RELATED PUBLICATIONS

The Applicants are authors or co-authors of several articles directed to the- subject matter of the present invention. These articles supplement those articles listed in U.S. Patent No. 4,603,106, which earlier articles are incorporated herein by reference. (1) [Applicant Cerami co-authored with B. Beutler, J.

Mahoney, N. Le Trang and P. Pekala] "Purification of Cachectin, a Lipoprotein Lipase-Suppressing Hormone Secreted By Endotoxin-Induced RAW 264.7 Cells", J. EXP. MED. 161:984-995 (May, 1985) ; (2) [Applicant Cerami co-authored with M. Kawakami, J.R. Mahoney, N. Le Trang, W. Vine, and Y. Ikeda] "Lipopolysaccharide-Treated RAW 264.7 Cells Produce a Mediator Which Inhibits Lipoprotein Lipase in 3T3-L1 Cells", J. IMMUNOL. 13A (3) :1673-1675 (March, 1985) ; (3) [Applicant Cerami co-authored with P.J. Hotez, N. Le Trang, and A.H. Fairlamb] "Lipoprotein Lipase Suppression in 3T3-L1 Cells by a

Haematoprotozoan-Induced Mediator From Peritoneal Exudate Cells." PARASITE IMMUNOL. (Oxf.) 6:203 (1984) ; (4) [Applicant Cerami co-authored with B. Beutler, D. Greenwald, J. D. Hulmes, M. Chang Y.-C.E. Pan, J.

Mathison and R. Ulevitch] "Identity of Tumor Necrosis Factor and Macrophage-Secreted Factor Cachectin", NATURE 316:552-554 (1985) ; (5) [Applicant Cerami co-authored with B. Beutler, F.M. Torti, B. Dieck ann and G.M. Ringold] "A Macrophage Factor Inhibits Adipocyte Gene Expression: An In Vitro Model of Cachexia" SCIENCE ,229:867-869 (1985) ; (6) [Applicant Cerami co-authored with B. Beutler and I.w. Milsark] "Passive Immunization Against Cachectin/Tu or Necrosis Factor (TNF) Protects Mice From the Lethal Effect of Endotoxin", SCIENCE

229:869-871 (1985) ; (7) [Applicants Cerami, Wolpe and Sherry co-authored with B. Beutler, G. Davateliε, D. G. Hesse, H. T. Nbuyen, L. I. Moldawer, C. F. Nathan and S.

F. Lowry] , "Macrophages Secrete a Novel Heparin-Binding Protein with Inflammatory and Neutrophil Chemokinetic Properties", J. EXP. MED. 162:570-581 (1988); (8) [Applicants Wolpe, Cerami and Tekamp-Olson co-authored with G. Davatelis, K. Hermsen, C. Luedke, C. Gallegos, D. Coit and J. Merryweather] , "Cloning and Characterization of a cDNA for Murine Macrophage Inflammatory Protein (MIP) , A Novel Monokine with Inflammatory and Chemokinetic Properties", J. EXP. MED., 167:1939-1944 (June, 1988); (.9) [Applicants co-authored with C. Gallegos, D. Bauer, G. Davatelis, F. Maεiarz and D. Coit] , "Resolution of the Two Components of Macrophage Inflammatory Protein 1, and Cloning and Characterization of One of Those Components, MIP-1B", J. EXP. MED. 168:2251-2259 (December, 1988); and (10) [Applicants Wolpe, Cerami and Sherry co-authored with D. Juerε, G. Davatelis, and R. Yurt] , "Identification and Characterization of Macrophage Inflammatory Protein 2", PROC. NATL. ACAD. SCI. USA 86:612-616 (January, 1989).

The research leading to the present invention was funded in part by grants from the National Institutes of Health and the Rockefeller Foundation.

BACKGROUND OF THE INVENTION

The present invention is generally directed to materials and associated methods for the analysis and treatment of the effects and corresponding operation of invasive stimuli such as infection upon animal hosts, and in particular, is concerned with the identification of materials which may participate in the host response to such invasive stimuli.

Several common physiological and biochemical derangements have been observed in various mammalian hosts responding to a variety of invasive stimuli such as bacterial, viral, or protozoal infection; tumors; or endotoxemia; as

well as in idiopathic states. For example, these responses include fever, leukocytosis, hyperlipidemia, reduced food intake (anorexia) , reduced activity, wasting (cachexia) , and other modifications in muscle, white blood cell and liver metabolism. In particular, recent studies aimed at elucidating the biochemical mechanism of cachexia in rabbits infected with Trypanosoma brucei noted that animals with a minimal parasite burden became moribund and exhibited an extreme hypertriglyceridemia, with a marked elevation of plasma very low density lipoprotein (VLDL) . See CA. Rouser and A. Cerami, MOL. BIOCHEM. PARASITOL. 1:31-38 (1980). The hyper- triglyceridemic state was remarkable in view of the severe wasting diathesis that accompanied this experimental infection. The elevation of plasma VLDL was shown to result from a clearing ^* defect, caused by a loss of peripheral tissue lipoprotein lipase (LPL) activity.

Reduced LPL activity has been observed by others, and it has been noted that this condition has existed when the human body was in shock. See E.B. Man, et al., "The Lipids of Serum and Liver in Patients with Hepatic Diseases", CLIN. INVEST. 24. at 623, et seq. (1945); See also John I. Gallin, et al., "Serum Lipids in Infection", N. ENGL. J. MED. 281 at 1081-1086 (November 13, 1969); D. Farεtchi, et al., "Effects of Three Bacterial Infections on Serum Lipids of Rabbits", J. BACTERIOL. 9_5 at 1615, et seq. (1968); S.E. Grossberg, et al., "Hyperlipaemia Following Viral Infection in the Chicken Embryo: A New Syndrome", NATURE (London) 208 at 954, et seq. (1965); Robert L. Hirsch, et al., "Hyperlipidemia, Fatty Liver and Bromsulfophthalein Retention in Rabbits Injected Intravenously with Bacterial Endotoxin", J. LIPID. RES. at 563-568 (1964); and Osamu Sakaguchi, et al., "Alternations of Lipid Metabolism in Mice Injected With Endotoxins", MICROBIOL. IMMUNOL. 23. (2) at 71-85 (1979) ; R.F. Kampschmidt, "The Activity of Partially Purified Leukocytic Endogenous Mediator in Endotoxin Resistant

C3H/HeJ Mice", J. LAB. CLIN. MED. 95 at 616, et seq. (1980) ; and Ralph F. Ka pschmidt, "Leukocytic Endogenous Mediator", J. RET. SOC. 22 (4) at ^" 287-297 (1978).

Additionally, publications are known by the Applicants that discuss the identification and existence of "mediators" that appear to be involved in the host response to infection; and in particular, the following articles, the texts of which are incorporated herein by reference, are .listed: Sipe, J.D., et al., J. EXP. MED., 150:597-606 (1979); and Barney, C.C., et al., LIPTON, J.M. (Ed.), FEVER: INTERNATIONAL SYMPOSIUM, Dallas, Texas, April 11-12, 1979 XII + 263P. Raven Press: New York, Illus. ISBN 0-89004-451-1 (08877), 0 (0), pp.111-122 (1980); and Dinarello, C . , "Human Leukocytic Pyrogen: Purification and Development of a Radioim unoassay", PROC. NATL. ACAD. SCI. USA, 24 _. (10) at 4624-4627 (October, 1977). All of the factors identified and investigated by each of the authors in the above noted articles and the articles authored or co-authored by Kampschmidt have been determined to comprise a single grouping of factors which has been identified as interleukin-1 (IL-1) . This determination has been documented in an article by Charles A. Dinarello, published in REVIEWS OF INFECTIOUS DISEASES, at Volume 6, No. 1 (January-February, 1984) , the text of which is also incorporated herein by reference.

A similar deficiency of LPL activity was noted by Applicants in C3H/HeN mice after administration of

Escherichia coli lipopolysaccharide (LPS) . In contrast, the loss of LPL activity was not demonstrable in C3H/HeJ mice, which are genetically resistant to LPS. This resistance to endotoxin-induced LPL deficiency could be circumvented by the administration of serum obtained from endotoxin-sensitive animals that had been injected with LPS two hours previously. Similarly, resistance could be overcome by injecting conditioned medium from

endotoxin-stimulated thioglycollate-elicited peritoneal macrophages, obtained from sensitive mice. These findings were set forth in full detail in Application Serial No. 414,098, now U.S. Patent No. 4,603,106, the disclosure of which is incorporated herein by reference.

The above work was prompted by the belief that the "mediator" or "mediators" existed and were suspected to have a significant effect upon general anabolic activity of energy storage cells in the animal host. It was suspected that such "mediator" exerted a depressive effect upon the activity of certain anabolic enzymes, whose reduced activity was observed for instance, where the hosts enter the condition known as shock, as in response to infectious invasion. Resultingly, the relationship of the mediator produced by endotoxin-stimulated peritoneal mouse exudate cells, upon endotoxin-sensitive and endotoxin-inεenεitive mice alike, and the development through εuch inveεtigation of a reagent for the meaεurement of anabolic enzyme activity was set forth in first filed abandoned application Serial No. 299,932, incorporated herein by reference.

Further investigation of this system was made in conjunction with the 3T3-L1 "preadipocyte" model system, and the corresponding development of methods and associated materials for the development of antibodies to the "mediator" and other diagnostic procedures was then set forth in application Serial No. 351,290, also incorporated herein by reference and now abandoned.

Thereafter, in subsequent application Serial No. 414,09.8, now U.S. Patent No. 4,603,106, it was eεtablished that the mediator substance derived from the endotoxin stimulation of macrophage cells exhibited the activities of suppressing the anabolic enzymes lipoprotein lipase, acetyl Coenzyme A Carboxylaεe and fatty acid synthetase, and further, inhibited the growth and differentiation of erythroid-co mitted cells.

Additional work set forth in articles (1) and (4) by Beutler et al., and Application Serial No. 766,852, the disclosure of which is incorporated herein by reference, has resulted in the discovery that the earlier identified mediator substance contained a further protein component which possesses a number of activities, which distinguished it from both the mediator substance and the other factors identified in the art and known .as interleukin-1 and interleukin-2. Further work set forth in articles (7)-(9) and Parent Application Serial No. 104,827, the disclosure of which is incorporated herein by reference, established the presence of an additional factor (MIP-1) in the mediator substance which factor demonstrates a distinguishable profile of activities.

Subsequently, the present inflammatory cytokine MIP-2 was isolated and purified and its distinctive activities elucidated as set forth in immediate Parent Application Serial No. 240,079. Since that time, the complete sequence of the present cytokine has been determined following the cloning of its cDNA, and expreεεion of the cytokine has been pursued. The present application is intended to include the additional information regarding this cytokine that is now available as a result of the investigations of the inventors herein.

MIP-2 is a member of a homologous multigene family. Members of this family that have highest homology in protein sequence (generally predicted from cloned cDNA) include MGSA and KC. MGSA (Richmond et al., EMBO J. 2= 2025 (1988) is an autocrine growth factor with potent mitogenic activity secreted by human melanoma cells and is the product of the human qro gene (Anisowicz et al. , PROC NAT. ACAD. SCI. USA 81: 188 (1987). MGSA has 61.6% identity in amino acid sequence to MIP-2; the predicted protein sequence of the hamster homolog of MGSA has 68.4% identify to MIP-2. The murine KC gene product is induced

by PDGF and is thought to be the murine ho olog of the human MGSA/qro gene (66.3% amino acid identity to MIP-2).

The present applicants know of no prior art on the expression of recombinant MIP-2 although Lipes et al., (PROC NATL. ACAD. USA 85: 9704, 1988) described baculovirus expression of Act-2 cDNA, a putative human homolog of murine MIP-lB, to show that the protein encoded was secreted and to identify the mature N- terminal sequence.

Members of the MIP-1 and MIP-2 gene families have been expresεed but the pertinence of theεe results to MIP-2 expression is queεtionable. The literature iε summarized as follows. JE, a cDNA that encodes a protein with homology to MlP-lα and MIP-lB, has been expressed in COS- 1 cells to confirm that it encodes a polypeptide core of about 12 kDa (Rollins et al. , PROC. NATL. ACAD. SCI. USA 85: 3738, 1988). KC, a cDNA that encodes a protein with homology to MIP-2, has been expressed in COS-1 cells to show that it encodes a secreted protein by Oguendo et al., J. BIOL. CHEM. 26 : 4133 (1989). Connective tissue activating peptide-III (CTAP) reported by Mullenbach et al., J. BIOL. CHEM. 2j61: 719 (1986) and IP-10, reported by Luster and Ravetch, J. EXP. MED. Ij66: 1084 (1987) both members of the MIP-2 gene family, have been expressed as an α-factor fusion in yeast and in E. coli. , respectively. Finally, Lindley et al., PROC. NAT. ACAD. SCI. USA 8_5: 9199 (1985) have expressed NAF, a member of the MIP-2 family, in E. coli.. After purification and renaturation, this recombinant protein was found to have the same bioactivity identified for the native molecule.

SUMMARY OF THE INVENTION

In accordance with the first aspect of the present invention, the inflammatory cytokine MIP-2 isolated from the mediator substance iε disclosed, and compriseε a

protein that has been purified and is cationic under basic physiological conditions. The inflammatory cytokine of the present invention exhibits the ability to bind to heparin, even at high salt concentrations, to induce localized inflammation characterized by polymorphonuclear cell infiltration when administered subcutaneously and is an extremely active chemotactic agent while inducing little or no chemokinetic activity. The present inflammatory cytokine however, lacks certain activities common to other factors that have been isolated from the mediator substance disclosed in U.S. Patent No. 4,603,106.

In particular, the present inflammatory cytokine lacks the ability to suppress the activity of the anabolic enzyme lipoprotein lipase (LPL) , and is unable to cause the cytotoxicity of cachectin/TNF-sensitive L929 cells, to stimulate the blastogenesis of endotoxin-reεiεtant C3H/HeJ thymocytes, or to induce the production of cachectin/TNF by primary thioglycollate-elicited mouse macrophage cells. These latter characteristics all absent from the present inflammatory cytokine are exhibited by the known factors cachectin/TNF and interleukin-l (IL-1) , and thereby distinguish the present inflammatory cytokine therefrom.

The most significant affirmative activities exhibited by the present inflammatory cytokine appear to be the ability to bind to heparin and the ability to induce localized inflammation characterized by polymorphonuclear (PMN) cell infiltration. Accordingly, while the exact role that the present isolate plays in the cascade of reactions to host invasion is as yet undefined, its participation in the elicitation of certain of the activities and conditions associated with mobilization against host invasion is clear. Accordingly, the inflammatory cytokine possesses the potential for use as a diagnostic tool to identify and perhaps differentiate

between variouε stimuli whether invasive or idiopathic, by the activation of the present inflammatory cytokine that such stimuli may promote.

The present inflammatory cytokine was initially identified and characterized and found to contain a single chain, 6 kilodalton protein on sodium dodecyl sulfate (SDS-PAGE) which migrates on gel filtration as a monomer or di er. Partial N-terminal amino acid sequence _. data as depicted in FIGURE 2 revealed that MIP-2 is a member of a family of cytokines, the archetype of which iε platelet factor 4 (PF _A ) .

As set out above, the present inflammatory cytokine may be prepared by the stimulation of macrophage cells with a material that accompanies an invasive stimulus. In particular, a sample of macrophage cells which may be derived from a variety of sources may be incubated with a stimulator material such as endotoxin or trypanosomeε, to produce the mediator substance disclosed in U.S. Patent No. 4,603,106. Such incubation may take place for a period of time of up to twenty hours, and exact time limits will vary with the particular cells selected for incubation.

Further properties of the present inflammatory cytokine include its inability to induce fever in rabbits, or to induce superoxide formation or a respiratory burst in human neutrophils in vitro.

Following such incubation, the medium may be appropriately treated as by centrifuging, to recover a supernatant containing the crude mediator substance. The mediator substance may then be further treated as by filtration or precipitation. Thereafter, the crude mediator substance may be subjected to a serieε of known isolation techniques, whereupon the inflammatory cytokine may be recovered. The present invention naturally

contemplates alternate means for preparation of the inflammatory cytokine, including where applicable known genetic replicative techniques, arid the invention iε accordingly intended to cover such synthetic preparations within its scope.

As noted above, the present invention also includes a purified protein having the above-noted activities and characteristics, that displays the NH ₂ -terminal partial amino acid consensus sequence set forth in FIGURE 6, as determined in mice. The cDNA for MIP-2 haε been cloned and, aε set forth in FIGURE 7, predicts a mature protein of 74 amino acids in length with a molecular weight of 7908 and a translated molecular weight of 10,622.52 for the precursor peptide.

Accordingly, the present invention also includes the identification of the purified peptide comprising the present cytokine that exhibits the above noted activities and characteristics, and that displays the mature amino acid sequence set forth below and in FIGURE 7, as determined in mice.

GLY ALA VAL VAL ALA SER GLU LEU ARG CYS GLN CYS LEU LYS THR LEU PRO ARG VAL ASP PHE LYS ASN ILE GLN SER LEU SER VAL THR PRO PRO GLY PRO HIS CYS ALA GLN THR GLU VAL ILE ALA THR LEU LYS GLY GLY GLN LYS VAL CYS LEU ASP PRO GLU ALA PRO LEU VAL GLN LYS ILE ILE GLN LYS ILE LEU ASN LYS GLY LYS ALA ASN

As stated earlier, the foregoing sequence bears no striking similarity to any of the known factors and accordingly establishes that the present inflammatory cytokine is distinguishable therefrom. The isolation of the above cDNA amino acid sequence facilitates the reproduction of this cytokine by recombinant genetic techniques as discussed in detail hereinafter. Thus, the invention provides the DNA sequence encoding the present

inflammatory cytokine or analogs thereof, which may be used to construct vectors for expression in host εyεtemε by recombinant DNA techniqueε.

The invention further includes a method for detecting idiopathic or invasive stimuli on the basis of their ability to elicit the activities affected by the present inflammatory cytokine. In particular, invasive stimuli could be identified and detected by their ability to induce a material which is able to bind to heparin and to induce localized inflammation with neutrophil infiltration and chemotacticity. In this method, macrophage cellε derived for example, from the RAW 264.7 cell line could be treated with/expoεed to a number of known εtimulator materials such as endotoxin, trypanoso es or the like, as a control, while parallel cellular samples could be treated with or exposed to an extract of material from the presumed εituε of the infective εtimuluε. All εampleε could thereafter be incubated in accordance with the methodε deεcribed above, and thereafter subjected to the sequence of separation techniqueε alεo defined, whereupon testing of the resulting isolates derived from the control and unknown samples could be compared to determine whether the inflammatory cytokine, if any, developed iε identical or even similar.

In similar fashion, an aεεay system for screening of potential drugs effective to counteract the inflammatory cytokine may be prepared. In one instance, the test drug could be administered to a stimulated macrophage sample to determine its effect upon the production of the inflammatory cytokine. In an alternate procedure, the inflammatory cytokine may be introduced into a cellular test system in which the cytokine is known to be active, and the prospective drug may also be introduced to the same cell culture and the culture may thereafter be examined to observe any changes in the activity of the

inflammatory cytokine in comparison with the addition of the prospective drug alone, or the effect of added quantities of the known inflammatory cytokine.

The present invention also relates to a method for detecting the presence of stimulated, spontaneous, or idiopathic pathological states in mammals, by measuring the activity and presence of the inflammatory cytokine of the present invention. More particularly, the activity of the inflammatory cytokine may be followed directly by the assay techniqueε discussed later on, through the use of an appropriately labeled quantity of the cytokine. Alternately, the cytokine can be used to raise binding partners or antibodies that could in turn, be labeled and introduced into a medium such as serum, to test for the presence of inflammatory cytokine therein, and to thereby assess the state of the host from which the medium waε drawn.

Thuε, both the inflammatory cytokine and any antibodieε that may be raised thereto, are capable of use in connection with variouε diagnoεtic techniqueε, including immunoassayε, such as a radioimmunoasεay, using for example, an antibody to the inflammatory cytokine that has been labeled by either radioactive addition, reduction with sodium borohydride, or radioiodination.

In an immunoassay, a control quantity of the inflammatory cytokine, its antibody, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a blood sample of a mammal believed to be undergoing invasion. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.

In the instance where a radioactive label, such aε the isotopes ^U C, ¹³¹ I, ³ H, ¹²⁵ I and ³⁵ S are used, known currently available counting procedureε may be utilized. In the inεtance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectro- photometric or gasometric techniques known in the art. The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the inflammatory* cytokine. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniqueε discuεsed herein, coupling a label to the inflammatory cytokine; and one or more additional immunochemical reagents, at leaεt one of which iε a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s) .

In a further embodiment, the present invention relates to certain therapeutic methods which would be based upon the activity of the inflammatory cytokine, antibodieε to the inflammatory cytokine, or upon agents or other drugε determined to possesε the same or an antagonistic activity. A first therapeutic method is associated with the prevention of the manifestationε of the activitieε of the inflammatory cytokine in mammals, such aε inflammation and fever, and compriεes administering either an antibody to the cytokine, an agent capable of modulating the production and/or activity of the cytokine, or an agent not an antibody to the cytokine that is capable of acting as an antagonist to the cytokine, either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host.

More specifically, the therapeutic method generally referred to herein could include the method for the treatment of inflammation and fever by the administration of pharmaceutical compositions that may comprise effective quantities of antibodies to the inflammatory cytokine, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further- aspect of the present invention.

A variant embodiment of this therapeutic method could include initially detecting the presence and activity of the inflammatory cytokine and thereafter administering the appropriate pharmaceutical composition.

A second therapeutic method seeks to take advantage of the inflammatory activity of the cytokine and in particular, its ability to cause the movement and mobilization of neutrophils in response to invasive stimuli such aε infection. Accordingly, the inflammatory cytokine may be prepared in a suitable formulation for administration to the situs of infection which for example, may develop where tissue trauma has occurred. In such instance, the inflammatory cytokine may be prepared in a sterile solution and delivered to the trauma or wound as part of an irrigation fluid or by direct dosage such aε, in a pharmaceutical compoεition, the latter course of administration contemplating topical and parenteral routes. Naturally, the inflammatory cytokine may be used to raise equally effective agents or drugs by known methods that may then be formulated into pharmaceutical compositions suitable for administration in the same manner and for the same purpose aε for the inflammatory cytokine itself.

Accordingly, it iε a principal object of the present invention to provide an inflammatory cytokine in purified form that exhibits certain characteristics and activities

associated with the host response to invasive stimuli in mammals.

It is a further object of the present invention to provide methods for the preparation of the inflammatory cytokine, including recombinant means.

It is a further object of the present invention to provide a method for detecting the presence of. the inflammatory cytokine in mammals in which invasive, spontaneous, or idiopathic pathological states such aε infection are suspected to be present.

It is a further object of the present invention to provide a method and associated assay εyεtem for screening substances such as drugs, agents and the like, potentially effective in either mimicking the activity or combating the adverse affects of the inflammatory cytokine in mammals.

It iε a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the inflammatory cytokine, so as to alter the adverse consequenceε of such presence or activity.

It iε a still further object of the present invention to provide a method for the treatment of mammals to promote the amount or activity of the inflammatory cytokine, so as to treat or avert the adverse consequences of invasive, spontaneous or idiopathic pathological states.

It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methodε which comprise or are based upon the inflammatory cytokine or its binding partner(ε), or upon agents or drugs that control the production, or that

mimic or antagonize the activities of the inflammatory cytokine.

Other objectε and advantageε will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 is an electrophoretic gel depiction of the purification of MIP-2. The final positions of the molecular weight markers (in kilodaltons) are shown on the left along with the positionε of cachectin/TNF and MIP-1 in thiε 10-18% NaDodS0 ₄ -PAGE system. The four lanes show succeεεive stageε of purification of MIP-2: Lane 1 - concentrated and diafiltrated crude supernatant from RAW 264.7 cellε; Lane 2 - effluent from Mono Q (anion exchange) column chromatography of RAW 264.7 supernatant; Lane 3 - pooled peak fractionε after purification on heparin-Sepharoεe; Lane 4 - pure MIP-2 fractionε after purification on Superoεe 12 (gel filtration) .

FIGURE 2 depictε the partial NH ₂ -terminal amino acid εequenceε of MIP-2 and other members of the platelet factor 4 family. Sequences were obtained from the literature and aligned via a conserved cysteine reεidue. PBP (platelet baεic protein) iε the precursor for β-thromboglobulin and CTAP III. Amino acidε enclosed in boxes are conserved between the various members of the platelet factor 4 family. Lower case prefixeε refer to species: m = murine, h = human, b = bovine, r = rat, c = chicken, ham — hamster.

FIGURE 3 is a table showing a comparison of the percent sequence identity over the region corresponding to the partial NH ₂ -terminal amino acid sequence obtained for MIP-2. Sequences were compared using the FASTP program.

Comparisons are only for the portion of each sequence corresponding to that available for MIP-2 aε aligned in Fig. 2. Ins. = Insignificant.

FIGURE 4 depicts an immunoblot analysis characterizing the relationship between MIP-2 and other members of the platelet factor 4 family. (A) Silver stain of duplicate of gel used for immunoblot* of MIP-2. (B) Immunoblot of MIP-2: Lane 1 - purified MIP-2; Lane 2 - supernatant from endotoxin-stimulated RAW 264.7 cells; Lane 3 - supernatant from non-stimulated thioglycollate-elicited mouse peritoneal macrophages; Lane 4 - supernatant from endotoxin-stimulated, thioglycollate-elicited mouse peritoneal macrophages; Lane 5 - purified NAP-1 protein; Lane 6 - supernatant from COS cells tranεfected with plasmid alone; Lane 7 - supernatant from COS cells transfected with plasmid containing the human crro gene; Lane 8 - supernatant from COS cells transfected with plasmid containing the hamster gro gene. Lanes 7 and 8 are reversed in the immunoblot (4b) .

FIGURE 5 iε a graphical depiction of human polymorphonuclear chemotaxis and chemokinesis in reεponεe to MIP-2. Chemotaxiε (darkened barε) was measured by introducing MIP-2 into the lower well of the chamber only. Chemokineεiε (lightened barε) was measured by introducing the same concentration of MIP-2 into the upper and lower chambers. fMet-Leu-Phe (fMLP) was used at a concentration of 10 ^'8 M.

FIGURE 6 is a depiction of the partial N-terminal sequence of the inflammatory cytokine of the present invention.

FIGURE 7 depicts the complete nucleotide εequence of a cDNA clone for MIP-2. The predicted translated molecular weight of the precursor peptide is 10,622.52. The mature

peptide sequence, starting at position one, is 74 amino acids in length.

DETAILED DESCRIPTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g. , Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1982) ; "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985) ; "Oligonucleotide Synthesiε" (M.J. Gait ed. 1984) ; "Nucleic Acid Hybridization" (B.D. Hameε & S.J. Higginε edε. 1985) ; "Transcription And Translation" (B.D. Hameε & S.J. Higgins edε. 1984); "Animal Cell Culture" (R.I. Freshney ed. 1986) ; "Immobilized Cells And Enzymes" (IRL Presε, 1986) ; B. Perbal, "A Practical Guide To Molecular Cloning" (1984) .

Therefore if appearing herein, the following termε shall have the definitions set out below.

The term "stimuluε" and itε plural as used herein are intended to apply to invasive events εuch as infection, as well aε conditionε caused by wounding, and to idiopathic or spontaneous stateε that may for example, originate from cellular or metabolic derangementε or other causes.

The termε "inflammatory cytokine", "macrophage inflammatory protein 2" and "MIP-2 ¹¹ as used throughout the present application and claims refer to protein material having the partial N-terminal sequence data presented in FIGURES 2 and 6, the mature peptide sequence presented in FIGURE 7, and the profile of activities set forth herein and in the Claims. Accordingly, proteins having similar sequenceε to those set forth herein but

diεplaying substantially equivalent or altered activity are likewise contemplated. Theεe odificationε may be deliberate, for example, such as modificationε obtained through site-directed mutagenesiε, or may be accidental, such as those obtained through mutations in hosts that are MIP-2 producers. Also, the terms "inflammatory cytokine", "macrophage inflammatory protein 2" and "MIP- 2" are intended to include within their scope the proteins specifically recited herein aε well as all substantially homologous analogs and allelic variations.

A "replicon" is any genetic element (e.g. , plaεmid, chromosome, virus) that functionε aε an autonomous unit of DNA replication in vivo; i.e. , capable of replication under its own control.

A "vector" iε a replicon, εuch aε plaεmid, phage or cos id, to which another DNA εegment may be attached so aε to bring about the replication of the attached εegment.

A "DNA molecule" referε to the polymeric form of deoxyribonucleotideε (adenine, guanine, thymine, or cytoεine) in itε either εingle εtranded form, or a double-εtranded helix. This term referε only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, thiε term includeε double-stranded DNA found, inter alia, in linear DNA molecules (e.g. , restriction fragments) , viruses, plasmids, and chromosomeε. In discuεεing 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 5' to 3 ' direction along the nontranscribed strand of DNA (i.e. , the strand having a sequence homologous to the mRNA) .

A DNA "coding sequence" is a double-stranded DNA sequence which iε transcribed and translated into a polypeptide in

vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5* (amino) terminus and a translation stop codon at the 3* (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (-S-S- , mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3 ^■ to the coding sequence.

Transcriptional and translational control εequenceε are DNA regulatory sequences, such as promoters, enhancers, polyadenylation εignalε, terminatorε, and the like, that provide for the expreεεion of a coding sequence in a host cell.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymeraεe in a cell and initiating transcription of a downstream (3* direction) coding sequence. For purposes of defining the present invention, the promoter εequence iε bounded at itε 3* terminuε by the transcription initiation site and extends upstream (5 ¹ direction) to include the minimum number of baεeε or elements neceεεary to initiate tranεcription at levelε detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease SI) , as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

A coding sequence is "under the control" of transcriptional and translational control sequenceε in a

cell when RNA polymerase transcribeε the coding εequence into mRNA, which is then translated into the protein encoded by the coding sequence.

A "signal sequence" can be included before the coding sequence. This εequence encodes a signal peptide, N- teπninal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide .is clipped off by the hoεt cell before the protein leaveε the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes. For instance, alpha-factor, a native yeast protein, is secreted from yeast, and its signal sequence can be attached to heterologous proteins to be secreted into the media (See U.S. Patent 4,546,082, EPO 0 116 201, publication date 12 January 1983; U.S. Patent Application Ser. No. 522,909, filed 12 August 1983) . Further, the alpha-factor leader and its analogs have been found to secrete heterologous proteins from a variety of yeast, such as Saccharomyces and Kluyveromyceε, (EPO 88312306.9 filed 23 December 1988; U.S. Patent Application Ser. No. 139,682, filed 30 December 1987, and EPO Pub. No. 0 301 669, publication date 1 February 1989) .

A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The tranεforming DNA may or may not be integrated (covalently linked) into chromoεomal DNA making up the genome of the cell. In prokaryoteε, yeaεt, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such aε a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This εtability iε demonεtrated by the ability of the

eukaryotic cell to eεtablish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cellε derived from a εingle cell or common anceεtor by mitoεis. A "cell line" iε a clone of a primary cell that is capable of stable growth in vitro for many generations.

Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions aε defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g. , Maniatis et al. , supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.

A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that iε not found in asεociation with the larger molecule in nature. Thus, when the heterologous region encodeε a mammalian gene, the gene will uεually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence 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 aε defined herein.

A composition comprising "A" (where "A" iε a εingle protein, DNA molecule, vector, etc.) iε εubεtantially

free of "B" (where "B" compriseε one or more contaminating proteinε, DNA moleculeε, vectors, etc.) when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of εpecieε to which A and B belong) in the composition is "A". Preferably, "A" comprises at least about 90% by weight of the A+B species in the composition, most ^* preferably at least about 99% by weight. It is also preferred that a composition, which is substantially free of contamination, contain only a single molecular weight species having the activity or characteristic of the species of interest.

An "antibody" iε any immunoglobulin, including antibodieε and fragments thereof, that binds a specific epitope. The term encompasεeε, inter alia, polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Noε. 4,816,397 and 4,816,567.

Intron-free DNA provided by the preεent invention iε novel, εince it is believed that the naturally-occurring human genes contain introns. Hence, the term "intron- free" excludes the DNA εeguenceε which naturally occur in the chromosomes of human or bovine cellε. The preεent invention also encompaεseε the intron-free cDNA εequenceε derivable from the DNA εequenceε disclosed herein.

In itε primary aεpect, the present invention concerns the isolation and identification of a newly discovered particular factor hereinafter referred to aε the inflammatory cytokine, macrophage inflammatory protein-2 or MIP-2, that has been found to be present in macrophages or macrophage cell lines that are stimulated by materials referred to herein as stimulator materials, that characteriεtically accompany an invaεive εtimuluε, such as bacteria, virus, certain tumorε, protozoa and other toxins such as endotoxin, or an idiopathic state. As with the mediator substance disclosed in U.S.. Patent

No. 4,603,106, the preεent inflammatory cytokine, which has been determined to be a component of the former mediator substance, appears to be capable of causing certain conditions such aε inflammation to develop in the tissues of a mammal, which reflect the reaction of a mammal in a stimulated or spontaneous pathological state.

In particular, the inflammatory cytokine appears to be capable of inducing localized inflammation when administered subcutaneously which inflammation iε characterized by polymorphonuclear cell infiltration. Alεo, the cytokine iε a potent chemotactic agent for human polymorphonuclear leukocyteε while inducing little or no che okineεiε or an oxidative burεt in human neutrophilε in vitro, which conditions reflect the influence of a cytokine involved in mobilization by the mammalian hoεt against an invasive stimulus. While the full and exact role played by the preεent inflammatory cytokine is unclear, it iε theorized that the cytokine in conjunction with other factorε previouεly identified and those yet to be elucidated, functions aε part of a communication εyste between the immune system of the host and other body tissues and organs.

The ability of the preεent inflammatory cytokine to bind to heparin gave riεe to the conεideration that the cytokine might correspond to certain heparin-binding growth factors such as FGF or PDGF. However, data indicating that the inflammatory cytokine is not mitogenic for smooth muscle cellε εuggeεts a distinction from these known growth .factors. Accordingly, what iε certain at thiε time, iε that the cytokine of the present invention participates in the development of the inflammatory response that iε known to be a part of hoεt responεes such aε to invasion.

The present inflammatory cytokine has been confirmed to comprise a protein that posseεεeε a molecular mass of

approximately 6,000 daltons on NaDodSO-PAGE and fractionateε from a gel filtration column with an apparent molecular maεs of approximately 10,000 daltons. The cDNA for MIP-2 has been cloned and predictε a mature protein of 74 amino acids in length with a predicted molecular weight of 7,908. In contrast to MIP-1 and cachectin/TNF, MIP-2 is cationic and does not bind to an anion exchange column equilibrated at pH 8.0. In addition, the determination of the N-terminal partial amino acid sequence and the full sequence of the mature protein confirms that the specific protein structure of the present inflammatory cytokine differs from that of other known factors. Accordingly, both structural and functional distinctions between the present inflammatory cytokine and the known factorε of the prior art exists aε is confirmed by the data set forth in Example 1.

More particularly, the inflammatory cytokine of the present invention posεeεεes certain other characteristics in conjunction with those outlined above, in that it iε capable of binding to heparin at high εalt concentrations, e.g. approximately 0.7 M, and demonstrates colony stimulating factor activity. The cytokine is alεo diεtinctive in thoεe activitieε that it lacks, εuch aε its inability to suppreεs the anabolic enzyme lipoprotein lipaεe (LPL) , to cauεe the cytotoxicity of cachectin/TNF-senεitive L929 cells, stimulate the blastogenesiε of endotoxin-reεiεtant C3H/HeJ thymocyteε or to induce the production of cachectin/TNF by primary thioglycollate-elicited mouεe macrophage cellε. All of these latter activities are exhibited by the other known macrophage-derived mediator factors whoεe general characteristics and activities have identified them as participants in the hoεt reεponse to invasion. In addition, the present inflammatory cytokine is distinguishable from other factors such aε MIP-1 by its inability either to induce fever in rabbits, or to

induce superoxide formation or respiratory burst in human neutrophilε.

The activity profile presented above accordingly distinguishes the present .inflammatory cytokine from those known factors and confirms in conjunction with the amino acid sequencing data presented herein, that the present inflammatory cytokine is indeed distinct from the other macrophage-derived mediator factors.

As stated earlier, the primary amino acid sequence shown in FIGURE 2 and the full sequence shown in FIGURE 7 are only illustrative, and similar εequenceε may reεult in proteinε which have substantially equivalent or enhanced activity. These modifications may be deliberate, for example, εuch aε modificationε obtained through site-directed mutageneεiε, or may be accidental, εuch aε those obtained through mutations in hoεtε which are MIP-2 producerε. All of thoεe modifications are included in the present invention, aε long aε the MIP-2 activity, aε defined above, is retained. Accordingly, the definition of MIP-2 as εtated herein and elεewhere in the specification includes proteins having an amino acid sequence subεtantially equivalent to that in FIGURES 2 and 7 aε well aε other substantially homologcuε analogs and allelic variationε within itε εcope.

The preparation of the inflammatory cytokine waε discussed in brief earlier herein, and iε confirmed to be capable of proceeding in the inεtance of the native material by the initiation of the incubation of a variety of cells with stimulator materials from invasive stimuli. In particular, the cell line RAW 264.7 may be utilized to initiate the production of the material from which the inflammatory cytokine may be isolated. The murine macrophage cell line RAW 264.7 has facilitated the isolation of the inflammatory cytokine in quantities large enough to permit analyεiε and purification.

Naturally, other cell lines or other sources for the development of either the material from which the inflammatory cytokine is thereafter iεolated, or the inflammatory cytokine itεelf, are contemplated herein and the preεent invention iε accordingly not limited.

As discussed earlier herein, alternate means such as by genetic replication which may be conducted in accordance with many of the generic principles of recombinant technology that are well known in the art, are contemplated herein in accordance with the present invention.

Accordingly, MIP-2 nucleic acid sequences may be obtained from the deduced amino acid sequence by recombinant DNA methods, such aε by screening reverse tranεcriptε of mRNA, or by εcreening genomic libraries from any cell. The DNA may alεo be obtained by εyntheεizing the DNA using commonly available techniqueε and DNA synthesizing apparatus. Synthesiε may be advantageouε because unique restriction sites may be introduced at the time of preparing the DNA, thereby facilitating the use of the gene in vectors containing restriction εiteε not otherwise preεent in the native source. Furthermore, any deεired εite modification in the DNA may be introduced by synthesiε, without the need to further modify the DNA by mutageneεiε.

A general procedure for isolating DNA encoding the present inflammatory cytokine from human, murine, or other sourceε iε to conεtruct a cDNA library from mRNA isolated from the appropriate cells or tissue; and screen with labeled DNA probes encoding portions of the polypeptide chain in order to detect cloneε in the cDNA library that contain homologous sequences.

Alternatively, one may isolate the DNA by polymeraεe chain reaction (PCR) amplification of the cDNA (from mRNA) and εubclone and εcreen with labeled DNA probes;

and then analyze the cloneε by restriction enzyme analysis and nucleic acid sequencing so as to identify full-length clones. If full-length clones are not present in the library, appropriate fragments from the various clones may be recovered and ligated at restriction sites common to the clones to asεemble a clone encoding a full-length molecule.

A suitable and preferred DNA probe is set forth in the accompanying examples. Any sequences miεεing from the 5* end of the MIP-2 cDNA may be obtained by the 3 ^» extenεion of the synthetic oligonucleotides complementary to MIP-2 sequences using mRNA as a template (so-called primer extension) , or homologous sequences may be supplied from known cDNAs derived from murine sequences as shown in FIGURE 7. Thiε will be more particularly deεcribed in Example 2; however, it iε realized that once being provided with intron-free DNA encoding murine MIP-2 and itε leader sequences aε deεcribed herein, one of ordinary skill in the art would recognize that other precisely hybridizing probes may be prepared from the deεcribed sequenceε in order to readily obtain the deεired gene.

Vectorε are used to simplify manipulation of the DNA which encodeε the MIP-2 polypeptide, either for preparation of large quantitieε of DNA for further processing (cloning vectors) or for expresεion of the MIP-2 polypeptide (expresεion vectorε) . Vectorε compriεe plasmids, viruseε (including phage) , and integratable DNA fragments, i.e., fragmentε that are integratable into the host genome by recombination. Cloning vectorε need not contain expression control sequences. However, control sequences are needed in an expresεion vector, and these control sequences include transcriptional and translational control sequenceε εuch aε a tranεcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable riboεome binding sites (for prokaryotic expression) , and sequenceε

which control termination of transcription and tranεlation. The expression vector should preferably include a- selection gene to facilitate the stable expression of MIP-2 and/or to identify transformantε. However, the εelection gene for maintaining expreεsion can be supplied by a separate vector in cotransformation systems using eukaryotic host cells.

For expreεεion suitable vectors generally will contain replicon (origins of replication, for use in non- integrative vectors) and control sequences which are derived from specieε compatible with the intended expreεεion hoεt. By the term "replicable" vector as used herein, it is intended to encompass vectors containing such replicons as well as vectors which are replicated by integration into the host genome. Cellε are then tranεfor ed or tranεfected with vectors containing MIP-2 encoding DNA and are now identified as transformed host cells. MIP-2 expressed from these transformed hoεtε will be either deposited mtracellularly or secreted into the periplasmic εpace or the culture εupernatant, depending upon the hoεt cell εelected and the presence of suitable procesεing signals in the expressed peptide, e.g. homologous or heterologouε εignal εequenceε.

Suitable hoεt cellε for expresεion can be prokaryotic or eukaryotic cellε. Prokaryoteε include Gram negative or Gram positive organisms, for example E. coli or Bacillus subtilis. Eukaryotic cells include yeast, baculoviruε or higher eukaryotic cells such as establiεhed cell lineε of mammalian origin.

Expreεεion vectorε for hoεt cells ordinarily include an origin of replication (unlesε it iε an integrating vector) , a promoter located upεtream from the MIP-2 coding εequence, together with a ribosome binding site, a polyadenylation site, and a transcriptional termination sequence. Those of ordinary skill will appreciate that

certain of theεe sequenceε are not ^" required for expreεsion in certain hostε. A non-integrating expression vector for uεe with microbeε need only contain an origin of replication recognized by the hoεt, a promoter which will function in the host and a selection gene.

An expression vector is constructed according to the present invention so that the MIP coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed and translated under the "control" of the control sequenceε (i.e. , RNA polymerase which bindε to the DNA molecule at the control sequences transcribes the coding sequence) . The control sequences may be ligated to the coding sequence prior to insertion into a vector, such aε the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expreεsion vector which already containε the control sequenceε and an appropriate reεtriction εite. For expreεεion of MIP-2 in prokaryoteε and yeaεt, the control sequenceε will neceεsarily be heterologous to the coding sequence. If the hoεt cell iε a prokaryote, it iε alεo neceεεary that the coding sequence be free of introns (e.g. , cDNA) . If the selected host cell iε a mammalian cell, the control sequences can be heterologous or homologous to the MIP coding sequence, and the coding sequence can either be genomic DNA containing introns or cDNA.

Expression vectors must contain a promoter which is recognized by the host organism. Promoters commonly known and available which are used in prokaryotic recombinant DNA expression include the β-lactamase (penicillinase) and lactose promoter systems, a tryptophan (trp) promoter syεtem and the tac promoter.

While these are commonly used, other known microbial promoterε are εuitable.

In addition to prokaryotes, eukaryotic cellε such as yeast may be transformed with MIP-2 encoding vectors. Yeast vectorε generally will contain an origin of replication or an autonomouεly replicating sequence (ARS) , (if non-integrating) a promoter, DNA encoding MIP- 2, sequences for polyadenylation and transcription termination, and a selection gene.

Of particular interest to the present invention are yeast species within the genera Pichia, Kluyveromyceε, Saccharomyces, Schizoεaccharomyceε and Candida. Of particular interest are the Saccharomyces εpecieε S. cerevisiae. S. carlsbergensiε. S. diaεtaticus, S. douglasii. S. kluweri, S. norbensis. and S. oviformis. Species of particular interest in the genus Kluyveromyceε include K. lactis, and in the genus Pichia include P_i_ pastoriε. Since the claεεification of yeaεt may change in the future, for the purpoεeε of thiε invention, yeaεt shall be defined aε described in Biology and Activitieε of Yeast (F.A. Skinner, S.M. Paεεmore and R.R. Davenport, edε. 1980) (SOC. APP. BACTERIOL. SYMP. SERIES NO. 9). In addition to the foregoing, thoεe of ordinary εkill in the art are preεu ably familiar with the biology of yeaεt and the manipulation of yeaεt geneticε. See, e.g. Biochemiεtrv and Geneticε of Yeaεt (M. Bacila, B.L. Horecker and A.O.M. Stoppani edε. 1978) ; The Yeastε (A.H. Roεe and J.S. Harriεon eds., 2nd ed. , 1987); The

Molecular Biology of The Yeast Saccharomyces (Strathern et al. eds. 1981). The disclosures of the foregoing references are incorporated herein by reference.

Suitable promoter sequences in yeaεt vectors include the promoters for the glycolytic enzymes εuch aε enolaεe, 3- phosphoglycerate kinaεe, glyceraldehyde-3-phoεphate dehydrogenaεe, hexokinaεe, pyruvate decarboxylaεe _f

phosphofructokinase, glucoεe-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephoεphate iεomerase, phosphoglucoεe isomerase, and glucokinase.

Other yeast promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 1 or 2, isocytochrome C, acid phosphataεe, as well as enzymes responsible for maltose and galactose utilization.

Higher eukaryotic cell cultureε may be uεed, whether from vertebrate or invertebrate cellε, including inεects, and the procedures of propagation thereof are known. See, for example, Tiεsue Culture. Academic Preεε, Kruεe and Patterεon, editorε (1973) .

Suitable host cells for expressing MIP-2 in higher eukaryotes include: monkey kidney CVI line tranεformed by SV40 (C0S-7, ATCC CRL 1651); baby ha εter kidney cellε (BHK, ATCC CRL 10) ; Chinese hamster ovary-eells-DHFR (described by Urlaub and Chasin, PNAS (USA) 72: 4216 (1980)); mouεe Sertoli cellε (TM4, Mather, J.P., Biol. Reorod. 23: 243-251 (1980) ) ; monkey kidney cellε (CVI ATCC CCL 70) ; African green monkey kidney cellε (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cellε (HELA, ATCC CCL 2); canine kidney cellε (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442) ; human lung cells (W138, ATCC CCL 75); human liver cellε (Hep G2, HB 8065); mouse mammary tumor (MMT 060652, ATCC CCL 51); rat hepatoma cells (HTC, Ml, 54, Baumann, M. , et al., J. Cell Biol. 85: 1-8 (1980)) and TRI cells (Mather, J.P., et al. , Annals N.Y. Acad. Sci. 383: 44-68 (1982)). Commonly used promoters are derived from polyoma, adenovirus 2, and simian virus 40 (SV40) . It will be appreciated that when expresεed in eukaryotic rather than prokaryotic cellε, the recombinant MIP-2 may have higher molecular weight due to glycoεylation. It iε therefore

intended that partially or completely glycoεylated formε of MIP-2 having molecular weightε greater than the predicted molecular weight of 7,908 are within the εcope of thiε invention aε well aε itε unglycoεylated formε.

A number of prokaryotic expreεεion vectorε are known in the art. See, e.g., U.S. Patent Nos. 4,440,859; 4,436,815; 4,431,740; 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 2,008,123; GB 2,007,675; and European Pub. No. 103,395. Preferred prokaryotic expresεion εyεtems are in E. coli.

Other preferred expreεsion vectorε are- those for use in eukaryotic εyεtems. An exemplary eukaryotic expreεεion system is that employing vaccinia viruε, which iε well- known in the art. See. e.g. , Macket et al. (1984) J. Virol. .49:857; "DNA Cloning," Vol. II, pp. 191-211, supra; PCT Pub. No. WO 86/07593. Yeast expresεion vectorε are known in the art. See, e.g.. U.S. Patent

Noε. 4,446,235; 4,443,539; 4,430,428; see also European Pub. Noε. 103,409; 100,561; 96,491. Another preferred expresεion εyεte iε vector pHΞl, which tranεformε Chineεe hamεter ovary cellε. See PCT Pub. No. WO 87/02062. Mammalian tiεεue may be cotranεfor ed with DNA encoding a selectable marker εuch aε dihydrofolate reductaεe (DHFR) or thy idine kinaεe and DNA encoding MIP-2.

If wild type DHFR gene iε employed, it iε preferable to select a host cell which is deficient in DHFR, thus permitting the use of the DHFR coding sequence as marker for successful transfection in hgt ^" medium, which lackε hypoxanthine, glycine, and thymidine. An appropriate host cell in this case iε the Chineεe hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated aε deεcribed by Urlaub and Chaεin, 1980, Proc. Nat. Acad. Sci. (USA) 22: 4216. Expreεεion vectorε

derived from baculovirus for uεe in insect cells are known and available in the art. See Lucklow and Summers, Biotechnology. 6_, p. 47-55.

Depending on the expression system and hoεt selected,

MIP-2 is produced by growing host cells transformed by an exogenous or heterologous DNA construct, such as an expression vector described above and in Example 2 herein, under conditions whereby the MIP-2 protein is expressed. The MIP-2 is then isolated from the hoεt cells and purified. If the expression syεtem secretes MIP-2 into growth media, the protein can be purified directly from cell-free media. If the MIP-2 protein is not secreted, it is isolated from cell lyεateε. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.

The recombinantly-made MIP-2 may be recovered from transformed cells in accordance with known procedures. Preferably, an expreεεion vector will be uεed which provideε for secretion of MIP-2 from the transformed cellε; thus the cellε may be εeparated by centrifugation. MIP-2 iε typically purified by general protein purification techniqueε, including, but not limited to, εize excluεion, ion-exchange chromatography, HPLC, and the like.

Once a coding sequence for MIP-2 has been prepared or isolated, it can be cloned into any suitable vector and thereby maintained in a composition of cellε which is substantially free of cells that do not contain a MIP-2 coding sequence (e.g. , free of other library clones) . Numerous cloning vectors are known to those of skill in the art. Examples of recombinant DNA vectors for cloning and hoεt cells which they can tranεform include the various bacteriophage lambda vectors (E. coli) , pBR322 (E . coli) , pACYC177 (E_ _! _cgli) , pKT230 (gram-negative bacteria) , pGV1106 (gram-negative bacteria) , pLAFRl

(gram-negative bacteria) , pME290 (non-E. coli gram- negative bacteria) , pHV14 (E. coli and Bacilluε subtiliε) , pBD9 (Bacillus) , pIJ61 (Streptomyces) , pUC6 (Streptomyces) , actinophage, φC31 (Strepto yceε) , YIp5 (Saccharomyceε) , YCpl9 (Saccharomyces) , YEp24 (Saccharomyces) , pCl/1 (Saccharomyces) , XRpl7 (Saccharomyces) , and bovine papilloma viruε (mammalian cells) . See generally. DNA Cloning: Vols. I & II, supra; T. Maniatis -et al., supra; B. Perbal, supra; Botstein et al. (1979) GENE 8: 17-24; Brake et al. (1984) PROC NATL. ACAD. SCI. USA £1: 4642-4646; Stnichcomb et al. (1982) J. MOL. BIOL. 158.: 157.

Alternatively MIP-2 may be made by conventional peptide syntheεiε, for instance, by using the principles of the Merrifield syntheεiε and uεing commercial automatic apparatuε deεigned to employ the methodε of the Merrifield εyntheεiε. Peptideε prepared uεing conventional peptide εyntheεiε may be purified uεing conventional affinity chromatography, gel filtration and/or RP-HPLC

It iε further intended from the nucleotide εequenceε that MIP-2 analogε are within the εcope of the preεent invention. Analogε, εuch aε fragments, may be produced, for example, by pepsin digestion of MIP-2. Other analogs, such as muteins, can be produced by standard site-directed mutagenesiε of MIP-2 coding sequences. Analogs exhibiting "MIP-2 activity" may be identified by known in vivo and/or in vitro assayε.

As mentioned above, a DNA sequence encoding MIP-2 can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codonε for the MIP-2 amino acid εequence. In general, one will εelect preferred codons for the intended hoεt if the εequence will be uεed for expresεion. The complete sequence iε asεembled from overlapping oligonucleotideε

prepared by standard methodε and assembled into a complete coding sequence. See, e.g. , Edge Nature 292:756 (1981); Nambair, et al. Science 223:1299 (1984); Jay et al. J. Biol. Chem. 259_:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes which will express MIP-2 analogs or "muteins". Alternatively, DNA encoding muteins can be made by site- directed πtutagenesis of native MIP-2 genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.

Site-directed mutageneεiε iε generally uεed to create analogε from a complete coding εequence. Site-directed mutagenesiε is conducted using a primer synthetic oligonucleotide complementary to a single stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct εyntheεis of a strand complementary to the phage, and the resulting double-εtranded DNA is transformed into a phage-supporting host bacterium. Cultureε of the tranεfor ed bacteria are plated in top agar, permitting plaque formation from εingle cellε which harbor the phage.

Theoretically, 50% of the new plaqueε will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence. The reεulting plaqueε are hybridized with kinased εynthetic primer at a temperature which permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization. Plaques which hybridize with the probe are then picked, cultured, and the DNA recovered.

A general method for site-specific incorporation of unnatural amino acids into proteins is described in

Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, SCIENCE 244: 182-188 (April 1989). This method may be.used to create analogε with unnatural amino acids.

The inflammatory cytokine in accordance with the present invention was isolated and analyzed in mice as set forth in Example 1. Human inflammatory cytokine (MIP-2) is presumably similar to mouse MIP-2, since the mouse MIP-2 has an effect upon human neutrophilε. Aε disclosed herein, this activity of the inflammatory cytokine may be harnessed by administering the inflammatory cytokine to the εituε of tiεεue infection to promote the delivery of neutrophilε to that location.

Aε diεcuεεed earlier, the inflammatory cytokine or itε binding partner(ε) or other ligandε or agents exhibiting either mimicry or antagonism to the cytokine or control over ^" its production, may be prepared in pharmaceutical compositionε, with a εuitable carrier and at a εtrength effective for adminiεtration by various means to a patient having a tiεεue infection or other pathological derangement, for the treatment thereof. A variety of adminiεtrative techniques may be utilized, among them topical applications as in ointments or on εurgical and other topical applianceε such as, εurgical εpongeε, bandages, gauze pads, and the like. Alεo, such compositionε may be administered by parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, including delivery in an irrigation fluid used to wash body wound areas, catheterizations and the like. Average quantitieε of the inflammatory cytokine may vary and in particular εhould be baεed upon the recommendationε and preεcription of a qualified phyεician or veterinarian.

Alεo, antibodies including both polyclonal and monoclonal antibodieε, and drugε that modulate the production or

activity of the inflammatory cytokine may posεeεs certain therapeutic applications and may thus be utilized for the purpose of treating the effects of poεt infection attributable the action of the inflammatory cytokine, such as inflammation and fever. In particular, the inflammatory cytokine may be used to produce both polyclonal and monoclonal antibodies to itself in a variety of cellular media, by known techniques such aε the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cellε.

The general methodology for making monoclonal antibodieε by hybridomaε iε well known. Immortal, antibody- producing cell lineε can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epεtein-Barr viruε. See, e.g.. M. Schreier et al. , "Hybridoma Techniqueε" (1980) ; Hammerling et al. , "Monoclonal Antibodieε And T-cell Hybridomas" (1981) ; Kennett et al.. "Monoclonal Antibodieε" (1980); see alεo U.S. Patent Noε. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890.

Panels of monoclonal antibodieε produced againεt MIP-2 peptideε can be screened for various properties; i.e. , isotype, epitope, affinity, etc. Of particular interest are monoclonal antibodies that neutralize the activity of MIP-2. Such monoclonals can be readily identified in MIP-2 activity assays. High affinity antibodies are also useful in immunoaffinity purification of native or recombinant MIP-2.

The resulting antibodies could also be prepared in a suitable pharmaceutical composition and administered to avert or treat the undesired condition. The exact quantities, intervals of adminiεtration and administrative techniques respecting such pharmaceutical

compoεitionε may vary in accordance with thoεe known in the medical artε, and upon the εpecific inεtruction of a qualified physician or veterinarian.

The present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of invasive stimuli by reference to their ability to elicit the activities which are affected by the present inflammatory cytokine. As mentioned earlier, the inflammatory cytokine can be used to produce antibodies to itself by a variety of known techniques, and such antibodieε could then be isolated and utilized as in testε for the presence of the inflammatory cytokine in suεpect mammalian hoεtε.

Antibody(ieε) to the inflammatory cytokine can be produced and iεolated by εtandard methodε including the well known hybridoma techniques. For convenience, the antibody(ieε) to the inflammatory cytokine will be referred to herein aε Ab ₁ and antibody(ieε) raiεed in another species aε Ab ₂ .

The preεence of inflammatory cytokine activity in mammalε can be ascertained by the uεual immunological procedureε applicable to εuch determinations. A number of useful procedures are known. Three εuch procedureε which are eεpecially uεeful utilize either the inflammatory cytokine labeled with a detectable label, antibody Ab., labeled with a detectable label, or antibody Ab ₂ labeled with a detectable label. The procedureε may be summarized by the following equations wherein the asterisk indicates that the particle is labeled, and "Cyt" εtandε for the inflammatory cytokine: A. Cyt* + Ab ₁ = Cyt*Ab ₁ B. Cyt + Ab* = CytAb,*

C Cyt + Ab ₁ + Ab ₂ * = CytAb ₁ Ab ₂ *

The procedureε and their application are all familiar to thoεe εkilled in the art and accordingly may be utilized

within the scope of the present invention. The "competitive" procedure, Procedure A, is deεcribed in U.S. Patent Noε. 3,654,090 and 3,850,752. Procedure C, the "εandwich" procedure, is described in U.S. Patent Noε. RE 31,006 and 4,016,043. Still other procedures are known such as the "double antibody", or "DASP" procedure.

In each instance, the inflammatory cytokine formε complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable labei. The fact that a complex has formed and, if deεired, the amount thereof, can be determined by known methodε applicable to the detection of labelε.

It will be seen from the above, that a characteristic property of Ab ₂ is that it will react with Ab,. This iε becauεe Ab, raiεed in one mammalian species has been used in another εpecieε aε an antigen to raise the antibody Ab ₂ . For example, Ab ₂ may be raised in goats uεing rabbit antibodieε aε antigenε. Ab ₂ therefore would be anti-rabbit antibody raiεed in goatε. For purpoεeε of this description and claims, Ab, will be referred to aε a primary or anti-inflammatory cytokine antibody, and Ab ₂ will be referred to as a εecondary or anti-Ab, antibody.

The labelε most commonly employed for these εtudieε are radioactive elements,- enzymes, chemicals which fluoreεce when expoεed to ultraviolet light, and others.

A number of fluorescent materials are known and can be utilized aε labelε. These include, for example, fluorescein, rhodamine and auramine. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.

The inflammatory cytokine or itε binding partner(ε) can also be labeled with a radioactive element or with an

enzyme. The radioactive label can be detected by any of the currently available counting procedureε. The preferred iεotope may be selected from ^U C , ¹³¹ I, ³ H, ¹²⁵ I and ³⁵ S.

Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric or gasometric techniques. The enzyme iε conjugated to the selected particle by reaction with bridging molecules such as carbodiimideε, diisocyanateε, glutaraldehyde and the like. Many enzymeε which can be uεed in theεe procedureε are known and can be utilized. The preferred are peroxidaεe, β-glucuronidaεe, β-D-glucoεidaεe, β-D-galactoεidaεe, ureaεe, glucoεe oxidaεe plus peroxidase and alkaline phoεphataεe. U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their diεcloεure of alternate labeling material and methodε.

A particular aεεay system developed and utilized in accordance with the present invention, iε known aε a receptor aεεay. In a receptor aεεay, the material to be aεεayed iε appropriately labeled and then certain cellular test colonieε are inoculated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.

Accordingly, a purified quantity of the inflammatory cytokine may be radiolabeled, after which binding studies would be carried out using for example, recently differentiated neutrophils. Solutionε would then be prepared that contain variouε quantitieε of labeled and unlabeled inflammatory cytokine and cell εamples would then be inoculated and thereafter incubated. The

reεulting cell monolayerε are then waεhed, εolubilized and then counted in a gamma counter for a length of time sufficient to yield a standard error of <5%. This data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor aεεay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic.

In a further embodiment of this invention, commercial test kitε suitable for use by a medical εpecialiεt may be prepared to determine the preεence or abεence of inflammatory cytokine in a suspected mammalian host. In accordance with the testing techniques discusεed above, one class of εuch kitε will contain at leaεt the labeled inflammatory cytokine or itε binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g.,

"competitive", "sandwich", "DASP" and the like. The kitε may alεo contain peripheral reagents such as buffers, εtabilizerε, etc.

Accordingly, a teεt kit may be prepared for the demonεtration of the reaction of a mammalian host to invasive εtimuli, comprising:

(a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of the preεent inflammatory cytokine or a specific binding partner thereto, to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise: (a) a known amount of the inflammatory cytokine aε deεcribed above (or a binding partner) generally bound to

a solid phase to form an immunosorbent, or in the alternative, bound to a suitable tag, or plural εuch end productε, etc. (or their binding partnerε) one of each; (b) if neceεεary, other reagentε; and (c) directions for use of said teεt kit.

In a further variation, the test kit may be prepared and used for the purposeε stated above, which operates according to a predetermined protocol (e.g. "competitive", "sandwich", "double antibody", etc.), and comprises:

(a) a labeled component which has been obtained by coupling the inflammatory cytokine to a detectable label;

(b) one or more additional immunochemical reagentε of which at leaεt one reagent iε a ligand or an immobilized ligand, which ligand iε selected from the group consisting of:

(i) a ligand capable of binding with the labeled component (a) ; (ii) a ligand capable of binding with a binding partner of the labeled component (a) ;

(iii) a ligand capable of binding with at leaεt one of the component(ε) to be determined; and

(iv) a ligand capable of binding with at leaεt one of the binding partners of at least one of the component(ε) to be determined; and

(c) directionε -for the performance of a protocol for the detection and/or determination of one or more componentε of an immunochemical reaction between the inflammatory cytokine and a specific binding partner thereto.

In accordance with the above, an assay εyεtem for screening potential drugs effective to modulate the synthesiε, releaεe, or activity of the inflammatory cytokine may be prepared. In a firεt procedure, the test drug could be administered to a εtimulated macrophage εample to determine itε effect upon the production of the

inflammatory cytokine. In an alternate procedure, the inflammatory cytokine may be introduced into a cellular teεt system such as neutrophils, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter 'examined to observe any changes in the activity of the inflammatory cytokine, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known inflammatory cytokine.

The following examples set forth the details of the isolation and identification of the preεent inflammatory cytokine, the observations noted as to itε activity, defining both the distinctions and similarities in activity between the present inflammatory cytokine and thoεe factorε identified earlier both by applicants and by otherε in the field, and the cloning, sequencing and expreεεion of the cytokine MIP-2. Naturally, the εpecific materialε and techniqueε εet forth hereinafter are exemplary only and may vary, so that the following iε presented aε illuεtrative but not reεtrictive of the present invention.

EXAMPLE 1

The following experiments were conducted to identify and characterize the inflammatory cytokine of the present invention. Initially, the mediator subtance was cultured, the inflammatory cytokine waε iεolated and itε structure then partially determined, after which a battery of tests were conducted in an effort to elucidate its activities, and where possible, to establish or refute identity with other known macrophage-derived factorε.

MATERIALS AND METHODS

Materials - Supernatants from COS cellε transfected with plasmid containing the hamster or human gro gene or control plasmid alone were provided by A. Anisowicz and R. Sager. Partially purified human NAP-1 protein was a gift from J. Van Damme and purified human NAP-1 protein was given by T. Yoεhimura and E. Leonard. All other reagentε were obtained from Sigma (St. Louiε, MO) .

Animals - C3H/HeN mice were obtained from Charles River (Kingston, NY) . Mice of the endotoxin-reεiεtant C3H/HeJ strain were obtained from Jackson Laboratories (Bar Harbor, ME) .

Cell Culture - The mouse macrophage cell line RAW 264.7 and the cachectin/TNF-εenεitive cell line L929 were obtained from American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 and Dulbecco'ε modified MEM ((DMEM) GIBCO, Grand Island, NY), respectively. Both media were supplemented with 20mM Hepes and 10% fetal bovine serum (Hyclone, Logan, UT) . For the production of stimulated RAW 264.7 supernatants, cells were grown in 150 mm tisεue culture diεheε (Falcon) in RPMI plus 10% fetal bovine serum until they reached confluency. The cellε were waεhed five times in Hankε' balanced salt solution and the medium waε replaced with serum-free RPMI supplemented with 1 μg/ml of lipopolysaccharide (LPS W, E. coli 0127:B8, Difco, Detroit, MI). The cellε were incubated at 37°C for 16-18 hours and the εupernatantε filtered through 0.22 μm filters.

Purification of MIP-2 - MIP-2 was purified uεing methodology previouεly deεcribed for MIP-1 (S.D. Wolpe, G. Davatelis, B. Sherry, B. Beutler, D.G. Hesse, H.T. Nguyen, L.L. Moldawer, C.F. Nathan, S.F. Lowry, & A. Cerami (1988), J. EXP. MED., 162:570-581). The degree of

purification was followed by sodium dodecyl sulfate (NaDodS0 ₄ -PAGE) with silver staining. In brief, two liters of conditioned supernatant from endotoxin-stimulated RAW 264.7 cells were concentrated and diafiltrated againεt 20 mM Tris-HCl buffer, pH 8.0, and applied to a Mono Q 10/10 (anion exchange) column (Pharmacia LKB Biotechnology, Rahway, NJ) . Greater than 90% of the MIP-2 was observed not to bind to the column and was recovered in the effluent.

Peak MIP-2 containing fractions were applied to a heparin-conjugated Sepharose (Pharmacia LKB) column equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and eluted with a 0-2 M NaCl linear gradient in the same buffer. MIP-2 eluted at approximately 0.75 M NaCl. Peak fractions were concentrated in a Centricon ultrafiltration device with a 3,000 dalton molecular weight cutoff (A icon Corp. , Danvers, MA) and applied to a Superoεe 12 (gel filtration [Pharmacia LKB]) column equilibrated with 100 mM ammonium acetate. From two literε of RAW 264.7 conditioned medium (which equalled approximately 100 g total protein), a quantity of 0.5 g of MIP-2 waε generally iεolated aε aεεeεsed by Bradford protein aεεay (Biorad, Rockville Center, NY) using bovine gamma globulin as standard. By comparison, approximately 2 mg of MIP-1 and 1 mg of cachectin/TNF could be purified from a like batch of conditioned medium.

Immunoblot Analysis - Antiεera to MIP-2 were produced in rabbits injected once subcutaneouεly with 10 μg of purified protein emulsified in complete Freund's adjuvant, and once one month later with 10 μg of purified protein in incomplete Freund's adjuvant. Antisera were collected one week after the second immunization. Approximately 50 ng of pure MIP-2 or NAP-1 protein, or roughly equivalent amounts of human or hamster gro protein from the serum-free supernatantε of COS cellε tranεfected with the appropriate vector, were εubjected

to NaDodS0 ₄ -PAGE in 10-18% linear gradient gelε and tranεferred to nitrocellulose using a tranεblot apparatuε (Biorad) . Blotε were blocked in 5% dry milk (Alba) for 1-2 hrε. and incubated in antiεerum diluted 1:100 for 1 hr. at room temperature. The blots were washed three times in phosphate-buffered saline containing 0.05% Tween 20 and 0.05% thimerosal and bound antibody was detected with an alkaline phosphatase-conjugated second antibody (Promega Biotec, Madison, WI) .

PMN Chemotaxis and Activation - The aεεay for chemotaxiε waε conducted aε previously deεcribed (S.D. Wolpe, G. Davatelis, B. Sherry, B. Beutler, D.G. Heεεe, H.T. Nguyen, L.L. Moldawer, C.F. Nathan, S.F. Lowry, & A. Cerami (1988), J. EXP. MED., 162:570-581). In brief, chemotaxiε waε aεεayed by placing 25 μl of chemoattractant (fMet-Leu-Phe [10 ^"8 M] , MIP-2 or buffer], Gey'ε balanced salt solution, pH 7.4 and 2% BSA]) in the bottom wells, and the top wellε were filled with 45 μl of buffer containing 1.1 X 10 ⁴ PMN'ε (iεolated by

Ficoll-Hypaque denεity gradient centrifugation and dextran sedimentation) . The two wells were separated by a cellulose nitrate membrane with a 3 μM pore εize (SM 11302; Sartorius Balanceε, Weεtbury, NY). Chamberε were incubated at 37°C in a humidified 5% carbon dioxide, 95% room air chamber for 45 minuteε. Membranes were removed and stained and the number of PMN's migrating into the membrane was counted every 10 μM up to 130 μM uεing an automated Optomax Imaging Syεtem (Optomax, Inc., Holliε, NH) . Random migration waε alεo determined under conditions where the gradient of chemotactic agent was abolished by including equal concentrations in the upper and lower chambers.

The ability of MIP-2 to elicit the releaεe of H ₂ 0 ₂ from adherent PMN'ε was assayed as previouεly deεcribed (S.D. Wolpe, G. Davatelis, B. Sherry, B. Beutler, D.G. Heεεe,

H.T. Nguyen, L.L. Moldawer, C.F. Nathan, S.F. Lowry, & A. Cerami (1988), J. EXP. MED., 167:570-581) .

RESULTS

Purification of MIP-2 - As judged from silver-stained NaDodS0 ₄ -PAGE gels, more than 90% of MIP-2 remained in the effluent of the Mono Q column (FIGURE 1) . This one-step purification step was sufficient to remove contaminating MIP-1 and cachectin/TNF. As previously shown (Wolpe, et al. , supra ) , these two proteins bind to the Mono Q column under these loading conditionε and elute at approximately 0.37 M NaCl. Two succeεεive steps of heparin-affinity chromatography and gel filtration were sufficient to purify MIP-2 protein to homogeneity (FIGURE 1) . MIP-2 eluted from heparin-Sepharose column at approximately 0.75 M NaCl and migrated with an apparent molecular weight of 10,000 daltonε on gel filtration.

Analysiε of partial NH ₂ -terminal amino acid sequence data of purified MIP-2 (FIGURE 2) revealed a unique sequence. Comparison with other sequenceε preεent in the Dayhoff bank uεing the d-FAST-P program (D.J. Lip an and W.R. Pearεon (1985), SCIENCE, 212:1435-1441) revealed εimilarity to a family of proteins with sequence relatedneεε to platelet factor 4. FIGURE 2 depicts the partial NH ₂ -terminal amino acid sequences of MIP-2 and various members of this family aligned by a conserved cysteine residue. FIGURE 3 depicts a comparison of the percent sequence identify over the region corresponding to the partial amino acid sequence obtained for MIP-2. The closest relationship observed to MIP-2 was with the predicted amino acid sequence for the gro gene product. The MIP-2 sequence is 62.5% identical with human gro and 68.7% identical with hamster gro. This relationship increases to 88% in both caseε when amino acid changeε which could result from a single baεe change are taken into conεideration.

The similarity in amino acid εequence between MIP-2 and gro suggested that MIP-2 could be"the murine equivalent of gro. The predicted murine KC gene product ("KC"), however, showed a closer relationship (65.6% identity to human gro and 81.2% identity to hamster gro for KC versus 62.5% and 68.7% respectively for MIP-2) when compared over the same region as the partial sequence for MIP-2. Similar results were obtained when the comparisons were conducted over the entire sequence, KC was 68% identical to human gro and 85% to hamster gro. Human and hamster gro were 68% identical when compared to each other over their entire sequence.

Immunoblot Analysis - In order to further characterize the relationship between MIP-2 and the other members of the platelet factor 4 family, a rabbit polyclonal antiserum was raised against MIP-2. The antiserum reacted monospecifically againεt εerum-free εupernatantε from endotoxin-stimulated RAW 264.7 cellε or thioglycollate-elicited mouεe macrophages but did not recognize any proteins in supernatants from unstimulated cellε. An example of such a blot with antibody againεt MIP-2 is shown in parts a and b of FIGURE 4. Preimmune serum also showed no reactivity. In particular, antiεerum to MIP-2 did not cross-react with MIP-1 or cachectin/TNF (see, for example, lane 2 of FIGURE 4) . Rabbit anti-MIP-2 crosε-reacted weakly with human and hamster gro but did not crosε-react with purified human NAP-1 protein (FIGURE 4) . Similarly, no croεε-reaction waε seen with partially purified human NAP-1 from another laboratory (supplied by J. Van Damme) . In addition, the human myelomonocytic cell line HL60 secreted a crosε-reacting protein after εtimulation with 10 ^"7 M phorbol myristic acid, further suggesting that the lack of reactivity of anti-MIP-2 antibody with the human NAP-1 protein is due to lack of immunological relatednesε rather than to species specificity. The nature of the

cross-reacting material from HL60 cells iε under inveεtigation.

PMN Chemotaxiε and Activation - Becauεe other memberε of the PF4 family have been shown to be chemotactic for and to activate PMN's, the effect of MIP-2 on these cells was studied. MIP-2 was significantly chemotactic for human PMN's at 10 ng/ml and was ^" more chemotactic for PMN's than fMet-Leu-Phe at concentrations greater than 100 ng/ml (FIGURE 5) . When equal concentrations of MIP-2 were added to both sideε of the membrane, no increaεe in migration waε obεerved. Thuε, the activity of MIP-2 at. the concentrationε tested appears to be due to stimulation of directed migration of the cells rather than enhancement of random migration.

PMN's did not undergo an oxidation burst (aε measured by production of hydrogen peroxide) when treated with concentrationε of MIP-2 ranging from 10 ng to 1 μg per ml.

In addition to theεe in vitro asεays, MIP-2 waε tested in vivo by injection of 100 ng into the footpads of endotoxin-resiεtant C3H/HeJ mice. Thiε injection induced a leukocyte infiltrate similar in magnitude to that previously shown for MIP-1.

DISCUSSION

MIP-2 has a molecular asε of approximately 6,000 daltons on NaDodS0 ₄ -PAGE and fractionates from a gel filtration column with an apparent molecular mass of approximately 10,000 daltons. In contrast to MIP-1 or cachectin/TNF, MIP-2 is cationic and does not bind to an anion exchange column equilibrated at pH 8.0. This property made separation of these activitieε and subsequent purification of MIP-2 relatively straightforward. After removal of the majority of contaminating proteins* by

anion exchange, MIP-2 was purified to homogeneity by sequential heparin affinity chromatography eluting at approximately 0.75M NaCl and gel filtration.

MIP-2 is an extremely active chemotactic agent but induces little or no chemokinetic activity at the doses tested. At 10 ng/ml (1.7 x 10 ^"9 M) , MIP-2 iε significantly chemotactic for human PMN's and at concentrations greater than 100 ng/ml (1.7 x 1'0 ^'8 M) MIP-2 exhibits a higher leukotactic index than fMet-Leu-Phe at the latter's optimum concentration of 10 ^'8 M. Studieε indicate that MIP-2 can also induce degranulation of PMN'ε with releaεe of lysozyme but not β-glucuronidaεe. Murine MIP-2 did not, however, induce the reεpiratory burst in human PMN's. It is not yet clear whether this is due to εpecies specificity or is an inherent property of the molecule.

The above effects on PMN's are similar to observationε made with a protein iεolated from human mononuclear cellε by a number of investigators and variously referred to aε "310C", "MDNCF", "MONAP", "NAF" or "GCP" [Schmid, J. & Weisεmann, C (1987) J. IMMUN. 139.:250-256; Yoεhimura, T. , Matsushima, K. , Oppenheim, J.J. & Leonard, E.J. (1987) J. IMMUN. 139:788-793; Yoεhimura, T. , Matεushima, K. , Tanaka, S., Robinεon, E.A., Appella, E. , Oppenheim, J.J. and Leonard, E.J. 1987. PROC. NATL. ACAD. SCI. USA 84:923-9237; Schroder, J.M., Mrowietz, U. , Morita, E. & Christopherε, E. (1987) J. IMMUN. 119:3474-3483; Walz, A., Peveri, P., Aschauer, H. & Baggiolini, M. (1987)

BIOCH. BIOPHYS. RES. COMM. 149:755-761; Peveri, P., Walz, A., Dewald, B. and Baggiolini, M. (1988) J. Exp. Med. 167:1547-1559; Van Damme, J. , Beeu en, J.V. , Opdennakker, G. and Billiau, A. (1988) J. EXP. MED. 167:1364-1376.1 This protein is now known as "neutrophil activating protein-1" (NAP-1) . Because of the striking similarity in properties of MIP-2 and NAP-1, the possibility was considered that MIP-2 might be the murine equivalent of

NAP-1. Thiε relationship appears not to be the case baεed on both sequence and immunological analyεeε. Comparison of the N-terminal sequence of MIP-2 with the PF-4 family using the FASTP program εhowε a 47% identity with NAP-1 but a 63% and 69% identity with human and hamster gro. respectively. This relationship agrees well with the immunoblotting results which demonstrated cross-reactivity with hamster and human σro but not with NAP-1 from two different laboratories.

It also appears ^" Unlikely that MIP-2 iε the murine equivalent of gro becauεe the predicted murine KC gene product εhowε an even higher εequence identity to gro. especially in the case of hamεter gro. Applicants therefore conclude that MIP-2 iε a novel gene product closely related to, but separate from, gro or KC

It is of interest that the gro and KC genes were originally found in studies on the control of cell growth. The gro gene was isolated by differential hybridization of DNA from transformed cellε [Aniεowicz, A., Bardwell, L. and Sager, R. (1987) PROC NATL. ACAD. SCI. USA 84.:P7188-7192] ; the KC gene waε isolated by differential hybridization of DNA from cellε treated with platelet-derived growth factor [Cochran, B.H., Reffel, A.C and Stileε, CD. (1983) CELL 31:939-947] . Tranεfection of cellε. with the gro gene does not lead to transformation, however [Aniεowicz, A., Bardwell, L. and Sager, R. (1987) PROC. NATL. ACAD. SCI. USA 84.:P7188-7192] . Similarly, treatment of denεity-arreεted 3T3 fibroblaεtε with MIP-2 or MIP-2 plus limiting amounts of serum or plasma does not increase uptake of ³ H thymidine.

EXAMPLE 2

The following setε forth the cloning and expreεεion of MIP-2. The cloning of the cDNA for murine MIP-2 was aε

followε. A degenerate oligonucleotide probe pool correεponding to amino acids 9-14 of a partial NH ₂ - terminal sequence of MIP-2 was synthesized. This portion of the partial sequence waε chosen becauεe of its lower codon degeneracy when compared with the remainder of the sequence. The resulting probe was a 128 fold degenerate pool of oligomers 17 nucleotides in length.

A cDNA library was constructed from Poly(A) ⁺ RNA isolated from E. coli lipopolysaccharide-εtimulated RAW 264.7 cellε aε taught in Davateliε et al., J. EXP. MED. 167: 1939-1941 (1988) and Sherry et al. J. EXP. MED. 168: 2251-2259 (1988) . Duplicate nitrocelluloεe filter lifts of the plated library (5X10 ⁵ plaques) were hydbridized at 42°C in 5xSSC, lx Denhardt's, 20mm sodium phosphate buffer, pH6.5, 50% formamide, 10% dextran εulfate, 0.1% SDS, 0.1 mg/ml sonicated salmon sperm DNA and 5xl0 ⁴ cpm per ml per degeneracy of ³ P-ATP 5' end-labelled εynthetic oligonucleotide probe pool. Following hybridization the filterε were waεhed employing the TMAC waεhing procedure deεcribed by Wood et al. PROC. NATL. ACAD. SCI. USA 82: 1585 (1985) . Plaqueε that were poεitive on duplicate filterε were subjected to a second round of low density plating and screening. Four independent positive phage clones were isolated from which DNA waε prepared for further analysis. cDNA insertε were excised by digeεtion with EcoRI and subcloned into M13 phage vector. DNA sequencing was performed by the didioxy chain termination method of Sanger et al. PROC. NATL. ACAD. SCI. USA 21: 5463 (1977) . The nucleotide sequence of one of the inserts was determined and found to encode a secreted protein that includeε the amino acid sequence determined from amino terminal sequencing of purified native MIP-2. The sequence of the cDNA clone and the predicted protein sequence are shown in FIGURE 7.

Construction of Expression Plaεmid pYMIP400 This plasmid encodes an α-factor leader linked to the mature coding sequence of MIP-2. The MIP-2 mature coding sequence was derived from the MIP-2 cDNA determined above. The GAPDH promotor sequence, the α-factor leader sequence and the α-factor transcription terminator were derived from plasmid pGAIl, the construction of which is described in European Publication No. 324,274, published 19 July 1989, the disclosure of which is incorporated herein by reference.

A Bglll site waε introduced by in vitro mutageneεiε into the nucleotide εequence encoding the carboxyl terminuε of MIP-2 in order to facilitate cloning into the expreεεion vector. The mutagenic primer waε:

** 5' - CAAAAGATCTTGAACAAAG - 3 ' "(♦denotes baεe changeε from original cDNA sequence)

Following verification of the altered cDNA sequence, phage RF was prepared and digested with Ball and Bglll. A 1966 bp fragment encoding most of the mature MIP-2 (lacking the sequence encoding 2 N-terminal and 9 C- terminal amino acids) waε isolated. Plaεmid pGAIl deεcribed in European Publication No. 324,274, publiεhed 19 July 1989, waε digeεted with Kpnl and ligated to the following adaptor which encodes the 3 alpha factor leader carboxyl terminal amino acidε, the LyεArg proceεεing site and the first 3 amino acids of mature MIP-2.

Kpnl - Ball adaptor

5' CCTTGGATAAAAGAGCTGTTGTGG 3 ^« 3' CATGGGAACCTATTTTCTCGACAACACC 5'

Following digestion with Sail, the following Bglll - Sail adaptor waε added which encodes for the 8 carboxyl

terminal amino acidε of MIP-2 aε well aε translational stop codonε.

Bglll - Sail adaptor 5' GATCTTGAACAAAGGCAAGGCTAACTGATAGCGTCG 5'

3' AACTTGT.TTCCGTTCCGATTGACTATCGCAGCAGCT 3'

The modified vector is gel purified and ligated to the 1966 bp Ball-Bglll fragment described above, after which screening to isolate plasmid pMIP400 is conducted. The plasmid is then isolated, and its nucleotide sequence across the adaptors is verified, after which the BamHl expreεsion casεette from this plasmid is isolated and cloned into the BamHl site of pAB24 to give the expresεion plasmid pYMIP400.

Expresεion of MIP-2

S. ce ^' reviεaie strain MB2-1 (leu2-3, leu2-112, hiε3-ll, hiε3-15, ura3A, pep4*, CAN ^1" , cir ⁰ ) iε tranεformed with plaεmid pYMIP400 by standard procedures and tranεformantε selected for ura prototrophy. Expresεion is analyzed by inoculation of εingle colonieε of individual transformantε into leucine selective medium, and growing for -48 hr. Cultures are then centrifuged, cells resuspended in medium lacking uracil and diluted 20 fold into ura selective medium. Cultures are grown for approximately 72 hr, then harvested and cell-free supernatantε prepared. Conditioned medium iε analyzed for the preεence of MIP-2 by SDS-PAGE followed by coomaεεie εtaining and immunoblotting.

Thiε invention may be embodied in other formε or carried out in other wayε without departing from the εpirit or essential characteristics thereof. The preεent diεclosure is therefore to be considered as in all respectε illuεtrative and not restrictive, the scope of the invention being indicated by the appended Claims, and

all changes which come within the meaning and range of equivalency are intended to be embraced therein.