WO2001057087A2 - Nouvelle proteine du type sous-unite beta de recepteur de haute affinite pour l'immunoglobuline epsilon (fceb) et acides nucleiques codant pour celle-ci - Google Patents

Nouvelle proteine du type sous-unite beta de recepteur de haute affinite pour l'immunoglobuline epsilon (fceb) et acides nucleiques codant pour celle-ci Download PDF

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WO2001057087A2
WO2001057087A2 PCT/US2001/003564 US0103564W WO0157087A2 WO 2001057087 A2 WO2001057087 A2 WO 2001057087A2 US 0103564 W US0103564 W US 0103564W WO 0157087 A2 WO0157087 A2 WO 0157087A2
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fceb
polypeptide
nucleic acid
protein
amino acid
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PCT/US2001/003564
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WO2001057087A3 (fr
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Debasish Raha
John Carruli
Richard L. Cates
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Curagen Corporation
Biogen, Inc.
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Priority to AU2001234797A priority Critical patent/AU2001234797A1/en
Publication of WO2001057087A2 publication Critical patent/WO2001057087A2/fr
Publication of WO2001057087A3 publication Critical patent/WO2001057087A3/fr

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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 invention relates to novel high affinity immunoglobulin epsilon receptor beta- subunit polynucleotides and polypeptides, as well as vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides.
  • Immunoglobulin E is a major mediator of immediate hypersensitivity reactions that elicit atopic conditions such as urticaria, seasonal allergy, asthma and anaphylaxis.
  • IgE Factors that regulate IgE production are essential to understanding the pathogenesis of these disorders.
  • FcRI This high affinity receptor for immunoglobulin E is designated FcRI and is the member of the antigen (Ag) receptor superfamily which share conserved structural features and functions in initiating intracellular signalling cascades.
  • the FcRI is a tetrameric hetero-oligomer composed of an alpha chain, a beta chain, and two identical disulfide-lin ed gamma chains.
  • the beta chain contains four transmembrane (TM) segments and long cytoplasmic domains.
  • TM transmembrane
  • IgE-mediated antigen presentation Multivalent antigens bind and crosslink IgE molecules held at the cell surface by FcRI. Receptor aggregation induces multiple signalling pathways that control diverse effector responses, including the secretion of allergic mediators and induction of cytokine gene transcription, resulting in secretion of molecules such as mterleukin-4, interleukin-6, tumour-necrosis factor- and granulocyte-macrophage colony- stimulating factor.
  • the invention is based, in part, upon the discovery of a novel polynucleotide sequence encoding a novel high affinity immunoglobulin epsilon receptor beta-subunit-like protein (FCEB).
  • FCEB immunoglobulin epsilon receptor beta-subunit-like protein
  • the invention provides an isolated nucleic acid molecule that includes the sequence of SEQ ID NO:l, or a fragment, homolog, analog or derivative thereof.
  • the nucleic acid can include, e.g., a nucleic acid sequence encoding a polypeptide at least 85% identical to a polypeptide that includes the amino acid sequences of SEQ ID NO:2.
  • the nucleic acid can be, e.g., a genomic DNA fragment, or a cDNA molecule.
  • Also included in the mvention is a vector containing one or more of the nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein.
  • the invention is also directed to host cells transformed with a vector comprising any of the nucleic acid molecules described above.
  • the invention includes a pharmaceutical composition that includes an FCEB nucleic acid and a pharmaceutically acceptable carrier or diluent.
  • the mvention includes a substantially purified FCEB polypeptide, e.g., any of the FCEB polypeptides encoded by an FCEB nucleic acid, and fragments, homologs, analogs, and derivatives thereof.
  • the invention also includes a pharmaceutical composition that includes an FCEB polypeptide and a pharmaceutically acceptable carrier or diluent.
  • the invention provides an antibody that binds specifically to an FCEB polypeptide.
  • the antibody can be, e.g., a monoclonal or polyclonal antibody, and fragments, homologs, analogs, and derivatives thereof.
  • the invention also includes a pharmaceutical composition including FCEB antibody and a pharmaceutically acceptable carrier or diluent.
  • the invention is also directed to isolated antibodies that bind to an epitope on a polypeptide encoded by any of the nucleic acid molecules described above.
  • the invention also includes kits comprising any of the pharmaceutical compositions described above.
  • the invention further provides a method for producing an FCEB polypeptide by providing a cell containing an FCEB nucleic acid, e.g., a vector that includes an FCEB nucleic acid, and culturing the cell under conditions sufficient to express the FCEB polypeptide encoded by the nucleic acid.
  • the expressed FCEB polypeptide is then recovered from the cell.
  • the cell produces little or no endogenous FCEB polypeptide.
  • the cell can be, e.g., a prokaryotic cell or eukaryotic cell.
  • the invention is also directed to methods of identifying an FCEB polypeptide or nucleic acid in a sample by contacting the sample with a compound that specifically binds to the polypeptide or nucleic acid, and detecting complex formation, if present.
  • the invention further provides methods of identifying a compound that modulates the activity of an FCEB polypeptide by contacting an FCEB polypeptide with a compound and determining whether the FCEB polypeptide activity is modified.
  • the invention is also directed to compounds that modulate FCEB polypeptide activity identified by contacting an FCEB polypeptide with the compound and determining whether the compound modifies activity of the FCEB polypeptide, binds to the FCEB polypeptide, or binds to a nucleic acid molecule encoding an FCEB polypeptide.
  • the invention provides a method of determining the presence of or predisposition of an FCEB-associated disorder in a subject.
  • the method includes providing a sample from the subject and measuring the amount of FCEB polypeptide in the subject sample.
  • the amount of FCEB polypeptide in the subject sample is then compared to the amount of FCEB polypeptide in a control sample.
  • An alteration in the amount of FCEB polypeptide in the subject protein sample relative to the amount of FCEB polypeptide in the control protein sample indicates the subject has a tissue proliferation-associated condition.
  • a control sample is preferably taken from a matched individual, i.e., an individual of similar age, sex, or other general condition but who is not suspected of having a tissue proliferation- associated condition.
  • the control sample may be taken from the subject at a time when the subject is not suspected of having a tissue proliferation-associated disorder, hi some embodiments, the FCEB is detected using an FCEB antibody.
  • the invention provides a method of determining the presence of or predisposition of an FCEB-associated disorder in a subject. The method includes providing a nucleic acid sample, e.g., RNA or DNA, or both, from the subject and measuring the amount of the FCEB nucleic acid in the subject nucleic acid sample. The amount of FCEB nucleic acid sample in the subject nucleic acid is then compared to the amount of an FCEB nucleic acid in a control sample. An alteration in the amount of FCEB nucleic acid in the sample relative to the amount of FCEB in the control sample indicates the subject has a tissue proliferation-associated disorder.
  • a nucleic acid sample e.g., RNA or DNA, or both
  • the invention provides a method of treating or preventing or delaying an FCEB-associated disorder.
  • the method includes administering to a subject in which such treatment or prevention or delay is desired an FCEB nucleic acid, an FCEB polypeptide, or an FCEB antibody in an amount sufficient to treat, prevent, or delay a tissue proliferation-associated disorder in the subject.
  • the invention is based in part on the discovery of a novel nucleic acid sequence encoding a polypeptide having an amino acid sequence with significant similarities to the high affinity immunoglobulin epsilon receptor beta-subunit.
  • the nucleic acid sequences and polypeptides of the present invention are herein referred to as FCEB. Included in the invention is a 1268 nucleotide sequence encoding a novel high affinity immunoglobulin epsilon receptor beta-subunit like polypeptide. (see Table 1; SEQ ID NO:l).
  • the amino acid sequences of the encoded polypeptide is shown in Table 2 (SEQ ID NO:2).
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotide 144, and ending with a TAA codon at nucleotide 875.
  • the putative untranslated region upstream from the initiation codon is underlined in Table 1, and the start and stop codons are illustrated in bold.
  • the predicted open reading frame codes for a 244 amino acid long protein in which the initiation Met codon is preceded by Kozak sequence.
  • FCEB polypepeptide sequence is homologous to the high affinity immunoglobulin epsilon beta- subunit family of proteins.
  • the high affinity immunoglobulin epsilon beta-subunit family of proteins belong to the superfamily of four transmembrane proteins.
  • Four transmembrane proteins are characterized as having four transmembrane spanning helices with both N- and C- terminal domains located intracellularly.
  • FCEB nucleic acid sequence has 300 of 505 bases (59 %) identical, to a Rattus norvegicus IgE Fc receptor beta-subunit mRNA (GENBA -ID: M22923) Additionally, the FCEB nucleic acid sequence has significant nucleic acid idenity to other four transmembrane proteins. For example, the FCEB nucleic acid sequence has 350 of 379 (92%) identical, to Mus musculus membrane-spanning 4-domains (GenBank Ace. No. NM 022421.1), 350 of 379 (92%) identical to Mus musculus mRNA for MS4A11 (Gen Bank Ace. No.
  • FCEB polypeptide sequence has 64 of 207 amino acid residues (30%) identical to, and 102 of 207 residues (49 %) positive with, the 214 amino acid residue human high affimty immunoglobulin epsilon receptor beta subunit (SPTREMBL-ACC:G561639).
  • FCEB 4 2 KI ⁇ I ⁇ lS cg ⁇ v[ ⁇ L@l[JfiA ⁇ PSNV
  • FCEB 218 PHSSJS
  • FCEB 244 (SEQ ID NO:
  • FCEB polypeptide sequence has significant amino acid homology to other four transmembrane proteins.
  • the FCEB polypeptide sequence has 120 of 179 (67%) identical, to Mus musculus membrane-spanning 4-domains (GenBank Ace. No. P 071878.1), and 115 of 217 (52%) identical to Homo sapiens CD20-like precursor (GenBank Ace. No. NP 071744.1).
  • the FCEB polypeptide also shares secondary and tertiary structural characteristics with other four transmembrane proteins, i.e., four transmembrane spanning helices with both N- andC-terminal domains located intracellularly.
  • PHDhtm analysis confirmed the presence of four transmembrane spanning regions within the FCEB polypeptide sequence.
  • PHDhtm is a neural network system predicting locations of transmembrane helices based on evolutionary profiles. B Rost, P Fariselli & R Casadio (1996) Protein Science, 7:1704-1718. Table 5 summarizes the locations of the four transmembrane regions as well as the intercellular regions and the extracelluar regions.
  • Kyte-Doolite Mean Hydrophobicity Profiles were generated for FECB polypeptide and two other four transmembrane proteins CD20- like precursor (GenBank Ace. No. AAG27920.1) and membrane spanning 4 domains (GenBank Ass. No BAB 18759.1).
  • a graphical illustration of the hydrophobicity profiles is shown in Table 6.
  • the FCEB polypeptide of the invention has a substantially similar hydrophobicity profile as other known four transmembrane proteins demonstrating that the FCEB polypeptide is a member of the four transmembrane protein superfamily
  • PFAM HMM Domain analysis (E-value cutoff of 0.000001) of FCEB also demonstrated homology to the high affinity immunoglobulin epsilon beta-subunit family of proteins.
  • the length and complexity of the homology between the consensus FCEB sequence have a probability of less than 7.8e "16 that the conservation between these sequences occurred by chance > alone. See, Batemean et al, 1999 Nucl. Acids Res. 27: 260-262.
  • FCEB nucleic acid, and the encoded polypeptide, according to the invention are useful in a variety of applications and contexts.
  • FCEB nucleic acids and polypeptides can be used to identify proteins that are members of the high affinity immunoglobulin epsilon beta- subunit family of proteins.
  • the FCEB nucleic acids and polypeptides can also be used to screen
  • nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., cellular activation, allergic response, apoptosis mast cell degranulation and the release of mediators such as histamine.
  • small molecules that modulate or inhibit, e.g., cellular activation, allergic response, apoptosis mast cell degranulation and the release of mediators such as histamine.
  • mediators such as histamine.
  • these molecules can be used to treat, e.g. atopic disorders such as familial asthma, hay fever and eczemza, cancers such
  • FCEB nucleic acids and polypeptides according to the invention are useful, inter alia, as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins.
  • the FCEB nucleic acids and their encoded polypeptides include structural motifs that are characteristic of proteins
  • tetraspan proteins transmembrane proteins
  • tetraspan proteins such as the high affinity immunoglobulin epsilon beta-subunit, CD20, CD9, CD63, CD81 and CD82.
  • Tetraspan proteins mediate signal transduction events that play a role in the regulation of cell development, activation, growth, adhesion differentiation and motility and cancer.
  • tetraspan proteins act as "molecular facilitators," grouping specific cell-surface proteins and thus
  • the high affinity receptor for immunoglobulin E is the member of the antigen receptor superfamily responsible for linking pathogen-or allergen-specific IgEs with cellular immunologic effector functions such as urticaria, seasonal allergy, asthma and anaphylaxis.
  • genetics studies have identified the gene for the high-affinity IgE receptor beta subunit as a 3 candidate for gene therapy for the treatment of atopy.
  • a polymorphism in high- affinity IgE receptor beta subunit has been identified suggesting it as a risk factor for such disorders as atopic dermatitis and asthma.
  • CD20 is a regulator of transmembrane Ca +2 conductance and signals intracellularly through non-receptor tyrosine kinases. CD20 is involved in B cell activation, proliferation and differentiation. The presence of CD20 has been associated with lymphoma and treatment of animals bearing primary cutaneous B-cell lymphoma tumors with anti-CD20 antibodies decreases tumor burden and results in disease stabilization.
  • CD9, CD63, CD 81 and CD82 have various functions including platelet aggregation, cellular activation and proliferation.
  • FCEB nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications in the meditation of the immune response, cancer and atopy.
  • the FCEB nucleic acids and polypeptides, antibodies and related compounds according to the invention may be used to treat immune system disorders, e.g., cancer, immunodeficiency disorders, or autoimmune diseases and various bacterial and viral infections, asthma, anaphalxis, and urticaria.
  • FCEB nucleic acids and polypeptides according to the invention are disclosed herein.
  • nucleic acids of the invention include those that encode a FCEB polypeptide or protein.
  • polypeptide and protein are interchangeable.
  • a FCEB nucleic acid encodes a mature FCEB polypeptide.
  • a "mature" form of a polypeptide or protein described herein relates to the product of a naturally occurring polypeptide or precursor form or proprotein.
  • the naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an open reading frame described herein.
  • the product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the 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, myristoylation 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.
  • FCEB nucleic acids is the nucleic acid whose sequence is provided in SEQ ID NO: 1, or a fragment thereof. Additionally, the invention includes mutant or variant nucleic acids of SEQ ID NO: 1, or a fragment thereof, any of whose bases may be changed from the corresponding bases shown in SEQ ID NO: 1, while still encoding a protein that maintains at least one of its FCEB-like activities and physiological functions (i.e., modulating angiogenesis, neuronal development). The invention further includes the complement of the nucleic acid sequence of SEQ ID NO: 1, including fragments, derivatives, analogs and homologs thereof. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
  • nucleic acid molecules that encode FCEB proteins or biologically active portions thereof. Also included are nucleic acid fragments sufficient for use as hybridization probes to identify FCEB-encoding nucleic acids (e.g., FCEB mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of FCEB nucleic acid molecules.
  • 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.
  • Probes refer to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as about, e.g., 6,000 nt, depending on use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
  • an "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid.
  • isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
  • 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.
  • the isolated FCEB nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, or a complement of any of this nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • FCEB nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al, eds., MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • oligonucleotides corresponding to FCEB nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
  • an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at lease 6 contiguous nucleotides of SEQ ID NO: 1 , or a complement thereof. Oligonucleotides may be chemically synthesized and may be used as probes.
  • 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, or a portion of this nucleotide sequence.
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO: 1 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEQ ID NO: 1, thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Von der Waals, hydrophobic interactions, etc.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO: 1, e.g., a fragment that can be used as a probe or primer, or a fragment encoding a biologically active portion of FCEB.
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.
  • Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution.
  • Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
  • Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
  • Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions.
  • a “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above.
  • Homologous nucleotide sequences encode those sequences coding for isoforms of a FCEB polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • homologous nucleotide sequences include nucleotide sequences encoding for a FCEB polypeptide of species other than humans, including, but not limited to, mammals, and thus can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
  • homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the nucleotide sequence encoding human FCEB protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO:2, as well as a polypeptide having FCEB activity. Biological activities of the FCEB proteins are described below. A homologous amino acid sequence does not encode the amino acid sequence of a human FCEB polypeptide.
  • the nucleotide sequence determined from the cloning of the human FCEB gene allows for the generation of probes and primers designed for use in identifying and/or cloning FCEB homologues in other cell types, e.g., from other tissues, as well as FCEB homologues from other mammals.
  • the probe/primer typically comprises a substantially purified oligonucleotide.
  • 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 or more consecutive sense strand nucleotide sequence of SEQ ID NO: 1; or an anti-sense strand nucleotide sequence of SEQ ID NO: 1; or of a naturally occurring mutant of SEQ ID NO: 1.
  • Probes based on the human FCEB nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a FCEB protein, such as by measuring a level of a FCEB-encoding nucleic acid in a sample of cells from a subject e.g., detecting FCEB mRNA levels or determining whether a genomic FCEB gene has been mutated or deleted.
  • a "polypeptide having a biologically active portion of FCEB” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
  • a nucleic acid fragment encoding a "biologically active portion of FCEB” can be prepared by isolating a portion of SEQ ID NO: 1 that encodes a polypeptide having a FCEB biological activity (biological activities of the FCEB proteins are described below), expressing the encoded portion of FCEB protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of FCEB.
  • a nucleic acid fragment encoding a biologically active portion of FCEB can optionally include an ATP-binding domain.
  • a nucleic acid fragment encoding a biologically active portion of FCEB includes one or more regions.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NO: 1 due to the degeneracy of the genetic code. These nucleic acids thus encode the same FCEB protein as that encoded by the nucleotide sequence shown in SEQ ID NO: 1, e.g., the polypeptide of SEQ ID NO: 2.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2.
  • FCEB DNA sequence polymorphisms that lead to changes in the amino acid sequences of FCEB may exist within a population (e.g., the human population).
  • Such genetic polymorphism in the FCEB 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 a FCEB protein, preferably a mammalian FCEB protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the FCEB gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in FCEB that are the result of natural allelic variation and that do not alter the functional activity of FCEB are intended to be within the scope of the invention.
  • nucleic acid molecules encoding FCEB proteins from other species and thus that have a nucleotide sequence that differs from the human sequence of SEQ ID NO: lare intended to be within the scope of the invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the FCEB cDNAs of the invention can be isolated based on their homology to the human FCEB nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • a soluble human FCEB cDNA can be isolated based on its homology to human membrane-bound FCEB.
  • a membrane-bound human FCEB cDNA can be isolated based on its homology to soluble human FCEB.
  • an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid is at least 10, 25, 50, 100, 250, 500 or 750 nucleotides in length.
  • an isolated nucleic acid molecule of the invention hybridizes to the coding region.
  • 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 FCEB proteins derived from species other than human
  • other related sequences e.g., paralogs
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • Stringent conditions are known to those skilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.
  • a non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C. This hybridization is followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 corresponds to a naturally occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided.
  • moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C.
  • Other conditions of moderate stringency that may be used are well known in the art.
  • a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, or fragments, analogs or derivatives thereof, under conditions of low stringency is provided.
  • a non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C.
  • allelic variants of the FCEB sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence of SEQ ID NO: 1, thereby leading to changes in the amino acid sequence of the encoded FCEB protein, without altering the functional ability of the FCEB protein.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential", amino acid residues can be made in the sequence of SEQ ID NO: 1.
  • a "non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of FCEB without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the FCEB proteins of the present invention are predicted to be particularly unamenable to alteration.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO: 2.
  • the protein encoded by the nucleic acid is at least about 80% homologous to SEQ ID NO: 2, more preferably at least about 90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO: 2.
  • An isolated nucleic acid molecule encoding a FCEB protein homologous to the protein of SEQ H) NO: 2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into the nucleotide sequence of SEQ ID NO: 1 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in FCEB is replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a FCEB coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for FCEB biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
  • a mutant FCEB protein can be assayed for (1) the ability to form protei protein interactions with other FCEB proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant FCEB protein and a FCEB receptor; (3) the ability of a mutant FCEB protein to bind to an intracellular target protein or biologically active portion thereof; (e.g., avidin proteins); (4) the ability to bind FCEB protein; or (5) the ability to specifically bind an anti-FCEB protein antibody.
  • Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, or fragments, analogs or derivatives thereof.
  • An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire FCEB coding strand, or to only a portion thereof.
  • Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a FCEB protein of SEQ ID NO: 2, or antisense nucleic acids complementary to a FCEB nucleic acid sequence of SEQ ID NO: 1 are
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding FCEB.
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of human FCEB corresponds to SEQ ID NO: 2).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding FCEB.
  • 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).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of FCEB mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of FCEB mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of FCEB mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxy
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a FCEB protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al (1987) Nucleic Acids Res 15: 6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al (1987) FEBSLett 215: 327-330).
  • modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
  • FCEB Ribozymes and PNA moieties FCEB Ribozymes and PNA moieties
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave FCEB mRNA transcripts to thereby inhibit translation of FCEB mRNA.
  • a ribozyme having specificity for a FCEB-encoding nucleic acid can be designed based upon the nucleotide sequence of a FCEB DNA disclosed herein (i.e., SEQ ID NO: 1).
  • a derivative of a Tetrahymena L-19 INS R ⁇ A can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a FCEB-encoding mR ⁇ A. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al U.S. Pat. No. 5,116,742.
  • FCEB mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al, (1993) Science 261:1411-1418.
  • FCEB gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the FCEB (e.g., the FCEB promoter and/or enhancers) to form triple helical structures that prevent transcription of the FCEB gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the FCEB e.g., the FCEB promoter and/or enhancers
  • FCEB promoter and/or enhancers e.g., the FCEB promoter and/or enhancers
  • the nucleic acids of FCEB can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) BioorgMed Chem 4: 5-23).
  • the terms "peptide nucleic acids” or "PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • PNAs The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.
  • PNAs of FCEB 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 FCEB 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., SI 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 FCEB 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 FCEB 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.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases. and orientation ( ⁇ vrut) (1996) above .
  • the svnthesis of PNA-DNA chimeras can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA,
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al, 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al, 1987, Proc.
  • oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al, 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549).
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
  • a FCEB polypeptide of the invention includes the FCEB-like protein whose sequence is provided in SEQ ID NO: 2.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in SEQ ID NO: 2 while still encoding a protein that maintains its FCEB-like activities and physiological functions, or a functional fragment thereof. In some embodiments, up to 20% or more of the residues may be so changed in the mutant or variant protein.
  • the FCEB polypeptide according to the invention is a mature polypeptide.
  • a FCEB -like variant that preserves FCEB-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence.
  • Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
  • FCEB proteins and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof.
  • polypeptide fragments suitable for use as immunogens to raise anti-FCEB antibodies are provided.
  • native FCEB proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • FCEB proteins are produced by recombinant DNA techniques.
  • a FCEB protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the FCEB protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of FCEB protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of FCEB protein having less than about 30% (by dry weight) of non-FCEB protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-FCEB protein, still more preferably less than about 10% of non-FCEB protein, and most preferably less than about 5% non-FCEB protein.
  • non-FCEB protein also referred to herein as a "contaminating protein”
  • the FCEB protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%o, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of FCEB protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of FCEB protein having less than about 30% (by dry weight) of chemical precursors or non-FCEB chemicals, more preferably less than about 20% chemical precursors or non-FCEB chemicals, still more preferably less than about 10% chemical precursors or non-FCEB chemicals, and most preferably less than about 5% chemical precursors or non-FCEB chemicals.
  • Biologically active portions of a FCEB protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the FCEB protein, e.g., the amino acid sequence shown in SEQ ID NO: 2 that include fewer amino acids than the full length FCEB proteins, and exhibit at least one activity of a FCEB protein.
  • biologically active portions comprise a domain or motif with at least one activity of the FCEB protein.
  • a biologically active portion of a FCEB protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • a biologically active portion of a FCEB protein of the present invention may contain at least one of the above-identified domains conserved between the FCEB proteins, e.g. TSR modules.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native FCEB protein.
  • the FCEB protein has an amino acid sequence shown in SEQ ID NO: 2.
  • the FCEB protein is substantially homologous to SEQ ID NO: 2 and retains the functional activity of the protein of SEQ ID NO: 2, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below.
  • the FCEB protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO: 2 and retains the functional activity of the FCEB proteins of SEQ ID NO: 2.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in either of the sequences being compared for optimal alignment between the sequences).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the nucleic acid sequence homology may be determined as the degree of identity between two sequences.
  • the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch 1970 J Mol Biol 48: 443-453.
  • GAP software provided in the GCG program package. See, Needleman and Wunsch 1970 J Mol Biol 48: 443-453.
  • GAP creation penalty of 5.0 and GAP extension penalty of 0.3 the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NO: 1.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • 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.
  • percentage of positive residues is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical and conservative amino acid substitutions, as defined above, occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of positive residues.
  • FCEB polypeptide refers to a polypeptide having an amino acid sequence corresponding to FCEB
  • non-FCEB polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the FCEB protein, e.g., a protein that is different from the FCEB protein and that is derived from the same or a different organism.
  • FCEB polypeptide can correspond to all or a portion of a FCEB protein.
  • a FCEB fusion protein comprises at least one biologically active portion of a FCEB protein. In another embodiment, a FCEB fusion protein comprises at least two biologically active portions of a FCEB protein.
  • the term "operatively linked" is intended to indicate that the FCEB polypeptide and the non-FCEB polypeptide are fused in-frame to each other. The non-FCEB polypeptide can be fused to the N-terminus or C-terminus of the FCEB polypeptide.
  • a FCEB fusion protein comprises a FCEB polypeptide operably linked to the extracellular domain of a second protein.
  • FCEB polypeptide operably linked to the extracellular domain of a second protein.
  • Such fusion proteins can be further utilized in screening assays for compounds that modulate FCEB activity (such assays are described in detail below).
  • the fusion protein is a GST-FCEB fusion protein in which the FCEB sequences are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.
  • FCEB sequences are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.
  • fusion proteins can facilitate the purification of recombinant FCEB.
  • the fusion protein is a FCEB-immunoglobulin fusion protein in which the FCEB sequences comprising one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.
  • the FCEB-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a FCEB ligand and a FCEB protein on the surface of a cell, to thereby suppress FCEB-mediated signal transduction in vivo.
  • a contemplated FCEB ligand of the invention is the FCEB receptor.
  • the FCEB-immunoglobulin fusion proteins can be used to affect the bioavailability of a FCEB cognate ligand.
  • FCEB-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-FCEB antibodies in a subject, to purify FCEB ligands, and in screening assays to identify molecules that inhibit the interaction of FCEB with a FCEB ligand.
  • a FCEB chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, 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.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a FCEB-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the FCEB protein.
  • the present invention also pertains to variants of the FCEB proteins that function as either FCEB agonists (mimetics) or as FCEB antagonists.
  • Variants of the FCEB protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the FCEB protein.
  • An agonist of the FCEB protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the FCEB protein.
  • An antagonist of the FCEB protein can inhibit one or more of the activities of the naturally occurring form of the FCEB protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the FCEB protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • 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 FCEB proteins.
  • Variants of the FCEB protein that function as either FCEB agonists (mimetics) or as FCEB antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the FCEB protein for FCEB protein agonist or antagonist activity.
  • a variegated library of FCEB variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of FCEB variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential FCEB sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of FCEB sequences therein.
  • a degenerate set of potential FCEB sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of FCEB sequences therein.
  • methods which can be used to produce libraries of potential FCEB variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential FCEB 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 FCEB protein coding sequence can be used to generate a variegated population of FCEB fragments for screening and subsequent selection of variants of a FCEB protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a FCEB 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 SI 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 FCEB protein.
  • Recrusive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify FCEB variants (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).
  • antibodies to FCEB proteins are also included in the invention.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (lg) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin (lg) molecules i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F ab , F ab , and F (ab , )2 fragments, and an F ab expression library.
  • an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG l5 IgG 2 , and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
  • FCEB-related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.
  • An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NO: 2, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.
  • the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
  • At least one epitope encompassed by the antigenic peptide is a region of FCEB-related protein that is located on the surface of the protein, e.g., a hydrophilic region.
  • a hydrophobicity analysis of the human FCEB-related protein sequence will indicate which regions of a FCEB-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
  • hydropathy plots showing regions of hydrophihcity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat.
  • a protein of the invention may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • an appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.
  • the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by inrmunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • the antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen- binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature.
  • Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.. 222:581 (1991)).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
  • nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
  • the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
  • U.S. Patent No. 5,939,598 An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
  • a method for producing an antibody of interest such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778).
  • methods 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 a protein or derivatives, fragments, analogs or homologs thereof.
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, 1991 EMBOJ., 10:3655-3659.
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.
  • Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab') 2 molecule.
  • Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the N H and N L domains of one fragment are forced to pair with the complementary N L and N H domains of another fragment, thereby forming two antigen-binding sites.
  • V H heavy-chain variable domain
  • V L light-chain variable domain
  • Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIN infection (WO 91/00360; WO 92/200373; EP 03089).
  • the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crossliriking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992).
  • Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
  • a ricin immunotoxin can be prepared as described in Vitetta et al, Science, 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the antibody in another embodiment, can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
  • a "receptor” such streptavidin
  • a "ligand” e.g., avidin
  • vectors preferably expression vectors, containing a nucleic acid encoding a FCEB 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.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • 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.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., FCEB proteins, mutant forms of FCEB proteins, fusion proteins, etc.).
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 61: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N. J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • FCEB can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufinan, et al, 1987. EMBOJ. 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • promoters are also encompassed, e.g., the murine hox promoters (Kessel and Grass, 1990. Science 249: 374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to FCEB mRNA.
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • FCEB protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as human, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding FCEB or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) FCEB protein.
  • the invention further provides methods for producing FCEB protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding FCEB protein has been introduced) in a suitable medium such that FCEB protein is produced.
  • the method further comprises isolating FCEB protein 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.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous FCEB gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing FCEB-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Sequences including SEQ ID NO: 1 can be introduced as a transgene into the genome of a non-human animal.
  • a non-human homologue of the human FCEB gene such as a mouse FCEB gene, can be isolated based on hybridization to the human FCEB cDNA (described further supra) and used as a transgene.
  • a transgenic founder animal can be identified based upon the presence of the FCEB transgene in its genome and/or expression of FCEB mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding FCEB protein can further be bred to other transgenic animals carrying other transgenes.
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras.
  • an animal e.g., a mouse
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene.
  • transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI.
  • cre/loxP recombinase system See, e.g., Lakso, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236.
  • FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al, 1991. Science 251:1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • 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.
  • the antibodies disclosed herein can also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al ., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.
  • a chemotherapeutic agent such as Doxorubicin is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst, 81(19): 1484 (1989).
  • 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, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), 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, tbimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a FCEB protein or anti-FCEB antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a FCEB protein or anti-FCEB antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • 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.
  • Systemic administration can also be by fransmucosal or transdermal means.
  • penevers appropriate to the barrier to be permeated are used in the formulation.
  • Such penevers are generally known in the art, and include, for example, for fransmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form ofsuppositori.es (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositori e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Natl. Acad. Sci. USA 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.
  • Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions.
  • Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington : The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa. : 1995; Drag Absorption Enhancement : Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M.
  • the formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth- inhibitory agent.
  • cytotoxic agent such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth- inhibitory agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules
  • sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the isolated nucleic acid molecules of the invention can be used to express FCEB protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect FCEB mRNA (e.g., in a biological sample) or a genetic lesion in a FCEB gene, and to modulate FCEB activity, as described further, below.
  • the FCEB proteins can be used to screen drugs or compounds that modulate the FCEB protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of FCEB protein or production of FCEB protein forms that have decreased or aberrant activity compared to FCEB wild-type protein .
  • the anti-FCEB antibodies of the invention can be used to detect and isolate FCEB proteins and modulate FCEB activity.
  • FCEB activity includes cell activation, proliferation and tumor growth.
  • the invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.
  • 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 FCEB proteins or have a stimulatory or inhibitory effect on, e.g., FCEB protein expression or FCEB protein activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to FCEB proteins or have a stimulatory or inhibitory effect on, e.g., FCEB protein expression or FCEB protein activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to FCEB proteins or have a stimulatory or inhibitory effect on, e.g., FCEB protein expression or FCEB protein activity.
  • the invention also
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a FCEB protein or polypeptide or biologically-active portion thereof.
  • the test compounds of the invention can be obtained 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.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997 '. Anticancer Drug Design 12: 145.
  • a "small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD.
  • Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules.
  • Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
  • an assay is a cell-based assay in which a cell which expresses a membrane-bound form of FCEB protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a FCEB protein determined.
  • the cell for example, can be of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the FCEB 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 FCEB 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.
  • test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting a cell which expresses a membrane-bound form of FCEB protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds FCEB to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a FCEB protein, wherein determining the ability of the test compound to interact with a FCEB protein comprises determining the ability of the test compound to preferentially bind to FCEB protein or a biologically-active portion thereof as compared to the known compound.
  • an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of FCEB protein, or a biologically-active portion thereof, on the cell surface with a test compound and dete ⁇ nining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the FCEB protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of FCEB or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the FCEB protein to bind to or interact with a FCEB target molecule.
  • a "target molecule” is a molecule with which a FCEB protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a FCEB interacting 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.
  • a FCEB target molecule can be a non-FCEB molecule or a FCEB protein or polypeptide of the invention
  • a FCEB 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 FCEB.
  • Determining the ability of the FCEB protein to bind to or interact with a FCEB target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the FCEB protein to bind to or interact with a FCEB 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.
  • an assay of the invention is a cell-free assay comprising contacting a FCEB protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the FCEB protein or biologically-active portion thereof. Binding of the test compound to the FCEB protein can be determined either directly or indirectly as described above.
  • the assay comprises contacting the FCEB protein or biologically-active portion thereof with a known compound which binds FCEB to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a FCEB protein, wherein determining the ability of the test compound to interact with a FCEB protein comprises determining the ability of the test compound to preferentially bind to FCEB or biologically- active portion thereof as compared to the known compound.
  • an assay is a cell-free assay comprising contacting FCEB 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 FCEB protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of FCEB can be accomplished, for example, by determining the ability of the FCEB protein to bind to a FCEB 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 FCEB protein can be accomplished by determining the ability of the FCEB protein further modulate a FCEB target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described above.
  • the cell-free assay comprises contacting the FCEB protein or biologically-active portion thereof with a known compound which binds FCEB protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a FCEB protein, wherein determining the ability of the test compound to interact with a FCEB protein comprises determining the ability of the FCEB protein to preferentially bind to or modulate the activity of a FCEB target molecule.
  • the cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of FCEB protein.
  • FCEB protein or its target molecule it may be desirable to immobilize either FCEB protein 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 FCEB protein, or interaction of FCEB protein 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-FCEB 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 FCEB 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, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of FCEB protein binding or activity determined using standard techniques.
  • FCEB protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated FCEB protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with FCEB protein or target molecules can be derivatized to the wells of the plate, and unbound target or FCEB protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the FCEB protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the FCEB protein or target molecule.
  • modulators of FCEB protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of FCEB mRNA or protein in the cell is determined. The level of expression of FCEB mRNA or protein in the presence of the candidate compound is compared to the level of expression of FCEB mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of FCEB mRNA or protein expression based upon this comparison. For example, when expression of FCEB mRNA or protein is greater (i.e., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of FCEB mRNA or protein expression.
  • FCEB mRNA or protein when expression of FCEB 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 FCEB mRNA or protein expression.
  • the level of FCEB mRNA or protein expression in the cells can be determined by methods described herein for detecting FCEB mRNA or protein.
  • the FCEB proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent 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, 1993.
  • FCEB-binding proteins proteins that bind to or interact with FCEB
  • FCEB-bp proteins that bind to or interact with FCEB
  • FCEB-binding proteins are also likely to be involved in the propagation of signals by the FCEB proteins as, for example, upstream or downstream elements of the FCEB pathway.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity 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 FCEB.
  • a reporter gene e.g., LacZ
  • the invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents.
  • these sequences can be used to: (i) identify an individual from a minute biological sample (tissue typing); and (ii) aid in forensic identification of a biological sample.
  • sequences of the 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 FCEB sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of conesponding 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 invention can be used to obtain such identification sequences from individuals and from tissue.
  • the FCEB 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.
  • the noncoding sequences 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 those in SEQ ID NO: 1 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • disorders associated with aberrant FCEB expression of activity include, for example, cancer, immunodeficiency disorders, or autoimmune diseases and various bacterial and viral infections, asthma, anaphalxis, and urticaria.
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with FCEB protein, nucleic acid expression or activity. For example, mutations in a FCEB 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 FCEB protein, nucleic acid expression, or biological activity.
  • Another aspect of the invention provides methods for determining FCEB protein, nucleic acid expression or 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., drags) 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., drags, compounds) on the expression or activity of FCEB in clinical trials.
  • agents e.g., drags, compounds
  • the nucleic acid probe can be, for example, a full-length FCEB nucleic acid, such as the nucleic acid of SEQ ID NO: 1, 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 FCEB mRNA or genomic DNA.
  • FCEB nucleic acid probes for use in the diagnostic assays of the invention are described herein.
  • One agent for detecting FCEB protein is an antibody capable of binding to FCEB protein, preferably an antibody with a detectable label.
  • Antibodies directed against a protein of the invention may be used in methods known within the art relating to the localization and/or quantitation of the protein (e.g., for use in measuring levels of the protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • antibodies against the proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antigen binding domain are utilized as pharmacologically-active compounds.
  • An antibody specific for a protein of the invention can be used to isolate the protein by standard techniques, such as inimunoaffinity chromatography or immunoprecipitation. Such an antibody can facilitate the purification of the natural protein antigen from cells and of recombinantly produced antigen expressed in host cells. Moreover, such an antibody can be used to detect the antigenic protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the antigenic protein. Antibodies directed against the protein 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.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamme fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 1,
  • 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 FCEB mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of FCEB mRNA include Northern hybridizations and in situ hybridizations.
  • in vitro techniques for detection of FCEB protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
  • In vitro techniques for detection of FCEB genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of FCEB protein include introducing into a subject a labeled anti-FCEB 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.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting FCEB protein, mRNA, or genomic DNA, such that the presence of FCEB protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of FCEB protein, mRNA or genomic DNA in the control sample with the presence of FCEB protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of FCEB in a biological sample can comprise: a labeled compound or agent capable of detecting FCEB protein or mRNA in a biological sample; means for determining the amount of FCEB in the sample; and means for comparing the amount of FCEB 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 FCEB 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 FCEB 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 FCEB protein, nucleic acid expression or activity.
  • disorders include for example, cancer, immunodeficiency disorders, or autoimmune diseases and various bacterial and viral infections, asthma, anaphalxis, and urticaria.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder.
  • the invention provides a method for identifying a disease or disorder associated with aberrant FCEB expression or activity in which a test sample is obtained from a subject and FCEB protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of FCEB protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant FCEB expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
  • 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 abenant FCEB 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
  • the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant FCEB expression or activity in which a test sample is obtained and FCEB protein or nucleic acid is detected (e.g., wherein the presence of FCEB protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant FCEB expression or activity).
  • the methods of the invention can also be used to detect genetic lesions in a FCEB gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and or differentiation.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a FCEB-protein, or the misexpression of the FCEB gene.
  • such genetic lesions can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from a FCEB gene; (ii) an addition of one or more nucleotides to.
  • a prefereed 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. Patent 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. Proc. Natl. Acad. Sci. USA 91 : 360-364), the latter of which can be particularly useful for detecting point mutations in the FCEB-gene (see, Abravaya, et al, 1995. Nucl.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al, 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Q ⁇ Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a FCEB gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • 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 indicates mutations in the sample DNA.
  • sequence specific ribozymes see, e.g., U.S. Patent No. 5,493,531
  • sequence specific ribozymes can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the FCEB gene and detect mutations by comparing the sequence of the sample FCEB with the coreesponding wild-type (confrol) sequence.
  • sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al, 1995.
  • Biotechniques 19: 448 including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen, et al, 1996. Adv. Chromatography 36: 127-162; and Griffin, et al, 1993. Appl Biochem. Biotechnol 38: 147-159).
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the FCEB gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, e.g., 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 FCEB 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.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S x nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al, 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al, 1992. Methods Enzymol 217: 286-295.
  • control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in FCEB cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g., Hsu, et al, 1994. Carcinogenesis 15: 1657-1662.
  • a probe based on a FCEB sequence e.g., a wild-type FCEB sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in FCEB genes.
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA fragments of sample and control FCEB 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.
  • RNA rather than DNA
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al, 1991. Trends Genet. 7: 5.
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al, 1986. Nature 324: 163; Saiki, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 6230.
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • 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; see, e.g., Gibbs, et al, 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11 : 238).
  • 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 a FCEB gene.
  • any cell type or tissue preferably peripheral blood leukocytes, in which FCEB 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.
  • 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 FCEB protein, expression of FCEB nucleic acid, or mutation content of FCEB 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 drags due to altered drag disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. din. Exp. Pharmacol. Physiol, 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drag action) or genetic conditions transmitted as single factors altering the way the body acts on drags (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms.
  • FCEB protein activity of FCEB protein, expression of FCEB nucleic acid, or mutation content of FCEB 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 polymorphic alleles encoding drag-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drag selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a FCEB modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • FCEB e.g., the ability to modulate abenant cell proliferation
  • agents e.g., drags, compounds
  • FCEB e.g., the ability to modulate abenant cell proliferation
  • the effectiveness of an agent determined by a screening assay as described herein to increase FCEB gene expression, protein levels, or upregulate FCEB activity can be monitored in clinical trails of subjects exhibiting decreased FCEB gene expression, protein levels, or downregulated FCEB activity.
  • the effectiveness of an agent determined by a screening assay to decrease FCEB gene expression, protein levels, or downregulate FCEB activity can be monitored in clinical trails of subjects exhibiting increased FCEB gene expression, protein levels, or upregulated FCEB activity.
  • FCEB and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell.
  • genes, including FCEB that are modulated in cells by treatment with an agent (e.g., compound, drag or small molecule) that modulates FCEB activity (e.g., identified in a screening assay as described herein) can be identified.
  • an agent e.g., compound, drag or small molecule
  • FCEB 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 FCEB 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 FCEB 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, freatment of the individual with the agent.
  • the 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 drag 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 a FCEB protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the FCEB protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the FCEB protein, mRNA, or genomic DNA in the pre-administration sample with the FCEB 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., an agonist
  • the 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 abenant FCEB expression or activity.
  • Disorders associated with abenant FCEB expression of activity include for example, cancer, e.g., hairy cell leukemia, multiple myeloma, Kaposi's sarcoma, various lymphomas, renal cell carcinoma, automimmune diseases, e.g. , cancer, immunodeficiency disorders, or autoimmune diseases and various bacterial and viral infections, asthma, anaphalxis, and urticaria. These methods of freatment will be discussed more fully, below.
  • Therapeutics that antagonize activity 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
  • Therapeutics that increase (i.e., axe agonists to) activity may be administered in a therapeutic or prophylactic manner.
  • Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
  • Increased or decreased levels can be readily detected by quantifying peptide and/or 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).
  • tissue sample e.g., from biopsy tissue
  • assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide).
  • the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of FCEB protein activity associated with the cell.
  • An agent that modulates FCEB protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a FCEB protein, a peptide, a FCEB peptidomimetic, or other small molecule.
  • the agent stimulates one or more FCEB protein activity.
  • 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., up-regulates or down-regulates) FCEB expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering a FCEB protein or nucleic acid molecule as therapy to compensate for reduced or abenant FCEB expression or activity.
  • FCEB activity is desirable in situations in which FCEB is abnormally downregulated and/or in which increased FCEB activity is likely to have a beneficial effect.
  • a subject has a disorder characterized by abenant cell proliferation and/or differentiation (e.g., cancer or immune associated ).
  • a subject has an immunodeficiency disease (e.g., AIDS).
  • Antibodies of the invention may be used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology in a subject.
  • An antibody preparation preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target.
  • Such an effect may be one of two kinds, depending on the specific nature of the interaction between the given antibody molecule and the target antigen in question.
  • administration of the antibody may abrogate or inhibit the binding of the target with an endogenous ligand to which it naturally binds.
  • the antibody binds to the target and masks a binding site of the naturally occurring ligand, wherein the ligand serves as an effector molecule.
  • the receptor mediates a signal transduction pathway for which ligand is responsible.
  • the effect may be one in which the antibody elicits a physiological result by virtue of binding to an effector binding site on the target molecule.
  • the target a receptor having an endogenous ligand which may be absent or defective in the disease or pathology, binds the antibody as a sunogate effector ligand, initiating a receptor-based signal transduction event by the receptor.
  • a therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, and in other cases, promotes a physiological response.
  • the amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered.
  • Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.
  • suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its admimstration 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 system known in the art may be used prior to admimstration to human subjects.

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Abstract

L'invention concerne de nouveaux polynucléotides de FCEB isolés et des polypeptides codés par les polynucléotides de FCEB. L'invention concerne aussi les anticorps qui se lient spécifiquement, d'un point de vue immunologique, à un polypeptide de FCEB et tout dérivé, variant, mutant ou fragment du polypeptide, du polynucléotide ou de l'anticorps de FCEB. L'invention concerne de plus des procédés dans lesquels un polypeptide, un polynucléotide et un anticorps de FCEB sont utilisés pour détecter et traiter une large gamme d'états pathologiques, et pour d'autres applications.
PCT/US2001/003564 2000-02-02 2001-02-02 Nouvelle proteine du type sous-unite beta de recepteur de haute affinite pour l'immunoglobuline epsilon (fceb) et acides nucleiques codant pour celle-ci WO2001057087A2 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005481A1 (fr) * 1993-08-18 1995-02-23 Isis Innovation Limited Procede de diagnostic et therapie
US5807988A (en) * 1992-04-16 1998-09-15 The United States Of America As Represented By The Department Of Health And Human Services Isolation, characterization, and use of the human and subunit of the high affinity receptor for immunoglobulin E
WO1999062550A1 (fr) * 1998-06-04 1999-12-09 Michael Caplan Therapie anti-allergique pan-specifique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5807988A (en) * 1992-04-16 1998-09-15 The United States Of America As Represented By The Department Of Health And Human Services Isolation, characterization, and use of the human and subunit of the high affinity receptor for immunoglobulin E
WO1995005481A1 (fr) * 1993-08-18 1995-02-23 Isis Innovation Limited Procede de diagnostic et therapie
WO1999062550A1 (fr) * 1998-06-04 1999-12-09 Michael Caplan Therapie anti-allergique pan-specifique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] Accesssion Number: AI391038, 4 February 1999 (1999-02-04) MARRA ET AL.: "High affinity immunoglobulin epsilon receptor bet-subunit" XP002177887 *
SUTTON B J ET AL: "The human IgE network" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 366, 2 December 1993 (1993-12-02), pages 421-428, XP000421323 ISSN: 0028-0836 *

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