WO1997022880A1

WO1997022880A1 - Use of rc-9 in diagnosis and treatment of proliferative arterial disease

Info

Publication number: WO1997022880A1
Application number: PCT/US1996/019671
Authority: WO
Inventors: Eliot Howard Ohlstein; Anthony Joseph Arleth; Michael Victor Autieri
Original assignee: SmithKline Beecham Corp
Current assignee: SmithKline Beecham Corp
Priority date: 1995-12-18
Filing date: 1996-12-13
Publication date: 1997-06-26
Anticipated expiration: 1998-06-18
Also published as: JP2000503764A; ZA9610584B; AU1413997A; EP0904543A1

Abstract

The present invention provides methods of using the polynucleotide sequences, recombinant RC-9 proteins encoded thereby, and RC-9 antibodies in diagnosis and treatment of atherosclerosis.

Description

USE OF RC-9 IN DIAGNOSIS AND TREATMENT OF PROLIFERATIVE ARTERIAL DISEASE

Field ofthe Invention

This application relates generally to proteins implicated in coronary artery disease.

Background ofthe Invention

The use of percutaneous transluminal coronary angioplasty (PTC A) as a treatment for advanced multi-vessel coronary artery disease has increased exponentially in the past decade. However, the long term efficacy of this procedure is significantly limited by the high incidence of vascular restenosis observed in as many as 40% of patients undergoing this procedure [P. Libby et al, Circulation. 86(Suppl. m):47-52 (1992)]. The resultant neointima formation associated with this procedure is a dynamic process actively involving several different cell types and occurring in several phases [A. N. Clowes et al. , Lab. Invest.. 49:208-215 (1983)]. The initial response is primarily inflammatory in nature involving T-lymphocytes and macrophages which secrete many different cytokines and growth factors seminal to the local inflammatory response. The second phase involves the principle cellular component ofthe restenotic lesion, the vascular smooth muscle cell (VSMC), which in response the these factors migrates into the intima, secrete soluble growth and chemotactic factors, and proliferate [Libby etal, (1992), cited above; Clowes et al, (1983), cited above; R. Ross, Nature. 362:801-809 (1993); and E. R. O'Brien et al, Circ. Res., 73:223-231 (1993)]. These activated VSMC also secrete extracellular matrix. The third phase may last several weeks post-injury and is characteristic ofa chronic fibroproliferative lesion formation [Ross (1993), cited above]. Although the cellular response to balloon angioplasty is fairly well characterized, there is still a lack of effective anti-restenotic pharmacological adjuncts to prevent neointima formation associated with PTCA.

An allograft inhibitory factor (ATF), has been described as useful in diagnosis and treatment of allograft rejection. The sequences of rat and human ALF are provided in WO 95/17506, published June 29, 1995.

What is needed in the art are methods of preventing such neointima formation, as well as better methods of diagnosing and monitoring same. Summary ofthe Invention

The present invention provides novel uses for the polynucleotide and arnino acid sequences of RC-9, which has been isolated from other cellular materials with which it is naturally associated. Also provided are novel uses for vectors containing these sequences, recombinantly produced RC-9 proteins, and anti-RC-9 antibodies.

Thus, in a first aspect, the present invention provides methods of diagnosing proliferative arterial disease and vascular restenosis. These methods utilize the nucleic acid and arnino acid sequences of RC-9 and anti-RC-9 antibodies described herein.

In a further aspect, the present invention provides methods of identifying compounds which specifically bind to a selected RC-9 sequence, including agonists and antagonists, using the RC-9 nucleic acid sequences, proteins, and antibodies described herein. In still a further aspect, the present invention provides for compounds or drugs produced by use ofthe above methods.

In yet another aspect, the present invention provides methods of treating proliferative arterial disease and vascular restenosis. These methods may utilize the compounds or drugs, nucleic acid and amino acid sequences of RC-9 and, preferably, the anti-RC-9 antibodies described herein.

Other aspects and advantages ofthe present invention are described further in the following detailed description ofthe preferred embodiments thereof.

Brief Description ofthe Drawings

Fig. 1 provides the partial DNA sequences [SEQ ID NO:l] of clone RC9 obtained from differential display. This sequence has been assigned GenBank Accession No. U10894.

Fig. 2 provides genomic DNA sequences [SEQ ID NO:2] of RC-9. This sequence has been assigned GenBank Accession No. U33471.

Fig. 3 illustrates the cDNA [SEQ ED NO:3] and putative amino acid sequences [SEQ EO NO:4] ofrat RC-9.

Fig. 4 illustrates the cDNA [SEQ ID NO:5] and amino acid sequences [SEQ ID NO:6] of human RC-9.

Detailed Description ofthe Invention

The present invention provides novel uses for a gene, termed RC-9, and the encoded protein, in detecting and treating proliferative arterial disease and vascular restenosis. The nucleotide and amino acid sequences and antibodies provided herein are useful in diagnosis and monitoring such vascular disease and damage. These sequences and antibodies are further useful in generating compounds useful in treatment of atherosclerosis and other conditions characterized by vascular stenosis.

The observation of RC-9 DNA cross-hybridization with DNA from a variety of animal species suggests substantial evolutionary conservation and a potentially important biologic function for the RC-9 encoded protein. The temporal expression of RC-9 mRNA in balloon angioplasty injured rat carotid artery indicates that its protein product plays a role in the early to middle stages of vascular restenosis.

I. RC-9 Polynucleotide Sequences

The methods ofthe invention may utilize the RC-9 polynucleotide sequences described herein. Figs. 1-4 provide DNA sequences ofthe novel transcript ofthe invention. Fig. 1 provides the nucleotide sequences of clone RC9 [SEQ ID NO:l], obtained as described in Example 1 below. This partial sequence is 424 bases in length and was used as a probe to obtain the longer sequence of Fig. 2. Fig. 2 provides genomic DNA sequences of RC-9 [SEQ ID NO:2], including two introns. Intron 1 spans base pairs 121-772 and intron 2 spans base pairs 1000-1276. PolyC tracts are located at base pairs 864-873 and 1254-1276. Branch point consensus sequences are located at base pairs 851-857 and 1212-1218 of Fig. 2. Fig. 3 provides the cDNA sequences of rat RC-9 [SEQ ID NO:3]. The transcription initiation codon is located at bp 143-145 and the stop codon is located at bp 581-583 of Fig. 3. Fig. 4 provides the 636 bp DNA sequences of human RC-9 [SEQ ID NO:5]. These sequences include a 444 bp open reading frame (bp 72-516), which encodes a 147 amino acid protein [SEQ ID NO :6], Fragments of these sequences may prove useful for a variety of uses. Desirably, these functional fragments are at least about 15 nucleotides in length and encode a desired amino acid sequence, e.g. an epitope, a therapeutically useful peptide desirably characterized by RC-9-like biological activity, or the like. These RC-9 nucleotide sequences may be isolated by conventional uses of polymerase chain reaction or cloning techniques. Alternatively, these sequences may be constructed using conventional genetic engineering or chemical synthesis techniques.

For use in the methods ofthe invention, the genomic RC-9 DNA sequence (Fig. 1 SEQ ID NO:l and Fig. 2 SEQ ID NO:2) or a cDNA sequence (Fig. 3 SEQ ID NO:3 and Fig. 4 SEQ U> NO:5) ∞ding for the encoded RC-9 protein or a functional fragment thereof may be modified. Utilizing the sequence data provided herein, it is within the skill ofthe art to obtain other polynucleotide sequences encoding the RC-9 proteins useful in the invention. Such modifications at the nucleic acid level include, for example, modifications to the nucleotide sequences which are silent or which change the amino acids, e.g. to improve expression or secretion.

In still another altemative, the polynucleotide sequences useful herein may be modified by adding readily assayable tags to facilitate quantitation, where desirable. Nucleotides may be substituted, inserted, or deleted by known techniques, including, for example, in vitro mutagenesis and primer repair. Also included are allehc variations, caused by the natural degeneracy ofthe genetic code.

In addition to isolated nucleic acid sequences encoding the RC-9 protein described herein, this invention also encompasses other nucleic acid sequences, including those complementary to the illustrated DNA sequences, such as antisense sequences. Useful DNA sequences also include those sequences which hybridize under high or moderately high stringency conditions [see, T. Maniatis et al., Molecular Cloning (A Laboratory Manual). Cold Spring Harbor Laboratory (1982), pages 387 to 389] to the DNA sequences illustrated in Figs. 1-4 [SEQ ED NO:l, 2, 3, 5]. An example ofa highly stringent hybridization condition is hybridization at 4XSSC at 65°C, followed by a washing in 0.1XSSC at 65°C for an hour. Alternatively, an exemplary highly stringent hybridization condition is in 50% formamide, 4XSSC at 42°C. Other, moderately high stringency conditions may also prove useful, e.g. hybridization in 4XSSC at 55°C, followed by washing in 0.1XSSC at 37°C for an hour. Alternatively, an exemplary moderately high stringency hybridization condition is in 50% formamide, 4XSSC at 30°C. As described in more detail below, the RC-9 nucleic acid sequences encoding these proteins are useful for a variety of diagnostic and therapeutic uses. Advantageously, the nucleic acid sequences are useful as diagnostic probes and antisense probes for use in the detection and diagnosis of proliferative arterial disease and vascular restenosis, among other conditions associated with undesirable RC-9 levels or expression. Oligonucleotide probes may be useful in such standard diagnostic techniques as Southern blotting and polymerase chain reaction.

Alternatively, the RC-9 nucleic acid sequences may be used to produce RC-9 proteins useful in the methods ofthe invention. Once constructed, or isolated, these DNA sequences or suitable fragments are preferably employed to obtain proteins of this invention, in vitro or in vivo.

π. RC-9 Amino Acid Sequences The methods ofthe invenuon may utilize RC-9 arnino acid sequences, including the

RC-9 proteins provided herein and suitable functional fragments thereof. The RC-9 rat protein [SEQ DD NO:4] is encoded by the cDNA sequences [SEQ ID NO:3] illustrated in Fig. 3 and the RC-9 human protein [SEQ ED NO: 6] is encoded by the cDNA sequences of Fig 4 [SEQ ID NO:5]. The rat protein [SEQ ID NO:4] is 147 amino acids in length and has a molecular weight of 16824.9 Daltons. The human protein is also 147 amino acids in length [SEQ ID NO: 6]. Also useful in the methods ofthe invention are biologically active fragments of RC-9. These functional fragments are desirably at least five amino acids in length and may encompass an epitope or other desired amino acid sequence.

Also useful in invention are analogs, or modified versions, ofthe RC-9 protein. Typically, such analogs differ by only 1, 2, 3 or 4 codon changes and are characterized by RC-9-like biological activity. Examples include polypeptides with minor arnino acid variations from the illustrated amino acid sequences of RC-9 (Figs. 3 and 4, SEQ TD NOs: 4 and 6); in particular, conservative amino acid replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains and chemical properties. Additionally, the RC-9 proteins useful in the invention may be modified, for example, to improve production thereof, to enhance protein stability or other characteristics, e.g. binding activity or bioavailability, to enhance its use for screening competitive compounds or to confer some other desired property upon the protein.

The RC-9 protein and RC-9 protein fragments described herein are useful in therapeutic compositions, as described in more detail below. The methods ofthe invention also include the use of these proteins in diagnostic applications, as well as for generation of other therapeutic and diagnostic reagents, such as anti-RC-9 antibodies. In common with other proteins generally, these RC-9 proteins may also serve in screening assays or as research tools. More desirably, the RC-9 proteins are also useful for the screening and development of chemical therapeutic agents useful for preventing the action of RC-9, e.g., in restenosis. m. Recombinant Expression of RC-9

The DNA sequences described herein may be used to produce recombinant RC-9 proteins. The resulting proteins may be used in the methods ofthe invention, or the method ofthe invention may involve in vivo expression ofthe proteins. To produce the RC-9 proteins, the RC-9 DNA sequences may be inserted into a suitable expression system.

Desirably, a recombinant molecule or vector is constructed in which the cDNA encoding RC- 9 is operably linked to a heterologous expression control sequence permitting expression of the RC-9 protein. Numerous types of appropriate expression vectors and host cell systems are known in the art for mammalian (including human) expression, insect, e.g., baculovirus expression, yeast, fungal, and bacterial expression, by standard molecular biology techniques. The transformation of these vectors into appropriate host cells can result in expression ofthe selected RC-9 proteins. Other appropriate expression vectors, of which numerous types are known in the art, can also be used for this purpose.

Suitable host cells or cell lines for transfection by this method include insect cells, such as Spodoptera frugipedera (Sf9) cells. Methods for the construction and transformation of such host cells are well-known. [See, e.g. Miller et al. , Genetic Engineering. 8:277-298 (Plenum Press 1986) and references cited therein].

Similarly, mammalian cells, such as Human 293 cells, rat aortic vascular cell lines [E. H. Ohlstein etal, Eur. J. Pharmacol. - Mol. Pharmacol. Section. 225:347-350 (1992)], Chinese hamster ovary cells (CHO), the monkey COS-1 cell line or murine 3T3 cells derived from Swiss, Balb-c or NTH mice may be used. Suitable mammalian host cells and methods for transformation, culture, amplification, screening, production and purification are known in the art. [See, e.g., Gething and Sambrook, Nature. 293:620-625 (1981), or alternatively, Kaufman etal., Mol. CeU. Biol.. 5(7): 1750-1759 (1985) or Howley et al, U. S. Patent 4,419,446]. Another suitable πiamrnalian cell line is the CV-1 cell line.

Similarly useful as host cells are bacterial cells. For example, the various strains of E. coli (e.g., HB101, MC1061, and strains used in the following examples) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli and the like may also be employed in this method. Many strains of yeast cells known to those skilled in the art are also available as host cells for expression ofthe polypeptides useful in the methods ofthe invention. Other fungal cells may also be employed as expression systems. Thus, the present invention provides a method for producing a recombinant RC-9 protein which involves transforming a host cell with at least one expression vector containing a recombinant polynucleotide encoding a RC-9 protein under the control ofa transcriptional regulatory sequence, e.g., by conventional means such as transfection or electroporations. The transformed host cell is then cultured under suitable conditions that allow expression of the RC-9 protein. For example, host cells, e.g., rat aortic vascular smooth muscle cells, may be transfected with sufficient vectors that they are capable of overexpressing the RC-9 protein, making them useful for screening compounds which inhibit RC-9 expression. In another embodiment, the expressed protein is recovered, isolated, and purified from the culture medium (or from the cell, if expressed intracellularly) by appropriate means known to one of skill in the art.

For example, the proteins may be isolated following cell lysis in soluble form, or extracted in guanidine chloride. If desired, the RC-9 proteins ofthe invention may be produced as a fusion protein. For example, it may be desirable to produce such RC-9 fusion proteins, to enhance expression ofthe protein in a selected host cell, or to improve purification. Suitable fusion partners for the RC-9 proteins described herein are well known to those of skill in the art and include, among others, b-galactosidase and poly-histidine.

IV. Production of Anti-RC-9 Antibodies The RC-9 proteins, as well as modified versions or analogs thereof, or cells expressing same, are useful as antigens for the development of antibodies to RC-9. Antibodies useful in the methods of this invention include monoclonal, polyclonal, chimeric, single chain and humanized antibodies, as well as Fab fragments. These antibodies may be produced by conventional methods, including the Kohler and Milstein hybridoma technique, recombinant techniques, such as described by Huse et al. , Science. 246: 1275-1281 (1988), or any other modifications thereof known to the art. Techniques described for the production of single chain antibodies (US Patent No. 4,946,778) can be adapted to produce single chain antibodies to the RC-9 proteins descried herein. Also, transgenic mice, or other organisms such as mammals, may be used to express humanized antibodies to a RC-9 protein or RC-9- derived protein. The antibodies of this invention may themselves be used to generate anti- idiotype antibodies. Techniques for generating such antibodies are well-known in the art. The antibodies ofthe invention may be utilized in protein form. Alternatively, the antibodies ofthe invention may be utilized in the form ofa polynucleotide, which expresses the antibody or a functional fragment thereof (e.g., a single chain or a Fab fragment) in vivo.

V. Diagnostic Reagents

The RC-9 proteins, antibodies, and polynucleotide sequences (including anti-sense polynucleotide sequences) described herein may be used as diagnostic reagents for diagnosing certain vascular disorders, e.g., atherosclerosis, associated with production or excessive production of RC-9. For example, a RC-9 protein, antibody, or polynucleotide may be utilized to diagnose vascular damage characteristic of such a condition. These reagents may optionally be labelled using diagnostic labels, such as radioactive labels, colorimetric enzyme label systems and the like conventionally used in diagnostic or therapeutic methods. The reagents may measure RC-9 levels in selected mammalian tissue in conventional diagnostic assays, e.g., Southern blotting, Northern and Western blotting, polymerase chain reaction and the like. For example, as diagnostic agents the polynucleotide sequences may be employed to detect or quantitate normal RC-9 mRNA or detect mutations in target gene RNA in a patient sample. Such a method may utilize PCR primers complementary to the nucleic acid sequence of Figs. 1-4 [SEQ ID NO:l, 2, 3, 5]. Alternatively, the detection of a specific DNA sequence (i.e., RC-9) may be achieved by metibods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, e.g., restriction fragment length polymoφhisms (RFLP) and Southern blotting of genomic DNA. Other suitable assays utilize a RC-9 protein, protein fragment, or anti-RC-9 antibody as a reagent. These assays include radioimmunoassays, competitive-binding assays, Western blot analysis and ELISA assays. The selection ofthe appropriate assay format and label system is within the skill of the art and may readily be chosen without requiring additional explanation by resort to the wealth of art in the diagnostic area.

Thus, the present invention provides methods for the use of these RC-9 protein- antibody or polynucleotide reagents in the diagnosis of disorders characterized by vascular restenosis, such as atherosclerosis. The methods may involve contacting a selected sample, e.g., blood, plasma, serum, or other suitable cells with the selected reagent, protein, antibody or DNA sequence, and measuring or detecting the amount of RC-9 present in the sample in a selected assay format based on the binding or hybridization or the reagent to the sample. VI. Therapeutic Reagents

The invention further provides methods for treatment of vascular restenosis. More particularly, this method involves administration of an anti-RC-9 antibody for blocking RC-9 activity in mammalian tissue. Also useful as therapeutic reagents of this invention are anti- idiotype antibodies, which can be used to block binding of RC-9 to its corresponding receptor. Although less desired, the therapeutic reagent may be a RC-9 nucleic acid sequence, a vector containing the nucleic acid sequences or an RC-9 protein. Alternatively, the therapeutic reagent may be a drug obtained using the methods described below.

As one example, the therapeutic reagents may be administered by appropriate routes in a pharmaceutical composition. Generally, the composition contains between about 10 mg to about 10 mg ofthe active agent (e.g., anti-RC-9 antibody, or a vector or polynucleotide described herein) per kg body weight. It will be appreciated that optimum dosage may be adjusted by one skilled in the art, taking into account the indication, its severity, route of administration, and the like. Suitable pharmaceutical carriers are well known to those of skill in the art and can be readily selected from among saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof, among others. The pharmaceutical compositions ofthe invention may optionally contain other active ingredients, or other desirable components, e.g., pH adjusters, preservatives, and the like. Appropriate routes may be readily determined by one of skill in the art and include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, interdermal, oral, vaginal, anal, intranasal, and topical routes. Currently, the preferred method of administration is intravenous. However, one of skill in the art can readily select another appropriate route of administration. Dosing may be repeated as needed or desired.

VII. Drug Screening and Development

The present invention also provides methods using the RC-9 proteins, antibodies and polynucleotide sequences described herein in the screening and development of chemical compounds or proteins which have utility as therapeutic drugs for the treatment of atherosclerosis and other vascular disorders.

As one example, such a compound is capable of binding to RC-9 and either enhancing (act as an agonist) or blocking (act as an antagonist) its biological activity. Such compounds are anticipated to be useful as a drug component for the treatment or prevention of vascular disorders, as described above. Presently, conventional assays and techniques exist for the screening and development of drugs capable of competitively binding to selected regions of RC-9. These include the use of phage display system for expressing the RC-9 proteins or portions thereof, and using a culture of transfected K coli or another microorganism to produce the proteins for binding studies of potential binding compounds . See, for example, the techniques described in G. Cesarini, FEBS Letters. 307(l):66-70 (July 1992); H. Gram et al. J. Immunol. Meth.. 161:169-176 (1993); C. Summer et al, Proc. Natl. Acad. Sci.. USA. 89:3756-3760 (May 1992), incoφorated by reference herein.

Other conventional drug screening techniques may be employed using the proteins, antibodies or polynucleotide sequences useful in this aspect ofthe invention. As one example, a method for identifying compounds which specifically bind to RC-9 DNA sequences can include simply the steps of contacting a selected RC-9 DNA fragment with a test compound to permit binding ofthe test compound to the DNA fragment; and determining die amount of test compound, if any, which is bound to the DNA fragment. Such a method may involve the incubation ofthe test compound and the RC-9 DNA fragment immobilized on a solid support.

Another method of identifying compounds which specifically bind to RC-9 sequences can include the steps of contacting a RC-9 DNA fragment immobilized on a solid support with both a test compound and the protein sequence which is a receptor for RC-9 to permit binding ofthe receptor to die RC-9 DNA fragment; and determining the amount ofthe receptor which is bound to ύie DNA fragment. The inhibition of binding ofthe normal protein by the test compound thereby indicates binding ofthe test compound to the RC-9.

RC-9-like effects of potential agonists, or the effects of potential antagonists, may be measured, for instance, by deterriiining activity ofa reporter system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of RC-9 or molecules that are found to elicit the same effects as RC-9. Reporter systems that may be useful in this regard include but are not limited to colorimetric labeled substrate converted into product, a reporter gene that is responsive to changes in RC-9 activity and binding assays known in the art. Other suitable methods are well known to those of skill in the art.

Thus, through use of such methods, the present invention provides compounds capable of interacting with RC-9 or portions thereof, and either enhancing or decreasing its biological activity, as desired. Such compounds are encompassed by this invention. The following examples which disclose the cloning and expression of RC-9 are for illustrative puφoses only, and should not be construed as limiting this invention in any way.

Example 1 - Isolation of RC-9 A. Rat Left Common Artery Balloon Angioplasty

Left common carotid artery balloon angioplasty was performed under aseptic conditions on male Sprague-Dawley rats [350 g; Charles River Breeding Laboratory Inc., Wihnington, MA] under sodium pentobarbital anesthesia [65 mg/kg, i.p.- Steris Laboratories, Phoenix, AZ] as described previously [E. H. Ohlstein et al. , Proc Natl Acad Sci USA. 90:6189-6193 (1993)]. Briefly, the left external carotid artery was identified and cleared of adherent tissue, allowing the insertion ofa 2-F Fogarty arterial embolectomy catheter [Model 12-060-2F; Baxter Healthcare, Santa Ana, CA)]. The catheter was guided a fixed distance down the common carotid artery to the aortic arch, inflated with a fixed volume of fluid, and withdrawn back to the site of insertion. This procedure was performed a total of three times. Once completed, the catheter was removed, and the wound was closed (9-mm Autoclips; Clay Adams, Franklin Lakes, NJ) and swabbed with Povadyne surgical scrub (7.5% Povidone-Iodine- Chaston Dayville, CT). Animals were housed in Plexiglass cages under a 12-hour light\dark cycle with access to standard laboratory chow and drinking water ad libitum until required for tissue collection. To isolate the carotid arteries, rats were exsanguinated via the vena cava under barbiturate anesthesia (100 mg/kg, i.p.). Left common carotid arteries were rapidly cleared of adherent tissue in situ, isolated, and placed directly in guanidine thiocyanate (Promega Company, Madison, WI). These vessels were then immediately flash frozen in liquid N₂ and stored at -80°C until required for RNA isolation. For subsequent Northern analysis, tissues were examined from naive animals (control) and from animals that had undergone angioplasty 6 hours, 3 days, and 14 days before to study die differential expression of transcripts in a more detailed, temporal fashion. Northern analysis was also performed on sham vessels.

B. RNA isolation and Northern analysis For each time point studied, four or five left carotid arteries were pooled, or

VSMC from culture isolated and total RNA obtained as described [P. Chomczynski and N. Sacchi, Ann. Biochem.. 162:156-159 (1987)]. Equal amounts of RNA were loaded and separated on a 1.3% agarose/formaldehyde gel, transferred to nitrocellulose, and hybridized (0.25MNaCl, 1% sodium dodecyl sulfite, 50% formamide, 2X Denhardt's solution, 25mg denatured salmon sperm DNA, 5% dextran sulphate 42°C overnight) with the indicated probe. Blots were washed under high stringency (0.1% sodium citrate, 0.1% sodium dodecyl sulfate, 65°C), and exposed to film for 6 - 48 hr. at -80°C. All probes were [a³²P]-labeled by the random priming method (Bciehringer Mannheim, Indianapolis, In.) (all isotopes were from Amersham Inc., Arlington Heights IL.). The same filter was stripped and subsequently hybridized with die various DNA probes. The glyceraldehyde-3 -phosphate dehydrogenase (G3PDH) probe was generated from PCR amplimers (Clonetech, Palo Alto, CA.). Relative intensities of hybridization signals were obtained by densiometric scanning (RFLP-Scan Software, Scanalytics, Inc.) of autoradiograms exposed widiin die linear range ofthe film (Kodak X-OMAT). Human and multiple tissue Northern blots were purchased from Clonetech, Inc. (Palo Alto, CA).

C. Differential Display of mRNA

Total RNA from control left common carotid arteries and diose diat had undergone balloon angioplasty 3 and 14 days earlier were isolated, and die differential display technique was used to examine changes in gene expression. DNA was removed from total RNA using a sequential procedure of DNA digestion, (1 U DNase; Gibco-BRL, Gaithersberg, MD), phenol/chloroform extraction, and ethanol precipitation according to the manufacturer's protocol. Purified total RNA (0.2 mg) was reverse transcribed using modified oligo dT primers (reverse transcriptase from GenHunter Coφoration, Brookline, MA) [P. Liang et al, Science. 257:967-971 (1992)]. cDNA was amplified and labeled by including 10% of die reverse transcription reaction widi the appropriate 3' oligo dT primer, dNTPs, [^ϊ5S]dATP, and an arbitrary 10-mer 5' primer (GenHunter). Forty PCR cycles were run widi die following parameters: 94°C for 30 seconds; 40°C for 120 seconds; 72°C for 45 seconds, widi die last cycle followed by a 72°C soak for 7 minutes. The labeled cDNAs obtained from this reaction were separated on a 6% acrylamide sequencing gel, which was dried and exposed to autoradiographic film 12 to 48 hours.

Gene expression in day 3 vessels represents early molecular events in the pauiogenesis of lesion formation (i.e., those events occurring before die physical formation of a neointimal lesion), whereas day 14 vessels represent later events (i.e., when approximately 80% of die final lesion volume has formed in ie neointima) [S. A. Douglas et al, Eur. J. Pharmacol.. 255:81-89 (1994)]. For diis study, RNA from diese samples was reverse transcribed using all four oligo dT anchor primers, and die resulting cDNA was amplified using two different 5' decamers. Attention was paid primarily to bands in die top third ofthe sequencing gel, because these samples are longer and therefore facilitate DNA sequence identification. Focusing on the top third of die sequencing gel, less uian 10% of die bands appeared to be differentially expressed (8 of 82). In dus initial study, two bands were selected diat displayed differential expression.

D. Recovery Reamplification and Cloning of cDNAs

Selected cDNAs were excised from die sequencing gel, eluted, and reamplified using d e same primers as previously described [Liang et al. , cited above] . The reamplified PCR product was visualized on 2.0% agarose gel, stained with ethidium bromide, and recovered by glass extraction (Bio 101, La Jolla, CA). Reamplified PCR products were labeled as probes as described above and were cloned into die pCRJJ vector using die Invitrogen TA cloning system (San Diego, CA), and DNA was sequenced on an applied biosystems model 373 A automated sequencing apparatus.

The DNA segments were recovered from die acrylamide sequencing gel by excision and elution and PCR amplified using the same primers and reaction conditions. Reamplification yielded single products, die size of which are consistent widi weir apparent sizes displayed on die acrylamide sequencing gel (data not shown). These reamplification products were tiien glass-purified from die gel and used as probes for Northern analysis.

Only two ofthe rat tissues examined expressed die RC9 transcript. Testes RNA showed a strong signal of approximately 1.35 kb, slighdy larger dian die 1.1 kb observed in carotid arteries. A second, much weaker signal of approximately 1.1 kb was detected in spleen. In addition to providing information on which cDNA library to choose, this data indicates that die expression of dus transcript is tissue specific, and die differences in size from testes to spleen suggest processing of ti is mRNA transcript, or die presence of similar, messages in different tissues.

Total RNA from naive rat carotid arteries and from carotid arteries isolated at tiiree timepoints after balloon angioplasty were probed widi tiiese DNA fragments in Northern analysis to confirm tiieir differential expression. Each of diese DNAs was found to represent a discrete gene transcript. Northern analysis verified dieir expression pattern. RC9 expression is undetectable in naive vessels, is detected at low levels 6 hours postsurgery, reaches rriaximal levels at day 3, and declines in day 14 vessels, cxmfirrning die pattern observed in die differential display gel. No expression of RC9 mRNA is detected in sham control vessels.

Each of these cDNAs were subcloned into die pCRJJ vector and dideoxy sequenced in both directions. The RC9 clone is 424 bases (Fig. 1, SEQ JJ) NO: 1) and displays no significant sequence homology to any previously characterized genes deposited in Genbank.

Example 2 - Isolation of RC-9

RC9 is a 424 bp partial genomic DNA fragment [SEQ ID NO:l] isolated from differential display analysis of rat carotid arteries post-balloon angioplasty, and identifies an mRNA of approximately 1.1 kb, as described in Example 1 above. To fully identify and characterize diis transcript, it was essential to obtain die full lengtii gene.

Since cDNA libraries prepared from restenotic rat carotid arteries are unavadable, it was necessary to identify a rat tissue in which this message was expressed and then screen a cDNA library prepared from mRNA from tiiis tissue. A. cDNA library screening

A rat testes cDNA library (Stratagene, Inc.) was screened at high stringency (0.1X SSPE at 65°C) with the 424 bp RC9 [SEQ ID NO:l]. This probe was used to screen approximately 72,000 plaques, of which diree were positive after diree rounds of screening. These diree clones, each containing an insert of approximately 1.68 kb flanked by EcoRI sites as part ofthe vector polylinker were obtained and shown to be identical by restriction analysis. One clone was chosen and shown to be 98% homologous to RC9 by dideoxy nucleotide sequencing using an RC9 specific primer and also used to probe rat carotid arteries subject to balloon angioplasty to confirm the identity of this clone by mRNA expression.

Total RNA from naive rat carotid arteries and from carotid arteries isolated at diree timepoints (1, 3 and 7 days) following balloon angioplasty were size fractionated and hybridized with a radiolabeled PCR product representing die insert of RC-9. More specifically, total RNA (10 mg) was separated on a 1.2% agarose/formaldehyde gel, transferred to nitrocellulose, hybridized, and washed as described above. A glyceraldehyde-3 -phosphate dehydrogenase (G3PDH) probe was used as a loading control. Scanning densiometric analysis of tiiis blot normalized to glyceraldehyde- 3 -phosphate dehydrogenase (G3PDH) content confirms a 9-fold increase in expression of tiiis message over basal levels 1 day post balloon angioplasty, 4-fold 3 days, and 2-fold 7 days post-injury. This expression pattern consistent widi tiiat observed for die RC9 differential display fragment, with die exception ofa basal level detected in naive vessels, and indicates tiiat similar to expression of tiiis gene is induced in rat carotid arteries in response to balloon angioplasty. Northern analysis of RC-9-1 mRNA from sham control vessels shows no change in basal expression (data not shown).

B. Sequence Analysis

The clone was sequenced on both strands in its entirety (Sequenase, United States Biochemical Coφoration) as previously described. DNA and protein sequences were analyzed using die Mac Vector software package (International Biotechnologies, Inc.).

Searches for sequence similarity were performed using die GenBank Nucleic Acid Database and the Genetics Computer Group program.

The 3' region of die 1.68 kb insert from this clone was identified as 98% homologous with die 424 bp fragment obtained from differential display, further corifirming die identity of tiiis clone as the sequence of RC9. The nucleic acid clone RC-9-1 is shown in Figure 2, SEQ ID NO:2. Subsequent analysis of die sequences of Fig. 2 [SEQ ID NO:2] revealed die presence of polyC tracts at base pairs 864-873 and 1254-1276 and branch point consensus sequences at base pairs 851-857 and 1212-1218. This indicated tiiat the sequence encompassed intronic sequences. Further analysis identified two introns. Intron 1 spans base paris 121-772 of Fig. 2 and intron 2 spans based pairs 1000-1276. The sequences of Fig. 2 [SEQ ID NO:2] were used to obtain a RC-9 cDNA clone, as described in Example 3 below.

C. Genomic DNA species analysis

To determine die evolutionary conservation of die RC-9 sequence of Fig. 2 SEQ ID NO:2, Southern blot analysis was carried out using EcoRI digested genomic DNA from a variety of animal species including human, monkey, rat, mouse, dog, cow, rabbit, chicken, and yeast {Saccharomyces cerevisiae). Genomic DNA Soudiem blots containing 4 mg of genomic DNA from nine different animal species digested was purchased from Clonetech, Inc. (Palo Alto, CA) and was hybridized at high stringency (5X sodium citrate, 10% Denhardt's solution, 100 mg/ml sheared salmon sperm DNA, 1% sodium dodecyl sulfate, 65 °C, overnight) witii die full-lengtii RC-9 1 cDNA clone according to manufacturers instructions. Blots were washed under low (2. OX sodium nitrate, 0.1% sodium dodecyl sulfate, 55°C), or high stringency (0.2X sodium citrate, 1% sodium dodecyl sulfate, 65°C), and audioradiographed. The RC-9-1 DNA of Fig. 2 [SEQ ID NO:2] hybridized to number of restriction fragments from various species under low stringency washing conditions (2X SSC, 0.1% SDS, 50°C), die most prominent being a 24 kb fragment in botii rat and cow, and a 4.4 kb fragment in mouse. Otiier bands were detectable in dog, mouse, rat, and cow. No bands were visible in rabbit, chicken, or yeast. A number of less prominent bands were also observable at tiiis stringency, including a band approximately 2.3 and 1.0 kb in length visible in human, rat, and cow. A strong signal at 2.5 kb in mouse is also prominent. Under higher stringency washing conditions, (0.2 X SSC, 1% SDS, 65°C), these bands are barely visible, and d e remaining prominent bands of 24 kb in rat and 4.4 kb in mouse did not change in intensity. The 2.5 kb band in mouse is no longer seen, suggesting that this band represents another gene that cross-hybridizes with RC-9-1 sequences of Fig. 2 [SEQ ID NO:2]. Thus it appears tiiat the RC-9-1 transcript is well conserved.

Example 3 - cDNA Cloning of RC-9 Rat tested mRNA was isolated as previously described and reverse transcribed to yield cDNA using oligo-dT primer. RC-9 primers were chosen from the extreme ends of die RC-9 genomic DNA (Fig. 2, SEQ ID NO:2). More particularly, the sense primer (25 mer ohgonucleotide) corresponds to nt 31- nt 55 of Fig. 2 [SEQ ED NO:2] and has a sequence of 5' - ACT TCA GAC TCT CTC TTC CCT ACG G - 3'; the anti-sense primer (28 mer ohgonucleotide) corresponds to nt 1651-1678 of Fig. 2 [SEQ ED NO:2] and has a sequence of

5' - GCA TCA GGG AGC ATT ATT TAT TTA GTT T - 3'. Polymerase chain reaction (PCR) was used to obtain the RC-9 cDNA. Forty PCR cycles were run using die following parameters: 94°C for 1 min; 53°C for 1 min; 72°C for 3 min, with die last cycle followed by a 4°C soak. The RC-9 cDNA was separated on a 1% agarose gel and purified using standard techniques. The RC-9 cDNA was ligated into die vector pCRII [InVitrogen], clones were isolated and restriction enzyme digested widi EcoRI to confirm they contained an insert ofthe correct size. Positive clones were sequenced and compared to the genomic sequence of Fig. 2. A partial cDNA sequence is provided in Fig. 3 [SEQ ID NO:3], togetiier w h die arnino acid sequence encoded tiiereby. The sequence of Fig. 3 is 696 basepairs in length [SEQ ID NO: 3]. A homology study performed with a cDNA sequence corresponding to nucleotides

72-696 of Fig. 3 revealed die cDNA sequence [SEQ ID NO:3] was 98% homologous to die RC-9 genomic sequence of Fig. 2 [SEQ ID NO:2]. SEQ ID NO:3 and 4 have been found to have substantial identity to the rat AD? sequences, previously described in WO 95/17506.

Example 4 - Function of RC-9 The RC-9 clone of Example 3 was cut out ofthe pCRII plasmid using die restriction endonuclease BstXl. The RC-9 DNA was purified and ligated into the expression vector pRc/CMV (Invitrogen Inc.) which had been pre-cut with BstXl and grown in E. coli. A single bacterial colony was chosen containing RC-9 in the correct orientation for propagation. Purified DNA was obtained with RC-9 in pRc/CMV. Rat aortic vascular smootii muscle (RAVSM) cells were transfected widi either pRc/CMV plasmid alone or with plasmid pRc/CMV containing RC-9. Transfection was done using LipofectAMJ-NE Reagent (Life Technologies Inc.) mixed widi die DNA to be transfected (pRc/CMV or pRc/CMV RC-9). The LipofectAMTNE Reagent allows the DNA to pass through die cell membrane, eventually permitting die DNA to be incoφorated into die host's chromosomes. Two days following transfection, the compound G418 (Geneticin) was added to the cells. G418 kills cells tiiat do not contain the pRc/CMV plasmid, leaving only cells containing pRc/CMV or pRc/CMV RC-9. Transfected RAVSM were grown in die presence of G418 for functional studies on RC-9.

RAVSM containing eitiier pRc/CMV or pRc/CMV RC-9 were seeded onto T-150 flasks at equal densities and left to grow over a period of 5 or 7 days in DMEM + 10% FBS + G418. The cells were then trypsinized and counted using a hemocytometer to look for differences in proliferation. At 5 days, cell counts for pRc/CMV were 4.55 x 10⁴ + 8.5 x 10³ cells/cm² (in a T-150 flask @ 150 cm²) and for pRc/CMV RC-9 1.22 x 10⁵ ± 5 x IO² cells/cm²; a change of 168%. The 7 day cell counts yielded similar results: cell counts for pRc/CMV were 5.93 x 10⁴ ± 5.5 x IO³ cells/cm² (in a T-150 flask @ 150 cm²) and for pRc/CMV RC-9 1.84 x IO⁵ ± 3.7 x 10⁴ cells cm²; a change of 210%.

The cell count data clearly shows tiiat RAVSM cells containing RC-9 proliferate at a higher rate tiian control cells.

Example 5 - Tissue Expression of Human RC-9

The human RC-9 sequences described herein, SEQ ID NO: 5 and 6, have substantial identity to the human AB? sequences described previously WO 95/17506. E. coli transformed with human RC-9 [SEQ DD NO 5], which was originally expressed in pBluescnpt, were grown and positive clones identified

Gel purified cDNA probe (generated by PCR), was used to assess human RC-9 mRNA expression by Northern "dot blot" analysis, as descnbed above Expression, normalized usmg cyclophihn, was relatively low (defined by hRC-9 cyclophihn ratios of < 1 3) m "normal" human tissue from vaπous regions ofthe CNS, GJΛirogenital tract and, suφπsingly, heart, aorta and testes (This last observation is particularly interesting smce rat RC-9 [SEQ DD NO.3] was cloned from a rat testes cDNA library) Expression was, however, elevated several-fold in organs associated with iriflammatory cell function (bone marrow > thymus > spleen > peπpheral blood leukocytes, e g., hRC-9 cyclophihn ratios of

>6 0) Fetal tissue showed a similar pattern of expression (e g , highest expression m tiiymus, spleen) In human "diseased" tissue blots, enhanced mRNA was present in mitogen (TGFb or angiotensin U) stimulated vascular smooth muscle cells demonstrating upregulation of human RC-9 [SEQ DD NO 6] in association wrth growtii factor stimuli Tissue analysis demonstrated elevated human RC-9 hybπdization (e g , in hearts and kidneys) m association wrth hypertension, congestive heart failure and myocardial infarction consistent wrth a role of human RC-9 [SEQ DD NO 5 and 6] in die control of cellular proliferation/differentiation and die pathogenesis of fibroprohferative disorders In accord, relative to medial expression, human RC-9 mRNA expression was also augmented in vascular smooth muscle cells deπved from the tunica neointima of blood vessels which had previously undergone angioplasty

All references cited herein are incoφorated by reference While certain embodiments of die mvention have been particularly descnbed, it will be apparent to tiiose stalled m the art that many modifications and vaπations may be made Therefore, the present mvention is not to be construed as limited by any of die particular embodiments shown, ratiier its scope will be defined only by the claims which follow SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: SmithKline Beecham Corporation Ohlstein, Eliot Arleth, Anthony Autieri, Michael V.

(ii) TITLE OF INVENTION: Use of RC-9 in Diagnosis and Treatment of

Proliferative Arterial Disease

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: SmithKline Beecham Corporation

(B) STREET: 709 Swedeland Road

(C) CITY: King of Prussia

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19406-2799

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: WO

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/008,801

(B) FILING DATE: 18-DEC-1995

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Schreck, Patricia A.

(B) REGISTRATION NUMBER: 33,777

(C) REFERENCE/DOCKET NUMBER: P50420-1

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 610-270-5031

(B) TELEFAX: 610-270-5090

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 424 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: AGCCAGCGAA GTACATGGAG TTTGATCTGA ATGGCAATGG AGATATCGAT ATTATGTCCT 60 TGAAGCGAAT GCTGGAGAAA CTTGGGGTTC CCAAGACCCA TCTAGAGCTG AAGAAATTAA 120 TTAGAGAGTT GTCCAGTGGC TCCGAGGAGA CGTTCAGTTA CTCTGACTTT CTCAGAATGA 180 TGCTGGGCAA GAGATCTGCC ATCTTGAGAA TGATTCTGAT GTATGAGGAG AAAAACAAAG 240

AACACCAGAA GCCAACTGGT CCCCCAGCCA AGAAAGCTAT TTCTGAGTTG GCCTAATGGA 300

GGTGGATATA ACACGGTGGG ACCGAGGACC TTCGTGTCAG CAGCATGGGA AGAGAAGAAT 360

CAGTTGCGAG CCAGACTGTC AATGCTCAAT AAATAATGCT CCCTAGTGCC TGAAAAAAAA 420

AAAA 424 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1678 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: DNA (genomic)

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

GTCTCACCCC ATTCTTGGAG CAGCCTGCAG ACTTCAGACT CTCTCTTCCC TACGGAGAAA 60

GTCAGCCAGT CCTCCTCAGC TGCTTCTTAA CCTGCACCAT GAAGCCTGAG GAAATTTCAA 120

GTAACCCTCC CAGCCCCCAC CCCAGGGTCT GGGGAAGAAG TCATTTTTGT GTTTCCTTGG 180

TTTGGGTTTG AGACAGGTTC TTACTGTGTA GGCCTGGCTG GTCTCCAACT CCCAGGATCC 240

TCTAGCCTCA ACCTCTCTTA GGTGCTAGCT TGCAGGCCTA GGGCATCACG ATAGTCCCCC 300

AGATCCTGGT GCCTTTCCAC GCCCCTCCCA TGTACAGTCT GCCTTTCTGA AGGAGCTGTG 360

TGGAGCTAGG AGTGTTACAG GGCTGCTGCT CGTTTGGGAG GCTGAGGCAG GACAATATGC 420

GCCTGGACAA TACGAGAACC TGCCCCCACC CTTCAAAAGT AGCTTTTCAG TATCTTACTT 480

TTCAACTTGG TCCGCCTTCT CCTGGGATGC TGGTGTCAGC AGAAGCTGAT GGGAAGTGAA 540

CCTGGGAAGT TAGCAAAGGA GGAATGGGTA GAAAGGGGAA GTGTGAGAGA AGTCCCAGAA 600

GGACTGGGGA GCTGGTGGAG AGAGGAGCCA GCCAACACAC TGCAGCCTCA TCGTCATCTC 660

CCACCTAAGG CCACCACGTC TGAGGAGCTA TGAGCCAGAG CAAGGATTTG CAGGGTAGGG 720

AAGGTGGACC GTACAGCACC GGAGACCAGC ATTGAATCAG GGAGCCAGAG GCGCTCCTGA 780

AGAATCTCAG AGGCCAGCCT TGGGCAACAC TTGGGCTGGG CACAGGGGAG GAGGAAAGGG 840

AGGGAGGGGT GGTTAACCTC TGTACCCCTC AGGATGAAAA GCTTTTGGAC TGCTGAAAGC 900

CCAGCATGGA GAGAGGTTGC TGGGATCAAC AAGCACTTCC TCGATGATTC CAAGTACAAC 960

AGTGATGAGG ATCTGCAGTC CAAACTGGAG TCCTCAAGAG TGAGGAGACT GGGGGGGGGG 1020

GGGAGCTGGA GTCTGGGGGC TCAAATCGTG GCAAGGCTGG CTGCTGTCCT GGGGAAGGAG 1080

GGAGGTGCCT TCAAGCTCCT CCTTCCCATG GTGGTCCTTT GTCCTGCTCT CCCCATCTTA 1140

GCATCCCCGC CCCCAGGCTC CCTCACAAGC TCTCTCCCCA TGCCCTCCTT CTGTATCTTC 1200

TACCCTGGCA TTGCTCAGCT GTCTCATACC GCAAACCCCA CAATGCAATT ATACTCTCTG 1260

CTCTCTTTCT ACCCAGCGAA GTACATGGAG TTTGATCTGA ATGGCAATGG AGATATCGAT 1320 ATTATGTCCT TGAAGCGAAT GCTGGAGAAA CTTGGGGTTC CCAAGACCCA TCTAGAGCTG 1380

AAGAAATTAA TTAGAGAGTT GTCCAGTGGC TCCGAGGAGA CGTTCAGTTA CTCTGACTTT 1440

CTCAGAATGA TGCTGGGCAA GAGATCTGCC ATCTTGAGAA TGATTCTGAT GTATGAGGAG 1500

AAAAACAAGA ACACCAGAAG CCAACTGGTC CCCCAGCCAA GAAAGCTATT TCTGAGTTGC 1560

CCTAATTGGA GGTGGATATA ACACGGTGGG ACCAGGACCT TCTAATGACA GCAGCATGGG 1620

AAAAGAAGAA GCAGTTGTGA GACCAGAGTC AAACTAAATA AATAATGCTC CCTAGTGC 1678 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 696 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 143..580

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

AGACGCACCC TCTGATGTGG TCTGCACAGG GCCCTGGGCT CAGCTCACCC CATTCTTGGA 60

GCAGCCTGCA GACTTCAGAC TCTCTCTTCC CTACGGGAGA AAGTCAGCCA GTCCTCCTCA 120

GCTGCTTGTC TTAACCTGCA CC ATG AAG CCT GAG GAA ATT TCA AGA GGA AAA 172

Met Lys Pro Glu Glu lie Ser Arg Gly Lys 1 5 10

GCT TTT GGA CTG CTG AAA GCC CAG CAG GAA GAG AGG TTG GAT GGG ATC 220 Ala Phe Gly Leu Leu Lys Ala Gin Gin Glu Glu Arg Leu Asp Gly lie 15 20 25

AAC AAG CAC TTC CTC GAT GAT CCC AAG TAC AGC AGT GAT GAG GAT CTG 268 Asn Lys His Phe Leu Asp Asp Pro Lys Tyr Ser Ser Asp Glu Asp Leu 30 35 40

CAG TCC AAA CTG GAG GCC TTC AAG ACG AAG TAC ATG GAG TTT GAT CTG 316 Gin Ser Lys Leu Glu Ala Phe Lys Thr Lys Tyr Met Glu Phe Asp Leu 45 50 55

AAT GGC AAT GGA GAT ATC GAT ATT ATG TCC TTG AAG CGA ATG CTG GAG 364 Asn Gly Asn Gly Asp lie Asp lie Met Ser Leu Lys Arg Met Leu Glu 60 65 70

AAA CTT GGG GTT CCC AAG ACC CAT CTA GAG CTG AAG AAA TTA ATT AGA 412 Lys Leu Gly Val Pro Lys Thr His Leu Glu Leu Lys Lys Leu lie Arg 75 80 85 90

GAG GTG TCC AGT GGC TCC GAG GAG ACG TTC AGT TAC TCT GAC TTT CTC 460 Glu Val Ser Ser Gly Ser Glu Glu Thr Phe Ser Tyr Ser Asp Phe Leu 95 100 105

AGA ATG ATG CTG GGC AAG AGA TCT GCC ATC TTG AGA ATG ATT CTG ATG 508 Arg Met Met Leu Gly Lys Arg Ser Ala lie Leu Arg Met lie Leu Met 110 115 120 TAT GAG GAG AAA AAC AAA GAA CAC CAG AAG CCA ACT GGT CCC CCA GCC 556 Tyr Glu Glu Lys Asn Lys Glu His Gin Lys Pro Thr Gly Pro Pro Ala 125 130 135

AAG AAA GCT ATT TCT GAG TTG CCC TAATTGGAGG TGGATATAAC ACGGTGGGAC 610 Lys Lys Ala lie ser Glu Leu Pro 140 145

CGAGGACCTT CTAATGACAG CAGCATGGGA AAAGAAGAAG CAGTTGTGAG CCAGAGTCAA 670

ACTAAATAAA TAATGCTCCC TGATGC 696

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 146 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Lys Pro Glu Glu lie Ser Arg Gly Lys Ala Phe Gly Leu Leu Lys 1 5 10 15

Ala Gin Gin Glu Glu Arg Leu Asp Gly lie Asn Lys His Phe Leu Asp 20 25 30

Asp Pro Lys Tyr Ser Ser Asp Glu Asp Leu Gin Ser Lys Leu Glu Ala 35 40 45

Phe Lys Thr Lys Tyr Met Glu Phe Asp Leu Asn Gly Asn Gly Asp lie 50 55 60

Asp lie Met Ser Leu Lys Arg Met Leu Glu Lys Leu Gly Val Pro Lys 65 70 75 80

Thr His Leu Glu Leu Lys Lys Leu lie Arg Glu Val Ser Ser Gly Ser 85 90 95

Glu Glu Thr Phe Ser Tyr Ser Asp Phe Leu Arg Met Met Leu Gly Lys 100 105 110

Arg Ser Ala lie Leu Arg Met lie Leu Met Tyr Glu Glu Lys Asn Lys 115 120 125

Glu His Gin Lys Pro Thr Gly Pro Pro Ala Lys Lys Ala lie Ser Glu 130 135 140

Leu Pro 145

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 635 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: cDNA ( i ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 72..512

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GGCACGAGAG CCTGCAGACA GAGGCCTCCA GCTTGGTCTG TCTCCCCACC TCTACCAGCA 60

TCTGCTGAGC T ATG AGC CAA ACC AGG GAT TTA CAG GGA GGA AAA GCT TTC 110 Met Ser Gin Thr Arg Asp Leu Gin Gly Gly Lys Ala Phe 1 5 10

GGA CTG CTG AAG GCC CAG CAG GAA GAG AGG CTG GAT GAG ATC AAC AAG 158 Gly Leu Leu Lys Ala Gin Gin Glu Glu Arg Leu Asp Glu lie Asn Lys 15 20 25

CAA TTC CTA GAC GAT CCC AAA TAT AGC AGT GAT GAG GAT CTG CCC TCC 206 Gin Phe Leu Asp Asp Pro Lys Tyr Ser Ser Asp Glu Asp Leu Pro Ser 30 35 40 45

AAA CTG GAA GGC TTC AAA GAG AAA TAC ATG GAG TTT GAC CTT AAT GGA 254 Lys Leu Glu Gly Phe Lys Glu Lys Tyr Met Glu Phe Asp Leu Asn Gly 50 55 60

AAT GGC GAT ATT GAT ATC ATG TCC CTG AAA CGA ATG CTG GAG AAA CTT 302 Asn Gly Asp lie Asp lie Met Ser Leu Lys Arg Met Leu Glu Lys Leu 65 70 75

GGA GTC CCC AAG ACT CAC CTA GAG CTA AAG AAA TTA ATT GGA GAG GTG 350 Gly Val Pro Lys Thr His Leu Glu Leu Lys Lys Leu lie Gly Glu Val 80 85 90

TCC AGT GGC TCC GGG GAG ACG TTC AGC TAC CCT GAC TTT CTC AGG ATG 398 Ser Ser Gly Ser Gly Glu Thr Phe Ser Tyr Pro Asp Phe Leu Arg Met 95 100 105

ATG CTG GGC AAG AGA TCT GCC ATC CTA AAA ATG ATC CTG ATG TAT GAG 446 Met Leu Gly Lys Arg Ser Ala lie Leu Lys Met lie Leu Met Tyr Glu 110 115 120 125

GAA AAA GCG AGA GAA AAG GAA AAG CCA ACA GGC CCC CCA GCC AAG AAA 494

Glu Lys Ala Arg Glu Lys Glu Lys Pro Thr Gly Pro Pro Ala Lys Lys 130 135 140

GCT ATC TCT GAG TTG CCC TGATTTGAAG GGAAAAGGGA TGATGGGATT 542

Ala lie Ser Glu Leu Pro 145

GAAGGGGCTT CTAATGACCC AGATATGGAA ACAGAAGACA AAATTGTAAG CCAGAGTCAA 602

CAAATTAAAT AAATTACCCC CTCCTCCAAA AAA 635

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 147 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Ser Gin Thr Arg Asp Leu Gin Gly Gly Lys Ala Phe Gly Leu Leu 1 5 10 15

Lys Ala Gin Gin Glu Glu Arg Leu Asp Glu lie Asn Lys Gin Phe Leu 20 25 30

Asp Asp Pro Lys Tyr Ser Ser Asp Glu Asp Leu Pro Ser Lys Leu Glu 35 40 45

Gly Phe Lys Glu Lys Tyr Met Glu Phe Asp Leu Asn Gly Asn Gly Asp 50 55 60

lie Asp lie Met Ser Leu Lys Arg Met Leu Glu Lys Leu Gly Val Pro 65 70 75 80

Lys Thr His Leu Glu Leu Lys Lys Leu lie Gly Glu Val Ser Ser Gly 85 90 95

Ser Gly Glu Thr Phe Ser Tyr Pro Asp Phe Leu Arg Met Met Leu Gly 100 105 110

Lys Arg Ser Ala lie Leu Lys Met lie Leu Met Tyr Glu Glu Lys Ala 115 120 125

Arg Glu Lys Glu Lys Pro Thr Gly Pro Pro Ala Lys Lys Ala lie Ser 130 135 140

Glu Leu Pro 145

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ACTTCAGACT CTCTCTTCCC TACGG 25

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GCATCAGGGA GCATTATTTA TTTAGTTT 28

Claims

What is claimed is:

1. A method of diagnosing proliferative arterial disease characterized by excessive production of RC-9, comprising die steps of:

(a) comprising contacting a sample from a patient with a diagnostic reagent selected from die group consisting of:

(i) an antibody to RC-9;

(ii) an RC-9 polynucleotide sequence; and

(iii) an RC-9 protein; said reagent associated widi a detectable label, and

(b) detecting the presence of RC-9 in said sample by the association between said reagent and RC-9 in said sample.

2. The method according to claim 1, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a Fab fragment, and a single chain antibody.

3. The metiiod according to claim 2, wherein die antibody is a monoclonal antibody.

4. The metiiod according to claim 1 wherein die polynucleotide sequence is selected from die group consisting of:

(a) a DNA sequence comprising SEQ DD NO: 1;

(b) a DNA sequence comprising SEQ DD NO: 2;

(c) a DNA sequence comprising SEQ DD NO: 3;

(d) a DNA sequence comprising SEQ DD NO: 4;

(e) an RNA sequence corresponding to the DNA sequence of (a) - (d) above;

(f) sequences capable of hybridizing to (a) - (d) under stringent conditions; and

(g) fragments and analogs of (a) - (d) having a RC-9-like biological activity.

. The method according to claim 1, wherein the RC-9 protein is selected from die group consisting of:

(a) die amino acid sequence of SEQ DD NO:4;

(b) the amino acid sequence of SEQ DD NO :6;

(c) analogs of (a) or (b); and

(d) a fragment of (a) or (b) comprising 5 amino acids and characterized by RC-9-like biological activity.

6. A method of treating proliferative arterial disease characterized by excessive production of RC-9, comprising die step of administering to a patient a pharmaceutical composition comprising an antibody to RC-9.

7. The metiiod according to claim 6, wherein the antibody is selected from the group consisting ofa polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a Fab fragment, and a single chain antibody.

8. The metiiod according to claim 7, wherein the antibody is a monoclonal antibody.

9. A metiiod of identifying compounds which specifically bind to a selected RC- 9 DNA sequence, comprising the steps of contacting said RC-9 DNA sequence with a test compound to permit binding ofthe test compound to the DNA sequence; and deterπiining the amount of test compound which is bound to the DNA sequence.

10. The method according to claim 10 wherein said RC-9 DNA sequence is immobilized on a sohd support.

11. A method of identifying compounds which specifically bind to a selected RC - 9 amino acid sequence, comprising the steps of contacting said RC-9 amino acid sequence with a test compound to permit binding of die test compound to the amino acid sequence; and determining die amount of test compound which is bound to die amino acid sequence.

12. The method according to claim 10 wherein said RC-9 amino acid sequence is immobilized on a sohd support.

13. A method of identifying compounds which specifically bind to a selected RC- 9 antibody, comprising the steps of contacting said RC-9 antibody with a test compound to permit binding ofthe test compound to die antibody; and determining die amount of test compound which is bound to the antibody.

14. The metiiod according to claim 10 wherein said RC-9 antibody is immobilized on a sohd support.

15. A compound capable of inhibiting the activity of RC-9, produced by screening a group of compounds with a RC-9 protein, RC-9 DNA sequence or anti-RC-9 antibody.

16. Use of an RC-9 antibody in die preparation of a medicament for the treatment of proliferative arterial disease characterized by excessive production of RC-9.