IE912192A1 - Human macrophage inflammatory protein 2-alpha - Google Patents

Abstract

An attempt to identify the human counterpart of murine macrophage inflammatory protein 2 unexpectedly resulted in not one but two candidate sequences, designated hu-MIP-2 alpha and hu-MIP- beta , respectively. By using the DNA sequences and recombinant systems provided by this invention, one can obtain hu-MIP-2 alpha , one of the two human counterparts to mMIP-2, substantially free of other human protein. This invention also provides antibodies reactive with hu-MIP-2 alpha polypeptides as well as immunoassay methods for determining hu-MIP-2 alpha and nucleotide probes for detecting nucleotide sequences encoding hu-MIP-2 alpha in biological samples.

Description

HUMAN MACROPHAGE INFLAMMATORY PROTEIN 2d BACKGROUND OP THB INVENTION Macrophage Inflammatory Proteins (MIPS) represent a class of proteins that are produced by certain cells (for example, macrophages or lymphocytes) in response to Invasive stimuli such as gram negative bacterial llpopolysaccharide. Thus, they may be important participants in the response of the cell (host organism) to such stimuli. As such, these molecules may have therapeutic potential in tbe treatment of infections, cancer, myelopotetic dysfunction and autoimmune diseases. Two distinct forms of MIP have been found In cultures of macrophage tumor cells from the mouse: MIP-1 and MIP-2. Murine MIP-1 Murine (m)MlP-i is a major secreted protein from llpopolysaccharide (LPS) - stimulated RAW 264.7 cells, a murine macrophage tumor ceil tine. It has been purified and found to consist of two related proteins, mMIP-Ια and mMIP-ls (Wolpe, et aL, 1967, J. Exo. Med.. 167-.570: Sherry, et al., 1988, J. Exp. Med.. 168:2251).

The.cDNA’s for both mMIP-ΐα and mMIP-ls have been cloned and sequenced (Davatetis, et aL, 1988, J, Exo. Med.. 162:1939; Sherry, et ah, op, ctt.) The cloning and sequencing of cDNAs corresponding to mMIP-Ια and mMIP-ls have been reported as well by Brown, et al. (1989, J. Immunol.. 142:679): Kwon and Weissman (L9B9, Proc, Natl. Acad, Set PSA. ££:1963) and by Brown, et aL, oo, dt.. respectively. Both groups isolated homologs of mMCP-la and/or mMIP-is from cDNA libraries prepared from RNA of murine helper T-cells that had been activated by treatment with conconavalln A. These results suggest that MIP-lc and MIP-ιβ may play a rote in T-cell activation. Λ Ο Ρ ΠΡ -2Human MIP-1 Homologs Several groups have cloned may be the human homologs of mMIP-ΐα and mMiP-ie. In all cases, cDNAs were isolated from libraries based on RNA from activated human T-cells. Thus Obaru et al., (1986, J, Biochem.. 23:885) and Zipfel, et aL (1939, J, Immunol.. 142:1582) have both reported cloning of a cDNA that predicts a protein with high homology to mMIP-la (76%). Similarly, Brown, et al., oo. dt.. Zipfel, et al., op, cit.. Llpes, et al. (1988, Proc, Natl, Acad. ScL PSA. ¢5:9704) and Miller, et al. (1989, J, ImmunoL. 143:2907) have reported the cloning and sequencing of human cDNAs, which predict a protein with high homology co mMiP-li (75%). MIP-la and MIP-ls belong to a newly described family of related proteins which have Immunomodulatory activities (see Sherry, et al., op. oit, for a review). Murine MIP-2 Murine MIP-2 (mMIP-2) Is an inducible protein that has also been purified from the conditioned medium of LPS-stimulated SAW 264.7 cells (Wolpe, et al., 1989, Proc. Natl. Acad, ScL USA. ¢£:3121). The cDNA encoding murine MIP-2 has been cloned and sequenced. mMIP-2 also is a member of a homologous multigene family. Members of this family include gro/MGSA. platelet factor 4, platelet basic protein, the precursor of connective tissue activating peptide (CTAP-HD and B-thromboglobulin, a gamma interferon Inducible protein, IP-Ιβ, and interleukin 8 CL-8), also known as 3-10C, MDNCF, NAP-1 and NAF. Members of this family that have highest homology in protein sequence (generally predicted from cloned cDNA) Include MGSA and KC. MGSA (Richmond, et al., 1988, EMBO J- 7:2025) is ao autocrine growth factor with mitogenic activity secreted by human melanoma cells and is the product of the human gro geoe (Anisowicz, et at, 1987, Proc. Nati, Acad. Sci, PSA. ¢4:7188). MGSA has 57.9% identity In amino acid sequence to murine MIP-2; the predicted protein sequence of the putative hamster homolog of MGSA (ibid.) has 68.3% identity to mMIP-2. The murine KC gene product (Oquendo, et aL, 1989, J. Biol, Chem.. ¢¢¢:4233) la induced by platelet-derived growth factor (PDGF) and is thought to be the murine homolog of the human MCSA/gro gene (63.0% amino acid identity to mMIP-2).

O C-T 3Recombinant Expression of MIP There Js no prior art on the expression of recombinant MIP-2, or human ΜΐΡ-2σ and Mlp-2s although members of tho MIP-2 gene family as well as members of the related KIP-1 gene family have been expressed. The pertinence of these results to the expression of murine or human MIP-2 Is questionable, given the Idiosyncratic nature ot expression in heterologous systems. The literature will be summarized nonetheless. mMIP-lo and mMIP-ls have been Independently expressed in COS cells (Graham, et aL, 1990, Nature, £££:442). LD78 cDNA (Obaru, et al., op. dt.) which encodes a protein that Is likely to be the human homolog of murine MIP-la has been expressed In £* coll as a carboxyl terminal fusion to human lntedeukin-2, as wen as in COS cells (Yamamura, et al., 1989, J, Clin. Invest.. ££:1707). Human 1-309, a cDNA that encodes a protein with homology to members of the MIP-1 family of proteins, has been expressed In COS-1 cells In order to confirm that it encodes a secreted protein (Miller, et al., siL). Lipes, et al., (op. dt.) described baculovirus expression of Act-2 cDNA, the human homolog ot murine MIP-lt, to show that the protein encoded was secreted and to identify the mature N-terminal sequence. JE, a murine cDNA that encodes a protein with homology to ΜΙΡ-1» and MIP-lt, has been expressed in COS-1 ceils to confirm that it encodes a polypeptide core of about 12 kDa (Rollins, et al., 1983, Proc, Natl, Acad. Sci. PSA. ££:3738).

KC, a murine cDNA that encodes a protein with homology to mMIP-2, has been expressed in COS-1 cells to show that it encodes a secreted protein (Oquendo, et al., op, dtJ Connective tissue activating peptide-m (CTAP, Mullenbach, et al., 1988, J. Biol Chem., 2()1,:71.9) and IP-ID, (Luster and Ravetch (1987, jJ, Bzd. Med,. l£8:1084) both membera of tbe M1P-2 gene family, have been expressed in yeast as an «factor fusion and in it coll, respectively. Malone, et aL (1990, Science. 247:77) expressed human platelet factor 4, (MIP-2 family) in E, coll as a peptide fused to a 35 amiao acid peptide fragment cf Ct goll β-glucurooidase. The insoluble fusion protein must be cleaved with cyanogea bromide in order to generate bioactive material.

Lindley, et Studies on the btoactivities of MIP-l and -2 are in progress and have utilized native murine MIP-l or MIP-2 and very recently recombinant murine (rm) MiP-i«, rmMlP-ie and rmMIP-2. Among the bloactivides defined for both recombinant and native mMIP-i and mMIP-2 is colony-stimulating-factor promoting activity as well as roles in inflammation.

Murine MIP-2 has been shown to elicit a localized inflammatory response when injected subcutaneously into the footpads of C3H/HeJ mice, to have potent chemotactic activity for human polymorphonuclear leukocytes (PMN), and to induce PMN degranulation of lysozyme but not of β-glucuronidase (Wolpe, et aL, 1989). IO addition, mMIP-2 has been shown to have myelopoteflc enhancing activities for CFO-GM (Broxmeyer, et al., 1989, J. Exp, Med,. 115:1583). Given the various biological activities of these factors, it b desirable to isolate their human homologs. Due to the necessity for quantities of purified factors to pursue definittoo of bioactivities and the expense of isolating these factors from ceil culture fluids it b desirable to produce these materials by employing recombinant DNA technology.

SOMMART OF THE INVENTION An attempt to Identify the human counterpart of mMIP-2 unexpectedly resulted in not one, but two candidate sequences. These two sequences were designated bu-MIP-2» and hu-MlP-21. The more abundant of the two homologs is hu-M!P-2e. Λβ,ΑΓΑΓΓ t A o no -sIt Is «η object (tf this invention to provide, a human MlP-2o (hu-MIP-2a) polypeptide substantially free from other human proteins and to provide a nucleic acid coding sequence for hu-MiP-2e. it is yet another object of this invention to provide antibodies reactive with hu-MlP-2a.

It la stiU Another object uf this Invention to provide diagnostic methods for detection of hu-MiP-2a or of nucleic acid coding sequences for hu-MlP-2o. to accordance with these and other objects, which will become apparent from the description herein, one aspect of this invention contemplates a protein composition comprising hu*MiP-2a polypeptides wherein the composition is substantially free of human tissue and preferably is substantially free of other human proteins.

In another aspect, the Invention contemplates a ONA molecule comprising a heterologous region that eocodes hu-MIP-2a polypeptide. In yet another aspect, the Invention contemplates a recombinant production system comprising ceils transformed with a DNA molecule comprising a heterologous region that encodes hu-M!P-2e polypeptide.

The invention also contemplates antibodies which are reactive with hu-MlP*2a polypeptides. Still further aspects of the Invention relate to immunoassay methods for determination of hu-MIP-2a in tissues or fluids and nucleic acid probe methods for detecting polynucleotide sequences coding for hu-MIP-2e in biological samples.

Another aspect of the invention contemplates polypeptides and small organic molecules that interact with the hu-MDP-2c receptoris) and possess agonistic or antagonistic activities.

Yet another aspect of the invention contemplates hu-MIP-2a polypeptides that exhibit agonist and antagonist activities to any of the members of the homologous multigene family.

Further, the invention contemplates a method of Inducing wound healing, a method of modulating myelopoelsis, and a method of inducing adjuvant activity, by administering an effective amount of hu-MIP-2a polypeptides.

The invention also contemplates a method of treating malignancy, autoimmune and Inflammatory diseases, such as, pathological n -β infiltration of neutrophils including psoriasis, rheumatoid arthritis, and diseases of the lungs, by administering an effective amount of hu-MIP-2« antagonist or hu-MlP-2o polypeptides with antagonistic activity to the members the homologous multigene family.

By using the DNA sequences and the recombinant system pro* Vided by this invention, it is possible to produce large quantities of one of the two human homologs of mMIP-2 substantially free of other human protein, because only one human protein is expressed in the recombinant system. Compositions such as this, containing only one MIP-2 homolog, would be difficult to obtain by purification from natural sources.

BRIEF DESCRIPTION QF THE DRAWINGS Figure 1. Nucleotide sequence of murine MIP-2. sequence and predicted protein Figure 2. Nucleotide sequence of hu-MiP-2o. sequence and predicted protein Figure). Nucleotide sequence of hu-MlP-28. sequence and predicted protein Figure 4. Nucleotide sequence homology of MIP-2 homologs. Percentages of nucleotide sequence identity between mMIP-2, hu-M!P-2a, hu-MIP-2s, hu-gro/MGSA and murine XC in the cDNA (A), coding region (B) and S' untranslated region (C).

Figures. Amino acid homology.and alignment of MlP-2 homologs and human tL-8. (A) Percentages of Identity between the predicted amino acid sequences ct MIP-2 homologs as well as human IL-8. (B) The aligned amino add sequences.

Figures. Southern analysis of genomic DNA with murine and human MIP-2 cDNA cflgested with BamHI, T‘; EcoRI, Έ* or EeoRV, Έ*. (A) Murine DNA hybridized with mMIP-2 cDNA. A blot or restricted human DNA was hybridized first with labelled hu-MIP-Ss cDNA (C) then the filter was Stripped and rehybricflzed with labelled hU-M2P-2a CDNA (B>. n e e -7* DETAILED DESCRIPTION The practice ot the present invention employs, unless otherwise indicated, conventional molecular biology, microbiology, and recombinant DNA techniques within the sWil ot the art. Such techniques are explained fully in the literature. See. e.g., Maniatis, Fritsch A Sambrook, Molecular Cloning: A Laboratory Manual <1982); DNA Cloning: A Practical Approach, Volumes I and Π (D.N. Glover, ed., 1983); Oligonucleotide Synthesis (M.J. Cait, ed., 1984k Nucleic Add Hybridization (B.D. Hames & &J. Higgins, eds., 1986); Transcription and Translation (B.D. Hames tc S4. Higgins, eds., 1984); Animal Cell Culture (RJ, Freshney, ed., 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. PerbaL A Practical Guide to Molecular Cloning (1980.

Definitions In describing the present Invention, the following terminology is used in accordance with the definitions set out below.

A repllcon la any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication jp vivo: Le., capable of replication under its own control.

A vector is a repllcon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A double-stranded DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its normal, double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes doublestranded DNA found, Inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. Ia discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 3* to 8* direction aloog the nontranscrlbed stand of DNA (Le., the strand having a sequence homologous to the mRNA). n a A DMA coding sequence-’ is a DMA sequence which is transcribed and translated Into a polypeptide In vivo when placed under the control ot appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5’ (amino) termin'is and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can Include, but is not limited to, procaryotic sequences, cDNA from eucaryotie mRNA, genomic DNA sequences from eucaryotie (e<„ mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located X to the coding sequence.

A promoter sequence Is a DNA regulatory region capable of binding RNA polymerase io a ceil and initiating transcription of a downstream (X ifirection) coding sequence. For purposes of defining the present Invention, the promoter sequence 1$ bounded at its X terminus by the translation start codon of a coding sequence and extends upstream (X direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease $1), as well as protein binding domains (consensus sequences) responsible for the bimfing of RNA polymerase. Eucaryotie promoters will often, but not always, contain TATA” boxes and CAT boxes. Procaryotic promoters contain Shine-Dtigarno sequences in* addition to the -10 and -35 consensus sequences.

A coding sequence is under the control of the promoter sequence In a cell when RNA polymerase which binds the promoter sequence transcribes the coding sequence into mRNA which is then in turn translated into the protein encoded by the coding sequence.

A cell has been transformed* by exogenous DNA when such exogenous DNA has been introduced inside the cell wall. Exogenous DNA may or may not be integrated (covalently linked) to chromosomal DNA making up the genome ot the cell, in procaryotes and yeast, for example, the exogenous DNA may be maintained on an episomai element such as a plasmid. With respect to eucaryotie cells, a stably transformed cell is one in which the exogenous DNA has become integrated Into a chromosome so that It is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population ot daughter cells containing the exogenous DNA. A clone” is a population of cells derived from a Single cell or common ancestor by mitosis. A cell line is a clone of a primary cell that Is capable ot stable growth in vitro for many generations.

Two DNA sequences are substantially homologous when at least about 8S% (preferably at least about 90%, and mo6t preferably at least about 95%) of the nucleotides match over the defined length of the Dna sequences. Sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See e.g., Maniatis et al., suora: DNA Cloning, vols. 1 and Π supra; Nucleic Add Hybridization, supra.

A heterologous region or domain of a DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found In association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA In tbe genome of the source organism. Another example of a heterologous region Is a construct where the coding sequence Itself Is not found In nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally occurring mutational events do not give rise to a heterologous region of dna as defined herein.

Tbe terms analyte polynucleotide and analyte strand refer to a single- or double-stranded nueleio acid molecule which is suspected ot containing a target sequence, and which may be present In a biological sample.

A« used herein, the term probe refers to a structure comprised of a polynucleotide which forms a hybrid structure with a -10target sequence, due to complementarity at at least one sequence in the probe with a sequence In the target region. The polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.

As used herein, the term target region refers to a region of file nucleic acid which is to be amplified and/or detected. The term target sequence” refers to a sequence with which a probe or primer will form a stable hybrid under desired conditions.

The term targeting polynucleotide sequence” as used herein, refers to a polynucleotide sequence which Is comprised of nucleotides which are complementary to a target nucleotide sequence; the sequence is of sufficient length and complementarity with tbe target sequence to form a duplex which has sufficient stability for the purpose intended.

The term binding partner as used herein refers to a molecule capable of binding a ligand molecule with high specificity, as for example an antigen and an antibody specific therefor. In general, the specific binding partners must bind with sufficient affinity to immobilize the analyte (or the copy/eomptementary strand duplex, in the case of capture probes) under the isolation conditions. Specific binding partners are known in the art, and include, for example, biotin and avidin or streptavidin. IgG and protein A, the numerous known receptor-ligand couples, and complementary polynucleotide strands. In the case of complementary polynucleotide bintfing partners, the partners are normally at least about 15 bases in length, and may be least 40 bases in length; in addition, they generally have a content of Ge and Cs of at least about 40% and as much as about 00%. The polynucleotides may he composed of DNA, RNA, or synthetic nucleotide analogs.

Tbe term coupled as used herein refers to attachment by covalent bonds or by strong non-covalent interactions (e.g.f hydrophobic interactions, hydrogen bonds, etc J. Covalent bonds may be, for example,, ester, ether, pbosphoester, amide, peptide, imide, carbon-sulfur bonds, carbon-pbosphorus bonds, and the like.

The term support refers to any solid or semisolid surface to which a desired binding partner may be anchored. Suitable supports include glass, plastic, metal, polymer gels, and the like, and may take the form of beads, wells, dipsticks, membranes, and the like.

The term label as used herein refers to any atom or moiety which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to polynucleotide or polypeptide.

As used herein, the term label probe* refers to a polynucleotide which is comprised of targeting polynucleotide sequence that is complementary to a target sequence to be detected in the analyte polynucleotide. This complementary region Is of sufficient length and complementarity to the target sequence to afford a duplex comprised of the label probe and the target sequence to be detected by the label. It is coupled to a label either directly, or indirectly via a set of ligand molecules with high specificity for each other. Sets of ligand molecules with high specificity are described supra (see binding partners).

As used herein, a biological sample? refers to a sample of tissue or fluid isolated from a individual, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of tbe skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vivo cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally Infected cells, recombinant cells, and cell components).

Human tissue* is an aggregate of human cells which may constitute a solid mass. This term also encompasses a suspension of human cells, such as blood cells, or a human cell line.

A composition comprising a selected compooent A is substantially free” of another component B when component A makes up at least about 75% by weight of the combined weight of components A and B. Preferably, selected component A comprises at least about 90% by weight of the combined weight, most preferably at least about 99% by weight of the combined weight. In ti» case of a composition Γ» 4 - 12comprising a selected biologically active protein, which is substantially free of contaminating proteins. It is sometimes preferred that the composition having the activity of the protein or interest contain species with only a single molecular weight (Le., a homogeneous” composition}.

Two amino acid sequences are ^substantially homologous** when at least about 90% of the amino ac’ds match over the defined length of the amino acid sequences, preferably a match of at least about 92%, more preferably a match of at least about 95%.

Human Homologs of Murine MIP-2 The cDNA for murine MIP-2 has been cloned using a degenerate oligonucleotide probe pool corresponding to a portion of the N-terminal amino acid sequence determined on the purified protein. When nucleotide probes representing the coding region of murine MIP-2 cDNA were used to probe a cDNA library from human macrophage ceil line stimulated with LPS (see Example 2} two different canddate cDNA sequences were identified. The proteins encoded by these two cDNA sequences have beea designated hu-MiP-2» and hu-sop-25, respectively. Examples of the nucleic acid and amino acid sequences of these three proteins are shown in the Figures: Figure 1 = murine MIP-2, Figure 2 = human MIP-2®, Figure S human MIP-2|.

Human macrophage inflammatory protein 2«. polypeptides (hu-MIP-2a polypeptides) encompass hu-MIP-2a and hu-MIP-2e analogs.

Hu-MIP-2tt is human macrophage inflammatory protein 2a, a naturally occurring, mature human protein secreted, Inter glia, by LPS-stimulated macrophages, and further encompasses all precursors and allelic variations of hu-MlP-2«, and as well as including forms of heterogeneous molecular weight that may result from inconsistent processing Jn vivo. An example of the hu-MTP-2a sequence 1$ shown In Figure 2.

Hu-MIP-2a analogs are a class tit peptides which includes: l) Hu-MlP-2c muteins, which are polypeptides substantially homolgous to hu-JUP-Jo. Preferably the amino add sequence of the mutelo differs from that of n 11 -13hu-MIP-2« by 8 or fewer amino acid residues, more preferably, 7 or fewer residues, even more preferably about 3 or fewer residues aad most preferably about 2 or fewer residues. It is sometimes preferred that any differences In the amino acid sequences of the two proteins involve only conservative amino acid substitutions. Conservative amino acid substitutions occur when an amino acid has substantially the same charge as the amino acid for which it is substituted and the substitution has so significant effect on the local conformation of the protein. Alternatively, changes such as the elimination of cysteine which alter the activity or stability of the protein may be preferred. 2) Truncated hu-MlP-2a peptides, which include fragments of either hu-MIP-2e or hu-MiP-2e mutelns that preferably retain either (0 an amino add sequence unique to bu-MIP-2», (ii) an epitope unique to hu-MIP-2a or (Iii) MIP-2 activity, 3) Hu-MlP-2a fusion proteins,· which include heterologous polypeptides, are made up of one of the above polypeptides (hu-MIP-2«, hu-MlP-2» mutelns or truncated bu-MIP-2» peptides) fused to any heterologous amino acid sequence. Preferably such heterologous sequences are fused to the N-terminal end of the hu-MIP sequence and comprise a leader sequence to direct secretion.

Unique hu-MIP-2e sequences, either amino acid sequences or nucleic acid sequences which encode them, are sequences which are identical to a sequence of a hu-MIP-2» polypeptide, but which differ in at least one amino add or nucleotide residue from the sequences of hMGSA, hu-MIP-2s, mMIP-2 and murine KC, and preferably, are not found elsewhere in the human genome. Similarly, an epitope is unique to hu-MIP-2» polypeptides if it Is found oo bu-MIP-2a polypeptides but not found on any members at the homologous gene family. n an -14Features of the Predicted Amino Acid Sequence The open reading frames of mMIP-2, hu-MIP-2a and hu-MIP-23 encode polypeptides of 100, 107 and 107 amino acids, respectively. The initial approximately 30 amino acids of each of the three polypeptides have characteristic features or a signal sequence (Perlman, et al., 1983, J. Mol. Biol.· 167:391: von Heljne, 1984, J, Mol, Blob. 173:243: von Heljne, 1988, Nucleic Acids Res.. ££:4883). The N-terminal amino acid sequence determined for secreted mMIP-2 purified from the conditioned medium ot LPS-sdmuIated RAW 284.7 cells (Woipe, et al., 1989) determined the start of tbe mature mMIP-2 protein in the predicted amino acid sequence. The mart of the predicted mature peptide sequence tor hu-M!P-2a and hu-MIP-2e aligns with that of mMIP-2 and has a consensus signal peptide cleavage site (Perlman, et al. 1983; von Heljne, 1984,1986). Tbe predicted length of the mature peptide sequence for all three proteins 1$ 73 amino acids. Murine MIP-2 Is a basic protein, and the human MJP-2 polypeptides are basic as well, based on predicted Isoelectric points of 9.9 and 9.7 for hu-MlP-2a and bu-MIP-2s, respectively. The native or recombinant proteins may be O-giycosylated. None of the three predicted polypeptides has a consensus signal for N-linked glycosylation. Features of Murine and Human MIP-2 oDN As The nucleotide sequences in cDNA*s for mMIP-2, hu-aup-2a and hu-MIP-2B each encode a single open reading frame. The nucleotide sequence environment of the initiating ATG codoo of mMIP-2 conforms to the consensus sequence Shared by many mRNAs of higher eucaryotes (Kozark, 1986, Cell. ££:283; Kozark, 1987, Nucleic Acid Res.. ££:8125); these of human Μ1Ρ-2α and MZP-2e lack the highly conserved purine at position -3 but posses many features of the consensus sequence including C residues at positions -l, -2, and -4 and a G residue at position *4.

The S untranslated region of mMIP-2 Includes the eucaryotic consensus polyadenylation signal AATAAA (Btrnstlel, et al., 1985, Cell. £1:349) at position *719-724 followed by a poly-A string beginning at nucleotide *735. No AATAAA polyadenylation signal was found in the 3* untranslated region of clones hu-MiP-2-4a or IS hvMlP-2-7d of bu-MlP-28 cDNA or in clones at hu-JCP-2» cDNA. This is most likely due to the fact that these clones have a truncated 3' untranslated region, since no poly-A string was present.

The consensus sequence TTATTTAT found In the f untranslated region of many cytokine genes (Caput, et at, 1986, Proc. Natl, Acad. Sci. PSA. 13:1670) and implicated in mRNA stability (Shaw, et al., 1986, Cell. 4§:659) and efficiency of translation (Kroys, et al., 1987, Proc. Natl. Acad. Set, USA. $4:6030·, Han. et aL, 1990, £ Exo. Med,. 111:465) is present io multiple oopies in all three cDNAs. This sequence is present at four positions, two overlapping, in the 3’ untranslated region of mMTP-2 (positions *122, *126, *142, *146k and is present two times (positions *158 and *471) and five times, two overlapping, (positions *148, *152, *156, *160 and *492} in the 9 untranslated regions of human MIP-2S and Μ1Ρ-2α, respectively. Homologs of Murine and Human ΜΙΡ-3 The percentage of nucleotide sequence identity among the three mip-2 cDNA's, one murine and two human, as well as that of the human gro/MGSA cDNA (Anisowlcz, et al., 1987; Richmond, et aL, . 1988) and the murine KC cDNA (Oquendo, et al., 1989; Cochran, et al., 1983, Cell. 22:939) is displayed in Figure 4A. Tbe human gro/MGSA cDNA encodes a protein with melanoma growth stimulating activity (Richmond, et ah, 1986, J. Cell· Physiol.. 129:375); murine KC is a platelet-derived growth factor (PDGFHnducible gene presumed to be the murine homolog of human MGSA. Noteworthy is the high degree of nucleotide homology among tbe three human cDNAs, particularly between hMGSA and hu-MiP-2a.. There is an even more striking degree ai nucleotide sequence Identity among the three human homologs in tbe coding region as shown in Figure 4B. The nucleotide sequence identity to the 3* untranslated regions or the human MIP-2 homologs Is considerably less than that observed in tbe coding regions, with the exception of the respective regions of hu-MIP-2a and hMGSA. These two cDNAs show a high percentage of * sequence homology throughout the 9 untranslated region as wen.

Homology comparisons and alignments of the predicted amino acid sequences of the precursor proteins of MIP-2 homologs including Λ ΛΑ 16hu-MIP-2a, hu-M!P-2e, human gro/MGSA, the hamster homolog of gro/MGSA (he-gro), mMIP-2 and mKC are presented in Figure 5(AB). The three human proteins are highly homologous (87-90%) but amino acid differences occur throughout the predicted sequences, particularly at the carboxy termini of the mature protein sequences. Based on predicted amino acid homologies alone, it Is not possible to assign hu-MIP-2a, hu-MIP-23 or human gro/MGSA as the human homolog of mMIP-2 or murine KC.

The percentage of nucleotide sequence Identity is greatest between hu-MIP-2a and hMGSA and it extends throughout the entire cDNA. The presence of both hu-M!P-2e and hu-MLP-28 in a U937 cDNA library, prepared using poly-A+ RNA from cells stimulated with both LPS and phorbol myristyi acetate (PMA), prompted us to screen for hMGSA as welL Screening of SxlO5 plaques from the library after amplification with oligonucleotides specific for hMGSA (and not hu-MiP-2a/a) gave no positive signals; in contrast 56 hu-M£P-2« positive signals were detected. This suggests that gro/MGSA transcription Is not induced In Π937 cells stimulated by PMA and LPS, in contrast to transcription of the hu-MIP-2« gene.

Activities of MIP-2 Murine MIP-2 purified from the conditioned medium of LPSstimulated RAW 264.7 cells has diverse activities including CSFdepenoent myeiopoletic enhancing activity for CFU-GM (Bruxmeyer, et al., 1989), elicitation of a localized inflammatory response following subcutaneous administration and a potent chemotactic activity (or human PMN (Wolpe, ee at, 1989). The latter activity Is characteristic of human IL-8 (hu-iL-8). Based on this functional equivalence alone it was suggested that mMIP-2 could be the murine homolog of hu-IL-8. However, given that the amino add homology of mMIP-2 to hu-iL-8 is low, relative to mMIP-2 homology to bu-MIP-2a, hu-MlP-20 or hu-MGSA (Figure 8), mMIP-2 and hu-IL-8 do not appear to be murine/ human homologs. Redundancy of function among cytokines is not uncommon; cachectin/TNF-a and Interleukin 1 have an overlapping activity profile (Manogue, et al., 1988, in Cellular and Molecular Aspects of inflammation, Poste, et <1., eds., Plenum, NY, p. 123).

O 01 17MIP-2 and IL-8 may be another example of this functional redundancy.

Based on its cDNA sequence, hu-MIP-2a can be classified as a member of tbe homologous multigene family which Includes murine Mip-2. The members of this gene family possess in vitro biological activities including neutrophil activation, neutrophil chemotaxis, MCSA-Ufce mitogenic activity, fibroblast mitogenic activity. CSF co-factor activity, monocyte chemotaxis, angiogenic activity, and inflammatory activity. Hu-MIP-3a polypeptides may be selected which possess one or more of the biological activities exhibited by any member of the multigene family. Hu-MIP-2a polypeptides may be utilized therapeutically for stimulation of myelopotesls, as an adjuvant in vaccine formulations, and for wound healing.

Hu-MIP-2a biological activity can be measured by assays described in Wolpe, et aL (1989) or Broxmeyer, et al., (1989) (each incorporated herein by reference). Other assays for hu-MIP-2a activities such as neutrophil activation or chemotaxis can be measured iD vitro as described In Waltz et al., (1989), 3, Exo. Med.. 173:1745, Clark-Lewis et al., (1991), Biochem.. ¢3:8128, and Dernyck et al., (1990), Biochem.. 2$-.10225 (alt incorporated herein by reference). Dernyck et al. also describes a MGSA bioassay. Assays for CSF co-factor activity are described in Broxmeyer et al., 1990, Blood, 73:1110 and Broxmeyer et al., 1989,3, Exp, Med.. 123:158 (all incorporated herein by reference).

Individual hu-10P-2a polypeptides serve as agonists or antagonists of one or more of the activities of any of the members of the above-mentioned multigene family. The biological activity assays described above, and receptor binding assays with receptors for MIP-2 tnultigene family members, are used to screen hu-MEP-2o polypeptides for antagonistic or agonistic activities. Hu-M3P-2a polypeptides exhibiting antagonistic activity will compete with the desired member of the multigene family for binding to a receptor but will also Inhibit a biological activity of the desired member. Hu-MIP-2c polypeptides with agonistic activity will possess enhanced biological activity or activities comparable to the desired multigene family member. Some D 90 -18hu-MIP-2e polypeptides with agonistic activity act as co-factors to increase the activity of another multigene family member. Other hu-MiP-2e polypeptides exhibit two or more non-overlapping activities from different members of the multigene family. Some hu-MiP-2o polypeptides act as agonists for one activity and antagonists for another activity, e<<, where one polypeptide Inhibits neutrophil activation and Increases monocyte activation activity.

As therapeutics, hu-MlP-2a polypeptides with agonistic activity are effective for the same therapeutic applications as hu-MIP-2o, as well as therapeutic applications of other members of the multigene family. Htt-MIP-2a polypeptide with antagonistic activity will suppress malignancy and will prevent Inflammatory conditions and autoimmune diseases, such as pathological Infiltration of neutrophils, including psoriasis, rheumatoid arthritis, aod lung diseases.

An example of a set of hu-MlP'2a polypeptides of Interest are the polypeptides which bind to the receptor binding site of IL-8. Hu-MlP-2o can compete effectively with Π.-8 for the DL-8 receptor. Hu-MiP-2c polypeptides that are IL-8 agonists will have a threedimensional structure similar the portion of IL-8 that hinds to the receptor. Recent studies of the NMR and x-ray structures for tt-8 are useful for determining the receptor binding site. (Clore et ah, 1989. J, Biol, Chem.. 28«;18907; Clore et aL, 1998, glochem.. 2S:1689; Clore et al., 1991, J. Mol. Biol.. 212:811, and Baldwin et al., 1991. Proc, Natl, Acad, Sci, PSA, 53^02).

An amount of bu-MIP-2o polypeptide which Is an effective amount for the stimulation of myelopoiesis In myelopoletlc cells is that amount which produces a detectable increase in myelopoiesis (see, e.g., assay in Broxmeyer, et aL, 1989). An effective amount of hu-MIP-2a polypeptide relative to the other activities of such peptides Is likewise the amount which produce a detectable response in the appropriate assay as described above.

Production of Compositions Containing hu-M!P-2a Compositions containing hu-MIP-2e polypeptides substantially free oi other human protein can be prepared by purification from natural sources, by chemical synthesis or by recombinant DNA -19methods. A crude extract of naturally-produced hu-MiP-2» can be prepared from any convenient source of the native protein. A preferred source is a human macrophage ceil line which has been stimulated with LPS. A cell-free extract containing hu-MlP-2«, as determined by, eg., immunoassay as described below, serves as the starting material for further purification. This purification can be accomplished according to techniques which are well-known in the protein purification art. For example, various types of chromatography may be used. Columns which may be used include a DEAE cellulose column, or an anion exchange column, as well as a gel permeation column. A preferred purification method follows that taught in wolpe, et ai. (1989). The purification process may be monitored by for example immunoassay as described below or by MIP-2 activity assays according to Wolpe, et ai. (1989), or Broxmeyer, et al. (1989). which are incorporated herein fay reference.

The hu-M!P-2a or peptide fragments thereof can also be purified using Immunoafflnity techniques. The use or the antibodies of the present invention to purify the proteins of the invention allows good separation from those proteins which are most similar to them. Of course, other techniques of purification are known in the art and can be used to purify the peptides of the inventloo.

Alternatively, the hu-MIP-2a polypeptides of this invention may he prepared by chemical synthesis. For example, the Merrifield technique (Journal of American Chemical Society, vol. 8S, pp. 2149— 2154,1988), can be used.

It is possible to purify hu-MIP-2e from an appropriate tissue/fluid source; however, it is preferred to produce it by recombinant methods from a DNA sequence encoding hu-MiP-2a, which can be synthesized chemically or isolated by one of several approaches. The DNA sequence to be synthesized can be designed with the appropriate codons for the hu-MIMa amino acid sequence. In general, one will select preferred codons for the Intended host, if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled Into a complete coding sequence. See. e^., Edge (1981) D ΟΛ -20Nature 222:756; Nambair, et al. (i984) science 223:1299; Jay, et al. (1984) J, Biol. Chem.. 253:6311. The isolation methods will rely in part on nucleic acid hybridization using appropriate oligonucleotide probes. Such probes can be constructed synthetically, based on the DNA or amino acid sequences disclosed herein, or isolated from genomic or cDNA clones also described herein.

Preparation of Nucleic Add Libraries The basic strategies for preparing oligonucleotide probes and DNA libraries, as well as their screening by nucleic add hybridization, are well known to those of ordinary skill in the art. See, e.g., DNA Cloning VoL I (D.P. Glover, ed., 1985); Nucleic Add Hybridization (B.D. Hames fc S.J. Higgins, eds., 1985b Oligonucleotide Synthesis” (M.J. Gate, ed., 1984b T. Maniatis, et at, Molecular Cloning: a Laboratory Manual (1982b B. Perbal, A Practical Guide To Molecular Cloning (1984). First, a DNA library is prepared. The library can consist of a genomic DNA library from a human source. Human genomic libraries are known in the art. See, «<., Madatis, et al. (1928) Cell. 15:687-701; Uwn, et al. (1978) Cell. 15:1157-1174. More preferred are DNA libraries constructed of cDNA, prepared from poly-A RNA (mRNA) by reverse transcription. See, e<·, C.S. Patent Nos. 4,446,325; 4,440,859; 4,443,140; 4,431,7400; 4.370,417; 4,383,877. The mRNA is isolated from a cell line or tissue believed to express hu-MJP-2o, such as a macrophage ceil line. A suitable source of mRNA for cDNA library constructions is the cell line U93?. The genomic DNA or cdna Is cloned into a vector suitable for construction of a library. A preferred vector is a bacteriophage vector, such as any of the phage lambda. The construction of an appropriate library is within the skill of the art. See. e^„ B. Perbal, supra. Preparation of Nucleic Add Probes Once the library is constructed, oligonucleotides are used to probe the library to identify the segment carrying a sequence encoding hu-MZP-2a polypeptide. In general, the probes are synthesized chemically, preferably based upon known nucleic add sequences, or alternatively, on predcted amino add sequences from cDNA clones. η nr '21Altemately, in tbe absence or a good tissue source for the mRNA, it may become necessary to obtain sequences from the protein. The N-terminal sequence can be obtained by N-terminal sequence analysis. Determination of Internal sequence can be done, for example, by Staph-V8 proteolysis of protein puriTied in the usual way, followed by reductive alkylation and separation by HPLC of the digestion products. Elution peaks corresponding to discrete enzyme fragments can then be sequenced by standard methods. From the amino acid sequence, oligonucleotides can be designed and produced for use as hybridization probes to locate either cdna sequences or the exons in genomic DNA. Ultimately, the isolated exons are ligated together in such a way that they correspond to the nucleic acid sequence which encodes the mature protein.

Nucleotide sequences are selected so as to correspond to the codons encoding tbe amino add sequence. Since the genetic code is redundant, it will usually be necessary to synthesize several oligonucleotides to cover all, or a reasonable number, of the possible nucleotide sequences which encode a particular region of the protein. Thus, it is generally preferred, in selecting a region of the sequence upon which to base the probes, that the region not contain amino adds whose codons are highly degenerate. It may not be necessary, however, to prepare probes containing codons whose usage Is rare in humans (rrom which the library was prepared).

Antibodies thereto can be used to lmmunopredpltate any of the desired protein present in a selected tissue, ceil extract, or body fluid. Purified VHP-2 from this source can then be sequenced and used as a basis for designing specific probes as described above.

One of skill in the art may find it desirable to prepare probes that are fairly long and/or encompass regions of the amino acid sequence which would have a high degree of redundancy in the corresponding nucleic acid sequences. Probes covering tbe complete gene, or a substantial part of the gene, may also be appropriate, because of the expected degree of homology. The sequence is highly conserved across species lines, and so probes containing the coding sequence from another species, such as the mouse, can be reacflly used to screen P 9R -22libraries prepared from human DNA. Ed other cases, it may be desirable to use two sets of probe simultaneously, each to a different region of the gene. While the exact length of any probe employed 1$ not critical, typical probe sequences are no greater than 1000 nucleotides in length, more typically they are not greater than 300 nucleotides, even more typically they are no greater than 250 nucleotides; they may be no greater than 100 nucleotides, and also may be no greater than 75 nucleotides In length. Generally It Is recognized in the art that probes from about 14 to about 20 base pairs are usually effective. Because hu-MIP-2a belongs to a group of highly homologous proteins, probes containing sequences unique to hu-MIP-ia are preferred in order to discriminate between the related sequences· Longer probe sequences may be necessary to encompass unique polynucleotide regions with differences sufficient to allow related target sequences to be distinguished. For this reason, probes are preferably from about 10 to about 100 nucleotides in length and more preferably from about 20 to about 50 nucleotides. using Probes to Select Clones As is known In the art, oligonucleotide probes are labeled with a marker, such as a radionucleotide or biotin, using standard procedures. The labeled set of probes Is then used in the screening step, which consists of allowing the single-stranded probe to hybridize to isolated ssDNA from the library, according to standard techniques. Either stringent or permissive hybridization conditions could be appropriate, depeocfing upon several factors including, but not limited to, tha length of the probe, whether the probe and library are from tbe same species, and whether.tbe species are evolutionarily close or distant. It is within the skill of the art to optimize hybridization conditions so that homologous sequences* are isolated and detectable above background hybridizations. The basic requirement is that hybridization conditions be of sufficient stringency so that selective hybridization occurs; Le., hybridization is due to a minimum degree of nucleic acid homology (e.g., at least about 75%), as opposed to nonspecific binding or hybridization due to a lower degree of homology. See generally. Nucleic Acid Hybridization,” supra. Because of the ο ητ -23number of sequences closely related to mMlP-2, both a unique probe sequence and stringent hybridization conditions are preferred. Once a clone from the screened library has been identified by positive hybridization, it can be further characterized by restriction enzyme analysis and DNA sequencing to confirm that the particular clone contains a coding sequence for the desired protein.

Partial genomic clones can be extended into complete clones by one of several techniques. A done can be extended in either the S’ or S' direction using chromosome walking techniques to ensure Inclusion of the entire gene coding region. Restriction fragments of the» clones can then be probed with, for example, cDNA encoding the desired protein. If sufficient homology exists within these exons, other exons could be identified with the same cDNA done.

Other coding regions in genomic clones may be rapidly identified by direct sequencing of the DNA downstream of a cloned exon using modern Mi3-tfideoxy sequencing techniques. The sequence Is then inspected in all three reading frames to reveal an open reading frame. Other exons will also be apparent, since they will be bounded on both sides by iniron-spticing signals and should encode amino acids that aro common to MlP-2*s from different species.

Moro specifically, once the correct gene coding sequence for at least one exon of hu-MIP-2a is known, it can be used to obtain the entire protein coding region of the enzyme by one or more of the following means. First, the desired sequence fragment can be trimmed from the clone and placed in a more convenient vector, such as pBR322, so that large quantities of DNA containing only the fragment itself can be obtained and used as a hybridization probe. Alternately, a oligonucleotide corresponding to unique regions of the coding region can be synthesized. Either can be used as a hybridization probe for a geoomic DNA library. cpna Clones Mammalian genomic clones (partial or full-length) containing the longest Inserts of the gene can be co-transfected into Chinese hamster ovary (CHO) cells with plasmid DNA containing a marker, n oo 24such as neomycin and metallothtonine resistance genes, surviving cells, selected in the presence of antibiotic G418 and Cd**, can be «radyzed for (he presence of RNA transcripts which hybridize to the sequence encoding the protein by Northern blot of extracted RXA. Clones containing the desired transcripts can then be used as an mRNA source for a cDNA library construction. Alternatively, northern blots of mRNA obtained from various sources, such as peritoneal cells and pus, eodotheiial tissue, and peripheral blood leukocytes, lymphocytes, and macrophages can be tested for hybrkfization to the probe. In addition, mRNA from various cell lines such as LPS-stimulated 0937 and KL60 can also be tested. Any tissue or cell source' containing detectable levels of hybridizing mRNA is then used to produce a cDNA library which will be screened with the same probes in order to detect a full-length cDNA encoding the desired protein. Site-directed Mutagenesis As mentioned above, a DNA sequence encoding ftGP-2 can be prepared synthetically rather than cloned, synthetic dna sequences allow convenient construction of genes which will express hu-MIP-2a analogs, particularly muteins. Alternatively, DNA encoding muteins can be made by site-directed mutagenesis Ot native MIP-2 genes or cDNAs, an) muteins can be made directly using conventional polypeptide synthesis.

Site-directed mutagenesis is preferably conducted by polymerase chain reaction methodology using a primer synthetic oligonucleotide complementary to a single stranded phage DNA comprising the sequence to be mutated, except for limited mismatching representing tbe desired mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage. Theoretically, 50% of tbe new plaques will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence. The resulting plaques are hybridized with kinased synthetic primer at a temperature -25' which permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybrid zation. Plaques which hybridize with the probe are then picked, cultured, and the DNA recovered.

Cloning for Expression Once a coding sequence for hu-MIP-2o has been prepared or Isolated, it can be cloned into any suitable vector or repllcon and thereby maintained io a composition which Is substantially free of vectors that do not contain a MIP-2 coding sequence (e.g., free of other clones from the library). Numerous cloning vectors are known tp those of skin in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning, and host cells which they can transform, include the various bacteriophage lambda vectors CE. «aD. pBR322 (E. coil). pACYC 177 (E. coll). pkT230 (gram-negative bacteria), pGVUOT (gram-negative bacteria), pLAFRl (gram-negative bacteria), pHVi4 (E, coll and Bacillus subtUis). pBD9 (Bacillus). pIJ6l (Streptomyces). pDC6 (Streptomyces). actlnophage, dC3l (Streptomyces). Yip5 (Saccharomyces). YCpi9 (Saccharomyces). and bovine papilloma virus (mammalian cells). See generally. DNA Cloning, vols. I & a, supra: T. Maniatis, et al., supra: B. Perbal, suora.

The DNA sequences and DNA molecules of tbe present Invention may be expressed using a wide variety of bost/vector combinations. For example, useful vectors may comprise segments of chromosomal, don-chromosomal (such as various known derivatives of SV40 and known bacterial plasmids, e<., plasmids from E.colt Including COlEl, peRl pBR322, pMB9 and RP4), or synthetic DNA sequences, phage DNAs (M13) including derivatives of phage (e.g., NM 989) and filamentous single-stranded DNA phages, vectors useful in yeasts (such as the 2 micron plasmid), vectors useful In eukaryotic cells (such as vectors useful in animal cells, ag. those containing SV-40 adenovirus and retrovirus derived DNA sequences) and vectors derived from combinations of plasmids and phage DNAs (such as plasmids which have been modified to employ phage DNA), or other derivatives thereof. -2ίAccording to the present Invention, the coding sequence for hu-MIP-2» polypeptide is placed under the control of a promoter, ribosome binding site (for bacteria} expression) and, optionally, an operator (collectively referred to herein as control elements), so that the DNA sequence encoding hu-MlP-2e polypeptide is transcribed Into RNA in the host cell transformed by a vector containing this expression construct. The cotDng sequence may or may not contain a signal peptide or leader sequence. If the coding sequence contains a signal peptide, it may or may not be the signal sequence naturally associated with hu-MIP-2a. In bacteria for example, mature hu-MIP-2a is preferably made by the expression of a coding sequence which does not contain the mammalian signal peptide, but rather by expression of a coding sequence containing a leader sequence which is removed by the bacterial host In post-translational processing. See e.g.. U J. Patent Nos. 4,431,739; 4,425,437; 4,338,397.

Preferably the expression vector already contains at least one expression control sequence that may be operatively linked to the DNA coding sequence when it Is Inserted in the vector In order to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the Jag system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the glycolytic promoters of yeast (·<.. the promoter for 3phosphoglycerate kinase), the promoters of yeast acid phosphatase (e<., PhoS), the promoters at the yeast alpha mating factors, and promoters derived from polyoma, adenovirus, retrovirus, or simian virus (e<„ the early and late promoters of SV40), and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.

Ao expression vector Is constructed according to the present invention so that the hu-MIP-2e coding sequence is located in the vector with the appropriate regulatory sequences such that the coding sequence is transcribed under the ’’control’’ of the control sequences (Le., RNA polymerase which binds to the DNA molecules at the control sequences transcribes the coding, sequence). The control P -27sequeoces may be ligated to tbe coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site. For expression of the desired protein In procaryotes and yeast, tbe control sequences win necessarily be heterologous to the coding sequence, ff the host cell is a procaryote, it is also necessary that tbe coding sequence be free of introns (e<.. CDNA). If the selected host cell is a mammalian cell, the control sequences can be heterologous or homologous to the hu-MIP-2a coding sequence, and the coding sequence can either be genomic DNA (containing introns) or cDNA. Either genomic or cDNA coding sequences can be expressed in yeast Furthermore, within each specific expression vector, various sites may be selected for insertion of the DNA sequences of this invention. These sites are usually designated by tbe restriction endonuclease which cuts them. They are well recognized by those at skill in the art. tt is, of course, to be understood that an expression vector useful in this invention need not have a restriction endonuclease site for insertion of the chosen dna fragment. Instead, the vector can be joined to the fragment by alternative means. The expression vector, and in particular tbe site chosen therein for insertion of a selected DNA fragment and its operative linking therein to an expression control sequence, Is determined by a variety of factors, eg., number of sites susceptible to a particular restriction enzyme, size at tbe protein and expression characteristics, such as the location of start and stop codons relative to the vector. An insertion site for a DNA sequence is determined by a balance of these factors, not all selections being equally effective for a given case.

A number of procaryotic expression vectors are known in tbe art. See, eg., U.S. Patent Νοβ. 4,440,359; 4,436,815; 4,431,749; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; see also U.K. pub. NOS. GB 2,121,054; GB 2.093,123; GB 2,007,675; and European Pub. No. 103,395. Preferred procaryotic expression systems are In E, coll. Other preferred expression vectors -2« are those for use in eucaryotic systems. A preferred eucaryotic expression system is that employing vaccinia virus, which is wellknown in the art. See, e.g., Mackett, et al. (1984) J, Virol. 49:857: DNA Cloning, vol. Π, pp. 191-211, supra: PCT Pub. No. WO 88/07593. Yeast expression vectors are known in the art. See, e.g.. U J. Patent Kos. 4,446,235; 4,443,539; 4,430,428; see also European PUO. Nos. 103,409; 100,561; 96,491. Another expression system is vector pHSi, which transforms Chinese hamster ovary cells. The use of the vector is described in PCT Pub. Ko. wo 87/02062.

Useful expression hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli. such as E.coU SG-938, E.coli HB 101, E.coa w3110, E.COtl X1776, B.coli X2282, E.coli DHI, and E.coli MRCl, Pseudomonas. Bacillus, such as Bacillus suhttfe. Streptomyces. yeasts and other fungi, animal cells, such as COS cells and CHO cells, and human ceils and plant cells in tissue culture.

Of course, not all host/expression vector combinations function with equal efficiency in expressing the DNA sequences of this invention or in producing the polypeptides of this Invention. However, a particular selection of a host/expression* vector combination may be made by those skilled in the art For example, the selection should be based oo a balancing of a number of factors. These Include compatibility of the host and vector, toxicity of the proteins encoded by the DNA sequence to the hoist, ease of recovery of the desired protein, expression characteristics of the DNA sequences and the expression control sequences operatively linked to them, biosafety, costs and the folding, form or any other necessary post-expression modifications of the desired protein. Preferably, the host ceQ will not express proteases which degrade hu-MIP-2*.

Cloning in a Yeast Expression System A preferred expression system is yeast. hu-MIP-2e can be expressed by a yeast cell transformed with an expression vector containing DNA coding sequence for hu-MIP-2a under the control of a yeast promoter. Such expression vectors may be constructed as follows.

A. A, -j»A yeast promoter b any DNA sequence capable cf binding yeast RNA polymerase and Initiating the downstream (X) transcription of a coding sequence (e.g„ structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5’ end of the. coding sequence. This transcription initiation region typically includes an RNA polymerase binding site (the TATA Boar) and a transcription Initiation site. A yeast promoter may also hare a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.

Yeast b a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (E.P.O Pub. No. 284044), enolase, glucoidnase, glueose-6-phosphate Isomerase, glyceraldehyde3-phosphate-dehydrogenase (Gap or GAPDH), hexokinase, phosphorruetokinase, 3-phospbogtyeerate mutase, and pyruvate kinase (PyKXE.P.O. Pub. No. 329203). The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences (Miyanohara, et at, (1983) Proc. Natl, Acad. Sci, USA. ¢¢:13.

In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (u.S. Patent Nos. 4,876,197; 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2. GAL4. GAL10, or PHOS genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (E-P.O- Pub. No. 164886). Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that Ο 04 -Μhave the ability to bind yeast RNA polymerase and initiate transcription. See, e<„ Cohen, et al. (1980) Proc. Natl. Acad, Sci, PSA. 77:1078; HenBcoff, et bL, (1981) Nature. 288:835; Hollenberg, et al., (1981) Curr, Topics Microbiol. Immunol.. S&H9; Hollenberg, et al., The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids of Medical Environmental and Commercial Importance (eds., K.N. Timmls and A. Puhler); Mercereau-Puigalon, et al. (1980) Gene U:183; Panthier, et al. (1980), Cwr. Genet.. £:109.

A DNA molecule may be expressed tntracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein win always be a methionine, which is encoded by the atg start codon. If desired, methionine at the N-terminus may be cleaved from the protein by 1q vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative to direct expression. Typically, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 8* end ot heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dlsmutase (SOD) gene, can be linked at the 5* terminus of a foreign gene and expressed in yeast. The DNA sequence at the junctloo of the two amino acid sequences may or may not encode a cleavable site. See, e.g., S.P.O. Pub. No. 198088. Another example Is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin leader or pro-* region that preferably retains a site for a processing enzyme (e.g. ubiqultln-speclflc processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated (P.C.T. WO 83/024088; commonly owned U.S. Patent Application Serial No. 390,599, filed 7 August 1989, or foreign pateots or applications claiming priority therefrom (as listed in World Patents Index produced by Derwent Publications, Ltd.), the disclosure of which Is incorporated herein by reference). -31Alternatively foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast and the foreign gene. Preferably, there are processing sites Qn vivo or in vitro) encoded between the leader fragment and tbe foreign gene. The leader sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (E.P.O. Pub. No. 12,873; J.P.O. Pub. No. 82,096,086) and the A-factor gene (U.S. Patent No. 4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (E.P.O. Pub. No. $0087).

A preferred class of secretion leaders are those that employ a fragment of the yeast «-factor gene, which contains both a pre” signal sequence, and a pro region. The types of α-factor fragments that can be employed Include the full-length pre-pro α-factor leader (about 83 amino acid residues) as well as truncated α-factor leaders (typically about 25 to about 80 amino acid residues) (U.S. Patent Nos. 4,846,083 and 4,870,008; E.P.O. Pub. No. 324274). Additional leaders employing an α-factor leader fragment that provides for secretion include hybrid α-factor leaders made with a pre-sequence of a first yeast, but a pro-region from a second yeast α-factor. (See, e.g., P.C.T. KO 89/02463.) Typically, transcription termination sequences recognized by yeast are regulatory regions located S' to the translation stop codon, and thus, together with the promoter, flank the coding sequence. These sequences (flrect the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples ot transcription terminator sequence include the wild-type a-factor transcription termination sequence and other yeast-recognized termination sequences, such as those for glycolytic enzymes.

Typically the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and a. ,·«, a, mt , a . onnerncr-io D CC -32transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg., plasmids) capable of stable maintenance in a host, such as-yeast or bacteria. The replicon may have two replicatioo systems, thus allowing it to be maintained, for example, in yeast for expression and in a procaryotic host for ekxiing and amplification. Examples at such yeast-bacteria shuttle vectors include YEp24 (Botsteln, et al., (1979) Gene £:17-24), pCl/1 (Brake, et al., (1984), Proc, Natl, Acad, Sd, USA. £1:4642-4648), and TRpl7 (Stinchcomb, et aL, (1982), J. MoL Biol.. 155:157). to addition. a replicon may be either a high or low- copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to About 200, and typically about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See, e.g., Brake, et aL. supra.

Alternatively, the expression constructs can he integrated into the yeast genome with an integrating vector, integrating vectors typically contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome (Orr-Weaver, et aL 1983), Methods to EnxvmoL £21:228-243). An integrating vector may be directed to a Specific locus to yeast by selecting the appropriate homologous sequence for.inclusion to the vector. See Orr-weaver, et al., supra. One or more expression constructs may integrate, possibly affecting levels of recombinant protein produced [Rim, et aL, (1983), Proc, Natl. Acad. Sci, USA. £$6750). The chromosomal sequences Included In the vector can occur either as a single segment to the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments to the chromosome and flanking the ΛΑΜΓΑΓΡ 4Λ η λτ -33expreesion construct In the vector, which can result In the stable Integration of only the expression construct.

Typically, extrachromosomal and Integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed. Selectable markers may include biosynthetic genes such as ADE2. Hist. LEU2. TRPl. and UR A3. Selectable markers may a’so Include drug resistance genes such as ALG7 and the G418 resistance gene, which confer resistance in yeast cells to tunicamyein and G418, respectively. in addition, a suitable selectable marker may also provide yeast with the ability to grow In the presence of toxic compounds, such as metaL For example, the presence of COPi allows yeast ω grow In the presence of copper ions (Butt, et al. (1987), Microbiol, Rev.. £1:381].

Expression vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for. Inter aUa, the following yeasts: Candida albicans [Kurtz, et al., {1986), Mol, Celt Biol·. £: 142 ], Candida maltosa (Xunze, et al., (1985). J. Basic Mlcrbbtol., 2£:141], Hansenula polymorpha [Gleeson, et al., (1986), J, Gen, Microbiol.. 182:3459: Roggenkamp, et al. (1986), Mol, Gen. Genet.. 202:3021. Kluyveromyces fragills [Das, et al., (1984), j. Bacteriol.. 158:11651. Kluyveromycoes lactis [De Louvencourt et al., (1983), J. BacterioL. 154:737; Van den Berg, et aL, (1990) Bio/Technologv. £:135), fisJaia gvHlerimondtl [Xunze et al.. (1985), £ Basic Microbiol.. 2£:141), Plchla pastoris [Cregg, et al., (1985), Mol, Cell Biol.. £:3876: U.S. Patent Nos. 4,887,148 and 4,929,555), Saccharomyces cerevisiae [Hlnnen et aL, (1978), Proc, Natl, Acad. Sd, USA, 25:1929; Ito, et at, (1988) 3, Bacteriol.. l££:163], Schlxosaccharomyces pombe [Beach and Nurse (1981), Nature. 300:706). and Yarrowia lipolytica [Davidow, et aL, (1985), Cjjjx. Genet.. l£:39-48; CalUardin, et at (1985), Curr. Genet., 1£:49]. · Methods of Introducing exogenous DNA into yeast hosts are well-known In the art, and typically Include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast spedee to be ΛΑΑΛΓΑΓΓtn n on -34transformed. See, eg., Kurtz, et at (1986), MoL Cell, Biol.. £:142. Kunze, et al. (1985), J. Basic Microbiol.. ££:141, for Candida: Gleeson, et al., (1986), J. Gen. Microbiol.. 182:8499. Roggenkamp, et al. (1986), Mol, Gen. Genet.. £02:302, for Hansenula; Das, et al., (1984), £ Bacteriol.. 158:1165. De Louvencourt et aL, (1993), j. Bacteriol.. £££:1169, Van den Berg, et al., (1990), gto/Technoioyv. £:133, for Kluweromvces: Cregg, et al„ (1985), Moi, Cell Biol. £:3376, Kuoze, et al. (1985), J, Basic Microbiol., ££:141, U4. Patent Nos. 4,837,148 and 4,929,555, for Pichia: Klnoen, et at (1978), Proc, NatL Acad, Sci, BSA. ££:1929, Ito, et al. (1983), J Bacteriol.. 1££:163, for Saccharomyces: Davldow, et al., (1988) Curr, Genet,. £fi:39, Gsdllardin, et al. (1985), Curr, Genet.. ££:49, for Yatrowia. Expresslooct the Protein Depending on the expression system and host selected, the protein is produced by growing host cells transformed by an expression vector containing the coding sequence for hu-MIP-2o under conditions whereby the protein is expressed. The protein is then isolated from tbe host cells and purified. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.

One preferred means of obtaining the preparations of this invention is to culture cells transfected with ah expression vector comprising the intron-free DNA corresponding to hu-MfP-2«, using appropriate culture conditions. The cells are then harvested and the cell fraction may be separated by standard separation procedures, such as centrifugation. If the expression system secretes the polypeptide Into growth media, the polypeptide can be purified directly from cell-free media, if the protein is not secreted, it is isolated from cell lysates. Once a crude extract containing hu-MIP-2a is obtained, purification can be accomplished as described above, using standard techniques. in general, recombinant production of hu-MIP-2a polypeptide can provide compositions of this cytokine substantially tree of other human proteins. The ability to obtain high levels of purity 1$ one result of using recombinant expression systems which also can produce hu-MIP-2e polypeptides in substantial quantities vis-a-vis In vivo Λ · -35* sources. Thus, by applying conventional techniques to recombinant cultures, hu-MIP-2» polypeptide compositions can be produced more readily. Those ot ordinary skill in the art can select among the above techniques to prepare the substantially pure peptides of this invention.

Pools of hu-HrP-2» Analogs Compositions containing pools of hv-MIP-2a analogs substantially free of other human proteins can also be prepared, preferably by recombinant DNA methods or chemical synthesis. These pools can be screened using a receptor binding assay or any of the biological activity assays described above for the hit-Mip-2a analogs with the desired activities.

Por example, Parmley and Smith, 1988, Gene. 22:305 {incorporated herein by reference), describe a protocol for producing and screening pools of proteins using recombinant DNA methods. This protocol, with a receptor to a MIP-2 multigene family member, can be used to screen pools of hu-MIP-2a mutelns, truncated hu-MIP-2a peptides, or hu-MiP-2o fusion proteins. First, pools of DNA fragments encoding different hu-MIP-2a analogs can be made. Next, the pool of DNA fragments can be inserted into the geoome of the fiiametnous phage described in Parmley and Smith. This library of phages will produce colonies, whose expression products can be sviwtiwl. The DNA frutn the desired euluiil» cau lie bulaled and used to identify the desired hu-MIP-2a analogfe).

Pools of hu-MIP-2e analogs can also be synthesized. For convenience in a receptor binding assay, hu-MiP-2a analogs can be synthesized on polyethylene pins arranged in a 96-pio array on a block (matching the spacing of a standard 96-weil microtiter plate). See for example Geyesen, U.S. Patent No. 4,708,871 (incorporated herein by reference in full), which describes such a process of synthesizing polypeptides applied to the vpi protein of foot and mount disease virus. Other methods for producing libraries of polypeptides and selecting tbe polypeptides with specific properties are described in U.S. Patent No. 8,010,175 (Incorporated herein by reference in full), and U.S. Patent Application Ko. 07/652,194 filed 6 Pebruary 1991 -3β (Incorporated herein by reference), or foreign patents or applications claiming priority therefrom (as listed in World Patents Index produced by Derwent Publishing Ltd.).

Pools of polypeptides unrelated to the hu*MIP-2o can be produced, using the recombinant DNA and chemical synthesis methods above, and screened for their ability to bind receptors for members of the MIP-2 gene family. Next, the polypeptides that do bind the receptorCs) can then be assayed in the biological activity assays described above to determine their agonistic or antagonistic activities. Such polypeptides are effective for the same therapeutic applications as hu-MlP-2a polypeptides. Another method of screening pools of peptides Is described in Cwirla et aL, 1990, Proc. Natl. Acad. Sci. OSA, £7:6378 (Incorporated herein by reference).

For ease of pharmaceutical administration, small organic molecules may be preferred over these polypeptide agonists and antagonists. From the structures of the polypeptides, small organic molecules that mimic .the shape and activities of these polypeptides may be found. Methods of determining the structures or these hu-MIP-2e agonists and antagonists are known in the art and Include x-ray crystallography and 2-dlmenslonal nuclear magnetic resonance.

Production of Antibodies Native, recombinant or synthetic hu-MIP-2« polypeptide (full length oc fragments containing one or more epitopes) can be used to produce both polyclonal and monoclonal antibodies, a polyclonal antibodies are desired, bu-MIP-2· polypeptide is used to immunize a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) and serum from the immunized animal later collected and treated according to known procedures. The hu-MIP-2· polypeptide of the present Invention can be used to stimulate production of antibodies In a mammal by immunizing the mammal with the preparation. Such immunization may optionally employ coupling or the polypeptide to a larger Immunogenic substance such as keyhole limpet hemocyanin. immunization of mammals, such as rabbits, mice, goats, etc., to produce antibodies is well known in the art. -37Polydonai antibody preparations can be partially, purified using immucoaffinity techniques employing a synthetic or recombinant polypeptide or the present invention. Such purification methods are well-known in the art. in particular, compositions containing polyclonal antibodies to a variety of antigens in addition to the desired protein can be made substantially free of antibodies which are directed to other antigens by immunoaffinlty chromatography. The substantially pure preparation of polypeptide of the present invention can be used to affinity purify antibodies reactive with hu-MIP-2» polypeptide. For affinity purification of antibodies, the polypeptide can be coupled to an inert matrix, such ac agaroco beads. Techniques for such coupling are well known in the art.

A preparation of the polypeptide can also be used to detect or quantitate antibodies specific for hu-Ml?-2a in an antibody preparation or other biological sample. In such a case, the synthetic peptide will usually be coupled to a larger substance, such as bovine serum albumin or a solid support. Ocoe again, tbe techniques for coupUcg polypeptides to such matrices are well known In the art.

Monoclonal Antibodies Monoclonal antibodies reactive with hu-MiP-2e are also readily produced byone skilled in the art following the disclosure herein. The general methodology for making monoclonal antibodies by hybridomas is well known. Monoclonal antibodies can be raised which are reactive with unique hu-MIP-2e epitopes. Generally, a rat or mouse Is immunized with the polypeptide of the present invention, and the rodent will later be sacrificed and spleen cells recovered for fusion with myeloma cells. Hybrid cells can be selected according to techniques known in the art. Antibody production or each hybrid cell can be screened individually to select antibodies which bind to epitopes on hu-MIP-2a.

In order to screen for antibodies which are immtraoreactive with epitopes unique to hu-MIP-2a polypeptides, a simple battery of tests can be performed. Antibodies can be tested for immunoreactivity with hu-MZP-2a polypeptide using a substantially pure preparation or the hu-MiP-2e polypeptide of the present Invention. The desired specific antibodies should be positive in this test. The antibody can then be tested for immuno reactivity with other members of the MIP-2 multigene family. Generally, antibodies which are negative in this test react with unique hu-MIP-2« epitopes.

Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA. or transfection with Epstein-Barr virus. See, e.g., M. Schreter, et aL, Hybridoma Techniques (1989); Hammerllng, et aL. Monoclonal AntSxxfies and T-Ceil Hybridomas” (1981); Kennett, et al., Monoclonal Antibodies (1980); see also U.S. Patent Nos. 4,841,781; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,488,917; 4,472,500; 4,491,832; 4,493,890.

Panels of monoclonal antibodies produced against hu-M!P-2a can be screened for various properties; LeM isotype, epitope, affinity, etc. Of particular Interest are monoclonal antibodies that neutralize the activity of MIP-2 or hu-MIP-2a peptides. Such monoclonals can be readily identified using MIP-2 activity assays such as those taught in Wolpe, et al. (1989) and Broxmeyer, et aL (1989) which are incorporated herein by reference. High affinity antibodies are also useful in Immunoaffinity purification of oative or recombinant hu-MIP-2a.

The types of antibodies contemplated by the present invention include, vertebrate antibodies, particularly mammalian (polyclonal and monoclonal), hybrid antibodes, chimeric antibodies, humanized antibocfies, altered antibodies, univalent antibodies, single domain antibodies, as weal as functional antibody fragments, such as Fab proteins, so long as they bind selectively to an epitope. For a general review see Winter A MUsteln, 1991, Nature. 349:293: Rlechmann et al. 1988, Nature, 332:323: Ward et al., 1989, Nature 341:544: U.S. Patent No. 4,816,457; and Glennie et aL 1982, Nature 295:712.

Epitopes are antigenic determinants of a polypeptide. An epitope could comprise 3 or more amino adds in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 5 such amino acids and, more isually, consists of at least 8-10 such amino adds. Methods of determining spatial conformation of amino -39acids are knows In the art and Include, for example, x-ray crystallography and 2-dlm«nsiooal nuclear magnetic resonance.

Diagnostic Assays for hu-K CP-2» Detection of hu-MIP-2# may be on the nucleotide or peptide level. Antibodies can be prepared by immunizing mammals with peptides expressed from the sequences corresponding to hu-MiP-2a polypeptides, as Indicated above, and selecting those antibodies specific to hu-MIP-2» using techniques that are well known to those skilled in the art. The particular procedures for gene probe assays and immunoassays will be well-known to those skilled in the art. immunoassay Methods Antibodies are useful In «Bagnostic'appllcatlons. Antibody specific for hu-MIP-2e can he formulated into any conventional immunoassay format; e<^ homogeneous or heterogeneous, radioimmunoassay or ELBA. The various formats are well known in those skilled in the art. See, e<., Immunoassay: A Practical Guide” (D.w. Chan and M.T. Perlsteln eds. 1987) the disclosure of which la incorporated herein by reference..

Antibodies of the invention are capable of binding to hu-MIP-Sa, and preferably they will not bind to hu-MIP-2e. These antibodies also permit the use of imaging analysis with isotopes, conjugated aotltxxfies, or other ligands. One example of a suitable imaging material is l25l conjugated to the antibodies specific for hu-MIP-2a.

The antibodies of the present invention can be used to detect hu-MiP-2» epitopes in histological sections of tissue as well as in serum. One can detect antibody binding to tissue sections by any detection means known in the art, for example, radioimmunoassay, enzyme linked immunoadsorbent assay, complement fixation, nephelometric assay, imaiunodSffuslon, lmmunoelectropboretic assay and the like.

A particularly useful stain employs peroxidase, hydrogen peroxide and a chromogenlc substance such as aminoethyl carbazole. The peroxidase (a well known enzyme, available from many sources) can be coupled to the antibody specific for hu-M!P-2o or merely a a -«j. complexed to It via one or more antibodies. Other chromogenic substances and enzymes may also be used. Such techniques are well known in the art. Radio-labeled antibodies may be specific for hu-MIP-2e or may be second antibodies immunoreactive with antibodies specific for hu-MIP-2e. Again, such techniques are well known. The precise technique hy which hu-MlP-2e is detected is not critical co the invention.

One particularly preferred method of detecting and/or quantitating hu-MIP-2a in biological samples employs a competitive assay. An antibody immunoreactive with an epitope found oo hu-MlP-2« but not found on hu-MIMe is attached to a solid support such as a polystyrene microtiter dish or nitrocellulose paper, using techniques known in the art. The solid support is then incubated In the presence of the sample to be analyzed under conditions where antibody-antigen complexes form and are stable. Excess and unbound components of the sample are removed, and the solid support is washed so that antlbody-andgen complexes are retained on the solid support. A fixed amount of a labelled polypeptide, containing an epitope found on hu-MlF-2o but not found on hu-MIR-2s, is then Incubated with the solid support. The labelled polypeptide has been coupled to a detectable moiety, such as biotin, peroxidase or radiolabel, by means well known in the art. The labelled polypeptide binds to an antibody immunoreactive with hu-MIP-2» which is attached'to the solid support. Excess and unbound polypeptide is removed and the solid support 1$ washed, as above. The detectable moiety attached to the solid support is quantitated. Since the hu-MIP-2e and the polypeptide have competed for the same antibody binding sites, the hu-M!P-2o in the sample can be quantitated by its diminution of the binding of the polypeptide to the solid support. Alternatively, the sample and the labelled polypeptide may be incubated with the solid support at the same time.

Nucleotide Probe Assays The nucleotide sequences provided by. the invention can be used to form gene probes in accordance with any of the standard techniques. The size of a probe can vary from less than approximately 20 -41oucteotides co hundreds of nucleotides. Tbe probe may be radiolabeled, labeled with a fluorescent material, or the like. Procedures for the preparation and labeling of nucleotide probes are well known in the art.

The diagnostic test employing a nucleotide probe will employ a biological sample from an IndivkJjaL Nucleic acids are recovered from the sample employing standard techniques well known to those skilled in the art. The nucleic acid then is Incubated with the probe and hybridization is thereafter detected.

Described below are examples of the present invention which ere provided only for illustrative purposes. They are not intended to limit the scope of tbe present invention in any way, as numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art in light of the present disclosure. Those of ormnary sxui in tne art are presumen to be laminar wim (ur iu have ready access to) the references cited in the application, and the disclosures thereof are Incorporated by reference herein. £Ζ2Φ£&1 Isolation and Clonlng_of cDNA Enwxflng Murine MIP-2 cDNA Library Construction The Isolation of poly-A+ RNA from E, coll LP5*stimulated murine raw 264.? cells and the construction of a cDNA library have been described previously (Davatelis, et al., 1988).

Murine MIP-2 cDNA Isolation A degenerate ollgooucleotide probe pool corresponded to amino adds 9-14 of the Nttg-terminal sequence of MIP-2 {wolpe, et aL, 1989) was synthesized. This portion of the partial sequence was chosen because it was predicted to be in a highly conserved coding region end because of Its lower codon degeneracy when compared to the other parts of the partial sequence. The resulting probe was a 128-fOld degenerate pool of oligomers IT nucleotides in length.

Duplicate oitroceQuioee filter lifts of the plated RAW 264.7 cDNA library (5x10s plaques) were prehytMdized at 42’C in 5xSSC, 2x Denhardrs, 80 mM sodium phosphate buffer, pH 6.S, 50% formamlde, 0.2% SDS and 0.28 mg/ml sonicated salmon sperm DNA and then were hybridized overnight at 42*C in SxSSC, lx Denhardvs, 20 mM sodium phosphate buffer, pH 6.5,50% formamide, 10% dextran sulfate, 0.1% SDS, 0.1 mg/ml sonicated salmon sperm DNA and 5xl04 cpm per ml per degeneracy of *2P-aTP S' end-labelled synthetic oligonucleotide probe pool. Following hybridization the filters were washed employing TMAC (wood, et al., 1985, Proc. Natl. Acad, sci. OSA. $2:1585). Plaques that were positive on duplicate filters were subjected to a second round of low density plating and screening. Positive phage clones were isolated from which DNA was prepared for further analysis.

Cloning of Murine MIP-2 cDNA Screening of the cDNA library derived from poly-A* RNA from RAW 264.7 ceils with a degenerate oligonucleotide pool specific for the N-terminal sequence of murine MIP-2 (Wolpe, et aL, 1989) resulted in the isolation of clone MIP-2-20a. Insert cDNA (approx. 1100 bp) was isolated, cloned into MI3 and the nucleotide sequence determined. The nucleotide sequence and predicted protein sequence are shown in Figure l. The predicted mature protein sequence starting at position l exactly matches the N-termlnal peptide sequence determined previously for purified MIP-2 (wolpe, et al., 1989). Srampiej isolation and Cloning of cDNA Encoding Hyman MiP-2o and t In order to isolate tbe human homologCs) of murine MiP-2 cDNA, a fragment encoding most of the mature mMlP-2 protein was isolated and used to probe a 0937 cDNA library prepared from poly-A* RNA of PMA-treated and LPS-stlmulated cells. DNA from plaques positive oo low stringency wash was Isolated and subjected to restriction endonuclease analysis which suggested tbe presence of two classes of clones. Insert cDNA from representative clones of each class was subcloned into MIS and the nucleotide .sequences were determined. cDNA library Construction The stimulation of the human monocytic-like cell line 0937 {Sundstrora, et aL, 1976, lot. J, Cancer. 12:565), tbe isolation of total and poly-A + rna and the construction of a CDNA library were r\ -43«a IvUv^a. 990T cell* (Aitt«,te«a Type Culture CcUcctloA· Rockville, MD) were grown to confluence and stimulated to differentiate by the addition of PMA to a final concentration of 5xlOeM. After 24 hours in the presence of PMA, LPS (LPS W, E. colt 0127:68; Difco Laboratories, inc., Detroit, MD was added to a final concentration of lug/ml and the cells were incubated for an additional 3 hours at 37 *C. Total RNA was prepared essentially, as described (Cathala, et al., 1983, DNA. £:329). Poly-A+ RNA was prepared by a single passage over oligo dT cellulose essentially as described (Manlantis, et aL, 1982). Double-stranded cDNA was prepared using a Ut for cDNA synthesis (Pharmacia LKB Biotechnology, Inc., Pleasant Hill, CA) and cloned and packaged Into igtlO. isolation of human homologs of murine ΜΓΡ-2 Plating of the U937 cDNA library, nitrocellulose filter prehybridization and hybridization of the filters were performed as described in Example l for the screening of the Raw 264.7 CDNA library. The probe DNA was a 186 bp Bal f-BgU7 fragment isolated from the mMIP-2 cDNA. Tbe Bgin site was introduced by in vitro mutagenesis using the mutagenic primer 5'CAAAACATCTTGAACAAAG-8'. The Ball-Bgin fragment encodes most of the mature mMIP-2 amino acid sequence, lacking tho6e base pairs encoding the 3 N-terminal and 8 C-terminal amino acids. This fragment was nick-translated and approximately 500,000 cpm per ml included in the hybridization solution.

After hybridization, filters were subjected to three low stringency washes at room temperature for 30 minutes each in 2xSSC, 0.1* SDS. Plaques positive on duplicate filters were subjected to a second round of low density plating and screening. Positive phage clones were isolated from which DNA was prepared for further analysis.

Cloning and DNA Sequence Analysis cDNA inserts were subcioned into MIS phage vectors and DNA sequencing was performed by tbe dOdeoxy chain termination method of Sanger et aL (1977, Proc, Natl. Acad. ScL PSA. ££:5463). *v 4 A . αλλλγλγγ sa π in “44The nucleotide sequence and predicted amino acid sequence or hu-MIP-2a is presented in Figure 2. This sequence was confirmed on four independent clones and is representative of the more abundant of the two classes of human cDNA homologous to mMIP-2. The nucleotide sequence and predicted amino acid sequence of hu-MIP-2i, representative of a second class of human cDNAs homologous to mMIP-2 is shown in Figure 3. This sequence was confirmed on two independent clones.

Example 3 Restriction Fragment Analysis Genomic DNA from RAW 284.7 cells was isolated as described by DiLella, et al. (1987, in Guide to Molecular Cloning Techniques, Berger, et al, eds., Academic Press, Orlando, p. 199). Human genomic DNA and murine CSH/HeN genomic DNA were purchased from Clontech (Palo Alto, CA).

Genomic dna was digested with restriction enzymes according to the suppliers specifications. Digested dna was separated on 1% agarose gels and then transferred to HyBond nylon membranes (Amersham, Arlington Heights, DU. Filters were prehybridized and ·» hybridized in 50 mM sodium phosphate, pH 8.5, 5XSSC, 1 mM sodium pyrophospate, 40% formamkle, 10% dextran sulfate, 5x Denbardfs solution, 0.1% SDS aod 100 mg/ml sonicated salmon sperm DNA. DNAs used tor Southern analysis were the l.i kb mMIP-2 cDNA (done mMlP-2-20a), the 0.98 kb bu-MiP-28 cDNA (clone bu-MiP-2-4a) and a LOS kb hu-MIP-2» cDNA (clone hu-MIP-2-5a). All cDNAs were labelled by random priming with *3P-CTP employing a Multiprimer DNA Labelling System (Amersham, Arlington Heights, IL). Following prehybridlzation for 2-4 hours at S7*C, labelled cDNA was added at 1x11)8 Cpm pgr πβ Hybridization was for 18-18 hr at S7*C. Filters were rinsed at room temperature for 10 min in 2xSSC, 0.1% SDS, then washed 8 times at 85*C for 45 min each in O.lxSSC, 0.1% SDS. In some cases hybridized probe was stripped from the blot by treatment for 45 min at 85’C in OJxSSC. 0.1% SDS and 50% formamide to allow re-hybri η ιλ Southern Analysis RAW 244.7 DNA was digested with each of three restriction enzymes: BamHI, EcoRI and EcoRV, separated by agarose gel electrophoresis and probed with 32P-labeIled mMIP-2 cDNA. The results, shown in Figure 6A are consistent with mMIP-2 cDNA defining a single gene. The same results were obtained when mouse C3N/HeJ dna was similarly analyzed (date not shown}.

A southern analysis or human genomic DNA was performed with hu-MiP-2» and hu-MIP-2s cDNA probes. Hybridization and wash eooditions were determined that made It possible to distinguish between hu-MlP-2» and bu-MIP-2S when probed with their respective cDNas. The conditions used, however, do not distinguish between hu-MIP-2» and hu-gro/MGSA specific sequences. The results of Southern analysis of genomic human DNA restricted with BamHI, EcoRI, or EcoRV and probed with hu-MIP-2e or hu-MIP-28 cDNA are presented in Figures SB and C, respectively. The patterns of hybridization obtained with each cDNA clearly differ. MIP-2e cDNA hybridized strongly to EcoRV fragments of approximately 23 and t.l kb, whereas MIP-2e cDNA hybridized to a 7.0 kb EcoRV fragment. Similarly, hu-MIP-2s cDNA hybridized to 1.8 kb And 3.3 kb (weakly) EcoRI fragments whereas hu-MIP-2e hybridized .strongly co 3.3 and an approximately l.l kb EcoRI DNA fragments and weakly to 4.6 and 3.8 kb EcoRI fragments. Finally, MIP-2B cDNA hybridizes strongly to a 2.4 kb BamHI fragment and very weakly to a 4.3 Kb BamHI fragment, whereas human ΜΙΡ-2» cDNA hybridizes strongly to 20,2.0 and 1.1 kb BamHI fragments and less strongly to a 4.3 kb BamHI fragment. The greater complexity of the hybridization patterns obtained with hn-MIP-2» cDNA probe, especially from BamHI digested DNA, relative to that Obtained with hu-MEP-20 cDNA probe, suggest that the hu-MJP-2» probe is detecting more than one gene, presumably hu-gro as wen as hu-MlP-2». We can conclude from the data Shown here that hu-MIP-20 and hu-MIP-2e are two distinct genes. n rr. 46Example 4 Cloning and Expression in Prokaryotes Plasmid ARV-2 p2fi gag (deposited with the American Type Culture Collection, Rockville, Maryland, on 27 August 1983, accession no. 53246) is a derivative of pBR322 containing the tao promoter, Shine Delgxrnc sequences, aod a polylinker as a substitution ot the original pBR322 sequences comprised between the EcoRI and Pwn restriction sties. Soe also EP 181,150.

Clone hu-M!P-2-5a (Example 3) is cleaved with PvuB and Ban, and ligated with a linker having the sequence: AATTATGCCCCCCCTGG.

TACCOCOGCGACC The plasmid ARV-2 p25 gag is then cleaved in EcoRI and Pvun, and the ligated hu-MlP-2-5a Inserted. The resulting construct is then transformed into competent 2» coll D1210 cells, following the protocol of Cohen, et al., Proc, Natl, Acad, ScL USA (1972) £2:2110). The cells are transformed with 25-50 ng ef the construct, aod the transformation mix plated on agar plates made in L-broth containing 100 ug/mL ampicillin. Plates are incubated at 87’C for 12 hours, and ampicillin-reristant colonies transferred into ι mL L-broth containing 100 ug/mL ampicillin. Cells are grown at 37*C, and expression induced by adding 10 vL of 100 mM 1PTG to a final concentration of 1 mM, followed by incubation at 37*c for 2 hours. The cells are then lysed, and the product purified.

Example 5 Plasmid pGAIl contains DNA encoding the yeast «-factor leader, a gene for human proinsulln, and a β-factor terminator, under transcriptional control of the yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter. The vector is fully described In EP 324,274.

The coding sequence for hu-MIP-2a was obtained by polymerase chain reaction (PCR) amplification of a fragment derived from a xgtlO clone (xgtiO-bu-MlP-2-6a) using the following primers; *.« r.n. Λ, mt ,λ,γγ Ρ ΚΙ ' Priaer: ’ - 3'Srlxr: ''•qAGTGCG7CGACI'CATCAGTIOGAITTGOCATTTITCAGCATCTTrrcGATG-3 * ♦ -- (♦hU-HIP-2e SAlI stop After 30 cycles of PCR amplification, the DNA was digested with Kpnl and Sail, and the 244 bp fragment encoding the < carboxyterminal amino adds of the «-factor leader, the dibasic processing site, and the entire 73 amino acid mature hu-MfP-2a was isolated by acrylamide gel electrophoresis. This fragment was then ligated into pGAfl that had been digested with Kpol and San and purified on an agarose gel. Following bacterial transformation and screening, plasmid pMIPSOO was obtained which upon DNA sequencing was found to have the predicted nucleotide sequence. This plasmid was digested with BamHI and the resulting 1163 pb fragment including the OAPDH promoter sequence, the a-factor lea Saccharomyces cerevisiae strain MB2-1 Qeu2-3, Ieu2-ll2. his3-ll, hls3-lS, ura34, CAN, Cit9) was transformed with plasmid pYMIPSOO by standard procedures and transformants selected for ura prototrophy. Expression was analyzed hy inoculation of single colonies of Individual transformants into leucine selective medium and growing for about 43 hours. Cultures were then centrifuged, cells resuspended in medium lacking uracil, and diluted 20x into ura selective medium. Cultures were then grown for about 72 boors, then harvested and cell-free supernatants prepared, conditiooed medium was analyzed for the presence of hu-MEP-2a by SDS-PAGE followed by Coomassie staining. A band was observed on SDS-PAGE which migrated similar to a native murine MIP-2 standard (provided by B. Sherry, Rockefeller University). The protein was expressed as >5% of the secreted protein. co -48Examole 6 Expression In Mammalian Cells (A) Expression in COS-7 Cells Plasmid pSV7tPA2I (ATCC Accession No. 40163, deposited 14 February 1985) Is a mammalian expression vector containing DNA encoding full-length human tPA in a polylinker sequence flanked by the SV40 origin, early promoter, and polyadenylation site. This plasmid Is also described in PCT Application wo 86/05514.

The sequence encoding hu-MIP-2a is amplified from plasmid PYM1P500 by PCR and inserted into a site of expression vector pSV7tPA2I, having an SV40 origin and early promoter 5' of the rite and a polyadenylation site. 3* of the insertion site, to form plasmid pSV-MlP2a. COS-7 cells are transfected with pSV-MIPla using a modification of the procedure described by Graham and van der Eb., Virol· (1973), ¢2:436-67. The sample are added to the dishes in duplicate and allowed to settle onto the cells for 6 hours in a COj incubator at 37*C. After culturing, the cells are rinsed gently with calcium- and magnesium-free PBS. The dishes are then exposed to glycerol as an adjuvant for 3-4 minutes, rinsed, and fed with DMEM medium supplemented with 4.5 mg/mL glucose, 3.7 mg/mL sodium bicarbonate, 292 Ng/ml glutamine, 110 ug/ml sodium pyruvate, 100 σ/ml penicillin, 100 U/ml streptomycin, and 10% fetal calf serum (FCS). After allowing the culture to recover from the glycerol shock, the medium Is replaced with serum-free me _Expression in CHO Cells CHO dhlr cells are plated at a density of 5x10s to l0e ceHsAHsh (10 cm) the day prior to transfection in F12 mecfium supplemented with 1.18 mg/mL NaHCOj, 292 ug/ml glutamine, 110 ug/ml sodium pyruvate, 100 U/mL penicillin, 100 U/raL streptomycin, 130 mg/mL proline, and 10% FCS. The cells are then transfected as described in part (A) above, further including a marker plasmid bearing a dhfr gene linked to the adenovirus major late promoter (coprecipitated in calcium phosphate). After culture for 48 hours, the ΛΛΑΓΑΓΓfΛ n -iacells are split 1:20, grown in selective medium (DMEM, ISO ug/mL proline, and 10% PCS), and cultured for 1-2 weeks.

BaapteT A. Preparation of a plasmid encoding a selectable marker The plasmid pAd-DHFR, bearing the murine DHFR cDNA, was constructed hy fusing the major late promoter rrom adenovirus-2 (AdMLP, map units 16-27.3) to the 3’ untranslated sequences of the mouse DHFR cDNA (J.H. Nunberg et al, Cell (1980) $$:355-64). SY40 DNA encoding part of the early transcription unit, including the intron of the small t antigen gene, and having the SV40 early region transcriptional termination region, was obtained from pSV2-neo (Southern and Berg, J. Mol. AppI, Gen. (1982) 1:327-41) and fused to the 8* untranslated end of tbe DHFR cDNA. These three segments were subcloned into pBR322 to obtain plasmid pAD-DHFR.

B. Preparation of Mammalian Expression Vector 1. gSVTd: The expression cassettes were prepared using the mammalian cell expression vector pSV?d (2423 bp).

The plasmid p$v7d (see Truett et al, 1985, DNA. $:333) was Λ constructed as follows: The 400 bp Bamffl/HindHI fragment containing the SV40 origin of replication and early promoter was excised from pSVgtl (obtained from Paul Berg, Stanford University, California) and purified. The 240 bp SV40 BeU/Bamtn fragment containing the SV40 polyA addition site was excised from pSV2/DKFR (Subramant et al, Mol. Cell. Biol. (1981) $:854-864) and purified. The fragments were fused through the following linker: Stop Codons i 2 3 S ’ -AGCTAGATCTCCCOGGTCrAGATAAgrAAT-3 ’ I III Hindlll Ball! Soal Xbal Bell overhang.

This linker contains five restriction sites, as wea as stop codons in ail three reading frames. The resulting 679 bp fragment containing the -aSV40 origin of replication, the SV40 early promoter, the polylinker with atop codons and the SV40 polyadenylation site was cloned into the BamHl site or pML, a pBR322 derivative having about 1.5 Kb deleted (lusty and Botchan, Ceil (1984) 2§:39l), to yield pSVS. The EcoRI and EcoRV sites in the pML sequences of psVS were eliminated by digestion with EcoRI and EcoRV, treated with Bai3t nuclease to remove about 200 bp on each end, and finally religated to yield p$V7a. The Ba 131 resection also eliminated one BamHl restriction site flanking the SV40 region, approximately 200 bp away from the EcoRV site. To eliminate the secood BamHl site flanking the SY40 region, pSV7a was digested with Nrul which cuts in the pML sequence upetream from the origin of replication. This was recircularized by blunt end ligation to yield pSV7b. pSVTc and pSVTd represent successive polylinker replacements. First, psv?b was digested with Stul and Xbal. Then, the following linker was ligated into the vector to yield pSVfc: ΒφΙΙΖ ScoM Seal Xpol XbeS ill I I 5' -aOMCTCeAATKCeCGGGOttACCT TCTKSAflCrrMdOGOCCOCCATOGftOMC Thereafter, pSV7c was digested with Bgin and Xbal, and thea ligated with the following linker to yield psV7d: Bgl 11 Bc>-ac Seal Xbal BamHl Sail I I I I I I * -&iTCTCGAATTCcccQtx7tcTA5MjGATCC&£CGAC JlQCTTJUtOOGOCCCAaaTCTCCTAOOCACSTGGAK 2. fiSfiffiga In an effort to improve the level ot transcription and stability of the messenger RNA of hetereologous proteins, the Svtfl early transcriptional initiation region was replaced by sequences from the human cytomegalovirus immediate early region (Boshart et al, £gU (1955) £521-530). In addltioo, 5’ untranslated sequences contributed by the SV40 early region to the messenger RNA were replaced with the 5* untranslated sequences of the HCMV 1E1 gene, including its first intron. This intron is Included on the assumption that spliced transcripts lead to faster processing and more stable mRNA. The expression vector also has an SV40 origin of replication to permit transient expression in COS7 cells, and a bacterial β-lactatnase gene to permit DNA cloning by selection for ampleillin resistance.

Tbe plasmid was constructed from a 700 bp Sall-Pvul fragment of pSV7d (described in Example 7, Section B, 1) containing tbe SV40 polyadenylation retfoa, a 1400 bp Pvul-EcoRI (filled in with Klenow polymerase) fragment of pSVT2 (Myers et al.. Cell (1981) 25:373-84; Rio et at, £$U (1983) £2:1227-40) providing the SV40 origin of replication and the rest of the 8-lactamase geoe, a 1700 bp Sspi-Sall fragment derived from a plasmid subclone of the human cytomegalovirus (Towne strain) in which the Sail site was Introduced by iQ vitro mutagenesis near the translational start site for the IE 1 protein, and the <300 bp Sall-Sall fragment of pSVF8-92C containing the cDNA encoding the Factor VTIfcC 92 X M, glycoprotein. pCMVSal20-SF2 plasmid is similar to pCMV6a described above except that the Sail-Sail fragment encoding the Factor VHT:C 92K M, protein was replaced with an expression cassette containing the 5' untranslated and signal sequence of human tPA fused to a heterologous gene. This vector was transformed into E.coli cells that are deposited with the ATCC, accession number 68249. Digestion of pCMV6al20-SF? with Nhel and Sail will remove the heterologous gene. Any hu-MIP-2a polypeptide gene can be ligated to linkers to be inserted into the mammalian expression vector pCMV6ai20-SF2. For example, the hu-MIP-2e gene is obtained from the yeast plasmid by polymerase chain reaction (PCR) amplification. Primers to direct the PCR reaction can easily be constructed based os the nucleic acid sequence of hu-MlP-2o shown in Figure 2, and the primers can be constructed to Include appropriate restriction sites, such as Nbel and Sail, for inserting the amplified gene Into the vector. For convenience, the DHFR gene can be Inserted into this new hu-MIP-2a expression cassette. This new vector is called pCMV-MIP2o. *>ΑΛΑΓΛΓΓ <Λ D CC -saC. Preparation of a Mip-2o Mammalian Expressing Ceil tine This example describes the preparation at a stable CHO cell line that produces the native hu-MIP-2a.

The DHFR* CHO cell line DG44 (G. Urlaub et al., Som, Celt. Moi, Genet (1986) 12:553-66) is first transfected with the plasmid pCMV-MIP-2o. In this plasmid the CMV promoter (described In Example 78-2) regulates MIP-2o gene derived from pYMIPSOO (described In Example 3), the tPA leader (described In Example 7B-2) directs the secretion of the encoded hu-MIP-2a, and contains downstream the Ad-MLP/dbfr cassette derived from pAd-DHFR (described in Example 7a). The cells arc transfoetod using the polybrene method described by w. Chaney et al., Som. Cell. MoL Genet. (1986) 12:237-44. By selecting for DHFR* cells (in DMEM + 10% DFSC) several hu-MIP-2a producers are isolated.

Deposit information The following materials were deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20832: Name Deposit Date Acces. No. 8 March 1990 CRL 10378 8 March 1990 88249 20 June 1990 74003 Chinese Hamster Ovary Cells CHO-DG44 6. coli HB101 pCMV6al20-SF2 S. cerevisiae MB2-1 (pYMIP300) ___________ CfcrWViSiae HU-l LpVHlPS4^) ip JmwC These materials were deposited under the terms of the Budapest Treaty on the International Recognition or the Deposit of Micro-organisms for Purposes of Patent Procedure. These deposits are provided as a convenience to those at skill in the art, and do not represent an admission that a deposit is required under 33 0.S.C. Section 112. The sequence of the polynucleotides contained in the deposited materials, es 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 the written description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

Claims (31)

1. A composition comprising human MIP-2e polypeptide substantially free of human tissue.

2. The composition of claim l which is substantially free or other human proteins.

3. The composition of claim 1 which is substantially free ot other protein.

4. The composition of claim 1 wherein said hu-MlP-2· polypeptide is hu-M2P-2a.

5. A composition comprising DNA molecules comprising a heterologous region that encodes hu-MIP-2e polypeptide wherein the composition Is substantially free of other DNA molecules.

6. The composition of claim 5 wherein said region encoding hu-MIP-2o polypeptide is an lntron-free DNA sequence. T. a dna molecule comprising an lntron-free DNA sequence encoding an amino acid sequence of hu-MlP-2e polypeptide. 3. A DNA molecule according to claim ? wherein said hu-MIP-2« polypeptide Is hu-&OP-2«.

7. 9. A cell population transformed with the DNA molecule of claim S, said population being substantially free of cells not transformed with said DNA molecule.

8. 10. The method of producing a hu-MtP-2a polypeptide, comprising: providing a population of transformed cells of claim 9; growing said population under conditions whereby said polypeptide is expressed; and recovering said polypeptide.

9. 11. The method of claim 10 where said polypeptide is excreted by said cell.

10. 12. a single stranded DNA molecule comprising et least 10 sequential nucleotides, wherein said sequential nucleotides comprise a subsequence unique to hu-MlP-2a or a DNA sequence complementary thereto. D CO -5»

11. 13. A method for determining the presence of a polynucleotide substantially homologous to a coding sequence for human MIP-2a comprising: providing a sample suspected of comprising said polynucleotide; 1 Incubating the sample with a nucleotide probe having a sequence complementary to the single stranded DNA of Claim 12, under conditions where said probe will form hybrids with nucleic acid from the sample; and detecting nucleic acid hybrids.

12. 14. An antibody reactive with an epitope on a hu-MIP-2e polypeptide but not reactive with murine MIP-2, human gro protein or the protein encoded by the murine KC gene.

13. 15. The antibody of claim 14 wherein said antibody is monoclonal

14. 16. A composition comprising the antibody of claim 14 wherein said composition is substantially free of other Immunoglobulin molecules.

15. 17. A hybridoma cell line which produces the monoclonal antibody of claim 15.

16. 18. A method for determining the presence of human Μ1Ρ-2α in a sample comprising: incubating said sample with an antibody reactive with hu-MIP-2» polypeptide; and . detecting Immunocomplex.

17. 19. A method for determining the presence of antl-hu-MIP-2tt antibodies in a sample, comprising: incubating said sample with the composition of claim 2; and detecting immunocomplex.

18. 20. A pharmaceutical composition stimulating myelopoietfs - in myelopolettc cells comprising an effective amount of hu-MlP-2o polypeptide.. -5521. A composition according to claim 1, substantially as hereinbefore described and exemplified.

19. 22. A composition according to claim 5, substantially as hereinbefore described and exemplified.

20. 23. A DNA molecule according to claim 7, substantially as hereinbefore described and exemplified.

21. 24. A cell population according to claim 9, substantially as hereinbefore described.

22. 25. A method according to claim 10 of producing a hu-MIP-2ot polypeptide, substantially as hereinbefore described and exemplified.

23. 26. A hu-MIP-2ct polypeptide, whenever produced by a method claimed in any one of claims 10, 11 or 25. z

24. 27. A single stranded DNA molecule according to claim 12, substantially as hereinbefore described and exemplified.

25. 28. A method according to claim 13 for determining the presence of a polynucleotide substantially homologous to a coding sequence for human MIP-2ot z substantially as hereinbefore described and exemplified.

26. 29. An antibody according to claim 14, substantially as hereinbefore described and exemplified.

27. 30. A composition according to claim 16, substantially as hereinbefore described.

28. 31. A hybridoma cell line according to claim 17, substantially as hereinbefore described and exemplified.

29. 32. A method according to claim 18 for determining the presence of human MIP-2ot in a sample, substantially as hereinbefore described.

30. 33. A method according to claim 19 for determining the presence of anti-hu-MIP-2ot antibodies in a sample, substantially as hereinbefore described.

31. 34. A pharmaceutical composition according to claim 20, substantially as hereinbefore described.