WO1998054326A1 - Human chemokine zchemo-8 - Google Patents

Abstract

Ligand polypeptides, polynucleotides encoding the ligand polypeptides, and related compositions and methods are disclosed. The ligand polypeptide is a member of the chemokine beta subfamily. The polypeptides may be used within methods relating to regulation of acute and chronic inflammatory disease conditions, lymphocyte migration and ischemia/reperfusion injury.

Description

DESCRIPTION HUMAN CHEMOKINE ZCHEMO-8

BACKGROUND OF THE INVENTION

Chemotactic cytokines were originally identified in supernatants of stimulated leukocyte cell cultures and were initially characterized through their chemotactic effects on a variety of leukocytes. Subsequent isolation of several chemotactic cytokines has led to the discovery of a family of structurally and functionally homologous molecules now known as "chemokines" (see Schnell, The Cytokine Handbook, Academic Press, 419-60, 1994). In vivo, chemokines have pro-inflammatory, pyrogenic, chemokinetic, myelosuppressive or hematopoietic effects; primarily, chemokines regulate inflammatory and immunoregulatory processes through their selective recruitment and activation of leukocytes. Chemokines are 8 to 16 kDa soluble basic proteins that are produced and released by a variety of cell types during an acute inflammatory response to injury, allergens, or microbial and viral infections. Moreover, chemokine involvement is apparent in some chronic inflammatory states such as arthritis, asthma, and eczema.

The chemokine family contains over 20 members sharing 20-70% amino acid sequence homology. Currently, most isolated chemokines are of human origin, with a few known orthologues reported. Chemokines are mainly divided into two subclasses (alpha (α) and beta (β) ) based on a four-cysteine motif contained within their protein structure. The first pair of cysteines are either separated by an intervening residue (the α subfamily, or "C-X-C" chemokines) or are adjacent (the β subfamily, or "C-C" chemokines) . In general, the C-X-C chemokines are involved in neutrophil recruitment and activation and are implicated in acute inflammatory diseases. The C-C chemokines exert their effect on other leukocyte populations such as, monocytes, T cells, eosinophils and basophils and are implicated in chronic inflammatory conditions. However, this functional distinction between the families is not exclusive; for example, the C-X-C chemokine, platelet factor 4 (PF4) , induces both neutrophil and monocyte migration. Lymphotactin is a prototype of a third class of chemokines ("C" chemokines) which contain only two of the four cysteines. Further investigation has elucidated other common characteristics of each family.

Most known members of the C-X-C family of chemokines are found clustered on human chromosome 4 between ql2 and q21. The well known members of this family are PF4 and interleukin 8 (IL-8) . The most extensively studied chemokine, PF4 , released from blood platelets, is thought to play a role in inflammation and wound healing. PF4 may provide a model for linking the physiologically coordinated processes of thrombosis, inflammation and wound healing. IL-8, which attracts neutrophils, may influence neutrophil -mediated damage during inflammation. Moreover, IL-8 is implicated in other acute immune reactions (e.g., response to bacterial endotoxins) . In fact, anti-I -8 antibodies prevent the migration of neutrophils to injured lung tissue and protect it from IL- 8 -induced lung injury in vivo, proving a causal role of locally-produced IL-8 in reperfusion injury in a rabbit lung model (Sekido et al . , Nature

365:654-57, 1993) .

The C-C family of chemokines appears to be clustered on human chromosome 17 between qll and q21.

Thus far, all have a three exon, two intron genomic arrangement. The biological activities of the C-C group appear more diverse than those of the C-X-C chemokines, indicating that the C-C chemokines may serve as links between monocytes, lymphocytes, eosinophils and basophils in immune and inflammatory responses. Some of the well known members of this family are macrophage inflammatory protein (MIP-1) , monocyte chemotactic protein (MCP-1) , RANTES, and eotaxin. Unlike nearly all other chemokines, MIP-1 is an acidic protein. It is expressed primarily in mitogen-stimulated T-cells, B-cells and monocytes. MIP-1 has been shown to have hematopoietic effects and pyrogenic effects. Interestingly, MIP-l's fever- inducing properties are independent of the prostaglandin pathway since they are unaffected by aspirin or ibuprofen (which block the cyclooxygenase pathway) . Studies show that homozygous MlP-lα null mutant mice are resistant to Coxsackievirus- induced myocarditis, and exhibit reduced pneumonitis and delayed viral clearance during influenza virus infection

(Cook et al., Science 269 : 1583-85, 1995). Thus, murine

MlP-lα is required for inflammatory response to viral infection in vivo . Although most C-C chemokines induce a spectrum of biological effects, eotaxin acts very specifically. Eotaxin exclusively attracts eosinophils both in vivo and in vi tro, with no effects on neutrophils or monocytes. Eosinophils are recruited in allergic reactions leading to eczema and asthma as well as in response to certain parasites. Human eotaxin is expressed at high levels in the small intestine and colon, and appears to have a specific interaction with only one member of the chemokine receptor family (CC CKR3 ) that is selectively expressed on human eosinophils (Kitaura et al . , J. Biol. Chem. 271 :7725-30, 1996). This evidence of narrow specificity for a chemokine, eotaxin, implies the existence of undiscovered chemokines with specific biological activities . Although chemokines exhibit a wide variety of biological activities that affect inflammatory cells, they appear to share interaction with only a few receptors. The specific effects of chemokines are mediated by a family of 7-transmembrane domain G-protein coupled receptors. How chemokines exhibit specific and varied effects via their receptors is generally unknown. For instance, MIP-1, MCP-1 and RA TES all bind C-C chemokine receptor 1 (CC CKR1) , but how the biological specificity is imparted by each is undefined. Several groups have shown that chemokine receptors react to different combinations of chemokines in different ways. For example, Ca⁺⁺ signaling desensitization studies suggest an array of shared and specific receptors; moreover, the desensitization pattern depends on the order of chemokine involvement and the cell type to which they are exposed. Moreover, differences in binding affinities and signaling activities with shared receptors are exhibited. In vivo, it is likely that both chemokine and specific chemokine receptor expression are regulated temporally and spatially. Because chemokines exhibit diverse biological activities, they are believed to be involved with several pathological conditions. Chemokines are implicated in inflammation, ischemia and reperfusion injury, wound healing, allergies, bacterial and viral pathogenesis . Moreover, chemokines may be involved in chronic diseases such as arthritis, asthma and atherosclerosis (migration of monocytes and arterial damage) . For example, MCP-1 mRNA is detected in a variety of conditions where monocytes predominate in the pathology; moreover, high levels of chemokines have been detected in certain disease states. Many tumor cell lines and some primary tumors produce chemokines. Whether chemokines are active in tumor biology is unknown, but the migratory and immune effects of these molecules may implicate a role in tumor regression or growth. Although there are correlations between chemokines and disease, the causal relationship and roles are not well defined. DARC, a promiscuous chemokine receptor is also a receptor for the malarial parasite (Horuk et al . , Science 261.1182-84, 1993) and the chemokine receptor CCCK5 is involved with HIV. Animal studies in which antibodies are used to neutralize the activity of individual members of the chemokine family confirm that these mediators contribute to the development of both acute and chronic inflammatory disease conditions. Because of their association with certain disease states, lymphocyte migration, immune and inflammatory regulation, chemokines and their inhibitors may prove useful as therapeutics .

The demonstrated in vi tro and in vivo activities of these chemokine family members illustrate the enormous clinical potential of, and need for, other chemokine ligands, chemokine receptors and chemokine agonists and antagonists. Therapeutics that target chemokines directly or enhance the body's mechanisms for controlling their actions may prove to be reasonable approaches for treatment of a number of disease states. The present invention addresses this need by providing a novel human chemokine and related compositions and methods.

SUMMARY OF THE INVENTION Within one aspect, the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 60% identical in amino acid sequence to residues 23-94 of SEQ ID NO: 2, said polypeptide comprising cysteine residues at positions corresponding to residues 33, 34, 57 and 73 of SEQ ID NO: 2. Within one embodiment the polypeptide is at least 80% identical in amino acid sequence to residues 23-94 of SEQ ID NO: 2, the polypeptide comprising cysteine residues at positions corresponding to residues 33, 34, 57 and 73 of SEQ ID NO : 2. Within other embodiments the polypeptide comprises residues 23-94 of SEQ ID NO : 2. The polypeptide comprises residues 1-94 of SEQ ID N0:2. Within another embodiment the polypeptide is covalently linked to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores . Within a related embodiment is further comprised a proteolytic cleavage site between the polypeptide and the moiety.

Within another aspect, the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described above; and a transcriptional terminator. Within one embodiment the DNA segment further encodes a secretory signal sequence operably linked to said polypeptide. Within a related embodiment the secretory signal sequence encodes residues 1-22 of SEQ ID NO: 2.

Within another aspect is provided a cultured cell into which has been introduced an expression vector as described above, wherein said cell expresses said polypeptide encoded by said DNA segment.

Within another aspect is provided a method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector as described above, whereby said cell expresses said polypeptide encoded by said DNA segment; and recovering said expressed polypeptide.

Within other aspects are provided, a pharmaceutical composition comprising a polypeptide as described above in combination with a pharmaceutically acceptable vehicle. An antibody that specifically binds to an epitope of a polypeptide of SEQ ID NO: 2. A binding protein that specifically binds to an epitope of a polypeptide of SEQ ID NO: 2.

Within another aspect is provided an isolated polynucleotide encoding a polypeptide as described above. Within one embodiment the polynucleotide is DNA. Within a related aspect is provided an isolated polynucleotide, wherein said polynucleotide is selected from the group consisting of, a) a sequence of nucleotides from nucleotide 103 to nucleotide 319 of SEQ ID NO:l; b) a sequence of nucleotides from nucleotide 38 to nucleotide 319 of SEQ ID NO : 2 ; c) orthologs of a) or b) ; d) degenerate nucleotide sequences of a) , b) or c) ; and f) nucleotide sequences complementary to a) , b) , c) or d) . Within another embodiment is provided a polypeptide having the nucleotide sequence of SEQ ID NO: 33.

Within another aspect is provided an oligonucleotide probe or primer comprising 14 contiguous nucleotides of a polynucleotide of SEQ ID NO: 33 or a sequence complementary to SEQ ID NO: 33. Within a further aspect is provided a DNA construct encoding a polypeptide fusion, said fusion comprising a secretory signal sequence having the amino acid sequence of residues 1-22 of SEQ ID NO: 2, wherein said secretory signal sequence is operably linked to an additional polypeptide.

Within yet another aspect is provided a method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO:l or the complement of SEQ ID NO : 1 , under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; comparing said first reaction product to a control reaction product, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient .

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows a comparison of the deduced amino acid sequence of ZCHEMO-8 (SEQ ID NO: 2) with the deduced amino acid sequences of SISD_HUMAN (SEQ ID NO: 36) (Skelton et al., Biochemistry 3_4:5329-42, 1995), MI10_HUMAN (SEQ id NO:37) (Irving et al . , Nucleic Acids Res. 18:3261-70, 1990), MI1A_HUMAN (SEQ ID NO:38) (Obaru et al . , i_- Biochem. JL9:885-94, 1986) and CCC3_HUMAN (SEQ ID NO:39) (Pardigol et al . , submitted MAY, 1996 to EMBL/GENBANK/DDBJ DATA BANKS) .

Figure 2A-D shows results from administration of ZCHEMO-8, IL-8 and vehicle to a mouse air pouch model. Figure 3A-D shows a Hopp/Woods hydrophilicity profile for ZCHEMO-8.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter:

Affinity tag: is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al . , EMBO J . 4:1075, 1985; Nilsson et al . , Methods Enzymol . 198 :3 , 1991), glutathione S transferase

(Smith and Johnson, Gene £7:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al . , Proc . Natl . Acad. Sci . USA £2:7952-4, 1985), substance P, Flag™ peptide (Hopp et al . , Biotechnology 6_..1204-10, 1988), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al . , Protein Expression and Purification 2 : 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ) .

Allelic variant : Any of two or more alternative forms of a gene occupying the same chromosomal locus . Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (i.e., no change in the encoded polypeptide) , or may encode polypeptides having altered amino acid sequence. The term "allelic variant" is also used herein to denote a protein encoded by an allelic variant of a gene. Also included are the same protein from the same species which differs from a reference amino acid sequence due to allelic variation. Allelic variation refers to naturally occurring differences among individuals in genes encoding a given protein.

Amino-terminal and carboxyl-terminal : are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide .

Complements of a polynucleotide molecule: is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3 ' is complementary to 5 ' CCCGTGCAT 3 ' .

Degenerate nucleotide sequence: denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide) .

Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp) . Expression vector: A DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and optionally one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like . Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

Isolated: when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 ' and 3 ' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985) .

Isolated polypeptide or protein: is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.

Operably linked: As applied to nucleotide segments, the term "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.

Ortholog: denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.

Polynucleotide : denotes a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double- stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.

Polypeptide : is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides" .

Promoter: is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes .

Protein : is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non- peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

Receptor : A cell-associated protein, or a polypeptide subunit of such protein, that binds to a bioactive molecule (the "ligand") and mediates the effect of the ligand on the cell. Binding of ligand to receptor results in a change in the receptor (and, in some cases, receptor multimerization, i.e., association of identical or different receptor subunits) that causes interactions between the effector domain (s) of the receptor and other molecule (s) in the cell. These interactions in turn lead to alterations in the metabolism of the cell. Metabolic events that are linked to receptor- ligand interactions include gene transcription, phosphorylation, dephosphorylation, cell proliferation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids .

Secretory signal sequence: A DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. Splice variant : is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene . Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to +10%.

The present invention is based in part upon the discovery of a novel DNA sequence (SEQ ID NO:l) and corresponding polypeptide sequence (SEQ ID NO: 2) which have homology to members of the β chemokine family, in particular, to Act-2 (SEQ ID NO: 3) . This ligand has been designated ZCHEMO-8.

Novel ZCHEMO-8 ligand-encoding polynucleotides and polypeptides of the present invention were initially identified by querying an expressed sequence tag (EST) database. Using this information, a novel 510 bp human cDNA fragment (SEQ ID NO:l) was obtained. Sequence analysis of a deduced amino acid sequence of ZCHEMO-8, as represented by SEQ ID NO: 2, indicates the presence of a 22 amino acid residue signal sequence from MET, amino acid residue 1 to THR, amino acid residue 22, and a 72 amino acid residue mature polypeptide from ALA, amino acid residue 23 to LEU, amino acid residue 94. Those skilled in the art will recognize that these domain boundaries are approximate, and are based on alignments with known proteins and predictions of protein folding. Those skilled in the art will recognize that predicted secretory signal sequence domain boundaries are approximations based on primary sequence content, and may vary slightly; however, such estimates are generally accurate to within ± 4 amino acid residues. Therefore the present invention also includes the polypeptides having amino acid sequences comprising amino acid residues 19-94 of SEQ ID NO : 2 , residues 20-94 of SEQ ID NO : 2 , residues 21-94 of SEQ ID NO: 2, residues 23-94 of SEQ ID NO: 2, residues 24-94 of SEQ ID NO: 2, residues 25-94, and residues 26-94 of SEQ ID NO : 2 as well as the polynucleotides encoding them.

A comparison of the ZCHEMO-8 deduced amino acid sequence (as represented in SEQ ID NO: 2) with the deduced amino acid sequence of SISD_HUMAN (SEQ ID NO: 36) (Skelton et al., Biochemistry 34=5329-42, 1995), MI10_HUMAN (SEQ id NO:37) (Irving et al . , Nucleic Acids Res. 18 :3261-70, 1990), MI1A_HUMAN (SEQ ID NO:38) (Obaru et al . , J_^ Biochem. 9:885-94, 1986) and CCC3_HUMAN (SEQ ID NO:39) (Pardigol et al . , submitted MAY, 1996 to EMBL/GENBANK/DDBJ DATA BANKS) is shown in Figure 1. ZCHEMO-8 shares 38.3% identity at the amino acid level with SISD_human, 42% identity with MI10_human, 37.5% identity with MIlA_human and 41.7% identity with CCC3_human .

One characteristic of the chemokine family is a motif of four conserved cysteine residues in the mature protein. In the β chemokine family, the first two of these cysteines are adjacent. ZCHEMO-8 shares this β chemokine four-cysteine motif; the first two cysteines are adjacent at residues 33 and 34, and the other two cysteine residues are at residues 57 and 73 of the deduced amino acid sequence represented in SEQ ID NO: 2. In addition, ZCHEMO-8 shares other conserved or shared residues that have been identified for the C-C chemokine group (Zipfel et al., J. Immunol. 14_2: 1582-90 , 1989; Schall, ijbid) including, a proline, at residue 43 of SEQ ID NO: 2, and a Trp-Val-Gln motif at residues 80-82 of SEQ ID NO : 2 , of which the Val is conserved and Trp and Gin shared by many members, a tyrosine (residue 50 of SEQ ID NO: 2) seven residues from proline at residue 43, a phenylalanine (residue 64 of SEQ ID NO: 2) nine residues preceding the fourth cysteine and a Leu at residue 88 of SEQ ID NO: 2. ZCHEMO-8 has a threonine (residue 74 of SEQ ID NO: 2) following the fourth cysteine, where other members have alanine . These features indicate that the polypeptide encoded by the DNA sequence as represented by SEQ ID NO : 1 is a member of the β chemokine family. Highly conserved amino acids, both within and without regions of high identity, can be used as a tool to identify ZCHEMO-8 polypeptides or ZCHEMO-8-like proteins. For instance, reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify sequences encoding the conserved motifs suggested by the multiple alignment from RNA obtained from a variety of tissue sources. In particular, the following primers are useful for this purpose :

ZCHEMO-8 residues 79-84 of SEQ ID NO : 2 K W V Q K Y

AAR TGG GTN CAR AAR TA ZCHEMO-8 (SEQ ID NO: 40)

RAR TGG GTN MRN RAN TA CONSENSUS (SEQ ID NO: 41)

YTY ACC CAN KYN YTN AT COMPLEMENT (SEQ ID NO: 42)

ZCHEMO-8 residues 33-38 of SEQ ID NO : 2 C C F Q Y S

TGY TGY TTY CAR TAY WS ZCHEMO-8 (SEQ ID NO: 43) TGY TGY TTY NVR TAY WB CONSENSUS (SEQ ID NO: 44) ACR ACR AAR NBN ATR WV COMPLEMENT (SEQ ID NO: 45)

ZCHEMO-8 residues 71-76 of SEQ ID NO : 2 K V C T H P

AAR GTN TGY ACN CAY CC ZCHEMO-8 (SEQ ID NO: 46)

HVN GTN TGY RCN VAY CC CONSENSUS (SEQ ID NO: 47)

DBN CAN ACR YGN BTR GG COMPLEMENT (SEQ ID NO: 48)

ZCHEMO-8 residues 62-67 of SEQ ID NO : 2 V I F T T K

GTN ATH TTY ACN ACN AA ZCHEMO-8 (SEQ ID NO: 49)

RTN RTN TTY NYN ACN MR CONSENSUS (SEQ ID NO: 50) YAN YAN AAR NRN TGN KY COMPLEMENT (SEQ ID NO: 51)

ZCHEMO-8 residues 49-54 of SEQ ID NO : 2 S Y E F T S

WSN TAY GAR TTY ACN WS ZCHEMO-8 (SEQ ID NO: 52) DVN TAY KWN KWN ACN WV CONSENSUS (SEQ ID NO: 53) HBN ATR MWN MWN TGN WB COMPLEMENT (SEQ ID NO: 54)

Northern blot analysis of various human tissues was performed using a 510 bp DNA probe (SEQ ID NO: 4) . A 0.8 kb transcript was detected in ovary, testis, heart, spinal cord, lymph node, trachea and adrenal gland. An additional 1.6 kb transcript was also detected in trachea.

Chromosomal localization of ZCHEMO-8 to chromosome 7qll.21 was determined using radiation hybrid chimeras. Other members of the C-C chemokine family have mapped to human chromosome 17 between qll and q21 or to chromosome 19pl3.3.

The present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the ZCHEMO-8 polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO: 33 is a degenerate DNA sequence that encompasses all DNAs that encode the ZCHEMO-8 polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO: 33 also provides all RNA sequences encoding SEQ ID NO : 2 by substituting U for T. Thus, ZCHEMO-8 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 282 of SEQ ID NO: 33 and their RNA equivalents are contemplated by the present invention. Table 1 sets forth the one- letter codes used within SEQ ID NO: 33 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide (s) . For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.

TABLE 1

Nucleotide Resolution Nucleotide Complement

A A T T

C C G G

G G C C

T T A A

R A|G Y C|T

Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T A|T H A|C|T D A|G|T B C|G|T V A|C|G

V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO: 33, encompassing all possible codons for a given amino acid, are set forth in Table 2.

TABLE 2

One

Amino Letter Codons Degenerate

Acid Code Codon

Cys C TGC TGT TGY

Ser S AGC AGT TCA TCC TCG TCT WSN

Thr T ACA ACC ACG ACT ACN

Pro P CCA CCC CCG CCT CCN

Ala A GCA GCC GCG GCT GCN

Gly G GGA GGC GGG GGT GGN

Asn N AAC AAT AAY

Asp D GAC GAT GAY

Glu E GAA GAG GAR

Gin Q CAA CAG CAR

His H CAC CAT CAY

Arg R AGA AGG CGA CGC CGG CGT MGN

Lys K AAA AAG AAR

Met M ATG ATG

He I ATA ATC ATT ATH

Leu L CTA CTC CTG CTT TTA TTG YTN

Val V GTA GTC GTG GTT GTN

Phe F πc III TTY

Tyr Y TAC TAT TAY

Trp W TGG TGG

Ter TAA TAG TGA TRR

As |Asp B RAY

Glu|Gln Z SAR

Any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR) , and the degenerate codon for arginine

(MGN) can, in some circumstances, encode serine (AGY) . A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO : 2. Variant sequences can be readily tested for functionality as described herein.

One of ordinary skill in the art will also appreciate that different species can exhibit

"preferential codon usage." In general, see, Grantham, et al., Nuc. Acids Res. £:1893-912, 1980; Haas, et al . Curr. Biol. _.6:315-24, 1996; Wain-Hobson, et al . , Gene 13:355-64, 1981; Grosjean and Fiers, Gene l£:199-209, 1982; Holm, Nuc. Acids Res. 14_:3075-87, 1986; Ikemura, J. Mol. Biol. 158 :573-97, 1982. As used herein, the term "preferential codon usage" or "preferential codons" is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2) . For example, the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO: 33 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein. Within preferred embodiments of the invention, isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, or to a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.

As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. It is generally preferred to isolate RNA from heart, although DNA can also be prepared using RNA from other tissues or isolated as genomic DNA. Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry l£:52-94, 1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder

(Proc. Natl. Acad. Sci. USA £9:1408-12, 1972).

Complementary DNA (cDNA) is prepared from poly (A) ⁺ RNA using known methods. Polynucleotides encoding ZCHEMO-8 polypeptides are then identified and isolated by, for example, hybridization or PCR. Those skilled in the art will recognize that the sequence disclosed in SEQ ID NO : 1 represents a single allele of the human gene, and that allelic variation is expected to exist. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO : 1 , including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO : 2.

The present invention further provides counterpart ligands and polynucleotides from other species (orthologs) . These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are ZCHEMO-8 ligand polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate receptors. Orthologs of human ZCHEMO-8 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the ligand. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A ligand- encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequence. A cDNA can also be cloned using the polymerase chain reaction (PCR) (Mullis, U.S. Patent No. 4,683,202), using primers designed from the sequences disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the ligand. Similar techniques can also be applied to the isolation of genomic clones. The present invention also provides isolated chemokine ligand polypeptides that are substantially homologous to the ligand polypeptide of SEQ ID NO : 2 and its orthologs. By "isolated" is meant a protein or polypeptide that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated protein or polypeptide is substantially free of other proteins or polypeptides, particularly other proteins or polypeptides of animal origin. It is preferred to provide the proteins or polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. The term "substantially homologous" is used herein to denote proteins or polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequence shown in SEQ ID NO : 2 or its orthologs. Such proteins or polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO: 2 or its orthologs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull. Math. Bio. 48 : 603-16, 1986 and Henikoff and Henikoff, Proc . Natl. Acad. Sci . USA £9:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one-letter codes) . The percent identity is then calculated as : Total number of identical matches x 100

[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences] Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above .

Table ! 3

A R N D C Q E G H I L K M F P S T W Y V

A 4

R -1 5

N -2 0 6

D -2 -2 1 6

C 0 -3 -3 -3 9

Q -l 1 0 0 -3 5

E -1 0 0 2 -4 2 5 o

G 0 -2 0 -1 -3 -2 -2 6

H -2 0 1 -1 -3 0 0 -2 8

I -1 -3 -3 -3 -1 -3 -3 -4 -3 4

L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4

K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5

M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5

F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6

P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7

S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4

T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5

W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11

Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7

V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1

Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. The present invention thus includes polypeptides of from 68 to 94 amino acid residues that comprise a sequence that is at least 60%, preferably at least 80%, and more preferably 90% or more identical to the corresponding region of SEQ ID NO: 2. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the ZCHEMO-8 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.

Table 4

Conservative amino acid substitutions

Basic : arginine lysine histidine Acidic : glutamic acid aspartic acid

Polar: glutamine asparagine Hydrophobic : leucine isoleucine valine Table 4 cont

Aromatic phenylalanine tryptophan tyrosine

Small: glycine alanine serine threonine methionine

The present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions. For example, a ZCHEMO-8 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos . 5,155,027 and 5,567,584. Preferred dimerizing proteins in this regard include immunoglobulin constant region domains. Immunoglobulin-ZCHEMO- 8 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric ZCHEMO- 8 analogs. Auxiliary domains can be fused to ZCHEMO-8 polypeptides to target them to specific cells, tissues, or macromolecules . For example, a ZCHEMO- 8 polypeptide or protein could be targeted to a predetermined cell type by fusing a ZCHEMO- 8 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A ZCHEMO-8 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al . , Connective Tissue Research 34:1-9, 1996. The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, traπs-3-methylproline, 2 , 4-methanoproline, cis-4-hydroxyproline, tra.ns-4-hydroxyproline, N-methyl - glycine, allo-threonine, methylthreonine, hydroxyethyleysteine, hydroxyethylhomocysteine, nitro- glutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3 , 3-dimethylproline, tert-leucine, norvaline, 2-aza- phenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine . Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vi tro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRΝAs . Methods for synthesizing amino acids and aminoacylating tRΝA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al . , J. Am. Chem. Soc . 113 : 2722 , 1991; Ellman et al . , Methods Enzvmol . 202 :301, 1991; Chung et al . , Science 259 :806-9, 1993; and Chung et al . , Proc . Νatl. Acad. Sci . USA _.90:10145-9, 1993) . In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRΝA and chemically aminoacylated suppressor tRΝAs (Turcatti et al . , J. Biol. Chem. 271:19991-8, 1996). Within a third method, E . coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine) . The non- naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 3_3_: 7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vi tro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci . 2=395-403, 1993) .

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for ZCHEMO- 8 amino acid residues .

Essential amino acids in the ligand polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244 : 1081-85, 1989; Bass et al . , Proc . Natl. Acad. Sci. USA 88 : 4498-502, 1991; Bajorath et al . , Biochemistry 34 : 1833-44, 1995; and Bajorath et al . , Biochemistry 34 : 9844-92, 1995). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g., receptor binding and signal transduction) to identify amino acid residues that are critical to the activity of the molecule. Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity; in conjunction with mutation of putative contact site amino acids. See, for example, Jones et al . , Nature 388 :225-8 , 1989; de Vos et al., Science 255 :306-12 , 1992; Eck et al . , J. Biol. Chem. 267:2119-22, 1992; Smith et al . , J. Mol. Biol. 224:899-904, 1992; Wlodaver et al . , FEBS Lett. 309:59-64, 1992 and Banner et al . , Cell 22:431-5, 1993. The identities of essential amino acids can also be inferred from analysis of homologies with related chemokine ligands .

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA £ :2152-56, 1989) . Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. £0:10832-37, 1991; Ladner et al . , U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al . , Gene 4_6:145, 1986; Ner et al . , DNA 7:127, 1988) .

Variants of the disclosed ZCHEMO- 8 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 3_70_: 389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vi tro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.

Mutagenesis methods as disclosed above can be combined with high-throughput screening methods to detect activity of cloned, mutagenized ligands. Mutagenized DNA molecules that encode active ligands or portions thereof (e.g., receptor-binding fragments) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

A Hopp/Woods hydrophilicity profile for ZCHEMO- 8 is shown in Figure 3. The top antigenic sites indicated were at residues 66-71 of SEQ ID NO: 2, residues 65-70 of SEQ ID NO: 2, residues 74-79 of SEQ ID NO : 2 , residues 89-94 of SEQ ID NO: 2 and residues 88-93 of SEQ ID NO: 2.

The ligand polypeptides of the present invention, including full-length ligand polypeptides, ligand fragments (e.g., receptor-binding fragments), and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al . , Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor, NY, 1989; and Ausubel et al . , eds . , Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987. In general, a DNA sequence encoding a ZCHEMO- 8 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers. Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway. The present invention provides for such fusion polypeptides. A signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid residues 1-22 of SEQ ID NO : 2 is be operably linked to another polypeptide using methods known in the art and disclosed herein. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway. Such constructs have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. Such fusions may be used in vivo or in vi tro to direct peptides through the secretory pathway.

Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al . , Cell 14_.: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 2:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al . , EMBO J . 1:841-45, 1982), DEAE-dextran mediated transfection (Ausubel et al . , ibid) , and liposome-mediated transfection (Hawley-Nelson et al . , Focus 15:73, 1993; Ciccarone et al . , Focus 15:80, 1993) . The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al . , U.S. Patent No. 4,713,339; Hagen et al . , U.S. Patent No. 4,784,950; Palmiter et al . , U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al . , J. Gen. Virol. £6:59-72, 1977) and Chinese hamster ovary (e.g., CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus . See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos . 4,579,821 and 4,601,978) and the adenovirus major late promoter. Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants" . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin- type drug, such as G-418 or the like. Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate . Other drug resistance genes (e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4 , CD8 , Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J. Biosci . (^"Bangalore ) H:47-58, 1987.

Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al . , U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) . DNA encoding the ZCHEMO- 8 polypeptide is inserted into the baculoviral genome in place of the AcNPV polyhedrin gene coding sequence by one of two methods. The first is the traditional method of homologous DNA recombination between wild-type AcNPV and a transfer vector containing the ZCHEMO- 8 flanked by AcNPV sequences. Suitable insect cells, e.g. SF9 cells, are infected with wild-type AcNPV and transfected with a transfer vector comprising a ZCHEMO- 8 polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences. See, King and Possee, The Baculovirus Expression System: A Laboratory Guide, London, Chapman & Hall; O'Reilly et al . , Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, (Ed.), Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. Natural recombination within an insect cell will result in a recombinant baculovirus which contains ZCHEMO- 8 driven by the polyhedrin promoter. Recombinant viral stocks are made by methods commonly used in the art .

The second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow et al . , J Virol £2:4566-79, 1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD) . This system utilizes a transfer vector, pFastBacl™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the ZCHEMO- 8 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." The pFastBacl™ transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case ZCHEMO-8. However, pFastBacl™ can be modified to a considerable degree . The polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins and Possee, J. Gen. Virol. 21:971-6, 1990; Bonning et al . , J. Gen. Virol. 75:1551-6, 1994; and, Chazenbalk, and Rapoport, J. Biol. Chem. 270 : 1543-9 , 1995. In such transfer vector constructs, a short or long version of the basic protein promoter can be used. Moreover, transfer vectors can be constructed which replace the native ZCHEMO- 8 secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from

Ecdysteroid Glucosyltransferase (EGT) , honey bee Melittin

(Invitrogen, Carlsbad, CA) , or baculovirus gp67

(PharMingen, San Diego, CA) can be used in constructs to replace the native ZCHEMO- 8 secretory signal sequence. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed ZCHEMO- 8 polypeptide, for example, a Glu- Glu epitope tag (Grussenmeyer et al . , Proc. Natl. Acad. Sci. £2:7952-4, 1985) . Using a technique known in the art, a transfer vector containing ZCHEMO-8 is transformed into E. coli , and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g.

Sf9 cells. Recombinant virus that expresses ZCHEMO- 8 is subsequently produced. Recombinant viral stocks are made by methods commonly used the art . The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda . See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High FiveO™ cell line (Invitrogen) derived from Trichoplusia ni (U.S.

Patent #5,300,435). Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa, KS) or Express FiveO™ (Life Technologies) for the T. ni cells. The cells are grown up from an inoculation density of approximately 2-5 x 10 cells to a density of 1-2 x 10⁶ cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. The recombinant virus-infected cells typically produce the recombinant ZCHEMO- 8 polypeptide at 12-72 hours post- infection and secrete it with varying efficiency into the medium. The culture is usually harvested 48 hours post- infection. Centrifugation is used to separate the cells from the medium (supernatant) . The supernatant containing the ZCHEMO-8 polypeptide is filtered through micropore filters, usually 0.45 μm pore size. Procedures used are generally described in available laboratory manuals (King and Possee, ibid. ; O'Reilly et al . , ibid. ; Richardson, ibid. ) . Subsequent purification of the ZCHEMO- 8 polypeptide from the supernatant can be achieved using methods described herein.

Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica . Methods for transforming S . cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al . , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al . , U.S. Patent No. 5,037,743; and Murray et al . , U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine) . A preferred vector system for use in Saccharomyces cerevisiae is the P0T1 vector system disclosed by Kawasaki et al . (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S.

Patent No. 4,599,311; Kingsman et al . , U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos .

4,990,446; 5,063,154; 5,139,936 and 4,661,454.

Transformation systems for other yeasts, including

Hansenula polymorpha , Schizosaccharomyces pombe, Kluyveromyces lactis , Kluyveromyces fragilis , Ustilago maydis , Pichia pastoris , Pichia methanolica , Pichia guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al . , J. Gen. Microbiol . 132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al . , U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al . , U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S.

Patent No. 4,486,533. The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming P . methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in P . methanolica, it is preferred that the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene {AUG1 or AUG2) . Other useful promoters include those of the dihydroxyacetone synthase (DHAS) , formate dehydrogenase (FMD) , and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine . For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes { PEP4 and PRBl ) are preferred.

Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P . methanolica cells. It is preferred to transform P . methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (τ) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichia coli , Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al . , ibid. ) . When expressing a ZCHEMO- 8 polypeptide in bacteria such as E. coli , the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant , such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell. P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors . A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto™ Peptone (Difco Laboratories, Detroit, MI), 1% Bacto™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine) .

ZCHEMO- 8 polypeptides or fragments thereof may also be prepared through chemical synthesis. ZCHEMO-8 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.

It is preferred to purify the polypeptides of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules , particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.

Expressed recombinant ZCHEMO- 8 polypeptides (or chimeric ZCHEMO-8 polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia) , Toyopearl butyl 650 (Toso Haas, Montgomeryvilie, PA) , Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity

Chromatography : Principles & Methods, Pharmacia LKB

Biotechnology, Uppsala, Sweden, 1988. The invention also provides ZCHEMO-8 polypeptides with N- and/or C-terminal extensions. To facilitate purification of the ZCHEMO- 8 polypeptide, a C- terminal extension, such as a poly-histidine tag, substance P, Glu-Glu tag (Grussenmeyer et al . ibid.), Flag ™ peptide (Hopp et al . , Biotechnology 6_.:1204-10, 1988; available from Eastman Kodak Co., New Haven, CT) or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the mature polypeptide.

The activity of molecules of the present invention can be measured using a variety of in vi tro and in vivo assays. These assays can also be used to identify cells expressing a ZCHEMO- 8 associated receptor. Competitive binding of ZCHEMO-8 ligand to receptors on purified normal leukocyte cell populations isolated from human peripheral blood can be done to confirm expression of a receptor. Transient elevation of Ca²⁺ upon chemokine binding to a cell surface receptor can be used to monitor receptor activation. Measurement of intracellular cytosolic free calcium can be done by adding ZCHEMO- 8 to leukocyte cell populations loaded with CA ⁺ indicator dyes such as, Indo-1 or Fura-2 (Kitaura et al . , J. Biol. Chem.

271:7725-30, 1996 and Ponath et al . , J. Clin. Invest.

£2:604-12, 1996). Transendothelial chemotaxis assays

(Ponath et al . , ibid) can be used to determine the ability of ZCHEMO- 8 to induce a chemotactic response in vi tro . This can also be done to identify receptor-bearing cells from leukocyte subpopulations .

To determine if ZCHEMO- 8 is a chemotractant in vivo , ZCHEMO- 8 can be given by intradermal or intraperitoneal injection. Characterization of the accumulated leukocytes at the site of injection can be determined using lineage specific cell surface markers and fluorescence immunocytometry or by immunohistochemistry (Jose, J . Exp . Med . 129:881-87, 1994). Release of specific leukocyte cell populations from bone marrow into peripheral blood can also be measured after ZCHEMO-8 injection.

ZCHEMO- 8 is homologous to Act-2, the human homolog of mouse MIP-1 which is an LPS-induced monocyte- derived murine factor leading to local inflammatory response when subcutaneously in mice (Irving et al . , Nuc . Acid Res. l£:3261-70, 1990) . Act-2 is expressed in activated but not resting T and B cells and monocytes stimulated with mitogens (Napolitano et al . , J. Biol. Chem. 266 :17351-6, 1991). In vi tro studies have shown that members of the C-C family of chemokines are chemoattractive to monocytes (Yu et al . , Clinc. Exp . Immunol . 109 :143-8, 1997). Some C-C chemokines, including monocyte chemotactic protein-4, are selective chemoattractive to eosinophils (Stellato et al . , J. Clinc. Invest . 9j):926-36, 1997) Administration of C-X-C chemokines, IL-8 or CINC into mouse air skin pouches induces selective migration of neutrophils at four hours after instillation (Harris et al . , Biochem. Biophys . Res. Comm. 221 : 962-6 , 1996) . Such an air pouch model could be used to determine if ZCHEMO- 8 induces differential infiltration of leukocytes.

The role of ZCHEMO- 8 in inflammation and respiratory hypersensitivity can be measured using known animal models. For example, models of allergic inflammation are performed in guinea pigs sensitized with ovalbumin by aerosol challenge. Bronchoaleolar lavage (BAL) fluid containing chemotractant activity from the sensitized animals is then injected intradermally into unsensitized animals and the accumulation of cells, such as eosinophils, at the site of injection is measured (Jose et al., ibid) . The affects of ZCHEMO-8 or ZCHEMO-8 antagonists can be measured to determine their ability to exacerbate or reduce cell infiltration after respiratory inflammation. A common biological response to cytokines and chemokines is fever. In vivo pyrogenicity studies can be done to determine if ZCHEMO-8 can elicit fever. ZCHEMO-8 is administered by either systemic or intracerebroventricular routes to mice and core body temperature is measured (Poltorak et al . , J. Inflamm. 4_5: 207-19, 1995) . An increase in temperature would indicate that the compound is pyrogenic . Measurements can also be taken to determine the increase in ZCHEMO- 8 expression following injury, such as intestinal injury, and subsequent increase in leukocytes at the site of injury. ZCHEMO-8 can also be tested in models of ischemia/reperfusion injury.

An alternative in vivo approach for assaying proteins of the present invention involves viral delivery systems . Exemplary viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno- associated virus (AAV) . Adenovirus, a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see Becker et al . , Meth. Cell Biol. 4_.3:161-89, 1994; and Douglas and Curiel, Science & Medicine 4_.:44-53, 1997). The adenovirus system offers several advantages : adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.

By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary) . When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system -has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.

The adenovirus system can also be used for protein production in vi tro . By culturing adenovirus- infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum- free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al . , Cvtotechnol . 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293S cell production protocol, non-secreted proteins may also be effectively obtained. ZCHEMO-8 polypeptides can also be used to prepare antibodies that specifically bind to ZCHEMO-8 epitopes, peptides or polypeptides. Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al . (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R. , Ed.,

Monoclonal Hybridoma Antibodies : Techniques and

Applications, CRC Press, Inc., Boca Raton, FL, 1982.

ZCHEMO- 8 ligand polypeptides and soluble ZCHEMO- 8 ligands may be used to identify and characterize receptors in the chemokine receptor family. Proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column

(Immobilized Affinity Ligand Techniques, Hermanson et al . , eds., Academic Press, San Diego, CA, 1992, 195-202). Proteins and peptides can also be radiolabeled (Methods in Enzymol . , vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or photoaffinity labeled (Brunner et al . , Ann. Rev. Biochem. £2:483-514, 1993 and Fedan et al . , Biochem. Pharmacol. £3:1167-80, 1984) and specific cell-surface proteins can be identified. The soluble ligand is useful in studying the distribution of receptors on tissues or specific cell lineages, and to provide insight into receptor/ligand biology. The polypeptides of the present invention can be isolated by exploitation of unique properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. £:l-7, 1985) . Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol . , Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification.

ZCHEMO- 8 polypeptides can also be used to prepare antibodies that specifically bind to ZCHEMO- 8 epitopes, peptides or polypeptides. Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see, for example, Sambrook et al . , Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982) . As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, hamsters, guinea pigs and rats as well as transgenic animals such as transgenic sheep, cows, goats or pigs. Antibodies may also be expressed in yeast and fungi in modified forms as well as in mammalian and insect cells. The ZCHEMO- 8 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal or elicit an immune response. Suitable antigens would include the ZCHEMO- 8 polypeptide encoded by SEQ ID NO : 2 from amino acid residue 23-94 of SEQ ID NO : 2 , or a contiguous 9-94 amino acid residue fragment thereof. The immunogenicity of a ZCHEMO- 8 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of ZCHEMO-8 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like" , such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid) for immunization. As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments thereof, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fc fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included. Non-human antibodies may be humanized by grafting only non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a humanlike surface by replacement of exposed residues, wherein the result is a "veneered" antibody) . In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. Human antibodies can also be made in mice having a humanized humoral immune system (Mendez et al., Nat. Genet. ^4_:146- 56, 1997). Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to ZCHEMO-

8 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled ZCHEMO- 8 protein or peptide) . Mutagenesis methods discussed herein, in particular domain shuffling, can be used to generate and mature antibodies.

The antibodies of the current invention, or fragments thereof, can be used to direct molecules to a specific target. For example, as T-bodies, chimeric receptors combining antibody recognition with T cell effector function, (Eshhar et al., Springer Semin I munopathol . _18_: 199-209, 1996; Eshhar, Cancer Immunol. Immunother . 4_5: 131-6, 1997). Intrabodies, engineered single-chain antibodies expressed inside the cell and having high affinity and specificity for intracellular targets. Such molecules have use in gene therapy and treatment of infectious diseases (Marasco, Immunotechnology Xl-19, 1995; Marasco et al., Gene Ther. 4_:ll-5, 1997; Rondon and Marasco, Annu . Rev. Microbiol. _51.:257-83, 1997 and Mhashilkar et al., J. Virol. 7_l^{:β 86_} 94, 1997). Diabodies, bispecific non-covalent di ers of scFv antibodies useful for immunodiagnosis and therapeutically . In addition they can be constructed in bacteria (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993) .

Antibodies herein specifically bind if they bind to a ZCHEMO-8 polypeptide, peptide or epitope with a binding affinity (K_a) of 10 6 M-1 or greater, preferably

7 —1 8 —1

10 M or greater, more preferably 10 M or greater, and most preferably 10 9 M-1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) .

Genes encoding polypeptides having potential

ZCHEMO-8 polypeptide binding domains, "binding proteins", can be obtained by screening random or directed peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli . Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. Alternatively, constrained phage display libraries can also be produced. These peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such peptide display libraries are known in the art (Ladner et al., US Patent NO. 5,223,409; Ladner et al . , US Patent NO. 4,946,778; Ladner et al . , US Patent NO. 5,403,484 and Ladner et al . , US Patent NO. 5,571,698) and peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA) , Invitrogen Inc. (San Diego, CA) , New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ) . Peptide display libraries can be screened using the ZCHEMO- 8 sequences disclosed herein to identify proteins which bind to ZCHEMO-8. These "binding proteins" which interact with ZCHEMO- 8 polypeptides can be used essentially like an antibody, for tagging cells; for isolating homolog polypeptides by affinity purification; directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. To increase the half- life of these binding proteins, they can be conjugated. Their biological properties may be modified by dimerizing or multimerizing for use as agonists or antagonists. A variety of assays known to those skilled in the art can be utilized to detect antibodies and/or binding proteins which specifically bind to ZCHEMO-8 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA) , dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant ZCHEMO-8 protein or polypeptide.

Antibodies and binding proteins to ZCHEMO- 8 may be used for tagging cells that express ZCHEMO-8; for isolating ZCHEMO- 8 by affinity purification; for diagnostic assays for determining circulating levels of ZCHEMO- 8 polypeptides; for detecting or quantitating soluble ZCHEMO- 8 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti- idiotypic antibodies; and as neutralizing antibodies or as antagonists to block ZCHEMO- 8 polypeptide adhesion modulating or anti-microbial or like activity in vi tro and in vivo . Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates. Moreover, antibodies to ZCHEMO-8 or fragments thereof may be used in vi tro to detect denatured ZCHEMO- 8 or fragments thereof in assays, for example, Western Blots or other assays known in the art .

Antibodies or polypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For instance, polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance) . More specifically, ZCHEMO-8 polypeptides or anti-ZCHEMO-8 antibodies, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti- complementary molecule.

Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like. Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like) , as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance) . Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion. For these purposes, biotin/streptavidin is an exemplary complementary/ anticomplementary pair.

In another embodiment, polypeptide-toxin fusion proteins or antibody-toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues) . Alternatively, if the polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus a targeting domain) , a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest. In instances where the domain only fusion protein includes a complementary molecule, the anticomplementary molecule can be conjugated to a detectable or cytotoxic molecule. Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue-specific delivery of generic anti- comple entary-detectable/cytotoxic molecule conjugates. The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially, intraductally with DMSO, intramuscularly, subcutaneously, intraperitoneally, also by transdermal methods, by electro-transfer, orally or via inhalant.

Antibodies may be made to ZCHEMO- 8 polypeptides containing C-terminal extensions. In particular, antiserum containing polypeptide antibodies to His-tagged soluble ZCHEMO- 8 can be used in analysis of tissue distribution of ZCHEMO-8 or receptors that bind ZCHEMO-8 by immunohistochemistry on human or primate tissue.

ZCHEMO-8 polypeptides can also be used to immunize mice in order to produce monoclonal antibodies to a human ZCHEMO- 8 polypeptide. Monoclonal antibodies to a human ZCHEMO- 8 polypeptide can also be used to mimic ligand/receptor coupling, resulting in activation or inactivation of the ligand/receptor pair. Monoclonal antibodies to ZCHEMO- 8 can be used to determine the distribution, regulation and biological interaction of the ZCHEMO- 8 receptor/ZCHEMO-8 ligand pair on specific cell lineages. The seven known chemokine receptors have limited expression patterns which are restricted to monocytes, neutrophils, T cells, B cells, basophils, eosinophils, monocytes, macrophages and in one case endothelial cells (Power and Wells, Trends in Pharm. Sci. 12:209-13, 1996). Antibodies to ZCHEMO-8 can also be used to detect secreted soluble ZCHEMO- 8 in biological samples. The ZCHEMO- 8 polynucleotides and polypeptides of the present invention may be used as tools for research and for discovery of therapeutic and diagnostic uses of chemokines. Numerous chemokine proteins, polypeptides, antibodies and polynucleotides for use in such methodologies are available for research use from many commercial sources, such as R & D Systems, Minneapolis, MN. ZCHEMO- 8 polypeptides can be used as standards to calibrate in vi tro chemokine assay systems or as standards within such assay systems. In addition, antibodies to ZCHEMO- 8 polypeptides could be used in assays for neutralization of bioactivity, and as negative controls for a variety of applications, such as ELISA and ELISPOT assays, Western blot, immunohistochemistry, and intracellular staining.

The invention also provides isolated and purified ZCHEMO-8 polynucleotide probes and primers. Such polynucleotide probes and primers can be ribonucleic acid

(RNA) or deoxyribonucleic acid (DNA) . DNA can be either complementary DNA (cDNA) or genomic DNA. Polynucleotide probes and primers are single or double-stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences . Analytical probes will generally comprise at least 16 nucleotides, more often from 17 nucleotides to 25 or more nucleotides, sometimes 40 to 60 nucleotides, and in some instances a substantial portion or even the entire ZCHEMO- 8 gene or cDNA. Primers, such as PCR primers, are at least 5 nucleotides in length, preferably 15 or more nucleotides, more preferably 20-30 nucleotides. The synthetic probes and primers of the present invention have at least 80% identity to a representative ZCHEMO-8 DNA sequence (SEQ ID NO:l) or its complements. Preferred regions from which to construct probes include the 5' and/or 3' coding sequences, receptor binding regions, signal sequences and the like. Techniques for developing polynucleotide probes and hybridization techniques are known in the art, see for example, Ausubel et al . , eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1991. For use as probes, the molecules can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art . Such probes can also be used in hybridizations to detect the presence or quantify the amount of ZCHEMO- 8 gene or mRNA transcript in a sample. ZCHEMO-8 polynucleotide probes could be used to hybridize to DNA or RNA targets for diagnostic purposes, using such techniques such as fluorescent in si tu hybridization (FISH) or immunohistochemistry .

Polynucleotide probes could be used to identify genes encoding ZCHEMO-8 -like proteins. For example, ZCHEMO-8 polynucleotides can be used as primers and/or templates in PCR reactions to identify other novel members of the chemokine family.

Polynucleotide probes can also be used to screen libraries for related sequences encoding novel chemokines. Screening would be carried out under conditions of low stringency which would allow identification of sequences which are substantially homologous, but not requiring complete homology to the probe sequence. Such methods and conditions are well known in the art, see, for example, Sambrook et al . , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989. Low stringency conditions could include hybridization temperatures less than 42°C, formamide concentrations of less than 50% and moderate to low concentrations of salt. Libraries may be made of genomic DNA or cDNA.

Polynucleotide probes are also useful for Southern, Northern, or slot blots, colony and plaque hybridization and in si tu hybridization. Mixtures of

ZCHEMO- 8 polynucleotide probes can be prepared which would increase sensitivity or the detection of low copy number targets, in screening systems. Probes based on the polynucleotide sequence of

SEQ ID N0:1 can be used to localize the ZCHEMO-8 gene to a particular chromosome. Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250 :245-50 , 1990). Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL) . These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence- tagged sites (STSs) , and other nonpolymorphic and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers. The precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.

Sequence tagged sites (STSs) can also be used independently for chromosomal localization. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome. An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described within an electronic database, for example,

Database of Sequence Tagged Sites (dbSTS) , GenBank,

(National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm. nih.gov) , and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences .

ZCHEMO- 8 polypeptides may also be used within diagnostic systems. Chemokines have been detected in a wide variety of tissues and fluid samples, such as those associated with many inflammatory conditions. For example, chemokines have been detected in asthmatic reaction lavage fluid, wound healing site tissue, arteriosclerosis tissue, inflamed gastrointestinal tissue, rheumatoid arthritis synovial fluid and psoriatic scale tissue. Antibodies or other agents that specifically bind to ZCHEMO- 8 may be used to detect the presence of circulating ZCHEMO- 8 polypeptides. Such detection methods are well known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay . Immunohistochemically labeled antibodies can be used to detect ZCHEMO-8 polypeptides in tissue samples. ZCHEMO- 8 levels can also be monitored by such methods as RT-PCR, where ZCHEMO-8 mRNA can be detected and quantified. Such methods could be used as diagnostic tools to monitor and quantify receptor or ligand polypeptide levels. The information derived from such detection methods would provide insight into the significance of ZCHEMO-8 ligand polypeptides in various diseases, and would serve as an early and accurate diagnostic marker for diseases for which altered levels of ZCHEMO-8 are significant. In particular, ZCHEMO- 8 may be an indicator for treatment in early stages of disease development before clinical symptoms manifest, such as in the case of tumor development. Altered levels of ZCHEMO-8 ligand polypeptides may be indicative of pathological conditions, including infections, cancer, myelopoietic disorders, autoimmune disorders and immunodeficiencies.

The ZCHEMO- 8 polypeptides disclosed herein are contemplated to be useful as therapeutics for treating mammals in need of a ZCHEMO-8 polypeptide. ZCHEMO-8 is a member of the chemokine family whose known pro- inflammatory, pyrogenic, chemokinetic, myelosuppressive or hematopoietic effects are being exploited in therapeutic regimes. Depending on the amount, mode of administration and site of administration, chemokines may be made to act in an agonist or antagonist manner. For example, a chemokine may be used in a pro- inflammatory or anti- inflammatory fashion, depending on the mode of administration. ZCHEMO- 8 polypeptides, agonists and antagonists could modulate one or more biological processes in cells, tissues and/or biological fluids.

Therapeutic use may be made of ZCHEMO-8 polypeptides to reduce the damage in ischemic and reperfusion injuries. Such applications would include surgical use. In particular, ZCHEMO-8 could be used to reduce damage to the muscle of the heart associated with coronary occlusion, such as that done during a coronary occlusive heart attack. One factor that influences such injury is neutrophil accumulation in the area. The chemoattractant properties of ZCHEMO- 8 could be exploited in a wound healing regime to stimulate an infiltration of immune cells (monocytes, neutrophils, T lymphocytes, basophils and the like) to a wound site to facilitate healing. ZCHEMO-8 may be used to mobilize progenitor cells from the marrow into the peripheral blood for transplants .

ZCHEMO- 8 polypeptides could be used to further define the role of chemokines in mediating suppression of HIV replication in CD4⁺ T-cells and limiting progression of HIV infection to AIDS. Members of the β chemokine family, RANTES, MlP-lα and MlP-lβ, and IL-16, have been shown to be involved in HIV control. Also, ZCHEMO- 8 and its associated receptor could be used to further characterize chemokine-HIV inhibitory activity and for analysis of the pathogenesis of HIV.

Use may be made of ZCHEMO- 8 polypeptides during chemotherapy or radiation therapy, to protect hematopoietic cells. For instance, chemokine myelosuppressive properties would enhance recovery of progenitor cells following chemo- or radiation therapies, by myelosuppressing normal cells so that only the cancerous cells are sensitive to the therapy.

The invention also provides antagonists, which either bind to ZCHEMO-8 polypeptides or, alternatively, to a receptor to which ZCHEMO-8 polypeptides bind, thereby inhibiting or eliminating the function of ZCHEMO-8. Such ZCHEMO-8 antagonists would include antibodies, oligonucleotides, natural or synthetic analogs of ZCHEMO- 8 polypeptides, which bind either to the ZCHEMO-8 polypeptide or to its associated receptor, but do not result in receptor signaling. Such natural or synthetic analogs could be peptides or peptide-like compounds. Natural or synthetic small molecules which bind to receptors of ZCHEMO- 8 polypeptides and prevent signaling are also contemplated as antagonists. As such, ZCHEMO-8 antagonists would be useful as therapeutics for treating certain disorders where blocking a signal derived from ZCHEMO-8 would be beneficial.

ZCHEMO- 8 can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of ZCHEMO- 8. In addition to those assays disclosed herein, samples can be tested for inhibition of ZCHEMO- 8 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of ZCHEMO- 8 -dependent cellular responses. For example, ZCHEMO- 8 -responsive cell lines can be transfected with a reporter gene construct that is responsive to a ZCHEMO- 8 -stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a ZCHEMO-8 -DNA response element operably linked to a gene encoding an assayable protein, such as luciferase. DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE) , hormone response elements (HRE) insulin response element (IRE) (Nasrin et al . , Proc. Natl. Acad. Sci . USA £2:5273-7, 1990) and serum response elements (SRE) (Shaw et al . Cell £ : 563-72, 1989). Cyclic AMP response elements are reviewed in Roestler et al . , _ Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec . Endocrinol . 4. (8):1087-94; 1990. Hormone response elements are reviewed in Beato, Cell £6: 335-44; 1989. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of ZCHEMO- 8 on the target cells as evidenced by a decrease in ZCHEMO-8 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block ZCHEMO-8 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of ZCHEMO- 8 binding to receptor using ZCHEMO-8 tagged with a detectable label (e.g., 125I, biotin, horseradish peroxidase, FITC, and the like) . Within assays of this type, the ability of a test sample to inhibit the binding of labeled ZCHEMO- 8 to the receptor is indicative of inhibitory activity, which can be confirmed through secondary assays. Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors . ZCHEMO- 8 antagonists would have beneficial therapeutic effect in diseases where the inhibition of activation of certain macrophages, neutrophils, basophils, B lymphocytes and/or T cells would be effective. In particular, such diseases would include autoimmune diseases, such as multiple sclerosis, insulin-dependent diabetes and systemic lupus erythematosus . Also, benefit would be derived from using ZCHEMO- 8 antagonists for chronic inflammatory and infective diseases. Antagonists could be used to dampen or inactivate ZCHEMO- 8 during activated immune response.

Chemokine antagonists are being exploited as therapeutics for treating chronic and acute inflammatory diseases. Bronchial epithelial cells produce chemokines which appear to play a role in local bronchial inflammation, such as asthma and other respiratory distress conditions. Antagonists to chemokines, such as ZCHEMO-8, could be used as therapeutic treatment for such bronchial inflammation. Chemokine antagonists would also be useful for modulating chemokine levels in the rheumatic joint and to inhibit influx of monocytes into the synovial fluid as a treatment for rheumatoid arthritis. In addition, antagonists may be used to treat some allergies by inhibiting the release of histamine.

ZCHEMO-8 antagonists would have therapeutic value for treating arteriosclerosis. Arterosclerotic lesions may be macrophage-derived. Use of a chemokine antagonist to block chemokine receptors in arterial smooth muscle cells would reduce the influx of macrophages to arterial walls.

Polynucleotides encoding ZCHEMO- 8 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit ZCHEMO-8 activity. If a mammal has a mutated or absent ZCHEMO- 8 gene, the ZCHEMO- 8 gene can be introduced into the cells of the mammal . In one embodiment, a gene encoding a ZCHEMO- 8 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV) , papillomavirus, Epstein Barr virus (EBV) , adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1

(HSV1) vector (Kaplitt et al . , Molec . Cell. Neurosci .

2:320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al . , L_ Clin. Invest. 9£:626-30, 1992; and a defective adeno- associated virus vector (Samulski et al . , J. Virol. £1:3096-101, 1987; Samulski et al . , J. Virol. 63 :3822-8, 1989) .

In another embodiment, a ZCHEMO-8 gene can be introduced in a retroviral vector, e.g., as described in Anderson et al . , U.S. Patent NO: 5,399,346; Mann et al . Cell ££:153, 1983; Temin et al . , U.S. Patent NO: 4,650,764; Temin et al . , U.S. Patent NO: 4,980,289; Markowitz et al . , J. Virol. £2:1120, 1988; Temin et al . , U.S. Patent NO: 5,124,263; International Patent Publication NO: WO 95/07358, published March 16, 1995 by Dougherty et al . ; and Kuo et al . , Blood £2:845, 1993. Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al . , Proc. Natl. Acad. Sci . USA £4:7413-7, 1987; Mackey et al . , Proc. Natl. Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides (e.g., hormones or neurotransmitters) , proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.

It is possible to remove the target cells from the body; to introduce the vector as a naked DNA plasmid; and then to re- implant the transformed cells into the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al . , J__^ Biol. Chem. 267:963-7, 1992; Wu et al . , J. Biol. Chem. 263 :14621-4, 1988.

Antisense methodology can be used to inhibit ZCHEMO-8 gene transcription, such as to inhibit cell proliferation in vivo . Polynucleotides that are complementary to a segment of a ZCHEMO-8 -encoding polynucleotide (e.g., a polynucleotide as set froth in SEQ ID N0:1) are designed to bind to ZCHEMO- 8-encoding mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides are used to inhibit expression of ZCHEMO-8 polypeptide-encoding genes in cell culture or in a subject.

Transgenic mice, engineered to express the ZCHEMO- 8 gene, and mice that exhibit a complete absence of ZCHEMO-8 gene function, referred to as "knockout mice" (Snouwaert et al . , Science 257 : 1083 , 1992), may also be generated (Lowell et al . , Nature ££ : 740-42 , 1993). These mice may be employed to study the ZCHEMO- 8 gene and the protein encoded thereby in an in vivo system.

The present invention also provides reagents for use in diagnostic applications. For example, the ZCHEMO-8 gene, a probe comprising ZCHEMO- 8 DNA or RNA, or a subsequence thereof can be used to determine if the ZCHEMO- 8 gene is present on chromosome 7 or if a mutation has occurred. Detectable chromosomal aberrations at the ZCHEMO- 8 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. These aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level.

In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient. Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID N0:1, the complement of SEQ ID N0:1, or an RNA equivalent thereof. Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications 1:5-

16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art

(Sambrook et al . , ibid. ; Ausubel et . al . , ibid. ; Marian,

Chest 108 : 255-65 , 1995). Ribonuclease protection assays (see, e.g., Ausubel et al . , ibid. , ch. 4) comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase . Hybridized regions of the RNA are protected from digestion. Within PCR assays, a patient's genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient. Another PCR-based technique that can be employed is single strand conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications 1:34-8, 1991) .

Pharmaceutically effective amounts of ZCHEMO- 8 polypeptides or ZCHEMO-8 agonists and antagonists of the present invention can be formulated with pharmaceutically acceptable carriers for parenteral, oral, nasal, rectal, topical, transdermal administration or the like, according to conventional methods. Formulations may further include one or more diluents, fillers, emulsifiers, preservatives, buffers, excipients, and the like, and may be provided in such forms as liquids, powders, emulsions, suppositories, liposomes, transdermal patches and tablets, for example. Slow or extended-release delivery systems, including any of a number of biopolymers (biological-based systems) , systems employing liposomes, and polymeric delivery systems, can also be utilized with the compositions described herein to provide a continuous or long-term source of the ZCHEMO- 8 polypeptide, agonist or antagonist. Such slow release systems are applicable to formulations, for example, for oral, topical and parenteral use. The term "pharmaceutically acceptable carrier" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient. One skilled in the art may formulate the compounds of the present invention in an appropriate manner, and in accordance with accepted practices, such as those disclosed in Remington's Pharmaceutical Sciences, Gennaro (ed.), Mack Publishing Co., Easton, PA 1990.

As used herein a "pharmaceutically effective amount" of such a ZCHEMO- 8 polypeptide or antagonist is an amount sufficient to induce a desired biological result. The result can be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an effective amount of a ZCHEMO-8 antagonist is that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer. In particular, such an effective amount of a ZCHEMO- 8 polypeptide results in reduction in leukocyte recruitment, edema, swelling, reduction in inflammatory cytokines, reduction in pyrogenicity or other beneficial effect. Effective amounts of the ZCHEMO- 8 polypeptides can vary widely depending on the disease or symptom to be treated. The amount of the polypeptide to be administered, and its concentration in the formulations, depends upon the vehicle selected, route of administration, the potency of the particular polypeptide, the clinical condition of the patient, the side effects and the stability of the compound in the formulation. Thus, the clinician will employ the appropriate preparation containing the appropriate concentration in the formulation, as well as the amount of formulation administered, depending upon clinical experience with the patient in question or with similar patients. Such amounts will depend, in part, on the particular condition to be treated, age, weight, and general health of the patient, and other factors evident to those skilled in the art. Typically a dose will be in the range of 0.1-100 mg/kg of subject.

The dosages of the present compounds used to practice the invention include dosages effective to result in the desired effects. Estimation of appropriate dosages effective for the individual patient is well within the skill of the ordinary prescribing physician or other appropriate health care practitioner. As a guide, the clinician can use conventionally available advice from a source such as the Physician's Desk Reference, 48 Edition, Medical Economics Data Production Co., Montvale, New Jersey 07645-1742 (1994) .

Preferably the compositions are presented for administration in unit dosage forms. The term "unit dosage form" refers to physically discrete units suitable as unitary dosed for human subjects and animals, each unit containing a predetermined quantity of active material calculated to produce a desired pharmaceutical effect in association with the required pharmaceutical diluent, carrier or vehicle. Examples of unit dosage forms include vials, ampules, tablets, caplets, pills, powders, granules, eyedrops, oral or ocular solutions or suspensions, ocular ointments, and oil-in-water emulsions. Means of preparation, formulation and administration are known to those of skill, see generally Remington's Pharmaceutical Science 15^th ed., Mack Publishing Co., Easton, PA (1990) .

When necessary, the dosage can be repeated daily, or sometimes twice a day, until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. Once a therapeutic effect is achieved, the dosage can be tapered or discontinued .

The invention is further illustrated by the following non-limiting examples.

EXAMPLES Example 1 Identification of ZCHEMO-8

Novel ZCHEMO-8 polynucleotides and polypeptides of the present invention were initially identified by querying an EST database. To identify the corresponding cDNA, a clone considered likely to contain the entire cDNA sequence was used for sequencing. Using a QIAwell 8 plasmid kit (Qiagen, Inc., Chatsworth, CA) according to manufacturer's instructions, a 5 ml overnight culture in LB broth + 50 μg/ml ampicillin was prepared. The template was sequenced on an Applied Biosystems TM model 377 DNA sequencer (Perkin-Elmer Cetus, Norwalk, CT) using the ABI

PRISM TM Dye Terminator Cycle Sequencing Ready Reaction Ki■t

(Perkin-Elmer Corp.) according the manufacturer's instructions. Sequencing reactions were carried out in a

Hybaid OmniGene Temperature Cycling System (National Labnet Co . , Woodbridge, NY). SequencherTM 3.0 sequence analysis software (Gene Codes Corporation, Ann Arbor, MI) was used for data analysis. The resulting 510 bp sequence is disclosed as SEQ ID NO:l. The sequence of the initially identified EST was confirmed within this sequence.

Example 2 Tissue Distribution

Human Multiple Tissue Northern Blots (MTN I, MTN

II, and MTN III; Clontech) were probed to determine the tissue distribution of human ZCHEMO-8 expression.

The 505 bp DNA fragment (SEQ ID NO: 4) was excised from the vector by restriction digestion using Sal I and Not I, the fragment was gel purified using a PCR purification kit (Qiagen Inc.), and then radioactively labeled with ³²P using a random priming MEGAPRIME DNA labeling system (Amersham, Arlington Heights, IL) according to the manufacturer's specifications.

The probe was purified using a NUCTRAP push

TM column (Stratagene, La Jolla, CA) . ExpressHyb (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 65°C using 1 x 10 cpm/ml of labeled probe. The blots were then washed at 25°C in 2X SSC, 0.05% SDS for 45 minutes followed by 1 hour at 50°C in 0. IX SSC, 0.1% SDS. Background was detected after exposure to film at -80°C for 4 hours so blots were washed at 55°C 0. IX SSC, 0.1% SDS for 90 minutes. A 0.8 kb transcript was detected in ovary and testis, heart, spinal cord, lymph node, trachea and adrenal gland. A 1.6 kb transcript was detected in trachea.

Example 3 Chromosomal Assignment and Placement of ZCHEMO-8

ZCHEMO- 8 was mapped to chromosome 7 using the commercially available GeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, AL) . The GeneBridge 4 Radiation Hybrid Panel contains PCRable DNAs from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient) . A publicly available WWW server (http://www-genome.wi.mit.edu/cgi-bin/contig/ rhmapper.pl) allows mapping relative to the Whitehead Institute/MIT Center for Genome Research's map of the human genome (the "WICGR" radiation hybrid map) which was constructed with the GeneBridge 4 Radiation Hybrid Panel.

For the mapping of ZCHEMO- 8 with the GeneBridge 4 RH Panel, 20 μl reactions were set up in a PCRable 96- well microtiter plate (Stratagene, La Jolla, CA) and used in a RoboCycler Gradient 96 thermal cycler (Stratagene) . Each of the 95 PCR reactions consisted of 2 μl 10X KlenTaq PCR reaction buffer (Clontech Laboratories, Inc., Palo Alto, CA) , 1.6 μl dNTPs mix (2.5 mM each, Perkin-Elmer, Foster City, CA) , 1 μl sense primer, ZC 13,649 (SEQ ID NO: 5), 1 μl antisense primer, ZC 13,637 (SEQ ID NO: 6), 2 μl i?ediLoad (Research Genetics, Inc.), 0.4 μl 50X Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH₂0 for a total volume of 20 μl . The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 95°C, 35 cycles of 1 minute at 95°C, 1 minute at 62°C and 1.5 minutes at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C. The reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD ) . The results showed that ZCHEMO-8 maps 405.45 cR_3000 from the top of the human chromosome 7 linkage group on the WICGR map of the human genome . The proximal and distal framework markers were D7S489 and D7S669, respectively. The use of the surrounding markers positions ZCHEMO-8 in the 7qll.21 region on the integrated LDB chromosome 7 map (The Genetic Location Database, University of Southhampton, WWW server: http : //cedar . genetics . soton. ac .uk/public_html/) .

Example 4

Construction of ZCHEMO- 8 Mammalian Expression Vectors ZCHEMO- 8CF/pZP9 and ZCHEMO- 8NF/pZP9

Two expression vectors were prepared for the

ZCHEMO- 8 polypeptide, ZCHEMO- 8CF/pZP9 and ZCHEMO- 8NF/pZP9 , wherein the constructs are designed to express a ZCHEMO- 8 polypeptide with a C- or N-terminal FLAG tag (SEQ ID NO: 7) . ZCHEMO- 8CF/pZP9

A 306 bp PCR generated ZCHEMO- 8 DNA fragment was created using ZC3549 (SEQ ID NO: 8) and ZC13550 (SEQ ID NO: 9) as PCR primers and colonies described above as a template. PCR amplification of the ZCHEMO- 8 fragment were

95°C for 90 seconds, 10 cycles of 94°C for 30 seconds and

50°C for 90 seconds, 25 cycles 94°C 30 seconds, 69°C 90 seconds, followed by a 10 minute extension at 72°C. A band of the predicted size, 306 bp, was visualized by 1%

TAE agarose gel electrophoresis, excised and the DNA was

® purified from the gel with a QUIAQUICK column (Qiagen) according the manufacturer's instructions. The DNA was digested with the restriction enzymes X o I and Bam HI, followed by extraction and precipitated.

The excised DNA was subcloned into plasmid CF/pZP9 which had been cut with Xho I and Bam HI . The ZCHEMO- 8/CFpZP9 expression vector uses the native ZCHEMO- 8 signal peptide, and the FLAG epitope (SEQ ID NO: 7) is attached at the C-terminus as a purification aid. Plasmid CF/pZP9 (deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD) is a mammalian expression vector containing an expression cassette having the mouse metallothionein-1 promoter, multiple restriction sites for insertion of coding sequences, a sequence encoding the flag peptide (SEQ ID NO: 7), a stop codon and a human growth hormone terminator. The plasmid also has an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene and the SV40 terminator.

ZCHEMO- 8NF/pZP9

A 236 bp PCR generated ZCHEMO- 8 DNA fragment was created in accordance with the procedure set forth above using ZC3547 (SEQ ID NO:10) and ZC13548 (SEQ ID NO:ll) as PCR primers. The purified PCR fragment was digested with the restriction enzymes BAM HI and Xho I, followed by extraction and precipitation. The excised ZCHEMO- 8 DNA was subcloned into plasmid NF/pZP9 which had been cut with Bam HI and Xho I . The ZCHEMO- 8/NFpZP9 expression vector incorporates the TPA leader and attaches the FLAG epitope (SEQ ID NO: 7) to the N-terminal of the ZCHEMO- 8 polypeptide-encoding polynucleotide sequence. Plasmid NF/pZP9 (deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD) is a mammalian expression vector containing an expression cassette having the mouse metallothionein-1 promoter, a TPA leader peptide followed by the sequence encoding the FLAG peptide (SEQ ID NO:7), multiple restriction sites for insertion of coding sequences, and a human growth hormone terminator. The plasmid also contains an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene and the SV40 terminator.

Ten nanograms of the restriction digested C- and N-terminal FLAG/ZCHEMO-8 inserts and 20 ng of the corresponding vectors were ligated at room temperature for 4 hours. One microliter of each ligation reaction was independently electroporated into DH10B competent cells (GIBCO BRL, Gaithersburg, MD) according to manufacturer's direction and plated onto LB plates containing 50 mg/ml ampicillin, and incubated overnight. Colonies were screened by PCR using primers ZC12641 (SEQ ID NO: 34) and ZC12642 (SEQ ID NO:35) . PCR screening was done at 94°C for 90 seconds, 35 cycles of 94°C for 30 seconds and 60°C for 90 seconds, followed by a 10 minute extension at 72°C. Positive clones were plated on to LB Amp plates as above. The insert sequence of positive clones, 200 bp fragment for each construct were verified by sequence analysis. A large scale plasmid preparation was done using a QIAGEN Maxi prep kit (Qiagen) according to manufacturer's instructions . Example 5 Mammalian Expression of ZCHEMO- 8

BHK 570 cells (ATCC No. CRL-10314) were plated in 10 cm tissue culture dishes and allowed to grow^" to approximately 50 to 80% confluency overnight at 37°C, 5% C0₂, in DMEM/FBS media (DMEM, Gibco/BRL High Glucose, (Gibco BRL, Gaithersburg, MD) , 5% fetal bovine serum (Hyclone, Logan, UT) , 1 μM L-glutamine (JRH Biosciences, Lenexa, KS) , 1 μM sodium pyruvate (Gibco BRL) ) . The cells were then transfected with the plasmid ZCHEMO- 8NF/pZP9 (N- terminal FLAG tag) or ZCHEMO- 8CF/pZP9 (C-terminal FLAG tag) , using Lipofectamine TM (Gibco BRL) , in serum free (SF) media formulation (DMEM, 10 μg/ml transferrin, 5 μg/ml insulin, 10 μg/ml fetuin, 2 ng/ml selenium, 1% L-glutamine and 1% sodium pyruvate) . Sixteen micrograms of ZCHEMO- 8NF/pZP9 and 16 μg of ZCHEMO- 8CF/pZP9 were separately diluted into 15 ml tubes to a total final volume of 640 μl SF media. In separate tubes, 35 μl of Lipofectamine TM (Gibco BRL) was mixed with 605 μl of SF medium. The

Lipofectamine TM mix was added to the DNA mix and allowed to incubate approximately 30 minutes at room temperature. Five milliliters of SF media was added to the DNA: Lipofectamine TM mixture. The cells were rinsed once with 5 ml of SF media, aspirated, and the

DNA: Lipofectamine TM mixture was added. The cells were incubated at 37°C for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSN media was added to the plate. The plate was incubated at 37°C overnight and the DNA: Lipofectamine mixture was replaced with fresh FBS/DMEM media the next day. On day 2 post-transfection, the cells were split into the selection media (DMEM/5% FBS media from above with the addition of 1 μM methotrexate (Sigma Chemical Co., St. Louis, Mo.)) in 150 mm plates at 1:10, 1:20 and 1:50. The plates were refed at day 5 post-transfection with fresh selection media. Approximately 10 days post- transfection, two 150 mm culture dishes of methotrexate resistant colonies, from each construct, were trypsinized and the cells were pooled and grown to confluence in a

T162 flask. The pools were subjected to Western blot analysis and transferred to large scale culture.

Example 6

Large Scale Culture of ZCHEMO- 8 FLAG tagged polypeptides

One T-162 flask, containing confluent cells expressing ZCHEMO-8/CF and ZCHEMO-8/NF obtained from the expression procedure described above, were expanded into six T-162 flasks. One of the six resulting flasks was used to freeze down four cryovials, and the other five flasks were used to generate a Nunc cell factory.

The cells from the five T-165 flasks were used to seed a Nunc cell factory (10 layers, commercially available from VWR) . Briefly, the cells from the T-162 flasks described above were detached using trypsin, pooled and added to 1.5 liters ESTEP 1 media (668.7g/50L DMEM

(Gibco), 5.5 g/50L pyruvic acid, sodium salt 96%

(Mallinckrodt) , 185.0 g/50 L NaHC0₃ (Mallinkrodt) , 5.0 mg/ml and 25 ml/50 L insulin (JRH Biosciences) , 10.0 mg/ml and 25 ml/50 L transferrin (JRH Biosciences), 2.5L/50L fetal bovine serum (characterized) (Hyclone) , 1 μM MTX, pH 7.05) prewarmed to 37°C. The media containing cells was poured into a Nunc cell factory via a funnel. The cell factory was placed in a 37°C, 5.0% C0₂ incubator.

At 80-100% confluence, a visual contamination test (phenol red color change) was performed on the Nunc cell factory. Since no contamination was observed, supernatant from the confluent factory was poured into a small harvest container, sampled and discarded. The adherent cells were then washed once with 400 ml PBS. To detach the cells from the factory, 100 mis of trypsin was added and removed and the cells were then incubated for 5 to 10 minutes in the residual trypsin. The cells were collected following two, 200 ml washes of ESTEP1 media. Forty milliliters of collected cells were then used to seed each of ten Nunc cell factories. To ten ESTEP1 media-containing bottles (1.5 liters each, at 37°C) was added 40 mis of collected cells. One 1.5 liter bottle was then used to fill one Nunc factory. Each cell factory was placed in a 37°C/5.0% C0₂ incubator.

At 80-90% confluence, a visual contamination test (phenol red color change) was performed on the Nunc cell factory. Since no contamination was observed, supernatant from the confluent factory was poured into a small harvest container, sampled and discarded. Cells were then washed once with 400 ml PBS. ESTEP2 media (1.5 liters, 668.7g/50L DMEM (Gibco), 5.5 g/50L pyruvic acid, sodium salt 96% (Mallinckrodt), 185.0 g/50L NaHC0₃

(Mallinkrodt), 5.0 mg/ml, 25 ml/50L insulin, 10.0 mg/ml and 25 ml/50 L transferrin) was added to each Nunc cell factory. The cell factory were incubated at 37°C/5.0% C0₂.

At approximately 48 hours (ZCHEMO- 8/NF, 15 L was obtained) and 72 hours (ZCHEM0-8/CF, 15 L was obtained) , a visual contamination test (phenol red color change) was performed on the Nunc cell factories . Supernatant from each factory was poured into small harvest containers . Fresh serum-free ESTEP 2 media (500 ml) was poured into each Nunc cell factory, and the factories were incubated at37°C/5.0% C0₂. One ml of supernatant harvest was transferred to a microscope slide, and subjected to microscopic analysis for contamination. The contents of the small harvest containers for each factory were pooled and immediately filtered. A second harvest was then performed, substantially as described above at 46 hours

(ZCHEMO-8/NF, 13.5 L were obtained) and 72 hours (ZCHEMO-

8/CF, 13.5 L were obtained) and the cell factories were discarded thereafter. An aseptically assembled filter train apparatus was used for aseptic filtration of the harvest supernatant (conditioned media) . Assembly was a follows: tubing was wire-tied to an Opti-Cap filter (Millipore Corp., Bedford, MA) and a Gelman Supercap 50 filter (Gelman Sciences, Ann Arbor, MI) . The Supercap 50 filter was also attached to a sterile capped container located in a hood; tubing located upstream of the Millipore Opti-cap filter was inserted into a peristaltic pump; and the free end of the tubing was placed in the large harvest container. The peristaltic pump was run between 200 and 300 rpm, until all of the conditioned media passed through the 0.22 μm final filter into a sterile collection container. The filtrate was placed in a 4 °C cold room pending purification.

Concentration and Western Blot

Conditioned media containing ZCHEMO- 8/CF and ZCHEMO-8/NF was collected for concentration at various time points (at the 5 T-162 flask stage; 1 factory, fetal bovine serum media; 10 factories, fetal bovine serum media; 10 factories, serum free media and a second 10 factory, serum free media time point) . Since the expected mass of the protein was in excess of 8 kDA, Millipore 5 kDa cut off concentrators were used. The starting volume for each sample was 15 ml, which was concentrated to a final volume of 1.5 ml. The concentrators were spun at 4°C in Beckman tabletop centrifuge at 2000 x g (3000 rpm) for 40 minutes. The concentrate was transferred to a 1.5 ml non-stick microfuge tube, and the volume was adjusted to 1 ml using flow through media to achieve a lOx concentration. To sterilize the media, the lOx concentrate was split into two Costar Spin-X tubes, and the tubes were spun at 8000 x g for two minutes in a Eppendorf 5415 microfuge (VWR, Seattle, WA) .

Western blot analysis was also conducted for the ZCHEMO-8/CF and ZCHEMO-8/NF samples described above. NuPage gel electrophoresis is conducted using 25 μl of conditioned media and 25 μl 2X reducing sample buffer, according to manufacturer's instructions, running the get at 150 volts for approximately one hour. The conditioned media sample lanes were loaded with 32.5 μl of sample. Following electrophoresis, the gels were transferred to 2 μm supported nitrocellulose (BioRad) at room temperature for 1 hour (500 mA) using a Hoeffer transfer tank unit (Hoeffer Scientific Instruments, San Francisco, CA) with stirring in accordance with the manufacturer's instructions. The transfer buffer contained 25 mM Tris- Base, 200 mM glycine, and 20% MeOH . Next, the nitrocellulose filters were blocked for 10 minutes at room temperature with 10% non-fat dry milk (NFDM) in Western A buffer (50 mM Tris, pH 7.4; 5 mM EDTA solution, pH 8.0; 0.05% Igepal (Sigma); 150 mM NaCl and 0.25% gelatin). The membrane was then rinsed with Western A buffer. The primary antibody, α-FLAG M2 (Kodak) was added at 0.5 ug/ml in Western A buffer containing 2.5% NFDM with shaking or rocking overnight at 4°C. The membrane was then washed three times for 5 minutes in Western A buffer. A secondary antibody, goat α-mouse IgG-HRP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added in Western A buffer containing 2.5% NFDM (10 μl of 400 μg/ml antibody solution in 20 mis Western A for a 1:2000 dilution) with shaking or rocking for one hour at room temperature. The membrane was then washed three times for 5 minutes in Western A buffer, and then rinsed in Milli-Q water. The membrane was then placed into a sheet protector (Avery Office Products, Gold Bar, CA) . A 1:1 solution of ECL Western Blotting Detection Reagents (Amersham Life Science., Buckinghamshire, England) was mixed and 500 μl of solution was added to the left edge of the membrane. The solution was then slowly spread over the blot and excess reagent was removed after one minute. The blot was then exposed to ECL Hyperfilm 8x10 (Amersham Corp., Arlington Heights, IL) for 10 seconds and 30 seconds. Example 7 Purification and Analysis of ZCHEMO- 8 /NF and ZCHEMO- 8 /CF o

All of the procedures are carried out at 4 C, unless otherwise noted. A total of 25 liters _ of conditioned ZCHEMO- 8CF and ZCHEMO- 8NF media from above was sequentially sterile filtered through a 4 inch, 0.2 μM Opti-cap filter (Millipore Corp., Bedford, MA) and a 0.2 μM Supercap 50 filter (Gelman Sciences, Ann Arbor, MI) . The material was then concentrated to about 1.3 liters using an Amicon DC 10L concentrator (Amicon, Beverly, MA) fitted with an A/G Tech hollow fiber cartridge (A/G Tech, Needham, MA) with a 15 sq. ft. 3000 kDa cutoff membrane. The concentrated material was again sterile filtered with a Gelman filter as described above. A 25 ml aliquot of anti-FLAG Sepharose (Kodak) was added to the concentrated material for batch absorption and the mixture was gently agitated on a Wheaton roller culture apparatus (Wheaton, Millville, NJ) for 18 hours at 4 C . The Sepharose mixture was then poured into a 5.0 x 20.0 cm Econo-Column (Bio-Rad Laboratories, Hercules, CA) and the gel was washed with 30 column volumes of phosphate buffered saline (PBS) . The unretained flow- through fraction was discarded. Once the absorbance of the effluent at 280 nM was less than 0.05, flow through the column was reduced to zero and the anti-FLAG Sepharose gel was washed with 2.0 column volumes of PBS containing 0.2 mg/ml of FLAG peptide (SEQ ID NO:20) . After 1.0 hour o at 4 C, flow was resumed and the eluted protein was collected. This fraction is referred to as the peptide elution. The anti-FLAG Sepharose gel was washed with 2.0 column volumes of 0.1 M glycine, pH 2.5, and the glycine wash was collected separately. The pH of the glycine- eluted fraction was adjusted to 7.0 by the addition of a small volume of 10X PBS and stored at 4 C .

The peptide elution was concentrated at 5.0 ml using a 5,000 molecular weight cutoff membrane concentrator (Millipore) according the manufacturer's instructions. The concentrated peptide elution was then separated from free peptide by chromatography on a 1.5 x 50 cm Sephadex G-50 (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) column equilibrated in PBS at a flow rate of 1.0 ml/minute using a BioCad Sprint HPLC system (PreSeptive BioSystems, Farmington, MA) . Two milliliter fractions were collected and the absorbance at 280 nM was monitored. The first peak of material absorbing at 280 nM and eluting near the void volume of the column was collected.

SDS-PAGE and Western Blot analysis using anti- FLAG M2 antibodies (Kodak) was done on the purified material. Purified ZCHEMO- 8NF was composed of approximately equimolar amounts of two Coomassie Blue- stained bands of apparent molecular weights 30,000 and 41,000, that also showed cross-reactivity with the anti- FLAG M2 monoclonal antibody. Each band exhibited slightly greater mobility on the SDS-PAGE gels in the absence of reducing agents. Purified ZCHEMO-8CF was composed of a single Commassie Blue-stained band of apparent molecular weight 41,000 that also cross-reacted with the anti-FLAG antibodies. This material also showed slightly greater mobility under non-reducing conditions on SDS-PAGE gels. The protein concentration of the purified proteins was determined by BCA analysis (Pierce, Rockford, IL) according to the manufacturer's instructions. The concentration of ZCHEMO-8CF was 1.05 mg/ml and ZCHEMO-8NF was 1.08 mg/ml. N-terminal sequence analysis and amino acid analysis confirm the identity of the purified material .

Example 8 Baculovirus Expression of ZCHEMO- 8

Two expression vectors were prepared to express ZCHEMO-8 polypeptides in insect cells: pCHNFδ, designed to express a ZCHEMO-8 polypeptide with an N-terminal FLAG tag and pCHCFδ, designed to express a ZCHEMO- 8 polypeptide with a C-terminal FLAG tag.

pCHNFδ

A ZCHEMO- 8 fragment having a N-terminal FLAG tag

(SEQ ID NO: 7) was generated by Nco I/Xba I restriction digest of ZCHEMO-8NF/pZP9 (described above) . The resulting 383 bp fragment was visualized by gel electrophoresis (1% SeaPlaque/l% NuSieve) . The band was excised, diluted to 0.5% agarose with 2 mM MgCl₂, melted at 65°C and ligated into a Nco I/Xba I digested baculovirus expression vector, pZBV3L (a modification of the pFastBac expression vector, the polyhedron promoter has been removed and replaced with the late activating Basic Protein Promoter) . 31.8 nanograms of the restriction digested ZCHEMO- 8 insert and ng of the corresponding vector were ligated overnight. The ligation mix was diluted 3 fold in TE (10 mM Tris-HCl, pH 7.5 and 1 mM EDTA) and 4 fmol of the diluted ligation mix was transformed into DHlOBac Max Efficiency competent cells

(GIBCO-BRL, Gaithersburg, MD) according to manufacturer's instruction, by heat shock for 45 seconds in a 42°C waterbath. The ligated DNA was diluted in 450 μl SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KC1, 10 mM MgCl₂, 10 mM MgS0₄ and 20 mM glucose) and plated onto LB plates containing 100 mg/ml ampicillin. Clones were analyzed by restriction digests and 1 ml of the positive clone was transformed into 20 ml DHlOBac Max Efficiency competent cells (GIBCO-BRL, Gaithersburg, MD) according to manufacturer's instruction, by heat shock for 45 seconds in a 42°C waterbath. The ligated DNA was diluted in 980 ml SOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KC1, 10 mM MgCl2, 10 mM MgS04 and 20 mM glucose) and plated onto Luria Agar plates containing 50 mg/ml kanamycin, 7 mg/ml gentamicin, 10 mg/ml tetracycline, IPTG and Bluo Gal. The cells were incubated for 48 hours at 37°C. A color selection was used to identify those cells having virus that had incorporated into the plasmid (referred to as a "bacmid"). Those colonies, which were white in color, were picked for analysis. Bacmid DNA was isolated from positive colonies using the QiaVac Miniprep8 system (Qiagen) according the manufacturer's directions. Clones were screened for the correct insert by amplifying DNA using primers to the Basic Protein Promoter and to the SV40 terminus via PCR. Those having the correct insert were used to transfect Spodoptera frugiperda (Sf9) cells.

PCHCF8

A 314 bp ZCHEMO-8 fragment having a C-terminal FLAG tag (SEQ ID NO: 7) was generated by Bsp Hl/Xba I restriction digest of zchemo8CF/pZP9 (described above) . The fragment was visualized by gel electrophoresis and ligated into the expression vector, pZBV3L, as described above. The vector pZBV3L was derived from pFastBac by replacing the polyhedron promoter with the Basic Protein promoter.

One microliter of pCHCFl was used to independently transform 20 μl DHlOBac Max Efficiency competent cells (GIBCO-BRL, Gaithersburg, MD) according to manufacturer's instruction, by heat shock at 42 °C for 45 seconds. The transformants were then diluted in 980 μl SOC media and plated on to Luria Agar plates as described above. Bacmid DNA was isolated from positive colonies and screened for the correct insert using the PCR method as described above. Those having the correct insert were used to transfect Spodoptera frugiperda (Sf9) cells.

Transformation

Sf9 cells were seeded at 5 x 10^s cells per 35 mm plate and allowed to attach for 1 hour at 27°C. Five microliters of bacmid DNA was diluted with 100 μl Sf-900 II SFM. Six μl of CellFECTIN Reagent (Life Technologies) was diluted with 100 μl Sf-900 II SMF . The bacmid DNA and lipid solutions were gently mixed and incubated 30-45 minutes at room temperature . The media from one plate of cells were aspirated, the cells were washed IX with 2 ml fresh media. Eight hundred microliters of Sf-900 II SFM was added to the lipid-DNA mixture. The wash media was aspirated and the DNA- lipid mix added to the cells. The cells were incubated at 27°C for 4-5 hours. The DNA- lipid mix was aspirated and 2 ml of Sf-900 II media containing penicillin/streptomycin was added to each plate. The plates were incubated at 27°C, 90% humidity, for 96 after which the virus was harvested.

Primary Amplification

Sf9 cells were grown in 50 ml Sf-900 II SFM in a 200 ml shake flask to an approximate density of 0.41-0.52 x 10 cells/ml. They were then transfected with 100 μl of the virus stock from above and incubated at 27°C for 2-3 days after which time the virus was harvested. The titer for AcCHCNδ was 2 x 10⁷ pfu/ml and for AcCHCFδ it wasl x 10⁷.

Example 9 Construction of ZCHEMO- 8 Amino Terminal Glu-Glu Tagged and Carboxy Terminal Glu-Glu Tagged Yeast Expression Vectors

Expression of ZCHEMO- 8 in Pichia methanolica utilizes the expression system described in co-assigned WIPO publication WO 97/17450. An expression plasmid containing all or part of a polynucleotide encoding ZCHEMO- δ is constructed via homologous recombination. An expression vector was built from pCZR203 to express C- terminal Glu-Glu-tagged (CEE) ZCHEMO-δ polypeptides. The pCZR204 vector contains the AUGl promoter, followed by the αFpp leader sequence, followed by a blunt-ended Sma I restriction site, a carboxy-terminal peptide tag (Glu- Glu) , a translational STOP codon, followed by the AUGl terminator, the ADE2 selectable marker, and finally the AUGl 3' untranslated region. Also included in this vector are the URA3 and CEN-ARS sequences required for selection and replication in S . cerevisiae, and the AmpR and colEl ori sequences required for selection and replication in E. coli . A second expression vector was built from zCZR191 to express a N-terminal Glu-Glu-tagged (NEE) ZCHEMO-8 polypeptides. The ZCZR191 expression vector is as described above, having an amino terminal Glu-Glu tag.

For each construct two linkers are prepared, and along with ZCHEMO-8, were homologously recombined into the yeast expression vectors described above. The untagged N- terminal linker (SEQ ID NO: 12) spans 70 base pairs of the alpha factor prepro (aFpp) coding sequence on one end and joins it to the 70 base pairs of the amino-terminus coding sequence from the mature ZCHEMO-8 sequence on the other. The NEE-tagged linker (SEQ ID NO: 13) joins Glu-Glu tag (SEQ ID NO: 14) between the aFpp coding sequence and the

ZCHEMO-8 sequence. The untagged C-terminal linker (SEQ ID NO: 15) spans about 70 base pairs of carboxy terminus coding sequence of the ZCHEMO- 8 on one end with 70 base pairs of AUGl terminator sequence. The CEE-tagged linker (SEQ ID NO:16) inserts the Glu-Glu tag (SEQ ID NO:14) between the C-terminal end of ZCHEMO- 8 and the AUGl terminator region.

Construction of the NEE-tagged-ZCHEMO- 8 plasmid An NEE-tagged-ZCHEMO- 8 plasmid was made by homologously recombining 100 ng of the Smal digested pCZR191 acceptor vector, 1 μg of Eco Rl-Xho I ZCHEMO-8 cDNA donor fragment, 1 μg NEE-tagged-ZCHEMO-8 linker (SEQ ID NO: 13) and 1 μg of C-terminal untagged linker (SEQ ID NO: 15) in S. cerevisiae .

The NEE-ZCHEMO- 8 linker was synthesized by a PCR reaction. To a final reaction volume of 100 μl was added 1 pmol each of linkers, ZC13731 (SEQ ID N0:17) and ZC14216 (SEQ ID N0:18), and 100 pmol of each primer ZC13497 (SEQ ID NO:19) and ZC14204 (SEQ ID NO:20), 10 μl of 10X PCR buffer (Boehringer Mannheim) , 1 μl Pwo Polymerase (Boehringer Mannheim), 10 μl of 0.25 mM nucleotide triphosphate mix (Perkin Elmer) and dH₂0. The PCR reaction was run 10 cycles at 30 seconds at 94°C, 1 minute at 50°C and 1 minute at 72°C, concluded with a 6 minute extension at 72°. The resulting 140 bp double stranded, NEE-tagged linker is disclosed in SEQ ID NO: 13.

The C-terminal untagged ZCHEMO- δ linker was made via a PCR reaction as described using 1 pmol of oligonucleotide primers ZC14349 (SEQ ID NO:21), ZC13734

(SEQ ID NO: 22) and 100 pmol oligonucleotide linkers ZC14323 (SEQ ID NO:23) and ZC14218 (SEQ ID NO:24) . The resulting 140 bp double stranded, C-terminal untagged linker is disclosed in SEQ ID NO: 15.

Construction of the CEE-ZCHEMO-8 plasmid A CEE-ZCHEMO- 8 plasmid was made by homologously recombining 100 ng of Sma I digested pCZR203 acceptor vector, the lμg of Eco Rl-Xho I ZCHEMO- 8 cDNA donor fragment, 1 μg of N-terminal untagged ZCHEMO- 8 linker (SEQ ID NO: 12) and 1 μg of CEE-tagged linker (SEQ ID NO: 16) in a S. cerevisiae .

The N-terminal untagged ZCHEMO-8 linker was made via a PCR reaction as described above using oligonucleotides linkers ZC14821 (SEQ ID NO:29), ZC14823

(SEQ ID NO:30), ZC14822 (SEQ ID N0:31) and ZC14204 (SEQ ID NO:32). The resulting 140 bp double stranded, N-terminal untagged linker is disclosed in SEQ ID NO: 12.

The CEE-tagged linker was made via a PCR reaction as described above using oligonucleotides linkers ZC14824 (SEQ ID NO:25), ZC14819 (SEQ ID NO:26), ZC14340 (SEQ ID NO:27) and ZC14820 (SEQ ID NO:28). The resulting approximately 140 bp double stranded, CEE-tagged linker is disclosed in SEQ ID NO: 16.

One hundred microliters of competent yeast cells ( S. cerevisiae) was independently combined with 10 μl of the various DNA mixtures from above and transferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixtures were electropulsed at 0.75 kV (5 kV/cm) , ∞ ohms, 25 μF. To each cuvette was added 600 μl of 1.2 M sorbitol and the yeast was plated in two 300 μl aliquots onto two URA D plates and incubated at 30°C.

After about 4δ hours the Ura⁺ yeast transformants from a single plate were resuspended in 2.5 ml H₂0 and spun briefly to pellet the yeast cells. The cell pellet was resuspended in 1 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH δ .0 , 1 mM EDTA) . Five hundred microliters of the lysis mixture was added to an Eppendorf tube containing 300 μl acid washed glass beads and 200 μl phenol-chloroform, vortexed for 1 minute intervals two or three times, followed by a 5 minute spin in a Eppendorf centrifuge as maximum speed. Three hundred microliters of the aqueous phase was transferred to a fresh tube and the DNA precipitated with 600 μl ethanol (EtOH) , followed by centrifugation for 10 minutes at 4°C. The DNA pellet was resuspended in 100 μl H₂0.

Transformation of electrocompetent E. coli cells (DH10B, Gibco BRL) was done with 0.5-2 μl yeast DNA prep and 40 ul of DH10B cells. The cells were electropulsed at 2.0 kV, 25 μF and 400 ohms. Following electroporation, 1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10 M NaCl, 2.5 mM KC1 , 10 mM MgCl₂,

10 mM MgS0₄, 20 mM glucose) was plated in 250 μl aliquots on four LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar

(Difco) , 100 mg/L A picillin) . Individual clones harboring the correct expression construct for NEE tagged ZCHEMO- 8 were identified by PCR to verify the presence of the ZCHEMO-8 insert and to confirm that the various DNA sequences had been joined correctly to one another. Correct expression constructs were identified by PCR as described above using oligos ZC13497 (SEQ ID N0:19) and ZC13734 (SEQ ID NO: 22) which gave a 300 bp fragment and oligos ZC14822 (SEQ ID NO:31) and ZC14820 (SEQ ID NO:2δ) which gave a 300 bp fragment. The insert of positive clones were subjected to sequence analysis. Larger scale plasmid DNA was isolated using the Qiagen Maxi kit (Qiagen) according to manufacturer's instruction and the DNA was digested with Not I to liberate the Pichia -ZCHEMO- δ expression cassette from the vector backbone. The Not I -restriction digested DNA fragment was then transformed into the Pi chia methanolica expression host, PMAD16. This was done by mixing 100 μl of prepared competent PMAD16 cells with 10 μg of Not I restriction digested ZCHEMO- 8 and transferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixture was electropulsed at 0.75 kV, 25 μF, infinite ohms. To the cuvette was added 1 ml of IX Yeast Nitrogen Base (Difco, Detroit, MI) and 500 μl aliquots were plated onto two ADE DS (0.056% -Ade -Trp -Thr powder, 0.67% yeast nitrogen base without amino acids, 2% D-glucose, 0.5% 200X tryptophan, threonine solution, and 18.22% D-sorbitol) plates for selection and incubated at 30°C. The transformed yeast cells were plated on ADE DS plates for selection. Clones were picked and screened via Western blot for high-level ZCHEMO- 8 expression. The resulting NEE-tagged-ZCHEMO-8 strain was designated PMAD16 : :pGMN5 and the CEE-tagged-ZCHEMO-8 strain was designated PMAD16 : :pGMN23.3.49. The above described strains were subjected to fermentation. 8 δ

Example 10 Leukocyte Infiltration using the Mouse Air Pouch Model

To determine if ZCHEMO- δ will selectively induce migration of neutrophils, a modified mouse skin air poμch model was used (Harris et al . , Biochem. Biophys . Res. Comm. 221:962-6, 1996). Twenty male Balb/c mice (Harlen Sprague Dawley, Indianapolis, IN) were divided into 4 groups. Group 1 received vehicle only (0.5% hydroxypropylmethylcellulose w/v in sterile phosphate buffered saline (PBS) ) . Group 2 received 100 ng/mouse recombinant human IL-δ. Group 3 received 100 ng/mouse ZCHEMO-δ. Group 4 received 1 μg/mouse ZCHEMO-δ. Air pouches were formed on day 0 by subcutaneous injection of 1.5 ml air on the dorsum of each mouse, just above the scapulae, under Metofane anesthetic. An injection of 1.5 ml air was repeated on day 3 into the same pouch on each mouse. On day 6, 0.5 ml of the vehicle or treatment solutions diluted into vehicle were injected into the pouches. Treatment solutions included vehicle alone (0.5% w/v hydroxypropylmethylcellulose in PBS) , 100 ng IL-δ, 100 ng ZCHEMO-δ or 1 μg ZCHEMO-δ in 0.5 ml vehicle. After 4 hours exposure the mice were sacrificed by anesthetic overdose following retro-orbital blood sampling for CBC's. The air pouches were lavaged with 2 ml 0.1% bovine serum albumin (BSA) and Haemaline 2 (BioChem ImmunoSystems Inc., Allentown, PA) with 50 U/ml heparin. The air pouch lavage solution (pouchate) was placed into an EDTA-coated tube and spun at 600 RPM at 10°C for 10 minutes. The cell pellet was resuspended in 200 ml Serono cell diluent (BioChem ImmunoSystems Inc.) with 0.1% BSA added and assayed for cell population distribution with an CellDyn 3500 hematology analyzer (Abbott Diagnostics, Abbott Park, ILL) . Manual differential white counts were also performed using CytoSpin slides (Shandon Lipshaw, Pittsburgh, PA) which were spun at 600 RPM for 6 minutes and stained with Wright-Giemsa stain (EM Diagnostic Systems, Gibbstown, NJ) following 2 minutes methanol fixation.

There was a statistically significant difference between the cytokine-treated groups and the vehicle treated groups (Figure 2A-D) . Manual differential white blood cell counts of the harvested pouchates indicated that the percentages of neutrophils and monocytes were both statistically significantly different in the three groups treated locally with the cytokines compared to vehicle. The average percentage of neutrophils in the WBC populations in the pouchates of the cytokine-treated groups were 20% greater than that in the vehicle-treated group. Monocytes accounted for a significantly greater

(~25%) average percantage of the population of WBCs in the pouchates of the vehicle-treated group than in the cytokine-treated groups. There was no significant difference between the three cytokine doses of 100 ng IL-8 and ZCHEMO-δ at 100 ng and 1 μg. Systemic circulating white blood cell populations were not different between the vehicle and cytokine-treated groups. No systemic effect of the subcutaneous cytokine treatment was anticipated at the 6 -hour timepoint.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. SEQUENCE LISTING

(1) GENERAL INFORMATION

(1) APPLICANT: ZymoGenetics, Inc.

1201 Eastlake Avenue East

Seattle

WA

USA

98102

(11) TITLE OF THE INVENTION: HUMAN CHEMOKINE ZCHEMO-8

(111) NUMBER OF SEQUENCES: 54

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: ZymoGenetics, Inc.

(B) STREET: 1201 Eastlake Ave. E.

(C) CITY: Seattle

(D) STATE: WA

(E) COUNTRY: USA

(F) ZIP: 98102

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(vlll) ATTORNEY/AGENT INFORMATION:

(A) NAME: Llngenfelter, Susan E

(B) REGISTRATION NUMBER: 41,156 (C) REFERENCE/DOCKET NUMBER: 97-23PC

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 206-442-6675

(B) TELEFAX: 206-442-6678

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 510 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: cDNA (ix) FEATURE:

(A) NAME/KEY: Coding Sequence

(B) LOCATION: 38...319 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GTCGACCCAC GCGTCCGAGG GCCTGATTTG CAGCATC ATG ATG GGC CTC TCC TTG 55

Met Met Gly Leu Ser Leu 1 5

GCC TCT GCT GTG CTC CTG GCC TCC CTC CTG AGT CTC CAC CTT GGA ACT 103 Ala Ser Ala Val Leu Leu Ala Ser Leu Leu Ser Leu His Leu Gly Thr 10 15 20

GCC ACA CGT GGG AGT GAC ATA TCC AAG ACC TGC TGC TTC CAA TAC AGC 151 Ala Thr Arg Gly Ser Asp He Ser Lys Thr Cys Cys Phe Gin Tyr Ser 25 30 35

CAC AAG CCC Cπ CCC TGG ACC TGG GTG CGA AGC TAT GAA TTC ACC AGT 199 His Lys Pro Leu Pro Trp Thr Trp Val Arg Ser Tyr Glu Phe Thr Ser 40 45 50

AAC AGC TGC TCC CAG CGG GCT GTG ATA TTC ACT ACC AAA AGA GGC AAG 247 Asn Ser Cys Ser Gin Arg Ala Val He Phe Thr Thr Lys Arg Gly Lys 55 60 65 70 AAA GTC TGT ACC CAT CCA AGG AAA AAA TGG GTG CAA AAA TAC ATT TCT 295 Lys Val Cys Thr His Pro Arg Lys Lys Trp Val Gin Lys Tyr He Ser 75 80 85

TTA CTG AAA ACT CCG AAA CAA TTG TGACTCAGCT GAATTTTCAT CCGAGGACGC 349 Leu Leu Lys Thr Pro Lys Gin Leu 90

TTGGACCCCG CTCTTGGCTC TGCAGCCCTC TGGGGAGCCT GCGGAATCTT TTCTGAAGGC 409 TACATGGACC CGCTGGGGAG GAGAGGGTGT TTCCTCCCAG AGTTACTTTA ATAAAGGTTG 469 TTCATAGAGT TGAAAAAAAA AAAAAAAAAA AAGGGCGGCC G 510

(2) INFORMATION FOR SEQ ID NO:2:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 94 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Met Gly Leu Ser Leu Ala Ser Ala Val Leu Leu Ala Ser Leu Leu

1 5 10 15

Ser Leu His Leu Gly Thr Ala Thr Arg Gly Ser Asp He Ser Lys Thr

20 25 30

Cys Cys Phe Gin Tyr Ser His Lys Pro Leu Pro Trp Thr Trp Val Arg

35 40 45

Ser Tyr Glu Phe Thr Ser Asn Ser Cys Ser Gin Arg Ala Val He Phe

50 55 60

Thr Thr Lys Arg Gly Lys Lys Val Cys Thr His Pro Arg Lys Lys Trp 65 70 75 80

Val Gin Lys Tyr He Ser Leu Leu Lys Thr Pro Lys Gin Leu

85 90

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 92 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

Met Lys Leu Cys Val Thr Val Leu Ser Leu Leu Met Leu Val Ala Ala 1 5 10 15

Phe Cys Ser Pro Ala Leu Ser Ala Pro Met Gly Ser Asp Pro Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ala Arg Lys Leu Pro Arg Asn Phe Val

35 40 45

Val Asp Tyr Tyr Glu Thr Ser Ser Leu Cys Ser Gin Pro Ala Val Val

50 55 60

Phe Gin Thr Lys Arg Ser Lys Gin Val Cys Ala Asp Pro Ser Glu Ser 65 70 75 80

Trp Val Gin Glu Tyr Val Tyr Asp Leu Glu Leu Asn 85 90

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 504 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

TCGACCCACG CGTCCGAGGG CCTGATTTGC AGCATCATGA TGGGCCTCTC CTTGGCCTCT 60

GCTGTGCTCC TGGCCTCCCT CCTGAGTCTC CACCTTGGAA CTGCCACACG TGGGAGTGAC 120

ATATCCAAGA CCTGCTGCTT CCAATACAGC CACAAGCCCC TTCCCTGGAC CTGGGTGCGA 180

AGCTATGAAT TCACCAGTAA CAGCTGCTCC CAGCGGGCTG TGATATTCAC TACCAAAAGA 240

GGCAAGAAAG TCTGTACCCA TCCAAGGAAA AAATGGGTGC AAAAATACAT TTCTTTACTG 300

AAAACTCCGA AACAATTGTG ACTCAGCTGA ATTTTCATCC GAGGACGCTT GGACCCCGCT 360

CTTGGCTCTG CAGCCCTCTG GGGAGCCTGC GGAATCTTTT CTGAAGGCTA CATGGACCCG 420

CTGGGGAGGA GAGGGTGTTT CCTCCCAGAG TTACTTTAAT AAAGGTTGTT CATAGAGTTG 480

AAAAAAAAAA AAAAAAAAAA GGGC 504

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS. single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (vn) IMMEDIATE SOURCE: (B) CLONE: ZC13.649

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5-

ATGATGGGCC TCTCCTTG 18

(2) INFORMATION FOR SEQ ID NO: 6-

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA (vn) IMMEDIATE SOURCE: (B) CLONE: ZC13.637

(xi) SEQUENCE DESCRIPTION. SEQ ID NO:6-

GGCTTGTGGC TGTATTGG 18

(2) INFORMATION FOR SEQ ID NO.7-

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION- SEQ ID NO:7

Asp Tyr Lys Asp Asp Asp Asp Lys 1 5

(2) INFORMATION FOR SEQ ID NO: 8.

(l) SEQUENCE CHARACTERISTICS. (A) LENGTH 13 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE cDNA (vi l) IMMEDIATE SOURCE (B) CLONE ZC3549

(xi) SEQUENCE DESCRIPTION SEQ ID NO 8

AGCTTTGGTC AAG 13

(2) INFORMATION FOR SEQ ID NO 9

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 30 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE cDNA (vn) IMMEDIATE SOURCE (B) CLONE ZC13550

(xi) SEQUENCE DESCRIPTION SEQ ID NO 9

TTAGGATCCC AATTGTTTCG GAGTTTTCAG 30

(2) INFORMATION FOR SEQ ID NO 10

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 9 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE cDNA (vn) IMMEDIATE SOURCE (B) CLONE ZC3547

(xi) SEQUENCE DESCRIPTION SEQ ID NO 10

AGCTCCCCG 9 (2) INFORMATION FOR SEQ ID NO 11

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 30 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE cDNA (vn) IMMEDIATE SOURCE (B) CLONE ZC13548

(xi) SEQUENCE DESCRIPTION SEQ ID NO 11

πACTCGAGG CTGAGTCACA AπGπTCGG 30

(2) INFORMATION FOR SEQ ID NO 12

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 137 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE cDNA

(xi) SEQUENCE DESCRIPTION SEQ ID NO 12

TACCCATCCA AGGAAAAAAT GGGTGCAAAA ATACATTTCT TTACTGAAAA CTCCGAAACA 60 ATTGGGTGGT GAAGAATACA TGCAATGGAA TAGAATTCCT AGTAπCTAG GGCTGCCTGT 120 TTGGATATTT πATAAT 137

(2) INFORMATION FOR SEQ ID NO 13

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 141 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE cDNA

(xi) SEQUENCE DESCRIPTION SEQ ID NO 13

AGCAπGCTG CTAAAGAAGA AGGTGTAAGC TTGGACAAGA GAGAAGAAGA ATACATGCCA 60 ATGGAAGGTG GTACACGTGG GAGTGACATA TCCAAGACCT GCTGCTTCCA ATACAGCCAC 120 AAGCCCCTTC CCTGGACCTG G 141

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:

Glu Glu Tyr Met Pro Met Glu 1 5

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 151 base pairs

(B) TYPE- nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY, linear

(n) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

TACCCATCCA AGGAAAAAAT GGGTGCAAAA ATACAπTCT TTACTGAAAA CTCCGAAACA 60 AπGTGAGAA TTCTAGTATT CTAGGGCTGC CTGTπGGAT AπTTTATAA πTTTGAGAG 120 πTGCCAACT AATGTππC TCTTCTATGA T 151

(2) INFORMATION FOR SEQ ID NO: 16-

(l) SEQUENCE CHARACTERISTICS-

(A) LENGTH. 146 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: 9 δ

ACGGTπATT GTπATCAAT ACTACTAπG CTAGCAπGC TGCTAAAGAA GAAGGTGTAA 60 GCπGGACAA GAGAGAAACA CGTGGGAGTG ACATATCCAA GACCTGCTGC πCCAATACA 120 GCCACAAGCC CCπCCCTGG ACCTGG 146

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA (vn) IMMEDIATE SOURCE: (B) CLONE: ZC13731

(xi) SEQUENCE DESCRIPTION. SEQ ID NO: 17:

GGTGTAAGCT TGGACAAGAG AGAAGAAGAA TACATGCCAA TGGAAGGTGG T 51

(2) INFORMATION FOR SEQ ID NO: 18

(l) SEQUENCE CHARACTERISTICS-

(A) LENGTH. 71 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (vn) IMMEDIATE SOURCE: (B) CLONE: ZC14216

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18.

TGGCTGTATT GGAAGCAGCA GGTCTTGGAT ATGTCACTCC CACGTGTACC ACCTTCCAπ 60 GGCATGTAπ C 71

(2) INFORMATION FOR SEQ ID NO: 19.

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (11) MOLECULE TYPE: cDNA (vn) IMMEDIATE SOURCE: (B) CLONE: ZC13497

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

AGCAπGCTG CTAAAGAAGA AGGTGTAAGC πGGACAAGA GAGA 44

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 44 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY- linear

(n) MOLECULE TYPE: cDNA (vn) IMMEDIATE SOURCE: (B) CLONE: ZC14204

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20.

CCAGGTCCAG GGAAGGGGCT TGTGGCTGTA πGGAAGCAG CAGG 44

(2) INFORMATION FOR SEQ ID NO: 21:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE- nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA (vn) IMMEDIATE SOURCE- (B) CLONE- ZC14349

(xi ) SEQUENCE DESCRIPTION. SEQ ID NO.21

TACCCATCCA AGGAAAAAAT GGGTGCAAAA ATACATTTCT TTACTGA 47

(2) INFORMATION FOR SEQ ID NO: 22:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 52 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n ) MOLECULE TYPE: cDNA (vn) IMMEDIATE SOURCE: (B) CLONE: ZC13734

(xi ) SEQUENCE DESCRIPTION: SEQ ID N0:22:

ATCATAGAAG AGAAAAACAT TAGTTGGCAA ACTCTCAAAA AπATAAAAA TA 52

(2) INFORMATION FOR SEQ ID N0:23.

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 71 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE- cDNA (vn) IMMEDIATE SOURCE- (B) CLONE: ZC14323

(xi) SEQUENCE DESCRIPTION. SEQ ID NO:23.

GGGTGCAAAA ATACATTTCT πACTGAAAA CTCCGAAACA AπGTGAGAA TTCTAGTAπ 60 CTAGGGCTGC C 71

(2) INFORMATION FOR SEQ ID N0:24.

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 60 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (vi i) IMMEDIATE SOURCE (B) CLONE: ZC14218

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:

TGGCAAACTC TCAAAAAπA TAAAAATATC CAAACAGGCA GCCCTAGAAT ACTAGAATTC 60 (2) INFORMATION FOR SEQ ID NO 25

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 69 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n ) MOLECULE TYPE cDNA (vn) IMMEDIATE SOURCE (B) CLONE ZC14284

(xi ) SEQUENCE DESCRIPTION SEQ ID NO 25

GGGTGCAAAA ATACATπCT πACTGAAAA CTCCGAAACA ATTGGGTGGT GAAGAATACA 60 TGCCAATGG 69

(2) INFORMATION FOR SEQ ID NO 26

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 58 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE cDNA (vn) IMMEDIATE SOURCE (B) CLONE ZC14819

(xi) SEQUENCE DESCRIPTION SEQ ID NO 26

AACAGGCAGC CCTAGAATAC TAGGAAπCT ATTCCATTGG CATGTAπCT TCACCACC 58

(2) INFORMATION FOR SEQ ID NO 27

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 47 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE cDNA (vn) IMMEDIATE SOURCE (B) CLONE ZC14349 (xi) SEQUENCE DESCRIPTION SEQ ID NO 27

TACCCATCCA AGGAAAAAAT GGGTGCAAAA ATACAπTCT TTACTGA 47

(2) INFORMATION FOR SEQ ID NO 28

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 39 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE cDNA (vn) IMMEDIATE SOURCE (B) CLONE ZC14820

(xi) SEQUENCE DESCRIPTION SEQ ID NO 28

AπATAAAAA TATCCAAACA GGCAGCCCTA GAATACTAG 39

(2) INFORMATION FOR SEQ ID NO 29

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 62 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE cDNA (vn) IMMEDIATE SOURCE (B) CLONE ZC14821

(xi) SEQUENCE DESCRIPTION SEQ ID NO 29

TCAATACTAC TAπGCTAGC AπGCTGCTA AAGAAGAAGG TGTAAGCπG GACAAGAGAG 60 AA 62

(2) INFORMATION FOR SEQ ID NO 30

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 72 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear (ii) MOLECULE TYPE: cDNA (vii) IMMEDIATE SOURCE: (B) CLONE: ZC14823

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:

TGGCTGTATT GGAAGCAGCA GGTCπGGAT ATGTCACTCC CACGTGTπC TCTCπGTCC 60 AAGCπAGAC CT 72

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (vii) IMMEDIATE SOURCE: (B) CLONE: ZC14822

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

ACGGπTATT GπTATCAAT ACTACTATTG CTAGCAπGC 40

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (vii) IMMEDIATE SOURCE: (B) CLONE: ZC14204

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:

CCAGGTCCAG GGAAGGGGCT TGTGGCTGTA TTGGAAGCAG CAGG 44

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 282 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE. cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:

ATGATGGGNY TNWSNYTNGC NWSNGCNGTN YTNYTNGCNW SNYTNYTNWS NYTNCAYYTN 60

GGNACNGCNA CNMGNGGNWS NGAYATHWSN AARACNTGYT GYTTYCARTA YWSNCAYAAR 120

CCNYTNCCNT GGACNTGGGT NMGNWSNTAY GARTTYACNW SNAAYWSNTG YWSNCARMGN 180

GCNGTNATHT TYACNACNAA RMGNGGNAAR AARGTNTGYA CNCAYCCNMG NAARAARTGG 240

GTNCARAART AYATHWSNYT NYTNAARACN CCNAARCARY TN 282

(2) INFORMATION FOR SEQ ID N0:34.

(i) SEQUENCE CHARACTERISTICS-

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA (vn) IMMEDIATE SOURCE: (B) CLONE: ZC12641

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:

GGGTACAGAC TπCTTGCCT 20

(2) INFORMATION FOR SEQ ID NO:35:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA (v ) IMMEDIATE SOURCE: (B) CLONE: ZC12642

(xi) SEQUENCE DESCRIPTION- SEQ ID NO:35:

TGATπGCAG CATCATGATG 20 (2) INFORMATION FOR SEQ ID N0:36:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 90 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:

Met Lys Val Ser Ala Ala Arg Leu Ala Val He Leu He Ala Thr Ala 1 5 10 15

Leu Cys Ala Pro Ala Ser Ala Ser Tyr Ser Ser Asp Thr Thr Pro Cys

20 25 30

Cys Phe Ala Tyr He Ala Arg Pro Leu Pro Arg Ala His He Lys Glu

35 40 45

Tyr Phe Tyr Thr Ser Gly Lys Cys Ser Asn Pro Ala Val Val Phe Val

50 55 60

Thr Arg Lys Asn Arg Gin Val Cys Ala Asn Pro Glu Lys Lys Trp Val 65 70 75 80

Arg Glu Tyr He Asn Ser Leu Glu Met Ser 85 90

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 93 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:

Met Gin Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gin Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gin He Pro Gin Asn Phe He 35 40 45 Ala Asp Tyr Phe Glu Thr Ser Ser Gin Cys Ser Lys Pro Ser Val He

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gin Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Val Gin Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala 85 90

(2) INFORMATION FOR SEQ ID N0:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 92 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE, protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

Met Gin Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala 1 5 10 15

Leu Cys Asn Gin Phe Ser Ala Ser Leu Ala Ala Asp Thr Pro Thr Ala

20 25 30

Cys Cys Phe Ser Tyr Thr Ser Arg Gin He Pro Gin Asn Phe He Ala

35 40 45

Asp Tyr Phe Glu Thr Ser Ser Gin Cys Ser Lys Pro Gly Val He Phe

50 55 60

Leu Thr Lys Arg Ser Arg Gin Val Cys Ala Asp Pro Ser Glu Glu Trp 65 70 75 80

Val Gin Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala 85 90

(2) INFORMATION FOR SEQ ID NO:39-

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 109 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Met Lys He Ser Val Ala Ala He Pro Phe Phe Leu Leu He Thr He 1 5 10 15

Ala Leu Gly Thr Lys Thr Glu Ser Ser Ser Gin Thr Gly Gly Lys Pro

20 25 30

Lys Val Val Lys He Gin Leu Lys Leu Val Gly Gly Pro Tyr His Pro

35 40 45

Ser Glu Cys Cys Phe Thr Tyr Thr Thr Tyr Lys He Pro Arg Gin Arg

50 55 60

He Met Asp Tyr Tyr Glu Thr Asn Ser Gin Cys Ser Lys Pro Gly He 65 70 75 80

Val Phe He Thr Lys Arg Gly His Ser Val Cys Thr Asn Pro Ser Asp

85 90 95

Lys Trp Val Gin Asp Tyr He Lys Asp Met Lys Glu Asn 100 105

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:

AARTGGGTNC ARAARTA 17

(2) INFORMATION FOR SEQ ID N0.41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:

RARTGGGTNM RNRANTA 17

(2) INFORMATION FOR SEQ ID N0:42: l O δ

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH 17 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE cDNA

(xi) SEQUENCE DESCRIPTION SEQ ID NO 42

YTYACCCANK YNYTNAT 17

(2) INFORMATION FOR SEQ ID NO 43

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 17 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE cDNA

(xi) SEQUENCE DESCRIPTION SEQ ID NO 43

TGYTGYπYC ARTAYWS 17

(2) INFORMATION FOR SEQ ID NO 44

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 17 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE cDNA

(xi) SEQUENCE DESCRIPTION SEQ ID NO 44

TGYTGYTTYN VRTAYWB 17

(2) INFORMATION FOR SEQ ID NO 45

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 17 base pairs

(B) TYPE nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45: ACRACRAARN BNATRWV 17

(2) INFORMATION FOR SEQ ID N0:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:46: AARGTNTGYA CNCAYCC 17

(2) INFORMATION FOR SEQ ID N0:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:

HVNGTNTGYR CNVAYCC 17

(2) INFORMATION FOR SEQ ID N0:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (11) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:

DBNCANACRY GNBTRGG 17

(2) INFORMATION FOR SEQ ID NO:49:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii ) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:

GTNATHTTYA CNACNAA 17

(2) INFORMATION FOR SEQ ID NO: 50:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:

RTNRTNXYN YNACNMR 17

(2) INFORMATION FOR SEQ ID NO: 51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: YANYANAARN RNTGNKY 17

(2) INFORMATION FOR SEQ ID N0:52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:

WSNTAYGART TYACNWS 17

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

DVNTAYKWNK WNACNWV 17

(2) INFORMATION FOR SEQ ID N0:54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

HBNATRMWNM WNTGNWB 17

Claims

CLAIMS What is claimed is:

1. An isolated polypeptide comprising a sequence of amino acid residues that is at least 60% identical in amino acid sequence to residues 23-94 of SEQ ID NO : 2 , said polypeptide comprising cysteine residues at positions corresponding to residues 33, 34, 57 and 73 of SEQ ID NO : 2.

2. An isolated polypeptide according to claim 1, wherein said polypeptide is at least ╬┤0% identical in amino acid sequence to residues 23-94 of SEQ ID NO : 2 , said polypeptide comprising cysteine residues at positions corresponding to residues 33, 34, 57 and 73 of SEQ ID NO : 2.

3. An isolated polypeptide according to claim 1, wherein said polypeptide comprises residues 23-94 of SEQ ID NO: 2.

4. An isolated polypeptide according to claim 1, wherein said polypeptide comprises residues 1-94 of SEQ ID NO: 2.

5. An isolated polypeptide according to claim 1, covalently linked to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores .

6. An isolated polypeptide according to claim 5 further comprising a proteolytic cleavage site between said polypeptide and said moiety.

7. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising a sequence of amino acid residues that is at least 60% identical in amino acid sequence to residues 23-94 of SEQ ID NO : 2 , said polypeptide comprising cysteine residues at positions corresponding to residues 33, 34, 57 and 73 of SEQ ID NO: 2; and a transcriptional terminator.

╬┤. An expression vector according to claim 7, wherein said DNA segment encodes a polypeptide according to claim 1, wherein said polypeptide is at least ╬┤O% identical in amino acid sequence to residues 23-94 of SEQ ID NO : 2 , said polypeptide comprising cysteine residues at positions corresponding to residues 33, 34, 57 and 73 of SEQ ID NO : 2.

9. An expression vector according to claim 7, wherein said polypeptide encoded by said DNA segment comprises residues 23-94 of SEQ ID NO: 2.

10. An expression vector according to claim 7, wherein said DNA segment encodes a polypeptide covalently linked to an affinity tag selected from the group consisting of polyhistidine, (SEQ ID NO:7), Glu-Glu, glutathione S transferase and an immunoglobulin heavy chain constant region.

11. An expression vector according to claim 7 wherein said DNA segment further encodes a secretory signal sequence operably linked to said polypeptide.

12. An expression vector according the claim 7, wherein said secretory signal sequence encodes residues 1-22 of SEQ ID NO: 2.

13. A cultured cell into which has been introduced an expression vector comprising the following operably linked elements : a transcription promoter; a DNA segment encoding a polypeptide comprising a sequence of amino acid residues that is at least 60% identical in amino acid sequence to residues 23-94 of SEQ ID NO : 2 , said polypeptide comprising cysteine residues at positions corresponding to residues 33, 34, 57 and 73 of SEQ ID NO: 2; and a transcriptional terminator; wherein said cell expresses said polypeptide encoded by said DNA segment .

14. A method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector comprising the following operably linked elements : a transcription promoter; a DNA segment encoding a polypeptide comprising a sequence of amino acid residues that is at least 60% identical in amino acid sequence to residues 23-94 of SEQ ID NO: 2, said polypeptide comprising cysteine residues at positions corresponding to residues 33, 34, 57 and 73 of SEQ ID NO: 2; and a transcriptional terminator; whereby said cell expresses said polypeptide encoded by said DNA segment; and recovering said expressed polypeptide .

15. A pharmaceutical composition comprising a polypeptide comprising a sequence of amino acid residues that is at least 60% identical in amino acid sequence to residues 23-94 of SEQ ID NO : 2 , said polypeptide comprising cysteine residues at positions corresponding to residues 33, 34, 57 and 73 of SEQ ID NO : 2 , in combination with a pharmaceutically acceptable vehicle .

16. An antibody that specifically binds to an epitope of a polypeptide of SEQ ID NO: 2.

17. A binding protein that specifically binds to an epitope of a polypeptide of SEQ ID NO: 2.

l╬┤. An isolated polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 60% identical in amino acid sequence to residues 23-94 of SEQ ID NO : 2 , said polypeptide comprising cysteine residues at positions corresponding to residues 33, 34, 57 and 73 of SEQ ID NO: 2.

19. An isolated polynucleotide according to claim l╬┤, wherein said polypeptide is at least 60% identical in amino acid sequence to residues 23-94 of SEQ ID NO: 2, said polypeptide comprising cysteine residues at positions corresponding to residues 33, 34, 57 and 73 of SEQ ID NO: 2.

20. An isolated polynucleotide according to claim l╬┤, wherein said polypeptide comprises residues 23-94 of SEQ ID NO: 2.

21. An isolated polynucleotide according to claim l╬┤, wherein said polynucleotide is DNA.

22. An isolated polynucleotide according to claim l╬┤, wherein said polynucleotide is selected from the group consisting of, a) a sequence of nucleotides from nucleotide 103 to nucleotide 319 of SEQ ID NO : 1 ; b) a sequence of nucleotides from nucleotide 3╬┤ to nucleotide 319 of SEQ ID NO: 2; c) orthologs of a) or b) ; d) degenerate nucleotide sequences of a) , b) or c) ; and f) nucleotide sequences complementary to a) , b) , c) or d) .

23. An isolated polynucleotide molecule according to claim l╬┤, having the nucleotide sequence of SEQ ID NO: 33.

24. An oligonucleotide probe or primer comprising 14 contiguous nucleotides of a polynucleotide of SEQ ID NO: 33 or a sequence complementary to SEQ ID NO: 33.

25. A DNA construct encoding a polypeptide fusion, said fusion comprising a secretory signal sequence having the amino acid sequence of residues 1-22 of SEQ ID NO : 2 , wherein said secretory signal sequence is operably linked to an additional polypeptide.

26. A method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO : 1 or the complement of SEQ ID N0:1, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; comparing said first reaction product to a control reaction product, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.