Polynucleotides encoding a human chemotactic cytokine I Number:7,393,943 from the United States Patent and Trademark Office (PTO) owispatent

Title: Polynucleotides encoding a human chemotactic cytokine I

Abstract: Human chemotactic cytokine I polypeptides and DNA (RNA) encoding such chemotactic cytokines and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such chemotactic cytokines for the treatment of leukemia, tumors, chronic infections, auto-immune disease, fibrotic disorders, wound healing and psoriasis. Antagonists against such chemotactic cytokines and their use as a therapeutic to treat rheumatoid arthritis, auto-immune and chronic and acute inflammatory and infective diseases, allergic reactions, prostaglandin-independent fever and bone marrow failure are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences and altered concentrations of the polypeptides. Also disclosed are diagnostic assays for detecting mutations in the polynucleotides encoding the chemotactic cytokines and for detecting altered levels of the polypeptide in a host.

Patent Number: 7,393,943 Issued on 07/01/2008 to Ni,   et al.

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Inventors:	Ni; Jian (Rockville, MD), Yu; Guo-Liang (Darnestown, MD), Alfonso; Pedro (Gaithersburg, MD), Gentz; Reiner (Silver Springs, MD), Su; Jeffrey Y. (Gaithersburg, MD)
Assignee:	Human Genome Sciences, Inc. (Rockville, MD)
Appl. No.:	08/761,289
Filed:	December 6, 1996

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Related U.S. Patent Documents

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	Application Number	Filing Date	Patent Number	Issue Date
	60008387	Dec., 1995

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Current U.S. Class:	536/23.5 ; 435/252.3; 435/320.1; 435/325; 435/471; 435/69.1
Current International Class:	C07H 21/04 (20060101); C12N 1/20 (20060101); C12N 15/00 (20060101); C12N 15/74 (20060101); C12N 5/00 (20060101); C12N 5/02 (20060101); C12P 21/06 (20060101)
Field of Search:	536/23.5,24.33 435/69.5,252.3,320.1

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References Cited [Referenced By]

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U.S. Patent Documents

5350836	September 1994	Kopchick et al.
5504003	April 1996	Li et al.
5525486	June 1996	Honjo et al.
5976832	November 1999	Hitomi et al.

Foreign Patent Documents

	731166		Sep., 1996		EP

Other References

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Primary Examiner: Landsman; Robert

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Parent Case Text

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This application claims benefit of priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Patent Application Ser. No. 60/008,387, filed Dec. 8, 1995.

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Claims

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What is claimed is:

1. An isolated polynucleotide comprising a nucleic acid sequence encoding at least 30 contiguous amino acid residues of SEQ ID NO:2.

2. The isolated polynucleotide of claim 1 comprising a nucleic acid sequence encoding at least 50 contiguous amino acid residues of SEQ ID NO:2.

3. The isolated polynucleotide of claim 2 comprising a nucleic acid sequence encoding amino acids 2 to 92 of SEQ ID NO:2.

4. An isolated polynucleotide comprising a nucleic acid sequence encoding amino acids 1 to 92 in SEQ ID NO:2.

5. An isolated polynucleotide comprising a nucleic acid sequence complementary to the polynucleotide of claim 1.

6. The isolated polynucleotide of claim 1 which is DNA.

7. The isolated polynucleotide of claim 1 further comprising a heterologous nucleic acid sequence.

8. An isolated polynucleotide comprising a nucleic acid sequence encoding at least 30 contiguous amino acid residues of the polypeptide encoded by the human cDNA contained in ATCC Deposit No. 97304.

9. The isolated polynucleotide of claim 8 comprising a nucleic acid sequence encoding at least 50 contiguous amino acid residues of the polypeptide encoded by the human cDNA contained in ATCC Deposit No. 97304.

10. The isolated polynucleotide of claim 9 comprising a nucleic acid sequence encoding the polypeptide encoded by the full-length human cDNA contained in ATCC Deposit No. 97304.

11. An isolated polynucleotide comprising a nucleic acid sequence complementary to the polynucleotide of claim 8.

12. The isolated polynucleotide of claim 8 which is DNA.

13. The isolated polynucleotide of claim 8 further comprising a heterologous nucleic acid sequence.

14. A vector comprising the polynucleotide of claim 1.

15. A vector comprising the polynucleotide of claim 2.

16. A vector comprising the polynucleotide of claim 3.

17. A vector comprising the polynucleotide of claim 4.

18. A vector comprising the polynucleotide of claim 8.

19. A vector comprising the polynucleotide of claim 9.

20. A vector comprising the polynucleotide of claim 10.

21. An isolated host cell comprising the polynucleotide of claim 1 operably linked to a heterologous regulatory sequence which controls gene expression.

22. An isolated host cell comprising the polynucleotide of claim 2 operably linked to a heterologous regulatory sequence which controls gene expression.

23. An isolated host cell comprising the polynucleotide of claim 3 operably linked to a heterologous regulatory sequence which controls gene expression.

24. An isolated host cell comprising the polynucleotide of claim 4 operably linked to a heterologous regulatory sequence which controls gene expression.

25. An isolated host cell comprising the polynucleotide of claim 8 operably linked to a heterologous regulatory sequence which controls gene expression.

26. An isolated host cell comprising the polynucleotide of claim 9 operably linked to a heterologous regulatory sequence which controls gene expression.

27. An isolated host cell comprising the polynucleotide of claim 10 operably linked to a heterologous regulatory sequence which controls gene expression.

28. A process for producing a polypeptide comprising culturing the host cell of claim 21 under conditions resulting in expression of the encoded polypeptide and recovering said polypeptide.

29. A process for producing a polypeptide comprising culturing the host cell of claim 22 under conditions resulting in expression of the encoded polypeptide and recovering said polypeptide.

30. A process for producing a polypeptide comprising culturing the host cell of claim 23 under conditions resulting in expression of the encoded polypeptide and recovering said polypeptide.

31. A process for producing a polypeptide comprising culturing the host cell of claim 24 under conditions resulting in expression of the encoded polypeptide and recovering said polypeptide.

32. A process for producing a polypeptide comprising culturing the host cell of claim 25 under conditions resulting in expression of the encoded polypeptide and recovering said polypeptide.

33. A process for producing a polypeptide comprising culturing the host cell of claim 26 under conditions resulting in expression of the encoded polypeptide and recovering said polypeptide.

34. A process for producing a polypeptide comprising culturing the host cell of claim 27 under conditions resulting in expression of the encoded polypeptide and recovering said polypeptide.

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Description

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BACKGROUND OF THE INVENTION

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention are human chemotactic cytokine I polypeptides. The invention also relates to inhibiting the action of such polypeptides.

The cytokine family of proteins exhibits a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including polymorphonuclear cells and macrophages. Many cytokines have proinflammatory activity and are involved in multiple steps during inflammatory reactions. In addition to their involvement in inflammation, cytokines have been shown to exhibit other activities. For example, interleukin-8 (IL-8) promotes proliferation of keratinocytes.

In light of the diverse biological activities, it is not surprising that cytokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis.

The S100 family of calcium binding proteins has chemotactic activity for polymorphonuclear cells, mononuclear cells and neutrophils and are calcium binding proteins. The S100 protein has been recently identified in cells of myeloid origin and consists of macrophage inhibitory factor-related protein (MRP-8), MRP-14, chemotactic protein 10 (CP-10) and calgranulin C.

MRP-8 and MRP-14 belong to the S100 family of proteins, which includes calbindin, and calcyclin (Kligman, D. and Hilt, D. C., Trends Biochem. Sci., 13:437-443 (1998)). This group of protein ranges in molecular size from 10 to 14 kd and are also expressed in a cell lineage-specific manner. Alignment of individual sequences shows that there is overall conservation of structure within the family, a notable feature being the two calcium binding sites, which are the "EF hand" type. Sequences at both the NH.sub.2- and COOH-terminal ends of MRP-8 and MRP-14 are relatively hydrophobic. An attractive hypothesis is that these regions of the molecule are buried until calcium binding brings about the conformational changes that cause their exposure, making them potentially available for interactions with other effector molecules. Because of the extended sequence of its COOH-terminal "tail" MRP-14 is the largest member of the S100 family. (Hessian, P., et al., J. Leuk. Bio., 53:197-204 (1993).

Each gene in the S100 family is composed of three exons with one intron interrupting the protein-coding sequence between the two EF hands. The MRP-8 and MRP-14 genes are both localized to chromosome 1Q12-Q21 with an undefined distance between them (Dorin J. R., et al., Nature, 326:614-617 (1987) and Lagasse, E. and Clerc, R. G., Mol. Cell. Biol., 8:2402, 2410 (1988)). Two other S100 family members 1882 (CAPL) and calcyclin/2A9 (CACY) also map to chromosome 1Q12-Q21 (Dorian, J. R., et al., Genomics, 8:420-426 (1990). It is probably that co-segregation of these five genes on chromosome 1 may represent an S100 family locus. However, this does not apply to all S100 homologs. MRP-8 and MRP-14 are restricted to cells of the monocytes/macrophage lineage, neutrophils, and under certain circumstance keratinocytes, suggesting that its expression is tightly regulated during differentiation (Hogg, N., et al., Eur. J. Immunol., 19:1053-1061 (1989)). Thus, monocytes and neutrophils in the circulation express MRP-8 and MRP-14, in contrast to other related cells such as lymphocytes, platelets, basophils and eocynophils which do not (Id.).

Resident tissue macrophages do not express MRP-8 and MRP-14, implying that differentiation of monocytes to macrophages is normally associated with loss of this molecule (Id.). Furthermore, immunohistochemical data show that at inflammatory sites MRP-8 and MRP-14 positive cells are associated with vessels but that the majority of monocytes already within the tissues at these sites have lost MRP-8 and MRP-14 expression (Id.). In keeping with these observations, tissue culture-matured monocytes down regulate this molecule (Zwadlo, G., et al., Clin. Exp. Immunol., 72:510-515 (1988)).

At sites of chronic inflammation in patients with diseases such as rheumatoid arthritis, sarcoidosis, tuberculosis or onchocerciasis macrophages express both MRP-8 and MRP-14 (Palmer, D. G., et al., Clin. Immunol. Immunopathol., 45:17-28 (1987)). In contrast, macrophages in acutely inflamed tissues may express only MRP-14 (Delabie, J., et al., Clin. Exp. Immunol., 81:123-126 (1990)). The expression of MRP-8 and MRP-14 by macrophages could be flecked exposure to tissue stimuli that either maintain expression or induce re-expression of the molecule (Palmer, D. G., et al., Clin. Immunol. Immunopathol., 45:17-28 (1987)).

In common with other members of the S100 family, MRP-8 and MRP-14 are found predominately in a cytosolic location in both monocytes and neutrophils (Dale, I., et al., Eur. J. Biochem., 134:1-6 (1983)). It is also possible that MRP-8 and MRP-14 can be expressed on the cell surface, although the majority of antibodies specific for these proteins do not react with circulating monocytes or neutrophils. There is also evidence that MRP-8 and MRP-14 exist extracellularly, however, neither protein has the signal peptide sequence for membrane translocation. Thus, MRP-8 and MRP-14 fall into the category of proteins, including interleukin-1 and basic fibroblast growth factor, that clearly have extracellular functions but about which little is known of their cellular release. Finally, MRP-8 and MRP-14 are found in the serum of patients with cystic fibrosis and other chronic inflammatory states such as rheumatoid arthritis and sarcoidosis (Bullock, S., et al., Clin. Genet., 21:336-341 (1982)).

CP-10 is one of the most potent chemotactic proteins of the S100 family. An extracellular function of the murine CP-10 includes a potent chemotactic agent involved in phagocyte recruitment during inflammatory reactions (Lackman, M., et al., J. Biol. Chem., 267:7499 (1992)). CP-10 has an apparent molecular weight of 10.3 kd and a complete sequence of 88 amino acids.

S100 proteins are characterized by two calcium binding regions, which are strongly conserved and are separated by an 8 to 12 amino acid hinge region (Kligman, D., Trends Biochem. Sci., 13:437 (1988)). Although the hinge region length is conserved, the amino acid sequences are widely divergent. This divergence led to the hypothesis that the hinge region may concur functional specificity by interaction with the actor proteins (Id.).

BRIEF SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there are provided novel polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding such polypeptides, including mRNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

In accordance with another aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressed by the DNA contained in ATCC Deposit No. 97304.

In accordance with another aspect of the present invention there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to sequences of the present invention.

In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques which comprises culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence of the present invention, under conditions promoting expression of said protein and subsequent recovery of said protein.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides for therapeutic purposes, for example, to treat tumors, chronic infections, leukemia, T-cell mediated auto-immune diseases, parasitic infections, psoriasis, asthma, allergy, to regulate hematopoiesis, to stimulate growth factor activity, to inhibit angiogenesis and to promote wound healing, to treat inflammatory disorders, to control cellular immune reactions, to treat malignant diseases, to inhibit casein kinase activity and to treat artherosclerosis.

In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides and a method of employing such antibodies to detect diseases related to an overexpression of the polypeptide of the present invention.

In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, in the treatment of certain auto-immune diseases, atherosclerosis, chronic inflammatory and infectious diseases, histamine and IgE-mediated allergic reactions, prostaglandin-independent fever, bone marrow failure, cancers, silicosis, sarcoidosis, rheumatoid arthritis, shock, hyper-eosinophilic syndrome and fibrosis in the asthmatic lung, cystic fibrosis, malignant diseases, psoriasis, diapedesis and urinary and kidney stones.

In accordance with another aspect of the present invention there is provided a method of diagnosing a disease or a susceptibility to a disease related to a mutation in the nucleic acid sequences and the protein encoded by such nucleic acid sequences.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings rein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

FIG. 1 displays the cDNA sequence SEQ ID NO:1 and corresponding deduced amino acid sequence SEQ ID NO:2 of the chemotactic cytokine I polypeptide of the present invention. The standard one-letter abbreviations for amino acids are used. Sequencing was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.).

FIG. 2 is an illustration of amino acid sequence homology between the polypeptide of the present invention and pig calgranulin C protein (SEQ ID NO:9).

FIG. 3 shows structural and functional features of the polypeptide of the present invention deduced by the indicated techniques, as a function of amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode for the mature polypeptides having the deduced amino acid sequences of FIG. 1 (SEQ ID No. 2).

Polynucleotides encoding the polypeptide of the present invention have been isolated from a human adult liver cDNA library. The polypeptide contains an open reading frame encoding a protein of 92 amino acids. The protein exhibits the highest degree of homology at the amino acid level to human MRP-14 with 46.739% identity and 67.391% similarity. The protein exhibits the highest degree of homology at the nucleotide level to SP100 protein with 56% identity and 56% similarity.

As seen in FIG. 2 the polypeptide of the present invention retains the calcium binding motifs present in all S100 protein members. For instance, amino acids, leucine and glutamine in amino acid position 4 and 5, the leucine at position 8, and the leucine and lyseine at position 28 and 30 are important for calcium binding activity and are shown to be preserved in the polypeptide of the present invention.

In accordance with another aspect of the present invention there are provided isolated polynucleotides encoding a mature polypeptide expressed by the DNA contained in ATCC Deposit No. 97304, deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA, on Sep. 25, 1995. The deposited material is a pBluescript SK (-) plasmid (Stratagene, LaJolla, Calif.) which contains the full-length chemotactic cytokine I cDNA cloned into the EcoRI, XhoI site.

The deposit(s) have been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. The strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 3.5 U.S.C. .sctn.112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptides may be identical to the coding sequences shown in FIG. 1 (SEQ ID NO:1) or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptides as the DNA of FIG. 1 (SEQ ID NO:1).

The polynucleotides which encode for the mature polypeptides of FIG. 1 (SEQ ID NO:2) may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptides.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes coding sequence for the polypeptide and may also include additional coding and/or non-coding sequence such as introns.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequences of FIG. 1 (SEQ ID NO:2). The variant of the polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleotides.

Further particularly preferred in this regard are polynucleotides encoding human chemotactic cytokine I variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments, which have the amino acid sequence of the polypeptide of FIG. 1 or of the deposit in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the human chemotactic cytokine I. Also especially preferred in this regard are conservative substitutions. Most highly preferred are polypeptides having the amino acid sequence of FIG. 1 or of the deposit, without substitutions.

Thus, the present invention includes polynucleotides encoding the same mature polypeptides as shown in FIG. 1 (SEQ ID NO:2) as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptides of FIG. 1 (SEQ ID NO:2). Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequences shown in FIG. 1 (SEQ ID NO:1). As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptides may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotides of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence).

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of FIG. 1 (SEQ ID NO:2).

Alternatively, the polynucleotide may have at least 20 bases, preferably at least 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:1, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 and polynucleotides complementary thereto as well as portions thereof, which portions have at least 30 consecutive bases and preferably at least 50 consecutive bases and to polypeptides encoded by such polynucleotides.

The present invention further relates to polypeptides which have the deduced amino acid sequences of FIG. 1 (SEQ ID NO:2), as well as fragments, analogs and derivatives of such polypeptides.

The terms "fragment," "derivative" and "analog" when referring to the polypeptides of FIG. 1 (SEQ ID NO:2), means polypeptides which retain essentially the same biological function or activity as such polypeptides. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

Among the particularly preferred embodiments of the invention in this regard are polypeptides having the amino acid sequence of FIG. 1, variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments. Alternatively, particularly preferred embodiments of the invention in this regard are polypeptides having the amino acid sequence of the human chemotactic cytokine I of the cDNA in the deposited clone, variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments.

Further particularly preferred in this regard are variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments, having the amino acid sequence of the polypeptide of FIG. 1 or of the cDNA in the deposited clone, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of human chemotactic cytokine I. Also especially preferred in this regard are conservative substitutions. Most highly preferred are polypeptides having the amino acid sequence of FIG. 1 or the deposited clone without substitutions.

The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.

The fragment, derivative or analog of the polypeptides of FIG. 1 (SEQ ID NO:2) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2, and which have at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P.sub.L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P.sub.R, P.sub.L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner to produce the gene products encoded by the recombinant sequences. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), .alpha.-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa, 293 and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease.

The human chemotactic cytokine I polypeptides may be employed to inhibit bone marrow stem cell colony formation as adjunct protective treatment during cancer chemotherapy and for leukemia.

The human chemotactic cytokine I polypeptides may also be employed to inhibit epidermal keratinocyte proliferation for treatment of psoriasis, which is characterized by keratinocyte hyper-proliferation.

The human chemotactic cytokine I polypeptides may also be employed to treat solid tumors by stimulating the invasion and activation of host defense cells, e.g., cytotoxic T cells and macrophages and by inhibiting the angiogenesis of tumors. They may also be employed to enhance host defenses against resistant chronic and acute infections, for example, mycobacterial infections via the attraction and activation of microbicidal leukocytes.

The human chemotactic cytokine I polypeptides may also be employed to inhibit T cell proliferation by the inhibition of IL-2 biosynthesis for the treatment of T-cell mediated auto-immune diseases and lymphocytic leukemias.

The chemotactic cytokine I polypeptides may also be employed to stimulate wound healing, both via the recruitment of debris clearing and connective tissue promoting inflammatory cells and also via its control of excessive TGF.beta.-mediated fibrosis. In this same manner, 1 may also be employed to treat other fibrotic disorders, including liver cirrhosis, osteoarthritis and pulmonary fibrosis.

The human chemotactic cytoKine I polypeptides of the present invention may also be employed as cytostatic agents for antibacterial and antimicrobial functions.

They may also be employed to regulate hematopoiesis, by regulating the activation and differentiation of various hematopoietic progenitor cells, for example, to release mature leukocytes from the bone marrow following chemotherapy.

The polynucleotides and polypeptides encoded by such polynucleotides may also be utilized for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors and for designing therapeutics and diagnostics for the treatment of human disease.

The polypeptides of the present invention and fragments and analogs and derivatives thereof may be identified by assays which detect chemotactic activity. One example of such an assay comprises testing such polypeptide for chemotactic activities toward murine polymorphonuclear cells or macrophages, human PMN (isolated on mono-poly-resolving medium; FLOW, McLean, Va.). Conditioned medium (diluted and fully supplemented Dulbecco's Modified Eagles medium) and cell lysates (diluted and supplemented Dulbecco's Modified Eagles medium containing 0.1% BSA instead of 10% FCS) from transiently transfected CV-1 cells are tested for chemotactic activities toward murine PMNs. Endotoxin content of media and all solutions to be tested are measured using a chromogenic limulus amoebocyte lysate assay (Cape Cod Associates, Woods Hole, Mass.), which was sensitive to 5 pg endotoxin/ml. Chemotactic activity is defined as the mean number of cells migrating through the pores of the membrane in 3 to 5 standard fields and quantitated by image analysis (Wild-Leitz, Rockly, N.J.) using planimetry measurements (magnification times 100) or by counting normally. Endotoxin-activated mouse serum (50%) or FMLP (10.sup.-7M) are used as positive controls.

This invention is also related to the use of the chemotactic cytokine I polypeptide gene as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the chemotactic cytokine I polypeptide nucleic acid sequences. Such diseases are related to under-expression of the human chemokine polypeptides, for example, tumors and cancers.

Individuals carrying mutations in the chemotactic cytokine I polypeptide gene may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding chemotactic cytokine I polypeptide can be used to identify and analyze chemotactic cytokine I polypeptide mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled chemotactic cytokine I polypeptide RNA or alternatively, radiolabeled chemotactic cytokine I polypeptide antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

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

Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA.

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

The present invention also relates to a diagnostic assay for detecting altered levels of chemotactic cytokine I polypeptide in various tissues since an over-expression of the proteins compared to normal control tissue samples may detect the presence of a disease or susceptibility to a disease, for example, cystic fibrosis or malignancies such as cancers and tumors. Assays used to detect levels of chemotactic cytokine I polypeptide in a sample derived from a host are well-known to those of skill in the art and include radioimmunoassays, competitive-binding assays, Western Blot analysis, ELISA assays and "sandwich" assay. An ELISA assay (Coligan, et al., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initially comprises preparing an antibody specific to the chemotactic cytokine I polypeptide antigen, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme. A sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein like BSA. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any chemotactic cytokine I polypeptide attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to chemotactic cytokine I polypeptide. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of chemotactic cytokine I polypeptide present in a given volume of patient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to chemotactic cytokine I polypeptide are attached to a solid support and labeled chemotactic cytokine I polypeptide and a sample derived from the host are passed over the solid support and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of chemotactic cytokine I polypeptide in the sample.

A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay chemotactic cytokine I polypeptide is passed over a solid support and binds to antibody attached to a solid support. A second antibody is then bound to the chemotactic cytokine I polypeptide. A third antibody which is labeled and specific to the second antibody is then passed over the solid support and binds to the second antibody and an amount can then be quantified.

This invention provides a method for identification of the receptors for the human chemotactic cytokine I polypeptides. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the labeled polypeptides. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and retransfected using an iterative sub-pooling and rescreening process, eventually yielding a single clones that encodes the putative receptor.

As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the p