (WO99/15
650) (“Random Activation of Gene Expr.
The process is described in the paper entitled "Regenerative Gene Discovery (RAGE-GD)," which involves the activation of endogenous gene expression or overexpression of genes by in situ recombination methods. For example, the expression of an endogenous gene is activated or increased by incorporating, by non-homologous or aberrant recombination, a regulatory sequence into the target cell that can activate the expression of the gene. The target DNA is first subjected to radiation and inserted into a gene promoter. The promoter is finally placed at the branch point in front of the gene to initiate transcription of the gene, which results in the expression of the desired peptide or polypeptide.

These methods can also be used to generate comprehensive ChMIrp polypeptide expression libraries, which can then be used in a variety of assays, such as biochemical assays, cellular assays and whole organism assays (e.g., plants, mice, etc.).
) can be used for high throughput phenotypic screening.

ChMIrp Proteins, Polypeptides, Fragments, Variants and Muteins Polypeptides of the present invention include isolated ChMIrp polypeptides and polypeptides related thereto, including fragments, variants, fusion polypeptides and derivatives, as described hereinabove.

[0203] The ChMIrp fragments of the invention may result from truncations at the amino terminus of the polypeptide (with or without the leader sequence), at the carboxy terminus, and/or internal deletions. Most deletions and insertions, and particularly substitutions, are not expected to result in drastic changes in the characteristics of the ChMIrp protein. However, when it is difficult to predict the exact effect of a substitution, deletion, or insertion prior to implementation, one of skill in the art will recognize that the effect is evaluated by routine screening assays. For example, variants are typically produced by site-directed mutagenesis of a nucleic acid encoding the ChMIrp polypeptide, expression of the variant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, e.g., by immunoaffinity adsorption on a polyclonal anti-ChMIrp antibody column (to adsorb the variant by binding to at least one remaining immune epitope). In preferred embodiments, the truncations and/or deletions comprise about 10 amino acids, or about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than about 100 amino acids. The polypeptide fragments so produced comprise about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids, or about 250 amino acids, or about 275 amino acids, or about 300 amino acids. Such ChMIrp polypeptide fragments may optionally include an amino-terminal methionine residue.

The ChMIrp polypeptide variants of the present invention contain one or more amino acid substitutions, additions and/or deletions compared to SEQ ID NO: 2. In preferred embodiments, the variants contain 1-3, or 1-5, or 1-10, or 1-15, or 1-20, or 1-25, or 1-50, or 1-75, or 1-
It has 100 or more than 100 amino acid substitutions, insertions, additions and/or deletions, where the substitutions may be conservative as described above or non-conservative, or a combination thereof.
The rp variant may comprise the amino acid sequence shown in SEQ ID NO:2, in which amino acids 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, 100, 101, 102, 103, 104, 105, 106, 107, 1
08, 109, 110, 111, 112, 113, 114, 115, 116, 1
17, 118, 119, 120, 121, 122, 123, 124, 125, 1
26, 127, 128, 129, 130, 131, 132, 133, 134, 1
35, 136, 137, 138, 139, 140, 141, 142, 143, 1
44, 145, 146, 147, 148, 149, 150, 151, 152, 1
53, 154, 155, 156, 157, 158, 159, 160, 161, 1
62, 163, 164, 165, 166, 167, 168, 169, 170, 1
71, 172, 173, 174, 175, 176, 177, 178, 179, 1
80, 181, 182, 183, 184, 185, 186, 187, 188, 1
89, 190, 191, 192, 193, 194, 195, 196, 197, 1
98, 199, 200, 201, 202, 203, 204, 205, 206, 2
07, 208, 209, 210, 211, 212, 213, 214, 215, 2
16, 217, 218, 219, 220, 221, 222, 223, 224, 2
25, 226, 227, 228, 229, 230, 231, 232, 233, 2
34, 235, 236, 237, 238, 239, 240, 241, 242, 2
43, 244, 245, 246, 247, 248, 249, 250, 251, 2
52, 253, 254, 255, 256, 257, 258, 259, 260, 2
61, 262, 263, 264, 265, 266, 267, 268, 269, 2
70, 271, 272, 273, 274, 275, 276, 277, 278, 2
79, 280, 281, 282, 283, 284, 285, 286, 287, 2
88, 289, 290, 291, 292, 293, 294, 295, 296, 2
97, 298, 299, 300, 301, 302, 303, 304, 305, 3
06, 307, 308, 309, 310, 311, 312, 313, 314, 3
One or more amino acids from the group consisting of up to 15, 316, or 317 are replaced with another amino acid. The variant may have the addition of amino acid residues at either the carboxy terminus or the amino terminus (with or without a leader sequence).

[0203] Preferred ChMIrp polypeptide variants include glycosylation variants in which the number and/or type of glycosylation sites is altered compared to the native ChMIrp polypeptide. In one embodiment, the ChMIrp variants include a greater or lesser number of N-linked glycosylation sites. The N-linked glycosylation sites are characterized by the sequence Asn-X-Ser, where the amino acid residue designated as X can be any type of amino acid except proline. Substitution of amino acid residues to create this sequence provides a possible new site for the addition of an N-linked glycosyl chain. Alternatively, substitution to remove this sequence removes an existing N-linked glycosyl chain. Also provided is a reconstitution of an N-linked glycosyl chain in which one or more N-linked glycosylation sites (typically naturally occurring sites) are removed and one or more new N-linked sites are generated.

[0203] Those skilled in the art can use well-known techniques to determine suitable variants of a native ChMIrp polypeptide. For example, they can predict suitable regions of the molecule that can be altered without destroying biological activity. They will also recognize that even regions that may be important for biological activity or structure can be subject to conservative amino acid substitutions without destroying the biological activity or adversely affecting the polypeptide structure.

To predict suitable regions of the molecule that may be altered without destroying activity, one of skill in the art may target regions believed to be important for activity. For example, if similar polypeptides with similar activity from the same or other species are known, one of skill in the art may compare the amino acid sequence of the ChMIrp polypeptide to such similar polypeptides. After making such a comparison, one of skill in the art may determine the residues and portions of the molecule that are conserved between the similar polypeptides. One of skill in the art may determine the residues and portions of the molecule that are conserved between the similar polypeptides.
One skilled in the art will also be aware that changes in regions of the hMIrp molecule are unlikely to adversely affect biological activity and/or structure, and may conceivably substitute chemically similar amino acids for naturally occurring residues (e.g., conservative amino acid residue substitutions) even in relatively conserved regions while retaining activity.

One of skill in the art may also consider structure-function studies to identify residues in similar polypeptides that are important for activity or structure. In light of such comparisons, one of skill in the art may identify ChM sequences that correspond to amino acid residues in similar polypeptides that are important for activity or structure.
The importance of amino acid residues in the Irp can be predicted. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues of ChMIrp.

[0201] If available, one of skill in the art can also analyze crystal structures of similar polypeptides and the amino acid sequences associated with those structures. Given that information, one of skill in the art can predict the alignment of amino acid residues of the ChMIrp polypeptide with respect to its three-dimensional structure. One of skill in the art can choose not to make drastic changes to amino acid residues predicted to be on the surface of the protein, because such residues may be involved in important interactions with other molecules.

[0202] Furthermore, one skilled in the art can generate test variants containing single amino acid substitutions at each amino acid residue. The variants can be screened using the activity assays disclosed herein. Such variants are used to gather information about suitable variants; for example, if it is found that a change to a particular amino acid residue results in the destruction of activity, variants containing such a change are avoided. Thus, when attempting to find acceptable additional variants based on the information gathered from such experiments, one skilled in the art will know the amino acids for which additional substitutions should be avoided, either alone or in combination with other mutations.

[0203] The ChMIrp fusion polypeptides of the present invention include ChMIrp polypeptides, fragments, variants or derivatives fused to heterologous functional portions of peptides or proteins. Heterologous peptides and proteins include, but are not limited to, epitopes that allow for detection and/or isolation of the ChMIrp fusion polypeptide, transmembrane receptor proteins or portions thereof (e.g., extracellular domains), or ligands or portions thereof that bind to transmembrane receptor proteins, catalytically active enzymes or portions thereof, proteins or peptides that promote oligomerization (e.g., leucine zipper domains) and proteins or peptides that increase stability or circulating half-life (e.g., immunoglobulin constant regions). The ChMIrp polypeptide may be fused to itself or to a fragment, variant or derivative thereof. Fusions may be performed with ChMIrp.
This can be at either the amino or carboxy terminus of the polypeptide and can be direct, without a linker or adapter molecule, or can be via a linker or adapter molecule (e.g., from one or more amino acid residues up to about 20 amino acid residues, or up to about 50 amino acid residues). The linker or adapter molecule can also be designed to include a cleavage site for a DNA restriction endonuclease or a cleavage site for proteolytic cleavage to allow separation and subsequent folding of the fused moieties.

[0204] In addition, the circular shunting of the ChMIrp polypeptide
rly permuted structural analogs are envisioned as part of the present invention.

The development of recombinant DNA methods has made it possible to study the effects of sequence rearrangements on protein folding, structure and function. The approach used in generating novel sequences involves linear rearrangements of the amino acid sequence.
This approach is similar to the approach of naturally occurring pairs of related proteins by r reorganization (Cunningham et al., Proc. Natl.
Acad. Sci. U. S. A. 76:3218-3222, 1979; Tea
Ther and Erfle, J. Bacteriol. 172:3837-38
41, 1990; Schimming et al., Eur. J. Biochem. 204
:13-19, 1992; Yamiuchi and Minamikawa, F.E.
BS Lett 260:127-130, 1991; MacGregor et al.
FEBS Lett. 378:263-266, 1996). The first in vitro application of this type of reconstitution to a protein was reported by Goidenberg and Creighton (J. Mol. Biol. 165:407-413, 1989).
The new N-terminus was introduced at an internal site (breakpoint (b)) of the original sequence.
A breakpoint (breakpoint) is selected such that the new sequence has the same amino acid order as the original from that breakpoint until an amino acid at or near the original C-terminus is reached. At this point, the new sequence can be added directly or by adding additional sequence segments (
The new sequence is joined to an amino acid at or near the original N-terminus, either through a linker or a linker, and the new sequence continues the same as the original until it reaches an amino acid at or near the amino acid that was N-terminal to the break in the original sequence, and this residue forms the new C-terminus of the chain.

This approach has been applied to proteins in the size range of 58 to 462 amino acids (Goldenberg & Creighton, J. Mol. Biol. 2002, 14:131-132).
ol. 165:407-413, 1983; Li & Coffino, Mol
Cell. Biol. 13:2377-2383, 1993). The proteins examined represent a wide range of structural classes, including primarily α-helical proteins (interleukin-4; Kreitman et al., Cytokine
7:311-318, 1995), proteins that contain primarily β-sheets (interleukin-1; Horlick et al., Protein Eng. 5:427-4
31, 1992) or a protein containing a mixture of the two (yeast phosphoribosylanthranilate isomerase; Luger et al., Science 243:20
6-210, 1989).

In a preferred embodiment, the ChMIrp polypeptide, fragment, variant and/or derivative is fused to the Fc region of human IgG. In one example, the hinge, CH2 and CH3 of human IgG are fused to the Fc region of human IgG using methods known to those of skill in the art.
The three regions may be fused to either the N-terminus or C-terminus of the ChMIrp polypeptide. In another example, a portion of the hinge region and the CH2 and CH3 regions may be fused. The ChMIrp Fc fusion polypeptide thus produced has the following structure:
Protein A affinity column (Pierce, Rockford,
The Fc region can be purified using a ELISA kit (IL) for purification. Furthermore, peptides and proteins fused to an Fc region have been found to exhibit half-lives in vivo that are substantially greater than their unfused counterparts. Fusion to an Fc region also allows for dimerization/multimerization of the fusion polypeptide. The Fc region can be a naturally occurring Fc region or can be altered to improve certain qualities, such as therapeutic qualities, circulation time, reduced aggregation, etc.

[0208] ChMIrp polypeptide derivatives are also included within the scope of the present invention. Covalent modifications of the ChMIrp proteins of the present invention are included within the scope of the present invention. Variant ChMIrp proteins can be conveniently prepared by in vitro synthesis. Such modifications can be introduced into the molecule by reacting targeted amino acid residues of purified or crude proteins with organic derivatizing agents capable of reacting with selected side chains or terminal residues. The resulting covalent derivatives are useful in programs to identify residues important for biological activity.

Cysteine residues are most commonly reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteine residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β(5-imidazoyl)propionic acid, chloroacetyl phosphate, N-alkalimaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoic acid, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidine residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidine side chain. p-Bromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysine and amino terminal residues are reacted with succinic anhydride or carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysine residues. Other suitable reagents for derivatizing α-amino-containing residues include imidoesters (e.g., methylpicoline imidate); pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;
O-methylisourea; 2,4 pentanedione; and transaminase (catalyzing reaction with glyoxylate).

Arginine residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions due to the high _pKa of the guanidine functional group. Furthermore, these agents derivatize the lysine functional group and the ε of arginine.
Can react with amino groups.

The specific modification of tyrosine residues has itself been extensively studied, with particular interest in the introduction of spectral labels into tyrosine residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosine species and 3-nitro derivatives, respectively. Tyrosine residues are iodized using ¹²⁵ I or ¹³¹ I to prepare labeled proteins for use in radioimmunoassays (the chloramine T method described above is suitable).

Carboxyl side groups (aspartic acid or glutamic acid) are selectively modified by reaction with a carbodiimide (R ¹ ) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3(4azonia-4,4-dimethylpentyl)carbodiimide. Additionally, aspartic acid and glutamine residues are converted to aspartic acid and glutamic acid residues by reaction with ammonium ions.

Derivatization with a bifunctional agent allows attachment of the ChMIrp polypeptide to a water-insoluble support matrix or surface for use in a method for cleaving a ChMIrp polypeptide fusion polypeptide to release and recover the cleaved polypeptide.
Commonly used teaching materials include 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, and N-hydroxysuccinimide esters (e.g., esters with 4-azidosalicylic acid).
, homobifunctional imidoesters (including disuccinimidyl esters such as 3,3'-dithio (including disuccinimidyl esters such as succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Methyl-
Derivatizing agents such as 3-[p-azidophenyldithio]propioimidate
Alternatively, photoactivatable intermediates can be produced that are capable of forming crosslinks in the presence of light.
and 4,330,440, reactive water-insoluble matrices (eg, cyanogen bromide activated carbohydrates) and reactive substrates are used for immobilizing proteins.

Glutamine and asparagine residues are frequently deamidated to the corresponding glutamyl (glutamic acid) and aspartyl (aspartic acid) residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues is within the scope of the invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of serine or threonine residues, methylation of the alpha amino acids of lysine, arginine, and histidine (Creighton, T. E. Proteins, 1999).
:Structure and Molecule Properties, W
．． H. Freeman & Co. , San Francisco, pp. 79
-86, 1983), acetylation of the N-terminal amine, and in some cases, amidation of the C-terminal carboxyl group. Such derivatizations result in chemically modified ChMIrp polypeptide compositions in which the ChMIrp polypeptide is conjugated to a polymer. The polymer selected is typically water-soluble so that the protein to which it is attached does not precipitate in an aqueous environment (e.g., a physiological environment). The polymer selected is usually a single reactive group (e.g., an active ester for acylation or an aldehyde for alkylation) so that the degree of polymerization can be controlled as provided in the methods of the invention.

The polymers can be of any molecular weight and can be branched or unbranched. The polymers are typically each from 2 kDa to about 100 kDa.
(wherein the term "about" refers to the fact that in a preparation of water-soluble polymers, some molecules may be more or somewhat less than the stated molecular weight.) The average molecular weight of each polymer is preferably between about 5 kDa and about 50 kDa, more preferably between about 12 kDa and about 40 kDa, and most preferably between about 20 kDa and about 35 kDa. Suitable water-soluble polymers or mixtures thereof include, but are not limited to, the following:
Linked or O-linked carbohydrates, sugars, phosphates, polyethylene glycols (PEG) (including forms of PEG used to derivatize proteins, including mono-(C1-C10), alkoxy-polyethylene glycol, or aryloxy-polyethylene glycol), monomethoxy-polyethylene glycol, dextran (e.g., low molecular weight (e.g., dextran of about 6 kD)), cellulose, or other carbohydrate-based polymers, poly-(N-vinylpyrrolidine) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), and vinyl alcohol. Also included in the invention are bifunctional crosslinking molecules that can be used to prepare covalently linked polypeptide multimers of a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a ChMIrp polypeptide variant.

In general, chemical derivatization can be performed under any suitable condition used to react an activated polymer molecule with a protein. The method for preparing a chemical derivative of a polypeptide generally includes the following steps: (a) reacting a polypeptide with an activated polymer molecule (e.g., a reactive ester or aldehyde derivative of the polymer molecule) under conditions in which the polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a ChMIrp polypeptide variant is conjugated to one or more polymer molecules, and (b) obtaining a reaction product. Optimal reaction conditions are determined based on known parameters and desired results. For example, the higher the ratio of polymer molecules to protein, the higher the percentage of conjugated polymer molecules. In one embodiment, a ChMIrp polypeptide derivative can have a single polymer molecule moiety at the amino terminus. See, for example, U.S. Patent No. 5,234,784.

Pegylation of the ChMIrp polypeptide can be specifically carried out using any pegylation reaction known in the art. Such reactions are described, for example, in the following references: Focus on Growth, 1999, 14:131-135, 19 ...0, 1990, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 199
h Factors 3:4-10, 1992; European Patent Nos. 0154316 and 0401384. For example, PEGylation can be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer), as described below.

A particularly preferred water-soluble polymer for use herein is polyethylene glycol (abbreviated PEG). As used herein, polyethylene glycol is meant to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy-polyethylene glycol or aryloxy-polyethylene glycol. PEG is a linear or branched, neutral polyether available in a wide range of molecular weights, and is soluble in water and many organic solvents. PEG is
when present in water, is effective in excluding other polymers or peptides;
This is mainly due to its high dynamic chain mobility and hydrophilic nature, and therefore
When attached to other proteins or polymer surfaces, it forms a water shell or hydration sphere. PEG is non-toxic and
It is non-immunogenic and is Food and Drug Administration (FDA) approved for internal consumption.

Proteins or enzymes when conjugated to PEG have demonstrated reduced bioactivity, non-antigenic properties and reduced clearance rates when administered to animals.
Veronese et al., Preparation and Properties
of Monomethoxypoly(ethylene glyco.)
-modified Enzymes for Therapeutic Ap
plications, J. M. Hariis, ed., Poly(Ethylene)
Glycol Chemistry--Biotechnical and
Biomedical Applications 127-36, 1992 (
(2003), incorporated herein by reference. This is due to the exclusion properties of PEG in preventing recognition by the immune system. In addition, PEG has been widely used in surface modification procedures to reduce protein adsorption and improve blood compatibility. S.
W. Kim et al., Ann. N. Y. Acad. Sci. 516:116-30 1
987; Jacobs et al., Artif. Organs 12:500-501,
1988; Park et al., J. Poly. Sci, Part A 29:1725
-31, 1991, herein incorporated by reference. Hydrophobic polymer surfaces (e.g., polyurethane and polystyrene) can be modified with PEG (MW 3,400).
and used as a non-thrombogenic surface. In these studies, the surface properties (contact angle) were more consistent with hydrophilic surfaces due to the hydration effect of PEG. More importantly, the adsorption of proteins (albumin and other plasma proteins) was greatly reduced, which results from the high chain mobility, hydration sphere and protein exclusion properties of PEG.

PEG (MW 3,4000) was used in surface immobilization studies (Park et al., J. Biol.
ed. Mat. Res. 26:739-45, 1992), whereas PEG (MW 5,000) was most advantageous in reducing protein antigenicity (F.M. Veronese et al., J.M. Harr
is edition, Poly (Ethylene Glycol) Chemistry--
Biotechnical and Biomedical Application
ions 127-36, supra).

In general, chemical derivatization can be carried out under any suitable conditions used to react a biologically active substance with an activated polymer molecule.
The process for preparing p polypeptide generally comprises the steps of: (a)
(b) reacting the polypeptide with polyethylene glycol (e.g., a reactive ester or aldehyde derivative of PEG) under conditions such that the ChMIrp polypeptide is conjugated to one or more PEG groups; and (b) obtaining a reaction product. In general, optimal reaction conditions for the acylation reaction are determined based on known parameters and desired results. For example, the higher the PEG:protein ratio, the higher the poly-
The percentage of PEGylated product is increased.

In a preferred embodiment, the ChMIrp polypeptide derivative has a single PEG moiety at the N-terminus, see US Patent No. 8,234,784, incorporated herein by reference.

In another embodiment, the ChMIrp polypeptide may be chemically linked to biotin. This biotin/Cdk11 polypeptide molecule may then be bound to avidin, resulting in a tetravalent avidin/biotin/Cdk11 polypeptide molecule. The ChMIrp polypeptide may also be linked to dinitrophenol (DN
The antibody can be covalently linked to anti-DNP or trinitrophenol (TNP) and the resulting conjugate precipitated with anti-DNP or anti-TNP-IgM to form a 10-valent decameric conjugate.

In general, conditions that may be alleviated or modulated by administration of the ChMIrp polypeptide derivatives of the invention include those conditions described herein for ChMIrp polypeptides. However, the ChMIrp polypeptide derivatives disclosed herein may have additional activity, enhanced or decreased biological activity, or other characteristics (e.g., increased or decreased half-life) compared to the underivatized molecule.
may have:

It will be apparent that DNA microarray technology may be utilized in accordance with the present invention. DNA microarrays are small arrays of DNA molecules arranged on a solid support (e.g., glass).
It is a tiny, high-density array of nucleic acids. Each cell or element in the array is
They have many copies of a single species of DNA, which acts as a target for hybridization to its cognate mRNA. In expression profiling using DNA microarray technology, mRNA is first extracted from a cell or tissue sample and then enzymatically converted to fluorescently labeled cDNA. This material is hybridized to the microarray, and the unbound cDNA is then removed.
The DNA is removed by washing. The expression of the individual genes represented on the array is then visualized by quantifying the amount of labeled cDNA specifically bound to each target DNA. In this way, the expression of thousands of genes can be quantified in a high-throughput parallel manner from a single sample of biological material.

This high throughput expression profiling has a wide range of applications for the ChMIrp molecules of the present invention, including, but not limited to, the identification and validation of ChMIrp disease-related genes as therapeutic targets, molecular toxicity studies of ChMIrp molecules and their inhibitors, the generation of surrogate markers for population stratification and clinical trials, and the enhancement of Cdk11-related small molecule drug discovery by aiding in the identification of selective compounds in high throughput screening (HTS).

Selective Binding Agents As used herein, the term "selective binding agent" refers to a selective binding agent that binds to one or more ChM
The term "selective binding agent" refers to a molecule having specificity for an Irp polypeptide. Suitable selective binding agents include, but are not limited to, antibodies and derivatives thereof, polypeptides, and small molecules. Suitable selective binding agents may be prepared using methods known in the art. An exemplary polypeptide selective binding agent of the present invention is a ChM
It may bind specific portions of the Irp polypeptide, thereby inhibiting the activity or function of the ChMIrp polypeptide.

[0231] Selective binding agents (e.g., antibodies and antibody fragments) that bind ChMIrp polypeptides are within the scope of the present invention. Antibodies can be polyclonal (including monospecific polyclonal), monoclonal (mAb), recombinant, chimeric, humanized (e.g., CDR-grafted), human, single chain and/or bispecific antibodies, as well as fragments, variants or derivatives thereof. Antibody fragments include those portions of antibodies that bind to an epitope of a ChMIrp polypeptide. Examples of such fragments include Fab fragments and F(ab') fragments generated by enzymatic cleavage of full-length antibodies.
) fragments. Other binding fragments include those produced by recombinant DNA techniques, such as the expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions.

Polyclonal antibodies directed against a ChMIrp polypeptide are generally raised in animals (e.g., rabbits or mice) by multiple subcutaneous or intraperitoneal injections of ChMIrp and an adjuvant. It may be useful to conjugate the ChMIrp polypeptide, or a variant, fragment or derivative thereof, to a carrier protein that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor).
Agglutinating agents, such as alum, are also used to enhance the immune response. After immunization, animals are bled and the serum is assayed for anti-ChMIrp antibody titer.

Monoclonal antibodies directed against ChMIrp may be produced using any method that provides for the production of antibody molecules by continuous cell lines in culture. Examples of suitable methods for preparing monoclonal antibodies include those described by Kohler et al., Natl. J. Clin. Immunol. 1999, 143:1311-1323, 1999.
The hybridoma method of J. Med. 256:495-497, 1975, and Ko
zbor, J. Immunol. 133:3001, 1984;
et al., Monoclonal Antibody Production Tec
hniques and Applications, pp. 51-63 (Marc
Examples of such methods include the human B cell hybridoma method by El Dekker, Inc., New York, 1987.

Hybridoma cell lines producing monoclonal antibodies reactive with ChMIrp polypeptides are also provided by the present invention.

The monoclonal antibodies of the present invention may be modified for use as therapeutic agents.
One embodiment is a "chimeric" antibody, in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass. Fragments of such antibodies are also included, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci., 2003, 143:1311-1315, 2003).
(See, e.g., J. Med. Acad. Sci. U.S.A. 81:6851-6855, 1985, incorporated herein by reference).

In another embodiment, the monoclonal antibody of the invention is a "humanized" antibody. Methods for humanizing non-human antibodies are well known in the art (see U.S. Pat. Nos. 5,585,089 and 5,693,762). Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. Humanization can be achieved by methods described in the art (Jones et al., Nature 321:522-52, 1986; Jones et al., Nature 321:522-52, 1986; Jones et al., Nature 321:522-52, 1986; Jones et al., Nature 321:522-52, 1986).
Riechmann et al., Nature 332:323-327, 1988;
erhoeyen et al., Science 239:1534-1536, 1988
)

[0237] Fully human antibodies that bind ChMIrp polypeptides, fragments, variants and/or derivatives are also included in the present invention. Such antibodies are produced by immunization with ChMIrp antigens (i.e., having at least six consecutive amino acids), optionally conjugated to a carrier. Transgenic animals (e.g., mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production are used. See, for example, Jakobovits et al., P.
roc. Natl. Acad. Sci. U. S. A. 90:2551-2555
Jakobovits et al., Nature 362:255-258
, 1993; Bruggermann et al., Year in Immuno. 7:
33, 1993. In one method, such transgenic animals are produced by disabling endogenous loci which encode therein immunoglobulin heavy and light chains and introducing loci which encode human heavy and light chain proteins into their genome. Partially modified animals (having less than the full complement of modifications) are then cross-bred to obtain animals which have all of the desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies with human (rather than, e.g., murine) amino acid sequences, including variable regions immunospecific for these antigens. PCT Application No. PCT/US
See, for example, U.S. Patent No. 5,545,807, PCT Application No. PCT/US91/96/05928 and PCT/US93/06926. Additional methods are described in U.S. Patent No. 5,545,807, PCT Application No. PCT/US91/96/05928 and PCT/US93/06926.
245, PCT/GB89/01207, and EP 546073 B1 and EP 546073 A1. Human antibodies can also be produced by the expression of recombinant DNA in host cells or in hybridoma cells as described herein.

Human antibodies can also be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein. In an alternative embodiment, human antibodies can be generated in phage display libraries (Hoogenboom et al., J. Mol. Biol.
227:381, 1991; Marks et al., J. Mol. Biol. 222:
581, 1991). These processes mimic immune selection by the display of antibody repertoires on the surface of filamentous bacteriophage, followed by selection of phage by their binding to selected antigens. One such technique is described in PCT Application No. PCT/US98/17364, which describes the isolation of high affinity and functional agonist antibodies to the MPL-receptor and msk-receptor using such an approach.

Chimeric, CDR-grafted, and humanized antibodies are typically produced by recombinant methods. Nucleic acids encoding these antibodies are introduced into host cells and expressed using the materials and methods described herein. In a preferred embodiment, the antibodies are expressed in mammalian host cells, such as CHO cells. Monoclonal (e.g., human) antibodies can be produced by expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.

For diagnostic applications, the anti-ChMIrp antibody will typically be labeled with a detectable moiety. The detectable moiety is any moiety that is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety can be a radioisotope (e.g., ^3H , ^14C , ^32P , ^35S or ^125I ), a fluorescent or chemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine or luciferin); or an enzyme (e.g., alkaline phosphatase, β
-galactosidase or horseradish peroxidase).
See, R. et al., Meth. Enz. 184:138-163,1990.

The anti-ChMIrp antibodies of the present invention can be used in any known assay method for detection and quantification of ChMIrp polypeptides, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Sola, Monoclonal Antibodies: A Manua
l of Techniques, pp. 147-158 (CRC Press
, Inc., 1987) The antibodies bind the ChMIrp polypeptide with an affinity appropriate for the assay method being used.

The activity of the cell lysates or purified ChMIrp protein variants is then screened in a suitable screening assay for the desired characteristic. For example, changes in binding affinity for a ligand or immunological characteristics of the ChMIrp protein (e.g., affinity for a given antibody) are measured by competitive-type immunoassays. Changes in immunomodulatory activity are measured by suitable assays. Such modifications of protein properties are assayed by methods well known to those skilled in the art, such as redox or thermal stability, hydrophobicity, susceptibility to proteolysis or tendency to aggregate with carriers or into multimers. Competitive binding assays involve the binding of a test sample analyte (
A labeled standard (e.g., ChMIrp polypeptide) is used to compete with the
The ability of the antibody to bind to the ChMIrp polypeptide (or an immunologically reactive portion thereof) is dependent on the ability of the antibody to bind to the ChMIrp polypeptide. The amount of ChMIrp polypeptide in a test sample is inversely proportional to the amount of standard that binds to the antibody. To facilitate measurement of the amount of standard that binds, the antibody is generally insolubilized before or after the competition, so that standards and analytes that bind to the antibody can be easily separated from those that remain unbound.

Sandwich immunoassays typically use two antibodies, one each to detect and one each to detect the antibody.
and/or involving the use of a distinct immunogenic portion, epitope, of the protein to be quantified. In a sandwich assay, the test sample analyte is typically bound to a first antibody immobilized on a solid support, after which a second antibody binds to the analyte, thus forming an insoluble tripartite complex. See, e.g., U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (e.g., a direct sandwich assay).
or anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is the enzyme-linked immunosorbent assay (ELISA), in which case the detectable moiety is an enzyme.

[0244] Selective binding agents, including anti-ChMIrp antibodies, are also useful for in vivo imaging. An antibody labeled with a detectable moiety can be administered to an animal (preferably into the bloodstream) and the presence and location of the labeled antibody in the host assayed. The antibody can be labeled with any moiety that is detectable in the animal (either by nuclear magnetic resonance, radiology, or other detection means known in the art).

[0245] Selective binding agents, including the anti-ChMIrp antibodies of the present invention, may be used as therapeutic agents. These therapeutic antibodies are generally agonists or antagonists, in that they either enhance or reduce, respectively, at least one of the biological activities of a ChMIrp polypeptide. In one embodiment, antagonist antibodies of the present invention are antibodies or binding fragments thereof that are capable of specifically binding to a ChMIrp polypeptide, fragment, variant and/or derivative, and that can inhibit or eliminate the functional activity of a ChMIrp polypeptide in vivo or in vitro. In a preferred embodiment, antagonist antibodies inhibit the functional activity of a ChMIrp polypeptide by at least about 50%, preferably at least about 80%, more preferably 90%, and most preferably 10%.
00% inhibition. Agonist anti-ChMIrp antibody and antagonist anti-ChM
Irp antibodies are identified by the screening assays described below.

The present invention also relates to kits containing ChMIrp selective binding agents (e.g., antibodies) and other reagents useful for detecting ChMIrp polypeptide levels in a biological sample, which may include the following: a second activity, a detectable label, blocking serum, positive and negative control samples, and detection reagents.

The ChMIrp polypeptide was synthesized using an "expression cloning" strategy:
Radiolabeled ( ¹²⁵ iodine) ChMIrp polypeptide or affinity/activity tagged ChMIrp polypeptide can be used to clone ChMIrp binding partners.
Irp polypeptides (e.g., fusions of Fc portions or alkaline phosphatase) can be used in binding assays to identify cell types or cell lines or tissues that express ChMIrp binding partners.
The NA can be converted to cDNA, cloned into a mammalian expression vector, and transfected into mammalian cells (e.g., COS or 293 cells) to generate an expression library. Radiolabeled or tagged ChMIrp polypeptides are then used as affinity ligands to detect C
A subset of cells in this library that express the hMIrp binding partner can be identified and isolated. DNA can then be isolated from these cells and transfected into mammalian cells to create a second expression library in which the proportion of cells expressing the ChMIrp binding partner is several-fold higher than in the first library. This enrichment process is carried out by enriching the cells in the library with the ChMIrp binding partner.
This can be repeated until a single recombinant clone containing the MIrp binding partner is isolated. Isolation of the ChMIrp receptor is useful for identifying or developing novel agonists and antagonists of the ChMIrp polypeptide signaling pathway. Such agonists and antagonists include, but are not limited to, CMIrp receptors.
hMIrp binding partners, cyclins, Cdk inhibitors, Cdk cofactors,
These include anti-CdkII binding partner antibodies, small molecules or antisense oligonucleotides.

Diagnostic Kits and Reagents The present invention contemplates the use of ChMIrp polypeptides, fragments thereof, peptides, binding compositions, and fusion products thereof in a variety of diagnostic kits and methods for detecting the presence of receptors and/or antibodies or ligands. Typically, the kits will have a compartment containing a ChMIrp peptide or gene segment, or a reagent that recognizes one or the other of them (e.g., a binding reagent).

Kits for determining the binding affinity of a receptor or test compound to ChMIrp will typically include a receptor test compound; a labeled compound (e.g., an antibody with known binding affinity for the protein); or a source of ligand (naturally occurring or recombinant); and a means for separating free labeled compound from bound labeled compound (e.g., a solid phase for immobilizing the ligand or its receptor). Once the compounds have been screened, compounds with suitable binding affinity for the ligand or its receptor are evaluated in a suitable biological assay (well known in the art) to determine whether these compounds bind to ChMIrp.
It can be determined whether a specific polypeptide can act on the receptor as an agonist or antagonist of Irp activity. The availability of recombinant receptor polypeptides also provides well-defined standards for calibrating such assays or as positive control samples.

For example, a preferred kit for determining the concentration of ChMIrp in a sample typically contains a labeled compound with known binding affinity for the target, such as
antibody), a source of ligand or receptor (naturally occurring or recombinant), and a means for separating bound from free labeled compound (e.g., a solid phase for immobilizing the ligand or receptor).
A compartment containing the reagents and instructions for use or disposal is provided.

Antibodies (including antigen-binding fragments specific for a ligand or receptor) or fragments are useful in diagnostic applications to detect the presence of elevated levels of the ligand, receptor and/or its fragments. Such diagnostic assays may use lysates, live cells, fixed cells, immunofluorescence, cell cultures, body fluids, and further include detection of antigens associated with the ligand or receptor in serum, etc. Diagnostic assays may be homogeneous (no separation step between free reagent and antigen complex is required) or heterogeneous (requiring a separation step). A variety of commercially available assays exist, including, for example, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), immunoassay ...
Enzyme immunoassay (EIA), enzyme-linked immunoassay technology
multiple immunoassay technique) (EM
These include the use of fluorescent immunoassays (SLFIA), substrate-labeled immunosorbent assays (FT), and substrate-labeled fluorescent immunoassays (SLFIA). For example, unlabeled antibodies can be used by using a secondary antibody that is labeled and recognizes the primary antibody to the ligand or receptor or a specific fragment thereof. Similar assays have also been extensively discussed in the literature (e.g., Ha et al., 2003).
rlow and Lane (1988) Antibodies: A Labo
ratory Manual, Cold Spring Harbor Lab
(See Oratory Press).

Anti-idiotypic antibodies may have similar uses for diagnosing the presence of antibodies to ligands or receptors, and thus may diagnose a variety of abnormal conditions. For example, overproduction of a ligand or receptor may result in the production of a variety of immunological responses which may be diagnostic of abnormal physiological conditions, particularly in various inflammatory or allergic conditions.

Frequently, reagents for diagnostic assays are supplied in kits to optimize the sensitivity of the assay. For the present invention, depending on the nature of the assay, the protocol, and the label, either labeled or unlabeled antibody or labeled ligand or receptor, provided. This is usually in association with other additives such as buffers, stabilizers, materials necessary for signal generation (e.g., enzyme substrates, etc.). Preferably, the kit also contains instructions for proper use and disposal of the contents after use. Typically, the kit has a compartment or container for each useful reagent. Desirably, the reagents are provided as lyophilized powders, where they can be reconstituted in an aqueous medium that provides the appropriate concentrations of reagents to perform the assay.

The aforementioned components of drug screening and diagnostic assays may be used unmodified or may be modified in various ways. For example, labeling may be achieved by covalently or non-covalently attaching moieties that provide a directly or indirectly detectable signal. In any of these assays, the ligand, test compound, receptor, or antibodies thereto may be labeled either directly or indirectly. Direct labeling possibilities include the following label groups: radioactive labels (e.g., ¹²⁵ I), enzymes such as peroxidase and alkaline phosphatase (U.S. Pat. No. 3,645,090), and fluorescent labels that allow monitoring of changes in fluorescence intensity, wavelength shifts, or fluorescence polarization (U.S. Pat. No. 3,940,475). Indirect labeling possibilities include:
Biotinylation of one component followed by binding to avidin coupled to one of the labeling groups described above.

There are also many ways to separate bound from free ligand, or free from bound test compound. The ligand or receptor can be immobilized on a variety of matrices, possibly with detergents or related lipids, and subsequently washed. Suitable matrices include ELISA,
Examples of suitable matrices include plastics such as A-plates, filters, and beads. Methods for immobilizing the ligand or receptor to the matrix include, but are not limited to, direct adhesion to the plastic, use of capture antibodies, chemical coupling, and biotin-avidin. The final step in this approach is to couple the antigen/receptor to the matrix by any of several methods, including those utilizing organic solvents such as polyethylene glycol or salts such as ammonium sulfate.
Other suitable separation techniques include, but are not limited to, the following: Rattles et al., Clin. Chem., 30:1.
457-1461, 1984, and the dual magnetic particle separation described in U.S. Pat. No. 4,659,6178, incorporated herein by reference.

Methods for linking proteins or their fragments to various labels have been extensively reported in the literature and need not be discussed in detail here. Many of the techniques involve the use of activated carboxyl groups, either through the use of carbodiimides or active esters to form peptide bonds, the reaction of sulfhydryl groups with activated halogens (e.g., chloroacetyl) to form thioesters, activated olefins (e.g., maleimides), linkages, etc. Fusion proteins also find use in these applications.

The nucleic acid molecules of the present invention can be used to map the location of the ChMIrp gene or related genes to chromosomes. Mapping can be performed using techniques known in the art, such as PCR amplification, in situ hybridization, and FIS.
H).

The present invention also relates to the use of ChMIrp as part of a diagnostic assay for detecting a disease or susceptibility to a disease associated with the presence of a mutated ChMIrp gene.
Such diseases relate to the abnormal expression of ChMIrp (eg, abnormal cell proliferation such as tumors and cancers).

Individuals carrying mutations in the human ChMIrp gene can be identified by DNA sequencing using a variety of techniques.
A level. Nucleic acids for diagnosis may be obtained from the patient's cells, for example from blood, urine, saliva, tissue biopsy and autopsy material. Genomic DNA may be used directly for detection or may be amplified enzymatically by PCR prior to analysis (see, for example, US Pat. No. 5,333,621).
Saiki et al., Nature, 324:163-166, 1986). RNA or cDNA can also be used for the same purpose. By way of example, PCR primers complementary to the nucleic acid encoding the ChMIrp polypeptide can be used to identify and analyze ChMIrp mutations. For example, deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be detected by PCR of the amplified DNA with radiolabeled ChMIrp RNA or radiolabeled ChMIrp RNA.
Perfectly matched sequences can be identified by hybridizing to MIrp antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences can be accomplished by detection of changes in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high-resolution gel electrophoresis. DNA fragments of different sequences can be differentiated to fragments on denaturing formamide gradient gels, where the mobility of different DNA fragments is hindered in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230:1242, 1985).
).

Sequence changes at specific positions can also be detected using nuclease protection assays (e.g., RNA
Se and S1 protection) or chemical cleavage methods (e.g.,
Cotton et al., Proc. Natl. Acad. Sci. , USA, 85:4
397-4401, 1985).

Thus, detection of specific DNA sequences can be accomplished by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes (e.g., restriction fragment length polymorphism (RFLP)) and Southern blotting of genomic DNA.

In addition to the more conventional gel electrophoresis and DNA sequencing, mutations can also be identified by
It can be detected by in situ analysis.

The present invention also relates to diagnostic assays for detecting altered levels of ChMIrp protein in various tissues, since overexpression of the protein, as compared to normal control tissue samples, can detect the presence of or susceptibility to disease, such as tumors, cerebral malaria, and hereditary periodic fever syndromes. Assays used to detect levels of ChMIrp polypeptide in samples obtained from a host are well known to those of skill in the art and include: radioimmunoassays, competitive binding assays, Western blot analysis, EL
ISA assay and "sandwich" assay. ELISA assay (Col
Igan et al., Current Protocols in Immunolog
(J. Immunol., 1(2), Chapter 6, 1991) involves, in part, the preparation of an antibody, preferably a monoclonal antibody, specific for the ChMIrp antigen. In addition, a reporter antibody is prepared against the monoclonal antibody. A detectable reagent (e.g., radioactive, fluorescent, or in this example, horseradish peroxidase enzyme) is attached to the reporter antibody. Here, a sample is removed from a host,
The sample is then incubated on a solid support (e.g., a polystyrene dish) that will bind the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin (BSA). A monoclonal antibody is then incubated in the dish for a period of time that allows the monoclonal antibody to bind to any ChMIrp protein bound to the polystyrene dish. Any unbound monoclonal antibody is washed away with a buffer solution. A reporter antibody linked to horseradish peroxidase is now placed in the dish,
This results in binding of the reporter antibody to any monoclonal antibody that binds to ChMIrp. Unbound reporter antibody is then washed away. A peroxidase substrate is then added to the dish and the amount of color developed in a given time is a measure of the amount of ChMIrp polypeptide present in a given dose of patient sample when compared against a standard curve.

A competitive assay may be used, in which an antibody specific for ChMIrp is bound to a solid support and labeled ChMIrp and a sample from a host are
The amount of label dispensed onto the solid support and detected (e.g., by liquid scintillation chromatography) can be correlated with the amount of ChMIrp in the sample. Furthermore, the above sandwich immunoassays can also be performed to quantitate the amount of ChMIrp ligand in a biological sample.

The sequences of the present invention are assessable for chromosome identification and mapping. The sequences can be specifically targeted to and hybridize to specific locations on individual human chromosomes. Furthermore, there is currently a need to identify specific sites on chromosomes where genes may be located. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are currently available for marking chromosomal locations. Mapping DNA to chromosomes according to the present invention is an important first step in correlating sequences with genes associated with disease.

Briefly, sequences are isolated from cDNA using PCR primers (preferably 15-25
The sequences can be mapped to chromosomes by preparing primers (3' bp) that span the 3' untranslated region of the sequence. Computer analysis of the 3' untranslated region of this sequence is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only hybrids containing the human gene corresponding to the primers will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning specific DNA to specific chromosomes. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved in a similar manner using panels of fragments from specific chromosomes or larger pools of genomic clones.
Other mapping strategies that can also be used to map ChMIrp to its chromosome include: in situ hybridization, flow sorted and labeled (flow sorted and labeled).
Preliminary screening on (over-sorted) chromosomes and chromosome-specific cDNAs
Pre-selection by hybridization to construct the A library.

Fluorescence in situ hybridization (FISH) of cDNA clones to metaphase chromosomal spreads can be used to provide a precise chromosomal location in one step. This technique can be used with cDNAs as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of being bound to a unique chromosomal location with sufficient signal intensity for easy detection. FISH is
This requires the use of genomic clones or clones from which expressed sequence tags (ESTs) are derived, and the larger the better. For example, 2,000 bp is preferred, 4,000 bp is more preferred, and anything over 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of this technique, see Verma et al., Human Chromosomes: A Manua.
l of Basic Techniques, Pergamon Press
, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data can be found, for example, in V. McKusick, Mendelian Inheritance, ed.
e in Man (Johns Hopkins University We
The association between genes and diseases that have been mapped to the same chromosomal region is then determined using linkage analysis (coinheritance of physically adjacent genes).
The fusion protein is identified through the fusion protein tance.

[0271] It is next necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

With the current resolution of physical and genetic mapping techniques, a cDNA precisely located in the chromosomal region associated with the disease could be one of between 50 and 500 potential causative factors. (This assumes a mapping resolution of 1 megabase and one gene per 20 kb.)

The nucleic acid molecules of the invention are also useful as antisense inhibitors of ChMIrp expression. Such inhibition can be achieved by blocking expression control sequences (triple helix formation) or C
The antisense probe may be provided by a nucleic acid molecule that is complementary to and hybridizes with hMIrp mRNA.
Using the sequence of p, antisense inhibitors can be designed by available techniques. Antisense inhibitors provide information relating to the reduction or absence of ChMIrp polypeptide in a cell or organism.

The nucleic acid molecules of the present invention can be used in gene therapy. Nucleic acid molecules that express ChMIrp in vivo provide information related to the effects of this polypeptide in a cell or organism. ChMIrp nucleic acid molecules, fragments, and/or derivatives that do not themselves encode a biologically active polypeptide can be used to detect, either qualitatively or quantitatively, the expression of ChMIrp in mammalian tissue or body fluid samples.
They may be useful as hybridization probes in diagnostic assays to test for the presence of the DNA or the corresponding RNA.

ChMIrp polypeptide fragments, variants, and/or derivatives include
It is useful to prepare antibodies that bind to ChMIrp polypeptides, whether biologically active or not, which can be used for in vivo and in vitro diagnostic purposes (e.g., in labeled form to detect the presence of ChMIrp polypeptides in body fluids or cell samples).
Ch to reduce or block at least one activity characteristic of the rp polypeptide.
It may bind to the MIrp polypeptide or may bind to the polypeptide in a manner that increases activity.

Genetically Engineered Non-Human Animals [0276] Further included within the scope of the present invention are non-human animals such as mice, rats, or other rodents, rabbits, goats, sheep, or other livestock, in which the gene encodes a ChMIrp polypeptide and either the mammal's native form of the gene or a heterologous ChMIrp polypeptide gene is overexpressed by the mammal, thereby creating a "transgenic" mammal. Such transgenic mammals may be prepared using well-known methods such as those described in U.S. Patent No. 5,489,743 and PCT Publication No. WO 94/28122.

Further included within the scope of the present invention are non-human animals, such as mice, rats, or other rodents, rabbits, goats, sheep, or other livestock, in which the gene encoding a native ChMIrp polypeptide has been disrupted ("knocked out") and the expression levels of this gene are significantly reduced or completely abolished.
Such mammals may be prepared using techniques and methods such as those described in US Pat. No. 5,557,032, incorporated herein by reference.

The present invention further includes non-human animals in which the promoter for one or more of the ChMIrp polypeptides of the present invention is either activated or inactivated (using homologous recombination methods as described below) to alter the level of expression of one or more of the native ChMIrp polypeptides.

These non-human animals can be used for screening drug candidates.
The effect of the drug candidate in an animal can be measured. For example, the drug candidate can be
The expression of the hMIrp polypeptide gene may be decreased or increased. In certain embodiments, the amount of ChMIrp polypeptide or fragment produced is
The drug candidate may be measured after exposing the mammal to the drug candidate. In certain embodiments, the actual effect of the drug candidate on the mammal may be detected. For example, the overexpression of a particular gene may cause or be associated with a disease state or pathological condition. In such cases, the ability of the drug candidate to reduce the expression of the gene or to prevent or inhibit the pathological condition may be tested. In another example, the production of a particular metabolite, such as a fragment of a polypeptide, may be tested.
It may result in or be associated with a disease or pathological condition, in which case the ability of a drug candidate to reduce the production of such metabolites or to prevent or inhibit the pathological condition may be tested.

Internalization Proteins The TAT protein sequence (from HIV) can be used to internalize proteins into cells by targeting the lipid bilayer component of the cell membrane.
See, e.g., Falwell et al., Proc. Natl. Acad. Sci. 91:6
See, e.g., HIV TAT protein 1
A single amino acid sequence (YGRKKRRQRRR; SEQ ID NO: 16), referred to as the "protein transduction domain," or TAT PDT, has been shown to mediate delivery of large bioactive proteins, such as β-galactosidase and p27Kip, across the plasma membrane and nuclear membrane of cells. Schwarze et al., Sc
ience 285:1569-1572, 1999; and Nagahara
See Schwartze et al., Nature Medicine, 4:1449-1452, 1998.
(1999) demonstrated that cultured cells acquired β-gal activity when exposed to a fusion of TAT PDT and β-galactosidase. Injection of TAT-β-gal fusion protein into mice resulted in the expression of β-gal in many tissues (liver, kidney, lung,
This results in the expression of β-gal in various tissues, including heart and brain tissue.

[0281] Thus, it will be appreciated that the TAT protein sequence can be used to internalize a desired protein or polypeptide into a cell. In the context of the present invention, the TAT protein sequence can be fused to another molecule, such as a ChMIrp antagonist (i.e., an anti-ChMIrp selective binding agent or small molecule),
and can be administered intracellularly to inhibit the activity of the ChMIrp molecule. If desired, the ChMIrp protein itself, or a peptide fragment or modified form of ChMIrp, can be fused to such a protein transducer for administration to a cell using the procedures described above.

Assays for Other Modulators of ChMIrp Polypeptide Activity In some situations, it may be desirable to identify molecules that are modulators of the activity of ChMIrp polypeptides (i.e., agonists or antagonists). Natural or synthetic molecules that modulate ChMIrp may be identified using one or more of the screening assays described below. Such molecules may be administered in either in vivo or in vitro modes, by topical or intravenous (iv) injection, or by oral delivery, implantation devices, etc. "Test Molecules"
refers to a molecule under evaluation for its ability to bind to the ChMIrp polypeptide and thereby modulate its activity. The test molecule binds to the ChMIrp polypeptide with an affinity constant of at least about 10 ⁻⁶ M, preferably about 10 ⁻⁸ M, more preferably about 10 ⁻⁹ M, and even more preferably about 10 ⁻¹⁰ M.
rp polypeptide.

Methods for identifying compounds that interact with a ChMIrp polypeptide are encompassed by the present invention. In general, a ChMIrp polypeptide is a compound that binds to the ChMI of a test molecule.
The test molecule is incubated with the ChMIrp polypeptide under conditions that allow binding to the ChMIrp polypeptide, and the degree of binding is measured. The test molecule may be screened in a substantially purified form or in a crude mixture. The test molecule may be a nucleic acid molecule, a protein, a peptide, a carbohydrate, a lipid, or a low molecular weight organic or inorganic compound. Once a set of test molecules is identified that bind to the ChMIrp polypeptide, the molecule may be further evaluated for its ability to increase or decrease ChMIrp activity.

Measurement of the interaction of a test molecule with a ChMIrp polypeptide can be performed in several formats, including cell-based binding assays, membrane binding assays, solution phase assays, and immunoassays. In general, the test molecule is allowed to bind to the ChMIrp polypeptide for a specific period of time.
The cells were incubated with hMIrp polypeptide, and the extent of binding to the hMIrp polypeptide was determined by filtration, electrochemiluminescence (ECL by IGEN, ORI).
GEN Systems), cell-based assays or immunoassays.

Homogeneous assay techniques for radioactivity (SPA; Amersham) and time resolved fluorescence (HTRF, Packard) are also performed. Binding is carried out using radioisotopes ( ¹²⁵ I, ³⁵ S, ³ H), fluorescent dyes (fluorescein), lanthanides (
For example, they may be detected by labeling with europium (Eu ³⁺ ) chelators or cryptates, or bipyridyl-ruthenium (Ru ²⁺ ) complexes. It will be appreciated that the choice of labeled probe will depend on the detection system used.
Alternatively, the ChMIrp polypeptide can be modified with an unlabeled epitope tag (e.g., biotin, peptide, His6, myc, Fc) and coupled to a protein bearing a detectable label as described above (e.g., streptavidin, anti-peptide or anti-protein antibody).

[0286] Interaction of a test molecule with a ChMIrp polypeptide can also be assayed directly using polyclonal or monoclonal antibodies in an immunoassay. Alternatively, modified forms of the ChMIrp polypeptide containing epitope tags as described above can be used in solution and immunoassays.

In one embodiment, agonists or antagonists of ChMIrp can be proteins, peptides, carbohydrates, lipids, or small molecular weight molecules that interact with ChMIrp to regulate its activity. Potential protein antagonists of ChMIrp include antibodies that bind to the active region of the polypeptide and inhibit or eliminate at least one activity of ChMIrp. Molecules that regulate expression of a ChMIrp polypeptide can include nucleic acid molecules that are complementary to a nucleic acid encoding a ChMIrp polypeptide or complementary to a nucleic acid sequence that directs or controls expression of the polypeptide, and nucleic acid molecules that act as antisense regulators of expression.

In cases where a ChMIrp polypeptide exhibits biological activity through interaction with a binding partner, such as a receptor or ligand, various assays can be used to measure the binding of the ChMIrp polypeptide to the corresponding binding partner.
In one assay, the ChMIrp polypeptide is immobilized by attachment to the bottom of a well of a microtiter plate. A radiolabeled ChMIrp binding partner (e.g., an iodized ChMIrp binding partner) and a test molecule can then be added to the well, either one at a time (in either order) or simultaneously. After incubation, the wells can be washed and counted for radioactivity using a scintillation counter to determine the extent to which the binding partner binds to the ChMIrp polypeptide. Typically, molecules are tested over a range of concentrations, and a series of control wells lacking one or more elements of the test assay can be used for accuracy in evaluating the results. An alternative to this method involves reversing the "location" of the proteins (i.e., immobilizing a ChMIrp polypeptide binding partner in the wells of a microtiter plate, incubating a test molecule with a radiolabeled ChMIrp polypeptide, and determining the extent of ChMIrp binding) (see, e.g., Ausubel et al., J. Immunol. 1999, 143:1311-1323).
Current Protocols in Molecular
Biology, John Wiley & Sons, New York, NY,
(See Chapter 18 of 1995).

As an alternative to radioactive labeling, the ChMIrp polypeptide or its binding partner can be conjugated with biotin and the presence of the biotinylated protein can then be detected using streptavidin linked to an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), which can be detected colorimetrically, or by fluorescent tagging of streptavidin.
An antibody directed against the p binding partner and conjugated with biotin can also be used, and this can be detected after incubation with enzyme-linked streptavidin linked to AP or HRP.

[0290] The ChMIrp polypeptide and the ChMIrp binding partner can also be immobilized by attachment to agarose beads, acrylic beads, or other types of such inert substrates. The substrate-protein complex can be placed in a solution containing the complementary protein and the test compound; after incubation, the beads can be precipitated by centrifugation, and the amount of binding between the ChMIrp polypeptide and its binding partner can be assessed using the methods described above. Alternatively,
The substrate-protein complex can be immobilized in a column, and the test molecule and complementary protein passed through the column. The formation of a complex between the ChMIrp polypeptide and its binding partner can then be assessed using any of the techniques described above (i.e., radiolabeling, antibody binding, etc.).

Another in vitro assay useful for identifying test molecules that increase or decrease the formation of a complex between a ChMIrp binding protein and a ChMIrp binding partner is a surface plasmon resonance detector system, such as the Biacore assay system (Pharmacia, Piscataway, NJ). The Biacore system can be performed using the manufacturer's procedures. The assay essentially involves attaching a dextran-coated sensor chip of either ChMIrp or a ChMIrp binding partner, which is located within the detector.
The test compound and other complementary proteins can then be injected, either simultaneously or sequentially, into a chamber containing the sensor chip, and the amount of complementary proteins that bind to each other can be assessed based on the change in mass of the molecules that physically associate with the dextran-coated side of the sensor chip; the change in mass of the molecules is measured by a detector system.

In some cases, it may be desirable to evaluate two or more test compounds together for their ability to increase or decrease the formation of a complex between a ChMIrp polypeptide and a ChMIrp binding partner complex. In these cases, the above assay can be easily modified by adding such additional test compounds either simultaneously with or subsequently to the first test compound. The remainder of the steps in the assay are as described above.

In vitro assays such as those described above can be used to detect ChMIrp and ChMIrp
They can be advantageously used to rapidly screen large numbers of compounds for their effect on the formation of complexes with binding partners. These assays can be automated to screen compounds generated in phage display, synthetic peptide, and chemical synthesis libraries.

Compounds that increase or decrease the formation of a complex between a ChMIrp polypeptide and a ChMIrp binding partner can also be screened in cell culture using cells and cell lines expressing either ChMIrp or a ChMIrp binding partner. The cells and cell lines can be obtained from any mammal, but are preferably from human, or other primate, canine or rodent sources.
Binding of the Irp polypeptide is assessed in the presence or absence of a test compound, and the extent of binding can be determined, for example, by flow cytometry using a biotinylated antibody to a ChMIrp binding partner. Cell culture assays can be advantageously used to further evaluate compounds that score positive in the above protein binding assays.

[0295] The cell cultures can also be used to screen the effects of drug candidates. For example, a drug candidate can decrease or increase expression of the ChMIrp polypeptide gene. In certain embodiments, the amount of ChMIrp polypeptide or fragment produced can be measured after exposure of the cell culture to the drug candidate.
In certain embodiments, the actual effect of drug candidates on cell culture can be detected.For example, the overexpression of a certain gene can have a certain effect on cell culture.In such a case, the ability of a drug candidate to increase or decrease the expression of this gene, or to prevent or inhibit a certain effect on cell culture can be tested.In other examples, the production of a certain metabolic product (e.g., a fragment of a polypeptide) can cause or be associated with a disease or pathological condition.In such a case, the ability of a drug candidate to reduce the production of such metabolic product in cell culture can be tested.

Yeast two-hybrid system (Chien et al., Proc. Natl. Acad. Sci.
ci. USA, 88:9578-9583, 1991) can be used to identify novel polypeptides that bind to or interact with the ChMIrp polypeptide. As an example, a yeast two-hybrid bait construct can be generated in a vector (e.g., pAS2-1 from Clontech) that encodes the yeast GAL4-DNA binding domain fused to a ChMIrp polynucleotide. This bait construct can be used to screen a human cDNA library, where the sequence of the cDNA library is a GALA4-binding domain.
The L4 activation domain is fused to the L4 activation domain. A positive interaction results in activation of a reporter gene, such as β-Gal. Positive clones resulting from the screen can be further characterized to identify the interacting protein.

Compositions and Administration of ChMIrp Polypeptides Members of the chondromodulin family are known to induce cartilage formation and bone growth. In addition, these proteins have been suggested to be inhibitors of angiogenesis. Angiogenesis mediates many pathological conditions.
These described biological activities suggest possible therapeutic applications for the administration of chondromodulin.

Pharmaceutical compositions of polypeptides of ChMIrp are useful for reducing levels of ChMIrp.
It is within the scope of the present invention to determine whether administration of a ChMIrp polypeptide results in the prophylactic and therapeutic treatment of humans and animals for indications resulting from p, or in the amelioration or cure of the indication. Such compositions may contain a therapeutically effective amount of a ChMIrp polypeptide and/or its binding partner, or a therapeutically active fragment, variant or derivative thereof, mixed with a pharmacologic additive and/or carrier. Suitable formulation materials or pharmacologic acceptable factors include, but are not limited to, antioxidants, preservatives, colorants, flavorings, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants. Typically, therapeutic compounds including ChMIrp polypeptides are administered in the form of a composition comprising a purified polypeptide, fragment, variant or derivative together with one or more physiologically acceptable carriers, excipients or diluents. For example, a suitable vehicle may be water for injection, physiological solution, or artificial cerebrospinal fluid, which may be supplemented with other substances common in compositions for parenteral delivery.

Neutral buffered saline or saline mixed with serum albumin are exemplary suitable carriers. Preferably, the product is formulated as a lyophilizate using appropriate excipients (e.g., sucrose). Other standard carriers,
Diluents and excipients may be included as desired. Other exemplary compositions include Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, which may further include sorbitol or a suitable substitute thereof. The pH of the solution should also be selected based on the relative solubility of the ChMIrp ligand at various pHs.

The primary solvent in the composition may be either aqueous or non-aqueous in nature. In addition, the vehicle may contain other formulation materials to modify or maintain the pH, osmolality, viscosity, clarity, color, sterility, stability, isotonicity, dissolution rate, or odor of the formulation. Similarly, the composition may contain additional formulation materials to modify or maintain the release rate of the ChMIrp polypeptide or to facilitate absorption or permeation of the ChMIrp polypeptide.

Compositions containing ChMIrp polypeptide compositions can be administered parenterally.
Alternatively, the compositions may be administered intravenously or subcutaneously. When administered systemically, the therapeutic compositions for use in the present invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such pharma- ceutically acceptable protein solutions is within the skill of the art, with due attention to pH, isotonicity, stability, and the like.

Therapeutic formulations of ChMIrp polypeptide compositions useful for practicing the present invention may be prepared by adding any physiologically acceptable carrier, excipient, or stabilizer (see Reming et al., J. Clin. Sci. 2003, 143:1311-1315).
ton's Pharmaceutical Sciences, 18th edition, A
R. Gennaro, ed., Mack Publishing Company
, 1990) with a selected composition having the desired degree of purity, in the form of a lyophilized cake or aqueous solution for storage.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients and preferably inert at the dosages and concentrations employed.
and include: buffers (e.g., phosphate, citrate, or other organic acids); antioxidants (e.g., ascorbic acid); low molecular weight polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents (e.g., EDTA); sugar alcohols (e.g., mannitol or sorbitol); salt-forming counterions (e.g., sodium); and/or non-ionic surface-active agents (e.g., Tween, Pluronic, or polyethylene glycol (PEG)).

An effective amount of a ChMIrp polypeptide composition to be used therapeutically will depend, for example, on the therapeutic purpose (e.g., the indication for which the composition is being used, the route of administration (e.g., whether it is administered locally or systemically), and the condition of the patient (e.g., the patient's general health, anaureuesis, age, weight, sex)). In determining a therapeutically effective dose, the amount of ChMIrp or other members of the secreted chondromodulin family responsible for the disease and the amount of endogenous ChMIrp or other members of the secreted chondromodulin family responsible for the disease will be taken into consideration.
It is essential to consider the amount of Irp. Thus, the therapist will need to titrate the dosage and/or modify the route of administration in vivo as necessary to obtain the optimal therapeutic effect. A typical daily dosage can range from about 0.1 mg/kg to 100 mg/kg or more, depending on the factors mentioned above. Typically,
The clinician administers the composition until a dosage is reached that achieves the desired effect. Thus, the composition may be administered as a single dose or as two or more doses over time (which may include
The compositions may be administered in a single dose (with or without the same amount of ChMIrp polypeptide) or as a continuous infusion via an implanted device or catheter.

[0305] The frequency of dosing will depend on the pharmacokinetic parameters of the ChMIrp molecule in the formulation used. Typically, the clinician will administer the composition until a dosage is reached that achieves the desired effect. Thus, the composition may be administered as a single dose, or as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely performed by, and falls within the scope of work routinely performed by, those of skill in the art. The appropriate dosage may be ascertained through the use of appropriate dose-response data.

As more research is conducted, more information will become available regarding appropriate dosage levels for the treatment of various conditions in various patients, and one of skill in the art will be able to assure appropriate dosing, taking into account the therapeutic context, the type of disorder being treated, the age and general health of the recipient.

[0307] ChMIrp polypeptide compositions to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization using these methods can be accomplished either prior to or after lyophilization and reconstitution. Compositions for parenteral administration are typically stored in lyophilized form or in a solution.

Therapeutic compositions generally are administered in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
You can be put inside.

Effective dosage forms (e.g., (1) slow-release formulations, (
(2) inhalant mists or (3) orally active formulations) are also envisioned. Pharmaceutical compositions containing a therapeutically effective dose of a ChMIrp polypeptide may also be formulated for parenteral administration. Such parenterally administered therapeutic compositions are typically in the form of a pyrogen-free, parenterally acceptable aqueous solution containing ChMIrp in a pharma- ceutically acceptable vehicle.
Pharmaceutical compositions of p may also include incorporation of ChMIrp into particulate preparations of polymeric compounds (eg, polylactic acid, polyglycolic acid, etc.) or into liposomes.
Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.

A particularly suitable vehicle for parenteral injection is properly preserved sterile distilled water in which the ChMIrp is formulated as a sterile isotonic solution. Yet another preparation may include formulation of ChMIrp with agents such as injectable microspheres, bio-erodible particles or beads, or liposomes that provide controlled or sustained release of the protein product and can then be delivered as a depot injection. Other suitable means for introduction of ChMIrp include implantable drug delivery devices that contain ChMIrp and/or its binding partner.

The preparations of the present invention may contain other ingredients, such as parenterally acceptable preservatives, tonicity agents, cosolvents, wetting agents, complexing agents, buffers, antibacterial agents, antioxidants, and surfactants, as is well known in the art. For example, suitable tonicity enhancing agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol, and the like. Suitable preservatives include, but are not limited to, benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, and the like. Hydrogen peroxide may also be used as a preservative. Suitable cosolvents are, for example, glycerin, propylene glycol, and polyethylene glycol. Suitable complexing agents are, for example, caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates (e.g.,
Polysorbate 80), tromethamine, lecithin, cholesterol, tyloxapal, etc. Buffers include conventional buffers (e.g., borate, citrate, phosphate, bicarbonate or Tris-H
Cl).

Formulation components are present in concentrations that are acceptable to the site of administration. For example, buffering agents may be used to maintain the composition at physiological pH or slightly lower (typically about 5 to about 8
may be used to maintain the pH of the solution within a pH range of 10-20.

When intended for parenteral administration, therapeutic compositions for use in the present invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired ChMIrp molecule in a pharma- ceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile, distilled water, properly preserved, in which the ChMIrp molecule is
The rp molecule is formulated as a sterile isotonic solution. Yet another preparation may include a formulation of the desired molecule with an agent, such as injectable microspheres, bioerodible particles, polymeric compounds (polylactic or polyglycolic acid) or beads or liposomes, that provides a controlled or sustained release of the product and can then be delivered via depot injection. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

In one embodiment, the pharmaceutical composition can be formulated for inhalation. For example, the ChMIrp molecule can be formulated as a dry powder for inhalation.
Inhalation solutions of rp polypeptides or ChMIrp nucleic acid molecules may also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution may be nebulized. Pulmonary administration is further described in PCT Application No. PCT/US94/00187.
No. 5. PCT Application No. PCT/US94/001875 described pulmonary delivery of chemically modified proteins.

It is also contemplated that certain formulations containing ChMIrp may be administered orally.
In one embodiment of the invention, the ChMIrp molecules administered in this manner may be formulated with or without carriers customarily used in solid dosage form mixtures, such as tablets and capsules. For example, capsules may be designed to release the active portion of the formulation at a point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of the ChMIrp molecules. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants,
Suspending agents, tablet disintegrating agents, and binders may also be used.

Another pharmaceutical composition may contain an effective amount of the ChMIrp molecule in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving tablets in sterile water or other appropriate vehicle, a solution may be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or acacia; or lubricants such as magnesium stearate, stearic acid or talc.

[0317] Additional pharmaceutical compositions of ChMIrp will be apparent to those of skill in the art, including formulations involving ChMIrp polypeptides in sustained or controlled delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those of skill in the art. See, for example, PCT Application No. PCT/US93/008, which describes controlled release porous polymeric microparticles for the delivery of pharmaceutical compositions.
See issue 29.

[0318] Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or a dry or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.

In certain embodiments, the invention relates to kits for producing single dose administration units. The kits may each include both a first container having a desired protein and a second container having an aqueous formulation. Also included within the scope of the invention are kits that include single and multi-chamber pre-filled syringes, such as liquid syringes and lyosyringes.

Regardless of the form of administration, the specific dose is calculated according to the weight, surface area or size of the organ. Further refinement of the calculations necessary to determine the dosage appropriate for treatment with each of the above-mentioned formulations is routinely made by those skilled in the art and is within the scope of work routinely performed by those skilled in the art. Appropriate dosages can be determined by use of appropriate dose-response data.

The route of administration of the pharmaceutical composition may be by known methods (e.g., oral, intravenous, intraperitoneal,
Through injection by intracerebral (intracerebral), intraventricular, intramuscular, intraocular, intraarterial or intralesional routes, or by sustained release systems, or by implantation devices, which may optionally include the use of a catheter. If desired, the compositions may be administered by bolus injection, or continuously by infusion, or by implantation devices. Alternatively, or in addition, the compositions may be administered by administration of ChMIrp
Local administration may be via implantation of a membrane, sponge or another suitable material into the affected area into which the polypeptide has been absorbed.

The pharmaceutical compositions of the present invention may further be administered by pulmonary administration. See, for example, International Publication No. WO 94/20069.
No. 9 discloses pulmonary delivery of chemically modified proteins. For pulmonary delivery, the particle size should be appropriate for delivery to the distal lung. For example, the particle size can be 1 mm to 5 mm, although larger particles can be used, for example, if each particle is fairly porous. Alternatively, or in addition, the composition can be administered locally via implantation of a membrane, sponge or other suitable material into the affected area in which the receptor polypeptide is absorbed or encapsulated. If an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery can be directly through the device, via bolus, or via continuous administration, or via a catheter using continuous infusion.

[0323] The ChMIrp-ligand polypeptide and/or its binding partner may also be administered in sustained release formulations or preparations. Suitable polymer compositions preferably have inherent and controllable biodegradability to: last from about 1 week to about 6 weeks; are non-toxic, not containing significant toxic monomers and not decomposing into non-toxic components; are biocompatible, chemically compatible with the material to be delivered, and do not tend to denature the active material; are sufficiently porous to permit uptake of biologically active molecules and their subsequent release from the polymer by diffusion, erosion, or a combination thereof; and are non-toxic, such that they are biodegradable by adhesion or geometrical actions (e.g., glycerol, glycerol, terpenes, etc.).
It may remain at the application site due to its geometric faction (e.g.,
They can be formed or softened in place and then molded and formed into microparticles which are captured at the desired location; they can be delivered by minimally invasive techniques (e.g., by catheter, laparoscope, or endoscope). Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,737,133), and the like.
73,919, EP 58,481), L-glutamic acid and γ-ethyl-L-
Copolymers with glutamate (Sidman et al., Biopolymers, 22
:547-556, 1983), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167
-277, 1981 and Langer, Chem. Tech., 12:98-
105, 1982), ethylene vinyl acetate (Langer et al., supra) or poly-D(-)-3-hydroxybutyrate (EP 133,988). The sustained release composition may also include liposomes. Liposomes may be prepared by any of several methods known in the art (see, for example, Eppstein et al., Proc.
c. Natl. Acad. Sci. USA, 82:3688-3692, 198
5; EP 36,676; EP 88,046; EP 143,949).

[0324] The ChMIrp polypeptides, variants, derivatives or fragments thereof may be used alone, together, or together or in combination with other pharmaceutical compositions. The ChMIrp polypeptides, fragments, variants and derivatives may be used in combination with cytokines, cytokine inhibitors, growth factors, antibiotics, anti-inflammatory agents and/or chemotherapeutic agents, as appropriate for the indication being treated.

In some cases, it may be desirable to use a pharmaceutical composition of ChMIrp in an ex vivo manner, in which cells, tissues or organs removed from a patient are exposed to a pharmaceutical composition of ChMIrp, after which the cells, tissues and/or organs are subsequently transplanted back into the patient.

In other cases, ChMIrp polypeptides can be delivered by implanting specific cells that have been genetically engineered, using methods as described herein, to express and secrete the polypeptide. Such cells can be animal or human cells, and can be of autologous, heterologous, or xenogeneic origin. Optionally, the cells can be immortalized. To reduce the chance of an immunological response, the cells can be encapsulated to avoid penetration of surrounding tissues. The encapsulating material is typically a biocompatible semipermeable polymer enclosure or membrane that allows release of the protein product but prevents destruction of the cells by the patient's immune system or other harmful factors from the surrounding tissues.

[0327] The methods used for membrane encapsulation of cells are familiar to those of skill in the art, and the preparation of encapsulated cells and their implantation into patients may be accomplished without undue experimentation (see, e.g., U.S. Pat. Nos. 4,892,538; 5,011,472;
and 5,106,627). A system for encapsulating viable cells is described in International Application No. WO 91/10425. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposomal carriers, bioerodible particles or beads, are also known to those of skill in the art, and can be found, for example, in
No. 5,653, 975. The cells, with or without encapsulation, can be implanted into suitable body tissues or organs of the patient.

Treating an isolated cell population (e.g., stem cells, lymphocytes, erythrocytes, chondrocytes, neurons, etc.) as described above; administering one or more ChMIs, as appropriate.
It may be desirable to add rp polypeptides, variants, derivatives and/or fragments, which can be accomplished by directly exposing the isolated cells to the polypeptides, variants, derivatives or fragments, where they are in a form that is permeable to the cell membrane.

Further embodiments herein relate to cells and methods for both in vitro production of therapeutic polypeptides and production and delivery of therapeutic polypeptides by gene therapy or cell therapy (e.g., homologous recombination and/or other recombinant production methods).
Regarding.

Homologous Recombination It is further envisioned that ChMIrp polypeptides may be produced by homologous recombination or using recombinant production methods that utilize control elements introduced into cells already containing DNA encoding a ChMIrp polypeptide. For example, homologous recombination methods may be used to modify cells that normally contain a transcriptionally silent ChMIrp gene (i.e., a gene that is silenced), thereby producing cells that express therapeutically effective amounts of ChMIrp. Homologous recombination is a technique originally developed for gene targeting to induce or correct mutations in transcriptionally active genes (Kucherla et al., 2003).
pati et al., 1989, Prog. in Nucl. Acid Res. &M
Ol. Biol. 36:301). The basic technique is to introduce specific mutations into specific regions of the mammalian genome (Thomas et al., 1986, Cell 44:
419-428; Thomas and Capecchi, 1987, Cell.
51:503-512; Doetschman et al., 1988, Proc. Nat.
l. Acad. Sci. U.S.A. 85:8583-8587), or to correct specific mutations in defective genes (Doetschman et al., 1987
Exemplary homologous recombination techniques are described in U.S. Pat. No. 5,272,071; European Patent No. 919024; and US Pat. No. 5,272,071.
3051; European Publication No. 505 500; PCT/US90/0764
2; International Publication No. WO 91/09955).

By homologous recombination, the DNA sequence to be inserted into the genome is inserted into the targeting DNA
Targeting DNA can be directed to a specific region of a gene of interest by attaching it to a nucleotide sequence that is complementary (homologous) to a region of genomic DNA. Small pieces of targeting DNA that are complementary to specific regions of the genome are called DNA fragments.
During the A replication process, the parental strand is contacted. This is a general property of DNA inserted into a cell to hybridize and thus recombine with other pieces of endogenous DNA through shared homologous regions. If this complementary strand is attached to an oligonucleotide containing a mutation or different sequence or additional nucleotides, it will also be incorporated into the newly synthesized strand as a result of this recombination. As a result of the proofreading function, the new sequence of DNA can act as a template. Thus, the transferred DNA is integrated into the genome.

D that can interact with or regulate the expression of ChMIrp polypeptide
Regions of the NA (e.g., flanking sequences) are attached to these pieces of targeting DNA. For example, a promoter/enhancer element, suppressor, or exogenous transcriptional regulatory element is inserted into the genome of the intended host cell proximal to the DNA encoding the desired ChMIrp polypeptide and in an orientation sufficient to affect transcription of the DNA. The control element is inserted into the genome of the host cell proximal to the DNA encoding the desired ChMIrp polypeptide and in an orientation sufficient to affect transcription of the DNA.
Thus, expression of the desired ChMIrp polypeptide is controlled by the
This can be achieved not by transfection of DNA encoding the hMIrp gene itself, but by the use of targeting DNA (containing a region of homology to the endogenous gene of interest) linked to a DNA regulatory segment that provides the endogenous gene sequence with a recognizable signal for transcription of the hMIrp gene.

In an exemplary method, expression of a desired target gene in a cell (i.e., a desired endogenous cellular gene) is altered by the introduction of DNA containing at least regulatory sequences, exons, and a splice donor site, via homologous recombination into the cell's genome at a preselected site. These components are introduced into the chromosomal (genomic) DNA in such a manner as to actually result in the production of a new transcription unit (where the regulatory sequences, exons, and splice donor site present in the DNA construct are operably linked to the endogenous gene). As a result of the introduction of these components into the chromosomal DNA, expression of the desired endogenous gene is altered.

As described herein, altered gene expression includes the activation (or expression) of genes that are normally silent (not expressed) in the as-obtained cell, as well as the increased expression of genes that are not expressed at physiologically significant levels in the as-obtained cell. This embodiment further includes changing the pattern of regulation or induction such that it differs from that which occurs in the as-obtained cell, and reduces (including eliminates) expression of genes that are expressed in the as-obtained cell.

One way in which homologous recombination can be used to increase or induce production of a ChMIrp polypeptide from the endogenous ChMIrp gene in a cell is to first use homologous recombination to extract recombination sequences from a site-specific recombination system (e.g., Cre/loxP, FLP/FRT) (Sauer et al., 1994, Curr. J. Clin. Exp. 1999, 143:1311-1323).
Ent Opinion In Biotechnology, 5:521-5
27; Sauer et al., 1993, Methods Enzymology, 22
5:890-900) upstream (i.e., 5') of the cell's endogenous genomic ChMIrp polypeptide coding region. A plasmid containing a recombination site homologous to the site located immediately upstream of the genomic ChMIrp polypeptide coding region is introduced into the modified cell line along with an appropriate recombinase enzyme. The recombinase causes integration of the plasmid through the recombination site of the plasmid into the recombination site located immediately upstream of the genomic ChMIrp polypeptide coding region of the cell line (Baubonis and Sauer, 1993, Nucleic Acids Res. 21:2025).
-29; O'Gorman et al., 1991, Science 251:1351-
1355). Any flanking sequences known to increase transcription (e.g., enhancers/promoters, introns, translational enhancers) are incorporated in such a manner as to create new or modified transcription units that, when appropriately positioned in this plasmid, result in new or increased ChMIrp polypeptide production from the cell's endogenous ChMIrp gene.

An additional method of using a cell line in which a site-specific recombination sequence has been placed immediately upstream of the cell's endogenous genomic ChMIrp polypeptide coding region is to use homologous recombination to introduce a second recombination site at another location in the cell line's genome. The appropriate recombinase enzyme is then introduced into the cell line at the two recombination sites, resulting in recombination events (deletions, inversions, and translocations) (Sauer et al., 1994,
Current Opinion In Biotechnology (ibid.)
Sauer, 1993, Methods In Enzymology, supra), which is a novel or increased C from the endogenous ChMIrp gene of the cell.
New or modified transcription units are created that result in the production of the hMIrp polypeptide.

A further approach to increase or induce expression of a ChMIrp polypeptide from the endogenous ChMIrp gene of a cell is to induce expression of the endogenous ChMIrp polypeptide of a cell by cloning the endogenous ChMIrp gene of the cell.
The method includes increasing or causing expression of a gene or genes (e.g., a transcription factor) and/or decreasing expression of a gene or genes (e.g., a transcription repressor) in a manner that results in new or increased production of a ChMIrp polypeptide from the MIrp gene. The method includes introducing a non-naturally occurring polypeptide (e.g., a polypeptide that includes a site-specific DNA-binding domain fused to a transcription factor domain) into a cell such that new or increased production of a ChMIrp polypeptide occurs from the cell's endogenous ChMIrp gene.

The present invention further relates to DNA constructs useful in methods of altering expression of target genes. In certain embodiments, exemplary DNA constructs include: (a) one or more targeting sequences; (b) a regulatory sequence; (c) an exon; and (d) a targeting sequence.
The targeting sequence in the DNA construct is
In another embodiment, the DNA construct directs integration of elements (b)-(d) into a target gene in a cell such that these elements (b)-(d) are operably linked to sequences of the endogenous target gene. In another embodiment, the DNA construct comprises: (a) one or more targeting sequences, (b) a control sequence, (c) an exon, (d) a splice donor site, (e) an intron, and (f) a splice acceptor site, wherein the targeting sequence is
Directing the integration of elements (a) to (f) so that these elements (
b)-(f) are operably linked to an endogenous gene. The targeting sequence is homologous to a preselected site in the cellular chromosomal DNA where homologous recombination occurs. In this construct, an exon is generally 3' to the regulatory sequence and a splice donor site is 3' to the exon.

If the sequence of a particular gene is known (e.g., the nucleic acid sequence of a ChMIrp polypeptide as set forth herein), a DNA fragment complementary to a selected region of the gene can be constructed.
A fragment of NA can be synthesized or otherwise obtained, for example, by appropriate restriction of native DNA at a specific recognition site that is linked to the region of interest. When this fragment is inserted into a cell, it acts as a targeting sequence and hybridizes to a homologous region in the genome. When this hybridization occurs during DNA replication, this fragment of DNA, and any additional sequences attached to it, acts as an Okazaki fragment and is incorporated into the newly synthesized daughter strand of DNA. Thus, the present invention includes a nucleotide that codes for a ChMIrp polypeptide, which can be used as a targeting sequence.

Alternatively, gene therapy can be used as described below.

ChMIrp Cell Therapy and Gene Therapy ChMIrp cell therapy (e.g., transplantation of cells that produce ChMIrp) is also contemplated. This embodiment involves transplanting cells into a patient that are capable of synthesizing and secreting a biologically active form of the ChMIrp polypeptide.
The hMIrp polypeptide producing cell may be a cell that is a natural producer of ChMIrp, or it may be a recombinant cell whose ability to produce a ChMIrp polypeptide has been increased by transformation with a gene encoding the desired ChMIrp polypeptide or a gene that increases the expression of the ChMIrp polypeptide. Such modification may be accomplished by a vector appropriate for delivering the gene and facilitating its expression and secretion.
In order to minimize potential immunological reactions in patients receiving the Irp polypeptide, which may involve the administration of a foreign polypeptide, it is preferred that the natural cells which produce the ChMIrp polypeptide are of human origin and produce a human ChMIrp polypeptide. Similarly, it is preferred that the recombinant cells which produce the ChMIrp polypeptide are transformed with an expression vector which contains a gene encoding a human ChMIrp polypeptide.

[0342] The implanted cells may be encapsulated to avoid infiltration of surrounding tissues. Human or non-human animal cells may be encapsulated in a biocompatible semipermeable polymer enclosure or membrane (such as C
The patient may be implanted in a vascular endothelial cell culture system (eg, a vascular endothelial cell culture medium) in a vascular endothelial cell culture medium that is encapsulated in a vascular endothelial cell culture medium ...

[0343] Techniques for encapsulating living cells are known in the art, and the preparation of the encapsulated cells and their implantation into a patient can be accomplished routinely.
For example, Baetge et al. (International Publication No. WO 95/05452; International Application No. P
No. 6,399,313, filed on 23 May 2002, describes a membrane capsule containing genetically engineered cells for efficient delivery of biologically active molecules. The capsule is biocompatible and easily retrievable. When implanted into a mammalian host, the capsule encapsulates cells transfected with a recombinant DNA molecule that contains a DNA sequence encoding a biologically active molecule operably linked to a promoter that is not subject to down-regulation in vivo. The device provides for delivery of molecules from living cells to specific sites within the recipient. In addition, U.S. Pat. Nos. 4,892,538 and 5,000,433, disclose a method for the delivery of a biologically active molecule to a specific site within the recipient.
See, for example, US Pat. Nos. 11,472 and 5,106,627. A system for encapsulating living cells is described in International Application WO 91/10425 (Aebischer,
WO 91/10470 (Aebischer et al.); Winn et al., 1
991,Exper. Neurol. 113:322-329; Aebisch
Neurol. 111:269-275; and Tresco et al., 1992, ASAIO 38:17-23.

In vivo and in vitro gene therapy delivery of ChMIrp is also included in the present invention. In vivo gene therapy can be achieved by introducing a gene encoding ChMIrp into cells by local injection of a polynucleotide molecule or other suitable delivery vector (Hefti, J. Neurobiol.
ogy, 25:1418-1435, 1994). For example, a polynucleotide molecule encoding ChMIrp can be included in an adeno-associated virus vector for delivery to targeted cells (International Publication No. WO 95/34670; International Application No. PCT/US95/07178). Recombinant adeno-associated virus (AAV
) The genome typically contains ChMIrp operably linked to a functional promoter.
It contains AAV inverted terminal repeats flanking the DNA sequence encoding the AAV nucleotide sequence, and a polyadenylation sequence.

Alternative viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, herpes simplex virus vectors, and papilloma virus vectors. US Patent No. 5,672,344 describes an in vivo viral-mediated gene transfer system, including a recombinant neurotrophic HSV-1 vector. US Patent No. 5,399,346 provides an example of a process for providing a therapeutic protein to a patient by delivery of human cells that have been treated in vitro to insert a DNA segment encoding the therapeutic protein. Additional methods and materials for the implementation of gene therapy techniques are described in US Patent No. 5,631,236; gene therapy involving adenoviral vectors is described in US Patent No. 5,672,510; and gene therapy involving the use of retroviral vectors is described in US Patent No. 5,635,399.

Non-viral delivery methods include liposome-mediated transfer, naked DNA delivery (direct injection), receptor-mediated transfer (ligand-DNA complexes), electroporation, calcium phosphate precipitation, and microparticle bombardment (e.g., gene guns).
Gene therapy materials and methods may also include inducible promoters, tissue-specific enhancer-promoters, DNA sequences designed for site-specific uptake, DNA sequences that may provide a selective advantage over parental cells, markers for identifying transformed cells, negative selection and expression control systems (measures of safety), cell-specific binding factors (for cell targeting), cell-specific internalization factors, transcription factors for enhancing expression by vectors, and methods of vector production. Such additional methods and materials for the implementation of gene therapy techniques are described in U.S. Patent No. 4,970,154, International Application No. WO 9640958; U.S. Patent No. 5,679,559; U.S. Patent No. 5,676,521; and U.S. Patent No. 5,676,521.
,954; U.S. Pat. No. 5,593,875; and U.S. Pat. No. 4,945,
The expression control techniques are described in the Japanese Patent Application Publication No. 20050.
For example, International Application Nos. WO 9641865 and WO 9731899), the use of progesterone antagonists in modified steroid hormone receptor systems (e.g., U.S. Pat. No. 5,364,791), the ecdysone control system (e.g., International Application No. WO 9637609) and positive tetracycline controllable transactivators (e.g., U.S. Pat. Nos. 5,589,362; 5,650,298; and 5,654,168).

In vivo and in vitro gene therapy delivery of ChMIrp polypeptides is also contemplated. One example of a gene therapy technique is to use a ChMIrp gene (either genomic DNA, cDNA, and/or synthetic DNA encoding a ChMIrp polypeptide, or a fragment, variant, or derivative thereof) that can be operably linked to a constitutive or inducible promoter to form a "gene therapy DNA construct." The promoter may be an endogenous C
The promoter may be homologous or heterologous to the hMIrp gene, provided that it is active in the cell or tissue type into which the construct is inserted.
Other components of the gene therapy DNA construct may optionally include DNA molecules designed for site-specific integration (e.g., endogenous flanking sequences useful for homologous recombination), tissue-specific promoters, enhancers, or silencers, DNA molecules that may provide a selective advantage over the parent cell, DNA molecules useful as targets to identify transformed cells, negative selection systems, cell-specific binding factors (e.g.,
For example, for cell targeting), cell-specific internalization factors, and transcription factors to enhance expression by the vector, as well as factors that allow for the manufacture of the vector.

[0348] A gene therapy DNA construct can then be introduced into cells (either ex vivo or in vivo) using viral or non-viral vectors.
One method for introducing gene therapy DNA constructs is by viral vectors. Suitable viral vectors are typically used in gene therapy for delivery of gene therapy DNA constructs by means of the viral vectors described herein. Certain retroviral vectors deliver DNA constructs to the chromosomal DNA of cells, and the DNA constructs can be integrated into the chromosomal DNA. Other vectors function as episomes, and the gene therapy DNA constructs remain in the cytoplasm.

In yet another embodiment, the control element is a ChMI in the target cell.
For controlled expression of the rp gene, such elements may be included. Such elements are turned on in response to appropriate effectors. In this way, a therapeutic polypeptide can be expressed when desired. One conventional means of control involves the use of small molecule dimerizers or rapalogs, which are used to dimerize chimeric proteins (e.g., DNA binding proteins or transcriptional activator proteins) that contain a small molecule binding domain and a domain that can initiate a biological process (PCT Publication WO 9641865 (PCT/US96/0994)).
86); WO9731898 (PCT/US97/03137) and WO9
731899 (PCT/US95/03157). Protein dimerization can be used to initiate transcription of a transgene.

An alternative modulation technique uses a method to store the protein expressed from a gene of interest inside the cell as aggregates or clusters. The gene of interest is expressed as a fusion protein containing a conditional aggregation domain, resulting in retention of the aggregated protein in the endoplasmic reticulum. The stored protein is stable and inactive inside the cell. However, the protein can be released by administering a drug (e.g., a small molecule ligand) that removes the conditional aggregation domain, thereby specifically disrupting the aggregates or clusters so that the protein can be secreted from the cell. Science 287:816-17 and 826-3
See 0.

Other suitable control means or gene switches include, but are not limited to, the following systems: Mifepristone (RU486) is used as a progesterone antagonist. Binding of the modified progesterone receptor ligand-binding domain to the progesterone antagonist activates transcription by forming a dimer of two transcription factors, which then travel to the nucleus and are expressed as D
The ligand-binding domain is modified to eliminate the ability of the receptor to bind to the natural ligand. Modified steroid hormone receptor systems are further described in U.S. Pat. No. 5,364,791, as well as WO 9640.
911 and WO9710337.

Yet another regulatory system binds to the ecdysone receptor (a cytoplasmic receptor)
It then uses ecdysone (a Drosophila steroid hormone) to activate it. The receptor then translocates to the nucleus and binds to specific DNA.
A response element (promoter from an ecdysone responsive gene).
The ecdysone receptor contains a transactivation domain/DNA binding domain/ligand binding domain and initiates transcription. The ecdysone system is further described in U.S. Patent No. 5,514,578, and WO9738117, WO9637609; and WO9303162.

Another means of control uses a positive tetracycline-controllable transactivator. This system generates a mutated tet repressor protein DNA binding domain (a reverse tetracycline-controlled transactivator protein) linked to a polypeptide that activates transcription (i.e., it inhibits the transcription of te in the presence of tetracycline).
Such systems include mutant tetR-4 amino acid changes that bind to the t operator. Such systems are described in U.S. Patent Nos. 5,464,758; 5,650,298, and 5,654,168.

Further expression control systems and nucleic acid constructs are described in U.S. Pat. Nos. 5,741,679 and 5,834,186 (Innovir Laboratories, Inc.
Inc.).

In vivo gene therapy involves the introduction of a gene encoding a ChMIrp polypeptide into a
This can be accomplished by introducing the hMIrp nucleic acid molecule into cells by local injection or by other suitable viral or non-viral delivery vectors. (Hefti, Neurobiology 25:1418-35, 1994)
For example, a nucleic acid molecule encoding a ChMIrp polypeptide can be included in an adeno-associated virus (AAV) vector for delivery to a target cell (e.g., J
ohnson, International Publication No. WO 95/34670; International Publication No. PCT/US
95/07178). The recombinant AAV genome typically comprises AAV inverted terminal repeats flanking a DNA sequence encoding a ChMIrp polypeptide, operably linked to a functional promoter and polyadenylation sequence.

[0356] Alternative suitable viral vectors include retroviruses, adenoviruses,
The vectors include, but are not limited to, herpes simplex virus, lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus, rhabdovirus, paramyxovirus and papillomavirus.US Patent No. 5,672,344 describes an in vivo virus-mediated gene transfer system, including recombinant neurotrophic HSV-1 vector.US Patent No. 5,399,346 provides an example of a process for providing therapeutic protein to patients by delivery of human cells that are treated in vitro to insert DNA segments that code for therapeutic protein.Additional methods and materials for carrying out gene therapy technology are described in US Patent No. 5,631,236 (for adenovirus vectors), US Patent No. 5,672,510 (for retrovirus vectors), US Patent No. 5,635,399 (for retrovirus vectors that express cytokines).

[0357] Non-viral delivery methods include, but are not limited to, liposome-mediated transfer, direct injection of naked DNA, receptor-mediated transfer (ligand-DNA complexes), electroporation, calcium phosphate precipitation, and microparticle bombardment (e.g., gene guns). Gene therapy materials and methods may also include inducible promoters, tissue-specific enhancer-promoters, DNA sequences designed for site-specific integration, DNA sequences that may provide a selective advantage over parental cells, markers to identify transformed cells, negative selection and expression control systems (safety indicators), cell-specific binding factors (for cell targeting), cell-specific internalization factors, and transcription factors that increase expression by vectors, as well as methods for producing vectors. Such additional methods and materials for the practice of gene therapy techniques can be found in U.S. Pat. Nos. 4,970,154 (relating to electroporation techniques); WO 96/40958 (relating to nuclear ligands); 5,679,559 (describing lipoprotein-containing systems for gene delivery); 5,676,954 (relating to liposome carriers); 5,59
No. 3,875 (which describes a method for calcium phosphate transfection), and No. 4,945,050 (which describes a process in which biologically active particles are sprayed onto cells at a constant rate, whereby the particles penetrate the surface of the cells and become incorporated inside the cells).

It is also contemplated that ChMIrp gene therapy or cell therapy may further include the delivery of one or more additional polypeptides in the same or different cell(s). Such cells may be introduced separately into a patient, or the cells may be contained in a single implantable device (e.g., the encapsulation membrane described above), or the cells may be separately modified by viral vectors.

Another means of increasing the expression of endogenous ChMIrp polypeptide in cells by gene therapy is to insert one or more enhancer elements into the promoter of the ChMIrp gene, where the enhancer element is
The enhancer element used is selected based on the tissue in which it is desired to activate the gene - an enhancer element known to confer promoter activation in that tissue is selected. For example, when the gene encoding the ChMIrp polypeptide is "turned on" in T cells, the enhancer element may act to increase the transcriptional activity of the ChMIrp polypeptide gene.
ck promoter enhancer element can be used, where functional portions of the transcriptional elements to be added can be cloned into the ChM promoter using standard cloning techniques.
A fragment of DNA containing the Irp polypeptide promoter can be inserted (
and optionally inserted into a vector and/or 5' and/or 3' flanking sequences, etc.). This construct, known as a "homologous recombination construct," can then be introduced into the desired cells, either ex vivo or in vivo.

Gene therapy may also be used to reduce ChMIrp polypeptide expression by modifying the nucleotide sequence of the endogenous promoter. Such modifications are typically accomplished by homologous recombination methods. For example, a DNA molecule containing all or a portion of the promoter of the ChMIrp gene selected for inactivation may be engineered to remove and/or replace a fragment of the promoter that regulates transcription. For example, the TATA box and/or binding site for a transcription activator of the promoter may be deleted using standard molecular biology techniques; such deletions may inhibit promoter activity, thereby reducing the expression of the corresponding ChMIrp polypeptide.
The deletion of a TATA box or transcription activator binding site in a promoter can be accomplished by generating a DNA construct containing all or a relevant portion of a ChMIrp polypeptide promoter (from the same or related species as the ChMIrp gene to be regulated), in which one or more nucleotides of the TATA box and/or transcription activator binding site are mutated by substitution, deletion, and/or insertion of one or more nucleotides. As a result, the TATA box and/or activator binding site have reduced activity or are completely inactivated. The construct also typically contains the native (endogenous) 5' and 3' ends adjacent to the modified promoter segment.
'contains at least about 500 bases of DNA corresponding to the DNA sequence. This construct can be introduced into appropriate cells (either ex vivo or in vivo), either directly or via a viral vector as described herein.
Typically, integration of the construct into the genomic DNA of the cell is by homologous recombination, where the 5' and 3' DNA sequences of the promoter construct can act to assist in integration of the modified promoter region by hybridization to endogenous chromosomal DNA.

Additional Uses of ChMIrp Nucleic Acids and Polypeptides The nucleic acid molecules of the present invention, including nucleic acid molecules that do not themselves encode biologically active polypeptides, can be used to map the chromosomal location of the ChMIrp gene and related genes. Mapping can be accomplished by techniques known in the art, such as by PCR.
This can be done by PCR amplification and in situ hybridization).

ChMIrp nucleic acid molecules (including nucleic acid molecules that do not themselves encode a biologically active polypeptide) can be used to detect ChMIrpD in mammalian tissue or body fluid samples.
It may be useful as a hybridization probe in diagnostic assays to test, either qualitatively or quantitatively, for the presence of NA or the corresponding RNA. ChMIrp may serve as a diagnostic/predictive marker or assay for a wide variety of human cancers. Monitoring changes in the expression of ChMIrp during cancer treatment may be used as a surrogate marker to monitor tumor growth and treatment success.

[0363] The ChMIrp polypeptides may be used in combination (concurrently or sequentially) with one or more cytokines, growth factors, antibiotics, anti-inflammatory agents and/or chemotherapeutic agents, as appropriate for the condition being treated. ChMIrp may be useful as small molecule inhibitors. Additionally, peptide inhibitors designed from the ChMIrp polypeptides may be used as therapeutic or identifying agents that modulate ChMIrp polypeptide activity.

Other methods may also be used when it is desired to inhibit the activity of one or more ChMIrp polypeptides. Such inhibition may be effected by nucleic acid molecules that are complementary to and hybridize to the expression control sequences (triple helix formation) or ChMIrp mRNA. For example, antisense DNA or RNA molecules (which may be
having a sequence complementary to at least a portion of a selected ChMIrp gene
can be introduced into cells. Antisense probes can be designed by available techniques using the sequences of the ChMIrp polypeptides disclosed herein. Typically, each such antisense molecule is designed to target the respective ChMIrp polypeptide selected.
The antisense molecule is complementary to the start site (5' end) of the rp gene. When this antisense molecule then hybridizes to the corresponding ChMIrp mRNA, it
The translation of A is prevented or reduced. Antisense inhibitors provide information relating to the reduction or absence of ChMIrp polypeptide in a cell or organism.

Alternatively, gene therapy may be used to create dominant-negative inhibitors of one or more ChMIrp polypeptides. In this situation, DNA encoding mutant polypeptides of each selected ChMIrp polypeptide may be prepared and introduced into the patient's cells using either the viral or non-viral methods described herein. Each such mutant may be
Typically, it is designed to compete with an endogenous polypeptide in its biological role. In particular, ChMIrp contains a kinase domain that may be useful for designing dominant-negative gene therapies for the treatment of a wide variety of tumors.

Additionally, ChMIrp polypeptides (whether biologically active or not)
Selective binding agents that bind to ChMIrp polypeptides (as described herein) can be used as immunogens, i.e., the polypeptides contain at least one epitope against which antibodies can be raised. Selective binding agents that bind to ChMIrp polypeptides (as described herein) can be used for in vivo and in vitro diagnostic purposes, including for the detection of
These antibodies can also be used to prevent, treat, or diagnose a number of diseases and disorders, including those listed herein. These antibodies can be used to bind to ChMIrp polypeptides to reduce or block at least one activity characteristic of the ChMIrp polypeptide.
It can bind to the MIrp polypeptide or can bind to the polypeptide to form ChMI
The compound may increase at least one activity characteristic of the rp polypeptide, including by increasing the pharmacokinetics of the ChMIrp polypeptide.

[0367] The subject agents of the present invention are further described by the following examples, which are intended for illustrative purposes only and should not be construed as limiting the invention in any way.

Example 1 Isolation of Mouse cDNA Encoding Chondromodulin-I Related Gene A commercially available RNA extraction kit (Pharmacia Biosciences, Inc.) was used according to the manufacturer's instructions.
Osteoprogerin (
Osteoprogerin) transgenic mice (Simonet et al.,
A mouse cDNA library was constructed from total RNA extracted from osteopetrotic bone of the mouse ovarian tumor (J. Cell. 89:309-319, 1997).
PolyA+ RNA was selected using ligo(dT)25 columns (Dynal, Oslo, Norway). Superscript Plasmid for cDNA synthesis and plasmid cloning was used according to the manufacturer's protocol.
The cDNA was synthesized using the ELISA Kit (Gibco-BRL, Rockville, MD). The resulting cDNA was digested with SalI and NotI restriction enzymes.
Restriction enzymes (Boehringer Mannheim, Indianapolis, Indiana
The designed cDNA was digested with T4 DNA ligase (Promega, Madison, IN) to generate sticky ends to aid in ligation to a vector.
) into the SALI/NOTI pre-designed pSPORT vector (
The ligated products were transformed into E. coli DB10B electrocompetent bacteria (Gibco-BRL) by electroporation.
The plasmid p53 was transformed into LB agarose plates containing 100 mg/ml ampicillin. Clones were randomly selected for sequencing.

Sequencing of clones generated from the mouse cDNA library described above revealed
DNA corresponding to the EST sequence smbo2-00029-h3 was identified.
It is now called chondromodulin-1 related peptide (ChMIrp). BLAST analysis of the SWISS-PROT database confirmed that the mouse ChMIrp cDNA, shown in SEQ ID NO:3, encodes a protein that shows 32% identity over 116 amino acids to the amino-terminal portion of the bovine chondromodulin-I polypeptide (SEQ ID NO:7). Homology to chondromodulin-I identifies ChMIrp as a chondromodulin-1 related peptide.
As shown in Figure 1, the polynucleotide sequence of mouse ChMIrp (SEQ ID NO:3) has an open reading frame of 951 nucleotides. The mouse ChIrp polynucleotide sequence was donated to the American Type Culture Collection, 10801 University Boulevard, on August 8, 2000, in accordance with the Budapest Treaty.
d Manassas, VA) and is assigned ATCC Accession No. PTA-2329.
was given.

Example 2 Identification of the Human Orthologue of Mouse ChMIrp Genetic analysis using the full-length mouse ChMIrp nucleotide sequence (SEQ ID NO:3)
BLAST analysis of the ank EST database identified seven human ESTs (Genebank accession nos.
297231, t121280, t12179, ai123839, ai146
280, 11453695, and ai147044). Full-length human cDNA was generated by 3'RACE and 5'RACE using the primers shown below in Table 1. The primer sequences are based on consensus sequences derived from a comparison of seven human ESTs.

[0371]

Table 3 Human Skeletal Muscle Marathon Preparation
arathon Ready cDNA and Human Heart Marathon Preparation (Human
ChMIrp (ChMIrp Heart Marathon Ready) cDNA was used as template DNA for 5′ RACE (Clontech, Palo Alto, Calif.) because ChMIrp was detected in human skeletal muscle and human heart by Northern blot analysis (see Example 3 below).
'AP-1 primer (SEQ ID NO: 15) as a PCR primer, and 3'P
Primer 2244-19 (SEQ ID NO: 13) was used as a PCR primer to amplify human skeletal muscle marathon-prepared cDNA.
A 5' RACE was generated from the Marathon Ready cDNA.
Human skeletal muscle marathon-prepared cDNA was amplified using AP1 (SEQ ID NO: 15) as the 5' PCR primer and primer 2244-23 (SEQ ID NO: 11) as the 3' PCR primer.
The 5'RACE was generated from the first Ready cDNA.
The first RACE reaction was carried out according to the protocol of Clontech. One milliliter of a 1:50 dilution of the first 5' RACE product was added to the nested 5' RACE product.
The human heart was used as a template for CE. The nested 5' RACE reaction primers for skeletal muscle were AP-2 (SEQ ID NO: 16) for the 5' primer and primer 2244-20 (SEQ ID NO: 14) for the 3' primer. In human heart nested 5' RACE, AP-2 (SEQ ID NO: 16) was used as the 5' PCR nested primer and 2244-22 was used as the 3' nested primer. The nested PCR reactions were performed according to the manufacturer's (Clontech) protocol.

Human Skeletal Muscle Marathon-Prepared cDNA
The cDNA was used as a template for 3'RACE. Primer 2311-20 was used as the 5' primer.
(SEQ ID NO: 14) was used as the 3' PCR primer and AP-1 (SEQ ID NO: 15) was used as the 3' PCR primer to generate the 3' RACE product. The initial 3' RACE reaction was carried out according to the manufacturer's (Clontech) protocol.
One microliter of a 1:50 dilution of the ACE product was used as template DNA for a nested 3' RACE reaction. Primer 2311-21 (SEQ ID NO: 18)
) was used as the 5′ PCR nested primer, and AP-2 (SEQ ID NO: 16
) was used as the 3′ PCR nested primer. Nested PCR reactions were carried out according to the manufacturer's protocol (Clontech).

The products of the 5'RACE and 3'RACE reactions were separated by electrophoresis on a 1% agarose gel. Appropriately sized bands were excised from the agarose gel and purified using a Qiaquick Gel Extraction Kit (Qiagen) according to the manufacturer's instructions.
The extracted DNA was purified using 10 mM PBS containing Taq polymerase (Boehringer Mannheim).
Tris-HCl (pH 8.3), 1.5mM _MgCl2 , 50mM KCl
, 0.2 mM dNTP's (dATP, dTTP, dGTP, dCTP) for 5 min at 37° C. The DNA was then ligated into PCR2.1 vector (Invitrogen, Carlsbad, Calif.) and transformed into bacteria according to the manufacturer's instructions. The size of the insert was determined by E. coli ELISA.
By digestion with the coRI restriction enzyme (Boehringer Mannheim),
The sequence was then determined by electrophoresis on a 1% agarose gel.
The identity of the 5'RACE and 3'RACE products expressed by the CR2.1 vector was confirmed.

As shown in FIG. 2, human ChMIrp cDNA (SEQ ID NO: 1) contains the 5'
In addition to 86 bp in the untranslated region and 182 bp in the 3' untranslated region, it constitutes an open reading frame of 953 nucleotides (SEQ ID NO: 2). The human (huChIrp) and mouse (muChIrp) amino acid sequences (SEQ ID NOs: 2 and 4, respectively) (FIG. 3) show 97% identity. The human ChMIrp polypeptide sequence has been donated under the terms of the Budapest Treaty to the American Type Culture Collection, 10801 University Boulevard, Cambridge, England.
The plasmid was deposited at the ATCC Headquarters, Manassas, Va., on Aug. 8, 2000, and assigned ATCC accession number PTA-2328.

As shown in FIG. 4, huChMIrp and muChMIrp were identified in mice (Genebank accession number: U43509), rats (Genebank accession number: AF051425), and bovine (Swiss-Prot accession number: CHM1 BOVIN), human (Genbank accession number: AB006000), and rabbit (Genebank accession number: AF072129) (SEQ ID NO: 5, respectively).
Chondromudulin-I from several species, including
The muChMIrp open reading frame was compared to the sequence of rat chondromudulin-I. BLAST analysis determined that the muChMIrp open reading frame exhibited 39% identity over 289 amino acids of rat chondromudulin-I. Additionally, matches with human, mouse, bovine and rabbit chondromudulin-I showed approximately 35% identity.
BLAST analysis revealed that the open reading frame of muChMIrp matches 4 sequences spanning 262 amino acids for the rat chondromudulin-I polypeptide.
1% identity to mouse chondromudulin-I polypeptide,
It shows 40% identity over 62 amino acids, 36% identity over 314 amino acids with bovine chondromudulin-I polypeptide, and 37% identity over 262 amino acids with rabbit chondromudulin-I polypeptide.
% identity to the human chondromudulin-I polypeptide,
It was determined that mouse ChMIrp exhibits 37% identity over 260 amino acids. Furthermore, mouse ChMIrp is 2.0 times more similar to chicken chondromudulin-I polypeptide.
The BLAST analysis data between huChMIrp and chondromudulin-I of other species shows 39% identity over 89 amino acids (not shown). Since the conservation between human ChMIrp and mouse ChMIrp is very high, the BLAST analysis data between huChMIrp and chondromudulin-I of other species shows 39% identity over 89 amino acids (not shown).
The MIrp identity was comparable.

Example 3 Tissue-specific Expression of ChMIrp The tissue-specific expression pattern of the ChMIrp gene was studied by Northern blot. PCR-generated ³² P-labeled probes were used to detect the presence of ChMIrp transcripts in various tissues from both human and mouse.
The mouse ChMIrp cDNA (SEQ ID NO:3) inserted into a vector (Gibco-BRL) was used as a template to PCR amplify the entire coding region and used as a probe for Northern blot analysis.
Primer 2245-78 was defined as having the sequence ATGGCAAA.
GAATCCTCCAGAGAAAC (SEQ ID NO: 19), and the 3′ primer was designated Primer2245-79 and consists of the sequence CTAT
TAGACTCTCCCAAGCATGCG (SEQ ID NO: 20).
mM Tris-HCl (pH 8.3), 1.5mM MgCl2, _50mM
KCl, 0.2 mM dATP, dTTP and dGTP, 0.01 mM dC
In a volume of 100 ml containing 0.17 mM (α- ^32P )dCTP, 0.4 mM of each primer, and 10 ng of muChMIrp template DNA,
PCR reactions were performed with the following parameters: 94°C for 30 seconds, 40 cycles for 2 minutes, a denaturation step at 94°C, annealing at 70°C for 30 seconds, and 1 cycle at 72°C.
The probe was purified by chromatography on a Sepharose G50 column (5 min extension).
The purified product was purified by ELISA (-3' Inc, Boulder, CO).

A commercially available multiple tissue Northern blot containing human, fetal mouse and adult mouse tissues (Clontech, Palo Alto, Calif.), as well as a commercially available Zoo B
lot (Clontech) was investigated using muChMIrp.
The ELISA was performed using standard techniques (Sambrook et al., Molecular Cloning, C
old Springs Harbor Laboratory Press,
RNA was extracted from osteoprotege
rin (OPG) transgenic mice, OPG knockout mice (Bu
Cay et al., Genes Dev., 12:1260-1268) and normal C
The tissue was isolated from D-1 mice. The tissue was then dissolved in 20 ml of TRIzol reagent (Gibco-
The cells were lysed with 100 ml of chloroform (BRL), homogenized for 30 seconds, and extracted with 40 ml of chloroform. The tubes were centrifuged at 4000 rpm for 30 minutes, and the aqueous phase was transferred to a new tube. 10 ml of isopropanol was added, mixed, and 4
RNA was precipitated by centrifugation at 200 rpm for 30 min. The RNA pellet was washed with 10 ml of 70% ethanol, gently dried, and resuspended in 0.5 ml of TE buffer. RNA was fractionated using a formaldehyde/agarose gel electrophoresis system. After electrophoresis, the gel was processed and the RNA was transferred to a nylon membrane (Sambrook et al., 2003).
See supra.

Northern blots were prepared using Rapid-hyb buffer (Amersham Life Technologies, Inc.).
e Sciences, Arlington Heights, IL), 65
The blots were prehybridized at 65° C. for 30 seconds. The blots were then hybridized in the same solution with the addition of ³² P-labeled muChMIrp probe for 2 hours at 65° C. The filters were first washed with 2×SSC/0.1% SDS at room temperature for 5 minutes, then washed twice with 0.1×SSC/0.1% SDS for 5 minutes.
Washed for 15 minutes at 5° C. The blots were exposed to X-ray film at −80° C. for 4 days.

Northern blot analysis of mouse fetal tissues under stringent conditions detected a 1.2 kb transcript in whole fetuses at days 15 and 17. Low levels of expression were detected in fetuses at day 11, but not at high levels in fetuses at days 15 and 17. Mouse multiple tissue blots detected a transcript of similar size in skeletal muscle.

The muChMIrp probe also showed
A 1.2 kb transcript was detected. Expression was detected in human ovary and, to a lesser extent, in skeletal muscle, prostate, and heart.

RNA from femurs and tibiae from control CD-1 mice and osteopetrotic femurs and tibiae from OPG transgenic mice expressed weak levels of muChMIrp, whereas no expression was detected in osteopetrotic femurs and tibiae from OPG knockout mice.

Zoo Blot (Clontech) was incubated with Rapid-hyb buffer (Am
The filters were prehybridized in 0.1×SSC/0.5% NaCl for 30 seconds at 65° C. The blots were then hybridized in the same solution with the addition of ³² P-labeled muChMIrp probe for 2 hours at 65° C. The filters were first washed twice with 2×SSC for 15 minutes at room temperature, then washed twice with 0.1×SSC/0.5% NaCl for 30 seconds at 65° C.
. Washed twice with 1% SDS for 15 min at 55° C. Blots were exposed to X-ray film at −80° C. for 3 days.

The muChMIrp probe detected two bands in Zoo Blots from mouse, rat, rabbit and bovine DNA. Southern blot analysis was performed using high stringency washes, which showed that muChMIrp
It was shown that there should be high homology between ChMIrp from rat, rabbit and bovine.

In situ hybridization was performed to determine the tissue site of ChMIrp expression. A panel of normal fetal (E8.5-E18.5) and adult mouse and rat tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 micrometers. Prior to in situ hybridization, tissues were permeabilized with 0.2 M HCl followed by 10 mg/ml
The sections were digested with proteinase K and acetylated with ^{triethanolamine} and acetic anhydride.
The antisense and sense ³³ P-labeled probes were hybridized with muChM A probe and sense (control) probe at 55° C. overnight.
The Irp cDNA was obtained by in vitro transcription of plasmid DNA. After hybridization, the sections were washed twice with 4×SSC at 55° C. and then transferred to a 20-μl plate.
100 μg/ml RNase A to remove hybridized probe;
Then, they were subjected to high stringency washes at 55°C in 0.1x SSC. Slides were immersed in Kodak NTB2 emulsion, exposed at 44°C for 2-3 weeks, developed, and concentrated. Sections were examined using dark field and standard illumination methods for simultaneous evaluation of tissue morphology and hybridization signals. The following tissues were then examined: brain, parotid, submandibular and sublingual glands, esophagus, stomach, duodenum, jejunum, proximal and distal colon, liver, pancreas, heart, lung, trachea, blood vessels, lymph nodes, spleen, thymus, bone marrow, kidney, bladder, thyroid, adrenal gland, testis, prostate, ovary, uterus, fallopian tube, placenta, bone, skeletal muscle, skin, and adipose tissue.

The muChMIrp antisense probe produced a clear signal detectable above the very low levels of background seen with the sense control probe. No signal was detected in E8.5, 10.5 or 11.5 sections. Expression in E13.5-adult sections was detected in tendons, presumably fascia. There was clear signal in all identifiable tendons, but no signal was detected in muscle, bone or cartilage. Signal was also detected in cells adjacent to hair follicles in the skin and in specialized epithelial cells present on lymphocytic patches in the gut known as M cells. Additional sites of expression were detected in the thymic medulla, the cerebral cortex just above the corpus callosum of the brain, and in the granule cells surrounding developing follicles in the ovaries.

Example 4 Characterization of ChMIrp Genomic DNA BLAST analysis revealed that Genbank accession number AL035608 is a human ChM
The sequence was identified as containing the Irp genomic sequence located on chromosome Xq21.33-
The human ChMIrp genomic DNA was derived from clone 479J7, which contains human genomic DNA from SEQ ID NO:10. This human ChMIrp genomic DNA (SEQ ID NO:10) contains seven exons and six introns and is shown in FIG.

Example 5 Production of ChMIrp Polypeptides A. Expression of ChMIrp Polypeptides in Bacteria PCR was used to amplify a template DNA sequence encoding a ChMIrp polypeptide using primers corresponding to the 5' and 3' ends of the sequence.
The amplified DNA product can be modified to contain restriction enzyme sites and inserted into an expression vector using standard recombinant DNA methods. A representative vector containing the lux promoter and a gene encoding chromosome resistance (e.g., pAM
G21 (ATCC NO: 98113) was digested with BamHI and NdeI for directional cloning of the inserted DNA. The ligation mixture was transformed into E. coli by electrophoresis and transformants selected for kannamycin resistance.
The plasmid DNA from selected colonies was isolated and subjected to sequencing to confirm the presence of the insert.

The transformed host cells were incubated in 2xYT medium containing 30 mg/ml kannamycin for 30 minutes prior to incubation.
Gene expression is induced by adding 3-oxohexanoyl)-D1-homoserine lactose to a final concentration of 30 ng/ml, followed by incubation for 6 hours at either 30° C. or 37° C. The cultures were centrifuged, the bacterial pellets were resuspended and lysed, and the host cell proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Expression of the hMIrp polypeptide is assessed.

Inclusion bodies containing ChMIrp were purified as follows: bacterial cells were pelleted by centrifugation and resuspended in water. The cell suspension was lysed by sonication and pelleted by centrifugation at 195,000×g for 5-10 min. The supernatant was discarded and the pellet was washed and transferred to a homogenizer. The pellet was diluted with 5 ml of Percoll solution (75%
The cells are homogenized in liquid Percoll/0.15M NaCl until uniformly suspended, then diluted and centrifuged at 21,600 x g for 30 minutes.
Gradient fractions containing inclusion bodies are collected and pooled. The isolated inclusion bodies are then analyzed by SDS-PAGE.

SDS-PAG corresponding to ChMIrp polypeptide produced by E. coli
The signal band of E was excised from the gel and the N-terminal amino acid sequence was determined essentially as described by Matsudaira et al. (J. Biol. Chem., 262:10-11).
The concentration is determined as described in (35, 1987).

B. Expression of ChMIrp Polypeptides in Mammalian Cells PCR is used to amplify a template DNA sequence encoding a ChMIrp polypeptide using primers corresponding to the 5' and 3' ends of the sequence. The amplified DNA product is modified to contain restriction enzyme sites and inserted into an expression vector. The PCR product is gel purified and inserted into an expression vector using standard recombinant DNA methods. A representative vector, pCEP4 (Invit
rogen) (which contains the Epstein-Barr virus origin of replication)
can be used for expression of ChMIrp in COS cells. The amplified and gel-purified PCR product is ligated into pCEP4 and lipofected into COS cells. The transfected cells are selected in 100 mg/ml hygromycin and the resulting drug-resistant cultures are grown to confluence. The cells are then cultured in serum-free medium for 72 hours, the conditioned medium is removed, and
ChMIrp polypeptide expression is then analyzed by SDS-PAGE.

Expression of the ChMIrp polypeptide can be detected, for example, by silver staining.
Alternatively, ChMIrp can be produced as a fusion protein with an epitope tag (eg, an IgG contact domain or a FLAG epitope), which can be detected by Western blot analysis using an antibody against the tag peptide.

[0393] The ChMIrp polypeptide can be excised from an SDS-PAGE gel or a ChMIrp fusion protein can be purified by affinity chromatography against its epitope tag and subjected to N-terminal amino acid sequence analysis as described above.

Example 6 Production of ChMIrp Antibodies Antibodies against the ChMIrp polypeptide can be produced by immunization with purified protein or by using ChMIrp antibodies produced by biological or chemical synthesis.
Substantially pure ChMI can be obtained by immunization with Irp peptide.
The rp polypeptide or polypeptides can be isolated from transfected cells as described in Example 5. The concentration of protein in the final preparation can be increased to a few μg/ml, for example by concentrating with an Amicon filter device.
Monoclonal or polyclonal antibodies to the protein can then be produced using procedures known in the art, such as those described in Hudson and Bay (Practical Methods, 2003).
Immunology 2nd Edition, Blackwell Scientific
The composition may be prepared by either USP or USP Productions.

A. Anti-ChMIrp Monoclonal Antibody Production Monoclonal antibodies against epitopes of any of the peptides identified and isolated above can be produced using the method of Kohler and Milstein (Nature, 2006).
The hybridoma cells can be prepared from mouse hybridomas according to the classical method of Roberts et al. (1975) or its derivatives. Briefly, mice are incubated with several μl of
The mouse is then sacrificed and
The splenic antibody-producing cells are then isolated. These spleen cells are fused with mouse melanoma cells (e.g., NS-1 cells) by polyethylene glycol, and excess unfused cells are destroyed by growing the line on a selection medium containing hypoxanthine; aminopterin; and thymidine (HAT medium). Successfully fused cells are diluted, and aliquots of the dilutions are placed into wells of microtiter plates where the cultures are allowed to continue growing. After selection, tissue culture supernatants are harvested from each fusion well and assayed for ChM by EIA.
The clones are tested for Irp antibody production. Selected positive clones can be expanded and their monoclonal antibody products harvested for use. Detailed procedures for monoclonal antibody production are described in Davis et al. (Basic Methods in
Molecular Biology, Section 21-2, Elsev
(Corporate Press, New York, NY).

B. Polyclonal ChMIrp Antibody Production Polyclonal antisera containing antibodies against heterologous epitopes of a single protein can be prepared by immunizing appropriate animals with the expressed protein, as described above. Effective polyclonal antibody production is influenced by many factors related to both the antigen and the host species. For example, small molecules tend to be less immunogenic than larger molecules and may require the use of a carrier adjuvant. Host animals also vary in response to the site and dose of inoculation, with either insufficient or excessive doses of antigen resulting in low titer antisera. Small doses (ng levels) of antigen administered at multiple intradermal sites appear to be most reliable. An effective immunization protocol for rabbits has been reported by Vaitukaitis et al.
(J. Clin. Endocrinol. Metab., 33:988-99
1, 1971).

Booster injections can be given at regular intervals and antisera can be withdrawn when the antibody titer begins to drop where it can be semiquantitatively determined (e.g., by double immunodiffusion in agar against known concentrations of antigen).
lony et al. (Chap. 19: Handbook of Experiments
al Immunology,D. Weir (ed.), Blackwell, 19
73). The plateau concentration of the antibody is usually 0.1-0.2 mg/ml.
The affinity of the antiserum for the antigen is determined by generating a competitive binding curve (see, e.g., Fischer, D., (Chap. 4
2: Manual of Clinical Immunology, 2nd Edition (
Amer. Soc. For Microb
The concentration of β-aminobutyric acid is determined as described in (J. Immunol., Washington, D.C., 1980).

[0413] Alternative procedures for obtaining anti-ChMIrp antibodies can also be used, such as immunization of transgenic mice carrying human IgG loci for the production of fully human antibodies, and screening of synthetic antibody libraries (as generated by mutagenesis of antibody variable domains).

Example 7 Functional Analysis of the Role of ChMIrp To determine the functional role of ChMIrp in vivo,
A gene is overexpressed in the germline of an animal or inactivated in the germline of a mammal by homologous recombination. An animal in which the gene is overexpressed under the regulatory control of an exogenous or endogenous promoter element is known as a transgenic animal. An animal in which an endogenous gene is inactivated by homologous recombination is also known as a "knockout" animal. Representative mammals include:
These include rabbits and rodent species such as mice. Exemplary procedures are described in U.S. Patent No. 5,489,743 and International Patent Publication No. WO 94/28122.

Transgenic animals demonstrate the potent role of ChMIrp in developmental and disease processes.
This allows the determination of the effects of over- or inappropriate expression of ChMIrp. ChMIrp transgenic animals can also be used as a model system to test compounds that can modulate receptor activity.

"Knockout" animals have been shown to reduce the expression of C-terminal deficiencies in embryonic development and immune and proliferative responses.
This allows the determination of the role of the hMIrp polypeptide in developmental and proliferative responses by analysis of the effects of gene knockouts on embryonic development and on the development and differentiation of various organs and tissues (e.g., skeletal components such as bone and cartilage) in these animals.

Example 8 Biological Activity of ChMIrp Polypeptides Growth and morphological assays can be performed to determine whether ChMIrp biological activity is similar to that of chondromodulin-I. For all assays, recombinant ChMIrp (prepared and purified as described in Example 5) was used.
(derived from OS cells) is used.

A. Stimulation of Chondrocytes The ability of ChMIrp to stimulate DNA and proteoglycan synthesis in chondrocytes can be examined as a chondromodulin family activity.
himomura et al. (Calcif. Tissue Res. 19:179-1
Chondrocytes are isolated from rib growth plate cartilage from young rabbits as described by Hiraki et al. (Eur. J. Biochem., 260, 87, 1975). Isolated chondrocytes are plated at 1× ¹⁰⁴ cells/well in 96-well microtiter plates and cultured in modified Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS). At confluence, proteoglycan synthesis is measured as described by Hiraki et al. (Eur. J. Biochem., 260, 87, 1975).
: 869-878, 1999). Briefly, chondrocytes are preincubated in 0.3% serum. After 24 hours, the medium is replaced with 0.3%
The medium is replaced with one containing serum and recombinant ChMIrp polypeptide (10-1000 ng/ml). After 3 hours, cells are labeled with 5 mCi/ml [ ^35S ]-sulfate for an additional 17 hours. Proteoglycans are then precipitated with 1% cetylpyridinium chloride and incorporated radioactivity is measured.
Insulin-like growth factor I or II may be used as a positive control.

To determine whether DNA synthesis is stimulated by ChMIrp polypeptide, isolated chondrocytes are plated at 1×10 ⁴ cells/well in 96-well microtiter plates in MEM medium containing 10% FBS. After 24 hours, the medium is changed to 0.3% serum plus recombinant ChMIrp polypeptide (10-1000 mM NaCl).
The medium is replaced with one containing [ ^3H ]-thymidine (5 mCi/ml) and the cells are incubated for 20 hours. The cells are then labeled with [3H]-thymidine (5 mCi/ml) for an additional 4 hours. Afterwards, the cells are washed with PBS and fixed with cold 100% methanol for 10 minutes. After fixation, incorporated radioactivity is precipitated with 10% trichloroacetic acid and counted.

Chondromodulin-I synergizes with FGF-2 to induce colony formation of chondrocytes in soft agar. To determine whether ChMIrp polypeptides exhibit this activity, isolated chondrocytes (5×10 ³ cells/well) were cultured in Ino
ue et al. (Biochem. Biophys. Res. Comm., 241:39
HAM F-12 supplemented with 5% FBS, 0.2 mM hydrocortisone, and 60 mg/ml transferrin as described in (5-400, 1997).
The cells are suspended in 0.5 ml of 0.41% agarose in medium.
. Pour onto a base layer of 72% agarose and incubate overnight. Increasing concentrations of recombinant ChMIrp polypeptide (1-1000 ng/ml) and 1 ng/ml
1 ml FGF-2 is diluted in serum-free F-12 medium containing 0.5% bovine serum albumin. The mixture is evenly added to the upper layer of the well. After 10 days, the colonies are counted under a phase contrast microscope.

B. Endothelial Cell Stimulation Endothelial cell proliferation and morphogenesis in the presence of ChMIrp polypeptides are assayed to determine whether inhibition of endothelial cell proliferation and tube formation occurs similarly to that in the presence of chondromodulin-I. Growth and tube morphogenesis in bovine carotid artery endothelial cells in vitro was measured as described by Hiraki et al. (FEBS, 415:32
1-324, 1997). Briefly, bovine carotid artery (BCAE) cells are isolated by gently scraping the inner surface of the carotid artery. The isolated BCAE cells are cultured in RPMI-1 medium supplemented with 10% FBS.
BCAE cells are grown and expanded in a 12-well plate containing 0.3% type I collagen gel diluted in ^{0.1 M NaOH and 10x MEM medium. After 24 hours, the medium is aspirated and the recombinant ChMIrp is added to BCAE cells. To determine whether the recombinant ChMIrp can inhibit the proliferation of BCAE cells, [3H ]} -thymidine incorporation is measured as described above. To test the effect of ChMIrp on endothelial cell tubular morphogenesis, BCAE cells (1x105 cells/well) are grown in 12-well plates containing 0.3% type I collagen gel diluted in 0.1 M NaOH and 10x MEM medium. After 24 hours, the medium is aspirated and the recombinant ChMIrp is added to BCAE cells.
An aqueous mixture (70 ml) containing Irp polypeptide (10-1000 ng/ml)
) is added. Then, the collagen solution is layered to prepare the upper layer. After 3 days,
The morphological changes of the cells into tube-like networks are observed under a phase contrast microscope. An in vivo assay for measuring endothelial cell angiogenesis is described by Hiraki et al. (Eur. J. Biol. 2002, 144:1311-1315).
The chicken chorioallantoic membrane assay is as described by (M. et al., 1999). Briefly, fertilized white leghorn chicken eggs are incubated for 3 h at 4 °C for 1 h.
Incubate at 7.8° C. On day 5, the air space is punctured and a 1 cm ² window is opened in the egg. Recombinant ChMIrp polypeptide (500 ng/ml
1) was diluted in 0.75% agarose. The fixed gel was placed on the chorioallantoic membrane in the egg for 24 hours. The membrane was examined under a dissecting microscope for fine capillary tube formation.

C. Osteoblast Stimulation The effect of ChMIrp on osteoblast proliferation was also reported by Mori et al. (FEBS 4
06:310-314, 1997) as an index of chondromodulin-I activity. The clonal osteoblastic cell line MC3T3-E1 is treated with increasing concentrations of recombinant ChMIrp (10-1000 ng/ml). DNA
As a measure of synthesis, osteoblastic [ ³ H]-thymidine incorporation is measured as described above.

This postulated biological function of the ChMIrp polypeptide is similar to that of chondromodulin-I. In particular, chondromodulin-I is known to stimulate the proliferation and differentiation of chondrocytes. Furthermore, chondromodulin-I
Chondromodulin-I has antiangiogenic activity, since it inhibits endothelial cell proliferation and fine capillary tube formation in vivo.
It may play a role in cartilage development and blood vessel formation.

It has been determined by Northern blot analysis and in situ hybridization that ChMIrp polypeptide is expressed in tendons, skeletal muscle, thymus, ovary, cerebral cortex of the brain, M cells of the intestine, and cells adjacent to hair follicles. Expression in tendons and muscles indicates that ChMIrp may play a role in tendon and muscle development and attachment of muscle to bone. Expression in thymus, hair follicles, and M cells of the intestine indicates a potential role for ChMIrp in immune function.
p may act as a growth factor involved in the regeneration (proliferation and development) of tissues and specific cell types present in tendons, skeletal muscles, thymus, ovaries, brain, gut and hair follicles.

Based on these potential functions, ChMIrp may be useful in the diagnosis and/or treatment of tendon diseases (e.g., tendonitis and tendon rupture), skeletal muscle diseases (e.g., cachexia and muscular dystrophies), immune system dysfunction diseases (e.g., inflammation and allergies, poor wound healing, arthritis and allergies), and infertility diseases.

While the present invention has been described in terms of preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. It is therefore intended that the appended claims cover all such equivalent variations that come within the scope of the invention as claimed.

[Sequence List]

[Brief description of the drawings]

FIG. 1 shows a mouse ChMI containing an open reading frame (SEQ ID NO:4).
1 shows the polynucleotide sequence shown as SEQ ID NO:3 which represents the entire coding region of the rp cDNA.

FIG. 2 shows human ChMIr containing an open reading frame (SEQ ID NO:2).
1 shows the polynucleotide sequence shown as SEQ ID NO:1 which represents the entire coding region of p cDNA.

FIG. 3 shows the human ChMIrp polypeptide sequence (SEQ ID NO:2) and mouse ChMIrp polypeptide sequence (SEQ ID NO:3).
1 shows the sequence of the MIrp polypeptide sequence (SEQ ID NO:4).

FIG. 4 shows human and mouse ChMIrp polypeptide sequences (SEQ ID NO:2, respectively).
and SEQ ID NO:4) and chondromodulin-I polypeptides from various mammalian species (SEQ ID NOs:5-9).

FIG. 5 shows the complete genomic DNA sequence of human ChMIrp, shown as SEQ ID NO: 10, with exons and splice acceptor/donor sites underlined in bold.

───────────────────────────────────────────────────────── Continued from the front page (51)Int.Cl. ⁷ Identification symbol FI Theme code (for reference) A61K 38/00 A61K 39/395 N 4B064 38/22 45/00 4B065 39/395 47/48 4C076 48/00 4C084 45/00 A61P 1/16 4C085 47/48 9/00 4C086 48/00 9/10 101 4C087 A61P 1/16 9/12 4H045 9/00 17/06 9/10 101 19/10 9/12 29/00 17/06 35/00 19/10 C07K 16/40 29/00 16/42 35/00 16/46 C07K 16/40 C12N 1/15 16/42 1/19 16/46 1/21 C12N 1/15 9/12 1/19 11/04 1/21 C12Q 1/02 5/10 1/48 Z 9/12 1/68 A 11/04 G01N 33/53 D C12Q 1/02 M 1/48 33/566 1/68 33/577 B G01N 33/53 33/58 A C12P 21/08 33/566 C12N 15/00 ZNAA 33/577 5/00 A 33/58 B // C12P 21/08 A61K 37/24 37/02 (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, G M, 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, BA, BB, BG, BR, BY, B Z, 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, R O, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Yun, Jeanie 5348 New Castle Avenue, Encino, California 91316, United States of America Apartment 227 (72) Inventor Clarkin, Christie 2318 Eagle Creek Lane, Oxnard, California 93030, United States of America F-term (reference) 2G045 AA34 AA35 BB07 BB20 BB50 CB01 DA13 DA36 FA16 FB02 FB03 FB08 4B024 AA01 AA11 BA10 BA41 CA04 CA07 CA09 CA11 DA02 DA06 EA02 EA04 GA01 GA11 GA18 GA19 HA03 HA14 HA17 4B033 NA16 NA25 NB58 NC06 ND12 NF06 4B050 CC01 CC03 DD11 FF13E LL03 4B063 QA01 QA18 QA19 QQ03 QQ27 QQ42 QR32 QR38 QR41 QR55 QR69 QR77 QR82 QS12 QS25 QS34 QS39 QX07 4B064 AG27 CA10 CA20 CC24 DA01 DA13 4B065 AA26X AA90X AA91X AA93Y AB02 AC14 BA02 BA08 CA25 CA29 CA44 CA46 4C076 AA94 BB11 BB16 BB32 CC04 CC11 CC27 EE59 4C084 AA01 AA02 AA03 AA06 AA13 AA16 BA01 BA02 BA08 CA17 CA23 DB52 DB60 MA17 MA22 MA24 MA27 MA37 MA43 MA44 MA55 MA56 MA65 MA66 MA67 NA14 ZA362 ZA422 ZA452 ZA752 ZA972 ZB112 4C085 AA13 AA14 AA15 AA16 BB41 BB43 BB44 CC22 CC23 EE01 GG02 GG04 4C086 AA02 AA03 EA16 MA01 MA05 MA17 MA22 MA23 MA24 MA35 MA37 MA44 MA52 MA55 MA56 MA65 MA66 MA67 NA14 ZA26 ZA36 ZA45 ZA75 ZA89 ZA97 ZB11 4C087 BB33 BC83 MA22 MA23 MA24 MA27 MA35 MA37 MA43 MA44 MA52 MA56 MA58 MA65 MA66 NA14 ZA26 ZA36 ZA42 ZA75 ZA89 ZA97 ZB11 4H045 AA10 AA20 AA30 BA10 CA40 DA75 DA76 DA86 DA89 EA50 FA72 FA74 [Summary continued]

Claims (73)

[Claims] [Claim 1] An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID NO:1; (b) a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO:2; (c) a nucleotide sequence that hybridizes under moderate or high stringency conditions to the complement of (a) or (b), wherein the encoded polypeptide has the activity of the polypeptide set forth in SEQ ID NO:2; and (d) a nucleotide sequence complementary to any of (a) to (c). [Claim 2] An isolated nucleic acid molecule, the nucleic acid molecule comprising: (a) a nucleotide sequence encoding a polypeptide which is at least about 70% identical to the polypeptide set forth in SEQ ID NO:2, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2; (b) a nucleotide sequence encoding an allelic or splice variant of the nucleotide sequence set forth in SEQ ID NO:1, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2; (c) a nucleotide sequence of SEQ ID NO:1, (a) or (b), which encodes a polypeptide fragment of at least about 25 amino acid residues, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2; (d) a nucleotide sequence of SEQ ID NO:1 or (a)-(c), which comprises a fragment of at least about 16 nucleotides; (e) a nucleotide sequence which hybridizes under moderately or highly stringent conditions to the complement of any of (a)-(d), wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2;
and (f) a nucleotide sequence selected from the group consisting of a nucleotide sequence complementary to any of (a) to (c). [Claim 3] An isolated nucleic acid molecule, the nucleic acid molecule comprising: (a) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO:2 having at least one conservative amino acid substitution, the polypeptide having an activity of the polypeptide set forth in SEQ ID NO:2; (b) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO:2 having at least one amino acid insertion, the polypeptide having an activity of the polypeptide set forth in SEQ ID NO:2; (c) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO:2 having at least one amino acid deletion, the polypeptide having an activity of the polypeptide set forth in SEQ ID NO:2; (d) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO:2 having a C-terminal and/or N-terminal truncation, the polypeptide having an activity of the polypeptide set forth in SEQ ID NO:2; (e) a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO:2 having at least one modification selected from the group consisting of an amino acid substitution, an amino acid insertion, an amino acid deletion, a C-terminal truncation and an N-terminal truncation, the polypeptide having
(f) a nucleotide sequence of (a) to (e) comprising a fragment of at least 16 nucleotides; (g) a nucleotide sequence that hybridizes under moderately or highly stringent conditions to the complement of any of (a) to (f), wherein the polypeptide has the activity of the polypeptide shown in SEQ ID NO:2;
and (h) a nucleotide sequence selected from the group consisting of a nucleotide sequence complementary to any of (a) to (e). 4. A vector comprising the nucleic acid molecule of claim 1, 2 or 3. 5. A host cell comprising the vector according to claim 4. 6. The host cell of claim 5, which is a eukaryotic cell. 7. The host cell of claim 5, which is a prokaryotic cell. 8. A process for producing a ChMIrp polypeptide, comprising culturing a host cell according to claim 5 under conditions suitable for expressing said polypeptide, and optionally isolating said polypeptide from said culture. 9. A polypeptide produced by the process of claim 8. 10. The process of claim 8, wherein the nucleic acid molecule comprises promoter DNA other than promoter DNA for a native ChMIrp polypeptide, the promoter being operably linked to DNA encoding the ChMIrp polypeptide. 11. The isolated nucleic acid molecule of claim 2, wherein
The percent identity may be determined by GAP, BLASTP, BLASTN, FASTA,
BLASTA, BLASTX, BestFit and Smith-Waterm
The nucleic acid molecule is determined using a computer program selected from the group consisting of an algorithm. 12. A process for identifying a candidate inhibitor of the activity or production of a ChMIrp polypeptide, the process comprising the steps of exposing a cell according to claim 5, 6 or 7 to said candidate inhibitor, detecting ChMIrp in said cell,
A process comprising measuring the activity or production of a ChMIrp polypeptide and comparing ChMIrp activity in cells exposed to the candidate inhibitor with activity in cells not exposed to the candidate inhibitor. [Claim 13] A process for identifying a candidate stimulatory factor for activity or production of a ChMIrp polypeptide, the process comprising the steps of exposing a cell described in claim 5, 6 or 7 to the candidate stimulatory factor, measuring activity or production of a ChMIrp polypeptide in the cell, and comparing the ChMIrp activity in the cell exposed to the candidate stimulatory factor with the activity in a cell not exposed to the stimulatory factor. 14. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2. [Claim 15] An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) the mature amino acid sequence set forth in SEQ ID NO:2, comprising a mature amino terminus at residue 1, and optionally further comprising an amino-terminal methionine; (b) an amino acid sequence of an ortholog of SEQ ID NO:2, wherein the encoded polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2; (c) an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in SEQ ID NO:2, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2; (d) a fragment of the amino acid sequence set forth in SEQ ID NO:2 comprising at least about 25 amino acid residues, wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2; (e) an amino acid sequence of an allelic variant or splice variant of SEQ ID NO:2, or any of the amino acid sequences set forth in at least one of (a)-(c), wherein the polypeptide has an activity of the polypeptide set forth in SEQ ID NO:2. [Claim 16] An isolated polypeptide, the polypeptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence as set forth in SEQ ID NO:2 with at least one conservative amino acid substitution, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2; (b) an amino acid sequence as set forth in SEQ ID NO:2 with at least one amino acid insertion, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2; (c) an amino acid sequence as set forth in SEQ ID NO:2 with at least one amino acid deletion, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2; (d) an amino acid sequence as set forth in SEQ ID NO:2 with a C-terminal truncation and/or an N-terminal truncation, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2; and (e) an amino acid sequence as set forth in SEQ ID NO:2 with at least one modification selected from the group consisting of an amino acid substitution, an amino acid insertion, an amino acid deletion, a C-terminal truncation and an N-terminal truncation, wherein the polypeptide has an activity of the polypeptide as set forth in SEQ ID NO:2. 17. The polypeptide according to claim 15 or 16, wherein the amino acid at position 276 of SEQ ID NO:2 is cysteine, serine or alanine. 18. The polypeptide according to claim 15 or 16, wherein the amino acid at position 280 of SEQ ID NO:2 is cysteine, serine or alanine. 19. The polypeptide according to claim 15 or 16, wherein the amino acid at position 281 of SEQ ID NO:2 is glutamic acid or aspartic acid. 20. The polypeptide according to claim 15 or 16, wherein the amino acid at position 285 of SEQ ID NO:2 is glycine, proline or alanine. 21. The polypeptide according to claim 15 or 16, wherein the amino acid at position 297 of SEQ ID NO:2 is arginine, lysine, glutamine or asparagine. 22. The polypeptide of claim 15 or 16, wherein the amino acid at position 300 of SEQ ID NO:2 is cysteine, serine, or alanine. 23. The polypeptide according to claim 15 or 16, wherein the amino acid at position 306 of SEQ ID NO:2 is cysteine, serine or alanine. 24. The polypeptide according to claim 15 or 16, wherein the amino acid at position 310 of SEQ ID NO:2 is valine, isoleucine, methionine, leucine, phenylalanine, alanine or norleucine. 25. An isolated polypeptide encoded by the nucleic acid molecule of claim 1, 2 or 3. 26. The isolated polypeptide of claim 15, wherein the percent identity is determined by GAP, BLASTP, BLASTN, FAS, or the like.
TA, BLASTA, BLASTX, BestFit and Smith-Wat
The polypeptide is determined using a computer program selected from the group consisting of the .erman algorithm. 27. An antibody produced by immunizing an animal with a peptide comprising the amino acid sequence of SEQ ID NO:2. 28. An antibody or a fragment thereof that specifically binds the polypeptide of claim 14, 15 or 16. 29. The antibody of claim 28, which is a monoclonal antibody. 30. A hybridoma which produces a monoclonal antibody that binds to a peptide comprising the amino acid sequence of SEQ ID NO:2. 31. A method for detecting or quantifying the amount of ChMIrp in a sample, comprising the steps of: adding to a sample suspected of containing a ChMIrp polypeptide, a compound according to claim 2,
30. A method comprising contacting an anti-h2520-109 antibody or fragment thereof of claim 7, 28 or 29 with said antibody or fragment, and detecting binding of said antibody or fragment. 32. A selective binding agent or fragment thereof that specifically binds at least one polypeptide, said polypeptide having: (a) the amino acid sequence set forth in SEQ ID NO:2; (b) at least one fragment of the amino acid sequence set forth in SEQ ID NO:2;
and (c) a selective binding agent comprising an amino acid sequence selected from the group consisting of naturally occurring variants of (a) or (b). 33. The selective binding agent of claim 32 which is an antibody or a fragment thereof. 34. The selective binding agent of claim 32 which is a humanized antibody. 35. The selective binding agent of claim 32 which is a human antibody or fragment thereof. 36. The selective binding agent of claim 32 which is a polyclonal antibody or fragment thereof. 37. The selective binding agent of claim 32 which is a monoclonal antibody or a fragment thereof. 38. The selective binding agent of claim 32 which is a chimeric antibody or fragment thereof. 39. The selective binding agent of claim 32 which is a CDR-grafted antibody or fragment thereof. 40. The selective binding agent of claim 32 which is an anti-idiotype antibody or a fragment thereof. Claim 41. The selective binding agent of claim 32 which is a variable region fragment. 42. A Fab fragment or a Fab' fragment.
42. The variable region fragment of claim 41. 43. A selective binding agent or fragment thereof, comprising at least one complementarity determining region having specificity for a polypeptide having the amino acid sequence of SEQ ID NO:2. 44. The selective binding agent of claim 32, having a detectable label attached thereto. 45. The selective binding agent of claim 32, which antagonizes the biological activity of a ChMIrp polypeptide. Claim 46. A method for treating, preventing or ameliorating a disease, condition or disorder comprising administering to a patient an effective amount of a selective binding agent of claim 32. 47. A selective binding agent produced by immunizing an animal with a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2. 48. A hybridoma producing a selective binding agent capable of binding the polypeptide of claim 14, 15 or 16. 49. A composition comprising a polypeptide according to claim 14, 15 or 16 and a pharma- ceutically acceptable formulation. 50. The composition of claim 49, wherein the pharma- ceutically acceptable formulation agent is a carrier, adjuvant, solubilizer, stabilizer, or antioxidant. 51. The composition of claim 50, wherein the polypeptide comprises the mature amino acid sequence set forth in SEQ ID NO:2. 52. A polypeptide comprising a derivative of the polypeptide of claim 14, 15 or 16. 53. The method of claim 5, which is covalently modified with a water soluble polymer.
3. The polypeptide according to claim 2. 54. The polypeptide of claim 53, wherein the water soluble polymer is polyethylene glycol, monomethoxy-polyethylene glycol,
A polypeptide selected from the group consisting of dextran, cellulose, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol, and polyvinyl alcohol. 55. A composition comprising the nucleic acid molecule of claim 1, 2 or 3 and a pharma- ceutically acceptable formulation. 56. The composition of claim 55, wherein the nucleic acid molecule is contained in a viral vector. 57. A viral vector comprising the nucleic acid molecule of claim 1, 2 or 3. 58. The polypeptide of claim 14, 15 or 1 fused to a heterologous amino acid sequence.
A fusion polypeptide comprising the polypeptide according to claim 6. 59. The fusion polypeptide of claim 58, wherein the heterologous amino acid sequence is an IgG constant domain or a fragment thereof. 60. A method for treating, preventing or ameliorating a medical condition in a mammal resulting from a decrease in ChMIrp polypeptide levels, comprising administering to the mammal a polypeptide described in claim 14, 15 or 16 or a polypeptide encoded by a nucleic acid described in claim 1, 2 or 3. [Claim 61] A method for diagnosing a pathological condition or a susceptibility to a pathological condition in a subject caused by or resulting from an abnormal level of ChMIrp polypeptide, comprising: (a) determining the presence or amount of expression of a polypeptide described in claim 14, 15 or 16 or a polypeptide encoded by a nucleic acid molecule described in claim 1, 2 or 3 in a sample; and (b) comparing the levels of ChMIrp polypeptide in biological, tissue or cell samples derived from normal or early stage subjects, wherein the susceptibility to the pathological condition is based on the presence or amount of expression of the polypeptide. 62. A device comprising: (a) a membrane compatible with implantation; and (b) cells encapsulated within said membrane, wherein said cells secrete a polypeptide according to claim 14, 15 or 16, and said membrane is permeable to said protein and impermeable to substances harmful to said cells. [Claim 63] A device comprising: (a) a membrane compatible with implantation; and (b) a ChMIrp polypeptide encapsulated within the membrane, wherein the membrane is permeable to the polypeptide. 64. A method for identifying a compound that binds to a polypeptide, comprising the steps of:
17. A method comprising: (a) contacting a compound with a polypeptide of claim 14, 15 or 16; and (b) determining the degree of binding of the polypeptide to the compound. 65. A method of regulating the level of a polypeptide in an animal, comprising administering to said animal a nucleic acid molecule according to claim 1, 2 or 3. 66. A transgenic non-human mammal comprising the nucleic acid molecule of claim 1, 2 or 3. 67. A diagnostic reagent comprising the amino acid sequence set forth in SEQ ID NO:2:
or a detectably labeled polynucleotide encoding a fragment, variant or homolog thereof, including allelic and splice variants thereof. 68. The diagnostic reagent of claim 67, wherein the labeled polynucleotide is a single-stranded cDNA. 69. A method for determining the presence of ChMIrp nucleic acid in a biological sample, comprising the steps of: (a) providing a biological sample suspected of containing ChMIrp nucleic acid; (b) contacting said biological sample with a diagnostic reagent according to claim 84, wherein said diagnostic reagent detects h2520 nucleic acid contained in said biological sample.
(c) performing a test under conditions in which the ChMIrp nucleic acid in the biological sample hybridizes with the diagnostic reagent; and (d) comparing the level of hybridization between the biological sample and the diagnostic reagent with the level of hybridization between a known concentration of ChMIrp nucleic acid and the diagnostic reagent. [Claim 70] A method for determining the presence of ChMIrp nucleic acid in a tissue or cell sample, comprising the following steps: (a) providing a tissue or cell sample suspected of containing ChMIrp nucleic acid; (b) contacting the tissue or cell sample with the diagnostic reagent of claim 68 under conditions in which the diagnostic reagent hybridizes to the ChMIrp nucleic acid; (c) detecting hybridization between the ChMIrp nucleic acid in the tissue or cell sample and the diagnostic reagent; and (d) comparing the level of hybridization between the tissue or cell sample and the diagnostic reagent with the level of hybridization between a known concentration of ChMIrp nucleic acid and the diagnostic reagent. 71. The method of claim 70 or 71, wherein the ChMIrp polynucleotide molecule is DNA. Claim 72. The method of claim 70 or 71, wherein the ChMIrp polynucleotide molecule is RNA. 73. An antagonist of ChMIrp polypeptide activity, said antagonist having specificity for a ChMIrp polypeptide.
The antagonist is selected from the group consisting of an hMIrp selective binding agent, a small molecule, an antisense oligonucleotide and a peptide or derivatives thereof.