WO2001092301A2 - Placc, nouvelle chimiokine c-c humaine isolee du placenta - Google Patents

Placc, nouvelle chimiokine c-c humaine isolee du placenta Download PDF

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WO2001092301A2
WO2001092301A2 PCT/US2001/016599 US0116599W WO0192301A2 WO 2001092301 A2 WO2001092301 A2 WO 2001092301A2 US 0116599 W US0116599 W US 0116599W WO 0192301 A2 WO0192301 A2 WO 0192301A2
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placc
activity
chemokine
protein
polynucleotide
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WO2001092301A3 (fr
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Robert A. Hromas
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Indiana University Advanced Research And Technology Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • PLACC A NOVEL HUMAN C-C CHEMOKINE ISOLATED FROM PLACENTA
  • the present invention relates generally to chemokines and more particularly to purified and isolated polynucleotides encoding a human C-C chemokine designated PLACC and analogs thereof. Methods and compositions for the purification, isolation and use of the polynucleotides and polypeptides are described.
  • Chemokines are a family of related proteins that regulate leukocyte migration and activation in the face of inflammatory stimulus (Oppenheim, Adv Exp MedBiol 351:183, 1993; Schall, Cytokine 3:165, 1991; Baggiolini et al, Adv Immunol 55:97, 1994; Schall and Bacon, Curr Biol 6:865, 1994; olpe and Cerami, FASEB J 3 :2565, 1989).
  • chemokines generally exhibit 20-70% amino acid identity to each other and contain four highly-conserved cysteine residues.
  • chemokines Based on the relative position of the first two of these cysteine residues, chemokines have been further classified into two subfamilies. In the “C-X-C” or “ ⁇ ” subfamily, encoded by genes localized to human chromosome 4, the first two cysteines are separated by one amino acid. In the “C-C” or “ ⁇ ” subfamily, encoded by genes which have been mapped to human chromosome 17, the first two cysteines are adjacent. X-ray crystallography and NMR studies of several chemokines have indicated that, in each family, the first and third cysteines form a first disulfide bridge, and the second and fourth cysteines form a second disulfide bridge, strongly influencing the native conformation of the proteins. In humans alone, nearly ten distinct sequences have been described for each chemokine subfamily. Chemokines of both subfamilies have characteristic leader sequences of twenty to twenty-five amino acids.
  • the C-X-C chemokines which include TL-8, GRO ⁇ / ⁇ / ⁇ , platelet basic protein, Platelet Factor 4 (PF4), neutrophil-activating peptide-2 (NAP-2), macrophage chemotactic and activating factor (MCAF), IP-10, and others, share approximately 25% to 60% identity when any two amino. acid sequences are compared (except for the GRO ⁇ / ⁇ / ⁇ members, which are 84-88% identical with each other). Most of the subfamily members (excluding IP-10 and Platelet Factor 4) share a common E-L-R tri-peptide motif upstream of the first two cysteine residues.
  • the C-X-C chemokines are generally potent stimulants of neutrophils, causing rapid shape change, chemotaxis, respiratory bursts, and degranulation. Specific truncation of the N- terminal amino acid sequence of certain C-X-C chemokines, including JX-8, is associated with marked increases in activity.
  • the C-C chemokines which include Macrophage Inflammatory Proteins MlP-l ⁇ (Nakao et al, Mol. Cell Biol, 10:3646, 1990) and MlP-l ⁇ (Brown et al, J. Immunol, 142:619, 1989), Monocyte Chemotactic Proteins MCP-1
  • the C-C chemokines generally activate monocytes, lymphocytes, basophils and eosinophils, but not neutrophils. Most of the reported C-C chemokines activate monocytes, causing calcium flux and chemotaxis. More selective effects are seen on lymphocytes, for example, T-lymphocytes, which respond most strongly to RANTES.
  • C-C chemokines can be further subdivided according to structural homologies and similar activities.
  • MlP-l ⁇ , MlP-l ⁇ and RANTES have closer homology and range of biological activities than the other members of the family.
  • Another subfamily within the C-C chemokine family are the monocyte chemotactic proteins (MCP), which are structurally more similar to each other than to other members of the C-C chemokine family, and which preferentially stimulate monocytes to migrate and respond to inflammatory stimuli.
  • MCP monocyte chemotactic proteins
  • chemokine receptors are seven-transmembrane-domain rhodopsin- like G protein-coupled receptors.
  • a receptor specific for IL-8 has been cloned by Holmes et al, Science, 253:1218-83 (1991), while a similar receptor (77% identity) which recognizes IL-8, GRO and NAP-2 has been cloned by Murphy and Tiffany,
  • CCR-1 C-C chemokine receptor-1
  • RANTES N-C chemokine receptor-1
  • CCR-4B MCP-1 receptor
  • CCR-3 eotaxin receptor
  • CCR-5 a receptor for MlP-l ⁇ , MlP-l ⁇ and RANTES (Raport et al, J. Biol. Chem., 271:11161-11166, 1996).
  • receptors tend to be multifunctional, and may bind a number of different chemokines.
  • the receptors themselves may play a role in human disease.
  • the Duffy antigen on human red blood cells also known as the erythrocyte chemokine receptor
  • chemokines including IL-8, NAP-2, GRO ⁇ , RANTES, MCP-1 is an invasion receptor for a malaria-causing parasite, Plasmodium knowlesi.
  • chemokines are believed to play a role in a wide variety of diseases involving inflammatory tissue destruction, such as rheumatoid arthritis, myocardial infarction, and adult respiratory distress syndrome.
  • C-X-C chemokine JX-8 has been well documented in various pathological conditions. See generally Baggiolini et al, supra, Table Nil.
  • IL-8 interleukin-8
  • Psoriasis has also been linked to over- production of JX-8.
  • C-C chemokines in pathological conditions has also been documented.
  • concentration of MCP-1 is higher in the synovial fluid of patients suffering from rheumatoid arthritis than that of patients suffering from other arthritic diseases.
  • the MCP-1 dependent influx of mononuclear phagocytes may be an important event in the development of idiopathic pulmonary fibrosis.
  • the role of C-C chemokines in the recruitment of monocytes into atherosclerotic areas is currently of intense interest, with enhanced MCP-1 expression having been detected in macrophage-rich arterial wall areas but not in normal arterial tissue.
  • MCPs may also be involved in induction of angiogenesis and tumor growth or metastasis. Expression of MCP-1 in malignant cells has been shown to suppress the ability of such cells to form tumors in vivo. (See U.S. Patent No. 5,179,078, incorporated herein by reference.)
  • chemokine activities include the ability to inhibit the proliferation of bone marrow progenitor cells.
  • Recombinant MlP-l but not MlP-l ⁇ , has been shown to suppress myelopoiesis of stem and progenitor cells, and appears to be selective in its ability to suppress growth factor-stimulated proliferation of multipotential progenitor cells (colony forming units of granulocyte-erythroid- macrophage-megakaryocytes, CFU-GEMM) and subpopulations of burst-forming units of erythroid (BFU-E) and colony-forming units of granulocytes-macrophages (CFU-GM) progenitor cells.
  • multipotential progenitor cells colony forming units of granulocyte-erythroid- macrophage-megakaryocytes, CFU-GEMM
  • BFU-E colony forming units of erythroid
  • CFU-GM colony-forming units of granulocytes-macrophages
  • MlP-l has the potential to protect multipotent hematopoietic cells from the cytotoxic effects of chemotherapeutic agents.
  • Clinical trials are reportedly under way for the use of a Mff-l ⁇ analog (designated BB10010, British Biotechnology) as a myeloprotective agent with Cytoxan® (cyclophosphamide from Bristol-Myers Squibb Oncology).
  • Cytoxan® cyclophosphamide from Bristol-Myers Squibb Oncology
  • HIN human immunodeficiency virus
  • the CCR-5 receptor which binds RANTES, MlP-l ⁇ and MTP-l ⁇ has been identified as the main co-receptor for most macrophage-tropic HIN strains (Deng et al, Nature, 381:661, 1996; Dragic et al, Nature, 381:661, 1996; Alkhatib et al, Science, 272:1955, 1996). It has been reported that occasional primary HTV-1 macrophage-tropic strains interact with the CCR-3 and CCR-2B receptors in vitro (Choe et al, Cell, 55:1135, 1996; Doranz et al, Cell, 55:1149, 1996).
  • a chemokine receptor designated “Fusin” (now known as the C-X-C chemokine receptor CXCR-4) has been identified as a receptor for T-cell tropic strains of HIN (Feng et al, Science, 272:812, 1996). These HIV co-receptors are in the chemokine receptor families, and appear to be cofactors with CD4 for the fusion and entry of HIV viruses into human target cells.
  • Chemokines of the C-C subfamily have been shown to possess utility in medical imaging, e.g., for imaging the site of infection, inflammation, and other sites having C-C chemokine receptor molecules. See, e.g., Kunkel et al, U.S. Patent No. 5,413,778, incorporated herein by reference. Such methods involve chemical attachment of a labeling agent (e.g., a radioactive isotope) to the C-C chemokine using art recognized techniques (see, e.g., U.S. Patent Nos.
  • a labeling agent e.g., a radioactive isotope
  • chemokines as mediators of chemotaxis and inflammation
  • the present invention is directed to providing methods, compositions and therapies relating to the identification of a novel C-C chemokine isolated from placental tissue.
  • the present invention provides an isolated and purified chemokine protein comprising an amino acid sequence as set forth in SEQ ID NO:2 or the mature protein portion thereof.
  • the present invention contemplates an isolated and purified chemokine protein comprising an amino acid sequence that is 90% identical to the sequence set forth in SEQ ID NO:2.
  • the invention further contemplates a peptide that comprises about 10 to about 50 contiguous amino acids of SEQ ID NO:2. This is an exemplary length of peptide amino acid residues and peptides of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
  • Another aspect of the invention provides an isolated and purified nucleic acid comprising a polynucleotide sequence encoding a chemokine having an amino acid sequence as set forth in SEQ ID NO:2.
  • the isolated and purified chemokine-encoding polynucleotide wherein the polynucleotide has a sequence as set forth in SEQ ID NO:l or a complement thereof.
  • the isolated polynucleotide may be a DNA.
  • the DNA may be genomic DNA or a cDNA.
  • the nucleic acid encodes a placental chemokine.
  • the polynucleotide encodes a chemokine and hybridizes under high stringency conditions to a polynucleotide set forth in SEQ ID NO:l.
  • Additional aspects of the present invention provide a purified antibody that is specifically immunoreactive with a protein comprising all or part of the amino acid sequence as set forth in SEQ ID NO:2.
  • the antibody is a monoclonal antibody.
  • Other embodiments contemplate a hybridoma cell line producing a monoclonal antibody that is specifically immunoreactive with a PLACC protein or peptide of the present invention.
  • the present invention further provides an expression construct comprising a vector having an isolated polynucleotide encoding a placenta-derived C- C chemokine having an amino acid sequence of SEQ ID NO:2 and a promoter operably linked to the polynucleotide.
  • the chemokine- encoding polynucleotide of the expression construct comprises a sequence as set forth in SEQ ID NO: 1.
  • the promoter may be selected from the group consisting of CMV IE, SN40 MLP, AdEl, keratin 10 promoter, baculovirus polyhedrin promoter, ⁇ -actin.
  • CMV IE CMV IE
  • SN40 MLP AdEl
  • keratin 10 promoter keratin 10 promoter
  • baculovirus polyhedrin promoter baculovirus polyhedrin promoter
  • ⁇ -actin ⁇ -actin
  • the expression construct may comprise a viral vector such as, for example, a retroviral vector, an adenoviral vector, a herpesviral vector, an adenoassociated viral vector, a lentiviral vector and a cytomegaloviral vector.
  • a viral vector such as, for example, a retroviral vector, an adenoviral vector, a herpesviral vector, an adenoassociated viral vector, a lentiviral vector and a cytomegaloviral vector.
  • the invention further provides a host cell stably transformed or transfected with a placenta-derived C-C chemokine-encoding polynucleotide of the present invention in a manner allowing the expression in the host cell of the placental derived C-C chemokine.
  • the host cell comprises a polynucleotide comprising a sequence as set forth in SEQ ID NO:l or the mature protein coding portion thereof.
  • the polynucleotide in the host cell encodes a placenta-derived C-C chemokine comprising a sequence as set forth in SEQ ID NO:2 or the mature protein portion thereof.
  • the present invention further provides a method of modulating an inflammatory response in an individual comprising modulating the activity of PLACC in the individual.
  • the various disorders that may be modulated by the present invention are described in further detail throughout the application and include but are not limited to asthma, adult respiratory distress syndrome, rheumatoid arthritis, lupus erythematosus, psoriasis, osteoarthritis, glomerulonephritis, osteoporosis, dermatomyositis, polymyositis, Addison's Disease, Graves Disease, Crohn's Disease, irritable bowel syndrome, atrophic gastritis, graft versus host disease, myasthenia gravis, multiple sclerosis, autoimmune thyroiditis, ulcerative colitis, atherosclerosis and pancreatitis.
  • the individual is immunocompromised.
  • the method comprises administering to the individual a modulator of PLACC activity identified by a method comprising the steps of contacting PLACC with the candidate modulator substance; monitoring the activity of PLACC; and comparing the activity of PLACC in the presence and absence of the candidate substance; wherein an alteration in the activity of PLACC activity indicates that the substance is an modulator of PLACC activity.
  • the modulator may be an antibody immunoreactive with PLACC; (e.g. a single chain antibody), a small molecule that binds to PLACC or its receptor; or a mutant PLACC.
  • An alternative embodiment comprises methods of increasing or augmenting an immune response in a subject comprising increasing the PLACC activity in the subject. Such methods may be particularly useful where the subject may be exhibiting signs of a malignancy or infection or have an immunodeficiency.
  • increasing PLACC is effected by administering to the individual a composition comprising PLACC, administering a stimulator of PLACC activity or by increasing the expression of PLACC in the subject.
  • increasing the PLACC activity in the cell population will serve to provide a method of stimulating cell proliferation, maturation or activity in the cell population.
  • aspects of this invention also disclose a method of screening for a modulator of C-C chemokine activity comprising contacting PLACC with the candidate modulator; monitoring the activity of PLACC; and comparing the activity of PLACC in the presence and absence of the candidate substance; wherein an alteration in the activity of PLACC activity indicates that the substance is an modulator of C-C chemokine activity.
  • compositions comprising an expression construct comprising a vector having an isolated polynucleotide encoding a placenta-derived C-C chemokine and a promoter operably linked to the polynucleotide; and a pharmaceutically acceptable carrier excipient or diluent.
  • Additional pharmaceutic compositions comprise an isolated and purified PLACC polypeptide and a pharmaceutically acceptable carrier, diluent or excipient.
  • the PLACC chemokine compositions of the present invention may be employed in tumor cell vaccines and other therapeutic compositions either alone or in combination with other chemokines.
  • FIG. 1 Sequence alignment of the sequence of PLACC (SEQ ID NO:3); EX-3 (SEQ ID NO:4); mEX-2(SEQ ID NO:5); hEx-2 (SEQ ID NO:6); Exodus-1 (SEQ ID NO:7); MTP-la (SEQ ID NO:8); MTP-lb (SEQ ID NO:9); RANTES (SEQ ID NO: 10); MCP-1 (SEQ ID NO.T 1); MCP-2 (SEQ ID NO: 12); MCP-3 (SEQ ID NO:13); MCP-4 (SEQ ID NO:14); 1-309 (SEQ ID NO:15).
  • PLACC denotes CF KE ⁇ HGKRNSNPvAHQGKHETYGHKTPY (SEQ ID NO: 18); # in mEX-2 denotes GKGSKGC RTEQTQPSRG (SEQ ID NO: 19); # in hEX-2 denotes RKGSKGCKRTERSQTPKGP (SEQ ID NO:20)
  • Chemokines are believed to play a role in a wide variety of diseases.
  • substances that promote the immune response may promote the healing of wounds or the speed of recovery from infectious diseases such as pneumonia.
  • chemokines inliibit the pro-inflammatory effects of chemokines maybe useful for treating pathological conditions mediated by inflammation. Additionally, the established correlation between chemokine expression and inflammatory conditions and disease states provides diagnostic and prognostic indications for the use of chemokines, as well as for antibody substances that are specifically immunoreactive with chemokines; a need exists for the identification and isolation of new chemokines to facilitate such diagnostic and prognostic indications.
  • the present invention is related to the identification of a new chemokine isolated from human placenta and termed PLACC.
  • PLACC a new chemokine isolated from human placenta and termed PLACC.
  • the polynucleotides encoding PLACC and the use of these compositions for the diagnosis, prevention and intervention of inflammation, auto-immune disorders, respiratory distress syndromes, infection, tumorigenisis and other disorders associated with chemokines is described herein below.
  • chemokine is well-known to those of skill in the art.
  • chemokine genes include but are not limited to Exodus, macrophage inflammatory proteins (MTP-la, MJT-l ⁇ ), RANTES, macrophage chemotactic proteins (MCPs), 1309, and TCA3. While these molecules are structurally distinct, they form a group of functionally-related molecules, of which PLACC is a member. The uses in which these other chemokines now are being exploited are equally applicable to PLACC.
  • PLACC maybe used in combination with other chemokine-based therapies to mediate a beneficial intervention in, for example, myeloproliferative disorders and myelosuppression, inhibition of HIV production, therapeutic intervention of cancers and neoplasia among others.
  • chemokine related applications those of skill in the art are referred to for example U.S. Patent Nos. 6,015,883; 5,981,230 (each incorporated herein by reference)
  • Human PLACC has been cloned by the present inventors and has a nucleic acid sequence as shown in SEQ ID NO: 1.
  • the coding region of the PLACC gene encodes a PLACC protein of SEQ ID NO:2.
  • the present invention also relates to fragments of the polypeptide that may or may not retain the leukocyte migration, myelosuppression, anti-hyperproliferative or other activity of PLACC.
  • Fragments including the N-terminus or C-terminus of the molecule may be generated by genetic engineering of translation start or stop sites within the coding region (discussed below).
  • treatment of the PLACC molecule with proteolytic enzymes, known as proteases can produce a variety of N-terminal, C-terminal and internal fragments. Examples of fragments may include contiguous residues of the
  • Such fragments preferably retain one or more of the biological activities of PLACC and/or retain an immunological (antigenic) property of PLACC.
  • These fragments may be purified according to known methods, such as precipitation
  • the gene for PLACC encodes a 127 amino acid polypeptide.
  • the predicted molecular weight of this molecule is 14 kDa including a signal peptide which is cleaved to an 1 IkDa mature protein.
  • this molecule may be used as standards in assays as a molecular weight marker.
  • identification or confirmation of the mature protein can be carried out by expressing e.g., the full length cDNA in a host cell that properly processes signal sequences and sequencing the processed expression product.
  • the PLACC protein of the present invention has a novel chemokine protein structure in that it has six cysteine residues.
  • PLACC is a novel C-C chemokine and as such will have properties analogous to those of other members of this family of intercrines.
  • PLACC likely will have significant chemotactic activity, may be myelosuppressive, may inhibit HIN proliferation, have anti-tumor properties and the like. Exemplary chemotaxis, chemoattractant, HIN proliferation, myeloproliferation and anti-tumor assays are described in further detail herein below.
  • PLACC analogs and variants may be prepared and will be useful in a variety of applications.
  • Amino acid sequence variants of the polypeptide can be substitutional, insertional or deletion variants.
  • Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity.
  • a common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell.
  • Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactive epitope or simply a single residue. Terminal additions, also called fusion proteins, are discussed below .
  • Substitutional variants typically exchange one amino acid of the wild- type for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • a particular aspect of the present invention contemplates generating PLACC mutants in which each of the six cysteine residues has individually been mutated to for example a serine. Such mutants will yield important information pertaining to the biological activity, physical structure and receptor binding potential of the PLACC molecule.
  • An alternative approach employs alanine scanning in which residues throughout molecule are randomly replaced with an alanine residue.
  • other mutants of the PLACC peptide may be generated that retain the biological activity but have increased or decreased receptor binding activity.
  • the receptor binding regions of C-C chemokines are generally located in the N-terminal region of the protein. As such, mutations of this region are particularly contemplated in order to generate protein variants with altered PLACC activity, hi addition mutants may be generated that have a greater homology to other chemokines, such as MlP-l ⁇ ,
  • the sequence of the PLACC peptide is compared to that of the other chemokines and selected residues are specifically mutated.
  • exemplary mutations that may be useful include, for example, deletion of the amino terminal 8 amino acids in an attempt to create a dominant inhibitor; deletion of the carboxy terminal extension past the last CYS at position 78 to establish function of this extension; deletion of the extra 2 CYS residues at position 28 and 78 which are not usually conserved in most CC chemokines; mutation of the NAL at position 25 to TYR, which is seen much more frequently in CC chemokines; mutation of the THR at position 59 to TRP seen much more frequently in CC chemokines; mutation of the LEU at position 44 to PHE seen in all other CC chemokines; and mutation of the NAL at 13 to TYR seen much more frequently in CC chemokines.
  • mutants such as those described above, one of skill in the art may employ well known standard technologies. Specifically contemplated are ⁇ -terminal deletions, C-terminal deletions, internal deletions, as well as random and point mutagenesis.
  • N-terminal and C-terminal deletions are forms of deletion mutagenesis that take advantage for example, of the presence of a suitable single restriction site near the end of the C- or N-terminal region.
  • the DNA is cleaved at the site and the cut ends are degraded by nucleases such as BAL31, exonuclease HI, DNase I, and SI nuclease. Rejoining the two ends produces a series of DNAs with deletions of varying size around the restriction site.
  • Proteins expressed from such mutant can be assayed for appropriate chemokine function as described throughout the specification. Similar techniques may be employed for internal deletion mutants by using two suitably placed restriction sites, thereby allowing a precisely defined deletion to be made, and the ends to be religated as above.
  • a random insertional mutation may also be performed by cutting the DNA sequence with a DNase I, for example, and inserting a stretch of nucleotides that encode, 3, 6, 9, 12 etc., amino acids and religating the end. Once such a mutation is made the mutants can be screened for various activities presented by the wild-type protein.
  • Point mutagenesis also may be employed to identify with particularity which amino acid residues are important in particular activities associated with PLACC.
  • one of skill in the art will be able to generate single base changes in the DNA strand to result in an altered codon and a missense mutation.
  • the amino acids of a particular protein can be altered to create an equivalent, or even an improved, second-generation molecule.
  • Such alterations contemplate substitution of a given amino acid of the protein without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules or receptors.
  • amino acids Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte & Doolittle, J. Mol. Biol, 157(1):105-132, 1982, incorporated herein by reference). Generally, amino acids may be substituted by other amino acids that have a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein.
  • Exemplary amino acid substitutions that may be used in this context of the invention include but are not limited to exchanging arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Other such substitutions that take into account the need for retention of some or all of the biological activity whilst altering the secondary structure of the protein will be well known to those of skill in the art.
  • polypeptide mimetics Another type of variant that is specifically contemplated for the preparation of polypeptides according to the invention is the use of peptide mimetics.
  • Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al, "Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al, Eds., Chapman and Hall, New York (1993).
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen.
  • a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
  • PLACC which belongs to a family of C-C chemokines. Various portions of these proteins possess a great deal of sequence homo logy.
  • mutations may be identified in PLACC which may alter its function. These studies are potentially important for at least two reasons. First, they provide a reasonable expectation that still other homologs, allelic variants and mutants of this gene may exist in related species, such as rat, rabbit, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep and cat. Upon isolation of these homologs, variants and mutants, and in conjunction with other analyses, certain active or functional domains can be identified. Second, this will provide a starting point for further mutational analysis of the molecule as described above. One way in which this information can be exploited is in "domain switching.”
  • Domain switching involves the generation of chimeric molecules using different but related polypeptides. For example, by comparing the sequence of
  • PLACC with that of a similar chemokine from another source and with mutants and allelic variants of these polypeptides, one can make predictions as to the functionally significant regions of these molecules. It is possible, then, to switch related domains of these molecules in an effort to determine the criticality of these regions to PLACC function. These molecules may have additional value in that these "chimeras" can be distinguished from natural molecules, while possibly providing the same or even enhanced function.
  • Exemplary domain switched chimeras can be performed in which the first eight amino acids of PLACC may be exchanged for the first seven amino acids of
  • Exodus-2/6Ckine/SLC to see if this allows for activation of CCR7 by PLACC.
  • Exchanging the carboxy terminal extension of PLACC (past amino acid 78) for the carboxy terminal extension of Exodus-2/6Ckine/SLC (past amino acid 79) maybe used to characterize the function of the carboxy terminal of PLACC and test CCR 7 activation by PLACC.
  • Another embodiment contemplates switching the amino terminal eight amino acids of PLACC for the first ten amino acids of the mature Mip-l ⁇ to determine whether the function of Mip-l ⁇ can be mimicked by PLACC.
  • exchanging amino acids 11 to 51 of PLACC for amino acids 10 to 50 of the mature Exodus-2/6Ckine/SLC could be used to assess the functional role the internal motif between the second and third CYS of C-C chemokines.
  • the present invention further contemplates the generation of a specialized kind of insertional variant known as a fusion protein.
  • This molecule generally has all or a substantial portion of the native molecule, linked at the N- or C-terminus, to all or a portion of a second polypeptide.
  • fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
  • Another useful fusion includes the addition of a immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification.
  • fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions. Fusion to a polypeptide that can be used for purification of the receptor-PLACC complex would serve to isolate the receptor for identification and analysis.
  • GST glutathione S-transferase
  • NEB maltose binding protein
  • FLAG U3L New Haven, CT
  • 6xHis system Qiagen, Chatsworth, CA
  • a particularly useful fusion construct may be one in which a PLACC peptide is fused to a hapten to enhance immunogenicity of a PLACC fusion construct.
  • fusion constructs to increase immunogenicity are well known to those of skill in the art, for example, a fusion of PLACC with a helper antigen such as hsp70 or peptide sequences such as from Diptheria toxin chain or a cytokine such as JX-2 will be useful in eliciting an immune response.
  • fusion construct can be made which will enhance the targeting of the PLACC-related compositions to a specific site or cell.
  • fusion constructs including a heterologous polypeptide with desired properties, e.g., an Ig constant region to prolong serum half life or an antibody or fragment thereof for targeting also are contemplated.
  • Other fusion systems produce polypeptide hybrids where it is desirable to excise the fusion partner from the desired polypeptide.
  • the fusion partner is linked to the recombinant PLACC polypeptide by a peptide sequence containing a specific recognition sequence for a protease. Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, MD) or Factor Xa (New England Biolabs, Beverley, MA). f . Purification of Proteins.
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
  • Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide.
  • the term "purified protein or peptide" as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.
  • a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity.
  • substantially purified is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
  • Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Various techniques suitable for use in protein purification will be well known to those of skill in the art.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • PLACC-related peptides may be useful in various embodiments of the present invention.
  • Such peptides or indeed even the full length protein, of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co., (1984);Tam et al, J. Am. Chem.
  • PLACC active protein or portions of the PLACC which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides.
  • Such chemical peptide synthesis techniques have been used successfully for the production of chemokines such as JX-8 (Clark-Lewis et al, J. Biol Chem., 266:23128-34; 1991) and MCP-1.
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below.
  • U.S. Patent 4,554,101 (incorporated herein by reference) also teaches the identification and preparation of epitopes from primary amino acid sequences on the basis of hydrophilicity. Thus, one of skill in the art would be able to identify epitopes from within any amino acid sequence encoded by any of the DNA sequences disclosed herein. h. Antigen Compositions.
  • PLACC proteins or peptides may be useful as antigens for the immunization of animals relating to the production of antibodies. It is envisioned that either PLACC, or portions thereof, may be coupled, bonded, bound, conjugated or chemically-linked to one or more agents via linkers, polylinkers or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced. It is further envisioned that the methods used in the preparation of these compositions will be familiar to those of skill in the art and should be suitable for administration to animals, i.e., pharmaceutically acceptable. Preferred agents are the carriers are keyhole limpet hemocyannin (KLH) or bovine serum albumin (BSA).
  • KLH keyhole limpet hemocyannin
  • BSA bovine serum albumin
  • the present invention also provides, in another embodiment, a gene encoding PLACC.
  • the gene for the human PLACC molecule has been identified.
  • the present invention is not limited in scope to the particular gene(s) identified herein, however, seeing as one of ordinary skill in the art could, using the nucleic acids corresponding to the PLACC gene, readily identify related homologs in various other species (e.g., rat, rabbit, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species).
  • PLACC gene may contain a variety of different bases and yet still produce a corresponding polypeptide that is functionally indistinguishable, and in some cases structurally, from the human gene disclosed herein.
  • the term “PLACC gene” may be used to refer to any nucleic acid that encodes PLACC peptide or polypeptide and as such is intended to encompass both genomic DNA and cDNA.
  • any reference to a nucleic acid should be read as encompassing a host cell containing that nucleic acid and, in some cases, capable of expressing the product of that nucleic acid.
  • cells expressing nucleic acids of the present invention may prove useful in the context of screening for agents that induce, repress, inhibit, augment, interfere with, block, abrogate, stimulate or enhance the function of PLACC or its receptor.
  • Nucleic Acids Encoding PLACC The human PLACC gene is disclosed in SEQ ID NO: 1.
  • Nucleic acids according to the present invention may encode an entire PLACC protein, polypeptide, or allelic variant, a domain of PLACC that expresses a chemokine activity, a C-C chemokine receptor binding function, a leukocyte migration activity, a myelosuppressive activity or any other fragment or variant of the PLACC sequences set forth herein.
  • the nucleic acid may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the nucleic acid would comprise complementary DNA (cDNA). Also contemplated is a cDNA plus a natural intron or an intron derived from another gene; such engineered molecules are sometime referred to as "mini-genes.” At a minimum, these and other nucleic acids of the present invention maybe used as molecular weight standards in, for example, gel electrophoresis.
  • cDNA is intended to refer to DNA prepared using messenger RNA (mRNA) as template.
  • mRNA messenger RNA
  • a given PLACC from a given species may be represented by degenerate variants that have slightly different nucleic acid sequences but, nonetheless, encode the same protein (see Table 1 below).
  • PLACC refers to a nucleic acid molecule that has been isolated from total cellular nucleic acid.
  • the invention concerns a nucleic acid sequence essentially as set forth in SEQ ID NO:l.
  • the term "functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine (Table 1, below), and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages.
  • nucleotide sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotides of SEQ ID NO:l are nucleic acids encoding a PLACC chemokine. Sequences that are essentially the same as those set forth in SEQ ID NO:l may also be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO: 1 under standard conditions.
  • the DNA segments of the present invention include those encoding biologically functional equivalent PLACC proteins and peptides as described above. Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through any means described herein or known to those of skill in the art.
  • the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO: 1
  • nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementary rules.
  • complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:l under relatively stringent conditions such as those described herein. Such sequences may encode the entire PLACC protein or functional or non-functional fragments thereof.
  • the hybridizing segments may be shorter oligonucleotides. Sequences of about 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Nucleotide sequences of this size that specifically hybridize to SEQ ID NO:l are useful as probes or primers.
  • an oligonucleotide that "specifically hybridizes" to SEQ ID NO:l means that hybridization under suitably (e.g., high) stringent conditions allows discrimination of SEQ ID NO:l from other chemokine genes. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization.
  • oligonucleotide Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that exemplary oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used, although others are contemplated. Longer polynucleotides encoding 250, 500, or 1000 bases and longer are contemplated as well. Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions.
  • Suitable hybridization conditions will be well known to those of skill in the art. In certain applications, it is appreciated that lower stringency conditions may be required. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
  • hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 3 mM MgCl 2 , 10 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C.
  • Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl 2 , at temperatures ranging from approximately 40°C to about 72°C.
  • Formamide and SDS also may be used to alter the hybridization conditions.
  • One method of using probes and primers of the present invention is in the search for genes related to PLACC, more particularly, homologs of the proteins from other species.
  • the target DNA will be a genomic or cDNA library, although screening may involve analysis of RNA molecules. By varying the stringency of hybridization, and the region of the probe, different degrees of homology may be discovered.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids also are routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
  • site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein.
  • An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared.
  • This primer is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization conditions, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment.
  • This heteroduplex vector is then used to transform appropriate cells, such as ⁇ . coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
  • site-directed mutagenesis is not the only method of generating potentially useful mutant PLACC species and as such is not meant to be limiting.
  • the present invention also contemplates other methods of achieving mutagenesis such as for example, treating the recombinant vectors carrying the gene of interest mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • Antisense treatments are one way of addressing this , situation.
  • Antisense technology also may be used to "knock-out" function of PLACC in the development of cell lines or transgenic mice for research, diagnostic and screening purposes. Further, antisense techniques may prove useful in reducing PLACC activity in subjects bearing a functional wild-type gene.
  • Antisense methodology takes advantage of the fact that nucleic acids tend to pair with “complementary" sequences.
  • complementary it is meant that polynucleotides are those which are capable of base-pairing according to the standard
  • the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.
  • G:C cytosine
  • A:T thymine
  • A:U uracil
  • Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
  • ds double-stranded
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions.
  • a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected. As stated above, “complementary” or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches.
  • sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions.
  • sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches.
  • Other sequences with lower degrees of homology also are contemplated.
  • an antisense construct which has limited regions of high homology, but also contains a non-homologous region e.g., ribozymes
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • a genomic clone will need to be used.
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • PLACC PLACC peptides and PLACC- encoding nucleic acid constructs
  • a variety of expression vector/host systems may be utilized to contain and express a
  • PLACC coding sequence 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 (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.
  • 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 (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or
  • Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RLN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for the recombinant expression of PLACC in bacteria, yeast and other invertebrates are described herein below.
  • the DNA sequence encoding the mature form of the protein is amplified by PCR and cloned into an appropriate vector for example, pGEX-3X (Pharmacia, Piscataway, NJ).
  • the pGEX vector is designed to produce a fusion protein comprising glutathione-S-transferase (GST), encoded by the vector, and a protein encoded by a DNA fragment inserted into the vector's cloning site.
  • the primers for the PCR may be generated to include for example, an appropriate cleavage site. Treatment of the recombinant fusion protein with thrombin or factor
  • pGEX-3X/PLACC construct is transformed into E. coli XL-1 Blue cells (Stratagene, La Jolla CA), and individual transformants were isolated and grown. Plasmid DNA from individual transformants is purified and partially sequenced using an automated sequencer to confirm the presence of the desired PLACC gene insert in the proper orientation.
  • the cells can be modified (heterologous promoter is inserted in such a maimer that it is operably linked to, e.g., by homologous recombination) to provide increase PLACC expression by replacing, in whole or in part the naturally occurring PLACC promoter with all or part of a heterologous promoter so that the cells express PLACC at higher levels.
  • the heterologous promoter is inserted in such a manner that it is operably linked to PLACC-encoding sequences, (e.g., PCT International Publication No. WO96/12650; PCT International Publication No. WO 92/20808 and PCT International Publication No. WO 91/09955). It is contemplated that, in addition to the heterologous promoter
  • amplifiable marker DNA e.g., ada, dhfr and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase and dihydroorotase
  • intron DNA may be inserted along with the heterologous promoter DNA. If linked to the PLACC coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the PLACC coding sequences in the cells.
  • PLACC peptide compositions For example, induction of the GST/PLACC fusion protein is achieved by growing the transformed XL-1 Blue culture at 37°C in LB medium (supplemented with carbenicillin) to an optical density at wavelength 600 nm of 0.4, followed by further incubation for 4 hours in the presence of 0.5 mM Isopropyl ⁇ -D-Thiogalactopyranoside (Sigma Chemical Co., St. Louis MO).
  • the fusion protein expected to be produced as an insoluble inclusion body in the bacteria, may be purified as follows. Cells are harvested by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/ l lysozyme (Sigma Chemical Co.) for 15 minutes at room temperature. The lysate is cleared by sonication, and cell debris is pelleted by centrifugation for 10 minutes at 12,000 X g. The fusion protein-containing pellet is resuspended in 50 mM
  • Tris Tris, pH 8, and 10 mM EDTA, layered over 50% glycerol, and centrifuged for 30 min. at 6000 X g.
  • the pellet is resuspended in standard phosphate buffered saline solution (PBS) free of Mg 4"1" and Ca *1 ⁇ .
  • PBS standard phosphate buffered saline solution
  • the fusion protein is further purified by fractionating the resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook et al , supra). The gel is soaked in 0.4 M KC1 to visualize the protein, which is excised and electroeluted in gel-running buffer lacking SDS. If the GST/PLACC fusion protein is produced in bacteria as a soluble protein, it may be purified using the GST Purification Module (Pharmacia Biotech).
  • the fusion protein may be subjected to thrombin digestion to cleave the GST from the mature PLACC protein.
  • the digestion reaction (20-40 ⁇ g fusion protein, 20-30 units human thrombin (4000 U/mg (Sigma) in 0.5 ml PBS) is incubated 16-48 hrs. at room temperature and loaded on a denaturing SDS-PAGE gel to fractionate the reaction products. The gel is soaked in 0.4 M KC1 to visualize the protein bands.
  • the identity of the protein band corresponding to the expected molecular weight of PLACC may be confirmed by partial amino acid sequence analysis using an automated sequencer (Applied Biosystems Model 473A, Foster City, CA).
  • the DNA sequence encoding the predicted mature PLACC protein may be cloned into a plasmid containing a desired promoter and, optionally, a leader sequence (see, e.g., Better et al, Science, 240:1041-43, 1988).
  • the sequence of this construct maybe confirmed by automated sequencing.
  • the plasmid is then transformed into E. coli strain MCI 061 using standard procedures employing CaCl 2 incubation and heat shock treatment of the bacteria (Sambrook et al, supra).
  • the transformed bacteria are grown in LB medium supplemented with carbenicillin, and production of the expressed protein is induced by growth in a suitable medium. If present, the leader sequence will effect secretion of the mature PLACC protein and be cleaved during secretion.
  • the secreted recombinant protein is purified from the bacterial culture media by the method described herein throughout or, e.g. , by adapting methods previously described for the purification of recombinantly produced RANTES chemokine (Kuna et al, J. Immunol, 149:636-642, 1992), MGSA chemokine (Horuk et al, J. Biol. Chem. 268: 541-46, 1993), and IP-10 chemokine (expressed in insect cells) (Sarris et al, J. Exp. Med., 178:1121-1132, 1993).
  • yeast system may be employed to generate the recombinant peptide.
  • the coding region of the PLACC cDNA is amplified by PCR.
  • a DNA encoding the yeast pre-pro-alpha leader sequence is amplified from yeast genomic
  • the pre-pro-alpha leader coding sequence and PLACC coding sequence fragments are ligated into a plasmid containing the yeast alcohol dehydrogenase (ADH2) promoter, such that the promoter directs expression of a fusion protein consisting of the pre-pro-alpha factor fused to the mature PLACC polypeptide.
  • ADH2 yeast alcohol dehydrogenase
  • the vector further includes an ADH2 transcription terminator downstream of the cloning site, the yeast "2-micron" replication origin, the yeast leu-2d gene, the yeast REP1 and REP2 genes, the E. coli beta-lactamase gene, and an E. coli origin of replication.
  • the beta- lactamase and leu-2d genes provide for selection in bacteria and yeast, respectively.
  • the leu-2d gene also facilitates increased copy number of the plasmid in yeast to induce higher levels of expression.
  • the REP1 and REP2 genes encode proteins involved in regulation of the plasmid copy number.
  • the DNA construct described in the preceding paragraph is transformed into yeast cells using a known method, e.g., lithium acetate treatment (Stearns et al. , Meth. Enz., 185: 280-297, 1990).
  • the ADH2 promoter is induced upon exhaustion of glucose in the growth media (Price et al, Gene, 55:281, 1987).
  • the pre-pro-alpha sequence effects secretion of the fusion protein from the cells.
  • yeast KEX2 protein cleaves the pre-pro sequence from the mature PLACC chemokine (Bitter et. al, Proc. Natl. Acad. Sci. USA, 5 :5330-5334, 1984).
  • PLACC is recombinantly expressed in yeast using a commercially available expression system, e.g., the Pichia Expression System (Invitrogen, San Diego, CA), following the manufacturer's instructions.
  • This system also relies on the pre-pro-alpha sequence to direct secretion, but transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction by methanol.
  • AOX1 alcohol oxidase
  • the secreted recombinant PLACC is purified from the yeast growth medium by, e.g., the methods used to purify PLACC from bacterial and mammalian cell supernatants.
  • the cDNA encoding PLACC may be cloned into the baculovirus expression vector pVL1393 (PharMingen, San Diego, CA). This PLACC-containing vector is then used according to the manufacturer's directions (PharMingen) to infect Spodopterafrugiperda cells in sF9 protein- free media and to produce recombinant protein.
  • the protein is purified and concentrated from the media using a heparin-Sepharose column (Pharmacia, Piscataway, NJ) and sequential molecular sizing columns (Amicon, Beverly, MA), and resuspended in PBS.
  • SDS- PAGE analysis shows a single band and confirms the size of the protein, and Edman sequencing on a Porton 2090 Peptide Sequencer confirms its N-terminal sequence.
  • the PLACC may be expressed in an insect system.
  • Insect systems for protein expression are well known to those of skill in the art.
  • Autographa calif ornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the PLACC coding sequence is cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of PLACC 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 PLACC is expressed (Smith et al, J Virol 46: 584, 1983; Engelhard EK et al, Proc Nat Acad Sci 91: 3224-7, 1994).
  • Mammalian host systems for the expression of the recombinant protein also are well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. 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 of the protein may also be important for correct insertion, folding and/or function.
  • Different host cells such as CHO, HeLa, MDCK, 293, WI38, and the like 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. It is preferable that the transformed cells are used for long-term, highryield protein production and as such stable expression is desirable.
  • the selectable marker is designed to confer resistance to selection and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell.
  • selection systems may be used to recover the cells that have been transformed for recombinant protein production.
  • selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively.
  • anti-metabolite resistance can be used as the basis of selection for dhfr, that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; als which confers resistance to chlorsulfuron; and hygro, that confers resistance to hygromycin.
  • Additional selectable genes that may be useful include trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine.
  • Markers that give a visual indication for identification of transformants include anthocyanins, ⁇ -glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.
  • expression vectors are employed to express the PLACC polypeptide product, which can then be purified and, for example, be used to vaccinate animals to generate antisera or monoclonal antibody with which further studies may be conducted.
  • expression vectors may be used in gene therapy applications to introduce PLACC-encoding nucleic acids into cells in need thereof and/or to induce PLACC expression in such cells.
  • the present section is directed to a description of the production of such expression vectors.
  • Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.
  • Elements designed to optimize messenger RNA stability and translatability in host cells also are defined.
  • the conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products also are provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
  • expression construct or "expression vector” is meant to include any type of genetic construct containing a nucleic acid coding for gene products in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • the transcript may be translated into a protein, but it need not be.
  • expression includes both transcription of a gene and translation of mRNA into a gene product.
  • the nucleic acid encoding a gene product is under transcriptional control of a promoter.
  • a "promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • the term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase JJ.
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the S V40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalo virus (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
  • CMV cytomegalo virus
  • the SV40 early promoter the Rous sarcoma virus long terminal repeat
  • ⁇ -actin rat insulin promoter
  • the phosphoglycerol kinase promoter and glyceraldehyde-3-phosphate dehydrogenase promoter can be used to obtain high-level expression of the coding sequence of interest.
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product.
  • inducible promoter systems are available for production of viral vectors.
  • One such system is the ecdysone system (Invitrogen, Carlsbad, CA), which is designed to allow regulated expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechamsm that allows virtually no basal level expression of the transgene, but over 200-fold inducibility
  • Tet-OffTM or Tet-OnTM system (Clontech, Palo Alto, CA) originally developed by Gossen and Bujard (Gossen and Bujard, Proc Natl Acad Sci USA. 15;89(12):5547-51, 1992; Gossen et al, Science, 268(5218):1766-9, 1995).
  • a transgene in a gene therapy vector.
  • different viral promoters with varying strengths of activity may be utilized depending on the level of expression desired.
  • the CMV immediate early promoter is often used to provide strong transcriptional activation. Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired.
  • retroviral promoters such as the LTRs from MLV or MMTV are often used.
  • viral promoters that may be used depending on the desired effect include SV40, RSV LTR, HIV-1 and HTV-2 LTR, adenovirus promoters such as from the EIA, E2A, or MLP region, AAV LTR, cauliflower mosaic virus, HSV-TK, and avian sarcoma virus.
  • tissue specific promoters may be used to effect transcription in specific tissues or cells so as to reduce potential toxicity or undesirable effects to non-targeted tissues.
  • promoters such as the PSA, probasin, prostatic acid phosphatase or prostate-specific glandular kallikrein (hK2) may be used to target gene expression in the prostate.
  • promoters as those that are hormone or cytokine regulatable.
  • promoters that are hormone regulatable include MMTV, MT-1, ecdysone and RuBisco.
  • Other hormone regulated promoters such as those responsive to thyroid, pituitary and adrenal hormones are expected to be useful in the present invention.
  • Cytokine and inflammatory protein responsive promoters that could be used include K and T Kininogen (Kageyama et al, JBiol Chem.
  • fibrinogen inducible by phorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide
  • collagenase induced by phorbol esters and retinoic acid
  • metallothionein heavy metal and glucocorticoid inducible
  • Stromelysin inducible by phorbol ester, interleukin-1 and EGF
  • alpha-2 macroglobuiin and alpha-1 antichymotrypsin inducible by phorbol ester, interleukin-1 and EGF.
  • cell cycle regulatable promoters may be useful in the present invention.
  • a strong CMV promoter to drive expression of a first gene such as pl6 that arrests cells in the Gl phase could be followed by expression of a second gene such as p53 under the control of a promoter that is active in the Gl phase of the cell cycle, thus providing a "second hit" that would push the cell into apoptosis.
  • Other promoters such as those of various cyclins, PCNA, galectin-3, E2F1, p53 and BRCA1 could be used.
  • Tumor specific promoters such as osteocalcin, hypoxia-responsive element (HRE), MAGE-4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase may also be used to regulate gene expression in tumor cells.
  • Other promoters that could be used according to the present invention include Lac-regulatable, chemotherapy inducible (e.g. MDR), and heat (hyperthermia) inducible promoters, radiation-inducible (e.g., EGR (Joki et al, Hum Gene Ther.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities.
  • Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • Enhancers useful in the present invention are well known to those of skill in the art and will depend on the particular expression system being employed (Scharf D et al Results Probl Cell Differ 20: 125-62, 1994; Bittner et al Methods in Enzymol 153: 516-544, 1987).
  • Polyadenylation Signals Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human or bovine growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator.
  • IRES internal ribosome entry site
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, Nature, 334:320-325, 1988).
  • IRES elements from two members of the picomavirus family polio virus and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988 supra), as well an IRES from a mammalian message (Macejak and Sarnow, Nature, 353:90-94, 1991). IRES elements can be linked to heterologous open reading frames.
  • each open reading frame can be transcribed together, each separated by an IRES, creating polycistronic messages.
  • IRES element By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
  • Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker. b. Delivery of Expression Vectors.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • non- viral delivery is contemplated.
  • the ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, In: Rodriguez R L, Denhardt D T, ed.
  • Vectors A survey of molecular cloning vectors and their uses.
  • Stoneham Butterworth, 467-492, 1988; Nicolas and Rubenstein, In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez & Denhardt (eds.), Stoneham: Butterworth, 493-513, 1988; Baichwal and Sugden, hi: Gene Transfer, Kucherlapati R, ed., New York, Plenum Press, 117-148, 1986; Temin, In: gene Transfer, Kucherlapati (ed.), New York: Plenum Press, 149-188, 1986).
  • the first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988 supra; Baichwal and Sugden, 1986 supra) and adenoviruses (Ridgeway, 1988 supra; Baichwal and Sugden, 1986 supra). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kb of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988 supra; Temin, 1986 supra).
  • expression constructs comprising viral vectors containing the genes of interest may be adenoviral (see for example, U.S. Patent No. 5,824,544; U.S. Patent No. 5,707,618; U.S. Patent No. 5,693,509; U.S. Patent No. 5,670,488; U.S. Patent No. 5,585,362; each incorporated herein by reference), retroviral (see for example, U.S. Patent No. 5,888,502; U.S. Patent No. 5,830,725; U.S. Patent No. 5,770,414; U.S. Patent No. 5,686,278; U.S. Patent No. 4,861 ,719 each incorporated herein by reference), adeno-associated viral
  • DNA constructs of the present invention are generally delivered to a cell, and in certain situations, the nucleic acid or the protein to be transferred may be transferred using non-viral methods.
  • the nucleic acid encoding the therapeutic gene may be positioned and expressed at different sites.
  • the nucleic acid encoding the therapeutic gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct ma be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • the lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, In: Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker, pp. 87-104, 1991).
  • the addition of DNA to cationic liposomes causes a topological transition from liposomes to optically birefringent liquid-crystalline condensed globules (Radler et al, Science, 275(5301):810-4, 1997).
  • These DNA-lipid complexes are potential non-viral vectors for use in gene therapy and delivery.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, Science, 243:315-318, 1989).
  • HVJ hemagglutinating virus
  • the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al, J. Biol. Chem., 266:3361-3364, 1991).
  • the liposome may be complexed or employed in conjunction with both HNJ and HMG-1.
  • expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a therapeutic gene into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993, supra).
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a D ⁇ A-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987, supra) and transferrin (Wagner et al, Proc. Nat'l Acad Sci. USA, 87(9):3410-3414, 1990).
  • ASOR asialoorosomucoid
  • transferrin Wang and transferrin
  • the delivery vehicle may comprise a ligand and a liposome.
  • a ligand for example, Nicolau et al. (Methods Enzymol, 149:157-176, 1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
  • nucleic acid encoding a therapeutic gene also may be specifically delivered into a particular cell type by any number of receptor-ligand systems with or without liposomes.
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically pe ⁇ neabilize the cell membrane. This is applicable particularly for transfer in vitro, however, it may be applied for in vivo use as well.
  • Dubensky et al. Proc. Nat. Acad. Sci. USA, 81 :7529-7533, 1984
  • polyomavirus DNA in the form of CaPO 4 precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection.
  • Benvenisty and Neshif Proc. Nat. Acad.
  • Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, Nature, 327:70-73, 1987).
  • Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, Proc. Natl. Acad. Sci USA, 87:9568-9572, 1990).
  • the microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • gene transfer may more easily be performed under ex vivo conditions.
  • Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues.
  • the present invention contemplates an antibody that is immunoreactive with a PLACC molecule of the present invention, or any portion thereof.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library, bifunctional/bispecific antibodies, humanized antibodies, CDR-grafted antibodies, human antibodies and antibodies which include portions of CDR sequences specific for PLACC.
  • Neutralizing antibodies i.e., those which inhibit chemokine activity, are especially preferred for therapeutic embodiments, hi a preferred embodiment, an antibody is a monoclonal antibody.
  • a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal.
  • an animal used for production of anti-antisera is a non-human animal including rabbits, mice, rats, hamsters, goat, sheep, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • adjuvants may be used to increase immunological response.
  • 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
  • Corynebacterium parvum are potentially useful human adjuvants.
  • Antibodies both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
  • the term "specific for” is intended to mean that the variable regions of the antibodies recognize and bind PLACC and are capable of distinguishing PLACC from other antigens, for example other chemokines.
  • a composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention.
  • Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
  • Monoclonal antibodies to PLACC 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 and Milstein ( Nature 256: 495-497, 1975), the human B-cell hybridoma technique (Kosbor et al, Immunol Today 4:72, 1983 ; Cote et al, Proc Natl Acad Sci 80: 2026-2030, 1983) and the EBN-hybridoma technique (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96, (1985).
  • myeloma cell lines may be used.
  • Such cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • the immunized animal is a mouse
  • rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210
  • U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions.
  • the hybridomas and cell lines produced by such techniques for producing the monoclonal antibodies are contemplated to be novel compositions of the present invention.
  • PLACC in an exemplary method for generating a polyclonal antisera immunoreactive with the PLACC related chemokines, 50 ⁇ g of PLACC is emulsified in Freund's Complete Adjuvant for immunization of rabbits. At intervals of for example, 21 days, 50 ⁇ g of PLACC are emulsified in Freund's Incomplete Adjuvant for boosts.
  • a mouse is injected periodically with recombinant PLACC (e.g., 10-20 ⁇ g emulsified in Freund's Complete Adjuvant).
  • recombinant PLACC e.g. 10-20 ⁇ g emulsified in Freund's Complete Adjuvant.
  • the mouse is given a final pre-fusion boost of PLACC in PBS, and four days later the mouse is sacrificed and its spleen removed.
  • the spleen is placed in 10 ml serum-free RPMI 1640, and a single cell suspension is formed by grinding the spleen between the frosted ends of two glass microscope slides submerged in serum-free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 ⁇ g/ml streptomycin (RPMI)
  • the cell suspension is filtered through sterile 70-mesh Nitex cell strainer (Becton Dickinson, Parsippany, New Jersey), and is washed twice by centrifuging at 200 g for 5 minutes and resuspending the pellet in 20 ml serum-free RPMI.
  • Splenocytes taken from three naive Balb/c mice are prepared in a similar manner and used as a control.
  • NS-1 myeloma cells kept in log phase in RPMI with 11% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three days prior to fusion, are centrifuged at 200 g for 5 minutes, and the pellet is washed twice as described in the foregoing paragraph.
  • FBS fetal bovine serum
  • PLACC as follows. Immulon 4 plates (Dynatech, Cambridge, MA) are coated for 2 hours at 37°C with 100 ng/well of PLACC diluted in 25mM Tris, pH 7.5. The coating solution is aspirated and 200 ul/well of blocking solution (0.5% fish skin gelatin (Sigma) diluted in CMF-PBS) is added and incubated for 30 min. at 37°C. Plates are washed three times with PBS with 0.05% Tween 20 (PBST) and 50 ⁇ l culture supernatant is added.
  • blocking solution (0.5% fish skin gelatin (Sigma) diluted in CMF-PBS)
  • Selected fusion wells are cloned twice by dilution into 96-well plates and visual scoring of the number of colonies/well after 5 days.
  • the monoclonal antibodies produced by hybridomas are isotyped using the Isostrip system (Boehringer Mannheim, Indianapolis, IN).
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screenmg recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (Proc Natl Acad Sci 86: 3833-3837; 1989), and Winter G and Milstein C (Nature 349: 293-299, 1991).
  • the antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and
  • both polyclonal and monoclonal antibodies against PLACC may be used in a variety of embodiments.
  • the antibodies may be employed for therapeutic purposes in which the inhibition of PLACC activity is desired (e.g., to reduce inflammation).
  • Antibodies may be used to block PLACC action and receptor function thereby reducing tissue destruction and autoimmune diseases such as for example, Crohn's disease, ulcerative colitis, graft versus host disease, Seogram's syndrome and other autoimmune diseases listed herein throughout. Of particular interest is the fact that this chemokine is produced in the mucosal lining.
  • Antibodies of the present invention also may prove useful in diagnostic purposes in order, for example, to detect increases or decreases in PLACC proteins in tissue samples including samples for sites of inflammation, or fluid samples including blood serum, plasma and exudate samples. Additional aspects will employ the antibodies of the present invention in antibody cloning protocols to obtain cDNAs or genes encoding other PLACC proteins. They may also be used in inhibition studies to analyze the effects of PLACC related peptides in cells or animals. Anti-PLACC antibodies will also be useful in immunolocalization studies to analyze the distribution of PLACC during various cellular events, for example, to determine the cellular or tissue-specific distribution of PLACC polypeptides under different points in the cell cycle. A particularly useful application of such antibodies is in purifying native or recombinant PLACC, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
  • chemokine-like activity of PLACC it may be necessary to determine the chemokine-like activity of PLACC and any variants thereof.
  • the assays for chemokine activity are well known to those of skill in the art, however, certain such exemplary assays are described in this section.
  • transwell insert 100 ⁇ l of this cell suspension is added to each transwell insert (Costar). DMEM with antibiotics and 0.2% BSA with or without PLACC is added to the lower wells in the 24 well plate. Transwell inserts are placed into the lower walls, and incubated at 37° C for 90 mins. At the completion of the incubation period inserts are removed and the adherent cells are removed. The entire insert is then stained with Wright-Giemsa. Cells adherent to the lower surface of the insert and those that migrated to the lower well are counted under microscope, and added together to obtain a total number of migrating cells.
  • the effects of PLACC upon human monocvtes/macrophages or human neutrophils may be evaluated, e.g., by methods described by Devi et al, J. Immunol, 753:5376-5383 (1995) for evaluating murine TCA3-induced activation of neutrophils and macrophages. Indices of activation measured in such studies include increased adhesion to fibrinogen due to integrin activation, chemotaxis, induction of reactive nitrogen intermediates, respiratory burst (superoxide and hydrogen peroxide production), and exocytosis of lysozyme and elastase in the presence of cytochalasin B.
  • PLACC In vivo Activity Assay.
  • the chemoattractant properties of PLACC may be assayed in vivo by adapting the protocol described by Meurer et al, J. Exp. Med., 175:1913-1921 (1993).
  • Recombinant PLACC (10-500 pmol/site) is injected intradermally into a suitable mammal, e.g., dogs or rabbits. At times of 4 to 24 hours, cell infiltration at the site of injection is assessed by histologic methods.
  • the presence of PLACC is confirmed by immunocytochemistry using antibodies directed against PLACC. The nature of the cellular infiltrate is identified by staining with Baxter's Diff Quick kit.
  • GNHD characterized by hepatic and small bowel inflammation and increased skin collagen content in the recipient mice.
  • the potential suppression of hematopoiesis by the PLACC chemokines may be tested in assays of stem/progenitor cell function and number, including LTC- IC, CFU-GEMM, CFU-GM, BFU-E. This will be an appropriate indicator of whether PLACC will have a suppressive effect on hematopoiesis on myeloproliferative disorders such as for example, chronic myelogenous leukemia
  • CML chronic myelogenous Leukemia
  • PN polycythemia vera
  • bone marrow cells are collected from human donors after obtaining informed consent.
  • Low density human bone marrow cells at 5 x 10 4 /ml are plated in 1% methylcellulose in Iscove's Modified Essential Medium (Biowhitaker, Walkersville, MD) supplemented with 30%> FCS (Hyclone), recombinant human erythropoietin (EPO, 1 U/ml, Amgen, Thousand Oaks, CA), recombinant human interleukin-3 (IL-3, 100 U/ml, Immunex, Seattle, WA), and recombinant human stem cell factor (SCF, 50 ng/ml, Amgen) for colony forming unit granulocyte/macrophage (CFU-GM), colony forming unit granulocyte/erythrocyte/macrophage/megakaryocyte (CFU-GEMM) or blast forming unit-erythrocyte (BFU-E) analysis. Cultures are incubated
  • the in vivo effect of PLACC on hematopoiesis in mice may be monitored using an assay essentially as described in Broxmeyer et al, Ann. Hematol, 71:235-246 (1995), in which untreated pure synthetic PLACC and PLACC treated with a 30% acetonitrile/1% trifluoroacetic acid (ACN) solution as described in Mantel et al, Proc. Natl. Acad, Sci, USA, 90:2232-2236 (1993) are used.
  • ACN trifluoroacetic acid
  • chemokines which exist in solution as multimers treatment with ACN stimulates formation of monomers, which are the active form in vivo, and thus enhances chemokine activity.
  • ACN-treated chemokines can be active at concentrations 200-fold lower than untreated chemokines.
  • solutions of untreated PLACC or ACN-treated PLACC are prepared at desired concentrations, for example, varying from 0.001 to 50 ng/ml PLACC.
  • ACN-treated or untreated diluent may be used as a control to show that the ACN is not toxic.
  • Normal C3H/HeJ mice are injected intravenously with a 0.2 ml dose of each solution and sacrificed after 24 hours.
  • Unseparated bone marrow cells are retrieved from femurs of these animals and plated in either agar with 10% v/v pokeweed mitogen mouse spleen cell-conditioned medium (PWMSCM), for CFU- GM assessment, or in methylcellulose with human ervthropoietin (Epogen®, Amgen, Thousand Oaks, CA), PWMSCM and hemin (Eastman Kodak Co., Rochester, NY), for BFU-E/CFU-GEMM assessment. Colony counts are determined after seven days of incubation in a humidified environment in an ESPEC N 2 -O 2 -CO 2 incubator BNP-
  • mice are treated with varying concentrations of untreated PLACC
  • PLACC PLACC cell cycle progression of bone marrow progenitor cells. If cell cycle arrest is seen it will be possible to use PLACC to protect normal bone marrow against S-phase cytotoxic chemotherapy.
  • the effect of PLACC protein products on the proliferation of cytokine dependent myeloid cell lines also may be a useful test of functional activity.
  • the human myeloid cell lines TF-1 and MO7E (Avanzi et al, Brit. J. Haematol, 69:359; 1988) require GM-CSF and SCF for maximal proliferation:
  • the cytokine-dependent primitive acute myeloid leukemia cell lines TF-1 and MO7E may be cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) plus 10% FCS (Hyclone) and 100 U/ml penicillin and 100 ⁇ g/ml streptomycin (tissue culture antibiotics, Life Technologies, Gaithersburg, MD).
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • SCF stem cell factor
  • PLACC produced according to the methods described herein may be added to log phase MO7E cells, in the presence of GM-CSF and SCF, to determine its effects on proliferation. It is expected that PLACC will produce inhibition of such proliferation and will therefore be useful in treating myeloprohferative disorders such as chronic myelogenous leukemia.
  • CML chronic myelogenous leukemia
  • colony formation assays as described in Hromas et al, Blood, 89:3 15-3322 (1997). Briefly, bone marrow cells are collected from six CML patients in chronic phase. Low density marrow cells at for example, 5 x 10 4 cells/mL are plated in 1% methylcellulose in Iscove's modified Dulbecco's medium supplemented with 30% fetal calf serum, 1 U/mL human erythropoietin (Epogen®, Amgen), 100 U/mL human interleukin-3 (Genetics
  • PLACC human stem cell factor
  • BFU-E Colony counts for cultures treated with chemokines are compared to colony counts of the control cultures and were expressed as a percentage of control CFU or BFU. g. In Vivo Tumor Growth Inhibition Assay.
  • PLACC Tumor growth-inhibition properties of PLACC are assayed, e.g. , by modifying the protocol described by Laning et al, J. Immunol, 753:4625-4635 (1994) for assaying the tumor growth-inhibitory properties of murine TCA3.
  • a PLACC-encoding cDNA may be transfected by electroporation into the myeloma-derived cell line J558 (American Type Culture Collection, Rockville, MD). Transfectants are screened for PLACC production by standard techniques such as ELIS A (enzyme-linked immunoadsorbant assay) using a monoclonal antibody generated against PLACC as detailed herein below.
  • a bolus of 10 million cells from a PLACC-producing clone is injected subcutaneously into the lower right quadrant of BALB/c mice. For comparison, 10 million non-transfected cells are injected into control mice. The rate and frequency of tumor formation in the two groups is compared to determine efficacy of PLACC in inhibiting tumor growth.
  • recombinant PLACC (20 ng) is mixed with non-transfected J558 cells and injected (20 ng/day) into tumors derived from such cells, to assay the effect of PLACC administered exogenously to tumor cells.
  • the cells which respond to PLACC in vivo may be determined through injection of 1-100 ng of purified PLACC into the intraperitoneal cavity of mice, as described by Luo et al, J. Immunol, 753:4616-4624 (1994).
  • leukocytes are isolated from peripheral blood and from the peritoneal cavity and identified by staining with the Diff Quick kit (Baxter, McGraw, IL). The profile of leukocytes is measured at various times to assess the kinetics of appearance of different cell types.
  • neutralizing antibodies directed against PLACC may be injected along with PLACC to confirm that the infiltration of leukocytes is due to the activity of PLACC.
  • a given chemokine protein product to inhibit HIV proliferation may be measured e.g., by HIV production of p24 protein.
  • Such a test may employ a standard p24 ELIS A assay as previously described in Cocchi et al, Science, 270:1811 (1996). Briefly, normal volunteer human peripheral blood mononuclear cells are isolated on a Ficoll gradient. These cells are activated with 1 ng/ml PHA (Sigma, St. Louis, MO) in RPMI 1640 (Life Technologies, Gaithersburg,
  • TCID 50 5000 of the HIV strains BAL (from ATCC) or A018-H112-2 (from ATCC) for 1 hour in complete media at 37° C. Cells are then washed three times in media to remove excess virus, and resuspended at
  • the assays described above are intended to exemplify the types of assays that may be conducted to determine the in vitro and in vivo effects of PLACC. These are by no means the only assays known to be used for determine chemokine activity. Those of skill in the art will know of other assays that may be substituted for these described above but nonetheless measure similar parameters of function and activity.
  • the present invention also contemplates the use of PLACC and active fragments thereof in the screening of compounds that modulate (increase or decrease activity) of PLACC.
  • PLACC and active fragments thereof in the screening of compounds that modulate (increase or decrease activity) of PLACC.
  • These assays may make use of a variety of different formats and may depend on the kind of "activity" for which the screen is being conducted.
  • Contemplated functional "read-outs" include PLACC binding to a substrate; PLACC binding to a receptor, leukocyte migration assays or any other functional assay normally employed to monitor chemokine activity.
  • Functional assays for chemokines are well known to those of skill in the art and some exemplary assays have been described elsewhere in this document.
  • the present invention provides methods of screening for stimulators of PLACC activity by monitoring PLACC activity in the presence and absence of the candidate substance and comparing such results. It is contemplated that this screening technique will prove useful in the general identification of a compound that will serve the purpose of promoting, augmenting or increasing the therapeutic effects of PLACC.
  • the present invention is directed to a method for dete ⁇ ining the ability of a candidate substance to stimulate the PLACC of cells that either naturally express PLACC or have been engineered to express PLACC as described herein.
  • the method includes generally the steps of:
  • step (iii) comparing the PLACC activity of the composition in step (iii) with the
  • PLACC activity observed in the absence of the candidate substance wherein an alteration in PLACC activity indicates that said candidate substance is a modulator of said activity.
  • a candidate substance as being capable of stimulating PLACC chemokine activity in the assay above, one would measure or determine the activity in the absence of the added candidate substance. One would then add the candidate substance to the cell and determine the activity in the presence of the candidate substance. A candidate substance which increases the activity relative to that observed in its absence is indicative of a candidate substance with stimulatory capability.
  • candidate substance may be an agent that alters the production of PLACC, thereby increasing or decreasing the amount of PLACC present as opposed to the per unit activity of the PLACC.
  • Inhibitors of PLACC activity or production may identified in assays set up in much the same manner as those described above in assays for PLACC stimulators, hi these embodiments, the present invention is directed to a method for determining the ability of a candidate substance to have an inhibitory or even antagonistic effect on PLACC activity.
  • a candidate substance as being capable of inhibiting PLACC activity, one would measure or determine PLACC activity in the absence of the added candidate substance.
  • a candidate substance which is inhibitory would decrease the PLACC activity, relative to the amount of PLACC activity in its absence.
  • the term "candidate substance” refers to any molecule that is capable of modulating chemokine activity.
  • the molecule is one which modulates PLACC activity.
  • the candidate substance may be a protein or fragment thereof, a small molecule inhibitor, or even a nucleic acid molecule. It may prove to be the case that the most useful pharmacological compounds for identification through application of the screening assay will be compounds that are structurally related to other known modulators of chemokine activity.
  • the active compounds may include fragments or parts of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive. However, prior to testing of such compounds in humans or animal models, it will be necessary to test a variety of candidates to determine which have potential.
  • the active compounds may include fragments or parts of naturally-occurring compounds or may be found as active combinations of known compounds which are otherwise inactive. Accordingly, the present invention provides screening assays to identify agents which stimulate or inhibit cellular chemokines. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents.
  • the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds.
  • the candidate substance identified by the present invention may be polypeptide, polynucleotide, small molecule inhibitors or any other inorganic or organic chemical compounds that may be designed through rational drug design starting from known stimulators or inhibitors of chemokines.
  • the candidate screening assays are simple to set up and perform. Thus, in assaying for a candidate substance, after obtaining a cell expressing functional PLACC, one will admix a candidate substance with the cell, under conditions which would allow measurable PLACC activity to occur. In this fashion, one can measure the ability of the candidate substance to stimulate the activity of the cell in the absence of the candidate substance. Likewise, in assays for inhibitors after obtaining a cell expressing functional PLACC, the candidate substance is admixed with the cell. In this fashion the ability of the candidate inhibitory substance to reduce, abolish, or otherwise diminish a biological effect mediated by PLACC from said cell may be detected.
  • Effective amounts in certain circumstances are those amounts effective to reproducibly stimulate a given PLACC mediated event from the cell in comparison to their normal levels. Compounds that achieve significant appropriate changes in activity will be used.
  • PLACC activity or function e.g., as measured using leukocyte migration assays, myeloid, lymphoid or erythroid proliferation assays, HIV proliferation assays, receptor binding, and the like are represented by an increase/decrease in activity of at least about 30%-40%, and most preferably, by changes of at least about 50%, with higher values of course being possible.
  • the active compounds of the present invention also may be used for the generation of antibodies which may then be used in analytical and preparatory techniques for detecting and quantifying further such inhibitors.
  • PLACC polypeptides of the invention are amendable to numerous high throughput screening (HTS) assays known in the art, including melanophore assays to investigate receptor-ligand interactions, yeast based assay systems and mammalian cell expression systems.
  • HTS high throughput screening
  • Combinatorial libraries are composed of large numbers of peptides oligonucleotides or organic compounds as a mixture. They are relatively simple to prepare by traditional automated synthesis methods, PCR cloning or other synthetic methods. Of particular interest will be libraries that include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial and polypeptide libraries. A review of combinatorial libraries and libraries created therefrom, see Myers Curr. Opin. Biotechnol. 8: 701-707 (1997). A candidate modulator identified by the use of various libraries described may then be optimized to modulate activity of PLACC through, for example, rational drug design. It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them.
  • the invention encompasses various binding assays. These can include screening for iiihibitors of PLACC-receptor complexes or for molecules capable of binding to PLACC, as a substitute of the receptor function and thereby altering the binding of the chemokine to its receptor and affecting its activity.
  • PLACC or a fragment thereof may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the polypeptide or the binding agent may be labeled, thereby permitting determination of binding.
  • Such assays are highly amenable to automation and high throughput.
  • High throughput screening of compounds is described in WO 84/03564.
  • Large numbers of 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 PLACC and washed.
  • Bound polypeptide is detected by various methods. Combinatorial methods for generating suitable peptide test compoimds are specifically contemplated.
  • Of particular interest in this format will be the screening of a variety of different PLACC mutants. These mutants, including deletion, truncation, insertion and substitution mutants, will help identify which domains are involved with the PLACC/receptor interaction. Once this region has been determined, it will be possible to identify which of these mutants, which have altered structure but retain some or all of the chemokine related functions of PLACC.
  • Purified PLACC or a binding agent can be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies to the polypeptide can be used to immobilize the polypeptide to a solid phase.
  • fusion proteins containing a reactive region may be used to link the PLACC active region to a solid phase.
  • in vitro assays include those in which functional readouts are taken.
  • cells in which a wild-type or mutant PLACC polypeptide is expressed can be treated with a candidate substance.
  • the substance would be formulated appropriately, given its biochemical nature, and contacted with the cell.
  • culture may be required.
  • the cell may then be examined by virtue of a number .of different physiologic assays, as discussed above.
  • molecular analysis may be performed in which the cells characteristics are examined. This may involve assays such as those for protein expression, enzyme function, substrate utilization, mRNA expression (including differential display of whole cell or polyA RNA) and others.
  • the present invention also encompasses the use of various animal models. Given the disclosure of the present invention, it will be possible to identify non-human counterparts of PLACC. This will afford an excellent opportunity to examine the function of PLACC in a whole animal system where it is normally expressed. By developing or identifying mice with aberrant PLACC functions (overexpression of PLACC, constitutively activated PLACC, PLACC negative), one can provide models that will be highly predictive of disease in humans and other mammals, and helpful in identifying potential therapies.
  • Another form of in vivo model is an animal with a chemokine mediated disorder, e.g., an autoimmune disease, an inflammation and the like. In this model, the model is treated with PLACC in combination with other agents to determine the effect on PLACC function in vivo. Similarly, in tissues exhibiting overexpression of PLACC, it is possible to treat with a candidate substance to determine whether the PLACC activity can be down-regulated in a manner consistent with a therapy.
  • Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal.
  • Administration will be by any route that can be utilized for clinical or non-clinical purposes, including but not limited to oral, nasal, buccal, rectal, vaginal or topical.
  • administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • systemic intravenous injection regional administration via blood, cerebrospinal fluid (CSF) or lymph supply and intratumoral injection.
  • CSF cerebrospinal fluid
  • Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Such criteria include, but are not limited to, survival, reduction of tumor burden or mass, inhibition or prevention of inflammatory response, increased activity level, improvement in immune effector function and improved food intake.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides or compounds with which they interact (agonists, antagonists, inhibitors, peptidomimetics, binding partners, etc.). By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules.
  • drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules.
  • An alternative approach, "alanine scan” involves the random replacement of residues throughout molecule with alanine, and the resulting affect on function determined.
  • Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.
  • Such rational drug design may start with lead compounds identified by the present invention, or may start with a lead compound known to be a modulator of chemokine function.
  • lead compounds identified by the present invention or may start with a lead compound known to be a modulator of chemokine function.
  • the present invention deals with the treatment of disease states that involve activation, recruitment or excessive proliferation of leukocytes, particularly monocytes, macrophages, eosinophils, basophils, mast and T cells.
  • leukocytes particularly monocytes, macrophages, eosinophils, basophils, mast and T cells.
  • Such disease states include inflammatory diseases, autoimmune disorders, allergic responses, and the like.
  • Inflammatory disease states include systemic inflammatory conditions and conditions associated locally with the migration and attraction of monocytes, leukocytes and/or neutrophils. Inhibition of chemotaxis or activity may be useful to ameliorate pathologic inflammatory disease states. Inflammation may result from infection with pathogenic organisms (including Gram positive bacteria, Gram- negative bacteria, viruses, fungi and parasites such as protozoa and helminths) transplant rejection including rejection of solid organs such as kidney liver heart lung or cornea as well as rejection of marrow transplants including graft versus host disease (GVHD) or from localized chronic or acute autoimmune or allergic reactions.
  • pathogenic organisms including Gram positive bacteria, Gram- negative bacteria, viruses, fungi and parasites such as protozoa and helminths
  • transplant rejection including rejection of solid organs such as kidney liver heart lung or cornea as well as rejection of marrow transplants including graft versus host disease (GVHD) or from localized chronic or acute autoimmune or allergic reactions.
  • GVHD graft
  • Autoimmune diseases include acute glomerulonephritis, rheumatoid or reactive arthritis, chronic glomerulonephritis, inflammatory bowel diseases such as Crohn's disease, ulcerative colitis and necrotizing entercolitis, Addison's disease, Grave's disease, granulocyte transfusion associated syndromes, inflammatory dermatoses such as dermatitis, atopic dermatitis, psoriasis, systemic lupus erthyromatosus (SLE), autoimmune thyroiditis, psoriasis, dermatomyositis, polymyositis, osteoarthritis, osteoporosis, atrophic gastritis, myasthenia gravis, multiple sclerosis, some forms of diabetes, pancreatitis or any other autoimmune states where attack by the subject's own system results in pathological tissue destruction.
  • inflammatory bowel diseases such as Crohn's disease, ulcerative colitis and necrotizing entercolitis
  • Allergic reactions include allergic asthma chronic bronchitis, allergic rhinitis, acute and delayed hypersensitivity.
  • Systemic inflammatory disease states include inflammation associated with trauma, burns, reperfusions following ischemic events (e.g. , thrombotic events in heart, brain, intestines or peripheral vasculature, including myocardial infarction and stroke), sepsis, adult respiratory distress syndrome or multiple organ dysfunction syndrome. Inflammatory cell recruitment also occurs in atherosclerotic plaques.
  • chemokine activities include the ability to inhibit the proliferation of bone marrow progenitor cells and essentially arrest bone marrow activity.
  • MJP-l , MIP-2 ⁇ , IL-8, PF4 and MCAF have been shown to suppress myelopoiesis of some stem and progenitor cells. Effect on bone marrow progenitor cells can be assessed by measurement of CFU-GEMM, BFU-E and CFU-GM progenitor cells after treatment with chemokine. Such myelosuppression may protect the stem/progenitor cells against cytotoxic effects during chemotherapy or radiotherapy.
  • Inhibition of hematopoietic or leukocyte proliferation by chemokine administration may also be useful in the treatment of blood cell malignancies including myeloprohferative diseases, such as chronic myelogenous leukemia, essential thrombocytosis, myelofibrosis and polycythemia vera, acute or chronic lymphocytic leukemia, multiple myeloma, Hodgkins disease, and non-Hodgkin's lymphoma.
  • PLACC administration may also reduce the growth of solid tumors/malignancies including breast cancer, ovarian cancer, other estrogen- responsive tissue cancers, non-estrogen responsive cancers, lung cancer, brain cancer, prostate cancer, colon cancer, kidney cancer, stomach cancer, liver cancer or melanoma.
  • PLACC chemokine compositions may also enhance the immune response which may be desirable to e.g., promote wound healing, enhance response to infectious diseases or improve immune response of immunocompromised patients including patients suffering from AIDS, patients undergoing chemotherapy, patients with cancer, or patients with other defects in their immune systems).
  • Specifically contemplated is the treatment of patients recently exposed to HIV, but not yet tested for or confirmed to be HIV-positive by standard diagnostic procedures (e.g., neonates from HIN-positive mothers, medical personnel expositive to HIN positive blood and the like), patients at risk of exposure to HIN or patients already infected with HIN (i.e., HIN positive patients).
  • Additional immunodeficiency diseases include but are not limited to X-linked agammaglobulinemia, ataxia telangiectasia, cirrhosis, cystic fibrosis, diabetes mellitus, hepatitis, and sickle cell diseases.
  • Purified nucleic acid sequences, antisense molecules, P ⁇ As, purified protein, antibodies, antagonists or inhibitors can all be used as pharmaceutical compositions. Delivery of these molecules for therapeutic purposes is further described below. The most appropriate therapy depends on the patient, the specific diagnosis, and the physician who is treating and monitoring the patient's condition.
  • One of the therapeutic embodiments contemplated by the present inventors is intervention, at the molecular level, using the PLACC compositions described herein.
  • the present inventors intend to provide, to a given cell or tissue in patient or subject in need thereof, an expression construct capable of providing PLACC to that cell in a functional form.
  • the genes disclosed herein will be employed in human therapy, as could any of the gene sequence variants discussed above which would encode the same, or a biologically equivalent polypeptide.
  • Particularly preferred expression vectors are viral vectors such as adenovirus, adeno-associated virus, herpesvirus, vaccinia virus and retrovirus. Also preferred is liposomally-encapsulated expression vector.
  • routes are contemplated for delivery.
  • the section below on routes contains an extensive list of possible routes.
  • systemic delivery is contemplated.
  • the tumor may be directly injected with the expression vector.
  • a tumor bed may be treated prior to, during or after resection. Following resection, one generally will deliver the vector by a catheter left in place following surgery.
  • One may utilize the tumor vasculature to introduce the vector into the tumor by injecting a supporting vein or artery.
  • a more distal blood supply route also may be utilized.
  • ex vivo gene therapy is contemplated. This approach is particularly suited, although not limited to, treatment of bone marrow.
  • ABMT Autologous bone marrow transplant
  • the patient will serve as his/her own bone marrow donor.
  • a normally lethal dose of irradiation or chemotherapeutic maybe delivered to the patient to kill tumor cells, and the bone marrow repopulated with the patients own cells that have been maintained (and perhaps expanded) ex vivo. Because, bone marrow often is contaminated with tumor cells, it is desirable to purge the bone marrow of these cells.
  • PLACC may be utilized according to the present invention.
  • PLACC polypeptide active fragments, synthetic peptides, mimetics or other analogs thereof.
  • the protein may be produced by recombinant expression means or, if small enough, generated by an automated peptide synthesizer.
  • Formulations would be selected based on the route of administration and purpose including, but not limited to, liposomal formulations and classic pharmaceutical preparations.
  • the PLACC compositions of the present invention may be used as part of a tumor cell vaccine. Due to their chemo-attractant and/or chemo-stimulatory properties, chemokines have generated interest as tumor vaccines. Tumor vaccines may prove useful in the gene therapy of malignancy for a number of reasons. Only a fraction of tumor cells need to be transduced for a therapeutic effect, as such, plasmid expression vectors may be used, reducing the risk of unwanted retro viral or adeno viral potential side effects. Additionally there are very few side effects, thus this therapy would not preclude other aggressive standard treatment of the disease. Further, this therapy has long lasting effects beyond the initial exposure.
  • the C-C chemokine TCA3/I-309 can induce tumor specific and long lasting immunity in mice that have been immunized with tumor cells expressing TCA3 (Laning et al, J Immunol 153: 4625-4635, 1994.). Such mice completely reject further challenges with live tumor cells.
  • Tumor cells transduced with the C-C chemokine JE/MCP-1 also stimulated a specific immune response that protects immunized rats against further tumor cell challenge (Manome et al. Cancer Immunol Immunother 41 :227-235, 1995.) Lymphotactin when transduced with IL-2 as a T-cell- stimulatory molecule also proved to be an effective tumor vaccine (Dilloo et al.
  • PLACC therapy could be used similarly in conjunction with other agents for inhibiting the proliferation of HIN, other anti-inflammatory agents, or those used in the therapy of the disorders enumerated herein.
  • compositions of the present invention To achieve the appropriate therapeutic outcome, be it a decrease in viral load, a reduction in tumor size or growth, myelosuppression or any other use for the chemokines discussed herein, using the methods and compositions of the present invention, one would generally contact a "target" cell with a PLACC expression construct and at least one other therapeutic agent (second therapeutic agent). These compositions would be provided in a combined amount effective to produce the desired therapeutic outcome.
  • This process may involve contacting the cells with the expression construct and the agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the second therapeutic agent.
  • the PLACC treatment may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the second therapeutic agent and expression construct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and expression construct would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both modalities within about
  • PLACC expression constructs or proteins may be a very efficient method for delivering a therapeutically effective gene to counteract a clinical disease.
  • the second therapeutic agent may be directed to a particular, affected region of the subject's body. Altematively, systemic delivery of expression construct and/or the second therapeutic agent may be appropriate in certain circumstances, for example, where extensive metastasis has occurred.
  • the polynucleotide sequences encoding PLACC may be used for the diagnosis of conditions or diseases with which the expression of PLACC is associated.
  • polynucleotide sequences encoding PLACC may be used in hybridization or PCR assays of fluids or tissues from biopsies to detect PLACC expression.
  • Such methods may be qualitative or quantitative in nature and may include Southern or northern analysis, dot blot or other membrane-based technologies; PCR technologies; dip stick, pin, chip and ELISA technologies. All of these techniques are well known in the art and are the basis of many commercially available diagnostic kits.
  • Such assays may useful in evaluating the efficacy of a particular therapeutic treatment regime in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for PLACC expression needs to be established. This generally involves a combination of body fluids or cell extracts taken from normal subjects, either animal or human, with PLACC, 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 PLACC run in the same experiment where a known amount of purified PLACC is used. Standard values obtained from normal samples maybe compared with values obtained from samples from cachectic subjects affected by PLACC expression. Deviation between standard and subject values establishes the presence of disease.
  • a therapeutic agent is administered; and a treatment profile is generated.
  • Such assays may be repeated on a regular basis to evaluate whether the values in the profile 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 U.S. Patent Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the PLACC sequence.
  • Such ohgomers are generally chemically synthesized, but they may be generated enzymatically or produced from a recombinant source as described herein above.
  • Ohgomers generally comprise two nucleotide sequences, one with sense orientation and one with antisense, employed under optimized conditions for identification of a specific gene or condition.
  • the same two ohgomers, nested sets of ohgomers, or even a degenerate pool of ohgomers maybe employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
  • methods to quantitate the expression of a particular molecule include radiolabeling (Melby et al, J Immunol Methods 159: 235-44, 1993) or biotinylating (Duplaa et al, Anal Biochem 229-36, 1993) nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated. Quantitation of multiple samples may be speeded up by running the assay in an ELIS A format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation. For example, the presence of PLACC in extracts of biopsied tissues may indicate the onset of a particular disease. A definitive diagnosis of this type may allow health professionals to begin aggressive treatment and prevent further worsening of the condition. Similarly, further assays can be used to monitor the progress of a patient during treatment.
  • a cDNA library is prepared, preferably from cells that respond to PLACC by chemotaxis and activation.
  • Radiolabeled PLACC can also be used to identify cell types which express high levels of receptor for PLACC. Cells which do not respond to MIP-1 ⁇ or RANTES, or cells which show a different pattern of receptor desensitization in response to these ligands are of particular interest. Pools of transfected clones in the cDNA library are screened for binding of radiolabeled PLACC by autoradiography. Positive pools are successively subfractionated and rescreened until individual positive clones are obtained.
  • a degenerate PCR strategy may be used in which the sequences of the PCR primers are based on conserved regions of the sequences of known chemokine receptors.
  • the template DNA used in the reaction may be cDNA derived from a cell type responsive to PLACC. These candidate receptor clones can be transduced into 293 cells and the calcium flux is measured to assess PLACC activation of the receptor as described (Myers et al. Biol Chem 270:29671, 1994.).
  • Stably transfected 293 cells are incubated in 1 ml RPMI + 10% FCS containing 1 ⁇ M Fura-2/AM (Molecular Probes) for 30 minutes at room temperature, washed once, and resuspended in PBS at one million/ml. Two ml of cells will be placed in a stirring cuvette at 37°C in a fluorometer. PLACC is added to the labeled cells, and the intracellular free calcium concentration is indicated by fluorescence at 510 nm, when excited by switching 340/380 nm.
  • compositions where clinical applications are contemplated, it will be necessary to prepare the viral expression vectors, antibodies, peptides, nucleic acids and other compositions identified by the present invention as pharmaceutical compositions, i.e., in a form appropriate for in vivo applications. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
  • pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • the active compositions of the present invention include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route.
  • the pharmaceutical compositions may be introduced into the subject by any conventional method, e.g., by intravenous, intradermal, intramusclar, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., term release); by oral, sublingual, nasal, anal, vaginal, or transdermal delivery, or by surgical implantation at a particular site, e.g., embedded under the splenic capsule, brain, or in the cornea.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • the active compounds may be prepared for administration as solutions of free base or pharmacologically acceptable salts in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions, h all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
  • Supplementary active ingredients also can be incorporated into the compositions.
  • the polypeptides of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (DobeiTs Solution).
  • the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups also can be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethyl
  • compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • Unit dose is defined as a discrete amount of a therapeutic composition dispersed in a suitable carrier.
  • PLACC polypeptide compositions are generally injected in doses ranging from l ⁇ g/kg to lOOmg/kg body weight/day, preferably at doses ranging from 0.1 mg/kg to about 50 mg/kg body weight/day.
  • Parenteral administration may be carried out with an initial bolus followed by continuous infusion to maintain therapeutic circulating levels of drag product.
  • the frequency of dosing will depend on the pharmacokinetic parameters of the agents and the routes of administration.
  • the optimal pharmaceutical formulation will be determined by one of skill in the art depending on the route of administration and the desired dosage. See for example Remington's Pharmaceutical Sciences, 18th Ed. (1990, MackPubl. Co, Easton PA 18042) pp 1435-1712, incorporated herein by reference. Such formulations may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the administered agents.
  • a suitable dose may be calculated according to body weight, body surface areas or organ size.
  • the unit dose may be calculated in terms of the dose of viral particles being administered.
  • Viral doses include a particular number of virus particles or plaque forming units (pfu).
  • particular unit doses include 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10", 10 12 , 10 13 or 10 14 pfu.
  • Particle doses maybe somewhat higher (10 to 100-fold) due to the presence of infection defective particles.
  • the present invention is directed at the chemokine mediated treatment of human disorders.
  • a classic and typical therapy will involve direct, injection of a discrete area of inflammation.
  • the discrete tumor mass may be injected.
  • the injections may be single or multiple; where multiple, injections are made at about 1 cm spacings across the accessible surface of the tumor.
  • targeting the tumor vasculature by direct, local or regional intra-arterial injection are contemplated.
  • the viral gene therapy may precede or following resection of the tumor.
  • the gene therapy may, in fact, permit tumor resection where not possible before.
  • a particularly advantageous embodiment involves the prior resection of a tumor (with or without prior viral gene therapy), followed by treatment of the resected tumor bed. This subsequent treatment is effective at eliminating microscopic residual disease which, if left untreated, could result in regrowth of the tumor. This may be accomplished, quite simply, by bathing the tumor bed with a viral preparation containing a unit dose of viral vector.
  • Another preferred method for achieving the subsequent treatment is via catheterization of the resected tumor bed, thereby permitting continuous perfusion of the bed with virus over extended post-operative periods.
  • compositions and treatment methods of the invention may be useful in fields of human medicine and veterinary medicine.
  • the subject to be treated may be a mammal, preferably human or other animal.
  • subjects include for example, farm animals including cows, sheep, pigs, horses and goats, companion animals such as dogs and cats, exotic and/or zoo animals, laboratory animals including mice rats, rabbits, guinea pigs and hamsters; and poultry such as chickens, turkey ducks and geese.
  • transgenic animals are produced which contain a functional transgene encoding wild-type or mutant PLACC polypeptides.
  • Transgenic animals expressing PLACC transgenes, recombinant cell lines derived from such animals and transgenic embryos may be useful in methods for screening for and identifying agents that induce or repress function of PLACC.
  • Transgenic animals of the present invention also can be used as models for studying indications of abnormal PLACC expression.
  • a PLACC transgene is introduced into a non-human host to produce a transgenic animal expressing a human PLACC gene. The transgenic animal is produced by the integration of the transgene into the genome in a manner that permits the expression of the transgene. Methods for producing transgenic animals are generally described by Wagner and Hoppe (U.S.
  • transgenic animals may be produced from the fertilized eggs from a number of animals including, but not limited to reptiles, amphibians, birds, mammals, and fish. Within a particularly preferred embodiment, transgenic mice are generated which overexpress
  • PLACC or express a mutant form of the polypeptide.
  • "knock-out" mice permits the study of the effects that loss of PLACC protein has on a cell in vivo.
  • Knock-out mice also provide a model for the development of PLACC-related abnormalities.
  • transgenic animals and cell lines derived from such animals may find use in certain testing experiments.
  • transgenic animals and cell lines capable of expressing wild-type or mutant PLACC may be exposed to test substances. These test substances can be screened for the ability to enhance wild-type PLACC expression and/or function or impair the expression or function of mutant PLACC .
  • the present Example describes the identification of a putative CC chemokine exon using BlastN searching, the subsequent use of the exon to identify a partial cDNA from placenta and the use of the partial cDNA to amplify the entire coding region of PLACC.
  • CC chemokine ALP Hromas et al. BBRC 258:737, 1999
  • CC chemokine ALP Hromas et al. BBRC 258:737, 1999
  • exons had homology to, but were not identical with, CC chemokines.
  • the exons were contained in Homo sapiens chromosome 5, clone CTD-2202K16 that has a Genbank accession number AC025457. This clone is 140839 bases in length and the potential coding exons were from 26,783 to 27021 (Exon 1), 31934 to 32185 (exon 2) 38483 to 38611 (exon 3).
  • the EST has a Genbank accession number of R38459 and is defined as the yh89dl2.rl Soares placenta Nb2HP Homo sapiens cDNA clone. This EST was obtained and sequenced. The sequencing the 769 nucleotides revealed an extra sequence outside the coding region that was a fourth exon in the above genomic clone. This extra cDNA sequence is included in the PLACC sequence shown in SEQ ID NO:l.
  • PLACC was expressed in the entire Gl tract, trachea, placenta, pancreas, thyroid gland, salivary glands (highest expression), and mammary glands.
  • immunohistology as previously described (Hromas, et al, Blood 82:2998-3004, 1993), PLACC was found to be present in the human colon and induced in the colon of patients who had developed graft versus host disease after allogeneic bone marrow transplantation.
  • PLACC expression was monitored in pancreatic and Gl mucosal cells. Radiolabeled PLACC cRNA was used for in situ immunohistology of formalin-fixed and paraffin-embedded tissue as previously described in Bavisotto et al., ( J. Exp. Med. 174: 1097-1101, 1991). These studies showed that PLACC is highly expressed in these tissues, and will stimulate T-cell chemotaxis into these organs.

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Abstract

L'invention concerne un polynucléotide (PLACC) isolé du placenta qui identifie et code pour une nouvelle chimiokine humaine. L'invention concerne également des compositions et des procédés relatifs à des vecteurs d'expression et des cellules hôtes comprenant la séquence d'acide nucléique codant pour PLACC. En outre, l'invention se rapporte aux utilisations de compositions de peptide PLACC associées dans l'intervention thérapeutique contre des maladies. L'invention concerne, enfin, l'utilisation de compositions PLACC associées dans des applications diagnostiques.
PCT/US2001/016599 2000-05-26 2001-05-23 Placc, nouvelle chimiokine c-c humaine isolee du placenta WO2001092301A2 (fr)

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Publication number Priority date Publication date Assignee Title
WO2019057808A1 (fr) * 2017-09-21 2019-03-28 University Of Copenhagen Peptides potentialisant la chimiotaxie et leurs utilisations

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WO2000038713A1 (fr) * 1998-12-24 2000-07-06 Schering Corporation Agonistes ou antagonistes de la chemokine d'attraction des lymphocytes t (ctack) ou des chemokines des contracteurs intestinaux vasoactifs (vic)
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WO1999006439A2 (fr) * 1997-08-01 1999-02-11 Genset Est 5' pour proteines secretees exprimees dans l'endoderme
WO1999036540A1 (fr) * 1998-01-20 1999-07-22 Codon Diagnostics, Llc Chemokine humaine et son utilisation dans le traitement d'une pathologie mammaire
WO2000024774A2 (fr) * 1998-10-28 2000-05-04 Incyte Pharmaceuticals, Inc. Molecules apparentees au facteur de croissance
WO2000038713A1 (fr) * 1998-12-24 2000-07-06 Schering Corporation Agonistes ou antagonistes de la chemokine d'attraction des lymphocytes t (ctack) ou des chemokines des contracteurs intestinaux vasoactifs (vic)
WO2001007482A1 (fr) * 1999-07-27 2001-02-01 Smithkline Beecham Corporation Gpr27, un recepteur couple a la proteine g
WO2001053455A2 (fr) * 1999-12-23 2001-07-26 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides associes

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019057808A1 (fr) * 2017-09-21 2019-03-28 University Of Copenhagen Peptides potentialisant la chimiotaxie et leurs utilisations

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