EP1230370A2 - Polypeptide du type cytokine regule par la proteine wnt et acides nucleiques codant ce polypeptide - Google Patents

Polypeptide du type cytokine regule par la proteine wnt et acides nucleiques codant ce polypeptide

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Publication number
EP1230370A2
EP1230370A2 EP00978769A EP00978769A EP1230370A2 EP 1230370 A2 EP1230370 A2 EP 1230370A2 EP 00978769 A EP00978769 A EP 00978769A EP 00978769 A EP00978769 A EP 00978769A EP 1230370 A2 EP1230370 A2 EP 1230370A2
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EP
European Patent Office
Prior art keywords
fctrx
polypeptide
nucleic acid
protein
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00978769A
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German (de)
English (en)
Inventor
Luca Rastelli
David Lewin
Bruce Taillon
David P. Andrew
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Genentech Inc
CuraGen Corp
Original Assignee
CuraGen Corp
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Filing date
Publication date
Application filed by CuraGen Corp filed Critical CuraGen Corp
Publication of EP1230370A2 publication Critical patent/EP1230370A2/fr
Withdrawn legal-status Critical Current

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is based in part upon the discovery of a nucleic acid sequence encoding a novel member of the Wnt signaling pathway.
  • the novel member encodes a S100 cytokine-like polypeptide.
  • the S100 cytokine-like nucleic acids and polypeptides encoded thereby are collectively referred to herein as "FCTRX".
  • Figure 1 is a ClustalW alignment of the human polypeptide of SEQ ID NO:6 with known S100 cytokine proteins.
  • Figure 7 is a CAP alignment and consensus of human assembly 65677221-3-frag (SEQ ID NO:5) compared to human EST AA315020 (SEQ ID NO:4).
  • the murine sequence shown in Table 2 includes an open reading frame ("ORF") from nucleotide 73 to nucleotide 465. The beginning of this ORF is indicated in bold in Table 2. The polypeptide encoded by this ORF is shown in Table 3 (SEQ ID NO:3).
  • the murine sequence shown in Table 1 is related to human EST AA315020. This EST originates from human cells forming a metastatic tumor when implanted in mice. The sequence of this EST is shown in Table 4 (SEQ ID NO:4).
  • MRP- 14 Macrophage Migration Inhibition Factor
  • the serum concentration of a S100 cytokine according to the invention can be used as a marker for the clinical predictive value for metastatic tumors.
  • a S100 cytokine according to the invention e.g., a polypeptide having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:6
  • the mouse gene fragment disclosed in Table 1 (SEQ ID NO:l) was originally identified in the tumors of Wnt-1 transgenic mice.
  • two of the human fragments used to produce the sequence shown in Table 5 (SEQ ID NO:5) were isolated from tumors.
  • the sequence shown in Table 5 (SEQ ID NO: 5) is highly expressed in several tumor cell lines, especially in colon cancer, breast cancer and ovarian cancer.
  • the nucleic acids and their gene products identified herein are useful as diagnostic markers for such cancers, and as targets for the treatment of cancer in a subject.
  • the targeting may especially be carried out by generating antibodies that specifically bind a protein encoded by SEQ ID NO: 5, and administering such antibodies to a subject suffering from a cancer such as colon cancer, breast cancer or ovarian cancer. These antibodies will likewise serve as diagnostic tools useful in detecting the presence or amount of the proteins disclosed herein.
  • the FCTRX polypeptides are homologous to members of the S100 cytokine family.
  • the S100 group of proteins are calcium-binding molecules with cytokine and chemokine activity.
  • the murine protein S100A8 is a potent chemoattractant for neutrophils and monocytes in vivo and in vitro (Xu K and Geczy CL, J Immunol 2000 May l;164(9):4916-23).
  • S 100B and S 100A1 activate RAGE (the receptor for an advanced glycation end product (AGE)) occurring in diabetes in concert with amphoterin. This interaction is reported to induce neurite outgrowth and activation of transcription factor NF-kappaB.
  • Amphoterin is a protein that enhances process extension and migration in embryonic neurons and in tumor cells through binding to RAGE, a multiligand transmembrane receptor.
  • activation of RAGE by amphoterin and S100B promotes cell survival through increased expression of the anti-apoptotic protein Bcl-2. Higher concentrations of S100B induce apoptosis in an oxidant-dependent manner.
  • mice overexpress a human beta- amyloid precursor protein (betaAPP) minigene encoding a familial Alzheimer's disease mutation. These mice develop Alzheimer- type neuritic beta-amyloid plaques surrounded by astrocytes. SlOObeta is an astrocyte-derived cytokine that promotes neurite growth and promotes excessive expression of betaAPP. SlOObeta overexpression in Alzheimer's disease correlates with the proliferation of betaAPP- immunoreactive neurites in beta-amyloid plaques.
  • betaAPP beta- amyloid precursor protein
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • an “isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • a "mature" form of a polypeptide or protein is the product of a naturally occurring polypeptide or precursor form or FCTRX-protein.
  • the naturally occurring polypeptide, precursor or FCTRX-protein includes, by way of non-limiting example, the full length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or FCTRX-protein encoded by an open reading frame described herein.
  • the product "mature" form arises, again by way of non-limiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises.
  • Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N- terminal methionine residue encoded by the initiation codon of an open reading frame, or the proteolytic cleavage of a signal peptide or leader sequence.
  • a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine would have residues 2 through N remaining after removal of the N-terminal methionine.
  • a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining.
  • a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristylation, or phosphorylation.
  • a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO:l, 2, 4, or 5 .
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO: 1, 2, 4, or 5 , or a portion of this nucleotide sequence.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID NO: 1 , 2, 4, or 5 or an anti-sense strand nucleotide sequence of SEQ ID NO:l, 2, 4, or 5 or of a naturally occurring mutant of one or more of these sequences.
  • FCTRX DNA sequence polymorphisms that lead to changes in the amino acid sequences of FCTRX may exist within a population (e.g., the human population).
  • Such genetic polymorphism in the FCTRX gene may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and "recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding an FCTRX protein, preferably a mammalian FCTRX protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the FCTRX gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in FCTRX that are the result of natural allelic variation and that do not alter the functional activity of FCTRX are intended to be within the scope of the invention.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • Homologs i.e., nucleic acids encoding FCTRX proteins derived from species other than human
  • other related sequences e.g., paralogs
  • hybridizes under stringent conditions can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
  • nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 2, 4, or 5 or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of FCTRX without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the FCTRX proteins of the present invention are predicted to be particularly unamenable to alteration.
  • FCTRX proteins that contain changes in amino acid residues that are not essential for activity. Such FCTRX proteins differ in amino acid sequence from SEQ ID NO:3 or 6, yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:3 or 6.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding FCTRX.
  • coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding FCTRX.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • PNAs of FCTRX can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs of FCTRX can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., Sl nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).
  • PNAs of FCTRX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of FCTRX can be generated that may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • the fusion protein is an FCTRX protein containing a heterologous signal sequence at its N-terminus.
  • the native FCTRX signal sequence can be removed and replaced with a signal sequence from another protein.
  • expression and/or secretion of FCTRX can be increased through use of a heterologous signal sequence.
  • the fusion protein is an FCTRX-immunoglobulin fusion protein in which the FCTRX are fused to sequences derived from a member of the immunoglobulin protein family.
  • the FCTRX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an FCTRX ligand and an FCTRX protein on the surface of a cell, to thereby suppress FCTRX-mediated signal transduction in vivo.
  • the FCTRX-immunoglobulin fusion proteins can be used to affect the bioavailability of an FCTRX cognate ligand.
  • FCTRX-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-FCTRX antibodies in a subject, to purify FCTRX ligands, and in screening assays to identify molecules that inhibit the interaction of FCTRX with an FCTRX ligand.
  • An FCTRX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g. , by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the FCTRX proteins.
  • Variants of the FCTRX protein that function as either FCTRX agonists (mimetics) or as FCTRX antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the FCTRX protein for FCTRX protein agonist or antagonist activity.
  • a variegated library of FCTRX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential FCTRX sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.
  • libraries of fragments of the FCTRX protein coding sequence can be used to generate a variegated population of FCTRX fragments for screening and subsequent selection of variants of an FCTRX protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an FCTRX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the FCTRX protein.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of FCTRX.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular FCTRX protein with which it immunoreacts.
  • any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized.
  • techniques can be adapted for the production of single-chain antibodies specific to an FCTRX protein (see e.g., U.S. Patent No. 4,946,778).
  • methodologies can be adapted for the construction of F ab expression libraries (see e.g., Huse, et al, 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F ab fragments with the desired specificity for an FCTRX protein or derivatives, fragments, analogs or homologs thereof.
  • Non-human antibodies can be "humanized” by techniques well known in the art. See e.g., U.S. Patent No. 5,225,539.
  • recombinant anti-FCTRX antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application No.
  • methodologies for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art.
  • ELISA enzyme-linked immunosorbent assay
  • Anti-FCTRX antibodies may be used in methods known within the art relating to the localization and/or quantitation of an FCTRX protein (e.g., for use in measuring levels of the FCTRX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • FCTRX proteins or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds [hereinafter "Therapeutics"].
  • An anti-FCTRX antibody e.g., monoclonal antibody
  • FCTRX can be used to isolate FCTRX by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-FCTRX antibody can facilitate the purification of natural FCTRX from cells and of recombinantly produced FCTRX expressed in host cells. Moreover, an anti-FCTRX antibody can be used to detect FCTRX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the FCTRX protein. Anti-FCTRX antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • vectors preferably expression vectors, containing a nucleic acid encoding FCTRX protein, or derivatives, fragments, analogs or homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector is a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Plasmid which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • a viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. , bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • expression vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al.
  • mammary gland-specific promoters e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166.
  • Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev 3:537-546).
  • a host cell can be any prokaryotic or eukaryotic cell.
  • FCTRX protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) FCTRX protein.
  • the invention further provides methods for producing FCTRX protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding FCTRX has been introduced) in a suitable medium such that FCTRX protein is produced.
  • the method further comprises isolating FCTRX from the medium or the host cell.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • the vector can be designed such that, upon homologous recombination, the endogenous FCTRX gene is mutated or otherwise altered but still encodes functional protein (e.g. , the upstream regulatory region can be altered to thereby alter the expression of the endogenous FCTRX protein).
  • the altered portion of the FCTRX gene is flanked at its 5' and 3' ends by additional nucleic acid of the FCTRX gene to allow for homologous recombination to occur between the exogenous FCTRX gene carried by the vector and an endogenous FCTRX gene in an embryonic stem cell.
  • the additional flanking FCTRX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • FCTRX nucleic acid molecules FCTRX proteins, and anti-FCTRX antibodies
  • compositions suitable for administration typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. 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 active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, infradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • 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), and suitable mixtures thereof.
  • 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.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) PNAS 91 :3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • This invention further pertains to novel agents identified by the above described screening assays and uses thereof for treatments as described herein. Screening Assays
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to FCTRX proteins or have a stimulatory or inhibitory effect on, for example, FCTRX expression or FCTRX activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to FCTRX proteins or have a stimulatory or inhibitory effect on, for example, FCTRX expression or FCTRX activity.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a membrane-bound form of an FCTRX protein or polypeptide or biologically active portion thereof.
  • test compounds of the present invention can be obta4ined using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • biological libraries are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des 12: 145).
  • Determining the ability of the test compound to bind to the FCTRX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the FCTRX protein or biologically active portion thereof can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 125 1, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
  • an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of FCTRX protein, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the FCTRX protein or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of FCTRX or a biologically active portion thereof can be accomplished, for example, by determining the ability of the FCTRX protein to bind to or interact with an FCTRX target molecule.
  • a "target molecule” is a molecule with which an FCTRX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an FCTRX protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • An FCTRX target molecule can be a non-FCTRX molecule or an FCTRX protein or polypeptide of the present invention.
  • an FCTRX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g.
  • the target for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with FCTRX.
  • Determining the ability of the FCTRX protein to bind to or interact with an FCTRX target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the FCTRX protein to bind to or interact with an FCTRX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e.
  • Binding of the test compound to the FCTRX protein can be determined either directly or indirectly as described above.
  • the assay comprises contacting the FCTRX protein or biologically active portion thereof with a known compound which binds FCTRX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an FCTRX protein, wherein determining the ability of the test compound to interact with an FCTRX protein comprises determining the ability of the test compound to preferentially bind to FCTRX or biologically active portion thereof as compared to the known compound.
  • an assay is a cell-free assay comprising contacting FCTRX protein or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the FCTRX protein or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of FCTRX can be accomplished, for example, by determining the ability of the FCTRX protein to bind to an FCTRX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of FCTRX can be accomplished by determining the ability of the FCTRX protein further modulate an FCTRX target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.
  • the cell-free assay comprises contacting the FCTRX protein or biologically active portion thereof with a known compound which binds FCTRX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an FCTRX protein, wherein determining the ability of the test compound to interact with an FCTRX protein comprises determining the ability of the FCTRX protein to preferentially bind to or modulate the activity of an FCTRX target molecule.
  • the cell-free assays of the present invention are amenable to use of both the soluble form or the membrane-bound form of FCTRX.
  • solubihzing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton ® X-100, Triton ® X-l 14, Thesit ® , Isotridecypoly(ethylene glycol ether) n , 3-(3-cholamidopropyl)dimethylamminiol-l-propane sulfonate (CHAPS), 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-l-propane sulfonate (CHAPSO), or N-dodecyl ⁇ N,N-dimethyl-3-ammonio-l-propane sulfonate.
  • non-ionic detergents such as n-octy
  • FCTRX FCTRX
  • its target molecule it may be desirable to immobilize either FCTRX or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.
  • Binding of a test compound to FCTRX, or interaction of FCTRX with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix.
  • GST-FCTRX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or FCTRX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
  • glutathione sepharose beads Sigma Chemical, St. Louis, MO
  • glutathione derivatized microtiter plates glutathione derivatized microtiter plates
  • the complexes can be dissociated from the matrix, and the level of FCTRX binding or activity determined using standard techniques.
  • Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention.
  • either FCTRX or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated FCTRX or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • modulators of FCTRX expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of FCTRX mRNA or protein in the cell is determined.
  • the level of expression of FCTRX mRNA or protein in the presence of the candidate compound is compared to the level of expression of FCTRX mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of FCTRX expression based on this comparison. For example, when expression of FCTRX mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of FCTRX mRNA or protein expression.
  • FCTRX mRNA or protein when expression of FCTRX mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of FCTRX mRNA or protein expression.
  • the level of FCTRX mRNA or protein expression in the cells can be determined by methods described herein for detecting FCTRX mRNA or protein.
  • the FCTRX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
  • the gene that codes for FCTRX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait” and the “prey” proteins are able to interact, in vivo, forming a FCTRX-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close FCTRXimity.
  • This FCTRXimity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with FCTRX.
  • a reporter gene e.g., LacZ
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the FCTRX, sequences, described herein, can be used to map the location of the FCTRX genes, respectively, on a chromosome. The mapping of the FCTRX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • human and mouse cells As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the FCTRX sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle.
  • FISH Fluorescence in situ hybridization
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the D ⁇ A sequences between individuals affected and unaffected with a disease associated with the FCTRX gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that D ⁇ A sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • sequences of the present invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the FCTRX sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the FCTRX sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).
  • SNPs single nucleotide polymorphisms
  • RFLPs restriction fragment length polymorphisms
  • each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. For example, the noncoding sequences of SEQ ID NO:l, 2, 4, or 5 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as the nucleic acid seqences that code for the amino acid sequences of SEQ ID NO:3 or 6 are used, a more appropriate number of primers for positive individual identification would be 500-2,000. Predictive Medicine
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with FCTRX protein, nucleic acid expression or activity. For example, mutations in an FCTRX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with FCTRX protein, nucleic acid expression or activity.
  • Another aspect of the invention provides methods for determining FCTRX protein, nucleic acid expression or FCTRX activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics").
  • Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent).
  • Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of FCTRX in clinical trials.
  • agents e.g., drugs, compounds
  • the nucleic acid probe can be, for example, a full-length FCTRX nucleic acid, such as the nucleic acid of SEQ ID NO:l, 2, 4, or 5 , or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to FCTRX mRNA or genomic DNA.
  • FCTRX nucleic acid such as the nucleic acid of SEQ ID NO:l, 2, 4, or 5
  • a portion thereof such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to FCTRX mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein.
  • An agent for detecting FCTRX protein is an antibody capable of binding to FCTRX protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab') ) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect FCTRX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of FCTRX mRNA include Northern hybridizations and in situ hybridizations.
  • FCTRX protein In vitro techniques for detection of FCTRX protein include enzyme linked immunosorbent assays (ELIS As), Western blots, immunoprecipitations and immuno fluorescence.
  • In vitro techniques for detection of FCTRX genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of FCTRX protein include introducing into a subject a labeled anti-FCTRX antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • kits for detecting the presence of FCTRX in a biological sample can comprise: a labeled compound or agent capable of detecting FCTRX protein or mRNA in a biological sample; means for determining the amount of FCTRX in the sample; and means for comparing the amount of FCTRX in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect FCTRX protein or nucleic acid.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant FCTRX expression or activity.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with FCTRX protein, nucleic acid expression or activity such as cancer or fibrotic disorders.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant FCTRX expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder, such as cancer or preclampsia.
  • such genetic lesions can be detected by ascertaining the existence of at least one of (1) a deletion of one or more nucleotides from an FCTRX gene; (2) an addition of one or more nucleotides to an FCTRX gene; (3) a substitution of one or more nucleotides of an FCTRX gene, (4) a chromosomal rearrangement of an FCTRX gene; (5) an alteration in the level of a messenger RNA transcript of an FCTRX gene, (6) aberrant modification of an FCTRX gene, such as of the methylation pattern of the genomic DNA, (7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of an FCTRX gene, (8) a non- wild type level of an FCTRX-protein, (9) allelic loss of an FCTRX gene, and (10) inappropriate post-translational modification of an FCTRX-protein.
  • a preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
  • any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
  • detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran,et al. (1988) Science 241 :1077-1080; and Nakazawa et al. (1994) PNAS
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an FCTRX gene under conditions such that hybridization and amplification of the FCTRX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic, mRNA or both
  • mutations in an FCTRX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicate mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Pat. No. 5,493,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in FCTRX can be identified by hybridizing a sample and control nucleic acids, e.g. , DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al. (1996) Human Mutation 7:
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the FCTRX gene and detect mutations by comparing the sequence of the sample FCTRX with the corresponding wild-type (control) sequence.
  • sequencing reactions include those based on techniques developed by Maxim and Gilbert (1977) PNAS 74:560 or Sanger (1977) PNAS 74:5463.
  • any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve et al, (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publ. No. WO 94/16101; Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38:147-159).
  • FCTRX gene Other methods for detecting mutations in the FCTRX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA DNA heteroduplexes (Myers et al. (1985) Science 230:1242).
  • the art technique of "mismatch cleavage” starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type FCTRX sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • alterations in electrophoretic mobility will be used to identify mutations in FCTRX genes.
  • single strand conformation polymorphism SSCP
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA fragments of sample and control FCTRX nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of apFCTRXimately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc Natl Acad Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an FCTRX gene.
  • any cell type or tissue preferably peripheral blood leukocytes, in which FCTRX is expressed may be utilized in the prognostic assays described herein.
  • any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
  • Agents, or modulators that have a stimulatory or inhibitory effect on FCTRX activity can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., cancer or immune disorders, neurological disorders, muscular dystrophy, or epidermolysis bullosa simplex) associated with aberrant FCTRX activity.
  • disorders e.g., cancer or immune disorders, neurological disorders, muscular dystrophy, or epidermolysis bullosa simplex
  • the pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of FCTRX protein, expression of FCTRX nucleic acid, or mutation content of FCTRX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons.
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations.
  • the gene coding for CYP2D6 is highly polymo ⁇ hic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme is the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • FCTRX protein activity of FCTRX protein, expression of FCTRX nucleic acid, or mutation content of FCTRX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • pharmacogenetic studies can be used to apply genotyping of polymo ⁇ hic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an FCTRX modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • FCTRX e.g., the ability to modulate aberrant cell proliferation and/or differentiation
  • agents e.g., drugs, compounds
  • FCTRX e.g., the ability to modulate aberrant cell proliferation and/or differentiation
  • the effectiveness of an agent determined by a screening assay as described herein to increase FCTRX gene expression, protein levels, or upregulate FCTRX activity can be monitored in clinical trails of subjects exhibiting decreased FCTRX gene expression, protein levels, or downregulated FCTRX activity.
  • genes, including FCTRX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates FCTRX activity can be identified.
  • an agent e.g., compound, drug or small molecule
  • FCTRX activity e.g. , identified in a screening assay as described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of FCTRX and other genes implicated in the disorder.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of FCTRX or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (/) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an FCTRX protein, mRNA, or genomic DNA in the preadministration sample; (Hi) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the FCTRX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the FCTRX protein, mRNA, or genomic DNA in the pre-administration sample with the FCTRX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent e.g.,
  • increased administration of the agent may be desirable to increase the expression or activity of FCTRX to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of FCTRX to lower levels than detected, i.e., to decrease the effectiveness of the agent.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant FCTRX expression or activity.
  • Therapeutics that antagonize may be administered in a therapeutic or prophylactic manner.
  • Therapeutics that may be utilized include, but are not limited to, (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989.
  • modulators i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention
  • modulators i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention
  • RNA by obtaining a patient tissue sample (e.g. , from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and or activity of the expressed peptides (or mRNAs of an aforementioned peptide).
  • Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • immunoassays e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.
  • hybridization assays to detect expression of m
  • the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant FCTRX expression or activity, by administering to the subject an agent that modulates FCTRX expression or at least one FCTRX activity.
  • Subjects at risk for a disease that is caused or contributed to by aberrant FCTRX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the FCTRX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • FCTRX agonist or FCTRX antagonist agent can be used for treating the subject.
  • the appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the present invention are further discussed in the following subsections.
  • the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of FCTRX protein activity associated with the cell.
  • An agent that modulates FCTRX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of an FCTRX protein, a peptide, an FCTRX peptidomimetic, or other small molecule.
  • the agent stimulates one or more FCTRX protein activity. Examples of such stimulatory agents include active FCTRX protein and a nucleic acid molecule encoding FCTRX that has been introduced into the cell.
  • the agent inhibits one or more FCTRX protein activity.
  • inhibitory agents include antisense FCTRX nucleic acid molecules and anti-FCTRX antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an FCTRX protein or nucleic acid molecule.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) FCTRX expression or activity.
  • the method involves administering an FCTRX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant FCTRX expression or activity. Stimulation of FCTRX activity is desirable in situations in which FCTRX is abnormally downregulated and/or in which increased FCTRX activity is likely to have a beneficial effect.
  • a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer).
  • Another example of such a situation is where the subject has a gestational disease (e.g., preclampsia).
  • suitable in vitro or in vivo assays are utilized to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.
  • in vitro assays may be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s).
  • Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects.
  • suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects.
  • any of the animal model systems known in the art may be used prior to administration to human subjects. Malignancies
  • Therapeutics of the present invention are useful in the therapeutic or prophylactic treatment of diseases or disorders that arc associated with cell hypcrproli fetation and/oi loss of control of cell proliferation (e.g., cancers, malignancies and tumors).
  • diseases or disorders that arc associated with cell hypcrproli fetation and/oi loss of control of cell proliferation (e.g., cancers, malignancies and tumors).
  • hypcrprolifcration disorders sec e.g., Fishman, el al, 1985. Mi.[)iciNi>, 2nd cd.,
  • Therapeutics of the present invention may be assayed by any method known within the art for efficacy in treating or preventing malignancies and related disorders.
  • assays include, but arc not limited to, in vitro assays utili/ing transformed cells or cells derived from the patient's tumor, as well as in vivo assays using animal models of cancer or malignancies.
  • Potentially effective Therapeutics are those that, for example, inhibit the proliferation of tumor-derived or transformed cells in culture or cause a regression of tumors in animal models, in comparison to the controls.
  • a malignancy or cancer has been shown to be amenable to treatment by modulating (i.e., inhibiting, antagonizing or agonizing) activity, that cancer or malignancy may subsequently be treated or prevented by the administration of a
  • Hypcrplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in its structure or function. For example, it has been demonstrated that endomctrial hypcrplasia often precedes cndomctrial cancer.
  • Metaplasia is a form of controlled cell growth in which one type of mature or fully differentiated cell substitutes for another type of mature cell. Metaplasia may occur in epithelial or connective tissue cells.
  • Dysplasia is generally considered a precursor of cancer, and is found mainly in the epithclia. Dysplasia is the most disorderly form of non-ncoplastic cell growth, and involves a loss in individual cell uniformity and in the architectural orientation of cells.
  • a patient that exhibits one or more of the following predisposing factors for malignancy is treated by administration of an effective amount of a Therapeutic: (/) a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome (bcr/abl) for chronic myelogenous leukemia and t(14;18) for follicular lymphoma, etc.); (ii) familial polyposis or Gardner's syndrome (possible forerunners of colon cancer); (Hi) monoclonal gammopathy of undetermined significance (a possible precursor of multiple myeloma) and (iv) a first degree kinship with persons having a cancer or pre-cancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, Peutz-Jeghers syndrome, neurona chromosomal translocation
  • a Therapeutic is administered in the therapeutic or prophylactic treatment of hype ⁇ roliferative or benign dysproliferative disorders.
  • the efficacy in treating or preventing hype ⁇ roliferative diseases or disorders of a Therapeutic of the present invention may be assayed by any method known within the art.
  • Such assays include in vitro cell proliferation assays, in vitro or in vivo assays using animal models of hype ⁇ roliferative diseases or disorders, or the like.
  • Potentially effective Therapeutics may, for example, promote cell proliferation in culture or cause growth or cell proliferation in animal models in comparison to controls.
  • Specific embodiments of the present invention are directed to the treatment or prevention of cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes); treatment of keloid (hypertrophic scar) formation causing disfiguring of the skin in which the scarring process interferes with normal renewal; psoriasis (a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination); benign tumors; fibrocystic conditions and tissue hypertrophy (e.g., benign prostatic hypertrophy).
  • FCTRX has been implicated in the deregulation of cellular maturation and apoptosis, which are both characteristic of neurodegenerative disease. Accordingly, Therapeutics of the invention, particularly but not limited to those that modulate (or supply) activity of an aforementioned protein, may be effective in treating or preventing neurodegenerative disease. Therapeutics of the present invention that modulate the activity of an aforementioned protein involved in neurodegenerative disorders can be assayed by any method known in the art for efficacy in treating or preventing such neurodegenerative diseases and disorders. Such assays include in vitro assays for regulated cell maturation or inhibition of apoptosis or in vivo assays using animal models of neurodegenerative diseases or disorders, or any of the assays described below.
  • Therapeutics for example but not by way of limitation, promote regulated cell maturation and prevent cell apoptosis in culture, or reduce neurodegeneration in animal models in comparison to controls.
  • a neurodegenerative disease or disorder Once a neurodegenerative disease or disorder has been shown to be amenable to treatment by modulation activity, that neurodegenerative disease or disorder can be treated or prevented by administration of a Therapeutic that modulates activity.
  • Such diseases include all degenerative disorders involved with aging, especially osteoarthritis and neurodegenerative disorders. Disorders related to organ transplantation
  • FCTRX has been implicated in disorders related to organ transplantation, in particular but not limited to organ rejection.
  • Therapeutics of the invention particularly those that modulate (or supply) activity, may be effective in treating or preventing diseases or disorders related to organ transplantation.
  • Therapeutics of the invention (particularly Therapeutics that modulate the levels or activity of an aforementioned protein) can be assayed by any method known in the art for efficacy in treating or preventing such diseases and disorders related to organ transplantation.
  • Such assays include in vitro assays for using cell culture models as described below, or in vivo assays using animal models of diseases and disorders related to organ transplantation, see e.g., below.
  • Potentially effective Therapeutics for example but not by way of limitation, reduce immune rejection responses in animal models in comparison to controls.
  • diseases and disorders related to organ transplantation are shown to be amenable to treatment by modulation of activity, such diseases or disorders can be treated or prevented by administration of a Therapeutic that modulates activity.
  • a limited and non-exclusive list of animal models includes knockout mice for premature atherosclerosis (Kurabayashi and Yazaki, 1996, Int. Angiol. 15: 187-194), transgenic mouse models of atherosclerosis (Kappel et al, 1994, FASEB J. 8: 583-592), antisense oligonucleotide treatment of animal models (Callow, 1995, Curr. Opin. Cardiol.
  • in vitro cell models include but are not limited to monocytes exposed to low density lipoprotein (Frostegard et al, 1996, Atherosclerosis 121 : 93-103), cloned vascular smooth muscle cells (Suttles et al, 1995, Exp. Cell Res. 218: 331-338), endothelial cell-derived chemoattractant exposed T cells (Katz et al, 1994, J. Leukoc. Biol.
  • an atherosclerosis-associated disease or disorder has been shown to be amenable to treatment by modulation of activity or formation, that disease or disorder can be treated or prevented by administration of a Therapeutic that modulates activity.
  • the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
  • Assays for T-cell or thymocyte proliferation include without limitation those described in: CURRENT PROTOCOLS IN IMMUNOLOGY, Ed by Coligan et al, Greene Publishing Associates and Wiley-Interscience (Chapter 3 and Chapter 7); Takai et al, J Immunol 137:3494-3500, 1986; Bertagnoili et al., J Immunol 145:1706-1712, 1990; Bertagnolli et al, Cell Immunol 133:327-341, 1991; Bertagnoili, et al, J Immunol 149:3778-3783, 1992; Bowman et al, J Immunol 152:1756-1761, 1994.
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described by Bottomly et al, In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al, eds., Vol 1, pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto 1991; deVries et al, JExp Med 173:1205-1211, 1991; Moreau et al, Nature 336:690-692, 1988; Greenberger et al, Proc Natl Acad Sci U.S.A. 80:2931-2938, 1983; Nordan, In: CURRENT PROTOCOLS IN IMMUNOLOGY.
  • Coligan et al Coligan et al, eds., Vol 1, pp. 6.6.1-5, John Wiley and Sons, Toronto 1991; Smith et al, Proc Natl Acad Sci U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11 -Bennett, et al. In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al, eds., Vol 1, pp. 6.15.1 John Wiley and Sons, Toronto 1991; Ciarletta, et al, In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al, eds., Vol 1, pp. 6.13.1, John Wiley and Sons, Toronto 1991.
  • Assays for T-cell clone responses to antigens include, without limitation, those described In: CURRENT PROTOCOLS IN IMMUNOLOGY.
  • An FCTRX protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a protein are useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SCID severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.
  • infectious diseases caused by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, he ⁇ esviruses, mycobacteria, Leishmania species, malaria species, and various fungal infections such as candidiasis.
  • a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitus, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
  • a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems.
  • Other conditions, in which immune suppression is desired may also be treatable using a protein of the present invention.
  • Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.
  • the functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
  • Tolerance which involves inducing non-responsiveness or energy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon re-exposure to specific antigen in the absence of the tolerizing agent.
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody
  • B7 lymphocyte antigen e.g., B7-1, B7-3 or blocking antibody
  • Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
  • the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al, Science 257:789-792 (1992) and Turka et al, Proc Natl Acad Sci USA, 89:11102-11105 (1992).
  • murine models of GVHD can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
  • Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and auto- antibodies involved in the pathology of the diseases.
  • Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • Administration of reagents which block costimulation of T cells by disrupting receptor: ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of auto-antibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythematosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., FUNDAMENTAL IMMUNOLOGY, Raven Press, New York, 1989, pp. 840-856).
  • Upregulation of an antigen function may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function are useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
  • anti- viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
  • up regulation or enhancement of antigen function are useful in the induction of tumor immunity.
  • Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
  • a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject.
  • the tumor cell can be transfected to express a combination of peptides.
  • tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and/or B7-3-like activity.
  • the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I a chain protein and b 2 microglobulin protein or an MHC class II a chain protein and an MHC class II b chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I a chain protein and b 2 microglobulin protein or an MHC class II a chain protein and an MHC class II b chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al, eds.
  • T-cell-dependent immunoglobulin responses and isotype switching include, without limitation, those described in: Maliszewski, J Immunol 144:3028-3033, 1990; and Mond and Brunswick In: CURRENT PROTOCOLS IN IMMUNOLOGY. Coligan et al, eds., Vol 1, pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto 1994.
  • lymphocyte survival/apoptosis which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis
  • assays for lymphocyte survival/apoptosis include, without limitation, those described in: Darzynkiewicz et al, Cytometry
  • Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al, Blood 84:111-117, 1994; Fine et al, Cell Immunol 155: 111-122, 1994; Galy et al, Blood 85:2770-2778, 1995; Toki et al,
  • FCTRX protein of the present invention are useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • Assays for proliferation and differentiation of various hematopoietic lines are cited above.
  • Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al, Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al, Blood 81:2903-2915, 1993.
  • Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G.
  • a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone- forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
  • a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
  • tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation.
  • a protein of the present invention which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.
  • Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
  • compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.
  • the compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament- forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions of the invention may also be useful in the treatment of tendonitis, ca ⁇ al tunnel syndrome and other tendon or ligament defects.
  • the compositions may also include an appropriate matrix and/or sequestering agent as a career as is well known in the art.
  • Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
  • a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothehum), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothehum) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate.
  • a protein of the invention may also exhibit angiogenic activity.
  • a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
  • a protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothehum).
  • FCTRX protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin a family, are useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals.
  • FSH follicle stimulating hormone
  • the protein of the invention as a homodimer or as a heterodimer with other protein subunits of the inhibin-b group, are useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, U.S. Pat. No. 4,798,885.
  • a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for activin inhibin activity include, without limitation, those described in: Vale et al, Endocrinology 91:562-572, 1972; Ling et al, Nature 321:779-782, 1986; Vale et al, Nature 321:776-779, 1986; Mason et al, Nature 318:659-663, 1985; Forage et al, Proc Natl Acad Sci USA 83:3091-3095, 1986.
  • a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.
  • Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
  • the activity of a protein of the invention may, among other means, be measured by following methods:
  • Assays for chemotactic activity consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.
  • Suitable assays for movement and adhesion include, without limitation, those described in: CURRENT PROTOCOLS IN IMMUNOLOGY, Coligan et al, eds. (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28); Taub et al. J Clin Invest 95:1370-1376, 1995; Lind et al.
  • a protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.
  • a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al, J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al, Thrombosis Res. 45:413-419, 1987; Humphrey et al, Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988. Receptor/Ligand Activity
  • the activity of a protein of the invention may, among other means, be measured by the following methods :
  • Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. coligan, A. M. Rruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et ⁇ l, Proc N ⁇ tl Ac ⁇ d Sci USA 84:6864-6868, 1987; Bierer et ⁇ l, J. Exp. Med. 168:1145-1156, 1988; Rosenstein et /. , J. Exp. Med. 169:149-160 1989; Stoltenborg et ⁇ l, J Immunol Methods 175:59-68, 1994; Stitt et ⁇ l., Cell 80:661-670, 1995.
  • Anti-Inflammatory Activity Proteins of the present invention may also exhibit anti-inflammatory activity.
  • the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell — cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
  • Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • infection such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • ischemia-reperfusion injury such as endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting
  • a protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth
  • Example 1 Identification of murine clone 7971c.7-r0s0-212.2-EXT (SEQ ID NO:2).
  • Fig. 6 Traces for two animals are shown in Fig. 6 for each of the two sets comparing transgenic mice to wild type mice. A series of transcripts overexpressed in the transgenic mice, including one at position 212.2 (vertical marker) was found. This band was designated as 7971c.7 (SEQ ID NO:l).
  • the sequence of murine clone 7971c.7 was extended by assembly with other murine nucleic acid fragments.
  • the resulting assembly provided the sequence of clone 7971c.7-r0s0- 212.2-EXT (SEQ ID NO:2).
  • Murine clone 7971c.7 was found to have high similarity to the human EST AA315020 (SEQ ID NO:4). This sequence includes two unspecified bases designated "N" in SEQ ID NO:4. EST AA315020 was assembled with human sequences present in CuraGen Co ⁇ oration's sequence database obtained by its SeqCalling technology. SeqCalling is a differential expression and sequencing procedure that normalizes mRNA species in a sample, and is disclosed in U. S. Ser. No. 09/417,386, filed Oct. 13, 1999, inco ⁇ orated herein by reference in its entirety.
  • the quantitative expression of various clones was assessed in about 41 normal and about 55 tumor samples by real time quantitative PCR (TAQMAN ® ) performed on a Perkin- Elmer Biosystems ABI PRISM® 7700 Sequence Detection System.
  • RNA samples were normalized to ⁇ -actin and GAPDH.
  • RNA 50 ng total or ⁇ 1 ng polyA+
  • TAQMAN ® Reverse Transcription Reagents Kit PE Biosystems, Foster City, CA; Catalog No. N808-0234
  • random hexamers random hexamers according to the manufacturer's protocol. Reactions were performed in 20 ul and incubated for 30 min. at 48°C.
  • cDNA (5 ul) was then transferred to a separate plate for the TAQMAN® reaction using ⁇ -actin and GAPDH TAQMAN® Assay Reagents (PE Biosystems; Catalog Nos.
  • the average CT values obtained for ⁇ -actin and GAPDH were used to normalize RNA samples.
  • the RNA sample generating the highest CT value required no further diluting, while all other samples were diluted relative to this sample according to their ⁇ -actin GAPDH average CT values.
  • primer concentration 250 nM
  • primer melting temperature (T m ) range 58°-60° C
  • primer optimal Tm 59° C
  • maximum primer difference 2° C
  • probe does not have 5' G probe T m must be 10° C greater than primer T m , amplicon size 75 bp to 100 bp.
  • the probes and primers selected were synthesized by Synthegen (Houston, TX, USA). Probes were double purified by HPLC to remove uncoupled dye and evaluated by mass spectroscopy to verify coupling of reporter and quencher dyes to the 5' and 3' ends of the probe, respectively. Their final concentrations were: forward and reverse primers, 900 nM each, and probe, 200nM.
  • the primers and probe used are designated Probe Name Agl526 and are shown in Table 7.
  • PCR conditions Normalized RNA from each tissue and each cell line was spotted in each well of a 96 well PCR plate (Perkin Elmer Biosystems). PCR cocktails including two probes (SEQX-specific and another gene-specific probe multiplexed with the SEQX probe) were set up using IX TaqManTM PCR Master Mix for the PE Biosystems 7700, with 5 mM MgC12, dNTPs (dA, G, C, U at 1:1:1:2 ratios), 0.25 U/ml AmpliTaq GoldTM (PE Biosystems), and 0.4 U/ ⁇ l RNase inhibitor, and 0.25 U/ ⁇ l reverse transcriptase. Reverse transcription was performed at 48° C for 30 minutes followed by amplification/PCR cycles as follows: 95° C 10 min, then 40 cycles of 95° C for 15 seconds, 60° C for 1 minute. The expression results are shown in Table 8.
  • the results in Table 8 indicate that the clone of SEQ ID NO:5 is very strongly expressed in several tumor derived cell lines compared with normal tissue, especially colon tumor cells, breast tumor cells and ovarian tumor cells. This observation indicates that the clone of SEQ ID NO: 5 has a role in cell proliferation and potential utility as a blood marker to identify and/or stage tumors. It may furthermore be a target for a specific monoclonal antibody that could be used to treat various cancers, especially colon cancer, breast cancer and ovarian cancer.
  • matched margins are evaluated by two independent pathologists (the surgical pathologists and again by a pathologists at NDRI or CHTN). This analysis provides a gross histopathological assessment of tumor differentiation grade. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical stage of the patient. Additional RNA or cDNA samples were obtained from various human tissues derived from autopsies performed on elderly people or sudden death human victims. These tissue were ascertained to be free of disease and were purchased from various commercial sources such as Clontech (Palo Alto, CA), Research Genetics, and Invitrogen (Carlsbad CA).
  • RNA integrity from all samples was determined by visual assessment of agarose gel electrophoresis using 28s and 18s ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s). Integrity was also determined by verifying the absence of low molecular weight RNAs, which is indicative of degradation products. Samples were examined for genomic DNA contamination by reactions performed in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
  • RNA or cDNA isolated from various human cell lines or tissues exposed to inflammatry agents were examined in a 96 well plate (2 control wells, 94 test samples) composed of RNA or cDNA isolated from various human cell lines or tissues exposed to inflammatry agents.
  • Total RNA from normal tissues colon and lung, purchased from Stratagene (La Jolla, CA), and thymus and kidney purchased from Clontech (Palo Alto, CA)
  • Total RNA from liver tissue from cirrhosis patients and kidney RNA from Lupus patients were obtained from Biochain (Hayward, CA).
  • Intestinal tissue for RNA preparation from tissues Crohns disease and ulcerative colitis patients was obtained from the National Disease Research Interchange (NDRI) (Philadelphia, PA).
  • cytokines were used; IL-1 beta at approximately 1-5 ng/ml, TNF alpha at approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml, IL-4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml, IL-13 at approximately 5-10 ng/ml.
  • IL-1 beta at approximately 1-5 ng/ml
  • TNF alpha at approximately 5-10 ng/ml
  • IFN gamma at approximately 20-50 ng/ml
  • IL-4 at approximately 5-10 ng/ml
  • IL-9 at approximately 5-10 ng/ml
  • IL-13 at approximately 5-10 ng/ml.
  • Mononuclear cells were prepared from blood of human subjects using Ficoll.
  • LAK cells were prepared from these cells by culture in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco/Life Technologies, Rockville, MD), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco) and Interleukin 2 for 4-6 days.
  • Cells were then either activated with 10-20 ng/ml PMA and 1-2 ⁇ g/ml ionomycin, IL-12 at 5-10 ng/ml, IFN gamma at 20-50 ng/ml and IL-18 at 5-10 ng/ml for 6 hours.
  • mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco) with PHA or PWM at approximately 5 ⁇ g/ml.
  • MLR samples were obtained by taking blood from two donors, isolating the mononuclear cells using Ficoll and mixing the isolated mononuclear cells 1 :1 at a final concentration of approximately 2xl0 6 cells/ml in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol (5.5 x 10 "5 M) (Gibco), and 10 mM Hepes (Gibco).
  • the MLR was cultured and samples taken at various time points ranging from 1- 7 days for RNA preparation.
  • monocytes were isolated from mononuclear cells using CD 14 Miltenyi Beads, positive VS selection columns and a Vario Magnet as per the manufacturer's instructions. Monocytes were differentiated into dendritic cells by culture in DMEM 5% FCS (Hyclone, Logan, UT), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco), 50 ng/ml GMCSF and 5 ng/ml IL-4 for 5-7 days.
  • Macrophages were prepared by culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), 10 mM Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately 50 ng/ml.
  • Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with LPS at 100 ng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at 10 ⁇ g/ml for 6 and 12-14 hours.
  • CD4 lymphocytes, CD8 lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and a Vario Magnet as per the manufacturer's instructions.
  • CD45RA and CD45RO CD4 lymphocytes were isolated by depleting mononuclear cells of CD8, CD56, CD 14 and CD 19 cells using CD8, CD56, CD14 and CD19 Miltenyi beads and +ve selection. Then CD4RO beads were used to isolate the CD45RO CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes.
  • the isolated NK cells were cultured in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.
  • B cells were obtained from tonsils. Tonsils were cut with sterile dissecting scissors and then passed through a sieve.
  • Thl, Th2 and Trl lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), 10 mM Hepes (Gibco) and IL-2 (1 ng/ml).
  • Thl, Th2 and Trl lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti- CD95L (1 g/ml) to prevent apoptosis.
  • the Thl, Th2 and Trl lymphocytes were washed and then expanded again with IL-2 for 4-7 days. Activated Thl and Th2 lymphocytes were maintained in this way for a maximum of three cycles.
  • RNA was prepared from primary and secondary Thl, Th2 and Trl after 6 and 24 hours following the second and third activations with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures in Interleukin 2.
  • leukocyte cell lines were obtained from the ATCC (Manassas, VA):
  • EOL cells were further differentiated by culture in 0.1 mM dbcAMP at 5 xlO 5 cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to 5 xlO 5 cells/ml.
  • the cells were cultured with DMEM or RPMI (as recommended by the ATCC), with the addition of 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), 10 mM Hepes (Gibco).
  • RNA was either prepared from resting cells or cells activated with PMA at 10 ng/ml and ionomycin at 1 ⁇ g/ml for 6 and 14 hours.
  • a keratinocyte line CCD106 and an airway epithelial tumor line NCI-H292 were also obtained from the ATCC. Both were cultured in DMEM 5% FCS (Hyclone), 100 ⁇ M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10 "5 M (Gibco), and 10 mM Hepes (Gibco).
  • CCD1106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and 1 ng/ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13 and 25 ng/ml IFN gamma.
  • RNA was prepared by lysing approximately 10 7 cells/ml using Trizol (Gibco). Briefly, 1/10 volume of bromochloropropane (Molecular Research Co ⁇ oration) was added to the RNA sample, vortexed and after 10 minutes at room temperature, the tubes were spun at 14,000 ⁇ m in a Sorvall SS34 rotor. The aqueous phase was removed and placed in a 15 ml Falcon Tube. An equal volume of isopropanol was added and left at -20 degrees C overnight. The precipitated RNA was pelleted at 9,000 ⁇ ra for 15 min in a Sorvall SS34 rotor and washed in 70% ethanol.
  • Trizol Trizol
  • bromochloropropane Molecular Research Co ⁇ oration
  • inflammatory conditions can be treated or prevented by antagonizing expression or activity of a FCTRX nucleic acid or polypeptide, respectively.
  • inflammatory conditions in the lung such as asthma, as well as other lung-inflammation diseases, can be prevented or treated by inhibiting expression or activity of a FCTRX nucleic acid or polypeptide.

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Abstract

Cette invention a trait à de nouveaux polypeptides, dénommés polypeptides FCTRX, ainsi qu'à des polynucléotides codant ceux-ci et à des anticorps se fixant de manière immunospécifique à un polypeptide FCTRX ou à un dérivé, un variant, un mutant ou encore à un fragment dudit polypeptide, polynucléotide ou anticorps. L'invention qui concerne, de surcroît, des méthodes de détection et de traitement d'un large éventail d'états pathologiques, lesquelles méthodes utilisent les polypeptides FCTRX ainsi que les polynucléotides et anticorps susmentionnés, porte également sur d'autres emplois de ceux-ci.
EP00978769A 1999-11-18 2000-11-17 Polypeptide du type cytokine regule par la proteine wnt et acides nucleiques codant ce polypeptide Withdrawn EP1230370A2 (fr)

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