AU7735696A - Chemokine from niddm pancreas - Google Patents

Description

CHEMOKINE FROM NIDDM PANCREAS

TECHNICAL FIELD

The present invention relates to nucleic acid and amino acid sequences ofa novel chemokine from the pancreas of a patient with non-insulin dependent diabetes mellitus (NIDDM) and to the use of these sequences in the diagnosis, study, prevention and treatment of disease.

BACKGROUND ART The chemokines are small polypeptides, generally about 70-100 amino acids (aa) in length, 8-1 1 kD in molecular weight, and active over a 1 -100 ng/ml concentration range. Initially, chemokines were isolated and purified from inflamed tissues and characterized relative to their bioactivity. More recently, chemokines have been discovered through molecular cloning techniques and characterized by structural as well as functional analysis. Closely related polypeptides currently assigned to a chemokine family also display definitive spacing of the first two cysteine residues in the mature molecule and act on a diverse group of target cells which include monocytes/macrophages, basophils, eosinophils, T lymphocytes and others. The known chemokines and their functions are reviewed by Thomson in The Cytokine Handbook (1994, Academic Press, New York NY). Relatively few C-C chemokines have been described, and they appear to have less

N-terminal processing than the nearest related family, the C-X-C chemokines. Known C-C chemokines include the monocyte chemotactic proteins (MCP), macrophage inflammatory proteins (MIP), 1-309, TCA3, and RANTES.

Monocyte chemotactic protein (MCP-1) is a 76 amino acid mature protein which appears to be expressed in almost all cells and tissues upon stimulation by a variety of agents. According to Charo (personal communication) the targets of MCP- 1 may be limited to monocytes and basophils in which MCP-1 induces calcium flux through a G protein-linked receptor. Shyy Y-J et al (1990;Biochem Biophys Res Commun 169:346-351) reported the induction of MCP- 1 in endothelial cell cultures by phorbol ester treatment and its possible implication in atherogenesis. Two other related proteins (MCP-2 and MCP-3) have been purified from a human osteosarcoma cell line. MCP-2 and MCP-3 have 62% and 73% amino acid identity, respectively, with MCP-1 and both share MCP- l's chemoattractant specificity for monocytes. MlP-lα and MIP-lβ were first purified from a stimulated mouse macrophage cell line and elicited an inflammatory response when injected into normal tissues. At least three distinct and non-allelic genes encode human MlP-lα, and seven genes, MIP-1B. MlP-lα and MIP-l β consist of 68-69 amino acids which are about 70% identical in their acidic, mature secreted forms. They are both expressed in stimulated T cells, B cells, and monocytes in response to mitogens, anti-CD3 and endotoxin; and both polypeptides bind heparin. Whiie both molecules stimulate monocytes, MIP- l et chemoattracts the CD-8 subset of T lymphocytes and eosinophils, while MIP-1B chemoattracts the CD-4 subset of T lymphocytes. In mouse, these proteins are known to stimulate myelopoiesis. 1-309 was cloned from a human γ-δ T cell line and shows 42% amino acid identity to T cell activation gene 3 (TCA3) cloned from mouse. There is considerable nucleotide homology between the 5' flanking regions of these two proteins, and they share an extra pair of cysteine residues not found in other chemokines. Such similarities suggest 1-309 and TCA3 are species homologs which have diverged over time in both sequence and function. RANTES is another C-C chemokine which is expressed in T cells (but not B cells), in platelets, in some tumor cell lines, and in rheumatoid synovial fibroblasts. In the fibroblasts, it is regulated by interleukins- 1 and -4, transforming nerve factor and interferon-γ. The cDNA cloned from T cells encodes a basic 8 kD protein which lacks N-linked glycosylation and is able to affect lymphocytes, monocytes, basophils and eosinophils. The expression of RANTES mRNA is substantially reduced following T cell stimulation.

Leukocytes including monocytes, macrophages, basophils, and eosinophils infiltrate inflamed areas ofthe pancreas, as is the case in inflammation of other tissues. While their primary role is to clean up inflammation, leukocytes also secrete a range of cytokines which recruit other cells to the area. Monocytes and macrophages may produce powerful oxidants and proteases which contribute to unwarranted tissue destruction.

The various chemokines possess the cellular specificity required to explain leukocyte trafficking under different abnormal, inflammatory or diseased situations. First, chemokines mediate the expression of particular selectin, integrin or other adhesion molecules on endothelial cells which precipitate diapedesis and extravasation; and second, they generate gradients of chemoattractant factors which activate specific cell types. In addition, the chemokines stimulate the proliferation and activation of cells which bear specific receptors, activities which demonstrate a high degree of target cell specificity. The chemokines are reviewed in Schall TJ (1994) Chemotactic Cytokines: Targets for Therapeutic Development. International Business Communications, Southborough MA, pp 180-270; and in Paul WE (1993) Fundamental Immunology. Raven Press, New York NY, pp 822-826. Pancreas, the tissue from which the chemokine of the present invention was derived, consists of both exocrine and endocrine tissues. In descending order, the exocrine portion is divided into lobes, lobules, and functional secretory units known as acini. All acini drain into the pancreatic duct which eventually empties into the duodenum. Acinar cells comprise 80% of the pancreas and secrete enzymes which assist digestion. Epithelia! cells of the ductules secrete large amounts of bicarbonate ions and water as well as the enzymes for digesting protein, carbohydrates, and fats. The most important and abundant proteolytic enzymes are trypsin, chymotrypsin, carboxypeptidase, elastase, amylase. pancreatic lipase, cholesterol esterase, and phospholipase. Acetylcholine, gastrin, cholecystokinin (CCK), and secretin control acinar secretion. The endocrine pancreas consists of islets of Langerhans which are distributed throughout the pancreas and secrete the hormones which participate in the metabolism of proteins, carbohydrates, and fats. The major endocrine cells are a cells which secrete glucagon, β cells which secrete insulin, and d cells which secrete somatostatin and vasoactive intestinal peptide; minor endocrine cells include C cells whose function is unknown, EC cells which secrete serotonin and PP cells which secrete pancreatic polypeptide.

Inflammation ofthe pancreas or pancreatitis may be classified as either acute or chronic by clinical criteria. With treatment, acute pancreatitis can often be cured, and normal function restored. Chronic pancreatitis often results in permanent damage. The precise mechanisms which trigger inflammation are not understood; however, some causes are alcohol ingestion, biliary tract disease, post-operative trauma, and hereditary disorders. Premature activation of proteolytic enzymes or blockage of pancreatic ducts may activate autodigestion resulting in grave cellular damage.

Diabetes mellitus is the most significant pancreatic disease. The clinical symptoms of non-insulin dependent diabetes (NIDDM) progress through three different phases. In the first phase, plasma glucose levels remain normal despite demonstrable insulin resistance in the presence of elevated insulin. Second, insulin resistance worsens, and glucose intolerance manifests as postprandial hyperglycemia. Finally, insulin resistance stabilizes, insulin secretion declines, and fasting hyperglycemia and overt diabetes become obvious. Patients with NIDDM often manifest no clinical symptoms until the later stages of disease development. long after the insidious onset of the underlying autoimmune destruction.

Current techniques for diagnosis of NIDDM or other abnormalities of the pancreas mainly rely on observation of clinical symptoms or serological analyses of body fluids or tissues.

Unfortunately, these symptoms and analyses are not present at early stages or may not differentiate between invasive diseases and other disorders which have overlapping or very similar ranges. The nucleic acid and amino acid sequences of a new chemokine associated with NIDDM pancreas would provide the means by which to diagnose incipient pathology and to develop drugs which specifically disrupt that pathology.

Anatomy, physiology, and diseases of the pancreas are reviewed, inter alia, in Guyton, AC (1991 Textbook of Medical Physiology. WB Saunders Co, Philadelphia PA; Isselbacher, KJ et al. (1994) Harrison's Principles of Internal Medicine. McGraw-Hill, New York NY; Johnson KE ( 1991 ) Histology and Cell Biology. Harwal Publishing, Media PA; and The Merck Manual of Diagnosis and Therapy (1992) Merck Research Laboratories, Rahway NJ.

DISCLOSURE OF THE INVENTION

The present invention relates to a novel chemokine identified among the cDNAs from a non-insulin dependent diabetes mellitus (NIDDM) pancreas library and to the use of the nucleic acid and amino acid sequences of this novel chemokine in the study, diagnosis, prevention and treatment of pancreatic disease. The novel chemokine of the present invention was first identified within Incyte Clone 273061 through a computer generated search for amino acid sequence alignments. The chemokine has a length of 99 amino acids, definitive cysteine residues at C₃₄ and C₃₅, and approximately 66% amino acid similarity to the deduced sequence of human mcp-1 (GenBank GI 487124; Shyy Y-J et al. (1990) Biochem Biophys Res Commun

169:346-351 ). The nucleic acid sequence of the novel chemokine designated herein as mcp-4, SEQ ID NO: 1 , encodes the amino acid sequence for MCP-4, SEQ ID NO: 2. The present invention is based, in part, on the homology between MCP-4 and the monocyte chemotactic protein, MCP-1 , and the definitive chemokine residues, C₃₄ and C₃₅, of MCP-4. The complete polynucleotide encoding mcp-4 provides the basis for diagnosis of pancreas related diseases, including NIDDM, before clinical symptoms or tissue destruction become obvious. It also provides for the design of antisense molecules useful in diminishing or eliminating expression of the genomic nucleotide sequence in individuals with overactive immune response, specifically to prevent destruction of pancreatic tissue. The present invention also relates, in part, to the inclusion of the polynucleotide in an expression vector which can be used to transform host cells or organisms. Such transgenic hosts are useful for production and recovery of the encoded MCP-4.

The invention further comprises administration of purified MCP-4 to immune-compromised individuals for the purpose of inducing leukocyte proliferation. It also provides for the use of MCP-4 to produce antibodies or to identify other antagonists or inhibitors which bind MCP-4. Antibodies, antagonists or inhibitors can be used in hyperresponsive individuals to prevent MCP-4 from inducing proliferation of leukocytes or from attracting them to a particular inflamed site thereby downregulating the immune response and preventing monocytes, macrophages and T cells from secreting proteolytic enzymes which cause damage. The invention also comprises pharmaceutical compositions containing the polypeptide, antibodies, antagonists or inhibitors for the diagnosis, prevention or treatment of NIDDM or conditions with similar biochemical or immunological properties such as viral, bacterial, fungal or helminthic infections; allergic or asthmatic responses; mechanical injury associated with trauma; arteriosclerosis, atherogenesis or collagen vascular diseases; hereditary diseases such as rheumatoid arthritis; leukemia, lymphomas or carcinomas; or other conditions which involve leukocytes, particularly monocytes, macrophages and T cells. The mcp-4 polynucleotide sequence, oligonucleotides. fragments, portions or antisense thereof, may be used in diagnostic assays to detect and quantify levels of mcp-4 mRNA in cells and tissues. For example, mcp-4 may be used for hybridization or amplification in solution-, membrane-, or tissue-based technologies to diagnose abnormalities in gene expression. The invention further provides diagnostic assays for the detection of native MCP-4 comprising purified polypeptide to be used as a positive control and anti-MCP-4 antibodies. Such antibodies may be used in solution-, membrane-, or tissue-based technologies to detect any disease state or condition related to the aberrant expression of MCP-4.

MCP-4, antisense molecules, antibodies, antagonists or inhibitors may be used for therapeutic purposes, for example, in stimulating leukocyte proliferation or in neutralizing the aberrant activity of MCP-4 associated with, for example, inflammation of pancreatic tissue. The present invention also provides for pharmaceutical compositions for the treatment of disease states associated with aberrant expression of mcp-4 comprising antisense molecules capable of inhibiting transcription and translation of mcp-4, and recombinant MCP-4 as well as antibodies, antagonists or inhibitors capable of binding to native MCP-4.

BRIEF DESCRIPTION OF DRAWINGS Figure 1 displays the nucleic acid and amino acid sequences of the novel human chemokine from NIDDM pancreas. The alignment was produced using MacDNAsis PRO™ software (Hitachi Software Engineering Co. Ltd,. San Bruno CA).

Figure 2 shows the amino acid sequence similarity between MCP- 1 , (GenBank GI 487124; Shyy Y-J et al. (1990) Biochem Biophys Res Commun 169:346-351) and MCP-4. The alignment was produced using the multisequence alignment program of DNASTAR software

(DNASTAR Ine, Madison WI)

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel chemokine which is expressed in pancreatic tissue. As used herein, the abbreviation for the novel macrophage chemotactic protein in lower case (mcp-4) refers to a nucleic acid sequence while the upper case (MCP-4) refers to an amino acid sequence.

Nucleic acid sequence as used herein refers to an oligonucleotide, nucleotide or polynucleotide sequence, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand. Similarly, amino acid sequence as used herein refers to an oligopeptide, peptide, polypeptide or protein sequence.

As used herein, MCP-4 refers to MCP-4 from any species, including, bovine, ovine, porcine, equine, murine and preferably human, in naturally occurring or in variant form, or from any source, whether natural, synthetic, semi-synthetic or recombinant. A preferred MCP-4 variant is one having at least 80% amino acid sequence similarity, another preferred MCP-4 variant is one having at least 90% amino acid sequence similarity and another preferred MCP-4 variant is one having at least 95% amino acid sequence similarity to the MCP-4 amino acid sequence illustrated in Figure 1. MCP-4 is a chemokine which contains the definitive double cysteine motif, C₃₄ and C₃₅, shown by MCP-1 and MCP-4 in Figure 2. Chemokines are involved in leukocyte proliferation and trafficking and are known to be produced by damaged or stressed cells as well as cells ofthe immune system.

As used herein, "naturally occurring" refers to an MCP-4 with an mRNA sequence found in nature. Similarly, the term "biologically active" refers to an MCP-4 having structural, regulatory or biochemical functions of the naturally occurring MCP-4. Likewise, "immunological activity" is defined as the capability of the natural, recombinant or synthetic MCP-4, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

The term "derivative" as used herein refers to the chemical modification of an mcp-4 or the encoded MCP-4. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. An mcp-4 derivative would encode a polypeptide which retains essential biological characteristics of a C-C chemokine such as, for example, the chemoattraction of monocytes.

As used herein, the term "purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment and isolated or separated from at least one other component with which they are naturally associated.

The MCP-4 Coding Sequences

The nucleic and deduced amino acid sequences of MCP-4 are shown in Figure 1. In accordance with the invention, any nucleotide sequence which encodes the amino acid sequence of MCP-4 can be used to generate recombinant molecules which express MCP-4. In a specific embodiment described herein, mcp-4 was first isolated and identified within Incyte Clone 273061 from a non-insulin dependent diabetes mellitus (NIDDM) pancreas library.

Methods for DNA sequencing are well known in the art and employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase^® (US Biochemical Corp, Cleveland OH)), Taq polymerase (Perkin Elmer, Foster City CA), thermostable T7 polymerase (Amersham,

Chicago IL), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg MD) Methods to extend the DNA from an oligonucleotide primer annealed to the DNA template of interest have been developed for both single- and double-stranded templates. Chain termination reaction products were separated using electrophoresis and detected via their incoφorated, labeled precursors. Recent improvements in mechanized reaction preparation, sequencing and analysis have permitted expansion in the number of sequences that can be determined per day. Preferably, the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton. Reno NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA) and the Applied Biosystems (Foster City CA) Catalyst 800 and 377 and 373 DNA sequencers.

The quality of any particular cDNA library may be determined by performing a pilot scale analysis ofthe cDNAs and checking for percentages of clones containing vector, lambda or E. coli DNA, mitochondrial or repetitive DNA, and clones with exact or homologous matches to public databases. Extending mcp-4 Polynucleotide Sequence

The polynucleotide sequence of mcp-4 may be extended utilizing partial nucleotide sequence and various methods known in the art to detect upstream sequences such as promoters and regulatory elements. Gobinda et al. (1993; PCR Methods Applic 2:318-22) disclose "restriction-site polymerase chain reaction (PCR)" as a direct method which uses universal primers to retrieve unknown sequence adjacent to a known locus. First, genomic DNA is amplified in the presence of primer to a linker sequence and a primer specific to the known region. The amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR can be used to amplify or extend sequences using divergent primers based on a known region (Triglia T et a!.(1988) Nucleic Acids Res 16:8186). The primers may be designed using Oligo 4.0 (National Biosciences Ine, Plymouth MN), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68°-72° C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Capture PCR (Lagerstrom M et al. (1991) PCR Methods Applic 1:11 1-19) is a method for

PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome (YAC) DNA. Capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double-stranded sequence into an unknown portion of the DNA molecule before PCR. Parker JD et al. (1991 ; Nucleic Acids Res 19:3055-60), teach walking PCR, a method for targeted gene walking which permits retrieval of unknown sequence. PromoterFinder™ a new kit available from Clontech (Palo Alto CA) uses PCR, nested primers and PromoterFinder libraries to walk in genomic DNA. This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

Another PCR method, "Improved Method for Obtaining Full Length cDNA Sequences " by Guegler et al. Patent Application Serial No 08/487,1 12, filed June 7, 1996. and hereby incorporated by reference, employs XL-PCR^{I M} (Perkin Elmer, Foster City CA) to amplify and extend nucleotide sequences.

Preferred libraries for screening for full length cDNAs are ones that have been size-selected to include larger cDNAs. Also, random primed libraries are preferred in that they will contain more sequences which contain the 5' and upstream regions of genes. A randomly primed library may be particularly useful if an oligo d(T) library does not yield a full-length cDNA. Genomic libraries are useful for extension 5' ofthe promoter binding region.

A new method for analyzing either the size or confirming the nucleotide sequence of sequencing or PCR products is capillary electrophoresis. Systems for rapid sequencing are available from Perkin Elmer, Beckman Instruments (Fullerton CA), and other companies. Capillary sequencing employs flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled devise camera. Output/light intensity is converted to electrical signal using appropriate software (eg. Genotyper™ and Sequence Navigator™ from Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data display is computer controlled. Capillary electrophoresis is particularly suited to the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample. The reproducible sequencing of up to 350 bp of Ml 3 phage DNA in 30 min has been reported (Ruiz-Martinez MC et al. (1993) Anal Chem 65:2851 -8).

Expression of mcp-4

In accordance with the present invention, mcp-4 polynucleotide sequences which encode MCP-4, fragments ofthe polypeptide, fusion proteins or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of MCP-4 in appropriate host cells. Due to the inherent degeneracy ofthe genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used to clone and express MCP-4. As will be understood by those of skill in the art, it may be advantageous to produce MCP-4-encoding nucleotide sequences possessing non-naturally occurring codons. Codons preferred by a particular prokaryotic or eukaryotic host (Murray E et al. (1989) Nuc Acids Res 17:) can be selected, for example, to increase the rate of MCP-4 expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence. Also included within the scope of the present invention are polynucleotide sequences that are capable of hybridizing to the nucleotide sequence of Figure 1 under conditions of intermediate to maximal stringency. Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Wahl and Berger (1987, Methods Enzymol 152:399-407) and Kimmel (1987. Methods Enzymol 152:507-1 1) incoφorated herein by reference, and confer a defined "stringency" as explained below.

"Maximum stringency" typically occurs at about Tm-5°C (5°C below the Tm of the probe); "high stringency" at about 5°C to 10°C below Tm; "intermediate stringency" at about 10°C to 20°C below Tm; and "low stringency" at about 20°C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences. The term "hybridization" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictionary of Biotechnology. Stockton Press, New York NY) as well as the process of amplification has carried out in polymerase chain reaction technologies as described in Dieffenbach CW and GS Dveksier

(1995, PCR Primer, a Laboratory Manual. Cold Spring Harbor Press, Plainview NY) and incoφorated herein by reference.

As used herein a "deletion" is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent. As used herein an "insertion" or "addition" is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring mcp-4.

As used herein "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. Altered mcp-4 polynucleotide sequences which may be used in accordance with the invention include deletions, insertions or substitutions of different nucleotide residues resulting in a polynucleotide that encodes the same or a functionally equivalent MCP-4. The protein may also show deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent MCP-4. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of MCP-4 is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine, phenylalanine, and tyrosine.

Included within the scope of the present invention are alleles of mcp-4. As used herein, an "allele" or "allelic sequence" is an alternative form of mcp-4. Alleles result from a mutation, ie, a change in the nucleic acid sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

The nucleotide sequences ofthe present invention may be engineered in order to alter an mcp-4 coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, eg, site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns, to change codon preference, etc.

In another embodiment of the invention, an mcp-4 natural, modified or recombinant sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for inhibitors of MCP-4 activity, it may be useful to encode a chimeric MCP-4 protein expressing a heterologous epitope that is recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between an MCP-4 sequence and the heterologous protein sequence, so that the MCP-4 may be cleaved and purified away from the heterologous moiety.

In an alternate embodiment ofthe invention, the coding sequence of mcp-4 could be synthesized, whole or in part, using chemical methods well known in the art (See Caruthers et al.

(1980) Nuc Acids Res Symp Ser 7:215-233; Crea and Horn (1980) Nuc Acids Res 9:2331 ; Matteucci and Caruthers (1980) Tetrahedron Lett 21 :719; and Chow and Kempe (1981) Nuc Acids Res 9:2807-2817). Alternatively, the protein itself could be produced using chemical methods to synthesize an MCP-4 amino acid sequence, whole or in part. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (eg, Creighton (1983) Proteins Structures And Molecular Principles. WH Freeman and Co, New York NY). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (eg. the Edman degradation procedure; Creighton, supra).

Direct peptide synthesis can be performed using various solid-phase techniques (Roberge JY et al. ( 1995) Science 269:202-204) and automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer in accordance with the instructions provided by the manufacturer. Additionally the amino acid sequence of MCP-4, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with other chemokine sequences, or any part thereof, to produce a variant polypeptide.

Expression Systems

In order to express a biologically active MCP-4, the nucleotide sequence coding for MCP-4, or a functional equivalent, is inserted into an appropriate expression vector, ie, a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing an MCP-4 coding sequence and appropriate transcriptional or translational controls. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination or genetic recombination. Such techniques are described in Maniatis et al. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Plainview NY and Ausubel FM et al. (1989) Current Protocols in Molecular Biology. John

Wiley & Sons, New York NY.

A variety of expression vector/host systems may be utilized to contain and express an mcp-4 coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (eg, baculovirus); plant cell systems transfected with virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (eg, Ti or pBR322 plasmid); or animal cell systems.

The "control elements" or "regulatory sequences" of these systems vary in their strength and specificities and are those nontranslated regions of the vector, enhancers, promoters, and 3 ^' untranslated regions, which interact with host cellular proteins to carry out transcription and translation. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the pBluescript^® phagemid (Stratagene, LaJolla CA) and ptφ-lac hybrids and the like may be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (eg, heat shock, RUBISCO; and storage protein genes) or from plant viruses (eg, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from the mammalian genes or from mammalian viruses are most appropriate. If it is necessary to generate a cell line that contains multiple copies of mcp-4, vectors based on SV40 or EBV may be used with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selected depending upon the use intended for MCP-4. For example, when large quantities of MCP-4 are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable. Such vectors include, but are not limited to. the E. coli cloning and expression vector pBluescript^® (Stratagene), in which the mcp-4 coding sequence may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster (1989) J Biol Chem 264:5503-5509); and the like. pGEX vectors (Promega. Madison WI) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsoφtion to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems are designed to include heparin, thrombin or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will. In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544. In cases where plant expression vectors are used, the expression of an MCP-4 coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al. (1984) Nature 310:51 1-514) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al. (1987) EMBO J 6:307-31 1). Alternatively, plant promoters such as the small subunit of RUBISCO

(Coruzzi et al. (1984) EMBO J 3: 1671-1680; Broglie et al. ( 1984) Science 224:838-843); or heat shock promoters (Winter J and Sinibaldi RM (1991 ) Results Probl Cell Differ 17:85-105) may be used. These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. For reviews of such techniques, see Hobbs S or Murry LE in McGraw Yearbook of Science and Technology ( 1992) McGraw Hill New York NY, pp 191 - 196 or Weissbach and Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, New York NY, pp 421-463.

An alternative expression system which could be used to express mcp-4 is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The mcp-4 coding sequence may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control ofthe polyhedrin promoter. Successful insertion of mcp-4 will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which MCP-4 is expressed (Smith et al. (1983) J Virol 46:584; Engelhard EK et al.

(1994) Proc Nat Acad Sci 91 :3224-7).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, an mcp-4 coding sequence may be ligated into an adenovirus transcription/translation complex consisting ofthe late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region ofthe viral genome will result in a viable virus capable of expressing MCP-4 in infected host cells. (Logan and Shenk (1984) Proc Natl Acad Sci 81 :3655-3659). In addition, transcription enhancers, such as the rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. Specific initiation signals may also be required for efficient translation of an inserted mcp-4 coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where mcp-4, its initiation codon and upstream sequences, are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system (Scharf et al. (1994) Results Probl Cell Differ 20:125-62: Bittner et al. (1987) Methods Enzymol 153:516-544).

In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to. acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "prepro" form ofthe protein may also be important for correct insertion, folding and/or function. Different host cells such as CHO. HeLa, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express mcp-4 may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media, before they are switched to selective media. The selectable marker confers resistance to selection and allows identification of cells which have stably integrated the introduced sequences into their DNA. Resistant clumps of cells can be proliferated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed cell line. These include, but are not limited to, the heφes simplex virus thymidine kinase (Wigler et al. (1977)

Cell 1 1 :223) and adenine phosphoribosyltransferase (Lowy et al. (1980) Cell 22:817) genes which can be employed in tk^" or aprt^" cells, respectively. Also, antimetabolite antibiotic or herbicide resistance can be used as the basis of selection; for example, dhfr confers resistance to methotrexate (Wigler et al. (1980) Natl Acad Sci 77:3567); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colberre-Garapin et al. ( 1981 ) J Mol Biol 150: 1) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase (Murry, supra). Additional selectable genes have been described, for example, tφB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman and Mulligan (1988) Proc Natl Acad Sci 85:8047). Recently, the use of visible markers has gained popularity with such markers as β glucuronidase. anthocyanin, and luciferin being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes CA et al.

(1995) Methods Mol Biol 55: 121-31).

Identification of Transformants Containing mcp-4

Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed. For example, if the mcp-4 is inserted within a marker gene sequence, recombinant cells containing mcp-4 can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with an MCP-4 sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of mcp-4 as well. Alternatively, host cells which contain the coding sequence for mcp-4 and express MCP-4 may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques which include membrane-, solution-, or chip-based technologies for the detection and/or quantification of the nucleic acid or protein. The presence ofthe mcp-4 polynucleotide sequence can be detected by DNA-DNA or

DNA-RNA hybridization or amplification using probes, portions or fragments of mcp-4. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the mcp-4 sequence to detect transformants containing mcp-4 DNA or RNA. As used herein "oligonucleotides" or "oligomers" refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides which can be used as a probe or amplimer.

The expression of an MCP-4 protein product can be assessed biologically in a chemotaxis or Ca ++ mobilization assay or immunologically in Western blot, enzyme-linked immunoassays (ELISA) and the like. Falk WR et al. (1980, J Immunol Methods 33:239) first described the assessment of chemotactic activity using 48-well microchemotaxis chambers. In this assay, the expressed chemokine is placed in media on one side of a polycarbonate filter and a particular population of cells is suspended in the same media on the opposite side ofthe filter. Sufficient incubation time allows the cells to traverse the filter in response to the chemokine concentration gradient. Filters are recovered from each well, and the cells adhering to the side of the filter facing the chemokine are typed and quantified. Populations of cells used in such assays may include blood cells obtained from venipuncture or enriched populations of neutrophils, peripheral blood mononuclear cells, monocytes and lymphocytes obtained by density gradient centrifugation and/or negative selection using antibodies specific for surface molecules of the nondesired population. For example, incubating a population of T cells with CD4+ and separating out CD4+bound T cells may result in a CD8+ enriched T-cell population.

To assay non-chemotactic activity of neutrophils and monocytes, testing may involve measurement of actin polymerization, increase in respiratory burst activity, degranulation ofthe azurophilic granule or mobilization of Ca" and comparison of the results with standard measurements. The assay for mobilization of Ca" as part of the signal transduction pathway requires preloading neutrophils with a fluorescent probe whose emission characteristics have been altered by Ca" binding. When the cells are exposed to an activating stimulus, Ca" flux is determined by observation of the cells in a fluorometer. The measurement of Ca" mobilization has been described in Grynkievicz G et al. (1985) J Biol Chem 260:3440, and McColl S et al. (1993) J Immunol 150:4550-4555, incoφorated herein by reference. Degranulation and respiratory burst responses are similarly measured in monocytes

(Zachariae COC et al. (1990) J Exp Med 171 : 2177-82). Further measures of monocyte activation are regulation of adhesion molecule expression in lymphocytes (Jiang Y et al. (1992) J Immunol 148: 2423-8; Taub D et al. (1993) Science 260: 355-358).

A variety of protocols for detecting and measuring the expression of MCP-4, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on MCP-4 is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R et al. (1990, Serological Methods, a Laboratory Manual. APS Press, St Paul MN) and

Maddox DE et al. (1983, J Exp Med 158:121 1).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to mcp-4 include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the mcp-4 sequence, or any portion of it, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art. are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway NJ), Promega (Madison WI), and US Biochemical Coφ (Cleveland OH) supply commercial kits and protocols for these procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US Patents 3,817,837; 3,850.752; 3.939,350; 3,996,345; 4,277,437; 4,275.149 and 4,366,241. Also, recombinant immunoglobulins may be produced as shown in US Patent No. 4,816,567 incoφorated herein by reference.

Purification of MCP-4

Host cells transformed with an mcp-4 nucleotide sequence may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein produced by a recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing mcp-4 can be designed with signal sequences which direct secretion of MCP-4 through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join mcp-4 to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et ai. (1993) DNA Cell

Biol 12:441-53; see also above discussion of vectors containing fusion proteins).

MCP-4 may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Coφ, Seattle WA). The inclusion of a cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and MCP-4 is useful to facilitate purification.

Uses of MCP-4 MCP-4 would appear to play a role in systemic defense based on its sequence similarity to MCP-1 and its expression in NIDDM pancreas. Therefore, anti-MCP-4 antibodies or other polypeptides, proteins or organic molecules that modulate the activity of MCP-4 can be used therapeutically in the treatment of disease states resulting from aberrant expression of MCP-4. A therapeutic molecule may find application in a disease state associated with MCP-4 such as NIDDM. In another embodiment of the present invention. anti-MCP-4 antibodies capable of neutralizing the activity of MCP-4 may be used to prevent or treat conditions or disease states which display biochemical similarities to NIDDM. The ability of antibodies, peptides, or other molecules to modulate the effect(s) of MCP-4 may be measured using the microchemotaxis or Ca++ flux assays described above. In fact, comparing the standard response with the amount of modulation in vitro may be used to rank the usefulness of various therapeutic molecules.

Procedures well known in the art may be used for the production of antibodies to MCP-4. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Neutralizing antibodies, ie, those which inhibit biological activity of MCP-4, are especially preferred for diagnostics and therapeutics.

For the production of antibodies, various hosts including goats, rabbits, rats, mice. etc. may be immunized by injection with MCP-4 or any portion, fragment or oligopeptide which retains immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants which may be employed if purified MCP-4 is administered to immunologically compromised individuals for the puφose of stimulating leukocyte proliferation. Monoclonal antibodies to MCP-4 may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler et al. (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kozbor et al. (1985) J Immunol Methods 81 :31 -42; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al (1984) Mol Cell Biol 62: 109- 120 ). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et al (1984) Proc Natl Acad Sci 81 :6851 -6855; Neuberger et al ( 1984) Nature 312:604-608; Takeda et al (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies (US Patent No. 4,946,778) can be adapted to produce MCP-4-specific single chain antibodies. Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991 ; Nature 349:293-299).

Antibody fragments which contain specific binding sites for MCP-4 may also be generated. For example, such fragments include, but are not limited to, the F(ab')_: fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al. (1989) Science 256: 1275- 1281 ). MCP-4-specific antibodies are useful for the diagnosis of conditions and diseases associated with expression of MCP-4. A variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the formation of complexes between MCP-4 and its specific antibody (or similar MCP-4-binding molecule) and the measurement of complex formation. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two noninterfering epitopes on a specific MCP-4 protein is preferred, but a competitive binding assay may also be employed. These assays are described in Maddox DE et al. (1983, J Exp Med 158: 121 1).

Diagnostic Assays Using MCP-4 Specific Antibodies

Particular MCP-4 antibodies are useful for the diagnosis of conditions, disorders or diseases characterized by aberrant expression of MCP-4. Diagnostic assays for MCP-4 include methods utilizing the antibody and a label to detect MCP-4 in human body fluids, cells, tissues or extracts of such tissues. The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule. A wide variety of reporter molecules are known, several of which were described above.

A variety of protocols for measuring MCP-4, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked irnmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on MCP-4 is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, DE et al. (1983. J Exp Med 158: 121 1).

In order to provide a basis for the diagnosis of disease, normal or standard values for MCP-4 expression must be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with antibody to MCP-4 under conditions suitable for complex formation which are well known in the art. The amount of standard complex formation may be quantified by comparing it with a dilution series of positive controls where a known amount of antibody is combined with known concentrations of purified MCP-4. Then, standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by a disorder or disease related to

MCP-4 expression. Deviation between standard and subject values establishes the presence of disease state.

Drug Screening MCP-4, its catalytic or immunogenic fragments or oligopeptides can be used for screening therapeutic compounds in any ofa variety of drug screening techniques. The fragment employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of catalytic activity or the formation of binding complexes, between MCP-4 and the agent being tested, may be measured. Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the MCP-4 polypeptides and is described in detail in European Patent Application 84/03564, published on September 13, 1984, incoφorated herein by reference. In summary, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with MCP-4 fragment and washed. Bound MCP-4 is then detected by methods well known in the art. Purified MCP-4 can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding MCP-4 specifically compete with a test compound for binding MCP-4. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with MCP-4.

Uses of mcp-4 Polynucleotide

An mcp-4 polynucleotide, or any part thereof, may be used for diagnostic and/or therapeutic puφoses. For diagnostic puφoses, the mcp-4 of this invention may be used to detect and quantitate aberrant gene expression in conditions, disorders or diseases in which mcp-4 activity may be implicated. These specifically include, but are not limited to, NIDDM or conditions with similar biochemical or immunological properties such as viral, bacterial, fungal or helminthic infections; allergic or asthmatic responses; mechanical injury associated with trauma; arteriosclerosis, atherogenesis or collagen vascular diseases; hereditary diseases such as rheumatoid arthritis; leukemia, lymphomas or carcinomas; or other conditions which involve leukocytes, particularly monocytes, macrophages and T cells. Included in the scope of the invention are oligonucleotide sequences, antisense RNA and DNA molecules and ribozymes, which function to inhibit translation of an MCP-4.

Another aspect ofthe subject invention is to provide for hybridization or PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding

MCP-4 or closely related molecules. The specificity ofthe probe, whether it is made from a highly conserved region, eg, 10 unique nucleotides in the 5' regulatory region, or a less conserved region, eg, 20 nucleotides downstream from the definitive CC motif, and the stringency ofthe hybridization or amplification (high, intermediate or low) will determine whether the probe identifies only native mcp-4. related mcp sequences, or other chemokine molecules.

2? Diagnostic Uses of mcp-4 Polynucleotide

An mcp-4 polynucleotide sequences may be used for the diagnosis of diseases resulting from aberrant expression of mcp-4. For example, polynucleotide sequences encoding MCP-4 may be used in hybridization or PCR assays of tissues from biopsies or autopsies to detect abnormalities in mcp-4 expression. The form of such qualitative or quantitative methods may include Southern or northern analysis, dot blot or other membrane-based technologies; PCR technologies; dip stick, pin or chip technologies; and ELISA or other multiple sample format technologies. All of these techniques are well known in the art, and are in fact the basis of many commercially available diagnostic kits. Such assays may be tailored to evaluate the efficacy of a particular therapeutic treatment regime and may be used in animal studies, in clinical trials, or in monitoring the treatment of an individual patient. In order to provide a basis for the diagnosis of disease, a normal or standard profile for mcp-4 expression must be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with mcp-4 or a portion thereof, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained for normal subjects with a dilution series of positive controls run in the same experiment where a known amount of purified mcp-4 is used. Standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by a disorder or disease related to mcp-4 expression. Deviation between standard and subject values establishes the presence of disease state.

If disease is established, an existing therapeutic agent is administered, and treatment profile or values may be generated. Finally, the assay may be repeated on a regular basis to evaluate whether the values progress toward or return to the normal or standard pattern. Successive treatment profiles may be used to show the efficacy of treatment over a period of several days or several months.

PCR as described in US Patent Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the mcp-4 sequence. Such oligomers are generally chemically synthesized, but they may be generated enzymatically or produced from a recombinant source. Oligomers generally comprise two nucleotide sequences, one with sense orientation (5'->3") and one with antisense (3'<-5') employed under optimized conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.

Additionally methods to quantitate the expression ofa particular molecule include radiolabeling (Melby PC et al. 1993 J Immunol Methods 159:235-44) or biotinylating (Duplaa C et al. 1993 Anal Biochem 229-36) nucleotides, coamplification ofa control nucleic acid, and standard curves onto which the experimental results are inteφolated. Quantitation of multiple samples may be speeded up by running the assay in an ELISA format where the oligomer-of-interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation. For example, upregulation of mcp-4 may result in an inflammatory response, resulting in swelling and discomfort. In like manner, underexpression of mcp-4 may result in an insufficient immunological response. In either case, a definitive diagnosis may allow health professionals to treat the patient and prevent further worsening of the condition. Similarly, assays known to those of skill in the art can be used to monitor the progress ofa patient displaying an mcp-4 associated disease state during therapy.

Therapeutic Uses of an mcp-4 Polynucleotide

An mcp-4 sequence may be useful in the treatment of various abnormal conditions. By introducing mcp-4 sequence into cells, gene therapy can be used to treat conditions characterized by underexpression of mcp-4. In some instances, the sequence encoding an MCP-4 is intended to replace or act in the place of functionally deficient endogenous gene.

Expression vectors derived retroviruses, adenovirus, heφes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of recombinant mcp-4, sense or antisense molecules, to the targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant vectors containing mcp-4. See, for example, the techniques described in Maniatis et al. (supra) and Ausubel et al. (supra). Alternatively, recombinant mcp-4 can be delivered to target cells in liposomes.

Abnormal conditions characterized by overexpression of mcp-4 can be treated by using the same gene therapy techniques to introduce recombinant antisense constructs. The successful delivery and expression of such sequences will modulate or inhibit the transcription of mcp-4 mRNA.

The full length cDNA sequence and/or its regulatory elements enable researchers to use mcp-4 as a tool in sense (Youssoufian H and HF Lodish 1993 Mol Cell Biol 13:98-104) or antisense (Eguchi et al. (1991) Annu Rev Biochem 60:631-652) investigations of gene function. Oligonucleotides. designed from the cDNA or control sequences obtained from the genomic DNA can be used in vitro or in vivo to inhibit expression. Such technology is now well known in the art. and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions.

Mcp-4 can be turned off by transfecting a cell or tissue with expression vectors which express high levels of an mcp-4 fragment. Such constructs can flood cells with untranslatable sense or antisense sequences. Even in the absence of integration into the DNA, such vector may continue to transcribe RNA molecules until all copies ofthe vector are disabled by endogenous nucleases. Such transient expression may last for a month or more with a non-replicating vector

(Mettler I, personal communication) and even longer if appropriate replication elements are part ofthe vector system.

On the other hand, stable transformation of appropriate germ line cells, or preferably a zygote, with a vector containing the mcp-4 fragments may produce a transgenic organism (US Patent No. 4,736,866. 12 April 1988), which produces enough copies ofthe sense or antisense sequence to significantly compromise or entirely eliminate activity ofthe endogenous mcp-4 gene. Frequently, disruption of such genes can be ascertained by observing behaviors such as reduced inflammatory response or reduced leukocyte proliferation.

As mentioned previously, modifications of gene expression can be obtained by designing antisense sequences to the control regions of the mcp-4 gene— the promoters, enhancers, and introns. Oligonucleotides derived from the transcription initiation site, eg, between -10 and +10 regions ofthe leader sequence, are preferred. Antisense RNA and DNA molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Similarly, inhibition can be achieved using Hogeboom base-pairing methodology, also known as "triple helix" base pairing. Triple helix pairing compromises the ability ofthe double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. Within the scope ofthe invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of mcp-4 RNA sequences.

Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences. GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide sequence inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Both antisense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incoφorated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues. DNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences ofthe 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone ofthe molecule.

Methods for introducing vectors into cells or tissue include those methods discussed in Section IV. In addition, several of these transformation or transfection methods are equally suitable for the ex vivo therapy, the introduction of vectors into stem cells taken from the patient and clonally propagated for autologous transplant as in US Patent Nos. 5,399,493 and 5,437,994, disclosed herein by reference.

Furthermore, the mcp-4 polynucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including but not limited to such properties as the triplet genetic code and specific base pair interactions.

Detection and Mapping of Polynucleotide Sequences Related to mcp-4 The nucleic acid sequence for mcp-4 can also be used to generate hybridization probes as previously described, for mapping the native genomic sequence. The sequence may be mapped to a particular chromosome or to a specific region ofthe chromosome using well known techniques. These include in situ hybridization to chromosomal spreads (Verma et al. (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York City), flow-sorted chromosomal preparations, or artificial chromosome constructions such as YACs, bacterial artificial chromosomes (BACs), bacterial Pl constructions or single chromosome cDNA libraries.

In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers are invaluable in extending genetic maps. Examples of genetic maps can be found in Science ( 1995; 270:41 Of and 1994; 265: 1981 f). Often the placement of a gene on the chromosome of another mammalian species may reveal associated markers even if the number or arm of a particular human chromosome is not known.

New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable infoπnation to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once a disease or syndrome, such as ataxia telangiectasia (AT), has been crudely localized by genetic linkage to a particular genomic region, for example, AT to 1 lq22-23 (Gatti et al. (1988) Nature 336:577-580), any sequences mapping to that area may represent associated or regulatory genes for further investigation. The nucleotide sequence ofthe subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. between normal, carrier or affected individuals.

Pharmaceutical Compositions

The active compositions ofthe invention, which may comprise all or portions of MCP-4 or inhibitors binding MCP-4 including antibodies and antagonists, alone or in combination with at least one other agent, such as stabilizing compound, may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.

MCP-4 can be administered to a patient alone, or in combination with other agents, drugs or hormones or in pharmaceutical compositions where it is mixed with excipient(s) or pharmaceutically acceptable carriers. In one embodiment ofthe present invention, the pharmaceutically acceptable carrier is a pharmaceutically inert. Depending on the condition, disorder or disease being treated, these pharmaceutical compositions may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack Publishing Co, Easton PA). Suitable routes may, for example, include oral, transvaginal, or transmucosal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. The preferred route for MCP-4 or its inhibitors is intravenous administration.

For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. For tissue or cellular administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such caπiers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral or nasal ingestion by a patient to be treated. Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended puφose. Determination of effective amounts is well within the capability of those skilled in the art, especially in light of he disclosure provided below.

In addition to the active ingredients these pharmaceutical compositions may contain suitable pharmaceutically acceptable earners comprising excipients and auxiliaries which facilitate processing ofthe active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

The pharmaceutical compositions ofthe present invention may be manufactured in a manner that is itself known, eg, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions ofthe active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility ofthe compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, etc.; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, poiyvinylpyπolidonc, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, ie, dosage.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

Compositions comprising a compound ofthe invention formulated in a pharmaceutical acceptable carrier may be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition. For MCP-4 inhibitors, conditions indicated on the label may include treatment of inflammation.

The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder in 1 mm-50 mm histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.

For any compound used in the method of the invention. the therapeutically effective dose can be estimated initially from cell culture assays. Then, preferably, dosage can be formulated in animal models to achieve a desirable circulating concentration range that adjusts MCP-4 levels. Such information can be used to determine useful doses in humans. Examples of animal models useful for studying therapeutic applications of MCP-4 or its inhibitors include those described in Hutz (1989) Biol Reproduction 40:709-713; Hutz et al. (1990) J Med Primatol 19:553-571 ; Kitzman et al. (1992) Cell Tissue Res 268: 191 -196; and Quandt et al. (1993) Biol Reprod 48:1088-1094. A therapeutically effective dose refers to that amount of MCP-4 or its inhibitor which ameliorates symptoms, eg, reduces inflammation and pain. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, eg, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio

LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and additional animal studies can be used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity ofthe patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity ofthe disease state; age, weight, and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate ofthe particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms. up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature. See US Patent No. 4,657,760; 5,206,344; or 5,225,212. Those skilled in the art will employ different formulations for MCP-4 than for the inhibitors of MCP-4. Administration to the pancreas may necessitate delivery in a manner different from that to lung, kidney, or stomach.

It is contemplated that conditions associated with altered MCP-4 expression are treatable with either MCP-4 or inhibitors of MCP-4. These conditions, specifically include, but are not limited to, NIDDM or conditions with similar biochemical or immunological properties such as viral, bacterial, fungal or helminthic infections; allergic or asthmatic responses; mechanical injury associated with trauma; arteriosclerosis, atherogenesis or collagen vascular diseases; hereditary diseases such as rheumatoid arthritis; leukemia, lymphomas or carcinomas; or other conditions which involve leukocytes may be specifically diagnosed by the assays previously discussed. In addition, these assays may be used to monitor treatment.

The examples below are provided to illustrate the subject invention. These examples are provided by way of illustration and are not included for the puφose of limiting the invention.

EXAMPLES I NIDDM Pancreas cDN A Library Construction

The full length gene mcp-4 from Incyte Clone No 273061 was identified among the cDNAs isolated from the NIDDM pancreas library. A pancreas tissue sample (Lot GHL-645) from a 57 year old Caucasian male diagnosed with NIDDM who died from a basal cell ganglia bleed was obtained from the International Institute for Advanced Medicine (Exton PA). The patient had hypertension and cardiovascular disease and had been treated with Micronase® tablets (Pharmacia-Upjohn, Kalamazoo MI).

The tissue was flash frozen in liquid nitrogen, ground in a mortar and pestle, and lysed immediately in a buffer containing guanidinium isothiocyanate. Lysates were then loaded on a 5.7 M CsCl cushion and ultracentrifuged in a SW28 swinging bucket rotor for 18 hours at 25.000 φm at ambient temperature. The RNA was precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in water and DNase treated for 15 min at 37°C. The RNA was isolated using the Qiagen Oligotex kit (QIAGEN Ine, Chatsworth CA) and sent to Stratagene for the construction ofa custom cDNA library.

First strand cDNA synthesis was accomplished using an oligo d(T) primer/linker which also contained an Xhol restriction site. Second strand synthesis was performed using a combination of DNA polymerase I, E. coH ligase and RNase H, followed by the addition of an EcoRI adaptor to the blunt ended cDNA. The EcoRI adapted, double-stranded cDNA was then digested with Xhol restriction enzyme and fractionated to obtain sequences which exceeded 800 bp in size. The cDNAs were inserted into the LambdaZap^® vector system (Stratagene); then the vector which contains the pBluescript™ phagemid (Stratagene) was transformed into E. coli host cells strain XLl-BlueMRF™ (Stratagene). The phagemid forms of individual cDNA clones were obtained by the jn vivo excision process. Enzymes from both pBluescript and a cotransformed fl helper phage nicked the DNA, initiated new DNA synthesis, and created the smaller, single-stranded circular phagemid molecules which contained the cDNA insert. The phagemid DNA was released, purified, and used to reinfect fresh host cells (SOLR, Stratagene). Presence of the phagemid which carries the gene for β-lactamase allowed transformed bacteria to grow on medium containing ampicillin.

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was purified using the Miniprep Kit (catalog # 77468, Advanced Genetic Technologies Coφoration, Gaithersburg MD), a 96-well block kit with reagents for 960 purifications. The recommended protocol included with the kit was employed except for the following changes. Each of the 96 wells was filled with only 1 ml of sterile Terrific Broth (Catalog #2271 1 , LIFE TECHNOLOGIES, Gaithersburg MD) with carbenicillin at 25 mg/L and glycerol at 0.4%. After the wells were inoculated, the bacteria were cultured for 24 hours and lysed with 60 μl of lysis buffer. A centrifugation step (Beckman GS-6R @2900 φ for 5 min; Beckman Instruments) was performed before the contents of the block were added to the primary filter plate. The optional step of adding isopropanol to TRIS buffer was not routinely performed. After the last step in the protocol, samples were transferred to a Beckman 96-well block for storage.

The cDNAs were sequenced by the method of Sanger F and AR Coulson (1975; J Mol Biol 94:44 If), using a Hamilton Micro Lab 2200 (Hamilton, Reno NV) in combination with four

Peltier Thermal Cyclers (PTC200 from MJ Research, Watertown MA) and Applied Biosystems 377 or 373 DNA Sequencing Systems (Perkin Elmer), and reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins Each cDNA was compared to sequences in GenBank using a search algorithm developed by Applied Biosystems and incoφorated into the INHERIT^™ 670 Sequence Analysis System. In this algorithm, Pattern Specification Language (TRW Ine, Los Angeies CA) was used to determine regions of homology. The three parameters that determine how the sequence comparisons run were window size, window offset, and error tolerance. Using a combination of these three parameters, the DNA database was searched for sequences containing regions of homology to the query sequence, and the appropriate sequences were scored with an initial value. Subsequently, these homologous regions were examined using dot matrix homology plots to distinguish regions of homology from chance matches. Smith- Waterman alignments were used to display the results of the homology search.

Peptide and protein sequence homologies were ascertained using the INHERIT^™ 670 Sequence Analysis System in a way similar to that used in DNA sequence homologies. Pattern Specification Language and parameter windows were used to search protein databases for sequences containing regions of homology which were scored with an initial value. Dot-matrix homology plots were examined to distinguish regions of significant homology from chance matches.

BLAST, which stands for Basic Local Alignment Search Tool (Altschul SF (1993) J Mol Evol 36:290-300; Altschul, SF et al. (1990) J Mol Biol 215:403- 10), was used to search for local sequence alignments . BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologs. BLAST is useful for matches which do not contain gaps. The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is inteφreted as the upper bound ofthe expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

IV Extension of mcp-4 to Recover Regulatory Elements

The nucleic acid sequence of full length mcp-4 (SEQ ID NO: l ) is used to design oligonucleotide primers for obtaining 5' sequences from genomic libraries. One primer is synthesized to initiate extension in the antisense direction (XLR) and the other is synthesized to extend sequence in the sense direction (XLF). The primers allowed the known mcp-4 sequence to be extended "outward" generating amplicons containing new, unknown nucleotide sequence for the control region of interest. The initial primers are designed from the cDNA using Oligo

4.0 (National Biosciences Inc. Plymouth MN), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68°-72° C. Any stretch of nucieotides which could result in haiφin structures and primer-primer dimerizations is avoided. A human genomic library is used to extend and amplify 5' upstream sequence. If necessary, a second set of primers is designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix, high fidelity amplification is obtained. Beginning with 40 pmol oi^' each primer and the recommended concentrations of all other components ofthe kit, PCR is performed using the Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA) and the following parameters:

Step 1 94° C for 1 min (initial denaturation)

Step 2 65° C for 1 min

Step 3 68° C for 6 min Step 4 94° C for 15 sec

Step 5 65° C for 1 min

Step 6 68° C for 7 min

Step 7 Repeat step 4-6 for 15 additional cycles

Step 8 94° C for 15 sec Step 9 65° C for 1 min

Step 10 68° C for 7: 15 min

Step 1 1 Repeat step 8-10 for 12 cycles

Step 12 72° C for 8 min

Step 13 4° C (and holding)

A 5-10 μl aliquot ofthe reaction mixture is analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. The largest products or bands were selected and cut out ofthe gel.

Further purification involves using a commercial gel extraction method such as QIAQuick™ (QIAGEN Ine). After recovery ofthe DNA, Klenow enzyme was used to trim single-stranded, nucleotide overhangs creating blunt ends which facilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl of ligation buffer, lμl T4-DNA ligase (15 units) and lμl T4 polynucleotide kinase are added, and the mixture is incubated at room temperature for 2-3 hours or overnight at 16° C. Competent Fi, coli cells (in 40 μl of appropriate media) are transformed with 3 μl of ligation mixture and cultured in 80 μl of SOC medium (Sambrook J et al, supra). After incubation for one hour at 37° C, the whole transformation mixture is plated on Luria Bertani (LB)-agar (Sambrook J et al, supra) containing

2xCarb. The following day, several colonies are randomly picked from each plate and cultured in 150 μl of liquid LB/2xCarb medium placed in an individual well of an appropriate, commercially-available, sterile 96-well microtiter plate. The following day, 5 μl of each overnight culture is transferred into a non-sterile 96-well plate and after dilution 1 : 10 with water, 5 μl of each sample is transferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerase, a vector primer and one or both of the gene specific primers used for the extension reaction are added to each well. Amplification is performed using the following conditions: Step l 94° C for 60 sec

Step 2 94° C for 20 sec

Step 3 55° C for 30 sec

Step 4 72° C for 90 sec

Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6 72° C for 180 sec

Step 7 4° C (and holding)

Aliquots ofthe PCR reactions are run on agarose gels together with molecular weight markers. The sizes of the PCR products are compared to the original partial cDNAs, and appropriate clones are selected, ligated into plasmid and sequenced.

V Labeling of Hybridization Probes

Hybridization probes derived from SEQ ID NO: l are employed to screen cDNAs, mRNAs or genomic DNAs. Although the labeling of oligonucleotides, consisting of about 20 base-pairs, is specifically described, essentially the same procedure is used with larger cDNA fragments. Oligonucleotides are labeled by combining 50 pmol of each oligomer and 250 mCi of

[γ-³²P] adenosine triphosphate (Amersham, Chicago IL) and T4 polynucleotide kinase (DuPont NEN^®, Boston MA). The labeled oligonucleotides are purified with Sephadex G-25 super fine resin column (Pharmacia). A portion containing IO⁷ counts per minute of each is used in a typical membrane based hybridization analysis of human genomic DNA digested with one of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1. or Pvu II; DuPont NEN^®).

The DNA from each digest is fractionated on a 0.7 percent agarose gel and transfeπed to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film (Kodak, Rochester NY) is exposed to the blots in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale CA) for several hours, hybridization patterns are compared visually.

VI Antisense Molecules

The mcp-4 sequence, or any part thereof, is used to inhibit in vivo or in vitro expression of native mcp-4. Although use of antisense oligonucleotides, consisting of about 20 base-pairs, is specifically described, essentially the same procedure is used with larger cDNA fragments. An oligonucleotide based on the coding sequence of mcp-4 is used to inhibit expression of native mcp-4. The complementary oligonucleotide is designed from the conserved 5' sequence and used either to inhibit transcription by preventing promoter binding to the upstream nontranslated sequence or translation of an mcp-4 transcript by preventing the ribosome from binding to the -10 to +10 region ofthe leader sequence.

VII Expression of MCP-4

Expression ofthe MCP-4 is accomplished by subcloning the cDNAs into appropriate vectors and transfecting the vectors into host cells. In this case, the cloning vector, pBluescript, previously used for the generation ofthe cDNA library is used to express MCP-4 in E. coli.

Upstream ofthe cloning site, this vector contains a promoter for β-galactosidase, followed by sequence containing the amino-terminal Met and the subsequent 7 residues of β-galactosidase.

Immediately following these eight residues is a bacteriophage promoter useful for transcription and a linker containing a number of unique restriction sites.

Induction of an isolated, transfected bacterial strain with IPTG using standard methods produces a fusion protein which consists ofthe first seven residues of β-galactosidase. about 5 to 15 residues of linker, and the full length MCP-4. The signal sequence of directs the secretion of

MCP-4 into the bacterial growth media which can be used directly in the following assay for activity. VIII MCP-4 Activity

Chemokine chemotactic activity is usually measured in 48-well microchemotaxis chambers. In each well, two compartments are separated by a filter that allows the passage of cells from one compartment into the other in response to a chemical gradient. Cell culture medium into which MCP-4 has been secreted is placed on one side of a polycarbonate filter, and peripheral blood cells are suspended in the same media opposite side ofthe filter. Sufficient incubation time is allowed for the cells to traverse the filter in response to diffusion and resulting concentration gradient of MCP-4. Filters are recovered from each well, and specific cell types, eg, monocytes, adhering to the side ofthe filter facing the chemokine are identified and counted.

Specificity of the chemoattraction is determined by performing the assay on fractionated populations of cells such as enriched populations of neutrophils, mononuclear cells, monocytes or lymphocytes obtained by density gradient centrifugation. Specific T cell populations are purified using CD8+ and CD4+ specific antibodies for negative selection.

IX Production of MCP-4 Specific Antibodies

Although MCP-4 purified using PAGE electrophoresis (Maniatis, supra) may be used to immunize rabbits using standard protocols, a monoclonal approach is more commonly employed. The amino acid sequence translated from mcp-4 is analyzed using DNASTAR software (DNASTAR Ine) to determine regions of high immunogenicity and a coπesponding oligopolypeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Analysis to select appropriate epitopes, such as those near the C-terminus or in adjacent hydrophilic regions is described by Ausubel FM et al. (supra).

Typically, the oligopeptides are 15 residues in length, synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel FM et al, supra). Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. The resulting antisera are tested for antipeptide activity, for example, by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radioiodinated, goat anti-rabbit IgG. X Purification of Native MCP-4 Using Specific Antibodies

Native or recombinant MCP-4 is purified by immunoaffinity chromatography using antibodies specific for MCP-4. An immunoaffinity column is constructed by covalently coupling MCP-4 antibody to an activated chromatographic resin such as CnBr-activated Sepharose (Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing MCP-4 is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of MCP-4 (eg, high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/MCP-4 binding (eg, a buffer of pFI 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and MCP-4 is collected.

XI Identification of Molecules Which Interact with MCP-4

MCP-4, or biologically active fragments thereof, are labeled with ¹²1 Bolton-Hunter reagent (Bolton, AE and Hunter, WM (1973) Biochem J 133: 529). Candidate molecules previously arrayed in the wells ofa 96 well plate are incubated with the labeled MCP-4, washed and any wells with labeled MCP-4 complex are assayed. Data obtained using different concentrations of MCP-4 are used to calculate values for the number, affinity, and association of MCP-4 with the candidate molecules. All publications and patents mentioned in the above specification are herein incoφorated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connection with specific prefeπed embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of he described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope ofthe following claims. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(l) APPLICANT: INCYTE PHARMACEUTICALS, INC. (il) TITLE OF INVENTION: NOVEL CHEMOKINE FROM NIDDM PANCREAS (iii) NUMBER OF SEQUENCES: 3

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: INCYTE PHARMACEUTICALS, INC.

(B) STREET: 3174 Porter Drive

(C) CITY: Palo Alto

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 94304

(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) PCT APPLICATION NUMBER: To Be Assigned

(B) FILING DATE: Herewith

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION SERIAL NUMBER: US 60/006,809

(B) FILING DATE: 15-NOV-1995

(vn) PRIOR APPLICATION SERIAL NUMBER:

(A) APPLICATION SERIAL NUMBER: US 08/56 ,334

(B) FILING DATE: 01-DEC-1995

(vm) ATTORNEY/AGENT INFORMATION:

(A) NAME: Billings, Lucy J.

(B) REGISTRATION NUMBER: 36,749

(C) REFERENCE/DOCKET NUMBER: PF-0050 PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 415-855-0555

(B) TELEFAX: 415-845-4166

(2) INFORMATION FOR SEQ ID NO:l:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 820 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vn) IMMEDIATE SOURCE: (A) LIBRARY: PANCREAS

(B) CLONE: 273061

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

GTGAAATCTC CAACTCTTAA CCTTCAACAT GAAAGTCTCT GCAGTGCTTC TGTGCCTGCT 60

GCTCATGACA GCAGCTTTCA ACCCCCAGGG ACTTGCTCAG CCAGATGCAC TCAACGTCCC 120

ATCTACTTGC TGCTTCACAT TTAGCAGTAA GAAGATCTCC TTGCAGAGGC TGAAGAGCTA 180

TGTGATCACC ACCAGCAGGT GTCCCCAGAA GGCTGTCATC TTCAGAACCA AACTGGGCAA 240

GGAGATCTGT GCTGACCCAA AGGAGAAGTG GGTCCAGAAT TATATGAAAC ACCTGGGCCG 300

GAAAGCTCAC ACCCTGAAGA CTTGAACTCT GCTACCCCTA CTGAAATCAA GCTGGAGTAC 360

GTGAAATGAC TTTTCCATTC TCCTCTGGCC TCCTCTTCTA TGCTTTGGAA TACTTCTACC 420

ATAATTTTCA AATAGGATGC ATTCGGTTTT GTGATTCAAA ATGTACTATG TGTTAAGTAA 480

TATTGGCTAT TATTTGACTT GTTGCTGGTT TGGAGTTTAT TTGAGTATTG CTGATCTTTT 540

CTAAAGCAAG GCCTTGAGCA AGTAGGTTGC TGTCTCTAAG CCCCCTTCCC TTCCACTATG 600

AGCTGCTGGC AGTGGGTTTG TATTCGGTTC CCAGGGGTTG AGAGCATGCC TGTGGGAGTC 660

ATGGACATGA AGGGATGCTG CAATGTAGGA AGGAGAGCTC TTTGTGAATG TGAGGTGTTG 720

CTAAATATGT TATTGTGGAA AGATGAATGC AATAGTAGGA CTGCTGACAT TTTGCAGAAA 780

ATACATTTTA TTTAAAATCT CCAAAAAAAA AAAAAAAAAA 820

(2) INFORMATION FOR SEQ ID NO:2:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 98 amino acids

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

(i ) MOLECULE TYPE: protein

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

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

Phe Asn Pro Gin Gly Leu Ala Gin Pro Asp Ala Leu Asn Val Pro Ser 20 25 30

Thr Cys Cys Phe Thr Phe Ser Ser Lys Lys Ile Ser Leu Gin Arg Leu 35 40 45

Lys Ser Tyr Val Ile Thr Thr Ser Arg Cys Pro Gin Lys Ala Val Ile 50 55 60

Phe Arg Thr Lys Leu Gly Lys Glu Ile Cys Ala Asp Pro Lys Glu Lys 65 70 75 80

Trp Val Gin Asn Tyr Met Lys His Leu Gly Arg Lys Ala His Thr Leu 85 90 95

Lys Thr

(2) INFORMATION FO? SEQ ID NO: 3: ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 99 ammo acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein

(vn) IMMEDIATE SOURCE:

(B) CLONE: GI487124

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

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

Phe Ile Pro Gin Gly Leu Ala Gin Pro Asp Aid Ile Asn Ala Pro Val 20 25 30

Thr Cys Cys Tyr Asn Phe Thr Asn Arg Lys Ile Ser Val Gin Arg Leu 35 40 45

Ala Ser Tyr Arg Arg Ile Thr Ser Ser Lys Cys Pro Lys Glu Ala Val 50 55 60

Ile Phe Lys Thr Ile Val Ala Lys Glu Ile Cy Ala Asp Pro Lys Gin 65 70 75 80

Lys Trp Val Gin Asp Ser Met Asp His Leu Asp Lys Gin Thr Gin Thr 85 90 95

Pro Lys Thr

Claims (20)

1. A purified polynucleotide comprising a nucieic acid sequence encoding a polypeptide of

SEQ ID N0:2.

2. The polynucleotide of claim 1 comprising the nucleic acid sequence for monocyte chemotactic protein (mcp-4) of SEQ ID NO: 1.

3. An antisense molecule comprising the complement of the polynucleotide of claim 2 or any portion thereof.

4. A pharmaceutical composition comprising the antisense molecule of claim 3 and a pharmaceutically acceptable excipient.

5. A method of treating a subject with a condition associated with altered mcp-4 expression comprising administering an effective amount of the pharmaceutical composition of claim 4 to the subject.

6. A diagnostic composition comprising an oligomer of the polynucleotide of claim 2.

7. A diagnostic test for a condition associated with altered mcp-4 expression comprising the steps of: a) providing a biological sample and combining the biological sample and the diagnostic composition of claim 6; b) allowing hybridization to occur between the biological sample and the diagnostic composition under suitable conditions; c) measuring the amount of hybridization to obtain a sample value; and d) comparing the sample value with standard values to determine whether mcp-4 expression is altered.

8. An expression vector comprising the polynucleotide sequence of claim 1.

9. A host cell transformed with the expression vector of claim 8.

10. A method for producing a polypeptide, said method comprising the steps of: a) culturing the host cell of claim 9 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.

1 1. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable excipient.

12. A method of treating a subject with a condition associated with altered MCP-4 expression comprising administering an effective amount ofthe pharmaceutical composition of claim 1 1 to the subject.

13. An antibody specifically binding the purified polypeptide of claim 1. or any portion thereof.

14. A diagnostic composition comprising the antibody of claim 13.

15. A diagnostic test for a condition associated with altered MCP-4 expression comprising the steps of: a) providing a biological sample and combining it with the diagnostic composition of claim 14 under conditions suitable for complex formation; b) measuring the amount of complex formation between MCP-4 and the diafnostic composition to obtain a sample amount; and c) comparing the amount of complex formation in the sample with standard amounts of complex formation, wherein a variation between the sample amount and standard amounts of complex formation establishes the presence of the condition.

16. A method of screening a plurality of compounds for specific binding affinity with the polypeptide of claim 1 or any portion thereof comprising the steps of: a) providing a plurality of compounds and combining MCP-4 with each of the plurality of compounds for a time sufficient to allow binding under suitable conditions; and b) detecting binding of MCP-4 to each ofthe plurality of compounds, thereby identifying the compounds which specifically bind MCP-4.

17. A pharmaceutical composition comprising the compound identified in claim 16 and a pharmaceutically acceptable excipient.

18. A method of treating a subject with NIDDM comprising administering an effective amount ofthe pharmaceutical composition of claim 17 to the subject.

19. A method of treating a subject with an immunological disease associated with altered

MCP-4 expression comprising administering an effective amount of the pharmaceutical composition of claim 17 to the subject.

20. A method of treating a subject with an infectious disease associated with altered MCP-4 expression comprising administering an effective amount of the pharmaceutical composition of claim 17 to the subject.